Biotechnology of Anti-Diabetic Medicinal Plants: Saikat Gantait Sandeep Kumar Verma Amit Baran Sharangi Editors

Saikat Gantait
Sandeep Kumar Verma

Amit Baran Sharangi Editors
Biotechnology
of Anti-diabetic
Medicinal Plants
Biotechnology of Anti-diabetic Medicinal
Plants
Saikat Gantait • Sandeep Kumar Verma •
Amit Baran Sharangi
Editors
Biotechnology of
Anti-diabetic Medicinal
Plants
Editors
Saikat Gantait Sandeep Kumar Verma
Crop Research Unit (Genetics and Plant Institute of Biological Science
Breeding) SAGE University
Bidhan Chandra Krishi Viswavidyalaya Indore, Madhya Pradesh, India
Mohanpur, West Bengal, India
Amit Baran Sharangi

Plantation Spices Medicinal & Aromatic
Crops
Bidhan Chandra Krishi Viswavidyalaya
Mohanpur, West Bengal, India
ISBN 978-981-16-3528-1 ISBN 978-981-16-3529-8 (eBook)

https://doi.org/10.1007/978-981-16-3529-8
# The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Singapore
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Foreword
Medicinal plants contain active phyto-pharmaceuticals aiding to cure many

disorders and diseases and are used to relieve pain and discomfort since ancient
times. The use of phytochemicals from medicinal plants as therapeutic agents is
widely acclaimed in many of the developing and the least developed countries for
the maintenance of good health and wellness. The World Health Organization
(WHO) estimated that 80% of the population of developing countries rely on
traditional folklore medicines, mostly plant drugs, for their primary health care
needs. New lifestyle diseases like diabetes, obesity and cardiovascular defects are
emerging as threat to human health and economic comfort. Demand for medicinal
plants having anti-diabetic properties is increasing in both developing and developed
countries. Surprisingly, bulk of the herbal materials harvested and traded is still from
wild sources like forests and tribal lands, and only a few species are cultivated. The
expanding trade in medicinal plants has serious implications on the survival of
several plant species, with many under threat to become extinct. There is a great
need to conserve medicinal plants as they contain highly bioactive components
which can be developed into pharmacologically active compounds. Biotechnologi-
cal advances—rapid multiplication, in vitro conservation, gene editing and use of
omics—permit us to tap into genetic resources of medicinal plants to fulfil health and
nutritional security in the face of environmental degradation, urbanization, popula-
tion growth and climate change. Biotechnology is the use of living systems and
organisms to develop or make useful products or “any technological application that
v
vi Foreword
uses biological systems, living organisms or derivatives thereof, to make or modify

products or processes for specific use”. The use of controlled environments can
overcome difficulties in cultivation and could be a means to manipulate phenotypic
variation and expression of bioactive compounds. In vitro techniques have found
increasing uses in the conservation of threatened plants in recent years as well as
enhancing the active ingredients, and this trend is likely to continue as more species
face risk of extinction.
The present book Biotechnology of Anti-diabetic Medicinal Plants is an excellent
compilation related to new insights into medicinal plants of anti-diabetic properties
with respect to in vitro culture techniques, metabolites engineering, gene-mediated
genetic transformation of anti-diabetic plants, tissue culture-mediated biotechnolog-
ical intervention, biotechnological improvement strategies, bioactive secondary
metabolites, cryopreservation biotechnology, artificial seed development, etc. The
book is expected to meet the long-felt demand for a compilation dealing with
biotechnological advances in medicinal plants towards a holistic understanding of
anti-diabetic properties, uses and practical applications. The editors Dr. Saikat
Gantait, Dr. Sandeep Kumar Verma and Dr. Amit Baran Sharangi are well-known
scientists and respected professors in the area of biotechnology with specialization in
medicinal plants. In this book, 15 learned chapters are contributed by established
working scientists from India, Turkey, Poland, Italy, Cuba, Belgium, Pakistan,
France, Morocco, United Kingdom and Thailand. The publisher Springer Nature is
to be appreciated for publishing this book of high demand and scientific content.
Kerala Agricultural University K. V. Peter

Thrissur, Kerala, India
06 December 2020
Preface
Biotechnology is technology based on biology. It harnesses cellular and biomolecu-

lar processes to develop technologies or products that can be translated for human
welfare. The anti-diabetic drugs from plants and their herbal components are pre-
ferred mainly due to lesser side effects and low cost. This book provides a unique
overview of anti-diabetic medicinal plant biotechnology, describing the whole
spectrum of biotechnological tools from micropropagation towards large-scale mul-
tiplication, cell culture techniques, to the biosynthesis and enhancement of pharma-
ceutical compounds in plants, genetic transformation as well as short- to long-term
conservation of plant genetic resources. Enriched with potential contributors across
the globe, this volume will satisfy the long-felt need of this kind of unique, pertinent
and much awaited topic to be useful for researchers in the pharmaceutical and
biotechnological industries, medicinal chemists, biochemists, botanists, molecular
biologists, academicians and students.
We would like to convey our deep appreciation to all the contributors and well-
wishers, especially Prof. K V Peter, Former Vice Chancellor, Kerala Agricultural
University, and Former Director, Indian Institute of Spices Research, who has
penned the foreword for the book. Springer Nature, USA deserves all praise for
the best efforts in publishing the book.
Saikat Gantait
Sandeep Kumar Verma
Amit Baran Sharangi
vii
Contents
1 In Vitro Approach and Quantification of “Puerarin and

Genistein”: Valuable Antidiabetic Compounds from Pueraria
tuberosa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Illa C. Patel, Masuma Hakim, Riddhi Prajapati, Anita Solanki,
and Jitendriya Panigrahi
2 In Vitro Exploitation of Medicinal Plants for Continuous Supply
of Antidiabetic Bioactive Compounds . . . . . . . . . . . . . . . . . . . . . . . 25
Subrata Kundu, Sk Moquammel Haque, and Biswajit Ghosh
3 Lower Plants as Potential Source of Antidiabetic Compounds:
The Present Knowledge and Future Prospects . . . . . . . . . . . . . . . . 65
Bhuvnesh Sareen and Amita Bhattacharya
4 Tissue-Culture-Mediated Biotechnological Intervention
in Centella asiatica: A Potential Antidiabetic Plant . . . . . . . . . . . . . 89
Priyanka Mohapatra, Asit Ray, I. Sriram Sandeep, Sanghamitra
Nayak, and Sujata Mohanty
5 In Vitro Approaches for Mass Propagation of Stevia rebaudiana . . . 117
Kasireddy Sivasankarreddy, K. Abha Manohar, Gopal Shukla,
Vineeta, Mohamad Maqbool Rather, and Sumit Chakravarty
6 Swertia chirayita, an Endangered Anti-diabetic Plant: Trends
in Biotechnological Interventions . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
Praveen Dhyani, Lalit Giri, Eshita Sharma, and Priyanka Sati
7 In Vitro Propagation and Biotechnological Improvement
Strategies of Plants with High-Intensity Sweetener and
Anti-Diabetic Activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
Elizabeth Kairuz, Alán Rivero-Aragón, and Geert Angenon
8 In Vitro Culture Techniques and Metabolite Engineering
for Enhanced Antidiabetic Secondary Metabolite Production . . . . . 211
Israt Jahan, Ahmet Onay, Mahmut Ozan Toksoy, and Sedat Kaya
ix
x Contents
9 Saffron: A Prized Herb with Therapeutic Potential Against

Diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263
Nitu Gautam and Amita Bhattacharya
10 New Insights to Enhance the Desired Anti-Diabetic Compounds
in Medicinal and Aromatic Plants Exposed to Abiotic
Stress Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285
Canan Gulmez and Muhittin Kulak
11 Boosting of Bioactive Secondary Metabolites in Anti-Diabetic
Plants Through Elicitation: A Simple Technology for
Better Future . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 307
Anamika Paul, Nehan Shamim, Anik Sarkar, Krishnendu Acharya,
and Nilanjan Chakraborty
12 Rhizobium rhizogenes-Mediated Genetic Transformation
of Antidiabetic Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341
Marta Libik-Konieczny, Żaneta Michalec-Warzecha, Ireneusz Ślesak,
and Laura Pistelli
13 Production of Antidiabetic Lignans in Flax Cell Cultures . . . . . . . . 383
Lucija Markulin, Samantha Drouet, Laurine Garros, Sumaira Anjum,
Duangjai Tungmunnithum, Bilal Haider Abbasi,
Randolph R. J. Arroo, Eric Lainé, Mohamed Addi,
and Christophe Hano
14 Artificial Seed Development of Selected Anti-Diabetic Plants,
Their Storage and Regeneration: Progress and Prospect . . . . . . . . . 409
Anamika Das, Manisha Mahanta, Biswajit Pramanik,
and Saikat Gantait
15 Cryopreservation of Anti-Diabetic Plants . . . . . . . . . . . . . . . . . . . . 437
M. R. Rohini, Marcos Edel Martinez Montero, and P. E. Rajasekharan
Editors and Contributors
About the Editors
Saikat Gantait Dr. Gantait is working as an Assistant

Professor in Genetics and Plant Breeding as well as a
Plant Breeder in Crop Research Unit, Directorate of
Research, Bidhan Chandra Krishi Viswavidyalaya
(BCKV) (State Agricultural University), India. Follow-
ing completion of his PhD in Biotechnology from
BCKV, he gained advanced research experience leading
a group to conduct fruitful research while working as
Research Associate in DBT (Government of India)-
funded project, followed by working as a Post-Doctoral
Researcher in Universiti Putra Malaysia (UPM). He has
guided/co-guided multiple MSc and PhD students. His
15-year research works, so far, include molecular
markers, plant tissue culture, polyploidy induction,
short-term (encapsulation/synthetic seed) and long-
term (cryopreservation) conservation of medicinal and
ornamental plant germplasms. In addition, he is acting
as an Section Editor of 3Biotech and Consulting Editor
of Sugar Tech (journals from Springer Nature).
Dr. Gantait has reviewed more than 150 manuscripts
as an ad-hoc reviewer in more than 50 different eminent
journals from Springer, etc. Dr. Gantait was awarded
with Post-Doc fellowship from UPM, Malaysia, and
he successfully completed 2 years of advanced training
on plant tissue culture and cryopreservation. To date,
Dr. Gantait has published 125 articles/chapters in peer-
reviewed journals and books (with cumulative SCI
Impact Factor of 195), mostly as the first/senior author.
xi
xii Editors and Contributors
Sandeep Kumar Verma Dr. Verma is working as an

Associate Professor in Institute of Biological Science,
SAGE University, Indore India. He has more than
13 years’ research and teaching experience in the field
of biotechnology in India, Italy, South Korea and Tur-
key, and he has published more than 45 papers in SCI
journals to date. He is a recognized expert in the use of
biotechnology, molecular biology and nanotechnology
techniques, giving many invited talks and presented
papers at international conferences and symposia around
the world. He has undergone advanced training courses
on “A practical course in mammalian cell biology”
(MAM-TUBITAK, Turkey), “RNA structure and func-
tion” (ICGEB, Italy), and “Quorum sensing in plant-
associated bacteria” (ICGEB, Italy). Furthermore, he
has received two best oral paper presentation awards in
national conferences and one young scientist award in
an international conference. Besides, he has also
received a memento for scientific achievement from
the Rector, BAIBU, Bolu, Turkey. He is also a reviewer
of many international-peer reviewed journals.
Amit Baran Sharangi Dr. Sharangi is a Professor of

BCKV (Agricultural University) attached with teaching,
research and extension for about 25 years. He completed
his doctoral degree from BCKV and post-doctorate from
the University of Melbourne, Australia. He is specially
trained from IISR, IIHR and ARO (Tel Aviv, Israel) and
is experienced in herbs, spices and MAPs. He spent
some productive times in several laboratories of
Australia, USA and UK for continuing research on
those crops as well as teaching post-graduate students
in universities abroad. Prof. Sharangi has several inter-
national awards including FULBRIGHT (USA), INSA-
RSE Visiting Scientist (UK), ENDEAVOUR Award
(Australia), Marquis Who’s Who (USA), Young
Achievers Award (SADHNA), Higher Education Lead-
ership Award, Outstanding Scientist Award, Bharat
Ratna Mother Teresa Gold Medal Award, etc. He is a
life member of several academic bodies and fellow of
eight prestigious academies/societies. He has published
about 90 peer-reviewed papers, 25 books from Springer
Nature, etc. and numerous book chapters within his
subject area. He is also associated with 50 journals
Editors and Contributors xiii
worldwide as editor-in-chief, regional and technical edi-

tor, editorial board member and active reviewer. He has
administrative experience for 4 years as HoD, chaired
several sessions of national and international
conferences, executed six projects so far as PI and
Co-PI, developed as well as restructured PG courses as
BSMA committee member (DG, ICAR, Government of
India) at national level.
Contributors
Bilal Haider Abbasi Department of Biotechnology, Quaid-i-Azam University,

Islamabad, Pakistan
K. Abha Manohar Uttar Banga Krishi Viswavidyalaya, Cooch Behar, West
Bengal, India
Krishnendu Acharya Molecular and Applied Mycology and Plant Pathology
Laboratory, Department of Botany, University of Calcutta, Kolkata, West Bengal,
India
Mohamed Addi Laboratoire d’Amélioration des Productions Agricoles,
Biotechnologie et Environnement, Faculté des Sciences, Université Mohammed
Premier, Oujda, Morocco
Geert Angenon Laboratory of Plant Genetics, Vrije Universiteit Brussel (VUB),
Brussels, Belgium
Sumaira Anjum Department of Biotechnology, Kinnaird College for Women,
Lahore, Pakistan
Randolph R. J. Arroo Faculty of Health and Life Sciences, De Montfort Univer-
sity, Leicester, UK
Amita Bhattacharya Division of Biotechnology, CSIR-Institute of Himalayan
Bioresource Technology, Palampur, Himachal Pradesh, India
Nilanjan Chakraborty Department of Botany, Scottish Church College, Kolkata,
West Bengal, India
Sumit Chakravarty Uttar Banga Krishi Viswavidyalaya, Cooch Behar, West
Bengal, India
Anamika Das Crop Research Unit (Genetics and Plant Breeding), Bidhan Chandra
Krishi Viswavidyalaya, Mohanpur, Nadia, West Bengal, India
Praveen Dhyani Department of Biotechnology, Kumaun University, Bhimtal,
Nainital, Uttarakhand, India
xiv Editors and Contributors
Samantha Drouet Laboratoire de Biologie des Ligneux et des Grandes Cultures,

INRAe USC1328, Eure et Loir Campus, University of Orleans, Orléans Cedex 2,
France
Bioactifs et Cosmetiques, CNRS GDR 3711 Orleans, Orléans Cedex 2, France
Saikat Gantait Crop Research Unit (Genetics and Plant Breeding), Bidhan
Chandra Krishi Viswavidyalaya, Mohanpur, Nadia, West Bengal, India
Laurine Garros Laboratoire de Biologie des Ligneux et des Grandes Cultures,
France
Nitu Gautam Division of Biotechnology, Council of Scientific and Industrial
Research (CSIR)-Institute of Himalayan Bioresource Technology, P.O. Box 6,
Palampur, Himachal Pradesh, India
Department of Biotechnology, Guru Nanak Dev University, Amritsar, Punjab, India
Biswajit Ghosh Plant Biotechnology Laboratory, Post Graduate Department of
Botany, Ramakrishna Mission Vivekananda Centenary College, Rahara, Kolkata,
India
Lalit Giri G.B. Pant National Institute of Himalayan Environment, Ladakh
Regional Centre, Leh, Ladakh, India
Canan Gulmez Department of Pharmacy Services, Tuzluca Vocational High
School, Igdir University, Igdir, Turkey
Masuma Hakim Department of Life Sciences, Hemchandracharya North Gujarat
University, Patan, Gujarat, India
Christophe Hano Laboratoire de Biologie des Ligneux et des Grandes Cultures,
France
Sk Moquammel Haque Plant Biotechnology Laboratory, Post Graduate Depart-
ment of Botany, Ramakrishna Mission Vivekananda Centenary College, Rahara,
Kolkata, West Bengal, India
Department of Botany, East Calcutta Girls0 College, Lake Town, Kolkata, West
Bengal, India
Israt Jahan Department of Molecular Biology and Genetics, Faculty of Science,
Dicle University, Diyarbakır, Turkey
Elizabeth Kairuz Departamento de Biología, Facultad de Ciencias Agropecuarias,
Universidad Central “Marta Abreu” de Las Villas (UCLV), Santa Clara, Villa Clara,
Cuba
Laboratory of Plant Genetics, Vrije Universiteit Brussel (VUB), Brussels, Belgium
Sedat Kaya Department of Organic Farming, Vocational School of Technical
Sciences, University of Muş, Muş, Turkey
Editors and Contributors xv
Muhittin Kulak Department of Herbal and Animal Production, Vocational School

of Technical Sciences, Igdir University, Igdir, Turkey
Subrata Kundu Plant Biotechnology Laboratory, Post Graduate Department of
Botany, Ramakrishna Mission Vivekananda Centenary College, Rahara, Kolkata,
West Bengal, India
Eric Lainé Laboratoire de Biologie des Ligneux et des Grandes Cultures, INRAe
USC1328, Eure et Loir Campus, University of Orleans, Orléans Cedex 2, France
Marta Libik-Konieczny The Franciszek Górski Institute of Plant Physiology,
Polish Academy of Sciences, Kraków, Polska
Manisha Mahanta Crop Research Unit (Genetics and Plant Breeding), Bidhan
Lucija Markulin Laboratoire de Biologie des Ligneux et des Grandes Cultures,
France
Żaneta Michalec-Warzecha The Franciszek Górski Institute of Plant Physiology,
Polish Academy of Sciences, Kraków, Polska
Sujata Mohanty Ramadevi Women’s University, Bhubaneswar, Odisha, India
Priyanka Mohapatra Centre for Biotechnology, Siksha‘O’Anusandhan (Deemed
to be University), Bhubaneswar, Odisha, India
Marcos Edel Martinez Montero Centro de Bioplantas Universidad de Ciego de
Ávila Máximo Gómez Báez, Ciego de Ávila, Cuba
Sanghamitra Nayak Centre for Biotechnology, Siksha‘O’Anusandhan (Deemed
to be University), Bhubaneswar, Odisha, India
Ahmet Onay Department of Biology, Faculty of Science, Dicle University,
Diyarbakır, Turkey
Jitendriya Panigrahi Department of Biotechnology, Shri A. N. Patel P.G. Institute
of Science and Research, Anand, Gujarat, India
Illa C. Patel Department of Life Sciences, Hemchandracharya North Gujarat
Anamika Paul Department of Botany, Scottish Church College, Kolkata, West
Bengal, India
Laura Pistelli Department of Agriculture, Food and Environment, University of
Pisa, Pisa, Italy
Riddhi Prajapati Department of Life Sciences, Hemchandracharya North Gujarat
xvi Editors and Contributors
Biswajit Pramanik Crop Research Unit (Genetics and Plant Breeding), Bidhan
P. E. Rajasekharan Indian Institute of Horticultural Research, Hessaraghatta Lake
PO, Bengaluru, Karnataka, India
Mohamad Maqbool Rather Division of Forest Biology and Tree Improvement,
SKUAST-K, Jammu & Kashmir, India
Asit Ray Centre for Biotechnology, Siksha‘O’Anusandhan (Deemed to be Univer-
sity), Bhubaneswar, Odisha, India
Alán Rivero-Aragón Departamento de Biología, Facultad de Ciencias
Agropecuarias, Universidad Central “Marta Abreu” de Las Villas (UCLV), Santa
Clara, Villa Clara, Cuba
M. R. Rohini Indian Institute of Horticultural Research, Hessaraghatta Lake PO,
Bengaluru, India
Sriram Sandeep Centre for Biotechnology, Siksha‘O’Anusandhan (Deemed to be
University), Bhubaneswar, Odisha, India
Bhuvnesh Sareen Division of Biotechnology, CSIR-Institute of Himalayan
Bioresource Technology, Palampur, Himachal Pradesh, India
Anik Sarkar Molecular and Applied Mycology and Plant Pathology Laboratory,
Department of Botany, University of Calcutta, Kolkata, West Bengal, India
Priyanka Sati Graphic Era University, Dehradun, Uttarakhand, India
Nehan Shamim Department of Botany, Scottish Church College, Kolkata, West
Bengal, India
Eshita Sharma CSIR-Institute of Himalayan Bioresource Technology, Palampur,
Himachal Pradesh, India
Gopal Shukla Uttar Banga Krishi Viswavidyalaya, Cooch Behar, West Bengal,
India
Kasireddy Sivasankarreddy Uttar Banga Krishi Viswavidyalaya, Cooch Behar,
West Bengal, India
Ireneusz Ślesak The Franciszek Górski Institute of Plant Physiology, Polish Acad-
emy of Sciences, Kraków, Polska
Anita Solanki Department of Life Sciences, Hemchandracharya North Gujarat
Mahmut Ozan Toksoy Department of Pharmaceutical Technology, Faculty of
Pharmacy, Dicle University, Diyarbakir, Turkey
Duangjai Tungmunnithum Department of Pharmaceutical Botany, Faculty of
Pharmacy, Mahidol University, Bangkok, Thailand
Vineeta Uttar Banga Krishi Viswavidyalaya, Cooch Behar, West Bengal, India
In Vitro Approach and Quantification
of “Puerarin and Genistein”: Valuable 1
Antidiabetic Compounds from Pueraria
tuberosa
Illa C. Patel , Masuma Hakim , Riddhi Prajapati, Anita Solanki,

and Jitendriya Panigrahi
Abstract
To produce elite clones of plants, in vitro approach is most acceptable. Plants with
susceptible germination rates and dependency on a specific plant part with
medicinal values are more vulnerable. Pueraria tuberosa (Willd.) DC is one
such perennial wild climber with poor seed germination and the only mode of
propagation is its tuber which has medicinal values such as the puerarin and
genistein with antidiabetic potential. To nullify the overexploitation of it, the
in vitro shooting was accomplished by inoculating the earlier generated nodal
parts from the in vitro seedling in benzyl adenine plus naphthalene acetic acid
(NAA) in Murashige and Skoog medium. The combination of indole butyric acid
and NAA makes it possible for successful induction of roots from the excised
in vitro shoot. The genetic fidelity of the mother plant and in vitro-raised plant
was assessed by inter simple sequence repeat analysis. It depicted true-to-type
plants. The extracts of in vivo and in vitro parts were taken for the quantification
of puerarin and genistein by following the International Council of Harmony
guidelines in high-performance thin-layered chromatography instrument. Both
the in vitro and in vivo parts substantially counted for the genistein and puerarin
content. Thus, this chapter gives an insight into the establishment of true-to-type
plants with antidiabetic potential compounds.
Keywords
Antidiabetic · Genetic fidelity · Genistein · HPTLC · Pueraria tuberosa · Puerarin
I. C. Patel · M. Hakim · R. Prajapati · A. Solanki

Department of Life Sciences, Hemchandracharya North Gujarat University, Patan, Gujarat, India
J. Panigrahi (*)
Department of Biotechnology, Shri A. N. Patel P.G. Institute of Science and Research, Anand,
Gujarat, India
# The Author(s), under exclusive license to Springer Nature Singapore Pte 1

Ltd. 2021
S. Gantait et al. (eds.), Biotechnology of Anti-diabetic Medicinal Plants,
https://doi.org/10.1007/978-981-16-3529-8_1
2 I. C. Patel et al.
1.1 Introduction
1.1.1 Plant Description
Pueraria tuberosa belongs to the family Fabaceae, usually named as “Kudzu” and
“Vidarikand”. P. tuberosa prefers to grow in shade and warm humid climate. The
soil must be sandy loam and rich in organic content, commonly found in Central
India and near the hills of Western Himalaya (Keung 2002). The twinning shrub
produces pinnately compound trifoliate hairy leaf (Fig. 1.1a). This Leguminous plant
is a woody perennial climber that produces several underground tuberous roots. The
mature tuber may attain a weight up to 10 kg, but it may vary according to the soil
type and environment (Fig. 1.1b). Seeds are the mode of propagation but the
germination rate of seed is low. That’s why the tuber is another mode of propagation
of the P. tuberosa plant. The tubers of P. tuberosa are treated as food both by
humans and animals. It is known for its nutritional as well as medicinal importance.
Thus, one of the important reproductive parts “tuber” is exploited highly as a source
of the drug, which restricted its regeneration, multiplication, and survival in nature.
1.1.2 Medicinal Properties of P. tuberosa
The rejuvenating drug “Vidari” is prepared from the tuberous roots. Vidari acts as a
galactagogue, stimulant, and emollient (Warrier et al. 1995). It is counted in Rasayan
drugs in Ayurveda. The tubers possess an extensive range of therapeutic features and
prescribe in spermatorrhea, bleeding in menopause, leprosy, tuberculosis, cough,
etc. P. tuberosa is highly valued in Chinese to the Indian system of Ayurveda
(Keung and Vallee 1998; Dev 2006). Pueraria species are known for its
Fig. 1.1 (a) Pueraria tuberosa plant growing in wild, (b) growing tubers of P. tuberosa. (Source:
R. Prajapati)
1 In Vitro Approach and Quantification of “Puerarin and Genistein”: Valuable. . . 3
hypolipidemic (Tanwar et al. 2008), antimicrobial (Venkata Ratnam and Venkata

Raju 2009), and hypoglycemic (Raghuwanshi and Jain 2012) puerarin (PRN) as
antioxidant activity (scavenging reactive oxygen species), genistein (GEN) as an
antifertility activity, eicosanoic acid as antimicrobial, 4-methoxypuerarin as
antidiabetic activity (Satpathy et al. 2018), etc. Apart from this, other constituents
such as tectoridin (androgenic activity), puerarone (cardiovascular activity),
tuberosin (neuroprotective activity), lupinoside (nephroprotective), robinin (antihy-
pertensive), and daidzin (anticonvulsant) are considered to possess the important
therapeutic properties (Satpathy et al. 2018). This plant contains different types of
isoflavonoids, namely, PRN, daidzein, GEN, and genistin (Goyal and Ramawat
2007) which are used in various Ayurvedic formulations. Isoflavonoid from a
group of distinct secondary metabolites produced predominantly in leguminous
plant. This isoflavonoid plays a great role in plant microbial interaction. It also
acts as chemoattractant for the rhizobial bacteria and as inducers of nod gene
expression in legumes. Among them, PRN, that is, daidzein 8-C-glucoside, has
been recommended against diabetes mellitus. It also shows antihyperglycemic
tendencies with enhancement in glucose tolerance level when rats were injected
with streptozotocin. This was also observed in the animal model of type 2 diabetes
mellitus (Hsu et al. 2003; Meezan et al. 2005). It has been found that PRN supports
the GLUT4 expression in the muscle that induces glucose utilization and also inhibits
sodium-dependent glucose transporter in the intestine (Xu et al. 2005). Pueraria spp.
augments the soils by biological nitrogen fixation (Selvakumar et al. 2008). Among
the isoflavonoids listed above, GEN and PRN are the most important in terms of
their immunomodulatory functions (Sawale et al. 2013). In addition to showing anti-
hypercholesterolemic activity, isoflavonoid has been associated with cancer and
osteoporosis prevention (Yan et al. 2006). Diabetes is a metabolic dysfunction,
which is connected with prolonged hyperglycemia by a distortion in lipid, protein,
and carbohydrate metabolism (Kim and Lim 2013). Mostly leguminous plants have
isoflavonoids, like PRN and GEN, which have advantageous effect on the inhibition
of metabolic dysfunction related to cancer, diabetes, and obesity (Gencel et al. 2012;
Stephenson et al. 2005; Ørgaard and Jensen 2008). Particularly, GEN and other
isoflavons do share the structural similarity with the human estrogen, thereby
competing for the estrogen receptor, and consequently take part in health benefit
(Bhathena and Velasquez 2002). PRN plays an effective antidiabetic activity in vivo
by insulin expression enhancing and metabolism-promoting attributes. Also, PRN
works as an assuring applicant that maintains the potential diagnostic for diabetes
treatment (Wu et al. 2013). The modern initiation of various phytoestrogen databases
clarifies the growing demand for food production in phytoestrogens from biological
resources (Schwartz et al. 2009). The isoflavonoid obtained from Pueraria lobata
(Matkowski 2004), Pueraria mirifica (Udomsuk et al. 2009) calli and via suspension
culture in Glycine max (Gueven and Knorr 2011). In the above three cases, the
alterations in the physical environment give a boost up to the production of
isoflavonoids. The extensive use of the tuber of P. tuberosa in pharmaceutical
companies makes it vulnerable. Therefore, there is an urgent need for its mass
propagation where in vitro propagation technique can fulfill the demand, as well as
its vulnerability can be lessened.
1.2 Micropropagation of P. tuberosa
Micropropagation protocol offers through tissue culture technique presently help a

lot in many medicinal plants to produce elite new clones in a short period, without
destroying the natural population (Rathore and Shekhawat 2009). Periodic work on
micropropagation of many species of Pueraria was reported. In vitro culture in
P. lobate and P. candolli var. mirifica was evidenced (Thiem 2003; Thanonkeo and
Panichajakul 2006). Successful micropropagation work on P. tuberosa using MS
medium with benzyl adenine (BA) and kinetin for shooting and indole butyric acid
(IBA) and naphthoxyacetic acid for rooting was reported (Rathore and Shekhawat
2009; Sharma et al. 2009; Bindu et al. 2017). In P. tuberosa, still detail reports on the
shoot multiplication rate, root forming ability of in vitro shoots, survival of
regenerated plantlets in soil, hardening procedure to bring regenerated plants from
the laboratory (in vitro) to field (in vivo) condition as well as in vitro tuberization
were not emphasized. As compared to in vivo production of bioactive principles,
in vitro products are more demanding and worthy. Ample research has already been
done on the in vivo toxicological and phytochemical profile of in vivo plant parts of
P. tuberosa. Thus, the present chapter introduces an ideal micropropagation protocol
which focuses on each step of shooting, rooting, tuberization, and hardening of
plantlet along with genetic fidelity assessment of clone using inter simple sequence
repeat (ISSR) molecular marker and give a comparative analysis of two important
isoflavonoids, that is, GEN and PRN, from in vivo and in vitro plant parts of
P. tuberosa.
1.2.1 Plant Material and Seed Germination
Mostly, during plant tissue culture, explant selection and collection time period from
the source and laboratory are important. In vivo plant samples and mature pods were
collected from the forest region of Rajpipla, Narmada District, and Gujarat, India.
Before in vitro culture, the healthy plant material needs to be sterilized to avoid any
unwanted contaminations. Healthy seeds were sterilized using two drops of Tween-
20 in 200 ml autoclaved water for 5 min. Then it was rinsed 2–3 times only with
autoclaved water for 5 min, followed by rinsing with 0.01% (w/v) Bavistin for 1 h. It
again rinsed 3 times with autoclaved water for 5 min, surface sterilized with 0.06%
(w/v) HgCl2 for 6 min. The final cleaning was done 2–3 times with autoclaved water
for 5 min. The sterilized seed was inoculated in MS (Murashige and Skoog)
medium (Murashige and Skoog 1962) containing 3% (w/v) sucrose, 2% (w/v)
activated charcoal, and prestandardized hormone combination of 4.44 μM BA
(Benzyladenine) and 2.83 μM GA (Gibberellic acid), pH 5.8, and autoclaved at
120 C for 30 min. The culture was incubated at 26 2 C under 16-h dark and 8-h
light photoperiod at a light intensity of 3000 Lx with the support of cool white
fluorescent tubes. Inoculated seeds were germinated after 15 days of inoculation.
Nodal segments from the seedling were used as explants for in vitro culture.
1.2.1.1 Seed Germination

As already mentioned, due to poor seed germination in wild conditions, to get
healthy plantlets it has to grow under in vitro conditions. The healthy, mature,
ellipsoid P. tuberosa seeds that are 2–3 mm in size were germinated in an MS
medium containing 4.44 μM BA and 2.83 μM GA (Fig. 1.2). In this hormonal
combination, the seedling has a broad, dark green leaf and shown approximately
10 cm of seedling length within 15 days of inoculation. When used, other hormonal
combination seedlings have a narrow leaf, less seedling length, and poor germina-
tion (data not shown). It was observed that dark brown seeds showed good germina-
tion compared to light brown/white seeds. Similarly, seed germination of
Fig. 1.2 In vitro seed germination in MS medium supplemented with 4.44 μM BA along with
2.83 μM GA. (a) Initiation of seed germination, (b) opening of cotyledonary leaf, (c) initiation of
shoot elongation, (d) well-developed seedling. (Source: M. Hakim)
P. tuberosa was done in MS medium supplemented with BAP + IAA

(4.43 + 1.43 μM) by Sharma et al. (2011).
1.2.1.2 Callus Development

In vitro leaf and cotyledonary nodes were collected from germinated seedling and
used for callus development while in vivo tubers were directly used for tuber callus
formation. All the explants were sterilized and inoculated in MS medium (pH ¼ 5.7
before the addition of gelling agent) supplemented with 3% (w/v) sucrose and 0.8%
(w/v) agar along with various concentrations. For different callus development,
different concentrations of BAP (4.44, 6.66, and 8.88 μM) and 2,4-D (4.55, 6.82,
and 9.10 μM) individual and combinations of BAP + 2,4-D (4.44 + 4.55,
6.66 + 6.82, and 8.88 + 9.10 μM) were used. Cultures were incubated at
26 2 C under 16-h dark and 8-h light photoperiod at a light intensity of
3000 lx provided by cool white fluorescent tubes and periodic data were recorded
for callus development. Then, callus developed in the best combination was selected
and used for phytochemical analysis.
As recorded in Table 1.1 and Fig. 1.3, the leaf callus reported after 8 weeks in MS
medium supplemented with 4.55 μM 2,4-D with 92.40 2.47 growth frequency,
whereas the maximum growth of tuber callus found in 6.66 μM 2,4-D + 6.82 μM
BAP with 82.38 3.57 and cotyledonary node callus was observed in 6.66 μM BAP
with 93.80 2.32 growth frequency. Initially, the leaf and node callus were
greenish-white, then turn dark green. Although tuber callus is a whitish compact
mass, this also turns brown after 8–10 weeks. Cytokinin and auxin are the important
plant hormones that control the cell division during tissue culture. Different
concentrations of auxin and cytokinin increase the production of callus. Callus
initiation and growth were best on media having 25 μM BA, respectively, in leaf
and stem callus of P. lobata (Matkowski 2004). Leaf callus was grown best in MS
medium supplemented with 6.6 μM BAP and 2.69 μM naphthalene acetic acid
(NAA) in leaf explants of P. tuberosa (Bindu et al. 2018). While studying the callus
formation from tubers of P. tuberosa collected during rainy and summer seasons, it
took 25 and 21 days for initiation of callus, respectively, when cultured in 2.27 μM
2,4-D fortified in MS media. But, in summer season collected tubers, with a
combination of BAP plus 2,4-D (4.4 μM + 4.12 μM) the tuber induction took only
13 days for initiation. Also, the calli appeared as greenish and hard in texture (Patel
et al. 2020).
1.2.2 Shoot Formation
Approximately 2-cm nodal segments were excised from in vitro seedling and were
used for shoot multiplication by inoculating in MS medium accompanied with 3%
(w/v) sucrose and 0.8% (w/v) agar along with the hormone (2.22, 3.33, 4.44, and
6.66 μM) 6-benzylaminopurine (BAP), (2.32, 4.65, and 6.97 μM) kinetin (Kn),
(1.44, 2.89, and 4.33 μM) GA, and (2.68, 5.37, and 8.05 μM) NAA individual as
well as combination of BAP + Kn (3.33 + 2.32 μM and 3.33 + 4.65 μM), BAP + GA
1
Table 1.1 Effect of different plant growth regulators (PGRs) on callus development of P. tuberosa
PGR Leaf callus Tuber callus Cotyledonary callus

Name concentration Number of days for % Growth Number of days for % Growth Number of days for % Growth
of PGR (μM) callus induction frequency callus induction frequency callus induction frequency
BAP 4.44 19.6 1.1 49.6 3.2 24.3 2.3 63.3 2.6 21.3 2.9 71.9 2.8
6.66 15.3 4.7 56.7 4.9 20.6 0.6 53.3 4.9 13.6 4.1 93.8 2.3
8.88 17.3 1.7 46.3 3.7 16.5 2.5 68.3 1.7 17.4 1.8 65.6 4.3
2,4-D 4.55 11.6 3.3 92.4 2.4 18.4 2.0 48.9 2.0 15.5 2.4 47.7 3.5
6.82 12.6 2.9 65.3 3.2 19.9 1.0 54.3 3.7 19.3 2.8 55.9 2.5
9.10 13.6 1.4 72.6 2.7 17.7 1.4 64.4 4.1 16.4 2.6 57.6 8.1
BAP+ 4.44 + 4.55 20.3 2.1 46.3 3.2 21.3 2.7 62.7 2.3 19.6 3.9 45.6 4.6
2,4-D 6.66 + 6.82 17.3 2.4 55.3 3.1 14.6 1.7 82.3 3.5 17.6 1.7 41.8 3.0
8.88 + 9.10 16.6 2.7 62.7 3.2 13.3 3.5 61.9 3.4 14.6 0.8 39.9 5.2
In Vitro Approach and Quantification of “Puerarin and Genistein”: Valuable. . .
7
Fig. 1.3 Callus development from different explants: (a) leaf callus, (b) tuber callus, and (c)
cotyledon callus. (Source: M. Hakim)
(3.33 + 2.89 μM and 3.33 + 4.33 μM), and BAP + NAA (3.33 + 2.68 μM and
3.33 + 5.37 μM). Cultures were incubated at 26 2 C under 16-h dark and 8-h light
photoperiod at a light intensity of 3000 Lx bestowed by cool white fluorescent tubes.
Periodic data on shoot induction and multiplication were recorded along with the
periodic transfer of each culture in the same media composition.
The process of shoot multiplication has been completed after three subculturing
cycles. In the first cycle of transfer, 2–3 cm of stem segment with 1–2 nodes was
collected from the above culture and inoculated in BAP + NAA (3.33 + 2.68 μM)
after 8 days. In the second cycle, 4–5 cm of stem segment with 2–4 nodes from first
cycle plantlets was again inoculated in the same media after 16 days and during the
third cycle again 4–5 cm of stem segment with 2–4 nodes from second cycles was
transferred in the same media after 24 days. After 3 weeks of culture/completion of
the third cycle, approximately 10–12 cm long multiple shoots produced in each set
were maintained. The results were presented as the mean value with standard error.
A three replica for each treatment was maintained and used for Duncan (1955)
multiple range test comparison (DMRT) ( p < 0.05).
Variable response of shoots formation in different PGRs in MS medium was
presented in Table 1.2. Results indicate that MS medium with a lower concentration
of 2.22 μM BA and 2.68 μM NAA showed 72.6 1.2 and 65.6 1.8% growth
frequency, respectively. It was observed that the shoot induction frequency was less
when using a higher concentration of PGRs. When MS medium was supplemented
with a hormonal combination, BA + NAA (2.22 + 2.68 μM) exhibited the highest
growth frequency of 95.3 2.0% with the highest number of shoots 17.8 0.4 and
maximum shoot length 11.8 0.1. In this hormonal combination, stem segments
from in vitro seedling initiated shoot induction within 8 days. After 8 days,
subculturing was operated by taking a sliced stem. After subculturing, the frequency
of the number of shoots production dramatically increased. After third subculturing,
the maximum number of shoots 17.0 0.4 with the highest shoot length was
recorded (Fig. 1.4).
Table 1.2 Effects of different concentration and combination of PGRs on shoot multiplication
from seedling node of P. tuberosa
Concentration of PGRs (μM) Frequency of shoot Number of shoots Average shoot
BAP Kn GA NAA regeneration (%) per explant length (cm)
2.22 72.6 1.2b 3.9 1.0cde 8.2 0.2b
3.33 64.0 2.8cd 2.1 0.6e 3.4 0.2k
4.44 48.6 4.6ghi 4.7 1.7cde 5.0 0.0gh
6.66 49.3 5.0ghi 4.5 0.7cde 3.9 0.5ijk
2.32 26.0 3.4l 6.7 0.9abc 7.1 0.2c
4.65 29.6 2.6l 4.5 0.8cde 6.2 0.3def
6.97 36.0 2.6k 3.5 0.8de 5.7 0.1defg
1.44 48.6 2.0ghi 3.0 0.9de 5.7 0.1efg
2.89 57.6 3.5def 3.0 1.1de 4.2 0.3ij
4.33 39.3 3.1k 2.9 1.1de 6.2 0.2de
2.68 65.6 1.8bc 5.5 0.7abcd 8.3 0.3b
5.37 39.3 1.4jk 3.9 1.7cde 3.7 0.2jk
8.05 55.3 2.1efg 2.7 0.8de 4.6 0.2hi
3.33 2.32 60.0 1.1cde 5.0 0.7bcde 7.1 0.2c
3.33 4.65 55.3 2.0efg 3.4 0.2de 6.5 0.2cd
3.33 2.89 49.6 1.2ghi 4.3 1.7cde 7.1 0.1b
3.33 4.33 43.0 1.5ijk 3.8 2.1cde 6.4 0.2gh
3.33 2.68 95.3 2.0a 7.8 0.4ab 11.8 0.1a
3.33 5.37 51.3 2.5fgh 3.5 0.8de 8.2 0.1b
Mean values followed by the different (in superscript) letters under different treatments within a
column are significantly different from each other at p < 0.05 according to Duncan’s multiple range
test (1955)
It has been recorded that repeated subculturing after regular intervals showed the
highest, healthy, and mature multiple shoots. Overall, it has been observed that in the
combination of cytokinin (BAP) and auxin (NAA) hormonal preparation, response
for shoot multiplication was higher as compared to other hormones. However,
delays in subculturing significantly turned the color of the shoot into yellow and
subsequent leaf fall. This limits the growth and multiplication of shoots. Therefore,
frequent subculturing must be followed. Similar results of multiple shoots were
obtained in the shoot tip of P. lobata using MS medium supplemented with
4.4 μM BA and 5.8 μM GA (Matkowski 2004). The nodal segment of P. tuberosa
produced maximum multiple shoots in MS medium having 4.4 μM BA, 1.16 μM Kn
and 0.57 μM IAA (Rathore and Shekhawat 2009).
1.2.3 Roots and Tuber Formation
After complete multiple shoot formation from the best treatment, 6–7-cm long stem
having 3–4 nodes was rooted in vitro in half MS medium supplemented with
different concentrations of (2.46, 4.92, and 7.38 μM) IBA and (2.68, 5.37, and
Fig. 1.4 Well-developed multiple shoots in MS medium supplemented with 3.33 μM BA+
2.68 μM NAA. (a) In vitro-germinated seedling in 4.44 μM BA +2.83 μM GA after 15 days, (b)
nodal explant inoculated in MS medium 3.33 μM BA+ 2.68 μM NAA, (c) shoot elongation after
12 days of growth, (d) nodal segment from first growth of cycle in above same media, (e) multiple
shoot formation after 30 days of inoculation, (f) multiple shoots are ready for third sub culturing, (g)
nodal explant inoculated in above media, (h) multiple shoots are ready after third subculturing.
(Source: M. Hakim)
8.05 μM) NAA individually and different combination of IBA + NAA (2.46 + 2.68,
4.92 + 2.68, and 7.38 + 2.68 μM). The culture was maintained at 12-h light and 12-h
dark regime with above all cultural conditions. After 22–25 days of inoculation, the
thin roots that were formed from the base of the shoots started to become thick and
tuberous at its tip parts confirms that the plantlet is ready for hardening. The rooting
under in vitro conditions definitely depends on both intrinsic and extrinsic factors
(Wilson and Van Staden 1990; Schiefelbein and Benfey 1991; Martin 2002). When
MS medium supplemented with 4.92 μM IBA and 2.68 μM, NAA exhibited
Table 1.3 Effect of different concentration and combination of PGRs on in vitro root formation
from shoots of P. tuberosa
Concentration
of PGRs (μM) Frequency of root Number of roots per Average root length
IBA NAA formation (%) shoot (cm)
2.46 36.6 2.9ef 1.6 0.6e 4.2 0.2c
4.92 64.6 2.7b 4.3 0.3abc 5.6 0.2a
7.38 44.3 3.5d 2.6 0.3cde 3.5 0.2cd
2.68 66.6 2.8b 5.3 0.6ab 5.3 0.1ab
5.37 41.6 6.3de 1.6 0.6e 2.9 0.3de
8.05 31.6 1.4f 3.3 0.3cde 4.3 0.3bc
2.46 2.68 22.6 3.1g 2.3 0.6de 2.4 0.6e
4.92 2.68 88.3 3.7a 5.6 0.3a 6.2 0.2a
7.38 2.68 54.6 3.4c 3.6 0.6bcd 3.0 0.4de
Mean values followed by the different (in superscript) letters under different treatments within a
column are significantly different from each other at p < 0.05 according to Duncan’s multiple range
test (1955)
frequency of root formation of 64.6 2.7% and 66.6 2.8%, respectively

(Table 1.3 and Fig. 1.5). The number of roots and root length was dramatically
increased when MS medium supplemented with IBA + NAA (4.92 + 2.68 μM) with
88.3 3.7 growth frequency. In this combination, the shoots produce 6.2 0.2 cm-
long roots and these roots are found to be very healthy and strong compared to the
other combinations. It was observed that after 22 days of transferring, the thin root is
swollen and appeared as a small tuber. A similar type of result was observed in
P. tuberosa containing MS medium with 9.84 μM IBA-generated in vitro roots with
98% rooting frequency (Rathore and Shekhawat 2009).
1.2.4 Acclimatization
Approximately 11–12 cm long in vitro shoots with tuberous roots were separated
carefully from each culture and removed from the bottle to avoid fungal contamina-
tion. The thick paste of wood ash by adding 6 μM IBA was prepared, the root tip of
each shoot was capped with this paste and transferred into a tray having 10:3:3 (soil:
NPK fertilizer: coco peat). Then after 15 days, these secondary rooted plantlets were
transferred into polythene begs with 10:3:3 (soil: NPP fertilizer: coco peat) and kept
in nursery condition. After 1 week, liquid NPK (19–19-19) spraying was done on
plantlets along with the watering till the field transferred. Approximately 11–12 cm-
long in vitro rooted shoots were removed from the media and the root portion was
covered with a thick pest of wood ash to avoid fungal contamination and then
transferred to the greenhouse with a tray having 10:3:3 (soil: NPK fertilizer: Coco
peat); this ratio is found to be best for planet growth. After 15 days, these plantlets
transferred in plastic beg having 10:3:3 (soil: NPP fertilizer: cocopeat). After
25–30 days of transferring in the field, it has been recorded that the tuber is becoming
Fig. 1.5 Root formation: (a) root initiation in MS medium supplemented with 4.92 μM IBA+
2.68 μM NAA, (b) mass of root after 30 days of growth, (c) in vitro-rooted shoots are ready for
hardening, (d) small tuber like structure in many primary roots. (Source: M. Hakim)
large approximately 3 cm in width and 5 cm in height (Fig. 1.6). The size of tubers
varies from soil to soil and environmental conditions.
1.3 Evaluation of Genetic Fidelity/DNA Isolation and ISSR-PCR

Amplification
Molecular markers are the basic need to register the genetic uniformity of in vitro
developed plants (Joshi and Vibha 2007). The genetic fidelity assessment of
micropropagated plants with its mother plant was still lacking in P. tuberosa.
DNA isolation was carried out from in vitro and in vivo young leaf tissues of
P. tuberosa using modified cetyl-trimethyl-ammonium-bromide protocol (Murray
Fig. 1.6 Acclimatization and tuber formation: (a) prepare polybags for acclimatization, (b)
acclimatization, (c) after 1 month of field transfer tube was formed, (d) young tuber. (Source:
M. Hakim)
and Thompson 1980). Each PCR reaction mixture possessed 2 μl of genomic DNA
(25 ng/ μl), 1 μl of primer (10 pmol/ μl), and 0.3 μl of dNTPs. This mixture was made
up of deionized water to a final volume of 25 μl. PCR reaction was run in Veriti™
96-Well Thermal Cycler following the optimal amplification conditions. For ISSR
amplification, PCR cycles consisted of DNA denaturation at 94 C for 4 min,
36 cycles at 92 C for 1 min, annealing at 45 C, and extension at 72 C for
2 min, followed by a final extension at 72 C for 10 min. All experiments were
repeated thrice. For complete separation of amplified PCR products, horizontal
agarose gel electrophoresis was operated using 1.5% agarose gel containing
ethidium bromide in 1 TBE buffer at 120 V for 80 min. A 100-bp DNA ladder
was run along with PCR products to determine the molecular weight of amplified
products. Ethidium bromide-stained gels were documented by Molecular Imager
Chemi Doc Doc XRS+ (Bio-Rad). Negative control was inserted in every PCR
reaction to crosscheck the contaminations of the samples if any. Twenty ISSR
primers were randomly selected for ISSR analysis, of which six primers were finally
Fig. 1.7 ISSR banding pattern of Pueraria samples with Pl-(AC)8T, P2-(AG)a CA, P3-(AG)sY C,
P4-(CA)r RG, P5-(CT)s RC, and P6-(GA)r RC (M-100 bp DNA ladder lane, 1–3: Pueraria
samples). (Source: I.C. Patel)
Table 1.4 List of primers, their sequences and total monomorphic number generated by ISSR
primers in P. tuberosa
50 –30 Total number of Total number of monomorphic Band length
Primers motif amplicons amplicons (bp)
ISSR- (AC)8T 6 6 350–2500
P1
ISSR- (AG)8CA 5 5 400–900
P2
ISSR- (AG)8YC 5 5 600–1700
P3
ISSR- (CA)8 3 3 600–1500
P4 RG
ISSR- (CT)8RC 4 4 700–1400
P5
ISSR- (GA)7RC 4 4 600–1600
P6
chosen for the genetic fidelity assessment as they were able to generate a reproduc-
ible amplification pattern. Six selected ISSR primers have generated a total of
27 distinct and scorable amplicons. The entire band across mother plant and
in vitro-raised plants were found to be monomorphic (Fig. 1.7 and Table 1.4). The
highest number of monomorphic bands (6) were observed in primer P1-(AC)8 T
with 350–2500 bp amplification range followed by primer P2-(AG)8 CA; primer
P3-(AG)8 YC produced five monomorphic bands; primer P5-(CT)8 RC and primer
P6-(GA)7 RC produced four monomorphic bands while primer P2-(CA)8 RG
produced only three scorable monomorphic bands (Table 1.4). Similarly, Martin
et al. (2004) reported genetic stability assessment of in vitro-raised clones of Prunus
dulcis by using ISSR analysis. As the ISSR technique is simple as well as cost-
effective, so, it has been widely used by many researchers in various plant species
such as Viola patrinii and Stevia rebaudiana (Chalageri and Babu 2012; Yadav et al.
2016).
1.4 High-Performance Thin-Layered Chromatography

Analysis
1.4.1 Preparation of Extracts
All air-dried in vivo leaf, stem young tuber, and old tuber along with in vitro leaf,
stem, root, tuber, node callus, leaf callus, and tuber callus were ground separately in
the mechanical grinder and the powdered material was kept in individual airtight
containers for further use. The extraction was carried out via the maceration method.
Each of the samples, weighing 1 g, was soaked in a conical flask containing 10 ml
methanol extract placed on a shaker with 150 rpm overnight. The extracts were
filtered through Whatman filter paper No. 1. The filtrates were collected, covered,
labeled, and stored at 4 1 C for the high-performance thin-layered chromatogra-
phy (HPTLC) screening.
1.4.2 High-Performance Thin-Layered Chromatography Method

Validation and Quantification
1.4.2.1 Sample Application

TLC plate silica gel 60 F254 (Merck) of size 20 cm 10 cm was used as a stationary
phase. Samples were put on the preactivated silica gel TLC plate. The standard and
samples were applied to the plates as 8-mm long bands, 8 mm apart by the use of a
Camag Linomat (V) sample applicator, programmed by Win CATS Planar Chroma-
tography Manager software equipped with a 100- μl microsyringe under a flow of
nitrogen gas.
1.4.2.2 Chromatogram Development and Scanning

The above dried sample-loaded HPTLC plate was placed in solvent-saturated twin
trough glass chamber of size 20 10 cm for the development of chromatogram. The
chromatogram was differentiated up to 80%. This distance covered by the chromato-
gram was marked at that time with a pencil, flowed by drying with a hairdryer, or
kept that on a hot plate to confirm the removal of any traces of solvent if at all by
evaporation. For HPTLC analysis, Toluene: Ethyl acetate: Formic acid (16:4:5 drops
v/v) was prepared as the mobile phase for standard GEN and the ratio of Toluene:
Methanol: ethyl acetate: Formic acid (14:4:2:5 drops) solution as mobile phase for
PRN. The developed chromatogram was scanned through Camag TLC Scanner 3 at
254 nm wavelength selecting the deuterium lamp, and 366 nm wavelength using
Mercury lamp, and 540 nm wavelength using the white lamp, respectively. Every
HPTLC procedure was operated at 25 2 C with 40% relative humidity. The
standard compound GEN and PRN were procured from Sigma-Aldrich and HPTLC
plates were purchased from Merck (Mumbai, India).
1.4.2.3 Standard Preparation for Quantification

GEN and PRN standards were purchased from Sigma, India, having a purity of 95%
(assessed by HPLC). Such 1 mg of GEN was added to 1 ml of HPLC grade methanol
to make it 1 mg/ml.
1.4.2.4 Method Validation

The HPTLC instrument was validated by using different parameters of ICH
guidelines (ICH 2005) such as linearity, LOD, LOQ, intraday, and interday precision
along with specificity and reproducibility. The standard curve for GEN and PRN was
obtained by performing linearity. The concentration range for GEN was 100–600 ng/
spot along with 0.95664 correlation coefficient (Fig. 1.8a) and the concentration
range for PRN was 100–1000 ng/spot along with 0.96074 correlation coefficient
(Fig. 1.9a). The method sensitivity for standard GEN and PRN, that is, LOD value,
was 36.15 and 356.8, respectively, while LOQ values were 109.5 and 1081.2,
respectively. Mostly the ratio of LOD/LOQ is 1:3 which was achieved in this case.
These data confirmed the sensitivity of the method. Similarly, the specificity of GEN
(Fig. 1.8b) and PRN (Fig. 1.9b) was clearly illustrated. The specificity was measured
for the purity of standard along with samples and mobile phase. The results of
Fig. 1.8 Method validation parameters for genistein at 254 nm: (a) standard curve of linearity, (b)
3D graph of specificity, (c) photoplate of reproducibility, (d) 3D graph for reproducibility. (Source:
I.C. Patel)
Fig. 1.9 Method validation parameters for puerarin at 254 nm: (a) standard curve of linearity, (b)
3D graph of specificity, (c) photoplate of reproducibility, (d) 3D graph for reproducibility. (Source:
I.C. Patel)
specificity were found to be specific. Besides this, the reproducibility curves

(Fig. 1.8c, d) for GEN and (Fig. 1.9c, d) for PRN showed a clear indication of the
acceptability of the protocol followed. The reproducibility data showed the perfect
Rf range for both standards, which displayed the accuracy of the instruments.
Precision was calculated by performing intraday and interday studies. The intraday
precision for GEN and PRN was 2.90% and 2.21%, respectively, while interday
precision was 1.98% and 1.88%, respectively (Table 1.5). These results were
recorded in terms of % relative standard deviation.
1.4.2.5 Quantification of GEN and PRN

The amount of isoflavonoids (GEN and PRN) was quantified in natural plant parts,
namely, leaf, stem, young tuber, old tuber as well as in vitro-developed plant parts,
namely, leaf, stem, root, tuber, leaf callus, node callus, and tuber callus. The
chromatogram was scanned at 254, 366, and 540 nm but the GEN and PRN were
observed clearly in UV light (254 nm) (Figs. 1.10, 1.11, 1.12, and 1.13). It has been
reported that both the isoflavonoids contents of different in vitro parts and in vivo
parts exhibit variation. Nevertheless, higher amount of GEN was found in in vitro
root (671.4 ng/ml) followed by in vitro tuber (442.9 ng/ml) and in vitro leaf
(426.2 ng/ml). The isoflavonoid GEN quantification represented the maximum
Table 1.5 Method validation parameters for the quantification of genistein and puerarin
Parameters Genistein Puerarin
Linearity range (ng/spot) 100–600 100–1000
Correlation coefficient (r) 0.95664 0.96034
Regression equation Y ¼ 5702.11 + 2.222X Y ¼ 343.557 + 31.866X
Sdv 8.67% 5.21%
LOD (ng/spot) (limit of detection) 36.15 356.8
LOQ (ng/spot) (limit of quantification) 109.5 1081.2
Intra assay precision (RSD [%] n ¼ 6) on 2.90% 2.21%
the same day
Intermediate precision (RSD [%] n ¼ 6) 1.88% 1.98%
after 3 days
Fig. 1.10 Photoplate of genistein quantification at 254 nm with all samples (1–6: genistein
standard, 7: in vivo leaf, 8: in vivo stem, 9: in vivo young tuber, 10: in vivo old tuber, 11:
in vitro leaf, 12: in vitro stem, 13: in vitro root, 14: in vitro tuber, 15: leaf callus, 16: node callus,
17: tuber callus). (Source: I.C. Patel)
amount in all in vitro parts rather than in vivo. Increased GEN-like compounds in
in vitro parts can be fruitful for its simultaneous and large-scale production in
Pueraria. In contrast to GEN, a higher amount of PRN was recorded in young
tuber (1371.5 ng/ml), followed by old tuber (1148.1 ng/ml) and leaf (294 ng/ml).
The higher quantity of GEN or PRN in particular part may be due to higher
expression of gene responsible for its biosynthesis in particular part. The lower
quantity of GEN or PRN may be due to unavailability of precursor needed during its
biosynthesis and also due to low gene expression. Additionally, the isoflavonoid
productions in specific tissue/organ/plant parts are again undercontrol of environ-
mental condition of the plant. The result of quantification showing high quantities of
PRN in the tuber extract along with the in vivo part extracts paves a way forward for
the standardization of herbal isoflavonoid drug material from this valuable plant. The
differences observed in in vivo plant parts and in vitro plant parts may be due to the
influence of metabolic pathway-mediated changes in the differentiation of parts
during the development. We can conclude that the degree of chemovariation in the
same plant was observed in its various parts in terms of GEN accumulation in both
in vivo and in vitro-developed controlled plant parts. The contents of the bioactive
compound in the plant are directly or indirectly related to gene expression for its
Fig. 1.11 (a) 3D graph of genistein in all samples, (b) standard curve with samples. (Source:
I.C. Patel).
synthesized pathway during the organogenesis processes. Here in P. tuberosa as

leguminous plant, the major pathway for isoflavonoids synthesis is shikimic acid and
phenylpropanoid pathway and the variation in its intermediates may lead variation in
isoflavonoids accumulation in plant parts. Complete HPTLC quantification of
important isoflavonoid GEN including in vivo and in vitro parts of P. tuberosa
will help to select the ideal part for phytochemical extraction. Thanonkeo and
Panichajakul (2006) studied the accumulation of daidzein and GEN in callus of
P. canollei var. mirifica and revealed that callus established from stem explant
produced the highest yield of daidzein and GEN. Similarly, HPLC analysis of the
P. tuberosa tuber extract was found to contain flavonoids such as PRN (Ae Park
et al. 2006), but complete HPTLC quantification of GEN including in vivo and
in vitro parts of P. tuberosa which will help to select the ideal part for phytochemical
extraction.
Fig. 1.12 Photoplate of puerarin quantification at 254 nm with all samples (1–6: puerarin standard,
7: in vivo leaf, 8: in vivo stem, 9: in vivo young tuber, 10: in-vivo old tuber, 11: in vitro leaf, 12:
in vitro stem, 13: in vitro root, 14: in vitro tuber, 15: leaf callus, 16: node callus, 17: tuber callus.
(Source: I.C. Patel).
Since P. tuberosa plant possesses these two antidiabetic compounds (GEN and
PRN), it has to be more exploited under in vitro conditions and the maximum output
can be exponentially extracted via the introduction of various elicitors. It can also be
noticed that in the present experiment, without any elicitor it provided an ample
amount of GEN that can be an eye opener for the pharma industry and exploited this
protocol for its higher production.
1.5 Conclusion
About 500 million people suffered from the deadly disease of diabetes and medicinal
plants are the major contributory partner that provides antidiabetic drugs. Several
antidiabetic plant products were identified and tried to extracted and make the drugs.
But these plant products are secondary metabolites and plant produced it secondar-
ily. In other words, very few amounts are intact in plants. To get these products, the
wild plants are being exploited too much that leads to the vulnerability of the
respective plants. To avoid these issues, the alternative method is to go for plant
tissue culture. Thus, this chapter provides a well-standardized, rapid, and reliable
protocol for micropropagation of this valuable plant, which can be applied for the
mass multiplication and large-scale plantation. Also to avoid the redundancy of the
in vivo and in vitro plants, the DNA profiling of the in vitro-developed plant and
mother plant by ISSR analysis was analyzed and has shown monomorphic banding
patterns. Hence, it may be predicted that the tissue culture-raised plants are true-to-
Fig. 1.13 (a) 3D graph of puerarin in all samples, (b) standard curve with samples. (Source:
I.C. Patel)
type of the mother plant and qualified genetic fidelity test. Thus, these genetically
uniform clones can be adapted for large-scale plantation and medicinal purposes.
Further, to know about the quantity of GEN and PRN as extracted from both in vivo
and in vitro samples of P. tuberosa, HPTLC quantification of these two antidiabetic
compounds was performed. The validated method generated a clear picture of their
quantitative presence in each part, which can offer a new avenue to greatly impact
the removal of chronic diseases, oxidant stress, aging, etc. from human life. The
intermediates of glycolytic and tricarboxylic pathways, such as phosphoglyceric
acid, phosphoenolpyruvate, erythrose 4-phosphate, and acetyl Co-A, are later on
entering into the secondary metabolite production. Particularly isoflavonoids like
GEN and PRN can be synthesized from the acetyl Co-A via malonic acidic pathway.
Further, it can be presumed that deciphering the pathway of GEN and PRN
production in P. tuberosa makes it possible for any biotechnological transformation

in the plant. Also, the quantified values of these two antidiabetics can be enhanced.
Acknowledgment Authors are thankful to the Gujarat State Biotechnology Mission (GSBTM,
Gandhinagar, Gujarat, India) for funding this research as a major project.
Author’s Contributions ICP was associated with supervising, designing, and guiding the exper-
imental work, analyzed all the data, and helped in writing the manuscript; MH carried out all the
phytochemical studies, generated all the data and figures, and wrote the initial draft of the
manuscript; RP performed experimental work of plant tissue; AS carried out review work and
helped in sampling; JP participated in planning and gave significant contribution in writing and
editing the manuscript. All the authors read and approved the final manuscript.
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In Vitro Exploitation of Medicinal Plants
for Continuous Supply of Antidiabetic 2
Bioactive Compounds
Subrata Kundu , Sk Moquammel Haque , and Biswajit Ghosh
Abstract
Diabetes mellitus, a complex metabolic disorder with multiple etiology, is one of

the key health problems among different countries all over the world. The
disruption of blood sugar homeostatic enforces profound consequences and
affects different organs including heart, eye, and kidney. Numerous classes of
drugs associated with antidiabetic potential with diverse mode of actions are
available in the market. The inexhaustible range of secondary metabolites of
medicinal plants has been extensively used as natural remedies against different
ailment. Regardless of the remarkable advancement in antidiabetic drugs, accep-
tance of medicinal plants as an alternative source has increased worldwide due to
comparative inexpensive nature and less adverse side effect. Plant tissue culture
techniques serve as substitution to entire plants for large-scale synthesis of
medicinally important secondary metabolites round the year regardless of the
geographical and environmental barriers. These advantages open an avenue for
the implementation of callus culture to induce the accumulation of therapeutic
compounds. Nevertheless, enrichment of secondary compounds with antidiabetic
activity in suspension cultures through elicitation is also a beneficial approach.
The advent of high throughput genomics and metabolomics tools, along with
S. Kundu · B. Ghosh (*)

Plant Biotechnology Laboratory, Post Graduate Department of Botany, Ramakrishna Mission
Vivekananda Centenary College, Rahara, Kolkata, India
S. M. Haque
Plant Biotechnology Laboratory, Post Graduate Department of Botany, Ramakrishna Mission
Vivekananda Centenary College, Rahara, Kolkata, India
Department of Botany, East Calcutta Girls’ College, Lake Town, Kolkata, India
e-mail: moquammel@ecgcollege.org

Ltd. 2021
https://doi.org/10.1007/978-981-16-3529-8_2
26 S. Kundu et al.
upgraded recombinant technology, explored exciting possibilities for elicitation

of antidiabetic therapeutic compounds. The present chapter enlightened the
current progress of plant tissue culture, specifically callus culture for the scaled-
up accumulation of antidiabetic compounds.
Keywords
Antidiabetic compounds · Callus culture · Cell suspension culture · Elicitor ·

Medicinal plants · Micropropagation · Hairy root culture · Plant biotechnology ·
Secondary metabolites
2.1 Introduction
Diabetes is a severe, metabolic disorder characterized by elevated amount of blood

glucose content that is a major public health concern in the twenty-first century
throughout the world due to its high occurrence, adverse side-effect on the quality of
life along with substantial economic penalties. It is a progressive disease due to
scarcity of either insulin or resistance to insulin. The interaction between numerous
genetic and ecological parameters resulted in variable grades of insulin resistance
and impaired function of pancreatic β cell (Stumvoll et al. 2005). Our current regime
with negligible physical activity along with lavishness of pleasurable foods
contributes to the amplified occurrence of this disease. Diabetes and fatness are
thoroughly connected with the expansion of severe impairment to the heart, nerves,
blood vessels, kidneys, and eyes. The disease is also connected with plethora of
symptoms such as polyuria, fatigue, weight loss, late wound healing, and upsurges in
urine glucose levels (Rahimi-Madiseh et al. 2017; Islam et al. 2018; Maideen and
Balasubramaniam 2018). The illness and death connected through diabetes will
continue to accelerate with our population ages and sedentary lifestyle. Diabetes
can execute a huge economic burden on patients suffering from diabetes along with
their relatives in terms of enormous health-care expenditures and damage of family
revenue connected with incapacity and early death. It has been reported that 463 mil-
lion persons are suffering from diabetes worldwide in 2019 and it is expected that
578 million people will be affected by diabetes in 2030 and the figure will rise by
51% (approximately 700 million) within 2045 (Saeedi et al. 2019).
There are miscellaneous treatments, such as adjustments of diet and exercise,
insulin replacement therapy along with glucose-lowering drugs (e.g., meglitinides,
metformin, sulphonylureas, α-glucosidase inhibitors) which are widely available to
manage diabetes. These drugs use antidiabetic effects by diverse mechanisms
including induction of insulin production by sulfonylurea and meglitinides, inducing
peripheral absorption of glucose through thiazolidinediones, interruption in the
assimilation of glucose from the intestine through alpha-glucosidase, and decrease
of hepatic production of glucose by biguanides (Hui et al. 2005; Bathaie et al. 2012).
Despite the advent of modern synthetic drugs for the management of diabetes, the
peoples in the developing countries are shifting toward the use of herbal medicine
2 In Vitro Exploitation of Medicinal Plants for Continuous Supply of. . . 27
due to the adverse effect of chemical drugs (Alarcon-Aguilara et al. 1998; Kar et al.
1999). Nevertheless, plant-based medicines are easily available as well as cost
effective compared to the traditional drug (Marles and Farnsworth 1995; Jafri
et al. 2000). According to the World Health Organization (WHO), up to 90% of
the peoples in underdeveloped countries are using medicinal plants as folk medicine
for wellness program (WHO 2002). Although doctors and consultants had used
diverse medicinal plants against diabetes, around 800 plants have been reported to
exhibit antidiabetic activity (Patil et al. 2011). However, among these medicinal
plants, a limited number was assessed in scientific laboratories for in vitro and
in vivo antidiabetic potential (Saad and Said 2011; Patil et al. 2011; Zaid and Saad
2013; Kadan et al. 2016; Subramoniam 2016; Krishna and Arun 2020). The medici-
nal plants with antidiabetic potential can be harvested from the wild source. This
practice ultimately resulted in habitat destruction along with reduction of the unique
valuable medicinal plant (Sivaraj et al. 2020). Hence, systematic commercial culti-
vation of medicinal plants can be implemented as alternative to collect from the
natural resource. Unfortunately, the diverse range of bioactive compounds
synthesized during cultivation were found to be reasonably altered compared to
wild populations due to diverse environmental conditions. Plant tissue culture
techniques serve as substitution to entire plants for commercial manufacture of
pharmaceutically vital metabolites round the year regardless of the variation in
environmental conditions (Saha and Pal 2020). The improvement accumulation of
antidiabetic compounds in cell suspension cultures through elicitation is also an
advantageous method. In this chapter, we have summarized the achievement
concerned with the in vitro culture established of various medicinal plants for
enhanced synthesis of antidiabetic compounds. Further, we have emphasized on
the efforts implemented by numerous scientists in the synthesis of antidiabetic
compounds by different biotechnological tactics, such as in vitro culture and elicita-
tion in cell culture.
2.1.1 Diabetes Mellitus: A Metabolic Disorder
Diabetes mellitus (DM) is a metabolic ailment consequential from insulin resistance

or the impaired secretion of insulin. This chronic disorder resulted in inaccurate
metabolism of carbohydrate, fat, and protein with concomitant enhanced levels of
serum glucose (Zaid et al. 2008). The major indications of diabetes comprise of
amplified thirstiness and appetite as well as recurrent urination. It can ultimately
result in serious problems including ketoacidosis, heart disease, impairment in
kidney function, eye damage, foot ulcer, and ultimately expiry (Kerner and Bruckel
2014). It has also been reported that diabetes is an outcome of inadequate regulation
of different genes as well as improper epigenetic controls. There are numerous genes
such as glucagon-like peptide-1 receptor (GLP1R) and paired box 4 (PAX4) that
regulate differentiation of pancreatic β cell. There are three main types of diabetes—
type 1 diabetes, type 2 diabetes, and gestational diabetes. Type 1 diabetes (insulin-
dependent or juvenile diabetes) that arises in children and teenagers is characterized
28 S. Kundu et al.
by inadequate insulin synthesis (Butalia et al. 2016; Krzewska and Ben-Skowronek

2016). Although precise reasons of Type 1 diabetes are unidentified, it is speculated
that complex interactions between genes and environmental factors are responsible
for the disease. It is related with the autoimmune impairment of pancreatic β cell
leading to insulin shortage (De Beeck and Eizirik 2016; Krzewska and
Ben-Skowronek 2016). The daily administration of insulin is prerequisite to main-
tain glucose homeostasis within the body. The major symptoms of type 1 diabetes
include frequent urination and thirstiness, continuous appetite, vision damage, and
tiredness. Type 2 diabetes (non-insulin-dependent) that is prevailing within enor-
mous number of people round the world is the concerns of body’s inadequate use of
insulin (WHO 1999; Bluestone et al. 2010). The major symptoms are alike to type
1 diabetes; however, they are generally not noticeable. Therefore, the ailment
remains untreated for numerous years. The occurrence of type 2 diabetes is expected
due to the complex interaction between genetic and metabolic factors. There are
several factors such as family history, earlier gestational diabetes combining with
oldness, weighty and fatness, improper diet, laziness, and smoking upsurging the
threat of incidence. Although type 2 diabetes was generally occurring in mature
persons, but nowadays it has started to be found in kids. Gestational diabetes is a
momentary ailment that happens during pregnancy and conveys longstanding threat
of type 2 diabetes (Bellamy et al. 2009). In this disorder, generally blood glucose
levels are found to be marginal above standard level but quiet under the investigative
of diabetes (WHO 2013). Pregnant women with gestational diabetes have higher
threat of certain problems during gestation. The occurrence of gestational diabetes is
growing universal and is the utmost general metabolic ailment throughout
pregnancies. The major causes of type 1 and type 2 diabetes are schematically
represented in Fig. 2.1.
2.1.1.1 Treatment of Diabetes in Conventional Medicine

The occurrence of diabetes is due to insulin resistance, insufficient insulin produc-
tion, dysfunction of pancreatic β cells, and extreme production of glucagon (Rasouli
et al. 2017). The prime threat for the prevalence of type 2 diabetes within adults
includes fatness, smoking, extreme drinking of alcohol, fatty food, and inactive
regime. The nonpharmacological approaches such as healthy diet, exercise, and
weight loss to maintain serum glucose homeostasis are some preventive approaches
to alleviate hyperglycemia and other associated symptoms. The implementations of
conventional drugs are prerequisite during failure of usual glucose metabolism. The
treatment of type 1 diabetes includes insulin replacement along with transplantation
of pancreatic islets (Poradzka et al. 2013). A plethora of drugs is available with
various mode of action such as induction of increased production and secretion of
insulin within pancreatic β cells, to treat the patients with type 2 diabetes (Bonora
et al. 2002). The most of the Food and Drug Administration (FDA)-approved drugs
are administrated to the patients in order to reduce blood glucose content by inducing
the production of insulin. The antidiabetic drugs are usually applied only or blended
with other antidiabetic compounds. The continuous use of synthetic drugs might not
happen due to its possible hazardous adverse effects. Nevertheless, the next
Fig. 2.1 The schematic representation of major causes of type 1 and type 2 diabetes
generation antidiabetic drugs mostly act through inducing production of insulin,

reducing release of glucose from liver, dropping the intestinal assimilation of
glucose, and successful disposal of peripheral carbohydrate (Prabhakar et al.
2014). The widely used drugs such as acarbose, voglibose, and miglitol hinder
activity of intestinal α-glucosidase while sulfonylurea-based drugs including
glimepiride and glyburide upsurge the accumulation of insulin. The principal draw-
back of the present antidiabetic drugs is their gastrointestinal penalties such as
stomach pain and diarrhea that are major adverse consequences of α-glucosidase
inhibitors. The abnormal lipolysis encourages hyperglycemia, decreased insulin
production, and glucose assimilation along with increased toxic lipid metabolites
30 S. Kundu et al.
in muscles, liver, and pancreas that ultimately resulted in cardiovascular diseases

(DeFronzo 2010). Similarly, overweight, heart attack, and anemia have been
described as substantial antagonistic consequences for thiazolidinediones. Addi-
tional impacts such as headache, faintness, vomiting, tiredness, indigestion, and
urinary tract infections also have been found for different antidiabetic drugs
(Dabhi et al. 2013). There are also certain drugs that are designed to inhibit lipolysis.
The utmost straight way to control hyperglycemic is through inhibition of glucose
assimilation in the intestine. Nowadays, biguanides, sulphonylurea with restricted
pharmacokinetic attributes, imperfect action, and less side effects have been
implemented for management of type 2 diabetes. However, the contemporary
implemented approaches for designing antidiabetic compounds unrevealed a novel
avenue for hunting natural and chemically manufactured compounds, but limited
invention has been found. The major obstacle in designing of drugs is due to their
improper interaction with several receptors associated with glucose transport. There-
fore, comprehensive understanding of molecular mode of action of antidiabetic
natural and synthetic compounds is very much needed to alleviate diabetic
difficulties.
2.2 Secondary Metabolites of Medicinal Plants: A Potential

Source of Antidiabetic Compound
Plants are the marvelous source for the detection of novel compounds with medicinal
importance in pharmaceutical industry. Plants that possess therapeutic metabolites
with valuable pharmacological effects are considered as medicinal plants. They are
the supreme versatile resource of drugs for the people worldwide. Miscellaneous
chemical compounds extracted from plants are rich source of vital drugs that are
presently used in different pharmaceutical organizations. The exploitation of plant
for the commercial synthesis of natural or reconstructed compounds has gained
extended interest in recent years (Canter et al. 2005). The medicinal plants are
receiving more consideration among the increasing population due to their
affordability, user-friendliness, environment-friendly, and encouraging effectiveness
compared to costly synthetic drug. Consequently, the prime consideration of
research on medicinally important plants has been intensive in the areas of pharma-
cognosy. The medicinal attributes of the plants are due to the presence of diverse
group of natural products termed as secondary metabolites that are diverse in their
chemical structure and in different metabolic pathways. These metabolites are not
vital for the growth and development but they exhibited key role in signal transduc-
tion pathways, adjustment of plants to their changing environment conditions, and
characterized due to the abundant source of pharmaceuticals (Rao and Ravishankar
2002; Kessler and Kalske 2018). They are synthesized by distracting energy-
generating paths in metabolic pathways such as glycolysis and Krebs cycle to
biosynthetic intermediates. They are classified in different groups based on their
biosynthetic origin, chemical structures, and functions (Kabera et al. 2014; Pagare
et al. 2015). The simplest classification includes three key categories: terpenoids
(polymeric isoprene derivatives), phenolics (comprising of hydroxylated aromatic

ring), and alkaloids (nonprotein nitrogen-containing compounds). The minor groups
include lipids, essential oils, carbohydrates, saponins, and ketones. They are
sequestered in particular tissues and organelle (e.g., vacuoles, trichomes), during
specified developmental stages and their accumulation is modulated by numerous
features such as genotype, climate, ecological conditions, and pathogen attacks
(Shitan 2016; Isah et al. 2018).
The extensive scientific reports have established that plant-based crude medicinal
preparations were applied to cope up with diabetes far before the implementation of
conventional therapies in rural and tribal area of the world (Marles and Farnsworth
1995; Modak et al. 2007; Campbell-Tofte et al. 2012; Khan et al. 2012; Raman et al.
2012; Behera and Yadav 2013; Gulshan and Rao 2013; Tripathi and Verma 2014).
Whereas the earliest approaches of management were initially based on speculative
and observed information, their longstanding presence verifies certain level of
effectiveness. It has been reported that over 1700 plants from different geographic
origin are used around the world to treat diabetes as traditional medicine within
diverse cultural groups (Marles and Farnsworth 1995). These traditional antidiabetic
plants were pharmacologically analyzed to identify specific bioactive compounds
associated with therapeutic value (Subramoniam 2016). Unfortunately, minimal
therapeutic potential as antihyperglycemic or hypoglycemic activities was observed
from most of these medicinal plants (Krishna and Arun 2020). Identification of
active principles and exploring their structure are prerequisite for development of
beneficial agents and learning their connections with another compounds in the host.
Numerous levels of research work including in vivo animal trials and in vitro assays
were executed on several medicinal plants for screening antidiabetic compounds.
The mode of actions of medicinal plants against diabetes are schematically
represented in Fig. 2.2. Nevertheless, mode of action was endeavored on limited
number of bioactive compounds. The antidiabetic compounds isolated from
corresponding medicinal plants with the mechanism of action are enlisted in
Table 2.1. The main secondary metabolites with antidiabetic activities are
polyphenols, terpenoids, nonstarch polysaccharides, and steroids. The chemical
structures of major antidiabetic compounds found within medicinal plants are
depicted in Fig. 2.3. The mechanisms of actions of active molecules of the
antidiabetic compounds are miscellaneous (Saravanamuttu and Sudarsanam 2012;
Chang et al. 2013; Singh et al. 2013; Arif et al. 2014; Gaikwad et al. 2014; Wang
et al. 2014a; Nazaruk and Borzym-Kluczyk 2015). Among them, antioxidant
polyphenols (flavonoids, anthocyanins, xanthones, tannins, etc.) decrease lipid per-
oxidation and oxidative stress in diabetic patients. The vital unrevealed mechanisms
of actions of active compounds include induction of insulin production from β cells,
decreasing resistance to insulin, mimicking insulin-like activity, triggering of aden-
osine monophosphate-activated protein kinase (AMPK) to reduce hepatic glucose
accumulation, activation of peroxisome proliferators-activated receptor-gamma
(PPAR-γ), inhibition of glucose absorption from intestine, and inhibiting glucose
reabsorption in the kidney. Consequently, modulation of specific immune response
by plant-based metabolites also delays or avoids the development of diabetes in
32 S. Kundu et al.
Fig. 2.2 Organ-specific effectiveness of antidiabetic medicinal plants
genetically susceptible persons. The summary of herbal along with non-herbal-based

treatment against diabetes is schematically depicted in Fig. 2.4.
2.3 Strategies for Continuous and Enhanced Production

of Antidiabetic Compounds
The commercial synthesis of plant secondary metabolites is very complicated due to

several reasons such as very low abundance, species- or genus-specific accumulation
along with growing phase-related constraint. Nevertheless, the accumulation of
numerous vital metabolites is also influenced by seasonal variation, biotic or abiotic
stress, or availability of nutrients (Verpoorte et al. 2002; Kala 2006). The chemical
syntheses of these compounds are also disadvantaged due to chemical intricacy,
stereo structure, and financial difficulties. To overcome these hindrances, a combi-
nation of traditional and modern technologies has been implemented as striking
substitution of the in planta manufacture of vital plant natural products. The major
benefit of current technologies is that it can provide uninterrupted, consistent source
of plant bioactive compounds and could be exploited for the wide-reaching culture
from which these metabolites can be efficiently extracted. Numerous biotechnologi-
cal approaches such as searching and collection of elite genotype, standardization of
media composition precursor supplementation, elicitation, commercial production in
bioreactor, hairy root culture, and metabolic engineering have been evaluated for
Table 2.1 The list of medicinal plants and their reported antidiabetic compounds along with mechanism of actions
2
Antidiabetic bioactive
Botanical names Family compounds Mechanisms of actions References
Alnusincana subsp. rugosa Betulaceae Oregonin Stimulates glucose transport and Eid and Haddad (2014)
(Du Roi) R.T. Clausen AMPK in cultured myocytes,
prevents differentiation and the
maturation of 3T3-L1 preadipocytes
Amorpha fruticosa L. Leguminosae Amorfrutins Activates of PPAR-γ Wang et al. (2014a), Weidner
et al. (2012)
Anthocleista Gentianaceae Bauerenone, bauerenol Inhibits α-glucosidase activity Mbouangouere et al. (2007)
schweinfurthii Gilg.
Artemisia dracunculus L., Compositae 6-Demethoxycapillarisin Inhibits the expression of Govorko et al. (2007), Wang
and 2,4-dihydroxy-4- phosphoenolpyruvate et al. (2008), and Obanda et al.
methoxy dihydrochalcone carboxykinase, activates PI3K (2012)
pathway; increases insulin
sensitivity, and attenuates the
FFA-induced upregulation of
protein tyrosine phosphatase
Azorella compacta Phil. Apiaceae Azorellanol Raises blood insulin level Fuentes et al. (2005)
Bauhinia forficata link. Leguminosae Kaempferitrin Induces insulin signaling pathway Tzeng et al. (2009), Ferreres
and stimulates production of et al. (2012)
adiponectin in 3T3-L1 adipocytes,
inhibits α-glucosidase activity
Berberis aristata DC Berberidaceae Berberine Enhances insulin action by Arif et al. (2014), Chen et al.
In Vitro Exploitation of Medicinal Plants for Continuous Supply of. . .
triggering AMPK, mimicks insulin (2010), Yu et al. (2010), and

function by inducing glucose uptake Cicero and Tartagni (2012)
by 3T3-L1 adipocytes and L6
myocytes in an insulin independent
manner, inhibits PTP1B activity and
enhanced phosphorylation of IR,
increases GLP-1 secretion in
streptozotocin-diabetic rats
33
(continued)
34
Table 2.1 (continued)

Caesalpinia sappan L. Leguminosae Brazilin Increases basal glucose transport in Kim et al. (1995), Liang et al.
3T3-L1 fibroblasts and adipocytes, (2013)
decreases the PKC activity in
3T3-L1 fibroblasts and adipocytes,
and inhibits adipocyte
differentiation
Cecropia peltata L. Urticaceae Chlorogenic acid, Blocks the hepatic glucose output, Andrade-Cetto and Wiedenfeld
isoorientin chlorogenic acid decreases glucose (2001), and Meng et al. (2013)
absorption from intestine
Commiphora mukul (hook. Burseraceae Commipheric acid Activates PPAR-γ and PPAR-α Wang et al. (2014b)
Ex stocks) Engl.
Curcuma longa Linn. Zingiberaceae Curcumin Exhibits PPAR-γ ligand-binding Lekshmi et al. (2012), Arun and
activity, inhibits α-glucosidase and Nalini (2002), and
α-amylase activities Chuengsamarn et al. (2012)
Glycine max (L.) Merr. Leguminosae Genistein Activates PPAR-γ Wang et al. (2014b)
Gymnema sylvestre R. Br. ex Apocynaceae Gymnemic acids Enhances accumulation of insulin Sugihara et al. (2000), Tiwari
Sm. from β-cells, inhibits absorption of et al. (2014)
glucose from intestine, modulates
incretin activity which triggered
insulin secretion
Hydrangea macrophylla Hydrangeaceae Hydrangeic acid Increases the amount of adiponectin Zhang et al. (2009), Arafa et al.
(Thunb.) Ser. released into the medium and (2017)
translocation of GLUT4 in 3T3-L1
cells
Lupinus mutabilis Sweet Leguminosae Conglutin-γ, lupanine, Increases glucose transport; García López et al. (2004),
multiflorine stimulates insulin signaling Terruzzi et al. (2011)
pathways
Morus nigra L. Moraceae Deoxynojirimycin Inhibits α-glucosidase activity Kumar and Chauhan (2008)
S. Kundu et al.
2
Paeonia suffruticosa Paeoniaceae Palbinone Activates AMPK, glycogen Ha et al. (2009)

Andrews synthase kinase-3β (GSK-3β), and
acetyl-CoA carboxylase (ACC)
phosphorylation in insulin resistant
human HepG2 cells
Phoradendron Santalaceae Oleanolic acid and ursolic Inhibits PTP 1B activity Nazaruk and Borzym-Kluczyk
reichenbachianum acid (2015)
(Seem.) Oliv.
Pseudolarix amabilis Pinaceae Pseudolaric acid Agonist activity against PPAR-γ Wang et al. (2014a)
(J. Nelson) Rehder
Pueraria montana var. Leguminosae Daidzein Activates PPAR-γ and PPAR-α Shen et al. (2006)
chinensis (Ohwi) Sanjappa &
Pradeep
Rheum palmatum Linn. Polygonaceae Emodin Induces accumulation of PPAR-γ in Xue et al. (2010)
liver and adipocytes
Robinia pseudoacacia var. Fabaceae Amorphastilbol Exhibits dual PPAR-γ/-α agonist Wang et al. (2014a)
umbraculifer DC. effects
Rosmarinus officinalis L. Lamiaceae Carnosic acid and carnosol Activates human PPAR-γ Wang et al. (2014b)
Salacia oblonga Wall. ex Celastraceae Salacinol; kotalanol Inhibits α-glucosidase activity, Matsuda et al. (1999)
Wight. & Arn. inhibits aldose reductase
Salacia reticulata Wight Celastraceae Salacinal and kotanol Inhibits α-glucosidase activity Ozaki et al. (2008)
Saururus chinensis (Lour.) Saururaceae Saurufuran A Exhibits agonist effect on PPAR-γ Wang et al. (2014b)
Baill
Symplocos paniculata Symplocaceae Ursolic acid Stimulates glucose uptake in L6 Jiang et al. (2012)
(Thunb.) Miq myotubes and facilitates GLUT4
translocation in CHO/hIR cells via
enhancing IR phosphorylation
Tabernaemontana bufalina Apocynaceae Canophylline Stimulates the differentiation of Chang et al. (2013)
Lour [synonym: Ervatamia progenitor cells into β cells
microphylla (Pit.) Kerr]
35
(continued)
36

Tabernaemontana divaricata Apocynaceae Conophylline Induces the differentiation of Fujij et al. (2009)
(L.) Roemer & Schultes pancreatic precursor cells to insulin-
producing cells in vitro
Tinospora cordifolia (Willd.) Menispermaceae Berberine Enhances insulin action by Arif et al. (2014), Chen et al.
Miers activating AMPK; mimicks insulin (2010), Yu et al. (2010), Cicero,
activity by enhancing glucose and Tartagni (2012)
uptake by 3T3-L1 adipocytes and
L6 myocytes in an insulin
independent manner; inhibits
PTP1B activity and increased
phosphorylation of IR, IRS1;
increases GLP-1 secretion in
streptozotocin-diabetic rats
Trifolium pratense L. Leguminoceae Genistein, biochanin A, Activates PPAR-γ ligand Wang et al. (2014a)
6-hydroxydaidzein
soflavones,
30 -hydroxygenistein
Trigonella foenum-graecum Leguminoceae Trigonelline Protects β cells from death and Puri et al. (2011), Hamden et al.
L. damage in diabetic rats; decreases (2013)
intestinal α-amylase, maltase and
lipase; increases serum insulin
levels; stimulates activity of
enzymes of glucose utilization in
diabetic rabbits
Vaccinium arctostaphylos L. Ericaceae Anthocyanin: Malvidin-3- Inhibits α-amylase, protects β-cells Nickavar and Amin (2004),
O-β-glucoside from oxidative stress and increased Sancho and Pastore (2012)
secretion of insulin
S. Kundu et al.
2
Zea mays L. Poaceae Hirsutrin Inhibits aldose reductase activity, Guo et al. (2009), Kim et al.
increases insulin level, and recovers (2013)
injured β-cells in diabetic rats
Zingiber officinale Roscoe Zingiberaceae 6-Shogaol and 6-gingerol Inhibits TNF-α-mediated Wang et al. (2014b)
downregulation of adiponectin
expression
37
38 S. Kundu et al.
Fig. 2.3 The chemical structures of major antidiabetic compounds found within medicinal plants
Fig. 2.4 The schematic representation of herbal along with non-herbal-based treatment against
diabetes
their efficacy to expand production of economically important metabolites through

in vitro plant cell and tissue cultures of different plant species. Plant cell and tissue
cultures contribute to a possible capacity for organized synthesis of innumerable
useful plant natural products.
2.3.1 Plant Cell, Tissue Culture, and Organ Culture for Antidiabetic
Compound Productions
The sessile plants synthesize versatile quantity of compounds not essential for
growth and development, but that exhibits a pivotal role in defense and adaptation
to the altering environment. The secondary metabolites, such as terpenes, steroids,
phenolics, and alkaloids, display varied array of activities and have huge possibility
in the pharmaceutical industries (Cusidó et al. 2013). Unfortunately, overexploita-
tion of these pharmaceutically important medicinal plants as a source of these
valuable compounds progressively amplified (Haque et al. 2017). However, the
accumulation of bioactive compounds is precisely confined to particular species or
genus and might be produced only within precise developmental stage or exact
environmental conditions. The medicinal plants difficult to commercially cultivate
are generally collected from its native source that ultimately raise the risk of
extinction (Yue et al. 2016; Kundu et al. 2017a, b). The in planta extraction of
pharmacologically important secondary metabolites is extremely challenging and
costly. Consequently, substantial work has been devoted in biotechnological manu-
facture of plant natural products through plant cell and tissue culture. The nutrition
enriched culture media augmented with several growth regulators along with con-
trolled aseptic conditions are used for in vitro growth of plant cells, tissues, and
organs (Murthy et al. 2014; Ochoa-Villarreal et al. 2016). Tissue culture has
developed as an imbedded method for culturing and understanding the functional
behavior of plant organs, tissues, and protoplasts in precise conditions.
Micropropagation is one of the utmost important approaches of plant tissue culture.
It offers copious benefits over traditional cultivation such as production of true-to-
type and aseptic plantlets of particular genotypes within reduced space in extremely
prompt way regardless of the seasonal and geographical variations (Haque et al.
2017; Haque and Ghosh 2020). Consequently, micropropagation methods are
advantageous not only for in vitro propagation but it also provides a dependable
approach for in vitro conservation of several rare and vulnerable germplasm (Haque
and Ghosh 2013; Kundu et al. 2015a, b). The plant tissue culture explored the
supreme region of application and gives a prominent lookout for near future.
However, quality control of tissue culture-raised plantlets is also very vital to
guarantee extensive plant secondary metabolites synthesis. The screening of explant,
establishment of sterile conditions, and stability of pharmaceutically important
compounds within the plants are certain vital parameters that must be assessed to
certify the superiority of the plants produced through tissue culture. Besides
micropropagation, plant tissue culture is comprehensively used for the commercial
production of plant natural products through callus and cell suspension culture
40 S. Kundu et al.
Fig. 2.5 Diagrammatic representation of different methods employed for commercial production
of secondary metabolites using plant tissue culture. (Source: authors)
(Alfermann and Petersen 1995; Rao and Ravishankar 2002; Verpoorte et al. 2002;
Kolewe et al. 2008; Lima 2009; Babu et al. 2016; Gantait et al. 2020). The
implementation of transgenic cultures and precise manipulation of biosynthetic
pathways permit extensive synthesis of pharmacologically important compounds.
The diverse approaches involved in commercial synthesis of secondary metabolites
through plant tissue culture are represented in Fig. 2.5.
2.3.1.1 In Vitro Propagation of Antidiabetic Plants

At the present time, in vitro plant cell and tissue culture technique is deliberated as
one of the rudimentary approach of modern plant biotechnology (Ghosh and Haque
2019). The remarkable advantages of in vitro mass propagation are that a huge
number of sterile and true-to-type plants can be regenerated within tiny space and
limited time (Haque and Ghosh 2016). In vitro mass propagation technique is also
independent of seasonal variation and continuous production can be possible
through this method (Haque and Ghosh 2018, 2020). Gymnema sylvestre is a
perennial woody plant with immense medicinal potential. It has been reported that
antidiabetic activities of G. sylvestre have been accredited due to the occurrence of
gymnemic acids (Liu et al. 1992; Parijat et al. 2007; Saha and Pal 2020). In vitro
shoot culture of G. sylvestre was developed by Syedy and Nama (2018) starting from
nodes in Murashige and Skoog (MS) medium (Murashige and Skoog 1962)
supplemented with 1.0 mg/L benzylaminopurine (BAP) and 2.0 mg/L 2.4-
dichlorophenoxyacetic acid (2,4-D). Zimare and Malpathak (2017) produced multi-
ple shoots in vitro and reported the accumulation of gymnemic acid in the extracts of
in vitro-cultured G. sylvestre. The multiple shoots were established from nodal

explants on MS medium supplemented with 5.70 μM of indole-3-acetic acid
(IAA), 4.44 μM of BAP, and 2.69 μM of naphthalene acetic acid (NAA). The low
temperature extraction of shoot powder in ethanol:water (1:1) was executed to
isolate gymnemic acid and measured through HPLC. Hence, antidiabetic properties
of Tinospora cordifolia have been documented in Ayurveda and even in current
modern researches. Among several phytochemicals, berberine has been reported
with potential antidiabetic activity (Cicero and Tartagni 2012). A proficient in vitro
regeneration protocol was developed for T. cordifolia using nodal explants on MS
medium supplemented with BAP (1.0 mg/L) and 2-iP (0.5 mg/L) (Mittal et al. 2017).
High frequency of multiple shoot formation with highest mean number of shoots
(7.9 0.45) per nodal explant was induced on the abovementioned media with
specified hormonal concentrations. Fenugreek (Trigonella foenum-graecum) is an
annual herb belonging to the family Leguminosae widely grown in Middle Eastern
countries (Alarcon-Aguilara et al. 1998). It has been reported that trigonelline, major
component of alkaloids in fenugreek, was responsible for reduction of blood glucose
level in human (Khosla et al. 1995; Al-Habori et al. 1998; van Dijk et al. 2009;
Olthof et al. 2011). Trigonelline guards β cells of the pancreas and rises insulin
sensitivity index as well as amount of insulin (Zhou et al. 2013). Optimized shoot
regeneration protocols from in vitro-grown cotyledon node explants was established
on MS medium supplemented with different concentrations of kinetin, BAP, NAA,
and thidiazuran (TDZ) with indole-3-butyric acid (IBA) (Aasim et al. 2010). Highest
shoot regeneration were found on MS medium supplemented with 0.40 mg/L TDZ
with or without IBA. In another study, plant regeneration via somatic embryogenesis
from cotyledonary node callus of T. foenum-graecum was also reported
(Al-Mahdawe et al. 2013). Callus cultures were derived from cotyledon node
segment excised from seedlings grown in MS medium supplemented with BAP
(2.0 mg/L) and NAA (1.0 mg/L). These cultures were induced to form somatic
embryoids in MS medium containing NAA (0.5 mg/L) with 15% v/v coconut water.
They were successfully rooted in PGR-free MS medium and transferred to soil. List
of important medicinal plants with antidiabetic compounds along with their estab-
lishment of optimized in vitro culture is tabulated in Table 2.2.
2.3.1.2 Callus and Cell Suspension Cultures

Callus are masses of undifferentiated fine-walled parenchyma cells that can be
friable or nonfriable (Frank et al. 2000; Evans et al. 2003; George et al. 2008;
Ikeuchi et al. 2013). The friable callus comprises of brittle cells and displays no
organ regeneration, whereas nonfriable nodular callus contains compactly wrapped,
unbreakable cells (Evans et al. 2003). The friable calli are mainly suitable for
development of cell suspension cultures, populations of quickly developing undif-
ferentiated cells cultured in liquid media (Chourey and Sharpe 1985; Evans et al.
2003). The fast rates of multiplication, shorter life span, and simplicity of mainte-
nance by subculturing makes cell suspension cultures convenient experimental
systems (Cai et al. 1987). The improvement of plant natural products synthesis in
cell suspension cultures over biotic or abiotic elicitation has established a
42
Table 2.2 The list of important medicinal plants with antidiabetic properties and their optimized in vitro propagation
Botanical names Family Mode of micropropagation Optimum PGRs for micropropagation References
Alnus incana Betulaceae Axillary propagation (shoot tip culture) 2.5 μM BA Périnet et al. (1988)
Amorpha fruticosa Leguminosae Axillary propagation (shoot tip and node 8.0 mg/L BA Gao et al. (2003)
L. culture)
Artemisia Compositae Axillary propagation (shoot tip culture) 0.6 mg/L BA and 0.05 mg/L NAA Kitto and Mackay
dracunculus L. (1992)
Callus-mediated regeneration For callus induction 0.1 mg/L 2,4-D; for Ibrahim et al. (2011)
shoot regeneration 0.5 mg/L BA and
0.05 mg/L NAA
Axillary propagation (shoot cutting) 0.1 mg/L IAA plus 0.5 mg/L KN Fernández-Lizarazo
and Mosquera-
Vásquez (2012)
Axillary propagation (shoot tip culture) 1.8 μM BA, 0.3 μM NAA Türközü et al. (2014)
Bauhinia forficata Leguminosae Callus-mediated regeneration from 4.0 mg/L BAP Melo et al. (2000)
Link hypocotyl segment explants
Berberis aristata Berberidaceae Indirect regeneration via callus from leaf For callus induction 4.53 μM 2,4-D, 2.22 Brijwal et al. (2015)
DC. explants. μM BAP, and 2.68 μM NAA; for shoot
regeneration, 0.5 μM TDZ; for root
induction, 50 μM IBA
Cecropia peltata Urticaceae Axillary propagation (shoot tip culture) 26.64 μM KN combined with 0.57 μM Nicasio-Torres et al.
L. NAA (2009)
Commiphora Burseraceae Axillary propagation (node culture) 5.0 mg/L BAP and 1.0 mg/L gibberellic Chaudhary (2005)
mukul (Hook. ex acid
Stocks) Engl.
Curcuma Zingiberaceae Axillary propagation (culture of active 0.2 mg/L KN and 2 mg/L NAA for Hashemy et al.
longa Linn. vegetative buds of 1–2 cm) multiple shoot induction (2009)
Axillary propagation (terminal bud For axillary bud proliferation 8.17 μM Prathanturarug et al.
explants) TDZ (2003)
S. Kundu et al.
2
Axillary propagation (terminal bud Multiple shoots were induced from bud Prathanturarug et al.
explants) explants in MS liquid medium augmented (2005)
with 72.64 μM TDZ
Axillary propagation (young vegetative 10 μM/L kinetin-riboside along with 1.0 Salvi et al. (2002)
buds from sprouting rhizomes) μM/L NAA
Axillary propagation (shoots from 4.44 μM of BAP and 1.08 μM of NAA de Souza Ferrari et al.
sprouting rhizomes) (2016)
Axillary propagation (vegetative shoot For axillary bud induction 2 mg/L BA and Jie et al. (2019)
buds), and callus-mediated regeneration 0.1 mg/L NAA found best for callus
induction 0.5 mg/L 2,4-D, and 2.0 mg/L p-
chlorophenoxy acetic acid and 0.1 mg/L
KN; for shoot differentiation from callus
0.5 mg/L TDZ and 0.1 mg/L BA
Gymnema Apocynaceae Axillary propagation (node culture) For multiple shoot induction, 4.44 μM of Zimare and
sylvestre R. Br. ex BAP, 2.32 μM of KN, and 2.69 μM of Malpathak (2017)
Sm. NAA; for root induction, 4.92 μM IBA
and 5.70 μM IAA
Callus-mediated regeneration For callus induction, 1.5 mg/L 2,4-D; for Cyriac et al. (2020)
shoot differentiation from callus, 1.0 mg/L
BAP; for root induction, 0.5 mg/L IBA
Axillary propagation (node culture) For multiple shoot induction, 1.0 mg/L Komalavalli and Rao
BA, 0.5 mg/L KN, 0.1 mg/L NAA, (2000)
100 mg/L malt extract and 100 mg/L citric
acid; for root induction, 3.0 mg/L IBA

Axillary propagation (mature axillary For multiple shoot induction 1.0 mg/L of Thiyagarajan and
node explants) BAP, 0.5 mg/L of KN and 1.0 mg/L of Venkatachalam
gibberellic acid; for root induction 2.0 mg/ (2013)
L IBA
(continued)
43
44

Callus-mediated somatic embryogenesis For embryogenic callus, induction 2.0 μM Ashok Kumar et al.
2,4-D and 1.0 μM BA; for embryo (2002)
maturation, 0.5 μM BA
Axillary propagation (apical buds as an For multiple shoot induction, 4.44 μM of Sharma and Bansal
explant) BAP, 4.64 μM of KN (2010)
Hydrangea Hydrangeaceae Axillary propagation (shoot tip explant) 1.0 mg/L BAP + 0.1 mg/L NAA + 1 mg/L Malyarovskaya and
macrophylla gibberellic acid Samarina (2017)
(Thunb.) Ser. Adventitious propagation through direct For direct shoot organogenesis, 2.25 mg/L Liu et al. (2011)
shoot organogenesis BA and 0.1 mg/L IBA; for root induction,
0.5 mg/L NAA
Lupinus mutabilis Leguminosae Axillary propagation (cotyledonary node) Combination of BAP and IBA Pniewski et al. (2002)
Sweet. Adventitious propagation through shoot Shoot induction, BA and NAA; for root Mulin and Bellio-
organogenesis from hypocotyl thin cell induction, NAA Spataru (2000)
layers
Morus nigra L. Moraceae Axillary propagation (apical and axillary For multiple shoot induction, 1.0 mg/L Duarte et al. (2019)
buds) BA; for root induction, 0.25 mg/L IBA
Axillary propagation (shoot tip and nodal For multiple shoot induction from both Yadav et al. (1990)
explant) types of explants, 1.0 mg/L BA proved
better. For root induction, 0.25 mg/L IBA
Axillary propagation (node explant) For multiple shoot induction, BA (2.0 mg/ Desai et al. (2018)
L), NAA (0.2 mg/L); for root induction,
0.50 mg/L IBA
Paeonia Paeoniaceae Callus-mediated regeneration For callus induction, 2,4-D (0.2 mg/L) + Zhu et al. (2018)
suffruticosa NAA (0.2 mg/L) + TDZ (3.0 mg/L); for
Andrews shoot organogenesis from callus BA
(2.0 mg/L) + NAA (0.2 mg/L) + TDZ
(0.3 mg/L); for root induction, 0.1 mg/L
NAA and 0.05 mg/L IBA
S. Kundu et al.
2
Axillary propagation (node explant) For multiple shoot induction, 4.44 μM of Beruto et al. (2004)
BAP; for root induction 49.20 μM IBA
Pseudolarix Pinaceae Indirect somatic embryogenesis and shoot For somatic embryogenesis 2,4-D and Zican et al. (1995)
amabilis organogenesis via callus phase BA; for shoot organogenesis, BA and KN
(J. Nelson) Rehder
Pueraria Leguminosae Axillary propagation (shoot tip culture) For axillary bud proliferation, 0.1 mg/L Ma et al. (2013)
thomsonii Benth. BA and 0.5 mg/L 2,4-D; for in vitro
rooting, 0.5 mg/L NAA
Axillary propagation (shoot tip culture) For axillary bud proliferation, 1.0 mg/L Zhou et al. (1994)
BA, 1.0 mg/L 2,4-D and 0.2 mg/L NAA;
for in vitro root induction, 0.2 mg/L IBA
Salacia Celastraceae Axillary propagation (node culture) 3.5 mg/L BAP and 1.0 mg/L IBA Deepak et al. 2015
oblonga Wall.
Tinospora Menispermaceae Axillary propagation (nodal explant) For multiple shoot induction, 8.87 μM Raghu et al. (2006)
cordifolia (Willd.) BA; for shoot elongation, 2.22 μM BA and
Miers 4.65 μM KN; for root induction, 2.85 μM
IAA
Adventitious propagation through indirect For callus induction IAA (2.0 mg/L) same Mridula et al. (2017)
and direct shoot organogenesis PGRs combination was found best for
both indirect shoot organogenesis via
callus as well as direct shoot
organogenesis BA (2.0 mg/L) + KN
(1.0 mg/L) + IAA (0.5 mg/L) + gibberellic
acid (0.5 mg/L) + ascorbic acid (100 mg/

L), for root induction, 0.5 mg/L IBA and
0.5 mg/L NAA
Axillary propagation (nodal explant) For multiple shoot induction BA (4.44 Mittal et al. (2017)
μM) + N6-2-iso-pentenyl adenine (2.45
μM); for root induction IBA (2.45 μM)
(continued)
45
46

Trigonella Leguminoceae Adventitious regeneration through indirect Best result in 0.4 mg/L TDZ Aasim et al. (2010)
foenum-graecum shoot organogenesis
L. Callus-mediated somatic embryogenesis For embryogenic callus induction, 2.0 mg/ Al-Mahdawe et al.
L BAP and 1.0 mg/L NAA; for embryo (2013)
induction, 0.5 mg/L NAA and 15% v/v
coconut water
Callus-mediated shoot organogenesis For callus induction, 0.5 mg/L BAP and Burdak et al. (2017)
0.5 mg/L 2,4-D; for shoot induction from
callus, 0.5 mg/L BAP; for root induction,
0.2 mg/L IAA
Vaccinium Ericaceae Axillary propagation via lateral-bud For multiple shoot induction, 1.0 mg/L Cüce et al. (2013)
arctostaphylos L. cultures zeatin and 0.1 mg/L IBA; for root
induction, 0.5 mg/L IBA
Axillary propagation via node cultures For shoot proliferation, 2.0 ppm zeatin and Bakhshipour et al.
IBA 0.1 ppm; for root induction IBA, (2019)
1.0 ppm
Zingiber officinale Zingiberaceae Axillary propagation (vegetative buds 4.0–5.0 mg/L BAP Bhagyalakshmi and
Roscoe culture) Singh (1988)
Axillary propagation (rhizome buds 3.0 mg/L BAP Balachandran et al.
culture) (1990)
Callus-mediated regeneration from leaf For callus induction, 13.6 μM 2,4-D; for Babu et al. (1992a)
explants shoot organogenesis 0.9 μM 2,4-D and
44.4 μM BA
Axillary propagation (immature 10.0 mg/L BA and 0.2 mg/L 2,4-D Babu et al. (1992b)
inflorescence culture)
Callus-mediated regeneration from ovary For callus induction, 0.5 mg/L 2,4-D and Babu et al. (1996)
explants 1.0 mg/L BA; for shoot induction, 0.2 mg/
L 2,4-D and 10.0 mg/L BA
S. Kundu et al.
2
Axillary propagation (active vegetative 2.0 mg/L KN and 3.0 mg/L NAA Sharma and Singh
buds culture) (1997)
Callus-mediated regeneration from anther For callus induction, 3.0 mg/L 2,4-D; for Samsudeen et al.
explants shoot organogenesis, 0.2 mg/L 2,4-D and (2000)
10.0 mg/L BA
Axillary propagation (meristem culture) 26.6 μM BAP, 8.57 μM IAA, 1111.1 μM Rout et al. (2001)
adenine sulfate
Cell suspension culture and plant For Somatic embryogenesis, 0.2 mg/L Guo et al. (2005)
regeneration via somatic embryogenesis 2,4-D and 5.0 mg/L BA; For embryo
germination, 3.0 mg/L BA and 0.1 mg/L
NAA
Different methods Different PGRs for different methods Babu et al. (2005)
Somatic embryogenesis via callus culture, For somatic embryogenesis, 5.0 mg/L BA Guo et al. (2007)
protoplasts culture from embryonic cell and 0.2 mg/L 2,4-D; for conversion of
suspension and plant regeneration embryo to complete plantlet, 3.0 mg/L BA
and 0.1 mg/L NAA
Axillary propagation through 3.0 mg/L KN Zheng et al. (2008)
microrhizome formation
Axillary propagation (vegetative buds 0.57 μM BAP Kavyashree (2009)
culture)
Axillary propagation from sprouting bud 4.5 mg/L BAP Abbas et al. (2011)
explant
BAP 6-benzylaminopurine, BA benzyladenine, KN Kinetin, 2,4-D 2,4-dichlorophenoxy acetic acid, NAA α-naphthaleneacetic acid, IAA indole-3-acetic acid, IBA
indole-3-butyric acid, TDZ thidiazuron
47
48 S. Kundu et al.
prospective biotechnological method for commercial manufacture of medicinally

important molecules.
The enrichment of gymnemic acids in the suspension cultures of G. sylvestre by
heavy metal stress has been established (Ch et al. 2012). Likewise, the establishment
of cell suspension culture of G. sylvestre in liquid media augmented with kinetin and
different concentration of sucrose was also reported (Nagella et al. 2011). It was
found that addition of 3% w/v sucrose within culture media was found to be vital
factors for synthesis of gymnemic acids and biomass in suspension culture. Among
different metal salts, cadmium chloride at 2 mM concentration induces maximum
gymnemic acid accumulation after 12-day time period. The accumulation of
gymnemic acid from G. sylvestre callus upon salt stress has also been confirmed
(Kumar et al. 2010). The callus was developed using leaves as explant on MS
medium augmented with plant growth regulators. The highest gymnemic acid was
reported from cultures in MS medium fortified with 3 mg/L sucrose, 3.0 mg/L of
2,4-D, 1.0 mg/L of BAP, and 100 mM sodium chloride. In another study, cell
suspension cultures of G. sylvestre was optimized and elicited with methyl
jasmonate, yeast extract, chitin, and pectin for upgraded manufacture of gymnemic
acid (Veerashree et al. 2012). The 5.25-folds higher accumulation of gymnemic acid
was achieved after 20 days of elicitation supplemented with 0.5 g/L yeast extract
compared to control culture. The endophytic fungus was isolated from the leaves of
G. sylvestre and recognized as Polyancora globosa and Xylaria sp. based on 18S
rRNA signature sequence. It has been reported that the cultures treated with the dried
powder of both fungal mycelia exhibited noticeable effect on the synthesis of
gymnemic acid and 139.98 mg/g dry cell weight (DCW) were achieved (Netala
et al. 2016). The effect of 2,4-D and kinetin was studied in callus culture of
G. sylvestre (Ahmed et al. 2009). The authors described that blue light increases
up to 4.4-fold higher accretion of gymnemic acid compared to control (fluorescent
light). The effects of biotic elicitors (cell-free extracts of different fungus and
bacteria like Aspergillus niger, Saccharomyces cerevisiae, Agrobacterium
rhizogenes, Bacillus subtilis, and Escherichia coli) were examined in the accumula-
tion of plant natural products in cell culture of G. sylvestre (Chodisetti et al. 2013).
Among all these biotic elicitors, A. niger was most efficient in improving production
of gymnemic acid (98.65 0.93 mg/g DCW) compared to untreated control culture.
In another study, nine-fold upsurge of gymnemic acid was also reported in suspen-
sion culture using A. niger as biotic elicitor (Devi and Srinivasan 2011). The
enhanced accumulation of gymnemic acid (135.41 0.43 mg/g DCW) in cell
suspension culture was also reported upon elicitation of methyl jasmonate
(Chodisetti et al. 2015). The cell suspension culture of G. sylvestre for manufacture
gymnemic acid was also optimized through modulation of amount of macro
elements and nitrogen source (Praveen et al. 2011). The highest accumulation of
gymnemic acid (11.32 mg/g DCW) was recorded in the medium augmented with
2 amount of potassium dihydrogen phosphate. The cell suspension culture of
T. foenum-graecum was first successfully established for production of trigonelline
in 1980 (Radwan and Kokate 1980). They have reported that maximum levels of
trigonelline were accumulated in suspension cultures maintained for 1–2 months on
modified Gamborg B5 medium (Gamborg et al. 1968) augmented with 2,4-D as a

plant growth regulator. It was also found that precursor feeding of nicotinic acid in
culture medium raised some extent of trigonelline production. Later on, cell suspen-
sion cultures of fenugreek originated from the cotyledon of seeds were also
established and maintained on MS medium augment with 2,4-D acid and kinetin
(Abd-El Mawla and Osman 2011). The treatment of 100 μM methyl jasmonate in
suspension cultures exhibited observable increase in trigonelline content. The
antidiabetic properties of Cecropia obtusifolia are accredited due to the existence
of two phenolic compounds—chlorogenic acid and isoorientin (Rodriguez de Sotillo
and Hadley 2002). The cell suspension cultures were established using leaf as
explant and with augmentation of BAP and 2,4-D in mixture within culture medium
(Nicasio-Torres et al. 2012). In the present study, a significant increase in both these
antidiabetic compounds was verified with decreasing nitrate content in the medium.
Ginger (Zingiber officinale Roscoe) is one of the extensively used herbs that
comprise numerous bioactive constituents and owns different healthiness attributes.
The antidiabetic properties of 6-shogaol and 6-gingerol have also been reported
(Wang et al. 2014a). The callus cultures of ginger were established from shoot tip of
auxiliary buds collected from in vitro growing rhizomes (El-Nabarawy et al. 2015).
It was recorded that 6-gingerol production was meaningfully increased by the
supplementation of mevalonic acid in the medium. The callus culture of Curcuma
longa Linn. was also induced and established from leaf explants with a combination
of phytohormones (Gurav et al. 2020). The maximum callus induction was found
with MS medium supplemented with 3.0 mg/L 2,4-D and 0.8 mg/L kinetin. The
phytochemical corroborating with chromatographic analysis of callus extract
showed the existence of 36.1 mg/g curcumin dry weight. The callus culture of
T. cordifolia with higher berberine contents was established from nodal explants
on MS medium supplemented with different types and concentrations of auxins
(Pallia and Siril 2020). The optimized concentration was found to be 5 μM NAA for
both callus fresh weight and berberine content. A noteworthy upsurge (3.19-fold) in
berberine content was attained through adding of 40 μM tyrosine (precursor) in the
MS medium.
2.3.1.3 Elicitors: A Trigger for Enormous Production of Secondary

Metabolites in Cell Culture
Plant secondary biosynthetic pathways can be remarkably induced by “elicitors” to
enable boosted metabolite synthesis by cell tissue and organ cultures. Elicitors are
considered as molecules of biotic or abiotic source that create alike response as stress
within the plants (Zhao et al. 2005; Namdeo 2007; Ferrari 2010; Onrubia et al. 2013;
Baenas et al. 2014; Murthy et al. 2014). They can modify physiological or metabolic
events within the plants by modulating different signaling pathway thereby altering
the gene expression patterns. Elicitation is one the utmost effective methods pres-
ently applied for enhancing the biotechnological synthesis of plant natural
products (Gorelick and Bernstein 2017). The elicitors can be classified into two
different types—biotic and abiotic. Abiotic elicitors are defined as compounds of
nonbiological origin, primarily inorganic compounds such as salts, metal ions,
50 S. Kundu et al.
Fig. 2.6 Diagram of multitasking skill of the elicitors along with their mode of action. (Source:
authors)
hormone, or physical factors. Biotic elicitors are derived from living origin including
carbohydrates from plant cell walls (e.g. chitin, pectin, and cellulose) and signaling
or surface compounds from fungi and bacteria. Both biotic and abiotic elicitors have
the ability to enhance the gathering of terrestrial plant defense-related metabolites of
diverse chemical classes, such as sesquiterpene lactones, anthocyanins, and
flavonoids. The elicitors interact with the plasma membrane receptors of numerous
plant cells by inducing an array of defense responses. The multitasking skill of the
elicitors along with their mode of action is schematically represented in Fig. 2.6. The
signal perceived by the receptors is associated with the action of secondary
messengers that consequently intensify the signal for downstream pathways. The
consecutively happening molecular events in elicitor-induced resistance responses
can be briefed as follows: recognize the elicitors; reversible phosphorylation and
dephosphorylation of plasma membrane proteins and cytosolic proteins; induction of
cytosolic calcium ion concentration; activation of mitogen-activated protein kinase
(MAPK); induction of NADPH oxidase followed by production of reactive oxygen
species (ROS); expression of defense-related genes; and phytohormone gathering.
The outcome of this altered phenomenon is the increased in planta synthesis of
secondary metabolites. A huge number of chemical elicitors such as jasmonic acid,
methyl jasmonate, salicylic acid, acetyl salicylic acid, ethylene, heavy metals,
different natural, or synthetic chemical compounds alone or in different
combinations are applied for elicitation in plant cell suspensions and hairy roots
cultures (Oikawa et al. 2001; Mithofer et al. 2004; Savitha et al. 2006).
Subsequently, elicitors are found to be the utmost supportable and environment-

friendly system to obtain chemically complex secondary metabolites biosynthesized
in endangered plants that resist domestication.
2.3.1.4 Hairy Root Cultures

Hairy roots are induced from the wounding site of plantlets by the infection with
Gram-negative symbiotic soil bacterium Agrobacterium rhizogenes (taxonomically
renamed Rhizobium rhizogenes). During the infection, specific bacterial DNA
fragments (T-DNA) from their root-inducing plasmid (Ri plasmid) are relocated
into the genome of the infected plant (Chilton et al. 1982; Gelvin 2009). To prevent
the infection, plants activate the expression of numerous defense-related proteins.
R. rhizogenes has evolved apparatuses to exploit the defense-related proteins by
countering action that ultimately perturbs plant defense paths (Sevón et al. 1997;
Mauro et al. 2017). Likewise, hairy roots resulting from R. rhizogenes-mediated
transformation can be achieved from a vast range of plants and have permitted
fruitful accumulation of different secondary metabolites exhibiting fascinating
features (Ray et al. 1996; Ghosh et al. 1997; Mukherjee et al. 2000; Chandra and
Chandra 2011; Häkkinen and Oksman-Caldentey 2018; Gantait and Mukherjee
2021). Due to greater level of cellular differentiation, prompt growth compared to
native-grown plant roots, genetic and biochemical constancy, and better preservation
facility, the hairy root cultures have gained an increasing interest among biotechnol-
ogy companies and pharmaceutical industries. In opposing to cell suspensions, hairy
roots have the ability to secrete expressed transgenic proteins in culture medium
thereby opening an avenue for convenient method for target protein purification
(Wang and Wu 2013). Another striking feature of hairy roots is the competency to
synthesize the targeted phytochemicals concurrently with development. Therefore,
contrasting the accumulation being suppressed throughout the development phase of
dedifferentiated cell culture, it is capable to acquire continuous synthesis of the
preferred secondary metabolites from developing hairy roots. Hairy root cultures are
proficient for the large-scale manufacture of complex compounds in bioreactor in the
culture media under controlled and compact conditions (Häkkinen and Ritala 2010;
Stoger et al. 2014). Furthermore, hairy roots can be genetically modified, thus
permitting variation of metabolite accumulation through RNA interference or
genome editing, along with the supplementation of exogenous compounds or
elicitors. Elicitation is a potential approach to overcome several problems related
to the large-scale accumulation of industrially vital secondary metabolites from hairy
root cultures. Hairy root cultures are favored for the implementation of elicitation
owing to their genetic and biosynthetic constancy, higher growth rate, and uninter-
rupted manufacture in response to elicitor treatment. Standardization of diverse
parameters such as type, concentration, length, and treatment schedule of elicitors
is indispensable for the efficiency of the elicitation approaches. Collective imple-
mentation of diverse elicitors, addition of precursors, replacement of medium, or in
situ product retrieval from the liquid medium has exhibited better gathering of plant
natural compounds due to their synergistic effect.
52 S. Kundu et al.
Transformed hairy-root cultures of G. sylvestre have been established by

infecting cotyledons and leaf explants by Agrobacterium rhizogenes strain KCTC
2703 (Nagella et al. 2013). The highest accumulation of biomass was reported in
hairy root cultures in liquid medium augmented with 3.0% sucrose after 25 days.
The gymnemic acid was also induced in the hairy root cultures and found to be
11.30 mg/g dry mass at 20 days of culture. The polyunsaturated fatty acids (PUFAs)
such as oleic and linolenic acid was added in different concentrations as elicitors in
hairy root cultures of G. sylvestre on 15th day to improve biomass buildup and
gymnemic acid synthesis (Praveen et al. 2014). Significant improvement of the
gymnemic acid content was observed upon supplementation of linolenic acid and
it was reported that 7.78-fold escalation of gymnemic acid content with elicitation of
5.0 μM linolenic acid compared to control. Another elicitor oleic acid also amplified
the content of gymnemic acid but not in significant quantities compared with the
linolenic acid. Modulation in total phenolics, flavonoids, and antioxidant activity in
hairy root cultures was also corroborated in the elicitation of PUFAs. The improved
production of antidiabetic compound trigonellin through hairy root cultures of
T. foenum-graecum was reported in different Iranian masses (Raheleh et al. 2011).
The A. rhizogenes strains A4, 9126, and 15834 were tried to examine the capacity for
transformation and accumulation of trigonellin in T. foenum-graecum. Among them,
maximum growth index was recorded in hairy roots induced by strains 15834 and
9126, in Zanjan and Borazjan masses, respectively. The maximum amounts of
trigonellin in Borazjan and Zanjan hairy roots were found to be 14.89 and
14.03 mM/g DW after 28 and 7 days of culture, respectively. The hairy root cultures
of T. foenum-graecum were optimized by transformation of different strains of
A. rhyzogenes (Qaderi et al. 2016). Two different elicitor’s methyl jasmonate and
chitosan at various amount were added in liquid medium to induce trigonelline
content. The highest trigonelline (36.7 and 37.3 mM/g DW) was reported in
100 μM of methyl jasmonate and 150 mg/L of chitosan, respectively.
2.4 Conclusion
Diabetes mellitus is the utmost common endocrine ailment manifested by constant

hyperglycemia due to lessened insulin production or insulin resistance, impaired
function of pancreatic β cells, and extreme production of glucagon. The occurrence
of different types of diabetes is increasing worldwide due to sedentary lifestyle,
extreme ingesting of alcohol, and diet enriched with fat. Diabetes has been consid-
ered to be the main source for disturbing the economic conditions of patients and
their families. Additionally, unrestrained diabetes leads to chronic difficulties such
as impaired vision and severely disturbing kidney and heart. Irrespective of the
progresses in current therapies, diabetes is a main reason of illness and death
throughout the world populations. The modern medicine used for the treatment of
diabetes has severe consequences; therefore, there is unremitting hunt for more
effective and harmless hypoglycemic agents. Traditional herbal medicine and eth-
nobotany have played pivotal role in treatment and managing diabetes mellitus with
standard drugs for combinatorial therapies. These medicinal plants possess their
specific bioactive secondary metabolites that have potential to control blood sugar
levels as well as minimize the associated problems of diabetes. Therefore, extensive
research has been focused on extraction, purification, and functional characterization
of medicinally important metabolites in medicinal plants. Unfortunately, large-scale
extraction of these medicinally important phytochemicals from field grown plants
have several limitations such as difficulties in cultivation, lower productivity, geo-
graphical and seasonal disparity in accumulation, tissue/organ-specific sequestra-
tion, complications of extractions, and unevenness of impurities. Moreover,
chemical synthesis is economically not possible due their highly complex structures
and stereospecificity. However, in planta synthesis of pharmaceutically important
secondary metabolites is very much complicated due to participation of several
genes and multifaceted nature of biosynthetic pathways. Under such circumstances,
different plant biotechnological methodologies such as in vitro culture, callus cul-
ture, micropropagation, and hairy root culture have been implemented for commer-
cial production of pharmaceutically important plant natural products. The
optimization and enhancement in the synthesis of limited antidiabetic compounds
through elicitation in hairy root culture was effectively implemented. The improve-
ment strategies will also be more productive if transgenic approaches are applied on
productive chemotypes of medicinal plants with antidiabetic compounds. Targeted
metabolic engineering executed by high-throughput molecular biological methods
and manipulation of specific biosynthetic pathways explore new avenue to defeat the
insufficiency of bioactive metabolites at commercial levels. The employment of
multidisciplinary “omics” approach along with system biology will explore our
understanding of comprehensive biosynthetic pathways that would be very useful
to precisely build model toward enhanced and continuous supply of antidiabetic
compounds at industrial scale.
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Lower Plants as Potential Source
of Antidiabetic Compounds: The Present 3
Knowledge and Future Prospects
Bhuvnesh Sareen and Amita Bhattacharya
Abstract
The antidiabetic potentials of algae, bryophytes, and pteridophytes have been

reviewed in this chapter. The aim was to provide a compilation as well as a ready
reckoner of the available information for further exploitation. The pharmacologi-
cal studies conducted with these plants and their bioactive compounds have been
discussed. Various extracts of lichens, liverworts, and mosses, as well as a
number of algae and pteridophytes and their bioactive compounds, were found
to possess significant α-glucosidase and α-amylase inhibitory activities. Many of
these also exhibited blood glucose lowering activities. These indicated the
antidiabetic potential of these plants. The limitations of using these lower plants
for commercial production of antidiabetic therapeutics and their future prospects
have also been discussed.
Keywords
Algae · Bryophytes · Blood glucose lowering property · α-Glucosidase inhibitory
activity · Pteridophytes · Type 2 diabetes mellitus
3.1 Introduction
Diabetes mellitus (DM) is a chronic and complex metabolic disorder affecting 25%
of the world population with India, China, and the USA being the countries with
consistently maximum number of diabetic people (Fig. 3.1) (Wild et al. 2004;
B. Sareen · A. Bhattacharya (*)

Division of Biotechnology, CSIR-Institute of Himalayan Bioresource Technology, Palampur,
Himachal Pradesh, India
e-mail: amitabhatta@ihbt.res.in

Ltd. 2021
https://doi.org/10.1007/978-981-16-3529-8_3
66 B. Sareen and A. Bhattacharya
Fig. 3.1 Incidence of diabetes in different countries between 1995 and 2030
Fig. 3.2 Prognosis on the incidence of diabetes in different countries between 2000 and 2030
based on the data of Wild et al. (2004)
Fig. 3.3 Incidence of diabetes. (a) Global increase in diabetic patients. (b) Predicted increase in
diabetes in different countries between 2017 and 2045
Arumugam et al. 2013). The disease is increasing at such an alarming pace that it has
emerged as the fifth leading cause of death worldwide (Roglic et al. 2005). As per
prognoses, the number of diabetic people from top ten countries is about to increase
enormously by 2030 (Fig. 3.2) with almost 578 million diabetic people from mostly
China, USA, Indonesia, India, Brazil, Mexico, Japan, Pakistan, Thailand, and
Nigeria (International Diabetes Federation, IDF 2019; Tran et al. 2020). As per the
International Diabetes Federation report, however, diabetes will affect 221 countries
by 2045 (Fig. 3.3a), and South East Asian countries will record the highest incidence
3 Lower Plants as Potential Source of Antidiabetic Compounds: The Present. . . 67
of the disease (Fig. 3.3b). With China followed by India, fast becoming the diabetic
capitals of the world, the prevalence will be especially more in the Asian countries
(Yang et al. 2010; Chen et al. 2012; Hussain 2018). India with about 72 million of
already diabetic people in 2017 will emerge as a hot spot of diabetes with 134 million
affected people by 2045 (IDF Report 2017). The risk of heart diseases, kidney
failure, loss of eye sight, rates of amputation, and also death are associated with
diabetes (Hippisley-Cox and Coupland 2016). Thus, the disease became the fourth
prevalent disability in 2017 according to the report of “Global Burden of Disease,
Injuries and Risk Factors” (Institute for Health Metrics and Evaluation 2018). This is
directly and/or indirectly adding to the national health expenses of different
countries (Zheng et al. 2018). Since by 2030, the diabetic population above the
age of 64 is predicted to increase to more than 82 million in developing countries and
48 million in developed countries (Wild et al. 2004), the fear that diabetes will
impose a huge socioeconomic burden in a number of nations (including the poor and
developing ones) is looming large (Beagley et al. 2014; Rawshani et al. 2018). It is
estimated that the expenditure due to the disease alone will be about 776 USD by
2045.
The reasons for this consistent increase in diabetes have been attributed to
unhealthy eating habits, dependence on fast food, obesity, aging, economic devel-
opment, urbanization, and sedentary life style (Chatterjee et al. 2017; Zheng et al.
2018). As a result, antidiabetic medicines/drugs are increasingly becoming the
lifeline of diabetic patients, irrespective of their affordability.
3.2 Antidiabetic Drugs
Mainly, six different classes of drugs have been recommended for the prevention and
management of diabetes, namely, sulfonylureas, biguanides, thiazolidinediones,
meglitinides, α-glucasidase inhibitors, and dipeptidyl peptidase-4 inhibitors (Tahrani
et al. 2016). These function by lowering the blood glucose level and/or managing
associated complications. Their multiple mechanisms of action involve (1) stimula-
tion of pancreatic β cells, (2) improved performance of pancreatic tissues, (3) inter-
ference with hormones related to increased blood glucose, (4) increased sensitivity
of insulin receptors, (5) improved metabolism of macromolecules, (6) prevention of
glycogen hydrolysis, (7) modification of the lipid peroxidation process, (8) increase
in glucose transport activity, (9) reduced absorption of glucose, and (10) prevention
of gluconeogenesis (Bösenberg and Van Zyl 2008; Thilagam et al. 2013). Among
these, the thiazolidinediones activate the peroxisome-proliferator-activated receptors
(PPARs) for mitigation of insulin resistance (Monsalve et al. 2013; Lefterova et al.
2014; Shao et al. 2016). Hence, the PPARs, particularly, the PPAR-Υ, are the
preferred class of antidiabetics for lowering the blood glucose level. However,
most antidiabetic drugs impose various adverse effects on human health (Jain et al.
2005; Lee et al. 2014; Tran et al. 2020). Gastrointestinal problems such as dyspepsia,
nausea, diarrhea, and also weight gain and fluid retention are some of the common
symptoms. Often, these turn into serious medical complications such as
cardiovascular risks, liver problems, bladder cancers, toxicity, drug resistance, etc.,
in diabetics, particularly, the kidney-compromised patients (Rizos et al. 2009; Piper
and Saad 2017). Antidiabetic drugs also have limitations in terms of cost and
affordability. Therefore, more and more people are turning to natural products and
medicinal plants because of their lower cost, appreciable efficacy, and minimal to no
side effects. Since these have significantly lower costs than commercial preparations,
they often serve as the “most sought after and affordable alternatives” for diabetic
patients (Evans 1994; Arumugam et al. 2013).
3.3 Antidiabetic Medicinal Plants
Presently, about 80% of the world’s population (including the ones from developed
or developing countries) depend on “medicinal-plant-based traditional or alternative
medicine(s)” either directly or indirectly (Ekor 2014; Mishra et al. 2016). In particu-
lar, plant extracts have emerged as attractive and affordable treatment for different
ailments in traditional or alternative systems of medicine. The efficacy of these
phytomedicines is generally attributed to either singular or synergistic effects of an
array of bioactive components present in specific plant parts (Ignacimuthu and
Ayyanar 2006; Elujoba et al. 2005). Many of these bioactive molecules from plants
have been identified as “antidiabetic(s)” (Salehi et al. 2019). While some of these are
being used for their blood glucose-lowering and other properties (Ribnicky et al.
2006; Vinayagam et al. 2017; Singab et al. 2014), others are gaining importance as
therapeutics in various pharmaceutical industries (Zaid et al. 2015; Gothai et al.
2016). One such example of a plant-based therapeutic for prevailing Type 2 diabetes
is “metformin” derived from the plant, Galega officinalis (Hussain 2002; Oubre et al.
1997). While the original plant-derived medicine is effective with hardly any side
effects, its commercial but synthetic versions are considered to have significant side
effects (Patade and Marita 2014).
Since the identification of the antidiabetic potential of Galega officinalis in 1920,
a large number of flowering plants or angiosperms have been explored for
antidiabetic properties (Bailey and Day 2004). These properties are attributed to a
number of bioactive compounds belonging to almost all major classes of
phytocompounds (viz., alkaloids, flavonoids, polysaccharides, dietary fibers, pheno-
lic acids, saponins, sterols, carotenoids, terpenoids, glycosides, glycopeptides,
amino acids, etc.) (Sen et al. 2016). However, the lower plants such as algae,
bryophytes, and pteridophytes, despite being rich in almost all of these bioactive
constituents, have rarely been exploited for their antidiabetic potential (Ruocco et al.
2016; Saleh et al. 2016; Suleria et al. 2016). Thus, the entire range of lower plants
from unique aquatic algae to terrestrial pteridophytes still remains largely untapped.
A number of these have a rich reserve of known as well as unique alkaloids, phenolic
acids, flavonoids, saponins, terpenoids, as well as nutritional properties.
Diabetes is associated with altered carbohydrate, protein, and fat metabolism,
increased levels of glucose as well as glycogen in blood, and altered or insufficient
secretion of insulin leading to hyperglycemia and dysfunction of various organs
(Sonksen and Sonksen 2000; Joshi et al. 2007; Newsholme et al. 2014; Defronzo
et al. 2015). On the other hand, many alkaloids such as allylpropyl disulphide,
aegelin, berberine, castanospermine, cryptolepine, ginkgolides, lupanine, harmine,
marmesin, marmelosin, pinoline, quinolizidine, tecomine, etc., are known to initiate
insulin secretion. Even polysaccharides such as aconitans A-D, atractrans A, lami-
narin 13, exopolysaccharide of Porphyridium cruentum 18, Spirulina
polysaccharides 22, alginic sodium diester 25, and protein bound polysaccharides
assists in lowering the blood glucose level as well as increasing the level of insulin in
serum (Lin et al. 2015; Gaikwad et al. 2014; Bharti et al. 2018). The flavonoids such
as kaempferol, myricetin, epigallocatechin gallate, rutin, catechins, genistein, fisetin,
quercetin, isorhamnetin, morin, etc., assist in lowering glucose level and maintaining
plasma cholesterol and triglycerides (AL-Ishaq et al. 2019). These also elevate the
hepatic glucokinase activity while increasing the secretion of insulin from β cells of
pancreas (Chauhan et al. 2010). The phenolic acids such as ferulic acid, resveratrol,
gallic acid, and chlorogenic acid also stimulate insulin secretion, whereas saponins
release the insulin blockers (Mishra et al. 2010). The reduced absorption of glucose
or serum glucose by dietary fibers and resultant reduction in glucose concentrations
in the blood is also known.
3.4 Antidiabetic Potential of Algae
Algae are avascular, photosynthetic single cell to multicellular aquatic organisms.

They exhibit micro to macro complexity, diverse classification based on pigmenta-
tion, ubiquitous diversity, and habitat preferences that range from fresh to marine
water. Algae are emerging as “valuable bioresource organisms” because of their
faster growth, high biomass, high productivity, and, importantly, their ability to
synthesize complex metabolites. Many of these metabolites possess numerous
biological activities such as anticancer, antidiabetic, antihypertensive,
antihyperlipidemic, antioxidant, anticoagulant, anti-inflammatory, antiestrogenic,
antiviral, antifungal, antibacterial, immunomodulatory, neuroprotective, and tissue-
healing properties in vivo (Mohamed et al. 2012). Even the active ingredients of
several edible macroalgae are popularly used as foods with antidiabetic activity
(Pangestuti and Kim 2011). Some of these, such as the bioactive polyphenols from
edible seaweeds, serve as antioxidants and modulate oxidative stress caused due to
glucose and inhibitory effect of starch digestive enzymes (Lee et al. 2009).
Polysaccharides from marine alga have been shown to interfere with carbohydrate
metabolism and prevent the increase in glucose level. Studies have also shown that
the bioactive constituents from cyanobacteria inhibit α-glucosidase and α-amylase
activities. Microalgae such as Chlorella species also exhibit similar activities.
Importantly, extracts of some microalgae have been shown to inhibit the activity
of protein tyrosine phosphatase 1B, a negative regulator of insulin receptor signal
transduction (Gum et al. 2003). Some important algae with their bioactive
compounds and their role as antidiabetic are presented in Table 3.1.
70
Table 3.1 Important algal resources and their bioactive compounds having antidiabetic property
Phylum/
Algae class Active component/extract Mechanism Model for in vivo study References
Pelvitia siliquosa Phaeophyta Fucosterol 1. Decreases serum glucose Streptozotocin (STZ)- Lee et al. (2004)
(Brown alga) levels induced diabetic rats
2. Inhibits glycogen
degradation
Pelvitia Phaeophyta Extract 1. α-glucosidase inhibitory – Ohta et al. (2002)
babingtonii activity
(Brown alga) 2. Suppresses postprandial
hyperglycaemia
Ecklonia Phaeophyta Methanolic extract 1. Inhibitory effect on Diabetic KK-Ay mice Gouveia et al.
stolonifera α-glucosidase activity (2007), Iwai
(Brown alga) 2. Not only suppresses the (2008)
increase in glucose level of
plasma but also lipid
metabolism
Ecklonia kurome Phaeophyta Phlorotannins 1. Inhibitory activities against Decreased postprandial Yotsu-Yamashita
(Brown alga) carbohydrate-hydrolyzing blood glucose levels et al. (2013), Xu
enzymes in vivo et al. (2012)
2. Lowers blood glucose level
Eisenia bicyclis Phaeophyta Phlorotannins/ ethanolic extract 1. Antihyperglycemic activity Zebra fish for type Moon et al. (2011)
(Kjellman) with bioactive compounds such 2. α-Glucosidase inhibitory 1 and type 2 diabetes
Setchell (Edible as eckol, dieckol, and activity
brown algae) phlorofucofuroeckol-A 3. Protein tyrosine phosphatase
1B (PTP1B) inhibitory
activities
Ecklonia cava Phaeophyta Phlorotannins/ Bioactive 1. Inhibitory activities against Improves insulin Lee et al. (2010),
(Edible sea weed) compounds such as dieckol, α-amylase and α-glucosidase sensitivity in rat model Wijesekara et al.
7-phloroeckol, 2. Use as functional food for (2010), Pontiroli
phlorofucofuroeckol-A, diabetic patients (2004)
B. Sareen and A. Bhattacharya
6,6-bieckol, fucodiphloroethol-G
3
Polyphenolic compounds 1. Reduces glucose level of Streptozotocin-induced Kang et al. (2010)

plasma type 1 diabetes mellitus
2. Increases insulin rats and C2C12
concentration myoblasts
Laminaria Phaeophyta Butyl-isobutyl-phthalate 1. Hyper glycemic effect Hyper glycemic effect Liu et al. (2011b)
japonica (Brown 2. Inhibition of α- glucosidase in in vivo model
alga) Polysaccharides 1. Reduces fasting glucose Diabetic mice model Jia et al. (2014), Li
2. Increases insulin level et al. (2012)
Dried whole seaweed 1. Reduces fasting and Obese diabetic Kim et al.
postprandial blood glucose individuals (2008a, b)
2. Modifies lipid level
Sargassum Phaeophyta Sargaquinoic acid, 1. Peroxisome proliferator- – Kim (2008),
yezoense sargahydroquinoic acid activated receptors (PPARα/γ) Adeghate et al.
(Brown algae) antagonist (2011), Kim et al.
2. Improves lipid and glucose (2012)
metabolism
Sargassum Phaeophyta Hydroalcoholic extract 1. Reduces serum glucose Streptozotocin-induced Akbarzadeh et al.
oligocystum diabetic rat (2018)
(Brown alga)
Sargassum Phaeophyta Extracts containing polyphenols 1. Inhibitory activities against – Lee and Han
ringgoldianum α-amylase and α-glucosidase (2012)
(Brown alga) 2. Stimulates insulin secretion
Sargassum Phaeophyta Extracts containing bioactive 1. Inhibitory activities against – Hwang et al.
hemiphyllum compound fucoxanthin α-amylase (2014)
(Turner) 2. Activities against
C. Agardh α-glucosidase
Lower Plants as Potential Source of Antidiabetic Compounds: The Present. . .
(Brown alga) 3. Stimulates insulin secretion

Alaria Phaeophyta Extract rich in phenolics Inhibitory activities against – Nwosu et al.
(Brown alga) α-amylase (2011)
(continued)
71
72

Phylum/
Ascophyllum Phaeophyta Extract rich in phenolics 1. Activities against – Nwosu et al.
(Brown algae) α-glucosidase (2011)
Durvillaea Phaeophyta – 1. Inhibitory activities against – –
antarctica α-glucosidase
(Brown algae)
Brown seaweed – Fucoxanthin (Carotenoid) 1. Prevents diabetes through Obese/diabetic mice D’Orazio et al.
and microalgae down regulated expression of (2012)
TNF-α and IL-6
2. Upregulation of glucose
transporter 4
Enteromorpha Chlorophyta Polysaccharides 1. Decrease blood glucose Diabetic rat model Lin et al. (2015)
prolifera level
2. Regulate glucose
metabolism
Ulva lactuca L. Chlorophyta Polysaccharides 1. Decreases blood glucose – Belhadj et al.
(Green algae) 2. Inhibitory effect on key (2013)
enzymes of starch digestion
and absorption in both plasma
and small intestine
Ulva rigida Chlorophyta Ethanolic extract 1. Decreases blood glucose Diabetic rats Celikler et al.
(Green algae) concentrations (2009), Tas et al.
2. Decreases micronuclei (2011)
frequency
Ulva fasciata Chlorophyta Aqueous extract 1. Hypoglycemic effect Wistar albino rats Abirami and
(Green algae) Kowsalya (2013)
Rhodomela Rhodophyta Bromophenol, 1. PTP1B inhibitory action – Shi et al. (2012)
confervoides 3,4-dibromo-5-(2-bromo-3,4-
(Red algae) dihydroxy-6-
(ethoxymethyl)benzyl) benzene-
1,2-diol
3
3,4-Dibromo-5-(2-bromo-3,4- 1. Decreases plasma glucose, Mouse model Shi (2013)

dihydroxy-6-(isopropoxymethyl) serum triglycerides and total
benzyl) benzene-1,2-diol cholesterol
Grateloupia Rhodophyta 2,4,6-Tribromophenol and 1. Inhibits α-glucosidase – Kim et al. (2008b)
elliptica 2,4-dibromophenol activity
(Red algae)
Symphyocladia Rhodophyta Bromophenols 1. Inhibits aldose reductase – Liu et al. (2011a),
latiuscula activity Wang et al. (2005)
(Red algae) 2. Used for complications of
diabetes especially Type
2 diabetes patients
Palmaria sp. Rhodophyta Polyphenols 1. Inhibitory α-amylase – Nwosu et al.
(Red algae) activity (2011)
Palmaria palmate Rhodophyta Protein hydrolysates 1. Potential anti-diabetes – Harnedy and
(Red algae) properties FitzGerald (2013)
Chlorella sp. Microalgae Carotenoids 1. Strong anti-glycative – Sun et al. (2010),
capacities Sun and Chen
(2012)
Nitzschia laevis Diatom Linoleic, arachidonic and 1. Strong anti-glycative – Sun et al. (2010)
eicosapentaenoic (EPA) fatty capacities
acids
Chlorella Microalgae Extract rich in carotenoids 1. Anti-glycoxidative – Sun et al. (2011)
zofingiensis properties
2. Anti-glycative capacities
3. Prevents intracellular
oxidative stress
Chlorella Microalgae Extracts 1. Inhibitory activities against – Sun et al. (2011)

pyrenoidosa α-amylase
2. Glucosidase inhibition
(continued)
73
74

Phylum/
Isochrysis galbana Marine Soluble and insoluble 1. Regulates glucose and lipid Diabetic rats Nuño et al. (2013)
microalgae polysaccharides, proteins and metabolism
polyunsaturated fatty acids 2. Maintains intestinal and
(docosahexaenoic acid) epithelium lining
3. Regulates lipoproteins
Nannochloropsis Marine Soluble and insoluble 1. Regulates glucose and lipid Diabetic rats Nuño et al. (2013)
oculata microalgae polysaccharide, protein and metabolism
polyunsaturated fatty acids 2. Maintains intestinal and
(eicosapentaenoic acid) epithelium lining
3. Regulates lipoproteins
3.5 Bryophytes
Bryophytes are the small, nonvascular plants placed between the nonvascular algae
and vascular pteridophytes. These oldest land plants are also known as amphibians
of plant kingdom. These plants lack true leaves, stem, as well as the vascular system
and are classified into three different categories, viz., hornworts, liverworts, and
mosses. Although less studied, these plants are rich in metabolites with potential
biological activities. Particularly, the mosses and liverworts consist of many biolog-
ically novel compounds with therapeutic as well as pharmaceutical importance
(Asakawa 1990a, b; Frahm 2004; Nath et al. 2007; Singh et al. 2007; Xie and Lou
2009; Asakawa et al. 2013; Mishra et al. 2014). As per the traditional knowledge of
India and China, extracts of mosses have been also used for their antidiabetic as well
as antimicrobial properties (Singh et al. 2006; Sabovljević et al. 2010).
3.6 Properties of Bryophytes Suggesting Their Antidiabetic

Potential
Mosses
1. Fissidens species (family, Fissidentaceae): Ethanolic extract of this moss has
inhibitory activity against α-amylase thereby, suggesting its antidiabetic potential
(Krisanth et al. 2020).
2. Fissidens grandiflora (family, Fissidentaceae): Extract rich in phenolics and
flavonoids inhibit α-glucosidase and pancreatic α-amylase activities (Hieu et al.
2020).
3. Taxithelium nepalense: (family, Leucobryaceae): Ethanolic extract has antioxi-
dant and antidiabetic properties (Tatipamula et al. 2017).
Liverworts
4. Marchantia emaginata (family, Marchantiaceae): Extract rich in tannins shows
high α-glucosidase inhibitory activity.
5. Marchantia subintegra (family Marchantiaceae): Diethyl ether extract
possesses higher α-glucosidase and α-amylase inhibitory activities (Mukhia
et al. 2017).
6. Lunularia cruciate (family Lunulariaceae): Extract possess α-glucosidase and
α-amylase inhibitory activities (Mukhia et al. 2019).
3.7 Pteridophytes
The pteridophytes comprising of the ferns and fern allies are the most primitive,
vascular, spore-bearing land plants that inhabit different ecogeographical regions
ranging from sea level to the highest mountains. Ferns have been used as
ethnomedicines in folk as well as traditional systems of medicine, since times
immemorial and are well documented in ancient Ayurvedic scriptures such as

“Samhitas.” These state the use of ferns as medicine by ancient physicians such as
“Sushruta” and “Charaka.” Ferns have also been used in the “Unani” system of
medicine (Uddin et al. 1998). The use of about 300 edible fern species in the
treatment of a large number of diseases in the “Chinese system of medicine” has
also been documented (Kimura and Noro 1965). In all of these systems of medicine,
freshly harvested leaves and also their juices, dried powder, infusion, as well as
decoctions have been used in the treatment of various diseases including diabetes
(Table 3.2). Therefore, a comprehensive pharmacological validation of these medic-
inal as well as edible ferns was attempted by different workers. Their studies
revealed biological activities that ranged from antibacterial, antimicrobial,
antinociceptive, anti-inflammatory, antioxidant, and antidiabetic to cytotoxic and
apoptotic properties (Nguyen 2005; Paul et al. 2012a, b; Chai et al. 2013, 2015). The
ferns that have been most popularly used for the treatment of diabetes belong to the
genus Adiantum such as A. capillus-veneris, A. lunulatum, A. venustum,
A. caudatum, and A. incisum.
3.8 Medicinal Importance of Ferns Having Antidiabetic

Potential Versus Chemical Constituents
Among the three different types of diabetes, classified as Type 1 (insulin-dependent),

Type 2 (insulin-independent), and Type 3 or gestational diabetes,
phytotherapeuticals impose the maximum beneficial impact on Type 2 diabetes.
Type 2 diabetes is the most common ailment affecting majority of people and can be
prevented and also treated by changing ones’ lifestyle and/or consuming healthy
diet. On the other hand, deficiency of insulin due to the loss of insulin producing β
cells from islets of pancreas causes the partly inherited, autoimmune, Type 1 diabe-
tes. While Type 1 is incurable, Type 3, being gestational, is a short-term diabetes. It
closely resembles Type 2 diabetes in many aspects and is characterized by glucose
intolerance during pregnancy (American Diabetes Association 2010; Atkinson et al.
2014). Naturally occurring compounds having antiglucosidase and blood glucose
lowering activities can be effectively used as nutraceuticals and functional foods
against Type 2 diabetes. Since Type 2 diabetes can be managed, controlled, or even
prevented by inhibition of α-glucosidase activity, it can serve as an important
therapeutic target for phytopharmaceuticals. Hence, different workers attempted to
establish the glucosidase inhibitory activity of unexplored but potential resources of
ferns. These workers also used various solvent systems to extract ferns’ plant parts to
identify the extract having maximum antidiabetic potential. In this regard, in one of
the early studies, Soleimani et al. (2007) was successful in demonstrating the
antidiabetic property of the methanolic extract of Equisetum arvense in
stretozotocin-induced diabetic rats. Similarly, the aqueous extracts of Gleichenia
truncate and ethanolic extract of Actiniopteris radiata were found to exhibit strong
glucosidase inhibitory activity (Chai et al. 2013; Chand Basha et al. 2013). Later on,
Chai et al. (2015) evaluated the glucosidase inhibitory activity of a number of ferns
3
Table 3.2 Contemporary use of ferns for the treatment of diabetes

Local/vernacular
Fern name Family Part used Preparation/usage Region References
Adiantum – Adiantaceae Leaves – Philippines Nair (1959)
poiretii
Wikstr.
Adiantum Hansraj Adiantaceae Whole Juice from fresh fronds Mahadeo Hills Singh and Upadhyay
capillus- plant (2012)
veneris
Adiantum Hanspadi Adiantaceae Leaves Leaf powder mixed with Mahadeo Hills, Singh and Upadhyay
incisum Forsk. butter Himachal Pradesh (2012), Kumar and
(India) Kanwar (2020), Upreti
Kumaun Himalaya et al. (2009)
(India)
Adiantum Neelkantha Adiantaceae Whole Infusion of crushed and Himachal Pradesh Kumar and Kanwar
caudatum shikhaa, plant and boiled fronds for oral (India), Mountainous (2020), Joshi (2011),
Sahastrahi, leaves consumption on empty regions of Nepal, Baskaran et al. (2018)
Saaharsra, Unyu, stomach for 10 days Philippines
Seto sinki
Adiantum Maidenhair fern Adiantaceae Leaves – – Baskaran et al. (2018)
venustum and
rhizomes
Cyathea Tree fern Cyatheaceae Aerial Freshly harvested rhizome India Kumar et al. (2012),
gigantea part and Sathiyaraj et al. (2015)
(Wall. ex rhizome
Hook.)
Holttum
Cyathea Nilgiri tree fern Cyatheaceae Aerial Endemic species used as India Kumar et al. (2012),
nilgirensis part and antidiabetic Sathiyaraj et al. (2015)
Holttum rhizome
(continued)
77
78

Local/vernacular
Fern name Family Part used Preparation/usage Region References
Hemionitis Heart fern Hemionitidaceae Sterile – Kerala (India) Nair et al. (2006)
arifolia fronds
(Burm. f.)
T. Moore
)Lygodium Lai-uri Lygodiaceae Fronds Decoction of fronds used to Manipur (India) Yumkham and Singh
japonicum treat diabetes (2011)
(Thunb.) Sw.
Lygodium Climbing fern Lygodiaceae – – Brazil, India Reinaldo et al. (2015)
venustum
Pteridium Bracken fern Dennstaedtiaceae Plant Decoction of edible fern Nigeria Baskaran et al. (2018),
aquilinum (L.) and Dried rhizome mixed with Nwiloh et al. (2020)
Kuhn rhizome milk for relief of diabetic
disorders
Dicranopteris Drooping forked Gleicheniaceae – – Brazil, India Reinaldo et al. (2015)
flexuosa fern
such as Blechnum orientale, Davallia denticulata, Diplazium esculentum,

Nephrolepis biserrata, and Pteris vittata. Among these, the aqueous extract of
D. esculentum showed potent glucosidase inhibitory activity. Thus, D. esculentum
was established as a potential antidiabetic fern. Even recently, the methanolic extract
of Angiopteris helferiana rhizomes and their fractions were reported to exhibit
α-glucosidase inhibitory activity followed by significant reduction in the blood
glucose level in in vitro and in vivo mice model (Lamichhane et al. 2019), also
confirming the antidiabetic potential of the fern.
Just after taking any kind of meal, liver initiates the conversion of food into
glucose in a healthy person. The resultant glucose is then released into the blood
stream, where its level is then regulated by the pancreatic hormone insulin. Insulin
also eases the movement of glucose from bloodstream into different tissues and
organs for utilization as a source of energy. Impaired insulin action and movement of
glucose from bloodstream into insulin sensitive tissues such as adipose tissue,
muscle, and liver leads to increased accumulation of glucose in the blood stream
and resultant hyperglycaemic condition (Defronzo 2010). This condition if not
treated causes dysfunction of other organs. Thus, the hypoglycemic or blood glucose
lowering potential of ferns was also tested by different researchers. In this regard,
flavonoids were considered to increase the expression of “proteins of insulin signal-
ing pathway and glucose metabolism” such as the PPAR-γ and IRS-1 (Zheng et al.
2011b). Thus, two important flavonoids, i.e., violanthin and isoviolanthin of
Angiopteris evecta, exhibited significant hypoglycemic effect on Type 2 diabetics
(Nguyen 2005), thereby confirming the in vivo antihyperglycemic activity of these
flavonoids. The hypoglycemic activity of flavonoids rich extracts of Selaginella
tamariscina was also demonstrated on diabetic rats (Zheng et al. 2011a). In particu-
lar, the “flavonoids rich aqueous and ethanolic extracts of Pteris vitata and Adiantum
philippense” were found to significantly decrease the plasma glucose levels in
alloxan induced Sprague Dawley rats without affecting other organs (Paul et al.
2012a, b). Similarly, extracts prepared from leaves and rhizomes of P. triloba
showed significant in vitro glucose inhibitory activity.
Combined antidiabetic and antioxidant activities were also considered to be
beneficial for controlling various complications that arise due to diabetes (Aryangat
and Gerich 2010). Thus, Zheng et al. (2013) correlated the antidiabetic potential of
flavonoids from ferns with their antioxidant and anti-inflammatory activities. While
Tadera et al. (2006) established the antihyperglycemic as well as antioxidant
properties of P. triloba leaf extracts, phenolics such as myricetin and sinapic acid
were shown to be responsible for these effects by Cherng et al. (2013). In a parallel
study, ( )-epiafzelechin 3,5-di-O-β-D-apiofuranoside of cell based and biochemi-
cally tested Phymatopteris hastate and P. triloba were found to improve the glucose
consumption and translocation of GLUT4 to skeleton muscles (Ma et al. 2013a, b).
The antidiabetic potential of Pterosin A, a low-molecular-weight bioactive com-
pound from various fern species, was tested in diabetic mice showing glucose
intolerance, insulin resistance, dyslipidemia, and islet hypertrophy. Interestingly,
the administration of pterosin A in the diabetic mice restricted the fall in the body
weight, altered the GLUT-4 translocation, activated the adenosine
monophosphate-activated protein kinase, regulated blood glucose homeostasis,

reduced the reactive oxygen species production, helped in preventing cell death,
glucose absorption by peripheral tissues, and activated the signaling pathway to
finally alter the intracellular glycogen synthesis in liver cells (Chen et al. 2015).
Pterosin A was also found to inhibit gluconeogenesis in liver. Pterosins isolated from
methanolic extracts of bracken fern also exhibited glycophenotypic alterations in
gastric mucose of mice (Gomes 2012). Finally, pterosin A was identified to be the
most potent candidate for the treatment of diabetes (Hsu et al. 2013; Chen et al.
2015). Later, six other pterosins, i.e., [(2S)-pterosin A, (2R)-pterosin B, and trans-
pterosin C and their associated glycosides, namely, (2S)-pteroside A, (2R)-
pteroside B, and pteroside Z] were assessed and found to have antidiabetic activity
(Mohammad et al. 2016). Very recently, silver nanoparticles prepared from Equise-
tum arvense extract were also evaluated for the antidiabetic potential and found to
exhibit high degree of α-glucosidase inhibition (Das et al. 2020).
3.9 Limitations of Using Lower Plants
Despite the presence of an array of unique phytochemicals with high therapeutic

value, scientific exploitations of lower plants (known to be important in traditional
medicine) for their emergence as valuable bioresource have been limited. This is
probably because of the varied problems associated with the study of lower plants
and consequent evaluation of their market potential in the form products and
formulations.
Besides inhabiting strategic, specific, and not so easily accessible locales, lower
plants are moderately to highly impacted by seasonal variations. This makes them
unamenable for year-round studies. Lower plants such as bryophytes also exhibit
low existence. All these make in-depth studies in the biology and quantification of
species in terms of species richness extremely difficult in case of lower plants. Other
problems include availability of limited number of bryologists and pteridologists,
lack of thorough and systematic studies on identification of species and their specific
and distinguishing features, lack of sufficient literature, taxonomical confusions,
concerns regarding distribution and documentation of key species, limited charac-
terization regarding chemical and molecular signatures, absence of proper validation
of primary information, and remarkably low biomass. All these render
bioprospection of lower plants for commercial formulations and antidiabetic
medicines, quite problematic. Additionally, a serious lack of awareness regarding
the potential value of a number of lower plants has hampered their use as therapeu-
tics including antidiabetics. Acute absence of toxicity data on several
ethnomedicinally important algae and ferns is also affecting their applications in
modern drugs, medicines, and herbal formulations.
Isolation of phytochemicals from lower plants has also been difficult due to
limitations of raw material. This is not only affecting the process of bioprospection
but also the cost of raw material required for therapeutic preparations.
3.10 Future Prospects
Plant-based medicines have been serving as potential therapeutics for almost

70–90% of the population in developing countries (Chin et al. 2006); yet, the huge
diversity of lower plants and their unique phytochemicals still remains largely
untapped. Lower plants comprising algae, bryophytes, and pteridophytes constitute
approximately 21% of all plant species and hold immense antidiabetic potential for
humankind. These need to be exploited for the development of commercial products
and treatment of diabetes. While ethnopharmacological studies have revealed their
potential value as therapeutics, and also as antidiabetics, scientific validation of these
knowledge is required for the development of cost-effective and new therapeutics
and antidiabetics, particularly for the economically backward countries. Some phar-
macological and chemical studies have been conducted on lower plants based on
ethnobotanical knowledge. Many new bioactive compounds with glucosidase inhib-
itory and blood glucose lowering properties have also been isolated from different
algae and pteridophytes for the control of mainly Type 2 diabetes. Phenolics,
alkaloids, and their glycosides, triterpenoids, as well as pterosins, which are abun-
dant in ferns, have been shown to act as regulators of blood glucose to facilitate its
homeostasis. Moreover, species from important genus of ferns such as Adiantum and
other lower plants are now known to have strong antidiabetic potential. However,
these leads are yet to be converted into commercial products.
In order to convert the “various leads and knowledge generated for lower plants”
into “commercial therapeutics, nutraceuticals, and functional foods,” the strengths
and limitations of using lower plants as potential sources of antidiabetics need to be
first identified. In this regard, high-throughput profiling of source plants followed by
extensive screening of libraries of compounds and extracts for biological activities
against specific targets will be required. Toxicological profiling of the extracts from
lower plants as well as studies on their synergistic and antagonist effects will also be
required for their future use. Furthermore, investment in research and development,
verification of health claims of the products, and also market research on lower
plants will be required for their effective commercialization.
Acknowledgment The authors thank the Director, CSIR-IHBT, Palampur, India for infrastructure
and support. Bhuvnesh Sareen thanks the Indian Council of Medical Research, Govt. of India, for
providing Senior Research Fellowship (file No.: 3/1/2/33/2014-Nut., IRIS ID- 2014-23210). CSIR-
IHBT publication number 4767.
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Tissue-Culture-Mediated Biotechnological
Intervention in Centella asiatica: A Potential 4
Antidiabetic Plant
Priyanka Mohapatra, Asit Ray, I. Sriram Sandeep,

Sanghamitra Nayak, and Sujata Mohanty
Abstract
Centella asiatica L. (Apiaceae) commonly known as Indian Pennywort is a

medicinally important perennial herb widely used in traditional Indian and
Chinese systems of medicine for treating various disorders. Bioactive compounds
such as asiaticoside possess antidiabetic properties by regulating insulin sensitizer
receptors responsible for the treatment of diabetic mellitus. The conventional
mode of propagation of C. asiatica using stem cuttings is quite slow and is not
sufficient to meet the increasing market demands. Therefore, micropropagation is
being used as a reliable and alternative method for propagation. The success of
any tissue culture depends on the survival of plantlets after transfer to field
conditions. Another important factor that is vital for conservation is the regenera-
tion of plants with uniform genetic makeup. This chapter provides a comprehen-
sive analysis of the plant growth regulator combinations employed for culture
initiation, proliferation, and eventual protocol optimization required for success-
ful large-scale in vitro regeneration of C. asiatica. Besides, this chapter offers a
detailed analysis of various acclimatization strategies used till date for successful
establishment of in vitro regenerated C. asiatica plantlets under in vivo
conditions. This chapter also summarizes the employment of different molecular,
morphological, biochemical, and cytological markers used for assessing the
clonal fidelity among donor plants and tissue-culture-derived plantlets of
C. asiatica.
P. Mohapatra · A. Ray · I. S. Sandeep · S. Nayak

Centre for Biotechnology, Siksha‘O’Anusandhan (Deemed to be University), Bhubaneswar,
Odisha, India
S. Mohanty (*)
Ramadevi Women’s University, Bhubaneswar, Odisha, India

Ltd. 2021
https://doi.org/10.1007/978-981-16-3529-8_4
90 P. Mohapatra et al.
Keywords
Antidiabetic properties · Centella asiatica · Clonal fidelity · Micropropagation
4.1 Introduction
Diabetes is a metabolic disorder that is marked by a chronic increase in the level of

blood sugar that emerges when the pancreas fails to secrete adequate insulin or when
the body is unable to use it (Ramírez-Espinosa et al. 2011). According to an
estimation by the World Health Organization, approximately 439 million people
will be diabetic by the year 2030 (Wild et al. 2004). Diabetes may occur due to
various reasons such as heredity, increasing age, stress, sedentary lifestyle, poor diet,
and obesity (Hung et al. 2009). This may cause several problems that occur mostly in
the arteries and capillaries resulting in disability and hospitalization. Moreover,
those with type 2 diabetes have more chances to suffer from cerebrovascular,
cardiovascular, and peripheral vascular diseases leading to strokes, myocardial
infarction, and amputations (Watkins 2003). The cure for diabetes requires lowering
blood glucose through several mechanisms, such as insulin secretion, glucose
absorption, and metabolism (Grundy 2006). Currently, conventional antidiabetic
drugs such as insulin and other hypoglycemic drugs are regarded as the most
effective diabetes treatment, but they are accompanied by various drawbacks such
as increased lipid reserves, hypoglycemic shock, and shrinkage of lipid at the
injection site (Asadi-Samani et al. 2017). Nowadays, the use of plant-based medicine
has increased all over the world, mostly because of the supposedly less side effects in
comparison to modern medicines (Hu et al. 2003).
Various plant species are used in ancestral therapeutic practices across many
cultures as a parallel resource for the treatment of diabetes (Paula et al. 2017). In
countries that practice Ayurvedic traditional medicine systems such as India and
Bangladesh, Centella asiatica has been used to treat diabetes for a long time
(Rahman et al. 2012; Swargiary et al. 2013). The plant is known to be a rich source
of triterpenes, flavonoids, and volatile oil (Table 4.1) (Seevaratnam et al. 2012).
These triterpenes saponins are collectively known as centellosides and are believed
to confer C. asiatica with its medicinal properties (Hashim et al. 2011). The
asiaticoside present in this plant has been reported to have an antidiabetic property
(Fitrianda et al. 2017). The requirement of C. asiatica is currently met from the wild
to cope up with its high market demands. Though C. asiatica produces seeds, it has
Table 4.1 Secondary metabolite present in Centella asiatica

Classes Bioactive metabolites
Terpenoids Asiaticoside, asiatic acid, madecassoside, madecassic acid, centic acid, cenellic
acid, brahmic acid, brahminoside ,thankuniside, isothankunisode, and madasiatic
acid
Flavonoids Quercetin, kaempherol, apigenin, rutin, naringin, and catechin
Volatile oil Caryophyllene, farnesol, and elemene
4 Tissue-Culture-Mediated Biotechnological Intervention in Centella. . . 91
been reported that the plants propagated from the stem cuttings are superior to the
ones that are produced from the seeds (Peiris and Kays 1996; Wankhar and Tripathi
1990). The conventional mode of propagation of C. asiatica using stem cuttings is
quite slow and is not sufficient to meet the market demands (Nath and Burgohain
2005). Since the plant primarily depends on vegetative propagation,
micropropagation has proved to be an efficient tool, as it is a method of accelerating
asexual propagation (Akin-Idowu et al. 2009). The aim of this chapter is to present
an exhaustive account of the tissue-culture-mediated biotechnological intervention
in C. asiatica.
4.2 Centella asiatica at a Glimpse
C. asiatica L. commonly known as Indian Pennywort has various beneficial

properties (Padmalatha and Prasad 2008). It belongs to the family Apiaceae and is
indigenous to regions having tropical and subtropical climate (Singh et al. 2010). It
thrives well in swampy, damp, wet regions and prefers shady domains (Seevaratnam
et al. 2012). It grows up to a length of 15 cm. Stems are cylindrical, striated, and
hairless (Singh et al. 2010). The stems of the plant are green in color and have long
stalk with spheroid leaves. The flowers grow in tufts, each tuft containing three to
four flowers, white or pinkish purple (2 mm) (Bandara et al. 2011). The flowers are
pentamerous, with reduced calyx and an inferior ovary. Seeds are very small,
compressed, with curved mericarps having seven to eight ridges. The seeds have a
typical odor, grayish–green color, and taste bitter and sweet (Arora et al. 2002).
Other features include calcium oxalate crystals in the leaves and aerenchyma present
in the cortex of stolon and root (Sudhakaran 2017). The main morphological parts of
the plant are shown in Fig. 4.1.
4.2.1 Botanical Description and Taxonomy
C. asiatica derives its name from the Greek word Centum, which means hundred;
this is referred to the herbs profuse branching and prostrate pattern; the epithet
asiatica implied that it is native to Asia. Out of some 33 species of Centella,
C. asiatica is the most common one found in India (Bandara et al. 2011; Bhavna
and Jyoti 2011). The plant is taxonomically positioned as follows:
Kingdom: Planta
Sub-Kingdom: Tracheobionta
Division: Magnoliophyta
Class: Magnoliopsida
Order: Apiales
Family: Apiaceae
Genus: Centella
Species: asiatica
Fig. 4.1 Morphology of Centella asiatica. (a) Whole plant, (b) leaf, (c) flower, (d) roots. (Source:
Priyanka Mohapatra)
4.2.2 Origin, Wild Relatives, and Geographical Distribution
Centella comprises 20 species that are endemic to South Africa (Peiris and Kays
1996). The most prevalent species of Centella is C. asiatica, which is commonly
found in South East Asia, Sri Lanka, parts of China, Madagascar, Mexico,
Venezuela, Colombia, and Eastern region of South America. The other species of
Centella are predominantly found mostly in South Africa (Kartnig and Hoffmann-
Bohm 1992). But the species found in India, Sri Lanka, and Madagascar has more
medicinal properties than the others (Awang 1998; Bhavan 1992). In India, the plant
has been found to flourish at a height of 1200 m above sea level in states like Sikkim
and Rajasthan (Mount Abu) (Subban et al. 2008). It has also been found to grow in
walls, rocky areas with high humidity (Awang 1998; Kartnig and Hoffmann-Bohm
1992; Ohwi 1965). In South Africa, C. asiatica and C. glabrata are the two species
mostly used for natural medicines (Long et al. 2012). C. glabrata is generally used
for dysentery and diarrhea. According to the geographic origin, there are three
varieties of C. asiatica. These are identified by relating to the morphology of leaves
and chemical composition. The most common one is C. asiatica L. var typica, which
has weak hairy, kidney-shaped leaves with crenated margins mostly found in South
Asia. This variety is mostly used for traditional medicines. C. asiatica L. var.
abyssinica has spheroid hairy leaves with soft crenated margins and is native to
tropical and equatorial Africa. C. asiatica L. var. floridana having oval-shaped
leaves mostly found in the southern parts of the USA and in tropical Oceania
(medicinalplants.us/centella asiatica-l-urban-pennywort).
4.2.3 Traditional and Medicinal Uses
C. asiatica has been used in traditional methods of treatments for centuries (Peiris
and Kays 1996). It is known both in the Indian and Chinese systems of traditional
medicine (Bhavna and Jyoti 2011). In India, the powdered leaf of C. asiatica is often
consumed to revitalize the nerve and the brain cells and, therefore, is popularly
known as the Brain Food (Seevaratnam et al. 2012). The leave syrup when mixed
with black pepper and ginger is used to treat cough but when is mixed with jaggery is
given as a tonic to women after childbirth. On the other hand, the leaf extract in oil is
used to treat bone fractures (Gupta and Sharma 2007). Cold compress of the fresh
leaves is externally applied to treat rheumatism and elephantiasis. Paste made of
fresh leaves is applied to boils. Furthermore, the leaf syrup is rubbed on the forehead
to relieve severe headache, whereas when mixed with bath water, it is used to treat
eczema (Bhavna and Jyoti 2011). The whole plant or its aerial parts are used to treat
various diseases like skin disorders, asthma, leprosy, ulcers, body aches, stomach
disorders, and urethritis (Singh et al. 2010). In countries like Nepal, the juice
extracted from the leaves is taken orally for its cooling effect on the body (Mahato
and Chaudhury 2005). Leaves of this plant are eaten as leafy vegetables due to its
aromatic and slightly pungent flavor. In Sri Lanka, freshly chopped leaves mixed
with salt, chillies, dried fish, and lime juice are eaten as salad popularly known as
Gotu kola sambola (Bandara et al. 2011). Fresh juice extracted from the whole plant
including the roots is boiled with coconut milk and rice to prepare a porridge called
“Gotu kola Kenda” (Peiris and Kays 1996). The whole plant is used to treat dog
bites, asthma, malaria, and tumor in Bangladesh (Rahmatullah et al. 2010). In Brazil,
the leaves are used to treat elephantiasis and leprosy (Singh et al. 2010). People in
villages of Malaysia prepare a salad consisting of C. asiatica leaves, onion, coconut,
and shrimp popularly known as “Ulam.” Moreover, the locals prefer to drink freshly
squeezed out juice by grinding leaves and petioles of the herb, whereas dried leaves
are used to make herbal tea (Bandara et al. 2011; Ahmad and Ismail 2003).
Conventional Chinese medicine uses the aerial part of this herb to treat leprosy,
scabies, tuberculosis, measles, tonsillitis, and jaundice. Moreover, the herb was used
by a Chinese herbalist to increase virility and longevity (Singh et al. 2010). East
Africans use C. asiatica to treat tuberculosis that affects the lymph nodes (Watt and
Breyer-Brandwijk 1962). Topical application of an aqueous extract of the herb on
Fig. 4.2 Pharmacological properties of Centella asiatica
wounds promotes collagen synthesis at the wound site by enhancing the tensile
strength and collagen tissue (Shukla et al. 1999). The herb in the form of its poultice
has been used as an anecdote for mushroom and arsenic poisoning and snake bites
(Leung and Foster 1996). In Fiji, the herb is used for treating eye problems, fractures,
rib pain, swollen joints, childhood tidal fever, and unwanted pregnancy (Leonard
1998).
Studies conducted earlier have examined and validated the pharmacological
activities of C. asiatica. Various extracts of the herb have been reported to show
various medicinal properties (Fig. 4.2) like anticancer, antitubercular, antileprotic,
antiulcer, immunomodulatory, antidepressant, antifertility, antibacterial, and
antiprotozoal activities. Crude water extract of C. asiatica when combined with
Mangifera indica extracts showed antiviral activities against herpes simplex virus
(Yoosook et al. 2000). Alcoholic extracts of the herb are known to display
antiprotozoan activity (Dhar et al. 1968). The chloroform extract of the plant was
found to be active against wide variety of bacteria (Mali and Hatapakki 2008).
Triterpenes present in C. asiatica reduce the level of corticosterone and elevate the
level of monoamines, thus showing an antidepressant effect (Bhavna and Jyoti
2011). Both the ethanolic and methanolic extracts of this plant are known to control
diabetes by lessening the blood glucose levels, thus also reducing the healing time
(Arora et al. 2002; Bandara et al. 2011; Bylka et al. 2014).
4.3 Micropropagation
Micropropagation is a method used for clonal, true-to-type propagation of plants,

where a small segment of tissues and organs known as explants are cultured
aseptically in closed vessels with defined culture medium and under controlled
environmental conditions. Tissue culture protocols have been established for
C. asiatica using different explants and employing different developmental

pathways such as direct regeneration, indirect organogenesis, and somatic
embryogenesis.
4.3.1 Stages Involved in Micropropagation
There are several sets with precise specifications to establish a valid

micropropagation protocol. These are (1) initiation of aseptic cultures, (2) shoot
multiplication, (3) rooting of micro shoots, and (4) hardening and field transfer of the
in vitro regenerants. These stages have been shown in Fig. 4.3.
4.3.1.1 Initiation of Aseptic Cultures
Explant
The explant is a segment of plant tissue placed into tissue culture. There are various
factors that should be considered before selecting an explant such as physiological
age of the organ to be used as explant, the season in which it is acquired, size, site of
collection, quality, and genotype of the mother plant, and the ultimate motive of
tissue culture. The most commonly used explant for C. asiatica is the leaf, nodal
Fig. 4.3 Stages of micropropagation of Centella asiatica. (Source: Priyanka Mohapatra)

segments, and stem segments. Different factors influence the initial stage of
micropropagation. Banerjee et al. (1999) found that leaf sections lacking petioles
were more responsive than those with petioles. Mohapatra et al. (2008) observed that
leaf explants showed more number of shoots in comparison to the nodal segments.
Furthermore, nodal segments are difficult to initiate as they are prone to heavy fungal
and bacterial contamination (Tiwari et al. 2000). Nath and Buragohain (2003) used
shoot tips as explant to overcome the problem of contamination, which was the real
obstacle when nodes were used as explant. A single shoot tip is capable of generating
25,000 plantlets in a short span of 160 days (Sivakumar et al. 2006). Contradictory
findings were reported by Karthikeyan et al. (2009). According to their report, nodal
explants showed shoot initiation and shoot elongation. This report was supported by
the work of Naidu et al. (2010) and Moghaddam et al. (2011).
Sterilization
Explants require surface sterilization before placing them in culture on the nutrient
agar. In-depth knowledge of the susceptibility of the plant species to various
pathological contaminants is needed for initiating an aseptic culture. The commonly
adopted process, in majority of the tissue culture experiments for explant steriliza-
tion, involves proper washing of the explant under running tap water for 15–30 min
and then soaking in detergent like Teepol for 10–30 min and surface sterilization
with 0.1% mercuric chloride for 3–5 min and washing with sterile double-distilled
water (Karthikeyan et al. 2009; Mohapatra et al. 2008; Nath and Buragohain 2003).
But Sivakumar et al. (2006) used 70% (v/v) ethanol instead of mercuric chloride in
the surface sterilization process. However, Tiwari et al. (2000) sterilized the explants
using cetrimide solution (1%) containing 150-mg bavistin and 50-mg trimethoprim
for 25–30 min. This was followed by washing with 0.1% mercuric chloride and then
with autoclaved distilled water. Moghaddam et al. (2011) established a protocol for
sterilization of C. asiatica explants by using 1% cetrimide, 150-mg/L bavistin,
50-mg/L trimethoprim, 0.1% HgCl2, 2% plant preservative mixture. The use of
fungicide, bavistin, and antibiotics proved to be very effective against fungal and
bacterial contaminations (Moghaddam et al. 2011; Tiwari et al. 2000). The duration
of the treatment with mercuric chloride was another important condition in the
sterilization protocol as a treatment for a longer duration led to blackening and
subsequent death of the explant (Patra et al. 1998).
Media Composition
According to the literature survey, Murashige and Skoog’s (1962) (MS) medium
was commonly used for shooting in C. asiatica. But Kim et al. (2004b) had used
Gamborg medium (1968) (B5) for stimulating asiaticoside in whole plant cultures of
C. asiatica. But Mohapatra et al. (2008) have found MS medium to be superior to B5
for shoot regeneration using a leaf or nodal explant of C. asiatica. A comparative
study was undertaken by Moghaddam et al. (2011) to investigate the role of MS and
Duchefa semisolid media on shoot multiplication; they reported that Duchefa semi-
solid medium containing benzylaminopurine (BAP) 2 mg/L and naphthaleneacetic
acid (NAA) 0.1 mg/L was more suitable for shoot proliferation. Other media like
Nitsch (Nitsch and Nitsch 1956) was used by Roy and Bharadvaja (2017) and Solet
et al. (1998). Roy and Bharadvaja (2017) studied the role of three media (MS, Nitsch
and Gamborg’s B5) fortified with BA (1 mg/L) on shoot multiplication and stigmas-
terol content. Shoot was induced on all the three media, but maximum shoot
multiplication (19.6 0.57) was achieved on MS medium, followed by B5
(16.3 0.57) and Nitsch medium (8.6 0.57).
For in vitro rooting, the excised shoots failed to root on half- or full-strength MS
basal medium (Moghaddam et al. 2011). Banerjee et al. (1999) used both full- and
half-strength MS medium augmented with growth hormones for initiating roots but
found half-strength MS medium to be superior. Karthikeyan et al. (2009) reported
that full-strength MS medium augmented with indole-3-butyric acid (IBA) (1.5 mg/
L) produced the highest number of roots after 30 days of incubation. Similar
outcomes were endorsed by Moghaddam et al. (2011).
Carbohydrates
Sucrose is the most common carbohydrate in the phloem sap of various plant
species; therefore, it is known as the sugar of choice (Yaseen et al. 2013). Sucrose
(3%) was generally employed as a source of carbohydrate for shoot regeneration of
C. asiatica. Hossain et al. (2005) studied the role of numerous sources of carbon like
gur, sugar, glucose, sucrose, and maltose on in vitro plantlet formation of C. asiatica
from the nodal segment. Among the carbon sources, sucrose was concluded to be the
optimum carbon source for shoot formation. The longest shoots (2.67) were
achieved with sucrose-containing BAP (1.5 mg/L) in medium. The other carbon
sources were less efficient than sucrose. For root development, sucrose was found
superior to the other carbon sources, whereas maltose and gur did not result in root
formation. Kundu et al. (2016) compared the effectiveness of sucrose and fructose
and concluded that MS media supplemented with sucrose exhibited better result than
fructose. The maximum number (16 3.05) of shoots was achieved on the MS
medium fortified with BA (1.5 mg/L) and 3% sucrose. But contradictory results were
obtained by Panathula et al. (2014a, b), who tried to evaluate the effect of glucose,
fructose, sucrose, and maltose on shoot development from nodal explants. They
found MS medium augmented with BA (1.5 mg/L) and Kn (1 mg/L) and fructose
(4%) to give the best outcome in terms of shoot number (22), but maximum shoot
length (13.6) was attained on the MS medium supplemented with sucrose (4%) and
same phytohormones. Carbohydrates are also considered essential for the develop-
ment of roots (Corrêa et al. 2005). An investigation was done to study the role of
various concentrations of sucrose on root development. They observed that the MS
medium augmented with 3% sucrose resulted in the highest percentage of roots per
explant (43.3 27.3%), whereas with 4% sucrose, the maximum number of roots
was formed (19.5 5.7) with an average length of 1.6 cm. Furthermore, Ling et al.
(2009) found MS medium containing 2 and 7% sucrose to be inefficient for root
development. The effect of carbon on shoot development and the sucrose concen-
tration was manipulated (0–7%). It was observed that 3% sucrose was the best suited
as maximum shoots (36 1) were obtained (Roy and Bharadvaja 2017).
Physical Factors
The role of light intensity is very crucial role for proper shoot growth, and 3000 lux
of light has been stated to be sufficient for shoot regeneration in C. asiatica (Banerjee
et al. 1999; Kundu et al. 2016; Nath and Buragohain 2003). Mohapatra et al. (2008)
described that 35 μmol/m2/s for 16 h everyday was favorable for shoot multiplica-
tion. Tiwari et al. (2000), however, used higher light intensity (50 μmol/m2/s) for
shoot growth. Karthikeyan et al. (2009) reported that 50–60 μmol/m2/s1 for 12 h
daily was optimal for shoot proliferation. Generally, 16-h light duration from a
source of white fluorescent light is given during culture conditions.
In most of the studies, the optimal temperature for shoot formation of C. asiatica
was reported to be 25 C (Banerjee et al. 1999; Mohapatra et al. 2008; Patra et al.
1998), whereas Sivakumar et al. (2006) reported 24 C and Kundu et al. (2016)
reported 26 C as optimal for shooting, while relative humidity of 55–65% was
suitable for shoot formation (Karthikeyan et al. 2009; Mohapatra et al. 2008).
Effect of Growth Regulators on In Vitro Shoot Multiplication

In vitro shoot development mostly depends on media formulations enriched with
cytokinins as the primary plant growth regulator (PGR) (Table 4.2). Full-strength
MS was used as a basal medium mostly in majority of the micropropagation
protocols of C. asiatica. Banerjee et al. (1999) used the MS medium enriched with
BAP (3 mg/L) and NAA (0.05 mg/L) for shoot multiplication from leaf explants.
They noted 11 1.4 shoots within 21 days of subculture. Hossain et al. (2000)
obtained the highest shoots using BAP 1 mg/L and NAA 0.5 mg/L using stem node
as explants. Nath and Buragohain (2003) reported the maximum number of shoots
(3.38) using shoot tips on the MS medium augmented with BAP 4 mg/L and NAA
0.1 mg/L. BAP was found to be more efficient and superior than Kinetin (Kn) for
inducing shoots. Similar observations was reported by Kumari et al. (2018) and
Kumar (2018); they recorded a maximum of 92.64% shoot bud initiation on the MS
medium supplemented with BA (4 mg/L) and NAA (0.5 mg/L),and induced 8.7
shoots per explants. They also found that the MS medium containing BA and KN
gave inferior results (2.24 shoot buds) in comparison to medium enriched with BA
and NAA.MS media fortified with 2-mg/L BAP and 0.5-mg/L Kn resulted in a
maximum number of shoots from nodal explants. They recorded 18.12 0.60
number of shoots with an average shoot length of 2.46 0.24 cm. But the highest
shoot length 5.30 0.27 cm was observed when the MS medium contained only
BAP 2.5 mg/L (Karthikeyan et al. 2009). Earlier, Sivakumar et al. (2006) had also
reported BAP to be better than Kn for shoot induction. They reported the highest
shoot multiplication using shoot tips as explant on the MS medium supplemented
with 4-mg/L BAP and 0.5-mg/L Gibberellic acid (GA3). An average of 16.8 number
of shoots with a shoot length of 11 cm was recorded per explant. These findings were
supported by the findings of Alagumanian et al. (2015), who achieved a maximum of
(16.8) shoots on the MS medium supplemented with 4-mg/L BA and 0.5-mg/L GA3.
They also reported that a combination of BA and GA3 gave superior results than
GA3 alone or in combination with Kn. Leaf explants when inoculated on the MS
medium supplemented with 3-mg/L BAP and 0.05-mg/L NAA resulted in the
Table 4.2 Commonly used growth regulators for in vitro shoot multiplication in C. asiatica
Growth regulators (mg/L)
Explant Media BAP Ads NAA IAA GA3 Kn TDZ References
Stem, 4.0 20.0 0.25 2.0 Patra et al.
leaf (1998)
Leaf MS 3.0 0.05 Banerjee et al.
(1999)
Stem MS 1.0 0.5 Hossain et al.
node (2000)
Node MS 5.0 0.5 Tiwari et al.
(2000)
Shoot tip MS 4.0 0.1 Nath and
Buragohain
(2003)
Leaf, MS 1.0 0.5 Martin (2004)
internode
Shoot tip MS 4.0 0.5 Sivakumar
et al. (2006)
Leaf, MS 3.0 0.05 Mohapatra
node et al. (2008)
Node MS 2.0 0.5 Karthikeyan
et al. (2009)
Node Duchefa 2.0 1.0 Moghaddam
et al. (2011)
Shoot tip MS 1.0 Kaensaksiri
et al. (2011)
Node MS 1.0 0.2 Habib et al.
(2012)
Node MS 2.5 Prasad et al.
(2012)
Node MS 4.0 0.4 Tiwari et al.
(2013)
Node MS 2.0 0.5 Singh et al.
(2014)
Shoot tip MS 4.0 0.5 Alagumanian
et al. (2015)
Shoot MS 4.0 0.5 Kumari et al.
bud (2018), Kumar
(2018)
maximum shoot numbers per explant (8.3), the highest percentage of shoot devel-
opment (81.6%), and the longest shoots 2.1 cm after 27 days of inoculation.
Moreover, it was observed that subculturing periodically led to an increase in the
number of shoots (Mohapatra et al. 2008). Tiwari et al. (2000) used nodal explants
and reported 5-mg/L BAP and 0.5-mg/L NAA to be the best media for shoot
multiplication. MS media fortified with 1.5-mg/L BAP and 0.5-mg/L indole-3-acetic
acid (IAA) resulted in shoot elongation. Nodal explant was found to show better
response in comparison to shoot tip explants for multiple shoot development. When
nodal explants were inoculated on the MS medium supplemented with BA (1.0 mg/
L) and NAA (0.2 mg/L), an average of 18 shoots per explant was recorded. But when
shoot tip was used, then 16 shoots per explants were recorded. But for multiple shoot
regeneration, the MS medium containing BA (1.5 mg/L) and IAA (0.25 mg/L) was
found to be the best (Habib et al. 2012). The synergistic combination of BAP and
NAA in concentrations of 2 and 0.1 mg/L, respectively, in the Duchefa medium,
induced the maximum number of shoots (5.2 0.079), nodes (4.0 0.067), and leaf
(14 0.107) per nodal explant. On the other hand, when the full-strength MS
medium was supplemented with same concentrations of BA and NAA, it resulted
in (3.5 0.067) nodes and (12 0.067) leaf per nodal explant. The shoot length
achieved in Duchefa and MS was 4.1 0.67 and 4.0 0.67 cm, respectively,
depicting negligible difference. When the concentration of both phytohormones was
increased beyond 2-mg/L BA and 0.1-mg/L NAA in both the media, it resulted in
callus formation (Moghaddam et al. 2011). An efficient two-step protocol for
micropropagation of C. asiatica was developed, where the highest number of shoots
(1.2 0.38) per explant were induced on the MS medium containing 4-mg/L BA
and 0.5-mg/L NAA and the highest shoot multiplication rate (6.13 0.16) on the
medium supplemented with BA (4 mg/L) and NAA (0.4 mg/L) (Tiwari et al. 2013).
A study was conducted by Prasad et al. (2013) to find out the effect of fungal elicitors
on multiple shoot cultures of C. asiatica; for elicitation experiments, nodal shoot
explants were excised from 2-month-old stock cultures that were regularly
maintained and transferred in a 10-mL liquid medium. The elicitors were added
one by one in the culture medium in specific doses at several stages of growth. When
culture filtrate 1–4% (v/v) of Trichodrma harzianumin was added to the medium on
0th, 10th, and 20th day of the culture cycle, it did not affect the shoot growth
significantly. But in the treatment where shoots were treated with 3% culture filtrate,
there was 1.24-fold increase in shoot growth, and the growth index (GI) was
recorded to be 7.67. The growth index of control (untreated) was noted to be 6.21.
When T. harzianum was applied at the first (0th) day of culture, it proved inhibitory
for the culture (GI ¼ 4.9–5.38). Small doses of Colletotrichum lindemuthianum
mycelial extract (0.5% and 1% v/v) when applied at the start of the culture cycle
proved significantly inhibitory to shoot growth, and GI was observed to be 5.09 and
8.12, respectively, whereas the GI of the control cultures in this part of experiment
was 11.11. But when 1.5% v/v C. lindemuthianum was added on the 0th day of
culture, the GI of 16.11 was recorded. An improved in vitro propagation method was
established by Singh et al. (2014), where nodal segments were used as explants, and
the maximum number of shoots (16.3) was obtained on the medium supplemented
with BA (2 mg/L) and Kn (0.5 mg/L). A study was conducted by Panathula et al.
(2014a, b) to evaluate the effect of antimicrobial agents like cefotaxime, bavistin,
and kanamycin on shoot regeneration of C. asiatica from nodal explants. In case of
bavistin, the highest number of shoots (6.6) was formed on the medium
supplemented with BA (2 mg/L) and bavistin (150 mg/L). When 48.4-mg/L kana-
mycin and 47.7-mg/L cefotaxamin were incorporated individually in the medium, it
resulted in 5.12 0.23 shoots per explant and 5.8 0.38 shoots per explant,
respectively. Increasing the concentration of kanamycin and cefotaxamine reduced

the number of shoots. The highest shoot growth was achieved from nodal explants
when inoculated on the MS medium containing various concentrations of BA (1 and
1.5 mg/L) and 0.5-mg/L NAA. But the maximum shoot multiplication was achieved
on the MS medium supplemented with 1.5-mg/L BA and 0.5-mg/L NAA (Biradar
and Rachetti 2016). Prasad et al. (2012) achieved the maximum number of shoots
from nodal segments when inoculated on a liquid MS medium fortified with 2.5-mg/
L Kn. A maximum of 8.9 shoots per explants were induced from shoot tip explants
on the liquid MS medium supplemented with 1-mg/L thidiazuron (TDZ)
(Kaensaksiri et al. 2011). Similar observations were noted by Thong-on et al.
(2014), who multiplied shoots by transferring 4-week-old in vitro plants derived
from shoot tip (explant) on the MS liquid medium containing 1-mg/L TDZ. The
effect of the MS medium containing elicitors (malt extract, jasmonic acid, and
salicylic acid) and carbon sources (sucrose and fructose) was evaluated on shoot
multiplication of C. asiatica. Among the elicitors used, the highest number of shoots
(25 1.52) and the maximum shoot length (2.35 0.12) was obtained on the MS
medium containing 1.5-mg/L BA and 1-mg/L malt extract (Kundu et al. 2016). Roy
and Bharadvaja (2017) studied the effect of micro (manganese) and macro (nitrogen,
phosphorus, carbon, potassium, and magnesium) nutrients present in the MS
medium on shoot growth. The media were prepared in such a way that only a
particular nutrient was manipulated, and the others were maintained at a constant
concentration. The treatments used in the experiment were fortified with BA (1 mg/
L). In case of nitrogen, the concentration used varied (100, 60, 40, and 20 mM); it
was observed that with increase in nitrogen in media, there was an increase in the
number of shoots. The highest number of shoots (47.6 1.52) was obtained on the
medium containing 100 mM (40:60) of nitrogen (NH4NO3:KNO3). Several
concentrations (0%, 30%, 60%, and 100%) of KNO3 salt was employed to study
the effect of potassium in the medium. The highest number of shoots (31.66 1.52)
was obtained on 30% potassium. Similarly, the KH2PO4 salt concentration was
manipulated (0%, 30%, 60%, and 100%) to study the effect of phosphorous on
shoot development. The highest number of shoots (28 1) was obtained on 150%
phosphorus treatment. Various concentrations of magnesium (MgSO4) (0, 1, 1.5,
2, and 3 mM) were used to study the effect on shoot development, and the highest
number of shoots (31 1) was recorded on 1.5-mM concentration of magnesium.
Several concentrations of manganese (MnSO4) (0, 100, 200, and 300 μM) were used
to investigate its effect on shoot development of C. asiatica; the highest number of
shoots (43 2) was achieved on 200- μM concentration of manganese.
In case of indirect organogenesis, maximum shooting was observed when the
friable callus (from both leaf and stem explants) was transferred on the MS medium
supplemented with 4-mg/L BA, 2-mg/L Kn, 0.25-mg/L NAA, and 20-mg/L Ads
(Adenine sulfate) (Patra et al. 1998). Naidu et al. (2010) reported the highest number
of shoots when the callus was transferred to the MS medium containing 1.5-mg/L
BA and 1.5-mg/L Kn. Bibi et al. (2011) reported the maximum number of shoots
(10) per callus when callus was shifted to the MS medium supplemented with 1-mg/
L BA and 0.5-mg/L NAA.
Effect of Growth Regulators on In Vitro Rooting

In most of the reports, various concentrations of auxins were responsible for root
induction (Table 4.3). Full-strength and half-strength MS media supplemented with
IBA have been commonly used for rooting of shoots of C. asiatica. Banerjee et al.
(1999) achieved the highest rooting on half-strength MS with 1-mg/L IBA. Prostrate
stems with rooting at the nodes were also observed, after about 2 weeks of culture.
0.2-mg/L IBA was found to be effective in the production of roots (Hossain et al.
2000). Tiwari et al. (2000) reported the maximum rooting on full-strength MS
supplemented with 0.5-mg/L IBA. The average number of roots per shoot was
noted to be 8.3 with a mean length of 6.3 cm. Nath and Buragohain 2003 observed
profuse rooting on the MS basal medium fortified with 2.0-mg/L IBA; they noted the
highest number of roots 46.8 per shoot with the root length of 19.7 cm. Similar
results were achieved by Panathula et al. (2014a, b), who transferred tissue-culture-
generated microshoots that were treated with antimicrobial agents to the MS medium
fortified with 2-mg/L IBA for rooting. The highest number (14.2) of roots with the
maximum root length of 4.2 cm was recorded. Mohapatra et al. (2008) achieved the
maximum rooting on the half-strength MS medium supplemented with 0.5-mg/L
IBA. The highest number of roots was 3.6 per shoot with an average length of
3.9 cm. This finding was further endorsed by Habib et al. (2012), who reported half-
strength MS medium containing IBA (0.2 mg/L) to be the best combination for root
Table 4.3 Commonly used growth regulators for in vitro root multiplication in C. asiatica
Growth regulators (mg/L)
Explant Media BAP IBA NAA IAA GA3 Kn References
Stem, ½MS 0.5 Patra et al. (1998)
leaf
Leaf ½MS 1.0 Banerjee et al. (1999)
Stem MS 0.2 Hossain et al. (2000)
node
Node MS 0.5 Tiwari et al. (2000)
Shoot MS 2.0 Nath and Buragohain
tip (2003)
Leaf, ½MS 0.5 Mohapatra et al. (2008)
node
Node MS 1.5 Karthikeyan et al.
(2009)
Node MS 1.0 Naidu et al. (2010)
Leaf MS 1.0, 2.0 Bibi et al. (2011)
Node ½MS 0.2 Habib et al. (2012)
Node MS 5.0 0.5 Tiwari et al. (2013)
Node MS 1.0 1.0 Singh et al. (2014)
Shoot ½MS 2.0 Alagumanian et al.
tip (2015)
induction. But contradictory results were reported by Alagumanian et al. (2015),

who found NAA to be more superior for inducing roots than IBA. When half-
strength MS was supplemented with 2-mg/L NAA, a maximum of 28 roots were
formed after 20 days of inoculation, whereas the medium containing IBA failed to
induce roots even after 30 days of inoculation. Karthikeyan et al. (2009) achieved
root initiation from the completely formed shoots when transferred to the full-
strength MS medium supplemented with IAA, IBA, and NAA. But the maximum
rooting per explant (12) with an average root length of 6 cm was achieved on the full-
strength MS medium supplemented with 1.5-mg/L IBA. Similar observations were
reported by Moghaddam et al. (2011), who found the full-strength MS medium to be
more efficient than the half-strength MS medium for inducing roots. They achieved
the highest number of roots (7.5 0.1) on the full-strength MS medium
supplemented with 0.5-mg/L IBA. But contradictory results were reported by
Biradar and Rachetti (2016), as they found the MS medium supplemented with
0.5- and 1.5-mg/L IAA to be efficient for maximum rooting. The highest number of
roots (2.0 0.63) was obtained on full-strength MS media augmented with a
combination of 0.5-mg/L IBA and 5-mg/L BAP. This combination was effective
in inducing shoots as well as roots (Tiwari et al. 2013). A combination of NAA
(1 mg/L) and IBA (1 mg/L) was found to be ideal for inducing maximum number of
roots (16.5) per shoot from well-developed in vitro raised shoots (Singh et al. 2014).
When leafy shoots regenerated from both leaf and stem callus were transferred to
the rooting medium, roots failed to develop on the MS medium without growth
regulators. Roots were formed on excised shoots grown in MS media supplemented
with either NAA (0.1–1.0 mg/L) or IBA (0.1–1.0 mg/L). The maximum rooting with
an average of four roots per explant was achieved on the half-strength MS medium
fortified with 0.5-mg/L IBA. A well-developed root system was formed within
15 days (Patra et al. 1998). Callus-derived shoots when transferred to the MS
medium supplemented with 1-mg/L NAA formed a completely rooted plant
(Naidu et al. 2010). These findings were strengthened by the results obtained by
Bibi et al. (2011), who achieved the highest rooting (20) per explant on a medium
containing NAA (1 and 2 mg/L) to form complete plants after 20 days.
Effect of Growth Regulators on Callus Formation

The type and texture of callus greatly depended on the kind and concentration of the
growth regulators used. The culture medium augmented with a combination of
NAA- and Kn-induced rapid callus growth. Higher concentration of NAA produced
a compact calli, which was undesirable (Patra et al. 1998). These findings were
further endorsed by Nath and Burgohain (2005), where they have reported that BA
(1.0 mg/L) and NAA (1.0 mg/L) when incorporated in the culture medium were the
best for callus induction. Bibi et al. (2011) indicated that a combination of BA and
NAA was efficient in producing good-quality callus. Similar results were found by
Joshi et al. (2013). Naidu et al. (2010) reported that the culture medium
supplemented with NAA (4 mg/L) and 2,4-D (2 mg/L) resulted in the highest
frequency (92%) of callus formation. Lai et al. (2011) induced callus from leaf
explant on the MS medium containing 2 mg/L 2,4-D, and 1-mg/L Kn. Callus was
induced from young green leaves on the MS medium containing 2,4-D 1-mg/L, Kn
0.5 mg/L, and Tryptophan (4 mg/L TRY) (Palengara 2017). Leaf explants were
inoculated on the MS medium containing 3% sucrose, 0.8% agar, and various
concentrations of hormones like 2,4-D, 2.4,5-T (2,4,5-trichlorophenoxyacetic
acid), IBA, NAA, and BA to induce callus from C. asiatica. The highest percentage
of callus (73.5 6.1) was induced on the medium containing 1 mg/L 2,4-D, but the
maximum percentage roots (92.3 8.4) were induced on the medium containing
2,4,5-T. When these roots were cultured on the MS medium supplemented with BA
(2 mg/L) and NAA (0.5 mg/L), it led to shoot induction and multiplication (Kumar
2017).
Effects of Elicitors for Enhancing the Secondary Metabolites

Kim et al. (2004a) implemented elicitors like CuCl2, CdCl2, yeast extract (YE), and
methyl jasmonate (MJ) to investigate the accumulation of asiaticoside in the whole
plants of C. asiatica derived from nodes. Out of all the elicitors used MJ was found
to produce the highest asiaticoside 6.74-mg/g DW, followed by yeast extract 5.91-
mg/g DW. The optimum concentration of MJ was reported to be 0.1 mM. However,
MJ when applied exogenously inhibited plant growth and induced leaf senescence.
So, to overcome the above problem, TDZ, which is known to be an antisenescence
agent, was added along with MJ. It was observed that the negative effects of MJ were
inhibited by TDZ, and the whole plant treated with a combination of TDZ and MJ
grew better than when treated with MJ alone. In fact, the asiaticoside content in
whole plants treated with a combination of TDZ and MJ increased with the incuba-
tion time. But it was also found that both TDZ and MJ had inhibitory effects on the
roots in comparison to their controls. Mangas (2006) evaluated the effect of MJ on
the growth rate, secondary metabolites production, and sterol content of cultured
C. asiatica. At the end of the fourth week, the asiaticoside, madecassic acid,
madecassoside, and asiatic acid increased by two to three folds in the aerial part
and four to six folds in the roots. It was suggested that time of exposure of the roots
to the elicitor was directly proportional to the increase in the amount of tritepenoid
compounds in the cultured whole plantlets. The elicited C. asiatica plantlets suffered
from leaves and root necrosis at the end of the experiment. The amount of
asiaticoside and madecassoside in the roots was reported to be negligible in case
of C. asiatica. A study was conducted by Kim et al. (2007), where C. asiatica was
transformed with Agrobacterium rhizogenes strain R1000 to induce hairy roots.
Even though hairy roots developed abundantly, asiaticoside was not detected in
them. But when 0.1-mM MJ was added to the medium as an elicitor, large quantity
of asiaticoside was produced 7.12 mg/g DW. In vitro grown shoot tips of C. asiatica
were treated with colchicine to induce tetraploid plants. It was found that the total
triterpenoid content in tetraploid plants was 11% higher than their diploid control
(Kaensaksiri et al. 2011). A study was undertaken by Yoo et al. (2011) to investigate
the combining effect of TDZ and MJ on centelloside produced from whole plant
cultures of C. asiatica. Nodal explants were cultures on half MS medium containing
TDZ (0.025 mg/L) or MJ (0.01 mM) individually or in combination. It was found
that the combination of MJ and TDZ resulted in an increase in the centelloside
content in leaves and did not harm the whole plant cultures as in case of MJ alone.
Furthermore, there was a 2.40- and 2.44-fold increase in madecassoside and
asiaticoside content and 1.08-fold increase in phytosterol content when treated
with a combination of both TDZ and M.Ths. They concluded that both TDZ and
MJ combined together improved the centelloside and phytosterol content of
C. asiatica. An experiment was conducted by Prasad et al. (2013) to find out the
effect of fungal elicitors on production of asiaticoside of C. asiatica. It was observed
that fungal elicitors are culture age and dose dependent. The culture filtrate of fungal
elicitors like Trichodema harzianum (3%), Fusarium oxysporum (0.5–1.5% v/v),
and Colletotrichum lindemuthianum (1.5% v/v) were used to enhance the production
of secondary metabolites. A treatment of Trichodema harzianum was added to the
medium on the 10th and 35th day of the culture period and was found to enhance the
asiaticoside content by 2.53-fold and 2.35-fold, respectively, when compared to their
controls. The highest dose (1.5% v/v) of Colletotrichum lindemuthianum decreased
the content of asiaticoside to 1.10 and 1.95 mg/g on the 0th day and 30th day of the
culture period, respectively. The third treatment Fusarium oxysporum was added on
the 0th day and 30th day of the culture period. On the 0th day, it showed reduction in
the content of asiaticoside as well as the yield. But on the 30th day, it increased the
asiaticoside content 1.66-folds. But due to its negative impact on the shoot growth,
the asiaticoside yield per culture recorded low in comparison to shoots that were
treated with 3% Trichodema harzianum. An investigation was conducted by Loc and
Giang (2012) to study the effects of dosage and elicitation of various elicitors like
salicylic acid (SA) and yeast extract (YE) on the secondary metabolite accumulation
in plant cell cultures. The concentrations of salicylic acid given on the day of
inoculation ranged in between 50–200 and 100 μM on the 5th–15th day post
inoculation. The asiaticoside content when treated with 100 μM of salicylic acid
was enhanced in the treated cultures, and the highest asiaticoside content on the 10th
day of inoculation was found to be 229.83 mg/g of DW, which was five times more
than the untreated cells, while on the other hand when treated with 4-g/L yeast
extract in the same conditions, the highest asiaticoside content was reported to be
165.41 mg/g of DW, and the yield was 3.5 times more than the control. Thus, it was
concluded that salicylic acid was a better elicitor than yeast extract in enhancing
asiaticoside content of C. asiatica. The effect of methyl jasmonate in upregulating
the triterpene content of C. asiatica has been studied by James et al. (2013), who
added 0.02-mM methyl jasmonate to the suspension cultures on second, fourth, and
sixth days of cell resulting in an increase in the concentrations of asiaticoside,
madecassoside, asiatic acid, and madecassic acid. Piriformospora indica a growth
promoting fungus was used to colonize the roots of C. asiatica in culture. High-
performance liquid chromatography (HPLC) analysis revealed a two-fold increase in
asiaticoside in the leaves 0.53% w/w and whole plant 0.23% w/w over the control
leaves 0.33% w/w and whole plant 0.14% w/w (Satheesan et al. 2012). A field
evaluation was carried by Thong-On et al. (2014) to compare four major secondary
metabolites in in vitro induced tetraploid plants and diploid plants of C. asiatica.
Total triterpenoids in tetraploids were reported to be 15.38 0.76% DW, which was
higher when compared to the diploid lines. A study was conducted in an attempt to
investigate the production of asiatic acid in the tissue culture grown shoots and
callus. 1.02 0.03 mg/g FW and 0.47 0.08 mg/g FW asiatic acid was produced in
semisolid and liquid media, respectively; on the other hand, leaf callus resulted in a
maximum amount of 1.46 0.06 mg/gFW (asiatic acid) (Gandi and Giri 2013). The
role of various organic elicitors like casein hydrolyzate, proline, glycine,
myo-iniositol, yeast extract, and glutamine on the asiatic acid of leaf callus was
studied for C. asiatica. The concentration ranges used were 1, 5, 25, 50, 100, and
300 mg/L. The highest amount of asiatic acid 258.3 μg/g fresh weight was produced
by 200-mg/L yeast extract. On adding 30-ppm chitosan to the medium, the maxi-
mum yield of asiaticoside content in C. asiatica was found to be 5.97 mg/g fresh
weight (Kumari et al. 2018a, b). The effect of MJ on the production of asiaticoside
and asiatic acid in shoot, cell suspension cultures, and callus was evaluated. The
maximum number of shoots (24 1) resulted in MS medium supplemented with BA
(1 mg/L) and 22.43 mg/L MJ. There was an increase in asiaticoside by 69-fold
(callus cultures), 39-fold (shoot cultures), and 1.9-fold asiatic acid in cell suspension
cultures (Krishnan et al. 2019). Skrzypczak-Pietraszek et al. (2019) studied the effect
of elicitors like methyl jasmonate, ethephon, L-phenylalanine on the accumulation of
secondary metabolites (centellosides, flavonoids, and phenolic acids) in the biomass
of C. asiatica microshoots cultured in agitated cultures. UPLC-MS/MS
(centellosides) and HPLC-DAD (phenolics) was applied to the harvested biomass
to measure the amount of accumulated secondary metabolites. There was an increase
in the contents of the metabolites as follows: centellosides (5.6-fold), phenolic acid
(122-fold), and flavonoids (22.4-fold). MJ was found to be the most optimal and
shortlisted to be used for the experiment in the bioreactors. Two types of bioreactors
were used: Platform™ and RITA®. Platform™ bioreactor was found to be superior
for the production of secondary metabolites.
Effect of Growth Regulators on In Vitro Flowering

Leaf and nodal explants were used to induce in vitro flowering in case of C. asiatica.
It was noted that the explants cultured on the MS medium fortified with BA
(0–2.5 mg/L) and IAA (0–2.5 mg/L) resulted in inducing shoots and pink flowers.
In case of nodal explants, the medium augmented with 1-mg/L BA and 1.5-mg/L
IAA gave rise to maximum (6.55 0.52) floral buds and (3.85 0.32) shoots,
whereas in case of leaf explants, the medium containing 1-mg/L BA resulted in the
highest number of (6.2 0.54) florets and (3.15 0.34) shoots. They were
subcultured on the MS medium enriched with IBA for root initiation (Gaddaguti
et al. 2013).
Effect of Plant Growth Regulators on Bud Breaking

Sivakumar et al. (2006) observed an early bud–break within 8–10 days of culture on
the medium fortified with 4-mg/L BAP. But increasing the concentration of BAP
beyond 4 mg/L resulted in a decrease in the percentage of bud-break and shoot
number and length. Incorporation of Gibberellic acid (GA3) alone in the culture
medium led to reduction in bud-break percentage and shoot number per explant. But
when GA3 at low concentrations was incorporated in the BAP fortified medium, it
induced highest percentage of bud break (88%). Similarly, the role of BA and GA3
was also reported by Bhandari et al. (2013) for early bud breaking. A mixture of BA
(2 mg/L) and GA3 (0.5 mg/L) was observed to initiate bud-break within 8 h of
inoculation. On the other hand, the longest time for bud break 84 h was taken when
the medium was supplemented with combination of BA (0.5 mg/L) and NAA
(0.5 mg/L) and BA (1 mg/L) and adenine (0.5 mg/L), respectively. Alagumanian
et al. (2015) achieved an early bud break (88%) on the MS medium supplemented
with 4-mg/L BA and 0.5-mg/L GA3.
Browning of Medium and Its Control

Plant tissue contains bioactive components like tannins, polyphenols in different
compartments of a plant cell. During excision of explant, an oxidation process of
these compounds is initiated and makes them appear dark brown (Monaco et al.
1977). Shashikala et al. (2009) indicated the browning of C. asiatica callus on the
medium containing 2,4-D. This was due to the oxidation of phenolics compounds
that had accumulated in the cytoplasm. This problem could be controlled by
subculturing frequently, by using substances such as polyvinylpyrrolidone, ascorbic
acid, or citric acid (Wang et al. 2016).
Hardening and Acclimatization

Hardening and acclimatization are very vital and indispensable steps for
micropropagation. Well-developed plantlets of C. asiatica were taken out from the
rooting medium and washed with tap water to eliminate any remnant gel, and planted
into pots containing different potting mixtures like soil:sand:well-rooted cow dung
manure (1:1:1; v/v) (Patra et al. 1998), soil and sand mixture (2:1) (Banerjee et al.
1999), soilrite (Tiwari et al. 2000); vermiculite and soil (1:1) (Nath and Buragohain
2003), vermicompost with soilrite mix (Mohapatra et al. 2008), sterilized vermiculite
(Karthikeyan et al. 2009); peat moss and clay (1:1) (Bibi et al. 2011); this was
followed by transfer to soil with varying degree of 55–100% survival rate.
4.3.2 Somatic Embryogenesis
Paramageetham et al. (2004) inoculated leaf sections of C. asiatica in MS med

fortified with 2-mg/L Kn and 2-mg/L 2,4-D and observed the formation of abundant
somatic embryos. These somatic embryos matured and germinated when transferred
to an MS medium supplemented with 0.5-mg/L Kn and 1-mg/L GA3. 76% somatic
embryos were reported to develop into plantlets, and out of 259 plantlets,
256 plantlets survived. Martin (2004) reported callus development from the leaf
(petiole and Lamina) and internode explants of C. asiatica, which lead to an
immense rate of somatic embryogenesis as well as plant regeneration. Callus that
was superior for somatic embryogenesis, resulted when the MS medium was
supplemented with 1-mg/L 2,4-D or 1-mg/L NAA, combined with 0.5-mg/L
Kn. The NAA- and Kn-fortified medium was found superior to the medium
supplemented with 2,4-D, and Kn for induction and maturation of embryos. 80%
of the embryos matured and developed into plantlets when transferred to half MS
medium augmented with 0.009-mg/L NAA and 0.0009-mg/L BA or 0.009-mg/L
Kn. Joshee et al. (2007) induced somatic embryos in dark conditions by inoculating
stolon and leaf tip of C. asiatica in the MS medium augmented with 0.09-mg/L
2,4-D. Maturation and development of embryos into plantlet was achieved after
removing 2,4-D from the treatments. Maximum shoot regeneration is achieved on
Kn (1.5 mg/L) and BA (2 mg/L) with NAA (0.2 mg/L) and IAA (0.2 mg/L). MS
augmented with NAA (2 mg/L) and IAA (2 mg/L) was concluded to be the best for
rooting (Biradar 2017).
4.3.2.1 Role of Artificial Neural Network for Projection of Culture

Conditions
Prasad et al. (2017) devised an artificial-neural-network-based model to give
projections of the role of important compounds such as sucrose, magnesium
(MgSO4), copper (CuSO4), zinc (ZnSO4), and nitrate (NO3) on the biomass content
of shoot cultures of C. asiatica. Different concentrations of these compounds were
used as inputs. The projections were given with reference to their growth index using
back-propagation neural network. Mg (1.5 mM), NO3 (40 mM), Cu (0.1 μM), Zn
(30 μM), and sucrose (6%) were declared as the optimum concentrations of the
respective compounds for attaining the highest (1654.46) growth index.
4.4 Evaluation of Genetic Stability Among Micropropagated

Plants
The evaluation of genetic stability of the tissue-culture-derived plants using molec-

ular markers in C. asiatica to the best of our knowledge has not been reported till
date. But Prasad et al. (2014) conducted random amplification of polymorphic DNA
(RAPD) analysis of nine randomly selected plants that were generated from the
conserved synthetic seeds in comparison to in vitro regenerated mother plant. They
screened out 40 decamer primers out of which 19 primers responded and generated a
total of 60 distinct reproducible monomorphic bands with an average of 3.15 bands
per primer. The number of bands with each primer varied from one to six with the
band size ranging from 100 to 2027 kB. The primers MAP 3, MAP 7, MAP 12, MAP
19, and MAP 20 exhibited the lowest number (1), whereas OPA 8 exhibited the
highest (6) number of monomorphic bands. Thus, it was concluded that encapsulated
axillary buds/nodal segments of C. asiatica can be conserved for up to 200 days
without any risk of genetic instability.
4.5 Artificial Seed Technology and Germplasm Conservation
Seed propagation in certain crop species is not feasible, due to minuscule size,
heterozygosity of the seed, etc.; seedless varieties can be propagated only via
vegetative means. Development of artificial seed production technology is forthwith,
speculated as a practical, efficacious prospective of propagation of several commer-

cially far-reaching agrarian and horticultural crops (Gantait et al. 2015). Prasad et al.
(2014) accomplished the encapsulation of tissue-culture-derived axillary buds or
nodal sections of C. asiatica, using 4% sodium alginate, which can be endorsed for
in vitro preservation for short duration and rapid plantlet development from
encapsulated seeds. It was stored for a period of 200 days in petri plates enveloped
on both sides with moist Whatman filter paper at a 25 3 C, and the conversion
frequency on transfer to a hormone-free MS medium was 85%. Moreover, the
in vitro storage facility enforced for the artificial seeds did not affect the biochemical
stability of the herb in terms of their centellosides content and wound healing
potential.
4.6 Biochemical Markers
The plants regenerated through in vitro propagation techniques should be evaluated

for its biochemical stability with the mother plants before recommending them for
large-scale propagation. Govarthanan et al. (2015) analyzed the biochemical com-
position of tissue-culture-derived plants with that of the mother plants. They
observed a marked decrease in the bioactive compounds (total phenolic compounds
(6.2 mg/g), flavonoids (36 mg/g), and ascorbic acid (27 mg/g) of the
micropropagated plants when compared with the mother plant (total phenolic
compounds (10 mg/g), flavonoids (45 mg/g), and ascorbic acid (35 mg/g) that was
collected from wetland habitat. To strengthen their work, they also studied the
scavenging activity of micropropagated plants and the mother plant and reported
that the maximum activity was observed in the mother plant (plants collected from
wetland habitat) (81%), followed by in vitro propagated callus (76%).
4.7 Molecular Markers to Study Genetic Diversity
The evaluation of the genetic diversity of a medicinally important species is very

vital for the development of its conservation strategies. Padmalatha and Prasad
(2008) assessed the genetic diversity of C. asiatica by random amplification of
polymorphic DNA (RAPD) analysis. Samples from 16 accessions across Andhra
Pradesh were collected. A total of 128 bands were amplified using 16 primers.
Eighty-seven percent of polymorphism was achieved after analyzing 137 markers
that were polymorphic, and the rest were monomorphic. Thus, the results indicated a
marked variation within the population of the same plant species. Zhang et al. (2012)
conducted an investigation that could help conserve populations of high genetic
diversity and improve agricultural practices of C. asiatica by taking 162 individuals
from 14 natural populations of C. asiatica in China, for studying the genetic
variation by using inter-simple sequence repeats (ISSR) markers. The genetic
diversity at both species and population level was high for Jinhua, Zhejiang prov-
ince, and Jiujiang, Jiangxi Province. The inter-simple sequence repeats (ISSR)
clustered data were found to be identical to the chemical clustering results obtained
from high-performance liquid chromatography (HPLC). Genetic diversity of six
populations of C. asiatica in Madagascar using simple-sequence repeats (SSR)
markers was investigated and three separate groups were found to be distinct from
South African and Indian genotypes (Rodier-Goud et al. 2013). Alqahtani et al.
(2017) collected three different species of Centella from Australia, namely,
C. cordifolia, C. asiatica, and C. erecta to investigate the similarity among the
three based on the morphology, phytochemical content, and genetic biodiversity and
antioxidant properties. Out of 34 arbitrary ISSR primers used, 14 primers produced
115 reproducible, consistent bands that ranged between 200 and 2200 bp. Based on
ISSR analysis, the similarity coefficient was found to be in between 0.47 and 1.00.
C. cordifolia and C. asiatica were clustered at 0.61 similarity, but C. erecta had 0.47.
Thus, it was reported that C. cordifolia was more similar to C. asiatica genetically
and morphologically.
4.8 Conclusion
In recent years, medicinal plants have attained prominence due to their stimulated
demand from the pharmaceutical industries in the local, as well as national and
international markets. C. asiatica is a very popular leafy vegetable in several
countries. Its consumption as a functional food along with the normal diet is a
beneficial way to transfer its nutritional benefits. It has been used since ancient
times due to its immense medicinal properties. It produces highly valuable
triterpenoids used to treat various kinds of diseases. The requirement of
C. asiatica is currently met from the wild, and no effort has been taken for its
conservation. Moreover, so far, no structured program for its cultivation has been
established to meet the industrial demands. Though there have been advances in
developing rapid, reproducible, and reliable tissue culture protocols for C. asiatica,
but a lot could be done through biotechnological tools. Evaluation of genetic
stability is the need of the hour for commercialization of micropropagated plants.
Micropropagation has proved to be a reliable method to maintain homogenous
genetically stable C. asiatica to meet its high global demand. It offers an alternative
to endure exploitation of plant and enhance the production of triterpenoids. Although
various reports are available that have developed micropropagation protocol for
C. asiatica, we are aware of that none of the works have focused on the biochemical
and genetic stability of tissue-culture-derived C. asiatica plantlets. Efforts are to be
raised up for attaining high-centelloside-containing chemotypes for industrial appli-
cation. It is also crucial to standardize factors the physiological condition of the
donor plant, genotype, composition of the nutrient medium as the carbon source,
PGRs, and their concentrations in addition to in vitro environmental components,
specifically abiotic stress pretreatments. More studies may be done in this aspect
through functional genomics and metabolomics approaches.
In essence, this chapter was aimed to arrange and summarize the status of
micropropagation work done till date on C. asiatica, which could be helpful in
developing a cost-effective method for conserving and propagating a premium

quality plant with higher yield and improved nutritional content.
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In Vitro Approaches for Mass Propagation
of Stevia rebaudiana 5
Kasireddy Sivasankarreddy, K. Abha Manohar, Gopal Shukla,
Vineeta, Mohamad Maqbool Rather, and Sumit Chakravarty
Abstract
Stevia rebaudiana (Bertoni), commonly known as a natural sweetener plant or

sweet herb grabs the economic and scientific interests owing to its sweetness and
therapeutic properties present in its leaves mainly due to the presence of steviol
glycosides (SGs), with stevioside being the most abundant, followed by
rebaudioside. Stevioside, the most abundant active component, is greatly pre-
ferred by diabetic patients since it is a non-calorie sweetener and is approved by
the Food and Drug Administration as a dietary supplement. This shows the
benefits of stevia over other artificial sweeteners as an ingredient for the food
business, subsequently, making stevia an increasingly appropriate substitute for
saccharine in various drinks, beverages, and bakery products. Stevia also offers
therapeutic benefits having anti-hyperglycemic, anti-hypertensive, and immuno-
modulatory effects. These beneficial effects largely focused on the importance of
stevia. Sexually developed plants were not efficient because of low fertility and
reduced viability. However, quality planting material can be produced through
biotechnological approaches like micropropagation. To date, a great deal of
studies has been completed on the tissue culture intervened approaches for the
mass propagation of stevia. In this chapter, the above-mentioned approaches and
their significance are emphasized for the large-scale production of identical plant
materials and for the improvement of important calorie-free SGs.
K. Sivasankarreddy · K. Abha Manohar · G. Shukla (*) · Vineeta · S. Chakravarty

Uttar Banga Krishi Viswavidyalaya, Cooch Behar, West Bengal, India
M. M. Rather
Division of Forest Biology and Tree Improvement, SKUAST-K, Srinagar, Jammu & Kashmir,
India
Ltd. 2021
https://doi.org/10.1007/978-981-16-3529-8_5
118 K. Sivasankarreddy et al.
Keywords
Auxins · Cytokinin · Genetic fidelity · Micropropagation · Steviol glycosides
5.1 Introduction
Stevia (Sugar leaf), botanically known as Stevia rebaudiana Bertoni belongs to the
family Asteraceae, is an herb famous for its sweet leaves. It is generally known as
sweet leaf, honey leaf, sweet weed, and the sweet herb (Anbazhagan et al. 2010;
Gantait et al. 2015). The leaves of stevia are mild green and sweet. The leaves have
been utilized for centuries in Paraguay and Brazil to sweeten the traditional teas,
medicines, and in sweet dishes (Carakostas et al. 2008). Steviol glycoside is a more
exact term for a group of strongly sweet mixes extracted and purified from
S. rebaudiana. The major sweet compounds in the leaves are stevioside and
rebaudioside (Brahmachari et al. 2011; Lemus-Mondaca et al. 2012). Having been
approved in the country Japan, since 1970, it took long periods of research and
studies to approve the utilization of steviol glycosides in many parts of the world.
European Commission in November 2011 decided to allow utilization of steviol
glycosides for edible purpose, from that point forward Stevia rebaudiana has been
the plant of enthusiasm by numerous analysts (Yücesan et al. 2016).
Natural non-calorie sugar stevioside is 100–300 times better compared with
sucrose and has been broadly utilized as a non-caloric sugar substitute in numerous
sorts of foods, medicine, drink, cosmetics, winemaking, household chemical indus-
try, and other food ventures (Ahmad and Ahmad 2018). Manufactured medications
utilized for the diabetes treatments brings certain complications but utilization of
natural sources like stevia for the treatment of diabetes is protected and non-cancer-
causing (Brahmachari et al. 2011; Lemus-Mondaca et al. 2012). Stevia also has other
various therapeutic uses like anti-hyperglycemic, anti-carcinogenic (Jeppesen et al.
2000), and anti-hypersensitive (Chan et al. 1998), but it also has contraceptive
properties (Melis 1999) and prevents dental caries (Fujita and Edahiro 1978; Ziv
et al. 1998). Stevioside and rebaudioside initiate insulin emission. Tea made of stevia
leaves gives relief from an upset stomach. Additionally, stevioside has antitumor,
antifungal, and antibacterial properties (Hossain et al. 2010). Thus, the propagation
of new stevia varieties is the current concern of many research institutions (Yücesan
et al. 2016).
At present, three techniques have been predominantly used for development and
biomass creation of stevia, i.e., stem cuttings, seed germination, and
micropropagation. Stem cuttings is affected by natural conditions as these lines
show a variable survival rate. The seeds are very small in size with low germination
potential (Gantait et al. 2018). New seeds typically show great results in contrast
with old ones. Conventional propagation approaches like stem cuttings and seed
germination have their limitations. Biotechnological strategies through the founda-
tion of tissue culture frameworks can address the issues of restricted efficiency of
stevia plant material production through conventional methods and help for rapid
5 In Vitro Approaches for Mass Propagation of Stevia rebaudiana 119
clonal propagation to generate good quality planting material production, secondary

metabolite production, in vitro selection for biotic and abiotic stress resistance
(Gantait et al. 2015; Kazmi et al. 2019). The other applications of this method are
regeneration of plant through somatic embryogenesis, organogenesis of shoot from
the callus, production of haploids through androgenesis or gynogenesis, and plant
production (triploid) through the culture of endosperm (Abraham and Thomas
2016).
With micropropagation, there is a need to do the trait-specific improvement
program for best clones utilizing different biotechnology-oriented apparatuses.
Recent advancements in the area of plant biotechnology and incredible methods
are available to introduce the exotic genes for the enhancement of productivity
(Merkle and Dean 2000). Transgenic technology allows the introduction of foreign
genes for the improvement of plant traits. The fundamental essentials for plant
transgenic transformation are (1) gene constructs carrying the polynucleotide
sequence coding for wanted proteins, (2) methods to efficiently transform the
explants, (3) procedure to select the plant tissue with the transgene, and (4) the
efficient protocol for regeneration from desired explants (Brunner et al. 2007). This
chapter is an endeavor to gather all the accessible data on the in vitro approach
research focused worldwide for a large number of plant production, mechanism of
action, and effect of several auxins, cytokinins, testing of clonal genetic fidelity, and
transformation techniques for the improvement of Stevia rebaudiana.
5.2 In Vitro Propagation
Traditional development of Stevia rebaudiana is influenced by different ecological

and climatic elements, yet plant tissue culture guarantees standard supply of plants
utilizing least space and time. Micropropagation synonym of plant tissue culture is
the procedure of vegetative development and multiplication of plants from tissues or
seeds in aseptic and favorable conditions in artificial media (Chandana et al. 2018).
Tissue culture depends on the totipotency that is the capacity of plant cells and
tissues to develop into a full plant. Gottlieb Haberlandt—a German botanist consid-
ered as the father of plant tissue culture, was the first to get the test proof on
totipotency by refined plant cells on Knop’s salt in 1898 (Krikorian and Berquam
1969).
5.2.1 Plant Material Source and Explant Preparation
Tissue culture achievement primarily relies upon the genotype, age, types, and
position of explants of the mother plant (Chandana et al. 2018; Mitra et al. 2020).
To establish sound cultures, the selection of healthy plant source material that is
actively growing and free from disease and insects is an important step. The explants
are washed in running tap water for few minutes and afterward cleaned with few
drops of Tween 20 for 10 min by persistent shaking to remove the surface dust
particles and then washed with the disinfectant, 0.4% (w/v) Bavistin, an expansive
range fungicide, for 15 min with persistent swirling at 110 rpm on the shaker. Further
washing is done in the laminar airflow, where explants are treated with 0.1% (w/v)
newly prepared mercuric chloride (HgCl2) for 6 min and afterward rinsed with
double distilled water for 3–4 times (Mitra and Pal 2006; Singh et al. 2017a, b).
After cleansing, the explants are trimmed at both the ends to the fitting size of
1–1.5 cm and culturing is done on sterile media.
5.2.2 Culture Media Preparation and Sterilization
Culture media contains crucial supplements/components for in vitro development of

plant tissues. A vast range of media is accessible for plant tissue culture, yet most of
the published methods use Murashige and Skoog (1962) basal supplement medium.
MS medium contains macro and microelements, vitamins, other organic substances,
and growth regulators. MS medium containing 3% (w/v) sucrose and 0.8% (w/v)
agar have been utilized in the maximum experiments. By utilizing NaOH (1 N) or
HCl (1 N), the pH of the medium is adjusted to 5.8 (Anbazhagan et al. 2010; Das
et al. 2010; Kalpana et al. 2010). The media is sterilized by autoclaving at 15 lb or
1.06 kg/cm2 pressure and 121 C temperatures for 15 min. The cultures develop at
24 2 C under 16 h photoperiod with cool white fluorescent lights (Mitra and Pal
2006; Singh et al. 2017a, b).
5.2.3 Establishment of In Vitro Cultures
The aseptically prepared explants were inoculated into the cultures containing MS
medium supplemented with different plant growth regulators/hormones (PGRs). The
PGRs or phytohormones are known as the growth hormones that are used to regulate
the physiological and morphological processes in plants (Chandana et al. 2018). The
concentration of various cytokinins [6-benzylaminopurine (BA), Kinetin (kn),
2-isopentenyl adenine (2iP), Thidiazuron (TDZ)] either separately or in a mix with
auxins [2,4-dichlorophenoxyacetic acid (2,4-D), Indole-3-acetic acid (IAA), indole-
3-butyric acid (IBA), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), and
α-naphthalene acetic acid (NAA)] have been utilized to assess the morphogenic
capability of different explants for direct regeneration or organogenesis through
callus development or somatic embryogenesis (Shahzad and Parveen 2013). PGRs
can similarly be added into cultures to improve plant development and to upgrade
metabolite synthesis (Chandana et al. 2018). The in vitro propagation of the stevia
plants via direct and indirect approaches (Figs. 5.1 and 5.2) utilizing explants
attempted by various workers is presented in Table 5.1.
Fig. 5.1 Indirect organogenesis of Stevia rebaudiana. (a) Inoculation of shoot tip explant; (b)
induction of callus; (c) proliferation of calli; (d) plantlet regeneration from calli; and (e)
acclimatization of plantlets. (Source: Authors)
Fig. 5.2 Direct organogenesis of Stevia rebaudiana. (a) Inoculation of shoot tip explant; (b)
multiple shoots regeneration from explant; (c) mass multiplication of shoots; (d) rooting, growth
and elongation of plantlets; and (e) acclimatization of plantlets. (Source: Authors)
5.3 Plant Growth Regulators
5.3.1 Effect of Cytokinins
6-Benzyl aminopurine (BAP) and kinetin are the most widely used cytokinins in the
micropropagation of Stevia rebaudiana. The role of cytokinins in breaking dor-
mancy and multiplication of shoots is well documented by various researchers
(Sahoo et al. 1997). Kinetin has been found efficient cytokinin than BAP since it
helped in more shooting response without callusing (Das et al. 2010). Increasing the
concentration of BAP decreased shoot induction (Thiyagarajan and Venkatachalam
2012). But, Sivaram and Mukundan (2003) showed increased concentrations of
BAP concentrations resulted in more shoot formation from all explant types of
S. rebaudiana. PBZ (Paclobutrazol) enhanced cytokinin activities and induced
adventitious shoot proliferation in Araceae. The effects of PBZ and BA enhance
shoot proliferation (Ziv 1990; Tefera and Wannakrairoj 2006). Intensification of
cytokinin activity can result from the increase in growth retardants and contents of
endogenous cytokinin (Rademacher 2000). Fletcher et al. (2000) observed that PBZ
as one in triazole group enhances cytokinin synthesis that creates chlorophyll
biosynthesis, chloroplast differentiation, and prevents chlorophyll degradation. In
the presence of only cytokinins, stevia leaves showed callusing (Deshmukh et al.
2017). Cytokinin was reported an important factor in shoot multiplication (Singh
et al. 2017a, b). Kinetin is much more effective than BAP as various reports have
Table 5.1 Summary of some of the selected micropropagation studies on Stevia rebaudiana
Morphogenic
Explant response Media composition References
Callus Bud induction MS + 0.1 mg/L BAP + 3% Ferreira and
sucrose + 0.8% agar (pH 5.8) Handro (1988)
Callus Bud induction MS + 1 mg/L BAP + 3% sucrose Swanson et al.
(pH 5.8) (1992)
Shoot tip Shoot MS + 2 mg/L BAP + 3% sucrose + 0.7% Sivaram and
multiplication agar (pH 5.8) Mukundan
(2003)
Node Shoot MS + 2 mg/L IAA + 0.5 mg/L Kn + 3% Hwang (2006)
multiplication sucrose + 0.3% Phytagel (pH 5.8)
Node Shoot MS + 1 mg/L IAA + 10 mg/L Mitra and Pal
multiplication Kn + 30 mg/L adenine sulfate + 3% (2006)
sucrose + 0.9% agar (pH 5.8)
Node Shoot MS + 1.5 mg/L BA + 0.5 mg/L Kn + 3% Ahmed et al.
multiplication sucrose + 0.7% agar (pH 5.7) (2007)
In vitro Root MS + 2 mg/L IAA + 3% sucrose + 0.7% Ahmed et al.
proliferated multiplication agar (pH 5.7) (2007)
shoots
Shoot tip Shoot MS + 3% sucrose + 0.5% agar (pH 5.7) Ibrahim et al.
multiplication (2008)
Leaf Shoot MS + 2 mg/L BA + 1 mg/L Kn + 3% Sreedhar et al.
Callus Shoot MS + 1 mg/L BA + 0.2 mg/L NAA + 3% Janarthanam
multiplication sucrose + 0.9% agar (pH 5.6 0.2) et al. (2009)
Callus Shoot MS + 1.8 mg/L BA + 0.12 mg/L Moktaduzzaman
multiplication NAA + 3.5% sucrose + 0.7% agar and Rahman
(pH 5.7–5.8) (2009)
Node Shoot MS + 3.5 mg/L BA + 3% Sairkar et al.
Callus Shoot MS + 5 mg/L BA + 1 mg/L NAA + 3% Sairkar et al.
Shoot tip Shoot MS + 1 mg/L BA + 0.5 mg/L IAA + 3% Anbazhagan
multiplication sucrose + 0.8% agar (pH 5.8) et al. (2010)
In vitro Root Half-strength N6 medium + 1.0 mg/L Anbazhagan
proliferated multiplication NAA et al. (2010)
shoots
Shoot tip Shoot MS + 2 mg/L Kn + 3% sucrose + 0.7% Das et al. (2010)
multiplication agar (pH 5.6)
In vitro Root MS + 3% sucrose + 0.7% agar (pH 5.6) Das et al. (2010)
proliferated multiplication
shoots
Node Shoot MS + 0.5 mg/L Kn + 0.5 mg/L Kalpana et al.
multiplication NAA + 3% sucrose + 0.8% agar (pH 5.7) (2010)
Leaf Callus MS + 1.0 mg/L BAP + 0.5 mg/L 2, 4-D Ojha et al. (2010)
formation
Node Shoot MS + 2.0 mg/L BA + 0.5 mg/L Kn + 3% Alhady (2011)
multiplication sucrose + 0.7% agar (pH 5.7–5.8)
(continued)

Morphogenic
Explant response Media composition References
In vitro Root MS + 2.0 mg/L IBA + 3% Alhady (2011)
proliferated multiplication sucrose + 0.7% agar (pH 5.7–5.8)
shoots
Node Shoot Modified MS + 1% sucrose + 0.7% agar Modi et al.
multiplication (pH 6.2) (2012)
Node Shoot MS + 1.0 mg/L BA + 0.05 mg/L Soliman et al.
induction NAA + 3% sucrose + 0.8% agar (pH 5.8) (2014)
In vitro Shoot MS + 2.0 mg/L BA + 3% sucrose + 0.8% Soliman et al.
proliferated multiplication agar (pH 5.8) (2014)
axillary
shoots
In vitro Root MS + 0.5 mg/L IAA + 3% Soliman et al.
proliferated multiplication sucrose + 0.8% agar (pH 5.8) (2014)
shoots
Leaf Callus MS + 2.0 mg/L 2,4-D + 0.7% agar Karim et al.
induction (pH 5.7) (2015)
Callus Shoot MS + 1.0 mg/L BAP + 0.7% agar Karim et al.
multiplication (pH 5.7) (2015)
In vitro Root MS + 2.0 mg/L IBA + 0.7% agar Karim et al.
proliferated multiplication (pH 5.7) (2015)
shoots
Shoot tip, Shoot MS + 0.3 mg/L BAP + 0.3 mg/L Yadav et al.
Node multiplication Kn + 0.1 mg/L NAA (2016)
Node Shoot Semisolid MS + 1.0 mg/L Thilakarathne
multiplication BAP + 1.5 mg/L Kn + 3% sucrose et al. (2019)
(pH 5.8)
Node Root Half MS + 2.0 mg/L IBA + 5% sugar Thilakarathne
multiplication (pH 5.7) et al. (2019)
MS Murashige and Skoog, BAP 6-benzyl aminopurine, BA 6-benzyladenine, IAA indole-3-acetic
acid, IBA Indole-3-butyric acid, NAA α-naphthalene acetic acid, Kn kinetin, N6 furfuryladenine,
2,4-D 2,4-dichlorophenoxy acetic acid
claimed (Wang et al. 1986; Gantait et al. 2009). The large number of adventitious
shoot buds developed in the presence of kinetin because it helps in apical dominance
and releases lateral buds from dormancy and upholds shoot formation (George and
Sherrington 1984). Reports of progressive elevations of BAP concentrations resulted
in more shoot formation (Sivaram and Mukundan 2003) that have been contrasted
by Thiyagarajan and Venkatachalam (2012) showing increasing concentrations of
BAP decreased percentage of shoot induction.
5.3.2 Effect of Auxins
Auxins are among important plant growth regulators produced naturally in plants
which are seen in shoot–root tips and promote cell divisions, stem and root growth.
There are many studies on the effect of auxins alone or in combinations with other
plant growth hormones on the micropropagation of stevia. Uddin et al. (2006) in a
study showed the establishment of callus culture in S. rebaudiana with leaf, node,
and internode segments cultured on MS medium with different quantity of 2,4-D
hormone (2, 3, 4, and 5 mg/L). The higher callusing was in MS medium with 3.0 mg/
L 2,4-D, while MS medium with 5.0 mg/L 2,4-D gave the poorest callus. TDZ
impacts metabolism and auxin uptake in plant tissues (Murthy et al. 1995;
Hutchinson et al. 1996; Murch et al. 2001) while induced ethylene-like effects in
tissue culture (Mundhara and Rashid 2006). Increasing the concentration of auxins
improved growth in woody plants (Ali et al. 2009; Domãnguez et al. 2010; Borah
et al. 2017). Root induction was also reported to decrease gradually with increasing
concentration of auxin (Ahmed et al. 2007; Atalay et al. 2011). Combination of
NAA and BAP (1 mg/L each) was found best for callus induction and formation in
stevia with minimum time, whereas B5 medium on adding 1 mg/L 2,4-D + 1 mg/L
BAP for leaf explant and 0.25 mg/L 2,4-D + 0.1 mg/L BAP for internodal explant
gave the highest fresh and dry weight of callus (Keshvari et al. 2018). Proliferative
callus was formed in MS medium with 2 mg/L 2,4-D from leaf explants of stevia
while poor callus was recorded in 6 mg/L 2,4-D in MS medium and MS medium
without 2,4-D did not respond for callus (Karim et al. 2015). In Douglas-fir DCR
(cotyledon revised medium) exhilarated with 0.2 mg/L BA and 0.01 mg/L NAA and
the additive gave maximum shoot multiplication. However, maximum (85%) root
induction was observed in half-strength cotyledon revised medium supplemented
with 0.5 mg/L NAA and 0.5 mg/L IBA. A substrate mixture of sterile perlite and peat
soil (1:3; v/v) was used for acclimatization which recorded 90.20% survival,
whereas DNA marker confirmed the genetical uniformity and stability of plants
regenerated (M. sirindhorniae) significantly enhanced shoot growth and prolifera-
tion was observed in Ceratonia siliqua with new formulated “LA” mineral compo-
sition as compared to MS medium in both solid and liquid condition, whereas liquid
culture system gave good results for shoot induction, shoot length, and multiplica-
tion rate (Lozzi et al. 2019).
5.4 Acclimatization of In Vitro Plants
The pre-acclimatization period is the tenure between in vitro culture and in vivo
transfer phases for coping up of the micropropagated plants in the external environ-
ment and eventually to increase the survival rate. In other words, acclimatization is
the transition period before in vitro cultured plants transfer to ex vitro or field
conditions (Sinta and Amanah 2019). High mortality is generally experienced by
in vitro raised plants during transfer to ex vitro conditions (Chandra et al. 2010). In
vitro propagated plants are always exposed to optimum aseptic environment
conditions and low stress for their multiplication. The tissue culture condition
provides plants with low light, a constant amount of sugar, and other nutrients for
heterotrophic growth and high humid condition which change when plantlets are
directly exposed to field conditions resulting in mortality. Another major limitation
is the in vitro propagated plantlets have poorly developed cuticle, stomata, and root
system (Meera Manjusha and Sathyanarayana 2008). For fruitful acclimatization and
survival of micropropagated plants on being moved to ex vitro conditions, they are
exposed to modified temperature, light intensity, and water stress conditions
(Chandra et al. 2010).
5.4.1 Substrates and Their Role on Successful Acclimatization
The stage of the transplanted plant, type of hardening media, and conditions of soil
play an important role in the acclimatization of in vitro propagated plants. The good
growing medium should have properties such as resistance to decomposition and
have the optimum porosity to ensure adequate aeration for the respiration of roots. It
should allow drainage of excess amount of water which is not required for proper
growth and development of plantlets and should be available at a low price. The
survival of transplanted plants in different hardening media during the process of
acclimatization attempted by various workers in stevia is presented in Table 5.2.
5.5 Genetic Fidelity Assessment
Clonal fidelity is one among the most significant parts of micropropagation industry.
Major limitation in the tissue culture is arising from somaclonal variation as an
immediate outcome of in vitro culture of plant cells, tissues, or organs (Aggarwal
et al. 2016). The degree of somaclonal variety relies on the concentration of media
and the length of culture. The cultures regenerated through shoot organogenesis are
more susceptible to genetic variation as related to branching in the axillary region
(Shenoy and Vasil 1992). The degree of instability relies on the method of regenera-
tion, regardless of whether it is direct organogenesis from explants or indirect
organogenesis through the callus stage (Rani et al. 1995). Additionally, the genetic
variation is determined by the source of the explant source (Kawiak and Łojkowska
2004). The chance of incidence of genetic variability in in vitro propagated plants
Table 5.2 Summary of some of the acclimatization substrates composition of Stevia rebaudiana
Hardening media composition (v/v) Survival (%) References
Soil:Compost @ 2:1 70 Ahmed et al. (2007)
Sand:Soil:FYM @ 1:1:1 82.14 Das et al. (2010)
Soil:Sand:Peat moss @ 1:1:1 71 Ojha et al. (2010)
Sand:Peat moss @ 1:1 75 Alhady (2011)
Soil 83.3 Razak et al. (2014)
Soil:Compost @ 2:1 95 Majumder and Rahman (2016)
Sand:Soil:Vermicompost @ 1:1:1 100 Yadav et al. (2016)
Soil 84 Yesmin (2019)
was estimated over the years through morphological, cytological, biochemical, and
molecular approaches (Rani and Raina 2000).
5.5.1 Morphological Approaches
This is one of the most established old methods used most widely to distinguish the
variation in in vitro culture. Somaclonal variations can be easily recognized based on
characters like variety in plant tallness, leaf morphology, pigment variation, and
canopy structure (Israeli et al. 1991). However, the identification of phenotypic
off-types through morphological markers among the numerous micropropagated
plants is tedious, affected by the environment, and is more time consuming espe-
cially in the case of perennial crops because multiple observations must be made
until development. Additionally, the hereditary change might not be reflected in the
observed phenotypic changes (Rani and Raina 2000). Furthermore, now and then,
the watched variation may lead to the inappropriate understanding also.
5.5.2 Cytological Approaches
The genetic structure of a life-form changes with the structural or numerical changes
in chromosome. Examination of chromosome variations and further nuclear compo-
nent changes has been used by several workers to identify the variation in tissue
cultured plants (Fiuk et al. 2010). The cytological techniques dependent on conven-
tional staining of condensed somatic chromosomes (D’Amato and Bayliss 1985),
shortened somatic chromosomal aberration using light microscopy, oil immersion
methods used by many workers (Ryynänen and Aronen 2005). Though this kind of
approach has its own limits as a method for determining changes, particularly for
plant species in which chromosome number is high because of polyploid condition
and additionally the size of the chromosome is excessively little for distinguishing
structural changes (Rani and Raina 2000). The regular strategy for counting and
examining chromosomes has been replaced by advanced, precise technology, flow
cytometry (Doležel et al. 2004). This method includes the method of aqueous
suspensions of intact nuclei of which the DNA is stained using a DNA fluorochrome,
by suspending them in a stream of fluid followed by passing them by an electronic
detection apparatus. This strategy has been utilized effectively for the identification
of somaclonal variations (Nehra et al. 1992). This method is presently used for the
determination of the nuclear DNA content and DNA ploidy level in plants (Alan
et al. 2007).
Absence of any morphological and cytological varieties among tissue cultured
plants does not mean that absence of epigenetic changes. Sometimes due to point
mutations, alteration in methylation patterns shows the somaclonal changes may
exist. To identify these changes precise molecular techniques are required
(Venkatachalam et al. 2007).
5.5.3 Biochemical Approaches
The phenotypic variations in the organisms are due to the biochemical difference
which is exhibited as variation among proteins. Proteins are the direct results of
genes. Among the protein-based marker system, isozyme electrophoresis has been
identified as a promising strategy to decide the hereditary variation among
micropropagated plants (Rani and Raina 2000). The utilization of isozyme markers
for examining somaclonal variation has been proposed (Lassner and Orton 2018).
From that point onwards, many researchers studied somaclonal variations in plants
produced in in vitro conditions from different explant sources. The variation
described has been summed up into three classifications: (a) modified electrophoretic
mobility, (b) gain or loss of protein bands, and (c) modified level of a particular
protein. The additional merit of non-expensive isozyme investigation over morpho-
logical and cytogenetical markers incorporates co-dominant expression and simplic-
ity of execution. But, the constraints with this methodology are that it just gives data
for DNA areas coding for the soluble proteins, and it is sensitive to developmental or
environmental conditions (Rani and Raina 2000).
5.5.4 Molecular Approaches
In molecular biology, one of the recent developments is the capacity to analyze the
organellar as well as nuclear genomes with nearly an unlimited number of DNA
markers. The molecular makers appropriate for producing DNA profiles are
demonstrated as real tools for finding genetic regenerated plants stability (Martins
et al. 2004). The markers are not influenced by environmental factors and produce
reliable, reproducible outcomes (Li et al. 2011). At first, the restriction fragment
length polymorphisms were used for molecular characterization of in vitro
propagated plants. After the invention of polymerase chain reaction, a great revolu-
tion appeared in the original molecular techniques by modification of standard
procedures planned to suit many requirements. Numerous such alterations, for
example, random amplified polymorphic DNA (RAPD), amplified fragment length
polymorphism, DNA amplification fingerprinting), inter-simple sequence repeat
(ISSR), sequence-tagged sites, and numerous others (Rani and Raina 1996) produce
classes of markers which are found to be exceptionally sensitive for genetic investi-
gation of in vitro propagated plants (Rani and Raina 2000).
The RAPD and ISSR markers have demonstrated to be efficient in building up
clonal fidelity of recovered plants. Both RAPD and ISSR markers have been
effectively applied to identify the genetic similarities or dissimilarities in
micropropagated plants of Stevia rebaudiana in numerous examinations (Modi
et al. 2012; Lata et al. 2013; Singh et al. 2014, 2017a, b; Aggarwal et al. 2016;
Ramírez-Mosqueda et al. 2016; Aziz and Khaled 2017).
5.6 Conclusion
Stevia rebaudiana has generated interest among plant biotechnologists and

pharmacologists in terms of its industrial or commercial and pharmaceutical impor-
tance. Unsustainable harvesting is causing this medicinal plant species to deplete
rapidly. Stevia is generally multiplied by seeds or stem cutting but propagation
through seed is not efficient due to low fertility or viability of seeds, while stem
cuttings also has limitation of reduced leaf yield, low number of new plants,
destruction of donor plants as well as high labor input. Attempts to standardize the
propagation protocols of this species for the production of superior planting
materials for large-scale cultivation and conservation were successful through vari-
ous effective mass multiplication techniques of biotechnological application which
has overcome the barrier of cultivation difficulties and enhance ease of germination.
However, knowledge on naturally available, cost-effective rooting media,
bio-inoculants, and their interaction with the growing conditions for the multiplica-
tion of these medicinal plants is scarce. There are many reports on in vitro propaga-
tion of stevia using cytokinin or cytokinin–auxin combinations, yet there is a need of
refined in vitro protocol establishment. This is because in vitro protocols have been
reported for species and cultivars as well as region-specific. In addition, there are few
other in vitro biotechnological interventions that need to be utilized in stevia for
enhanced production of steviosides under in vitro conditions and the establishment
of a reproducible protocol would be the pre-requisite.
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Swertia chirayita, an Endangered
Anti-diabetic Plant: Trends 6
in Biotechnological Interventions
Praveen Dhyani, Lalit Giri, Eshita Sharma, and Priyanka Sati
Abstract
Swertia chirayita, a medicinal herb (critically endangered) of the Himalayan

region, occurring between 1200 and 2100 m above mean sea level from Kashmir
to Bhutan, has been used as a potent anti-diabetic plant in traditional as well as
modern medical systems. The anti-diabetic efficacy of Swertia chirayita is mainly
attributed to its compounds amarogentin, mangiferin, and swerchirin. Its preva-
lent curative uses in traditional and modern medical approaches have led to its
over-exploitation from its natural habitat, ultimately making it one of the most
traded plant species in South Asia. The excessive over-exploitation and unsolved
difficulties of seed viability and germination has lead S. chirayita to the verge of
extinction. Consequently, alternate propagation approaches are urgently required
to reduce industrial/market pressure on wild stocks. Therefore, for the harvest of
desirable medicinal active compounds of plants, many in vitro propagation efforts
are going on. Although the development of these effective micropropagation
protocols have ensured a satisfactory supply of S. chirayita plants to industrial/
commercial demands with a succeeding decrease in hysterical harvesting burden
on wild populations. Apropos, this chapter highlights trends in biotechnological
interventions such as synthetic seed technology, development of somatic
P. Dhyani
Department of Biotechnology, Kumaun University, Bhimtal, Nainital, Uttarakhand, India
L. Giri (*)
G.B. Pant National Institute of Himalayan Environment, Ladakh Regional Centre, Leh, Ladakh,
India
E. Sharma
CSIR-Institute of Himalayan Bioresource Technology, Palampur, Himachal Pradesh, India
P. Sati
Graphic Era University, Dehradun, Uttarakhand, India
Ltd. 2021
https://doi.org/10.1007/978-981-16-3529-8_6
134 P. Dhyani et al.
embryogenesis protocol from different explants with the effect of plant growth
hormones, followed by acclimatization, to produce true-to-type regenerates
throughout the year.
Keywords
Anti-diabetic · Biotechnology · In vitro propagation · Over-exploitation · Swertia

chirayita
6.1 Introduction
Diabetes, a chronic disease, affects 422 million people worldwide, with most low-
and middle-income countries (WHO 2020; https://www.who.int/health-topics/
diabetes). Over the past few decades, diabetes prevalence is steadily increasing,
which poses a global health concern. The concern also grows because 1.6 million
deaths worldwide are attributed directly to diabetes each year. According to the
Diabetes Foundation, India, around 50 million people have diabetes, and this
number is projected to reach 80 million by 2025 (http://www.
diabetesfoundationindia.org). Among the two types of diabetes (Type 1 and Type
2), Type 1 diabetes is usually treated through an external supply of insulin. In
contrast, Type 2 requires lifestyle changes alongside drug therapy. Although widely
used, anti-diabetic drugs pose several side effects, such as hepatic impairment, heart
failure, atherosclerosis, etc.; consequently, the usage of herbal medication to control
diabetes has augmented over the years.
The Himalayan region has been home to numerous medicinal plants, of which
many are reported for their anti-diabetic potential. Swertia chirayita, a native plant of
temperate Himalaya is one of the most critical species with high anti-diabetic
potential among 40 species found in Swertia’s genus in India and is commonly
used in many herbal formulations. The S. chirayita was first described as
Gentianachyrayta in 1814 by Roxburgh (Scartezzini and Speroni 2000). It is an
endangered annual/biennial 0.6–1.5 m tall herb with distinguishing characteristics
(Table 6.1), flower in September–October, with fruiting in November S. chirayita is
a dicot (2n ¼ 26) plant with both self and cross-pollination being reported (Khoshoo
and Tandon 1963; Soumendra et al. 2009).
Taxonomic Classification of S. chirayita

Kingdom: Plantae
Phyllum: Tracheophyta
Order: Gentianales
Family: Gentianaceae
Tribe: Gentianeae
Subtribe: Swertiinae
6 Swertia chirayita, an Endangered Anti-diabetic Plant: Trends in. . . 135
Table 6.1 Morphological characteristics of S. chirayita plant

Part Description References
Flower • Small, numerous, tetramerous, large leafy Scartezzini and Speroni (2000),
panicles, green-yellow Joshi and Dhawan (2005)
• Calyx: Gamophyllous with four lobes
• Corolla: Four (twisted and superimposed)
• Stamens: Four (opposite to the corolla
lobe)
• Ovary: Unilocular with ovules laminal
placentation
• Stigmas: Two
• Capsules: Two-valved with a transparent
yellowish pericarp, egg-shaped
Leaves • Lanceolate Scartezzini and Speroni (2000)
• In opposite pairs
• Acuminate
• Cordate at the base
• Sessile
• Five to seven nerved
• 4 cm long
Stem • Upright Bentley and Trimen (1880), Joshi
• 2–3 ft long and Dhawan (2005)
• Middle portion: Cylindrical
• Upper portion: Quadrangular
• Orange-brown or purplish
• Large continuous yellowish pith
Root • Simple Bentley and Trimen (1880),
• Yellowish Scartezzini and Speroni (2000)
• Oblique, or geniculate
• Short and tapering
• 7–8 cm long, 1–1.5 cm thick
Seeds • Numerous Chandra et al. (2012)
• Very small
• Dark brownish
Genus: Swertia
Species: chirayita
6.1.1 Habitat and Geographical Distribution
S. chirayita has a geographical distribution between 1200 and 3000 m amsl (Joshi
2012), ranging from Kashmir to Bhutan and Khasi hills. The wild populations are
usually patchy in occurrence and occur on the inclines of typically shaded places
(Gaur 1999). Nonetheless, it can be found in open grounds beside newly slash-and-
burn forests. Additionally, some studies have reported the populations of S. chirayita
from the South-East facing mixed forests of Acer and Quercus (Bhatt et al. 2006) and
north or North-West facing a mixed forest of Oxalis corniculata, Fragaria indica,
Digitaria ciliaris, Cynodon dactylon, Artemisia vulgaris, and Anaphalis triplinervis
(Joshi 2012). S. chirayita is frequently found in a diverse range of soils with sandy
loam enriched in humus (Barakoti 2004). The chirayita plants grow in temperatures
up to 15 C.
Vernacular names: Arabic: Qasab-uz-zarirah; Burma: Sekhagi; Nepal:
Chirratoor, Chiraita; Hindi: Chirata, Kalapnath, Kalmegh, Kirayatcharaytah; Per-
sian: Nenilawandi; Sanskrit: Anarya-tikta, Bhunimba, Chiratitka, Kairata; Urdu:
Chiaravata (Joshi and Dhawan 2005).
6.1.2 Medicinal Uses and Ethnobotanical Uses
S. chirayita is known as kairata, kirata, Ramasenka, Haima, Tikta, Nidrari, in

Sanskrit, Qasabuzzarirah in Arabic and Farsi, Chiravata in Urdu, Chireta in Bengali,
and market name as Chiretta. It is available in Indian Medical Conformity to remedy
a diverse range of disorders such as neurological disorders, urinogenital tract
disorders, liver disorders, gastrointestinal disorders, skin, liver, and kidney diseases.
S. chirayita is used as antipyretic, anthelmintic, anti-periodic, analeptic, anti-
inflammatory, relaxant for pregnant uterus and fever (Kirtikar and Basu 1935).
The astringent flavor of chirayita stops nausea (queasiness) by the production of
saliva and gastric enzymes; it helps to cure indigestion, bloating, and hiccups. The
decoction of chirayita is used as a breast milk cleanser and has laxative, appetizer,
and carminative activity. A hot infusion of chirayita mixed with dhaanyaka
(Coriandrum sativum) leaves alleviates fever immediately (Badola and Pradhan
2013). The S. chirayita as a whole plant has been extensively used in traditional
medication to treat diabetes for many centuries and treatment for hypertension, fever,
cough, cold, stomach-ache, asthma, malaria, hepatitis, ulcers, and diarrhea (Karan
et al. 1999; Banerjee et al. 2001; Saha et al. 2004; Chen et al. 2011; Kumar and van
Staden 2016). A paste of chirayita mixed with an equivalent quantity of honey is
useful to treat nausea and vomiting (Sapkota et al. 2019). Similarly, a paste of
chirayita and sunthi destroys chronic oedema. Generalized oedema is removed by
taking paste of chirayita and sunthi followed by intake of punarnava decoction.
Chirayata, kramuka, musta, etc., separately mixed with sandals, are beneficial in the
intrinsic hemorrhage. This curative value is well-documented in different ancient
and modern medical systems such as Ayurveda, Siddha, Unani, as well as in
American and British pharmacopeia (Joshi and Dhawan 2005). In addition to its
diverse activities to different ailments, S. chirayita extracts have also displayed anti-
hepatitis B virus (anti-HBV) activities (Zhou et al. 2015).
S. chirayita is one of the most frequently singly or in combination used herb
(higher use value) by many tribal communities of India in their folk medicine such as
Limboo tribe from Khangchendzonga Biosphere Reserve, Sikkim (Badola and
Pradhan 2013), Lepcha tribe from Dzongu valley in North Sikkim (Pradhan and
Badola 2008), Bhotiya tribal communities from Niti valley in Uttarakhand
(Phondani et al. 2010), Apatani tribe from Ziro Valley in Arunachal Pradesh,
Tangkhul tribe in Manipur (Salam et al. 2014), Gaddi and Gujjar tribes in Himachal
Pradesh (Guleria and Vasishth 2009). The whole herb is immersed in water for
overnight and decoction is then taken for treatment to different diseases (Kirtikar and
Basu 1935).
Earlier, the medicinal plants were taken in the form of a crude drug (tinctures,
powder) or combination with other herbs as a herbal formulation; this indigenous
knowledge of herbal formulations now serves as the primary basis of the discovery
of novel lead molecules which can serve as a novel drug. Likewise, chirayita has
also provided many lead molecules for drug development against various diseases.
Some of the popular chirayita containing formulations are Melicon V, Diabecon,
Ayush-64, used as anti-pyretic, anti-bacterial, anti-fungal, and anti-diabetic
(Tabassum et al. 2012).
Moreover, S. chirayita is one of the constituents in important ayurvedic
preparations such as Dermafex oil (to treat various skin and hair fall problems
ms), Kabdeen (treatment for viral hepatitis), Sudarshan Churna, Mahasudarshan
Churna, Kiratatikta Dai Kvatha, Bhunimbadi Kwatha, Kiratadi Taila (treatment for
fever), Chandraprabha Vati (treatment for cancer), Palasahabeejadi Churna (anthel-
mintic activity) (Pradhan and Badola 2015).
The wide range of biological activity of S. chirayita mainly attributes to a varied
group of pharmacologically bioactive compounds of diverse classes such as
xanthones and their derivatives, lignans, alkaloids, flavonoids, iridoids, terpenoids,
secoiridoids, chiratin, ophelic, oleic, palmitic, and stearic acid.Chowdhury et al.
(1995) proposed the anti-inflammatory activity of an aqueous suspension of
S. chirayita attributable to total xanthones present in S. chirayita. The pharmacolog-
ical effectiveness of S. chirayita is mainly accounted to the biological efficacy of key
phytoconstituents, i.e., swertiamarin, amarogentin, swerchirin, Mangiferin,
sweroside, gentiopicrin, and amaroswerin.
Amarogentin has been reported to anti-diabetic (Phoboo et al. 2013), anti-
cancerous (Saha et al. 2006; Pal et al. 2012), anti-proliferative, anti-leishmanial
(Ray et al. 1996; Medda et al. 1999), and pro-apoptotic actions (Saha et al. 2006;
Khanal et al. 2014). Swertiamarin has been shown to exhibit anti-hepatitis (Wang
et al. 2001), anti-diabetic (Vaidya et al. 2013), anti-cancer (Kavimani and
Manisenthlkumar 2000), and anti-arthritic activities (Saravanan et al. 2014). The
anti-helminthic activity reported by S. chirata has been attributed to the presence of
amarogentin and other secoiridoid glycosides like amaroswerin and sweroside (Iqbal
et al. 2006). Sweroside has been recommended as an excellent natural osteoporosis
therapeutic product, anti-bacterial, hepatoprotective, and for hyper-pigmentation
treatment (Jeong et al. 2015; Luo et al. 2009; Šiler et al. 2010; Sun et al. 2013).
Additionally, amaroswerin has demonstrated the gastroprotective properties of bitter
values (Niiho et al. 2006; Kumar and Van Staden 2016).
Mangiferin has been described to possess anti-diabetic, anti-atherosclerotic, anti-
inflammatory, anti-oxidant, anti-cancer, anti-HIV, anti-parkinson, chemopreventive,
and immunomodulatory activities (Pardo-Andreu et al. 2008; Saha et al. 2004; Guha
et al. 1996; Kavitha et al. 2013; Yoshimi et al. 2001). A xanthone isolated from
S. chirata plant, recognized as 1, 8-dihydroxy-3, 5-dimethoxyxanthone (swerchirin)
is known to be anti-malarial, hypoglycemic, hepatoprotective, pro-hematopoietic
with blood-glucose-lowering, and chemopreventive pharmacological activity
(Hirakawa et al. 2005; Saxena et al. 1996; Sekar et al. 1987; Ya et al. 1999).
Swertiamarin, a secoiridoid glycoside extracted from S. chirayita, has been proved
to be a potential anti-depressant agent (Sapkota et al. 2019).
6.2 Anti-diabetic Activity: Clinical and Preclinical Studies
A varied range of in vitro and in vivo experiments have been used to assess the
pharmacological properties of S. chirayita. The hypoglycemic and anti-diabetic role
of S. chirata is the most investigated action of this plant, which is well accepted in
the literature (Kumar and Van Staden 2016; Phoboo et al. 2013; Singh et al. 2012).
S. chirayita several pharmacologically bioactive compounds of diverse classes such
as xanthones (and their derivatives), terpenoids, alkaloids, lignans, flavonoids,
secoiridoids, iridoids, chiratin, ophelic, palmitic, oleic, and stearic acids contribute
to wide-range biological activities. The biological activity of crucial
phytoconstituents mainly accounts the pharmacological efficacy of S. chirayita,
i.e., amarogentin, mangiferin, swerchirin, and swertiamarin (Fig. 6.1).
Amarogentin: Amarogentin has been informed to possess anti-diabetic, anti-
proliferative, anti-cancerous, anti-leishmanial, and pro-apoptotic activities (Phoboo
Fig. 6.1 Chemical structures of anti-diabetic and hypoglycemic phytochemicals found in Swertia
chirayita. (Source: https://pubchem.ncbi.nlm.nih.gov/compound)
et al. 2013; Saha et al. 2006; Pal et al. 2012; Ray et al. 1996; Medda et al. 1999;
Khanal et al. 2014). The amarogentin ethanolic fraction from S. chirata showed an
anti-diabetic effect by inhibiting an angiotensin-1-converting enzyme in Type-2-
diabetes. Inhibition of enzymes such as α-glucosidase delays the digestion of
carbohydrates by slowing down the systemic circulation’s glucose release, allowing
the β-cell adequate interval to adjust the insulin discharge in reaction to the upsurge
in plasma glucose level (Phoboo et al. 2013).
Mangiferin: Mangiferin has anti-atherosclerotic, anti-diabetic (Pardo-Andreu
et al. 2008), anti-oxidant, anti-inflammatory (Saha et al. 2004), anti-HIV, anti-cancer
(Guha et al. 1996), anti-parkinson (Kavitha et al. 2013), chemopreventive, and
immunomodulatory actions (Yoshimi et al. 2001). The action mechanism included
that mangiferin directly stimulates insulin production, enhances glycolytic enzymes,
inhibits α-glucosidase and other enzymes such as maltase, sucrose, isomaltase, and
aldose reductase (Phoboo et al. 2010).
Swerchirin: The hexane fraction of S. chirata plant containing swerchirin lowered
blood sugar level (40%) significantly in fasted, fed, glucose loaded, and tolbutamide
pre-treated male albino rat models (bodyweight 140–165 g) with an ED50 value
23.1 mg/kg/oral (Bhat and Surolia 2001; Gupta et al. 2008). Investigation on the
mechanism revealed that the effect was associated with marked depletion of beta-
granules and pancreatic islets (Saxena et al. 1991, 1993). In vivo studies showed that
serum of swerchirin significantly enhanced the glucose uptake and glycogen synthe-
sis by muscle cells in treated rats, stimulating insulin release from isolated islets of
Langerhans, indicating that swerchirin lowers the blood sugar level (Saxena et al.
1993). The swerchirin ethanolic fraction of S. chirata plant (250 mg/kg) potentially
contributes to reducing blood sugar in fasted, glucose fed, and tolbutamide
pre-treated animals (Sekar et al. 1987; Kar et al. 2003). Swerchirin (50 mg/kg,
b.w. orally) isolated from S. chirata lowered the blood glucose up to 60% after 7 h
post-treatment (Chandrasekar et al. 1990). The swerchirin containing fractions from
S. chirata showed a better glucose-lowering influence in normal and streptozotocin-
induced rats than other structurally different hypoglycemic agents, i.e., tolbutamide
and centpiperalon (Singh et al. 2012).
Swertiamarin: Swertiamarin has displayed anti-hepatitis, anti-diabetic, anti-
cancer, and anti-arthritic activities (Vaidya et al. 2013; Kavimani and
Manisenthlkumar 2000; Wang et al. 2001; Saravanan et al. 2014). Swertiamarin
significantly blocks HMG-CoA reductase and impedes hepatic cholesterol biosyn-
thesis. The obstruction of cholesterol synthesis results in a lesser intracellular
quantity of cholesterol, thus activating hepatic LDL receptors’ over-expression and
increasing clearance of circulating LDL particles. Swertiamarin stimulates the
sterol’s elimination by improving cholesterol’s metabolism proportion and increas-
ing bile acid and sterol elimination in fecal matter.
6.3 Conservation Status and Challenges
Due to the remarkable medicinal properties and high demand, the S. chirayita has
high trade value and is one of the “most extensively traded” in a 150 species genus.
This trade is composed of produce in the form of the whole dried plant or plant
extract from wild populations with some cultivated populations imported and
exported between cross-borders of Nepal-Bhutan-India. As the entire plant has
been attributed to its charismatic medicinal properties in Ayurveda, India has been
a center of the trade for import/export of S. chirayita (S. chirayita is among the
32 utmost prioritized medicinal plants of India, as recognized by the National
Medicinal Plant Board, Government of India (https://www.nmpb.nic.in/). Between
the years 2015 and 2020, India exported 2.22–27.32 MT of S. chirayita, primarily to
European nations such as France and Italy. However, in the same period, India
imported 99.35–348.53 MT under HS 12119091 commodity (Ministry of Com-
merce & Industry, Government of India; https://commerce.gov.in/), mostly from
Nepal, as Nepal has extensive cultivation in Eastern Nepal (Cunningham et al.
2018). Despite legal trade options, a significant section of S. chirayita wild
populations is traded illegally through local trade paths accompanied by improper
descriptions and tariff codes on export statements and misbranding/misrepresenta-
tion of other plants (Andrographis paniculata) with S. chirayita, leading to under-
representation in the trade value of S. chirayita (Cunningham et al. 2018).
The illegal harvest of wild populations coupled with low seed regeneration rates,
injudicious premature harvesting before seed development (Joshi 2011) in natural
habitats, and clearing of forests for road or other constructions Purohit et al. (2013),
has also posed a high degree of threat to the S. chirayita to meet the commercial
demand leading to declining population density and distribution. Although the
S. chirayita is the most explored and traded, it has not been accessed by the global
IUCN Red List (The IUCN Red List of Threatened Species; https://www.iucnredlist.
org/) for threat category. However, the Conservation Action and Management Plan
(CAMP) workshops have been accessed in India and Nepal with Chirayita as
“vulnerable” in Arunachal Pradesh, Assam, Meghalaya, and Sikkim (Ved et al.
2003) and “critically endangered” in Himachal Pradesh (Goraya et al. 2013). In
Nepal, S. chirayita was considered to be VU (Tandon et al. 2001). Nevertheless, the
perceptions of threats are so profound that the Government of India has enforced an
overall prohibition on the gathering or removal of planting materials of S. chirayita
from wild populations; in Nepal, there is a recurrent ban on harvest and trade
between May to September (Pyakurel and Oli 2013). To meet the growing demand
and increasing threat to its conservation, Biotechnology offers various approaches
such as micropropagation techniques to improve biodiversity.
6.4 Biotechnological Interventions for Conservation
Under biotechnical intervention, the micropropagation technique is being used

widely for threatened and high-value plants for the large-scale production of quality
planting materials (Bhojwani and Razdan 1996; Ravishankar and Venkatraman
1997). In micropropagation, many factors such as concentration and composition
of nutrient medium and plant growth regulators the environmental setting in culture
growth, donor plant’s genotypic and physiological status along with others play a
critical role in the development of an efficient protocol for the establishment of
quality planting material. An extensive review reflects only few studies exist on
in vitro propagation of S. chirayita (Table 6.2).
6.4.1 In Vitro Seed Germination
Regeneration from seeds is the usually used propagation technique in numerous

plant species (Sharma et al. 2006). Very few reports are available on studies
unfolding to in vitro seed germination and viability of S. chirayita. Among these,
Raina et al. (1994) obtained 91% in vitro seed germination in contrast to 3.4%
germination in control seeds in MS medium of chilling seeds at 3 C for 15 days.
Moreover, Joshi and Dhawan (2007a, b) obtained 80% in vitro seed germination
when pre-treated seeds (soaked overnight in 1.146 M GA3 solution) were cultured in
½ MS medium. The seeds, nevertheless, were unsuccessful in germinating when not
pre-treated by GA3.
6.4.2 Shoot Multiplication
6.4.2.1 Shoot Multiplication Through In Vitro Roots

A study by Wawrosch et al. (1999) was perhaps the earliest finding on in vitro
propagation of S. chirayita through the root as explants. In this study, an average of
1.9 shoots/explant was attained by following a two-step system comprising of
primary 3-week cultivation on modified MS medium augmented with 3 μM BA
followed by additional 3 weeks in medium free from plant growth regulator. Rooting
was induced by dipping the sterilized shoots in an autoclaved aqueous NAA solution
(15 ppm) for a short period (2 s) followed by 3–4 weeks growth on hormone-free
half MS medium, proving to be the best capable process for rooting of S. chirayita.
6.4.2.2 Shoot Multiplication Through In Vitro Nodal Explant/Shoot Tip

Chaudhuri et al. (2007) have established genetically identical plants from nodal
explants of S. chirayita. Aseptic nodal explant culture on MS medium augmented
with BA (0.44 μM) 6-furfurylaminopurine (4.65 μM), KNO3 (10 mM), and casein
hydrolysate (75 mg/L) obtained a maximum of 18 shoots/explant. Regenerated
shoots proliferated when shifted to half MS medium augmented with 10 mM of
KNO3. Consequently, fibrous, adventitious roots advanced from 20 to 30% of the
Table 6.2 A comprehensive description of work carried out on in vitro propagation of S. chirayita
142
Media + plant growth regulators (PGRs)

Callus induction/
Explant used organogenesis Shoot multiplication Rooting Results References
Radicle from Not carried out MS + 3 μM BA MS medium without Multiple shoot formation (1.9 Wawrosch
in vitro grown plant PGRs shoots/explant) and rooting et al.
(1999)
Aseptic nodal Not carried out MS + 0.44 BA μM + 4.65 μM ½ MS + 10 mM Multiple shoot formation Chaudhuri
segment kinetin +10 mM KNO3 + 75 mg/L KNO3 (18 shoots/explant) and rooting et al.
casein hydrolysate (30%) (2007)
Aseptic axillary Not carried out MS + 4 μM BAP + 1.5 μM 2iP ½ MS + 1 μM Multiple shoot formation (4.5- Joshi and
shoots NAA + 500 mg/L fold) and rooting (7.7 roots per Dhawan
activated charcoal shoot) (2007a, b)
Shoot tip derived Not carried out MS + 1 mg/L BA + 0.1 mg/L ½ MS + 0.1 mg/L Multiple shoot formation (42.16 Balaraju
from in vitro grown kinetin IBA shoots/explant) and rooting (4.22 et al.
seedlings roots) (2009)
Aseptic leaf Not carried out MS + 2.22 μM BA +11.6 μM – Direct organogenesis (seven shoot Chaudhuri
kinetin +0.5 μM NAA buds/explant) et al.
(2008)
Sterile leaf explant Not carried out 190–2 basal medium +13.32 μM – Indirect organogenesis (94.57% Wang et al.
BA +0.54 μM NAA multiple shoot formation with 4.81 (2009)
roots/shoots)
In vitro regenerated MS + 13.32 μM MS + 13.32 μM BA +0.90 μM MS + 13.32 μM BA Indirect organogenesis (callus Pant et al.
roots BA +0.90 μM 2,4-D +0.90 μM 2,4-D 581 mg, callus multiplication (2012)
2,4-D 11.63-fold, multiple shoot (8.10
shoots/callus)
P. Dhyani et al.
6
Sterile leaf explant MS + 15.0 μM MS + 10 μM BA +5.0 μM GA3 MS + 5.0 μM BA Callus induction (100%), multiple Kumar
2,4-D IBA shoot (32 shoots/callus piece), et al.
rooting (50 roots/shoots) (2018)
Shoot tip Not carried out MS + 1.5 mg/L BAP + 0.1 mg/L ½ MS + 1.5 mg/L Multiple shoot (16.53 shoots/ Kapoor
KIN +0.2 mg/L NAA IBA + 0.02% explant) and rooting (6.26 roots) et al.
activated charcoal (2019)
Swertia chirayita, an Endangered Anti-diabetic Plant: Trends in. . .
143
shoots in the same medium. The plantlets were efficaciously moved to the field and
subsequently produced viable seeds.
Joshi and Dhawan (2007a, b) have developed a micropropagation protocol of
S. chirayita from axillary shoot derived from nodal explants. A maximum of 4.5-fold
shoot proliferation was obtained when the axillary shoot tips were cultured on MS
medium augmented by 4 μM BAP and 1.5 μM 2ip. Maximum roots (7.7 roots per
shoot) were obtained on ½ MS medium augmented 1 μM NAA and 500 mg
activated charcoal with 100% rooting. In vitro hardening in the growth chamber
and ex vitro hardening in the greenhouse resulted in a success rate of 94%. Shoot
multiplication through shoot tip from in vitro developed seedlings has been reported
by Balaraju et al. (2009). The highest number of shoots (42.16 shoots/explant) was
obtained in MS medium supplemented with 1.0 mg/L BA and 0.1 mg/L kinetin
along with 2% sucrose induced highest number. The highest frequency of rooting
(66.6%) with a root number of 4.22 was obtained in ½ MS medium supplemented
with 0.1 mg/L IBA.
6.4.3 Callus Induction and Organogenesis
6.4.3.1 Direct Organogenesis Through In Vitro Leaf

Chaudhuri et al. (2008) have formulated a protocol for direct organogenesis through
the in vitro leaf. MS medium supplemented with 2.22 μM BA, 11.6 μM kinetin, and
0.5 μM NAA was the best combination for direct organogenesis and obtained a
maximum of seven shoots buds/explant. Moreover, the plants raised through organ-
ogenesis were found to have no dissimilarity at the chromosomal level when tested
for genetic fidelity by DNA fingerprinting and chromosomal analysis.
6.4.3.2 Callus Culture and Indirect Organogenesis

Callus culture is an excellent alternate method for the production of in vitro plants.
Wang et al. (2009) obtained embryogenic callus from leaf explants of S. chirayita
when cultured in three medium types included MS (Murashige and Skoog 1962), B5
(Gamborg et al. 1968), and 190–2 (Zhuang et al. 1984). All mediums were aug-
mented with varying NAA concentrations (0, 0.54, and 1.07 μM) and BA (4.44,
8.88, and 13.32 μM). The best result of shoot regeneration (25.13% with 4.30 shoots/
explant) was achieved on a 190–2 basal medium supplemented with 13.32 μM BA
and 0.54 μM NAA. Maximum rooting was obtained with 94.57% with 4.81 roots/
shoots on MS medium supplemented with 5.40 μM NAA. An effective procedure for
developing the plantlets of S. chirayita via indirect organogenesis was also devel-
oped by Pant et al. (2012). Callus was attained from in vitro regenerated roots on MS
medium supplemented with different concentrations of BA and 2,4-D. Among
various BA and 2,4-D concentration, 13.32 μM BA, and 0.90 μM 2,4-D was
noted for the best combination for callus induction (581 mg), multiplication
(11.63-fold), and adventitious shoot regeneration (8.10 shoots/callus). Kumar et al.
(2018) induced the callus from leaf explant of S. chirayita in MS medium containing
different concentrations of 2,4-D, BA, and GA3. The best callus induction was
Fig. 6.2 Different stages of in vitro propagation of S. chirayita. (a) Nursery established mature
plants collected from wild; (b) establishment of leaf explants in culture media; (c) swelling in leaf
explants after 7 days; (d, e) conversion of leaf explants in green–yellow callus after 60 days; (f, g)
initiation of microshoots from calli after 15 days; (h, i) different stages of shoot development and
growth after 30 days; (j) root formation in microshoots after 45 days; (k) transfer of rooted
microshoots for hardening in mixture of soil, sand, and FYM (2:1:1); (l) well-grown plants in
nursery/field condition. (Source: Kumar et al. (2018); http://creativecommons.org/licenses/by/4.0/)
obtained in 15.0 μM 2,4-D after 60 days of the culture period. Further, the callus was
cultured for shoot induction; a maximum of 32 microshoots was obtained in MS
medium supplemented with 10 μM BA and 5.0 μM GA3. Root induction was
obtained in all microshoots cultured in various concentrations of auxins (IBA and
IAA), maximum rooting (50 roots per shoot) was observed in MS medium
supplemented with 5.0 μM BA IBA (Fig. 6.2).
6.4.4 Synthetic Seed Production
A synthetic seed or artificial is a crucial application of somatic embryo and consid-

ered related to the natural seeds (Gantait et al. 2015). The synthetic seed of
S. chirayita through somatic embryogenesis was first established by Kumar and
Chandra (2014). In this study, embryogenic callus (76%) was induced on MS
medium supplemented with 0.5 mg/L 2,4-D and 0.5 mg/L kinetin from in vivo
leaf explants. After that, the torpedo stage embryos attained were encapsulated in 4%
w/v sodium alginate gel and dipped into 100 mM calcium chloride (CaCl2 ∙ 2H2O)
solution. A maximum frequency of synthetic seed germination (i.e., 84%) was
achieved on MS medium supplemented with 1.0 mg/L BA and 0.5 mg/L NAA.
The regenerates effectively acclimatized under ex vitro condition.
6.4.5 Hairy Root Culture (Agrobacterium rhizogenes-Mediated

Transformation)
Hairy root cultures of S. chirayita via Agrobacterium rhizogenes-mediated transfor-

mation were first successfully established by Samaddar et al. (2019). A. rhizogenes
strain LBA9402 was obtained as the best strain for inducing the highest root
induction frequency (25 4.4%) with 2–9 roots node/intermodal cut site. Analysis
of characteristic secondary metabolites, especially the anti-diabetic compounds, i.e.,
amarogentin, swertiamarin, and mangiferin, has also been assessed in the present
study through High Performance Thin Layer Chromatography (HPTLC). The
swertiamarin content varied between 0.042 and 0.207%; amarogentin content varied
from 0.019 to 0.012% whereas mangiferin content was recorded at 0.017% in the
assessed root lines.
6.4.6 Assessment of Genetic Fidelity
Micropropagation is widely used as a mass multiplication tool for the germplasm

conservation of high value and threatened plant species. The most precarious
objective of tissue culture is to achieve true-to-type plants to preserve the germ-
plasm. However, there is still a possibility of genetic aberration during tissue culture,
regularly known as “somaclonal variations.” Thus, the in vitro raised plantlets
obtained from micropropagation be checked for their clonal fidelity. The application
of DNA-based markers like Restriction Fragment Length Polymorphism (RFLP),
Random Amplified Polymorphic DNAs (RAPDs), Inter Simple Sequence
Repeats (ISSRs), Single Nucleotide Polymorphisms (SNPs), Cleaved Amplified
Polymorphic Sequence (CAPS), Simple Sequence Repeats (SSR), Amplified Frag-
ment Length Polymorphism (AFLP), etc., offers more dependable tools for the
determination of somaclonal variations or genetic variability. The genetic variability
of tissue culture raised plants has been evaluated in an inclusive range of plants for
estimating clonal fidelity and genetic stability among tissue cultured plants and
donor plants (Bekheet et al. 2007; Giri et al. 2012; Hirai and Sakai 1999; Mishra
et al. 2011; Purohit et al. 2017).
Genetic fidelity assessments of S. chirayita have also been assessed through
RAPD based markers (Chaudhuri et al. 2007, 2008). Chaudhuri et al. (2008) used
29 RAPD primers to fingerprint the tissue culture-raised plants and their respective
donor plants. The RAPD profile comprised a total of 83 alleles, ranging from 1 to
8 per primer. The RAPD profile was monomorphic and thus indicated homogeneity
among the tissue culture raised plants and confirmed a genetic homogeneity to the
donor plants.
6.5 Conclusion and Way Forward
Published literature affirms that S. chirayita is a high-value medicinal plant used in

traditional and modern medicine systems for curing various diseases since ancient
times. Due to its high extraction from the natural habitat and another anthropogenic
factor, the species is considered under different threatened conditions. To date, less
effective work has been done on the conservation and sustainable use of S. chirayita
neither in situ nor ex situ conditions. Therefore, biotechnological mediations such as
micropropagation, callus culture, synthetic seed, hairy root culture, etc., are required
and can prove an effective strategy for planting material preparation, thereby reduc-
ing harvest pressure on natural populations of S. chirayita. Among others, more
detailed research is required in synthetic seed technology, especially in the improve-
ment of its germination frequency so that it can be used on a commercial scale.
Additionally, hairy root culture technology can be improved for harnessing the
active constituents on a large scale, and the demand of various pharmaceutical
companies can be fulfilled adequately.
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In Vitro Propagation and Biotechnological
Improvement Strategies of Plants 7
with High-Intensity Sweetener
and Anti-Diabetic Activities
Elizabeth Kairuz , Alán Rivero-Aragón , and Geert Angenon
Abstract
Diabetes mellitus is an endocrine and metabolic disease with high prevalence

worldwide. Moreover, sugar intake has increased in the last decades leading to a
higher risk of obesity and type 2 diabetes. Therefore, new compounds have been
prospected for the food industry to produce very low-calorie and intense
sweeteners from natural sources. Besides, medicinal plants with anti-diabetic
activities have become popular as an alternative to drug treatments. This study
aimed to perform a systematic review of plants that combine these two properties:
sweeteners and anti-diabetic activity. Four plant species were identified following
these features, one widely known as a sweetener (Stevia rebaudiana Bertoni) and
three less common high-potency sweeteners (Cyclocarya paliurus (Batal.)
Iljinsk, Glycyrrhiza glabra L., and Siraitia grosvenorii (Swingle) C. Jeffrey ex
A. M. Lu and Zhi Y. Zhang). First, the bioactive compounds and the pathways
involved in their biosynthesis are summarized. Afterwards, we describe the anti-
diabetic action and other medicinal effects of these species. Moreover, we
E. Kairuz (*)
Departamento de Biología, Facultad de Ciencias Agropecuarias, Universidad Central “Marta
Abreu” de Las Villas (UCLV), Santa Clara, Villa Clara, Cuba
e-mail: kairuzhd@uclv.edu.cu
A. Rivero-Aragón
Departamento de Biología, Facultad de Ciencias Agropecuarias, Universidad Central “Marta
Abreu” de Las Villas (UCLV), Santa Clara, Villa Clara, Cuba
e-mail: alanra@uclv.edu.cu
G. Angenon
e-mail: Geert.Angenon@vub.be
Ltd. 2021
https://doi.org/10.1007/978-981-16-3529-8_7
154 E. Kairuz et al.
reviewed the principal in vitro propagation studies and biotechnological strategies

reported so far on these species. Finally, the most promising biotechnological
approaches and future perspectives for the industrial exploitation of these species
are discussed.
Keywords
Glycyrrhizin · Licorice · Medicinal plant · Mogrosides · Monk fruit · Stevia ·

Steviol glycosides · Sweet tea tree
7.1 Introduction
Dietary sugar intake through food, beverages and additives has become a health
issue to address in nutritional policies worldwide (Wittekind and Walton 2014). The
tendency to a sedentary lifestyle and to consumption of junk food or excessive
amounts of sugar has promoted a higher incidence of deadly diseases like obesity,
hypertension, cardiovascular problems, and diabetes (Khan et al. 2021). Diabetes
mellitus is a chronic endocrine syndrome that provokes metabolic disorders of
carbohydrates, lipids, proteins, electrolytes and water, as a consequence of a mal-
function in insulin production or action (Chukwuma et al. 2019). In 2019, approxi-
mately 463 million of adults, mainly between 20 and 79 years, were living with
diabetes. If this pattern continues by 2045 this will rise to 700 million (International
Diabetes Federation, www.idf.org).
Insulin resistance in target tissues is the main cause of type 2 diabetes, which
represent around 90% of cases reported worldwide (Khan et al. 2019). Moreover,
type 2 diabetes can also affect the immune system responses to infections and
generate oxidative stress as further complications (Ferlita et al. 2019). The manage-
ment of diabetes includes sustained glycaemic control, a restricted diet, drugs and
self-controlled lifestyles (Gomes et al. 2019). Nevertheless, society is becoming
increasingly aware of the importance of consuming a healthy diet and doing exer-
cise. Therefore, the consumers and the food industry are looking for natural nutrients
and sweeteners (Saraiva et al. 2020).
Numerous high-intensity sweeteners compounds derived from plants have been
analysed (Świąder et al. 2019; Çiçek 2020). In general, these sweet taste molecules
produce an effect more than 50 times higher than sucrose without affecting the
calories intake. Moreover, a potential anti-hyperglycemic action and beneficial
effects against several kinds of diseases have been also identified in these sweet-
tasting plants (Świąder et al. 2019; Mustafa et al. 2019). However, scientific
evidence of the potency in the treatment of diabetes is not provided for much of
these plants. In a preliminary study, we identified four plant species with anti-
hyperglycemic effect supported by scientific research, one widely known as a
sweetener (Stevia rebaudiana Bertoni) and three less common high-potency
sweeteners (Cyclocarya paliurus (Batal.) Iljinsk, Glycyrrhiza glabra L. and Siraitia
grosvenorii (Swingle) C. Jeffrey ex A. M. Lu and Zhi Y. Zhang). Hence, in this
7 In Vitro Propagation and Biotechnological Improvement Strategies of Plants. . . 155
chapter, we aimed to perform a systematic review on these four high-intensity

sweetener plants with concomitant anti-diabetic effect. The information summarized
in this work will provide a clear overview of the nutraceutical potential of these
medicinal plants for the food industry and of the biotechnological approaches for
their propagation and improvement.
7.2 Search Strategy
Reviewing of the literature was conducted using Google Scholar and the following
Boolean search terms:
Stevia OR steviol glycosides AND (Sweeten* OR antidiabetic OR anti-diabetic);
Siraitia grosvenorii OR Monk fruit OR Momordice grosvenorii OR mogrosides
AND (Sweeten* OR antidiabetic OR anti-diabetic); Cyclocarya paliurus OR
Pterocarya paliurus AND (Sweeten* OR antidiabetic OR anti-diabetic);
Glycyrrhiza glabra OR licorice OR mulaithi OR glycyrrhizin AND (Sweeten*
OR antidiabetic OR anti-diabetic).
Stevia OR steviol glycosides AND (micropropagation* OR genetic transformat*
OR culture OR cultiv* OR in vitro OR elicit* OR metabol* OR engineer* OR
biosynthe*); Siraitia grosvenorii OR Monk fruit OR Momordice grosvenorii OR
mogrosides AND (micropropagation* OR genetic transformat* OR culture OR
cultiv* OR in vitro OR elicit* OR metabol* OR engineer* OR biosynthe*);
Cyclocarya paliurus OR Pterocarya paliurus AND (micropropagation* OR genetic
transformat* OR culture OR cultiv* OR in vitro OR elicit* OR metabol* OR
engineer* OR biosynthe*); Glycyrrhiza glabra OR licorice OR mulaithi OR
glycyrrhizin AND (micropropagation* OR genetic transformat* OR culture OR
cultiv* OR in vitro OR elicit* OR metabol* OR engineer* OR biosynthe*).
Additional intensive search capturing everything that might be relevant was
conducted for the species Cyclocarya paliurus because only two papers about
biotechnological techniques were recovered on the first attempts. In this case, the
search term was Cyclocarya paliurus.
7.3 Stevia rebaudiana (Bert.) Bertoni
Synonyms: Eupatorium rebaudianum Bertoni, Stevia rebaudiana (Bertoni) Hemsl.

https://www.gbif.org/es/species/3125557).
The genus Stevia Cav. contains about 150–200 species of herbaceous, shrub and
sub-shrub plants. Its distribution ranges from the Southwest of the USA to Central
America and from non-Amazonian South America to Central Argentina (Yadav
et al. 2011; Gantait et al. 2018). The species S. rebaudiana is originally from
Paraguay, South America, in the highland regions on the Brazilian border between
latitudes 23 and 24 S. The exceptional sweetening effects of its leaves and its
medicinal properties have been recognized by the Guarani tribe for centuries (Yadav
et al. 2011). The Guarani people used it as sweetener for their herbal infusion ‘mate’
Superorder: Asteranae
Order: Asterales
Family: Asteraceae
Genus: Stevia Cav.
Species: Stevia rebaudiana
Fig. 7.1 Taxonomic hierarchy and specimen of S. rebaudiana
and as a flavour enhancer (Soejarto et al. 1983). The plant, which is easy to grow, is
small and perennial, reaches a height of 65–80 cm and has sessile, oppositely
arranged leaves (Goyal et al. 2010). It has a widespread root system and fragile
stems producing small elliptical leaves (Fig. 7.1). Stevia plants naturally grow in
semi-humid subtropical conditions with temperatures ranging from 6 C to 43 C
and an annual rainfall fluctuating from 1500 to 1800 mm (Yadav et al. 2011). Saline
soils are not suitable for this species and the pH of the soil should be in the range of
6.5–7.5 (Goyal et al. 2010). In the native regions, it grows on the edges of marshes or
in grassland communities on soils with shallow superficial water. The first seeds of
stevia were exported to the United Kingdom back in 1943. Later, in 1968, the species
was exported to Japan and from this country the plant spread around the world
(Yadav et al. 2011). The suitable part of this species is the leaf. The leaves are known
to contain a combination of sweet-tasting compounds known as steviol glycosides
(Yadav et al. 2011; Šic Žlabur et al. 2013).
Common names: Guaraní: Kaa he-he (sweet herb) (Soejarto et al. 1983), English:
Stevia, Paraguay-Stevie, Candyleaf, Sweet-leaf-of-Paraguay, Azucaca, Swedish:
Sötflockel (Yadav et al. 2011) (https://gbif.org/es/species/3125557).
7.4 Cyclocarya paliurus (Batal.) Iljinsk
Synonyms: Cyclocarya paliurus var. micropaliurus (Tsoong) P.S. Hsu, X.Z. Feng &
L.G. Xu, Pterocarya paliurus Batalin (https://www.gbif.org/es/species/7310524).
C. paliurus is the single species in its genus (Xie et al. 2015b). Even though it is
now recognized as native from China, Cyclocarya has an extended fossil record of
fruits, from eastern Asia, Europe and North America (Crane and DuVal 2013). The
plant has been known as a natural ‘sweetener’ which gave rise to its Chinese
Superorder: Asteranae
Order: Fagales
Family: Juglandacea
Genus: Cyclocarya Iljinsk
Species: Cyclocarya paliurus
Fig. 7.2 Taxonomic hierarchy and specimen of C. paliurus
common name ‘tian cha shu’ (sweet tea tree). The leaves of this species have been
traditionally utilized by native people for the treatment of hypertension and diabetes
(Jiang et al. 2006). Its leaves have been a food resource for maritime people for a
long time.
The Chinese people have also used the plant as a drug in traditional medicine and
as a food ingredient (Xie et al. 2015b). On the basis of the ancient Chinese
pharmacopeia, C. paliurus is a medicinal herb to clear heat and toxin and to help
produce saliva and satiate thirst. The traditional Chinese medicine theory recognizes
‘damp’ and ‘heat’ as key pathological issues related to overweightness and diabetes.
In China, C. paliurus leaves have been extensively used in folk medicine for treating
hyperlipidemia and diabetes. New epidemiological data exposed that diabetes and
hyperlipidemia are very infrequent in the Xiushui County of Jiangxi Province where
C. paliurus leaves are used in beverages in a daily basis (Wang et al. 2013). The
plant is also employed for landscaping because of its attractive leaves and seeds
(Fang et al. 2006). C. paliurus is a deciduous tree about 30 m high. Its bark is thick,
grey, fibrous and deeply fissured on old trees. Other features are: (1) branches with
chambered pith; (2) leaves imparipinnate with a longitude of 20–25 cm; (3) 7–9
leaflets are sub-opposite or alternate, sessile or with a short stalk, rounded or cuneate
at the base, broadly acute at the apex, with finely serrate margins and the veins
beneath (Fig. 7.2) (Crane and DuVal 2013).
The plant is widely distributed in mountainous regions of subtropical China (Liu
et al. 2018b). It is typically grown at riparian regions at 420–2500 m in elevation in
mountainous regions of Anhui, Fujian, Hubei, Hunan, Jiangsu, Jiangxi, Sichuan,
Guizhou and Zhejiang provinces (Fang et al. 2006). C. paliurus is distributed in
China from about 24 to 40 N and from about 105 to122 E. The average annual
temperature in this area fluctuates considerably from 7.8 to 19.9 C and the mean
annual precipitation varies from 825.9 to 2394.5 mm. However, regardless of this
extensive distribution, there is little natural regeneration and C. paliurus has been
designated as endangered in China (Crane and DuVal 2013).
The suitable parts of this species are the leaves and bark to a lesser extent (Fang
et al. 2006). Studies have proved that C. paliurus has a diversity of biological
activities and anti-hypertensive, hypoglycaemic, mental efficiency improving, anti-
carcinogenic, anti-HIV-1, and antioxidant properties have been ascribed to it. All
these effects have been attributed, to some extent, to its diversity of chemical
compounds, including polysaccharides, proteins, triterpenoids, flavonoids, steroids,
saponins, phenolic compounds, and minerals.
Common names: English: wheel wingnut (Hu et al. 2017); Chinese: tian cha shu
(Jiang et al. 2006).
7.5 Glycyrrhiza glabra L.
Synonyms: Amorpha fruticosa var. typica (L.) C. K. Schneid., Glycyrrhiza

brachycarpa Boiss., Glycyrrhiza glabra subsp. glandulifera (Waldst. & Kit.) Ponert,
Glycyrrhiza glabra var. caduca X.Y. Li, Glycyrrhiza glabra var. Glabra,
Glycyrrhiza glabra var. glandulifera (Waldst. & Kit.) Boiss., Glycyrrhiza glabra
var. glandulifera (Waldst. & Kit.) Galushko, Glycyrrhiza glabra var. glandulifera
(Waldst. & Kit.) Regel & Herder, Glycyrrhiza glabra var. glandulosa X.Y. Li,
Glycyrrhiza glabra var. laxifoliolata X.Y. Li, Glycyrrhiza glabra var. typica L.,
Glycyrrhiza glabra var. violacea (Boiss. & Noe) Boiss., Glycyrrhiza glandulifera
Waldst. & Kit., Glycyrrhiza hirsuta L., 1753, Glycyrrhiza hirsuta Pall., Glycyrrhiza
pallida Boiss. & Noe, Glycyrrhiza violacea Boiss. & Noe (https://www.gbif.org/es/
species/2965732).
Glycyrrhiza plants are tall shrubs; the generic name Glycyrrhiza has its origin in
the ancient Greek words ‘glykos’ (sweet) and ‘rhiza’ (root), which was Latinized to
‘liquiritia’ and eventually to ‘licorice’. The genus comprises about 30 described
species of licorice that are natives of warmer regions. Among the most studied
species are G. uralensis, G. inflata, and G. glabra. The last species is the most
commonly used in the food industry (Quirós-Sauceda et al. 2016).
G. glabra is originally from Eurasia. It is native from Central and Southwestern
Asia and the Mediterranean region. However, it grows in South Europe (Spain,
Italy), Central Asia (Turkey, Iran, Iraq) and the North-western part of China. Tree
varieties of G. glabra are generally recognized: G. glabra var. typica (Spanish
licorice), G. glabra var. glandulifera (Russian licorice), and G. glabra var. violacea
(Persian and Turkish licorice). The plant is a herbaceous perennial from 50 to 150 cm
high. The stem is woody at the base, densely scaly and spotted with glands. The roots
are stoloniferous. The leaves are imparipinnate, ovate-oblong, oblong-lanceolate or
elliptic, from 7 to 15 cm long and 9 to 17 cm in width (Lim 2016) (Fig. 7.3).
The plants develop well in a moderate, warm and subtropical climate. They prefer
limed, well-drained, composted, friable, deep soil and grow better in full sun.
Licorice is not affected by frosts because it is dormant in winter and can benefit
Superorder: Rosanae
Order: Fabales
Family: Fabaceae
Genus: Glycyrrhiza L.
Species: Glycyrrhiza glabra
Fig. 7.3 Taxonomic hierarchy and specimen of G. glabra
from a defined cold period. Such a cold period induces the translocation of
substances to the underground rhizomes. The plants can be propagated without
difficulty from root cuttings (Lim 2016).
The licorice root contains the saponin glycyrrhizin, a substance that is 50 times
sweeter than sucrose (Lim 2016). The roots of G. glabra are used as an additive and
sweetening agent for tobacco (Sterry 1875), chewing gums, candies, toothpaste and
drinks. This plant is also a component in over 70% of Chinese medicines and has
been used for at least 4000 years. It has been employed as an analgesic, anti-
spasmodic, expectorant, for the treatment of tuberculosis, infectious hepatitis, and
bronchitis (Świąder et al. 2019). Fresh licorice root is outside of a bright yellowish-
brown dye and can be chewed as a mouth freshener and for teething in children. The
desiccated root can be chewed as a sweet. The extract of licorice is shiny black. In
Calabria, Italy, a liqueur is elaborated from the pure extract. The plant is also very
popular in Syria and Egypt, in these countries it is sold as a drink. It is employed to
flavour and colour beers, the enzymes contained in the root also stabilize the foam
heads. The leaves are used in Mongolia to make an infusion alternative to tea (Lim
2016).
G. glabra contains flavonoids and isoflavonoids, which display medicinal
properties. Its extract also modifies gene expression in a unique form, indicating
the presence of potentially useful components other than glycyrrhizin. The
phytoestrogens contained in the roots can be used to reduce the effects of postmeno-
pausal osteoporosis. Glabridin is the principal isoflavan known in the root of
G. glabra (Świąder et al. 2019).
Common names: G. glabra has many names such as English: Black Sugar,
Common Liquorice, Licorice, Licorice-Root, Liquorice, Liquorice Root; Spanish:
Alcazuz, Orozus, Paloduz, Regaliz; Arabic: Irq As-Sus, Irqu As-Sus, Irqu Al-Sus,
Sous, Sus; Armenian: Madoodag, Matutak; Chinese: Gān Cǎo, Gam Chou,
Guangguo–Gancao, Kan Tsau, Xi-Bei, Yang Gan Cao; Hindi: Atimadhura,

Jestamadu; Gujarati: Jetimadh, Mulaithi (Lim 2016).
7.6 Siraitia grosvenorii (Swingle) C. Jeffrey ex A. M. Lu & Zhi

Y. Zhang
Synonyms: Momordica grosvenorii Swingle, Siraitia grosvenorii (Swingle)

C. Jeffrey, Thladiantha grosvenorii (Swingle) C. Jeffrey (https://www.gbif.org/es/
species/3623059).
Siraitia grosvenorii is originally from China and Indonesia, it is one of the seven
species belonging to the genus Siraitia Merr. S. grosvenorii has been cultivated for
more than 200 years in Guangxi province of China which harvests more than 90% of
world production (Pandey et al. 2019). The plant is a perennial vine and it is an
endemic species from China (Fig. 7.4). The vine spreads a length of 3–5 m,
ascending over other plants using its creepers, which wrap around the shapes
encountered. The leaves are about 10–20 cm long and slender, heart-shaped. The
fruits are round, 5–7 cm in width, smooth, yellow-brown or green-brown with hard,
thin skin covered with tiny hairs. The pulp of the fruit is edible and after drying,
produces a thin, light-brown, fragile shell of about 1 mm thickness. The seeds are
elongated and almost spherical. The first mention of this species appeared in the
thirteenth century, once monks started consuming it for the first time. Because of the
restricted natural range of cultivation (mostly the mountain in Guangxi and
Guangdong) and the trouble in its cultivation, this plant is not included in the general
Chinese herbal tradition. S. grosvenorii became more popular in the twentieth
century. Its fruit is frequently used as the core ingredient in cold drinks. The inside
part of the fruit is eaten fresh and the bitter skin is used to make tea (Li et al. 2014;
Świąder et al. 2019). The fruit of S. grosvenorii has been used for several hundred
years as a sweetener and in Chinese traditional medicine to treat pharyngitis and as
an anti-tussive. In the year 1987, it was established having medicinal properties by
Order: Cucurbitales
Family: Cucurbitaceae
Genus: Siraitia Merr.
Species: Siraitia grosvenorii
Fig. 7.4 Taxonomic hierarchy and specimen of S. grosvenorii

the Chinese Ministry of Health (Li et al. 2014). The culture of S. grosvenorii is
restricted to only some areas of China and Indonesia; consequently, the production is
insufficient and only a small volume as dried fruits and extract are supplied to other
countries. The low production and the high market demand led to a higher price of
these products (Pandey et al. 2019).
As previously said the useful part of the plant is the fruit. Its sweet flavour derives
primarily from mogrosides, comprising around 1% of the fleshy portion of the fruit.
Both fresh and dried fruits are processed to produce a powder with 80% or more of
mogroside (Świąder et al. 2019). In recent pharmacological studies, several health-
protective properties are reported for S. grosvenorii, for instance, liver protection,
anti-oxidative, anti-hyperglycemic, anti-asthmatic, anti-carcinogenic and anti-
inflammatory action (Pandey et al. 2019).
Common names: English: monk fruit, Arhat, Buddha (Świąder et al. 2019);
Chinese: Luo Han Guo (Pandey et al. 2019), Luo han kuo; Vietnamese: la han qua
(Świąder et al. 2019).
7.7 High-Intensity Sweetener Effect
Sweet-tasting natural compounds alternative to sucrose have been studied for more
than five decades (Çiçek 2020). In 1967, Shallenberger and Acree proposed a
molecular theory of sweet taste known as the AH-B model (Shallenberger and
Acree 1967). This model suggested a common structural unit involved in the
recognition of the molecule by a chemoreceptor. This unit was formed by an acidic
proton (AH) and an electronegative atom (B) separated by an average distance of
3 Å. Moreover, the sweetness of numerous compounds like sugars varies with the
conformation of vicinal hydroxyl groups. For instance, hydroxyl groups in the glycol
units in gauche (60 ) or staggered conformation are sweet, but not in anti-clinal
(180 ) or eclipsed (0 ) conformations (Shallenberger and Acree 1969). However,
these authors concluded that AH-B could be a necessary condition for sweetness but
not sufficient to predict sweetness or potency levels (Acree et al. 1998).
Acree et al. followed the structure-activity research into sweetness and published
two follow-up reviews discussing the impact of the AH-B theory 30 and 40 years
later after its publication in 1967 (Acree et al. 1998; Acree and Lindley 2008). New
insights were oriented to describe the structure of the sweet receptor proteins and to
unravel the structure-function relationship of this molecule using sweetness
inhibitors. Nonetheless, the finding of natural sweeteners with highly variable
chemical structures refuted the idea of creating a model to predict the taste of a
molecule from its structure. So far, numerous plant-derived compounds have been
identified as high-intensity sweeteners with an effect considerably higher than
sucrose (Çiçek 2020). These active compounds might be classified as carbohydrates,
amino acids, phenols, monoterpenes, sesquiterpenes, diterpenes and triterpenes as
previously reviewed in Çiçek (2020). The sweet-tasting compounds obtained from
the four species reviewed in this chapter are an example of this structural variability.
Steviol glycosides are highly popular natural sweeteners isolated from

S. rebaudiana (Fig. 7.5a). These Kauran-type diterpenoids are found in the leaves
up to 15% of the chemical content. Stevioside, steviolbioside, rebaudioside A, B, C,
D, E, F and dulcoside content depend on growing conditions and cultivars (Šic
Žlabur et al. 2013). Stevioside is the most common sweet taste compound produced
in the leaves (4–13% w/w). Nevertheless, S. rebaudiana Morita is a cultivar devel-
oped by selective breeding to produce a considerably higher amount of rebaudioside
A (Ohta et al. 2010). Moreover, 10 new steviol glycosides were isolated from this
cultivar.
Rebaudioside A is the sweetest among the glycosides with a lower bitter aftertaste
(Çiçek 2020). Glycosylation at C13 and C19 of steviol is determining for sweetness.
Besides, sweetness intensity increases by extension of the ester sugar chain at the
C19 position (Ohta et al. 2010). Furthermore, relative sweetness might be also
altered by branching and type of sugar moieties (Çiçek 2020).
On the other hand, stevioside and rebaudioside A are very stable at high
temperatures (up to 198 C) and a wide range of pH. Moreover, steviol glycosides
extracted from S. rebaudiana leaves form a white, crystalline powder,
non-fermentable, non-caloric sweetener (Goyal et al. 2010; Šic Žlabur et al. 2013).
It is highly soluble in water and does not change in colour after cooking (Świąder
et al. 2019). Therefore, S. rebaudiana has become a very popular and promising
source of sweet compounds for the food and pharmaceutical industry (González
et al. 2014; Espinosa-Leal et al. 2018).
C. paliurus leaves yield potently sweet dammaran-type triterpenoids named
pterocaryosides (A, B) and cyclocaryosides (A, B, C, I, II, III) (Fig. 7.5b) (Kennelly
et al. 1995; Kinghorn and Soejarto 2002; Jiang et al. 2006). These compounds are at
least 50 times sweeter than sucrose, but also had a persistent off-taste (Siems et al.
2018). Cyclocaryoside I have been proposed as the main sweet-tasting principle in
this species (Çiçek 2020). Moreover, five new triterpene glycosides have been
isolated from leaves of C. paliurus, with sweetener activity comparable to
rebaudiosides and considerably lower negative aftertaste than pterocaryoside A
(Siems et al. 2018). These compounds differ from the previously described
pterocaryosides in aspects like (1) additional hydroxyl groups in the side chain,
(2) a ketone instead of the lipophilic alkyl side chain. These structural modifications
improve the solubility and influence the bitter taste without affecting the sweetening
power. Therefore, Siems et al. (2018) endorsed their industrial application as
sweeteners or sweetener enhancer.
Unlike stevia and the ‘sweet tree tea’, the active compounds of G. glabra are
contained in the roots of the plant (Świąder et al. 2019). The glycyrrhizin is an
oleanan-type triterpenoid, also named glycyrrhizic acid or glycyrrhizinic acid, found
in concentrations of 6–10% (Fig. 7.5c) (Murray 2020). This compound is formed by
glycyrrhetinic acid and O-β-D-glucuronosyl-(10 -2)-β-D-glucuronic acid (Çiçek
2020). It might be hydrolysed and absorbed by the intestinal flora. However, the
Committee of Food in the EU suggests not consuming more than 100 mg per day
(Çiçek 2020). The excessive consumption of licorice-derived sweetener can cause
7
In Vitro Propagation and Biotechnological Improvement Strategies of Plants. . .
Fig. 7.5 Chemical structures of selected high-intensity sweeteners from Stevia rebaudiana (a), Cyclocarya paliurus (b), Glycyrrhiza glabra (c), and Siraitia
grosvenorii (d) Pterocaryosides, cyclocaryosides, glycyrrhizin, mogrosides and siamenosides are triterpenes, while steviol glycosides are diterpenes
163
high blood pressure, low potassium levels, retention of fluids and edema due to the
indirect increase of cortisol levels (pseudohyperaldosteronism) (Świąder et al. 2019).
Glycyrrhizic acid is a potent sweetener with a characteristic licorice aftertaste.
Therefore, it is more frequently used as a flavour enhancer than as a pure sweetener
in the tobacco, pharmaceutical and confectionery industries (Lim 2016). Nonethe-
less, glycyrrhizin hides bitter flavours, increases the sweetness of sucrose and is
beneficial for a wide range of diseases as we will discuss later in this manuscript
(Lim 2016; Świąder et al. 2019).
As mentioned before, the source of sweeteners and medicinal compounds in
S. grosvenorii are the fruits. Mogrosides are the major sweetening compounds in
this species (Fig. 7.5d) (Pandey and Ayangla 2018; Świąder et al. 2019). Some
authors have recognized these cucurbitane-type triterpenoids as the second more
notorious sweetening after steviol glycosides (Wang et al. 2019; Çiçek 2020).
However, even when more than 60 triterpenoids have been isolated so far from
monk fruit, only those with certain structural features are sweet. A hydroxy-group in
position 11 and at least 3 sugar moieties (positioned in C3 and C24) are determining
for sweetness; otherwise, these compounds are bitter. Besides, in those compounds
with higher sweet potency such as mogroside V and siamenoside I (Fig. 7.5d), a
branched trisaccharide at position C24 has been identified (Çiçek 2020). Hence, this
particular feature might be directly linked to the lingering of the sweet taste, due to
the interaction of the 11-hydroxy-group, but further studies should be conducted to
confirm this hypothesis.
Other sweet compounds isolated from S. grosvenorii are mogrosides I, II, IVA,
IVE and 11-oxomogroside V (Pandey et al. 2019; Świąder et al. 2019; Gong et al.
2019). Extracts from fresh or dried fruits are used as sweeteners and medicinal food
additives (Pandey et al. 2019; Gong et al. 2019). Moreover, ripe fruits have no
aftertaste, while immatures are bitter (Jia and Yang 2009). Therefore, the combina-
tion of mogrosides extract and steviol glycosides allows obtaining a non-caloric
sweetener with enhanced taste and reduced aftertaste (Pandey et al. 2019).
The relative sweetness of some of the previously mentioned compounds isolated
from C. paliurus, G. glabra, S. grosvenorii, and S. rebaudiana are summarized in
Table 7.1. The safe application of these compounds (or their combination) is a
promising solution to reduce high-caloric sweeteners consumption. Moreover,
their medicinal effect is an advantage to reduce high-incidence diseases as Diabetes
mellitus.
7.7.1 Biosynthesis of High-Intensity Sweeteners
Three of the four plant species studied in this chapter produce sweet taste compounds
classified as triterpenes. However, this is not surprising since the triterpenes are the
most diverse and numerous groups of bioactive compounds produced by plants
(Thimmappa et al. 2014). Therefore, these compounds share the initial precursors of
biosynthesis through the mevalonate pathway from acetyl-CoA (Thimmappa et al.
2014; Pandey and Ayangla 2018; Wang et al. 2019; Murray 2020). This metabolic
Table 7.1 Relative sweetness of natural high-intensity sweeteners produced by plants with anti-
diabetic activity
Active Relative
Plant source compounds Aftertaste References sweetnessa
Stevia Stevioside Slightly bitter Bawane et al. (2012) 210–300
rebaudiana and Çiçek (2020)
Steviolbioside Slightly bitter Çiçek (2020) 100
Rebaudioside A Least bitter of Bawane et al. (2012) 242–400
steviol glycosides and Çiçek (2020)
Rebaudioside B Slightly bitter Çiçek (2020) 150
Rebaudioside C Slightly bitter Bawane et al. (2012) 30–120
and Çiçek (2020)
Rebaudioside D Slightly bitter Çiçek (2020) 221
Rebaudioside E Not reported Puri et al. (2011) 250–300
Rebaudioside F Slightly bitter Çiçek (2020) 200
Dulcoside Slightly bitter Bawane et al. (2012) 30–120
and Çiçek (2020)
Cyclocarya Cyclocaryoside Not reported Çiçek (2020) 200
paliurus A
Cyclocaryoside Not reported Çiçek (2020) 250
I
Pterocaryoside Slightly bitter Çiçek (2020) 50
A
Pterocaryoside Slightly bitter Çiçek (2020) 100
B
Glycyrrhiza Glycyrrhizic Bitter Çiçek (2020) 93–170
glabra acid
Siraitia Mogroside IVE No bitter taste Çiçek (2020) 392
grosveronii Siamenoside I No bitter taste Çiçek (2020) 563
Mogroside V No bitter taste Çiçek (2020) 425
a
Compared to an equal amount of sucrose
route diverges after the production of 2,3-oxidosqualene (the last common interme-
diate of sterols and triterpenes) into a broad range of triterpenes of diverse skeletal
types by cyclization (Thimmappa et al. 2014). To our knowledge, there is no report
of the biosynthetic pathways of pterocaryosides and cyclocaryosides of C. paliurus.
Nevertheless, the subsequent reactions to produce glycyrrhizin and mogrosides have
been accurately described.
In G. glabra, the 2,3-oxidosqualene is converted to ß-amyrin by a ß-amyrin
synthase. Hereafter, several oxidation steps, catalysed by cytochrome P450
monooxygenases, yield the glycyrrhetinic acid. Finally, glycyrrhizin is synthesized
by glycosylation of this aglycone by UDP-glucuronosyltransferases (Lim 2016;
Pandey and Ayangla 2018).
Similar reactions occur in S. grosvenorii to obtain the aglycone mogrol. Here,
2,3-oxidosqualene is transformed in cucurbitadienol by the cucurbitadienol synthase
and this triterpenoid skeleton is converted to mogrol by subsequent reactions
catalyzed by cytochrome P450 monooxygenases. Different mogrosides are thereaf-

ter produced by UDP-glucosyltransferases (Itkin et al. 2016; Wang et al. 2019; Shi
et al. 2019).
Steviol glycoside biosynthesis is completely different as these sweetener
compounds are structurally dissimilar from the previously described compounds.
The pathway has 16 steps divided into three phases: (1) seven reactions common
with the methylerythritol-4-phosphate pathway to synthesize isopentenyl diphos-
phate and dimethylallyl diphosphate, (2) four steps involving the synthesis of
kaurenoic acid from geranylgeranyl diphosphate (similar to gibberellic acid biosyn-
thetic pathway), (3) and five reactions specific to steviol glycoside biosynthesis
including hydroxylation of the kaurenoic acid to produce steviol. The initial steps
until the production of the kaurene skeleton take place in the chloroplast and the
conversion of kaurene to steviol is performed in the endoplasmic reticulum. After-
ward, steviol might be transformed in the cytosol of the leaf to steviol glycosides by
the subsequent transference of sugar moieties by UDP-glucosyltransferases.
7.8 Anti-diabetic Activity
Diabetes mellitus is a complex and heterogeneous disease associated with a decline

in insulin activity and subsequent chronic metabolic alterations (Wang et al. 2013;
Xiao et al. 2017). Besides the production of high-intensity sweeteners, the four
plants described in this chapter have a confirmed anti-diabetic effect, among other
medicinal properties. In this section, we will remark on the published studies
investigating this therapeutic activity and the bioactive molecules identified in
each of these plant species.
7.8.1 Stevia rebaudiana
For centuries, Paraguayans held a hypoglycemic effect of stevia associated with a

beneficial action on the pancreas (Singh and Rao 2005). However, this effect is not
produced in normal subjects. Steviol glycosides have been reported to have an
influence on ß-cells in the pancreas inducing insulin secretion (Goyal et al. 2010).
This anti-hyperglycemic effect combined with their non-caloric sweetness makes the
steviol glycosides a promising candidate to treat type 2 diabetes without limiting the
consumption of sweet compounds (Puri et al. 2011; Yadav and Guleria 2012;
González et al. 2014). Moreover, this action is not mediated by K+ ATP sensitive
channels such as sulfonylurea (Puri et al. 2011). Instead, these compounds trigger an
insulin-like effect on glucose transport activity (GLUT4) and activate the insulin
receptor (Prata et al. 2017).
To examine whether steviol glycosides and insulin share the same transduction
pathway, Prata et al. (2017) designed research using neonatal rat cardiac fibroblasts.
The sweetener compounds and insulin are both able to activate the glucose intake
into the cells through the phosphatidylinositol 3-kinase/Protein kinase B (PI3K/Akt)
pathway, but not by AMP-activated protein kinase (AMPK). Therefore, these

authors confirmed that steviol glycosides mimic the insulin action (Prata et al.
2017). Nonetheless, there are other actions of stevia-derived compounds able to
enhance this anti-diabetic influence such as the hypolipidemic and antioxidant effect
(discussed in Sect. 7.9.1).
7.8.2 Cyclocarya paliurus
Multiple studies have been conducted to demonstrate the anti-diabetic effect of

C. paliurus in different models. In 2003, Kurihara et al. studied the hypoglycemic
action of aqueous extracts from leaves in normal and diabetic mice. The elevation of
glucose level followed by a sucrose intake was increased after a unique administra-
tion of the extract in both groups. Moreover, this extract also inhibited the
α-glucosidase activity, therefore controlling the glucose adsorption (Kurihara et al.
2003b).
The anti-hyperglycemic effect of ethanol and aqueous extracts was also evaluated
in diabetic rats (Wang et al. 2013). Both extracts exhibit a similar action restoring the
insulin and glucose levels and the disorder of lipid metabolism. Moreover,
C. paliurus bioactive compounds significantly decreased the appearance of early
diabetic nephropathy in diabetic-induced rats. Several substances such as flavonoids,
polysaccharides, triterpenoids, sterols and carotenoids might produce these positive
effects. This would explain the similar bioactivity of the aqueous and ethanol
extracts (Wang et al. 2013).
New positive effects of aqueous leaf extracts were confirmed in pancreatic ß-cells
of diet-induced diabetic mice. The pancreatic ß-cells were protected by C. paliurus
through the inhibition of apoptosis and regulation of caspase-3 and caspase-9.
Moreover, MAPK and Akt pathways were affected inducing the anti-apoptotic
effect. Hence, diabetes-related complications (i.e. loss of weight, hepatic steatosis,
nephropathy, cardiac hypertrophy) were also considerably diminished (Xiao et al.
2017). However, the bioactive compounds responsible for these beneficial effects
were not identified in this study.
In 2018, Liu et al. evaluated the effect of aqueous and ethanol extract of
C. paliurus collected in five geographical locations. Both extracts were beneficial
to reverse body weight loss and to reduce glucose levels. However, these actions
were significantly different among the regions of collection and also the chemical
compositions of the extracts were dissimilar. The anti-hyperglycemic effect was
correlated with the total content of flavonoids, mainly quercetin-O-glucuronide and
kaempferol-3-O-glucuronide, in ethanol extract. Moreover, triterpenoids showed an
anti-hyperlipidemic effect. These authors concluded that the geographical locations
and extraction solvents might modify the biological activities of C. paliurus extract
(Liu et al. 2018b).
Yao et al. (2020) isolated the polysaccharides of leaf dried extracts to study its
impact on type 2 diabetic rats. These compounds decreased insulin resistance,
prevent hyperglycemic and improve dyslipidemia. Moreover, the composition of
the gut microbiota was altered and short-chain fatty acids production was increased
in rats and human gut after polysaccharide ingestion. Finally, these authors
concluded that C. paliurus polysaccharides improve type 2 diabetes through the
regulation of gut microbiota and increase of short-chain fatty acids (Yao et al. 2020).
The therapeutic potential of triterpenic acid-enriched fraction from C. paliurus
was analysed by Zheng et al. (2020). These compounds reduced hepatic steatosis,
blood glucose concentration, insulin levels and insulin resistance. These effects were
triggered by phosphorylation activating the PI3K, Akt and glycogen synthase-3ß
pathways.
7.8.3 Glycyrrhiza glabra
Licorice roots have a confirmed anti-hyperglycemic activity in male rats of Wistar

strain. Glycyrrhizin and 18-ß-glycyrrhetinic acid, the compounds also responsible
for the sweet taste, produce this effect (Kaur et al. 2013). Moreover, the hypoglyce-
mic action of glabridin was studied in male Kunming mice. This polyphenolic
flavonoid is a main active component of licorice roots with multiple therapeutic
effects (Wu et al. 2013). Particularly in this study, the body weight, glucose tolerance
and antioxidant activity were significantly improved in diabetic mice after glabridin
treatment (40 mg/kg dose). The molecular basis of these actions might be a mimetic
effect of insulin through the AMPK-dependent GLUT4 translocation pathway (Lim
2016). Nonetheless, to promote a feasible anti-diabetic treatment with licorice roots
should be considered that the production of this and other bioactive compounds of
G. glabra is influenced by several factors including the harvest time (Cheel et al.
2013). Moreover, licorice consumption might reduce diabetes symptoms like poly-
dipsia, polyuria and postprandial blood glucose rise (Pastorino et al. 2018).
Mustafa et al. (2019) evaluated the anti-hyperglycemic activity of several
hydroalcoholic extracts from roots of medicinal plants, including G. glabra and
their combination in alloxan-induced diabetic mice. All the extracts diminished
significantly the blood glucose concentrations compared with the diabetic control
treatment. Moreover, the combination of plant extracts produced a higher hypogly-
cemic effect. Curiously, the phytochemical analysis of G. glabra extract confirmed
the presence of steroids, terpenoids, saponins and tannins, but not alkaloids and
flavonoids. Hence, in this case, the anti-diabetic action is not mediated by flavonoids.
However, as this group of compounds were present in the rest of the extracts, the
higher effect of their combination might be related to a synergistic effect of all these
phytoconstituents (Mustafa et al. 2019).
7.8.4 Siraitia grosvenorii
The anti-diabetic effect of monk fruit extracts has been widely evaluated in different
models. The main sweet taste molecules produced by this plant are also responsible
for the anti-hyperglycemic action reported in these works (Zhang et al. 2020).
However, S. grosvenorii polysaccharide might influence likewise glucose levels (Lin

et al. 2007).
Cucurbitane-type triterpene glycosides of this plant inhibit maltase activity
reducing glucose intake. Therefore, similarly to steviol glycosides, these sweetener
compounds are not only non-caloric but also display an anti-hyperglycemic effect
(Suzuki et al. 2005). Moreover, fruit extract with a high content of mogrosides
(94%), mainly mogroside V (69.24%), reduces the blood glucose, biochemical
abnormalities and pathological damage of the islet ß-cells of diabetic mice
(Xiangyang et al. 2006). Thus, it was recommended to treat type 1 diabetes. On
the other hand, this therapy has no effects on normal mice.
In 2007, Lin et al. isolated S. grosvenorii polysaccharide from dry fruit and
evaluated its effect in male New Zealand white rabbits fed with high-fat/high-
sucrose chow. The blood glucose and insulin levels on diabetic rabbits treated
with polysaccharide were not statistically different from the normal and control
groups. Moreover, this compound also reduced the metabolic lipid disorder
associated with diabetes (Lin et al. 2007).
Suzuki et al. (2007) also investigated Monk fruit extract in type 2 diabetic Goto-
Kakizaki rats. Similarly, this extract improved insulin response and reduced the
plasma glucose level. Moreover, this effect was combined with an antioxidant
activity that attenuates complications related to type 2 diabetes such as oxidative
stress and kidney damage (Suzuki et al. 2007).
Furthermore, the anti-hyperglycemic action of extracted mogrosides was con-
firmed in alloxan-induced diabetic mice (Qi et al. 2008). Besides, the crude extract
and mogroside V isolated from fruits are both able to stimulate insulin secretion by
the ß-cells (Zhou et al. 2009). To identify the anti-diabetic principle of this crude
extract, mogrosides were treated by acid hydrolysis. Nine compounds were deter-
mined, five new cucurbitane triterpenoids, two known ones, the aglycone mogrol
and mogroside V. The main aglycone mogrol and two other cucurbitane
triterpenoids were found to be potent AMPK activators in vitro, but mogroside V
showed no effect (Chen et al. 2011). Therefore, this action might contribute, at least
partially, to the hypoglycemic effect previously reported.
Li et al. (2017) conducted similar studies years later. Eighteen mogrosides were
isolated and all produced effects on the glucose uptake in HepG2 cells (Li et al.
2017). The underlying mechanism of this anti-hyperglycemic action was further
studied using induced type 2 diabetic mice (Liu et al. 2019). These authors con-
firmed that a mogroside extract activates the hepatic AMPK signalling pathway
in vivo. Moreover, it produces multiple beneficial effects on type 2 diabetes includ-
ing inhibition of gluconeogenesis and insulin resistance, enhancement of hepatic
fatty acid oxidation and improvement of serum lipid profiles. These results were
supported a few years later by transcriptome analysis of type 2 diabetes rats
nourished with symbiotic yogurt fortified with monk fruit extract (Ban et al. 2020).
7.9 Other Medicinal Effects
In 2005, Singh and Rao named in their paper Stevia as the ‘herbal sugar of the 21st
Century’ because of its many advantages as a sweetener and also for its potential
medicinal and nutritive properties. As they explained, for a long time users found
countless reliable reasons to use stevia as a medicinal food. Besides the celebrity
stevia has gained as a sweetener, it contains a variety of other active compounds
different from the steviol glycosides, including triterpenes, sterols, tannins,
flavonoids and a volatile oil comprising high quantities of aromatics, aldehydes,
monoterpenes, and sesquiterpenes. These and other unidentified compounds could
have some effect on human physiology and may explain the reported therapeutic
effects of stevia (Singh and Rao 2005).
7.9.1.1 Cardiovascular Effect

At the time of the mentioned paper, a significant amount of research was
accumulated on the cardiovascular effects of stevia and stevioside in human and
animals, but no significant properties were found. Just a small lowering of arterial
blood pressure at low and normal doses and a slight rise at very high doses. Hence,
the continuous use of stevia would probably have a cardiotonic action (Singh and
Rao 2005).
In later trial, the effect of consumption of the stevia extract on
20 hypercholesterolemic women. The authors observed that the consumption of
the extract diminishes significantly the content of cholesterol, triglyceride and
low-density lipoprotein-cholesterol. On the other hand, a desirable increase in
high-density lipoprotein-cholesterol was obtained. The researchers concluded that
Stevia extract had a hypolipidemic effect beneficial to reduce the risk of cardiovas-
cular disease (Goyal et al. 2010). In other research, stevioside has shown a hypoten-
sive effect in strains of hypertensive rats (Puri et al. 2011).
7.9.1.2 Anti-Microbial Effect

Some authors have reported the capacity of stevia to inhibit the growth and repro-
duction of bacteria. Singh and Rao (2005) reported that Streptococcus mutants,
Pseudomonas aeruginosa, Proteus vulgaris and other microbes do not grow in the
presence of the non-nutritive stevia constituents.
The anti-microbial activity of various extracts of S. rebaudiana (with water,
acetone, chloroform, methanol, ethyl acetate or hexane as solvents) has been studied.
The effect of such extracts on microorganisms such as Salmonella typhi, Aeromonas
hydrophila, Vibrio cholerae, Bacillus subtilis, Staphylococcus aureus, and others
has been determined. In the same way, a fermented hot water extract from
S. rebaudiana was found to be bactericidal towards enterohaemorrhagic Escherichia
coli and other food-borne pathogenic bacteria. Other microorganisms like Salmo-
nella typhimurium, B. subtilis, and S. aureus have also been found to be inhibited by
the fermented leaf extract (Lemus-Mondaca et al. 2012).
7.9.1.3 Anti-Carcinogenic Effect

In mouse skin cancer models, stevioside inhibited the tumour-promoting activities
and inflammatory ear edema promoted by TPA (12-O-tetradecanoylphorbol-13-
acetate). Such a result is very relevant from the viewpoint of the chemoprevention
of cancer. The inhibitory effects of stevioside were greater than those of glycyrrhizin
(Puri et al. 2011).
7.9.1.4 Antioxidant Effect

The use of stevioside has been associated with an antioxidant defence in adipose
tissue and in the vascular wall. This effect leads to inhibition of atherosclerotic
plaque development and inducing plaque stabilization (Puri et al. 2011).
In another study, the antioxidant capacities of S. rebaudiana were explored,
together with five other plant species (Pueraria mirifica, Curcuma longa Linn.,
Andrographis paniculata (Burm. f.) Nees., and Cassia alata Linn.). The medicinal
plants were extracted with ethanol, methanol, acetone, acetic acid or distilled water.
The highest antioxidant capacity was found in S. rebaudiana, extracted with acetone
and methanol, followed by S. rebaudiana extracted with ethanol and C. alata
extracted with ethanol. The leaf extract of stevia has inhibited hydroperoxide
formation in sardine oil, with a power higher than DL-α-tocopherol or the green tea
extract. The antioxidant activity of stevia leaf extract has been attributed to the
scavenging of free radical and superoxides. Other studies assessing the in vitro
potential of the ethanol extract of S. rebaudiana shows that it has a significant
potential for use as a natural antioxidant (Lemus-Mondaca et al. 2012).
In 2017, Sánchez-Aceves et al. evaluated the antioxidant activity of steviol
glycosides on diverse tissues of Cyprinus carpio by means of oxidative stress
biomarkers. The antioxidant capacity was proved by exposing C. carpio specimens
to four systems: (1) steviol glycosides free control, (2) CCl4 0.5 mL/kg, (3) steviol
glycosides 1 g/L and (4) CCl4 0.5 mL/kg + steviol glycosides 1 g/L at 96 h. The
biomarkers used were: lipoperoxidation (LPX), hydroperoxide content (HPC), pro-
tein carbonyl content (PCC) and antioxidant activity provided superoxide dismutase
(SOD) and catalase (CAT). It was found that treatments 3 and 4 decrease LPX, CHP,
PCC, SOD and CAT with respect to the CCl4 system. As the results of this study, an
antioxidant effect appears to be induced by steviol glycosides. Furthermore, in 2017,
Prata et al. proved the potential antioxidant activity of four different combinations of
steviol glycosides rich in stevioside and rebaudioside A. As a result, steviol
glycosides counteracted oxidative stress by increasing intracellular levels of reduced
glutathione and by upregulating expression and activity of the antioxidant enzymes
SOD and CAT.
7.9.1.5 Anti-Amnesia Properties

In addition, anti-amnesia properties of stevioside have been reported. The treatment
with stevioside was able to invert the scopolamine-induced learning and memory
deficits, together with the reduction of the scopolamine-induced increase in brain
acetylcholine esterase activity and brain oxidative stress. The authors determined
that stevioside produce a memory preservative effect in the cognitive deficits of rats
(Puri et al. 2011).
7.9.1.6 Safety of Use

Pomaret et al. in South America performed the first reported research on the
toxicology of Stevia in 1930. Their tests were negative (Singh and Rao 2005).
After that, in the opinion of the same authors, almost every possible toxicity test
has been performed on Stevia extract or stevioside and the result was continuously
negative. The single associated effect detected was the inhibition of cell respiration
(oxidative phosphorylation) in particular isolated cell components, but never in
whole cells.
Nevertheless, several studies suggest a risk of mutagenicity. In two studies,
steviol caused a concentration-related positive mutagenic result. In the same trials,
stevioside does not show this effect. Other studies revealed the absence of mutage-
nicity of both compounds. As a result of these contradictory reports, the Food and
Drug Administration is still thoughtful in introducing stevia as sweetener in the
United States of America (Goyal et al. 2010).
Because the Stevia products are widely used as sweeteners, research was
conducted to test the possible cariogenic effect of stevioside and rebaudioside.
Rats were nourished with a diet containing 0.5% stevioside or 0.5% rebaudioside
A for a period of 5 weeks. Neither stevioside nor rebaudioside exhibited an increased
risk of producing dental caries (Goyal et al. 2010).
Many studies pointed to a variety of biological active effects produced by the extract
of C. paliurus. Previous investigations indicated that the leaf of this plant contains
high amounts of physiologically active compounds such as triterpenoids, flavonoids,
phenolic acids, and polysaccharide (Liu et al. 2018d).
7.9.2.1 Anti-hyperlipidemic Effect

In 2003, Kurihara et al. found an inhibitory activity of a C. paliurus extract against
pancreatic lipase, a very important enzyme of dietary triglyceride absorption. The
IC50 of the extract in vitro was 9.1 mg/mL. Therefore, the hypolipemic action is
probably interrelated with this suppression. The study indicated that a single oral
administration of C. paliurus extract (250 mg/kg) could avoid dyslipidemia in rats
and hamsters after a long-lasting lard and olive oil diet treatment (Kurihara et al.
2003a).
In other tests, several C. paliurus polar extracts reduced the total cholesterol in
serum, triglycerides, low-density lipoprotein cholesterol and hepatic cholesterol.
These extracts also increase the level of high-density lipoprotein and cholesterol in
serum. Moreover, they restored the function and histomorphology of hepatic and
renal tissues. HMG-CoA reductase activity and mRNA expression were also
decreased after the oral administration of extracts to diet-induced hyperlipidemic
mice. As a result of the same treatment, the activity of 7α-hydroxylase and mRNA
expression and the level of faecal and hepatic bile acid increased (Jiang et al. 2015).
Similar results were obtained by Yang et al. (2016) using C. paliurus polysac-
charide. Its activity decreases the total cholesterol in plasma, triglycerides and
low-density lipoprotein cholesterol levels, while it increases the high-density lipo-
protein cholesterol level of rats fed with a diet rich on fat. This work also showed
higher expression of liver adipose triglyceride lipase and peroxisome proliferator-
activated receptor, together with a relative decrease in liver fatty acid synthase and
HMG-CoA expression.
In addition, Yang et al. (2018) found in the analysis of liver from mice fed with a
high-fat diet and treated with C. paliurus polysaccharide, that this component has the
potential for decreasing the risk of fatty liver and other hyperlipidemia
complications. In the same paper, the preventive effect of C. paliurus polysaccharide
was closely associated with its antioxidant ability.
In 2010, Zhang et al. (2010) observed an inhibition against PTP1B using phenolic
compounds extracted from the leaves of C. paliurus. These authors suggested that
the extract could be a potential source for an anti-obesity ingredient. Other authors
also reported decreased body loss and food intake in diabetic mice as a result of the
treatment with C. paliurus extract (Xiao et al. 2017).
7.9.2.2 Anti-inflammatory Effect

In lipopolysaccharide-induced macrophage cells, sulphated C. paliurus extract
attenuated inflammatory mediators by inhibiting the phagocytosis of macrophages,
nitric oxide production, and the release of IL-6 and IL-1β. In the
lipopolysaccharides-injected liver damage model, liver function was significantly
reduced in mice, indicating the occurrence of acute inflammation. The treatment
with sulphated C. paliurus extract decreased the alanine aminotransferase activity in
the serum and improved the thymus and spleen indexes. They also increased the
SOD activity and total antioxidant capacity while decreasing the malondialdehyde
(MDA) level. Combined with previous structural information, this effect could be
attributed to the change in chemical components, molecular weights and the degree
of substitution caused by sulphated modification. In summary, sulphated modifica-
tion enhanced the anti-inflammatory effect of polysaccharides and had potential
value to develop non-toxic and cheap health food supplements and drugs (Wang
et al. 2017).

In research conducted by Xie et al. (2010), a notable antioxidant effect of a
polysaccharide from aqueous extract of C. paliurus was demonstrated. In this
work, the free radical scavenging activity was measured. At a concentration of
400 μg/mL, a scavenging effect around 91.4% on the DPPH radical was observed,
compared with 98.9% of ascorbic acid and 91.2% of butylated hydroxytoluene
actions. The tested polysaccharide contains 8.44% protein and has an average
molecular weight of 1167 kDa. A similar result was obtained by Xie et al. (2015a)
but in this case using flavonoids of C. paliurus. In vitro antioxidant tests showed a
strong DPPH radical scavenging capability (IC50 ¼ 0.146 mg/mL). The DPPH free
radical scavenging of C. paliurus flavonoids was less than that of ascorbic acid but it
was stronger than that of butylated hydroxytoluene.
As a result of experiments conducted by Xie et al. (2016), four compounds
obtained by sulphated modification of a polysaccharide extracted from C. paliurus
showed distinctive antioxidant effect. The physicochemical and antioxidant
characteristics changed because of the sulphation. The trials showed a significant
effect of the sulphated derivatives on scavenging DPPH free radicals, especially at
high concentrations.
7.9.2.4 Immunomodulatory Effect

Acetylated C. paliurus polysaccharide derivative showed stronger immunomodula-
tory activities than the polysaccharide in its original state. The activity was evaluated
in terms of stimulating the production of pro-inflammatory cytokines, including
TNF-α, IL-1β, and IL-6 in macrophage cells. No cytotoxic effect on macrophage
cells was found. Moreover, the results suggested that the introduction of acetyl
groups could lead to the change of bioactivities and acetylation can increase the
immune activity of the polysaccharide (Liu et al. 2017). In other work, sulphated
C. paliurus polysaccharide stimulated macrophage cells to secrete cytokines and
enhanced phagocytic activities. Nitric oxide release was significantly improved by
the treatment containing sulphated C. paliurus polysaccharide in contrast with native
C. paliurus polysaccharide. In the author’s opinion, sulphated C. paliurus polysac-
charide might be a promising immuno-potentiating agent (Yu et al. 2017).

Evaluations on the mouse acute toxicity and bacterial mutagenesis determinations
supported the safety of two highly sweet secodammarane-type glycosides from C.
paliurus obtained from the leaves and stems. The compounds were: (23E)-
(12R,20S)-12,20,25-trihydroxy-3,4- secodammara-4(28),23-dien-3-oic acid
12–0-β-D-quinovopyranoside and (23E)-(12R,20S)-12,20,25-trihydroxy-3,4-
secodammara-4(28),23-dien-3-oic acid 12-O-α-arabinopyranoside (Kennelly et al.
1995).
No genotoxic effect caused by the leaves extract of C. paliurus was demonstrated
in the study of Deng et al. (2014). In a teratogenicity assay, the same authors proved
that aqueous extract of C. paliurus leaves has no reproductive toxicity on rats at the
highest dosage of 20.0 g/kgbw, equivalent to 150 times the recommended human
dosage. The study included the weights of pregnant rats, the indexes of
embryotoxicity, the sexuality, foetal body weight, body length, tail length of the
live foetuses, the external deformities and deformity in viscera and skeletal of the
live foetuses (Deng et al. 2014).
As early as April 10, 1875, in the county and State of New York, George E. Sterry
declared the invention of a new and useful improvement in the compound for
sweetening, seasoning, flavouring and preserving tobacco. The invention consisted

of powdered salt with a powdered licorice root (Sterry 1875). This is an example of
the long history of use of G. glabra due to the numerous bioactive ingredients
contained in the plant. The principal compounds of licorice extracts are triterpenoids,
polyphenols and polysaccharides (Wang and Nixon 2001). Other active constituents
include flavonoids, isoflavonoids, chalcones, coumarins, sterols, amino acids,
amines, gums, and volatile oils (Murray 2020).
7.9.3.1 Anti-asthmatic Effects

The anti-allergenic activity of the glycyrrhizic acid leading to anti-asthmatic effects
was evaluated in mice. Glycyrrhizic acid down-regulated the Th2 cytokines
interleukin-4, 5 and 13 and increases the Th1 cytokines IFN-γ. It also prevented
the recruitment of eosinophils and mucus abundance. Moreover, the inflammation of
the airway was reduced. Besides, it promoted the proliferation of regulatory T cells
(Ma et al. 2013).
7.9.3.2 Anti-inflammatory Effect

The anti-inflammatory activity of glycyrrhizin and glycyrrhetinic acid was proved
for the first time in formaldehyde-induced arthritis in rats. Glycyrrhizin,
glycyrrhetinic acid, hydrocortisone, and butazolidine were equally effective with
the reference standards (Gujral et al. 1959). In vitro, glycyrrhetinic acid, as aldoste-
rone, was capable to regulate the intracellular concentration of electrolytes and the
cellular volume by genomic and non-genomic pathways (Sabbadin et al. 2019). The
research suggests that licorice can induce pseudohyperaldosteronism and an inflam-
matory reaction related to the binding to mineralocorticoid receptor and to the
blocking of 11-HSD2. Besides, it can also show anti-inflammatory and anti-
atherosclerotic effects modulating cellular membrane fluidity and oxidative stress.
Moreover, estrogen-like and glucocorticoid-like activities have been confirmed for
this plant.
The proinflammatory effect in erythrocytes has been studied via the augmented
response to diamide, an oxidant compound capable to activate the tyrosine phos-
phorylation of erythrocyte membrane proteins. The increased tyrosine phosphoryla-
tion levels are related to the failure of cellular anti-oxidative defences. It has been
suggested that glycyrrhetinic acid prevents alteration of erythrocyte caused by
diamide, avoiding the oxidative and proteolytic damage of the membrane (Sabbadin
et al. 2019).
7.9.3.3 Anti-microbial Effect

Several studies have been conducted to probe the efficacy of licorice extracts to act
as antimicrobial agents. Kaur et al. (2013) mentioned antimicrobial activity of ether,
chloroform, and acetone extracts against Escherichia coli, Bacillus subtilis, Pseudo-
monas aeruginosa and Staphylococcus aureus. In another trial, Ajagannanavar et al.
(2014) obtained good results using aqueous and ethanolic licorice root extract
against Streptococcus mutans and Lactobacillus acidophilus. The inhibitory effect
reported was higher than with chlorhexidine. Likewise, different kinds of
compounds of licorice have shown antimicrobial activity against Helicobacter

pylori, Mycobacterium smegmatis, Bacillus subtilis, S. pyogenes, Haemophilus
influenzae, Moraxella catarrhalis, Enterococcus faecalis, Micrococcus luteus, the
yeast Candida albicans, Candida utilis, Saccharomyces cerevisiae, and Pichia
nakazawae; and the fungi Trichophyton mentagrophytes, Aspergillus niger, Asper-
gillus fumigatus, Mucor sp., Penicillium marneffei, and Pythium ultimum (Lim 2016;
Murray 2020). Additionally, some antibiotic-resistant bacteria strains have been
inhibited using licorice derivatives. It has been established that isoflavonoids have
the main antimicrobial effects whereas saponins only contribute in a minor way
(Murray 2020).
7.9.3.4 Anti-mutagenic Effect

Glycyrrhetinic acid has shown an anti-mutagenic effect, α-glycyrrhetinic acid and β-
glycyrrhetinic acid inhibit the mutagenic effect of benzo[α]pyrene, 2-aminofluorene,
and aflatoxin B1 in strains of Salmonella typhimurium. The β- glycyrrhetinic acid
appeared more effective. In the same model, the extract of licorice controlled the
mutagenetic effect of furapromidum, zhengdingmycin hydrochloride, N-Methyl-N0 -
nitro-N-nitrosoguanidine, and methyl-methane-sulfonate. This extract was also
capable to protect mice against the mutagenesis induced by cyclophosphamide.
Moreover, the β-glycyrrhetinic acid has a dose-related effect, capable to inhibit
epidermal aryl-hydrocarbon hydroxylase, 7-ethoxycoumarin-O-deethylase and
7-ethoxyresorufin-O-deethylase in rats. Licorice extract also reduced the formation
of micronuclei in mouse bone marrow cells treated with cyclophosphamide (Wang
and Nixon 2001).
7.9.3.5 Anti-carcinogenic Effect

The anti-carcinogenic effect of G. glabra has been confirmed in several reports
(reviewed in Wang and Nixon 2001). It is the most widely studied therapeutic effect
in licorice. In 2015, Tang et al. made a comprehensive systematic review of this
particular topic. A summary of prevailing knowledge is presented in Table 7.2
(adapted from the formerly mentioned paper).
7.9.3.6 Antioxidant Activity

A group of substances from G. glabra were proved to have a potent antioxidant
effect (European Medicines Agency 2013). The antioxidant activities of several
compounds isolated from licorice (5-(1,1-dimethylallyl)-3,4,40-trihydroxy-2-
methoxychalcone (DTM), licochalcone B, licochalcone A, echinatin, glycy-
coumarin and glyurallin B) were evaluated. The treatment with licochalcone B,
echinatin and glycy-coumarin resulted in a high antioxidant activity expressed as
ABTS (2,2-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) radical scavenging
capacity. All the compounds, except echinatin, produced potent inhibition of lipid
peroxidation in rat liver microsomes (Fu et al. 2013).
Table 7.2 Anti-carcinogenic compounds from licorice, its impacts, and potential action
mechanisms (modified from Tang et al. 2015)
Compounds Studied relevant anti-cancer effects Potential mechanisms
Mixed extracts Cell cycle arrest, apoptosis, Inhibition in TOR signalling,
authophagy, anti-angiogenesis induction of p53 and Bax genes
Mixed extracts Apoptosisa Expression of HSP90 gene, growth
and apoptosis in the HT-29 colon
cancer cell linea
Triterpenoids
Glycyrrhizic Cell cycle arrest, apoptosis, anti- Inhibition NF-κB, E-selecton,
acid angiogenesis TNF-α and HMGB1
Glycyrrhetinic Cell cycle arrest, apoptosis, Inhibition of NF-κB, PKC and GSH
acid autophagy, anti-metastasis,
sensitization
Flavonoids
Isoliquiritigenin Cell cycle arrest, apoptosis, Inhibition of NF-κB and JNK
autophagy, anti-metastasis, anti-
angiogenesis, differentiation,
sensitization
Licochalcone A Cell cycle arrest, apoptosis, Blockage of ERK, NF-κB and
autophagy, anti-metastasis, anti- STAT3 signalling
angiogenesis, sensitization
Licochalcone B Cell cycle arrest, apoptosis Inhibition of Bcl-2 gene and
surviving, induction of Bax
Licochalcone E Cell cycle arrest, apoptosis, anti- Inhibition of DNA topoisomerase
metastasis, anti-angiogenesis 1, NF-κB and Ki67 (marker of
proliferation)
Liquiritigenin Apoptosis, anti-metastasis, anti- Retardation of Akt, activation of
angiogenesis, p53, production of ROS
Isoangustone A Cell cycle arrest, apoptosis Blockage of mTOR and JNK
signalling, activation of DR4
Glabridin Anti-metastasis, anti-angiogenesis Down-regulation of RhoA, FAK,
Akt and Src
Isoliquiritin Anti-angiogenesis Down-regulation of tube formation
Licoricidin Anti-metastasis Inhibition of uPA, MMP-9 and
ICAM
Phenolic compounds
Licocoumarone Apoptosis Up-regulation of DNA
fragmentation
Glycyrol Cell cycle arrest, apoptosis, Induction of caspase 8, caspase
autophagy 9 and Fas
β-Hydroxy- Apoptosis Induction of Bcl-2 phosphorylation
DHP
β-hydroxy-DHP 1-(2,4-dihidroxyphenyl)-3-hydroxy-3-(40 -hydroxyphenyl)1-propanone, TOR tar-
get of rapamycin, NF-κB nuclear factor-κB, TNF-α tumour necrosis factor-α, HMGB1 high-
mobility group box 1, PKC protein kinase C, GSH glutathione, JNK c-Jun N-terminal kinase,
ERK extracellular regulated protein kinases, STAT3 signal transducer and activator of transcription
3, ROS reactive oxygen species, mTOR mammalian target of rapamycin, DR4 death receptor
4, RhoA Ras homolog family member, FAK focal adhesion kinase, uPA urokinase-type plasmino-
gen activator, MMP-9 matrix metalloproteinase-9, ICAM intercellular adhesion molecule, Src Src
kinase, Fas Fas receptor
a
Reported by Nourazarian et al. (2016)
7.9.3.7 Anti-tussive Activity

The effect of arabinogalactan protein-enriched polymeric fraction extracted from
G. glabra was probed in guinea pigs. The therapeutic compound was applied orally
in response to a cough effect induced by an aerosol of citric acid. The treatment
achieved the cough suppression and, after 300 min, the response was better than the
resultant of codeine (Saha et al. 2011).
7.9.3.8 Antiviral Activity

In several studies, antiviral activity of different extracts and substances of G. glabra
have been demonstrated. This kind of activity has been reported against herpes
simplex, varicella-zoster, influenza, severe acute respiratory syndrome virus, vesic-
ular stomatitis virus, Kaposi sarcoma-associated herpesvirus and duck hepatitis virus
(Pastorino et al. 2018). The compounds most widely associated with antiviral
activity are glycyrrhizin and 18β-glycyrrhetinic acid. Interestingly glycyrrhizin
inhibits replication of SARS-associated coronavirus FFM-ic (Cinatl et al. 2003).
The mentioned active compounds are capable to inhibit gene expression and repli-
cation of the virus. This effect produces a reducing of the adhesion force and
restrains high mobility of group box 1 (Pastorino et al. 2018).
Other antiviral effects reviewed by Lim (2016) include interfered transport to the
membrane, sialylation of virus antigen, reduction of membrane fluidity, induction of
interferon-gamma in T cells, inhibition of phosphorylating enzymes in vesicular
stomatitis virus infection and reduction of viral latency. Furthermore, the activity of
some substances from licorice has been probed against the human immunodefi-
ciency virus (HIV). Several of these compounds proved to have therapeutic effects
on this particular virus. Glycyrrhizin, licochalcone A, isolicoflavonol,
glycycoumarin, glycyrrhisoflavone and licopyranocoumarin inhibited the cytopathic
effect of HIV. Glycyrrhizin also suppressed the HIV-specific antigen expression in
MT-4 cells and giant cell formation of HIV-infected human cells (Molt-4). This
activity of glycyrrhizin has been attributed to its inhibition of protein kinase and
partly to interference in the binding of the virus and host cells. Other compounds,
such as 18α-glycyrrhizic acid, also proved to be active against HIV (Lim 2016).
7.9.3.9 Cardiovascular Effect

The licorice act as a potent antioxidant and a moderate reducer of blood pressure in
hyper-cholesterolemic patients. These effects result beneficial to restrain cardiovas-
cular disease (Fuhrman and Aviram 2004). Nevertheless, according to Deutch et al.
(2019), the boundaries of beneficial and harmful effects of licorice are not always
evident. According to the same authors, cardiac arrhythmias are the main secondary
effect caused by licorice. Other described actions include coronary artery spasm and
vasospastic related to interference with endothelin and nitric oxide systems. On the
other hand, the beneficial effects of licorice compounds are usually linked to their
antioxidant properties. Some of the beneficial cardiovascular effects registered in
experiments with animals include interruption of the evolution of ischemic heart
disease, protection against induced oxidative stress, and inhibition of oxidation of
low-density lipoprotein (Deutch et al. 2019).
7.9.3.10 Hepatoprotective Effect

According to a comprehensive review performed by Pastorino et al. (2018), the
hepatoprotective effect of G. glabra extracts have been widely reported. This action
is linked to the inhibition of free radicals and lipid peroxidation. Glycyrrhizin and
18β-glycyrrhetinic acid have beneficial effects against carbon tetrachloride-induced
hepatotoxicity in the liver of mice, prevention of drug-induced liver injury in humans
and interruption of bile acid metabolism. Moreover, they reduce the blood
aminotransferases levels and improve the liver histology, with concomitant reduc-
tion of serum enzymes, bilirubin and pro-inflammatory cytokines in the liver,
decreasing the expression of P450 E1 (Pastorino et al. 2018).
7.9.3.11 Other Registered Effects

Other therapeutic actions, supported in scientific research, reported for G. glabra or
its components are anti-ulcerative, sedative, neuroprotective, anti-depressive, and
skin effects (Pastorino et al. 2018).

The Council of Europe Report on Flavouring Substances and Natural Sources of
Flavourings (1981) classifies licorice as a ‘plant or part thereof, including herbs,
spices and seasonings commonly added to foodstuffs in small quantities, the use of
which is considered acceptable with a possible limitation of an active principle in the
final’. Glycyrrhizin is also comprised as a ‘flavouring substance subject to a limit in
foods and beverages of 50 ppm’. The UK Food Additives and Contaminants
Scientific Committee’s report stated in 1985 that the available data were inadequate
for a toxicological evaluation of the substance as a sweetener, but the findings from
clinical toxicology revealed a possible need to restrict consumption of licorice.
Fatalities and illness have been reported in humans after excessive or prolonged
consumption of licorice-containing products (Fenwick et al. 1990).
In 1946, the application of licorice extracts for the treatment of peptic ulcers was
considered but the use was interrupted in a short time because of a side effects
leading to edema, hypertension, and hypokalemia. Glycyrrhetinic acid and
glycyrrhizin induce a mineral corticoid-like action, known as pseudoaldosteronism,
which retains Na+ and water, and excludes K+. This side effect appears due to an
elevated intravenous dose of glycyrrhizin administrated for an extended period of
time or more easily after oral administration of glycyrrhetinic acid (Shibata 2000).
The latter causes pseudoaldosteronism via two different ways 1) block
11-β-hydroxysteroid dehydrogenase type 2, 2) act as an agonist joining directly to
the mineralocorticoid receptor recognition site. Nevertheless, its affinity is around
5000–fold lower than aldosterone. Hence, long-time overconsumption is necessary
to reach a direct effect of glycyrrhetinic acid at the mineralocorticoid receptor level.
Besides, it has been confirmed in human mononuclear leukocytes that the mineralo-
corticoid effector mechanism of the glycyrrhetinic acid might be reversed by
co-incubation with canrenone (Sabbadin et al. 2019).
The effect on blocking 11-β-hydroxysteroid dehydrogenase type 2 starts with
relatively low concentrations of glycyrrhetinic acid in serum and the binding to
mineralocorticoid receptor starts later when the compound is accumulated in the

blood (Sabbadin et al. 2019). In other observations, Harada et al. (2002) reported a
drug interaction of licorice and Digitalis sp., the combination causing an electrolyte
imbalance accompanied of congestive heart failure.
Finally, there is no evidence of carcinogenic effect produced by G. glabra.
Several experiments have been performed, but tumorigenicity or toxicity were
never found in mice treated with disodium glycyrrhizinate applied in the drinking
water (Wang and Nixon 2001).
According to Gong et al. (2019), more than 100 active compounds have been
isolated from S. grosvenorii. The obtained substances include about 46 triterpenoids,
7 flavonoids, 19 amino acids, and 2 polysaccharides. The same authors remarked this
plant is one of the first approved medicine food homology species in China, which
follow the theory establishing food that can also be used as drugs.
7.9.4.1 Anti-fatigue Effect

Extract of the fruit of S. grosvenorii was capable to prolong the swimming time of
mice. Besides, the muscle and liver glycogen quantity increased. Additionally, the
lactic acid in blood decreased in the same way than serum urea nitrogen levels. These
effects were dose-response dependent (Liu et al. 2013).
7.9.4.2 Anti-hyperlipidemic Effect

Mogrosides from S. grosvenorii proved to have inhibitory effects on pancreatic
lipase activity in vitro. In mice, total mogrosides cancelled the increase in body
weight, abdominal, and epididymal fats, triglycerides, and total cholesterol in the
liver. Moreover, triglycerides in plasma were reduced after treatment with oral
administration of a high-fat diet and mogrosides. The effects may be partially due
to the inhibitory actions of mogrosides on pancreatic lipase activity. The results
suggested that mogrosides are effective as a therapy for obesity and fatty liver
produced by a high-fat diet (Bai-Shen 2012).
Another study investigated the activity of mogrol on adipogenesis in 3T3-L1
pre-adipocytes. The data showed that mogrol suppressed adipogenesis by reducing
activation of cAMP response element-binding protein in the initial phase of cell
differentiation and by activating AMPK signalling (Harada et al. 2016).
7.9.4.3 Anti-microbial Effect

Some research led to the conclusion that extracts of the fruits, leaves, and stems of
S. grosvenorii have inhibitory action against several bacterial species. Some of the
probed bacteria include Pseudomonas aeruginosa, Escherichia coli, Streptococcus
mutans (Li et al. 2014; Gong et al. 2019), and Micrococcus luteus (Li et al. 2014). In
one trial, 50 compounds extracted from S. grosvenorii were tested against bacteria;
however, only four of them showed strong inhibitory effect. The rest resulted in a
weak or no activity at all. Besides, mogroside V had no antibacterial activity (Gong

et al. 2019). On the other hand, the bacteriostatic properties of ethanol extract to
inhibit P. aeruginosa and E. coli reached a 90.9% and 76.7%, respectively, when the
concentration was 50 mg/mL. The effect on S. aureus, M. luteus, and C. albicans
was under 50% (Li et al. 2014).

Mogroside V and 11-oxomogroside V from the fruits of S. grosvenorii showed a
notable reactive oxygen species scavenging capacity. Besides, 11-oxo-mogroside V
has a stronger scavenging activity towards hydrogen peroxide and constraining
effect on DNA damage. On the other hand, mogroside V is more efficient in
scavenging the hydroxyl radical. Therefore, these two compounds could act as free
radical scavenging agents (Chen et al. 2007).
In a study conducted by Liu et al. (2018a), mogroside extract proved to be a
potent antioxidant. The activity was demonstrated in vitro, by the capacity of the
compound to capture peroxyl radicals and alleviate glucose-mediated protein
glycoxidation and cross linking. Moreover, the anti-glycative activity was alike to
aminoguanidine at equal concentration (Liu et al. 2018a).
The crude extract of S. grosvenorii has also shown high scavenging effect and an
antioxidant power comparable to ascorbic acid. As previously described, in this
work, cucurbitane glycosides mogroside V and 11-oxo-mogroside produced a nota-
ble reactive oxygen species scavenging ability. In the same way, flavonoids pro-
duced in the leaves can promote blood circulation and have a strong antioxidant
activity (Gong et al. 2019).
7.9.4.5 Anti-tussive Effect

S. grosvenorii has been traditionally used in Chinese medicine as a pulmonary
demulcent and for the treatment of coughs, sore throats, and constipation (Jin and
Lee 2012). In studies using rats, mogrosides were capable to increase significantly
the excretion of phlegm. The response was dose depending. Besides, the sputum-
reducing action was equal to that of ammonium chloride. Other results indicated that
the anti-tussive effect changes with the growing habitats of the plants and the fruit
growth period. Its efficacy as an expectorant is produced by the joint action of
several components such as oxomogroside V and mogroside V (Gong et al. 2019).
On the other hand, to drink an aqueous extract of the fruit reduces significantly the
cough induced by ammonia-water or sulphur-dioxide in mice. Moreover,
mogrosides displayed a similar effect (Li et al. 2014).
7.9.4.6 Hepatoprotective Effect

Some studies have shown a hepatoprotective effect of S. grosvenorii compounds
(Gong et al. 2019). The fruit extracts of S. grosvenorii fruit might reduce the
biological activities of transaminase in the serum of mice with liver injury induced
by carbon tetrachloride or thioacetamide (Li et al. 2014). Similarly, mogrosides
showed a protective effect against carbon tetrachloride-induced acute liver injury,
tuberculosis vaccine combined with lipopolysaccharide-induced immunological
liver injury and carbon tetrachloride-induced chronic liver injury. Besides,

mogrosides have no effect on enzyme activities in normal liver (Gong et al. 2019).
7.9.4.7 Immunomodulatory Effect

In research conducted by Zhang et al. (2011) it was concluded that S. grosvenorii
polysaccharide produces an increase in the content of interleukin-2 at 50 μg/mL and
down-regulated the secretion of interleukin-1β in the thymus at 400 μg/mL. The
results indicate that the compound promoted the immunity activity of T and B
lymphocytes by inducing interleukin-2 production and consequently reducing inter-
leukin-1β release in mice.
7.9.4.8 Anti-Carcinogenic Effect

Several compounds isolated from S. grosvenorii, 18 triterpenoids, 11-oxomogrol,
and 11-oxomogroside A1 were tested for their potential anti-carcinogenic effect. The
response was determined by their inhibitory effects on the induction of Epstein-Barr
virus early antigen (EBV-EA) by 12-O-tetradecanoylphorbol-13-acetate (TPA) in a
line of human lymphoblast cells. This response is known to be a primary screening
test for anti-tumour promoters. All the compounds produced potent inhibitory effects
on EBV-EA induction (70–100% inhibition at 1 103 mol ratio/TPA) (Ukiya et al.
2002).

According to the review of Gong et al. (2019), S. grosvenorii is considered to have
low toxicity. In a study evaluating the effect of a compound containing mogrosides
in dogs, no changes in body weight, organ weight or food consumption were
detected after a dose of 3000 mg/kg bw/day. Besides, no significant effects on
blood chemistry analysis and urinalysis were detected. No systemic or organ toxicity
were founded in these studies (Gong et al. 2019).
Similar negative results were obtained in a study treating rats with the same
compound. The body weight and feed efficiency were not affected, but a decrease in
bilirubin level and higher total protein content were determined. Moreover, in mice
administered with an extract of S. grosvenorii (7200 mg/kg bw) for 12 h, no changes
in organ morphology nor short-term toxicity were observed. Additionally,
mogroside did not show a genotoxic effect in Salmonella typhimurium test (Gong
et al. 2019).
7.10 Biotechnological Approaches
Several biotechnological tools have been applied to improve the clonal propagation
and secondary metabolite production of these four species. Multiple in vitro culture
techniques (direct regeneration, indirect regeneration, somatic embryogenesis, tem-
porary immersion system culture, etc.) were focused on more efficient and homoge-
neous plant propagation. Moreover, the effect of elicitor substances on accumulation
of bioactive compounds has been determined. Besides, genetic transformation of
these medicinal plants was explored with different goals. In this section, we will
condense some of the most promising studies performed so far in these species.
S. rebaudiana is the most intensively investigated high-intensity sweet-tasting plant.

Dozens of reports on stevia improvement, in vitro culture and other biotechnological
strategies have been published in the last 30 years (reviewed in Yadav et al. 2011;
Luwańska et al. 2015; Gantait et al. 2015; Modi and Kumar 2018; Khan et al. 2021).
Therefore, we will mostly focus on some interesting investigations published
after 2000.
7.10.1.1 In Vitro Culture

The widespread in vitro culture of stevia arises as a response to a need for massive
propagation maintaining the genetic identity and secondary metabolite production of
selected lines. In natural conditions, low seed germination and the small number of
individuals obtained by vegetative propagation limit its proliferation. For in vitro
propagation, several types of explants have been studied such as shoot tips (1–3 cm),
leaves, roots, nodal, and internodal segments (Singh et al. 2008; Gantait et al. 2015).
In general, these are cultured on MS medium (Murashige and Skoog 1962) with
different concentrations of growth regulators for regeneration. Afterwards, shoots
might be subcultured to obtain higher propagation (mainly after the second subcul-
ture). Later, individual shoots are transferred to new media for rooting and finally
established. If the starting material are plants or seeds grown in the greenhouse or
outside, an additional step for disinfection should be performed (i.e. Singh et al.
2008; Janarthanam et al. 2009; González-Hernández et al. 2019b).
Steviol glycosides production in plant, organ and tissue cultures of stevia was
analysed by Bondarev et al. (2001). The content in intact plants (35.4–43.7 mg/
gDW) was five or six times higher than in plants grown in vitro (6.1–7.3 mg/gDW),
in spite of its similar qualitative composition. On the other hand, these compounds
were found in very low amounts in suspension cultures and calli. Higher stevioside
and rebaudioside (A, C) biosynthesis was confirmed in morphogenic callus and
during shoot regeneration from morphogenic callus. Hence, the steviol glycosides
biosynthetic pathway was inhibited in non-differentiated cells, since functional
chloroplasts are necessary for this metabolic process. Nonetheless, the production
of sweetener compounds during prolonged in vitro culture was stable after 5 years
(Bondarev et al. 2001).
In 2006, multiple shoots of stevia were regenerated from nodal segments on MS
medium with benzyladenine (BA) or kinetin. A 100% of induction and an average
regeneration of 13 shoots per explant were obtained with 2.66 μM BA concentration
after 25–30 days. Next, rooting of regenerated shoots was performed in MS medium
containing 4.92 μM indole-3-butyric acid (IBA). Finally, the micropropagated plants
were established in a soil and sand mixture (1:1 w/w) with a survival rate of 70%
(Tadhani et al. 2006).
Direct shoot regeneration from Stevia leaves was first reported by Sreedhar et al.
(2008). Furthermore, the regenerated shoots were transferred to a bioreactor to
produce biomass and subsequently established in field conditions after rooting. In
this study, five growth regulators were evaluated: indole-3-acetic acid (IAA), IBA,
2,4-dichlorophenoxy-acetic (2,4D), BA and kinetin. Shoot buds were obtained only
in the presence of BA (8.88 μM), and kinetin (4.65 μM). The best overall response
was produced with immature leaves of 0.6–1 cm (93%, 4.93 shoot buds per explant)
after 7 weeks. Next, shoots were transferred to MS medium with 4.92 μM IBA for
elongation and rooting for 3 weeks. Moreover, the culture conditions were optimized
for shoot culture in a liquid medium, and biomass was increased 8.87-fold in a
bioreactor with 2x MS salts and sucrose 60 g/L. Rooted plants were successfully
established in a substrate with a composition of sand: vermicompost: garden soil
(1:1:1 w/w) (Sreedhar et al. 2008). This scale-up step allowed producing 590 micro-
cuttings in a short time.
Regeneration of multiple shoots through indirect organogenesis from leaves and
node explants was reported by Janarthanam et al. (2009). Leaf segments cultured on
MS medium supplemented with 11.31 μM 2,4D and 2.22 μM 6-BAP yielded the
highest response (80%). Callus segments (around 0.5 g) on MS medium with
4.44 μM BA and 1.34 μM naphthaleneacetic acid (NAA) produced on average
14 well-developed shoots in 80% of the explants. Moreover, 100% of the shoots
produced roots after 25 days in basal medium with 2.46 IBA and 90% survived the
acclimatization phase. These authors estimated production of 50,000 plants after five
subcultures. They also recommended to perform genetic stability and phytochemical
analysis of the in vitro propagated plants in further studies (Janarthanam et al. 2009).
In 2010, Thiyagarajan and Venkatachalam developed multiple shoot in vitro
propagation strategy also using nodal explants. Similarly, MS medium
supplemented with different concentrations of growth regulators were studied for
shoot bud induction and multiplication (6-BAP, kinetin), and rooting (NAA, IAA).
As result, 6-BAP at 4.44 μM was found to be the best concentration for multiple
shoot regeneration (12.21 shoots per explant, 100% response). Moreover, rooting
was achieved in 91% of the shoots after 3 weeks on half-strength MS medium with
2.198 μM NAA. The micropropagated plants were established on a mixture of sand
and soil (1:2 w/w) (Thiyagarajan and Venkatachalam 2010).
Ghauri et al. (2013) achieved micropropagation of S. rebaudiana from roots.
First, the sterilization conditions were selected (0.025% of HgCl2). Thereafter,
maximum multiple shoot induction (89.5%) and proliferation (40 shoots per explant)
were achieved on basal medium supplemented with 5.55 μM 6-BAP and 2.275 μM
thidiazuron (TDZ). Finally, the rooting and acclimatization were successfully
performed using IBA (0.492 μM) and a mixture of sand, loamy soil, and farmyard
manure (1:1:1, w/w), respectively (Ghauri et al. 2013). This is a fast and easy
protocol for mass propagation of stevia plants. However, a morphological compari-
son of the micropropagated plants and genetic stability evaluation should be
performed to confirm a homogenous propagation of plants through this
methodology.
Furthermore, Pawar et al. (2015) conducted in vitro regeneration studies on direct

organogenesis from nodal segments and shoot tips. After optimization of plant
sterilization conditions, eight node-derived shoots and six shoots tip-derived shoots
were obtained on MS medium with 8.88 μM 6-BAP. A rooting percentage of 62.3
was achieved with 5.37 μM NAA. Later, the rooted plantlets were established on
cocopeat achieving a 70% of survival (Pawar et al. 2015).
Propagation from shoot tips produced lower shoot regeneration efficiencies.
However, this is a rapid and simple method for plant propagation with lower risk
of somaclonal variability. González et al. performed a similar work in 2019. In this
report, the shoot propagation was achieved without growth regulators and rooting
was produced in the same basal medium after several subcultures for proliferation.
The survival of rooted plants was between 80 and 90% during acclimatization on
different combinations of zeolite and compost. Moreover, the morphological
features of plants cultured in vitro were less variable and significantly higher than
plants propagated by cuttings (González-Hernández et al. 2019b).
Lopez-Arellano et al. (2015) achieved somatic embryogenesis and plant regener-
ation from protoplast culture of S. rebaudiana. Protoplast isolation and culture
conditions were optimized to produce high yield and viability. For this, different
factors were studied such as combination of cell wall degrading enzymes,
concentrations of mannitol, incubation time, type of explants, age of the explant,
and culture method. Protoplasts were successfully isolated from leaves of 4-week old
in vitro-grown plants using an enzyme mixture comprising four hydrolytic enzymes,
in 0.5 M mannitol. Protoplast suspension was cultured in KM8P liquid medium (Kao
and Michayluk 1975) and 98.8% viable protoplasts were obtained after 4 h of
incubation in the dark. Protoplast derived micro-calli were transferred to MS
medium containing 6-BAP (4.44 μM) within 4 weeks. Bipolar embryos were
isolated on MS medium supplemented with 5.37 μM NAA for rooting and plantlets
were obtained in this medium within a month (Lopez-Arellano et al. 2015). This is
the first report of stevia regeneration from protoplasts. It might be applied in other
studies for the improvement of this medicinal plant.
Alternatives to diminish the cost of plant propagation from nodal explants were
proposed, like the use of sodium hypochlorite as sterilization technique
(Thilakarathne et al. 2019) or highly efficient micropropagation using RITA biore-
actor without growth regulators (Bayraktar 2019). Moreover, preliminary studies of
medium composition and pre-treatments to achieve androgenesis were published
(Uskutoğlu et al. 2019). On the other hand, in vitro culture studies in S. rebaudiana
were oriented to improve the culture conditions and steviol glycosides contents with
additives, elicitors, nanoparticles and genetic transformation.
7.10.1.2 Influence of Additives on the Enhancement of In Vitro Culture

Conditions and Steviol Glycoside Content
Other studies were oriented to boost some phases of the in vitro culture through the
application of growth additives, amino acids, micronutrients or macronutrients to the
medium (i.e. Das and Mandal 2010; Jain et al. 2012; Fakhrul et al. 2014; Sridhar and
Aswath 2014; Ghorbani et al. 2017). Das and Mandal (2010) studied the effect of
amino acids (glutamine, tryptophan) and additives (casein hydrolysate, yeast extract,
potato extract) on the embryogenic callus. For callus induction, the explants were
cultured on MS medium with 9.1 μM 2,4D and 2.22 μM 6-BAP. Higher callus
response (percentage of the explants with callus induction) and fresh weight were
obtained on medium supplemented with glutamine at 50 mg/L. On the other hand,
the effect of tryptophan was negative for all parameters evaluated. Casein hydroly-
sate (100 mg/L) produced a higher response than the other growth modulators
evaluated and glutamine. Unfortunately, these authors did not present the results
of the control (explants cultured in basal medium with growth regulators). Moreover,
the addition of potato extract produced brown and loose calli. Finally, these authors
recommended the use of a medium supplemented with glutamine and casein hydro-
lysate at 50 and 100 mg/L, respectively (Das and Mandal 2010).
Jain et al. (2012) improved the direct plant regeneration from nodal explants
modifying the concentrations of micronutrients usually used in MS medium (cobalt,
iron, boron, manganese, potassium iodide and copper). Higher shoot induction and
response were achieved with 300 μM MnSO4, 2 μM CoCl2, and 10 μM KI. A
medium with these micronutrient levels was further evaluated. The chlorophyll
content and biomass were higher than control, but no significant increase was
observed in shoot regeneration. These authors endorsed that individual nutrient
composition should be optimized for each culture medium to achieve higher effi-
ciency (Jain et al. 2012).
Similarly, Fakhrul et al. (2014) studied the effect of potassium on plant growth
and steviol glycoside production. One-month-old plantlets regenerated from nodal
segments (0.5 cm) and transferred to MS media with different concentrations of this
macronutrient. All the morphological parameters, fresh and dry biomass were
significantly increased at four times higher potassium concentrations. Addition of
potassium had a differential effect on the content of steviol glycosides. Higher
potassium concentration diminished the accumulation of stevioside. In contrast,
the production of rebaudioside A was enhanced with potassium and the best result
was obtained at twice the concentration (Fakhrul et al. 2014). Therefore, medium
conditions could be adjusted to optimize not only the plant regeneration but also the
secondary metabolite production.
As well in 2014, Sridhar and Aswath analysed the influence of additives on an
optimized direct regeneration protocol from nodal explants. First, the combination of
growth regulators was improved to achieve higher shoot multiplication. Then, basal
medium containing 8.88 μM 6-BAP, 2.325 μM kinetin and 0.537 μM NAA were
selected to study the effect of casein hydrolysate, coconut water, malt extract, and
yeast extract. All the complex organic extracts induced a higher number of shoots
per explant. Casein hydrolysate at 0.05% (w/v) was the most effective treatment with
an average of 15 plantlets per explant and a 90% response. These authors proposed a
two-step protocol, multiple shoot induction using the combination of growth
regulators previously selected during 4 weeks, followed by a subculture to the
same medium supplemented with casein hydrolysate (Sridhar and Aswath 2014).
More recently, the influence of sucrose concentrations (50, 100, 150 mM) on
morphological features and on expression of steviol glycosides biosynthetic genes
was assessed. Similarly, nodal explants were cultured on MS medium for 28 days. A
significant correlation (0.01) was founded between sucrose concentration and all the
growth parameters analysed. The best result for all traits was obtained at 100 mM.
Moreover, gene expression was also modified by sucrose concentrations, but not in a
predictable way (Ghorbani et al. 2017). This and other carbon sources could be
studied in follow-up studies. Furthermore, the steviol glycosides content should be
determined.
7.10.1.3 Elicitation
The effect of several elicitors in S. rebaudiana in vitro cultures has been studied with
a positive overall result on secondary metabolite production. Salinity and/or drought
stress were evaluated during shoot proliferation (Pandey and Chikara 2015; Rameeh
et al. 2017), in callus and suspension cultures (Gupta et al. 2015) and in hairy roots
(Libik-Konieczny et al. 2020). Pandey and Chikara (2015) reported the effect of
salinity and drought stress on plant growth, glycoside content and the expression
level of genes involved in the steviol glycosides biosynthetic pathway. All the
concentrations of sodium chloride and mannitol decreased the growth parameters
evaluated, mainly at concentrations higher than 25 mM. However, stevia plant
showed some tolerance to salinity and drought stress (50 and 75 mM). On the
other hand, sodium chloride increased the expression of the kaurenoic acid
13-hydroxylase and UDP-glycosyltransferases genes, while mannitol repressed
expression of the genes. This effect was confirmed by steviol glycoside content
quantification. Stevioside and rebaudioside A accumulation was significantly
enhanced in plants cultured with sodium chloride at 50, 75, and 100 mM, but
accumulation of these compounds was negatively affected by mannitol induced
drought stress. Therefore, plants cultured under salinity stress might produce higher
amounts of sweetener compounds (Pandey and Chikara 2015).
Gupta et al. (2015) studied the effect of proline and polyethylene glycol (PEG) on
biomass yield and steviol glycoside production from callus and suspension cultures.
Proline produces an osmotic adjustment in stressed plants, while PEG induces
drought stress. Proline (5 mM) and PEG (5%) increased the biomass of both cultures,
while higher concentrations affected this parameter. Moreover, proline (7.5 mM) and
PEG (5%) increased steviol glycoside content in callus (4 and seven-fold, respec-
tively) and suspension culture (3.7 and 4.7-fold, respectively) (Gupta et al. 2015).
The effect of osmoprotectant glycine betaine was evaluated in shoot tip cultures
under salinity stress produced by sodium chloride (Rameeh et al. 2017). In this
study, sodium chloride concentrations of 50, 75, and 100 mM significantly reduced
the growth parameters of plantlets. Moreover, total chlorophyll, rebaudioside A, and
stevioside contents were also diminished under salinity stress. In contrast, glycine
betaine produced a positive effect on all the parameters studied. Thereafter, the
interaction of both compounds was analysed and glycine betaine counteracted the
negative effect of the salinity stress.
More recently, Libik-Konieczny et al. (2020) hypothesized that the application of
stress factors (NaCl, PEG, sucrose, and light) during in vitro culture of hairy roots
would modify the biosynthetic pathway and the accumulation of steviol glycosides.
A stronger increase in weight and length of hairy roots was obtained in the treatment
with higher osmotic potential (175 mM sucrose cultured in dark conditions). More-
over, oxidative stress induction and gene expression was confirmed in the culture
media with continuous light or application of osmotic active substances. Likewise,
upregulated gene expression was detected for kaurenoic acid 13-hydroxylase and
UDP-glycosyltransferases genes under the formerly mentioned conditions. Finally,
hairy roots were able to accumulate steviol glycosides in different concentrations as
a result of variable culture conditions.
In 2016, Ahmad et al. evaluated the effect of various light spectral compositions
in callus cultures. White fluorescent tube lights (16/8 h) produced higher callogenic
response and biomass content, while blue light increased the production of total
phenolics and flavonoids as well as the antioxidant capacity (Ahmad et al. 2016).
Hence, light conditions during in vitro culture should be optimized for each culture
phase to enhance biomass and secondary metabolite production.
Tavakoli et al. (2019) reviewed the effect of abiotic elicitors (methyl jasmonate,
salicylic acid, auxins, cytokinins, and gibberellins) and two inhibitors (paclobutrazol
and chloroquate) on expression of steviol glycoside biosynthetic genes. Methyl
jasmonate, abscisic acid, gibberellic acid, and salicylic acid produced a positive
effect on genes expression. Cytokinin, IAA, and both inhibitors had the opposite
effect on gene expression and steviol glycoside biosynthesis (Tavakoli et al. 2019).
Green root cultures were also elicited with hydrogen peroxide and methyl
jasmonate (Alvarado-Orea et al. 2020). Steviol glycoside production was enhanced
in 2.4-fold with the addition of 250 μM hydrogen peroxide. Moreover, both elicitors
at 250 and 500 μM increased the content of phenolic and flavonoid compounds
improving the antioxidant activity.
Very recently, Rasouli et al. (2021) studied the effect of various elicitors
(chitosan, yeast extract, and methyl jasmonate) on in vitro shoot multiplication.
All of them produced a positive effect on growth parameters and steviol content. In
summary, chitosan (200 mg/L), methyl jasmonate (100 mg/L), and yeast extract
(50 mg/L) could be effectively used for the production of biomass and sweeteners in
S. rebaudiana.
7.10.1.4 Effect of Nanoparticles

In recent years, the application of nanoparticles in plant tissue culture has
demonstrated to produce positive effects on in vitro growth, genetic transformation
and secondary metabolism (reviewed by Kim et al. 2017). This approach has been
also applied to S. rebaudiana during direct organogenesis from nodal segments
using copper oxide (Javed et al. 2017a) and zinc oxide (Javed et al. 2017b)
nanoparticles. In both cases, the concentrations of nanoparticles assayed ranged
between 0.1 and 1000 mg/L. The physiological parameters of regenerated shoots,
secondary metabolite content, and oxidative stress induced by nanoparticles were
evaluated. Copper oxide and zinc oxide nanoparticles induced shoot organogenesis
and positively influenced all the physiological parameters at concentrations of
10 mg/L and 1 mg/L, respectively. Moreover, steviol glycoside production was
enhanced in both studies using these concentrations. The promoting effect of
nanoparticles was associated with the induction of oxidative stress response (Javed
et al. 2017a, b). These results might be further applied to establish in vitro cultures
with increased accumulation of sweetener compounds.
In 2019, Rezaizad et al. performed another study of the effect of titanium dioxide
nanoparticles (20–400 ppm) in potted-plants. The fresh and dry weight of shoots was
increased by these nanoparticle concentrations until reaching the highest values at
400 ppm. Steviol glycoside content was also modified and better results were
observed when using concentrations of 200 ppm. On the other hand, this treatment
also produced lower lipid peroxidation (Rezaizad et al. 2019).
7.10.1.5 In Vitro Culture and Genetic Improvement

The opportunities of transgenic breeding for obtaining a high and uniform yield of
sweeteners from stevia plant were discussed before by Singh et al. (2008). In
numerous publications, the optimization of in vitro culture conditions combined
with the genetic transformation to produce hairy roots, secondary metabolites, gene
overexpression or efficient protocols was studied. A summary of these works is
presented in Table 7.3.
Guleria and Yadav (2013) reported novel insights into the regulation of steviol
glycoside biosynthesis after Agrobacterium tumefaciens-mediated transient silenc-
ing. The genes coding for the ent-kaurenoic acid-13 hydroxylase (SrKA13H) and
three UDP glycosyltransferases (SrUGT85C2, SrUGT74G1, and SrUGT76G1) were
significantly silenced (up to 63%) using syringe agroinfiltration method. Leaves
infiltrated with RNAi construct produced considerably lower steviol glycosides. On
the other hand, gibberellin accumulation was significantly enhanced. These authors
concluded that silencing of SrKA13H and SrUGT85C2 genes affects the metabolite
flux in the steviol glycoside pathway and promotes gibberellin biosynthesis (Guleria
and Yadav 2013).
Khan et al. (2013) developed direct and indirect shoot regeneration protocols
from nodal segments. Leaf segments from regenerated shoots were transformed via
A. tumefaciens with a genetic construction containing ß-glucuronidase reporter gene
(uidA) and hygromycin phosphotransferase selectable marker gene (hpt). Shoot bud
selection was performed on selective medium with hygromycin 50 mg/L. GUS assay
and PCR analysis for selective amplification of the uidA gene allowed to confirm the
genetic modification of hygromycin-resistant shoots. The transgenic plants were
phenotypically indistinguishable from non-transformed plants. However, their
inter-simple sequence repeat (ISSR) profiling and steviol glycosides content were
different. Out of 13 primers evaluated, only 8 produced banding patterns. Low
amplification of random amplified polymorphic DNA markers of S. rebaudiana
plants was reported before in other studies (González-Hernández et al. 2019a).
Somaclonal variations among the mother plant, transgenic line and four somaclones
were detected. Variable steviol glycosides profiles were also identified.
Several studies of hairy roots culture were achieved after A. rhizogenes-mediated
transformation from leaves, internodes and hypocotyls using different bacterial
strains and culture conditions (Fu et al. 2015; Michalec-Warzecha et al. 2016;
Pandey et al. 2016; Sanchéz-Cordova et al. 2019). Fu et al. (2015) proposed the
Table 7.3 Summary of the research on genetic transformation of Stevia rebaudiana

Transformation
mediated by Explant Molecular analysis Aim Reference
A. tumefaciens Leaves Semi-quantitative Evaluate the Guleria
strain LBA4404 PCR analysis of regulation of steviol and
SrKA13H, glycoside Yadav
SrUGT85C2, biosynthesis and its (2013)
SrUGT74G1 and relation with
SrUGT76G1 genes gibberellic acid
pathway by transient
gene silencing using
RNAi
A. tumefaciens Leaf GUS assay, PCR Development of Khan et al.
strain EHA-105 segments analyses and ISSR regeneration and (2013)
from profiling transformation
in vitro protocols and
grown somaclones via
shoots indirect regeneration
A. rhizogenes Young PCR analyses of rolB Production of Fu et al.
leaves from and rolC genes chlorogenic acid and (2015)
seedlings its derivatives in
hairy root cultures
and optimization of
culture conditions
A. rhizogenes Leaves and PCR analyses of rolB Obtention of hairy Michalec-
strains ATCC internodes and rolC genes root cultures from Warzecha
15384 and LBA different kinds of et al.
9402 explants induced by (2016)
A. rhizogenes
transformation for
further modification
to produce secondary
metabolites
A. rhizogenes Leaf and PCR analyses of rolB To explore the Pandey
strain A4 nodal and rolC genes. biosynthetic potential et al.
segments qRT-PCR analysis of of hairy roots culture (2016)
SrUGT85C2 gene to produce steviol
glycosides
Biolistic Calli GUS assay, PCR Production of Mubarak
bombardment analyses of uidA and transgenic plants et al.
bar genes resistant to bialaphos (2015)
herbicide
A. rhizogenes Hypocotyls PCR analysis of Comparison of Sanchéz-
strain K599 of in vitro CaMV 35S promoter transformed plants Cordova
plantlets with a wild et al.
phenotype cultivated (2019)
in vitro in terms of
SG production, total
phenols and growth
parameters
(continued)

Transformation
mediated by Explant Molecular analysis Aim Reference
A. tumefaciens Leaf PCR analysis of Development of an Zheng
strain AGL2 segments T-DNA fragment, efficient et al.
Southern blot transformation (2019)
analysis to detect method using GFP.
nptII gene, qRT-PCR Overexpression of
of SrDXS1 and SrDXS1 and SrKAH
SrKAH genes genes for higher
steviol glycoside
production in vitro
A. tumefacies Leaf PCR, southern blot, Development of Kim et al.
strain AGL2 segments qRT-PCR analyses of transgenic Stevia (2019)
SrUGT76G1 gene plants overexpressing
the SrUGT76G1 gene
A. tumefaciens Nodal PCR, Southern blot, Optimization of Taak et al.
strain GV1301 sections and RT-PCR and A. tumefaciens- (2020)
calli qRT-PCR analyses of mediated
bar gene. transformation of
stevia
A. tumefaciens Dark grown PCR and southern Development of a Wu et al.
strain EHA-105 axillary blot analyses of hpt, stable A. tumefaciens- (2020)
shoots relative expression of mediated
SrUGT76G1 transformation
system using
hygromycin B as
selective agent.
Overexpression of
SrUGT76G1 gene
production of chlorogenic acid and its derivatives form hairy roots optimized culture
conditions. These bioactive compounds produce several beneficial effects such as
anti-hypertensive, anti-microbial, and antiviral. While Michalec-Warzecha et al.
(2016) described the optimization of inoculation and hairy roots culture conditions.
On the other hand, the research published by Pandey et al. (2019) was focused on
the production of secondary metabolites in hairy roots cultured under dark or light
conditions. Steviol glycosides were quantified in a green hairy root clone under light
condition, but not in another green and two non-green rizoclones. A positive
correlation between the steviol glycosides content and SrUGT85C2 gene expression
pattern was confirmed by qRT-PCR. These results demonstrate the regulatory
influence of expression of this gene on the biosynthetic pathway of steviol
glycosides and its potential for metabolic engineering of Stevia to enhance sweetener
compound production (Pandey et al. 2016). In another study, higher phenotypical
traits and steviol glycosides yield were achieved in transgenic line modified by
A. rhizogenes (Sanchéz-Cordova et al. 2019).
A different transformation method was applied to stevia calli to obtain herbicide-

resistant plants. The biolistic bombardment method allowed producing transgenic
lines expressing uidA and bar genes. Shoot survival was completely inhibited after
culture with 4 or 5 mg/L bialaphos in non-transformed explants, while 52 and 44%
of the transformed explants survived under the same culture conditions (Mubarak
et al. 2015).
Further studies were focused on the enhancement of steviol glycosides accumu-
lation. Acute gamma irradiation was applied to shoot tip for inducing mutations.
Stevioside, rebaudioside A, and rebaudioside B contents were increased in irradiated
plantlets compared to control. The results summarized in this publication might be
used to develop new stevia cultivars with desirable features (Chiew et al. 2019).
More recently, A. tumefaciens-mediated transformation protocols were oriented
to develop efficient transformation protocols (Zheng et al. 2019; Taak et al. 2020;
Wu et al. 2020). These protocols are promising tools for functional studies and
metabolic engineering of S. rebaudiana.
Zheng et al. (2019) obtained transgenic stevia plants expressing the green fluo-
rescent protein (GFP) and neomycin phosphotransferase gene (nptII) with selection
on 50 mg/L kanamycin. Moreover, these authors overexpressed the SrDXS1 and
SrKAH genes without affecting the normal growth and development of the plants. As
consequence, steviol glycoside content was increased up to 54 and 88%,
respectively.
This protocol was applied a year later to overexpress Stevia
UDP-glycosyltransferase 76G1 (SrUGT76G1) gene that catalyses the conversion
of stevioside to rebaudioside A. Therefore, rebaudioside A content was enhanced,
but total steviol glycosides accumulation was not modified. Moreover, the
rebaudioside A to stevioside ratio was considerably increased from 0.30 in
non-transformed plants up to 1.55 in transgenic lines. Besides, these authors proved
that rebaudioside C can be produced from dulcoside A through glycosylation
catalysed also by this enzyme. In addition, a comprehensive phenotypical analysis
of the transgenic lines was performed. The internode length, stem thickness, and leaf
size of the lines overexpressing the SrUGT76G1 gene were similar to those of wild-
type. Furthermore, no significant differences between both groups were found after
analysing chlorophylls and total carotenoids content (Kim et al. 2019). This work
demonstrated that genetic engineering is a suitable approach to create new cultivars
of S. rebaudiana with favourable traits for its industrial application.
Taak et al. (2020) reported optimization of the transformation protocol studying
the effect of several parameters such as explant type, pre-incubation duration,
acetosyringone concentration, Agrobacterium cell density, inoculation duration
and co-culture duration. After selection of the transformed tissues with
phosphinothricin, an average transformation efficiency of 40.48% was achieved
using nodal explants. The stable integration of the transgene (bar) was confirmed
by PCR, RT-PCR, qRT-PCR and Southern blot hybridization. Moreover, the herbi-
cide tolerance of the transgenic lines sprayed with glufosinate ammonium (8 mg/L)
was demonstrated in greenhouse-grown plants.
Wu et al. (2020) reported new interesting insights on an enzyme involved in the

steviol glycoside biosynthetic pathway. In this protocol, highly efficient hygromycin
selection was performed during callus induction and proliferation (6 mg/L), and in
shoot induction and rooting media (4 mg/L). Stevia plants overexpressing a specific
isoform of SrUGT76G1 were generated and its expression influenced steviol
glycosides accumulation. Moreover, several isoforms of this enzyme were identified
in this work. Therefore, the authors suggested alternative splicing might produce
enzyme variants in this medicinal plant.
C. paliurus is a relict tree considered an endangered species in its natural habitat

(Crane and DuVal 2013; Hu et al. 2017). Despite numerous studies supporting the
therapeutic effects of C. Paliurus, the limited area planted with this species restrict
its use (Zhao et al. 2020). C. paliurus has an extended range of distribution in China.
Notwithstanding this widespread distribution, its natural regeneration is very low.
Germination of seeds in this species can take about 2 years under natural conditions
mainly because of the thick and impermeable wall of the nutled (Crane and DuVal
2013). In this context, biotechnology offers invaluable tools allowing wider appli-
cation of this species and its therapeutics properties (Zhao et al. 2020).

There are very few papers reporting results on in vitro culture of C. paliurus. The
first report we found about the in vitro culture of C. paliurus was a preliminary study
using mature embryos of the species as initiating material for tissue culture (Xie et al.
2009). The authors indicate that the success rate of excised embryos in a culture
medium with 30 g/L sucrose and 6.5 g/L agar reached 72% after 60 days. On the
other hand, the determined best culture medium for multiple shoots induction was
composed of woody plant media (WPM) supplemented with, 2.22 μM 6- BAP, plus
0.049 μM IBA, plus 30 g/L sucrose. The proposed subculture cycle was 40 days, in
which the induction rate of multiple shoots was 93.33% and the highest multiplica-
tion coefficient was 4.75. Besides, the addition of uniconazole (0.1 mg/L) and the
rare-earth lanthanum (5 mg/L) in culture medium was positive for strong seeding and
the development of plantlets. Finally, the recommended rooting medium was com-
posed of WPM plus 0.984 μM IBA and 20 mg/L sucrose. Using this medium the
rooting rate was 16.67% (Xie et al. 2009).
In another research, the sterilization conditions, kind of explant, and basic culture
medium were optimized for the culture of calli from C. paliurus. The suggested most
appropriate explant for the callus formation was young stem sections under a year of
age. These authors proposed MS plus 8.88 μM 6-BAP and 0.537 μM NAA as the
best culture medium for callus induction. The percentage of explants with calli was
83.33 (Cheng et al. 2009). Furthermore, another culture medium combination
recommended in this case for the formation of C. paliurus leaves-derived callus
was MS supplemented with 4.44 μM 6-BAP, 2.685 μM NAA, and 4.57 μM 2,4-D.
The selection of this culture medium combination was performed with attention to
the overall effect on the induction rate, size and colour of callus (Hu et al. 2010). The
same authors proposed the combination 1/4 Driver and Kuniyuki Walnut medium
(DKW) supplemented with 2.22 μM 6-BAP and 4.57 μM 2,4-D for callus prolifera-
tion. Besides, a dark period of 10 days was recommended as appropriate when calli
were induced from C. paliurus leaves (Hu et al. 2010).
With the objective to expand the opportunities of using the potential therapeutic
properties of C. paliurus, Yin et al. (2013) developed during years stable and
uniform cell suspension cultures. The culture medium used for callus establishment
was liquid MS medium supplemented with 2,4-D (2.285 μM), NAA (1.611 μM), and
kinetin (4.65 μM). The optimum time for subculture and harvest was 8–10 day and
14 day after inoculation, respectively. Besides, culture media MS and WPM with
additives proved to be appropriate for both cell growth and triterpenic acid accumu-
lation. The proposed supplementation for the media was 3% sucrose (w/v), 60 mM
total nitrogen (NO3/NH4+ ¼ 2/1), 1.25 mM KH2PO4, 2 mM CaCl2, and 2 mM
MgSO4. Using this medium total triterpenic acid, ursolic acid and oleanolic acid
concentration was higher in the suspended cultured when compared to leaves and
calli (Yin et al. 2013).
Furthermore, for several years Zhao et al. (2020) selected callus lines to improve
the yield of natural secondary metabolites in cell culture. They selected five
promising callus lines with different characteristics. Two distinctive lines were
light yellow-green calli and extremely rare red calli. Both kinds of calli proved to
be significantly different not only morphologically but also in secondary metabolites
content. The total triterpenoids and total polyphenols contents in red calli were 1.65
and 1.71 times higher, respectively, than in yellow-green calli. Besides,
anthocyanins were only found in red calli. Additionally, the expression patterns of
genes involved in the triterpenoid and polyphenol biosynthesis pathways were
studied (Zhao et al. 2020). Bioactive compounds concentration was explored as a
key factor for commercial production. The establishment of high-producing cell
lines is an important step for improving the output of target metabolites (Yin et al.
2013).
Finally, Huang et al. (2020) proposed a rapid propagation technology for
C. paliurus. According to this research, the best initial material for tissue culture is
stem segments slightly lignified and the optimal moment for collecting them is from
April to June. These authors also recommended soaking the stem segments in 0.1%
HgCl2 for 5–7 min as a surface disinfection method. Besides, for the induction of
primary buds MS medium supplemented with 6-BAP 8.88 μM, IBA 0.984 μM, and
sucrose 30.0 g/L was recommended. The reported bud induction rate was 80.5%. For
multiplication, MS medium supplemented with 6-BAP 2.22 μM, IBA 0.246 μM,
2,3,5 trilodobenzoic acid (TIBA) 0.02 mg/L, and sugar 30.0 g/L. A proliferation
coefficient of 7.0 after 35 days was achieved. Other media recommended in this
publication was MS plus 6-BAP 2.22 μM, IBA 0.246 μM, and sugar 30.0 g/L for
obtaining strong shoots before rooting, and 1/2WPM supplemented with IBA
7.38 μM, 5-NGS 4.5 mg/L, and sugar 20.0 g/L as the rooting medium. Besides,
35 days cultivation in the medium for strong shoots and later 40 days in rooting
medium were recommended. The highest reported rooting rate was 83.3%. Hereaf-
ter, the rooting seedlings were grown for 40 days in greenhouse with a shade of 70%
using calcareous soil as substrate. The reaching survival rates were 54.3–65.6%
(Huang et al. 2020).
Additionally, Liu et al. (2018c) investigated the responses of C. paliurus to
differential light conditions in controlled environment. In the trial, LED light of
different colour was used to influence leaf biomass production, flavonoid accumula-
tion, and related gene expression of C. paliurus young seedlings. As a result, the
highest leaf biomass was produced using white light, while the lowest leaf biomass
was found in the treatment with green light. On the other hand, the content of total
flavonoids was higher under blue, red and, green light in contrast to white light.
While, the highest content of flavonoids kaempferol, isoquercitrin and quercetin
were observed in the treatment with blue light. Moreover, a correlation was found
between the content of leaf flavonoids and the expression of flavonoid biosynthesis-
related genes. The exposure to green and blue light resulted in greater transcript
levels of genes related to flavonoid biosynthesis (Liu et al. 2018c).
Finally, another research for the characterization of the complete chloroplast
genome of C. paliurus was conducted (Hu et al. 2017). This result does not match
into the in vitro culture designation but must be useful for subsequent research such
as the comprehension of metabolic pathways and the genetic transformation.
As far as we know, no research has been conducted on the production of
sweetening or anti-diabetic compounds in vitro or its induction by biotic or abiotic
stress. Subsequent research might be conducted for developing high productive
method for in vitro culture including temporal immersion system culture and
scale-up propagation using bioreactors. With the development of such as techniques,
more studies could be conducted for genetic improvement. Moreover, several biotic
and abiotic effects could be tested for the enhancement of secondary metabolite
production in vitro. In addition, genetic transformation could be used for elucidation
of metabolic pathways, functional studies, and engineering higher accumulation of
bioactive compounds.
7.10.2.2 Other Techniques

Furthermore, other studies have been conducted in the field to better understand the
response of C. paliurus to different culture conditions. These works were focused on
breaking seed dormancy (Fang et al. 2006), evaluating the effect of light intensity
and fertilization on growth and flavonoid levels (Deng et al. 2012), and determining
the effects of cerium nitrate on the growth and physiology of seedlings (Xie et al.
2015a).
Licorice traditional propagation is restricted by a low frequency of germination.

Therefore, in vitro mass propagation techniques have been developed in several
studies (reviewed by Jaiswal et al. 2017; Pandey and Ayangla 2018). Moreover, to
produce maximum hairy root biomass yield, several parameters for the optimization
of culture conditions were predicted using neural networks (Prakash et al. 2010) and
studied by Yadav and Singh (2012).
In vitro propagation of licorice has been performed with two main approaches
(1) shoot proliferation and the subsequent establishment of the regenerated plants,
(2) callus induction to develop suspension cultures. Both strategies pursue a homog-
enous production of biomass and glycyrrhizin (summarized in Table 7.4). Callus
induction has been achieved using seedlings (Shams-Ardakani et al. 2007), leaf
segments (Sharma et al. 2008), axillary buds (Parsaeimehr and Mousavi 2009),
nodal segments (Sharma et al. 2010), hypocotyl (Fu et al. 2010; Safari et al.
2013), and cotyledons (Safari et al. 2013). Direct shoot regeneration was effectively
performed from nodal explants (Kukreja 1998; Yadav and Singh 2012), single node
stems (Kojoma et al. 2010), and leaf segments (Gupta et al. 2013). Indirect organo-
genesis has been obtained as well by Sharma et al. (2008, 2010) and Fu et al. (2010).
The highest propagation frequency (20–25 plants per stolon) was showed by
stolon proliferation from leaf explants after 3 months (Gupta et al. 2013). Moreover,
these authors used ISSR markers to confirm the fidelity of the micropropagated
plants. RAPD and AFLP (amplified fragment length polymorphism) markers have
been effectively applied in this species as well (Pandey and Ayangla 2018). Finally,
the regenerated plants were successfully established with a 70% survival.
The production of glycyrrhizin was also evaluated in several studies. In callus
tissues, the levels of glycyrrhizin decreased after several subcultures and it was
lacking in non-differentiated cells (Shams-Ardakani et al. 2007). On the other hand,
Kojoma et al. (2010) studied glycyrrhizin content in cultured stolons. This and other
related triterpenoids and sterols were detected in cultured stolons and in intact field-
grown stolons. The levels of glycyrrhizin of the former were considerably higher
(36,500 μg/g DW) than in vitro grown stolons (14 μg/g DW). In contrast, betulinic
acid and oleanolic acid accumulation were 3.4-fold and 18-fold higher than in intact
stolons. The regenerated plants produced 7600 μg/g DW of glycyrrhizin in their
roots after acclimatization (Kojoma et al. 2010).
Gupta et al. (2013) detected dissimilar contents of glycyrrhizin in root tuft,
immature and matures stolons (4.21, 4.0, and 4.7 μg/g DW, respectively). Higher
content was quantified in mature brown stolon. These results would be applied in
further studies to understand the biosynthesis of terpenoids and sterols in G. glabra.
Moreover, glycyrrhizin content should be enhanced by other biotechnological
strategies such as elicitation and metabolic engineering.
7.10.3.1 Elicitation
Elicitation of G. glabra to increase secondary metabolites through abiotic and biotic
elicitors has been reported in several studies (Jaiswal et al. 2017). Abiotic elicitors
such as methyl jasmonate and salicylic acid enhanced soyasaponin
5-deoxyflavonoid and glycyrrhizin in cells or plants cultured in vitro. While yeast
extract promoted betulinic acid and soyasaponin accumulation in cell cultures,
chitosan stimulated higher levels of other important licorice components such as
licoisoflavone, liquirtigenin, and licochalcone in callus cultures. Arbuscular
7
Table 7.4 In vitro culture of Glycyrrhiza glabra for mass propagation and glycyrrhizin production
Propagation response and/or
Aim Explant Medium and growth regulators rate Establishment Reference
Standardization of culture Nodal MS medium 6–8 shoots per explant Sand, soil and Kukreja
conditions for rapid multiple shoot explant Shoot proliferation: 6-BAP organic manure (1998)
regeneration and establishment of (8.88 μM) and IAA (5.71 μM) (1:1:1, w/w)
in vitro regenerated plants Rooting: IAA (5.71 μM) Survival
90–95%
Production of glycyrrhizin in Seedlings MS medium – – Shams-
callus and suspension cultures of Callus formation: 2,4-D (4.55 μM) Ardakani
G. glabra var. glandulifera and kinetin (0.93 μM) et al. (2007)
Development of a protocol for Leaf disc Gamborg B5 medium callus Response 35.28% Sand, soil and Sharma et al.
plant regeneration from foliar (6–8 mm) induction: 2,4-D (4.55 μM) and farmyard (2008)
explants kinetin (2.325 μM) manure (1:1:1,
Plantlet regeneration: 6-BAP w/w)
(4.44 μM) and NAA (2.685 μM)
Rooting: IBA (2.46 μM)
Producing a friable callus to be Axillary MS medium – – Parsaeimehr
used for a suspension culture buds Callus induction: 2.275 μM 2,4-D and Mousavi
(5 mm) and 8.88 μM 6-BAP (2009)
Development of a stolon culture Single MS medium Stolon proliferation rate: Up – Kojoma
system and shoot regeneration. node stem Stolon induction and regeneration: to 6.58-fold; shoot et al. (2010)
Evaluation of glycyrrhizin and 0.01 μM NAA regeneration response: 93%
other related triterpenoids and
sterols
Develop an effective protocol for Nodal Gamborg B5Callus induction: Direct regeneration: Sand, soil and Sharma et al.
shoot proliferation from nodal segments 2,4-D (9.1 μM) 94.12%, 9.32 shoots per farmyard (2010)
In Vitro Propagation and Biotechnological Improvement Strategies of Plants. . .
segments Shoot induction: 6-BAP (2.22 μM) explant manure (1:1:1,

and NAA (2.685 μM) w/w)
Rooting: IBA (2.46 μM)
(continued)
197
198
Propagation response and/or

Aim Explant Medium and growth regulators rate Establishment Reference
Study on callus induction and Hypocotyl MS medium Callus induction rate: 93.3% – Fu et al.
embryogenesis Callus induction: 8.88 μM 6-BAP embryo multiplication (2010)
and 2.285 μM 2,4-D index: 8.24
Embryogenesis: 6-BAP (8.88 μM),
kinetin (2.325 μM), IBA
(0.492 μM) and 1000 mg/L malt
extract
Establishment of an efficient Nodal MS medium 86.6%, 3 shoots per explant Sand: Soil, (1:1, Yadav and
protocol on micropropagation by explants Shoot induction: 6-BAP (4.44 μM) w/w) Singh
manipulating growth regulators, and NAA (2.685 μM) (2012)
culture conditions and other
external factors influencing in vitro
multiplication
Utilization of root culture and Leaf MS medium 20–25 plants per stolon Sand: Soil, (2:1, Gupta et al.
stolon proliferation for plant explants Root induction: Combination of w/w) survival (2013)
regeneration kinetin (4.65 μM) with NAA 70%
(5.37 μM) or IBA (4.92 μM) and
IAA (5.71 μM) alone
Stolon proliferation: Liquid
medium with 0.537 μM NAA
Determine the role of different Cotyledon B5 medium Callus formation response: – Safari et al.
combinations of NAA and BAP on or Callus induction: 10.74 μM NAA, 100% and 96.29, (2013)
callus induction and to select an hypocotyl 2.22 μM 6-BAP respectively
explant for in vitro multiplication
E. Kairuz et al.
mycorrhizal fungi (Glomus mosseae and Glomus intraradices) also increased

glycyrrhizin and total phenolic contents in G. glabra roots (Jaiswal et al. 2017).
However, further studies should be conducted to establish a suitable method to
enhance glycyrrhizin production in vitro.
7.10.3.2 Genetic Transformation

In 2006, Tenea et al. achieved G. glabra and Morus nigra L. genetic transformation
mediated by A. rhizogenes by infecting internodes and root segments. The presence
of the rolB gene was confirmed by PCR. The expression of root-inducing genes
allowed to obtain hairy roots biomass containing glycyrrhizin (Tenea et al. 2006).
Mehrotra et al. (2008) also induced hairy roots from leaf segments with a similar
strategy. However, hairy roots were not produced with leaf explants of 2 to 5 weeks
old. Leaf explants from 3 to 4 weeks old were able to produce hairy roots and
showed a transformation efficiency of 46.6 and 35.0%. Transgenic hairy roots were
confirmed by opine and PCR analyses (rolA gene). Moreover, higher biomass
production was obtained using a bioreactor. The optimization of these scale-up
culture conditions would allow producing licorice biomass at an industrial level
(Mehrotra et al. 2008).
More recently, metabolic engineering of licorice to overexpress the ß-amyrin
11-oxidase (CYP88D6) in transgenic roots was performed. Positive selection was
achieved using kanamycin (20 mg/L) after the emergence of hairy roots. CYP88D6
was significantly overexpressed in one transgenic hairy root line and was reduced in
two others. However, the glycyrrhizin production in this transgenic line was not
significantly enhanced compared to control hairy roots without CYP88D6 gene. This
result might be related to the activation of certain mechanism(s) of control over this
or other enzymes along the pathway. Therefore, these authors recommended to
overexpress multiple genes involved in glycyrrhizin biosynthetic pathway or to
up-regulate its expression using transcription factors (Shirazi et al. 2018).
S. grosvenorii has a long history of folk use. Nowadays, products derived from this
species have an increasing demand from international markets (Gong et al. 2019).
On the other hand, its traditional propagation by means of cuttings takes a long time
and can spread viral diseases. These infections weaken the cultivars and produce
considerable reduction of the harvest (Yan et al. 2010). Furthermore, propagation of
S. grosvenorii from seeds yields progeny with a higher proportion of male than
female individuals (Lu et al. 2011). Therefore, more efficient cultivation systems are
needed for fulfil the continuously increasing international demand for high quality
products (Gong et al. 2019). Biotechnological strategies are an excellent way to
address such issues.

The first successful example of S. grosvenorii micropropagation was made as early
as 1980 (Yan et al. 2010). Nevertheless, the regenerated plantlets exhibited some
disadvantages when taken to the field such as low transplant survival and lower
yield. This weakness raised doubts about the quality of the micropropagated plants.
In addition, as another undesirable effect, massive callus formation at the base of
shoots is produced on both proliferation and rooting media. With the intention of
solving this problem, callus proliferation was probed using sugar-free medium and
ex vitro rooting methods. The application of ex vitro rooting methods allowed
obtaining plantlets with an extensive root system and without callus proliferation
at the base. The root system developed in these conditions was similar to the natural
root system of S. grosvenorii (Yan et al. 2010).
Furthermore, S. grosvenorii temporary immersion system culture was also stud-
ied (Yan et al. 2010). The best physiological parameters were obtained on MS
medium supplemented with 0.444 μM 6-BAP, 0.0537 μM NAA, and 3% (w/v)
sucrose. The explants were immersed for 4 min every 4 h. As a result, the shoots
cultured in temporary immersion systems showed a higher length, weight, and total
biomass compared to cultures on semisolid or in liquid media. In the same way, the
proliferation was higher under these culture conditions. No hyperhydricity or mor-
phological changes were observed between these plants and those grown on liquid or
semisolid medium. Additionally, the culture in the temporary immersion system also
reduces callus proliferation at the base of shoots (Yan et al. 2010).
S. grosvenorii have been also successfully regenerated by direct organogenesis.
Several explant sources such as leaf, petiole and stem were probed for shoot
induction (Lu et al. 2011). The authors referred to a high frequency of regeneration
observed using MS medium supplemented with TDZ, 6-BAP, and IAA. In contrast,
when using MS medium supplemented with TDZ or combined with IAA a moderate
shoot initiation was achieved. Besides, using a combination of BA and IAA
produces a very low or no shoot induction. The highest shoot regeneration frequency
and the number of shoots per explant were 100% and 15.3, respectively. These
values were reached when using MS medium supplemented with 2.275 μM TDZ,
8.88 μM BA, and IAA 2.875 μM. While the best shoot induction efficiency was
obtained when using petiole and stem with 0.91 μM TDZ, 8.88 μM 6-BAP, and
2.855 μM IAA. The shoot regeneration efficiency was higher with leaf than with
petiole or stem explants (Lu et al. 2011).
7.10.4.2 Other Techniques

Other interesting studies have been conducted in this species leading to new knowl-
edge that might be applied for genetic improvement, optimization of in vitro culture
and secondary metabolite production. Some examples are the assessment of genetic
diversity of cultivars and wild populations of S. grosvenorii (Tang et al. 2007), the
use of RNA-seq and digital gene expression profile analysis to identify candidate
genes encoding enzymes responsible for the biosynthesis of novel secondary
metabolites (Tang et al. 2011), and the analysis of gene expression for the production
of secondary metabolites (Chen et al. 2011; Wu et al. 2014; Zhao et al. 2018; Qiao
et al. 2019). Moreover, the cloning and expression of genes (Zhao et al. 2017) and
elicitation studies by the exogenous application of different compounds like methyl
jasmonate (Zhang et al. 2016) and forchlorfenuron (Shi et al. 2019) in fruits have
been performed. Besides, some genomic studies have been conducted (Xia et al.
2018) and the complete chloroplast genome sequence of S. grosvenorii has been
determined (Zhu et al. 2019).
7.11 Conclusions and Future Perspectives
Natural high-intensity sweeteners derived from plants are a feasible alternative for
reducing sugar intake worldwide without renouncing a sugary flavour. The potent
sweet taste compounds produced by S. rebaudiana, C. paliurus, G. glabra, and
S. grosvenorii are non-caloric, non-cariogenic and thermostable sucrose substitutes.
These molecules are commonly obtained from licorice-roots, Siraitia-fruits and
leaves from stevia or C. paliurus. The extracts of these plant sections are also
responsible for multiple therapeutic properties including a marked anti-
hyperglycemic action. Moreover, scientific evidence of this anti-diabetic effect has
been confirmed in compounds also responsible for the sweet taste such as steviol
glycosides, glycyrrhizin, and mogrosides. Therefore, these plants are highly valuable
for the food and pharmaceutical industries as nutraceuticals.
However, plant propagation through conventional techniques is constrained.
Furthermore, S. grosvenorii and C. paliurus are local species with severe restrictions
for multiplication or categorized as endangered plants. Hence, in vitro culture
techniques have been developed as a promising alternative to reduce the overexploi-
tation of natural populations. Several investigations have been performed for mass
propagation and secondary metabolite production in these four species. Stevia and
licorice have been more widely studied than C. paliurus and S. grosvenorii. More-
over, in vitro culture is not only useful for plant propagation; new strategies for the
conservation of these species might be achieved without any environmental or
seasonal limitations. Besides, plant regeneration protocols should be assessed to
confirm the genetic stability, phenotypical features, and metabolic profile of the
clones.
In vitro culture studies combined with other biotechnological approaches such as
elicitation and genetic transformation has been extensively applied in S. rebaudiana.
Amongst others, this has led to new insights in steviol glycoside biosynthesis and its
regulation. Similarly, but to a lesser extent, reports of the application of these
techniques have been published for G. glabra. In contrast, there is a lot to be
investigated regarding the stress response, genetic modification, and secondary
metabolite production of C. paliurus and S. grosvenorii.
The combination of these and other biotechnological tools would allow develop-
ing commercial strategies for obtaining high-yield and uniform sweeteners from
these species in a sustainable way. These sweet-tasting compounds will be used to
treat Diabetes mellitus and to revert the complications associated with this disease.
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In Vitro Culture Techniques and Metabolite
Engineering for Enhanced Antidiabetic 8
Secondary Metabolite Production
Israt Jahan , Ahmet Onay , Mahmut Ozan Toksoy , and

Sedat Kaya
Abstract
Diabetes is characterized by hyperglycemia, a serious chronic disease, which has

become an important public health problem worldwide and affecting human
health severely. These days, the use of modern drugs for the control of glycemia
is causing many negative side effects, and therefore, the demand for safer and
more affordable treatments is increasing day by day. Most of the antidiabetic
plants contain phyto-components, capable of exhibiting strong antihyperglycemic
activity. Studies have reported that alkaloids interact with proteins involved in
glucose homeostasis and can be used as an alternative in the treatment of diabetes.
Based on the previously published studies, this chapter mentioned and discussed
various significant antidiabetic plant species with their active ingredients. Fur-
thermore, biotechnological methods were reviewed in details that were used for
increasing the production of antidiabetic bioactive compounds using these plants.
Moreover, these biotechnological methods can be categorized into two groups:
(1) plant cell, tissue, and organ cultures (callus, cell and root cultures, suspension
I. Jahan
Department of Molecular Biology and Genetics, Faculty of Science, Dicle University, Diyarbakır,
Turkey
A. Onay (*)
Department of Biology, Faculty of Science, Dicle University, Diyarbakır, Turkey
e-mail: ahmeto@dicle.edu.tr
M. O. Toksoy
Department of Pharmaceutical Technology, Faculty of Pharmacy, Dicle University, Diyarbakir,
Turkey
S. Kaya
Department of Organic Farming, Vocational School of Technical Sciences, University of Muş,
Muş, Turkey
e-mail: s.kaya@alparslan.edu.tr
Ltd. 2021
https://doi.org/10.1007/978-981-16-3529-8_8
212 I. Jahan et al.
cultures, and single-cell culture lines) and (2) metabolic engineering (-omics
analysis, gene-editing techniques, and synthetic biology). The outcomes of pre-
vious studies discussed in this chapter suggested that biotechnological methods
could be potential, profitable, and novel for large-scale production of effective
active ingredients from several plant species for the purpose of diabetes mellitus.
Keywords
Biotic/abiotic elicitors · Bioreactor · Cell suspension culture · Diabetes mellitus ·
Hairy root culture · Temporary immersion systems · Transcription factors
8.1 Introduction
Diabetes mellitus (DM) is a serious chronic metabolic disorder characterized by a

state of hyperglycemia, i.e., a condition of high blood sugar level due to decreased or
absent insulin activity or malfunction in insulin secretion or both (Ota and Ulrih
2017). Beta cells of the pancreatic islets produce insulin into the blood in response to
high level of glucose (Koeslag et al. 2003). It is the key anabolic hormone that
reduces the blood sugar level by stimulating the absorption of glucose from the
blood, and helping it to deposit into liver, fat cells, and different adipose tissue in the
form of glycogen or fats (Sonksen and Sonksen 2000). Increased appetite and thirst
and/or frequent urination are the common symptoms of diabetics (Sonksen and
Sonksen 2000). In addition, increased sugar level in the blood or chronic hypergly-
cemia for a long time is the general reason of uncontrolled diabetes which can cause
many complications such as dysfunction and even malfunction of various organs,
damages in the blood vessels heart and cardiovascular disease, damage to the eyes,
damage to the nerves, chronic kidney disease and damages in kidneys, foot ulcers,
cognitive impairment, and stroke (Kitabchi et al. 2009; Saedi et al. 2016). DM can be
categorized into two groups: (1) type 1 diabetes or juvenile or childhood-onset
diabetes or insulin-dependent diabetes that occurs when pancreatic islets fail to
secret adequate amount of insulin due to the destruction of beta cells by an autoim-
mune reaction (Sonksen and Sonksen 2000) and (2) adult-onset diabetes or type
2 diabetes mellitus (T2D) or noninsulin-dependent diabetics is the condition where
cells fail to respond to insulin properly due to insulin resistance (Pasquel and
Umpierrez 2014). T2D, which accounts for around 90% of DM cases all over the
world, mainly develops as a result of insufficient physical activity, poor diet,
overweight or obesity, genetic factors, hypertension, etc. (Chehade et al. 2013).
In 2020, the World Health Organization estimates that about 422 million people
worldwide have diabetes, and 1.6 million deaths each year are directly linked to
diabetes, many of whom live in low- and middle-income countries (WHO 2020). At
the moment, existing treatment for type 1 diabetes is the direct intake of dose-
dependent insulin, whereas for T2D various monotherapic or combined oral
antidiabetic agents, like dipeptidyl peptidase IV inhibitor, thiazolidinediones,
meglitinides, sulfonylureas, biguanides, and α-glucosidase inhibitors, are very
8 In Vitro Culture Techniques and Metabolite Engineering for Enhanced. . . 213
popular to achieve better glycemic regulation (Pozzilli et al. 2014). In the early
twentieth century, the only insulin available for the treatment of diabetes was the
pure type of insulin of animal origin. Afterward, in 1978, the first synthetic human
insulin was invented from Escherichia coli via genetic engineering, and both E. coli
and yeast, especially Saccharomyces cerevisiae, are used to synthesize biosynthetic
recombinant human insulin or its analogs for worldwide usages (Johnson 1983).
More recently, however, much cheaper insulin production has been achieved by
another approach (biofarming) that introduces the human insulin gene into safflower
which can reduce the production cost (Moon et al. 2019). On the other side, many
available synthetic oral antidiabetic agents have serious side effects such as hypo-
glycemia, lactic acid intoxication, and gastrointestinal upset occur (Pozzilli et al.
2014). To control diabetes without any side effects therefore, different types of
similar antidiabetic medication can also be obtained from several sources, especially
from medicinal plants (Ota and Ulrih 2017).
Medicinal plants have been used all over the world for therapeutic purposes since
ancient times (Anand et al. 2019). The pharmacological properties of plants are
based on their phytochemical components, especially alkaloids, terpenes, phenolic
compounds, steroids quinones, phenylpropanoids, rare amino acids, plant amines,
glycosides, etc., which are the outstanding sources of significant bioactive
compounds (Grover et al. 2002). Plants produce a large, diverse array of secondary
metabolites from the metabolic pathways that derived from the primary metabolic
systems, i.e., photosynthesis, glycolysis, and the Krebs cycle by modifying primary
metabolite synthases (Chandran et al. 2020). These secondary metabolic pathways
are known as mevalonic acid (MVA), shikimic acid, acetyl coenzyme A,
deoxyxyllulose 5-phosphate, or combined pathways (Fig. 8.1) (Thirumurugan
et al. 2018).
Plants accumulate these secondary metabolites with different structures and
functions necessary for plant defense mechanisms. But these bioactive compounds
also exhibit some biological activity and protective functions in terms of human
health (Thirumurugan et al. 2018). These compounds can be directly extracted from
plants as the source of potential raw materials for various scientific, technological,
and commercial applications, especially in medicine, industry, and agricultural
sectors (Thirumurugan et al. 2018). Besides, these chemical compounds are rich in
natural antioxidants (vitamins C and E, flavonoids, tannins, etc.) and can also
demonstrate their antidiabetic activity by prohibiting diabetes-induced reactive
oxygen species (ROS) formation (Sahu et al. 2015). Nevertheless, the extraction,
identification, and purification of hypoglycemic compounds from medicinal plants
by conventional methods are not economically attractive, so the search for more
effective and safer hypoglycemic agents is still in the dark and needs to be explored
for research purposes. Therefore, this chapter was aimed to provide an updated
overview of the important plant metabolites with antidiabetic agents for the treat-
ment of diabetes and their production by biotechnological approaches.
In this chapter, after briefly mentioning the information of traditional drugs and
currently available therapeutic methods in the treatment of DM, current biotechno-
logical techniques for the production of antidiabetic compounds using some plant
214 I. Jahan et al.
Fig. 8.1 Basic biosynthetic pathways leading native plant secondary metabolites (Ghasemzadeh
and Jaafar 2011)
species possessing active antidiabetic ingredients were reviewed based on previ-

ously published literatures. The phytochemical constituents of thousands of well-
known plant species with antidiabetic potential and their experimental results as well
as methods of commercially producing phytochemicals for treating diabetic are
reported by ethno-botanical studies (Anand et al. 2019; Ota and Ulrih 2017; Sahu
et al. 2015). Information of antidiabetic metabolite production through plant, cell,
tissue and organ cultures and metabolite engineering, and the impacts of these
biotechnological methods for the production of phytochemicals have been derived
from hundreds of published articles. Brief interpretations of some of these compre-
hensive reviews were presented here for interpreting the significance of these
biotechnological methods for commercial applications. Antidiabetic secondary
metabolites produced from different plant species through in vitro plant tissue
culture methods were listed in Table 8.1. Metabolite engineering studies for the
production of some antidiabetic secondary metabolites by microorganisms were also
represented in Table 8.5. Keywords such as “diabetes mellitus,” “antidiabetic
plants,” “hypoglycemic plants,” “organ culture,” “callus culture,” “suspension cul-
ture,” “use of in Bioreactors,” “optimization approaches to increase metabolite
production,” “metabolic engineering,” etc. were used in different databases such as
Science Direct, PubMed, Springer, Scopus, and Web of Science to search the most
suitable and relevant research works. Experimental studies and clinical studies for
8
Table 8.1 Partial list of plants and their constituents having insulin mimetic activity and their production via plant cell, tissue, and organ cultures
Plant species Active ingredient Culture medium and growth regulator(s) Culture type References
Adhatoda vasika Vasicine, vasicinone Murashige and Skoog medium (MS) + 2.2 μM Shoot Dinesh and
6-benzyladenin (BA) + 10.7 μM indole-3- Parameswaran
acetic acid (IAA) (2009)
Allium cepa Quercetin MS + 1 mg/L 2,4-D + 200 μM methyl Callus Iqbal et al. (2019)
jasmonate (Me-JA)
Allium sativum Allin, alliinlyase MS + 11.4 μM IAA, 10.8 μM 1-naphthalene Callus Malpathak and
acetic acid) (NAA) + 9.3 μM David (1986)
N6-furfuryladenine (kinetin)
Aloe saponaria Glucosides MS + 2,4-dichlorophenoxyacetic acid (2,4-D) Cell culture Yagi et al. (1983)
+ kinetin
Arachis hypogaea L. Resveratrol Hormone-free MS 200 mg/L timentin Hairy root Kim et al. (2008)
Artemisia annua Artemisinin MS + 0.1 mg/L of each of NAA and kinetin Cell suspension Baldi and Dixit
(2008)
Artemisia vulgaris Inulin Hormone-free half-strength MS Hairy root Drobot et al.
and Artemisia (2017)
dracunculus
Aspidosperma Ramiflorine A and Ramiflorine MS + 1 mg/L 2,4-D Callus Oliveira et al.
ramiflorum Muell. B (2001)
Arg.
Azadirachta indica Azadirachtin MS + 0.2 mg/L BAP + 0.2 mg/L kinetin; callus Suspension Sujanya et al.
on MS + 1 mg/L NAA + 3 mg/L BAP (2008)
Beta vulgaris L. Betanin, rutin Linsmeier–Skoog medium containing Suspension Križnik and
In Vitro Culture Techniques and Metabolite Engineering for Enhanced. . .
ammonium to nitrate ions (1:14) Pavoković

(2010)
Brucea javanica Cathin MS +1 mg/L 2,4-D, 1 mg/L NAA, 0.1 mg/L Suspension Wagih et al.
kinetin and 10 mg/L tryptophan (2008)
Camellia sinensis Epigallocatechin gallate and MS + 2 mg/L 2,4-D + 1 mg/L kinetin + 1 mg/L Callus Chevala et al.
other polyphenols BAP (2016)
215
(continued)
216
Camellia sinensis Flavan-3-ol MS + 3 mg/L NAA + 2.0 mg/L kinetin Callus and suspension Sutini et al.
(2017)
Capsicum annuum Capsiacin MS + 4.52 μM 2,4-D + 0.44 μM BA Callus Santos and Souza
(2018)
Catharanthus roseus Catharanthine and vindoline MS+ 2 mg/L NAA, 0.2 mg/L kinetin + Suspension Ramani and
irradiated with UV-B Jayabaskaran
(2008)
Cayratia trifolia (L.) Stilbenes MS+ 0.25 mg/L 1 NAA + 0.2 mg/L kinetin Suspension Roat and
Domin Ramawat (2009)
Cichorium endivia L. Phenolic and flavonoids MS + NAA + IAA Adventitious root Ibrahim et al.
(2019)
Citrus aurantium Naringin Murashige and tucker medium 6 μM kinetin + Callus Del Río et al.
3 μM 2,4-D (1992)
Coffea arabica L. Caffeine MS + 13.6 μM 2,4-D + 4.4 μM 6-BAP Suspension Pech-Kú et al.
(2018)
Coscinium Berberine MS + 0.5 to 2 mg/L 2,4-D + 0.5 to 2 mg/L BAP Callus Khan et al.
fenustratum L. (2008)
Curcuma longa Kaempferol and curcumin Zenk suspension media + 5 mg/100 ml of Immobilized Chaturvedi et al.
cinnamic acid suspension (2014)
Eclipta alba L. Coumarin, eclalbatin, MS + 3 mg/L BAP and 1 mg/L NAA Callus Khurshid et al.
wedelolactone, and (2020)
demethylwedelolactone
Ephedra distachya L-Ephedrine MS + 0.25 μM kinetin + 5.0 μM 2, 4-D or Callus O’Dowd et al.
1-NAA (1993)
Eriobotrya japonica Tormentic acid MS + 2.5 mg/L 6-BA, 1 mg/L NAA Suspension Ho et al. (2010)
Eryngium alpinum L. Phenolic acids and flavonoids MS medium with BAP, IAA, and GA3 (each Shoot Kikowska et al.
1.0 mg/L) (2020)
I. Jahan et al.
8
Gentiana clusii Perr. Secoiridoid and xanthone WPM + 0.5 mg/L kinetin; ½ MS + 1. mg/L Shoot and root Krstić-Milošević
and Song IBA et al. (2020)
Gentianella austriaca Xanthone MS + 2.22 μM BA + 0.54 μM NAA Multiple shoot Vinterhalter et al.
(2008)
Glycyrrhiza glabra Glycyrrhizin (glycoside) MS + 0.1 mg/L IAA Hairy root Mehrotra et al.
(2008)
Glycyrrhiza glabra L. Cinnarnaldehyde MS+ 1 mg/L 2,4-D + 0.2 mg/L, kinetin + Suspension Shams-Ardakani
1 mg/L NAA et al. (2005)
Gymnema sylvestre Gymnemic acids and Liquid MS+ 5 μM linolenic acid Hairy roots Praveen et al.
gymnemagenin (triterpenoid (2014)
glycosides)
Hemidesmus indicus Rutin, quercetin MS+ 2 μM IAA Shoot Pathak et al.
(L.) (2020)
Momordica charantia Charantin Hormone-free MS Hairy roots Thiruvengadam
et al. (2014)
Momordica charantia Phenolic compounds Liquid MS + Agrobacterium rhizogenes strains Hairy roots Thiruvengadam
LK. et al. (2014)
Morus nigra (black Quercetin, rutin, morusin, and MS + 2 mg/L NAA + 0.2 mg/L kinetin Suspension ABD El-Mawla
mulberry) cyclomorusin et al. (2011)
Olea europaea L. Oleanolic acid Liquid MS Cell cultures Saimaru et al.
(2007)
Panax ginseng Saponin MS + 2,4-D + mevalonic acid Callus culture Furuya et al.
(1983)
Panax ginseng Ginsenoside MS + 0.1 mg/L kinetin + 2 mg/L 2,4-D + Callus culture Bonfill et al.
In Vitro Culture Techniques and Metabolite Engineering for Enhanced. . .
9.98 mM IBA + 10.74 NAA (2002)

Panax ginseng Ginseng, saponin (glycoside) 1/2 MS medium + selenium Hairy root Jeong and Park
(2006)
Pluchea lanceolata Quercetin MS + 1.0 mg/L BAP + 1.0 mg/L kinetin Callus Arya et al. (2008)
(continued)
217
218
Psoralea drupacea Bakuchiol MS+ 1 mg/L 2,4-D + 1 mg/L BA Callus, cell Lystvan et al.
Bge suspensions, and (2010)
transgenic hairy root
Psoralea drupacea Bakuchiol MS+ 1 mg/L BA + Me-JA The aerial parts of Lystvan et al.
Bge P. drupacea grown (2010)
in vitro
Rheum emodi Wall. Emodin MS + 5.0 μM NAA + 10.0 μM BAP Callus Singh and
Chaturvedi
(2019)
Salvia leriifolia The rosmarinic acid, caffeic MS + 5% polyethylene glycol (PEG) 6000 Embryo and callus Hosseini et al.
acid, and salvianolic acid B (2020)
Swertia paniculata Xanthone glycosides and 1/2 MS + 2.22 mM each of BA and kinetin + Shoot Kaur et al. (2020)
secoiridoid 2.54 mM NAA
Teucrium polium L. Apigenin MS + BAP + kinetin + NAA + 2,4-D Callus Meguellati et al.
(2019)
Tinospora cordifolia Berberin Linsmaier and Skoog's medium Suspension Rao et al. (2008)
Miers
Trigonella foenum- Trigonelline MS + Me-J + chitosan Hairy root Qaderi et al.
graecum (2016)
Urena lobata Flavonoids and phenolics WPM + 10 mM phenylalanine (PA) or 50 mM Hairy root Cao et al. (2020)
chitosan
Zingiber Gingeroland zingiberene MS + 1.0 to 3.5 mg/L 2,4-D + 0.2 mg/L kinetin Callus Anasori and
officinale Rosc. Asghari (2009)
I. Jahan et al.
evaluating the antidiabetic effects of these bioactive component on patients or

diabetic-induced animals were included in this study.
8.2 Plants with Antidiabetic Potentials
Medicinal plants or medicinal herbs have been exploited as traditional curative

agents since ancient times. This is because of least side effects, raw consumption,
inexpensive sources, and easy accessibility of medicinal herbs which lead their
widespread use as available therapies in traditional medicine. Due to the side effects
of synthetic drugs and the health benefits of natural herbal remedies in terms of
safety and cost, the intention of using medicinal plants as therapeutic drugs has
increased considerably in recent years (Anand et al. 2019). Therefore nowadays,
large-scale production of herbal medicine has become one of the fastest growing
industries worldwide. The global pharmaceutical market is about US $1.1 trillion
from which about 35% of the drugs are approached from natural-based raw materials
(Calixto 2019). According to the 2017 record of “World Health Organization
(WHO) 2019 global traditional and complementary medicine report,” more than
60,000 traditional herbal medicines at commercial level were permitted in China
alone, and permission with production licenses was provided to 4424 pharmaceuti-
cal enterprises (Calixto 2019).
Plants produce hundreds of secondary metabolites for defending themselves, i.e.,
self-defense mechanisms from pathogenic microbes, pests, and herbivorous
mammals, adapting to different stress conditions, and the sustainability of generation
(Grover et al. 2002). These chemical compounds are rich in natural antioxidants
(vitamins C and E, flavonoids, tannins, etc.), and many of these phytocompounds are
capable of retaining ß-cell activities and, thus, can prohibit diabetes-induced ROS
formation (Sahu et al. 2015). Moreover, these phytocompounds can also demon-
strate their antidiabetic activity by repairing pancreatic beta cells, stimulating insulin
secretion, reducing insulin resistance or inhibiting insulin degradative processes, and
improving digestion system that could reduce blood sugar (Sahu et al. 2015). So far,
almost 800 plant species from different families such as Moraceae, Lamiaceae,
Fabaceae, Euphorbiaceae, Asteraceae, etc. were found to exhibit antidiabetic
potentials (Patel et al. 2012). For instance, leaves and other plant parts of Momordica
charantia L. are able to control the plasma glucose levels dramatically by inhabiting
α-glucosidase activity and restraining postprandial hyperglycemia (Uebanso et al.
2007).
Flavonoids glycosides, i.e., isostrictinin, pedunculagin, and strictinin from
Psidium guajava, have the capacity to improve insulin sensitivity (Chauhan et al.
2010). Seed-crude protein and rhizome extract of Nelumbo nucifera Gaertn are
capable of reducing blood sugar level significantly, which have been used in
traditional Chinese medicine for a long time to treat diabetes (Lee et al. 2001).
Moreover, bioactive compounds containing extract of Anisodus tanguticus, and
Agrimonia pilosa Ledeb. (Weilin et al. 2002), alkaloid (vindolinine, vleurosine,
and vindoline) containing leaf extract of Catharanthus roseus (Chattopadhyay
220 I. Jahan et al.
1999), triterpenes (geranic acid, geranyl acetate, citral, squalene, terpineol, sesqui-
terpene), tannins (tannic acid, hydrolysable, and condensed varieties of tannins),
flavonoids (rutin, epicatechin, quercetin), phenolic compounds (proanthocyanidin,
catechin, epigallocatechin, epigallocatechin-3-O-gallate, epicatechin, epicatechin-3-
O-gallate) from Eucalyptus citriodora (Patra et al. 2009), mineral composition, i.e.,
manganese of Eucalyptus globules (Pinto et al. 2007), root extract of Andrographis
paniculata (Rao 2006), and flavonoids (30 -formyl-40 ,60 ,4-trihydroxy-20 -methoxy-5-
0
-methylchalcone, 30 -formyl-60 ,4-dihydroxy-20 -methoxy-50 -methylchalcone 40 -O-
beta-D-glucopyranoside, (2S)-8-formyl-6-methylnaringenin, and (2S)-8-formyl-6-
methylnaringenin 7-O-beta-D-glucopyranoside) from flower buds of Cleistocalyx
operculatus (Tuyet et al. 2013) proved to be extremely effective to decrease blood
glucose levels.
On the other hand, gallic acids, chlorogenicacids, quercetins, and rutins from
Campomanesia xanthocarpa can reduce liver glycogen (12–20% reduction compare
to the control) which helps to suppress blood glucose and glycosylated hemoglobin
A1c (HbA1c) levels (Cardozo et al. 2018). It was observed that phenolic compounds
(rutin, gallic acid, and ellagic acid) and tannins (hydrolysable) from the leaf extract
of Eugenia uniflora can reduce lipid peroxidation, sustain hepatic glutathione, retain
insulin levels, and decrease inflammation of pancreatic islets (Schumacher et al.
2015). Furthermore, Solanum xanthocarpum extract was observed to be capable of
increasing serum insulin level (8 times more than the control) and reducing uric acid,
urea, creatinine, and glucose levels in blood, i.e., 200 mg/kg extraction concentration
could decrease 25.77% blood glucose level in glucose-loaded hyperglycaemic rats
(Sahu et al. 2015). S-methyl cysteine sulfoxide, the precursor of garlic oil and allicin,
was found to be the most effective antidiabetic and antihyperlipidemic component
available in garlic (Allium sativum L.) and onion (Allium cepa L.) bulbs (Sheela et al.
1995). Ursolic acid and oleanolic acid-containing pulp extract of Cornus officinalis
is capable of enhancing the expression of glucose transporter type 4 (GLUT4)
mRNA which helps to persuade the production of pancreatic islets, which increase
the speed of glucose transport by raising insulin secretion, and thus, shows
antidiabetic activity (Weilin et al. 2002). Furthermore, Several studies confirmed
that aegeline 2, i.e., an alkaloidal amide from Aegle marmelos leaf extract (Narender
et al. 2007); ephedrans A, B, C, D, E, and glycans from Ephedra distachya (Xiu et al.
2001); gymnemosides b and gymnemic acids III, IV, V, VII from Gymnema
sylvestre (Miyatake et al. 1994); and triterpene dehydrotrametenolic acid secluded
from desiccated and hardened mycelia (sclerotia) of Poria cocos mushroom (Sato
et al. 2002) have the hyperglycemic potentials as insulin sensitizer.
8.3 Production of Secondary Metabolites
Plants are the great source of raw materials for most therapeutic drugs vital for
human health. These therapeutics are a wide variety of natural compounds known as
primary and secondary metabolites, i.e., amino acids, peptidoglycans,
polysaccharides, carbohydrates, glycopeptides, hypoglycans, inorganic ions,
glycosides, guanidine, terpenoids steroids, alkaloids, galactomannan, gum, etc.

(Grover et al. 2002). In the previous section, several plants are mentioned capable
of producing antidiabetic metabolites; however, the large-scale production of these
metabolites requires an incomprehensible collection of the plant materials from the
nature, which can cause severe endangerment of many species. Therefore, it is
necessary to adopt other approaches for confirming a secure and efficient production
of secondary metabolites at commercial level. In this context, biotechnological
techniques could be the potential development to overcome the problems of
extracting natural products from plants. Applications of these techniques not only
produce these pharmacologically active antidiabetic compounds but also offer new
opportunities to increase their quantities. Moreover, different biotechnological
techniques for producing antidiabetic metabolites can be categorized into two
groups: (1) plant tissue cell, tissue, and organ cultures (cell, callus, and shoot or
root cultures and suspension cultures and single-cell culture lines); and (2) metabolic
engineering (omics analysis, gene-editing techniques, and synthetic biology).
8.4 Plant Cell, Tissue and Organ Cultures, and Secondary

Metabolite(s) Production
Plant metabolites are usually isolated from naturally growing plant materials using
different traditionally available extraction methods; however, the commercial pro-
duction of these metabolites could be limited in those areas where cultivation is
carried out under natural conditions due to environmental constrictions such as
extreme climate conditions, high or low precipitation and temperature, and some-
times, existence of genetic variability for some plant species as a result of cross-
pollination (Anand et al. 2019). In addition, conventional methods for plant
metabolites isolation are often time-consuming since some plant species take several
years to grow for reaching the required developing stage for desirable metabolite
productions. Hence, an alternative method of surpassing these challenges is to
produce these desired commercially metabolites through optimized plant cell, tissue,
and organ cultures of pharmaceutically significant plant species in controlled envi-
ronmental conditions without any seasonal constraints (Hussain et al. 2012a, b).
Plant cell, tissue, and organ cultures are the collection of in vitro procedures that
are exploited for the development of identical entire plants or organs, tissues, and
cells on a nutrient culture medium of known composition under sterile and optimized
physical and environmental conditions (Hussain et al. 2012a, b). Controlled
conditions during these processes help to provide a favorable environment for an
ideal growth and proliferation of explants (viable plant parts used for initiating the
growth in culture medium) (Onay et al. 2019). The basis of plant tissue culture
studies that initiated by a German physiologist, Gottlieb Haberland, in the early
twentieth century (Haberlandt 1902) and the successes achieved today using these
techniques at basic and applied levels have reached beyond his imaginations. In
recent years, plant tissue culture techniques have been extensively utilized all over
the world, especially in the major industrial areas for the purpose of plant
222 I. Jahan et al.
Fig. 8.2 Schematic diagram of some existing methods used for the large-scale production of
bioactive compounds using in vitro plant tissue culture techniques (Lu et al. 2016)
propagation and multiplication, micrografting, rapid proliferation of rare plant

genotypes, plant improvement, plant-derived secondary metabolites production,
cryopreservation of plants, disease elimination and production of disease-free plants,
plant genome transformation, and large-scale production in bioreactors of plant-
derived metabolites (Fig. 8.2) (Espinosa-Leal et al. 2018).
Furthermore, finding the alternative approaches for producing therapeutically
important phytocompounds, plant cell, tissue, and organ culture are found to com-
prise great opportunities for an efficient production of secondary metabolites with
significant commercial values (Hussain et al. 2012a, b). Therefore, a group of plant
researchers all over the world have been trying to explore biosynthetic capacities of
various plant-based cell cultures. Using plant tissue culture, scientists are focusing
mainly on identifying biosynthetically active cultured cells in order to gain high
production of secondary metabolites for profitable exploitation, which can be
obtained by screening high-producing strains, standardizing the cultural conditions,
improving transformation, and immobilization techniques (DiCosmo and Misawa
1995). In a comparison with in vivo medicinal herbs for producing valuable plant
metabolites, plant cell culture also possesses a series of distinctive benefits (Hussain
et al. 2012a, b) as follows:
• Plant metabolites fabrication is more predictable, facile, and more consistent due
to controlled physical and environmental conditions.
• Phytocompounds extraction from specific cultured tissues or cell clusters could
be quicker and more proficient than extraction from entire plants collected from
fields.
• Artificially added nutrients in the tissue culture medium influence the production
of significant and standard amount of phytocompounds in large volumes which
could be parallel to the amount from a whole plant.
• Unexpected and hampering compounds can be eliminated from the cell cultures
using different techniques and modifications, which is relatively difficult for
in vivo plants.
• For testing elicitation for increasing metabolites synthesis, plant tissue, and cell
cultures are considered as an impending and prospective model.
• Phytocompounds accumulated from in vitro tissue cultures are able to be
radiolabeled easily, and hence, they can be monitored and detected metabolically
while applied for further laboratory-based utilization such as feed to laboratory
animals for applied research.
Secondary metabolites can be produced by following a two-step in vitro process:

(1) accumulation of biogenic materials or biomass, and (2) fabrication or synthesis of
secondary metabolites (Murthy et al. 2016; Yue et al. 2016). In vitro
micropropagated plantlets or differentiated tissues or organs through organogenesis
such as roots, shoots, and embryos or undifferentiated cells such as callus and cell
suspensions (initiated from single-cell lines) from regenerable explants, i.e., plant
roots, stems, leaves, and meristems, can be utilized for secondary metabolites
production (Atanasov et al. 2015; Morales-Rubio et al. 2016). The quantity and
quality of metabolites can vary based on the methods used for plant tissue culture,
such as high hypericin content was found in differentiated leaves of Hypericum
triquetrifolium Turra, while undifferentiated callus and suspension cells lack the
ability to produce hypericin or are found in traceable amounts (Karakaş et al. 2015).
8.4.1 Secondary Metabolite Production from Differentiated Tissues

(Shoot, Root, Adventitious Root, and Embryo Cultures)
For the production of the desirable metabolites, selection of suitable explants is the
prerequisite condition since plant secondary metabolites are tissue-specific like some
metabolites are produced in the roots, some in the stem or shoots, and some in the
embryo (Karuppusamy 2009).
8.4.1.1 Root Cultures

In this culture, radical tip of the germinated seeds or lateral tip containing roots is
chosen and transferred aseptically into a liquid medium and let them grow autono-
mously; the main axes of the growing roots in the medium are then cut into small
pieces (each bearing four or five young laterals) and transferred for subculturing
(Steffens and Rasmussen 2016). Moreover, adventitious roots are the special type of
roots that grow from nonroot tissues like nodal area (Steffens and Rasmussen 2016).
In tissue culture, adventitious root can be produced by stimulating different plant
tissues such as leaf, nodes, or stems (Inabuy et al. 2017). Organized cultures,
especially all methods for various root cultures, are capable of making a substantial
224 I. Jahan et al.
role in the production of some specific metabolites that mainly synthesized in the
roots, and therefore, these methods are chosen for producing selective
phytocompounds from some plant.
Several studies have already been reported the use of root cultures for antidiabetic
metabolites production from different medicinal plants. For instance, the root of
Panax ginseng contains many bioactive compounds, including saponin, triterpene
glycosides, ginsenosides, panaxans, vanillic acid, and salicylates that possess strong
antidiabetic potentials; hence, many researchers have been trying to increase the
quantity and quality of these metabolites through the root culture of P. ginseng
(Jeong and Park 2006; Ratan et al. 2020). Similarly, root culture of Stevia
rebaudiana (Kazmi et al. 2019), Salvia miltiorrhiza (Dreger et al. 2010), and
Rubia tinctorium (Biçer et al. 2017) showed significant possibilities of antidiabetic
secondary metabolites production. Adventitious root cultures of S. rebaudiana
cultured in MS medium supplemented with methyl jasmonate (Me-Ja),
phenylvacetic acid, and melatonine (Mel) have been noted to produce higher levels
of stevioside compared to other cell cultures in response to Me-Ja (Kazmi et al.
2019). From the root cultures of S. miltiorrhiza, it has been determined that the
cultured roots contain two main active substance groups including phenolic
components, namely, rosmarinic acid, salvianolic acid, lithospermic acid, caffeic
acid (CA), protocatactic acid, abietane-type diterpenoids, etc. and Me-Ja as elicitor
increased the phenolic compound ratios (Dreger et al. 2010). In vitro root culture of
Rubia tinctorium was investigated for estimating secondary metabolite content
where MS medium was supplemented with Me-Ja and CA (Biçer et al. 2017). In
this study, Me-Ja slowed increased root growth compared to the control group, and
CA also effected root growth positively (Biçer et al. 2017). Moreover, 2 mM CA +
100 μM Me-Ja combination was reported to show maximum increased level of total
anthraquinones (AQ; 39.45 mg/g) in the roots (Biçer et al. 2017).
Different concentration of indole butyric acid (IBA) or naphthalene acetic acid
(NAA) were supplemented with half-strength MS nutrient medium for root prolifer-
ation of Cichorium endivia L. where reflective responses were observed on the
growth of adventitious roots under dark conditions with the highest concentration
of IBA (Ibrahim et al. 2019). A considerable amount of mass yield obtained from
these adventitious roots also contained high contents of antidiabetic activity
exhibiting phenolics and flavonoids (Ibrahim et al. 2019).
8.4.1.2 Shoot Cultures

Shoot tip cultures or shoot cultures are the methods used for initiating the terminal
portion of a shoot, which consists of meristem tissues with primordial leaves and
adjacent stem tissue (Georgiev et al. 2009). Similar to the root culture, shoot culture
can also be used directly for secondary metabolite production. Besides, regenerated
nodal buds and shoot tips can be retransferred into a special medium to initiate callus
and suspension cultures for secondary metabolite synthesis (Georgiev et al. 2009).
By inducing in vitro somaclonal variation, shoot cultures are often used to select
clonal lines to produce large amounts of secondary metabolites along with the aim of
preventing genetic erosion from the natural vegetation (Piątczak et al. 2015). In case
of a few medicinal plant species, secondary metabolite accumulation has been

achieved by the shoot cultures in a close or much greater amount than their natural
forms. Compared to the parent plants, shoot cultures of Swertia paniculata,
Eryngium alpinum L., and Digitalis purpurea L. were found to accumulate higher
amount of antidiabetic compounds such as bitter secoiridoid and xanthone
glycosides, several phenolics and flavonoids, and ecoiridoids and xanthone, respec-
tively (Kaur et al. 2020; Kikowska et al. 2020; Krstić-Milošević et al. 2020). A
strong antidiabetic compound named meroterpene bakuchiol was trying to synthe-
size aseptically using Psoralea drupacea Bge (Fabaceae) explants through in vitro
micropropagated method, transgenic hairy root cultures, diverse origin-based callus
cultures, and cell suspensions culture (Lystvan et al. 2010). However, only shoot tip
cultures were able to accrue significant amount of bakuchiol (up to 11% dry weight)
which could be considered as a prospective approach for producing this metabolite
(Lystvan et al. 2010). Moreover, addition of certain substances to the culture
medium also optimistically influences metabolite synthesis. It has been observed
that insulin production enhancing and blood glucose level reducing bioflavonoid,
such as rutin and quercetin synthesis, can be initiated in the shoot culture of
Hemidesmus indicus (L.) after adding yeast extract or salicylic acid in medium as
elicitors (Pathak et al. 2020).
Furthermore, AQ are basically polycyclic aromatic organic hydrocarbon
compounds, and more than 25 AQ were identified having promising antidiabetic
potentials since they are capable of exhibiting blood glucose-lowering ability
(Mohammed et al. 2020). An experiment was conducted to evaluate the effects of
various physical and chemical factors regarding the ability of AQ production in
shoot cultures of Frangula alnus Mill., Frangula rupestris (Scop.) Schur., and
Rhamnus catharticus L. (Kovačević and Grubišić 2005). It was observed that the
highest amount of anthraquinone (1731 mg/100 g of total AQ aglycones) was
obtained from F. alnus shoots cultured on MS medium supplemented with
0.1 mg/L NAA and 0.1 mg/L thidiazuron (Kovačević and Grubišić 2005). Besides,
the highest anthraquinone production (451 mg/100 g of total AQ aglycones) from
F. rupestris shoots was confirmed in a nutrient medium supplemented with
6-benzylaminapurine (0.5 mg/L; BAP) and 2,4-dichlorophenoxyacetic acid
(0.1 mg/L; 2,4-D) (Kovačević and Grubišić 2005). Moreover, R. catharticus shoots
on MS medium supplemented with 2,4-D (0.1 mg/L) accumulated the maximum
amount of AQ (1205 mg/100 g of total AQ aglycones) (Kovačević and Grubišić
2005).
8.4.1.3 Embryo Culture

It is a part of in vitro fertilization where embryos from seeds and ovules are used as
explant for cultivating on a nutrient medium under aseptic conditions (Swain and
Smith 2011). In this plant tissue culture process, the micropropagation can expand
either simply from the growth of embryos or indirectly through callus formation
which consequently converts into shoots and roots (Swain and Smith 2011). Long
dormancy period of a seed can be reduced effectively by embryo culture, which
helps to shorten the breeding cycle of a plant; therefore, this technique is frequently
226 I. Jahan et al.
utilized for haploid plants and rare species production, testing seed vitality, breaking
seed dormancy, and growing embryos that might terminated otherwise, like an
embryo developed from interspecific or intergeneric cross (Burun and Poyrazoglu
2002; Chioma Com et al. 2017; Kundu et al. 2017). Moreover, embryo culture can
also be used for the production of secondary metabolites for some plant species. In
this context, an alkaloid named neferin that is significant therapeutic secondary
metabolites and potential for inhibiting lung cancer was able to synthesize success-
fully with the help of embryo cultures of N. nucifera seed embryo (Bhatia 2015).
Roots, leaves, and other plant parts of Salvia lerifolia contain many unique
phenolic compounds such as rosmarinic acid, CA, salvianolic acids A–K, etc.,
which possess many medicinal properties including treating inflammation and
decreasing blood sugar (Modarres et al. 2014). From a study, it was observed that
the whole plantlets of S. lerrifollia derived from embryo culture medium
supplemented with very low concentration of polyethylene glycol (5% PEG) showed
an increased antioxidant activity followed by a significant increase in the production
of the rosmarinic acid, CA, and salvianolic acid B (Hosseini et al. 2020).
8.4.1.4 Hairy Root Culture

Sometimes a special type of hairy roots can be developed in the culture media with
genetic engineering called hairy root culture or transformed root culture potential for
the modification of secondary metabolites production (Sevón and Oksman-
Caldentey 2002). The hairy roots are basically developed by the infection of gram-
negative soil bacteria, Agrobacterium spp. in roots that cause hairy root disease in
plants (Shanks and Morgan 1999). Moreover, Agrobacterium-mediated infection in
tissue cultures is usually identified by observing genetic stability and high growth
rate in media with no supplementation of hormones. Hairy roots formation in the
medium can help to increase the quantity of root-specific metabolites by increasing
the biomass of roots (Hu and Du 2006). Distinct morphological and dynamic
features as well as high genetic stability of hairy roots permit their utilization for
genetic modification by transferring any foreign gene of interest after incorporating
with root-inducing plasmids, also called Ri plasmids through Agrobacterium
(Palazón et al. 2008). These genetically modified hairy root cultures are capable of
producing target-specific secondary metabolites, useful for producing recombinant
proteins and valuable secondary metabolites or studying plant metabolic processes
(Palazón et al. 2008). In the last two decades, Agrobacterium-mediated hairy root
culture for the production of root-specific secondary metabolites has become quite
popular (Pistelli et al. 2010).
Moreover, hairy root cultures are vital for achieving high secondary metabolites
production by optimizing nutrient content and adding elicitors in the medium
(Hu and Du 2006). The response of hairy roots of Artemisia annua to five main
hormones such as abscisic acid, gibberellins (GA), ethylene, cytokinins, and auxins
was studied by measuring growth, development, and production of antidiabetic
activity containing artemisinin compound (Weathers et al. 2005). It was observed
that 2-isopentenyladenine only induced the production of artemisinin production
which was more than two times of the B5 controls, even more than any other
hormone combinations. This experiment demonstrated the significance of hairy root

growth with hormone combination for increasing artemisinin production (Weathers
et al. 2005).
The roots and other plant parts of bitter gourd (M. charantia), well-known herbal
medicine, are exploited in Indian traditional medicine for treating diabetes with
hypoglycemic activity (Wang et al. 2014). In a human study, preliminary clinical
evidence was reported that M. charantia is beneficial on glycemic control and
potential systemic complications of T2D (Wang et al. 2014). A comparative study
of transgenic Agrobacterium rhizogenes-mediated hairy roots of Korean
M. charantia and Indian M. charantia was performed where the highest accumula-
tion of biomass and phenolic compounds (a total of 27) production for both type
plants were observed in Murashige and Skoog (1962) liquid medium supplemented
with 3% sucrose (Thiruvengadam et al. 2014). Compared to the untransformed
control roots, transgenic hairy roots produced increased amount of phenolic acids
(salicylic acid, gentisic acid, chlorogenic acid, and gallic acid) and flavonoids
(quercetin, myricetin, and catechin) (Thiruvengadam et al. 2014).
For the patients with DM, ginseng saponin (glycoside) containing red ginseng
(P. ginseng) root extracts are applied clinically for repairing intractable skin ulcers
(skin sores) (Kimura et al. 2006). In a study on the hairy root of P. ginseng, it was
observed that increased amount of ginseng saponin within a shorter time can be
initiated by applying suitable elicitors (Jeong and Park 2006). The results suggested
that the amount of ginseng saponin (glycoside) content was increased 1.31 and 1.15
times than the control levels within a short time period after adding 0.5 mM selenium
and 0.1% (w/v) sodium chloride in the culture medium as elicitors, respectively
(Jeong and Park 2006). Similarly, exposure of C. roseus hairy root cultures to
pectinase or jasmonic acid also resulted in an increased accumulation of terpenoid
indole alkaloid which was 150–370% higher compared with untreated controls
(Goklany et al. 2013; Narayani and Srivastava 2017).
Plant extracts of G. sylvestre (Asclepiadaceae) are traditionally used for the
treatment of diabetes in India which pharmacologically contain active triterpenoid
glycosides such as gymnemic acids and gymnemagenin (Ulbricht et al. 2011). By
interacting with taste receptors on the tongue, gymnemic acids restrain the taste of
sweetness for a short period of time, and therefore, known as sweetness inhibitors, or
antisweet compounds (Stoecklin 1969; Gent 1999). These sweetness inhibitors are
capable of reducing blood sugar level by decreasing the absorption of sugar
molecules in the intestinal gut since they can bind with the receptors on the intestinal
walls, and thereby, influence insulin secretion (Tiwari et al. 2014). Through hairy
root cultures of G. sylvestre, a study demonstrated that as elicitor, different polyun-
saturated fatty acids such as linolenic acid at 5 μM were able to increase gymnemic
acid content (87.99 0.53 mg/g DW) which was 7.78-fold higher than the control
(Praveen et al. 2014).
228 I. Jahan et al.
8.4.2 Secondary Metabolite Production from Undifferentiated

Cells (Callus and Suspension Cultures)
Callus and suspension cultures are more prominent over other tissue culture
methods, i.e., soot, root, and embryo cultures regarding the biosynthesis of second-
ary metabolites.
8.4.2.1 Callus Culture

Plant calli (singular callus) are a cluster of active and undifferentiated cells which
can be induced through in vitro tissue culture using any suitable explants, i.e., plant
tissue samples (Takeda and Katoh 1981). All terrestrial plant species are found to
have the capacity to produce callus in tissue culture (Beutelmann and Bauer 1977;
O’Dowd et al. 1993; Jeon et al. 1995; Chavez et al. 1998; Atmane et al. 2000;
Petersen 2003; Yang et al. 2007). Callus cultures are widely used for bioactive
secondary metabolites production and also for the purpose of any biological and
biotechnological research. With genetic modification, it can be utilized for
incorporating desirable features, i.e., resistance against diseases, and pests, salt,
draft in crop plants (Efferth 2019). Several previous studies have also been reported
the use of calli for in vitro antidiabetic secondary metabolites fabrication (Malpathak
and David 1986; del Río et al. 1992; Chevala et al. 2016; Iqbal et al. 2019).
Allicin (allyl 2-propenethiosulfinate or diallyl thiosulfinate), alliin, and allinase
enzyme of garlic (A. sativum L.) extract found have significant efficiency to control
improved blood glucose as well as influence improving blood liquid regulation
(Wang et al. 2017). Both differentiated and undifferentiated tissue cultures of
A. sativum have the capacity to increase the quantity and quality of alliin and
alliinlyase (Malpathak and David 1986). On the other hand, plant parts of Indian
rhubarb (Rheum emodi Wall.), especially the rhizome, are rich of AQ, i.e., emodin,
rhein, aloe-emodin, physcion, emodin glycoside, chrysophanol glycoside, and
chrysophanol (Malik et al. 2010), which display many pharmacological activities
such as laxative and neuroprotective effects, antidiabetic, antibacterial, antiviral,
antifungal, anti-inflammatory, anti-arthiritic, etc. (Chihara et al. 2015). Gas
chromatographic–mass spectroscopy was used in a study for exploring and
characterizing the bioactive constitutions available in rhizome, fruit, leaf, and callus
extracts of R. emodi Wall., in which callus was found to possess maximum number
of metabolites, i.e., 31, 38, 27, and 47 compounds were identified from rhizome,
fruit, leaf, and callus extract, respectively (Singh and Chaturvedi 2019).
Moreover, almost 4000 bioactive compounds, mostly polyphenolics/flavonoids,
have been identified from green tea (Camellia sinensis) extract which exhibit many
medicinal values including antimicrobial, antidiabetic, anti-inflammatory, and
anticarcinoma potentials (Lambert 2003; Chattopadhyay et al. 2004; Bansal et al.
2013; Tang et al. 2013). Different polyphenol compounds were able to be obtained
from the tea anther-derived calli cultured on half-strength MS media complimented
with 1 mg/L BAP, 1 mg/L Kinetin and 2 mg/L 2,4-D after 9:15 h photoperiodic
cycle (Chevala et al. 2016). Furthermore, callus culture initiated from petiole explant
of tree turmeric (Coscinium fenestratum (Goetgh.) Colebr) on MS medium
complemented with 2,4-D, BAP and kinetin was reported to enhance the production
of berberine from 1.124% to 1.788% compared to the control (Khan et al. 2008).
Berberine is one of the most pharmaceutically active isoquinoline alkaloids which
can be extracted from many plants, such as tree turmeric (C. fenestratum (Goetgh.)
Colebr.), phellodendron (Phellodendron amurense), Oregon grape (Mahonia
aquifolium), goldthread (Coptis trifolia), goldenseal (Hydrastis canadensis), and
European barberry (Berberis vulgaris) (Yin et al. 2008). Berberine as trade name
is generally recommended as oral medication for the patients with T2D helpful for
controlling blood sugar levels (Yin et al. 2008).
In the callus culture, stress stimulation or elicitation using different chemicals is
also found to be very effective like other tissue culture methods for secondary
metabolites production. For instance, methyl jasmonate (100 μM) as an elicitor
played a vital role for increasing a flavonoid named as quercetin (0.81 0.03 mg/
g dry cell weight) content in the leaf callus culture of onion (A. cepa L.) (Iqbal et al.
2019). Different types of flavonoids and other biologically active compounds are
available in fresh onions which are reducing blood glucose levels (Sheela et al.
1995). Like many other chemical elicitors, extreme physical stress and environmen-
tal fluctuations can play a vital role for increasing the production of secondary
metabolites (Yang and Stöckigt 2010). Multispectral monochromatic lights in
many studies were found to have the capacity to elicit metabolic pathways, and
thus, stimulate primary as well as secondary metabolites production (Tariq et al.
2014). This antagonistic/synergistic effect of these monochromatic lights however
can display diverse influence depending on the plant species, culture type, exposure
time, and ultimately, intensity and quality of light (Khurshid et al. 2020). Cnidium
officinale Makino extract is capable of prohibiting high glucose-induced glomerular
mesangial cells proliferation and, therefore, could be a potential source for early-
stage treatment of kidney-related complication of type 1 and T2D, i.e., diabetic
glomerulopathy (Jeong et al. 2005). The callus culture of this species on MS medium
supplemented with 0.5 mg/L of each BAP and 2,4-D was used to observe the
influence of different lights (red, red: blue, blue, white, and dark) on secondary
metabolites production (Adil et al. 2019). The results showed that white light-grown
callus contained higher 3-butylidenephthalide content (0.43 μg/g DW), whereas dark
condition influenced phthalide (28.3 μg/g DW) accumulation (Adil et al. 2019).
Similarly, antidiabetic and hepatotoxic activities exhibiting Eclipta alba L. were also
used to evaluate the effect of monochromatic lights where significant and positive
effect of red light on increasing phenolic (TPC: 57.8 mg/g) and flavonoid (TFC:
11.1 mg/g) contents were observed in callus cultures (Khurshid et al. 2020).
8.4.2.2 Suspension Culture

Suspension cultures are also called adherent cultures that can be expanded by
transferring the comparatively soft, uncompacted, loose, and crumbly calli into a
disconcerted liquid medium under suitable aseptic conditions where small
aggregated cells are permitted to proliferate and function separately and lead to a
semiliquid cell suspension formation (Eibl et al. 2018). Cell suspension culture
remains the most suitable for the production of active metabolites, especially natural
230 I. Jahan et al.
products, and the most important example is Taxol and baccatin III from Taxus
baccata (Cusidó et al. 2007). Similarly, bioactive and phytochemical properties of
callus suspension cultures of Haworthia angustifolia have been reported to produce
larger amounts of total saponins (29.21 mg OAE/g), flavonoids (56.15 mg RE/g),
and phenolics (17.75 mg GAE/g) with antidiabetic effect than equivalent wild roots
(Yang et al. 2019). Nowadays, cell suspension cultures at large scale are utilizing for
the purpose of extracting commercially valuable secondary metabolites since this
process not only offers a constant and consistent source of natural raw materials but
also helps to exterminate interfering and unwanted compounds (Curtis and Emery
1993).
Neem (Azadirachta indica A. Juss) is one of the most versatile and well-known
Indian medicinal plants that exhibits antidiabetic, antimicrobial, antipyretic, anti-
inflammatory, and many other therapeutic properties (Satyanarayana et al. 2015).
The cell suspension culture of neem was used to evaluate the composition of nutrient
composition of culture medium by observing biomass content and azadirachtin
production (Sujanya et al. 2008). Reduction of phosphate was able to increase the
amount of intracellular azadirachtin (6.98 mg/L), and on the other hand, azadirachtin
was traced in the medium with reduced sucrose (15 mg/L) content (Sujanya et al.
2008).
Although artemisinin and its derivatives are well known worldwide for treating
Plasmodium falciparum malaria, several studies have indicated that they are capable
of protecting pancreatic beta cells by dropping the apoptosis which can enhance
insulin secretion, hence could be a promising and perspective drug component for
T2D treatment (Guo et al. 2018). At optimized concentrations, methyl jasmonate
(Me-JA) as elicitor and MVA lactone as precursor in the cell suspension culture of
A. annua increased artemisinin synthesis, which was 5.93 times higher than its
control cultures (Baldi and Dixit 2008). Similarly, elicitation by any of the elicitors,
i.e., yeast extract, ethrel, Me-JA, and/or salicylic acid on the cell suspension culture
of Cayratia trifolia, was found to be capable of enhancing (3–6 times higher
compared the control) the accumulate of stilbenes (piceid, resveratrol, viniferin,
ampelopsin) (Roat and Ramawat 2009). Naturally occurring stilbenes and their
derivatives can stimulate the regulation of carbohydrate-metabolizing enzymes and
enhance glucose transporter type 4 (GLUT4) translocation, increase glucose uptake
potency of insulin, and, thereby, exhibit promising mechanisms for improving
insulin sensitivity (Ito-Nagahata et al. 2013; Bagul and Banerjee 2015). Likewise,
cell cultures of Morus nigra (black mulberry) treated with 100 μM Me-JA
demonstrated a significant increase in level of flavonoids with enhanced and more
effective antidiabetic and hypoglycemic activities than that for extract from leaves
(El-Mawla et al. 2011).
Different alkaloids of C. roseus leaf extract demonstrate many antidiabetic
potentials (Chattopadhyay 1999). Therefore, a number of approaches have been
tried on C. roseus cell cultures to amplify the synthesis of alkaloids; however, the
obtained results were not sufficient enough for producing these compounds at
commercial level (Siu-Leung et al. 1981; Simpson and Kelly 1989). Moreover,
use of UV-B irradiated on leaf-derived cell suspension cultures not only synthesized
increased amounts of catharanthine but also of accumulate vindoline which was

absent in control suspension, i.e., without any irradiation (Ramani and Jayabaskaran
2008). On the other hand, different doses of gamma irradiation, i.e., 2, 16, and
32 gray (Gy) on the cell suspension culture of Lithospermum erythrorhizon, were
significant enough to boost up both intracellular and extracellular shikonin
productions by 240%, 400%, and 180%, respectively (Chung et al. 2006).
Moreover, a partial list of some important medicinal plants capable of producing
therapeutic compounds reported on different tissue culture techniques for
antidiabetic metabolite production are listed in Table 8.1.
8.4.3 Coculture Systems
Coculture methods are mainly intended to increase the amount of the metabolite
involved by culturing the host plant culture (often suspension cultures) with another
living culture, e.g., plant, bacterial, or fungal cultures (Narayani and Srivastava
2017). These methods are more appropriate for producing bioactive secondary
metabolites and vital for progressing synthetic biology (Tan et al. 2019). According
to the culture type, these systems are categorized into three major groups, i.e., plant–
plant (Verma 2012), plant–fungus (Srivastava et al. 2016), and plant–bacteria
interactions (Liu et al. 2010). Co-existence of two different cells offers some
indications essential for association, interaction, differentiation, and a balanced
chemical, physical, and internal condition in the living system; and bioactive
secondary metabolites contribute a vital role throughout these interactions
(Wu et al. 2008). Several mechanisms in a coculture can induce the synthesis of
diverse bioactive secondary metabolites, such as symbiotic association, parasitism,
and the stimulation of plant defense responses, aggression for space/nutrients, and
antagonistic interaction (Vinale et al. 2017).
P. ginseng C.A. Meyer, the ginseng, one of the most popular herbal remedies,
possesses many bioactive compounds, and in a study, adventitious root coculture of
P. ginseng and Echiancea purpurea (L.) Moench coculture was established to
increase the production of plant secondary metabolites on a large scale (Wu et al.
2008). On the other hand, advantageous microorganisms such as endophytic fungus
are capable of colonizing inside the plant system which offers numerous advantages
for the host, i.e., enhanced growth regulation, disease resistance stimulation,
increased defense mechanisms against pests and pathogens, and bioactive
compounds production with antibiotic, antidiabetic, anti-inflammatory, antitumor,
etc. properties (Hardoim et al. 2015). Several experiments have suggested that
endophytic and symbiotic interactions between plants and microbes imply promising
sources for novel bioactive compounds production, and many potential drugs have
already been synthesized from plants–microbe or microbe–microbe interactions
(Hardoim et al. 2015). Taxus chinensis var. mairei and its endophytic fungi Fusar-
ium mairei were successfully cocultured using their cell suspension cultures, and it
was observed that this coculture system increased the production of paclitaxel,
which was 38 times higher than that by uncoupled culture (Li et al. 2009). Likewise,
232 I. Jahan et al.
coculture system of the cell culture of C. roseus (L.) G. Don and its endophytic
fungus were able to increase alkaloid production, 48% higher than the control group
(Tang et al. 2009).
Unlike a batch culture, a flawless solution throughout the constant and uninter-
rupted exchange of metabolites is provided in a coculture where some of these
metabolites as elicitors help to enhance the accumulation of desired
phytocompounds (Tan et al. 2019), and hence, it has become one of the most
attractive approaches in the recent past. However, there are many difficulties
associated with establishing common culture systems. Containing different
morphologies of more than one organism and exhibiting different growth conditions
(as pH, temperature, photoperiodic cycle, media composition, etc.) and accommo-
dating and adjusting with these factors at the same time in the same culture vessels
are the most vital challenges faced in the coculture systems (Wu et al. 2008). In
addition, the growth rate of an organism using these methods can be different
sometimes, and therefore the ratio of vaccine densities based on growth indices as
well as the duration of vaccination must be optimized to ensure successful concur-
rency (Wu et al. 2008).
8.4.4 Production in Bioreactors
In laboratories, cell suspensions are first grown in shake flasks and then transferred
into a large-scale liquid phase bioreactor. Bioreactors have been developed since
their first use for the reproduction of Hiemalis begonia in 1981 (Klöckner et al.
2013). Nowadays, the use of bioreactors has become suitable propagation systems
for large-scale cell culture and industrial plant tissues (Valdiani et al. 2019). There
are many different types of bioreactors, including airlift bioreactors, stirred tank
reactors, helical ribbon impeller bioreactors, membrane bioreactors, tubular mem-
brane bioreactor, rotating wall vessel bioreactors, bubble beds, tank reactors,
photobioreactors, TIS/temporary immersion bioreactors, super-spinner bioreactors,
etc. (Jolicoeur et al. 1992; Miao et al. 2008; Valdiani et al. 2019). Moreover, the
prospects and key achievements, including the use of metabolites for diabetic
treatment regarding the utilization of various bioreactors, are presented in Fig. 8.3.
Although development of bioreactors has several disadvantages, like lack of
protocols and production procedures, contamination problem, foaming and shear
stress, increased hyperhydricity, accumulation of inhibiting metabolites, following
the scale-up engineering, and high cost, this system is increasingly preferred in plant
tissue culture studies over traditional tissue culture methods due to their numerous
advantages, such as full control of culture conditions, better growth of some plant
species in liquid vigorous media rather than solid media, easier to scale up of a
bioreactor system, and rapid transfer of new technologies related to bioreactors from
one area to another (Shuler 1988; Valdiani et al. 2019).
The first saleable and large-scale application of cell suspension cultures for
secondary metabolites was for producing shikonin from suspension culture of
L. erythrorhizon, accomplished by using stirred tank reactors of 200 L and 750 L
Fig. 8.3 Graphical illustration of the application of bioreactors in plant cell and tissue culture
studies, their main achievements, and expectations (Valdiani et al. 2019)
234 I. Jahan et al.
capacities (Chattopadhyay et al. 2002). Similarly, various bioreactors have been

utilized for the production of secondary plant products using the cell suspension
culture of Taxus wallichiana, Panax notoginseng, Dioscorea deltoidea, Digitalis
lanata, C. roseus, and Podophyllum hexandrum (Chattopadhyay et al. 2002). The
production of three promising molecules (corosolic acid, resveratrol, and
ginsenosides) with adventitious and hairy root cultures in the liquid-phase airlift
bioreactors has been demonstrated for developing therapeutic approaches to treat
diabetes and obesity with natural biocompounds instead of synthetic chemicals
(Sivakumar et al. 2011). Corosolic acid is a chemical compound that has efficiency
in the treatment of diabetes without induction of anti-insulin antibodies (Sivakumar
et al. 2011). A high-tech in vitro tissue culture platform has been developed using a
bioreactor system to produce plant-based, cost-effective corosolic acid for large-
scale diabetes treatment in which it was observed that liquid suspension cultures of
cells originating from Eriobotrya japonica and Lagerstroemia speciosa callus hold
great promise for the production of corrosolic acid (Eibl et al. 2018). The production
of corosolic acid in the cell suspension cultures of L. speciosa was 56 times higher
than the natural leaf content. Root cultures were used for the industrial scale
production of biopharmaceuticals with antidiabetic effect such as ginsenosides,
and the feasibility of a root culture system was established as a powerful commercial
platform (Sivakumar et al. 2011). Using different modified bioreactor systems
moreover, several studies reported the production of α-tocopherol from hazelnut
(Sivakumar et al. 2011), rosmarinic acid from lavender (Georgiev et al. 2009), and
basil (Kintzios et al. 2004), betalain from the hairy root culture of sugar beet (Pavlov
et al. 2007), and artemisin from the hairy root culture of A. annua (Patra and
Srivastava 2016).
Furthermore, there are several renown worldwide companies, especially in Japan
(Mitsui Petrochemical Industries), South Korea (CBN Biotech Company), and the
USA (Phyton Biotech Itnaca) that produce plant-based bioactive molecules such as
corosolic acid, berberine, ginseng, chikonin, and paclitaxel at saleable levels using in
bioreactors (Choi et al. 2008). Thus, it is possible to increase these therapeutic
bioactive compounds in plant cells and tissues by utilizing this in vitro bioproduction
platform at commercial production levels.
8.4.5 Shoot Teratoma Cultures
Another culture obtained by genetic transformation is shoot teratomas, and these

cultures are also considered as interesting methods for the production of valuable
secondary metabolites. Genetically transformed shoots called “shoot teratomas”
were developed by infecting plants with Agrobacterium tumefaciens bacteria (Ray
and Jha 1999). Although the molecular basis of shoot teratomas is not fully
explained, it was observed that production of various secondary metabolites, i.e.,
terpen from Mentha citrate (Banthorpe 1996), steroidal glycoalkaloid from Solanum
dulcamara (Ehmke et al. 1995), and vithanolit from Withania somnifera (Ray and
Jha 1999), can be produced using shoot teratomas. Steroidal glycoalkaloids and
terpenes are new and promising antidiabetic therapeutics and are used as drug for
antidiabetic treatments (Panigrahy et al. 2020).
8.4.6 Optimization Approaches for Increasing Antidiabetic

Metabolite Production
Metabolic efficiency at the tissue or cellular level often fails to meet commercial
expectations regarding the production of desirable secondary metabolites. Therefore,
optimization of cultural conditions is required to obtain high yields that could meet
the suitability of commercial application. For this reason, the cells, tissues, or
organisms with high efficiency or containing the desired metabolite in high amounts
are needed to be selected firstly. Afterward, the focuses are kept on cell or precursor
feeding, use of elicitors, application of stress, immobilization, synchronization of
culture, use of two-stage culture systems, hairy root cultures, stem teratoma cultures,
and the use of bioreactors in large-scale production (Hussain et al. 2012a, b; Lu et al.
2016).
8.4.6.1 Precursor Feeding

Plants use specific organic compounds as building blocks known as precursors for
synthesizing primary and secondary metabolites. Sometimes, the nonexistence of
these precursors can attend to low yields of secondary metabolites. Besides, these
organic precursors can be supplied in the culture medium externally and artificially
as the plants synthesize themselves. In both in vivo and in vitro conditions, therefore,
it is possible to provide these precursor molecules for feeding different biosynthetic
pathways, which would be an efficient approach to enhance the bio-production of
desired metabolites (Shinde et al. 2009).
Moreover, the amount of the desired antidiabetic compounds can be increased by
adding the precursors to the culture medium at different concentrations in different
plant species (Table 8.2). The first attempts were on the addition of amino acids
(arginine, cinnamic acid, glutamine, naringenin, phenylalanine, proline, tryptophan,
tyrosine, etc.), the most used precursors in cell suspension cultures for alkaloid
biosynthesis, especially for tropane and indole alkaloids production (Ellis and
Towers 1970). For example, the addition of phenylalanine to Salvia officinalis cell
suspension cultures stimulated rosmarinic acid production (Ellis and Towers 1970).
According to a recent reported study, rosmarinic acid is effective against hyper-
glycemia and is possibly a potential therapeutic agent against diabetes (Ngo et al.
2018). In a study conducted with Scutellaria baicalensist, L-phenylalanine was used
as a precursor in hairy root cultures, and increased flavonoid production was
confirmed (Kuzovkina et al. 2001). Similarly, addition of 5 mg/L cholesterol as a
precursor to Vitex glabrata R. Br. cell cultures caused significant increases in cell
growth and 20-hydroxycyclone production (Chamnipa 2012). Furthermore,
chalcones were used as precursor compounds for flavonoids biosynthesis in plants,
and therefore, they can also be synthesized in laboratory (Yerragunta et al. 2013).
Chalcones are the precursors for natural flavonoid biosynthesis found in most
236 I. Jahan et al.
Table 8.2 Applications of various precursors for antidiabetic compound production in plant cell
and tissue cultures
Plant
culture Antidiabetic
Plant species Precursor type metabolites References
Gurmar Isopentenyl pyrophosphate Leaves, Gymnemic acid Nikalje
(Gymnema (IPP), 2,3-oxidosqualene, stem, and et al.
sylvestre) and squalene roots (2013)
Origanum Proline, proline analogue Shoot Rosmarinic acid Yang and
vulgare cultures Shetty
(1998)
Psoralea Phenylalanine Hairy root Phytoestrogenic Shinde
corylifolia L. cultures isoflavones et al.
(2009)
Salvia Phenylalanine Suspension Rosmarinic acid Ellis and
officinalis Towers
(1970)
Scutellaria L-Phenylalanine Hairy root Flavonoid Kuzovkina
baicalensist et al.
(2001)
Solenostemon L-Phenylalanine, L-tyrosine Whole- Rosmarinic acid Dewanjee
scutellarioides plant et al.
culture (2014)
Vitex glabrata Cholesterol Cell Hydroxycyclone Chamnipa
cultures (2012)
terrestrial plant species, and different flavonoids are used in traditional medicine for
therapeutic purposes due to their wide range of biological activities including
antidiabetic potentials (Rocha et al. 2020).
G. sylvestre, locally called gurmar (sugar destroyer) and commonly known as
Australian cow plant, and its stem are rich in gymnemic acid, a triterpenoid saponin
used in the traditional treatment system to control DM (Nikalje et al. 2013). In a
study investigating the effects of three different precursors, namely, isopentenyl
pyrophosphate, 2,3-oxidosqualene, and squalene, in order to enhance the production
of gymnemic acid in in vitro shoot cultures of G. sylvestre, the gymnemic acid ratios
were 66.56%, 146.43%, and 90.18%, respectively, as compared to the control
(Nikalje et al. 2013). Therefore, these precursors have the potential for a continuous
production of gymnemic acid.
8.4.6.2 Use of Elicitors and Their Mode of Action

Elicitors can be defined as a physical or chemical agent that initiates or increases the
biosynthesis of specific compounds when introduced into a living cell system at a
small concentration (Ramirez-Estrada et al. 2016). Depending on the types of elicitor
used, they are capable of activating various biosynthetic pathways in plants. This
agent can be classified as abiotic and biotic elicitors according to their structure; as
exogenous (e.g., glucans and glycoproteins) and endogenous (e.g., alginate
oligomers and hepta-glucosides) elicitors according to their origin (Namdeo 2007;
Naik and Al-Khayri 2016). Elicitors that are originated from biological source, i.e.,
bacteria, fungi, virus, and herbivore infections, are called biotic elicitors whereas UV
(UV-A, B, C), temperature (cold, hot), osmotic stress, drought (lack of water), heavy
metals, high salinity, toxic gases, mineral substances, and hormones (BBD) that can
stimulate bioactive compounds accumulation are known as nonbiological or abiotic
elicitors (Naik and Al-Khayri 2016). Elicitation, one of the most effective tools,
means the application of biotic and abiotic factors on plant cells for increasing the
production of secondary metabolites through plant tissue cultures (Narayani and
Srivastava 2017). The elicitors cause many changes in the primary metabolism of the
plant, i.e., photosynthesis, respiratory growth cycle, food intake; tissue differentia-
tion; homeostasis; biosynthesis of carbohydrates, proteins, and lipids as well as
cause changes in gene expression (Baenas et al. 2014). Although these changes
show negative impact on growth and reduce biomass yield in plant cells and tissues
cultures, they increase the production of secondary metabolites. This is due to the
fact that elicitors induce stress which inactivate nondefense genes or activate several
defense-related genes, following the expression of enzymes whose transient phos-
phorylation/dephosphorylation information of proteins can be used to establish the
biosynthetic pathways of many secondary metabolites (Narayani and Srivastava
2017). Moreover, elicitor-induced signal transduction pathways in plants for pro-
ducing secondary metabolites are shown in Fig. 8.4.
All types of in vitro tissue culture such as cell, callus, root, hairy root, shoots,
seedling, and whole plant can be subjected to elicitor treatments. Different
polysaccharides (alginate, chitin, chitosan, dextran, and pectin), or other compounds
like salicylic acid, jasmonic acid, and methyl jasmonate, are the most effective
elicitors often used to induce secondary metabolism, especially antidiabetic bioac-
tive compounds in plant cell and tissue cultures (Nartop 2018). Among all, yeast-
based elicitors, i.e., yeast extract or polysaccharide fraction of yeast extract, are one
of the widely used elicitors that have enhancing capacity to produce many
antidiabetic metabolites, including betulin and betulinic acid (Park et al. 2017),
rosmarinic acid (Sahu et al. 2013), cardenolide (Sun et al. 2012), camptothecin
(Deepthi and Satheeshkumar 2016), andrographolide (Gandi et al. 2012), and
Cryptotanshinone and Tanshinone IIA (Zaker et al. 2015). For instance, Astragalus
membranaceus, a well-known herbal medicine, and its dried root are well-known
herbal remedies for treating chronic diseases. Astragalosides are natural triterpenoid
saponins that can be extracted from the roots of A. membranaceus and useful for the
treatment of diabetes (Park et al. 2020). It was observed that pretreatment of hairy
root cultures of A. membranaceus with yeast extract (3.65 mM) for 72 h provides an
influential strategy for increasing astragaloside levels (Park et al. 2020).
Similarly, in a study of three A. rhizogenes strains (R1601, LBA9402, and
R1000) for hairy root induction in Morus alba L., it was revealed that silver nitrate
as elicitor confirmed the highest yield of betulinic acid while yeast extract showed
the highest induction of betulin accumulation (Park et al. 2017). Additionally, other
important examples related to the utilization of in vitro elicitation for extracting
important plant secondary metabolites with hypoglycemic and antidiabetic effects
are azadirachtin from A. indica (Rodrigues et al. 2014), clalbatin, wedelolactone,
238 I. Jahan et al.
Fig. 8.4 Graphical representation of production of plant secondary metabolites by elicitor-induced

signal transduction pathway (adapted from Narayani and Srivastava (2017) and redrawn by authors)
dimethyl wedelolactone, and stigmasterol from E. alba (Khurshid et al. 2020),

atrorrhizine and palmatine from Tinospora cordifolia (Kumar et al. 2017), and
gymnemic acid from G. sylvestre (Zimare and Malpathak 2020).
Synthesis of antidiabetic secondary metabolites such as accumulation of different
flavonoids, phenolics, and glycosides can be elicited by using nanoparticles as
elicitors. Silver is the most commonly used nanoparticles, but gold, zinc, and copper
nanoparticles have also been used as elicitors (Nartop 2018). Silver nanoparticles as
elicitors were noticed to have inducing capacity for inducing antidiabetic
compounds synthesis (Zhang et al. 2013). Silver nanoparticles as elicitors are
capable of accumulating increased amount of artemicin and capsaicin from hairy
root cultures of A. annua L. and the suspension cultures of Capsicum species,
respectively (Zhang et al. 2013; Bhat and Bhat 2016). However, a partial list of
application of biotic or abiotic elicitors on plants for increasing in vitro production of
antidiabetic metabolites in plant cell/tissue culture is given in Table 8.3.
Table 8.3 Partial list of plants reported to have increased in vitro production of antidiabetic
compounds by the application of elicitation
Potentially targeted
antidiabetic
Plant species Culture type Elicitor compound References
Aloe vera Cell cultures Nano-Ag Aloin Raei et al.
(2014)
Andrographis Suspension Yeast Andrographolide Gandi
paniculata cultures et al.
(2012)
Artemisia annua Hairy roots Ag–SiO2 Artemisinin Zhang
et al.
(2013)
Astragalus Hairy root Yeast extract Triterpenoid Park et al.
membranaceus cultures saponins (2020)
(Astragalosides)
Azadirachta indica Callus Me-JA Azadirachtin Rodrigues
et al.
(2014)
Capsicum sp. Suspension Silver Capsaicin Bhat and
culture nanoparticles Bhat
(2016)
Eclipta alba Callus Red light Clalbatin, Khurshid
wedelolactone, et al.
dimethyl (2020)
wedelolactone,
stigmasterol
Gymnema sylvestre Cell Endophytic Gymnemic acid Netala
suspension Xylaria sp. and et al.
Phaeocystis (2016)
globosa
Hypericum Suspension Aspergillum Naphthodianthrones Xu et al.
perforatum cultures niger cell wall (hypericin) (2005)
Melissa officinalis Shoot Ozone Phenolics, rosmarinic Tonelli
acid et al.
(2015)
Morus alba L. Hairy root Yeast extract, Betulin, betulinic Park et al.
silver nitrate acid (2017)
Morus alba L. Hairy root Yeast extract, Betulin, betulinic Park et al.
silver nitrate acid (2017)
Perovskia Adventitious Yeast extract Cryptotanshinone Zaker et al.
abrotanoides root Tanshinone IIA (2015)
Karel
Pueraria candollei Hairy root Yeast Isoflavonoids Udomsuk
cultures et al.
(2011)
Tinospora Cell Methyl Jatrorrhizine and Kumar
cordifolia (Willd.) suspension jasmonate palmatine et al.
Miers ex (2017)
Hook. F. and Thoms
240 I. Jahan et al.
Table 8.4 Few examples of immobilized plant cell cultures used for the production of antidiabetic
compounds
Antidiabetic
Plant species metabolites Immobilization agents References
Catharanthus Ajmalicine and Alginate Lee and Shuler
roseus catharanthine (2000)
Coleus blumei Rosmarinic acid Loofa sponge Park and Martinez
(1994)
Cruciata Anthraquinone Calcium chloride Dörnenburg and
glabra Knorr (1996)
Curcuma Curcumin Calcium alginate Chaturvedi et al.
longa (2014)
Juniperus Podophyllotoxin Calcium alginate Premjet and
chinensis Tachibana (2004)
Plumbago Plumbagin Calcium alginate Bansal and Bharati
rosea (2016)
Taxus baccata Paclitaxel and Calcium alginate Bentebibel et al.
baccatin III (2005)
Tinospora Arabinogalactan Sodium alginate and Roja et al. (2005)
cordifolia calcium chloride
Tylophora Kaempferol Calcium alginate Chaturvedi et al.
indica (2014)
8.4.6.3 Immobilization of Cells

Indeed, immobilization systems have been used to stabilize amino acids, sugars, and
especially enzymes used in the industrial production of pharmaceutical products
(Brena et al. 2013). These systems are more suitable for bioreactors, especially in
mechanically agitated bioreactors, where hydrodynamic shear stress caused by
dispersion or shaking can be reduced and cell removal by continuous cultures can
be prevented (Ochoa-Villarreal et al. 2016). In addition, this method provides
appropriate cell recovery from the culture medium without any requirement of
inoculation for repeated use as well as improves the synthesis and accumulation of
metabolites by preventing the natural tendency of cells to aggregate (Tyler et al.
1995; Xu et al. 2011). Calcium alginate, sodium alginate, agar, agarose, hybrid sol–
gel matrices, glass fibers, reticulate polyurethane foam polyester, loofa sponge, and
jute and sisal fibers are frequently utilized for immobilization of plant cell cultures
(Bentebibel et al. 2005; Nartop 2018). Although ideal immobilization matrices of
cell cultures of each plant species have not been reported yet, most of the reports
have however shown that more antidiabetic metabolites production can be induced
from immobilized of cells, even more than cell suspension cultures (Brena et al.
2013). A short list of plants that produced antidiabetic active metabolites by
immobilized cultures is given in Table 8.4.
8.5 Production of Secondary Metabolites Through Metabolic

Engineering
Plants produce a wide range of secondary metabolites with specific and significance
usages for mankind, particularly related to the healthcare sectors; and consequently,
commercial production of these phytocompounds as well as maintaining their
sufficient supply is vital to meet the global market demand (Peña et al. 2018).
However, traditional methods for isolating these chemicals are not sufficient enough
at commercial level due to their inadequate synthesis by their natural hosts. For that
reason, metabolic modification in plants through metabolic engineering techniques
could be a powerful and applicable tool to increase the yield/production of secondary
metabolites, especially antidiabetic pharmaceuticals (Grover et al. 2002). Metabolic
engineering is a powerful tool in which recombinant DNA technology is used to alter
different metabolic pathways for producing fuels, desirable metabolites,
pharmaceuticals, and medicine (Stephanopoulos et al. 1998). This technology can
optimize genes and genetic regulation processes within cells which are able to
change enzyme(s) or regulatory protein(s), eliminate harmful byproducts or prohibit
certain competitive pathways, bring overexpression or downregulation of certain
enzymes and proteins in a metabolic pathway, and thereby, modify cellular and
metabolic characteristics of a host organism and enhance the cells’ production of a
certain substance (Stephanopoulos et al. 1998). Recently, metabolic engineering is
applied in bacteria, fungi, plant, and animal cells as host organisms to amplify the
synthesis of distinctive secondary metabolites, especially with the continuous devel-
opment of new generation sequencing technologies and new algorithms for bioin-
formatics analysis (Siddiqui et al. 2012). In most of these studies, the main research
focus was designing heterologous biosynthetic gene clusters using synthetic biology
techniques that increase the expression of targeted genes (Farhi et al. 2011). For
applying engineered metabolism in plants, it is necessary to expand fundamental
biomolecular tools like cloning, promoter analysis, protein targeting, plant transfor-
mation, biochemical genetics, and other areas of plant science (DellaPenna 2001). In
this section, a brief discussion is brought to review the metabolic engineering
techniques applied for increasing the production of secondary metabolites, espe-
cially antidiabetic pharmaceuticals. There are five important strategies used for
studying metabolic engineering in plants (DellaPenna 2001):
1. Transfer of genes to plants encoding with new regulatory proteins or enzymes,

2. Use of transcription factors (TF: transcriptional factors) that control specific
metabolic pathways,
3. Application of gene-editing techniques (GET): CRISPR/Cas, TALEN, and
ZFN-based methods,
4. Applications of synthetic biology,
5. Omic technologies (genomics, transcriptomics, proteomics, and metagenomics).
242 I. Jahan et al.
8.5.1 Transfer of Genes Encoding New Enzymes to Plants
Several model plants have been used for transferring genes that are encoded with
new regulatory proteins or enzymes; for instance, CRYP716A47 and pgDDS genes
from P. ginseng were transferred in transgenic Nicotiana spp. for dammarene
sapogenin (protopanaxadiol) production (Chun et al. 2015). Similarly, recombinant
DNA technology was also applied for producing antimalaria drug artemycin and its
precursors in tobacco plants (Farhi et al. 2011). Nevertheless, impenetrability in
determining the rate of limiting strategies in a pathway as well as multi-enzymatic
effects on a single biosynthetic pathway may prohibit the process of transferring
genes in a species, for example, increasing the activity of the phyton synthase
enzyme in tomato did not increase the carotenoid content proportionally (Fraser
et al. 2002). Similarly, increased expression of the geraniol synthase gene of
Lippiadulcise did not increase geranic acid content after transferred to maize plant
(Yang et al. 2015). This was due to the fact that the metabolite of interest was
metabolized to another form by another enzyme (Yang et al. 2015).
Due to the strong and specific inhibition capacity of montbretin A (MbA) on
human pancreatic α-amylase, this plant metabolite is utilized as an anti-obesity and
antidiabetic agent for a long time (Irmisch et al. 2020). MbA is a complex acylated
flavonol glycoside that accumulates in Montbretia (Crocosmia crocosmiiflora)
onion in small quantities during the early summer (Irmisch et al. 2020). After
discovering the full biosynthetic pathway for this antidiabetic metabolite, it was
observed that stimulation of some enzymes (the CcUGT5, CcUGT4, and
UDP-glycosyltransferases) in this pathway is often capable of ensuring high accu-
mulation of target metabolites (Irmisch et al. 2020).
8.5.2 Use of Transcription Factors (TF: Transcriptional Factors) That

Control Specific Metabolic Pathways
TFs are involved as the important tools to regulate the production of secondary
metabolites in plants by possessing the significant roles in controlling the transcrip-
tion of biosynthetic genes (Vom Endt 2002). It was observed that the use of TFs for
activating the pathways and synthesizing increased amount of antidiabetic secondary
metabolites is more effective since they can increase the expression of structural
genes encoding biosynthetic enzymes. Different types of TFs were found to exhibit
significant regulatory functions of pharmaceutical terpenoids (Lu et al. 2016),
flavonols (Luo et al. 2008), anthocyanins (Butelli et al. 2008), glycosinolates
(Kumar et al. 2017), dimericindole alkaloids (Pan et al. 2012), etc. A flavonol-
specific transcriptional activator (AtMYB12) was isolated from Arabidopsis thaliana
which were observed to be capable of accumulating very high levels of flavonols by
inducing the expression of genes in transgenic tobacco plants (Luo et al. 2008).
Similarly, several examples of TFs were utilized to monitor the regulatory
functions of different terpenoids. For instance, AP2/ERF TFs (hold a binding domain
of 57–66 amino acids) are involved in metabolic regulation and can response to
biotic and abiotic stresses in plants (Agarwal et al. 2006). In case of transgenic
A. annua plants’ enhanced amount of artemisinin and artemisinic acid were observed
with the overexpression of two AP2 family TFs, i.e., AaERF1 and AaERF2 (Yu et al.
2012). Furthermore, WRKY TFs (attach to the W-box (TTGACC/T) of promoters)
are also engaged in the regulation of developmental, morphological, and physiologi-
cal processes as well as defense mechanisms of plants (Rushton et al. 2010). Since
these factors are involved in trichome initiation, it was observed that transcription of
CYP71AV1 was increased up to 33 times more than the wild-type plants with the
trichome-specific overexpression of AaWRKY1 (Lu et al. 2016). Isoprenoids or
terpenoids are one of the largest, diverse, and most common biocompounds that
biosynthesized by a wide variety of plants and animals via MVA pathway and
2C-methyl-D-erythritol-4-phosphate pathway (Brahmkshatriya and Brahmkshatriya
2013). These naturally occurring hydrocarbons play a vital role in traditional herbal
remedies since they exhibit anticancer, immunomodulatory, antioxidants,
antiparasitic, anti-inflammatory, antihyperglycemic, antiviral, antibacterial, antifun-
gal potentials (Brahmkshatriya and Brahmkshatriya 2013). Nowadays, different
terpenoids are widely used as important chemotherapy drug (taxol derivative, i.e.,
paclitaxel, and docetaxel) for treating and preventing cancer as well as utilized for
skin permeation enhancer (Brahmkshatriya and Brahmkshatriya 2013).
8.5.3 Gene-Editing Techniques; ZFN, TALEN, and CRISPR/Cas-Based

Methods
Several transformative tools such as Cas-based RNA-guided DNA endonucleases/

emergence of the clustered promoter-spaced short palindromic repeat (CRISPR),
zinc-finger nucleases (ZFNs), and transcription activator-like nucleases (TALENs)
can affect the personalization of medicine and also restructure the margins of
biological research (Gaj et al. 2013). Lately, CRISPR/Cas system however has
appeared to be a dexterous, prospective, and simpler tool for inducing targeted
genetic alterations that could be an impending replacement of site-specific nucleases,
i.e., TALENs and ZFNs (Gaj et al. 2013). This is due to the fact that the production
of secondary metabolites can only be accumulated in enhanced levels by utilizing
tissue-specific promoters (Xu et al. 2020) or involving RNAi of redundant and
branched pathways (Zhang et al. 2015) or applying CRISPR/Cas-based transforma-
tive tools that can correct genomic structure helps to reduce the competition of other
metabolites from the desired metabolites in a particular metabolite pathways.
CRISPR/Cas-mediated genome editing has exhibited its flourishing demonstration
for bacterial metabolic engineering (Mougiakos et al. 2018), lovastatin, and taxol
synthesis (El-Sayed et al. 2017), specifying genome engineering in vegetable crops
(D’Ambrosio et al. 2018), discovering natural products (Tong et al. 2019), and
finally, persisting advanced plant breeding techniques in agriculture (Chen et al.
2019). Fenugreek (Trigonella foenum-graecum) seeds contain an amino acid known
as 4-hydroxyisoleucine (4-HIL) which is a vital drug compound used for preventing
type 1 and T2D (Fowden et al. 1973). The production of this phytocompound
244 I. Jahan et al.
through traditional ways is very low, and hence, a faster switching of multiple genes
was promoted via CRISPR-Cas9 editing strategy for l-isoleucine dioxygenase
enzyme, i.e., a Fe(II)/α-ketoglutarate (α-KG)-dependent dioxygenase stimulation
which can alter (2S,3R,4S)-4-HIL from l-isoleucine (l-Ile) and, thereby, optimize
the production of this compound at commercial level (An et al. 2020). However,
extensive studies are needed to contribute to the supply of natural antidiabetic
compounds of this system, including the potential for off-target effects.
8.5.4 Applications of Synthetic Biology
Synthetic biology methods are used to increase biological diversity and produce
more natural products by adding assessable and prevailing mechanisms to the
biological systems which has expanded the possibilities to use living organisms
for a variety of applications (Rahmat and Kang 2020). To achieve a successful
approach of synthetic biology, it is necessary to understand the sequencing pattern
and synthesis strategies of cost-effective genome, the roles of both genes and gene
expression products, functions of the latest sensor-reporter techniques, and
standardizing process of gene-splicing methodologies (Nielsen and Moon 2013).
Yeast (S. cerevisiae) has been widely used in the field of genetic and metabolic
engineering since 1973 after the successful genetic modification of this species (Lian
et al. 2018), and a variety of optimization techniques of advanced genetic engineer-
ing have been applied on this species developed for the modification and recombi-
nation of DNA (Lian et al. 2018). Along with S. cerevisiae, other microbial
platforms such as bacterial strains, i.e., Streptomyces species (Lee et al. 2019) and
E. coli (Xu et al. 2020), have been utilized for secondary metabolites production
within the scope of synthetic biology, whereas N. benthamiana (Gülck et al. 2020)
has been used as herbal host organisms for the same purpose.
Each of these hosts has various advantages and limitations compared to each
other. For example, the production and genetic manipulation of microbial–host
interaction is relatively rapid. With the first studies using synthetic biology in this
sense, artemisinic acid was produced in E. coli (Keasling 2012) and the role of
cytochrome P450 in terpenoid metabolism was revealed in yeast (Ro et al. 2005).
Use of Pseudomonas putida was a prominent example in synthetic biology studies
due to its relatively wider metabolic versatility than E. coli (Akkaya et al. 2018).
Streptomyces, one of the most common gram-positive bacterial strains, have been
utilized for recombinant DNA studies and synthetic biology in almost two-third
commercial production of medically and agriculturally important secondary
metabolites such as antidiabetic, anticancer, immune suppressive, antiparasitic,
antibiotic, and antifungal compounds (Khan et al. 2011). Synthetic biology
strategies applied for the production of secondary metabolites are mostly conducted
with Streptomyces rather than highly organized plants. In this context, synthetic
biology strategies are classified into three stages, as they are defined in engineering
biology with a repetitious “design build-test” cycle (Lee et al. 2019). In the design
phase, new Secondary Metabolism Biosynthetic Gene Clusters (SM-BGCs) are
identified through genome mining, and then SM-BGCs are designed using various
synthetic genetic parts developed for Streptomyces spp. Afterward in the second
step, the process of generating the redesigned SM-BGCs is started, including
genome engineering of the original host and transformation of the SM-BGC assem-
bly into plasmids for heterologous expression. In the last stage, high efficiency test
methods are used to measure the activation of target SM-BGCs for the next “design-
build-test” cycle (Lee et al. 2019).
Heterologous synthesis of dihydrocalones (DHCs) such as phlorizin and
nothofagin, which also exhibit antidiabetic activities, is one of the mentionable
other examples of synthetic biology where metabolically modified yeast strain was
used for increasing these compounds (Rahmat and Kang 2020). Overexpression of
the gene (ScTSC13) in genetically modified engineered S. cerevisiae can produce
DHCs through de-novo synthesis, i.e., biosynthesis of a complex molecules or
chemical group from dissimilar simple molecules or groups (Eichenberger et al.
2017). Moreover, a partial list of previous studies of metabolic engineering for
producing secondary metabolites with antidiabetic effects is presented in
Table 8.5, in which most of these studies were targeted on designing heterologous
biosynthetic gene clusters, i.e., capable of enhancing desirable metabolites via
synthetic biology techniques with the aim of increasing the expression of these
target genes (Frias et al. 2018).
8.5.5 Omics Technologies (Genomics, Transcriptomics, Proteomics,

and Metagenomics)
Omics technology is the high-dimensional biology which is developed basically for

detecting mRNA (transcriptomics), genes (genomics), proteins (proteomics),
glycomics, and metabolites (metabolomics) in a nontargeted and nonbiased manner
within a particular biological specimen (Horgan and Kenny 2011).
The Latin word “-ome” is used as suffix for referring the objects of study of
biological fields, for example, genome, proteome, or metabolome. This suffix was
coined for the first time in 1920 with the term “genome” by Professor Hans Winkler,
a botanist at the University of Hamburg addressing the whole of the hereditary
material located on different chromosomes (Winkler 1920). In the following years,
this simple Latin supplement, which is frequently used in defining different levels of
cellular activities such as transcriptome, proteome, and metabolome as a whole, has
been transformed into an “-omic” state in a way that allows the expression of a large
number of data obtained through high-output technologies together with the
advances in sequencing technologies with the human genome project (Venter et al.
2001). Although omics technologies appear to consist mainly of genomics,
transcriptomics, proteomics, and metabolomics, these days, epigenome, ionome,
lipidome, glycome, hormonome, cytome, metallome, ribonom, regulom, and even
at the phenomenal level it has become a field of study that combines all the obtained
information under the name of system biology with bioinformatics approaches
(Sánchez-Vidaña et al. 2017; Fig. 8.5).
246 I. Jahan et al.
Table 8.5 Production of some secondary metabolites with major antidiabetic potentials through
metabolite engineering of microorganisms
Host
Secondary metabolites microorganism Engineering strategy References
Amorphadiene Saccharomyces Overexpressing every Westfall et al.
cerevisiae enzyme of the mevalonate (2012)
pathway and focusing on
amorpha-4,11-diene
production
Phlorizin S. cerevisiae In S. cerevisiae, de-novo Eichenberger
production of phlorizin by et al. (2017)
the overexpression of a
native gene (ScTSC13)
Anthocyanins (ACNs; S. cerevisiae In a single yeast cell, Eichenberger
delphinidin 3-O-glucoside, reconstruction of a full- et al. (2018)
cyanidin-3-O-glucoside, length metabolic pathway
pelargonidin 3-O- which leads to the de-novo
glucoside) biosynthesis of each of the
three basic ACNs
Taxifolin Yarrowia For hydroxylated Lyu et al.
lipolytica (a yeast flavonoid production, (2019)
from the family cytochrome P450
Dipodascaceae) reductases (CPR) and
chalcone synthase (CHS)
were identified. Targeted
gene segments (copy
number of CHS and CPR)
were determined and
genes allied with malonyl-
CoA supply and
chorismate expressed for
the biosynthesis of
taxifolin
Kaempferol, naringenin S. cerevisiae Precursor PEP/E4P and Lyu et al.
F3H were supplemented in (2019)
yeast cell and
mitochondrial DNA
engineering was followed
by F3H and FLS. This
genetic recombination
optimized the core
flavonoid synthetic
pathway and eliminated
the biosynthetic branch of
phenyl ethanol
Squalene Y. lipolytica Squalene production was Liu et al.
improved by categorizing (2020)
the mevalonate pathways
and determining substitute
reduction equivalents
(NADPH) pathways
(continued)

Host
Secondary metabolites microorganism Engineering strategy References
Curcuminoids Escherichia coli Genes capable of altering Rodrigues
pathways were optimized et al. (2020)
which separately
converted tyrosine to
ferulic acid and
hydroxycinnamic acids to
curcuminoids
Naringenin S. cerevisiae Complex biosynthetic Du et al.
pathways of flavonoid (2020)
were expressed by
engineering microbial
consortia
Ent-kaurene (steviol E. coli Reconstructing an Kong et al.
glycoside) ent-kauren biosynthetic (2015)
pathway in recombinant
E. coli
Polyketides and the Y. lipolytica Employing a β-oxidation- Palmer et al.
diketide intermediate based engineering strategy (2020)
for the production of an
expanded palette of
polyketides and
establishing biosynthesis
of the 4-coumaroyl-CoA-
derived polyketides
Valienamine Streptomyces Suitable candidate of Cui et al.
hygroscopicus amino transferases was (2020)
identified for stimulating
valienone, a ketone
substrate
The fundamental of these approaches is to understand the complex structure of an

organism more systematically, in details by considering as an entire system. Tradi-
tional biological study is largely hypothesis-driven or reductionist, biology, and
omics technologies are different from the conventional methods. Regarding the
antidiabetic potential secondary metabolites research, these advance technologies
are mainly utilized to understand and predict the mechanisms of action of medicinal
plants by using technologies from systems biology (proteomics, pharmacogenomics,
and metabolomics) (Ulrich-Merzenich et al. 2007). Since omics technologies evalu-
ate the universal profiles of genes, proteins, and metabolites, each molecule plays a
critical role in the biology of the organism; the synthetic biology approach simplifies
and makes the discovery of innovative antidiabetic treatment solutions affordable
(Campbell-Tofte et al. 2012). This technology is not only significant for a vast and
wider conceptualization of physiological processes but also for screening,
processing, diagnosis, and prognosis of diseases, and also for assisting our under-
standing of the etiology of diseases (Horgan and Kenny 2011). Since omic strategies
248 I. Jahan et al.
Fig. 8.5 Different omic techniques and their functions in systems biology (Yan et al. 2018)
are capable of investigating multiple molecules simultaneously, they can lead

themselves to biomarker discovery, therefore are progressively utilized for screening
and assessment processes of drug detection as well as evolution of their (drugs)
efficacy and toxicity (Horgan and Kenny 2011).
8.6 Conclusions and Perspectives
Although there are many medicinal plants available in various geographies around
the world, many biologically active molecules from these plants with antidiabetic
effect have not been discovered. Therefore, there must be some novel mechanism to
select such active ingredients such as terpenoids, alkaloids, flavonoids, and phenolic
compounds among all secondary metabolites found in plants. The biotechnological
tools we have today might be the right choice for discovering, identifying, utilizing,
and conserving these plants and plant-based phytocompounds. However, sometimes
conventional experiments on many medicinal plants fail to produce the desired
products in high quantities, and often basic plants tissue culture methods are not
sufficient to fulfill this desire. In such situation, different advanced strategies such as
adventitious root cultures, genetic transformation and hairy root cultures, use of
biotic and abiotic elicitors, cell-suspension cultures with immobilization techniques
in bioreactors as well as metabolic engineering through recombinant DNA technol-

ogy can be utilized for improving the production of secondary metabolites, espe-
cially antidiabetic bioactive compounds. Moreover, it is also necessary to understand
the basic knowledge of the biosynthesis pathways and mechanisms responsible for
the production of plant metabolites, and physiological processes of diseases for
successful applications of these advanced technologies.
Acknowledgments Funding: This research has used no external funding.

Conflicts of Interest: The authors do not declare a conflict of interest.
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Saffron: A Prized Herb with Therapeutic
Potential Against Diabetes 9
Nitu Gautam and Amita Bhattacharya
Abstract
The exponential rise in the incidence of diabetes mellitus worldwide is driving the
current search for antidiabetic phyto-therapeutics as cheaper and safer alternative/
adjunct to existing chemical/synthetic drugs. Based on folk and traditional medi-
cine, a number of target plants have been identified. Amongst these, saffron has
been established as a plant with potential ‘antidiabetic’ properties. Its bioactive
compounds, namely, crocin, crocetin and safranal have the capacity to improve
the sensitivity as well as the production of insulin for maintenance of hypoglyce-
mic levels via the AMPK/ACC and AKT kinase-mediated pathways. These
bioactive compounds can also overcome oxidative stress-mediated tissue
damages and ameliorate patho-physiological conditions associated with diabetes.
The different studies that have demonstrated these antidiabetic properties of
saffron, its extracts and bioactive compounds in in-vivo and in-vitro models
have been reviewed in this chapter. The aim is to collate available information
for the future development of safe and efficacious nutraceuticals, dietary
supplements and therapeutics for use against diabetes.
N. Gautam
Division of Biotechnology, Council of Scientific and Industrial Research (CSIR)-Institute of
Himalayan Bioresource Technology, Palampur, Himachal Pradesh, India
Department of Biotechnology, Guru Nanak Dev University, Amritsar, Punjab, India
A. Bhattacharya (*)
Division of Biotechnology, Council of Scientific and Industrial Research (CSIR)-Institute of
Himalayan Bioresource Technology, Palampur, Himachal Pradesh, India
e-mail: amitabhatta@ihbt.res.in; amitabhatta@yahoo.co.uk
Ltd. 2021
https://doi.org/10.1007/978-981-16-3529-8_9
264 N. Gautam and A. Bhattacharya
Keywords
Antioxidant · Crocus sativus · Crocin · Crocetin · Hyperglycemia · Insulin

sensitivity and resistance · Safranal
9.1 Introduction
The magical herb, ‘saffron’ (Crocus sativus L.) is acclaimed for its immense
medicinal properties. The vivid red-coloured trifid stigmata (the spice) has the
potential to treat various diseases including diabetes. This property is attributed to
more than 150 volatile, non-volatile and aroma-yielding lipophilic and hydrophilic
compounds present in the stigmata. The stigmata also contain vitamins (especially,
riboflavin and thiamine), gums, starch, sugars (63%), proteins (12%), moisture
(10%), fat (5%), minerals (5%), crude fiber (5%; w/w), flavonoids and pigments
like lycopene, anthocyanins, α- and beta-carotenes, zeaxanthin, etc. Amongst these,
crocin, picrocrocin and safranal are the major bioactives responsible for the charac-
teristic colour, taste and odiferous properties of saffron. Recognized by various
names such as ‘Zafran’, ‘Kesar’, ‘Kumkuma’, ‘autumn crocus’ and ‘red gold’,
saffron is collected manually from highly attractive, bright-purple coloured flowers
of the herb; and constitutes the costliest spice on earth.
Domestication of saffron and its use in food, medicine and dyes date back to
2000–1500 years BC, and is well documented in ancient literature and historical
records (Sharafzadeh and Alizadeh 2012). The wide range of health benefitting
attributes of ‘saffron’ and its use in the treatment of various ailments is also recorded
in Iranian medical books. Although various floral parts are rich in bioactive
compounds (Popovic-Djordjevic et al. 2021), yet, mainly, stigmata of saffron
flowers have been used in the traditional preparations and medications against
depression, cardiovascular disease, menstruation disorders, asthma, insomnia, diges-
tive ailments, etc.
Presently, traditional herbal medicines (including those containing saffron) are
serving as the first line of treatment for various ailments for almost 60% of the world
population (Tilburt and Kaptchuk 2008). This growing craze for herbals and tradi-
tional medicines is driving a robust and ever-increasing market across the world. The
demand for these products has been so high, that presently, the herbals market in
both developing and developed countries is constituting about 60 billion US$
(World Health Organization, http://www.who.int/topics/traditional_medicine/en/).
As per the survey conducted by National Center for Complementary and Alternative
Medicines, India, herbals or traditional medicines have become extremely popular
because of their higher affordability, safety and non-toxic characteristics. Medicinal
herbs and their bioactive compounds are also gaining recognition as important and
useful constituents of modern medicine, particularly for the treatment of diseases
like cardiovascular and auto-immune disorders, prostate problems, depression,
inflammation and diabetes (Atanasov et al. 2015). As a result, several countries
have started making huge investments on the development of herbals. This in turn
9 Saffron: A Prized Herb with Therapeutic Potential Against Diabetes 265
has fueled research on basic aspects like phytochemical validation, development of

herbals/formulations and most importantly, regulatory studies on the delivery,
safety, toxicity and bioavailability of phyto-molecules and or bioactive compounds
in in vitro as well as in vivo systems. The anti-cancer, anti-inflammatory, anti-
oxidant or radical scavenging, anti-allergic, anti-arthritic, anti-genotoxic, anti-
aging, anti-hypertensive, anti-angiogenesis, anti-atherogenic, antibacterial,
antidiabetic, anti-obesity, neuro-protective, hepato-protective and nephro-protective
properties of these bioactive compounds/extracts/saffron were established through
such studies (Table 9.1). Many of these properties can be potentially harnessed for
treating various life-style diseases like diabetes.
Lifestyle disorders have become a matter of great concern for people across the
world. This is because of their increasing incidences, irrespective of age and/or
livelihood. Being directly influenced by one’s habits as well as dietary and living
patterns, life-style disorders are cumulative effects of a person’s physical capacity
and psychological functioning. Diabetes mellitus (DM) is one such complex lifestyle
disorder that is increasingly affecting millions of people worldwide. Interestingly,
the number of cases as well as the prevalence of the disease has been more in
developing or poor countries as compared to the developed ones. DM is also called
as ‘chronic endocrine disease’ or ‘metabolic disorder’ and it affects the metabolism
of macromolecules such as carbohydrates, proteins and lipid leading to hyperglyce-
mic condition. Under this condition, the body either secretes lesser amounts of
insulin (a blood glucose regulator) or fails to utilize the insulin produced by the
body. As a result, there is drastic alteration of sugar/glucose metabolism in the body,
which, if not controlled or managed leads, to heart disease, kidney failure, stroke,
foot ulcers and even damage of eyes/retina. Lifestyle, food habits, physical work,
environment, ethnicity and genetic factors govern the occurrence of DM.
Management of the hyperglycemic condition and the consequent complications
that arise with the progression of diabetes is, therefore, an important strategy
employed for the treatment of the disease. Retinopathy, nephropathy, neuropathy
and impaired antioxidant defense mechanism are general complications associated
with diabetes. The enzymatic and non-enzymatic pathways that scavenge toxic
reactive oxygen species (ROS) in healthy beings are impaired in diabetics. This in
turn leads to numerous diabetes-related pathophysiological processes. This is the
consequence of auto-oxidation of glucose in diabetics leading to a shift in redox
balance followed by a decrease in the concentration of low molecular weight
antioxidants, apoptosis of pancreatic beta-cells and also insulin resistance. High
levels of glucose in blood stimulate cytochrome P450-like activity by excessive
production of nicotinamide adenine dinucleotide phosphate-oxidase (NADPH oxi-
dase), and this leads to ketosis and increased susceptibility of pancreatic and liver
cells to oxidative damage. In this regard, the mechanism of alloxan and
streptozotocin (STZ) induced diabetes has been the subject of many investigations
and it is now accepted that the destruction of β-cell pancreatic islets is strictly
associated with the systemic and/or local induction of oxidative and nitrosative
stress. This stress makes the pancreatic tissues especially susceptible to the action
Table 9.1 Hypothetical mechanism of action of saffron bioactive compounds (crocin, crocetin and safranal) against diabetes mellitus
266
Class of
compound Extract Experimental model Modulates Mechanism of action Reference
Carotenoid Aqueous Alloxan and STZ Serum glucose, MDA, TG, TC and Corrects insulin resistance by He et al. (2005)
extract of inducible diabetic rat LDL-C balancing the oxidant/antioxidant
crocin GSH and HDL C system and inhibiting the
pancreatic lipase activity
Carotenoid Aqueous Rats fed with (i) fructose Serum glucose, TG and TC, serum Promotes regeneration of β-cells of Xi et al. (2005)
extract of (ii) high-fat diet and (iii) insulin islets of Langerhans and also
crocetin exposed to dexamethasone corrects insulin resistance by
balancing the oxidant/antioxidant
system
Carotenoid Aqueous Bovine aortic endothelial – Balances the oxidant–antioxidant Xiang et al.
extract of cells system, inhibits adhesion of (2006)
crocetin leukocytes to aortic endothelial
cells and prevents the progression
of atherosclerosis
Carotenoid Crocetin Male Wistar rats Improve of insulin sensitivity Significantly reduces the Xi et al. (2007)
powder expression of proteins as well as
mRNA of insulin-sensitizing
adipocytokine (an adiponectin)
Carotenoid Crocin Male Sprague–Dawley Levels of serum, LDL and VLDL Promotes hypolipidemic effect by Sheng et al.
powder rats cholesterol and also triglyceride. inhibiting pancreatic lipase and (2006)
also leads to the mal absorption of
fats and cholesterol
– Stigma Alloxan inducible diabetic Serum glucose, TG and TC serum Increases the number of β-cells in Mohajeri et al.
extract rat insulin pancreas, balances the oxidant- (2009)
antioxidant system
– Ethanolic Male Wistar rats – Scavenges ROS, regenerates Mohajeri et al.
extract of endocrine cells of pancreas leading (2009)
stigma to hypoglycemic and anti-
N. Gautam and A. Bhattacharya
hyperglycemic effects
9
– Hydro- Male Sprague–Dawley Serum glucose without affecting Hypoglycemic and hypo-lipidemic Arasteh et al.
methanolic rats serum cholesterol and insulin effects (2010)
extract levels
– 75% Crocin 5 and 25 mg/mL) Serum glucose Scavenges ROS, suppresses Mousavi et al.
ethanolic and GSH(10lM) glucose-related cell toxicity, (2010)
extract of pretreatment of PC12 cells decreases excessive glucose
stigma inducible neurotoxicity
Carotenoid Aqueous Alloxan and STZ Serum glucose, total lipids, TC, Balances the oxidant–antioxidant Kianbakht and
extract of inducible diabetic rat TG, LDL-C, MDA and NO system Hajiaghaee
stigma Serum insulin, HDL, GSH, CAT (2011)
(safranal and SOD
picrocrocin)
– Saffron tablet Students Immuno-modulatory activities – Kianbakht and
Ghazavi (2011)
– Ethanolic Male Wistar rats Blood glucose Reduces blood glucose and Mohammad
extract of attenuates oxidative stress of et al. (2011)
stigmata hepatic tissue through ROS
scavenging activities leading to
anti-hyperglycemic effect
– Extract of Skeletal muscle cells Blood glucose Phosphorylates AMPK muscle Kang et al.
stigmata cells leading to glucose uptake in (2012)
muscle cells, regenerates β-cells of
islets of Langerhans
Saffron: A Prized Herb with Therapeutic Potential Against Diabetes
Carotenoid Crocin Male Wistar rats – Reduces lipid peroxidation leading Rajaei et al.
powder to hypoglycemic and antioxidative (2013)
effect
Carotenoid Crocin Adult male Wistar rats Blood glucose, serum insulin Improves learning and reduces Tamaddonfard
powder memory impairment, leads to et al. (2014)
neuroprotective activities and anti-
hyperglycemic and also anti-hypo
insulinemic effects
267
(continued)
268
Class of
– Aqueous neonatal male Wister rats Blood glucose, HbA1c, TG, total Decreases body weight of diabetic Shirali et al.
extract of cholesterol, LDL and HDL in rats and reduces mortality rate (2013)
stigmata fasting serum
– 90% ethyl Sprague-Dawley albino Blood glucose, serum TL, TG and Increases the levels of blood insulin Elgazar et al.
alcohol rats TC, AST, ALT and ALP, serum in diabetic rats, prevents liver (2013)
extract BUN, UA and Cr necrosis, maintains normal kidney
function and protects from other
hyperglycemia related
complications
– Aqueous Adult male rats – Decreases insulin resistance, Hosseini and
extract of lowers glycemic indices Azarbayjani
stigmata (2013)
– 1.0 g saffron 204 T2D patients Total cholesterol, LDL, and HDL – Azimi et al.
levels (2014)
– DMSO Mouse myoblast cell line Glucose in myoblasts Induces a ligand-independent Maeda et al.
extract of (C2C12) insulin signaling in cultured (2014)
stigmata myotube, covalent modification of
catalytic cysteinyl thiol, improves
impaired glucose tolerance and
glucose uptake through
translocation of glucose transporter
4 and inhibits PTP1B activity
– Aqueous Wistar albino rats ROS scavenging enzymes SOD, Significantly increases ROS Samarghandian
extract of CAT and GSH, serum TNF-α and scavenging enzyme activity, et al. (2014)
stigmata nitric oxide synthase (iNOS) decreases cognitive deficit, reduces
hyperglycemia and hyperlipidemia
9
Carotenoid Saline 30 female Wistar albino – Protects kidney hemodynamics, Altinoz et al.
solution of rats decreases plasma Cr and BUN (2015)
crocin levels and reduces diabetes-
inducible renal injury
– Hydro- 65 adult male Wistar- ROS scavenging enzymes, lipids, Ameliorates hyperglycemia- Hemmati et al.
alcoholic derived rats total cholesterol mediated oxidative stress, (2015)
extract of improves antioxidant capacity,
stigmata reduces lipid peroxidation,
improves lipid profile and reduces
total cholesterol levels
– Oral capsules 50 diabetic patients – Antispasmodic, pain-killing, Dehghan et al.
of saffron carminative and appetite (2016)
stimulating and anxiety relieving
effects
– Aqueous 24 men – Decreases plasma levels of Barari et al.
extract malondialdehyde (MDA) and GPX (2017)
activities and inhibits tissue
damages
Carotenoid Crocin extract Type 2 diabetic male Blood glucose and HbA1c Reduces apoptosis of pancreas Ghorbanzadeh
Wistar rats tissue through reduction of p53 et al. (2017)
levels and decreases diabetes
related suffering
Carotenoid Crocin BV-2 and N9 cells PI3K/AKT inhibitor LY294002 Prevents oxidative stress through Yang et al.
digentiobiose PI3K/AKT signaling, suppresses (2017)

ester pro-inflammatory responses and
imparts neuroprotective and anti-
inflammatory effects
– Safranal STZ inducible diabetic Blood glucose, serum SOD, Improves blood glucose Delkhosh-
adult male Wistar rats catalase (CAT) and MDA concentration leading to metabolic Kasmaie et al.
and behavioral alterations, repairs (2018)
histopathological and biochemical
269
outcomes, improves learning and

corrects memory impairments
(continued)
270
Class of
– Hydro- 54 type 2 diabetic patients Blood glucose, triglycerides, uric Controls blood glucose, improves Milajerdi et al.
alcoholic acid and blood urea nitrogen blood and lipid profile components (2016)
extract of
saffron
Carotenoid Crocin in Wistar albino rats Plasma TNF-α, IL-1β and IFN-γ Lowers the plasma TNF-α and Hazman et al.
saline water IL-1β levels and also TNF-α and (2016)
IFN-γ levels in pancreatic tissue,
reduces oxidative stress, relieves
from hyper-insulinemia, hyper-
leptinemia, insulin resistance and
weight gain
– Hydro- RIN-5F and L6 myotube Serum glucose, cholesterol, Decreases serum glucose, Dehghan et al.
alcoholic cells triglycerides, low-density and very cholesterol, triglycerides, (2016)
extract of low-density lipoproteins, GLUT4 low-density and very low-density
stigmata and AMPKα genes lipoproteins, decreases insulin
resistance and glycated
hemoglobin levels but increases
GLUT4 and AMPKα expression
– Hydro- 54 type 2 diabetic patients Uric acid and blood urea nitrogen Lowers uric acid and blood urea Milajerdi et al.
alcoholic nitrogen and protects kidney (2017)
extract of
stigmata
– Hydro- 54 T2D patients – Improves blood glucose level, Milajerdi et al.
alcoholic induces anti-inflammatory and (2018)
extract of antioxidant properties
stigmata
– Hydro- 64 patients with type FPG, HbA1c, cholesterol, LDL-c Decreases FPG, HbA1c, Moravej Aleali
alcoholic 2 diabetes and LDL/HDL cholesterol, LDL-c and LDL/HDL et al. (2019)
extract of ratio and improves hyperglycemia

stigmata and lipid profile significantly
9
– Dried powder 80 T2D patients SBP Reduces SBP significantly, Ebrahimi et al.
of stigmata protects kidney (2019)
– 80% 64 T2DM patients FPG, HbA1c Reduces FPG, HbA1c Shahbazian
ethanolic et al. (2019)
extract of
stigmata
Carotenoid Crocin Adult male Sprague- – Reduces blood glucose levels, Samaha et al.
Dawely rats enhances pancreatic insulin (2019)
expression and secretion,
suppresses glucagen and oxidative
burden, modulates apoptotic
pathway and attenuates pancreatic
inflammation
– Methanolic Fifty Sprague-Dawley rats Blood sugar, lipids and Reduces blood sugar level, Ahmad et al.
extract of antioxidants improves lipid profile, enhances (2020)
stigmata antioxidant profile, exerts
antidiabetic and anti-
hyperlipidemic effect
– Dried powder 60 T2DM patients FBG and serum TNF-α Decreases FBG levels and serum Mobasseri et al.
of stigmata TNF-α, modulates glucose levels, (2020)
reduces inflammation through
reduced expression of
inflammatory mediators
271
of STZ and alloxan-induced free-radical damage and causes diabetes in experimental

animals/models (Jörns et al. 1999).
More recently, the role of oxidative stress in micro- and macro-angiopathy during
hyper-glycemia, hyper-insulinemia and dyslipidemia was investigated in-depth
(Matough et al. 2012). It was found that enhanced oxidative stress caused macro-
angiopathy wherein beta and endothelial cell were damaged and there was athero-
sclerosis. The beta cells have very low free radical quenching ability and are, thus,
very sensitive to ROS. When prolonged exposure to high glucose, leads to beta cells
dysfunction, the levels of free fatty acids (FFA) becomes elevated and cause mild-to-
severe damage of mitochondria. This then blunts the secretion of insulin. Moreover,
the over-expression and activity of ‘mitochondrial inner membrane uncoupling
proteins (UCPs)’ further contribute to an increase in superoxide radical formation
under diabetic conditions. NADPH oxidase is also a major source of ROS produc-
tion during diabetes and causes damage of all major groups of biomolecules (mainly,
proteins and lipids) resulting in mild to severe cardiovascular disorders. In short, the
free-radicals overload in a diabetic disrupts the normal glucose homeostasis resulting
in insulin resistance in peripheral tissues and consequent complications (Maritim
et al. 2003). A major therapeutic aim during the management of diabetes and
associated complications is, therefore, to improve the redox state in a being
(Yaribeygi et al. 2018a, b). Many experimental studies have been conducted to
this effect; and the management of ROS production in different vascular cells under
hyperglycemic condition has been demonstrated.
9.2 Prevalence of Diabetes and Saffron as a Potential

Antidiabetic Phyto-pharmaceutical
With the progressive rise in diabetic populations across the world, more than
366 million people are feared to become afflicted by the disease by 2030. India
and China are primarily at risk of becoming an epicenter of the global DM pandemic,
where every year, 62 million patients may become diabetic. In view of the
complications, potential risk and mortalities associated with diabetes, the need to
address the disease prophylactically and therapeutically has become imperative.
Since it is feared that most people will be affected by the type 2 diabetes mainly, a
combination of therapies are being envisaged for maximum efficacy. Different
researchers worldwide are striving towards the identification of phyto-therapeutic
targets capable of reducing complications such as oxidative stress-induced tissue
damages, insulin resistance and insulin deficiency for use as safe and efficacious
adjuncts to routine antidiabetic medication. Molecules capable of targeting diabetes
such as ‘Metformin’, ‘Januvia’ and ‘Orlistat’ have been identified and approved by
FDA in this regard. However, many more plant-based bioactives with hypoglycemic
effects are known in folk medicine and traditional system of healing; and are
languishing to be exploited. It is known that more than 1200 medicinal plants are
used around the globe to control DM. In traditional Indian system alone, records
reveal Asparagus racemosus, Butea monosperma, Coccinia indica, Catharanthus
roseus, Gymnema sylvestre, Syzygium cumini, Momordica charantia and Crocus

sativus as plants species with antidiabetic properties. It can be confidently endorsed
that the particular phyto-constituents (either many or few of them) present in these
medicinal plants are responsible for their antidiabetic properties. Although it would
be of interest to identify and study these in depth, yet, only a few of these (30%) have
been validated chemically and pharmacologically.
Amongst the different antidiabetic plant species known till date, saffron has been
recognized as an ‘adaptogen’ in Indian Ayurvedic medicine (Kianbakht and
Mozafari 2009). Mainly, the stigmata and their extracts have been employed by
different researchers to establish the therapeutic properties of saffron. For example,
the methanolic extract of saffron was found to have significant free radical scaveng-
ing activity (Kurechi et al. 1980) wherein the active ingredients were considered to
donate the hydrogen atoms for 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical stabi-
lization. In another study, Mohajeri et al. (2009) compared the glycemic properties
of ethanolic extract of stigmata (20, 40 and 80 mg/kg) versus the standard hypogly-
cemic drug, ‘tolbutamide’, in alloxan-induced mild and severely diabetic rats.
Regeneration of pancreatic islet cells and healing of damaged pancreas leading to
significant hypoglycemic effects were recorded after 14 days of intra-peritoneal and
oral administration of 40 mg/kg of the ethanolic extract. Significant reduction in
fasting blood glucose level but increase in the number of β-cells of pancreas, serum
insulin level and insulin immune-reactivity were also recorded in the diabetic rats
after 14 days. Importantly, regeneration of pancreatic islets with normal appearance
was recorded in the diabetic-rats after 14 days of ethanolic extract administration.
9.3 Antioxidant Activity of Saffron Towards Management

of Diabetes Mellitus
Oxidative stress leads to severe complications in a diabetic patient, whereas,

antioxidants offer a promising therapy for ROS-induced complications in diseases
like cancer, diabetes and cardio-vascular disorders (Seifried et al. 2007). Therefore,
the search for naturally available antioxidants has been gaining momentum in
different systems of medicine.
The antioxidant defense mechanisms in plants include the enzymatic pathway
involving ROS scavenging enzymes and also the non-enzymatic ROS scavengers
such as vitamins, carotenoids, flavonoids, etc. (Maritim et al. 2003).
ROS-scavenging enzymes are universally present in all plants but the
non-enzymatic plant antioxidants vary from species to species (Ahmad et al.
2010). In case of saffron, a number of antioxidant compounds have been identified
and shown to prevent oxidative damages in different tissues (Nikbakht-Jam et al.
2015; Yang et al. 2017; Rahiman et al. 2018; Yaribeygi et al. 2018a, b). The various
ingredients and antioxidants of saffron cannot only scavenge ROS but also stabilize
bio-membranes by decreasing the peroxidation of unsaturated membrane-lipids
(Papandreou et al. 2011). Crocin, crocetin and safranal, in particular, have hydroxyl
radical scavenging activity. These can also impose an inhibitory effect on the free
radical chain reactions produced during diabetes (Assimopoulou et al. 2005; Kanakis
et al. 2007; Halataei et al. 2011; Hariri et al. 2010). These facts were further endorsed
by a study of Samarghandian et al. (2013), where the levels of glucose,
malondialdehyde (MDA) and nitric oxide (NO) decreased while the glutathione
(GSH) content and catalase (CAT) and superoxide dismutase (SOD) activities
increased in the plasma after oral administration of safranal (0.25, 0.5 and
0.75 mg/kg/day) to STZ-induced diabetic rats for 4 weeks. The fact that safranal
possessed distinct anti-oxidant and hypoglycemic properties were evident from the
study. Yaribeygi et al. (2018a, b) also studied the effect of oral administration of
crocin (50 and 150 mg/kg), safranal (0.25 and 0.5 mL/kg) and also methanolic
extract of stigmata (80 and 240 mg/kg) on alloxan-induced diabetic rats. Their
findings, mainly, demonstrated the insulin-sensitizing activity of crocin after
6 weeks. Even crocetin was found to reduce the fasting blood glucose and HbA1c
levels, significantly while elevating the levels of blood insulin without hepatic and
renal toxicities as evident from no significant effects on blood serum glutamic
oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT)
and creatinine levels. In contrast, the untreated alloxan-inducible diabetic control rats
exhibited significant increase in fasting blood glucose and HbA1c levels but
decreased levels of blood insulin.
Modulation of the genes involved in the scavenging of free radicals in the
mitochondria was considered to be the underlying mechanism for the earlier-
mentioned properties of bioactive compounds of saffron. This upregulation of
antioxidant genes was also probably responsible for reduction in oxidative stress,
hyperglycemia and hyperlipidemia in experimental diabetic models (Hosseinzadeh
and Sadeghnia 2005), thereby, corroborating a variety of other studies, where saffron
bioactive compounds were shown to impart protection against oxidation-induced
tissue injuries (Premkumar et al. 2003; Samarghandian et al. 2010, 2011; Farahmand
et al. 2013).
Besides free radical scavenging, saffron also have the potential to treat many
complications associated with diabetes such as neurodegenerative disorders, mem-
ory impairment, ischemic retinopathy, age-related macular degeneration, coronary
artery disease, abnormalities in blood pressure, acute and/or chronic inflammatory
diseases, mild-to-moderate depression, seizure, perkinsonism, etc. In addition to
these, several other health-benefitting attributes of saffron have been demonstrated
in various animal models by different researchers (Table 9.1). These studies suggest
the role of bioactive compounds of saffron in the alteration of various molecular
mechanisms through transcription factors, growth factors and diverse intracellular
signaling pathways leading to prevention of diabetes-induced apoptosis in pancreatic
β cells and modulation of glycemia in diabetics (Samarghandian et al. 2016; Yang
et al. 2017; Yaribeygi et al. 2018a, b).
9.4 Prevention of Diabetes-Induced Apoptosis in Pancreatic

b Cells
Crocin is now known to suppress the tumor necrosis factor-α (TNF-α) and interleu-
kin-1β (IL-1β) levels in plasma and the TNF-α and interferon-γ (IFN-γ) levels in
pancreatic tissues to significantly improve insulin sensitivity (Li et al. 2018). Impor-
tantly, it was shown to prevent diabetes-induced apoptosis in pancreatic β cells via
the down regulation of the p53 protein and inhibition by B-cell lymphoma 2 (BCL2)
(Elsherbiny et al. 2016). BCL2 inhibits P53-induced apoptosis in pancreatic β-cells
and is thus identified as a modifier of p53 function (Chiou et al. 1994). BCL2 also
activates cytochrome c family (Cyt-c) proteins and plays a major role in cell
apoptosis (Garrido et al. 2006). Saffron bioactive compounds also inhibit the
production and activity of caspases family of proteins involved in various forms of
cellular death, apoptosis, necrosis, pyroptosis and, finally, β-cell destruction and islet
failure (Rojas et al. 2018). These bioactive compounds also downregulate
adiponectin, TNF-α and leptin expression in insulin-dependent cells both at the
protein and mRNA levels (Yaribeygi et al. 2019). Moreover, the bioactive
compounds of saffron are now known to serve as antagonist of a regulator of
pro-inflammatory mediator, that is, a nuclear transcription factor, κB (NF-κB) and
agonist of peroxisome proliferator-activated receptor gamma (PPAR-γ) (Zeinali
et al. 2019). These also downregulate the key pro-inflammatory enzymes and
monocyte chemo-attractant protein 1 (MCP) and IL-6 (Jungbauer and Medjakovic
2012).
9.4.1 Induction of Insulin Sensitivity and Improvement in Insulin

Signaling
During diabetes, the peripheral insulin-dependent cells (adipocytes, muscular tissues

and cardiomyocytes) become incapable of taking up circulating glucose. As a result,
there is development of insulin resistance (Röder et al. 2016). On the other hand,
saffron bioactive compounds have been shown to markedly increase the peripheral
insulin sensitivity by phosphorylation of acetyl-CoA carboxylase (ACC) and
mitogen-activated protein kinases (MAPK) (Kianbakht and Ghazavi 2011). It has
been also shown to lower free fatty acids and triglycerides in plasma through
downregulation of TNF-α to overcome the dexamethasone-induced insulin resis-
tance (Xi et al. 2005; Dehghan et al. 2016).
9.4.2 Glycemic Control and Use of Saffron in the Management

of Diabetes
The concentration of blood glucose after a meal or in other words, ‘glycemia’

distinguishes a diabetic from a non-diabetic. Hyper-glycemic levels in a diabetic
are a major health issue. Hence, bringing down the level of glucose in the
bloodstream through the administration of insulin is an important strategy for

controlling diabetes. In addition to insulin, several natural compounds are gaining
importance for their ability to control blood sugar or, in other words, ‘hypoglyce-
mia’. In this regard, the bioactive compounds as well as extracts of saffron stigmata
were reported to have strong hypoglycemic effects (Yang et al. 2003; Shirali et al.
2013). Studies also showed that these bioactive compounds have the ability to
stimulate the uptake of glucose by peripheral tissues while inhibiting its intestinal
absorption (Youn et al. 2004). Their ability to inhibit insulinase activity in both liver
and kidney (Achrekar et al. 1991), inhibit the production of endogenous glucose
(Eddouks et al. 2003), correct the problem of insulin resistance (Xi et al. 2005; Hu
et al. 2018), inhibit the re-absorption of renal glucose (Maghrani et al. 2005),
stimulate the β-cells of islets of Langerhans for the release of more insulin while
increasing its sensitivity to insulin (Xi et al. 2007) and also promote the regeneration
of healthy β-cells islets of Langerhans (Elgazar et al. 2013) were demonstrated in
various studies. Xi et al. (2007) further showed that crocetin can increase insulin
sensitivity and ameliorate abnormalities related to impaired glucose tolerance and
high-fructose diet-induced hyperinsulinemia in rats injected with dexamethasone.
Lowering of plasma glucose and insulin levels and improvement of serum glycemic
profile by crocin in diabetic animals (Rajaei et al. 2013; Shirali et al. 2013;
Tamaddonfard et al. 2014) and by crocin and safranal in alloxan diabetic rats
(Kianbakht and Ghazavi 2011) were also reported (Mohajeri et al. 2009; Arasteh
et al. 2010; Shirali et al. 2013). These studies demonstrated the positive effect of
ethanolic extract of saffron (20, 40 and 80 mg/kg) on alloxan induced diabetic or
healthy male rats without inducing any hepatic or renal toxicities. The extract not
only increased the number of β-cells in pancreas but also improved the secretion of
insulin from pancreatic β-cells. In addition, the extract (600 mg/kg) cured hypertro-
phy and hyperplasia in cells of islets of Langerhans in alloxan-induced diabetic rats
(Elgazar et al. 2013).
Furthermore, in a recent study, saffron-mediated enhanced glucose uptake cou-
pled with phosphorylation of AMP-activated protein kinase (AMPK)/ACC and
MAPKs leading to increase in insulin sensitivity in skeletal muscle cells were
demonstrated by Kang et al. (2012). However, possibilities that other mechanisms
by which saffron-bioactive compounds induce a hypoglycemic effect cannot be
overlooked.
9.5 Proposed Mechanisms by Which Bioactive Compounds

Impose Hypoglycemic Effects
9.5.1 Induction of Glucose Transporter Type 4 (GLUT-4)

Expression/Localization
Glucose enters into a cell through a membrane-bound receptor and the energy-
sensing AMP-activating protein kinase (AMPK)/ACC pathway. AMPK is a
phylogenetically conserved intracellular energy sensor that plays a central role in
the regulation of glucose and lipid metabolism to produce ATP through glucose
transport and glycolysis. It also maintains the energy balance in muscles by

suppressing glycogen synthase (GS) activity and inhibiting the anabolic synthesis
of glycogen. The AMPK/ACC pathway is activated with the phosphorylation of
AMPK followed by the phosphorylation of a number of downstream targets of
diverse pathways as mediated by ACC. The activated AMPK also phosphorylates
the MAPK family components for full activation of insulin-stimulated glucose
uptake via GLUT4 translocation and also for insulin entry through insulin receptors.
These then activate the PI3-kinase through the phosphorylation of protein kinase B
(AKT) to promote the translocation of more and more GLUT-4 into the insulin-
dependent tissues from an intracellular pool through IRs and PI3- kinase pathways.
These pathways, in turn, are negatively regulated by protein tyrosine phosphatase 1B
(PTP1B) to induce de-phosphorylation of insulin receptors substrate1 (IRS1) and
AKT to stop the entry of glucose into the cells but to enhance its circulation in
skeletal muscles. It is known that safranal acts as a potent inducer of insulin signal
transduction (IST) by inhibiting a negative modulator of IST, that is, the protein
tyrosine phosphatase 1B (PTP1B) via tyrosine-mediated de-phosphorylation of
insulin receptors (IRs). In fact, insulin-stimulation of PI3-kinase/AKT is a key step
in insulin-signaling mechanism and is negatively regulated by the mammalian
targets of rapamycin (mTOR) pathway. The negative regulation of the phosphoryla-
tion of mTOR and its downstream targets (p70S6K and 4EBP1) lead to protein
synthesis. In a parallel route, ACC is phosphorylated to activate the adipose triglyc-
eride lipase (ATGL)-mediated lipolysis and down-streaming of fatty acid metabo-
lism. As a result, the AMPK signaling pathway leads to more and more translocation
of GLUT-4 into the cell membrane and, thereby, improves the β-cell function for
stimulation of insulin secretion (Fig. 9.1). AMPK also phosphorylates ULK1 protein
kinase, a key initiator of the autophagic process.
Based on the aforementioned discussion, a hypothetical mechanism for the
hypoglycemic effect of bioactive compounds of saffron is being proposed on the
basis of ‘available reports’ and also ‘mechanism-of-action of standard antidiabetic
drugs in human beings. As per this mechanism, during DM and related
complications, bioactive compounds from saffron improve insulin signaling and
sensitivity by activating the AMPK/ACC pathway. This, in turn, controls a number
of processes like the inhibition of apoptosis by downregulation of P53 and
adiponectin, translocation of more GLUT 4 towards membrane for optimal glucose
uptake via TBC1D1, negative regulation of AKT by phosphorylation,
downregulation of TNF-α for suppression of inflammation pathway, inhibition of
ROS production by antioxidant enzyme activities, suppression of caspases activity,
inhibition of HbA1C and finally downregulation of leptin expression for elevating
insulin level (Fig. 9.2).
Fig. 9.1 Insulin production versus diabetes mellitus. (a) During low circulating glucose, there is
low ATP production but enhanced potassium efflux, thereby, causing hyperpolarization of
membranes due to low calcium influx and low to no release of insulin. This ultimately causes
hyperglycemia and type 1 DM. (b) In the presence of high circulating glucose, ‘glucose’ enters into
beta pancreatic cells and mitochondria through GLUT 2 receptors and is metabolized into ‘pyru-
vate’ by pyruvate kinase. The mitochondrial pyruvate is used for ATP/ADP biosynthesis to block
the inflow of potassium into cells by the ATP-sensitive potassium channels. This results in
decreased hyperpolarization of membranes but increase in calcium inflow into cells through
voltage-gated calcium channels. Next, insulin is released into blood stream through exocytosis or
fusion of insulin vesicles with membranes. The released insulin then lowers the blood sugar levels
and causes ‘hypoglycemia’
9.6 Conclusion
In conclusion, the stigmata of saffron have all the requisite properties of an

antidiabetic. According to the findings of different phytochemical and pharmacolog-
ical studies, these properties are mainly due to the active components, that is, crocin,
crocetin and safranal. Different studies have shown that these bioactive compounds
of saffron induce insulin sensitivity, improve insulin signaling and prevent β-cell
failure to exert hypoglycemic effects with almost no side effects. Even its aqueous,
ethanolic and methanolic extracts can significantly overcome oxidative stress-
mediated tissue damages, while improving the sensitivity as well as production of
insulin for the maintenance of hypoglycemia. In fact, these bioactive compounds in
Fig. 9.2 Hypothetical schematic diagram proposing the action of saffron bioactive compounds on
hyperglycemia during diabetes mellitus and related complications. (1) Bioactive compounds from
saffron improves insulin signaling and sensitivity by activation of AMPK/ACC pathway that
controls many other related metabolism; (2) inhibition of apoptosis by downregulation of P53
and adiponectin; (3) translocation of more GLUT 4 towards membrane for optimal glucose uptake
via TBC1D1; (4) negative regulation of AKT by phosphorylation; (5) downregulation of TNF-α so
as to reduce inflammation pathway; (6) inhibition of ROS production via increase in antioxidant
enzymes and reduced caspases activity (7) inhibition of HbA1C and downregulation of leptin
expression for elevating insulin level where AMP adenosine monophoshphate, AMPK/ACC
AMP-activated protein kinase/acetyl-CoA carboxylase, mTOR mammalian target of rapamycin,
ULK1 ULK1 protein kinase, ATGL adipose triglyceride lipase, HMG-Co A reductase, TBC1D1,
Rab GTPase-activating proteins; GS glycogen synthase, GLUT 4 glucose transporter type 4, P13K
phosphoinositide 3-kinase, AKT protein kinase B, IRs insulin receptor substrate, TNF-α tumor
necrosis factor-α, NF-KB nuclear transcription factor κB, IL-6 interleukin 6, MCP monocyte
chemo-attractant protein 1, PTP1B protein tyrosine phosphatase 1B, PPAR γ peroxisome
proliferator-activated receptor gamma, BCL2 B-cell lymphoma 2, Cyt-c cytochrome c, ROS reactive
oxygen species, HbA1C hemoglobin A1C, P phosphorylation, JNK c-Jun N-terminal kinase. (The
green-highlighted text in circles represents possible mechanisms by which saffron bioactive
compounds function during DM; red-highlighted text represents studies confirmed/reported by
different researchers)
saffron increase insulin transport into insulin-dependent cells through enhanced

GLUT-2 receptor translocation from the intracellular pool to the plasma membrane
and suppress insulin resistance, oxidative stress, inflammation and associated patho-
physiological conditions by activating various AMPK/ACC and AKT kinase-
mediated pathways. In addition, saffron is widely consumed as a spice and a
condiment. Therefore, it can be developed into safe and efficacious nutraceuticals for
use as adjuncts to routine antidiabetic medication; and also, as dietary supplements
to mitigate insulin resistance in pre-diabetic individuals. It is envisaged that this
information will pave the way for future research on the development of natural and
safe phyto-pharmaceuticals having strong antidiabetic properties.
Acknowledgements The authors thank the Director, CSIR-institute of Himalayan Bioresource

Technology, Palampur, India for infra-structure and support. Nitu Gautam thanks the Indian
Council of Medical Research (ICMR), Govt. of India, for providing Senior Research Fellow-
ship. The IHBT publication number 4767.
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IJBMS 22(4):334
New Insights to Enhance the Desired
Anti-Diabetic Compounds in Medicinal 10
and Aromatic Plants Exposed to Abiotic
Stress Factors
Canan Gulmez and Muhittin Kulak
Abstract
Diabetes mellitus is at the top of the twenty-first century world health agenda as a
hazard to human health and the worldwide economy. According to the latest
reports of the International Diabetes Federation, diabetes is affecting over
425 million people in 2017 and is expected to affect about 629 million people
by 2045. Diabetes prevalence varies depending on the development level of
countries, socio-economic status, life style, and eating habits of people. Due
to the side effects of synthetic or semi-synthetic drugs and limited accessibility
to proven drugs by rural people and people suffering from poverty, a tendency to
natural products has been significantly preferred. Hereby, the plants used for
treatments were then assayed for their pharmacological properties in order to
verify or test the “assumed” activities, which were then clearly reported to be
correlated or dependent on the secondary metabolites available. The revealed
findings pioneered the plant biologists to figure out the ways to enhance the
metabolites using manipulative abiotic stress treatments. Due to their sessile and
open system nature, plants cannot escape from stress and evolve sophisticated
adaptations. The received stimuli or signals cause significant changes in their
pharmacological properties and subsequently activities along with the alterations
in their metabolites. Herewith we sorted the chapter into two main sub-topics
including plant health and human being health, finalizing the chapter with the
Canan Gulmez and Muhittin Kulak contributed equally to the chapter.
C. Gulmez
Department of Pharmacy Services, Tuzluca Vocational High School, Igdir University, Igdir, Turkey
M. Kulak (*)
Department of Herbal and Animal Production, Vocational School of Technical Sciences, Igdir
University, Igdir, Turkey
Ltd. 2021
https://doi.org/10.1007/978-981-16-3529-8_10
286 C. Gulmez and M. Kulak
unclear responses for the two-faced questions regarding manipulating with the
abiotic stress factors for enhancing specific components or yielding high second-
ary metabolites, aiming at pioneering further studies.
Keywords
Abiotic stress · Anti-diabetic drugs · Diabetes mellitus · Exogenous treatments ·

Phenolics · Therapeutic approaches
10.1 Introduction: An Overview of Diabetes
Diabetes mellitus (DM) is at the top of the twenty-first century world health agenda
as a hazard to human health and the worldwide economy. DM is a chronic metabolic
disorder of the endocrine system with rapidly increasing prevalence worldwide. In
2015, 415 million people worldwide, 215.2 million men and 199.5 million women,
were DM with a prevalence of 8.8% (IDF 2015). According to the latest reports of
the International Diabetes Federation, in 2017, diabetes is affecting over 425 million
people and is expected to affect about 629 million people by 2045. Around 90% of
people with DM worldwide have Type 2 DM (T2DM) (WHO 1999), and 87–91% of
people with DM will be diagnosed as T2DM by 2045 (IDF 2017).
Diabetes prevalence varies depending on the development level of countries,
socio-economic status, lifestyle, eating habits, age, physical activity, and genetic
characteristics of people. While the worldwide prevalence of diabetes for all ages is
2.8%, it is estimated to be 4.4% by 2030 (Wild et al. 2004). In 2011, the prevalence
of diabetes for adults aged between 20 and 69 was 7.7%; it is thought that this will
rise to 9% by 2030 (Wou et al. 2019). The majority of people (269.7 million) with
DM live in urban areas (IDF 2015). DM will affect low- and middle-income
countries (77% of the diabetics) more in terms of treatment and prevention of the
disease as they have limited healthcare opportunities. By 2030, the prevalence of
diabetes is estimated to be 4.1% in Africa, 8% in Europe, 10% in Southeast Asia,
11.8% in North America, and 9.4% in South Central America (Wou et al. 2019). In
Turkey, while about three million patients with diabetes in 2000, in 2010 reached
3.679 million. Two community-based diabetes studies in 1998 and 2010, in the adult
population, diabetes increased by about 90% during this period and continues to
increase (WHO 2011). Turkey in the 20–79 age range is approximately seven
million diabetics in 2013 and has been reported to be about 15% of the total adult
population (Satman et al. 2002). The country carries almost 13% of the diabetes
burden in Europe (IDF 2013).
Undiagnosed cases cause more financial loss as well as serious complications
(IDF 2014). In 2015, globally US$ 673 billion, 12% of all health expenditure, was
spent on treating DM, the majority of which was the USA (US$ 320 billion) (IDF
2015). It is estimated that this economic burden will be 776 billion USD by 2045
10 New Insights to Enhance the Desired Anti-Diabetic Compounds in Medicinal. . . 287
(IDF 2017). In the USA, the total losses in Gross Domestic Product from 2011 to
2030 due to diabetes are estimated to be US$ 1.7 trillion. US$ 900 billion of this is
high-income countries and US$ 800 billion are middle- and low-income countries
(WHO 2016). In addition to the human burden, all these numbers represent a
significant economic burden for healthcare systems and countries, especially for
middle- and low-income countries.
Diabetes is caused by insufficient or no insulin production in the pancreas or
insulin not being used effectively by the body. Even though the classification of
diabetes is deemed to be quite complex, four major types of diabetes have been
reported and they are type 1 DM (T1DM), T2DM, gestational DM, and specified
types that depend on other causes (monogenic diabetes syndromes, exocrine pancre-
atic diseases, drug- and chemical-related diabetes, etc.). T1DM, which occurs as a
result of the effect of genetic, environmental and immunological factors, and insulin
hormone deficiency, is frequently seen in childhood and adolescence. T1DM, which
accounts for 5%–10% of diabetes cases, is characterized by T cell-mediated destruc-
tion of pancreatic β-cells and has autoimmune background. T2DM, the most com-
mon type of diabetes, accounts for approximately 90% of all diabetic cases. It occurs
when the pancreas does not produce enough insulin or body cells do not respond to
insulin. Also, T2DM has a high prevalence in both children and adults, and is highly
associated with lifestyle and many risk factors such as hypertension, dyslipidemia,
and obesity (Kharroubi and Darwish 2015). If this disease is left untreated, acute
fatal complications can occur in parallel with the characterized by diabetic ketosis
and coma depend on the increase in blood glucose. In addition to the fatal
consequences of macro and microvascular complications, dementia, sexual dysfunc-
tion, depression, and lower limb amputations are seen (Forbes and Cooper 2013).
10.2 Approaches to Current Anti-Diabetic Therapy Routes
Diabetes causes impairment in carbohydrate, fat, and protein metabolism

characterized by persistent hyperlipidemia (IDF 2017). Anti-diabetic agents control
the high circulating glucose levels by increasing insulin secretion and the use and
intake of glucose and decreasing glucagon release, glucose absorption from gastro-
intestinal system, and appetite (Choudhury et al. 2018). Various signal paths,
including nuclear factor-κβ (NF-κβ), transforming growth factor β (TGF-β), poly
ADP-ribose polymerase, interleukin-6 (IL-6), mitogen-activated protein kinase,
interleukin-1beta (IL-1β), and vascular endothelial growth factor play an important
role in diabetic complication processes (Chen et al. 2019; Fernandes et al. 2012;
Laddha and Kulkarni 2019; Papaetis et al. 2015).
There are three main classes of drugs in the control of diabetes. The first group
increases endogenous insulin availability, the second group increases insulin sensi-
tivity, and the third group decreases the digestion and bioavailability of
polysaccharides (Stranks and Meyer 2016). Recently, anti-diabetic drugs such as
metformin, sulfonylureas, glinides, thiazolidinediones, α-glucosidase, dipeptidyl
peptidase-4 (DPP-4) and sodium-glucose co-transporter 2 (SGLT2) inhibitors,
incretin-based agent-like glucagon-like peptide-1 (GLP-1) receptor agonists, and

insulin have been used (Hu and Jia 2019).
Many drugs such as acarbose, miglitol, and voglibose are used where cells are
resistant to insulin and these drugs reduce glucose absorption by disrupting carbo-
hydrate metabolism (Modak et al. 2007). Biguanides like metformin increase glu-
cose uptake from peripheral cells and decrease hepatic glucose production. It shows
its effect by inhibiting mitochondrial complex I, preventing ATP production, thus
activating AMP-activated protein kinase, related to the energy metabolism.
Sulfonylureas work as an insulin secretagogue for pancreatic cells and its action is
mediated by adenosine triphosphate-dependent potassium channels (Hu and Jia
2019; Turner 1998; Yokoshiki et al. 1998). The GLP-1 receptor agonists play an
important role in glucose homeostasis. The incretin mediates the effects of decreased
glucose by increasing glucose-dependent insulin secretion and decreasing glucagon
secretion (Neumiller 2011). Since endogenous GLP-1 is rapidly degraded by DPP-4,
DPP-4 inhibitors prevent this degradation and show a hypoglycemic effect. Another
hypoglycemic effect is demonstrated by SGLT2 inhibitors by increasing the glucose
excretion from the kidney (Kanai et al. 1994).
Diabetes relates to increased cardiovascular mortality with long-term
macrovascular consequences. The other morbidity and mortality result from
nephropathy (kidney damage), neuropathy (nerve damage), retinopathy (blindness)
that develop in diabetes (Atalay and Laaksonen 2002; Memisogullari et al. 2003).
The most important cause of cardiovascular deaths, 30%–45% of diabetic nephrop-
athy cases result in end-stage renal disease, renal failure, and coma (Pugliese 2014).
In almost half of the patients with diabetes, the disease is associated with hyperten-
sion, heart, and kidney disease (Dubois and Bankauskaite 2005). In this disease,
hyperglycemia generates superoxide radicals, increasing oxidative stress, and
initiating the pathogenesis of diabetes. This active oxygen species trigger the lipid
peroxidation and the formation of reactive products, causing serious damage to the
molecules and structure of the cell (Atalay and Laaksonen 2002; Lipinski 2001;
Memisogullari et al. 2003).
Although the oral hypoglycemic agents or insulin used in the treatment are
effective in controlling hyperglycemia, they have serious side effects and signifi-
cantly change the course of complications. Common effects include hypoglycemia,
weight gain, gastrointestinal disorders, peripheral edema, and liver disease (Mallare
et al. 2005). Treatment of diabetes using synthetic drugs (sulphonylurea derivatives
and glitazone compounds, etc.) can frequently fail to restore the glycemic index
(Iwaki et al. 2003). These drugs have adverse side effects such as inhibiting hepatic
regeneration, inducing obesity and osteoporosis (de souza et al. 2001; Iwaki et al.
2003; Meier et al. 2016; Turmelle et al. 2006). Again, although thiazolidinediones
(especially pioglitazone), which control the blood glucose level, prominently reduce
the risk of impaired glucose tolerance, they increase the risk of weight gain and
edema and cardiovascular disease in long-term use (Kole et al. 2016; Tripathy et al.
2016). In the light of all this information, considering the serious side effects and
efficacy of existing anti-diabetic agents, it is a fact that new treatment options and the
development of existing treatments are needed. In this context, plant-derived
products and supplements are of the treatments with reported less side-effect. In the
sections ahead, we will discuss the plants and their metabolites in the therapy of DM,
keeping with the alterations of secondary metabolites under abiotic stress conditions.
10.3 What Makes a Plant to be Medicinal and Aromatic

or Natural Product to be Evaluated for Therapy
of the Disorders?
Thousands of medicinal plants are used in the treatment of a range of diseases and
are aimed to treat many complications. The traditional utilization fields of herbal
medicines are cancer, musculoskeletal disorders, infectious diseases, metabolic
disorders, dermatological affections, gastrointestinal tract diseases, articular
problems, and other diseases related to urinary tract, respiratory tract, and central
nervous system (Choudhury et al. 2018). Plant-derived products with hypoglycemic
properties have been used as a folk remedy around the world since ancient times
(Eisenberg et al. 2003). There are many reasons for this, but perhaps the most
important; patients began to prefer natural products because of their limited
affordability and availability access to medicines facilities (Gong et al. 2018). Herbal
remedies attract considerable attention in the treatment of diabetes as a complemen-
tary or alternative. Although it has few side effects and provides good glycemic
control, it may depend on more than one factor in the selection of the herbs for
treatment. These include the plant’s safety, availability, affordability, and stage of
disease (Choudhury et al. 2018). Until today, some diabetic drugs known as
polyherbal formulations have been preferred by patients as herbal supplements.
As the prevalence of DM increases worldwide, a number of therapy programs
such as diet, exercise programs, oral anti-hyperglycemic agents, and insulin are
being implemented to improve the quality of life of people (Warren 2004). To
date, promising new therapeutic routes for diabetes include oral anti-diabetic peptide
delivery, nano-carrier delivery system, implantable drug delivery system and islet
cell transplantation (Hu and Jia 2019). The convenient, effective, safe, and econom-
ical therapeutic innovations for diabetes are important in controlling the disease and
improving the quality of care. Natural products are very inspiring resources for
treating diabetes. New anti-diabetic drugs called natural products (NPs) have been
emerged, which have relatively less toxicity and adverse effects. The World Health
Organization has determined that approximately 20% of the plants are used for
medical purposes universally. More than 1200 herbs have been used to control of
diabetes, and only 30% of them have been studied for their chemical and pharmaco-
logical effects (Alarcon-Aguilar et al. 2002; Bartolome et al. 2013).
Certain medicinal herbs like curcumin are used in pre-diabetes therapy
(Chuengsamarn et al. 2012) while cinnamon is a good option for diabetic patients
with hypertension (Howard and White 2013). On the other hand, cumin and Nigella
sativa regulate insulin secretion and have both anti-diabetic and anti-hyperlipidemia
properties (Meddah et al. 2009). In one review, herbs for diabetes are divided into
four different classes. These plants act in the following ways: regulate insulin
release, control insulin resistance, affect glucose absorption, and have multiple
effects on glucose regulation (Choudhury et al. 2018). Medicinal plants and their
components in vegetables, herbs, fruit, and other plant-based foods such as
phenolics, alkaloids, flavonoids, terpenoids, coumarins, glycosides which occupy
an important place in the literature; they act as modulators of β-cell proliferation,
survival, sedatives of the over-reactive immune response, insulin production
promoters, and regulators of β cells and immune cells (Chang et al. 2013). In a
review, clinical studies, pharmacological mechanisms, structure–activity relation-
ship, and phytochemistry of 90 phytochemicals and plant extracts published between
2004 and 2019 were investigated. In the study, the multiple action mechanisms of
plant compounds and extracts in the treatment of T1D were classified as immune
intervention, regulation of β-cell homeostasis, and simultaneous regulation of immu-
nity and β-cell homeostasis (Apaya et al. 2020). Likewise, flavonoids, terpenoids,
polyphenols, alkaloids, saponins, and quinones present are increasingly used in the
treatment of TD2. The potential mechanism of natural products against T2D is
through regulating the expression of various key proteins such as peroxisome
proliferator-activated receptor gamma, Janus kinase-dependent forkhead box protein
O1, extracellular signal-regulated protein kinase, glucose 6-phosphatase and NF-κβ,
and targeting specific proteins (Hu and Jia 2019; Kube et al. 2018; Xu et al. 2018:
Vezza et al. 2016).
When the data to date were examined, it was seen that polyphenol metabolites,
especially plant-derived phytochemical-compounds, were used in the treatment of
diabetes. Anti-diabetic activities of flavanol and flavan-3-ols metabolites, which are
among these metabolites, are to moderate glucose homeostasis by inhibiting key
enzyme activities related to diabetes, regulation of intracellular signaling pathways
and genes involved in glycogenesis and gluconeogenesis (Swen 2007).
Protocatechuic acid, an anthocyanin metabolite, is a well-known hypoglycemic
drug, which when given 2% for 12 weeks to diabetic mice, plasma sorbitol, fructose,
and carboxymethyl lysine levels were decreased. At a concentration of 4% were
reduced renal levels of type-IV collagen, fibronectin, protein kinase C activity,
enhance renal glyoxalase I and TGF-β1 (Chia-Yu et al. 2011). Ellagic acid had a
renal protective effect in diabetic rats by regulating the inflammation processes
through inhibition of NF-κB pathway (Ahad et al. 2014). Intravenous isoferulic
acid injection in streptozotocin-induced diabetic rats decreased plasma glucose level
due to anti-diabetic and anti-glycation effects via mediation β-endorphin (Aramsri
et al. 2015). Resveratrol has been shown to have an anti-diabetic effect in animal
models. It has been reported to show this effect by stimulating intracellular glucose
transport, increasing glucose uptake from various cells (Vallianou et al. 2013) and
insulin sensitivity in insulin resistance animal model (Szkudelski and Szkudelska
2015).
10.4 Not to Be Only Abided by the Modern Drugs But We Have

the Natural Products for Therapy of Diabetes Mellitus
Since Time Immemorial!
Most plants have adapted to land, namely terrestrial, aided by the seed and pollen
adaptations, being appeared between about 480 and 360 million years ago in the
mid-Palaeozoic era (Cheynier et al. 2013). Herewith the chapter, we mainly
concerned with terrestrial plant species, not aquatic ones. Among the biodiversity,
plants including angiosperms, gymnosperms, ferns, lycophytes, and bryophytes are
presented with 500,000 species (Corlett 2016). Of those plant species, it is estimated
that between 50,000 and 80,000 flowering plants are used for medicinal and aro-
matic purposes worldwide, especially in developing countries (Naguib 2011). The
uses of plants for multiple purposes date back to the time immemorial, being
recorded at Mesopotamian civilizations and are old as 2600 BC (Gurib-Fakim
2006). Imadi et al. (2016) report the uses of plants among Assyrian, Babylonian,
Chinese, Greek, and Hebrew civilizations (citing: Hamayun 2007). The selection of
the plants to be used for those purposes was probably based on the heuristic
approach by our prehistoric ancestors, relying on the senses of testing plants and
efficacy of the plants in the treatment of diseases. Accumulated indigenous knowl-
edge regarding the importance of the plants has passed from past to present (Valussi
and Scirè 2012), leading to the discovery of new drugs. It is worthy to note that due
to socio-economic facts or accessibility to the health facilities, people in rural areas,
especially in developing countries (Imadi et al. 2016), plants still constitute the
significant parts of the rural health system, namely traditional medicine, ethnomedi-
cine, ethnopharmacology, indigenous knowledge, etc.
10.5 The Uses of Plants for Ethnomedicinal Purposes Depend

on Phytochemicals Available
The plant kingdom produces a wide array of low molecular weight organic
compounds with an estimated number from 100,000 to one million (Fang et al.
2019; Erb and Kliebenstein 2020). Until 2000s, approximately 50,000 organic
compounds have been structurally elucidated (De Luca and St Pierre 2000). Those
compounds, in accordance with their assumed functions, have been classified into
three major groups, viz. primary metabolites, secondary metabolites, and hormones.
Biosynthesis pathways leading to the formation of secondary metabolites and their
interconnection with primary metabolites were presented in Fig. 10.1. Primary
metabolites including nucleic acids, proteins, lipids, and carbohydrates are directly
required for the plant growth and subsequently its proper development, constituting
a few parts of the metabolite group of the plants. Secondly, secondary metabolites,
also regarded as specialized metabolites or natural products, are confined to specific
plant taxa, exhibiting mediator role between plant and environment interactions. The
last group is the hormones, out of which regulatory roles in metabolism are well-
documented (Erb and Kliebenstein 2020). Along with the following parts of the
H2O CO2
Glycolysis
Carbohdrate
Shikimate pathway
Primary carbon
(Phosphoenolpyruvic acid
metabolism
→ phenylalanine)
Cinnamic acids
Carbohydrate phenylpropanoid metabolism Benzoic acids
metabolism (cinnamate→4–coumarate →
4–coumaroyl–CoA Ester of organic
acid
Malonyl-CoA
Flavonoids
Fig. 10.1 Biosynthesis pathways leading to formation of secondary metabolites and their inter-
connection with primary metabolites (according to Ryan et al. 1999; Michalak 2006; Khare et al.
2020, modified)
chapter, we will be interested in secondary metabolites. Herewith primarily, we will

present the basic definitions of alkaloids, phenolics and terpenoids, respectively,
with some examples of plants used or known for their anti-diabetic properties.
10.5.1 Alkaloids: Toxic But Beneficial in the Case of Optimal Dose

Treatment
According to the chemical structure, secondary metabolites include two major

groups. Those are nitrogen-containing compounds (alkaloid) and nitrogen-deficient
compounds (terpenoid and phenolics) (Patra et al. 2013). Of those molecules,
alkaloids with an estimation number of 12,000 molecules are astonishing interests
of the researchers due to their toxicity and medicinal properties (Verma and Shukla
2015), being a class of amino acid (viz. tryptophan, tyrosine, phenylalanine, lysine,
and ornithine)-derived nitrogen-including low molecular weight molecules (Moreira
et al. 2018; De Luca and St Pierre 2000). It is quite interesting that those metabolites
have dual functions, out of which the first function is to protect plant health in
response to environmental stress modulators endowing structural diversity for
alkaloids, which in turn exhibit significant biological roles for human being health
(Taha et al. 2009; Peng et al. 2019). Alkaloids exhibit a broad spectrum of potential
in medicine, plant protection, veterinary, and toxicology (Adamski et al. 2020).
Several reports revealed anti-cancer, anti-bacterial, anti-viral, and anti-fungal

activities of alkaloids (Adamski et al. 2020; Thawabteh et al. 2019).
10.5.2 Phenolics: Significant Route Regarding Carbon Flow Sensu

Lato
Phenolics compounds, containing an aromatic ring with a hydroxyl group, represent

a wide array class of secondary metabolites, with an estimation number of 9000
compounds (Waśkiewicz et al. 2013), being mainly synthesized from cinnamic acid,
which is consequence of phenylalanine conversion by phenylalanine ammonia lyase
(Michalak 2006). The biosynthesis is based on the two pathways such as shikimic
acid pathway and the malonic acid pathway (Patra et al. 2013). The significance of
the biosynthesis route is supported by the fact that 20% of the fixed carbons flow
through this route in normal conditions (Michalak 2006). Multiple functions have
been attributed to the phenolics in plants; and an augmented yield of phenolics was
observed in those plants exposed to different environmental factors and stressful
conditions (Moradbeygi et al. 2020; Vafadar et al. 2020; Gharibi et al. 2016). As
previously reported, the alterations in total phenolics or their individual fractions are
deemed as the consequences of adaptation behavior of the plants against dynamic
environmental conditions. As a commensalistic approach, those produced
metabolites were then used in the context of multiple trophic purposes by human
beings, viz. anti-aging, anti-inflammatory, anti-oxidant, and anti-proliferative
agents. Significantly, in addition to the hindering progress of long-term diabetes
complications, the inhibitory roles of polyphenols involving phenolic acids,
flavanoids, and tannins against α-glucosidase and α-amylase, the key enzymes
responsive to digest the dietary carbohydrates to glucose, have been well-
documented (reviewed by Lin et al. 2016).
10.5.3 Terpenoid: Volatile to Be Easily Diffusible Compounds
Terpenoids, also known as isoprenoids, are synthesized from five-carbon units of

isopentenyl pyrophosphate and dimethylallyl pyrophosphate (Sangwan et al. 2001),
constituting the largest class with most structurally diversified class of secondary
metabolites with an estimation of >40,000 molecules (Aharoni et al. 2006; Yazaki
et al. 2017). Significant roles with respect to the plant–abiotic environment
interactions, plant–plant communication, and plant–biotic environment interactions
have been attributed to the terpenoids (Pichersky and Gershenzon 2002). In addition
to their overarching and signaling robustness acts against the surrounding stimuli,
the multiple biological properties to be considered against many disorders have been
clearly reported (Luchesi et al. 2020; DeCarlo et al. 2020; Abdelkhalek et al. 2020).
Of the essential oil-bearing plants exhibiting potential against DM, Melissa
officinalis (Chung et al. 2010), Pelargonium graveolens (Boukhris et al. 2012),
Foeniculum vulgare (El-Soud et al. 2011), Salvia officinalis (Eidi et al. 2005),
Piper guineense (Oboh et al. 2013), Nigella sativa (Sultan et al. 2014), and Carum
carvi (Abou El-Soud et al. 2014) are of the medicinal plants.
Nothing stays the same as its former form and the changes are the nature of all
living and non-living things (Cetinkaya et al. 2017). Due to the dynamic but
complicated nature of plants as all living organisms, for growth and differentiation
at the cellular level of the plants, a resource including precise requirements such as
minerals, temperature, photo-assimilates, and growth-promoting substances is sine-
qua-non. Since the environmental conditions and nature of the plants involving
allocation of the sources between the organs or energy demanding with the maturity
are constantly changing, those alterations or needs cause diversion of notable
quantities of substrates from primary to secondary metabolism, being assumed that
there might be a cost of production regarding defense system of the plants under
limited resources (Cheynier et al. 2013). Those result in trade-off between oxidative
stress, growth, and survival at cellular and organ levels (Morales and Munné-Bosch
2016) with respect to the regulation of carbon fluxes between primary and secondary
metabolism, which is triggered with environmental stimuli (Cheynier et al. 2013).
Medicinal plants are characterized with the biologically active constituents pres-
ent, which exhibit stimulatory or regulatory acts on the physiology of the ailments.
We should hereby point important matter that the content and composition of the
chemicals is organ-, ontogenetic-, and diurnal-specified (Imadi et al. 2016), also
indicating that the chemicals available exhibit dynamic responses along with the
“stress” factors.
10.6 How to Define and Categorize the Stress Factors
The term stress in plants is defined as any external factors that hamper or totally
cease the plant growth, development, reproductive success, or survival rate (Rhodes
and Nadolska-Orczyk 2001). The stress is also clearly defined as any deviation from
optimal conditions that negatively influences plant metabolism (Lichtenthaler 1996).
Of the living organism, due to their entities, plants are sessile and are often and
simultaneously exposed to various and multiple environmental stresses (Khare et al.
2020), which are classified into major categories, namely abiotic and biotic stress
factors. While abiotic stress signals are characterized with temperature (heat and cold
[chilling or frost], salinity, water [drought or flooding, radiation [light, UV, ioniza-
tion radiation], chemical stress [mineral salts, gaseous toxins, pollutants, heavy
metals, pesticides, and aerosols] and mechanical stress [wind, soil movement, and
submergence]) (Mahajan and Tuteja 2005; Akula and Ravishankar 2011) (Fig. 10.2)
and biotic stress signals include viruses, nematodes, and bacteria.
In response to stress factors, plants have developed sophisticated regulatory
mechanisms in order to combat with the stress and subsequently to allow adaptation
to changing environmental conditions by modulating the global gene expression
(Kim and Kim 2020), which in turn cause alterations in the biosynthesis of the yield,
growth, and development of the plants and metabolites (Wang et al. 2003; Hirt and
Shinozaki 2003). Those responses, aiming at restoring or reconstruction of the
Water stress
(drought and
flooding) Radiation
stress (light,
Temperature
UV,
(heat and cold
ionization
(chilling and
radiation)
frost)
Plant
metabolism
Salinity Chemical stress
stress (low (mineral salts,
or high salt gaseous toxins,
content pollutants etc.
Mechanical
stress (wind,
soil movement,
submergence
Fig. 10.2 Abiotic stress factors resulting stress in plants but in case of excessive amount than
required (according to Mahajan and Tuteja 2005; Akula and Ravishankar 2011, modified)
homeostasis of the cell, are crucial for completing the life cycle and reach the full
genetic potential of the plants themselves (Rejeb et al. 2014). In the current scenario
of the instantaneous climate change, plant scientists and biotechnologists come
across challenges with respect to the researches regarding elucidation and revealing
the stress-induced metabolic changes for adaptation to the abiotic stress conditions
(Ahanger et al. 2017). The fact remains that the responses exhibit quite differences in
accordance with genotypes, populations, varieties, developmental stages of the plant
species as well as duration, severity, frequency, and type of the abiotic stress factors.
We, hereby, also should note that the augmented and experienced reactions by the
plants might have cause unknown and unexpected alterations in the next generations
or even during the later developmental stages of the plants when the plants face the
extended same or severe environmental stress periods.
10.7 Changes in Secondary Metabolites: How to Decide

the Right Conditions for Manipulative Treatments
Along with this section, we summarized the researches relating with the interaction
of stress factors—plants known to have anti-diabetic properties. Of the secondary
metabolites, the alterations in alkaloids, phenolics, and terpenoids were noted,
respectively, in response to some abiotic stress factors.
10.7.1 Alkaloids
Of the plant species used for therapy of DM, Catharanthus roseus (L.) has been
evaluated in traditional Chinese medicine in treatment of DM (Li et al. 2004) and
alkaloid extracts and its isolated compounds (namely, indoline, vindolidine,
vindolicine, and vindolinine) of C. roseus were documented to possess anti-diabetic
properties (Tiong et al. 2013). Furthermore, anti-hyperglycemic of mahanimbine
(carbazole alkaloid from Murraya koenigii leaves) was revealed (Dineshkumar et al.
2010). With respect to the exogenous manipulations on C. roseus, treatment of
drought with CaCl2 increased total indole alkaloid in shoot and root in comparison
with those of drought and well-watered plants (Jaleel et al. 2007a), proposing a
possible feasibility of CaCl2 treatment in alleviating the deleterious impacts of
drought coupled with augmented content of total indole alkaloid. In the same plant
species but treated with salinity stress, Jaleel et al. (2007b) examined interaction of
NaCl with CaCl2, reporting an increase in total indole alkaloid content in comparison
with those of stressed from NaCl and control plants. With the increasing levels of
salinity in growing media, higher total indole alkaloid content was recorded in
C. roseus (Jaleel et al. 2008). Regarding with the individual alkaloid compounds,
drought increased the content of alkaloid ajmalicine but the treatment of drought
with ketoconazole yielded more ajmalicine in C. roseus and the accumulation was
attributed to the oxidative stress burst (Jaleel et al. 2007c). Not being only confined
to the drought and salinity stress studies, similarly, treatments with chemicals,
elicitors, earth elements, and bioregulators also improved the indole alkaloid pro-
duction in C. roseus (Zhao et al. 2001).
10.7.2 Phenolics
Of the plants, out of which have been documented to exhibit anti-diabetic properties,
drought stress increased the total phenolic content of Tridax procumbens
(Gnanasekaran and Kalavathy 2017). Similarly, drought but depending on the
level of severity increased total flavanoid and phenolic content in three species of
Achillea (Gharibi et al. 2016). Moreover, with the severity of the drought stress
(100, 90, 60, and 30 field capacity), total phenolic and flavanoid content increased in
Amaranthus tricolor. Also, hydroxybenzoic acid, hydroxycinnamic acid, and indi-
vidual flavonoids exhibited significant increments under drought stress (Sarker and
Oba 2018), suggesting and reporting that farmers of semi-arid and dry areas might
have opportunities of growing the plants due to improvement in nutritional and
bioactive compounds. We should hereby note that in those relevant studies, the
plants or their extracts or fractions have not been screened or assayed for their anti-
diabetic properties, hence assuming that significant rises in total metabolites, indi-
vidual fractions, or diversification in the composition cannot equally pay role against
diseases unless the in vitro or in vivo activities were not assayed.
10.7.3 Terpenoid
With respect to the manipulative attempts for alterations in chemical content and
composition, moderate and severe water deficit improved S. officinalis essential oil
coupled with the increments in monoterpene hydrocarbons, ethers, aldehydes,
oxygenated monoterpene, esters, sesquiterpene hydrocarbons, and oxygenated
sesquiterpenes (Bettaieb et al. 2009). Similarly, significant changes in the yield
and composition of S. officinalis essential oil were also noted (Corell et al. 2009;
Chrysargyris et al. 2016), suggesting that the deficit irrigation may increase the
quality of the essential oil of aromatic plants. Concerning with salinity and
S. officinalis, salt concentrations (50 and 75 mM) were reported to have stimulatory
impacts on the essential oil production, specifically oxygenated monoterpenes
1,8-cineole and α-thujone (Taarit et al. 2009). Regarding M. officinalis, there were
no large quantitative changes in essential oil content under salinity and deficit
watering conditions (Ozturk et al. 2004). Farahani et al. (2009) and Abbaszadeh
et al. (2009) reported increases in essential oil content of M. officinalis L under water
deficiency. Likewise, more stress manipulating researches including water stress and
salinity (Rosmarinus officinalis; Sarmoum et al. 2019), water stress (Lavandula
angustifolia; Chrysargyris et al. 2016), salt stress (Lavandula angustifolia;
Chrysargyris et al. 2018), etc., have been reported.
The growing media conditions related to the temperature, light regime, and
nutrient supplies exhibit significant influences on the biosynthesis and accumulation
of the secondary metabolites. Corresponding to the abiotic and biotic stress elicitors’
manipulation, most of the reports have been addressed on extracting phenolics,
volatile oils, and fixed oils, as well as identification of the compounds using
chromatographic techniques. Then, distilled or extracted compounds were assayed
for their some biological activities, mostly in vitro. As well and detailed explained in
the review by Kleinwächter and Selmar (2015), the responses against stress factors
are sophisticated and multifarious. In that context, inductive acts of stress factors as
elicitors are multi-layered, meaning that the stress factors can result in severe
repercussions.
For that reason, the points to be addressed are;
• What kind and which levels of the stress factors do favor for the anti-diabetic
compounds?
• Are exogenous treatments possible or applicable for the enhancement of the

desired compounds?
• At which developmental stages should stress be applied in order to obtain the
maximal quality?
• Decision regarding conflicts of yielding or content of secondary metabolites?
In addition to the given points to be clarified, Selmar and Kleinwächter (2013)

and Kleinwächter and Selmar (2015) proposed questions in mind but pioneering the
forthcoming researches. Those are as follows:
• What to do in order to obtain the enhanced accumulation of metabolites without

high losses in biomass?
• At what stage of cultivation should stress applied?
• Where the metabolite is synthesized or accumulated, source–sink, at generative or
vegetative organs?
10.8 Unclear Answers to the Two-Faced Questions
We should clearly note that the resistance of the plants against biotic stress factors
generally depends on monogenic traits but the mechanisms of the plants in response
to the abiotic stress factors are multigenic and more complex. For that reason,
controlling and manipulating the mechanisms of the plants under abiotic stress
conditions are more difficult (Wang et al. 2003; Borsai et al. 2018). The response
of each plant species exhibits significant differences against the same stress factor. It
is also worthy to utter herein that most of the experiments are carried out at
controllable growing conditions, viz. growth chambers, greenhouses. The limited
field studies are few in comparison to those of controlled studies. Furthermore, most
of the field studies are based on the 2-year trials, not reporting the behavior of the
plants after three or more years. In the researches, up to our best readings and
knowledge, the maturity processes are mostly ignored and is not searched in detail
even the developmental stage, as an independent factor, is considered to be influen-
tial in responses of plants against unfavorable conditions in various reports.
10.9 Darkness of the Stress Memory, Trans-Generational

Memory, and Priming of Abiotic Stress Elicitors: Getting
Blurry in the Dark!
We can begin the section with the phrase the gauntlet has been thrown, meaning that
any manipulative attempts aiming at enhancing the desired content and quality of the
metabolites might cause unforeseen consequences. No scientific data available can
certainly claim and report the possible outcomes of stress on the next period of the
plants, but only can hypothesize the outcomes in accordance with observations
obtained. In the literature, most of the studies, even might be all, investigated the
impacts of single abiotic stress on single plant species. In that context, the stress-
experienced plants were examined for their physiological, biochemical, or metabo-
lite changes, but not monitored for their next generations. The effects of the stress on
the plant system might depend on the imprint previous stress episodes. This imprint
is also known as stress memory, which is deemed as the structural, genetic, and
biochemical alterations as a consequence of first stress exposure. It is considered that
the stress exposure has potential to prepare the plant against the same stress in the
possible future exposure. However, we should note hereby that there is no certain
evidence of encountering the same stress factor but possible to meet other stress
factors in future (termed as cross-stress tolerance). Having a previous imprint does
not equally pay that plants would exhibit the same behavior in response to being
exposed to recurrent stress, making the plants more tolerant, or more sensitive in
some cases (Fleta-Soriano and Munné-Bosch 2016). Due to the fact that the envi-
ronment is continually changing, in order to ensure the food global security and
plant-based raw material to be used in industry for multiple purposes, joint efforts by
plant biologists and biotechnologists are required in contribution to our understand-
ing of how plants and crops propound behavior under global climate change
scenarios (Morales and Munné-Bosch 2016; Tombesi et al. 2018). Even though
Scheffer et al. (2001) reported the theory predicting that abiotic stress, a single stress,
adversely influences the resilience of ecosystems, highlighting that a possible recur-
rent stress would result in hampered performance and loss of resilience of the plants,
Walter et al. (2011) underlines that the consequences of the reiterated abiotic stress
in comparison with a single stress or post-stress recovery are not still well and
completely clarified.
The questions perplexing the minds can be noted as:
• What happens to seeds of the plants after the plants are exposed to single or
double (recurrent) stress?
• What kinds of alterations might emerge when plants are exposed to single or
double stress through the normal life cycle of themselves? Does single or double
stress make difference?
• How do stress-experienced plants exhibit behavior regarding metabolite, we
herein concern on the metabolites beneficial in the treatment of diabetes, in
their next generations?
• Or interestingly, can we utter that with a phrase Once bitten twice shy, meaning
that a stress-experienced plant can have traumatic behavior in down- or
upbiosynthesis of the metabolites?
• Does being exposed to stress conditions make plant resistant or sensitive in the
forthcoming generations?
10.10 Conflicts to Be Annihilated: Intelligence of the Plants but

for Themselves Not for Other Livings, Allowing
a Commensalistic Relationship with Others?
Ecologically, a commensalistic relationship might be attributed to the symbiosis of

the plants and human beings. To clarify that, symbiosis literally means living
together, being also defined as the close relationship or interaction between
organisms of different species for an extended period. Of those symbioses, commen-
salism is as the case of that one species always benefits in the relationship, but the
other species might be unaffected, harmed, or helped (Audesirk et al. 2005). We
herein acknowledge that human beings do not harm as “parasitic” actors on plants
but benefit from the plants for different purposes, viz. therapeutic, aromatic, cuisine,
and cosmetics, doing that with their intelligence. Trewavas (2005) defines the
intelligence as an insight of sophisticated behavior but not a term applied for the
plants. For the plants, the term network is applied instead of intelligence. In order to
be equally flexible for the fitness against competitive circumstances, plants have to
activate their networks after processing the signaled information required for the
ultimate goal regarding sustainability of their reproductive success, not success
concerned with the desired metabolite accumulation for others’ uses.
10.11 Conclusion and the Way Forward
Along with the chapter, the main conclusion and points to be considered for the
plausible future studies were listed as follows:
• Pros and cons balancing relating yield and content of the secondary metabolites
are required since abiotic stress factors adversely, in most cases, decelerate or
cease growth, yield, and development of the plant.
• The source–sink behavior of the plants with respect to the secondary metabolite
accumulation should be monitored.
• Since nearly all researches on stress-metabolite have been performed at a certain
developmental stage of a plant life-span, for that reason, trans-generational
behaviors of the plants should be also pursued for the long-term sustainability
of yielding or content of secondary metabolites but with desired content. We
mean that stress factors might not pay equal in accordance with recurrent stress
possible in the future.
• As well-known that the efficacy or pharmacological impacts of plant-derived
natural products not only depend on total content, but also the interaction of the
major and minor components available. For that reason, stress-experienced plants
might be assayed for their anti-diabetic activities in comparison to extracts of non-
stress-experienced plants.
• Plant researches inside of themselves are multifarious and require detailed clinical
experiments on this complex issue but with strong and joint efforts of scientists.
• Despite the rapid growth in ethnopharmacology field in controlling diabetes,

well-designed clinical trials are still needed to determine the quality of existing
active pharmaceutical and efficiency of the final product. It is true that NP is a
potentially promising therapeutic option for diabetes but with more studies, the
mechanism underlying its clinical healing effects should be explained and the
necessary technology should be developed.
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Boosting of Bioactive Secondary
Metabolites in Anti-Diabetic Plants 11
Through Elicitation: A Simple Technology
for Better Future
Anamika Paul, Nehan Shamim, Anik Sarkar, Krishnendu Acharya,

and Nilanjan Chakraborty
Abstract
Medicinal plants are the key source of important metabolites that have a wide
range of applications in not only just pharmacological industry but also in various
other commercial purposes. Constantly increasing demand of various secondary
metabolites acquired from diverse group of plants has made researchers concen-
trate on the most efficient strategy for obtaining superior amount of valuable plant
products. The effective biotechnological strategies that have been implemented to
fulfilling the desired demand include transgenesis, metabolic engineering and
elicitation. Out of these three strategies, this chapter deals with elicitation as a
biotechnological tool for its eco-friendly nature and cost-effectiveness, for
elevating the production of plant secondary metabolites. The plants taken into
consideration are anti-diabetic for the reason that diabetes is one of the major life-
threatening diseases and is the cause of nearly 3.2 million deaths every year. A
comprehensive account of several anti-diabetic plants and the strategies
employed for increasing the accumulation of their hypoglycaemic and other
medicinally important chemical constituents have also been mentioned in this
chapter.
Keywords
Anti-diabetic · Biotechnological tools · Elicitation · Secondary metabolites
A. Paul · N. Shamim · N. Chakraborty (*)

Department of Botany, Scottish Church College, Kolkata, West Bengal, India
A. Sarkar · K. Acharya
Molecular and Applied Mycology and Plant Pathology Laboratory, Department of Botany,
University of Calcutta, Kolkata, West Bengal, India
Ltd. 2021
https://doi.org/10.1007/978-981-16-3529-8_11
308 A. Paul et al.
11.1 Introduction
Diabetes is a chronic and non-communicable disease, which has become a matter of

concern specifically in developing countries. Every year nearly 3.2 million people
die as a result of diabetes. In 2016, the World Health Organization described about
diabetes in their Global Report, ~422 million adults were detected with diabetes in
respect to 108 million diagnosed with diabetes in 1980. In the year 2012, 1.5 million
people marked as died due to diabetes. Moreover, the global prevalence of diabetes
has increased from 4.7% to nearly 8.5% in the last 40 years. It has been speculated
that Indians are genetically more prone towards diabetes and as every fourth Indian
is diagnosed as diabetic. Although the discovery of insulin therapy reduces the rate
of mortality caused by diabetes, the main constraint is the correction of insulin
deficiency, though yet not in insulin resistance. Moreover, the rise in the incidence of
several complications associated with insulin therapy has led to an increased interest
in traditional medicines that possess active constituents with hypoglycaemic effects.
Thus presently more researchers are involved in searching for plant-based drugs that
exhibit potential anti-diabetic activity without having any side effects (Aggarwal and
Shishu 2011).
Plants have been exploited due to the curative properties of their high-value
products ever since the mankind came into being. In ancient history, the Indian
Vedas, the Ebers Papyrus written in 1550 BC, the Pen T’Sao, written by the Emperor
of China, Shen Nung, in 2500 BC, the Bible and the Talmud contain records of the
plant used for the treatment of poor health. Furthermore, all the traditional healing
systems such as Ayurveda, Unani, Homoeopathy, Siddha and Chinese traditional
medicine are broadly dependent on the utilization of secondary metabolites pro-
duced by the plants (Kowalczyk et al. 2020). Due to ever increasing importance and
demand of these plant-based products, a number of strategies are being employed for
large-scale production of metabolites within a short time period. The diverse groups
of plant secondary metabolites are normally produced at a low concentration but
increases gradually upon encounter with various stimuli such as adverse environ-
mental conditions (Halder et al. 2019). The secondary metabolites can be introduced
by its classification into nitrogenous compounds (cyanogenic glycosides, alkaloids,
alcamides. Glucosinolates non-protein amino acids and amines) and
non-nitrogenous compounds (saponins, flavonoids, steroids and coumarins,
monoterpenes, diterpenes, triterpenes, tetraterpenes, sesquiterpenes) (Matsuura and
Fett-Neto 2015). Another term for such stimulus that causes an increase in the
production of defence compounds that eventually assists the plants in overcoming
the effects of stressful conditions are known as elicitors (Chandra et al. 2014). This
knowledge has led to the establishment of a simple and efficient biotechnological
tool, elicitation, with several abiotic and biotic elicitors for increasing the secondary
metabolites production. Apart from being an easy, simple and a cost-effective
procedure, elicitation has also been helpful in understanding the regulatory mecha-
nism of biosynthetic pathway giving new insights for in vitro interference, alteration
or manipulation of that specific pathway as per the requirements (Halder et al. 2019).
After the discovery of cell culture, it has been extensively utilized in favour of
11 Boosting of Bioactive Secondary Metabolites in Anti-Diabetic Plants. . . 309
controlled secondary metabolites production. The in vitro culture from different

parts of plants provided us with a possibility for not just giving rise to genetically
similar plants but also a new approach for the production plant secondary metabolite.
Cell suspension culture, callus culture and organ culture are the various types of
culture that are very often employed for obtaining an increased concentration of
plant metabolites. The combination of cell suspension culture with elicitation has
been found to be potent or an efficient strategy for increasing the accumulation of
commercially valuable plant products. Moreover, the advancements in plant cell
cultures made it possible to perform in vitro genetic transformation in cultures for
increasing the accumulation of plant secondary metabolites (Debnath et al. 2006).
The various biotechnological approach has been elaborately discussed in this chap-
ter; moreover, the use of these techniques for increasing the production of not just
hypoglycaemic agent but also other metabolites found in the anti-diabetically used
plants that were explicitly used as traditional medicines have also been emphasized
in this study.
11.2 Biotechnological Approaches Required for Increasing

Secondary Metabolites Production
Recently, the worldwide demand for plant-based products is increasing day by day
(Debnath et al. 2006). As the production of secondary metabolites has a wide range
of applications in the pharmacological industry, perfumery, cosmetics, nutrition,
dietary supplements, dyes, fragrance, flavours and as a variety of commercial
preparations that are available in the market. On the other hand, these are safe and
more dependable, i.e. exactly opposed to expensive synthetic products, which may
have adverse side effects (Naik and Al-Khayri 2016). To meet the demand, net worth
of global herbal market is growing at a rate of 7% per annum and is expected to be at
$5 trillion by 2050. Therefore, to dominating the international herbal market, every
year India and China exports nearly 32,000 and 120,000 tonnes of medicinal plants,
respectively (Debnath et al. 2006). However, the increasing commercial value of the
secondary metabolites has led to the overexploitation and loss of biodiversity. Apart
from this, other factors hindering the use of plants for therapeutic purposes are
unavailability due to seasonal restriction of plants, low productivity of the novel
compound, difficult isolation and purification procedures that are uneconomic
(Hussain et al. 2012; Halder et al. 2019). Therefore, to meet the increasing demand
for plant-derived chemicals new biotechnological methods and strategies are being
employed for better and higher yield (Debnath et al. 2006). The biotechnology of
plant tissue culture is a vital in vitro tool for enhancement of calli and cell production
that simultaneously increases secondary metabolite production of different plants,
such as vinblastine, vincristine alkaloids of Catharanthus roseus (Naeem et al. 2017;
Barrales-Cureño et al. 2018). Figure 11.1 is giving an overview of the pathway
beginning from the identification of important secondary metabolites up to choosing
310 A. Paul et al.
Fig. 11.1 Extraction and biotechnological manipulation of secondary metabolite production in

medicinal plants. (Diagram by A. Paul)
a biotechnological tool for improving the production in a schematic representation.

This collaboration may help to select an effective biotechnological method for
implicating a successive result. The major biotechnological approaches that are
highlighted for increasing metabolite production are discussed below:
11.2.1 Genetic Transformation
Transgenesis refers to use of various systems that causes the integration of a foreign
gene into an organism’s genome (Jouanin et al. 1993). It can be achieved by direct
methods such as polyethene glycol mediated transformation, particle bombardment
and electroporation or by using vector-mediated transformation. The most simple
and efficient method of genetic transformation is the Agrobacterium-mediated
transformation. Agrobacterium tumefaciens is known to infect wounded plant tissue
and this causes the formation of tumours or galls. For manipulating the plant
genome, the oncogenes encoding for phytohormones such as opine which is respon-
sible for tumour induction are removed. This step is followed by the insertion of
gene of interest along with a suitable marker in the T-DNA (transfer DNA), which is
inherited and expressed in the subsequent generations (Kowalczyk et al. 2020).
Another important example, hairy root cultures, is one of the most broadly used
strategies for bulk amount of secondary metabolites production (Gantait and
Mukherjee 2021). For this purpose, Agrobacterium rhizogene is used that induces
the formation of hairy roots upon infecting the plant tissue. These hairy roots after
excision are transferred to a culture medium in which it can be grown indefinitely
and cause an increase in the biomass at a higher rate. In hairy root cultures, the stable
integration of genetic material into host cells DNA is facilitated by rol (rolA, rolB
and rolC) genes of root inducing (Ri) plasmid of A. rhizogene. Hairy root culture is
mostly preferred for in vitro elicitation as they produce relatively bulk amounts of
biologically active compounds, grow faster without the requirement of plant putres-
cine, is the precursor of nicotine growth regulators and possess genetic and biosyn-
thetic stability (Halder et al. 2019; Kowalczyk et al. 2020). Cui et al. (2015)
introduced Catharanthus roseus genes encoding for geraniol 10-hydroxylase
(G10H) and strictosidine synthase (STR) separately and simultaneously in the
hairy roots of Ophiorrhiza pumila. They observed that in individual lines G10H
significantly improved camptothecin production in comparison to STR over-
expressing lines, suggesting that camptothecin accumulation dependent more on
the expression of G10H than that of STR. Moreover, the co-overexpression of STR
and G10H genes in the hairy roots caused a 56% enhancement in the production of
camptothecin as compared to the non- transformed hairy roots lines. Furthermore,
camptothecin is a potent topoisomerase I inhibitor belonging to a monoterpene
alkaloids group. Its derivatives such as topothecan and irinothecan are commonly
used for the treatment of various human cancers (Kowalczyk et al. 2020). Besides all
major advantages, only disadvantage of Agrobacterium mediated transformation is
the lack of origin of replication, so that the gene cannot expressed for production in
bacterial cells. To overcome this problem, the use of binary vector system has been
implemented, which involves the separation of vir and T-DNA regions into separate
replicons. To meet this difficulty, two autonomously replicating plasmids are used,
one is a binary vector that contains the T-DNA and origin of replication for
Escherichia coli and Agrobacterium, and another is the helper plasmid that contains
the vir genes. An example is the introduction of 6-hydroxylase gene of Hyoscyamus
muticus in Atropa belladonna by a binary vector system using, which led to an
312 A. Paul et al.
increase in the enzyme activity and up to fivefold higher accumulation of scopol-

amine in the transformed roots (Yun et al. 1992). Although the major drawback of
genetic transformation is the requirement of expensive equipments and expertise
(Rivera et al. 2014).
11.2.2 Metabolic Engineering
Metabolic engineering refers to in vitro manipulation of the genes encoding the rate-
limiting enzymes in biosynthetic pathway by their over-expression or silencing. In
favour of in vitro manipulation of any one or more enzymatic reaction of the
metabolic pathway, a comprehensive study of the biosynthetic pathway at gene,
transcriptomic, and proteomic levels is required. This approach involves either
blocking or increasing the metabolic flux to introduce a new metabolic pathway in
the plant for improving the accumulation of secondary metabolite or reducing the
production of unwanted compounds (Kowalczyk et al. 2020). The most successful
event of metabolic engineering was increasing the biosynthesis of β-carotene (pro-
vitamin A) in rice endosperm by overexpression of phytoene desaturase, phytoene
synthase and lycopene β-cyclase to produce the Golden rice. Other examples include
over-expression of the deoxyxylulose phosphate reductoisomerase (DXR) in mint,
Petunia chalcone isomerise (CHI) gene in tomato, tobacco putrescine N-
methyltransferase (PMT) gene in Atropa belladonna and Nicotiana sylvestris, and
the expression of cyanogenic glycoside biosynthesis genes from Sorghum bicolor in
Arabidopsis (Verpoorte et al. 2002). Yun et al. (1992) successfully increased the
production of scopolamine in Atropa belladonna by introducing the hydroxylase
gene from Hyoscyamus niger under the control of cauliflower mosaic virus 35S
promoter using Agrobacterium-mediated transformation. Scopolamine which is a
tropane alkaloid with anti-cholinergic activity is synthesized from hyoscyamine with
the help of the enzyme hyoscyamine 6β-hydroxylase (H6H), which catalyzes the
hydroxylation of hyoscyamine to 6β-hydroxyhyoscyamine, as well as the epoxida-
tion of 6β-hydroxyhyoscyamine to scopolamine. Another important finding noted
was that the scopolamine is made up of more than 92% of the total alkaloid content
of the leaf in the progeny produced by self pollination of the transformed plant. Apart
from this, metabolic engineering has also been employed for improving flower and
fruit colour, taste and flavour of the fruit, fragrance of the flower (Verpoorte et al.
2000). Another alternative approach to metabolic engineering involves using
microbes such as Escheriachia coli or Streptomyces venezuelae as hosts for the
reasons that they grow rapidly, are trouble-free to cultivate and transform, and most
of them already generate precursors of secondary metabolites, which is yet to be
fully explored. Although the development of new methods for gene sequencing did
make it easy to identify genes encoding enzymes in metabolic pathway but complete
elucidation of plant metabolic pathway is a major challenge for large-scale applica-
tion of metabolic engineering (Miralpeix et al. 2013).
11.2.3 Elicitation
Elicitation is considered as the most effective and widely used biotechnological tool
for large-scale production of plant-derived secondary metabolites or enhanced
biosynthesis. Elicitors are low molecular weight inducer compounds that, when
applied in small quantities to plants, result in enhancement in the biosynthesis of
secondary metabolites and other defence-related compounds (Chakraborty et al.
2013). This enhancement in the production of metabolites upon application of
trace amounts of inducers is known as elicitation. Upon perception of elicitors by
specific receptors there is an increase in the synthesis and accumulation of secondary
metabolites through initiation of signal transduction pathway that causes an eleva-
tion in gene level expression of enzymes and factors associated with the biosynthetic
pathway of the metabolite like nitric oxide (NO), etc. (Chakraborty et al. 2015;
Chakraborty and Acharya 2016). Elicitors can be classified into two types, where
abiotic elicitors are of non-biological origin and are grouped into chemical, physical
and hormonal factors (e.g. are temperature, salinity, UV radiation, drought, heavy
metals, sodium chloride, potassium chloride, polyvinyl pyrrolidone, ethylene,
jasmonic acid, methyl jasmonate, salicylic acid, acetylsalicylic acid, etc.)
(Chakraborty et al. 2016) and the other type is biotic elicitors that are substances
of biological origin that consists of either crude extracts or partially purified products
of bacterial or fungal origin (e.g. are fungal homogenate, fragments of pectin, chitin,
glucans, glycoproteins and inactivated enzymes) (Chakraborty and Acharya 2016).
Furthermore, elicitors are classified into endogenous (originated inside the cell) and
exogenous (originated outside the cell) elicitors on the basis of their origin (Namdeo
2007). In Fig. 11.2, we can summarize the classification according to specific basis
and the mechanism of action of elicitors. After receiving the signal from the elicitor,
receptor present in the cell membrane can induce signal transduction cascade by
regulating the gene expression and the alteration of ion channels. On one hand, the
ion induces activation of several defence related pathways. On the other hand,
enzymes produced by altering or inducing gene expression can either catalyse
secondary metabolites biosynthetic pathways or enhances signalling molecules
(salicylic acid, jasmonic acid, methyl jasmonate). Though, these signalling
molecules and metabolites increase transcription factors synthesis that efficiently
regulate gene expression (Namdeo 2007; Baenas et al. 2014; Chakraborty et al.
2019; Halder et al. 2019). Whereas, in every elicitation reaction, the type of elicitors,
concentrations, time of exposure and treatment schedule are used as important
parameters. Elicitation has often been employed with cell suspension culture and
hairy root culture to increase the secondary metabolites accumulation in each of root
or cell obtained in the culture (Ramakrishna and Ravishankar 2011; Wang and Wu
2013; Halder et al. 2019). In vitro production of secondary metabolites can be
obtained by one-step or two-step culture systems. In a one-step system, the growth
of cells, as well as the production of secondary compounds, are carried out in the
same medium whereas in the two-step system the cells are grown in one medium,
which are later transferred into the production medium (Gonçalves and Ramano
2018). Shahin (2018) experiment the elicitation of vinblastine and vincristine on
314 A. Paul et al.
Fig. 11.2 Schematic representation of structural and functional understanding of elicitors. (OA
oxalic acid, AA arachidonic acid, SA salicylic acid, AS acetyl salicylate, INA isonicotinic acid, JA
jasmonic acid; MeJA methyl jasmonate, NO nitric oxide, ROS reactive oxygen species, RNS
reactive nitrogen species, MAPK mitogen-activated protein kinase, TFs transcription factors, ISR
induced systemic resistance, SAR systemic acquired resistance). (Diagram by A. Paul)
callus culture of Catharanthus roseus using methyl jasmonate and silver

nanoparticles, resulted in highest production of vinblastine was obtained in case of
75 mg L1 concentration of silver nanoparticles and 50 μM methyl jasmonate gives
best result for both vinblastine and vincristine.
These all are effective biotechnological methods for secondary metabolites
enhancement. Moreover, elicitation is an eco-friendly and cost-effective process
(Ramirez-Estrada et al. 2016). Among these all strategies, elicitation is such a
method where elicitors have ability to control the cellular activities by both bio-
chemical and molecular level by up-regulating gene expression. At the same time, it
is the specific method that triggers defence responses in plants against a range of
pathogens, Here, we are focusing on higher level of secondary metabolites accumu-

lation by biotechnological strategies, but the elicitation method is more acquainted
for plant secondary metabolite production enhancement and increasing defence
response (Zhao et al. 2005).
11.3 Relationship Between Diabetes and Medicinal Plant
Evidence exists that plants have been discovered and used on behalf of medicinal
purposes since prehistoric times. Ever since the first isolation of secondary
metabolites in 1803, efforts are being made to explicate the structure, function,
mechanism of action and the biosynthetic pathway of those compounds. Hence,
the compounds associated with the health benefits have been modified for exploita-
tion (Kowalczyk et al. 2020). Nearly, 422,000 flowering plants have been reported
from all over the world and ~ 50,000 of them are being used as medicinal plants.
Interestingly, among the total number of flowering plants found in India, 43% of
them have immense medicinal properties. Even to this date, a number of the tribal
communities depend extensively on the use of medicinal plants for treating ailments,
like the tribal communities of Chhota Bhangal, Western Himalaya reported the use
of Aconitum heterophyllum for stomach ache and fever, Artemisia sieversiana as
abortifacient, Bergenia ciliate to cure kidney stones and Rumex nepalensis as anti-
allergic (Uniyal et al. 2006). On the other hand, the previous acquaintance restricted
the secondary metabolites as low molecular weight compounds that were regarded as
waste products because they lack a fundamental role in the plants’ normal growth,
development, and reproductive cycle (Verpoorte 1998).
For studying the widely spreading non-communicable diseases, diabetes mellitus
is a major endocrine disorder characterized by increased blood sugar levels
(hyperglycaemia) due to defects in insulin action and secretion for majority of the
peoples in any countries (Govindappa 2015). The complication associated with
diabetes includes weight loss, blurred vision, polyuria, renal failure, ulceration,
cardiovascular disorder, neuropathy and nephropathy (Alberti and Zimmet 1998;
Bastaki 2005; Govindappa 2015). Diabetes can be categorized into two types:
1. Type 1 diabetes is insulin-dependent and occurs due to deficiency in the secretion

of insulin. This form of diabetes results from autoimmune destruction of β-cells of
islets of Langerhans. Nearly 5%–10% of diabetes occurring in a population is
type 1 diabetes.
2. Type 2 diabetes is non-insulin-dependent diabetes caused due to insulin resis-
tance. Impairment in the insulin secretion or insulin resistance along with increase
in production of hepatic glucose is observed. It does not involve autoimmune
destruction of β-cells and is the most common form of diabetes. About 90%–95%
cases of diabetes are type 2 diabetes and obesity is the major risk factor of it
(Govindappa 2015; Ota and Ulrih 2017; Gupta et al. 2018). Gestational diabetes
is another type of diabetes that arises due to glucose intolerance during preg-
nancy. Women with gestational diabetes are at a higher risk of developing type
316 A. Paul et al.
Fig. 11.3 Schematic representation of glucose uptake regulation in healthy person, type 1 and type
2 diabetes. (a) In healthy person, insulin normally produce binds with insulin receptor to activate
normal level of glucose uptake through glucose transporters (GLUT). (b) In type 1 diabetes patient,
pancreas fails to produce sufficient insulin, which in turn inactivate glucose transporters (GLUT).
(c) In type 2 diabetes patients, pancreas produces enough amount of insulin, but receiving cells fail
to response against insulin, resulting in inactivation of insulin receptor and GLUT. (Diagram by
A. Paul)
2 diabetes in future (Gupta et al. 2018). Difference between the mechanism of

type 1 and type 2 diabetes with the healthy cell are represented in Fig. 11.3.
The equilibrium between the uptake and release of the glucose is maintained by
the liver through the process of glycogenesis, glycogenolysis and gluconeogenesis.
Furthermore, the pancreatic β-cells of islets of Langerhans are responsible for
secreting insulin, a peptide hormone, essential for the transport and absorption of
glucose from the blood. Insulin promotes the rate of glycogenesis (synthesis of
glycogen) and increase the utilization of glucose by the tissues. Elevation in insulin
level is associated with a decrease in activity and mRNA levels of glucose-6-
phosphatase which is a key enzyme of gluconeogenesis. The transport of glucose
out of the liver is facilitated by glucose transporters (GLUT), an integral membrane
protein with 12 membrane-spanning domains. GLUT 2 is responsible for the glucose
transport out of the liver. As the affinity of GLUT 2 for glucose is lower than other
glucose transporters such as GLUT 1, GLUT 3 and GLUT 4, it is more suited for
maintaining glucose homoeostasis (Nordlie et al. 1999). Once glucose is transported
out of the liver, insulin mediates the translocation of GLUT 4 from the cytoplasm to
the plasma membrane (Zierath et al. 2000). The patients with type 1 diabetes need
insulin therapy for survival, whereas type 2 diabetes depends on the use of
hypoglycaemic agents for correction. Several complications are associated with
insulin therapy, hypoglycaemia and weight gain being the most common. Other
complications associated with these are insulin allergy, insulin resistance, atrophy
(decrease in cell size) at the site of insulin injection and insulin edema (Bastaki
2005).
Before the discovery of insulin therapy, the treatment of diabetes involved
maintaining the diet of the person and extracting the hypoglycaemic agents present
in the plants by crushing the different parts of plants in various solvents. The
traditional treatment involved consuming vegetables such as Momordica charantia,
Allium cepa, Brassica oleracea, Brassica rapa, Letuca sativa, Phaseolus vulgaris,
Pisum sativum, berries of Juniperus communis and using decoction of leaves Viscum
album, Medicago sativa, Eucalyptus globules, Agrimony eupatoria.(Swanston-Flatt
et al. 1991; Hussain et al. 2012). In addition to this, a number of mushrooms such as
Agaricus subrufescens, Agaricus bisporus, Pleurotus spp., Coprinus comatus,
Cordyceps sinensis, Ganoderma lucidum, Phellinus linteus, Inonotus obliquus,
Sparassis crispa and Poria cocos showed anti-diabetic and glucose-lowering effect.
Due to the ill effects of consumption of mushroom beyond the safe limit and
difficulties associated with mushroom cultivation, the use of plants for therapeutic
purposes has been favoured widely from many years (Bano et al. 1988; De Silva
et al. 2012). Along with these various other anti-diabetic plants and their extracts
have been found to effective in lowering blood sugar level has been shown in
Table 11.1.
11.4 Enhancement of Secondary Metabolites Production

of Anti-Diabetic Plants: At a Glance
After finding of the advantages of medicinal plants, commercial cultivation of

medicinal plants was extensively used prior to obtain uniform, high-quality plant
product. Although apart from being time consuming and laborious, the major
difficulties of systematic cultivation are the seasonal restriction of plants and large-
scale damage to the plants due to various fungal and bacterial diseases (Alamgir
2017). Nevertheless, plants cell and tissue culture are an alternative in vitro method
for producing useful secondary metabolites when the conventional and traditional
Table 11.1 Medicinally important anti-diabetic plants with their extracts containing anti-hyperglycemic activity
318
Name of the anti- Extract used against

Family diabetic plants Medicinal properties diabetes References
Acanthaceae Andrographis Anti-bacterial, anti-fungal, anti-viral, Hot water and Hossain et al. (2007), Mishra et al.
paniculata (Burm. F.) choloretic, hypoglycaemic, ethanolic extracts of (2007) and Ganeshkumar et al.
Wall ex. Nees hypocholesterolemic, adaptogenic, anti- plant (2019)
inflammatory, astringent, diuretic, anti-
helminthic, anti-pyretic
Barleria priontis L. Hepatoprotective, anti-fungal, anti-oxidant, Alcoholic extract of Dheer and Bhatnagar (2010) and
diuretic, anti-diabetic roots and leaves Gupta et al. (2018)
Amaranthaceae Aerva lanata (L.) Juss. Anti-parasitic, anti-microbial, anti- Alcoholic extract of Vetrichelvan and Jegadeesan
ex Schult. inflammatory, diuretic, hypoglycaemic whole plant (2002) and Gupta et al. (2018)
Apocynaceae Catharanthus roseus Anti-oxidant, anti-viral, anti-tumour, anti- Dichloromethane Tiong et al. (2013) and Das and
(L.) G. Don bacterial, anti-diabetic, anti-hypotensive, extract of leaves Sharangi (2017)
anti-helminthic, anti-neoplastic
Asteraceae Elephantopus scaber Diuretic, anti-diabetic, anti-cancer, anti- Aqueous extract of Daisy et al. (2007) and Gupta et al.
L. pyretic, astringent leaves (2018)
Smallanthus Anti-oxidant, anti-diabetic, anti- Methanolic extract of Genta et al. (2010) and Gupta
sonchifolius (Poepp.) inflammatory, hepatoprotectant leaves et al. (2018)
H. Rob.
Taraxacum officinale Anti-diabetic, anti-viral, anti-tumour, detox Aqueous and ethanolic Nnamdi et al. (2012) and Gupta
(L.) weber ex agent exatract of roots and et al. (2018)
F.H. Wigg leaves
Vernonia amygdalina Hypoglycaemic, hepatoprotectant, anti- Ethanolic extract of Ong et al. (2011) and Gupta et al.
Delile bacterial, anti-oxidant, anti-fungal leaves (2018)
Cucurbitaceae Citrullus colocynthis Anti-inflammatory, anti-bacterial, analgesic, Ethanolic extract of the Dallak et al. (2009) and Gupta
(L.) Schrad. anti-diabetic pulp et al. (2018)
Cucumis Diuretic, anti-diabetic, hepatoprotective, Aqueous extract of the Salahuddin and Jalalpure (2010)
trigonus Roxb. analgesic fruit and Gupta et al. (2018)
A. Paul et al.
11
Mukia maderaspatana Vasidilator, diuretic, anti-asthmatic, Methanolic extracts of Wani et al. (2011) and Gupta et al.
(L.) M..Roem. hypotensive, anti-inflammatory, anti- the root (2018)
diabetic
Euphorbiaceae Phyllanthus emblica L. Anti-mutagenic, anti-oxidant, anti- Aqueous extract of Khan (2009) and Gupta et al.
microbial, hepatoprotective, anti-diabetic fruit (2018)
Phyllanthus niruri L. Anti-oxidant, anti-microbial, anti-viral, anti- Aqueous extract of the Okoli et al. (2010) and Gupta et al.
cancer, hepatoprotective, hypoglycaemic leaf (2018)
Ricinus communis L. Analgesic, anti-inflammatory, anti-oxidant, Ethanolic extract of the Shokeen et al. (2008) and Gupta
anti-hyperglycaemic root et al. (2018)
Fabaceae Acacia auriculiformis Anti-bacterial, anti-fungal, anti-oxidant, Acetone extract of bark Sathya and Siddhuraju (2012) and
A.Cunn. Ex Benth. anti-diabetic Gupta et al. (2018)
Cassia auriculata Anti-arthritic, anti-fungal, anti-bacterial, Ethanolic extract of the Gupta et al. (2010, 2018)
(L.) Roxb. anti-inflammatory, anti-diabetic flower
Gentianaceae Swertia chirata Buch.- Anti-bacterial, anti-fungal, analgesic, anti- Ethanolic extract and Grover et al. (2002)
Ham. ex Wall. pyretic, hepatoprotective, anti-diabetic hexane fraction of the
plant
Graminaceae Zea mays L. Diuretic, anti-depressant, anti-fatigue, anti- Ethanolic extract of the Okokon and Nyong (2018)
hyperlipidemic, anti-diabetic grains
Lamiaceae Ocimum sanctum L. Analgesic, anti-diabetic, anti-microbial, anti- Alcoholic extract of Rizvi and Mishra (2013)
oxidant, anti-cancer leaves
Lilaceae Allium sativum L. Cardiovascular syndrome, hyperlipidemic, Ethyl acetate extract Younas and Hussain (2014) and
hypotensive, anti-diabetic root and leaf Gupta et al. (2018)
Allium cepa L. Anti-mutagenic, anti-cancer, anti-oxidant, Ether extract of the root Grover et al. (2002) and Gupta
Boosting of Bioactive Secondary Metabolites in Anti-Diabetic Plants. . .
hypoglycaemic, hypocholesterolemic tuber et al. (2018)

Menispermaceae Tinospora cordifolia Anti-oxidant, anti-inflammatory, Aqueous extract of root Rajalakshmi et al. (2009) and
(Thunb.) Miers hypoglycaemic, hepaprotective, Gupta et al. (2018)
cardioprotective, anti-diabetic
Moraceae Ficus amplissima Rees. Anti-diabetic, anti-oxidant, anti-bacterial, Methanolic extract of Arunachalam and Parimelazhagan
anti-inflammatory, hypolipidimic the bark (2013) and Gupta et al. (2018)
319
(continued)
320
Name of the anti- Extract used against

Family diabetic plants Medicinal properties diabetes References
Ficus religiosa (L.) Anti-bacterial, anti-viral, anti-diarrheal, anti- Ethanolic extract of Choudhary et al. (2011) and
diabetic, astringent fruit Gupta et al. (2018)
Myrtaceae Callistemon Anti-hyerglycaemic, astringent Dicholoro-methane Kumar et al. (2011) and Gupta
lanceolatus (Sm.) extract of leaf et al. (2018)
sweet
Psidium guajava L. Analgesic, anti-bacterial, anti-oxidant, anti- Ethanolic extract of the Mukhtar et al. (2006) and Gupta
hyperglycaemic, hepatoprotectant bark et al. (2018)
Syzygium jambolanum Anti-bacterial, anti-fungal, anti-oxidant, Ethanolic extract of Ravi et al. (2004) and Gupta et al.
(Lam.) DC. hypoglycaemic seed (2018)
Ranunculaceae Nigella sativa L. Hypocholesterolemic, anti-oxidant, anti- Oil of the plant Benhaddou-Andaloussi et al.
diabetic, anti-inflammatory effects (2011)
Rhamnaceae Ziziphus mauritiana Anti-microbial, diuretic, diuretic, anti- Ethanolic extract of Bhatia and Mishra (2010) and
Lam. inflammatory, sedative, anti-diabetic fruit pulp Gupta et al. (2018)
Rosaceae Agrimonia eupatoria Anti-oxidant, diuretic, astringent, anti- Aqueous extract of leaf Gray and Flatt (1998) and Gupta
L. diabetic et al. (2018)
Umbelliferae Coriandrum sativum L. Hypoglycaemic, anti-inflammatory, anti- Ethanolic extract of the Chithra and Leelamma (1999) and
oxidant seed Gupta et al. (2018)
Zingiberaceae Zingiber officinale Anti-bacterial, anti-viral, anti-fungal, anti- Aqueous extract of the Akhani et al. (2004) and Gupta
Roscoe oxidant, hypoglycaemic, cardiotonic root et al. (2018)
A. Paul et al.
methods turn out to be unfeasible. It refers to the mass production of plants under
aseptic conditions in a relatively short time period (Debnath et al. 2006; Hussain
et al. 2012). Furthermore, there are several plant secondary metabolites found with
their active medicinal activities, like Callistemon lanceolatus is known for its
anti-diabetic secondary metabolites such as three new flavones (5,7-dihydroxy-6,8-
dimethyl-40 -methoxy flavone, 8-(100 -hydroxyisopranyl)-5,6-dihydroxy-7,4-
0
-dimethoxy flavone and 8-(2-hydroxypropan-2-yl)-5-hydroxy-7-methoxy-6-
methyl-40 -methoxy flavone) and two phenolic esters, 2,3,4-trihydroxyphenethyl
tetracontanoate-4-β-xylopyranoside and 2,3,4-trihydroxyphenethyl tetracontanoate
(Nazreen et al. 2012, 2014). Whereas, some of the important plant-based drugs that
are frequently used by us includes caffeine with its stimulant properties; morphine to
treat severe pain; quinine used due to its antimalarial properties and bitter taste;
emetine and cephaeline as antidotes for intoxication; the anti-tumorals vincristine,
vinblastine and camptothecin; anti-arrhythmic ajmaline; antimicrobials berberine
and sanguinarine; antihypertensives serpentine and ajmalicine; vasodilator papaver-
ine; antitussive noscapine; the muscle relaxant tubocurarine and so on (Matsuura and
Fett-Neto 2015). On the basis of our extensive literature study, we have discussed
about anti-diabetic plant extracts previously in Table 11.1. Although many plants are
known to posses antihyperglycaemic activity but the standardization of their active
constituents is yet to be achieved (Aggarwal and Shishu 2011). Hence, below we
have emphasized on the plants with their hypoglycaemic constituents and mecha-
nism of action (Table 11.2).
Although, for escalating the plant secondary metabolites, the simplest method in
cell culture is to manipulate the culture conditions such as light intensity, tempera-
ture, pH of the medium, composition of the medium, agitation and aeration, i.e. is the
environmental conditions of the medium. Presently metabolites such as berberine,
ginsenosides, shikonin, scopolamine and rosmarinic acid are being produced at the
commercial scale and of these only shikonin and gingseng were the first two
secondary metabolites to be produced at the commercial level. The advantages of
plant cell and tissue culture mediated production enhancement include mass produc-
tion of plants within a short period from small amount of tissue, simple and efficient
extraction of metabolites, large scale multiplication of cell lines true-to-type with
higher yields of secondary compounds, isolation of novel compounds that are not
naturally found in the intact plant, virus-eradication using meristem culture, large
scale production of metabolites at an industrial level using bioreactors, metabolic
tracing of secondary metabolite through radiolabeling of cell cultures, preservation
of biodiversity, producing disease-free plants, propagation of seasonally restricted
plants all year round (Gonçalves and Ramano 2018). Moreover, biotechnological
tools are used to accumulate higher concentration of different secondary metabolites
of some anti-diabetic plants are discussed below:
322 A. Paul et al.
Table 11.2 The plants with their hypoglycaemic constituents and mechanism of action
Antidiabetic
secondary Mechanism of
Family Name of plants metabolites action Reference
Acanthaceae Andrographis Andrographolide Increases mRNA Yu et al.
paniculata and protein (2003)
(Burm. F.) Wall levels of GLUT 4
ex. Nees
Amaryllidaceae Allium cepa L. Quercetin Inhibits D- Daniega
glucose (2018)
liberation from
disaccharide and
oligosaccharides
by inhibiting
a-glucosidase,
cause delayed
absorption of
glucose from
intestine
Apocynaceae Catharanthus Vindolidine, Had a greater Tiong et al.
roseus (L.) vindolicine, suppressive (2013)
G. Don vindolinine activity against
protein tyrosine
phosphatase-1B
inhibitory
activity
Gymnema Gymnemic acid Increased Kanetkar
sylvestre R. Br. secretion of et al. (2007)
insulin, and Zimare
regeneration of et al. (2018)
β-cells and delay
in absorption of
glucose in the
blood
Araliaceae Panax ginseng Ginsenosides Increase in Ota and Ulrih
C.A. Meyer insulin (2017)
sensitivity and
transcription of
proteins involved
in glucose uptake
Asteraceae Stevia Stevioside Increased Aggarwal
rabaudiana secretion of and Shishu
(Bertoni) insulin along (2011)
Bertoni with decrease in
the gene
expression of
enzyme
catalyzing
gluconeogenesis
(continued)

Antidiabetic
secondary Mechanism of
Family Name of plants metabolites action Reference
Cucurbitaceae Momordica Charantin Prevents Aggarwal
charantia L. absorption of and Shishu
glucose by the (2011)
intestine and
inhibits the
enzymes of
catalyzing
glucogenesis and
guconeogenesis
Fabaceae Trigonella 4- Stimulates Abd-El
foenum- hydroxyisoleucine pancreatic cells Mawla and
graecum L. and trigonelline to secrete insulin Osman
and hampers (2011),
glucose transport Aggarwal
and Shishu
(2011) and
Zhou et al.
(2012)
Pterocarpus Epicatechin Promotes Aggarwal
marsupium regeneration of and Shishu
Roxburgh β-cells and (2011)
increases insulin
secretion
Gentianaceae Swertia chirata Mangiferin Stimulates the Kavitha and
Buch-Ham. ex β-cells to Dattatri
Wall produce insulin, (2013)
reduce the
intestinal
absorption of
glucose and
cause an
enhancement in
glycolytic
enzymes
Lauraceae Cinnamommum Procyanidin Suppressed gene Ota and Ulrih
Schaeff. expression of (2017)
major regulators
of
gluconeogenesis
Meliaceae Azadirachta Meliacinolin Increase in Perez-
indica A. Juss. hepatic glucose Gutierrez and
uptake and Damian-
decrease in Guzman
hepatic glucose (2012)
release
324 A. Paul et al.
11.4.1 Andrographis paniculata (Burm. f.) Wall ex. Nees
Andrographis paniculata is known to have antibacterial, antifungal, antiviral,

choloretic, hypoglycaemic, hypocholesterolemic, adaptogenic, anti-inflammatory,
astringent, diuretic, anti-helminthic and antipyretic activities. A. paniculata has
been used in traditional Chinese medicine and is still being used in Ayurveda for
the treatment of fever, sore throat, herpes, gastro-intestinal infections, and respira-
tory infections. Moreover, it is also recommended for leprosy, gonorrhoea, scabies,
boils and skin eruptions. Few of the alkaloids found in kalmegh are andrographolide,
andrographiside, andrographan, andrographon, andro-graphostenin,
deoxyandrographiside, homoandrogrpholide, stigmassterol, neoandrographolide
and deoxyandrographolide (Mishra et al. 2007; Ganeshkumar et al. 2019). Yu
et al. (2003) showed andrographolide that is a bitter diterpenoid possess anti-
hyperglycaemic activity. It reduced the plasma glucose concentration in
streptozotocin-induced diabetic rats in a dose-dependent manner upon oral treat-
ment. They suggested that andrographolide can promote glucose utilization in
peripheral tissue of skeletal muscles by increasing the mRNA and proteins levels
of GLUT 4, which is known to mediate glucose transport across the plasma
membrane. When glucose loaded rats were orally administered with hot water and
ethanolic extracts of this plant, a considerable decrease in the blood sugar level was
noted (Hossain et al. 2007). Elicitation of cell suspension cultures of A. paniculata
using arachidonic acid increased the production of andrographolide to five times
more than the control (Ganeshkumar et al. 2019). Furthermore, Gandi et al. (2012)
proved that biotic elicitors (Escherichia coli, yeast, Agrobacterium rhizogenes
532, Bacillus subtilis and Agrobacterium tumefaciens C 58) were found to be
more efficient to elicit andrographolide production in the cell suspension culture of
A. paniculata than abiotic elicitors (CdCl2, CuCl2, HgCl2 and AgNO3). Interest-
ingly, maximum andrographolide accumulation was observed in case of yeast,
i.e. about 8.82-fold higher than the untreated set. Another elicitation experiment in
A. paniculata culture in half MS medium containing 20 g/L sucrose and elicitors
such as copper sulphate (500 μM/L), chitin (500 mg/L), methyl jasmonate (25 mg/L)
or fungal mycelium (500 mg/L) added in different experimental set, resulted that
each of the sets showed increased in andrographolide biproduction at a rate of
29.42 0.31 mg/g dry cell weight (DCW), 19.45 0.68 mg/g DCW,
13.13 0.11 mg/g DCW and 13.629 1.12 mg/g DCW, respectively. It was
clear from the observations that the copper sulphate was potent than the others
(Dawande and Sahay 2020). Aspergillus niger and Penicillium expansum are two
other biotic elicitors that used to enhance andrographolide synthesis in cell suspen-
sion culture of A. paniculata (Vakil and Mendhulkar 2013).
11.4.2 Catharanthus roseus (L.) G. Don
The popularity of Madagascar periwinkle is accounted to the presence of its multi-

purposely usable alkaloids like vinblastine, vincristine (anticancer drugs), ajmalicine
(antihypertensive compound), serpentine (sedative activity), tabersonine,

venalstonine, vindoline, vindolidine, vindolicine, vindolinine. Leurosine,
lochnerine, tetrahydroalstonine, pericalline, perivine, carosine, vincolidine,
lochrovicine, etc. Although the plants have various therapeutic activities such as
anti-oxidant, antiviral, anti-tumour, antibacterial, anti-diabetic, anti-hypotensive,
anti-helminthic and anti-neoplastic activity, but in this study, we are going to
focus on the hypoglycaemic activity and elicitation of secondary metabolites pro-
duction of the plant (Das and Sharangi 2017). A dose-dependent lowering of blood
sugar was observed in comparable to the standard drug glibenclamide, when
ethanolic extracts of the leaves and flower of C. roseus were used for the treatment
of diabetes (Chattopadhyay 1994; Aruna et al. 2015). Furthermore, the aqueous
extract of C. roseus, was lower the blood glucose of about 20% in diabetic rats.
Whereas, dichloromethane and methanol extracts lowered the blood glucose level
about 49–58%. The increased glucose utilization in the liver is results to the
hypoglycemic effects. There are seven alkaloids that were isolated and identified
from the dichloromethane extract of Catharanthus leaves, such as vindoline,
vindolinine, vindolidine, serpentine, perivine and vindolicine along with a new
indole alkaloid vindogentianine. These alkaloids helped to increase the glucose
uptake in pancreatic β-TC6 or myoblast C2C12 cells, but vindolicine showed
relatively high activity. Besides that, experimental study revealed that vindolidine,
vindolicine and vindolinine could be used for diabetes because they had a greater
suppressive activity against protein tyrosine phosphatase-1B inhibitory activity. This
implicates their therapeutic potential against type 2 diabetes (Tiong et al. 2013).
C. roseus derived medicine has been marketed under the name Vinculin for treat-
ment of diabetes (Chattopadhyay 1994; Singh et al. 2001; Aruna et al. 2015).
Considerable anti-hyperglycemic and hypotensive activity of the leaf extracts
(dichloromethane-methanol or hydroalcoholic) were reported in laboratory animals
(Das and Sharangi 2017). There are several experimental data like dried leaves at a
concentration of 6.25% of the diet of male mice when applied (28 days) for inactive
vs streptozotocin-induced hyperglycaemia. In another case, about 60% of blood
sugar was notably decreased when water extract of fresh cells administered
intragastrically to male rats were treated as active vs streptozotocin-induced
hyperglycaemia (Benjamin et al. 1994; Aslam et al. 2010). There also have some
widely accepted experiments where the fresh leaf juice of C. roseus has been used to
reduce blood glucose level in normal and alloxan diabetic (caused by a beta-
cytotoxin, which destroy the β-cells) rabbits because extract exerts some insulin-
releasing effect that helps the survival of β-cells. On the other hand, leaves and twigs
of C. roseus have hypoglycaemic activities that have been reported in the experiment
of streptozotocin-induced diabetic rats (Singh et al. 2001; Nammi et al. 2003; Balaji
2014). Both biotic and abiotic elicitation experiment was observed in the experiment
of Abdel-Rahman et al. (2010). This experiment conducted by the elicitation with
three biotic and abiotic stress factors (100 mg/g dry cells Alternaria alternata elicitor,
275 mg/g dry cells KCl and 2.75 mg/g dry cells VaSO4) in hypocotyls suspension
culture of C. roseus. The result showed the enhanced alkaloid production in suspen-
sion cultures either tested singly or in combinations. They also observed that much
326 A. Paul et al.
higher quantity of vincristine was produced than that of vinblastine, in suspension

culture either for intracellular or for extracellular samples. Another renowned bio-
technological strategy of increasing terpenoid indole alkaloid (TIA) production is the
manipulation of TIA biosynthetic pathway mediated by TIA biosynthetic pathway
genes discovery, compartmentalization and their regulation in C. roseus hairy roots
(Sun and Peebles 2016). In another example, vinblastine and vincristine production
was experimented in C. roseus cell cultures, in MS liquid medium supplemented
with 1.5 mg/L 2,4-D, 30 g/L sucrose and 0.5 mg/L kinetin and addition of chitosan
as elicitor, where they can observe 100 mg/L of both medium molecular weight
chitosan and chitosan derived from shrimp shell enhanced the maximum vincristine
and vinblastine production within in vitro cell culture (Pliankong et al. 2018).
Application of the combination of auxin and cytokinin in vitro tissue culture
shows induction of vinblastine and vincristine production (Saiman et al. 2018;
Barrales-Cureño et al. 2018). In case of serpentine and ajmalicine production
enhancement, 10 and 100 μM concentration of methyl jasmonate showed twofold
and fivefold augmentation when introduce it to cell suspension culture on sixth day
of growth (Gaines 2004).
11.4.3 Azadirachta indica A.Juss
The traditional medicinal system of ayurveda has recorded the use of Azadirachta
indica as anti-arthritic, diuretic, anti-malarial, anti-diabetic, antibacterial, antifungal,
antipyretic, antipyretic, hypoglycaemic, spermicidal, vermicidal and anti-
inflammatory. Few of the bioactive compounds found in A. indica are nimbin,
nimbolide, gedunin, epicatechin, catechin, quercetin, 6-desacetylnimbinene
β-sitosterol, n-hexacosanol, non-acosane and azadiractin. Nimbin is majorly
extracted from the oil of seed kernels of the plant, may act as an antihistamine and
exhibit antiulcer property along with anti-fungal activity. The anti-malarial property
has been attributed to nimbolide. The leaf extract and seed oil of the plant dilated the
blood vessels and improved the circulation of blood and it was also helpful in
reducing the requirement for insulin. The ethanolic leaf extracts of the plant are
known to confer anti-diabetic properties. It is suggested that a reduction in peripheral
utilization of glucose is one of possible mechanisms by which the blood sugar level
is lowered upon oral administration of neem extract (Biswas et al. 2002; Aggarwal
and Shishu 2011). Perez-Gutierrez and Damian-Guzman (2012) upon chro-
matographic analysis of the plant’s chloroform extract identified a new compound
and named it meliacinolin. They concluded that meliacinolin acts as a potent
α-amylase and α-glucosidase inhibitor. They treated the suspension to streptozotocin
(STZ)-induced diabetic mice with meliacinolin and recorded a diminution in blood
sugar levels along with increase in insulin. A decrease in the activity glucose-6-
phosphatase, increase in the activity glucokinase was also observed, which suggests
that hepatic glucose uptake increased whereas hepatic glucose release decreased.
Although, the calli of A. indica grown in woody plant medium liquid medium,
supplemented with 2% glucose, 100 μM of methyl jasmonate and 500 mg/L
hydrolyzed casein showed the highest azadirachtin concentration, i.e. an average of

0.247 μg/g in first 2 weeks of culture (Rodrigues et al. 2014). Satdive et al. (2007)
reported elicitation with both abiotic and biotic elicitors for azadirachtin production
by hairy root cultures in three tested media (namely Skoog’s basal media, Ohyama
and Nitsch, and Gamborg’s and Murashige media) and they want to know which one
is the best for the elicitation. Addition of biotic elicitor (Claviceps purpurea) resulted
about fivefold increase of azadirachtin production, salicylic acid and jasmonic acid
showed about ninefold and sixfold enhancement of azadirachtin respectively, in the
culture containing on Ohyama and Nitsch medium as compared with the control.
Furthermore, suspension cultures of A. indica on exposure to DMSO, Triton X-100
and chitosan resulted in the maximum at 7.5% DMSO where higher biosynthesis of
azadirachtin observed and 4.2 mg/L azadirachtin found to be accumulated (Kuruvilla
et al. 1999).
11.4.4 Gymnema sylvestre R. Br
It is known to possess anti-microbial, anti-hypercholesterolemic and

hepatoprotective activity and is used to treat asthma, inflammation, and
snakebites. G. sylvestre is known to contain gymnemic acids, gymnemasaponins,
gymnemasides, anthraquinones, hentri-acontane, pentatriacontane, butyric acid,
formic acid, lupeol, tartaric acid and β-amyrin. The anti-diabetic property of the
plant is attributed to the presence of gymnemic acid, which is an oleanane type of
triterpenoid saponin. It is suggested that gymnemic acid delays the absorption of
glucose in the blood, increases the secretion of insulin and utilization of glucose,
promotes regeneration of β-cells and decreases the synthesis of glucose. Due to
analogy in the atomic arrangement of gymnemic acid with glucose, they fill the
receptor on the tongue and in the intestine, thereby blocking the absorption of the
glucose and resulting in low blood sugar levels. It was noted that biotic elicitation of
in vitro grown shoots of G. sylvestre with Alternaria alternata, Rhizoctonia solani
and Botrytis cinerea enhances the production of gymnemic acid (Kanetkar et al.
2007; Zimare et al. 2018). Netala et al. (2016) focused on an interesting experiment
with two endophytic fungi (Polyancora globosa and Xylaria sp.) isolated and
identified from the G. sylvestre leaves and their preliminary experiment proved
that the dried powder of fungal mycelia showed higher gymnemic acid accumulation
in cell suspension culture of G. sylvestre in a range of 2.57–10.45-fold as compared
to extracellular culture filtrate (ECF) (range of 2.39–7.8-fold), but the greatest
activity observed in case of P. globosa. They also showed that the cultures treated
with consortium of both DPEM and ECF give increased gymnemic acid content as
compared with the cultures treated by DPEM and ECF. Ch et al. (2012) focussed on
the abiotic elicitor mediated increase of gymnemic acid (saponin) production in
suspension culture of G. sylvestre. Result showed that among various metal salt
elicitors (silver nitrate, mercuric chloride, cobaltous chloride, cadmium chloride,
cupric chloride and calcium chloride), CdCl2 (2 mM concentration) gave maximum
gymnemic acid accumulation (59.97 mg/g dry cell weight) after 24-h time period
328 A. Paul et al.
and the lowest response was found in case of AgNO3 (1 mM concentration) gave less
gymnemic acid accumulation (18.35 mg/g dry cell weight) after 48 h of incubation
period.
11.4.5 Swertia chirata Buch-Ham. ex Wall
Medicinal properties that have been associated with Swertia chirata are anti-
helminthic, analgesic, hepatoprotective, hypoglycaemic and antipyretic (Kavitha
and Dattatri 2013; Gupta et al. 2018). Amarogentin, swerchirin, swertiamarin and
mangiferin are the most active photochemical found in the plant along with various
xanthones, secoiridoid glycoside and triterpenoid alkaloid (Kavitha and Dattatri
2013). Out of these, amarogentin is the bitterest compound known to mankind and
mangiferin is the principle anti-diabetic compound (Kumar et al. 2013). It has been
suggested that mangiferin directly stimulates the β-cells to produce insulin, reduce
the intestinal absorption of glucose and cause an enhancement in glycolytic
enzymes, which ultimately results in increased rate of glycogenesis in the liver,
thereby contributing to a reduction in blood sugar levels (Kavitha and Dattatri 2013).
Kumar et al. (2013) elicited the in vitro grown shoots of S. chirata with different
concentrations of elicitors, which are chitosan, methyl jasmonate, salicylic acid,
vanadylsulphate, biotic elicitors such as Agrobacterium rhizogenes and yeast extract
and growth hormones. They observed that the media containing A. rhizogenes at a
concentration of 10 μL showed significant enhancement in the production of
amarogentin, swertiamarin and mangiferin. Moreover, swertiamarin, amarogentin
and mangiferin production analysis of S. chirata shoot culture grown in MS media
were elicited by the application of sodium nitroprusside, seaweed and salicylic acid
(Kotvi et al. 2017).
11.4.6 Taraxacum officinale (L.) Weber ex F.H. Wigg
It is normally known as common dandelion and is used for its anti-inflammatory,

antioxidant, anti-rheumatic, anti-carcinogenic, diuretic, chloretic, antiulcer,
hypolipidemic, antiviral and anti-diabetic property (Sharma and Zafar 2016; Gupta
et al. 2018). The major metabolites found in the plant are taraxasterol, taraxerol,
oleanolic acid, aursolic acids, α- and β-amyrins and lupeol (Akashi et al. 1994;
Sharma and Zafar 2016). Taraxasterol, taraxerol are the tritepenes of pharmacologi-
cal important anticancer compunds (Sharma and Zafar 2016). It is clear that water
extract of all parts of this plant have shown highest anti-diabetic activity as compared
to the methanolic extract of any. Moreover, the highest inhibitory effect of alpha
amylase and alpha glucosidase were resulted in the experiment with in vitro anti-
diabetic studies with stem part (Amin Mir et al. 2015). Mahesh and Jeyachandran
(2011) induced the formation of hairy roots by infecting the leaf and petiole of
T. officinale with Agrobacterium rhizogenes to analyse the accumulation of
sesquiterpenes lactones (active compounds found in the roots of dandelion) in
transformed, non-transformed and wild plant roots. They reported that the accumu-
lation of sesquiterpene lactones was greater in hairy roots. Sharma and Zafar (2016)
elicited the callus culture of dandelion with methyl jasmonate and β-cyclodextrin for
enhancing the production of taraxsterol and taraxerol. On the basis of the results,
they concluded that β-cyclodextrin was more effective in increasing the yield of
taraxsterol and taraxerol. Furthermore, the cell culture raised from leaves of
T. officinale was elicited by Cladosporium herbarum, resulting in the maximum
lettucenin A production in 2–6 h. As well as similar production of lettucenin A in
similar time duration was observed when cell culture stressed with 1 mM cupric
chloride (Hanawa et al. 1995).
11.4.7 Trigonella foenum-graecum L
The fenugreek seeds contain galactomannans, sitosterol, 4-hydroxyisoleucine,

trigonelline, trigogenin, gitogenin, trigocoumarin, trigomethylcoumarin and
diosgenin. The compound sotolone is responsible for the characteristic smell of
the fenugreek. In the traditional medicine system chewing fenugreek seeds has been
recommended for reducing the blood sugar level. Furthermore, the seeds have rich
fibre content due to which lipid and carbohydrate metabolism is inhibited. The
extracts prepared from seeds and leaves of the plants reduced the incidence of
cataracts in diabetic mice. Its anti-diabetic activity of has been associated with
4-hydroxyisoleucine and trigoneline. It has been suggested that
4-hydroxyisoleucine stimulates the pancreatic cells (β-cells) to secrete insulin and
hampers the transport of glucose (Aggarwal and Shishu 2011). The other alkaloid,
trigonelline which is vitamin B6 derivative and its bitter taste suggested inhibiting
the intestinal uptake of glucose (Zhou et al. 2012). Abd-El Mawla and Osman (2011)
initiated the cell suspension culture of Trigonella foenum-graecum from the
cotyledons of the sterile germinated seeds. They elicited the suspension with methyl
jasmonate for 24 h and observed an increase in the production of
4-hydroxyisoleucine and trigonelline. Upon feeding the extracts of the elicited cell
STZ-induced diabetic rats a major reduction in the blood sugar level was noted. On
one hand, 100 μL/L (0.1%) of methyl jasmonate applied to seedlings of six fenu-
greek varieties, it shows increased diosgenin production, because methyl jasmonate
upregulated the expression of sterol-3-β-glucosyl transferase (STRL) and 3-hydroxy-
3-methylglutaryl-CoA reductase (HMG), two gene of mevalonate pathway that plays
important role in diosgenin metabolic route (Chaudhary et al. 2015). On the other
hand, out of the four abiotic elicitors (CdCl2, CoCl2, K2Cr2O7 and NiCl2), only
CdCl2 and CoCl2 increased the diosgenin production up to 40- and 41-fold, respec-
tively in seedlings. Whereas, toxic effects of K2Cr2O7 and NiCl2 were observed that
inhibited diosgenin production. In addition, their experiment with calcium (Ca2+)
results in the inhibition of diosgenin production when cytosolic Ca2+ concentration
increases, but a calcium chelator or Ca2+ channel inhibitor shows enhance diosgenin
production in treated plant (De and De 2011).
330 A. Paul et al.
11.4.8 Allium cepa L
Allium cepa shows anti-mutagenic, anticancer, antioxidant, hypoglycaemic,

hypocholesterolemic, antibacterial, antiviral, anti-parasitic, antifungal, antihyperten-
sive, antithrombotic and anti-inflammatory activities (Jose and Krishnakumar 2017;
Gupta et al. 2018). The main secondary metabolites of interest found in this plant are
flavonols, anthocyanin, phytosterols and saponins (Marrelli et al. 2018). Some of the
identified major secondary metabolites of onion are flavonols like quercetin
monoglucoside (quercetin 40-O-glucoside), quercetin-3,40-O-glucoside, quercetin
rhamnoside, kaempferol-O-glucoside, isorhamnetin (Rodriguez Galdon et al. 2008;
Farag et al. 2017), phenolic acid derivatives, i.e. ferulic and caffeic acids (Dehpour
et al. 2011; Farag et al. 2017) etc. Jose and Krishnakumar (2017) stated clearly that
allyl propyl disulphide have anti-diabetic activity, which helps to increase free
insulin. Moreover, quercetin of onion has an activity by inhibiting D-glucose
liberation from disaccharide and oligosaccharides by restrict a-glucosidase, causes
delayed absorption of glucose from intestine to reduce blood glucose (Daniega
2018). Iqbal et al. (2019a) focussed on eliciting the callus culture of A. cepa for
increasing the production of quercetin by using various concentration of methyl
jasmonate as elicitor. They observed nearly 2.31-fold increase in the antioxidant
activity and a 13.94-fold increase in quercetin production in the callus culture of the
plant upon using methyl jasmonate at a concentration of 100 μM.
11.4.9 Allium sativum L
Apart from being anti-diabetic, antioxidant, anti-inflammatory and antihypotensive

(Ota and Ulrih 2017; Gupta et al. 2018), garlic is also known to cure gout, fever,
cardiovascular syndrome and reduce the risk of cancer (Malik et al. 2020). The
medicinal properties of garlic are generally attributed to the presence of various
organosulphur compounds such as alliin (S ¼ allyl-l-cysteine sulfoxide, a flavour
precursor), allicin (responsible for the pungent odour), allylpropyl disulfide,
S-allylcysteine, diallyltrisulfide, S-allylmercaptocysteine, ajoene and vinyldithiins
(Malik et al. 2020), terpenes such as nerolidol, phytol, α-pinene, squalene, limonene,
terpinolene, 1,8-cineole and γ-terpinene (Pontin et al. 2015), phenolic acid
derivatives like caffeic and ferulic acids (Dehpour et al. 2011; Farag et al. 2017)
have also been found in garlic. Malik et al. (2020) used cadmium chloride for
elicitation of alliin production within in vitro grown tissues such as somatic embryos,
callus, roots and leaves of A. sativum. On the basis of their major findings, they have
concluded that low doses of CdCl2 were efficient in increasing the production of
alliin in all cultures, although maximum accumulation of alliin was found in leaves
probably because the metabolite is mostly synthesized in the leaves of garlic.
Sclerotium rolfsii is a fungus causing economic loss can also elicit the production
of secondary metabolites in garlic (Cavalcanti et al. 2018). Pontin et al. (2015)
focussed on infecting the in vitro grown garlic plant with Sclerotium cepivorum,
which is known to cause Allium White Rot disease for checking the fungistatic
properties of the terpenes found in garlic, resulting in significant inhibition of the

in vitro mycelium growth of the pathogen by nerolidol, α-pinene and terpinolene. In
another case, sulphur treated 16 mgl 1 gypsum showed maximum enhancement of
alliin production in all in vitro grown tissues and organs (Nasim et al. 2010).
11.4.10 Citrullus colocynthis (L.) Schrad
The traditional evidence showed that this plant has been used for the treatment of
asthma, diabetes, cancer, constipation, jaundice, joint pain, leprosy and mastitis
(Parveen et al. 2020). Few of the secondary metabolites found in C. colocynthis
are cucurbitacin C, p-Coumaric acid, quercetin, gallic acid, catechin,
colocynthoside-B, toluene, A-sphinosterol and vanillic acid (Tanveer et al. 2012;
Parveen et al. 2020). Whereas, it was specified by Jayaraman et al. (2009) that the
anti-diabetic activities of this plant observed due to presence of saponins, flavonoids
and glycosides. In preliminary study by Lahfa et al. (2015) showed aqueous, total
alkaloids, saponins and glycosidic extraction of colocynth seeds showed
antihyperglycemic effect in normal wistar rats and streptozotocin-diabetic rats.
Though, seeds are rich source of choline and three alkaloid derivatives of pyridine
and quinoline (Darwish-Sayed et al. 1973; Lahfa et al. 2015). On the other hand, the
fruit used widely in diabetes (Al-Snafi 2016; Kapoor et al. 2020) and colocynthoside
compounds, 4-methylqumoline, ursolic acids, cucurbitacin E,2–0-β-D-
glycopyranoside from methanolic extract and cucurbitacins D, E, F, G, some
flavonoids glucosides (isovitixin, isorintin and isosapanorin) were isolated from
butanolic fruit extracts (Jeon and Lee 2014; Dhakad et al. 2017; Kapoor et al.
2020). Furthermore, occurance of quercetin in C. colocynthis makes it a biochemi-
cally distinct cucurbitaceae plant (Tanveer et al. 2012). Tanveer et al. (2012)
focussed on increasing the yield of total quercetin content in in vitro cultures of
C. colocynthis by simply manipulating the culture conditions by different
concentrations and combinations of plant growth regulators where BAP and NAA
at a concentration of 2–3 mg/L showed significant quercetin production. Similarly,
Meena et al. (2014) observed a twofold to threefold increase in the production of
quercetin (anti-cancerous) by treating the callus culture of the plant with various
concentration of β-phenylalanine. At a concentration of 75 μM, methyl jasmonate,
precursors squalene, salicylic acid and mevalonic acid resulted highest cucurbitacin
E (anti-cancerous compound) production in in vitro suspension culture, though the
methyl jasmonate and precursors squalene were potent than others (Dasari et al.
2020).
11.4.11 Aerva lanata (L.) Juss. Ex Schult
Aerva lanata is well known anti-diabetic plant used to treat diabetes mellitus by its
alcoholic extract via reducing blood sugar level. Other than the hypoglycaemic
activity, it possess several other therapeutic properties such as anticancer,
332 A. Paul et al.
antioxidant, antitumor, fungicide, antimicrobial, anti-inflammatory, diuretic and

immunosuppressive activities (Vetrichelvan and Jegadeesan 2002; Surya et al.
2012; Gupta et al. 2018). Though its anti-diabetic secondary metabolites are not
known to us, but few of the various secondary metabolites found in A. lanata are
alkaloids like aervine, methylaervine, methylervine, ervine, ervoside, aervoside;
flavonoids like quercetin, persinol, persinosides A and B, kaempferol; phenolics
like vanillin and syringic acid (Goyal et al. 2011; Surya et al. 2012; Boobalan and
Kamalanathan 2020). Vanillin is an aromatic flavour compound with white to pale
yellow crystal structure, is known to posses inhibitory action against a range of
microorganisms and more than 10,000 tons of vanillin is produced every year.
Although, kinesin mediated elicitation in the hairy root culture showed an efficient
methodology for rapid in vitro propagation of A. lanata as well as in vitro production
of vanillin than that of the control (Surya et al. 2012). Similarly, Boobalan and
Kamalanathan (2020) confirmed an enhancement in the production aervine in hairy
root culture through Agrobacterium rhizogenes mediated Ri transformation (MTCC
352 strain) established via in vivo leaf, callus and node of A. lanata.
11.4.12 Nigella sativa L
Nigella sativa is known to possess anti-cancer, anti-inflammatory, anti-parasitic,

anti-diabetic, anti-analgesic, anti-microbial, anti-stress, antioxidant,
hypocholesterolemic and anti-asthmatic properties (Gupta et al. 2018; Iqbal et al.
2020). The leaves of the plant contain metabolites such as α-hederin and
kalopanaxsaponin I (KsI) (Scholz et al. 2009), whereas the seed oil showed the
existence of p-cymene monoterpenes, β-pinene, α-thujene, γ-terpinene, α-pinene,
nigellone, caracole, thymoquinone and thymol (Iqbal et al. 2020). The anti-tumour
and anti-proliferative property of N. sativa is attributed to the presence of most
important and the first secondary metabolite of this plant, i.e. thymoquinone (Iqbal
et al. 2020) but anti-diabetic activity has been shown by defatted extract and aqueous
extract (Mathur et al. 2011). Scholz et al. (2009) used methyl jasmonate to elicit the
production of saponins in the leaves of hydroponically cultivated N. sativa. They
observed a 12-fold increase in the production of kalopanaxsaponin I and α-hederin.
Moreover, the two saponins accounted for ~10% of the dry plant matter, of which
7% was α-hederin and 93% KsI, indicating that α-hederin is the immediate precursor
of the saponin biosynthetic pathway, whereas KsI might represent the end product of
the pathway in N. sativa. Iqbal et al. (2019b) sowed the seeds of N. sativa after
treating them different concentration of sodium azide. The stress response resulted in
increased antioxidant property in a concentration dependent manner with a substan-
tial decrease in the thymoquinone content of the plant. They suggested that the
reaction between sodium azide and thymoquinone results on formation of analogues
with potent antioxidant property. In the further experiment of Iqbal et al. (2020), they
showed that similar analogous results were obtained by them when they used sodium
azide within in vitro callus culture.
11.4.13 Coriandrum sativum L
The leaves of Coriandrum sativum are a rich source of flavonoids, β-carotene and
phenolic compounds. Moreover, various secondary metabolites such as ascorbic
acid, rutin, chlorogenic acid, p-coumaric acid, caffeic acid, ferulic acid, vanillic acid,
kaempferol, apigenin and luteolin are also found in the plant (Nguyen et al. 2020).
The antioxidant activity, anti-bacterial, anti-allergenic, anti-thrombotic and
vasodilatory effects have been attributed to the phenolic compounds produced by
the plant under stressful conditions. Furthermore, C. sativum also exhibit
cytoprotective, anti-diabetic, anti-inflammatory, anti-carcinogenic and
cardioprotective activities (Gupta et al. 2018; Nguyen et al. 2020). The aqueous
extract of coriander fruits showed anti-hyperglycaemic action by enhancing insulin
secretion, increasing glucose uptake and metabolism by muscle (Gray and Flatt
1999). Indicating the phenolic compounds of the plant, Jiménez-Gómez et al. (2020)
suggested that use of biofertilizer may improve the nutritional content and
phytochemicals found in the plant. They inoculated the leaves of in vitro produced
C. sativum with Bacillus halotolerans and observed a significant improvement in the
concentration of phenolic compunds such as dimethoxy-cinnamic acid hexoside,
5-O-caffeoylquinic acid, cinnamic acid derivate, 4-methoxy-cinnamic acid hexoside
and ferulic acid. Moreover, a substantial increase in the production of various
flavonoids was also noted upon application of B. halotolerans.
11.5 Conclusion
The demand for human life is increasing day by day, i.e. either for the development
of medicinal system or to meet their daily life needs. Although reports are showing
that nowadays nearly 3.2 million people die per year for the only reason of diabetes.
Hence, by giving enough respect for human needs, it is necessary to focus on
the production enhancement of those compounds required as curative agents. On
the other hand, plants are the only source of secondary metabolites that has the
properties to meet both sides of human needs. Moreover, secondary metabolites are
compounds having anti-diabetic, anti-cancerous, anti-allergic, sedative etc. medici-
nal activities, as well as these are used as perfume, flavouring agent, etc.
Anti-diabetic plants are not only the plants having anti-diabetic secondary
metabolites, but also they contain a large number of secondary metabolites with
several medicinal properties. Hence, it is important to isolation and proper identifi-
cation with clinical trials of those compounds, so that these can be further processed
towards the biotechnology to augment their production within short time and cost-
effective manner. For fulfilling the criteria, among the three biotechnology tools
(genetic transformation, metabolic engineering and elicitation), elicitation is chosen
as most efficient by many scientists for its cost-effective nature. In this book chapter,
we have tried to amalgamates the elicitor mediated all types of medicinally important
secondary metabolites production enhancement that found in conventionally and
scientifically used anti-diabetic plants. Here it is clear that the elicitations in in vitro
334 A. Paul et al.
cultures of plants are highly reliable process for the scientists, because it is
performed within inexpensive lab set up, as well as it helps to regulate the metabolite
biosynthesis pathway.
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Rhizobium rhizogenes-Mediated Genetic
Transformation of Antidiabetic Plants 12
Marta Libik-Konieczny , Żaneta Michalec-Warzecha,
Ireneusz Ślesak, and Laura Pistelli
Abstract
Rhizobium rhizogenes induces a disease syndrome in infected plants commonly

known as hairy roots (HR) production. These unique organs are considered of
transgenic nature since they derived after horizontal gene transfer from the
bacterial root-inducing (Ri) plasmid to the plant genome. The HR culture
represents a major biotechnological tool facilitating the production of plant-
derived compounds exhibiting interesting features for pharmaceutical, cosmetics
and food industries. Moreover, HR cultures can be used for studying the biosyn-
thetic pathways of plant-derived molecules or investigating interactions occurring
during phytoremediation. Among this wide range of HR applications, it is
important to point out the possibility to synthesize some antidiabetic compounds
which are of special interest because diabetes is one of the major and frequently
occurring debilitating diseases of the human body in the world. The use of
available synthetic antidiabetic drugs has several limitations, including drug
resistance, side effects, or toxicity. Therefore, the availability of new antidiabetic
drugs from a natural source is of big importance. Stevia rebaudiana Bertoni is one
of the promising plant species, constituting the source of desired plant molecules.
This plant accumulates steviol glycosides (SGs), which are responsible for the
sweet flavour of stevia leaves. These secondary metabolites are considered
metabolically inert and therefore could be a safe substitute for sugar for diabetic
people. In this chapter, we would like to present recent achievements in the range
of antidiabetic substances production via genetic transformation of plants using
M. Libik-Konieczny (*) · Ż. Michalec-Warzecha · I. Ślesak

The Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków, Polska
e-mail: libik@mail.ifr-pan.edu.pl
L. Pistelli
Department of Agriculture, Food and Environment, University of Pisa, Pisa, Italy
Ltd. 2021
https://doi.org/10.1007/978-981-16-3529-8_12
342 M. Libik-Konieczny et al.
R. rhizogenes strains. In this context, a key role of the so-called rol genes, carried
by Ri plasmids, in the biosynthesis of various secondary metabolites is also
discussed. Our aim is to bring closer the issue of HR cultures and their big
potential in the pharmacognosy of antidiabetic plants.
Keywords
Genetic manipulation · In vitro culture · Pharmacognosy · Plant biotechnology ·

Secondary metabolites · Transformed root culture
12.1 Introduction
The name “hairy roots (HR)” was first used in the early 1990s (Steward et al. 1900;
Riker et al. 1930) for a description of specific organs produced as a result of
pathogen infection in horticultural plants. Since the identification Phytomonas
rhizogenes renamed Agrobacterium rhizogenes and lately Rhizobium rhizogenes
as a microorganism that causes hairy root phenotype, many studies have been
undertaken to explain the processes underlying the occurrence of hairy root syn-
drome. HR are induced in the response of DNA segment transfer (T-DNA) from the
bacterial root-inducing (Ri) plasmid into the host cell and its integration with the host
genome. The gene (T-DNA-encoded) expression stimulates the HR induction at the
infection site (Fig. 12.1a). Morphologically, HR are considerably comparable to
normal roots of the infected plant (Fig. 12.1b), but they also exhibit some unique
features such as a greater frequency of horizontal branching, the abundance of root
hairs, rapid, plagiotrophic growth and fast increase in biomass without exogenously
supplied plant growth regulators, as well as genetic and biochemical stability over an
extended period of time (Hu and Du 2006). Considering the unique features of HR, it
Fig. 12.1 Hairy roots from S. rebaudiana. (a) Hairy roots emerging from wounded leaf of
S. rebaudiana infected with R. rhizogenes. (b) Isolated hairy roots line growing on solid medium.
(Source: unpublished photos by Marta Libik-Konieczny)
12 Rhizobium rhizogenes-Mediated Genetic Transformation of Antidiabetic Plants 343
is not surprising that they are widely used in both basic research and
agrobiotechnology as a material to study the effect of horizontal gene transfer
(HGT) in plants. Ackermann in 1977 was the first who performed directed transfor-
mation of higher plants using R. rhizogenes (Ackermann 1977). According to the
data provided by Christey and Braun (2005), successful R. rhizogenes-mediated
transformation was achieved in 89 different taxa, representing 79 species from
55 genera and 27 families of higher plants and since then more additions are still
being made to the list. Over the past three decades, the transformation of plants with
R. rhizogenes has been used to study various physiological processes and biosyn-
thetic pathways, as well as for the production of recombinant therapeutic proteins,
for phytoremediation and the synthesis and isolation of plant secondary metabolites
(Georgiev et al. 2012) (Table 12.1).
12.1.1 The Advantages of R. rhizogenes Transformation

for Secondary Metabolites Production
Due to the unusual ability to biosynthesize compounds naturally produced in the

native plant, HR are often called “phytochemical factories” (Majumder et al. 2016)
and utilized for the production of secondary metabolites with high pharmaceutical
properties on large scale (Srivastava and Srivastava 2007). Various methods have
been used to enhance secondary metabolite production in HR (Georgiev et al. 2012;
Hussain et al. 2012; Sharma et al. 2013) (Table 12.2). These include the increase of
substrate availability, the use of different elicitors, genetic modification of the major
genes engaged in the biosynthesis, regulation of transcriptional factors, as well as
introduction of new genes (Shi et al. 2021). The last one provides the possibility of
artificially designed HR to produce not naturally occurring compounds by modifica-
tion of naturally existing biosynthetic pathways (Sirikantaramas and Taura 2017;
Gutierrez-Valdes et al. 2020). All of the attempts resulted in many patents
concerning the possibility to use of HRs as a basic biotechnological means for
different secondary metabolites production (Datta et al. 2010; Talano et al. 2012).
Plant-derived compounds have been discovered and employed for medicinal
purposes since prehistoric times and they form the basis of modern medicine
(Vickers et al. 2001). Due to the vast range of positive properties like anti-
inflammatory, hemostatic, expectorant, antispasmodic or immunostimulatory, plants
extract provides multifaceted benefits on human health affecting multiple biological
pathways and preventing/treating many different health disorders. Therefore, it is not
unexpected that despite the great progress in modern medicine, herbal medicine is
still widely used by people in most developed and developing nations and has even
increased worldwide in the past three decades (Ekor 2014; Li and Weng 2017).
Meeting the requirements of modern medicine, pharmacognosy defined as an inter-
disciplinary science at the interface of chemistry and biology, the task of which is to
apply the research of natural products in medicine, is developing very fast (Jones
et al. 2006). New scientific achievements in this field, including the identification
and production of biologically active molecules and the development of new
Table 12.1 The list of selected, medicinal plant species and produced secondary metabolites in
hairy root cultures. (According to Giri and Narasu 2000; Guillon et al. 2006; Georgiev et al. 2007;
Chandra and Chandra 2011; Talano et al. 2012; Huang et al. 2014; Shi et al. 2021)
Plant species Produced metabolite Medical application
Ambrosia Thiarubrine A Antibiotic
artemisiifoia
Anisodus Hysocyamine, Anticholinergic effect
acutangulus Scopolamine
Arachis Resveratrol Antioxidant, anticancer and cardioprotective
hypogaea Piceatannol activity, implicated in leukemia, non-Hodgkin’s
lymphoma
Artemisia sp. Artemisin Antiparasitic activity, used to treat malaria
Astragalus Isoflavonoids Increase production of white blood cells, anti-
membranaceus aging and anti-inflammatory effects
Atropa Scopolamine Anticholinergic effects
belladonna
Beta vulgaris Dopa and dopamine Neurotransmiters
Brugmansia Hysocyamine Anticholinergic effects
candida
Camptotheca Camptothecin Anticancer activity
acuminata
Catharathus Ajmalicine/ Antihypertensive drug
roseus Catharanthine
Chicorium Esculin Vasoprotective agent
intybus
Datura innoxia Hysocyamine and Anticholinergic effects
Scopolamine
Echinacea Chicoric acid Inhibit the function of hyaluronidase (protect
purpurea collagen)
Erigeron Scutellarin Protective effects for nerve cells, prevent diabetic
breviscapus blindness
Fagopyrum Rutin Used in treatment allergies, viral infections and
tataricum arthritis
Ferula Farnesiferol B Protective effect in renal ischemia/reperfusion
pseudalliacea injury
Gentiana scabra Iridoids, Secoiridoids Anti-inflammatory; analgesic, antireumathic,
antidiuretic, antidiabetic
Gossypium Gossypol Male oral contraceptive, antimalarial properties
hirsutum
Gingko biloba Ginkgolide Carbovascular disease, reduce migraine frequency
Gmelina Verbascoside Anti-inflammatory properties
arborea
Gynostemma Saponin gypenoside Treatment of cardiovascular diseases
pentaphyllum
Harpagophytum Iridoid glycosides Antidiabetic, anti-inflammatory, analgesic
procumbens
Hyoscyamus Hysocyamine Anticholinergic effects
muticus
(continued)

Hyoscyamus
niger
Isatis indigotica Lariciresinol Anti-influenza and anti-inflammatory effects
Lithospermum Shikonin Antibacterial activity
erythrorhizon
Ophiorrhiza Camptothecin Anticancer drugs
pumila
Panax ginseng Ginsenoside antitumor, anti-inflammatory, antioxidation effect
Papaver Morphine Opioid analgesics, decrease moderate and severe
somniferum Codeine feeling of pain, used to treat coughing and diarrhea
Papaver
orientale
Peganum β-Carboline alkaloids Central nervous system stimulant and a reversible
harmala (harmine, harmal, inhibitor of MAO-A
harmaline and
harmalol)
Plumbago indica Plumbagin Anticancer, antimicrobial, antimalarial activity
Polygonum Rhein Vasorelaxant, anti-bacterial/fungal,
multiflorum neuroprotection, anticancer activities
Psoralea Daidzin Used in treatment of alcohol dependency
corylifolia
Pueraria Puerarin Antioxidant activity, hypothermic, spasmolitic,
phaseoloides hypothensive, antiarrhythmic
Rauvolfia Ajmalicine Antihypertensive effects
micrantha Ajmaline
Rhodiola rosea Salidroside Antidepressant and anxiolytic actions
Rubia tinctoria Anthraquinone Laxatives and antimicrobial and anti-inflammatory
agents
Salvia Tanshinone/phenolic Anticoagulation drug
miltiorrhiza acids
Saussurea Jaceosidin Used in cancer therapy
medusa
Scopolia Scopolamine Anticholinergic effect
parviflora
Scutellaria Flavone Cardiovascular effects
baicalensis
Solanum Saponin Cardiovascular disease, gastric pain, liver damage
chrystotrichum Solasodine
Solanum
khasianum
Stevia Steviol glycosides Antidiabetic
rebaudiana
Stizolobium 3,4-Dihydroxy-L- Parkinson’s disease treatment
hassjoo phenylalanine
Tanacetum Parthenolide Anti-inflammatory and anti-hyperalgestic effects
parthenium
Taxus media Taxol Cytotoxic agents use in chemotherapy for cancer
(continued)

Valeriana Valepotriates Regulatory effect on the autonomic nervous
officinalis system
Withania Withanoloide A Stimulatory effect on immune, endocrine and
somnifera central nervous system, neuroprotective function
Table 12.2 The list of plant species in which hairy roots synthesize specific metabolites at the
level higher than in untransformed host plant tissue. (According to Srivastava and Srivastava 2007;
Shi et al. 2021)
Produced secondary
Plant species metabolite/yield increase Strategy
Anisodus Hysocyamine, Scopolamine/2- Genetic transformation/overexpression
acutangulus 3-fold of TRI–PMT genes
Atropa Atropine and Scopolamine/5- Genetic transformation/insertion gene
belladonna fold h6h
Catharanthus Serpentine/20-fold Genetic transformation/overexpression
roseus Catharantine/7-fold of ORCA4 gene
Ajmalicine/2-fold
Isatis indigotica Lariciresinol/8-fold Genetic transformation/overexpression
of Ii049 gene
Lithospermum Menisdaurin/5-fold Genetic transformation/ insertion ubiC
erythrorizon gene
Lobelia Polyacetylenes/20-fold Growth in the light condition
sessilifolia
Ophiorrhiza Camptothecin/2-3-fold Genetic transformation/overexpression
pumila of several genes
Panax ginseng Ginsenoside/2,5x Addition of indole-3-butyric acid (IBA)
to the medium
Peganum harmala β-carboline alkaloids/3-5-fold Genetic transformation/ insertion TDC
gene
Salvia Tanshinone/15–21-fold Genetic transformation/overexpression
miltiorrhiza of several genes
technologies for obtaining drugs of plant origin, play a significant role in the fight
against chronic diseases, which are the predominant challenge to global health.
12.1.2 Possibilities of Diabetes Treatment with Plant-Derived

Products
Diabetes also known as diabetes mellitus is concerned to be one of the most critical
ailments of the twenty-first century. It is defined as a metabolic disease characterized
by hyperglycemia caused by disorders of insulin secretion or insulin malfunction, or
both. Insulin (a peptide hormone produced by the pancreas) regulates the
metabolism of carbohydrates, lipids and proteins by stimulating the assimilation of
glucose from the blood into cells of the liver, fat and skeletal muscle. Due to the
deficiency or malfunction of insulin, the blood glucose level increases leading to
hyperglycemia. The chronic increased level of glucose causes the damage of various
organs, especially the heart, kidneys, nerves, eyes and blood vessels. It is also
classified among the top ten leading causes of death worldwide (American Diabetes
Association 2013). It has been observed that both the number of cases and the
incidence of diabetes have been steadily increasing over the past few decades.
According to the International Diabetes Federation, more than 463 million people
worldwide were diagnosed with diabetes in 2019, and this figure might be doubled in
2045 (International Diabetic Federation (IDF) (2019). Thus, it is necessary to focus
the research efforts not only on the prevention but especially on effective treatment.
Nowadays, there are several types of oral glucose-lowering medicines that have an
antidiabetic effect through various mechanisms. These mechanisms include:
(a) reduction of hepatic gluconeogenesis by biguanides, (b) delay in the absorption
of carbohydrates from the intestine by alpha-glucosidase, (c) stimulation of insulin
secretion by sulfonylurea and non-sulfonylureas secretagogues drugs, or
(d) improvement of muscle and adipose tissue sensitivity to insulin and to a smaller
extent, reducing liver glucose production by thiazolidinediones (Salehi et al. 2019).
Treatments with synthetic drugs have many drawbacks, involving multiple side
effects, drug resistance and toxicity (Dey et al. 2002; Kooti et al. 2016). Therefore,
it is important to focus scientific efforts on discovering alternative sources of
diabetes medications. Currently, it is recommended to treat diabetes with medicinal
plants containing alkaloids, carotenoids, flavonoids, saponins, terpenoids and
glycosides (Salehi et al. 2019). Medicinal plants, apart from being highly effective,
have also fewer side effects, and the costs of obtaining potential drugs from them are
relatively low (Bansode and Salalkar 2017).
For these reasons, the current scientific approaches should concentrate on com-
bined research in the field of medicinal plants culture development aimed at the
synthesis of biologically active compounds in high concentration, characterization of
biologically active compound-rich extracts with particular emphasis on
phytochemicals interactions and the development of technologies for the recovery
of desired products. Plant cell suspension cultures technologies developed in the past
as possible tools for the production of plant bioactive substances have largely failed
due to their instability, non-uniformity of the produced compounds and too low yield
of these compounds synthesis to be commercialized (Chandra and Chandra 2011).
Hence, differentiated plant organ cultures, which give the possibility to control the
biosynthetic processes of specific compounds at the molecular level, such as
transformed HR, can be a useful tool for studies on bioactive plant-derived
substances.
The main purpose of this chapter is to bring closer the issue of HR induction from
different medicinal plant species in order to consider the possibilities of their use for
the search and production of new plant-derived antidiabetic drugs.
12.2 Overview of R. rhizogenes Plant Infection

and Transformation Process
12.2.1 Structure of Ri Plasmid
Rhizobiun rhizogenes is a gram-negative, rod-shaped soil bacterium, which can

transfer a portion of its DNA to a susceptible plant cell (Tzfira et al. 2004). All
R. rhizogenes strains are equipped with a large, root-inducing (Ri) plasmids
(Fig. 12.2). The transferable region of this plasmid is known as T-DNA (transfer
DNA). This region encodes the genetic information required for HR formation
(Veena and Taylor 2007). T-DNA genes have eukaryotic regulatory sequences
that enable their expression in infected plant cells (Giri and Narasu 2000; Mishra
and Ranjan 2008). “T-DNA is flanked by 24-bp direct repeats, known as border
sequences” (Yadav et al. 1982). Transfer of T DNA into the plant genome occurs
with the aid of virulence (vir) genes. R. rhizogenes strains carry also on their
plasmids genes essential for opines biosynthesis. They are used by the bacteria as
a nutrient source (Ozyigit et al. 2013). Based on the type of opines produced
Fig. 12.2 Scheme of the Ri-plasmid (agropine strain) structure. (Diagram prepared by authors)
R. rhizogenes strains can be classified into various sub-groups. One of them is

represented by the agropine-type strains producing agropine, mannopine, agropinic
acid and mannopinic acid. The other strains belong to mannopine-type producing all
agropine-type opines excluding agropine (Ozyigit et al. 2013; Vladimirov et al.
2015). The most common R. rhizogenes strains include agropine-type strains,
mannopine-type strains, cucumopine-type strains and mikimopine-type strains
(Veena and Taylor 2007).
There are two separate T-DNA regions known as left T-DNA (TL-DNA) and
right T-DNA (TR-DNA) in the agropine type of Ri plasmid (Fig. 12.2). “TL-DNA
and TR-DNA are separated by 15 kb of non-integrated DNA, which is represented
by T-Central DNA (TC-DNA)” (White et al. 1985; Sinkar et al. 1987). There is only
one T-DNA in mannopine type of Ri plasmid in contrary to the agropine type of Ri
plasmid (Rawat et al. 2019). Independent transformations of both TL-DNA and
TR-DNA to the plant genome termed as “split” T-DNA are carried out by agropine
type of Ri plasmid, whereas mannopine type transfers a single T-DNA (Vilaine and
Casse-Delbart 1987; Chandra 2012).
TR-DNA of agropine Ri-plasmid carries the genes responsible for opine synthesis
(ags, mas1, mas2) and the genes involved in auxin biosynthesis, designated as aux1
and aux2. Gene product encoded by the aux1 (tryptophan 2- monooxygenase)
catalyzes the conversion of tryptophan to indole-3-acetamide (IAM). Then, IAM is
converted to indole-3-acetic acid (IAA) by IAM hydrolase, the product of the aux2
gene (Ozyigit et al. 2013). The T-DNA of some R. rhizogenes strains (mikimopine,
cucumopine and mannopine strains) does not carry aux genes in Ri plasmids;
however, these strains are still capable to induce a “hairy-root” phenotype. There-
fore, it is supposed that the presence of the aux genes on TR-DNA is not a
prerequisite for the generation hairy root phenotype. It was demonstrated that the
aux gene function is rather to support the “hairy root” phenotype and to broaden the
host range of the bacterium (Nemoto et al. 2009). TR-DNA also contains a rolB
homolog (rolBTR). Bouchez and Camilleri (1990) found out that this gene exhibits
52.6 % nucleotide similarity with rolB gene presents on TL-DNA region and might
contribute to the activation of root-inducing genes from TL-DNA.
TL-DNA region of agropine Ri-plasmid houses four root loci (rol), namely rolA,
rolB, rolC and rolD. They modify phytohormones concentration and/or biosynthesis
and their expressions promote the formation of HR in transformed tissues and
biosynthesis of various secondary metabolites (for details see below).
12.2.1.1 rolA
The rolA gene is found in all known Ri plasmids. It encodes a protein with a high
isoelectric point and having molecular mass 11.4 kDa. Based on the deduced amino
acid sequence, it is concluded that rolA gene also contains a sequence motif common
in DNA-binding proteins (Suzuki 1989). These features might suggest that the
transcription product of rolA gene activity could be engaged in the regulation
of gene expression in plants (Levesque et al. 1988). It was shown that the expression
of rolA gene in transgenic Nicotiana tabacum line led to the reduction in the level of
auxin, cytokinin, gibberellic acid (GA) and abscisic acid (Dehio et al. 1993). It was
also demonstrated that in transgenic plants low level of auxin concentration

correlated with the enhancement in auxin sensitivity (Maurel et al. 1991). Ozyigit
et al. (2013) have shown that R. rhizogenes-transformed plant tissues are 100-times
extra auxin-sensitive to control).
12.2.1.2 rolB
rolB gene can be found in all Ri plasmids. It exhibits ~60% distinctiveness between
various strains (Meyer et al. 2000). rolB gene encodes 254–279 amino acid protein
of 30 kDa molecular weight, localised in the plasma membrane (Meyer et al. 2000;
Veena and Taylor 2007). The studies performed by Bellincampi et al. (1996) with
the use of auxin biosynthesis activators and inhibitors, demonstrated that rolB had
the most important role in the differentiation of transformed cells and induction of
HR. It was due to its function in the hydrolysis of bound auxins which ultimately
resulted in the higher intracellular level of IAA. Estruch et al. (1991a) have assigned
this function to the fact that “rolB encodes for a β glucosidase, which may release
active auxins through hydrolysis of inactive β-glucosides”. rolB-transformed cells
exhibit also variations in auxin signal transduction/reception, which probably arise
from tyrosine phosphatase activity of rolB, engaged in auxin signal transduction
pathway(s) (Filippini et al. 1996). Besides its crucial role in HR promotion, it was
revealed that rolB has the greatest stimulatory effect on secondary metabolism
(Bulgakov et al. 2002; Shkryl et al. 2008; Shoja 2010).
12.2.1.3 rolC
The rolC gene is present in all studied R. rhizogenes strains, and it is the most
conserved of all rol genes (Tzfira and Citovsky 2008). The size of rolC gene from
various Ri plasmid is similar consisting of 540 bp. rolC has a low contribution to HR
formation by itself, but it actively induces this process in combination with rolA and
rolB (Palazón et al. 1998). The rolC encodes 179- to 181-amino acid protein (Meyer
et al. 2000) and its function relies on the control of free and conjugated forms of
cytokinins balance as well as auxins and gibberellins, abscisic acid (ABA), poly-
amine and ethylene concentration in plant cells (Estruch et al. 1991b; Nilsson et al.
1993, 1996a, b; Martin-Tanguy 2001; Martin-Tanguy et al. 1996). Estruch et al.
(1991b) proposed that rolC gene product is a β-glucosidase—an enzyme catalyzing
the release of free active cytokinins from their inactive glucosidic conjugates ().
Unfortunately, the results of in vivo quantifications of the substrates or the products
of the suggested reaction did not confirm the stated hypothesis in transgenic rolC-
expressing plants (Mauro et al. 2017). It is rather supposed that rolC does not affect
the hormonal balance directly and it has rather an indirect effect on the development
program due to its potential ability to cleave oligosaccharides (Faiss et al. 1996). It
was also found out that rolC transformants are capable of actively accumulating
starch and active absorption of sucrose (Mohajjel-Shoja et al. 2011). The promoter
of rolC was found to be positively regulated by sucrose (Yokoyama et al. 1994; Faiss
et al. 1996), suggesting that rolC may participate in the regulation of sugar metabo-
lism and transport in the plant cell (Veena and Taylor 2007). Simultaneously,
sucrose may be a substrate for the rolC protein, and the metabolic products of
sucrose can be used to form new organs (Pavlova et al. 2014). The rolC gene is also
able to stimulate the production of the high level of secondary metabolites in
transgenic plant tissue (Bulgakov et al. 1998, 2002; Bonhomme et al. 2000; Shkryl
et al. 2008; Shoja 2010).
12.2.1.4 rolD
The rolD gene is present only in TL-DNA of agropine type Ri plasmids. It is also the
only rol gene that is not able to induce HR formation on its own (Mauro et al. 1996).
The rolD encodes an ornithine cyclodeaminase, converting ornithine to proline
(Trovato et al. 2001). Since proline is an osmoprotectant and its accumulation is
known to increase in plant cells as the response to some environmental stress factors
(Mauro et al. 1996; Trovato et al. 2001), hence the rolD might be considered as a
stress-inducible gene. Indeed, Bettini et al. (2003) have shown that tomato
transformants with rolD expression, exhibited increased level of PR (pathogen-
related) gene activity and they were more resistant to Fusarium oxysporum f. sp.
lycopersici, in comparison to the control plants.
Besides rol genes, there are some open reading frames (ORFs) positioned on
TL-DNA. The agropine type Ri T-DNA contains 18 ORFs nucleotide sequences
(Slightom et al. 1986) and few are conserved in all Ri plasmids. This can indicate
their important function in basic morphogenetic processes (Veena and Taylor 2007).
The results from the analysis of T-DNA region insertion mutants (White et al. 1985)
as well as transformation trials revealed that ORF10, 11, 12 and 15 coincided with
rolA, rolB, rolC and rolD, respectively, are able to promote the formation of HR
syndrome (Ozyigit et al. 2013).
12.2.2 Mechanism of the Plant Cell Infection and Transformation

Processes
R. rhizogenes-mediated plant cell infection and transformation is a multistage

process (Fig. 12.3). It starts from the perception of plant signals by bacterial strain
and the establishment of physical contact with plant cells. Multiple plant proteins
have been associated with the attachment of bacteria to plant cells. Among them,
arabinogalactan proteins localized in cell walls and secreted into the apoplast play an
important function (Gelvin 2010). The signals activating bacterial infection originate
from wounded plant cells that release phenolic compounds (e.g. acetosyringone)
inducing the activity of bacterial vir genes (Stachel et al. 1985) (Fig. 12.3, I). The
induction requires also proper environmental conditions like weakly acidic pH,
moderate temperature and the presence of monosaccharides (Suzuki et al. 2009).
vir gene products participate in the processing of T-DNA. The production of a
transfer intermediate begins with the generation of the T-strand, a single strand
(ss) copy of the T-DNA, (Fig. 12.3, II). Subsequently, the T complex is formed of
some vir proteins that become covalently attached to the 5 'end of the T-DNA strand.
Export of the T-complex from bacteria is not a well-known step in the transfer
process. It occurs via transfer channel apparatus generated by vir proteins activity
Fig. 12.3 Scheme of Rhizobium rhizogenes—mediated infection of the plant cell. Roman numbers
(I–VII) describe the subsequent stages of T-DNA transfection into the plant cell genome (for details
see the text). (Scheme prepared by authors)
(Fig. 12.3, III). The T-complex and several vir proteins are transferred to the
cytoplasm of host plant cells. In this step, mature T-DNA is formed as a result of
some plant proteins associated with T-complex to preserve its integrity during
cytoplasmic trafficking through the plant cell (Fig. 12.3, IV). Among the identified
class of plant proteins that mediate T-DNA complex targeting, importin and histone
proteins play an important function. After getting inside the plant cell, the T-complex
targets to the cell nucleus and crosses the nuclear membrane through the nuclear pore
(Zupan and Zambryski 1995; Gelvin 2010; Chandra 2012). Import of the mature
T-DNA complex to the cell nucleus is mediated by nuclear localization signals
(NLSs) carried by vir proteins (Suzuki et al. 2009). Integration of bacterial DNA
into a plant host chromosome is the next step of the T-DNA transfer process
(Fig. 12.3, V). In this step, some plant proteins, like vir-interacting proteins (VIPs)
are also involved (Anand et al. 2007). Before or during the process of T-DNA
integration to host plant genome both vir proteins and plant proteins are removed
from T-strand (Gelvin 2010; Chandra 2012). When T-DNA is integrated into the
plant genome, proteins encoded by genes localised on T-DNA are produced. Apart
from proteins responsible for HR induction and growth (Fig. 12.3, VI), enzymes that
direct the production of opines are synthesized, secreted by the transformed plant
cells and consumed by bacteria as a nutrient source (Dessaux et al. 1992). This
phenomenon is known as “genetic colonization” (Schell et al. 1979). Emerging HR
(Fig. 12.3, VII) is the morphological confirmation of the plant genome transforma-
tion with bacterial genes.
Various R. rhizogenes strains are classified into polar and non-polar types
depending on the position of the explant surface from which HR emerged. When
studied on carrot root discs, it was shown that some R. rhizogenes strains were
capable to induce HR growth only on the apical surface, whereas the other strains
induced HR growth on both apical and basal surfaces (Cardarelli et al. 1985; Ryder
et al. 1985). Considering these results, agropine type strains were assigned to
non-polar type, which induces the formation of HR regardless of the orientation of
the explant, whereas all other strains are polar, as they lead to the formation of HR
only when the plant explant is placed in the upturned orientation.
12.2.3 The Evolutionary Context
The genetic material between organisms can be transferred not only in a vertical but
also in a lateral (horizontal) manner, and this phenomenon is called the horizontal
gene transfer (HGT). HGT dominates as the main way of exchange of the genetic
material among prokaryotes, i.e. bacteria and archaea, resulting in their natural
genome variability. In the microbial world, multiple molecular mechanisms of
HGT occur, where free DNA in solution, plasmids and the genetic material of
viruses (bacteriophages) as “selfish” (or “parasitic”) genetic elements are involved.
As a result, HGT between prokaryotes may produce new features like antibiotic
resistance (Koonin and Wolf 2012; Koonin 2016; Lacroix and Citovsky 2016).
In contrast to prokaryotes, HGT between bacteria (or archaea) and eukaryotes is
much rarer (Lacroix and Citovsky 2016). As it was mentioned above (see Introduc-
tion), an example of HGT is the transfer of the bacterial (R. rhizogenes) T-DNA to
the plant cell. However, sequences homologous to the rol genes of T-DNA of the Ri
plasmid from R. rhizogenes have been found in the genome of uninfected plants that
belong to the members of Nicotiana genus, in Linaria vulgaris and sweet potato
(Matveeva and Lutova 2014; Kyndt et al. 2015; Mauro et al. 2017). This homolo-
gous T-DNA, naturally occurring in the genome of some plant species is referred to
as “cellular T-DNA” (cT-DNA) (Matveeva and Lutova 2014). The origin of these
naturally occurring rol genes in the plant genome is not entirely recognized yet.
Nevertheless, some of their features like the promoter structure typical for eukaryotic
genes, and the supposed expression in plants via RNA polymerase II suggest a
eukaryotic origin. Therefore, it cannot be excluded that cT-DNA and rol genes were
derived by bacteria from ancient plant genomes in both directions: from bacterium to
plant and vice versa (Mauro et al. 2017). As a result, such two-directional HGT
plausibly positively affected both plants and bacteria fitness.
The transfection of the T-DNA of Ri plasmids from R. rhizogenes to

S. rebaudiana is described here as the example of HGT from bacteria to a eukaryotic
organism and a promising source of antidiabetic compounds. However, it should be
emphasized that such a practical approach would not have been possible if HGT
from bacteria to plants was not the natural and an ancient evolutionary trait. In other
words, besides its significant application potential, the bacterium-to-eukaryote gene
transfer has a particular relevance for studies of modern eukaryotes evolution or
plant responses to various environmental stresses (Yue et al. 2012; Matveeva and
Lutova 2014; Lacroix and Citovsky 2016).
12.2.4 General Procedure of Plant Cell Transformation

and Establishment of Hairy Root Culture
Many factors influence the efficient transformation of plant tissue, including age,
type and species of the plant (tissue) involved (Sevon and Oksman-Caldentey 2002).
The most of plant organs, such as hypocotyl, leaf, stems, stalks, petioles shoot tips,
cotyledons, storage roots, or tubers, can be used to induce HR (Hu and Du 2006).
Explants collected from the plant are individually injured and co-cultivated with
R. rhizogenes strain. Processes of bacterial infection can be promoted by some
chemicals, e.g. phenols (Joubert et al. 2002) or by some techniques, like
sonication-assisted Agrobacterium-mediated transformation (SAAT), (Trick and
Finer 1997) Since there are strong differences in virulence, morphology, and growth
rate of Ri plasmids within various bacterial strains (Park and Facchini 2000), the type
of R. rhizogenes strain and the density of the bacterial suspension are very important
factors to succeed in transformation (Park and Facchini 2000). However, the choice
of an effective R. rhizogenes strain for the production of HR significantly depends on
the plant species and must be selected empirically. In transformation experiments
performed on S. rebaudiana plants, it was found out that the competence of
transformation was affected by the type of explant, time of inoculation, concentra-
tion of inoculum, along with the bacteria strain (Michalec-Warzecha et al. 2016)
(Fig. 12.4). More efficient transformation has been achieved following inoculation
of leaves explants from ex vitro plants with LBA9402 in high concentration when
compared with inoculation with ATTCC 15834 strain (Michalec-Warzecha et al.
2016).
HR growth from transformed tissue is usually visible within a relatively short
time, which varies from 7 to 30 days. The explants with visible HR can be
transferred onto solid media supplemented with antibiotics. After several subcultures
on the media with a gradually decreasing concentration of antibiotics, individual
lines of HR can be established from a single, isolated HR. Decontaminated HR can
be transferred to PGR-free medium. Factors such as ionic concentration and pH of
the medium, source and levels of carbon, light, etc., regulate the growth and
development of HR (Hu and Du 2006). The choice of medium depends on plant
species but its composition is usually based on Murashige and Skoog medium
(Christey and Braun 2005).
Fig. 12.4 Schematic protocol of hairy roots formation from S. rebaudiana leaves. (Scheme
prepared by authors)
Identification of HR transgenic nature by PCR study validates the origin of HR

culture as R. rhizogenes. Since HR production includes rol genes integration from Ri
plasmid with host plant genome, PCR analysis for the presence of rolB or rolC
transcripts is usually adopted to approve the activity of these genes in transformed
tissue. In addition, PCR for R. rhizogenes vir transcript can be used to exclude the
presence of bacterial contamination in cultured HR (Christey and Braun 2005).
For most of the scientific research works on secondary metabolites production or
studies on their biosynthetic pathways and the effect of additives on the HR cultures,
the growth of HR on solid media is sufficient. However, for production purposes,
HR must be grown on a large scale, making the entire production process economi-
cally viable. Accordingly, it may be required to establish liquid cultures or scale up
to larger bioreactors. It comprises relocating a portion of HR culture to the flask
containing liquid culture medium supplemented with antibiotic to suppress
R. rhizogenes growth since residual bacteria cells remain linked with the HR cultures
for an extended time.
Bioreactor cultures exemplify the “ultimate phase” during in vitro secondary
metabolite production (Bourgaud et al. 2001; Georgiev et al. 2007). Many types of
bioreactors (airlift bioreactors, rotating drum bioreactor, convective flow reactor,
bubble column reactor, stirred tank reactor, mist reactors, turbine blade reactor, etc.)
have been effectively utilized for the proliferation of HR cultures (Curtis 2000;
Srivastava and Srivastava 2007; Rawat et al. 2019). It must be however underlined
that for fruitful scale-up of HR-based procedures, regardless of bioreactor category,
several factors should be considered, among them high sensitivity of HR to stress is
of big importance (Wysokinska and Chmiel 1997).
12.3 Secondary Metabolism Regulation by R. rhizogenes

Transformation of Medicinal Plants
Metabolic activity of the plant cell leads to the production of primary metabolites
that are absolutely essential for survival and secondary metabolites that are not
mandatory for the growth and development but are produced in order to construct
a selective advantage to the organism. They were long considered as by-products of
metabolism. Nowadays it is commonly known that the term “secondary metabolites”
was used inequitably and the phrase “specialized metabolites” is emerging to
describe them, since they fulfil a multitude of important functions for plant surviv-
ability, including the interactions with the environment. They are often involved in
plant protection against biotic or abiotic stresses. Secondary metabolites consist of a
vast range of biologically active compounds differing in their function, structure and
biosynthetic pathways (Fig. 12.5). However, they all originate from the precursors
provided by primary metabolism, mainly from photosynthesis, glycolysis, tricarbox-
ylic acid (TCA) cycle and amino acids.
Plants that synthesise secondary metabolites with high pharmaceutical values are
called medicinal plants. However, there are several trouble shots that need to be
considered. Many of these pharmaceutically important compounds are present at a
low level in plants. Furthermore, many of them are synthesized only in specific
tissues like roots or secretory trichomes. Moreover, the plant organs from which
these compounds are obtained often grow slowly or produce these metabolites in
very small amounts over a long period of time, sometimes even several years, before
they can be harvested. In addition, medicinal plants grow in various ecological
environments and often have bacterial, fungi or pesticide residues, leading to the
degradation of their quality (Shi et al. 2021). Therefore, the production of a geneti-
cally modified HR culture is a good alternative approach to produce these valuable
compounds from medicinal plants. It represents several obvious advantages and one
Fig. 12.5 Main pathways of secondary metabolite biosynthesis in plants and their relationship
with the substrate of primary metabolism. Scheme prepared by authors
of them is a possibility of secondary metabolism regulation in order to maximize the

production of target compounds (Gantait and Mukherjee 2021). Moreover, the HR
culture system does not need many fields which can be used for crop growing and is
not limited by weather and season changes. Also, no herbicide and insecticide are
used during the maintaining of the system, and therefore, it is ecologically friendly.
After establishing a stable HR culture, the content of useful compounds is more
stable than in herbs harvested from different areas, which facilitates the quality
control of the desired products (Gaosheng and Jingming 2012).
Since the ability of HR to produce large amounts of plant secondary metabolites
has been described, a question has arisen about the involvement of specific bacterial
genes from T-DNA in this effect. While there is still insufficient understanding of the
role of the individual rolA, rolB, rolC and rolD genes or when they are integrated in
combinations, several reviews have been written explaining the “rol effect” on the
accumulation of secondary metabolites in medicinal plants (Hamill et al. 1987;
Tepfer 1990; Toivonen 1993; Costantino et al. 1994; Bourgaud et al. 2001; Rao
and Ravishankar 2002; Verpoorte et al. 2002; Guillon et al. 2006; Srivastava and
Srivastava 2007; Bulgakov 2008; Bulgakov et al. 2013; Karuppusamy 2009; Pistelli
et al. 2010; Chandra 2012; Sharma et al. 2013; Roychowdhury et al. 2013, 2015;
Matveeva et al. 2015; Parr 2017; Mitra et al. 2017; Salehi et al. 2018). After the
compilation of described knowledge, it can be assumed that rolC gene has been the
most popular choice for the studies on bacterial genes regulation of secondary
metabolites in plants, followed by rolB and then rolA. Surprisingly, the function of
rolD in the accumulation of secondary metabolites has remained not studied in detail
(Sarkar et al. 2018).
12.3.1 Function of rol Genes in Plant Secondary Metabolism
12.3.1.1 rolA Gene Function

From the very few data achieved so far it can be concluded that on one hand, rolA
exhibited a positive influence on secondary metabolites production as it enhanced
some secondary metabolites level like for example nicotine concentration in
transformed N. tabacum roots (Palazón et al. 1998) or anthraquinone accumulation
in transformed Rubia cordifolia cultures (Shkryl et al. 2008). However, on the other
hand, rolA gene did not cause significant differences in transformed culture of
Artemisia dubia maintaining the level of secondary metabolites production similar
to their content in non-transformed cultures (Amanullah et al. 2016).
12.3.1.2 rolB Gene Function

The rolB gene is considered as one exhibiting a positive effect on secondary
metabolite production in some medicinal plants (Kiselev et al. 2007; Shkryl et al.
2008; Dilshad et al. 2016). In R. cordifolia transformed culture, enhanced expression
of rolB correlated with increased activity of a key gene for anthraquinone biosyn-
thesis. Cultures of Artemisia carvifolia Buch. transformed with rolB gene showed
the increase in the level of quercetin, rutin and isoquercetin (Dilshad et al. 2016). In
rolB-transformed cultures of Maackia amurensis Rupr., the yield of isoflavonoids
has been found slightly higher compared to control samples (Grishchenko et al.
2016). Arshad et al. (2014) have also found the significant increase in the lycopene
content in rolB expressing plants compared to the non-transformed Solanum
lycopersicum L. (). The most spectacular example of the effectiveness of rolB
transformation was found in Vitis amurensis cultures, where transformation led to
more than a 100-fold increase in resveratrol production (Kiselev et al. 2007).
However, since rolB gene is also the most powerful suppressor of cell growth, the
practical application of rolB-transformed cells might be rather limited (Bulgakov
2008).
12.3.1.3 rol C Gene Function

From the available literature data, it can be concluded that rolC gene might exhibit
both stimulatory and inhibitory effects on the accumulated levels of target secondary
metabolites. The stimulatory effect of rolC gene was quite evident in nicotine
production by N. tabacum transformed cultures (Palazón et al. 1998); indole
alkaloids in Catharanthus roseus (Palazón et al. 1998), ginsenoside content after
transformation of Panax ginseng (Bulgakov et al. 1998), anthraquinone level in
transformed R. cordifolia cultures, (Shkryl et al. 2008), resveratrol in V. amurensis
(Dubrovina et al. (2010), hysocyamine and scopolamine in transformed cultures of
Atropa belladonna (Bonhomme et al. 2000), polyphenol levels in Cynara
cardunculus var. altilis (Vereshchagina et al. 2014), artemisin artesunate and
dihydroartemisin, caffeic acid quercetin isoquercetin and rutin in transgenic cultures
of Artemisia annua and Artemisia carvifolia (Dilshad et al. 2015a, b, 2016), as well
as flavonoid content in Lactuca sativa (Ismail et al. 2016). On the other hand, rolC
gene exhibited an inhibitory effect on the production of some secondary metabolites
in transformed cultures of Eritrichium sericeum and Lithospermum erythrorhizon
(Bulgakov et al. 2005). Transformed cultures of this plant species showed a lower
level of rhabdosin and rosmarinic acid when compared to non-transformed cultures.
Moreover, in rolC-transformed cultures of Maackia amurensis slightly higher level
of some isoflavonoid was found but together with a decreased production of genistin
(Grischenko et al. 2013). Therefore, it might be concluded that rolC gene is
responsible for differential regulation of secondary metabolites within the same
transformed culture causing an increase in the level of one compound and reduction
in the level of others simultaneously (Grishchenko et al. 2013).
Changes in rolC expression were also positively correlated with the higher
capacity of the transgenic tissue to grow, therefore it was hypothesized that rolC
gene stimulates the production of secondary metabolites not directly by increasing
the biosynthetic activity of transformed cells but rather by stimulating their growth
(Palazón et al. 1998). Considering all these data, one can conclude that rolC provides
a signal that activates secondary metabolic processes (Bulgakov 2008).
It is likely that rolC may participate in a wider spectrum of processes in addition
to secondary metabolite stimulation. As an example, it was noted that rolC expres-
sion in P. ginseng cells correlated with activation of β-1,3-glucanase, which belongs
to the PR-2 family of plant defence proteins (Kiselev et al. 2006). Thus, it may
suggest the engagement of rolC in plant defence mechanisms against stress factors.
It should also be emphasized that the rol genes show an antagonistic effect in
transformed plant cells. This was found both in the growth of transformed cells,
e.g. in the case of N. tabacum, where the expression of rolB inhibited the growth of
transformed cells and the rolC gene was able to attenuate this growth inhibition
(Schmülling et al. 1988), as well as at the level of biosynthesis of secondary
metabolites, where rolC (and possibly rolA) exerts an antagonistic effect on the
rolB-induced increase in anthraquinone. Bulgakov has concluded that rolB is a
major agent in the activation of secondary metabolism while rolC provides a signal
that tunes the action of rolB (Bulgakov 2008).
The results of the studies on the combinatory effect of rol genes in transformed
plant cells are also available and they confirmed the key role of rol genes in the
enhancement of secondary metabolites production; however, the level of the
increase is not as strong as it could be expected in all species that were investigated.
While the strong effect of rolABC on the enhancement of secondary metabolite
production in transformed N. tabacum cultures was noted (Palazón et al. 1998), the
stimulatory effect of rolABC gene on transformed R. cordifolia cultures (Shkryl et al.
2008) was relatively weaker than on N. tabacum as measured in terms of fold
increment of their respective target metabolites (Sarkar et al. 2018). Acting together,
these genes play an important role in the regulation of pathway leading to the
increase in secondary metabolites synthesis. The possibility that other T-DNA
genes may also have an effect on secondary metabolism in HR cannot be excluded,
although their influence seems to be less apparent (Bulgakov 2008).
12.3.2 Biotechnological Strategies to Improve Secondary

Metabolite Production in HR Cultures
A recent review on the available scientific literature regarding the strategies for
regulating secondary metabolism shows that several of them are already in use and
many new ones are being developed to maximize the production of target
compounds in the HR culture (Gantait and Mukherjee 2021). Earlier strategies to
alter secondary metabolite levels in HR cultures relied solely on manipulation of
culture conditions, the use of bioreactors and the use of elicitors (Banerjee et al.
2012). Currently, new strategies including multigene engineering, combined use of
elicitors and genetic engineering, use of newly identified key genes or transcription
factors in metabolic engineering, use of newly emerging clustered and regularly
interlaced short palindromic repeats (CRISPR)/CRISPR-related protein 9 technol-
ogy, and “omics-related techniques”, including genomics, transcriptomics, proteo-
mics and metabolomics, are being refined based on the HR culture technique from
medicinal plants (Shi et al. 2021).
The HR culture technique enables the genetic manipulation of key genes involved
in the biosynthesis of the desired secondary metabolites by enhancing or blocking
their transcriptional activity. It should be emphasized, however, that this type of
manipulation requires a thorough knowledge of the entire biosynthetic pathway for
each specific compound and a full understanding of the regulation of each pathway.
Therefore, this method might exhibit some limitations since the biosynthetic
pathways of many important secondary metabolites have not yet been fully
elucidated. Despite this, several samples of genetic manipulation for secondary
metabolites production have been published. For example, overexpression of the
valerendiene synthase gene in Valeriana officinalis HR resulted in a higher level of
valerenic acid compared with the control (Ricigliano et al. 2016). Genetic manipu-
lation of several key genes of tashinone biosynthetic pathway yielded a significant
increase of this metabolite in transgenic Salvia miltiorrhiza HR compared to the
control (Shi et al. 2016). In addition, suppression and/or blockade of a competitive
branching pathway was also tested to increase the production of plant-derived

secondary metabolites. For example, RNA interference (RNAi) was used to
downregulate 4-hydroxyphenylpyruvate dioxygenase (SmHPPD) gene activity in
homogentisate biosynthesis, which competes with rosmarinic acid biosynthesis in
Salvia multiorrhiza HR cultures (Xiao et al. 2011). In the same species, down-
regulation of copalyl diphosphate synthase gene (SmCPS1) by RNAi also resulted in
a substantial reduction of tanshinones in HR culture (Cui et al. 2015).
Another approach for increasing secondary metabolite production via manipula-
tion of genes responsible for their production involves the “push–pull” effect of
overexpressing several genes simultaneously (Shi et al. 2021). Cointroduction of
several genes (multigene engineering) in different combinations resulted in the
production of several-fold higher levels of tropane alkaloids in Anisodus
acutangulus HR than in the control (Kai et al. 2011). It was suggested that due to
the complexity of plant secondary metabolites biosynthetic pathways, multigene
engineering is a more effective strategy than overexpressing a single gene in HR (Shi
et al. 2020).
As various transcription factors have recently been identified in different medici-
nal plants, TF-targeted metabolic engineering that coordinates the regulation of
multiple genes in the biosynthetic pathway has gained big potential for increasing
secondary metabolite accumulation. Several attempts have been made to manipulate
some secondary metabolites biosynthetic pathways via overexpression of transcrip-
tional factors in transformed HR. Paul et al. (2017) observed a significant increase of
tabersonine in C. roseus HR overexpressing Octadecanoid-Responsive
Catharanthus AP2-domain protein 4 (CrORCA4). Similarly, overexpression of the
Apetala2 Ethylene Response Factor (AP2/ERFs) Ii049 in transgenic HR of Isatis
indigotica positively regulates lariciresinol biosynthesis (Ma et al. 2017). The
transcripts of genes responsible for the biosynthesis of tanshinone and salvianolic
acid as well as the level of these metabolites was enhanced in S. miltiorrhiza HR
overexpressing MYB98 or WRKY transcriptional factors (Cao et al. 2018; Deng et al.
2019; Hao et al. 2020). On the other hand, Rohani et al. (2016) found out that
overexpression of MYB1 transcriptional factor in Ophiorrhiza pumila HR reduced
camptothecin content. Similary, Sun et al. (2019) and Huang et al. (2019) described a
novel AP2/ERF transcription factor SmERF11 which overexpression in
S. miltiorrhiza HR increased salvianolic acid levels but reduced tanshinone produc-
tion. Thus, it has to be underlined that for manipulation of secondary metabolites
production a suitable target TF should be selected (Shi et al. 2021).
HR of several medicinal plants has been used to identify novel genes and their
function, transcriptional factors, biosynthetic pathways, or new secondary
metabolites via “omics-related techniques” (Shi et al. 2021). Genomics analysis
has recently led to discover a novel gene from A. belladonna HR (AbPPAR),
which encodes a phenylpyruvic acid reductase required for tropane alkaloids bio-
synthesis (Qiu et al. 2018). Transcriptomic analysis of S. miltiorrhiza HR allowed to
isolate many novel genes involved in tanshinone biosynthesis and new TFs that
regulate the tanshinone and/or salvianolic acid biosynthesis (Zhou et al. 2017).
Similarly, transcriptomic studies led to the identification of bHLH iridoid synthesis
1 (BIS1) transcription factor transactivated the expression of all genes encoding the
enzymes catalyzing the conversion of the terpenoid precursor geranyl diphosphate to
the iridoid organic acid in C. roseus (Van Moerkercke et al. 2015). Proteomics
analysis of S. miltiorrhiza HR revealed the presence of five novel cytochromes
P450s (CYP450) enzymes and five berberine bridge enzyme-like enzymes involved
in tanshinone biosynthesis (Wang et al. 2016; Contreras et al. 2019). Proteomic
analysis of Scutellaria baicalensis HR led to isolation and characterisation of two
novel CYP450 involved in baicalein biosynthesis (Zhao et al 2018). Metabolomics
analysis of S. miltiorrhiza HR allowed us to identify several dozen potential
intermediates in tanshinone biosynthesis providing useful data for the clarification
of this biosynthetic pathway (Cui et al. 2015). Nuclear-magnetic-resonance-based
metabolomics of HR derived from Verbascum nigrum revealed the presence of
glutamine that has never been found in mother plant tissues (Georgiev et al.
2015). Furthermore, a combination of metabolomic and transcriptomic analyses
allowed to indicate genes and their products that are master regulators of high-
value secondary metabolites produced in Ophiorrhiza pumila and S. miltiorrhiza
(Udomsom et al. 2016; Gao et al. 2014).
Currently, the CRISPR-associated protein has evolved as the most encouraging
tools with a great potential for identification of gene function, and for enzyme and
pathway (involved in secondary metabolite production) characterization in HR
culture (Hussain et al. 2018). The CRISPR-Cas9 system is based on a microbial
adaptive immune system (Jinek et al. 2012) that uses Cas9 endonuclease complexed
with a synthetic guide RNA (gRNA) to cleave foreign DNA at the desired location.
This technique is advantageous since it is simple, less expensive and it can target
multiple genes (Zhang et al. 2018).
Recently, CRISPR/Cas9 has been used to eliminate OpG10H and OpSLS genes in
Ophiorrhiza pumila HR, thus reducing the levels of camptothecin by 90% (Shi et al.
2020). and to knock out SmMYB98 in S. miltiorrhiza, which resulted in a decrease in
tanshinone and phenolic acid levels (Hao et al. 2020).
12.4 Medicinal Plants with Antidiabetic Potential
Traditional Chinese medicine, along with Ayurvedic therapy, has been offering
healthcare for diabetes for hundreds of years. Today, the use of many medicinal
plants remains widespread in developing countries due to the high cost-effective
budget needed for conventional medicine (Salehi et al. 2019).
In the past decades (around 30 years), several scientists have focused on renewed
interest in natural products extracted from medicinal plants to counteract diabetes
(Yeung et al. 2020). Several scientific reviews summarize the knowledge about
common natural products used in the treatment of diabetes (Shapiro and Gong
2002; Tiwari and Rao 2002; Rafiullah et al. 2006; Ríos et al. 2015; Alam et al.
2018; Choudhury et al. 2018; Salehi et al. 2019). The antidiabetic effects of herbal
extracts from several medicinal plants have been proved in some studies. However,
in many cases, the use of these extracts is not yet endorsed by sufficient clinical trials
according to the standards that are becoming established in the international scien-
tific community.
Salehi et al. (2019) summarized in a review a list of 194 individual species
showing hypoglycemic or antidiabetic activity, and 50 plant extracts with
antidiabetic potential. Among the various species, Ficus benahaliensis is one of
the most often used in traditional Indian medicine (Kar et al. 2003), while in
South Africa the most popular herbal product is the water extract of Artemisia afra
(Nedjimi and Beladel 2015). The leaf decoction from Bauhinia forficate is the most
often used as an antidiabetic herbal remedy in South America (Pepato et al. 2002).
Senna auriculata and Artocarpus heterophyllus leaves are frequently used to man-
age diabetes in Sri Lanka (Sathasivampillai et al. 2017).
12.4.1 Mechanism of Some Antidiabetic Compounds Action
Various mechanisms are known by which the plant extracts operate as antidiabetic
agents (Ríos et al. 2015; Choudhury et al. 2018). The antihyperglycemic effects of
herbal extracts are usually attributed to their ability to regulate insulin secretions, cell
sensitivity to insulin and/or to the regulation of glucose absorption and transport.
The α-glucosidase inhibitors are currently the most often used synthetic drugs to
treat type 2 diabetes. However, they cause adverse side effects. Many different
medicinal plants have been tested for α-glucosidase inhibitors activity (Yeung
et al. 2020) and it was revealed that some of them may produce substances with
inhibitory effect on the mentioned enzyme. The inhibition of α-glucosidase, as well
as α-amylase in the digestive tract, was documented in the extracts of Acacia
ligulatae, Beyeria leshnaultii, Catharantus roseus, Momordica charantia, Mucuna
pruriens, Ptecarpus marsupium, Rehmannia glutinosa and Urena lobata (Gulati
et al. 2012; Tran et al. 2007; Onoagbe et al. 2010; Purnomo et al. 2015; Babu et al.
2016). Urena lobata HR cultures have been successfully used to identify the
bioactive compound responsible for the α-glucosidase inhibitory activity (Cao
et al. 2020) and a positive correlation was found between the concentration of
phenolic compounds in HR extracts and the enhancement of α- glucosidase inhibi-
tory activity.
Some medicinal plant extracts stimulate insulin secretion and pancreatic β-cell
proliferation. Among them extracts from Cuminum cyminum, Nigella sativa, Aloe
vera, Chloroxylon swietenia, Forshytia suspense, Coccinia grandis and Gymnema
sylvestre are the most often listed (in review Choudhury et al. 2018).
A further mechanism by which plant extracts operate in the management of
diabetes is the modulation of glucose uptake and transport. It was shown that
bioactive compounds from Abelmoschus esculentus play a significant role in lower-
ing blood glucose levels in rats (Dubey and Mishra 2017; Durazzo et al. 2019).
Several studies have been published concerning multiple functions of some plant
extracts as Momordica charantia, Catharantus roseus and Aloe vera, exhibiting
different mechanisms of glucose level control. In addition to mentioned above plant
species, the multiple functions of extracts from Allium sativum, Camellia sinensis,
Corchorus olitorus, Ficus deltoidei, Glycine max, Ocimum basilicum, Olea europea,
P. ginseng and Punica granatum were observed (Choudhury et al. 2018; Salehi et al.
2019).
The other mechanisms of antidiabetic compounds from different medicinal plants
may rely on control of diabetes by regulation of oxidative stress level. Various plants
exhibiting antioxidant properties like A. sativum, Amacardium occidentale,
Helicterus angustifolia, Symplocos cochinensis, Uvaria chamae, Urtica dioica,
and Vitis vinifera were investigated and described as the important sources of
reactive oxygen scavenging compounds playing an indirect function in diabetes
management: (Ríos et al. 2015; Choudhury et al. 2018; Salehi et al. 2019).
12.4.2 Main Classes of Secondary Metabolites with Antidiabetic

Potential
The herbal extracts of medicinal plants may originate from the whole plant or from
separate plant organs, such as bark, flower, root, seeds, leaves, bulb and tubers,
where the desired compound is produced or accumulated with the highest level.
These extracts contain different classes of phytochemical compounds used as a
treatment due to their antidiabetic potential (Fig. 12.6). Alkaloids, polyphenols and
terpenoids are the most often cited compounds in the scientific literature concerning
the antidiabetic potential of plant-derived compounds. Polysaccharides, as
galactomannans and the fructan inulin, are also introduced as antidiabetic agents
however their function seems to be synergistic with other hypoglycemic substances
(Chen et al. 2009; Salehi et al. 2019).
The very first medicinal plant, Galega officinalis (Italian fitch), known as the
source of antidiabetic compounds since Middle age, contains alkaloid—galegine
which decreases blood sugar but was also discovered to be very toxic to mammals
and its use has been superseded by better alternatives. The research based on the
effects of galegin eventually led to the development of metformin, which is used
today for type 2 diabetes treatment (Bedekar et al. 2010; Ríos et al. 2015). Both
in vitro and in vivo studies still are providing evidence, that indicates the ability of
the alkaloid interference with the signal transduction pathway of insulin, for revers-
ing the molecular defects causing insulin resistance, and glucose intolerance, and to
relieve the complications of the disease. The alkaloids with antidiabetic activity have
been detailed reviewed by Christodoulou et al. (2019). They have listed several
compounds like berberine, trigonelline, piperine, oxymatrine, vindoneline,
evodiamine, neferine and serpentine which need to be urgently clinicallytried in
order to confirm their potential as antidiabetic agents. Among them, berberine is the
most studied compound, found in various antidiabetic plants and exhibiting multiple
actions to counteract diabetes. It decreases glucose absorption and transport or
inhibits α-glucosidase (Pan et al. 2003; Cicero and Baggioni 2016).
Several studies have also been carried out to investigate the antidiabetic
properties of various polyphenols. As a result of these analyzes, some compounds
were identified to be promising in the prevention of the harmful effects of diabetes.
12
Rhizobium rhizogenes-Mediated Genetic Transformation of Antidiabetic Plants
Fig. 12.6 Chemical structure of some secondary metabolites with hypoglycemic properties. Scheme prepared by authors
365
These compounds include catechins, kaempferol, naringenin, luteolin, quercetin,

fisetin, rutin, myricetin, silymarin, chrysin, baicalein, genistein, chlorogenic acid,
ellagic acid, curcumin and resveratrol (Salehi et al. 2019). Polyphenols can act as
hypoglycemic indirectly due to their antioxidant effect. However, in vitro and
in vivo studies performed on animals reported a direct effect of the most representa-
tive compounds. Their main functions rely on the resistance to lipid peroxidation and
the regulation of pancreatic β-cells proliferation. Kaempferol activity was
demonstrated to promote insulin sensitivity and preserve pancreatic β-cell
(Alkhalidy et al. 2015). Moreover, naringenin was found to reduce glucose absorp-
tion, myricetin was discovered to exhibit an inhibitory effect on α-amylase activity
(Wang et al. 2010) while luteolin was suggested as a therapeutic drug against
diabetic nephropathy (Wang et al. 2011). Due to these properties, the above-
mentioned compounds can be considered as the prime candidates for medicinal
use against insulin resistance and type 2 diabetes (Den Hartogh and Tsiani 2019).
Among terpenoids, triterpenes glycosides show biological activity hindering the
absorption of glucose. Their presence was confirmed in the leaves, roots and fruits of
G. sylvestre, Gynostemma pentaphyllum, P. ginseng and Momordica charantia
(Kanetkar et al. 2006; Alqahtani et al. 2013). Gymnemic acid extracted from
G. sylvestre is a biologically active compound which function relies on the
supporting the regeneration of pancreatic β-cells in rats (Ahmed et al. 2009
and 2010). Gypenoside, isolated from G. pentaphyllum was recognized as an
agent with a high hypoglycemeic effect on rats (Bao et al. 2018). It enhances insulin
secretion and protects against oxidative stress and lipotoxicity (Patibandla et al.
2015). HR cultures from Gynostemma pentaphyllum have been established as a
potential alternative source for the production of gypenosides. Chang and
collaborators (2005) presented several attempts for the enhancement of HR biomass
and gypenosides content. The increment of gypenosides concentration in HR has
been related to the medium compositions, culture conditions and elicitation. How-
ever, the transformed roots contained lower gypenosides content in comparison with
the maternal plant. Ginsenosides, a group of steroidal saponins isolated mainly from
roots and berries of P. ginseng, and of other ginseng species (e.g. Panax
quinquefolius) significantly improves systemic insulin sensitivity and glucose
homeostasis in mice (Attele et al. 2002).
Charantin, acquired from Momordica charantia fruits is a blend of two steroidal
saponins: β-sitosterol glucoside and stigmasterol glucoside. Its function relies on
increasing glucose uptake and glycogen synthesis in the liver, muscles and fat cells
leading to the improvement of blood sugar level i. Charantin acts also as an enhancer
of insulin release from pancreatic β-cells, and repairs or promotes new insulin-
secreting β-cells growth (Keller et al. 2011; Nagappan et al. 2018).
Different tissue cultures technologies were applied from Momordica charantia in
order to produce and isolate charantin (Agarwal 2015). Hairy roots were successfully
obtained from cotyledons, leaves and hypocotyls of Momordica charantia and the
production of charantin by these transformed organs was compared with the produc-
tion by fruits of the maternal plant, offering a new method to obtain a high amount of
valuable anti-diabetic compound (Swarna and Ravindhran 2012).
It should be emphasized that relevant clinical data should be collected to establish

the hypoglycemic potential of these compounds and support the emergence of a safe
and effective antidiabetic agent. Additionally, new techniques should be adopted for
the cultivation of plant materials that are rich in antidiabetic compounds. Effective
isolation techniques, analytical methods and novel drug delivery systems can bring
plant-based molecules to the lead of hypoglycemic agents for the management of
diabetes (Desai and Tatke 2015).
12.4.3 Stevia rebaudiana Bertoni as a Novel Source of Antidiabetic

Compounds
S. rebaudiana Bertoni is a perennial plant from Asteraceae family, naturally growing

in certain regions of South America. It is a small shrub that grows to a height up to
30 cm or even 65 cm, having upright woody stems with sessile and opposite
lanceolate to oblanceolate leaves (Gantait et al. 2018). The inflorescence is a
chyme of corymbs containing five small white tubular flowers. The fruit is achene
having a single seed with a feathery pappus (Pandey 2018) (Fig. 12.7a). The
uniqueness of this plant lies in the production of specific secondary metabolites
called steviol glycosides (SvGs). They are known as the compounds giving an
impression of sweetness 250–300 times higher than sucrose. Therefore, Stevia
Fig. 12.7 Morphology of Stevia rebaudiana plant (a) growing in temperate climate conditions of
Southern Poland in the open field (b) and in the tunel (c). (Source: unpublished photos by Marta
Libik-Konieczny)
rebaudiana is also named as sweet herb, sweet leaf, honey leaf, candy leaf and honey
yerba (Carakostas et al. 2008).
SvGs are entkaurenoid diterpene glycosides synthesized through the
methylerythritol phosphate pathway (MEP) from the isopentenyl diphosphate
(IPP) skeleton (Totté et al. 2000) (Figs 12.5 and 12.6). The biosynthetic pathway
is a multistep process SvGls, and some phases of the process are shared with the
biosynthetic pathway of gibberellins (Helliwell et al. 1999). Bifurcation of the two
pathways occurs after the step of hydroxylation of the ent-kaureonic acid for the
production of steviol catalyzed by 13-kaureonic acid hydroxylase (KAH). At this
stage, steviol is glycosylated by the uridine diphosphate-dependent (UDP)-depen-
dent glucosyltransferases (UGT) (Brandle and Telmer 2007; Ceunen and Geuns
2013).
More than 30 different SvGs have been found in leaves of S. rebaudiana. They
include steviol, stevioside, rebaudiosides (A, B, C, D, E, F), steviolbioside and
dulcoside A presented in varying concentrations (Gupta et al. 2013). All together
they can represent up to 20% of the leaf dry weight (Brandle et al. 1998). SvGls,
except that they are not metabolized in the human body, also do not exhibit
teratogenic, mutagenic, carcinogenic effects or allergic reactions (Geuns 2003;
Rank and Midmore 2006). Many studies concerning the possible action of stevia
extracts or individual steviol glycosides as antidiabetic agents have been performed
(for review Gupta et al. 2013; Mishra 2011; Abdel-Aal et al. 2021). The data
obtained showed that the compounds derived from the stevia provide a comprehen-
sive set of mechanisms to combat type II diabetes and its potential complications
(Gupta et al. 2013). Several studies performed on animals have reported that
compounds from stevia have antihyperglycemic, insulinotropic and glucagonostatic
actions in diabetic rats and mice (Ahmad et al. 2018). Stevioside and steviol enhance
insulin secretion by acting directly on pancreatic β-cells, which may indicate that
SvGs can be used as powerful antihyperglycaemic agents (Jeppesen et al. 2000,
2002, 2003). Shibata et al. (1995) and Chen et al. (2007) indicated that the
antidiabetic effect might be due to steviosides which counteract the glucotoxicity
in β-cells or also suppress the glucagon secretion by α-cell of pancreas. Stevioside
regulates blood glucose levels by enhancing insulin secretion and also insulin
utilization in insulin-deficient rats. It was found out that stevioside decreased
phosphoenolpyruvate carboxykinase (PEPCK) gene expression in rat liver and
slowed down gluconeogenesis (Chen et al. 2005). It was also stated that the extracts
of S. rebaudiana could decrease the blood glucose level in diabetic rats in a time-
dependent manner (Kujur et al. 2010). Moreover, extracts of S. rebaudiana produce
a delayed but significant decrease in the blood glucose level, without produc-
ing the condition of hypoglycemia (Misra et al. 2011). Most recently Abdel-Aal
et al. (2021) have proven that stevia extracts could enhance the antidiabetic activity
of saxagliptin—an oral hypoglycemic drug and may lead to the improvement of
insulin sensitivity in diabetic rats.
Unfortunately, only a few studies were conducted in diabetic humans; however,
they have also indicated beneficial effects of stevioside on the glucose metabolism.
Gregersen et al. (2004) have shown that stevioside reduces postprandial blood
glucose levels in type 2 diabetic patients, Misra et al. (2011) have found a potential
antidiabetic effect of stevia leaf powder influencing positively fasting blood sugar
level (FBS) and postprandial blood sugar (PPBS) level.
Due to above mentioned beneficial properties of stevia plant on diabetes preven-
tion and treatment, it is not surprising that this plant leaves, the extract of leaves and
purified SvGs are widely used in food products and as dietary supplements in many
countries. Today stevia is cultivated and used as a sweetener in East Asia, including
China, Korea, Thailand and Malaysia and Japan, as well as in USA and in South
America. There are very few reports concerning the successful growth of
S. rebaudiana plant under temperate climate conditions (Libik-Konieczny et al.
2018) (Figs 12.7b, c).
The propagation of stevia plants is labor-intensive and done by stem cuttings
mostly (Yadav et al. 2011). Therefore, a substitute micropropagation using in vitro
systems have been developed. This was started by Yang and Chang (1979) and since
then, this technique is effectively utilized to generate stevia plants on a large
scale since stevia seed germination rate is quite poor (Carneiro et al. 1997). How-
ever, different plant parts of stevia contain different levels of SvGs production (high
in leaves and low in roots) and that also varies depending on the stages of growth
(Kang and Lee 1981; Bondarev et al. 2003b; Yang et al. 2015; Libik-Konieczny
et al. 2018). Therefore, instead of whole plant regeneration, the other modes such as
cell suspension, multiple shoots, callus, including HR were rigorously attempted on
S. rebaudiana to date (Lee et al. 1982; Hsing et al. 1983; Swanson et al. 1992;
Bondarev et al. 1997, 2001, 2003a, b; Sivaram and Mukundan 2003; Jadeja et al.
2005; Ladygin et al. 2008; Janarthanam et al. 2010; Taware et al. 2010; Das et al.
2011; Mathur and Shekhawat 2013; Gupta et al. 2014; Khalil et al. 2014; Gantait
et al. 2015; Gupta et al. 2015; Michalec-Warzecha et al. 2016; Pandey et al. 2016;
Reis et al. 2017; Singh et al. 2017). However, the results achieved in some studies
concerning the content of SvGs in cultured samples are contradictory. Some authors
did not find any SvGs in callus or cell suspension cultures, while others described
that the concentration of SvGs in the callus cultures might even be higher than in
stevia leaves (Luwańska et al. 2015; Pandey et al. 2016). Similarly, different data on
SvGs biosynthesis by induced HRs after transformation of S. rebaudiana with
Rhizobium strains were published by (Yamazaki and Flores 1991; Pandey et al.
2016; Libik-Konieczny et al. 2020). In the first experiments on the formation of
hairy roots by stevia plants, SvGs were not detected in the transformed tissue
(Yamazaki and Flores 1991). However, later on in the studies performed by Pandey
et al. (2016), the presence of stevioside was noted in both HR and in the culture
medium, although the differences in the ability of stevioside production were noted
between hairy roots clones growing in the same conditions. On the other hand, in a
recently published report (Libik-Konieczny et al. 2020), the production of different
SvGs in HR cultured under various osmotic and light conditions was observed. The
authors suggested that the profile of SvGs depended on the level of oxidative stress
in HR tissues and applied stress factors in the culture medium. Due to these
discrepancies, the technique of producing transgenic hairy root cultures as a source
of S. rebaudiana secondary metabolites requires more scientific attention. The
general sequence of modifications in the steviol glucoside pathway has been already
determined and particular enzymes and genes have been identified by screening of
candidate genes from expressed sequence tags (EST) collections or RNAi based
Agrobacterium-mediated transient gene silencing (AMTS) (Brandle et al. 2002;
Richman et al. 2005; Guleria and Yadav 2013; Lee et al. 2019). This provides a
good insight into the process of glycosylation and creates a possible template for the
engineered biosynthesis of steviol glycosides (Lee et al. 2019).
12.5 Conclusions and Further Perspectives
Secondary metabolites may be useful as important compounds in the treatment of

various human diseases, including diabetes. Among many of them, flavonoids,
terpenoids, saponins, carotenoids, alkaloids and glycosides may have antidiabetic
effects. The production of plant secondary metabolites in in vitro culture provides a
safe and qualitative methodology for use in research, pharmaceutical and food
industries.
Hairy root induction (HR) is the result of the transfection of the T-DNA of the Ri
plasmid from R. rhizogenes into the plant cell. Several genes found in T-DNA,
so-called rol genes, influence the secondary metabolism of infected plants. There-
fore, HR culture is a key biotechnological technique intended for the production of
various biologically active and medicinally valuable pharmacological compounds.
Metabolomic engineering is developing very fast and to HR biotechnological tools
significant progress has been achieved to ameliorate plant secondary metabolite
production, however, biosynthetic pathways and their regulation in order to produce
SvGs are yet to be explored.
The sweet herb Stevia rebaudiana (Bertoni) has a precious prospect and is widely
utilized in several parts of the world. SvGs are commercially valuable antidiabetic
sugar-substitutes in food, drink and medicine. Steviol glycosides produced in stevia
leaves have the ability to enhance insulin action and its, production. They can also
stabilize glucagon secretion and improve glucose tolerance leading to the mainte-
nance of the proper blood sugar level in both animals and humans. Therefore, sugars
can be replaced with steviol glycosides to support healthy glucoregulation.
Here we demonstrated a novel plant-microbial interaction in which HR are
induced by R. rhizogenes in S. rebaudiana tissues. HR from S. rebaudiana may
be an efficient source for the production of SG as well as other potentially useful
antidiabetic drugs. However, more research is needed on S. rebaudiana to obtain
clinical evidence of the beneficial effects of steviol glycosides and to accurately
describe the biosynthetic pathways of these compounds. In this way, the potential of
stevia as an antidiabetes plant can be fully understood.
Acknowledgment Results concerning S. rebaudiana hairy roots culture were obtained as part of
experiments carried out in projects funded by the National Science Centre (no. 2012/05/B/NZ9/
01035 and no. 2013/09/N/NZ9/01650).
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Production of Antidiabetic Lignans in Flax
Cell Cultures 13
Lucija Markulin, Samantha Drouet, Laurine Garros, Sumaira Anjum,
Duangjai Tungmunnithum, Bilal Haider Abbasi,
Randolph R. J. Arroo, Eric Lainé, Mohamed Addi, and
Christophe Hano
Abstract
Lignans are a group of polyphenols that mimic endogenous estrogen in a struc-

tural context. Clear evidence was provided by animal studies and preclinical
models that phytoestrogens could have antidiabetic activity in both estrogen-
dependent and estrogen-independent pathways. A number of studies, including
epidemiological studies and clinical studies conducted in different populations,
link lignans, diabetes prevention, and management. Several lignans have been
reported as potent antidiabetic compounds, in particular, secoisolariciresinol
diglucoside (SDG), the major antidiabetic lignan from flax seeds. SDG have to
be converted by the human intestinal microbiota into the bioactive mammalian
L. Markulin · S. Drouet · L. Garros · E. Lainé · C. Hano (*)

Laboratoire de Biologie des Ligneux et des Grandes Cultures, INRAe USC1328, Eure et Loir
Campus, University of Orleans, Orléans Cedex, France
e-mail: hano@univ-orleans.fr
S. Anjum
Department of Biotechnology, Kinnaird College for Women, Lahore, Pakistan
D. Tungmunnithum
Department of Pharmaceutical Botany, Faculty of Pharmacy, Mahidol University, Bangkok,
Thailand
B. H. Abbasi
Department of Biotechnology, Quaid-i-Azam University, Islamabad, Pakistan
R. R. J. Arroo
Faculty of Health and Life Sciences, De Montfort University, Leicester, UK
M. Addi
Laboratoire dʼAmélioration des Productions Agricoles, Biotechnologie et Environnement, Faculté
des Sciences, Université Mohammed Premier, Oujda, Morocco
Ltd. 2021
https://doi.org/10.1007/978-981-16-3529-8_13
384 L. Markulin et al.
lignans enterodiol and enterolactone. Most of the published research indicate that
SDG may have a great potential to reduce type 1 diabetes mellitus incidence and
to delay the development of type 2 diabetes mellitus in humans. Flax (Linum
usitatissimum L.) cell cultures can provide an attractive and sustainable resource
for the production and extraction of antidiabetic lignans. The advantage of using
cell cultures as opposed to whole plants is that they can be used independently
of climatic or seasonal considerations. This ensures more reproducible production
of economically important bioactive extracts. This chapter provides an overview
of the biotechnological approaches to the production of antidiabetic lignans in
flax cell cultures.
Keywords
Callus · Cell suspension · Diabetes · Elicitation · Linum usitatissimum L. ·

Secoisolariciresinol diglucoside
13.1 Introduction: Antidiabetic Actions of Flax Lignans
13.1.1 Flax Is a Functional Food Due to Its Exceptional Capacity

to Produce Health Promoting Lignans
In recent decades, there has been a rising demand in the use of linseed (Linum
usitatissimum L., Linaceae) in the diet to improve nutritional and health status
(Oomah 2001; Lainé et al. 2007, 2009). Indeed, a number of biological activities
have been reported for lignans and many of them have a positive impact on human
health. Dietary lignans, especially flaxseed lignans (e.g., secoisolariciresinol;
Fig. 13.1), and their positive health benefits have been extensively investigated
making flax a functional food. Some of the favorable effects are a reduction of
blood pressure (Ursoniu et al. 2016), cardioprotection (Zanwar et al. 2011), and
prevention of hormone-dependent cancers because of their pronounced antioxidant
and phytoestrogenic features (Dikshit et al. 2016; Hano et al. 2017). The special
interest in flax is justified by the fact that its seeds are, by far, the major food source
for dietary lignans (Fig. 13.2; Milder et al. 2005; Lamblin et al. 2008; Lainé et al.
2009). In flaxseed, the main lignan is secoisolariciresinol diglucoside (SDG), stored
in the seedcoat part of the seed in the form of a macromolecular complex (Struijs
et al. 2007) together with flavonols such as herbacetin diglucoside (Fliniaux et al.
2014) and other phenolic acid glucosides such as ferulic acid, p-coumaric acid, or
caffeic acid (Corbin et al. 2015; Fig. 13.1). Following its ingestion, SDG is first
deglucosylated into secoisolariciresinol and then converted into the so-called
phytoestrogenic mammalian lignans (aka enterolignans), enterodiol and
enterolactone (Clavel et al. 2006a; Fig. 13.1), after conversion by the human
intestinal microbiota. These mammalian lignans have been shown to be responsible
for a significant part of the health benefits of linseed (Setchell et al. 1981; Clavel
et al. 2006a). It is therefore important to bear in mind that the biological function of
13 Production of Antidiabetic Lignans in Flax Cell Cultures 385
Fig. 13.1 Structure of phenolic compounds involved in the lignan macromolecular complex. (a)
Structure of the complex components: (1) secoisolariciresinol (R ¼ H), or secoisolariciresinol
diglucoside (SDG, R ¼ β-D-glucose), (2) herbacetin (R ¼ H) or herbacetin diglucoside (HDG,
R ¼ β-D-glucose), (3) p-coumaric acid (R ¼ H), or p-coumaric acid glucoside (CouG, R ¼ β-D-
glucose), (4) caffeic acid (R ¼ H) or caffeic acid glucoside (CafG, R ¼ β-D-glucose), (5) ferulic acid
(R ¼ H) or ferulic acid glucoside (FerG, R ¼ β-D-glucose), (6) hydroxymethylglutaric acid
(HMGA). (b) Schematic representation of lignan macromolecule, where a unit of SDG or HDG
is ester-linked to another unit, thanks to HMGA, which can be replaced by one hydroxycinnamic
acid glucoside (HCAG) unit (CouG, CafG, or FerG) in terminal position of the chain
Fig. 13.2 Flaxseed is the main natural food source of mammalian lignans (adapted from Milder
et al. 2005)
flaxseed lignans in humans is conditional on their conversion to mammalian lignans

through gastrointestinal microbiota. Main flaxseed lignan is secoisolariciresinol
(SDG), but it should be noted that lignans such as matairesinol, pinoresinol,
pinoresinol diglucoside, and isolariciresinol have also been detected in flaxseed
(Ramsay et al. 2017). All of these compounds, except isolariciresinol, can serve as
precursors to enterodiol and enterolactone biosynthesis (Heinonen et al. 2001; Xie
et al. 2003). The role of the bacteria in the production of mammalian lignans was first
demonstrated in germ-free rats where no lignans were detectable in the urine
(Axelson and Setchell 1981). Formation of enterodiol and enterolactone from
SDG requires O-deglycosylation, demethylation, dehydroxylation, and dehydroge-
nation (Fig. 13.3). Enterolactone can be produced from enterodiol by dehydrogena-
tion (Borriello et al. 1985; Meselhy et al. 2000) or in two-step conversion from
matairesinol (Heinonen et al. 2001). Some of the bacterial strains capable of
performing these conversions have been identified. They include members of the
genus Bacteroides and Clostridium (Clavel et al. 2006b) as well as the selected
strains of Bifidobacterium (Roncaglia et al. 2011) involved in SDG deglycosylation.
SECO demethylation is catalyzed by Butyribacterium methylotrophicum, Eubacte-
rium callanderi, Eubacterium limosum, and Peptostreptococcus productus and
SECO dehydroxylation is catalyzed by Clostridium scindens and Eggerthella lenta
(Clavel et al. 2006b). Enterodiol dehydrogenation to enterolactone is catalyzed by
Lactonifactor longoviformis and Clostridium saccharogumia (Clavel et al. 2006b,
2007). Additionally, bacterial strains able to catalyze pinoresinol to lariciresinol
(Eggerthella lenta and Enterococcus fecalis) and lariciresinol to secoisolariciresinol
Fig. 13.3 (a) Major pathways of bioactivation of plant lignans by gastrointestinal microbiota.
Pinoresinol (PINO), lariciresinol (LARI), secoisolariciresinol (SECO), SECO-diglucoside (SDG),
matairesinol (MATA), 2.3-bis(3,4-dihydorxybenzyl)butene-1,4-diol (dhEND), 2–3-bis
(3,4-dihydroxybenzyl)butyrolactone (dhENL); arrows—black: reduction, orange: O-
deglycosylation, blue: demethylation, green: dehydroxylation, purple: dehydrogenation (adapted
from Clavel et al. 2006b, 2007; Jin and Hattori 2010; Yoder et al. 2015). (b) Chemical structures of
plant phytoestrogens derived from plant lignans and human hormone 17-β-oestradiol
(Eggerthella lenta) have also been identified (Xie et al. 2003; Clavel et al. 2006a).
Matairesinol can be also converted to enterolactone by demethylation by Clostrid-
ium bacterium and Ruminococcus productus (Clavel et al. 2006a; Jin and Hattori
2010) and dehydroxylation by Eggerthella lenta (Jin et al. 2007). Bifidobacterium
adolescentis is the first probiotic bacterium able to form enterodiol from lignan
extracts (no conversion from pure secoisolariciresinol, matairesinol, or sesamin)
(Gaya et al. 2017).
13.1.2 Flax Lignans Actions Against Diabetes Mellitus
Diabetes mellitus is one of the most prevalent global diseases and type 2 diabetes
mellitus (T2DM) is the most common form. T2DM constitutes 90% of the ca
400 million people suffering from diabetes worldwide (WHO 2020). Diabetes is a
metabolic syndrome characterized by elevated central adiposity, serum tirglycerides,
serum glucose, blood pressure, inflammation, and reductions in HDL-cholesterol,
which raises the risk of insulin resistance. T2DM (formerly named noninsulin-
dependent or adult-onset) is linked to an inefficient use of insulin and characterized
by hyperglycemia, decreased glucose tolerance, insulin resistance, and hyperlipid-
emia (Prasad and Dhar 2016). T2DM is primarily due to excess body weight and
physical inactivity, and is connected to poor quality of life and increased risks of
death and/or morbidity. Diabetes and its effects also have a huge economic impact
on people, communities, health systems, and countries. Diverse mechanisms have
been proposed to explain the antidiabetic action of flax lignans (Prasad and Dhar
2016; Imran et al. 2015). Flaxseed and flax lignan complex improve glycemic
control (Prasad and Dhar 2016; Imran et al. 2015). Both animal and human studies
have shown that high fat diets supplemented by 0.5–1% SDG significantly reduce
liver triglycerides, serum triglycerides, and total cholesterol levels, as well as insulin
and leptin concentrations, resulting in a significant reduction in visceral fat produc-
tion relative to the control group (Fukumitsu et al. 2008). Studies have shown that
flaxseed lignan supplements have positive association with C-reactive protein
(CRP), and also indicate that lignans may have lipid-and blood pressure-reducing
associations (Peterson et al. 2010). Indeed, SDG have been proposed to decrease
CRP concentrations in association with insulin resistance in T2DM (Prasad 2009).
Daily consumption of SDG (500 mg/day) for 6 weeks can reduce CRP
concentrations (Prasad 2007). SDG supplementation has also been reported to
delay the development of T2DM in Zucker rat and to decrease the risk of glucosuria
in female rats receiving glucosuria induced diet by 80% (Prasad et al. 2000; Prasad
2001). In addition, its hypoglycemic effect in T2DM animal models has proposed to
be, at least in part, attributable to its ability to suppress the gene expression of
phosphoenolpyruvate carboxykinase observed in primary hepatocyte cell cultures
(Prasad 2002). However, a possible inhibitory action on intestinal α-glucosidase
(EC 3.2.1.20) and pancreatic α-amylase (EC 3.2.1.1) activities of SDG and its
mammalian-derivatives could not be ignored. In support of this hypothesis, it has

been documented that pinoresinol, which is a common precursor of SDG (Fig. 13.4),
as well as SDG, secoisolariciresinol, enterodiol, and enterolactone, can act as
α-glucosidase (Wikul et al. 2012) and α-amylase inhibitors (Hano et al. 2013).
Both enzymes are essential enzymes for carbohydrate digestion and absorption,
and have been identified as important therapeutic targets for the regulation of
postprandial pathologic hyperglycemia observed in patients with T2DM
(Adisakwattana et al. 2011). In addition, both type 1 (T1DM) and T2DM have
been associated with oxidative stress (Prasad and Dhar 2016), and flax lignans are
potent antioxidants (Kitts et al. 1999; Hano et al. 2017). Animal models of T1DM
involving streptozotocin administration or utilizing bio-breed diabetic prone (BBdp)
rats are associated with oxidative stress. SDG supplementation has decreased the
incidence of serum glucose diabetes by 75% in the streptozotocin model of diabetes
and 72% in the BBdp rat model of diabetes (Prasad and Dhar 2016). These
reductions were associated with decreases in serum oxidative stress and pancreatic
malondialdehyde (MDA) formation (Prasad and Dhar 2016). In the T2DM Zucker
rat model, the SDG antidiabetic effect was associated with reduced serum MDA and
decreased hemoglobin A1C glycation (Prasad and Dhar 2016).
Altogether these data suggest that SDG may have a great potential for reducing
the incidence of both T1DM and delaying the development of T2DM in humans
(Prasad and Dhar 2016). However, while both in vivo and in vitro results are
globally favorable to the chemopreventive action of lignans, epidemiological studies
are far less conclusive (Zamora-Ros et al. 2013). Moreover, the mechanism by which
flax lignans prevent diabetes also remains unclear and requires further elucidation.
For this purpose, the availability of purified flax lignans at a reasonable cost would
facilitate in vivo supplementation trials and elucidation of mechanisms.
13.2 Biosynthesis of Lignan in Linseed and Extraction

Complications
13.2.1 Overview of the Lignan Biosynthetic Pathway in Flax
The lignan derived from the phenylpropanoid pathway and from a structural point of
view are dimers of phenylpropanoid units called monolignols (aka hydroxycinnamic
alcohols), with a varying degree of oxidation and an aromatic moiety substitution
pattern (Ward 1999; Fang et al. 2013; Teponno et al. 2016). Structures are formed by
an oxidative dimerization occurring between the two central carbons (C-8 and C-80 )
of the side chains of the phenylpropanoid units. All other forms of linkages are
known as neolignans. In flax, both lignans and neolignans are biosynthesized by a
stereoselective oxidative dimerization of the two coniferyl-alcohol moieties
(Fig. 13.4; Ford et al. 2001; Corbin et al. 2018).
In flax seeds, the two coniferyl alcohol radicals are first converted to ()-
pinoresinol by the action of the dirigent proteins DIR5 or DIR6 guiding the phenoxy
radical coupling (Dalisay et al. 2015; Corbin et al. 2018). Through two successive
reduction steps involving the pinoresinol lariciresinol reductase PLR1, ()-
390
Fig. 13.4 Proposed biosynthetic pathway of lignans in flax (Linum usitatissimum L.) seeds and aerial parts (adapted from Ford et al. (2001), Ghose et al.
(2014), Dalisay et al. (2015). and Corbin et al. (2017)). LuPLR1 (pinoresinol-lariciresinol reductase 1) enzyme is responsible for the biosynthesis of the major
lignan (+)-secoisolariciresinol accumulated in the seedcoat in its diglucosylated form SDG, whereas LuPLR2 (pinoresinol-lariciresinol reductase 2), which is
expressed in leaves, leads to the accumulation of another type of lignans derived from the ()-secoisolariciresinol enantiomer, such as ()-yatein. Other
L. Markulin et al.
abbreviations: SDG secoisolariciresinol diglucoside, DIR dirigent protein oxidase complex, UGT UDP-glucosyl transferase
pinoresinol is converted into ()-lariciresinol and to (+)-secoisolariciresinol (Fang

et al. 2016; Corbin et al. 2017; Renouard et al. 2014; Garros et al. 2018; Markulin
et al. 2019a). Then, the C-9 and C-90 hydroxyl positions of (+)-secoisolariciresinol
are then successively glycosylated by the UDP-glycosyltransferase UGT74S1 to
form SDG (Ghose et al. 2014; Fofana et al. 2017). The main flax seed lignan SDG is
finally stored as a macromolecular hydroxymethyl glutaryl ester-linked complex
(aka SDG-HMG) by linking hydroxylmethylglutaryl (HMG) to C-6 and C-60
through the action of HMG CoA-transferase (Ford et al. 2001; Kamal-Eldin et al.
2001). The core of the problem faced during the extraction and purification of SDG
from flaxseeds is partly due to this uncommon storage form (Renouard et al. 2010;
Corbin et al. 2015).
13.2.2 Lignans and Neolignans Biosynthesis in Flax: A Tunable

Pathway
Flax is a unique model for the study of the biochemical and physiological functions
of the remarkable DIRs; since its seed coats accumulate (+)-secoisolariciresinol
lignan derivatives in high quantities, its leaves accumulate ()-yatein derivatives,
while its in vitro cultures have been reported to have a wide range of neolignans, all
derived from the activity of specific DIRs (Ford et al. 2001; Hemmati et al. 2010;
Renouard et al. 2014; Corbin et al. 2017; Bose et al. 2020). A genome-wide analysis
and characterization of the 44-membered multigene family of predicted flax DIR
was performed, and functions for specific DIRs in ()-pinoresinol formation in seed-
coats, as well as (+)-pinoresinol in vegetative organs and/or specific stress response
were proposed (Corbin et al. 2018). On the other hand, this study revealed that
neolignan formation, first proposed as a nonenzymatic dimerization process of two
coniferyl alcohol units, is connected with the expression in flax of some specific DIR
genes (Corbin et al. 2018). Considering this huge variety of lignan and neolignan
structures accumulated in elevated quantities, flax is also considered a unique
starting material for their production.
At least two PLRs with opposite enantiospecificity are also expressed in flax. A
clear correlation between lignan synthesis and gene expression of these two PLR
genes has been observed in the different flax organs. Although PLR1 transcript was
only detected in roots and seeds, PLR2 gene was expressed in stem, leaves, roots,
and seeds (Hano et al. 2006b; Hemmati et al. 2010; Renouard et al. 2012; Corbin
et al. 2017). PLR1 have been shown to convert ()-pinoresinol to (+)-
secoisolariciresinol via ()-lariciresinol (von Heimendahl et al. 2005). Interestingly,
though both pinoresinol enantiomers and pure (+)-SDG produced from (+)-
secoisolariciresinol are present in the flax cell culture (von Heimendahl et al.
2005), its seeds contain almost pure (+) SDG (99% e.e.) (Ford et al. 2001; Sicilia
et al. 2003). Transgenic plants producing seeds devoid of lignan through the
knocked down of the PLR1 gene by RNAi support the role of PLR1 in (+)-SDG
biosynthesis in the flax seed coat (Renouard et al. 2014). Interestingly, the absence of
active (+)-SDG biosynthesis in seed, resulting from this suppression of the PLR1
expression not only results in the accumulation of ()-pinoresinol, but also in the
production of neolignans with monolignols connected through 8-O-4 ‘or 8–5’
bounds (Renouard et al. 2014), thus supporting a possible control occurring at the
DIRs level.
On the contrary, flax aerial parts mainly contain levorotatory nonpolar
dibenzylbutyrolactone (DBL) lignans with absolute stereochemistry 8R, 80 R deriv-
ing from 8R, 80 R-secoisolariciresinol and 8R, 80 R-matairesinol (i.e., ()-
secoisolariciresinol and ()-matairesinol; Schmidt et al. 2006, 2010). This specific
configuration appeared to correspond to the transcriptional activity of PLR2 gene in
stem and leaves (Hemmati et al. 2010). PLR2 have been shown to form ()-
secoisolariciresinol by converting (+)-pinoresinol and (+)-lariciresinol (Hemmati
et al. 2010). Both flax PLRs have been found to be specific in the use and production
of each secoisolariciresinol enantiomers. In the case of flax, a possible explanation
results in gene duplication, enabling two concurrent biosynthetic pathways to
evolve. It was proposed in support of this hypothesis that gene duplication occurred
in the progenitor of flax (L. usitatissimum), called Linum bienne (Hemmati et al.
2010).
Flax in vitro cultures, in addition to the antidiabetic lignans SDG and LDG, have
been reported to accumulate high amounts of neolignans, in particular
dehydrodiconiferyl alcohol glucoside (DCG) and guaiacylglycerol-β-coniferyl
ether glucoside (Hano et al. 2006a; Attoumbré et al. 2006a, b; Beejmohun et al.
2007a, b; Anjum et al. 2017a, b, 2020 Markulin et al. 2019b; Fig. 13.5). In line with
this observation, a phenylcoumaran benzyl ether reductase (PCBER) gene poten-
tially involved in the metabolism of neolignans was isolated and its gene expression
characterized in flax in vitro cultures (Attoumbré et al. 2006a; Hano et al. 2006a).
Bose et al. (2020) recently further expanded the diversity of lignans and neolignans
in flax by identifying seven additional compounds from cell suspensions and callus
cultures (Fig. 13.5). Note that a vast array of both aglycone and glycosidic forms of
lignans and neolignans have been reported in flax stem tissues (Huis et al. 2012), but
at much lower concentrations than in in vitro systems.
Taken together, these data clearly identify flax as a tunable system, in particular
in vitro cultures, for the production of a wide array of lignans and neolignans,
including those with well-described antidiabetic actions.
13.2.3 The Troublesome Steps in the Extraction and Purification

of Antidiabetic Lignan from Flax Seeds
Optimization of lignan extraction (primarily SDG and aglycone) from linseed is of

considerable importance to human health. However, several parameters affect the
quality and yield of extraction, including the preparation of samples, the use, form,
and concentration of solvents, hydrolysis type, temperature, duration, and solid and
liquid ratio. The extraction of the lignans can be impeded by some of the seed
components and their modification, removal, and destruction can help facilitate the
extraction process.
Fig. 13.5 Chemical structures of the main lignans and neolignans accumulated in the callus and
suspension cell cultures of flax (L. usitatissimum) (adapted from Bose et al. 2020)
Flaxseed, along with phenolics, is rich in mucilage, a cell layer protecting the
outer surface of the flax seed coat. Mucilage can affect the extraction yield by
impeding access to the seed coat or by trapping lignans (Renouard et al. 2010).
Pretreatment of flaxseeds with cellulase can decrease mucilage thickness
(Wanasundara and Shahidi 1997; Renouard et al. 2010). Mucilage viscosity can
also be adjusted with the change of pH. High viscosity occurs in the pH range 5–9,
but at lower pH, the viscosity reduces because charge suppression could lead to
smaller polymer conformation and a high pH drop in viscosity may occur due to an
alkaline depolymerization reaction (Fedeniuk and Biliaderis 1994; Renouard et al.
2010).
The oil is another element present in a high quantity of linseed. Oil contamination
of extracts is avoided by defatting seeds with organic nonpolar solvents such as
petroleum ether, chloroform (Rickard et al. 1996), or n-hexane (Mazur et al. 1996;
Kraushofer and Sontag 2002). Removing oils helps alcoholic extraction of SDG
(Sainvitu et al. 2012).
The release of lignans can also be hindered by their linkage to other molecules in
the HMG macromolecular complex. Indeed, most flaxseed phenolics are
glucosylated and stored as hydroxymethyl glutaryl (HMG) in ester-linked complex
(SDG-HMG) (Ford et al. 2001). For this reason, the two main steps in the extraction
of lignans from flax seeds are: (1) the extraction of SDG-HMG polymer, and
(2) SDG-HMG polymer hydrolysis. A third optimal step can be the obtention the
aglycone forms. SDG-HMG polymer extraction is most commonly accomplished by
alcoholic extraction (methanol, ethanol) in combination with hydrolysis. Hydrolysis
can be alkaline, acidic, or enzymatic or any combination of those, but only acidic and
enzymatic hydrolysis can produce aglycones. In particular, a particular challenge in
lignan extraction is the wide variety of conjugated carbohydrates connected to
aglycone as well as to monosaccharides themselves. Often phenolic compounds
have several hydroxyl groups that could be involved in glycosidic bonding (Liggins
et al. 2000). The release of SDG from the SDG-HMG polymer is usually accom-
plished by alcoholic solid–liquid extraction and alkaline treatment (Eliasson et al.
2003), but this method could be time consuming. Acid hydrolysis is another
procedure that enables the recovery of the aglycone form of secoisolariciresinol,
but this approach may be destructive if too long a heating time or too high a
concentration of hydrochloric acid is used (Kraushofer and Sontag 2002; Li et al.
2008). Microwave-assisted extraction of SDG has also been described allowing time
to be gained, but no marked increase in yield has been achieved (Beejmohun et al.
2007a, b; Nemes and Orsat 2011; Zhang and Xu 2007).
In flax cell cultures, these impairing compounds are not present and lignans are
found in free form (i.e., not in the form of a macromolecular complex with HMG),
thereby making the hydrolysis steps unnecessary and thus simplifying their extrac-
tion and purification. For all these reasons, flax cell cultures may provide an
attractive alternative source for the sustainable production and extraction of
antidiabetic lignans.
13.3 Flax Cell Cultures: Promising Alternative Sources

of Antidiabetic Lignans
13.3.1 Plant Cell Culture as an Alternative Source of Bioactive

Metabolites
Cell in vitro cultures is an important alternative to whole plant cultivation for the
production of bioactive secondary metabolites (Chandran et al. 2020). Callus is a
solid and undifferentiated mass of plant cells that can also be obtained individually
or in combination by inoculating various plant sections on a controlled nutrient-rich
medium with different phytohormones such as cytokinin or auxin (Chandran et al.
2020). In order to increase the production of pharmaceutically active secondary
metabolites, many callus cultures of many plant species have been established so far
(Chandran et al. 2020), but their lack of uniformity and limited growth are generally
troublesome for industrial purposes. It is therefore possible to establish cell suspen-
sion cultures from callus cultures. Cell suspension cultures are small cell-aggregates
that are dispersed uniformly in a stirred liquid medium. These cell suspensions can
be cultivated in a bioreactor to allow the production of high biomass consistent with
industrial requirements, and can produce tremendous amount of medicinally
important secondary metabolites (Yue et al. 2016). A wide array of pharmaceutically

relevant secondary metabolites from various plant species have been produced by
cell suspension cultures under controlled conditions (Yue et al. 2016; Chandran et al.
2020). Plant cell suspension cultures have many advantages over traditional cultiva-
tion techniques for secondary metabolite processing and constitute an attractive
system for plant biosynthetic pathway metabolic engineering to increase the natural
and/or de novo synthesis of bioactive secondary metabolites (Chandran et al. 2020).
These advantages include: (a) independence from environmental, seasonal and
geographical constraints; (b) it is possible to multiply a single cell to generate unique
metabolites; (c) it is possible to ensure stable product quality due to genetic stability;
(d) automated cell growth control and metabolic process regulation; (e) it is possible
to discover a new biosynthetic route using mutant cell lines; and (f) it is simpler to
produce and subsequently to extract secondary metabolites under controlled chemi-
cal environment (Yue et al. 2016; Chandran et al. 2020). In addition, all safety and
regulatory requirements for food drug administration are fulfilled by these naturally
produced secondary metabolites (Yue et al. 2016). Many medicinally important
secondary plant metabolites are currently developed on a commercial scale through
cell suspension cultures, such as ginsenoids, shikonin, and paclitaxel (Yue et al.
2016; Chandran et al. 2020).
13.3.2 Establishment of Flax Callus and Cell Suspension Cultures

for the Production of Antidiabetic Lignans
Several studies are available on multiplication of flax in vitro (Nichterlein et al.

1991; Cunha and Ferreira 1999; Dedičová et al. 2000; Rutkowska-Krause et al.
2003; Salaj et al. 2005). Flax callus and cell suspension cultures were successfully
established from stem, hypocotyl, leaf, and cotyledon explants (Attoumbré et al.
2006a, b; Hano et al. 2006a, 2008; Beejmohun et al. 2007a, b; Corbin et al. 2013a, b;
Gabr et al. 2016; Anjum et al. 2017a, b; Nadeem et al. 2018, 2019; Ahmad et al.
2019; Markulin et al. 2019c; Zahir et al. 2018; Khan et al. 2020; Zaeem et al. 2020)
in Murashige and Skoog (MS) medium (1962). Flax cell suspensions grown in
Linsmaier and Skoog (LS) medium (Linsmaier and Skoog 1965) was also reported
(Attoumbré et al. 2006b). From the different starting tissues used for initiation, stem
and leaf constituted the best initial explants to establish calli that gave highest
biomass accumulation (Anjum et al. 2017b; Abbasi et al. 2019; Zahir et al. 2018;
Zaeem et al. 2020). Concerning the influence of phytohormone balance, highest
biomass production for flax cell suspensions (Anjum et al. 2017b; Abbasi et al.
2019) and callus (Zahir et al. 2018; Zaeem et al. 2020) was achieved in presence of
1-naphthaleneacetic acid (NAA) at 1 mg/L.
The presence of lignans and neolignans have been reported in both callus cultures
(Anjum et al. 2017a; Zahir et al. 2018; Khan et al. 2020; Zaeem et al. 2020) and cell
suspension cultures (Attoumbré et al. 2006a, b; Hano et al. 2006, 2008; Beejmohun
et al. 2007a, b; Corbin et al. 2013a, b; Gabr et al. 2016; Anjum et al. 2017b; Nadeem
et al. 2018, 2019; Ahmad et al. 2019; Markulin et al. 2019c; Bose et al. 2020). We
can also note that hairy root cultures (Gabr et al. 2016; Markulin et al. 2019b) and
adventitious root cultures (Anjum et al. 2020) have also been developed in the recent
years. Highest SDG accumulation was obtained for leaf-derived callus grown in B5
medium supplemented with NAA at 1 mg/L (Zahir et al. 2018) and stem-derived cell
suspension grown in MS medium supplemented with NAA at 1 mg/L (Abbasi et al.
2019). For LDG, maximum accumulation was reported for leaf-derived callus grown
in MS medium supplemented with NAA at 2 mg/L (Zaeem et al. 2020) and stem-
derived cell suspension grown in MS medium supplemented with NAA at 1 mg/L
(Anjum et al. 2017b).
The diverse publications on the establishment of flax cell cultures, culture
conditions, biomass, and the production of antidiabetic lignans SDG and LDG are
summarized in Table 13.1. The accumulation of antidiabetic lignans in in vitro cell
cultures is comparable for SDG and much better for LDG relative to those recorded
for flax seeds (Renouard et al. 2010; Ramsay et al. 2017). Lignan production tends to
be higher in callus cultures, but the production of biomass in cell suspension cultures
is much greater, and given the potential scale up in the bioreactor, it seems to be the
best option for future industrial production of antidiabetic lignan.
13.3.3 Elicitation of the Production of Antidiabetic Lignans in Flax

Cell Cultures
A classic biotechnological approach to increasing the production of secondary

metabolites is elicitors that activate secondary metabolic pathways to promote
plant defense (Chandran et al. 2020). Depending on their source, elicitors can be
separated into biotic and abiotic elicitors. Abiotic elicitors originate from nonbiolog-
ical sources, such as physical stresses (e.g., cold, UV, osmotic stress, and water
stress) or inorganic compounds (e.g., heavy metal salts, metal ions, or metal oxides).
Biotic elicitors, on the other hand, originate from biological origins and may either
be exogenous or endogenous. Microbial cell walls such as chitosan, chitin, or plant
cell wall components such as polysaccharides or oligosac-charides are considered as
exogenous biotic elicitors. Phytohormones such as salicylic acid (SA) and methyl
jasmonate generated by plant cells in response to stress or pathogen invasion have
been identified as endogenous signaling compounds in elicitation (Chandran et al.
2020).
In order to stimulate the production of antidiabetic lignans, both biotic and abiotic
elicitors have been applied to flax cell cultures. These different elicitation strategies
are summarized in Table 13.2. Most of the time, with the exception of SA applica-
tion to flax callus cultures (Nadeem et al. 2019), as well as yeast extract and chitosan
exogenous applications (Nadeem et al. 2018; Ahmad et al. 2019), elicitation with
zinc (ZnONP) and silver (AgONP) oxide nanoparticles (Abbasi et al. 2019; Zahir
et al. 2019), or UV-C application (Anjum et al. 2017b) in flax cell suspensions, these
approaches had a negative effect on biomass production. Interestingly, the highest
SDG and LDG productions were measured after SA elicitation of flax callus cultures
(Nadeem et al. 2019), while the highest SDG and LDG contents were reported after
13
Table 13.1 Establishment of flax cell cultures from various explants, culture conditions, biomass production, and accumulation of antidiabetic lignans SDG
and LDG
Lignans
Explant Medium PGR used (mg/L) Biomass (g) SDG LDG References
Callus
Shoot B5 2,4D: 1.0 1.0 (28)a 0.24 (28)1 – Gabr et al. (2016)
BAP: 0.1
BAP: 0.2
GA3: 0.5 0.31 (28)2
GA3: 1.0
Stem MS NAA: 2.0 15.2 (35)b 1.8 (35)2 7.8 (35) Anjum et al. (2017a)
Stem MS NAA: 5.0 3.5 (35)b 1.0 (35)2 0.8 (35) Anjum et al. (2017a)
TDZ: 1.0
Production of Antidiabetic Lignans in Flax Cell Cultures
Leaf MS NAA: 2.0 15.7 (35)b 2.2 (35)2 8.1 (35) Anjum et al. (2017a)
Leaf MS NAA: 5.0 3.7 (35)b 0.8 (35)2 1.2 (35) Anjum et al. (2017a)
TDZ: 1.0
Leaf MS NAA: 1.0 25.9 (42)b 23.3 (35)2 4.6 (35) Zahir et al. (2018)
Leaf B5 NAA: 1.0 27.3 (42)b 8.9 (35)2 1.6 (35) Zahir et al. (2018)
Leaf SH NAA: 1.0 26.9 (42)b 4.0 (28)2 2.9 (42) Zahir et al. (2018)
Stem MS NAA: 1.0 10.1 (30)b 2.9 (30)2 4.2 (30) Nadeem et al. (2019)
Stem MS NAA: 1.0 18.1 (30)b 2.3 (30)2 3.3 (30) Zaeem et al. (2020)
Root MS NAA: 1.0 13.3 (45)b 2.2 (30)2 7.1 (30) Anjum et al. (2020)
Cell suspension
Root MS NAA: 0.5 2.2 (15)b – – Attoumbré et al. (2006b)
397
(continued)
398
Lignans
Explant Medium PGR used (mg/L) Biomass (g) SDG LDG References
BAP: 0.5
Hypocotyl MS NAA: 2.0 PCV 0.36 (14)3 – Hano et al. (2006a)
BAP: 0.5
Hypocotyl MS NAA: 2.0 PCV 0.52 (14)3 Hano et al. (2008)
BAP: 0.5
Hypocotyl MS NAA: 2.0 ND 1.9 (14)2 – Corbin et al. (2013a)
BAP: 0.5
Stem MS NAA: 1.0 9.5 (30)b 4.6 (30)2 13.6 (30) Anjum et al. (2017b)
Stem MS NAA: 1.0 5.4 (15)b 4.1 (15) 8.9 (15) Nadeem et al. (2018)
Stem MS NAA: 1.0 8.6 (25)b 11.9 (25)2 3.9 (25) Abbasi et al. (2019)
Stem MS NAA: 1.0 5.5 (30)b 4.7 (30)2 8.9 (30) Ahmad et al. (2019)
Hypocotyl MS NAA: 2.0 ND 1.8 (14)2 – Markulin et al. (2019c)
BAP: 0.5
Stem MS NAA: 1.0 7.5 (30)b 11.9 (25)2 3.9 (20) Zahir et al. (2019)
a b
Biomass: g FW per culture starting from 0.5 FW initial biomass; g DW per liter of culture medium; ø: no PGR addition; ND not determined, PCV packed cell
volume, Lignans: 1SECO; 2SDG; not mentioned; The number in parentheses corresponds to the day on which each biomass or phytochemical concentration
value that preceded it was determined
L. Markulin et al.
13
Table 13.2 Influence of abiotic and biotic elicitors on biomass, accumulation of antidiabetic lignans SDG and LDG in flax cells cultures
Lignans
Elicitor name Elicitor type Concentration/other Biomass SDG LDG References
Callus
Ligth (MS)a Abiotic Photoperiod 16/8 27.1 (+4.6%) 1.1 (95.2%) 1.4 (69.6%) Zahir et al. (2018)
Ligth (B5)a Abiotic Photoperiod 16/8 26.0 (4.8%) 6.3 (28.9%) 5.2 (+226.2%) Zahir et al. (2018)
Ligth (SH)a Abiotic Photoperiod 16/8 27.3 (+1.5%) 7.6 (+90.0%) 1.9 (70.3%) Zahir et al. (2018)
Ligth (MS)a Abiotic Continuous light 24.9 (3.9%) 4.8 (79.3%) 2.3 (50.7%) Zahir et al. (2018)
Ligth (B5)a Abiotic Continuous light 23.8 4.8 (45.8%) 2.7 (+48.1%) Zahir et al. (2018)
(12.8%)
Ligth (SH)a Abiotic Continuous light 25.2 (7.1%) 2.0 (50.6% 2.1 (27.6%) Zahir et al. (2018)
ZnO NP Abiotic 25 mg/L 34.5 (+90.4%) 3.0 (+30.4%) 3.9 (+18.2%) Zaeem et al. (2020)
(metal)
SA Biotic 50 μM 11.0 (+8.7%) 7.9 (+174.1%) 7.5 (79.1%) Nadeem et al. (2019)
Chemical
(SM)
Cell suspension
Immobilization Abiotic Calcium-alginate beads (44.4%) – – Attoumbré et al.
Production of Antidiabetic Lignans in Flax Cell Cultures
([Ca2+] ¼ 0.45 M) (2006b)

UV-C Abiotic Continuous dark 8.8 (7.4%) 5.6 (+21.7%) 19.7 (+44.9%) Anjum et al. (2017b)
UV-C Abiotic Continuous light 6.3 (33.6%) 5.4 (+17.4%) 16.2 (+21.8%) Anjum et al. (2017b)
UV-C Abiotic Photoperiod 16/8 10.9 (+14.5%) 7.2 (+56.9%) 26.2 (+97.0%) Anjum et al. (2017b)
Fungal elicitors Biotic Fusarium oxysporum (44.4%) 0.05 – Hano et al. (2006a)
(87.2%)
Fungal elicitors Biotic Phoma exigua (35.6%) 0.4 (+13.9%) – Hano et al. (2006a)
Fungal elicitors Biotic Botrytis cinerea (31.1%) 0.4 (+2.7%) – Hano et al. (2006a)
Fungal spores Biotic (alive) Fusarium oxysporum (76.2%) 0.05 Hano et al. (2008)
(90.4%)
(continued)
399
400
Lignans
Elicitor name Elicitor type Concentration/other Biomass SDG LDG References
ABA Biotic 50 μM – 4.3 (+128.8%) – Corbin et al. (2013a)
Chemical
(DV)
GA Biotic 0.1–3.0 μM – 0.6 (69.5%) – Corbin et al. (2013b)
Chemical
(DV)
Yeast extract Biotic 200 mg/L 8.3 (+53.1%) 10.3 11.1 (+25.3%) Nadeem et al. (2018)
(+152.6%)
ZnO NP Abiotic 100 mg/L 13.6 (+58.1%) 25.0 7.7 (+97.4%) Abbasi et al. (2019)
(metal) (+110.1%)
Chitosan Biotic 10 mg/L 16.3 23.2 18.3 Ahmad et al. (2019)
(+196.4%) (+392.6%) (+105.2%)
ABA Biotic 100 μM – 2.9 (+61.1%) – Markulin et al. (2019c)
Chemical
(DV)
ABA + Ca2+ Biotic ABA: 100 μM – 5.9 (+210.5%) – Markulin et al. (2019c)
Chemical Ca2+: 5 mM
(DV)
AgO NP Abiotic NAA: 1.0 14.8 (+96.3%) 19.6 (+64.7%) 4.9 (+25.6%) Zahir et al. (2019)
(metal)
a
Different mineral base (MS Murashige and Skoog, B5 Gamborg B5, SH Schenk and Hildebrandt) were evaluated (continuous dark as control conditions); biotic
chemical (DV): phytohormone involved in developmental and environmental adaptations; biotic chemical (SM): phytohormone acting as stress mediator. The
observed differences in biomass and lignan content relative to the control condition are shown in parentheses
L. Markulin et al.
ZnONP application (Abbasi et al. 2019) and UV-C treatment (Anjum et al. 2017b),
respectively, in flax cell suspension, which can augur major productivity.
While these methods are still only on a laboratory scale, they point to the potential
for these elicitation strategies to yield large quantities of antidiabetic lignans.
13.4 Concluding Remarks and Future Directions
SDG is the major antidiabetic lignan, stored in the seed coat of flax in the form of a
macromolecular complex. SDG have to be converted by the human intestinal
microbiota into the bioactive mammalian lignans enterodiol and enterolactone. It
has been shown that these animals are responsible for a large part of the health
benefits of flax seed lignans, including antidiabetic action. To explain this
antidiabetic activity of flax lignans, different mechanisms have been suggested.
SDG improves glycemic control, delays the development of T2DM in Zucker rat
in association with decreases in oxidative stress, and inhibits the activity of pancre-
atic α-amylase. Most of the published research indicate that SDG may have a great
potential to reduce both T1DM incidence and delay the development of T2DM in
humans. However, although both in vivo and in vitro effects of SDG are globally
favorable to this chemopreventive action, epidemiological studies are much less
conclusive. This disparity may be attributed to the need for microbiotia to activate
lignans into mammalian lignans, which is highly individual dependent. Therefore,
the mechanism by which diabetes is prevented by flax lignans requires further
elucidation. To this end, the availability at a reasonable cost of purified flax lignans
would encourage in vivo supplementation trials and mechanism elucidation. Thus,
optimization of the extraction of flax lignans (primarily SDG and aglycone) is of
great importance. However, as it can be impeded by several seed components and
because of their complex storage form, the performance and quality of the extraction
and purification steps of lignans from flaxseeds are negatively affected. On the
opposite, these impairing compounds are not present in flax cell cultures and lignans
are found in free form (i.e., not in the form of a macromolecular complex with
HMG), thereby making the hydrolysis steps unnecessary and thereby simplifying
their extraction and purification. Thus, it is clear that flax cell cultures can provide an
attractive alternative and sustainable resource for the production and extraction of
antidiabetic lignans. Both callus and cell suspension have been established for the
production of high biomass in conjunction with high amounts of antidiabetic
lignans. Elicitation techniques have also shown their efficacy in increasing the
production of these antidiabetic lignans.
We are only at the onset of the development of antidiabetic lignans. Flax cell
cultures are a system that can be tuned to produce a wide variety of lignans and
neolignans, not only those with well-described antidiabetic actions. It is therefore
possible to expect new lignans or neolignans with antidiabetic activity. Metabolic
engineering approaches allowing a direct production of mammalian lignans could
also be considered and scale-up studies with bioreactors are also anticipated for
feasible industrial production.
Acknowledgments BHA, RRJA, and CH acknowledge research fellowship and Research Con-
sortium from Le Studium-Institute for Advanced Studies, Loire Valley, Orléans, France. SD
acknowledges research fellowship of Region Centre-Val de Loire. DT gratefully acknowledges
the support of French government via the French Embassy in Thailand in the form of Junior
Research Fellowship Program 2018. CH and DT gratefully acknowledge the support of Campus
France through the PHC SIAM (PNPIA, Project 44926WK). CH and BHA gratefully acknowledge
the support of Campus France through the PHC PERIDOT. This research was also supported by the
Region Centre-Val de Loire (Acti-LIN project, grant number LBL19035).
Conflict of Interest: The authors declare no conflict of interest. The funders had no role in the
design of the study; in the collection, analyses, or interpretation of data; in the writing of the
manuscript; or in the decision to publish the results.
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Artificial Seed Development of Selected
Anti-Diabetic Plants, Their Storage 14
and Regeneration: Progress and Prospect
Anamika Das, Manisha Mahanta, Biswajit Pramanik, and

Saikat Gantait
Abstract
Diabetes mellitus is becoming a prime area of public health concern around the
world that is shifting its focus toward plants having anti-diabetic properties,
which can be used for the treatment of diabetes. To meet the demand based on
the pharmaceutical importance, approaches on rapid propagation, conservation,
and germplasm exchange of anti-diabetic plant materials are need of the hour. In
this context, artificial seed production technique can be employed for large-scale
propagation, conservation of germplasms, and exchange of anti-diabetic plants
materials among laboratories to facilitate the production of secondary metabolites
for drug development. Artificial seed technology is mainly attempted for easier
exchange and short- to mid-term storage of germplasm. In addition, long-term
storage can be achieved through encapsulation-dehydration or encapsulation-
vitrification followed by liquid nitrogen exposure, i.e., cryopreservation (storing
at ultra-low temperature). The alginate encapsulation technology is being used in
various anti-diabetic plants to produce artificial seeds using different kinds of
explants depending on the plant species, which can be stored and regenerated
after definite storage duration. To attain uniform, isodiametric, firm beads, a range
of sodium alginate with calcium chloride was found to be effective across the
concerned anti-diabetic plant species. Relatively low progress has been made
regarding the long-term storage of artificial seeds retaining its viability. This
chapter primarily addresses the factors affecting the artificial seed production
and their storage of some anti-diabetic plants across various families.
A. Das · M. Mahanta · B. Pramanik · S. Gantait (*)

Crop Research Unit (Genetics and Plant Breeding), Bidhan Chandra Krishi Viswavidyalaya,
Mohanpur, Nadia, West Bengal, India
Ltd. 2021
https://doi.org/10.1007/978-981-16-3529-8_14
410 A. Das et al.
Keywords
Axillary bud · Calcium chloride · Nodal segment · Shoot tip · Sodium alginate ·
Somatic embryo
14.1 Introduction
In the present day, diabetes mellitus has become a story of every household all
around the world. According to an estimation, nearly one-fourth of the world’s
population is suffering from this endocrine metabolic disorder. Out of this huge
diabetic population, around 5–10% suffer from type 1 (insulin-dependent diabetes
mellitus) wherein the endocrine system of a person suffering from the disorder
that does not produce any insulin and the remaining 90–95% of the population
suffer from type 2 (insulin-independent diabetes mellitus) in which the person either
does not secrete enough insulin or unable to use the secreted insulin properly,
eventually leading to high sugar (glucose) level in the blood in both the cases. A
report by World Health Organization (WHO) has an estimation that by end of 2025,
the world’s diabetic population is expected to be around 300 million or above (Patel
et al. 2012). Presently, many oral hypoglycemic agents such as glinides, biguanides,
sulfonylureas, and α-glucosidase inhibitors along with insulin are being used for the
treatment of diabetes. There are many adverse effects of these treatments, hypogly-
cemia being the severe and the commonest one. Therefore, safer and effective
alternatives to these anti-diabetic agents have been always an important topic for
research. In the course of searching for better alternatives for conventional anti-
diabetic treatments, it was found that many plants possess anti-diabetic properties
that are being used for diabetes treatment in the traditional system of medicine. A
number of review reports are available in which many plants across different
families are listed out for their anti-diabetic activity (Bnouham et al. 2006; Jung
et al. 2006; Patel et al. 2012; Arumugam et al. 2013). The demand for herbal
medicines from anti-diabetic plants has increased in the recent past creating pressure
on such medicinal plants for enough supply. The rigorous exploitation of medicinal
plants for their pharmaceutical importance has to be anticipated with the rapid
propagation and effective conservation methods than the existing conventional
methods. The seed banks provide a better way for ex situ conservation of many
important plant species but some limitations are associated with it, such as loss of
viability and pathogen infestation during storage. Lately, the interest of researchers
has shifted to the encapsulation technology to produce and store artificial seeds of
many threatened and endangered species for in vitro conservation and large-scale
micropropagation (Gantait et al. 2015a; Gantait and Kundu 2017a). Artificial seed
consists of vegetative plant parts such as nodal segment, shoot tip, or somatic
embryo surrounded by a matrix, which forms after dropping the aliquots of sodium
alginate solution (carrying individual vegetative plant parts) in calcium chloride
solution (Fig. 14.1). In general, artificial seeds provide genetically identical virus-
free propagules for longer storage, which can be regenerated as and when required
14 Artificial Seed Development of Selected Anti-Diabetic Plants, Their. . . 411
Fig. 14.1 General concept of artificial seed development and their storage procedure (Source:
Unpublished diagram of Saikat Gantait)
(Gantait and Mitra 2019). This chapter basically focuses on the advancement of
artificial seed production of some anti-diabetic plants across various families. It
emphasizes the use of various explants such as shoot tips, nodal segments, calli,
somatic embryos, protocorm-like bodies (PLBs), etc., to produce artificial seeds,
their maximum possible storage duration, and respective regeneration at optimum
condition.
412 A. Das et al.
14.2 Selection of Explants
Production of artificial seeds requires explants to be encapsulated under in vitro

condition. A variety of explants, both unipolar propagules such as shoot tips, nodal
segments (Fig. 14.2a–d), microshoots, and bipolar propagules like somatic embryos
and PLBs were successfully utilized. Further development in the production and
extraction of secondary metabolites leads to the use of hairy roots as plant material
for artificial seed development. The size of explants was maintained at 3–5 mm for
the production of appropriate size of encapsulation bubble.
14.2.1 Shoot Tips
Shoot tips are unipolar (non-embryogenic) structure used for the production of
artificial seeds where somatic embryos are difficult to attain or do not produce
complete plantlet. They are considered to be uninvaded by pathogens due to active
mitotic activity and are reported to maintain the genetic stability of the plant (Bennici
et al. 2004). Shoot tips containing apical buds and leaf primordia are utilized for
encapsulation for the production of artificial seeds in some medicinal plants having
anti-diabetic properties (Table 14.1). The appropriate size of shoot tip for encapsu-
lation is reported to be 2–4 mm (Fig. 14.2d). Shoot tips are effectively used for
artificial seed production in various medicinal plants such as Ocimum sp. (Mandal
et al. 2000), Adhatoda vasica (Anand and Bansal 2002), Musa sp. (Ganapathi et al.
2002), Withania somnifera (Singh et al. 2006b), Curculigo orchioides (Nagesh et al.
2009), Stevia rebaudiana (Andlib et al. 2011), and Beta vulgaris (Rizkalla et al.
2012), etc.
14.2.2 Nodal Segments
Nodal segments (Fig. 14.2d) are the most extensively used non-embryogenic plant
propagule for the artificial seed production in most of the plants (Table 14.1). Nodal
segments with axillary buds are the most effective and preferred ones. They can be
encapsulated with or without inducing root primordia. Report is present where root
primordia are induced in nodal segments before encapsulation in Dalbergia sisso
(Chand and Singh 2004). Ideal size of the nodal segment for the artificial seed
production is maintained at 3–5 mm (Faisal and Anis 2007; Siddique and Anis 2009;
Lata et al. 2011; Varshney and Anis 2014). Use of nodal segment as explant for the
artificial seed production is reported in Punica granatum (Naik and Chand 2006),
Olea europaea (Micheli et al. 2007), Cannabis sativa (Lata et al. 2009), Withania
somnifera (Fatima et al. 2013), Bacopa monnieri (Gantait et al. 2015b), Althaea
officinalis (Naz et al. 2018), etc. Sharma et al. (2014) reported nodal segments of
Salvia splendens to be better explant for clonal propagation as compared to shoot
tips because of the higher shoot and root multiplication and survival rate after
hardening. Shoot tips contain a single apical bud whereas nodal segments tend to
Fig. 14.2 Different stages of synthetic seed development and its regeneration in Tylophora indica
(Burm. Fil.) Merrill., an important anti-diabetic plant species. (a) Multiple shoot culture, (b)
Isolation of in vitro shoots, (c) Trimming of shoots to prepare shoot tips and nodal segments, (d)
Shoot tips and nodal segments after trimming, (e) Preparation of different doses of sodium alginate
(SA) and calcium chloride (CC) solution, (f) Drenching of trimmed shoot tips and nodal segments in
SA solution, (g) Dropping aliquots of SA containing a single explant into CC solution, (h)
Incubation of droplets in CC solution for 30 min, (i) Well-formed artificial seeds from CC solution
after washing with sterile water, (j) Effect of SA and CC on shape and consistency of artificial seeds
(3% SA and 100 mM CC-isodiametric beads; 2% SA and 100 mM CC-deformed and soft beads;
414 A. Das et al.
contain two axillary buds leading to the development of two shoots at a time and
becoming a reason for opting of nodal segments for multiple shoot regeneration.
14.2.3 Calli
Callus is the undifferentiated mass of cells which start forming from the wound part
of the explant under in vitro condition. It is least exploited as explant for the artificial
seed production because of its undifferentiated nature resulting in complexity during
encapsulation. It was also reported that callus tips showed promising growth
characteristics as compared to callus segments (Choudhary et al. 2019). Successful
encapsulation of calli was seen in the case of Allium sativum and Rhodiola kirilowii
with 95% of regeneration frequency (Kim and Park 2002; Zych et al. 2005) and
around 88% regeneration frequency was obtained in the case of Allium sativum after
storage (Kim and Park 2002). Recently, callus as explant for the artificial seed
production was utilized in Plumbago zeylanica (Jain et al. 2018) and Annona
squamosa (Choudhary et al. 2019) and attained 76.6 and 70% of regeneration,
respectively.
14.2.4 Somatic Embryos
Somatic embryos are known to have both plumule and radicle axis and are, there-
fore, known as bipolar plant propagules that have the ability to develop into a
complete plantlet having shoots and roots in a single step. They are capable of
maintaining their regenerative potential for a long time along with multiplication
ability (Rihan et al. 2017). Somatic embryo is the pioneer explant exploited for the
production of artificial seeds. Over time, many plant species were seemed to be
regenerated via somatic embryos and were attempted for artificial seed technology.
The production of artificial seeds using somatic embryo as explant has been attained
in Musa sp. (Ganapathi et al. 2002), Rotula aquatic (Chithra et al. 2005), Cucumis
sativus (Tabassum et al. 2010), Rhinacanthus nasutus (Cheruvathur et al. 2013),
Swertia chirayita (Kumar and Chandra 2014), Bacopa monnieri (Khilwani et al.
2016), Eclipta alba (Salma et al. 2019), etc. In the case of Bacopa monnieri, artificial
seeds encapsulating somatic embryos were found to be viable for a longer period of
time as compared to apical shoot buds and showed higher regeneration percentage
(Khilwani et al. 2016). In Rhinacanthus nasutus (L.) Kurz. (Cheruvathur et al. 2013)
and Anethum graveolens (Dhir et al. 2014), somatic embryos at torpedo and cotyle-
donary stage are selected for the encapsulation as in mature torpedo stage, root and
Fig. 14.2 (continued) 5% SA and 100 mM CC-beads are hard with tail formation), (k) Storage of
artificial seeds (at 5 1 C), (l) Post-storage (after 60 days) regeneration of plantlet from artificial
seed in ½ Murashige and Skoog (MS) semisolid medium (figures are not in scale) (Source:
Unpublished photos of Saikat Gantait)
14
Table 14.1 Factors influencing artificial seed production of different anti-diabetic plant species, their storage, and regeneration (in chronological order and
alphabetical order within each year)
Conc. of
Sodium calcium Storage Storage
Explant alginate chloride condition duration Regeneration
Plant species type (%) (mM) ( C) (days) Regeneration medium (%) References
Actinidia AB 2.5 100 NP NP ½ MS + 0.7% agar +12.5 g/L 50–57 Adriani et al.
deliciosa sucrose (2000)
Morus sp. AB 4 75 – – MS + 4.4μM BA 78–98 Pattnaik and
4 60, 90 8–22, 13–18 Chand (2000)
Ocimum sp. ST 4 75 4 60 MS + 1.1–4.4μM BA 95–99 Mandal et al.
(2000)
Musa spp. AAB SE 5 NM NP NP MS 66 Ganapathi et al.
group (2001)
Adhatoda vasica ST 4 100 NP NP B5 + 4.65μM Kn + 50 mg/L 66.28 Anand and
PG Bansal (2002)
Musa sp. ST 3 NM NP NP White’s 100 Ganapathi et al.
(2002)
Ananas comosus Tiny 3 1.36 g/ 4 2 MS + 9.84μM IBA + 9.29μM 66.67 Soneji et al.
shoots 150 mL Kn (2002)
Allium sativum Ca 1.5 55 – – ½ MS 95 Kim and Park
4 40 ½ MS + 5μM NAA + 0.1μM 88 (2002)
Kn
Artificial Seed Development of Selected Anti-Diabetic Plants, Their. . .
Simmondsia AB 6 100 NP NP MS + 1 mg/L BAP + 3 mg/L 73 Hassan (2003)

chinensis IAA + 40 mg/L ads
Quercus robur SE 4 50 4 90 P24 + 0.1μM IBA 9 Prewein and
+ 0.9μM BAP Wilhelm (2003)
Dalbergia sissoo NS 3 75 – – ½ MS 85 Chand and
4, dark 15 80 Singh (2004)
(continued)
415
416
Conc. of
Ananas comosus Mst 2.5 – 8 45 MS 86.13 Gangopadhyay
et al. (2005)
Arnebia SE 3 100 NP NP MS 60.6 Manjkhola
euchroma et al. (2005)
Rotula aquatic SE 3 50 NP NP ½ MS 100 Chithra et al.
(2005)
Rhodiola kirilowii Ca 5 50 4 110 MS 95 Zych et al.
(2005)
Allium sativum Bulblets 3 2.5% 15 60 MS + 2 mg/L BA +2 mg/L 80 Bekheet (2006)
NAA
Vitis vinifera SE 2 100 NP NP ¼ MS + B5 + 2.9μM GA3 69.2 Das et al.
(2006)
Morus sp. AB 4 50 – – LSBM +8.88μM BAP + 2μM 48.2 Kavyashree
4 180 TIBA 29.2 et al. (2006)
Punica granatum NS 3 100 4 45 MS + 4.44μM BA +0.54μM 15.83 Naik and
NAA Chand (2006)
Chonemorpha ST 3 55 NP NP ½ MS 95 Nishitha et al.
grandiflora (2006)
Phyllanthus ST 3 75 – – MS 90 Singh et al.
amarus 4, dark 60 47 (2006a)
Withania ST 3 75 – – MS + 0.5 mg/L IBA 87 Singh et al.
somnifera 4, dark 30 63 (2006b)
Tylophora indica NS 3 100 – – MS + 2.5μM BA +0.5μM 91 Faisal and Anis
4 60 NAA 50.3 (2007)
A. Das et al.
14
Olea europaea NS 2.5 1.1% 4 30 ½ OMM + 1 mg/L zeatin – Micheli et al.

+50 g/L sucrose (2007)
Quercus suber SE 5 50, 100 4 60 MS + 0.2μM BA +0.5μM – Pintos et al.
IBA (2008)
Artemisia NS 2 50 5 450 MS + 4.9μM 2iP 85 Sujatha and
vulgaris Kumari (2008)
Pogostemon NS 4 100 25 180 MS + 0.5μM/L BA 91 Kumara
cablin Benth Swamy et al.
(2009)
Cannabis sativa NS 5 50 NP NP MS + 0.5μM TDZ + 0.075% 100 Lata et al.
PPM (2009)
Curculigo ST 2.5 100 4 50 ½ MS + 10% CW 64 Nagesh et al.
orchioides (2009)
Musa sp. cv. MSt 1 100 4 10 MS + 100 mM KNO3 76 Sandoval-
Grand Naine Yugar et al.
(2009)
Spilanthes NS 4 100 – – MS + 1μM BA +0.5μM 87.8 Sharma et al.
acmella 4 60 NAA 46 (2009a)
Spilanthes NS 4 100 4 30 ½ MS >80 Sharma et al.
mauritiana (2009b)
Ocimum NS 3 75 4 45 ½ MS + 5μM BA +0.5μM 71 Siddique and
basilicum IAA Anis (2009)
Cineraria ST, NS 3 3%, 272.7 25 2 180 MS 11.76 Srivastava et al.
maritima (2009)
Vitex negundo NS 3 100 – – MS + 2.5μM KIN +1μM 92.6 Ahmad and
4 60 NAA 50 Anis (2010)
Simmondsia ST 3 100 NP NP MS 66.6 Kumar et al.
chinensis (2010)
(continued)
417
418
Conc. of
Dalbergia sissoo SE 2.5 75 – – ½ MS 43.3 Singh and
4 15 29.4 Chand (2010)
Eclipta alba NS 3 100 4 60 MS + 0.88μM BAP 51.2 Singh et al.
(2010)
Zingiber MSt 4 100 – – MS 66 Sundararaj
officinale 25 90 MS + 2.5 mg/L BA 13 et al. (2010)
Cucumis sativus SE 3 100 4 7 MS + 14.4μM GA3 + 4.8μM 82 Tabassum et al.
BAP + 1μM NAA (2010)
Stevia ST 3 2.5% 48 30 MS + 2 mg/L BAP + 1 mg/L 70 Andlib et al.
rebaudiana IAA (2011)
Khaya ST, NS 3 100 25 60 ½ MS + 245μM IBA 52–98 Hung and
senegalensis Trueman
(2011)
Cannabis sativa NS 5 50 15 180 MS + 0.5μM TDZ (shoot 66 Lata et al.
induction), ½ MS + 2.5μM (2011)
IBA (rooting)
Pogostemon NS 4 – 25 180 MS + 0.5 mg/L BA 43.3 Patil et al.
cablin Benth. (2011)
Stevia ST, NS 3 100 4 15 MS + 1 mg/L BAP 100 Ali et al. (2012)
rebaudiana
Beta vulgaris ST 4 100 NP NP MS + 0.05 M mannitol – Rizkalla et al.
(2012)
Aristolochia MSt 3 1% NP NP MS + 3μM BAP + 0.5μM – Remya et al.
tagala KIN (2013)
A. Das et al.
14
Rhinacanthus SE 3 100 NP NP MS + 0.2 mg/L GA3 92 Cheruvathur

nasutus et al. (2013)
Ceropegia NS 3 100 – – MS + 8.88μM BA 100 Dhir and
bulbosa 4 60 50.7 Shekhawat
(2013)
Withania NS 3 100 4 60 MS + 2.5μM BA+ 0.5μM 50–52 Fatima et al.
somnifera NAA (2013)
Dendrobium PLB 3 100 NM 60 ½ MS + 1 mg/L 78.2 Mohanty et al.
nobile BAP + 0.1 mg/L NAA (2013)
Centaurium HR 3 50 – – WPM + 3% sucrose +0.5 mg/ 100 Piątczak and
erythraea 4 45 L BAP 100 Wysokińska
(2013)
Artemisia SE 2 75 – – – 90 Sudarshana
vulgaris L. 4 120 – et al. (2013)
Cucumis sativus ST 3 100 4 30 MS + 0.8% agar 82.1 Adhikari et al.
(2014)
Ochradenus NS, ST 3 100 4 15 MS + 1μM BA (shooting), 86 Al-Qurainy
baccatus MS + 100μM IBA (rooting) et al. (2014)
Cassia NS 3 100 4 30 ½ MS + 5μM TDZ + 1μM 93 Bukhari et al.
angustifolia IAA (2014)
Sterculia urens NS 4 100 – – MS + 0.2 mg/L TDZ 95 Devi et al.
4 180 73 (2014)
Anethum SE 3 100 – – MS + 0.8% agar 83 Dhir et al.
graveolens 4 60 49.3 (2014)

Terminalia ST 3 100 NP NP MS + 0.5 mg/L 91.6 Gupta et al.
arjuna BAP + 0.1 mg/L NAA (2014)
Swertia chirayita SE 4 100 NP NP MS + 1 mg/L BA +0.5 mg/L 84 Kumar and
NAA Chandra (2014)
(continued)
419
420
Conc. of
Stevia ST 4 100 NP NP ½ MS 69 Nower (2014)
rebaudiana
Centella asiatica AB, NS 4 75 – – MS 85.7 Prasad et al.
25 200 >85 (2014)
Cassia NS 3 100 – – MS + 2.5μM BA +0.4μM 94 Parveen and
angustifolia Vahl. 4 60 NAA 43.9 Shahzad (2014)
Ocimum MSt 3 75 – – MS + 1 mg/L BA 98.6 Saha et al.
gratissimum 25 90 61.6 (2014)
Salvia splendens NS 4 100 4 45 MS 63.6 Sharma et al.
(2014)
Sterculia urens NS 4 100 – – MS + 0.2 mg/L TDZ 95 Subhashini
4 180 73.3 Devi et al.
(2014)
Phyllanthus NS 3 100 – – ½ MS + 0.5 mg/L BAP 92.5 Upadhyay et al.
fraternus 4 90 47.3 (2014)
Balanites NS 3 100 – – MS + 12.5μM BA +1μM 80 Varshney and
aegyptiaca 4 60 NAA 60 Anis (2014)
Vitex trifolia L. NS 3 100 – – MS + 5.0μM BA +0.5μM 84.9 Ahmed et al.
4 60 NAA 42.5 (2015)
Bacopa monnieri NS 2.5 75 NP NP ½ MS – Gantait et al.
(2015b)
Bacopa MSt 3 3% (calcium 15 60 MS + 3 mg/L TDZ + 15 mg/ >60 Haque et al.
chamaedryoids nitrate) L AvG (2015)
A. Das et al.
14
Digitalis ST 4 75 4, dark 60 MS + 0.25 mg/L >25 Verma et al.

davisiana NAA + 1 mg/L BAP (2016)
Vitex trifolia L. NS 3 100 – – MS + 2.5μM kin +1μM NAA 89.6 Alatar et al.
4, dark 60 71.6 (2017)
Bacopa monnieri SE 3 125 25 140 MS + 12.5μM BA +1μM 41.66 Khilwani et al.
2,4-D (2016)
Tylophora indica NS 3 75 15 45 ½ MS liquid 70 Gantait et al.
(2017a)
Rauvolfia ST 3 75 NP NP ½ MS 88 Gantait et al.
serpentina (2017b)
Rauvolfia ST 3 75 8 60 ½ MS 52 Gantait and
serpentina Kundu (2017b)
Plumbago Ca – 50 NP NP MS + 0.25 mg/L 76.6 Jain et al.
zeylanica NAA + 1 mg/L BAP (2018)
Althaea officinalis NS 3 100 – – MS + 7.5μM BA +0.5μM 66 Naz et al.
4 45 NAA 16 (2018)
Gymnema NS 3 100 – – MS + 2.5μM BA +2.5μM 88 Saeed et al.
sylvestre 4 30 (GA3) + 50μM AdS 22 (2018)
Annona Ca 3 75 4 30 MS + 2 mg/L BAP + 0.5 mg/ 70 Choudhary
squamosa L NAA et al. (2019)
Olea europaea NS 2.5 11 g/L 4 45 PGR-free OMM 32.7 Micheli et al.
(2019)
Eclipta alba SE 2.5 75 8 90 ½MS 40–52 Salma et al.

(2019)
Simmondsia AB 3 100 NP NP MS 80 Bala et al.
chinensis (2020)
Hedychium ST 3 100 – – MS 90 Behera et al.
coronarium 4 60 44.4 (2020)
421
(continued)
422
Conc. of
Decalepis ST 3 100 4 90 MS + 2.2μM BAP + 5.7μM 53.33 Rodrigues et al.
salicifolia IAA (2020)
2,4-D 2,4-dichlorophenoxy acetic acid, 2iP isopentyl adenine, AB axillary bud, ABA abscisic acid, AC activated charcoal, Ads adenine sulfate, AvG Aloe vera
gel, BA benzyl adenine, BAP 6-Benzylaminopurine, Ca calli, CS cell suspension, CW coconut water, GA gibberellic acid, HR hairy root, IAA indole-3-acetic
acid, IBA indole-3-butyric acid, KIN kinetin, LSBM Linsmaier and Skoog’s basal medium, MSt microshoot, MS Murashige and Skoog, NAA naphthalene acetic
acid, NP not performed, NS nodal segment, OMM olive medium modified, PGR plant growth regulator, PG phloroglucinol, PLB protocorm-like body, PPM
plant preservative mixture, SE somatic embryo, ST shoot tip, TDZ thidiazurone, TIBA 2,3,5-Triiodobenzoic acid, WPM Woody plant medium. Blank spaces ( )
denote the unavailability of information
A. Das et al.
shoot poles can be seen after which they undergo maturation phase (post-embryonic
development) and two distinct cotyledons are developed leading to the regeneration
of complete plantlets.
14.2.5 Hairy Roots
Inoculation of Agrobacterium rhizogenes into the plants results in the induction of

hairy roots at the site of infection due to the transfer and integration of T-DNA from
Ri plasmid into the plant genome. They serve as a source for the extraction of useful
secondary metabolites and enzymes (Gantait and Mukherjee 2021). Hairy root as
explant for artificial seed production was attempted in Centaurium erythraea
(Piątczak and Wysokińska 2013) for large-scale regeneration of transformed
plantlets from which extraction of secondary metabolites can be attempted having
anti-diabetic properties and can be used for the manufacturing of anti-diabetic drugs.
Use of hairy root as explant requires frequent transfer of culture into fresh media,
which may lead to increase in the cost of production and labor-intensive.
14.2.6 Protocorm-like Bodies
PLBs are similar to somatic embryos in form and development. The term
“Protocorm-like bodies” remains confined to the in vitro culture of orchids (Ishii
et al. 1998). PLBs are considered as an important structure in the in vitro mass
propagation of orchids. Use of PLBs as explant for artificial seed production in
plants having anti-diabetic utilities was reported in Dendrobium nobile and nearly
80% conversion frequency was obtained (Mohanty et al. 2013) and reported that
incorporation of sucrose and mannitol in the encapsulation matrix, extend the
storage time (short-term storage) of artificial seeds. In lower sucrose (3–5%) and
mannitol concentration, the PLBs were seen to burst out from the capsule and at
higher sucrose (15%) and mannitol concentration, the PLBs died within 10 days
because of cell dehydration. At 7.5% sucrose and mannitol concentration, the PLBs
survived and did not burst out maybe due to the creation of osmotic stress resulting
in slower cell division. Therefore, the use of different osmotica during encapsulation
was found to be beneficial for short-term storage of PLBs.
14.3 Alginate Encapsulation
Artificial seeds can be of desiccated or hydrated type. In the case of most of the anti-
diabetic plant species, artificial seed production is based on hydrated technology in
which the explant is surrounded by a hydrogel providing protection and nutrition to
the plant propagules. The encapsulation provides a coating that mimics the seed coat
of a natural seed. For encapsulation, plant propagules are mixed with a mixture
prepared by mixing an encapsulating agent with double distilled water (or any liquid
nutrient medium), then the droplets containing propagules are incubated in a
424 A. Das et al.
complexing agent such as calcium nitrate or calcium chloride solution for an

incubation period of 20–40 min (usually 30 min) (Fig. 14.1). During the incubation,
the exchange of ions takes place, which eventually provides a firm coating around
the plant propagules. The main principle behind the encapsulation technique using
alginate is the ion-exchange phenomena in which sodium ions (Na+) present in
sodium alginate are replaced by calcium ions (Ca2+) of calcium chloride forming a
firm coating of calcium alginate.
14.3.1 Encapsulating Agent
A gelling agent along with a complexing agent is used to form soft hydrogel around
the explant. An ideal encapsulating agent should be non-toxic to the plant
propagules, gentle enough to allow the emergence of propagules, sufficiently tena-
cious for handling, storage, and transport (Nongdam 2016). There are several
encapsulating agents such as agar, gelrite, potassium alginate, sodium pectate,
sodium alginate with gelatin, carrageenan, carboxymethyl cellulose, gaurgum,
sodium alginate, etc., used for the gel matrix. However, among these, sodium
alginate is most commonly used for the gel matrix (Rai et al. 2009). This can be
because of its low cost, non-toxic nature, gelling property, and adequate sturdiness to
form a protective layer around the explant. Also, sodium alginate is a copolymer
having L-glucuronic and D-mannuronic acid units which is biocompatible, biode-
gradable, and capable of producing hydrogel when co-exist with divalent cations
(Gantait et al. 2015a). Moreover, the structure and pore size of this water-insoluble
gel allow easy exchange of elements from the surrounding medium. Depending on
the plant species and type of explant used for artificial seed production, the range of
sodium alginate concentration varies from 1 to 5% (w/v) (Fig. 14.2e). The most
commonly used concentration of sodium alginate is around 3% (w/v) irrespective of
species and choice of explant (Fig. 14.2f–i) (Table 14.1), as below this concentration
(1–2%), very soft, fragile, asymmetric capsules are produced (Fig. 14.2j) that are
difficult to handle and at higher concentrations of sodium alginate (4–5%), capsules
are very hard which makes it difficult for the plant propagules to break the coating
and germinate (Fig. 14.2j). The plant propagules along with a droplet of encapsula-
tion mixture (sodium alginate) are dropped into the complexing agent (calcium
chloride). During the incubation period, sodium ions are replaced by calcium ions,
which results in hard calcium alginate beads containing plant propagules. For
instance, in Vitex trifolia, different concentration of sodium alginate (1–5%) and
calcium chloride (25–200 mM) was tested and concluded that 3% sodium alginate
with 100 mM calcium chloride formed the firm and uniform artificial seeds (Ahmed
et al. 2015; Alatar et al. 2017). Gantait et al. (2015b) tried and tested various
concentrations of sodium alginate ranging from 1.5 to 4% with two concentrations
of calcium chloride (75 and 100 mM) and reported that 2.5% sodium alginate with
75 mM calcium chloride provides uniform artificial seeds in Bacopa monnieri.
Kavyashree et al. (2006) reported that firm and isodiametric artificial seeds by
using high sodium alginate (4%) with low calcium chloride (50 mM) in Morus
indica. The relative concentration of sodium alginate and calcium chloride varies
depending on the species. In some species such as Sterculia, sugarbeet, Salvia, etc.,
4% sodium alginate is used with 100 mM of calcium chloride (Table 14.1). In an
exception, Sandoval-Yugar et al. (2009) used 1% sodium alginate with 100 mM
calcium chloride to encapsulate microshoots of Grand Naine cultivar of banana.
14.3.2 Encapsulating Matrix
The concentration of sodium alginate and calcium chloride along with the incubation
period affects the texture (hardening) of calcium alginate beads, depending on
various species and plant propagules used for artificial seed production. The encap-
sulation involving calcium alginate is the most advantageous as it provides a firm
protective covering by enhancing capsule formation, which secures the plant
propagules from mechanical injuries. It also has moderate viscosity, low toxicity,
and low cost, which make it the most desired encapsulation matrix (Nongdam 2016).
In most of the anti-diabetic plant species, calcium chloride concentration ranging
from 50 to 100 mM is used for the encapsulation (Table 14.1). The ion-exchange
process is very slow and requires longer incubation to form firm artificial seeds in
case of lower concentration of calcium chloride, whereas at higher concentration the
matrix becomes very hard. However, in some reports, a low concentration of calcium
chloride (50 mM) was found to be effective to produce artificial seeds with a
prolonged incubation period around 30 min (Kavyashree et al. 2006; Lata et al.
2009). Artificial seeds lack an endosperm, which chiefly provides nutrition to the
germinating embryo in natural seeds. Therefore, some nutrients, plant growth
regulators, and carbon sources can be provided in an encapsulation matrix to play
the role of an artificial endosperm. Some other adjuvants such as pesticides,
fungicides, antibiotics, etc., also can be added to provide protection against various
microbial attacks during storage and handling. For instance, encapsulation matrix
was prepared by adding sodium alginate to full strength MS medium (Murashige
and Skoog 1962) containing 3% sucrose, 0.5μM thidiazuron (TDZ) and 2.5μM
indole-3-butyric acid (IBA) along with a fungicide plant preservative mixture
(PPM). Calcium chloride solution was also prepared similarly except the addition
of PPM for the encapsulation of Cannabis nodal segments (Lata et al. 2009). To get
uniform, isodiametric, firm beads, 3% sodium alginate with 100 mM calcium
chloride was found to be the most effective across the species (Table 14.1).
14.4 Storage
Artificial seeds serve as an alternative for large-scale propagation of identical plants

and exchange of germplasm between laboratories globally. For the exchange and
preservation of plant germplasm, storage plays a decisive factor in the successful
regeneration of artificial seeds (Fig. 14.2k). In the case of elite and endangered
cultivars, storage of germplasm becomes the utmost concern. Therefore, suitable
426 A. Das et al.
storage condition and optimized duration of storage are necessary to maintain the
viability of the artificial seed during the exchange and for commercial utility.
14.4.1 Storage (Physical) Condition
Storage condition generally defines the temperature, relative humidity, and illumi-
nation of the room in which the artificial seeds are stored. The most effective
temperature for storage of artificial seeds was reported to be 4 C in almost all the
plant species with few exceptions in which the artificial seeds were stored at 15 or
25 C (Table 14.1). Cold storage at 4 C results in slowing down of the metabolic
activities of the artificial seeds and they tend to remain in quiescent state and the
nutrient reservoir is preserved that helps the explant to regenerate under favorable
medium and conditions (Ikhlaq et al. 2010). Storing of artificial seeds in room
temperature resulted in comparatively lower conversion rate than cold storage and
sometimes necrosis of tissues was observed in room temperature. However, 25 C
(room temperature) was seemed to have worked best for storage in case of
Pogostemon cablin Benth (Kumara Swamy et al. 2009), Cineraria maritima
(Srivastava et al. 2009), Zingiber officinale (Sundararaj et al. 2010), Khaya
senegalensis (Hung and Trueman 2011), Centella asiatica (Prasad et al. 2014),
and Bacopa monnieri (Khilwani et al. 2016). This may be due to their
acclimatization to higher temperature and susceptivity toward chilling injury. In
the case of Cineraria maritima, moist environment was maintained during storage
by spraying of sterile distilled water at an interval of 15 days (Srivastava et al. 2009).
However, Andlib et al. (2011) reported that in Stevia rebaudiana as high as 48 C
was maintained for the storage of artificial seeds. Effect of light condition during
storage was studied and found that it may cause stressful impact on the explant
leading to increase in abscisic acid (ABA) level, thereby increasing the ABA:
gibberellic acid (GA) ratio resulting in physiological dormancy of tissues. On the
other hand, in dark condition, lower ABA:GA ratio was found that has a positive
impact on survival and proliferation of encapsulated tissues (Kamińska et al. 2018).
14.4.2 Storage Duration
Duration of storage is an important factor for the viability and regeneration fre-
quency of artificial seeds. Generally, regeneration ability is inversely proportional to
the storage duration, i.e., as the duration increases, the viability and regeneration
frequency of artificial seeds decreases. The main reason for this is the limited
availability of oxygen for the living tissues as in the process of respiration they
intake oxygen and releases carbon dioxide as a result of which oxygen in the
encapsulated environment becomes exhausted affecting the metabolic activities
that result in reduced germination rate upon prolonged storage (Ikhlaq et al. 2010).
As the storage duration increases, the time taken for germination of artificial seeds
also increases because of the time taken by the plant tissue to recover from the stress
condition (Ali et al. 2012). Artificial seeds of plant species such as Morus sp.,
Pogostemon cablin Benth., Cineraria maritima, Cannabis sativa, Sterculia urens,
Bacopa monnieri reported to be stored for 6 months after which viability was
reduced drastically resulting in poor germination percentage and storage duration
varies according to the plant species and storage condition (Table 14.1). Artificial
seeds in case of Artemisia vulgaris were reported to be stored for 15 months with
85% of conversion rate but took around 12 weeks of incubation under the
proliferating condition to obtain new shoots growth (Sujatha and Kumari 2008).
Short-term storage can be achieved by maintaining suitable storage conditions and
capsulation materials, while long-term storage can be carried out by applying
dehydration (physical or chemical) and cryopreservation (exposure to liquid nitro-
gen) techniques. Kaviani (2010) reported that increase in sucrose level in alginate
matrix can help during cryogenic storage (encapsulation-dehydration) as it helps in
improving the tolerance toward dehydration and maintaining the viability during
storage and post-storage germination, and other suitable cryoprotectants can also be
used during encapsulation. Long-term storage adds benefits in the preservation of
germplasm and exchange and transport of plant materials between laboratories.
14.5 Pre- and/or Post-Storage Regeneration
Regeneration or conversion refers to the percentage of artificial seeds performing

both sprouting and rooting in order to form entire plantlets (Fig. 14.2l). On the other
hand, the percentage of sown artificial seeds showing an increase in size with proper
breakage of capsule for extrusion of at least one shootlet or rootlet can be evaluated
as regrowth rate. Generally, the regeneration ability of artificial seeds shows a
gradual decrease with the increasing storage duration. Eventually, a huge difference
in the regeneration percentage can be observed between that pre- and post-storage
condition. There are some factors affecting the conversion percentage of the artificial
seeds toward complete plantlet development.
14.5.1 Basal Medium
Basal medium provides a balanced nutrition to the artificial seeds in vitro in order to
develop complete plantlets by breaking the alginate covering. This action resembles
the property of soil in the sense of providing nutrients (both macro and micro) to the
seeds in vivo. According to Mandal et al. (2000), the encapsulation method under
in vitro condition can be assigned to serve the most conventional means of plant
propagule development. Rather, it can be considered as the most efficient of artificial
seed development too. As a basal medium, full strength of MS medium was reported
to be the most preferred one (Table 14.1), though half strength of the same was
reported to induce roots in many species (Adriani et al. 2000; Lata et al. 2011;
Gantait et al. 2015b). Moreover, while using shoot tips and nodal segments as
explants for encapsulation, higher (>90%) regeneration or conversion efficiency
428 A. Das et al.
can be observed. Further to mention, the nutrient amended medium was always
considered over the nutrient deficient one (Table 14.1). In some species, specificity
can be noticed in the case of using particular medium. For instance, Linsmaier and
Skoog’s basal medium (LSBM) (Linsmaier and Skoog 1965) for Morus
sp. (Kavyashree et al. 2006), olive medium modified (OMM) for Olea europaea
(Micheli et al. 2007, 2019), woody plant medium (WPM) (Lloyd and McCown
1981) for Rauvolfia tetraphylla (Alatar and Faisal 2012) and woody plants like
Centaurium erythraea (Piątczak and Wysokińska 2013). On the contrary to all
published reports, Das et al. (2006) reported of using a combination of one-fourth
strength of MS medium with Gamborg’s B5 medium (Gamborg et al. 1968) for
artificial seed development. The unique composition of nutrients, inorganic salts,
vitamins, amino acids, etc., in each medium explicitly influences particular species
for attaining optimum growth and development.
14.5.2 Physical Environment
The presence of suitable physical environment has a beneficial effect on the regen-
eration of artificial seeds inoculated in the media. A suitable physical environment
comprises of some factors like temperature, photoperiod, light intensity, and relative
humidity (RH). Generally, for in vitro germination of explants or artificial seeds,
23–27 C temperature, 16 h photoperiod, 30–60μmol/m2/s photosynthetic photon
flux density and 50–70% RH are considered to be optimum. Majority of the
researchers have mentioned these requirements within the aforesaid ranges, though
some exceptions have been highlighted. For instance, Zych et al. (2005) have used
12 h photoperiod for artificial seed germination of Rhodiola kirilowii, 28–30 C
temperature was utilized by Singh et al. (2006a) during their research work using
Withania somnifera. On the other side, lower light intensity (25μmol/m2/s) was used
in Chonemorpha grandiflora by Nishitha et al. (2006), whereas a transition between
lower and higher light intensity was experimented by Gupta et al. (2014) in
Terminalia arjuna. On the contrary, Hung and Trueman (2011) reported of using
100μmol/m2/s intensity of light for the same purpose in Khaya senegalensis. Tem-
perature is a major factor to stabilize the metabolic activities along with the
functionalities of the enzymes inside the plant bodies (Gantait and Kundu 2017a).
Similarly, light intensity, as well as photoperiod, maintains various physiological
processes like photosynthesis and biological clock in the plant system. All these
factors also help in proper bud breakage and play their roles perfectly under in vitro
condition while being available in optimum concentrations.
14.5.3 Plant Growth Regulators
Plant growth regulators (PGRs) are those molecules that help in the development of a
plant while being present in a very much lower concentration. Various natural and/or
artificial PGRs are used in the regeneration media of artificial seed development. The
PGRs are the major factors to decide the fate of the artificial seeds, i.e., whether and
how it will form shoots and/or roots. PGR-added medium was often preferred over
the PGR-free one in order to achieve higher conversion proficiency. The concentra-
tion of the PGRs varied from species to species (Table 14.1). Apart from some
researchers, majority have used various combinations of PGRs for caulogenesis and
rhizogenesis from the inoculated artificial seeds. As a single cytokinin for shoot
initiation, use of 6-benzyladenine (BA) was the most recommended one followed by
6-benzylaminopurine (BAP) (Table 14.1). Composite use of both cytokinin and
auxin can also be seen in many published reports. Mainly higher ratio of cytokinin
and auxin was used for shoot induction while the opposite trend was reported to
induce roots in a larger amount. Singular use of auxin was also reported by Singh
et al. (2006b), Hung and Trueman (2011) and Saha et al. (2014) for successful
conversion of artificial seeds. In some reports, shoot and root induction were
experimented in different media using cytokinin and auxin separately (Lata et al.
2011; Al-Qurainy et al. 2014). Although a huge contrast can be noticed between the
artificial seed viability in both pre- and post-storage situation, the application of BAP
is said to have a major impact on the same with 100% post-storage regeneration in
Stevia rebaudiana (Ali et al. 2012) and Centaurium erythraea (Piątczak and
Wysokińska 2013). Nagesh et al. (2009) have conducted an experiment using
coconut water as PGR. Being a natural constituent, it has no detrimental effect.
Besides, higher level of germination percentage was also found if coconut water had
been added to MS salt for artificial seed germination (Nugrahani et al. 2018).
Enhancement of conversion efficiency has also been found through the addition of
different media additives like adenine sulfate (AdS), PPM, etc. The addition of
40–50 mg/L AdS was remarked as the considerable range for this (Hassan 2003;
Saeed et al. 2018). Ads, as an adjuvant, can reduce shoot tip necrosis and the
problems related to leaf abscission of the artificial seed regenerated plantlets (Naaz
et al. 2014). On the other hand, PPM is considered as a potential biocidal compound,
which is effective against a wide range of contaminants. Thus, it is having a positive
effect on the entire plantlet development (Lata et al. 2009) because artificial seeds are
very prone to microbial attack (Vij and Kaur 1994). Haque et al. (2015) have utilized
Aloe vera gel (AvG) for the same. AvG is an undefined complex consisting of
carbohydrates, lipids, proteins, amino acids, etc., (Habeeb et al. 2007; Hamman
2008) and, therefore, serving as an important source of functional food for in vitro
regenerants. In case of Musa sp. cv. Grand Naine, Sandoval-Yugar et al. (2009) have
used activated charcoal (AC) with the basal medium and achieved 76% conversion.
AC enhances respiration rate by breaking the alginate capsule, and thus extending
the storage duration of the propagules (Saiprasad 2001). Apart from this, toxic
substances like 5-hydroxymethylfurfural (formed from sucrose at the time of
autoclaving) are absorbed by AC too (Wang et al. 2007). Likewise, the promo-
tional effect of AC (that absorbs growth-hindering polyphenols from the
medium) during in vitro root induction was reported as well (Gantait et al. 2009).
430 A. Das et al.
14.6 Conclusion and Future Research Guidelines
The increasing diabetic population in the world has become a matter of concern to
find the safe and effective alternatives for conventional treatment of diabetes. The
long list of anti-diabetic plants is in focus of researchers to utilize them for the
diabetes treatment and, therefore, these plants need to be conserved along with mass
propagation. In this regard, artificial seed production of anti-diabetic plants is very
important. Various explants, unipolar and bipolar, can be used for artificial seed
production depending on the species. Low-temperature storage of artificial seeds
especially at 4 C result in better germination after a finite duration, though 25 C
was recommendable in some cases as well. Eventually, a trial of cryopreservation
can be a useful way out for conserving the artificial seeds for further germplasm
conservation and exchange as well. Effective regeneration of plants also depends on
the kind of explants used like in B. monnieri, somatic embryos were found to be
more effective than shoot buds (Khilwani et al. 2016) and in Salvia splendens, nodal
segments were effective in regenerating multiple plants than shoot tips (Sharma et al.
2014). Hairy root as explant has not been explored much for artificial seed produc-
tion in medicinal plants. This area can be explored in future for the production of
useful secondary metabolites. Cutting-edge nanotechnological approaches (such as
use of different metalic or non-metalic nanoparticles in the gelling agent or matrix)
may be another option in order to enhance the post-storage germination of artificial
seeds. The relative concentration of encapsulating agents in the matrix varies
depending on the explant and species. To date, calcium nitrate has not been explored
much as an encapsulation matrix. Also the effect of potassium nitrate for breaking of
the capsule can be explored in all the species having anti-diabetic properties. Limited
reports are available regarding the use of osmotica like mannitol, sorbitol, etc., that
help in extending the storage duration which can be employed in future research
works.
Acknowledgments The authors acknowledge the e-library assistance the Central Library, Bidhan
Chandra Krishi Viswavidyalaya (BCKV), West Bengal, India and the experimental assistance from
Plant Tissue Culture laboratory at Regional Nuclear Agricultural Research Centre, BCKV.
Conflict of Interest: The authors declare no conflict of interest.
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Cryopreservation of Anti-Diabetic Plants
15
M. R. Rohini, Marcos Edel Martinez Montero, and P. E. Rajasekharan
Abstract
Medicinal plant use and trade has seen a dramatic increase over the years owing to
the increased realization about its health benefitting effects. Limited commercial
cultivation has forced the industries and traders to rely on the wild collections to
meet the growing demand. The situation has threatened the survival of many
species including those having anti-diabetic potential. These difficult to conserve
species need biotechnological techniques for their long-term conservation and
sustainable utilization. For non-orthodox and vegetatively propagated species,
cryopreservation in liquid nitrogen (LN) at 196 C offers the most successful
and economical technique for long-term conservation. Traditional techniques
based on freeze-induced dehydration and recent techniques based on vitrification
have been efficiently used for cryopreservation of all types of the explants.
Cryopreservation offers multiple advantages over other conservation strategies
as it minimizes the risk of contamination, cost of maintenance and cost of labour.
The process of cryopreservation exposes the cell or tissue to various physical,
chemical and physiological stresses which may result in cryoinjury or genetic
level changes sometimes. The analysis of such morphological, structural, genetic
or functional changes is important to assess the genetic integrity of cryopreserved
germplasm to see if they are ‘true to type’ after cryopreservation. This can be
studied at the phenotypic, histological, cytological, biochemical and molecular
levels. The chapter will throw light upon the importance and status of cryopres-
ervation, different methods of cryopreservation adopted in major anti-diabetic
plants along with the pre and post cryotreatments and regeneration protocols,
M. R. Rohini · P. E. Rajasekharan (*)

Indian Institute of Horticultural Research, Hessaraghatta Lake PO, Bengaluru, Karnataka, India
M. E. M. Montero
Centro de Bioplantas Universidad de Ciego de Ávila Máximo Gómez Báez, Ciego de Ávila, Cuba
Ltd. 2021
https://doi.org/10.1007/978-981-16-3529-8_15
438 M. R. Rohini et al.
infrastructure requirements for cryobank. The concept of root cryobanking and

cryobionomics dealing with genetic stability and the reintroduction of
cryopreserved plants into the environment is also covered.
Keywords
Cryopreservation · Cryobank · Cryobionomics · Liquid nitrogen · Vitrification
15.1 Introduction
Efficient conservation of genetic resources is fundamental for achieving food and

nutritional security globally. Plant genetic resources are the reservoir of genes
essential for crop improvement programmes so as to develop varieties adapted to
socio-economic requirements and changed environments. Safeguarding these
genetic resources is an enormous task and will require prudent conservation strategy.
The traditional approaches of ex situ conservation through seed and field gene banks
have made remarkable contribution in the conservation of plant germplasm. But in
the case of vegetatively propagated plants and recalcitrant seed species, conservation
efforts has been hampered due to problems faced during the application of their
conventional method of ex situ conservation. Thus, efforts have been made to
develop biotechnological approaches for conservation. Biotechnological methods
of conservation include medium term conservation using in vitro slow growth
cultures and long-term conservation using cryopreservation. The complementary
application of the new and conventional approaches has increased the efficiency of
conservation system. The appropriate conservation method to be adopted depends
on the breeding nature of the species and their seed storage behaviour. For annuals,
which are propagated by seeds and have orthodox storage behaviour, seed gene bank
conservation is the most convenient and reliable method. For recalcitrant seeded
species, especially perennial tree species and vegetatively propagated species which
are considered as ‘difficult to conserve species’ cryopreservation is a promising
technique for long-term conservation.
15.2 Cryopreservation
Cryopreservation involves the conservation of biological tissue at ultra-low temper-

ature by immersing in liquid nitrogen (LN) at 196 C. Due to storage at such low
temperatures, cell divisions and metabolic activities are arrested and thus, plant
material can be stored for unlimited periods of time. Cryopreservation employs the
use of tissue culture techniques in conservation of germplasm. Different types of
explants can be used for cryopreservation like seeds, zygotic embryos, embryonic
axes, pollen, dormant buds, shoot apices etc. Cryopreservation is the most reliable
method of choice for ensuring the long-term storage of non-orthodox seeds, clonally
propagated species and biotechnologically important plant cell lines. Many studies
15 Cryopreservation of Anti-Diabetic Plants 439
have confirmed that it is economically more viable than other conservation methods
as the cost of maintaining an accession in LN for the long term (over 20 years) is
substantially lower than that of in the field or in vitro, particularly when dealing with
a large number of accessions. Cryopreservation is advantageous as it requires only
minimum space and also eliminates the need for frequent subculturing because of
which chance of somaclonal variation is reduced. In terms of simplicity and the
applicability to a wide range of genotypes, cryopreservation is always reported to be
a reliable, safe and cost-effective method over most other conservation methods used
for long-term conservation of germplasm.
15.2.1 Principles of Plant Tissue Cryopreservation
The most critical factor in cryopreservation is the removal of intracellular water thus
preventing the formation of ice crystals during freezing. According to Crowe et al.
(1988), 95% of the water present in biological tissues is free and will convert to ice
during freezing causing irreparable damage. There are chances that the cell may get
injured during both the processes, i.e., dehydration (removal of water) and intracel-
lular freezing. Too much removal of water results in cell injury, possibly as a result
of membrane stress and too little dehydration results in intracellular freezing. Thus,
the different techniques of cryopreservation are aimed at removing the intra cellular
water in such a way so as to prevent the dehydration injury and freezing injury. The
fundamental process occurring during cryopreservation, keeping in view of which
cryopreservation protocols should be optimized for better viability and survival of
tissues has been schematically presented in Fig. 15.1.
Removal of water to Tolerance to

avoid crystallization temperature
Excess Less
Slow freezing Fast freezing
dehydration dehydration
Physical damage Intracellular Vapour pressure Excessive No

to cell contents freezing equilibrium dehydration crystallization
Cell survival Cell injury Cell survival

Cell injury
Fig. 15.1 Schematic diagram showing principle of cryopreservation

Before implementation of any conservation method, the preliminary step is to

determine the seed storage behaviour of the species. Seed storage behaviour is
analysed by studying seed morphological and seed physiological features especially
desiccation and freezing sensitivity along with seed longevity. Roberts (1973)
classified seeds into two broad physiological categories; (1) Orthodox and
(2) Non-orthodox (recalcitrant and intermediate) based on storage characteristics.
Seed gene banks are generally used for the conservation of orthodox seeds and field/
in vitro or cryogene banks are used for the conservation of non-orthodox seed
species.
Orthodox Seeds Seeds that can tolerate low moisture and low temperature storage
without losing viability. Such seeds can be desiccated to low moisture contents, i.e.,
below 5%, and can be stored at sub-zero temperatures. Viability is prolonged in a
predictable manner by such reduction in moisture and storage temperature.
Recalcitrant Seeds Seeds that are desiccation sensitive, short lived and are gener-
ally intolerant to low temperatures. Seeds of recalcitrant species maintain high
moisture content at maturity (often more than 30–60% and are sensitive to desicca-
tion below 12–30%, depending on species. They rapidly lose viability under any
kind of storage conditions.
Intermediate Seed Seeds that can tolerate desiccation to some extent, i.e., up to
12–15% moisture content but are sensitive to low temperature.
Cryopreservation is achieved either through classical technique which is based on

freeze-induced cell dehydration and vitrification-based method. The techniques
employed and the physical mechanisms upon which they are based are different in
classical and new cryopreservation techniques (Withers and Engelmann 1998).
Classical cryopreservation-based techniques involve slow cooling down to a defined
pre-freezing temperature followed by rapid immersion in LN. They are generally
operationally complex since they require the use of sophisticated and expensive
programmable freezers. In some cases, their use can be avoided by performing the
freezing step with a domestic or laboratory freezer (Kartha and Engelmann 1994). In
the new vitrification-based procedures, cell dehydration is performed prior to freez-
ing by exposure of samples to concentrated cryoprotective media and/or air desicca-
tion. Vitrification can be defined as the transition of water directly from the liquid
phase into an amorphous phase or glass, while avoiding the formation of ice crystal
(Fahy et al. 1984). This is followed by rapid freezing. As a result, all factors which
affect intracellular ice formation are avoided. Vitrification-based procedures are
more appropriate for complex organs like shoot tips and embryos which contain a
variety of cell types.
15.2.2 Classical Techniques
In this method, the tissues are treated with cryoprotectants followed by gradual
cooling at a controlled rate (0.5–2 C/min), usually with a programmable freezing
apparatus, down to about 40 C. After holding at this temperature for a short time
(30 min), tissues are immersed in LN. The most commonly used cryoprotectants
such as Dimethyl Sulphoxide (DMSO), glycerol, Polyethylene glycol (PEG), sorbi-
tol etc. are added singly or in combination. To prevent excessive super cooling, the
ice inoculation step (seeding) is performed when temperature as low as 7 to 10 C
is reached. Seeding is performed either automatically if the programmable freezer
possesses such an automatic seeding facility or by touching the outside wall of the
cryovials with the forceps pre-cooled in LN. This method is applicable to a range of
in vitro plant culture system but is most successful with culture systems that consist
of small units of uniform morphology such as protoplast cultures, exponentially
growing cell suspension cultures or fragmented callus cultures.
15.2.3 Vitrification-Based Techniques
In this method, cell desiccation is performed either by exposure of samples to

concentrated cryoprotective solutions or by air desiccation. Cryoprotectants include
dimethyl sulfoxide (DMSO), glycerol, ethylene glycol, polyethylene glycol, sugars
and sugar alcohols used either alone or in combination to protect living cells against
damage during freezing and thawing. They act by lowering the freezing point and
also alter the crystalline structure of ice. They also prevent solute accumulation at a
given level of dehydration so that cell injury is prevented. Careful selection of
cryoprotectants is essential since most of the cryoprotectants exhibit varying degree
of cytotoxicity especially at higher concentration and temperature. This is followed
by rapid freezing through direct immersion of the samples in LN. Vitrification-based
procedures are simpler than classical technique. These methods are suitable to for
cryopreservation of organized structures such as shoot tips, axillary buds etc.
A range of protocols has been widely applied to cryopreserve plant species using
diverse cell and tissue types: cell cultures and suspensions, calluses, apices, somatic
and zygotic embryos, pollen and dormant buds. Some approaches are based on a
singular technique, whereas others use a combination of them. The majority of the
cryopreservation methods involves multi-stage steps. Their common essential steps
include preculture, cryoprotective treatment (under air or in a cryoprotectant solu-
tion), freezing, rewarming, unloading (removing of cryoprotectant solutions) and for
one to several days.
15.2.4 Preculture
Preculture of explants on medium with low concentration of cryoprotectants is

recommended before the freezing process. Process of preculture allows explants to
recover effectively from the dehydration stress. Preculture on medium with

cryoprotectants (often sucrose or sorbitol) also induces freezing tolerance through
sugar uptake and dehydration of explants (Sakai 2000).
15.2.5 Cryoprotective Treatment
In this process, explants are subjected to dehydration either by air desiccation or by

exposing to cryoprotectants. The nature as well as concentration of cryoprotectants,
time of treatment and temperature conditions needs to be optimized for specific
species. Numerous cryoprotective chemicals are used with plant tissues, commonly
used ones are sucrose, DMSO, ethylene glycol, PEG and glycerol. The concentration
of cryoprotectant solution depends on the cryopreservation technique used. It ranges
from 0.5 to 1 M in classical freezing protocols to up to 7 M with vitrification
protocols. The treatment duration also varies with the technique used, from
10–40 min in the case of vitrification (Sakai 2000) to around 1 h in classical freezing
protocols (Kartha and Engelmann 1994). In any case, the process of dehydration is
essential for successful cryopreservation to avoid intracellular freezing and irrevers-
ible injury of cells caused by the formation of ice crystals.
15.2.6 Freezing
Freezing rate and pre-freezing temperature are the two parameters which need to be
optimized in the freezing step. In vitrification-based methods, freezing is rapid by
direct immersion of explant in LN. For zygotic embryos or embryonic axes of
recalcitrant species, increasing the freezing rate will help to successfully cryopre-
serve samples with high water content. After freezing, explants should be stored at
sufficiently low temperature (vapours of LN at 150 C or immersed in LN at
196 C).
15.2.7 Rewarming
Rewarming of cryopreserved samples should be done as fast as possible to avoid the

possibility of damaging ice recrystallization which can cause irreversible damage to
the tissues. It is usually done by rapid immersion of cryotubes in water bath at
37–40 C. In air desiccation technique, rewarming of the samples may be done at
room temperature also.
15.2.8 Recovery
After rewarming, samples should be placed under optimal conditions for rapid and
direct growth. Samples are usually placed in the dark or under reduced light for
several days to reduce detrimental photo-oxidation phenomenon. The culture

conditions also need to be optimized for each species. Explants are transferred
back to standard culture conditions after a few days or weeks.
15.2.9 Air Desiccation
Cryopreservation using desiccation involves dehydrating the plant material down to

a suitable water content (critical moisture content) followed by rapid freezing
through direct immersion in LN. In desiccation, the physiological maturity of
seeds is an important factor for the successful cryopreservation of embryo/embry-
onic axes extracted from seeds. Dehydration of the plant material is done either by
exposing them to sterile air flow in a laminar flow chamber or over activated silica
gel for different duration of time until the critical moisture content is obtained. Air
desiccation or dehydration is considered as the simplest technique because it does
not require any cryoprotectant chemicals or expensive equipment’s like programma-
ble freezer. Plant material is usually recovered by the rewarming of samples under
ambient conditions. In samples with very high water content, ultra-rapid drying is
adopted which involves flash drying by exposing cells to a stream of compressed dry
air (Berjak et al. 1989; Wesley-Smith et al. 1992). As of now, this is the most
followed method of cryopreservation practiced for routine storage of germplasm in
gene banks handling a greater number of germplasms per day.
15.2.10 Encapsulation/Dehydration
In this method, alginate beads are used to encapsulate the plant material and then
partially desiccated in laminar flow or over silica gel followed by direct plunging in
LN. The process involves preculture of the explant material in sucrose overnight.
This is followed by encapsulation of the explant in calcium alginate beads, then
partially desiccated by pre-culturing in liquid MS medium with different sucrose
concentrations (0.3, 0.5 and 0.75 M) at 100 rpm for 20 or 40 h followed by
transferring the beads to cryovials and plunging the cryovials in LN. Rewarming
is done by exposing the cryopreserved samples at room temperature for 15 min to
half an hour and then cultured on the standard media. The technique has been
successfully applied to conserve the embryonic axes of Poncirus trifoliata, neem,
Citrus sinensis etc.
15.2.11 Vitrification
This process involves the treatment of tissues with cryoprotectants in vitrification

solution followed by fast freezing. In vitrification, initially the explant material is
precultured on basal media supplemented with 0.3 M sucrose and 2 M glycerol
overnight. Next day, the material is transferred to cryovials containing loading
Fig. 15.2 Schematic diagram showing the process of desiccation, encapsulation and vitrification
for embryonic axes
solution which is a combination of 0.4 M sucrose and 2 M glycerol. After 25 min, the
loading solution is replaced by plant vitrification solution (PVS2: 30% (w/v) glyc-
erol, 15% (w/v) ethylene glycol, 15% (w/v) dimethyl sulphoxide) with 0.3 M
sucrose for different duration of time followed by rapid immersion in LN. Rapid

thawing is done by immersing frozen cryovials in water bath (38 1 C) for 5 min.
After thawing, PVS2 solution is drained off and unloading solution is added (1.2 M
sucrose) for 20 min to remove the effect of PVS2. This is followed by culturing of
the explant material.
The above three process, i.e., desiccation, encapsulation-dehydration and
vitrification in the case of cryopreservation of embryonic axes of Citrus species
have been illustrated in Fig. 15.2.
15.2.12 Droplet Freezing
The technique combines the droplet-freezing method with the vitrification procedure
(Sakai et al. 1990). This method consists of pretreatment of the tissue in liquid
medium supplemented with cryoprotectants followed by transferring them into
drops of cryoprotective medium placed on aluminium foil followed by rapid freezing
in LN. It is of two types:
15.2.12.1 DMSO Droplet Freezing

This method was initially developed for the cryopreservation of shoot tips of cassava
by Kartha et al. (1982) and was later applied to conserve potato shoot tips (Schäfer-
Menuhr et al. 1996). The method involves the treatment of the explant material with
the cryoprotectant DMSO for 1–3 h and then transferring the explants to aluminium
foils containing a drop of DMSO solution (2.5 μL). The foil is then placed in the
cryovial and rapidly immersed in LN. This method also needs rapid thawing of
samples by exposing the cryovials in water bath at 38 1 C for 5 min followed by
culturing of the sample.
15.2.12.2 PVS2 Droplet Freezing

This is similar to the above method except that in place of DMSO, PVS2 solution is
used for cryoprotection. Rapid LN plunging is followed by rapid thawing and
culturing.
Droplet methods have the disadvantage of ultra-rapid freezing of the tissues
because aluminium foil permits heat transmission faster than normally used
cryovials and also because of the minute volume of cryoprotectant (Benson et al.
2013).
15.2.13 Encapsulation Vitrification
As the name suggests, this method combines two techniques of cryopreservation,

i.e., encapsulation/dehydration and vitrification. In this, first the explant material is
encapsulated in alginate beads and then exposed to cryoprotectant treatment by
PVS2 solution followed by immersion in LN. Rapid or slow thawing is done
followed by removing of vitrification solution and culturing of the beads
(Martinez-Montero et al. 2012).
15.2.14 Cryoplate Methods
This is the recently developed method of cryopreservation which makes use of the
cryoplates. Cryoplates are small aluminium plates with embedded wells for holding
the explant material. Cryoplates explant inoculation in the plates and its immersion
in LN is illustrated in the Fig. 15.3.
Fig. 15.3 Diagram showing

the cryoplate method
In V-cryoplate method, explant material is precultured in 0.3 M sucrose and

transferred to cryoplates after encapsulation in alginate beads and then treated with
loading solution (2 M glycerol +0.6–1 M sucrose) for 30 min. Dehydration is
performed by treating with PVS2 solution followed by rapid freezing. Frozen
cryoplates are rapidly thawed by transferring to 1 M sucrose (unloading solution)
for 15 min followed by culturing of the same. In D-cryoplate method, the steps
followed are similar to the ones in V-cryoplate method except that after treating with
loading solution, instead of PVS 2 solution explants are dehydrated in laminar flow
cabinet for suitable period. Cryoplate methods are advantageous because of their
user friendliness and ease of handling samples on the aluminium plates. Successful
cryopreservation using cryoplates methods has been reported for many plant species
including strawberry, sugarcane, date palm, mat rush and potato.
15.3 Root Cryobanking
Root cryobanking is emerging as a potential method of cryopreservation especially

for medicinal plants. Roots are uniquely suited for long-term conservation of plant
genetic resources due to their ease of excision and in vitro maintenance, and the
ability to produce new lateral roots and/or shoots. Cryopreservation and regeneration
of root tips in potato cultivars was first attempted by Bajaj (1978), but thereafter only
few studies are available on root cryopreservation when compared with cryopreser-
vation of other plant organs and tissues (Normah et al. 2019; Agrawal et al. 2019).
But, with the boom in the in vitro micropropagation of medicinal plants for second-
ary metabolite production, successful cryopreservation of root explants which are
isolated from in vitro cultures as well as adventitious and hairy roots has been
successfully attempted. Cryopreservation of adventitious and hairy roots offers
scientific as well as commercial advantage because they are considered as the
alternative sources for many important secondary metabolites thus having potential
application in pharmaceutical, cosmetic and natural health product industries
(Popova et al. 2020). The cryopreservation process does not affect the biosynthetic
or genetic stability of the roots keeping their potential same as that of field grown
roots. Root cryopreservation was successfully achieved using encapsulation as well
as vitrification methods. Encapsulation of root tips protects them from mechanical
damage and vitrification method provides fast cooling and warming.
15.4 Pollen Cryopreservation
A pollen cryobank with diverse pollen collections provide long-term security for
wild flora, ornamentals, fruit and vegetable crops, medicinal herbs and endangered
species. Recently for so many anti-diabetic species pollen has been successfully
cryopreserved, for example, Momordica doica Momordica sahyadrica. The tech-
nique of pollen cryopreservation has the potential to overcome different challenges
facing in crop breeding programs, such as flowering asynchrony between different
parent genotypes, and the production of insufficient pollen in the case of many wild
species. The technique of pollen cryopreservation is simple enough to be used
routinely in research, plant breeding and in complementary m conservation
strategies of plant genetic resources (Dinato et al. 2020). A pollen cryobank become
an important component of national gene banks can provide a constant supply of
viable and fertile pollen to allow supplementary pollinations for breeding
programmes. Pollen banks, where large quantities of pollen are cryopreserved in a
relatively small area, are available to breeders for use in crop improvement programs
and to supplement plant genetic resources (Rajasekharana and Ganeshan 2019). The
technology for the establishment of pollen cryobank has been optimized at the
in vitro Conservation and Cryopreservation Laboratory of Division of Plant Genetic
Resources, ICAR-IIHR, Bangalore. Cryopreservation is now an accessible
conservation option for a wide range of users and it has the potential to support
both small- and large-scale laboratories involved various research programmes and
centres involved in conservation of plant genetic resources.
15.5 Current Status of Cryopreservation
Cryopreservation is now considered as the safest, low-cost and efficient long-term

conservation strategy applied to seeds of many orthodox and recalcitrant species,
dormant buds and embryos of many tree species and pollen of several difficult to
conserve species. Cryopreservation protocols are now available for cell and tissue
types of around 200 species including both differentiated as well as undifferentiated
explants (Dulloo et al. 2010). Many economically important crops including root
and tuber crops, fruit crops, ornamentals and plantation crops of temperate and
tropical origin are successfully cryopreserved using various techniques (Engelmann
2000; Kaczmarczyk et al. 2012). Cryogene banks are now an integral part of many of
the institutes Consultative Group on International Agricultural Research (CGIAR)
like International Potato Centre (CIP) at Peru, International Transit Centre for Musa,
International Institute for Tropical Agriculture, International Centre for Tropical
Agriculture. Table 15.1 shows the germplasm holdings maintained at National
Plant Germplasm System of USDA-ARS. In India, major thrust on crop
Table 15.1 Status of germplasm in USDA-ARS, National Plant Germplasm System (NPGS), as
of 2017
Number Number of Total
Primary NPGS repositories of genera species accessions
National Arid Land Plant Genetic Resources, Parlier, 13 70 603
CA
National Clonal Germplasm Repository, Corvallis, OR 64 664 8754
National Clonal Repository for Citrus and Date, 9 10 1789
Riverside, CA
National Clonal Germplasm Repository for Tree Fruit/ 21 248 7257
Nut Crops and Grapes, Davis, CA
National Germplasm Repository, Brownwood, TX 1 12 2328
North Central Regional Plant Introduction Station, 152 622 2456
Ames, IA
Ornamental Plant Germplasm Centre, Columbus, OH 54 300 1013
Plant Genetic Resources Conservation, Griffin, GA 45 196 1823
Plant Genetic Resources, Geneva, NY 7 103 4410
Subtropical Horticulture Research Station, Miami, FL 327 802 2790
Tropical Agriculture Research Station, Mayaguez, PR 285 483 1152
Tropical Plant Genetic Resources Management, Hilo, 19 58 709
HI
United States Potato Gene Bank, Sturgeon, Bay, WI 1 92 836
Western Regional Plant Introduction, Pullman, WA 24 64 542
Woody Landscape Plant Germplasm, Washington, DC 172 701 1493
Table 15.2 Status of germplasm in cryogene bank of NBPGR (as on March 31, 2019)
Categories Total number of accessions
Recalcitrant and intermediate 6782
Fruits and nuts 3520
Spices and condiments 152
Plantation crops 88
Agroforestry, industrial crops, medicinal and aromatic plants 3022
Orthodox 3902
Dormant buds 387
Pollen grains 572
Genomic resources 1934
Total 13,577
cryopreservation activities is carried out at NBPGR, New Delhi maintaining approx-

imately 14,000 crop accessions in different forms (Table 15.2). Cryopreservation
protocols have been in anti-diabetic plant species like Allium, Bacopa, Dioscorea,
Morus, Musa, Picrorhiza etc. Major success has been achieved in two genera,
namely, Allium and Musa. The NBPGR cryogene bank (in vapour phase of LN)
has 13,363 accessions of diverse crops conserved in the form of seeds, embryos,
embryonic axes, pollen, budwood and DNA. Apart from NBPGR, Central Potato
Research Institute at Shimla holds pollen collection of potato germplasm (6 nos.) and
Indian Institute of Horticultural Research, Bangalore holds pollen collection of many
horticulture crops including medicinal plants (675 nos.).
15.6 Relevance of Cryopreservation in Medicinal Plants

with Special Emphasis on Anti-Diabetic Plants
Plants have been the only source of life saving drugs available to mankind since
ancient time until the modern system of medicine emerged. In developing countries
like India, plant-based medicines still hold a very significant place in the system of
medicine and now with the growing realization of the health benefitting properties of
medicinal plants there has been an increased demand for plant-based products over
the synthetic drugs. Plant-based drugs being safe with less side effects have expo-
nentially increased the global market for medicinal plants. Diabetes mellitus (DM) is
one of the most rapidly increasing lifestyle disorder affecting approximately 2.8% of
the global population, is expected to increase to 5.4% by 2025, and this number will
be ca. 440 million people worldwide in 2040. Large number of plants are reported to
have anti-diabetic or hypoglycaemic property and are used in ayurvedic and other
plant-based systems for the treatment of diabetes. The plants with anti-diabetic
property can be broadly classified as one which are purely medicinal possessing
anti-diabetic property like Gymnema sylvestre, Aloe vera, stevia, Dioscorea
bulbifera, neem etc. and the other category plants belong to fruit, vegetable or spices
group possessing anti-diabetic property like bitter gourd, jamun, fenugreek, garlic,
Citrus sinensis etc. In this chapter, we will be covering the cryopreservation status of
both these groups. More than 35% of compounds for diabetic treatments are
extracted from leaves of these anti-diabetic species. However, the fruits of many
plants with anti-diabetic potential can be consumed orally in the form of juices.
These plants contain various phytoconstituents such as flavonoids, terpenoids,
saponins, carotenoids, alkaloids and glycosides, which may possess anti-diabetic
activities. The most serious concern with majority of medicinal plants is that they are
accessed from their natural habitats such as forest or natural ecosystems. Unlike very
few commercially cultivated anti-diabetic medicinal species like Aloe vera, all others
are sourced from wild thereby threatening their survival in the natural habitats.
Unscrupulous collection from wild, destruction of natural habitats, climate change
scenarios has transformed the status of many native plants into rare, endangered and
threatened category. About 15,000 plant species are threatened with extinction by
the overharvesting of plant resources and their natural habitat destruction. In view of
this rapidly eroding native flora, conservation efforts have gained momentum
nationally and internationally to conserve them. For the conservation of wild genetic
resources like medicinal plants, in situ conservation through biosphere reserves are
advocated and adapted depending upon the need and situation. In situ conservation
efforts aims at the conservation of genetic resources in its natural habitats by
preventing encroachment activities, but of loss of plant populations due to factors
like biotic or abiotic stresses, climate change is inevitable in the in situ areas. Thus,
the in situ conservation strategies should be complemented with ex situ conservation
efforts for long-term conservation of the targeted species. Majority of the medicinal
plants are categorized under wild, rare, endangered and threatened group for which
cryopreservation is considered as the most viable long-term conservation strategy.
Moreover, seed banking cannot be applied to species that produce few or no seeds,
or for which the seeds are inaccessible for collecting which is the case with most of
the medicinal species.
Recently, Walters and Pence (2020) reviewed that lifespan of many samples is
reduced after storage. Triacylglycerols present in seed changes owning to different
storage conditions and thereby reducing their shelf life. This is the case with many of
the anti-diabetic plants (e.g. Juglans regia, Carya, Castaena and Corylus) and
tropical species (e.g. Cuphea, Elais and Hevea), as well as seeds from numerous
Hawaiian species. These seeds with high triacylglycerols become viscous and
crystallizes at low temperatures and therefore they require alternative conservation
approaches.
15.7 Infrastructure Requirements for Plant Cryopreservation
Cryopreservation techniques are now being used increasingly for the long-term
conservation of germplasm worldwide. Before adopting cryopreservation strategy
for any system, it is essential to examine the effects of different parameters on the
germplasm conserved. Cryopreservation protocols are already available for more
than 500 species of plant tissues, at the same time it is essential to develop and
optimize standard protocols for specific plant species and or tissue types for effective
conservation. Cryopreservation is labour and resource intensive procedure; there-
fore, prioritization of plant species for conservation is important. Secure storage
location and complete inventory of the germplasm stored is vital for the successful
recovery of conserved germplasm whenever needed.
Basic facilities required for initializing the experimental procedures in small scale
laboratories are:
• Tissue culture facility.

– Washing room: For washing and storage of glassware, plasticware and other
labware.
– Media preparation room: For preparation and storage of nutrient media.
– Autoclave room: For sterilization of media, water and instruments.
– Inoculation room: For aseptic manipulation of plant material.
– Culture room: For maintenance of cultures under controlled conditions of
temperature, light and humidity.
• Storage facility
LN tanks (small capacity 10–30 L). If the number of accessions is more, wide
mouth LN tanks of 1000 L capacity can also be used.
• Green house facility
Green house facility is essential for the initial establishment of dormant plant
propagules. It is also essential for acclimatization of in vitro grown plantlets as a
part of viability testing process.
15.7.1 Laboratory Requirements
Setting up of a long-term cryopreservation facility is a one-time investment and

involves the following laboratory equipment.
• Large capacity cryotanks ranging from 60–1000 L capacity with electronically

controlled filling system along with alarm.
• Transportation containers without canisters used for transporting LN.
• Diverse inventory system specific to the cryotanks.
• Pressurized LN vessel.
• Programmable freezer.
• Small biological containers with canisters.
• High Precision electronic balance.
• Controlled temperature water bath.
• Fan assisted hot air oven.
• Cryovials and cryomarkers.
• Safety gadgets—gloves, apron, face shield or goggles.
• Heavy duty trolleys for transportation of tanks.
15.7.2 Storage Containers
• All types of explants are to be finally packed in airtight containers before

cryostorage.
• Glass containers can shatter when warmed from sub-zero temperatures.
• Polypropylene screw cap cryovials of different sizes (1 mL, 2 mL, 5 mL, 50 mL)
depending upon the size as well as the quantity of the seeds/embryonic axes/
dormant buds/pollen grains are used.
• Pollen desiccated to suitable moisture content can be stored in aluminium packets
or gelatin capsules.
• Large seeds and twigs are to be stored in heat-sealable polyolefin tubing with cork
stoppers or in goblets in sleeves.
15.8 Maintenance of Cryotanks
Cryotanks are self-contained, vacuum-insulated double walled stainless steel

vessels. They essentially have a LN reservoir at the base and samples are suspended
on a stationary or rotating tray which partitions the tank. It is important to choose a
suitable storage system, i.e., material containers, holding supports etc. Careful
planning needs to be done taking into account:
(a) Explant size.

(b) Number of explants to be stored.
(c) Accessibility.
(d) Vapour or liquid phase storage.
If explants are of different sizes, then cryovials of different sizes (1–10 mL)
should be used. Small seeds, embryos, embryonic axes, shoot apices and meristems
can be easily stored in large numbers in cryovials of 5 mL and 10 mL capacity. Each
tank has a static holding time which is the maximum time for which a tank can hold a
particular quantity of LN, after which more LN have to be replenished. This is
dependent on the rate of evaporation and volume of the cryotank. Evaporation rate of
LN for most tanks is 0.5–1.5% of its capacity per day and accordingly, replenish-
ment of LN would be requires about 2 times per week. Equipment which are used
routinely for cryopreservation of plant material is depicted in the Fig. 15.4.
15.9 Management of Cryopreserved Collections: Practical

Issues
Adoption of cryopreservation techniques for conservation depends upon technical

feasibility, conservation procedures and relation between cost and benefits (Kartha
and Engelmann 1994). These all are in turn influenced by the objectives of conser-
vation, needs and types of materials to be conserved and available resources.
Fig. 15.4 Equipment used for cryopreservation
15.10 Prioritizing the Materials for Cryopreservation
Cryopreservation is often considered as a secondary storage strategy which is

complementary to the gene bank, field gene bank and in situ conservation. In such
a case, it is important to prioritize the materials, i.e., to be conserved, as it will
depend on the available infrastructures and national needs of the country and
institutions. The categories of anti-diabetic medicinal plant species or any plant
species for that matter which needs cryopreservation includes:
(a) Species producing intermediate and recalcitrant seeds: For example, citrus
species, neem and Jamun.
(b) Threatened and endangered plant species with critically small population size:
For example, Rauwolfia serpentina, Costus speciosus, Gymnema sylvestre etc.
(c) Wild species and wild relatives of crop plants: Almost all the anti-diabetic
medicinal plants will come in this category.
(d) Registered germplasm: Germplasm holding any unique value in terms of mor-
phological or economic traits.
(e) Released varieties and genetic stock.
For embryogenic cultures that may lose their capacity for embryo formation with
time, cryopreservation provides primary storage, with cultures revived and used to
produce more embryos at a later date.
15.11 Selection of Explants for Cryopreservation
Explants for cryopreservation may be either seeds, pollen, shoot tips, dormant buds,
embryos, embryonic axes, callus or cell cultures depending on the species. Seeds are
mostly used for seed propagated orthodox species, pollen is used when the plant
breeder wants to conserve and use the part of genetic diversity in easily accessible
form and also for recalcitrant seed species. Dormant buds are used mostly in the case
of temperate budded or grafted trees. Shoot tips can be used for any type of
temperate or tropical plants.
Whatever be the explant material to be used, it has to be freshly obtained and
processed fast to retain the viability. Seeds collected should be used within few hours
to few days, similarly shoot tips, embryos or embryonic axes should be freshly
extracted, in the case of pollen, it should be collected from flowers with freshly
dehisced anthers. For collection of buds 60 cm long twigs of last 1 year’s growth
having dormant buds are to be harvested from plants growing at the field gene bank
in winter seasons. Table 15.3 shows different types of explants which have been
used for the cryopreservation in some of the anti-diabetic species.
15.12 Size of Germplasm to be Cryopreserved
The optimum sample size for cryopreservation depends on many factors like the
reproductive biology and pollination behaviour, seed storage behaviour, availability
of the plant species, its recovery potential, size of the explant used etc. For self-
pollinating species, minimum of 2000 seeds may be stored and for cross-pollinating
species, a minimum of 4000 seeds needs to be stored. For embryos, embryonic axes,
shoot tips, meristems and pollen, there is no standard recommendation for minimum
number of explants to be stored. It depends on the availability of material and
percentage of survival. Storage should ensure the availability of enough propagules
to regenerate sufficient plants whenever required and also additional vials can be
used for periodic viability testing. The type of explant used also determines the size
because seeds and dormant buds need more space as compared to embryos and cell
Table 15.3 Different types of explants used for cryopreservation of anti-diabetic plants
Sl
no Species Explant Method Reference
1 Citrus Nuclear cells Classical slow freezing Kobayashi et al.
sinensis (1990)
2 Citrus Embryonic axes Air desiccation, vitrification, Malik et al.
sinensis encapsulation-dehydration (2012)
3 Dioscorea Somatic Encapsulation-dehydration Mandal et al.
bulbifera embryos (1999)
4 Dioscorea sp. Shoot tips Vitrification Mandal and Dixit
(2000)
5 Allium Shoot tips Vitrification Makowska et al.
sativum (1999)
6 Morus alba Winter hardy Desiccation Nino et al. (1992)
buds
7 Rauwolfia In vitro grown Vitrification Ray and
serpentina nodal segments Bhattacharya
(2008)
8 Azadirachta Embryonic axes Vitrification Malik and
indica (neem) Chaudhury
(2019)
9 Musa Zygotic embryo Desiccation freezing Abdelnour-
acuminata Esquivel et al.
(1992)
cultures. Recovery potential of a stored accession will also determine its storage size,
as species with high recovery can be stored less in number in fewer vials and difficult
species has to be conserved in more numbers in more vials. A formula proposed by
Dussert et al. (2003) can be used for determining the number of propagules needed
for long-term recovery of plant materials in culture based on binomial distribution
using the number of controls tested, the recovery of those control plants, the number
stored and the probability of recovering one growing sample. The number of
propagules stored may vary from fewer than 100 for dormant buds to many
thousands for pollen grains.
15.13 Optimization of Protocol
While developing cryopreservation protocols for species with orthodox storage

behaviour, the strategy would be to examine the response of seeds of different
genera, species and accessions to initial LN exposure. Once it is established that
survival values tested after 18–24 h of LN storage are fairly uniform, routine
application can be taken up. For orthodox seeds, simple desiccation of seeds to
5–7% moisture content followed by placing them in cryotanks in vapour phase of
LN will ensure their indefinite storage. For recalcitrant, intermediate and vegeta-
tively propagated species, either new cryopreservation protocols should be
developed or already existing protocols should be optimized for higher viability and
survival. It is possible to do preliminary tests with 3–5 genotypes and, if successful,
apply the technique to the remaining collection. Accessions with low recovery will
require improvement in the plant materials or modifications of the technique to
improve recovery following LN exposure (Reed 2008).
15.14 Germination/Viability Standards
In a cryobank, the accessions are checked for ensuring high germination and
viability. At least 80% germination should be ensured for the stored samples. This
is particularly important for genotypes where there is a chance to obtain rare genes or
alleles. The germination or viability standards can be set still high if the genes are
reported to be extremely rare in a population and even a slight deterioration of
viability can lead to loss of such potentially valuable genes. Lower germination rates
are acceptable for released varieties because they are genetically more homogenous
than landraces or wild species.
15.15 Recovery Growth of Cryopreserved Germplasm
The success of any cryopreservation process depends on the recovery of viable

propagules (growing shoots, germinating pollen, multiplying cells) after
cryostorage. Development of optimum rewarming protocol and selection of the
appropriate regrowth medium is very crucial in determining the recovery of the
propagules. Recovery of true-to-type plants and actively growing cultures requires
the best regrowth conditions. Regrowth of cryopreserved tissues in in vitro should be
without a callus phase to retain the genetic stability.
15.16 Sample Labelling and Data Storage
Proper and accurate labelling of the cryopreserved accessions is very important and
critical especially when the gene bank holds large collections. Appropriate labelling
should include accession number, name, date of storage and initial viability %.
Bar-coded labels are also now available for cryovials. Clear labelling and numbering
of canes or boxes is important for easy retrieval of specimens. Linking of
cryopreserved samples to its passport information is crucial for generating any
information pertaining to the sample. Complete protocol information for each of
the sample should be stored like the information on pre-growth of the plant material,
pre-treatments, cryopreservation protocol, thawing method and the recovery
medium because these are vital to recovering living plants. A computerized database
with the passport data and storage location is to be compulsorily maintained in the
cryogene bank for easy access and utilization of the cryopreserved germplasm.
Some items needed for the database:
• Accession identifying number or code.

• Accession name.
• Cane or rack location.
• Number of vials stored.
• Storage date.
• Standard culture medium (reference).
• Preconditioning treatments.
• Preculture conditions.
• Osmoprotection.
• Cryopreservation technique (reference to the technique).
• Cryoprotectant used.
• Rewarming protocol.
• Regrowth medium.
• Data from control regrowth.
Care should be taken to store the active collections or test samples which have to
be taken out at regular intervals separately from the base collections.
15.17 Monitoring Genetic Stability of Cryopreserved Germplasm
Plant cryopreservation is a complex, multistage process which requires optimization

of various steps involved right from the selection of quality donor material,
preculture process, cryoprotection technique to be used, thawing, recovery medium,
regrowth etc. Successful cryopreservation is the overall result of all these processes
to achieve a higher recovery along with genetic stability. Even though cryopreser-
vation has proved to be the most amenable way for the long-term conservation of
recalcitrant species, there are an increasing number of difficulties encountered in the
process to achieve better regrowth rate (Normah et al. 2012). The field of
cryobionomics deals with cryoinjury, genetic stability and regrowth studies of
cryopreserved tissues. Cryobionomics is an interdisciplinary subject covering the
morphological, biochemical, histological, cytological as well as molecular study of
an organism to identify any type of cryoinjury or loss of metabolic or reproductive
functions in them because of cryopreservation. It also studies the change in gene
expressions causing disruption of normal regulatory mechanisms, growth and devel-
opmental sequences (Harding 2004). Cryobionomics uses high-throughput omics
technologies such as genomics, transcriptomics, proteomics, metabolomics and
other omics platforms to study the potential impacts of cryoinjury on the genome,
transcriptome, proteome and metabolome of a species.
The development of advanced biomolecular or ‘omics’ technologies has enabled
a better understanding of the fundamental biological process underlying cryopreser-
vation system (Morrison et al. 2006). It can readily be applied to the same sample,
enabling a detailed investigation into biomolecular changes that accompany the
cryopreservation of plant materials (Martinez-Montero and Harding 2015). Thus,
cryobionomics provides a conceptual framework to examine the significance of cell
signalling mechanisms on cellular functions, the impacts of cryoinjury and

cryogenic/non-cryogenic stress factors on germplasm viability and the implications
for genetic stability following cryostorage.
Cryopreservation exposes the stored material to a variety of stresses because of
which the genetic stability of the conserved material will be hampered. Such stresses
may be due to the exposure of tissues to different chemicals and storage conditions.
It has been studied that in most of the cryopreserved tissues, very little or no change
is seen after short-term cryostorage, i.e., within 1-month duration (Wang et al. 2017).
But many workers like Castillo et al. (2010), Maki et al. (2015) and Pence et al.
(2017) showed that some genetic changes occur after long duration of storage. These
types of changes are found to be epigenetic. Genetic changes occur more frequently
due to DNA methylation rather than change in DNA sequences. DNA methylation-
induced changes are reversible also (Johnston et al. 2009; Peredo et al. 2008; Kaity
et al. 2013).
Most common type of variation, i.e., observed during the regrowth in in vitro
condition is somaclonal variation. These kinds of variations lead to the loss of true to
type character of stored germplasm and fail to implement the whole purpose of
cryopreservation. Different types of injuries or changes that an explant material
undergoes during different steps of cryopreservation have been depicted in Fig. 15.5.
Thus, it is important to ensure the genetic stability of the cryopreserved material.
Assessment of genetic integrity of conserved material is done by comparing it to the
same material before conservation (Engelmann 2011). Numerous studies on genetic
stability analysis have been already done in many crops and revealed that no
cytological or genetic alterations occur due to cryopreservation. Different techniques
including biochemical, molecular, cytological and morphological methods can be
used to assess the genetic stability of cryopreserved germplasm. Morphological or
phenotypic method is the traditional method of comparing the morphological traits
of conserved and the original plant to see if there is any variation. Any kind of
chromosomal changes during cryopreservation can be detected by cytological
methods and biochemical methods can be used to detect any changes in metabolite
or enzyme profiles. In medicinal plants, it is important to retain the quantity of
bioactive compounds in the cryopreserved samples as it is present in the original
sample. Biochemistry techniques involving HPLC, GC, LC, MS etc. can be used for
analysing the biochemical profile of medicinal plants. Molecular techniques employ
a range of molecular markers such as RAPDs, AFLPs, RFLPs, SSRs, SNPs etc. to
study the variations occurring at the DNA level. These conventional markers will
analyse only a small part of the genome, but with the advancement of plant genomics
and rapid progress in next generation sequencing (NGS) technology, more and more
plant genomes are getting sequenced. The availability of NGS has speeded up the
analysis of plant genomes and can be used to test the genetic stability of
cryopreserved germplasm effectively. Morphological and biochemical variations
should be supplemented with the latest molecular techniques for confirmatory
results. Overall, genetic changes from cryopreservation appear small and relatively
rare, and the value of holding a sample in a cryostorage facility to guard against
Fig. 15.5 The multistage process of cryopreservation signifying the possible events that leads to
cellular damage and genetic instability
extinction likely outweighs any risk of small genetic changes. Few of the genetic
stability studies carried out in some anti-diabetic plants are mentioned below:
Hasan et al. (2015) used AFLP technique to study the genetic stability in shoot
tips of Ziziphora tenuior L., before and after cryopreservation and found that there
were no genetic variations between the two. Agrawal et al. (2014) studied the genetic
stability of Musa plants recovered from cryopreserved and regenerated meristems
using 11 phenotypic (biometric) characters and 21 simple sequence repeat (SSR)
markers. Data on phenotypic traits revealed that cryopreserved plants were statisti-
cally comparable to the mother plants raised from suckers for all important growth
and yield parameters. SSR profiles of plants recovered from cryopreserved material
and control plants had a higher similarity coefficient concluding that Musa
meristems can be effectively cryopreserved for storage and regeneration of true-to-
type plants. Dixit et al. (2003) assessed genetic stability of plants regenerated from
cryopreserved embryogenic tissues of Dioscorea bulbifera using molecular, bio-
chemical and morphological analysis. RAPD markers used for molecular analysis
shows similar profile for both cryopreserved and control plants. Diosgenin content
analysed using HPLC also showed same content in cryopreserved and control plants.
Similarly, morphology and the ability to form microtuber were also found unaltered
in cryopreserved embryo-derived plantlets. Thus, it was concluded that the
D. bulbifera plants regenerated from cryopreserved embryogenic tissues were genet-
ically stable at the molecular, biochemical and morphological levels. Ravindran et al.
(2005) and Yamuna et al. (2007) reported genetic uniformity was observed in
cryopreserved and recovered plants of cardamom, ginger and black pepper based
on RAPD and ISSR profiling. Choudhary et al. (2013) studied genetic stability of
Morus alba in in vitro regenerated plants of mulberry (fresh, before and after
cryopreservation) using RAPD and ISSR markers. Dormant buds of Morus alba
were used for cryopreservation using two-step freezing. In this study, the plants
regenerated directly from dormant buds (before and after cryopreservation) without
intermediary callus phase were analysed. Both markers showed monomorphic
banding patterns and did not reveal any polymorphism among the mother plant
and in vitro regenerants before and after cryopreservation, suggesting that cryopres-
ervation, using two-step freezing, does not affect genetic stability of mulberry
germplasm.
15.18 Case Studies in Anti-Diabetic Plants
15.18.1 Syzigium cumini, Garcinia indica, Emblica officinalis

and Aegle marmelos
Malik et al. (2010) conducted seed storage and cryopreservation studies in

underutilized fruit crops having anti-diabetic property like Syzigium cumini,
Garcinia indica, Emblica officinalis and Aegle marmelos. Seed storage behaviour
studies showed that Garcinia indica and Syzigium cumini seeds were of highly
recalcitrant nature, whereas Aegle marmelos showed intermediate and Emblica
officinalis showed orthodox seed storage behaviour. Seeds, embryos as well as
embryonic axes were tested for viability as well as cryopreservation. In vitro culture
of embryos showed better germination than the seeds. This study showed that
A. marmelos and E. officinalis have orthodox seed storage behaviour as they were
viable even after desiccating to 5–7% moisture content whereas Garcinia indica and
Syzygium cumini lost the viability by 10–12% on desiccation showing intermediate
seed storage behaviour. Seeds of A. marmelos suffered from freezing sensitivity
after exposure to LN. Cryopreservation was attempted successfully in all these
species.
15.18.2 Aloe vera
Anti-diabetic activity of Aloe vera has been mentioned by Sharma et al. (2014).
Droplet-vitrification method has been developed by Josette et al. (2019) for
cryopreserving the clonal accession of Aloe vera. Apical shoot tips obtained from
in vitro grown plants was used as the explant. They were treated with loading
solution for 20 min followed by PVS2 treatment for different duration of time and
then transferred to aluminium foil strips for immersing in LN. Thawing was done
and PVS2 solution was replaced by unloading solution for 20 min followed by
culturing in regeneration media. Preculture in sucrose media prior to freezing also
improved the recovery growth.
15.18.3 Allium sativum
Anti-diabetic activity of Allium sativum has been mentioned by Grover et al. (2002).
Cryopreservation of Allium sativum (garlic) has been standardized using vitrification
and droplet-vitrification method (Singh et al. 2018). Droplet vitrification proved to
be more successful in terms of regeneration. In vitrification method, garlic cloves
were isolated from dormant bulbs and meristem tip was extracted. Extracted meri-
stem tips were surface sterilized and implanted in preculture medium (containing
0.3 M sucrose in MS basal medium) followed by treatment with PVS2 solution for
40 min. Meristems with PVS2 solution was plunged into LN for 1 h. Later, the
cryovials containing PVS2 solution is removed from LN and treated with unloading
solution for 20 min. For regrowth, the explant was removed from the unloading
solution and transferred to regrowth medium.
In droplet-vitrification method, after treatment with preculture media, explants are
transferred to aluminium foil strips containing PVS2 solution, waited for 40 min and
then plunged into the LN for rapid freezing. Remaining procedure was same as that
of vitrification.
15.18.4 Neem
Chaudhury and Chandel (1991) showed that seeds of neem show typical characters
of recalcitrant seeds like short life and high moisture content (about 46%) at the time
of shedding. But desiccating seeds to low moisture content of 4% and storing in LN
showed that the seeds are tolerant to desiccation and freezing showing their orthodox
storage behaviour. And possessing endocarp which played a significant role in
germination and desiccation responses, showed orthodox nature in terms of desicca-
tion and freezing sensitivity. Seeds showed desiccation tolerance up to 4% and
freezing tolerance at 196 C for seeds desiccated between 18.5% and 4% moisture
level. Thus, it was found that cryopreservation at the ultra-low temperatures of LN
(196 C) can be utilized for long-term conservation of neem germplasm.
15.18.5 Stevia
Shatnawi et al. (2011) used vitrification method for in vitro propagation and
cryostorage of shoot tips of Stevia rebaudiana. Shoot tips were used as the explant
for cryopreservation. Several pretreatment and preculture trials were conducted for
maximizing the recovery after cryostorage. Maximum recovery of 68% was

obtained by preculturing the plantlets in MS medium with 30 g/L sucrose and
preculture of shoot tips in 0.4 M sorbitol followed by 80–100% PVS2 treatment
and immersion in LN. Acclimatization and regrowth of plants showed that growth
parameters were not affected by the cryopreservation process even after 6 months.
Thus, it was concluded that the protocol is applicable for long-term storage of
S. rebaudiana germplasm in LN.
15.18.6 Musa spp.
Kaya et al. (2020) developed an efficient cryopreservation protocol for the long-term
conservation of economically important Musa species. Cryopreservation of seeds
was done using air desiccation method in which seeds were dehydrated in a sterile
laminar flow cabinet for different exposure times and then they were directly
immersed in LN. The critical point was to support the initial germination of
cryopreserved seeds and this was achieved by the excision of zygotic embryos
after LN treatment that allowed the seed germination. The best moisture content
for tolerance to cryopreservation ranged from 15.8% (M. acuminata spp. zebrina) to
17.1% (M. ornata) and the maximum post-cryopreservation germination rates varied
from 86.4% (M. velutina) to 55.0% (M. ornata).
Apart from the above well-studied species, Table 15.4 lists the cryopreservation
protocol standardized by different workers in many other anti-diabetic plants.
15.19 Conclusion
Long-term conservation of difficult to conserve species using cryopreservation has

rapidly been evolving since the past two decades. Since most of the medicinal plants
comes under the category of difficult to conserve species (clonally propagated and
recalcitrant seed species), cryopreservation offers the most reliable conservation
method for these species. Cryopreservation protocols are already in place for many
medicinal and anti-diabetic species employing different methods ranging from
simple desiccation, encapsulation and vitrification to most modern methods of
droplet freezing and cryoplate method. Development of protocols specific to species
needs to be developed in terms of selection of explant, size of germplasm to be
stored, freezing and rewarming methods, recovery media and recovery method etc.
Stability of the cryopreserved material in terms of its genetic and biochemical
content is most important in the case of medicinal or anti-diabetic plants. Morpho-
logical, biochemical, cytological and molecular methods are routinely employed for
the genetic stability studies to ensure the true-to-type nature of conserved material
and so far, no alterations have been observed after cryopreservation. Thus, cryopres-
ervation has evolved as the most promising method for the viable long-term conser-
vation of potential medicinal crops with intact genetic and biochemical stability.
Table 15.4 Cryopreservation of plants with anti-diabetic property

Anti-diabetic
Cryopreservation property
Species Explant method Reference Reference
Allium sativum Shoot tips Vitrification Niwata (1995) Ozougwu (2011)
Allium cepa Shoot tips Droplet Wang et al. Mootoosamy
var. vitrification (2020) and
aggregatum Mahomoodally
(2014)
Beta vulgaris Shoot tips Vitrification Vandenbussche Murthy and
et al. (2000) Manchali (2013)
Citrus sinensis Embryogenic Cryoplate method Souza et al. Shakthi Deve
calli (2017) et al. (2014)
Ipomea batatas Shoot tips Encapsulation Hirai and Sakai Akhtar et al.
vitrification (2003) (2018)
Picorhiza Shoot tips Vitrification Sharma and Joy and Kuttan
kurroa Sharma (2003) (1999)
Cicer arietinum Seeds Vitrification Bajaj (1995) Wei et al. (2017)
Arachis Seeds Vitrification Bajaj (1995) Akter et al.
hypogea (2014)
Asparagus Embryogenic Vitrification Nishizawa et al. Hafizur et al.
officinalis suspension (1993) (2012)
cells
Morrus sp. Dormant buds Droplet El-Homosany Hasani-Ranjbar
and shoot tips vitrification and Farag et al. (2008)
(2020)
Poncirus Seeds PVS2 vitrification Wang et al. Jia et al. (2016)
trifoliata (2002)
Dioscorea Shoot tips Vitrification Mukherjee et al. Ghosh et al.
bulbifera and (2009) (2012) and Wang
Dioscorea alata et al. (2011)
Carica papaya Shoot tips and PVS2 vitrification Azimi et al. Juárez-Rojop
seeds (2005) et al. (2012)
Acacia nilotica Seeds Desiccation, Jebelli et al. Saha et al. (2018)
vitrification (2015)
Rauwolfia In vitro PVS2 vitrification Ray and Azmi and
sepentina grown nodal Bhattacharya Qureshi (2016)
segment (2008)
Catharanthus Suspension DMSO + sorbitol Tony et al. Muralidharan
roseus cultured cell treatment (1984) and (2014)
lines Fatima et al.
Embryogenic (2009)
cell culture
Juglans regia Pollen Desiccation Luza and Polito Hosseini et al.
(1988) (2014)
Ficus carica Shoot tips Vitrification El-homosany Deepa et al.
and Sayed (2018)
(2020)
Psidium Pollen Desiccation Vishwakarma Oh et al. (2005)
guajava et al. (2020)
(continued)

Anti-diabetic
Cryopreservation property
Species Explant method Reference Reference
Momordica Pollen Desiccation Rajasekharan Sharma et al.
doica and et al. (2010) (2018) and Singh
M. sahyadrica et al. (2011)
15.20 Future Prospects
Several base collections using cryopreservation have already been established in

many national and international crop-based research institutes. Yet, their application
is still limited to few species. This is mainly because of the lack of infrastructure and
lack of expertise in developing the protocols. The success or recovery rate of
cryopreserved samples can still be improved by manipulating various parameters
such as the maturity stage of explant, duration and rate of desiccation, rate of
freezing and the recovery media. More and more protocols need to be developed
for more number of crops especially the wild medicinal species which are difficult to
conserve. Basic studies pertaining to biochemical and metabolic activities of the cell,
their response to dehydration and freezing stress should be carried out so as to
develop the cryopreservation protocols effectively. As per the previous reports,
success rate of cryopreservation is more in intermediate species as compared to
recalcitrant species. Much improvement is expected by exploring different technical
approaches to improve the efficiency and increase the applicability of cryopreserva-
tion techniques to recalcitrant species. To conclude, the development of cryopreser-
vation protocol should come out from the framework of academic purpose and
should become a routine programme of conservation projects so that it can be
implemented for all the species.
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Biotechnology of Anti-Diabetic Medicinal Plants: Saikat Gantait Sandeep Kumar Verma Amit Baran Sharangi Editors

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Saikat Gantait

Sandeep Kumar Verma

Amit Baran Sharangi

ISBN 978-981-16-3528-1 ISBN 978-981-16-3529-8 (eBook)

Medicinal plants contain active phyto-pharmaceuticals aiding to cure many

uses biological systems, living organisms or derivatives thereof, to make or modify

Kerala Agricultural University K. V. Peter

Biotechnology is technology based on biology. It harnesses cellular and biomolecu-

1 In Vitro Approach and Quantiﬁcation of “Puerarin and

9 Saffron: A Prized Herb with Therapeutic Potential Against

About the Editors

Saikat Gantait Dr. Gantait is working as an Assistant

Sandeep Kumar Verma Dr. Verma is working as an

Amit Baran Sharangi Dr. Sharangi is a Professor of

worldwide as editor-in-chief, regional and technical edi-

Bilal Haider Abbasi Department of Biotechnology, Quaid-i-Azam University,

Samantha Drouet Laboratoire de Biologie des Ligneux et des Grandes Cultures,

Muhittin Kulak Department of Herbal and Animal Production, Vocational School

Illa C. Patel , Masuma Hakim , Riddhi Prajapati, Anita Solanki,

I. C. Patel · M. Hakim · R. Prajapati · A. Solanki

# The Author(s), under exclusive license to Springer Nature Singapore Pte 1

1.1.1 Plant Description

1.1.2 Medicinal Properties of P. tuberosa

hypolipidemic (Tanwar et al. 2008), antimicrobial (Venkata Ratnam and Venkata

1.2 Micropropagation of P. tuberosa

Micropropagation protocol offers through tissue culture technique presently help a

1.2.1 Plant Material and Seed Germination

1.2.1.1 Seed Germination

P. tuberosa was done in MS medium supplemented with BAP + IAA

1.2.1.2 Callus Development

1.2.2 Shoot Formation

PGR Leaf callus Tuber callus Cotyledonary callus

1.2.3 Roots and Tuber Formation

frequency of root formation of 64.6 2.7% and 66.6 2.8%, respectively

1.3 Evaluation of Genetic Fidelity/DNA Isolation and ISSR-PCR

1.4 High-Performance Thin-Layered Chromatography

1.4.1 Preparation of Extracts

1.4.2 High-Performance Thin-Layered Chromatography Method

1.4.2.1 Sample Application

1.4.2.2 Chromatogram Development and Scanning

1.4.2.3 Standard Preparation for Quantification

1.4.2.4 Method Validation

speciﬁcity were found to be speciﬁc. Besides this, the reproducibility curves

1.4.2.5 Quantification of GEN and PRN

synthesized pathway during the organogenesis processes. Here in P. tuberosa as

production in P. tuberosa makes it possible for any biotechnological transformation

Subrata Kundu , Sk Moquammel Haque , and Biswajit Ghosh

Diabetes mellitus, a complex metabolic disorder with multiple etiology, is one of

S. Kundu · B. Ghosh (*)

# The Author(s), under exclusive license to Springer Nature Singapore Pte 25

upgraded recombinant technology, explored exciting possibilities for elicitation

Antidiabetic compounds · Callus culture · Cell suspension culture · Elicitor ·

Diabetes is a severe, metabolic disorder characterized by elevated amount of blood

2.1.1 Diabetes Mellitus: A Metabolic Disorder

Diabetes mellitus (DM) is a metabolic ailment consequential from insulin resistance

by inadequate insulin synthesis (Butalia et al. 2016; Krzewska and Ben-Skowronek

2.1.1.1 Treatment of Diabetes in Conventional Medicine

generation antidiabetic drugs mostly act through inducing production of insulin,

in muscles, liver, and pancreas that ultimately resulted in cardiovascular diseases

2.2 Secondary Metabolites of Medicinal Plants: A Potential

(polymeric isoprene derivatives), phenolics (comprising of hydroxylated aromatic

Fig. 2.2 Organ-speciﬁc effectiveness of antidiabetic medicinal plants

genetically susceptible persons. The summary of herbal along with non-herbal-based