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Scientific Cultivation of Ashwagandha (Withania somnifera)

Conference Paper · December 2014

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December 11, 2014

Message
It is a matter of happiness and satisfaction that the research scholars of CSIR-CIMAP are
organizing a two day symposium “JIGYASA”, which will provide them an opportunity of not
only sharing their experience on medicinal and aromatic plants, and brighten their
communication skills, it will also enable them to learn the basics as well as details of organizing
a scientific event. I trust that this event will promote interdisciplinary, better understanding and
togetherness among them and the scientists, which will help in scaling greater heights of
excellence and in fulfilling the mandate of CSIR-CIMAP with acceleration. I also sincerely hope
that this kind of event will become an annual feature in which all the research scholars of CSIR-
CIMAP will participate with full enthusiasm. I also take this opportunity to express my
thankfulness to the scientists and other support staff who have guided and facilitated the research
scholars in making this event a success.

My best wishes for the success of the symposium and for the bright future of the researchers of
CSIR-CIMAP. 

(Prof. Anil K. Tripathi)

Director, CSIR-CIMAP
Lucknow

 
 Preface
Healing with medicinal plants is as old as mankind itself, there use in healthcare dates back
practically to the existence of human civilization. Increased awareness and interest towards
medicinal plants usage is a result of impressive successes in botanical medicines and growing
incidences of side effects of conventional medicines. Although contemporary science has
acknowledged their active action, but very few drugs of plant origin have been included in
modern pharmacotherapy. With rapid increase in the incidence of cancer, malaria and stress
related health disorders, the demand of medicinal plants like Catharanthus, Withania and
Artemisia has increased manifold, attracting the attention of medicinal plants researchers’ world
over.

Catharanthus roseus (Sadabahar) is a plant belonging to dogbane family, Apocynaceae. Like

genus Vinca, they are known commonly as periwinkles. The name Catharanthus comes from the
Greek for "pure flower". The species has long been cultivated for herbal medicine and as an
ornamental plant. In Ayurveda (Indian traditional medicine) the extracts of its roots and shoots,
though poisonous, are used against several diseases. In traditional Chinese medicine, extracts
from it have been used against numerous diseases, including diabetes, malaria, and Hodgkin's
lymphoma. Vinblastine and vincristine extracted from the leaves are used in the treatment of
leukemia and Hodgkin's lymphoma while ajmalicine from roots is antihypertensive.

Withania somnifera (Ashwagandha) is an important medicinal plant of Indian origin. The

therapeutic use of this plant is reported in immunomodulation, hematopoiesis, anti-aging, chronic
stress, cardiovascular protection, hypothyroidism, anxiety and depression. The secondary
metabolites from the roots of this plant is not only a major source of several alkaloids viz.
tropine, pseudotropine & somniferine but also important steroids like withaferin A and
withanolides are useful for human being as pharmaceutical agents in curing various diseases.

Artemisia is a large, diverse genus of plants with more than 200 species belonging to the daisy
family Asteraceae. The name "artemisia" ultimately derives from the Greek goddess Artemis
(Roman Diana), the namesake of Greek Queens Artemisia I and II.Most species have strong
aroma and bitter taste derived from terpenoids and sesquiterpene lactones, which discourage
herbivory, and may have had a selective advantage. Of these, Artemisia annua is an important
medicinal plant, a source of artemisinin, widely used for the treatment of chloroquine resistant
and cerebral malaria.

It has been realized research awareness about these three important medicinal plants is of great
importance. So, a two day symposium JIGYASA-2014, is organized by research scholars of
CSIR-Central Institute of Medicinal and Aromatic Plants (CIMAP), Lucknow. Aiming to kindle
inquisitiveness (Jigyasa) and share the scientific advancements made in these plants viz.
Withania somnifera (Ashwagandha), Catharanthus roseus (Sadabahar), Artemisia annua
(Qinghao) and bring out a compendium, which would be useful for researchers. This symposium
will be beneficial in promoting awareness, capacity and interdisciplinary networking among the
students and scientists in the area of medicinal plants.

We are indebted to all speakers for their valuable time and effort to familiarize the participants
with basic research aspects of these three medicinal plants. We are very thankful to Prof.
A.K.Tripathi, Director, CSIR-CIMAP, Lucknow for his kind support and encouragements.
Administrative support is also acknowledged. We are also thankful to funding agency Council of
Scientific and Industrial Research, CSIR for their financial support.

We are thankful to HRD, CSIR-CIMAP for continuous support for organizing the symposium.
All the members of organizing committee deserve appreciation for successfully organizing the
whole symposium. We are also thankful to all volunteers and various committee members, who
did lot of hard work during preparation of symposium.

Editors
 Date of Symposium
13th and 14th December 2014.

Venue
CSIR-Central Institute of Medicinal and Aromatic Plants
Kukrail Picnic Spot Road, P.O. CIMAP, Lucknow – 226015

Core Organizing Team

Dr. Shubhra Rastogi Dr. Anupam Maurya
Ms. N. Manika Ms. Seema Dharni
Ms. Deepamala Maji Mr. Sougata Sarkar

Reviewing Team
Dr. Soni gupta
Dr. Rajeev Singh
Dr. Shalini Dixit
Ms. Deepti Yadav
Mr. Ashutosh Awasthi
Ms. Deepti
Mr. Surjeet Verma
Dr. Kripal Singh
Dr. Priyanka Verma
Mr. Avinash Rai
Ms. N. Manika
Ms. Sanchita
Mr. Lokesh Narnoliya
Mr. Sajendra Verma

Editing Team
Shubhandra Tripathi Akhil Kumar
Varun Dwivedi Garima Singh
Swati Srivastava Noopur S. Rajpoot
Kaneez Fatima Muktesh Chandra
Tanya Ray Shivangi Mishra
Nisha Raj

Chairman of Organizing Team

Prof. Anil K. Tripathi, Director
CSIR-CIMAP, Lucknow

Organizational Support
Dr. M.P. Darokar & Er. Manoj Semwal
HRD-Cell
Dr. Alok Kalra & Dr. Laiq-ur-Rahman
TABLE OF CONTENTS

S.No. THEMES Author Pg. No.

Biotechnology and Bioinformatics
1 Agrobacterium rhizogenes mediated transformation studies in Neha Verma 1
Catharanthus roseus – A status update
2 Agrobacterium tumefaciens mediated transformation studies in Abhishek 7
Catharanthus roseus: Sharma
Current status and future perspectives
3 Catharanthus: Exploration of Phytoremediation Potential Nidaf khan 12
4 Cell culture mediated biotransformations using Artemisia and Divya 14
Catharanthus Agnihotri
5 Emerging Roles of Protein Prenylation Mediated Signaling Avanish Rai 17
Networks in Regulation of
Plant Secondary Metabolism
6 Engineering metabolic pathways in microorganisms to produce Shivangi Mishra 21
higher levels of
semi-synthetic anti-malarial drug, artemisinin
7 Exploring the use of Catharanthus roseus in Alzheimer ’s Akhil Kumar 24
disease
8 Genus Artemisia: Available avenues to explore beyond Ritesh Kumar 27
“Artemisinin”
9 Hairy root mediated biotechnological intervention in Artemisia Sailendra 31
species for the
production of artemisinin- A mini review
10 In vitro production of Artemisinin acid and their derivatives Anand Mishra 38
11 Metabolic engineering approaches for enhanced artemisinin Sunita Jindal 41
production
12 Molecular marker study in Artemisia annua for high yielding Susheel Kumar 45
artemisinin content Singh
13 OMICS approaches for Withania somnifera: A need for Sanchita 47
exploration
14 Stumbling blocks and their prospective solutions in research on Maneesha Mall 51
Catharanthus roseus
15 Trans-acting regulators of gene expression in Catharanthus Pravin Prakash 57
roseus
16 Transcriptome Analysis of Indian ginseng Withania somnifera Swati Upadhyay 61
(Ashwgandha)
17 Vinca alkaloids: tubulin binding and cancer treatment Shubhandra 63
Tripathi
Plant Chemistry
18 A review of the extraction technologies for artemisinin ex Karishma 66
Artemisia annua Agarwal
19 A wonder plant Withania: Pharmacological and Chemical Surjeet Verma 72
 review
20 Anti-cancer properties of artemisinin and its derivatives Rashmi Gaur 81
21 Artemisinin and its derivatives as potent antimalarial agents Deepak Singh 87
Kapkoti
22 Semi-synthetic approach of withaferin A: Related steroidal Imran Ahmad 92
lactones as a potent anti proliferative agents
23 Substantiation of withanolides as anticancer agents Pallavi Joshi 98
24 Terpenoid Diversity in Artemisia annua VS Pragadheesh 104
25 Vinblastine, Vincristine and their derivatives as anti-cancer Yashveer 109
agents
26 Withania coagulans pharmacological and phytochemical Shalini Dixit 118
investigation
Biochemistry and Plant physiology
28 Dynamics of Secondary Metabolite Biosynthesis in Withania Jyoti Singh 120
somnifera Jadaun
29 Hairy Root Induction via Agrobacterium rhizogenes in Shilpi Bansal 123
Withania somnifera
30 Micropropagation of Withania somnifera Dunal Shiwani Maurya 126
31 Mining of BAHD superfamily alcohol acyl transferases from Lokesh K. 128
Artemisia annua trichome transcriptome Narnoliya
32 Modelling and Characterization of Tropine forming Tropinone Muktesh 131
reductase gene in Withania Chandra
33 ROS Homeostasis of Withania somnifera against Cadmium Bhawana 134
Stress Mishra
34 Withania somnifera Reverses Alzheimer’s Disease by Archana 136
Degrading-Amyloid β Peptides in Brain
35 Withanolides: The Steroidal Lactones Biosynthesis Yashdeep 141
Srivastava
Plant Systematics, Conservation and Prospection
36 A tale of three medicinal plants - Catharanthus roseus L., Amit Kumar 144
Artemesia annua L. and
Withania somnifera (L.) Dunal
37 Anti-mitotic Property of Natural Dimers from Catharanthus Vijaya Dubey 147
roseus
38 Biological activities of artemisinin and its derivatives Himanshu 152
Tripathi
39 Elucidating Anticancer Activity of Withaferin A, Major Lubna Siddiqui 158
Constituent of
Withania somnifera Through Suppression of NF-кB, a
Prominent Marker in Cancer
40 Role of Artemisinin and related compounds in cancer Ravinder Naik 163
prevention and treatment Dharavath
41 Role of Withania somnifera (L.) in management of Alzheimer Dhananjay 167
disease Kumar Singh
42 The Time Travel of Artemisia: An Ethno-Botanical Review Tanya Biswas 169
43 Therapeutic Potential of Artemisia against Malaria Archana Saxena 172
44 Toxicological safety trends of Withania somnifera Pone Kamdem 175
Boniface
Crop Production and Improvement
45 Artemisia annua, artemisinin and stresses Pragya Trivedi 179
46 Genetic Improvement for high yielding varieties in Rashmi Lahiri 182
Ashwagandha
47 Development of in-situ moisture conservation practices for Nilofer 185
enhancing productivity of
the crops under moisture stress conditions: A potential R & D
area for Ashwagandha
(Withania somnifera L. Dunal)
Optimization of plant population in Ashwagandha (Withania Rupal 188
somnifera L. Dunal) under rainfed conditions
48 Scientific Cultivation of Ashwagandha (Withania somnifera) Prawal 191
49 Unravelling the Periwinkle genetics and breeding efforts in last Pranshu 193
five decades
Microbial Technology and Crop Protection
50 A plethora of endophytic microorganisms interacting with Sucheta Singh 196
Catharanthus roseus
51 Viruses: Invaders of a high-valued alkaloid crop (Catharanthus Asifa Khan 200
roseus)
52 Current scenario of fungal diseases in Withania somnifera and Arvind Saroj 204
its sustainable management
53 Endophytes and PGPRs in artemisinin research: status and Vikas Kumar 207
potential Patel
54 Microbes mediated stress tolerance and plant growth promotion Tania Ray 215
in Withania somnifera
BIOTECHNOLOGY

AND

BIOINFORMATICS
Agrobacterium rhizogenes mediated transformation studies in Catharanthus
roseus – A status update

NehaVerma, Abhishek Sharma, PriyankaVerma and AK Mathur*

Plant Tissue Laboratory, Department of Plant Biotechnology, CSIR-Central Institute of
Medicinal and Aromatic Plants (CSIR-CIMAP), PO CIMAP, Lucknow-226015 (India)
Email: nehhaa_1987@yahoo.co.in

ABSTRACT

Catharanthus roseus (L).G. Don is a well-known important medicinal plant belonging to

family Apocyanaceae. The herb is commercially valued for harbouring >130 bioactive
terpenoid indole alkaloids (TIAs) including the anti-neoplastic (mitotic spindle disrupting)
drugs, vincristine and vinblastine, albeit at a very low productivity level. Genetic engineering
of TIAs pathway genes in cultured cells and tissues to provide alternate production platforms
with enhanced production of C. roseus phytoceuticals is therefore a widely pursued activity
today. Agrobacterium-mediated genetic transformation studies for up-regulating the
metabolic flux towards TIAs synthesis is one such preferred strategy in this pathway
modulation effort. A literature search on the subject suggests that C. roseus has so far been
proved rather non-tractable plant species from genetic engineering angle through
A.tumefaciens-mediated transformation. On the other hand, A.rhizogenes-mediated
transformations for hairy root production are a routine here. Regeneration of transgenic plants
from genetically engineered cells/tissues, however, remains a serious constraint in both cases.

A. rhizogenes is a natural plant genetic engineer. It is a gram negative soil bacterium that
induces hairy roots syndrome in majority of dicotyledonous plant species. Hairy roots are
generated by integration of Ri- plasmid of Agrobacterium rhizogenes into the plant host
genome.The conditions of transformation (nature and age of the explants, bacterial strain,
bacterial density, and the protocol of infection) regulate transformation efficiency as well as
the growth and productivity of the hairy roots.Since individual hairy root clone emerging
from different sites of infection generally represents an independent transformation event,
they mostly depict a diverse morphology, in vitro growth patterns and metabolites
productivity profiles. This necessitates that individual transformed clone be thoroughly
screened for desired productivity traits in vitro. Nevertheless, higher level of cellular
differentiation, cellular organization, genetic stability, up-scaling opportunity, hormone-
autotrophy and amenability towards biotic elicitation are some of the speciality features of
these transformed roots that qualify them to be better candidates for metabolite production
than undifferentiated cell cultures. More than 150 research papers on hairy root induction in
C. roseus have been published during last 20 years with varied level of success. In general,
the technology has been found more useful for the production of monomeric TIAs like
catharanthine, serpentine and ajmalicine. Synthesis of vindoline and vinblastine in induced
transformed hairy roots of C. roseus has been sporadic and rare. In the present review an
effort has been made to summarize an over view of hairy root technology development in C.
roseus with an attempt to identify the R&D gaps that are required to be focused and filled.

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INTRODUCTION

The Catharanthus roseus (Madagascar periwinkle) is a perennial tropical/sub-tropical plant

and is a rich source of pharmaceutically valuable secondary metabolite such as catharanthine,
ajmalicine, serpentine, vindoline, vincristine and vinblastine etc. (Zargar et al.,2010; Gayatri
et al.,2013; Koul et al.,2013;Heijden et al.,2004; Dutta et al.,2005).The more than 40
enzymatic steps associated with TIAs biosynthetic pathway are known today and about 12 of
the genes associated with this metabolic route are cloned. Involvement of at least four
different cell types (epidermis, internal phloem-associated parenchyma, laticifers and
idioblast) and five intracellular compartments (chloroplast, vacuole, nucleus, endoplasmic
reticulum and cytosol) has been implicated with this biogenetic route (Heijden et al.,2004;
Luca et al.,2008; Guirimand et al., 2010; Verma et al., 2012 a).However the yields of these
TIAs are low in the intact plants and their chiral complexity defies their chemical synthesis,
leading to their very high market costs. Metabolic engineering efforts to boost the TIAs
productivity levels either in plant or in cultured cells and tissue of C. roseus, therefore, is a
priority. A major line of Catharanthus research has been to identify the genetic and
biochemical organization and regulation of TIAs pathway, identification of limiting steps at
the level of constituent enzymes and genes and, then to strategize how to overcome these
limitations through modern tools of genetic engineering (Tripathi et al.,2003). Agrobacterium
rhizogenes-mediated hairy roots of C. roseus are in the centre of such investigations (Rizvi et
al., 2014). The special features of hairy root associated with these R&D endeavours includes
higher level of genetic stability and cellular differentiation, fast biomass generation in vitro,
possibility of easier up-scaling in bioreactors and hormone auxotrophy (Nader et al.,2006;
Pistelli et al.,2010). The technology has yielded very satisfactory outcomes in many other
medicinal plant species like Hypericum perforatum, Gentiana macrophylla, Papaver
somniferum, Bacopa monnieri, Panax ginseng,Glycyrrhiza glabra,Centella asiatica,
Withania somniferum,Tylphora indica,Saussurea involucrate etc.

Plant infection with Agrobacterium rhizogenes induces the formation of proliferative multi-
branched adventitious roots at the site of infection; the so-called‘hairy roots’. Hairy root
disease is induced by incorporation of root loci genes of root inducing (Ri) plasmid into the
host plant genome. The right and left T-DNA regions (TR-DNA and TL -DNA) of the Ri
plasmid, are delimited by their border sequences and are the regions that are transferred to the
plant.Within the TR section, loci involved in auxin biosynthesis are transferred to the plant
genome, thus increasing the auxin autotrophy to the transformed cells (Gartland.,1995).
Evidence suggests that rol genes mediate signal transduction pathways by acting
independently on phytolexin production and diverting flux towards alkaloid production.
A.rhizogenes strains have been categorised on the basis of the type of opines they
producesuch as octopine, mannopine, agropine, nopaline and cucumopine types.Agropine
strains (A4, 15834,1855, LBA 9402) induce agropine, mannopine and agropinic acid in the
host cell while the mannopine strains (8196) and the cucumopine (Petit et al., 1983) strains
induce the production of one single opine. Agropine strains transfer independently both the
TL -DNA and TR –DNA to the plant genome, while mannopine strains only transfer the TL –
DNA. Ri region of plasmid contains the four rolgenes A, B, C and D (Schmülling et al.,
1988; Petersen et al., 1989) which enhance the auxin and cytokininsynthesis (Estruch et al.,
1991) of plant cells and are responsible for the formation of roots by transformed tissues
(Bonhomme et al., 2000a ; Hong et al., 2006 ). Following Agrobacterium infection,rolA, rolB
rol C genes are transferred into plant genome and act as potential activators of secondary
metabolism in transformed cells. Hairy roots obtained from different bacterial strain infection
vary in their transforming abilities. The potential of hairy roots of C.roseus has been

2|P ag e
investigated in many studies with respect to the production of root specific indole alkaloids
(Toivonen et al., 1989; Verpoorte et al., 1991; Jung et al., 1992; Shanks et al., 1997). Hairy
root cultures produce secondary metabolites for longer duration while maintaining genetic
and biosynthetic stability. In hairy roots proliferating cell resides at apical meristem followed
by laterals forming elongation zone.This defined growth pattern leads to steady accumulation
of biomass in root cultures (Kumar et al., 2006; Giri et al., 2000). Hairy roots are able to
synthesize phytochemicals but desired compounds are not readily released into the medium
causing difficulty in their extraction during down-stream processing. Such efforts with hairy
root clones of C. roseus are few and scanty. The major phyto-molecules so far detected in
various hairy root lines of C. roseus in different studies include: serpentine, ajmalicine,
catharanthine, vindoline, tabersonine, lochnericine, horhammericine and tryptamine.

Elicitation studies and metabolic engineering of hairy roots culture of Catharanthus

roseus
Valuable secondary metabolites synthesized by plant for defence against pathogen and the
corresponding biosynthetic pathway are known to be induced by elicitors. Several elicitors
have been tested to increase the secretion of secondary metabolites in C. roseus hairy roots.
Methyl jasmonate (MeJA), an organic compound used in plant defence caused large increase
in the levels of transcript of TIAs pathway genes and the catharanthine concentration while
sodium nitroprusside alone or in combination with MeJA caused dramatic decrease of
catharanthine concentration and increase of transcripts of type-1 protein prenyltransferase
gene in C.roseus hairy roots (Zhou et al., 2010; May et al., 2011). Efforts were also made to
express genes coding enzymes of the TIA pathway with goal to circumvent a negative
feedback regulatory loop in the biosynthesis of tryptamine, the indole precursor of
TIAs.Tryptamine is synthesized from tryptophan by enzymatic reaction catalyzed by
tryptophan decarboxylase(TDC). The expression of Arabidopsis thaliana gene encoding a
feedback resistant anthranilate synthase(AS) α-subunit simultaneously with the gene
encoding AS β-subunit and TDC gene in the C. roseus hairy rootsimproved the metabolic
flux of the indole precursors tryptamine towards anti-hypertensive drug ajmalicine with a
corresponding decrease in lochnericine, horhammericine and tabersonine (Hughes et al.,2004
b; Hong et al.,2006; Peebles et al.,2005).In order to facilitate the expression of vindoline
pathway and hence possible synthesis of vincristine and vinblastine in hairy root system,
Sander (2009) attempted over-expression of tabersonine 16-hydroxylase (T16H) under the
control of gluco-corticoid inducible promoter and detected higher level of lochnericine in the
transformed roots. Deacetylvindoline-4-O-acetyltransferase (DAT) which is the last enzyme
in the vindoline biosynthetic pathway that converts de-acetylvindoline into vindoline, was
also over-expressed with cauliflower mosaic virus (CaMV) 35 S promoter in C.roseus hairy
roots (Magnotta et al., 2007). These workers also found no increment in the vindoline level in
the transformed roots but instead, observed 4-folds increase in horhammericine to control
lines.Enzymatic analysis revealed that the DAT protein inhibits activity of minovincinine-19-
O-acetyltransferase (MAT) that in turn favoured horhammericine accumulation. A methyl
jasmonate, gibberellic acid and ethylene-inducibleWRKY transcription factor, CrWRKY1
was also expressed in root and found to up-regulate key pathway gene like tryptophan
decarboxylase(TDC) as well as transcriptional repressors ZCT1, ZCT2 & ZCT3 for zinc
finger. Up-regulation of TDC increased TDC activity and tryptamine concentration compared
with control roots.CrWRKY1 hairy roots accumulated upto 3-fold higher level of serpentine
(Suttipanta et al.,2011).Similarity, hairy root lines were constructed with inducible expression
of DXS (1-deoxy-D-xylulose synthase) or geraniol-10-Hydroxylose (G10H) wherein DXS
over-expression resulted in significant increase in ajmalicine by 67%, serpentine by 29% and
lochnericine by 49% while co- over repressor of DXS and G10 H caused increase in

3|P ag e
ajmalicine by 16% and lochnericine by 31%.The over-expression of G10H or both G10H and
ORCA3 genes in C. roseus hairy roots increased catharanthine content by 6.5-fold when
compared with the control lines (Wang et al.,2010). Similarly the AP2/ERF domain
transcription factor ORCA2overexpression increased catharanthine and vindoline by 2.03 and
3.67 folds in comparison the control line (Liu et al. 2011). Over-expression and suppression
of a peroxidase gene CrPrx in C.roseus transgenic hairy root lines detected higher levels of
ajmalicine and serpentine accumulation in over-expressed lines (Jaggi et al., 2011).Recently,
(Rizvi et al.,2014) have reported an effective protocol for generation of transgenic C. roseus
hairy roots with estrogen-inducible GFP expression .The estrogen-inducible system provides
advantages as an additional inducible system for manipulating and understanding the
regulation of TIA biosynthesis. According to these workers the estrogen-inducible system
does not elicit ORCA and ZCT expression involved in the defense response of C. roseus.
Recent report (Verma et al., 2014) showed the better stability of rol gene than the GUS gene
in A. rhizogenes and A.tumefaciens mediated genetic transformations in C. roseus.

Plant regeneration studies from hairy roots in Catharanthus roseus

There are just two reports on regeneration of transgenic plants from transformed hairy roots
of C. roseus (Choi et al., 2004; Verma et al., 2012). The regeneration response was found to
be highly genotype-specific and varied with different root clones. The regenerated shoots
showed prolific rooting with extensive lateral branching and shortened inter-nodes in the
transgenic plants. PCR and Southern blotting analysis confirmed the retention of Ri-TL-DNA
in these regenerants. Verma et al., (2012 b) also found excessive in-vitro flowering in the
regenerated transgenic plants.

CONCLUSION

Hairy roots are at the centre stage of pharmaceutical technologies. Ability of A.rhizogenes to
transfer foreign genes into host genome helps to strategize metabolic pathway to produce
important drug molecules with economy space, lesser cost, better productivity and
sustainability of production system. Hairy roots because of their higher level of cellular
differentiation can not only carry out multi-step complex biogenetic pathways but are also
more robust to perform better post-translational protein modification and
sequestration/storage of synthesised molecules then single plant cells or microbial systems.
More concerted efforts towards industrial up-scaling of technology at bioreactor level are
required in this direction.

REFERENCES

Bhadra R, Shanks JV (1997) Transient studies of nutrient uptake, growth, and indole alkaloid
accumulation in heterotrophic cultures of hairy roots of Catharanthus roseus. Biotechnology
and Bioengineering.55: 527–534.
Bonhomme V, Laurain –Mattar D, Fliniaux MA (2000 a) Effects of the rol C gene on hairy root:
induction development and tropane alkaloid production by Atropa belladonna. Journal of
Natural Products.62: 1249-1252.
Choi PS, Kim YD, Choi KM, Chung HJ, Choi DW (2004) Plant regeneration from hairy-root cultures
transformed by infection with Agrobacterium rhizogenes in Catharanthus roseus. Plant Cell
Reports.22: 828–831.
Dutta A, Batra J , Rai SP, Singh D, Kumar S, Sen J(2005) Expression of terpenoid indole alkaloid
biosynthetic pathway genes corresponds to accumulation of related alkaloids in
Catharanthus roseus (L.) G. Don.Planta.220: 376- 383.

4|P ag e
Estruch JJ, Chriqui D, Grossmann K, Schell J, Spena A (1991) The plant oncogene rolc is responsible
for the release of cytokinins from glucoside conjugates. EMBO Journal.10: 2889- 2895.
Facchini PJ, De Luca V (2008) Opium poppy and Madagascar periwinkle: Model non-model systems
to investigate alkaloid biosynthesis in plants. Plant Journal.54: 763–784.
Gartland JS (1995) Agrobacterium virulence.In Gartland KMA, Davey MR, eds, Methods in
Molecular Biology 44 Agrobacterium protocols Humana press,Totowa New Jersey. 15-28.
Gayatri L, Chakravarthy R (2013) Micro Propagation in Catharanthus roseus. International Journal of
Innovative Technology and Exploring Engineering.2: 2278-3075.
Giri A, Narasu LM (2000) Transgenic hairy roots: recent trends and applications. Biotechnology
Advances.18: 1–22.
Guirimand G, Burlat V, Oudin A, Lanoue A, St-Pierre B, Courdevault V (2009) Optimization of the
transient transformation of Catharanthus roseus cells by particle bombardment and its
application to the sub-cellular localization of hydroxymethylbutenyl 4-diphosphate synthase
and geraniol 10-hydroxylase. Plant Cell Reports.28:1215–1234.
Heijden RV, Jacobs DI, Snoeijer W, Hallard D and Verpoorte R (2004) The Catharanthus Alkaloids:
Pharmacognosy and Biotechnology. Current Medicinal Chemistry.11: 607-628.
Hong SB, Peebles CA, Shanks JV, San KY, Gibson SI (2006) Terpenoid indole alkaloid production
by Catharanthus roseus hairy roots induced by Agrobacterium tumefaciens harboring rol ABC
genes. Biotechnology and Bioengineering.93: 386–390.
Hughes EH, Hong SB, Gibson SI, Shanks JV, San KY(2004b) Metabolic engineering of the indole
pathway in Catharanthus roseus hairy roots and increased accumulation of tryptamine and
serpentine. Metabolic Engineering.6: 268–276.
Hughes, EH, Hong SB, Gibson SI, Shanks JV, San KY (2004a) Expression of a feedback-resistant
anthranilate synthase in Catharanthus roseus hairy roots provides evidence for tight regulation
of terpenoidindole alkaloid levels. Biotechnology and Bioengineering. 86: 718–727.
Jaggi M, Kumar S, Sinha AK (2011) Over-expression of an apoplastic peroxidase gene CrPrx in
transgenic hairy root lines of Catharanthus roseus. Applied Microbiology and Biotechnology.
90:1005-16.
Jung KH, Kwak SS, Kim SW, Lee H, Liu JR (1992) Improvement of the catharanthine productivity in
hairy root cultures of Catharanthus roseus by using monosaccharides as a carbon source
.Biotechnology Letters.14: 695-700.
Koul M, Lakra NS , Chandra R and Chandra S (2013) Catharanthus roseus and prospects of its
endophytes: a new avenue for production of bioactive metabolites. International Journal of
Pharmaceutical Sciences and Research.4: 2705-2716.
Kumar V, Sharma A, Prasad NCB, Gururaj BH, Ravishankar AG (2006) Agrobacterium rhizogenes
mediated genetic transformation resulting in hairy root transformation is enhanced by
ultrasonication and acetosyringone treatment. Electronic Journal of Biotechnology.9: 349-357.
Liu H, Ren WW, Cui LJ, Zhang LD, Sun XF and Tang KX (2011) Enhanced accumulation of
catharanthine and vindoline in Catharanthus roseus hairy roots by over-expression of
transcriptional factor ORCA2. African Journal of Biotechnology.10: 3260-3268.
Magnotta M, Murata J, Chen J, De Luca V (2007) Expression of deacetylvindoline-4-O-
acetyltransferase in Catharanthus roseus hairy roots. Phytochemistry. 68:1922–1931.
May ER, Pena CDL, Avalos RMG, Watson BS , Sumner LW, Vargas VML (2011) Methyl
Jasmonate Induces ATP Biosynthesis Deficiency and Accumulation of Proteins Related to
Secondary Metabolism in Catharanthus roseus (L.) G. Hairy Roots.Plant and cell
physiology.52: 1401- 1421.
Nader BL, Taketa AT, Pereda-Miranda R, Villarreal ML (2006) Production of triterpenoids in liquid –
cultivated hairy roots of Galphima glauca. Planta Medica Journal.72:842-844.
Peebles CAM, Hong SB, Gibson SI,Shanks JV, and San KY (2005) Transient effects of Over-
expressing Anthranilate Synthase α and β Subunits in Catharanthus roseus Hairy
Roots.Biotechnology Progress.21:1572-1578.
Petersen SG, Stummann BM, Olesen P, Henningen KW (1989) Structure and function of root
inducing (Ri) plasmids and their relation to tumor inducing (Ti) plasmids. Physiologia
Plantarum.77:427-435.

5|P ag e
Petit A, David C, Dahl GA, Ellis JG, Guyon P, Casse- Delbart F, Tempe J (1983) Further extension of
the opine concept: plasmids in Agrobacterium rhizogenes cooperate for opine degradation.
Molecular Genetics and Genomics.190:204-214.
Pistelli L , Giovannini A, Ruffoni B ,Bertoli A ,Pistelli L (2010) Hairy Root Cultures for Secondary
Metabolites Production.Advances in Experimental Medicine and Biology.698: 167-184.
Rizvi NF, Cornejo M , Stein K , Weaver J , Cram EJ , Lee-Parsons CWT (2014) An efficient
transformation method for estrogen-inducible transgene expression in Catharanthus roseus
hairy roots. Plant Cell Tissue Organ Culture. DOI 10.1007/s11240-014-0614-1.
Ruiz-May E, De-la-Peña C, Galaz-Ávalos RM, Lei Z, Watson BS, Sumner LW, Loyola-Vargas VM
(2011) Methyl Jasmonate Induces ATP Biosynthesis Defeciency and Accumulation of proteins
related to secondary metabolism in Catharanthus roseus (L.) G. Hairy roots.Plant Cell
Physiology.52:1401-1421.
Sander G (2009) Quantitative analysis of metabolic pathways in Catharanthus roseus hairy roots
metabolically engineered for terpenoid indole alkaloid overproduction. Dissertation. Iowa State
University, Ames, Iowa.
Schmulling T, Schell J, Spena A (1988) Single genes from Agrobacterium rhizogenes influence plant
development.EMBO Journal. 7: 2621-2629.
Suttipanta N, Pattanaik S, Kulshrestha M, Patra B, Singh SK, Yuan L (2011) The transcription factor
CrWRKY1 positively regulates the terpenoid indole alkaloid biosynthesis in Catharanthus
roseus.Plant physiology 157: 2081-2093.
Toivonen L, Balsevich J, Kurz WGW (1989) Indole alkaloid production by hairy root cultures of
Catharanthus roseus. Plant Cell Tissue Organ Culture.18: 79–93.
Tripathi L and Tripathi JN (2003) Role of biotechnology in medicinal plants.Tropical Journal of
Pharmaceutical Research.2: 243-253.
Verma P, Mathur AK, Shanker K (2012 b) Growth, alkaloid production, rol genes integration,
bioreactor Up-scaling and plant regeneration studies in hairy root lines of Catharanthus roseus.
Plant Biosystems. 14: 27-40.
Verma P, Mathur AK, Srivastava A, Mathur A (2012 a) Emerging trends in research on spatial and
temporal organization of terpenoid indole alkaloid pathway in Catharanthus roseus: a literature
update. Protoplasma.249: 255-268.
Verma P, Sharma A, Khan SA, Mathur AM, Shanker A (2014) Morphogenetic and chemical stability
of long-term maintained Agrobacterium-mediated transgenic Catharanthus roseus
plants.Natural Product Research. dx.doi.org/10.1080/14786419.2014.940348.
Verpoorte R, van der Heijden R, van Gulik WM, ten Hoopen HJG (1991) Plant biotechnology for the
production of alkaloids: present status and prospects. In: Brossi A (ed) The alkaloids, 40.
Academic, San Diego 1–187.
Wang CT, Liu H, Gao XS, Zhang HX (2010) Over-expression of G10H and ORCA3 in the hairy
roots of Catharanthus roseus improves catharanthine production. Plant Cell Reports.29: 887-
894.
Zargar M, Farahani F, Nabavi T (2010) Hairy roots production of transgenic Catharanthus roseus L.
plants with Agrobacterium rhizogenes under in vitro conditions.Journal of Medicinal Plants
Research 4: 2199-2203.
Zhao L, Sander GW, Shanks JV (2013) Perspectives of the metabolic engineering of terpenoid indole
alkaloids in Catharanthus roseus hairy roots. Advances in Biochemical Engineering/
Biotechnology.134:23–54.
Zhou ML, Zhu XM, Shao JR, Wu, YM, Yi Xiang Tang- Appe (2010) Transcriptional response of the
catharanthine biosynthesis pathway in methyl jasmonate/nitric oxide elicitation in
Catharanthus roseus hairy root cultures .Applied Microbiology and Biotechnology.88:737-750.

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 Agrobacterium tumefaciens mediated transformation studies in
Catharanthus roseus – Current status and future perspectives

Abhishek Sharma, Neha Verma, Priyanka Verma and AK Mathur*

Plant Tissue Culture Laboratory, Department of Plant Biotechnology; CSIR-Central Institute
of Medicinal & Aromatic Plants (CSIR-CIMAP, PO CIMAP; Lucknow-226015 (India)
Email: abhi19ind@gmail.com

ABSTRACT

Agrobacterium tumefaciens (a gram negative-rod shaped-soil bacterium) - mediated genetic

transformation of plants is a popular and very useful strategy for foreign gene insertion into a
desired recipient host plant for transgene expression for higher productivity and other useful
agronomic traits. A. tumefaciens naturally infects the wound site of the dicotyledonous plants
and transfer its specific DNA segment (T-DNA) of a Tumor-Inducing (Ti) Plasmid into the
host genome to impart crown gall syndrome and therefore, rightly regarded as a Natural
Genetic Engineer. T-DNA contains two types of genes: a) Oncogenes- for auxins and
cytokinins synthesis for tumor development and; b) Opine Synthesis Genes- for the formation
of carbon and nitrogen containing compounds (opines) synthesized through condensation of
amino acid and sugar for meeting the nutrient requirement of the bacteria for its growth. For
plant genetic engineering work this T-DNA region is modified and loaded with foreign genes
of interest using molecular biology techniques, for their insertion, stable integration and
expression in the resultant transgenic plant progeny.

In this review presentation, an effort has been made to provide an over-view of A.

tumefaciens-mediated transformation studies so far carried out in the anti-neoplastic
medicinal herb – Catharanthus roseus, popularly known as Sadabahar or Madagaskar
Periwinkle. It is pertinent to recall here that C. roseus is known to produce > 150 types of
bioactive terpenoid indole alkaloids, many of which are already in clinical use. Extremely
low occurrence of these compounds in C. roseus plants make their extraction difficult and
costly and hence are sold in the drug market at an exorbitant price. Nearly 40 laboratories of
the world, including CSIR-CIMAP, are actively engaged in Catharanthus research with a
sole objective of increasing the content of TIAs metabolites through classical and modern
tools of plant biology including the transgenic approach. Landmark developments in
Catharanthus engineering with A. tumefaciens are discussed.

INTRODUCTION

C. roseus (Family: Apocynaceae; 2n=16) is a dicotyledonous medicinal herb of the genus

Catharanthus. There are eight known species of this genus out of which, seven
are endemic to Madagascar but are widely naturalized around the world today. The eighth
species, C. pusillus, is native to India and Sri Lanka. The name Catharanthus comes from
the Greek for "pure flower". C roseus is commercially valued for harboring a variety of
terpenoid indole alkaloids (TIAs), including the two high-value anti-neoplastic dimeric
molecules - vinblastine and vincristine in the aerial shoot tissues and, two antihypertensive
drugs - ajmalicine and serpentine in the roots. The more than 40 enzymatic steps associated
with TIAs biosynthetic pathway are known today and about 12 of the genes associated with
this metabolic route are cloned. The pathway has been shown to be strictly regulated by
environmental factors, pest and disease infestations and, most importantly by degree of tissue

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differentiation at the whole plant level. Involvement of at least four different cell types
(epidermis, internal phloem-associated parenchyma, laticifers and idioblast) and five
intracellular compartments (chloroplast, vacuole, nucleus, endoplasmic reticulum and
cytosol) has been implicated with this biogenetic route (Heijden et al. 2004; Facchini and De
Luca 2008; Guirimand et al. 2010; Verma et al. 2012). Search for alternate in vitro
production platforms of valuable TIAs or designing of speciality C. roseus plant types with
enhanced flux towards targeted phytomolecules through genetic modifications, is therefore, a
rigorously followed activity amongst Catharanthus community today (Zarate and Verpoorte
2007; Zhao and Verpoorte 2007; Verma et al. 2011, 2012; Hoopen et al. 1994). Current
experimental strategies in the direction of TIAs pathway engineering are mainly revolving
around the generation of cell and tissue types characterized by sufficient precursor
availability from terpene and indole pools, over-expression of key limiting pathway enzymes,
activation of regulatory transcription factors, silencing of competitive pathways to flux the
intermediates in desired direction and optimization of growth parameters for bioreactor based
cultivation (Canel et al. 1998; Zhao et al. 2001; Hughes et al. 2004; Seth and Mathur 2005;
Zarate and Verpoorte 2007; Zhao and Verpoorte 2007; Murata et al. 2008; Fits and Memelink
2000). Unfortunately majority of such efforts have so far been made at the level of cell
suspension and transformed hairy root cultures that do not represent cell and tissue
organization that is required for the execution of entire TIAs pathway. Regeneration of
complete plants from transgenic cells or tissues is a rarity and hence is a major bottleneck
problem to be resolved in realizing the full gains of a pathway modulation program.

C. roseus is largely considered as a genetically non-tractable plant species from a genetic

engineering point of view due to the non-availability of robust and repeatable regeneration
protocol for transgenic plant production (Fits et al., 2001; Di Flore et al., 2004). This
recalcitrancy is more pronounced in efforts made through A. tumefaciens-mediated
transformations than with A. rhizogenes.

First report on A. tumefaciens based transformations in C. roseus appeared in early 1980s

(Palni et al. 1983; 1984). These workers induced crown gall tissue of C. roseus to study
cytokinin metabolism in cultured gall cells. Soon after, a few reports on A. tumefaciens
mediated transformed callus and cell suspension appeared in literature wherein elevated
levels of ajmalicine and serpentine were observed in the transformed cultures (Tremouillaux-
Guiller et al. 1994; Garnier et al. 1996a, b; Godoy-Hernandz and Loyola-Vergas et al. 1997).
O’keefe et al. (1997) found stable vindoline production in two transformed lines of shooty
teratomas. They also found higher expression and activity of acetyl CoA DAT enzyme in the
cell free extracts of these cell lines. Directed metabolic engineering efforts using transgenic
approaches in C. roseus were initiated in late 1990s by Whitmer et al. (1998) who isolated a
transgenic cell line to study the influence of tryptamine and loganin availability on TIAs
synthesis and inferred that tryptamine utilization via its coupling with secologenin is a more
limiting step rather than its availability. Godoy-Hernandz & Loyola-Vergas et al. (1997) also
showed the positive influence of acetyl-salicylic acid feeding alkaloid synthesis in a C. roseus
tumor cell suspension culture transformed with Agrobacterium tumefaciens PC2500 strain.
The most explicit experimentation on TIAs engineering using A. tumefaciens mediated
transformation was carried out by Canal et al. in 1998. Using TIAs pathway specific gene
constructs of TDC (tryptophan decarboxylase) and/or STR (strictosidine synthase), these
workers found that 10 fold STR over-expression in the transformed cell suspensions resulted
in higher accumulation of strictosidine, ajmalicine, catharanthine, serpentine and tabersonine.

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Interestingly, the over-expression of TDC did not result into high levels of alkaloid
accumulation, and instead, showed a detrimental effect on cell growth. Employing the same
transgenic cell lines (S10), Whitmer et al. (2002a,b; 2003) also found that the transformed
line displayed a high tryptamine level in comparison to non-transformed cultures.
Strictosidine and catharanthine accumulation in the TDC over-expressing cells occurred
when fed with loganine and secologanin. These workers further confirmed that formation of
strictosidine was not only dependent on high TDC expression but also on effective coupling
of tryptamine with secologanin. These transformed cell lines showed variable degree of long-
term stability. In some of the lines the STR and TDC activities fall to levels similar to that of
the wild type cultures. Even the transformed lines wherein the STR and TDC activity levels
remained well above the wild type levels did not maintained their high TIAs production
levels in successive generations. Hilliou et al. (1999) developed a very efficient
transformation system for C. roseus callus using the particle bombardment technique. The
bombardment was carried out on leaf derived callus and 70-80% of cells in callus showed the
transgene expression. The transformation was carried out using three plasmids WRG1515
carrying hph and gusA genes, WRG 2426 carrying bar, hph and gusA genes and WRG 1610
carrying npt2 gene. The method developed by these workers demonstrated for the first time
that C. roseus cells could be transformed using single or two plasmid systems (Co-
transformation). Nine out of 10 analysed transgenic lines showed the insertion of both the
plasmids following a re-transformation strategy. This study has opened a fresh avenue for
engineering a complex multienzyme pathway (like that of TIAs in C. roseus) by possible
insertion of more than one limiting gene. Begum (2009) developed a procedure for
establishing hormone autotrophic shooty teratomas to study the production of dimeric
alkaloids using Agrobacterium tumefaciens strain C58. Dimeric alkaloid vincristine in the
transformed cultures as present at a concentration of 0.011 that was ten folds higher
compared to untransformed control cultures.

Development of complete A. tumefaciens-mediated transformation protocol to recover

complete transgenic plants in C roseus was published for the first time from CSIR-CIMAP in
2011 (Verma et al. 2011a,b). Agrobacterium tumefaciens (strain-LBA4404) harboring a
binary vector pBI121 (with GUS and ntp II genes) with p35SGUS-INT (having GUS Introns)
was used. Employing a direct regeneration method of adventives shoot bud formation from
leaf explant, transgenic plants with GUS expression were successfully retrieved by these
workers. The stability of marker genes inserted into transgenic plants was also deeply studied
for a period of 5 year and it was observed that the stability of GUS gene was gradually
reduced and eliminated when compared to rol genes (Verma et al. 2014). CIMAP laboratory
is presently using these protocols to over-express TIAs pathway genes (STR, TDC, G10H
etc) at whole plant levels (unpublished). Recently, Wang et al. (2012) has also reported
another efficient regeneration and transformation system for C roseus using A tumefaciens
and hypocotyls explants. EHA105 (having pCAMBIA2301) harboring a reporter GUS and a
selectable marker nptII gene was used in this study.

Details pertaining to some of these aforesaid landmark developments in C. roseus transgenics

will be presented to advance the discussion during the presentation.

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REFERENCES:

Begum F, Rao SSN, Rao K, Prameela DY, Giri A, Giri CC (2009) Increased vincristine production
from Agrobacterium tumefaciens C58 induced shooty teratomas of Catharanthus roseus G.
Don. Nat Prod Res., 23(11): 973-81
Canel C, Lopes-Cardoso MI, Whitmer S, Van-Der-Fits L, Pasquali G, Van-Der-Heijden R, Hoge JH,
and Verpoorte R (1998) Effects of over-expression of strictosidine synthase and tryptophan
decarboxylase on alkaloid production by cell cultures of Catharanthus roseus. Planta., 205(3):
414-419.
Facchini PJ, De Luca V (2008) Opium poppy and Madagascar periwinkle: model non-model systems
to investigate alkaloid biosynthesis in plants. Plant J., 54: 763–784
Fits LVD and Memelink J, (1987) Comparison of the activities of CaMV 35S and FMV 34S promoter
derivatives in Catharanthus roseus cells transiently and stably transformed by particle
bombardment. Pl. Mol. Bio., 33(5): 943-946.
Garnier F, Carpin S, Label P, Creche J, Rideau M, Hamdi S (1996) Effect of cytokinin on alkaloid
accumulation in periwinkle callus cultures transformed with a light inducible ipt gene. Pl. Sci.
Limerick., 120: 47–55
Godoy-Hernández G and Loyola-Vargas VM (1997) Effect of acetylsalicylic acid on secondary
metabolism of Catharanthus roseus tumor suspension cultures. Pl. Cel. Rep., 16(5): 287-290.
Guirimand G, Burlat V, Oudin A, Lanoue A, Pierre BS, Courdavault V (2009) Optimization of the
transient transformation of Catharanthus roseus cells by particle bombardment and its
application to the subcellular localization of hydroxymethylbutenyl 4-diphosphate synthase and
geraniol 10-hydroxylase. Pl. Cel. Rep., 28; 1215–1234.
Heijden RVD, Jacobs DI, Snoeijer W, Hallard D, Verpoorte R (2004) The Catharanthus alkaloids:
Pharmacognosy and biotechnology. Cur. Med. Chem., 11(5): 607-628.
Hilliou F, van der Fits L, Memelink J (2001) Molecular regulation of monoterpenoid indole alkaloid
biosynthesis. In: Romeo JT, Saunders JA, Matthews BF (eds) Recent advances in
phytochemistry: regulation of phytochemicals by molecular techniques. Els. Sci. Oxford, 35:
275–295
Hughes EH, Hong SB, Gibson SI, Shanks JV, San KY (2004) Metabolic engineering of the indole
pathway in Catharanthus roseus hairy roots and increased accumulation of tryptamine and
serpentine. Meta. Eng., 6(4): 268–276.
Murata J, Roepke J, Gordon H, De Luca V (2008) The leaf epidermome of Catharanthus roseus
reveals its biochemical specialization. Pl. Cel., 20: 524–542
O’Keef BR, Mahady GB, Gills JJ, Beecher CWW (1997) Stable vindoline production in transformed
cell culture of Catharanthus roseus. J. Nat. Prod. 60: 261–264
Palni LMS (1984) Cytokinin Accumulation in the Culture Medium of Vinca rosea L. Crown-Gall
Tissue: a Time-Course Study. Aus. J. of Pl. Phy., 11(3): 129 – 136.
Palni LMS and Horgan R (1983) Cytokinins in transfer RNA of normal and crown-gall tissue
of Vinca rosea. Planta.,159(2): 178–181.
Pietrosiuk A, Furmanowa M, Lata B (2007) Catharanthus roseus: micropropagation and in vitro
techniques. Phytochem. Rev., 6: 459–473
Seth R, Mathur AK (2005) Selection of 5-methyltryptophan resistant callus lines with improved
metabolic flux towards terpenoid indole alkaloid synthesis in Catharanthus roseus. Curr. Sci.
89: 544–548
Trémouillaux-Guiller J, Kodja H, JC Chénieux (1994) Single-cell cloning of a crown gall from
protoplasts regenerated in hormone-free medium. Establishment of pure transformed cell-lines
of Catharanthus roseus. Pl. Cel. Tis. Org. Cul.., 37(1): 25-30
Verma P and Mathur A K (2011b) Agrobacterium tumefaciens mediated transgenic plant production
via direct shoot bud organogenesis from pre-plasmolyzed leaf explants of Catharanthus roseus.
Biotech. Let., 33: 1053-1060
Verma P and Mathur AK (2011a) Direct shoot bud organogenesis and plant regeneration from leaf
explants in Catharanthus roseus. Pl. Cel. Tis. Org. Cul.,106: 401-408.

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Verma P, Mathur AK, Srivastava A, Mathur A (2012) Emerging Trends in Research on Spatial and
Temporal Organization of Terpenoid Indole Alkaloids Pathway in Catharanthus roseus: A
Literature Up-date. Protoplasma., 249: 255-268
Verma P, Sharma AS, Khan SA, Mathur A, Shankar K (2014) Morphogenetic and chemical stability
of long-term maintained Agrobacterium-mediated transgenic Catharanthus roseus plants. Nat.
Prod. Res., 10.1080/14786419.2014.940348
Wang Q, Xing S, Pan Q, Yuan F, Zhao J, Tian Y, Chen Y, Wang G, Tang K (2012) Development of
efficient Catharanthus roseus regeneration and transformation system using agrobacterium
tumefaciens and hypocotyls as explants. BMC Biotechnology, 12:34.
Whitmer S, Canel C, Hallard D, Gonçalves C, Verpoorte R (1998) Influence of Precursor Availability
on Alkaloid Accumulation by Transgenic Cell Line of Catharanthus roseus. Pl. Physio.,
116(2): 853–857.
Zárate R, Memelink J, Heijden RVD, Verpoorte R (2007) Genetic transformation via particle
bombardment of Catharanthus roseus plants through adventitious organogenesis of buds.
Biotech. Let., 21(11): 997-1002.
Zhao J, Verpoorte R (2007) Manipulating indole alkaloid production by Catharanthus roseus cell
cultures in bioreactors: from biochemical processing to metabolic engineering. Phytochem. Rev.
6:435–457
Zhao J, Zhu W, Hu Q (2001) Enhanced catharanthine production in catharanthus roseus cell cultures
by combined elicitor treatment in shake flasks and bioreactors. Enz. Micro. Tech., 28(7-8): 673-
681.

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 Catharanthus: Exploration of Phytoremediation Potential

Nidaf Khan
CSIR-Central Institute of Medicinal and Aromatic Plants, Lucknow 226015, UP, INDIA
E-mail - nidaf011khan@gmail.com

Extracts of entire dried plant of Catharanthus roseus contains many alkaloids of medicinal
use. This valuable plant produces a spectrum of terpenoid indole alkaloids (TIAs), vinblastine
and vincristine, the anticancer lead molecules in leaves and ajmalicine and serpentine in
roots. Contamination of soil and accumulation of heavy metals in environment is a serious
environmental concern, as the accumulated heavy metal ions can find their way into living
organisms via contamination of ground water or food chain. Medicinal and aromatic plants
appear to be a good choice for phytoremediation since these species are mainly grown for
secondary products (essential oil) thus the food chain with heavy metals is eliminated. In
natural ecosystems, plants act as filters and metabolize substances generated by nature.

Phytoremediation is a promising technology using plants to clean up contaminated soil and

groundwater in situ because of low cost of maintenance and operation with public acceptance
(Schnoor et al., 1995). The fact that plants are able to accumulate metals to high
concentrations in their tissues is well known (Salt et al., 1998). This technology presents
clear benefits over traditional methods, including wide applicability, ecological value and
cost-effectiveness. Whereas organic pollutants can be degraded to less toxic forms by plants,
or even mineralized, most research has focused so far on heavy metals, which are immutable
(reviewed recently by Campos et al., 2007). Phytoremediation of metal-contaminated soil
relies on the ability of plants to accumulate metals at concentrations substantially above those
found in the soil in which they grow. The most common heavy metal contaminants were: Cd,
Cr, Cu, Hg, Pb, and Zn. Their presence in toxic concentrations can result in the formation of
superoxide radicals (O2-), hydrogen peroxide (H2O2), hydroxyl radicals (OH-), etc., can cause
severe oxidative damage to biomolecules like lipids, proteins and nucleic acids. Cr, Cu and
Zn can induce the activity of various antioxidant enzymes and also non-enzymes like
ascorbate and glutathione (Varsha Mudgal et al., 2010).

This review summarises the phytoremediation potential of Catharanthus with particular

emphasis on phytoextraction of soil heavy metal contamination. Catharanthus is a fast
growing plant with high biomass and good metal uptake ability. Studies exposed C. roseus to
different concentrations of heavy metals to observe bioaccumulation efficiency. Recently,
this plant has also been used for the phytoremediation of radionuclides, particularly
radiocesium (137Cs) from low level nuclear waste (LLNW) without causing any negative
effects (Fulekar et al., 2010) .The plant species highly accumulated lead than nickel, show
better tolerance to Zn than Cu based on the bioconcentration factor and potential sources of
valuable bioactive metabolites. (V. Subhashini, A. V. V. S. Swamy., 2013). It can also well
tolerate low amounts of chromium and useful in the reclamation and remediation of
chromium contaminated soil (Ahmad, R and Misra, N., 2014). It was shown to absorb up to
about 38% of the amount of Cr present in primary and secondary sludge amended soil
through roots and accumulate it to about 22% in leaves. Effect of chromium concentration on
the status of antioxidant enzyme peroxidase (POD) and detoxification enzyme glutathione-S-
transferase (GST) from its leaves was also analyzed which showed increased expressions of
POD and GST on native PAGE under stress conditions as compared to control. Total alkaloid
was found decreased in the roots of CdCl2 treated plants. Analyses of leaves of treated plant

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showed 5-10 % accumulation of cadmium, but no accumulation of lead at all. Effect of heavy
metals, Cu and Zn on the plant growth parameters, fresh weight (FW), dry weight (DW) and
water content (WC), alkaloid content and antioxidant enzymes activity, peroxidase (POX),
catalase (CAT) was evaluated. The plant growth (FW and DW) and POX activity increased
in presence of low concentration (50 μM, 100 μM). But in presence of high concentration
(500 μM CuCl2), just opposite trend was observed (Yusuf et al, 2014). CAT activity was
found to decrease in presence of both metals. Root growth was more readily affected by the
heavy metals than seed.

Phytoremediation utilizes physical, chemical, and biological processes to remove, degrade,

transform, or stabilize contaminants within soil and groundwater. Hydraulic control, uptake,
transformation, volatilization and rhizodegradation are important processes used during
phytoremediation. Remediation of heavy metals occurs via phytoextraction,
phytodegradation, rhizofiltration, phytostabilization and phytovolatilization (Ghosh & Singh,
2005). Phytoextraction or phytoaccumulation is the process used by the plants to accumulate
contaminants into the roots and above ground shoots or leaves. Phytotransformation refers to
the uptake of organic contaminants from soil, sediments, or water and subsequently, their
transformation to more stable, less toxic, or less mobile form. Phytostabilization is a
technique in which plants reduce the mobility and migration of contaminants and
contaminated soil. Leachable constituents are adsorbed and bound into the plant structure so
that they form a stable mass of plant from which the contaminants will not reenter the
environment. Phytodegradation is the breakdown of contaminants due to presence of proteins
and enzymes produced by the plants. Based on the bioconcentration factor and translocation
factor the plant species showed higher accumulation of heavy metals and thus can be
explored in a prosperous way to remediate heavy metals contaminated environment.

REFERENCES

Ahmad R. and Misra N, (2014) Evaluation of Phytoremediation Potential of Catharanthus roseus

with Respect to Chromium Contamination. American Journal of Plant Sciences, 5,:2378-2388.
Campos VM, Merino I, Casado R, Pacios LF, Gómez L (2007). Review: Phytoremediation of organic
pollutants. Spanish Journal of Agriculture Research, 2008(6):38-47
Fulekar MH, Singh A, Thorat V, Kaushik CP, Eapen S, (2010) Phytoremediation of 137Cs from low
level nuclear waste using Catharanthus roseus. Indian Journal of Pure & Applied Physics,
48:516-519
Ghosh & Singh (2005). A review on phytoremediation of heavy metals and utilization of its
byproducts. Applied Ecology and Environmental Research, 3(1): 1-18.
Mudgal V, Madaan N, Mudgal A, (2010). Heavy metals in plants: Phytoremediation: Plants used to
remediate heavy metal pollution. Agriculture and Biology Journal of North America, 2010:40-
46
Salt DE et al., (1998). Phytoremediation. Annual Review of Plant Physiology and Plant Molecular
Biology, 49:643–668
Schnoor JL et al., (1995). Phytoremediation of organic and nutrient contaminants. Environmental
Science Technology, 29:A318–A323.
Subhashini V, Swamy AVVS, (2013). Phytoremediation of Pb and Ni contaminated soils using
Catharanthus roseus (L.). Universal Journal Environmental Research and Technology, 3 (4):
465-472
Yusuf K, Yusuf L Mishra N, (2014). Effect of heavy metals (Cu, Zn) on the growth of Catharanthus
roseus. International Journal of Global Science Research,1( 1):87-95.

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 Cell culture mediated biotransformations using Artemisia and
Catharanthus

Divya Agnihotri, Mamta Kumari , Archana Mathur

Plant Biotechnology Division; Central Institute of Medicinal & Aromatic Plants, Council of
Scientific & Industrial Research; PO CIMAP, Lucknow-226015 (India)

INTRODUCTION

Plant tissue culture has provided a conscientious platform for secondary metabolite
engineering and production. Plant cell suspension cultures have enormous biochemical
potential for in vitro production of various secondary metabolites as well as have interesting
ability to modify the chemistry of foreign compounds added externally. Since the 1960s plant
cell culture have been used for the extraction, modification and upscaling of the new/ an array
of metabolites (Mulabagal and Tsay, 2004). In recent years, this has revolutionised
biotechnological approach by exploiting the bioconversion potential of plant enzymes for the
production or value addition of pharmaceuticals (Pras et al., 1995). Biotransformation using
plant system is one of the splendid technique of plant biotechnology in which enzymatic
machinery of cultured plant cells are used for the production of pharmacologically more
active and stable compounds. It is a technique in which cells or enzymes of plant are used for
regio and stereo selective conversion of compound which is otherwise difficult to achieve
chemically. The plant cell cultures show regio and stereo-selective hydroxylation, oxido-
reduction, hydrogenation, glycosylation, methylation, aminoacylation, glucosylation,
hydrogenation and hydrolysis of various organic compounds (Ishihara et al., 2003). Plant
system is a manufactory of different metabolites as different enzymes catalyses different
reactions. The process of biotransformation can be of single step or multi step. Single step
biotransformation is more efficient than multi step as yield decreases with increase in number
of steps. It is superior to the de novo chemical synthesis of compound due to specificity of
plant enzyme system. A simple plant cell culture mediated biotransformation reaction
involves establishment of aspetic cultures, addition of substrate and harvesting with due
optimisation. Different media are used to bring about like biotransformation such as
immobilised cells (Zafar et al.,2012; Jyothishrawanm et al.,2007), hairy root culture (Yan et
al.,2006; Peng et al.,2007),cell suspension culture (Patel et al.,2010 and 2011), cell free
system (Furuya et al.,1982; Dhingra et al.,1999). The biotransformation of various organic
compounds has been investigated as a target in the biotechnological applications of plant cell
culture system (Giri et al., 2001; Shimoda et al, 2008). Cell suspension culture techniques are
preferred for biotransformation studies because they provide higher growth rate and greater
ease of substrate addition (Ardekani et al., 2007).

Catharanthus
Cells of Catharanthus roseus of Apocynaceae family, have been used for the
biotransformation to yield more potent and effective compounds like vinblastine, tabersonine,
hydroquinone, artemisinin, glychyrrhizin, 4-Pyridyl-1-ethanol, 2,4,6-Trinitrotoluene(TNT)
(Giri et al.,2001). It is a rich source of terpenoid indole alkaloids (TIAs), anti-hypertensive
compound like ajmalicine, sedative compound like serpentine, and the anticancer drugs like
vinblastine and vincristine. C. roseus cells from early log to stationary phase of cell
suspension culture were used for the production of Arbutin, a potent suppressor of melanin
synthesis in human skin (Yokoyama et al., 1996). Cell suspension culture of C. roseus has

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also been used for the in vivo formation of vanillin glucoside (Sommer et al., 1997).
Glycosylation of capsaicin and 8- nordihydrocapsaicin has been achieved by cell suspension
culture of C. roseus (Shimoda et al 2007). Biotransformation of monoterpenoids by C. roseus
cell suspension culture has proved the regioselectively of the plant system by its the ability to
hydroxylate regioselectively at 10th position of geraniol and C-4 and C-5 positions of
carvones (Hamada et al., 1997). A novel hydroxylase from C. roseus hydroxylates the 5th
position of 2 hydroxybenzoic acid to give gentisic acid, used for intoxication of aromatic
pollutants (Shimoda et al.; 2004). Another study reported that the cell suspension culture of
C. roseus stereoselectively reduced the carbonyl group of (+) and (-) camphorquinones (Chai
et al., 2001). This biological system has been reported copiously for their regio and
stereoselectivity. C. roseus cell suspension cultures have also been reported to facilitate
conversion of artemisinin into 3α-hydroxydeoxyartemisinin (Ning et al., 2003) and by using
5-methyltryptophan tolerant-N30 callus line of C. roseus (Seth & Mathur.,2005) to
deoxyartemisinin (Patel et al.; 2010). Cell suspension cultures of C. roseus have been shown
to perform biotransformation of various inexpensive molecules into more important and
expensive phytomolecules of interest (Ushiyama et al, 1989; Giri et al. 2001; Yang et al.,
2005; Ardekani et al., 2007) through regio-selective glycosylation, stereoselective oxidation
& hydrolysis, regio and stereoselective hydroxylation and hydrogenation (Ishihara et
al.,2003).

Artemisia
Artemisia (Asteraceae family,) is native to China, is a capacious source of variety of
secondary metabolite like flavonoids, coumarins, triterpenoids, steroids, phenolics, purines,
lipids, aliphatic compounds, monoterpenoids and a potent antimalarial sesquiterpene i.e
artemisinin. For many centuries in China it has been traditionally used for the treatment of
fever and malaria. Artemisinin is the most acive compound of A. annua but due to the low
content and uneconomical chemical synthesis, biotransformation studies have been
attempted. Apart from artemisinin there are some semisynthetic derivatives viz. artemether,
arteether, artesunate, arteannuin B, artemisinic acid,that are used as precursor in many
biotransformation and elicitation studies. Using cell suspension culture of A. annua three
biotransformed products of artemisinic acid have been reported namely artemisinic acid β-D-
glucopyranosyl ester, 9-β-hydroxyartemisinic acid β- D-glucopyranosyl ester and 3- β-
hydroxyartemisinic acid β- D-glucopyranosyl ester (Kawamoto et al., 1997). There is a recent
study on biotransformation of artemisinic acid using cell suspension culture of A.annua and
Cephalotaxus fortune ( Chinese Plum Yew) that resulted in 3-alpha-hydroxyartemisinic acid
as a product ( Hu et al 2010 ).

REFERENCES
Ardekani MRS, Linley PA, Harkiss KJ, Mohagheghzadeh A, Gholami A, Mosaddegh M.
Biotransformation of Monoterpenoides by suspension culture of Lavendula officinalis .
Iranian Journal Pharmaceutical Sciences 3(2007)93-100.
Chai W, Hamada H, Suhara J, Horiuchi A. Biotransformation of (+) and (-) camphorquinones by
plant cultured cells. Phytochemistry 57(2001) 669-673.
Dhingra V, Rajoli C, Narasu ML. Partial purification of proteins involved in the bioconversion of
Arteannuin B to Artemisinin. Bioresource Technology 73(2000) 279-282.
Furuya T, Yoshikawa T, Taira M. Biotransformation of codeinone to codeine by immobilized
cells of papaver somniferum. Phytochemistry 23(1984) 999-1001.

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Giri A, Dhingra V, Giri CC, Singh A, Ward O.P, Narasu MK. Biotransformations using plant
cells, organ cultures and enzyme systems: current trends and future prospects.
Biotechnology Advances 19(2001) 175-199.
Hu YS, Zhu JH, Jiang B, Yu RM. Biotransformation of artemisinic acid by cell suspension
cultures of Cephalotaxus fortunei and Artemisia annua. Journal of Chinese Medicinal
Materials 33(2010) 662-665.
Ishihara K, Hamada H, Hirata T , Nakajima N. Biotransformation using plant cultured cells .
Journal of Molecular Catalysis B: Enzymatic 23(2003) 145–170.
Jyothishwaran G, Seetharam Y N. Biotransformation of cholesterol to diosgenin by freely
suspendedand immobilised cells of Dioscorea bulbifera L. Journal of Asian Natural
Products Research 10(2008) 139-145.
Kawamoto H, Yoshihisa A, Sekine H, Furuya T. Biotransformation of Artemisinic Acid by
cultured cells of Artemisia annua. Phyochemistry 48(1998) 1329-1329.
Mulabagal V and Tsay H.S. Plant Cell Cultures - An Alternative and Efficient Source for the
Production of Biologically Important Secondary Metabolites. International Journal of
Applied Science and Engineering 2(2004) 1: 29-48.
Patel S, Gaur R ,Verma P, S.Bhakuni RS, Mathur A. Biotransformation of Artemisinin using cell
suspension cultures of Catharanthus roseus L. and Lavendula officinalis L. Biotechnology
Letters 32(2010) 1167-1171.
Patel S, Gaur R, Upadhaya M, Mathur A, Mathur AK., Bhakuni RS. Glycyrrhiza glabra L. and
Lavendula officinalis L. suspension cultures based Biotransformation of β-artemether.
Journal of Natural Medicines 65(2011) 646-650.
Peng CX., Gong JS, Zhang XF, Zhang M and Zheng SQ. Production of gastrodin through
biotransformation of p-hydroxybenzyl alcohol using hairy root cultures of Datura tatula L.
African Journal of Biotechnology 7(2008) 1684-5315.
Pras N, Woerdenbag HJ, Uden WV. Bioconversion potential of plant enzymes for the production
of pharmaceuticals. Plant Cell Tissue and Organ Culture 43(1995) 117-121.
Seth R, Mathur AK. Selection of 5-methyltrptophanresistant callus line with improved metallic
flux towards terpenoid indole alkaloid synthesis in C. roseus. Current Science
89(2005)544-548.
Shimoda K , Hamada H, Hamada H. Glycosylation of hesperetin by plant cell cultures.
Phytochemistry 69 (2008) 1135-1140.
Shimoda K, Kubolta N, Sano T, Hirakawa H ,Hirata T. A Novel Hydroxylase from Catharanthus
roseus participating in Hydroxylation of 2-Hydroxybenzoic Acid. Journal of Bioscience
and Bioengineering 98(2004)67-70.
Shimoda K, Kwon S, Utsuki A, Ohiwa S, Katsuragi S,Yamamoto N, Hamada H, Hamada H.
Glycosylation of capsaicin and 8-nordihydrocapsaicin by cultured cells of Catharanthus
roseus. Phytochemistry 68(2007) 1391-1396.
Sommer J, Schroeder C, Stockigt J. In vivo formation of vanillin glucoside .Plant Cell Tissue and
Organ Culture 50(1997) 119-123.
Ushiyama M, Kumagai S, Furuya T. Biotransformation of phenylcarboxylic acids by plant cell
cultures. Phytochemistry 28(1989) 3335-3339.
Yang CY, Yu RM., Zhang Z and Kong LY. Biotransformation of Hydroxybezen Derivatives by
Hairy Root Cultures of Polygonum multiflorum Thunb. Journal of Integrative Plant Biology
49(2007)207-212.
Yokoyama M, Inomata S, Yanagi M, Wachi Y. Change of Maximal Cellular Productivity of
Arbutin by Biotransformation Depending on the Culture stage of Catharanthus roseus
cells. Plant Tissue Culture Letters 13(1996) 285-290.
Zafar RU, Kausar A. Biotransformation of limonene by freely suspended and immobilised cells
of Nigella sativa. International Journal of Pharmacy and Pharmaceutical Sciences 5(2013)
0975-1491.

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 Emerging Roles of Protein Prenylation Mediated Signaling Networks in
Regulation of Plant Secondary Metabolism

Avanish Rai and Dinesh A. Nagegowda*

Biotechnology Division, CSIR-Central Institute of Medicinal and Aromatic Plants, Lucknow,
India
Email: rai.avanish002@gmail.com

ABSTRACT

Protein geranylgeranylation is an important post-translational modification in eukaryotic cells

that mediates protein membrane and, in many cases, protein–protein interactions. It is carried
out by gernylgeranyl transferase, which catalyzes the covalent, thioether linkage of a C 20
(geranylgeranyl) group from geranylgeranyl diphsophate (GGPP) to a C-terminal “Cys”
residue of specific proteins. In plants, geranylgeranylation occurs in the cytosol: however, the
origin of GGPP is still not clear whether it is derived from plastids or the cytosol. The
biosynthesis of natural products is under the control of complex regulatory mechanisms.
Because of this complexity, increased production of pharmacologically important natural
products by metabolic engineering has met with little success. It has been shown that
inhibition of geranylgeranylation leads to downregulation of pathway genes and reduced
accumulation of terpenoid natural products in Catharanthus roseus and Nicotiana tabacum.
Prenylated (particularly geranylgeranylated) proteins in which prenyl moieties derived from
MVA/MEP pathways seems to be involved in this regulation possibly by modulation of
GTPases (G/ROP/Rac proteins), which could act as part of regulatory mechanism
coordinating exchange of metabolites between compartmentalized metabolic pathways.

Keywords Catharanthus roseus, geranylgeranylation, geranylgeranyl diphosphate, GTPases

INTRODUCTION

Plants produce vast array of natural products also called “secondary metabolites” as means of
self-defense against herbivores and pathogens, and as ecological mediators. Among them,
isoprenoids (or terpenoids) constitute the largest and most diverse class of secondary
metabolites. Several of these terpene compounds produced in plants as means of defense
possess important therapeutic and commercial values. For instance, the sesquiterpene
compound artemisinin produced by Artemisia annua has antimalarial properties; similarly
monoterpene indole alkaloids vinblastine and vincristine from C. roseus, and diterpene
compound taxaol from Taxus brewifolia, are known anticancer agents used in medicine. Due
to their extremely low accumulation in plants, production of these compounds is very
expensive. Moreover synthetic or semisynthetic routes to produce these compounds exceed
the cost of natural sources. Therefore, better understanding of the regulation of biosynthetic
pathways could facilitate metabolic engineering efforts to increase the production of
important natural products in the host plant or in the heterologous system. In addition to
providing backbone to various terpene compounds, FPP and GGPP also serve as precursors
for protein prenylation, a post-translational modification of certain proteins carried out by
prenyltransferases. Prenylation is a kind of protein lipidation and is essential for translocation
of inactive small GTPase from cytosol to membrane (Crowell 2000). The protein prenylation
reactions are catalyzed by geranylgeranyltransferase type 1 (GGTase-I) and
farnesyltransferase (FTase). Protein prenylation can be regulated by controlling the
expression of GGTase-I and FTase gene which has not been studied much. Similar to
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Arabidopsis, C. roseus possess several GGPPS enzymes and based on the prediction program
all identified GGPPS except GGPPS2 have a putative localization signal which directs their
translocation into plastids, ER and mitochondria (Rai et al., 2013). In plants, GGPP, the
substrate for GGTase-I is proposed to be either transported from plastids to cytosol, or could
be made in the cytosol itself. geranylgeranylated proteins are involved in abscisic acid
signaling, meristem development, plant stress responses, and regulation of cell cycle and also
directs the subcellular localization of signaling proteins, such as ROP/RAC small G-proteins,
transcription factors, cell cycle regulators for proper functioning (Sorek et al., 2009;
Hemmerlin 2013). Plant Rac/ROP small GTPases constitute subfamily of Rho family of
small GTPases which are highly conserved in plants (Kawasaki et al., 2005).

Geranylgeranylation dependent regulation of plant secondary metabolism

In few studies, prenyltransferase inhibitors like S-perillyl alcohol, GGTI-2133 and S-Carvone
have been used to prevent geranylgeranylation affecting the secondary metabolite production
(Courdavault et al., 2009; Huchelmann et al., 2014). Recently, involvement of a MEP
pathway-derived geranylgeranylated protein in the regulation of cellulase-induced capsidiol
biosynthesis from Nicotiana tabacum has been suggested (Huchelmann et al., 2014).
Moreover, it has been reported that the regulation Octadecanoid-responsive Catharanthus
AP2-domain ORCA3 and geraniol 8-hydroxylase (G8H) gene expression depends on protein
geranylgeranylation in C. roseus (Courdavault et al., 2005) and understanding the molecular
mechanism is a key aim to unravel the signaling pathways mediated through
geranylgeranylation. Based on previous study, plastid membrane possesses a unidirectional
proton symport system in which GGPP has not been shown to be transported (Bick and
Lange, 2003). For this reason, the possible existence of cytosolic form of GGPPS in C.
roseus could provide GGPP as a substrate for protein geranylgeranylation. We have
hypothesized that the presence of cytosolic GGPPS may affect the availability of GGPP pool,
which would be utilized as substrate in protein geranylgeranylation occurring in cytosol
thereby influencing the amount of geranylgeranylated proteins. Although it has been shown
that blocking GGTase-I activity affects MIA biosynthesis in C. roseus (Courdavault et al.,
2005), it is not known whether altering pool of isoprenoid intermediates in planta would
exert certain effect on plant secondary metabolism through protein prenylation. In light of
these observation, silencing of cytosolic GGPPS by virus induced gene silencing (VIGS)
decreases the production of cytosolic GGPP pool which hampers transfer of GGPP by the
GGTase-I on to the cysteine residue of C-terminal CAAX motif of putative small GTPase
proteins crucial for signaling cascade (Rai et al., unpublished results). This resulting loss of
geranylgeranylation could leads to the inactivation of small GTPase proteins as normal
functioning of these proteins involves their correct subcellular localization leading to the
abolishment of certain signaling events which downregulate expression of transcription
factors like ORCA3, MYC2 and CrWRKY1 (Rai et al., unpublished results). In another
study, specific inhibition of geranylgeranylation by inhibitors resulted in the inhibition of
capsidiol (a sesquiterpene involved in defense) production in tobacco (Huchelmann et al.,
2014). Geranylgeranylation dependent regulation of secondary metabolite formation might be
present in other plants, for instance, the production of the sesquiterpenoid artemisinin in
Artemisia annua was shown to be MVA derived (Schramek et al., 2010), but its biosynthesis
is inhibited by treatments with mevinolin or fosmidomycin (Towler and Weathers, 2007). In
a similar manner, the biosynthesis of the diterpene taxadiene in Taxus species is dependent
upon the biosynthesis of MEP (Eisenreich et al., 1996), but compactin (an analog of
mevinolin) and fosmidomycin both blocks the accumulation of this product (Soliman et al.,
2011. Furthermore, it is also suggested that disrupting protein geranylgeranylation leads to
the accumulation of unfolded/misfolded small GTPase proteins and induce ER stress which

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in turn activates unfolded protein response (UPR). The ER stress induces and repress
different set of UPR genes in which induced genes are chaperones, vesicle transport protein
and ER-associated degradation proteins and downregulated genes functions as extracellular
proteins like ER-located HMGR enzyme involved in sterol biosynthesis (Martinez and
Chrispeels, 2003). It is possible that unfolded protein response is strongly activated in
response to impairment of geranylgeranylation due to blocking of GGPP biosynthesis which
would lead to the downregulation of MIA biosynthesis-related genes and thus MIAs content
during UPR and this phenomenon needs to be further investigated in C. roseus.

Plant Rac/Rop GTPases

Rac/Rop GTPases are signaling molecules which are shown to be associated with lipid rafts
in plasma membrane containing signal perception assembly for diverse stimuli which
includes elicitors like hormones, pathogens and stress (Nibau et al., 2006). Rac/ROP GTPases
acts as the integrator or coordinator of diverse signals which regulates plant growth and
defense. Cellulase induced capsidiol biosynthesis in pepper has shown to be regulated by
Rop/Rac small GTPases (Ma, 2008) which actively coordinates diverse stimuli including
stress responses (Moeder et al., 2005; Nibau et al., 2006). Moreover, Oryza sativa Rac1
(OsRac1), a member of the Rac/Rop family of small GTPases, regulates lignin biosynthesis
through NADPH oxidase and cinnamoyl-CoA reductase which acts as an effector of OsRac1
(Kawasaki et al., 2005). In C. roseus, inhibition of protein geranylgeranylation caused by
GGPPS deletion could directly control MIA biosynthetic pathway. After GGPPS deletion, the
geranylgeranylation of small GTPases would also be affected owing to alteration of GGPP
pool (Rai et al., unpublished results). The cellular substrate pool of prenylation target small
GTPase proteins could also be a crucial factor as low concentration results in imapaired
modification. It is known that plant contains single 7TM receptor GAP instead of typical G
protein coupled receptors (GPCRs). Hence, geranylgeranylation modulates multitude of
signaling pathways as inactivation of G proteins leads to the modification but not always loss
of the signaling and this indicates that diverse stimuli channeled through small GTPases after
their prenylation and many signals merged upstream of G protein complexes.

CONCLUSIONS
In C. roseus and A. annua, specific geranylgeranylated protein/s is/are involved in signaling
cascade that regulates the production of terpenoid compounds. Although it is speculated that
G/ROP/Rac proteins could be involved, so far there is no clear evidence to show their direct
role in this signaling. However, the exact class of GTPase involved in this regulation is yet to
be identified. This would not only improve the basic understanding of regulation of
secondary metabolism, but also will facilitate in improved production of important
metabolites by metabolic engineering. Impairment of geranylgeranylation might affect
certain signaling networks and it is interesting to know that how interaction between them or
their targets fine tuned the activation or repression of metabolic processes. In this perspective,
study of their subcellular localization and protein interaction may unravel their mode of
action. The role of geranylgeranylation in secondary metabolism reprogramming in plants
should be investigated by transcriptome, proteome, and metabolome approach from which
one could get information about GGPP/GTPases/signaling networks (TOR, Receptor kinases
etc) regulating central metabolic pathways. The regulation of terpenoid biosynthesis in plants
is controlled by several regulatory mechanisms depending on various elicitations, so it is
convenient to assume that some of the mechanism like protein geranylgeranylation regulating
secondary metabolite biosynthesis could be widespread in plants.

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REFERENCES

Courdavault V, Thiersault M, Courtois M, Gantet P, Oudin A, Doireau P, St-Pierre B, Giglioli-

Guivarc’h N (2005) CaaX-prenyltransferases are essential for expression of roseus cells. Plant
Mol Biol 57: 855–870
Crowell, DN (2000) Functional implications of protein isoprenylation in plants. Prog Lipid Res 39:
393–408
Eisenreich W, Menhard B, Hylands PJ, Zenk MH. Bacher A. (1996) Studies on the biosynthesis of
taxol: the taxane carbon skeleton is not of mevalonoid origin. Proc Natl Acad Sci USA. 25,
6431-6436.
Hemmerlin A (2013) Post-translational events and modifications regulating plant enzymes involved in
isoprenoid precursor biosynthesis. Plant Sci 203–204: 41–54
Huchelmann A, Gastaldo C, Veinante M, Zeng Y, Heintz D, et al. (2014) S-Carvone suppresses
cellulase-induced capsidiol production in Nicotiana tabacum by interfering with protein
isoprenylation. Plant Physiol 164: 935–950. doi: 10.1104/pp.113.232546
Kawasaki T, Koita H, Nakatsubo T, Hasegawa K, Wakabayashi K, Takahashi H, Umemura K,
Umezawa T, Shimamoto K (2006) Cinnamoyl-CoA reductase, a key enzyme in lignin
biosynthesis, is an effector of small GTPase Rac in defense signaling in rice. Proc Natl Acad
Sci USA 103: 230–235
Ma CJ (2008) Cellulase elicitor induced accumulation of capsidiol in Capsicum annumm L.
suspension cultures. Biotechnol Lett 30: 961–965
Martınez IM, and Chrispeels MJ (2003) Genomic analysis of the unfolded protein response in
Arabidopsis shows its connection to important cellular processes. Plant Cell 15: 561–576
Moeder W, Yoshioka K, Klessig DF (2005) Involvement of the small GTPase Rac in the defense
responses of tobacco to pathogens. Mol Plant Microbe Interact 18: 116–124
Nibau C, Wu HM, Cheung AY (2006) RAC/ROP GTPases: ‘hubs’ for signal integration and
diversification in plants. Trends Plant Sci 11: 309–315
Rai A, Smitha SS, Singh AK, Shanker K, Nagegowda DA (2013) Homomeric and heteromeric
geranyl diphosphate synthases from Catharanthus roseus and their involvement in
monoterpene indole alkaloid biosynthesis. Molecular Plant, 6 (5): 1531-1549
Schramek N, Wang H, Römisch-Margl W, Keil B, Radykewicz T, Winzenhörlein B, Beerhues L,
Bacher A, Rohdich F, Gershenzon J, Liu B. and Eisenreich W. (2010). Artemisinin
biosynthesis in growing plants of Artemisia annua. A 13CO2 study. Phytochemistry. 71, 179-
187.
Soliman SSM, Tsao R, Raizada MN (2011) Chemical inhibitors suggest endophytic fungal paclitaxel
is derived from both mevalonate and nonmevalonate- like pathways. J Nat Prod 74: 2497–2504
Sorek N, Bloch D, Yalovsky S (2009) Protein lipid modifications in signaling and subcellular
targeting. Curr Opin Plant Biol 12: 714–720
Towler MJ, Weathers PJ (2007) Evidence of artemisinin production from IPP stemming from both the
mevalonate and the nonmevalonate pathways. Plant Cell Rep 26: 2129–2136

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 Engineering metabolic pathways in microorganisms to produce higher
levels of semi-synthetic anti-malarial drug, artemisinin

Shivangi Mishra and Anil Kumar Tripathi

CSIR-Central Institute of Medicinal and Aromatic Plants (CSIR-CIMAP), PO CIMAP,
Lucknow-226015 (India)
Email:shivangimishra1610@gmail.com

ABSTRACT

More than 200 million cases of malaria were registered in the year of 2012, and
approximately, 627,000 deaths reported due to malaria caused by Plasmodium falciparum
(World Malaria Report; 2013). WHO has recommended artemisinin-based combination
therapies (ACTs) for the treatment of malaria caused by the multi-drug resistant strains of P.
falciparum. Artemisinin, a highly effective compound against multi-drug resistant P.
Falciparum, is a sesquiterpene lactone endoperoxide derived from the plant, Artemisia annua
(sweet wormwood) (Meshnick et al; 1991). Total production of artemisinin from A. annua is
not sufficient to meet its ever growing demand. Therefore, its semi-synthetic production
using an alternative host has become important and cost effective. This review focuses on the
use of synthetic biology for the production of artemisinic acid, a precursor of artemisinin,
through engineering of terpenoid biosynthetic and mevalonate-dependent (MEV) pathways in
microorganisms like Escherichia coli, Saccharomyces cerevisiae and Aspergillus nidulans. It
also outlines the expression of A. annua gene encoding amorphadiene synthase in a suitable
microorganism, and development of fermentation process to achieve high levels of amorpha-
4, 11-diene.

Terpenoids constitute a highly diverse group of natural compounds, many of which have
commercial importance as anti-cancer and anti-malarial drugs. Artemisinin is one of the
terpene-based compounds having a potent anti-malarial action, and is highly effective against
Plasmodium falciparum caused malaria, when used as ACTs (Jean Robert et al; 2009). Level
of artimisinin production is very low in A.annua (0.1-0.8% of dried biomass) and hence, is
not very economical to produce from the plant. As its production is limited and very costly
from the plant, its semi-synthetic production has become promising. Advances in the area of
genetic manipulation of microorganisms for the production of higher level of small molecules
had shown the feasibility of increasing the yield in the range of gL-1.

Terpenoids are derived from a five carbon compound, Isopentenyl pyrophosphate (IPP)
(Gershenzon et al, 2007; McGarvy et al, 1995). IPP and its isomer, dimethyl allyldiphosphate
(DMAPP) are converted by condensation reactions into geranyldiphosphate (GPP),
farnesyldiphosphate (FPP), and geranylgeranyldiphosphate (GGPP) through GPP, FPP and
GGPP synthases, respectively. These derivatives of IPP finally lead to the production of
various monoterpenes, sesquiterpenes and diterpenes. FPP is a substrate for amorphadiene
synthase (ADS) for the production of amorpha-4, 11-diene, which is a precursor of
artemisinin (Kirby et al).

The first successful attempt for amorpha-4, 11-diene production was done in E. coli by
introducing a heterologous mevalonate-dependent pathway from Saccharomyces cerevisiae
and codon-optimised variant of the gene encoding amorphadiene synthase (ADS) from A.
annua (Martin et al, 2003). This engineered strain of E. coli contained a biosynthetic pathway

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consisting of eight genes divided into two operons. The first operon converts acetyl-CoA into
mevalonate in three enzymatic steps, and the second operon converts mevalonate to IPP or
FPP. Amorphadiene synthase (ADS) finally converts FPP into amorphadiene. This engineered
strain of E. coli produces amorphadiene concentration of 3.1 µg caryophyllene
equivalents/ml/OD600 after 9 h of culture. However, addition of 0.8% glycerol to the LB
medium leads to an increased production of amorphadiene up to 24µg caryophyllene
equivalents/ml (Martin et al, 2003).

This strain was further engineered along with improvements in fermentation techniques for
higher amorphadiene production. The engineered strain of E. coli contained two genes from
the heterologous mevalonate pathway of S. cerevisiae i.e. HMG-CoA synthase (HMGS) and
the catalytic domain of HMG-CoA reductase (tHMGR). More active enzymes from
Staphylococcus aureus were used to replace the above genes doubling the amorpha-4, 11-
diene production (Tsuruta et al, 2009). Glucose and ammonia restricted high cell density
bioprocessing was employed with ammonia maintained between 30-60mM (Tsuruta et al,
2009). The production of amorphadiene, initiated by adding IPTG, which yielded 90g/L dry
cell weight and 27.4g/L amorpha-4, 11-diene (Tsuruta et al, 2009). Achievement of this level
of amorphadiene has led to the production of large amount of artemisinin.

Aspergillus nidulans has been an organism of interest for the cloning heterologous genes. It
possesses a sesquiterpenoid biosynthesis pathway that is similar to that of A. annua. In A.
nidulans, similar to plants, eukaryotes and some bacteria, IPP is derived from the mevalonate
pathway. The committed step towards terpenoid production is the condensation of IPP and
DMAPP to produce GPP, which again condenses with another IPP to produce FPP. The FPP
is a substrate for ADS leading to the production of amorphadiene, and eventually artemisinin.
Therefore, it was also explored as another potent host for amorphadiene production
(Lubertozzi et al, 2008). Although the gene encoding amorpha-4, 11-diene synthase was
cloned and expressed in A. nidulans the amount of amorphadiene produced was very small
(Lubertozzi et al, 2008).

S. cerevisiae has also been engineered for the production of artemisinic acid, the precursor for
artemisinin. Artemisinic acid can be converted into artemisinin using a chemical source of
singlet oxygen (Newman et al, 2013). This was done by using engineered mevalonate
pathway, ADS and a novel cytochrome P450 monooxygenase (CYP71AV1) from A. annua.
The cytochrome P450 oxidizes amorphadiene into artemisinic acid via three oxidation steps.
Upregulation of tHMGR and downregulation of ERG9 (squalene synthase) increases FPP
production (Keasling et al, 2006). Therefore, more conversion of FPP into amorphadiene by
ADS, and subsequent P450 mediated higher production of artemisinic acid. This strain
produces about 100mg/L of artemisinic acid. Further, interaction with cytochrome b5
(CYB5) enhances the reaction rate of cytochrome P450 (Schenkman et al, 2003, Zang et al,
2007). It was also found that CYB5 expression increases and almost doubles the artemisinic
aldehyde production. As artemisinic aldehyde is found to be highly toxic to the culture,
artemisinic aldehyde dehydrogenase (ALDH1) was expressed to enhance artemisinic acid
production (Tech et al, 2009). Hence, it shows that all the five enzymes CYP1AV1, CPR1,
CYB5, ALDH1 and ADH1 (NAD-dependent alcohol dehydrogenase, specific towards
artemisinic alcohol) are involved in artemisinic acid production from amorphadiene.
Improvements in fermentation methodology led to further increase in artemisinic acid levels
reaching upto 25g/L, which is considerably higher than the earlier levels of 1.6g/L of
artemisinic acid (Newman et al, 2013).

22 | P a g e
These attempts of developing an alternative source for artemisinin for ACTs have paved the
way for efficient and less expensive alternative source, independent of the fluctuations
attached with the plant-derived artemisinin. The limited plant-derived yield poses a
significant challenge for the large scale industrial production of this kind of essential drugs.
Hence, these significant developments in pathway engineering, fermentation technology and
chemistry related with the production of artemisinin have brought forth an industrial process
capable of supplementing the present day needs of artemisinin.

REFERENCES

Gershenzon J, Dudareva N: The function of terpene natural products in the natural world. Nat
ChemBiol 2007, 3:408-414.
Jean-Robert Ioset, Kaur H.; Simple Field Assays to Check Quality of Current Artemisinin-Based
Antimalarial Combination Formulations; PlosOne(2009).
Keasling Jay D et al; Production of the antimalarial drug precursor artemisinic acid in engineered
yeast; Nature letters; 440 (2006).
Kirby J. and Keasling,Jay D. ; Biosynthesis of Plant Isoprenoids: Perspectives for Microbial
Engineering
Lubertozzi David, Keasling Jay D; Expression of a synthetic Artemisia annuaamorphadiene
synthase in Aspergillusnidulans yields altered product distribution; JInd Microbial
Biotechnology; (2008) 35.
Martin Vincent JJ, Pitera J Douglas, Withers T Sydnor, Newman D Jack, Keasling Jay D;
Engineering a mevalonate pathway in E. coli for production of terpenoids; Nature
Biotechnology 21 number 7(2003)
McGarvey DJ, Croteau R: Terpenoid metabolism. Plant Cell 1995,7:1015-1026.
Meshnick S.R., Abraham T., Ranz A., Cai-Min Xu,Hua-Zhen Pan; Artemisinin (qinghaosu): the
role of intracellular hemin in its mechanism of antimalarial action. Mol. And Biochem.
Parasitology; 49; (1991).
Newman J.D. et al; Nature Letters(2013)
Schenkman J.B. and Jansson I. The many roles of cytochrome b5.Pharmocol. 97(2003)
Tech K.H. ,Polichuk D.R. , Reed D.W., and Covello P.S.; Molecular cloning of an aldehyde
dehydrogenase implicated in artemisinin biosynthesis in A. annua; Botany; 87 (2009)
Tsuruta H, Paddon C.J., Eng D., Lenihan J. R., Horning T., Anthony L.C., Regentin R., Keasling
J. D. , Renninger N.S., Newman J.D.; Plos one; 4:2 (2009).
WHO, World Malaria Report (2013)
Zhang H.,Im S.C. and Waskell L. Cytochrome b5 increases the rate of productformation by cyt
P450, J. of Biochem282; (2007)

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 Exploring the use of Catharanthus roseus in Alzheimer ’s disease

Akhil Kumar, Ashok Sharma*

CSIR-Central Institute of Medicinal and Aromatic Plants, P.O. – CIMAP, Near Kukrail
Picnic Spot, Lucknow – 226 015, Uttar Pradesh, India
Email: akhil2687@gmail.com

INTRODUCTION

The Alzheimer’s disease (AD) is most common form of dementia. AD is progressive

neurodegenerative disorder and rapidly growing as number of patient increases per year
drastically. In one study it’s estimated that AD [1]. Most of the FDA approved drug or based on
natural scaffold, moiety, analogue or derivative. The first two drug approved by FDA i.e. tacrine
(1993) and donepezil (1996) were synthetic in origin [2] Drug rivastigmine (2000), galatine and
FDA approved dietary supplement for memory support Huperzine were respectively derived from
alkaloid physostigmine, obtained from Galanthus spp. and Huperzia spp. [2,3] The natural leads
have fewer side effects in term of ADME/toxicity if given in correct doses form. Natural
molecules also have extra advantage such as vast chemical diversity. They are more readily
absorbed than synthetic drugs and are suitable for multifactorial disease like cancer and
Alzheimer’s. Taking this into consideration current scenario is shifting towards natural drug
discovery.

Many medicinal and aromatic plant extract and compound were reported for Alzheimer’s disease
[4] One example of an Amaryllidaceae alkaloid already used medically to treat AD is
galanthamine. Galanthamine is an alkaloid discovered in 1953 that is produced by members of
the Amaryllidaceae family [5]. We present the current literature on the potential therapeutic
properties of medicinal plants Catharanthus roseus against AD. The well known medicinal plant
C. roseus is used in treatment for diabetes, breast cancers, leukemia and Hodgkin’s lymphoma
[6]. Some of the alkaloids found in this medicinal plant are effective in tubulin depolymerisation
and inhibiting microtubule assembly [7]. C. roseus was found to have, AChE inhibitor activity,
similar to that of galantamine and other currently licensed AD treatments [4] Leaf and stem
extract of C. roseus was found to be more potent than the petal extract to inhibit AChE [8].
Further analysis showed that the roots of the C. roseus had 16.5 times more anticholinesterase
inhibitory activity than the leaves and over 100 time’s higher potency than the petals. [9] This
increased effect was found to be due the presence of serpentine that served as a contributory
factor. The IC50 of serpentine alone shows approximately 100 times increased potency compared
to the roots [4]. Ajmalicine, the precursor for serpentine does not inhibit AChE and can be used
as control [10].

Other compound Vinpocetine (semisynthetic derivative of the vinca alkaloid vincamine) was
studied on two hundred and three patients suffering from mild to moderate organic
psychosyndromes. The study included, placebo-controlled, randomized double-blind, multicentre
trial and received different doses of Vinpocetine. Further patients were assessed on different
parameters likes clinical global impression, cognitive performance and on measures of the quality
of life including depressive illness. This study demonstrated the usefulness and efficacy of
vinpocetine in the management of patients with moderate organic psychosyndromes. Vinpocetine
was also superior to placebo in ratings of the "severity of illness". [11] Another study on human
trial was conducted to investigate the therapeutic efficacy of vincamine in the treatment of
primary degenerative and vascular dementia. They suggested that the therapeutic effect of
vincamine is superior to placebo in patients with mild to moderate dementia of degenerative and
vascular etiologies [8, 12]

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In another study, Vinorelbine and paclitaxel that target microtubules have been reported to inhibit
STAT3 phosphorylation in breast cancer cells. STAT activation by phosphorylation is mediated
by growth factor receptor tyrosine kinases, and the cytoplasmic kinases, such as cytokine
receptor-associated Janus kinases (JAKs), and Src family kinases. These two agents modulate
pathway via interacting with these kinases [13]. Vinorelbine is also reported as a microtubule
destabilize. Similar studies were performerd by the other group supported the study and confirm
the sensitivity of the bovine brain tubulin polymerization assay to the inhibitory effects of a
known polymerization inhibitor, the vinca alkaloid vincristine sulfate, derived from C. roseus.
Vincristine sulfate binds to αβ-tubulin dimers to prevent formation of microtubules, thus leading
to mitotic arrest and apoptosis [14, 15, 16]. In AD Tau as well as amyloid fibril polymerization
are directly linked to disesse pathology and progression. Tau polymerization is directly related to
hyper phosphorylation of tau protein via different kinase such as glycogen synthase kinase-3β
(GSK-3β), cyclin-dependent kinase 5 (cdk5), and other tau kinases [17]. As these molecules
interacting with kinases and have polymerization inhibiton effect thus may be used to inhibit
glycogen synthase kinase-3β and to test for disaggregating properties for AD.

It is generally believed that the alkaloids derived from the plant are the key to their efficacy, It’s
reported that C. roseus produce around 130 alkaloid [18]. The two classes of active compounds in
Vinca are alkaloids and tannins. The major alkaloid is vincamine and its closely related semi-
synthetic derivative widely used as a medicinal agent, known as ethyl-apovincaminate or
vinpocetine, has vasodilating, blood thinning, hypoglycemic and memory-enhancing actions. Its
vasodilating and memory-enhancing properties have been shown to alleviate vascular dementia
and Alzheimer's. Jung et al. reported that three major alkaloids in Coptidis Rhizoma-
groenlandicine, berberine, and palmatine can potentially exhibit anti-AD effects through both
ChEs and Aβ pathways and antioxidant capacities to inhibit ROS and RNS [19] C. roseus may
possess free radical scavenging ability. The petals were found to be the most potent free radical
scavengers [20]. The alkaloid from C. rosues, which have antioxidant properties directly tested
against AD. As antioxident property gives extra advantage as therapeutic molecule because its
deal with oxidative stress caused in AD patients during the development of the disease.

Other plant like Voacanga africana and Hunteria zeylanica form family Apocynacea were earlier
reported for AD treatment. C. rouses belong to Apocynacea Here we tried to correlate the
current treatments for AD and therapeutic benefit of C. roseus supported with the available
literature. Different alkaloids are and derivatives as well as analogue were tested as against AD
with the help of computational techniques like 3D QSAR, pharamcophore, docking and
dynamics. This approach explores the potential of C. rosues in AD and also save time and money.

REFERENCE

1. Thies W., Bleiler L., Report Alzheimer Disease facts and figures. Alzheimers Dement. 2013 9
: 208-45
2. Murray A. P., Faraoni M. Be., Castro M. J., Alza N. P., Cavallaro V., Natural AChE Inhibitors
from Plants and their Contribution to Alzheimer’s Disease Therapy Curr Neuropharmacol. Jul
2013; 11(4): 388–413.
3. Chopra K, Misra S, Kuhad A. Current perspectives on pharmacotherapy of Alzheimer’s.
Expert. Opin. Pharmacother. . 2011; 12:335–350.
4. Shakir T., Coulibaly A. Y., Kehoe P. G., An exploration of the potential mechanisms and
translational potential of five medicinal plants for applications in Alzheimer’s disease Am J
Neurodegener Dis. 2013; 2(2): 70–88
5. Uyeo S, Kobayashi S (1953) Lycoris alkaloids. XXIV. Isolation and characterization of
lycoremine. Pharm Bull 1: 139–142

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6. The discovery of the vinca alkaloids--chemotherapeutic agents against cancer. Noble RL
Biochem Cell Biol. 1990 Dec; 68(12):1344-51.
7. Plant-derived anticancer agents. Pezzuto JM Biochem Pharmacol. 1997 Jan 24; 53(2):121-33.
8. Pereira D. M, Ferreres F, Oliveira J, Valentão P, Andrade P .B, Sottomayor M. Targeted
metabolite analysis of Catharanthus roseus and its biological potential. Food Chem Toxicol.
2009;47:1349–1354.
9. Pereira D. M., Ferreres F, Oliveira J. M, Gaspar L, Faria J, Valentão P, Sottomayor M, Andrade
P. B. Pharmacological effects of Catharanthus roseus root alkaloids in acetylcholinesterase
inhibition and cholinergic neurotransmission. Phytomedicine. 2010;17:646–652
10. Zenk M. H. Enzymatic synthesis of ajmalicine and related indole alkaloids. J Nat Prod 1980;
43: 438-451.
11. Hindmarch I1, Fuchs H. H, Erzigkeit H. Efficacy and tolerance of vinpocetine in ambulant
patients suffering from mild to moderate organic psychosyndromes. Int Clin Psychopharmacol.
1991 Spring;6(1):31-43.
12. Fischhof P. K1, Möslinger-Gehmayr R, Herrmann W. M, Friedmann A, Russmann D. L.
Therapeutic efficacy of vincamine in dementia. Neuropsychobiology. 1996;34(1):29-35.
13. Walker S. R, et al. Microtubule-targeted chemotherapeutic agents inhibit signal transducer and
activator of transcription 3 (STAT3) signaling. Mol Pharmacol. 2010;78:903–908.
14. Jordan A, Hadfield J. A, Lawrence N. J, et al. Tubulin as a target for anticancer drugs: agents
which interact with the mitotic spindle. Med Res Rev. 1998;18:259–296.
15. Wang LG, Liu XM, Kreis W, et al. The effect of antimicrotubule agents on signal transduction
pathways of apoptosis: a review. Cancer Chemother Pharmacol. 1999;44:355–361.
16. Butler TR, Smith KJ, Self RL, Braden BB, Prendergast MA. Neurodegenerative effects of
recombinant HIV-1 Tat(1–86) are associated with inhibition of microtubule formation and
oxidative stress-related reductions in microtubule-associated protein-2(a,b) Neurochemical
Research. 2011;36:819–828
17. Gong C. X., Iqbal K. Hyperphosphorylation of microtubule-associated protein tau: a promising
therapeutic target for Alzheimer disease. Curr. Med. Chem. 2008. 15, 2321–2328
18. Van Der Heijden R, Jacobs D. I, Snoeijer W, Hallard D, Verpoorte R.. The Catharanthus
alkaloids: pharmacognosy and biotechnology. Current Medicinal Chemistry 2004. 11: 607–628.
19. H. A. Jung, B. S. Min, T. Yokozawa, J. H. Lee, Y. S. Kim, and J. S. Choi, “Anti-Alzheimer and
antioxidant activities of coptidis rhizoma alkaloids,” Biological and Pharmaceutical Bulletin,
2009. 32,(8) 1433–1438.
20. Ferreres F, Pereira D. M, Valentão P, Andrade P. B, Seabra R. M, Sottomayor M. New
phenolic compounds and antioxidant potential of Catharanthus roseus. J Agric Food Chem.
2008;56:9967–9974

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 Genus Artemisia: Available avenues to explore beyond “Artemisinin”

Ritesh Kumar, Shubhra Rastogi and Ajit Kumar Shasany*

CSIR-Central Institute of Medicinal and Aromatic Plants, Lucknow 226015, UP, INDIA
Email: riteshbiochem2006@gmail.com

ABSTRACT

Genus Artemisia belongs to family Asteraceae and comprises of about 481 species which
have an enormous diversity of bioactive secondary metabolites. One of the publically
available databases, Pubmed, indicates 2666 published research articles on genus Artemisia
which are represented by just 26% of its species. The highest contributing species, A. annua,
is represented by 668 articles which are equally distributed in 4 major research categories viz.
Chemistry, Biological Activity, Molecular and Others. Further, a large number of research
publications particularly on resistance to artemisinin and other areas narrow down the
spectrum of work done and published on A. annua. Fortunately, the genus comprises of about
352 (76%) unexplored species with new promising scopes in the areas of phytochemistry,
biological activity, molecular approaches and ecological adaptations. This review
summarizes the potential of this genus Artemisia beyond artemisinin and A. annua which
may pave the path of future research.

INTRODUCTION

Artemisia is well known genus of family Asteraceae and Artemisia annua is one of its
popular species mainly due to the antimalarial sesquiterpene lactone, “artemisinin”,
biosynthesized by the plant (Zhang et al., 2014). Projections of Artemisia beyond the
artemisinin dependent popularity are the areas to be explored. There are several generic
questions such as- “Are there any unexplored avenues of genus Artemisia beyond
artemisinin? Whether it will be obsolete or there are new horizons for further explorations?”
Fortunately, the answer to the above questions is “Yes”. According to Bora and Sharma
(2011) genus Artemisia contains over 500 species and “The Plant List”
(http://www.theplantlist.org/1.1/browse/A/Compositae/Artemisia/) reports 1,598 scientific
plant names of species rank. Of these 481 are accepted species names and a further 867
scientific plant names of intraspecific rank are listed which are not intended to be complete
names of intraspecific rank. These are primarily included because names of species rank are
synonyms of accepted intraspecific names.

A large number of different members of this genus contribute to a wide range of traditional
and modern usages in the form of folk medicine, ornamental plants and essential oils for
pharmaceutical and cosmetic industries (Abad et al., 2012). The wide geographical
distribution of this genus from North America to Asia and Europe with huge biochemical
diversity of secondary metabolites make it very interesting both for basic research such as
molecular regulations of carbon partitioning between different secondary metabolites as well
as applied research such as identification of commercially important phytomolecules and
their medicinal applications.

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Published research work on genus Artemisia
A
3% 2% 1%
B
Species with 7% 21% Only 1
publication record
26% Only 2
9%
3 to 5
6 to 10
11 to 20
Species without 21 to 50
19% 21% 51 to 100
publication record
74% 100 to 200
Above 200
17%

Figure 1: (A) Representation of Artemisia species (%) in Pubmed. (B) Distribution of

number of articles in recorded species of Artemisia in Pubmed.

Total number of research articles available on Artemisia species in Pubmed was found to be
2666 which are surprisingly the records of only 122 species (26%). Out of these recorded
species, 1% species have more than 200 articles documented whereas about 21% are
represented by one or two articles only (Figure 1B). The data suggests that the documented
literature ranges from 1 to over 650 published works in the different species of this genus.
Not surprisingly, A. annua, with 668 article hits is much ahead than the other species, A.
vulgaris (176 records) and A. absinthium (173 article records) (Figure 2).

800

700 668
No. of available publication records

600

500

400

300

200 176
130
107
88 79
100 58 56
38 36 34 34 28 23 22 21 20 18 17 17 17 13 12 11 11
0

Figure 2: The documented records of top 25 species of genus Artemisia

Categories of Articles

Articles of each species were separately classified into four categories namely (i) Chemistry,
(ii) Biological activity, (iii) Molecular and (iv) Others. Category “Chemistry” includes the
articles concerned with the isolation and/or identification of their chemical constituents.
Articles covering biological activities viz. antioxidant, anti-bacterial, anti-cancerous and
immunomodulatory effects, etc. are included in the “Biological Activity” category.
Publication on molecular approaches like cloning, characterization or expression of genes
were classified in “Molecular” category while the articles related to ecological,

28 | P a g e
environmental and adaptation of particular plant which could not be placed in the
aforementioned three categories were included in “Others” category.

A substantial work has been carried out under the category “Biological activity” as suggested
by 1052 research articles representing 43% of the total publications. Conversely, work
documenting modern molecular approaches under the category, “Molecular”, is represented
by least number of articles (283 records; 12% of the total publications) (Figure 3A).

Current status of records on Artemisia annua

The articles of A. annua are almost equally divided among the above mentioned categories
(Figure 3B). Along with its artemisinin based anti-malarial activity, A. annua has been taken
up for a wide range of studies such as phytochemistry (Ulubelen A and Halfon B, 1976),
biological activities [anti-microbial (Bilia et al., 2014), anti-cancer, anti-viral, and anti-
inflammatory activities (Lee et al, 2014)], cloning and characterization of pathway genes
(Rydén et al, 2010 and Wang et al. 2011) and other regulatory genes (Ji et al., 2014 and
Mishra et al.). Currently, with reports of artemisinin resistant malaria (Ashley et al., 2014),
the scope of this genus in relation to its artemisinin related activities seems to diminish.

Figure 3B shows the status of published records in other species of this genus which looks
encouraging from the scientific perspective. The other species wait to be explored and
subsequently commercially utilized.

A B % Chemical % Biological % Molecular % Others

100.00
90.00
% Others % 80.00
22% Chemical
23% 70.00
60.00
50.00
%
40.00
Molecular
12% 30.00
% 20.00
Biological 10.00
43%
0.00

Figure 3: (A) Categories of published articles in genus Artemisia and their distribution (B)
Category wise distribution of publications of top ten recorded species of genus Artemisia.

Future prospects of genus Artemisia

Above statistics gives an insight for the exploration of probable research areas among the
different species of genus Artemisia. Only 1% species had been investigated in all probable
aspects while about 74% of species are awaiting exploration in chemical, biological,
molecular as well as ecological areas. The family Asteraceae is known for its enormous
diversity of secondary metabolite and other phytomolecules (Abad et al., 2012) and the
chemical constituents of Artemisia have versatile activities. This review provides an overview

29 | P a g e
about the research aspects that have been explored in the different species of the genus
Artemisia. The information provided could be a stepping stone for unraveling other
innumerable properties of the genus which may tend to us to think of Artemisia beyond
“artemisinin”.

REFERENCES

Abad M.J., Bedoya L.M., Apaza L. and Bermejo P. (2012). The Artemisia L. Genus: A Review of
Bioactive Essential Oils Molecules; 17: 2542-2566.
Ashley E.A., Dhorda M., Fairhurst R.M. and Amaratunga C. et al. (2014). Spread of Artemisinin
Resistance in Malaria. N Engl J Med.; 371(5):411-23.
Bilia A.R., Santomauro F., Sacco C., Bergonzi M.C. and Donato R. (2014). Essential Oil of Artemisia
annua L.: An Extraordinary Component with Numerous Antimicrobial Properties. Evid Based
Complement Alternat Med.; 2014:159819. doi: 10.1155/2014/159819. Epub 2014 Apr 1.
Bora K. S. and Sharma A. (2011).The genus Artemisia: A comprehensive review. Pharm. Biol.; 49,
101–109.
Ji Y., Xiao J., Shen Y., Ma D., Li Z., Pu G., Li X., Huang L., Liu B., Ye H. and Wang H. (2014).
Cloning and characterization of AabHLH1, a bHLH transcription factor that positively
regulates artemisinin biosynthesis in Artemisia annua. Plant Cell Physiol.; 55(9):1592-604.
Lee S.H., Cho Y.C., Kim K.H., Lee I.S., Choi H.J. and Kang B.Y. (2014). Artesunate inhibits
proliferation of naïve CD4(+) T cells but enhances function of effector T cells. Arch Pharm Res.
[Epub ahead of print].
Misra A., Chanotiya C.S., Gupta M.M., Dwivedi U.N. and Shasany A.K. (2012). Characterization
of cytochrome P450 monooxygenases isolated from trichome enriched fraction
of Artemisiaannua L. leaf. Gene.; 510(2):193-201.
Rydén A.M., Ruyter-Spira C., Quax W.J., Osada H, Muranaka T., Kayser O. and Bouwmeester H.
(2010). The molecular cloning of dihydroartemisinic aldehyde reductase and its implication in
artemisinin biosynthesis in Artemisia annua. Planta Med.; 76(15):1778-83.
The Plant List (2010). Version 1. Published on the Internet; http://www.theplantlist.org/ (accessed 1st
January).
Ulubelen A. and Halfon B. (1976). Phytochemical investigation of the herba of Artemisia annua.
Planta Med.; 29(3):258-60.
Wang Y., Yang K., Jing F., Li M., Deng T., Huang R., Wang B., Wang G., Sun X. and Tang K.X.
(2011). Cloning and characterization of trichome-specific promoter of cpr71av1 gene involved
in artemisinin biosynthesis in Artemisia annua L. Mol Biol (Mosk).; 45(5):817-24.
Zhang X.G., Li G.X., Zhao S.S., Xu F.L., Wang Y.H., and Wang W (2014). A review of
dihydroartemisinin as another gift from traditional Chinese medicine not only for malaria
control but also for schistosomiasis control. Parasitol Res.; 113(5):1769-73.

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 Hairy root mediated biotechnological intervention in Artemisia species for
the production of artemisinin- A mini review

Sailendra Singh, Pallavi Pandey, Harshita Pandey, Ruby Gupta, Suchitra Banerjee*

Department of Plant Biotechnology, Central Institute of Medicinal and Aromatic Plants (CSIR-
CIMAP), PO CIMAP, Kukrail Picnic Spot Road, Lucknow 226015, India

E-mail: sk_rmsu@yahoo.com

ABSTRACT

Artemisia annua an important medicinal herb of Asteraceae family is the major producer of
artemisinin, which has exhibited diverse pharmaceutical efficacies in treating cerebral malaria and
various other diseases, such as cancer, HIV, hepatitis-B and leishmaniasis. The lower yield of
artemisinin from the natural source and difficulty in its chemical synthesis have led to the
advancement of Agrobacterium rhizogenes mediated “hairy root” cultures to act as a feasible
alternative production source of this bioactive molecule. Different methods have been adopted
worldwide for the enhancement of artemisinin biosynthesis in hairy root cultures, evaluation of
which in a comprehensive manner highlights the significance of such endeavors. This review
summarizes the overall reported progress made in the area of hairy root mediated production of
artemisinin / or its derivatives in combination with the reported efforts to enhance the yield through
elicitations. Bacterial strain specificity and influence of medium composition played a crucial role on
the overall production efficiency of the hairy root cultures of A. annua. Explorations of other species
of this genus in terms of HR technology also gained momentum in recent years and six new species
have made promising entry in this global endeavor.

Keywords: Artemisia annua, Artemisinin, Bioreactor, Elicitation, Hairy root culture

INTRODUCTION

Artemisinin, a lactone endoperoxide sesquiterpene, isolated from the trichomes of chinese herb,
Artemisia annua (Asteraceae), has been used extensively for treating malaria, specially cerebral
malaria & drug resistance malaria (Parshikov et al., 2012). Moreover, this nature derived antimalarial
molecule has been recently recognized by WHO of having excellent anticancerous activity. (Duffy et
al., 2012). Besides artemisinin, its derivatives such as artemether, artesunate, artemotil,
dihydroartemisinin are the other leading Artemisia based molecules having anticancer properties

31 | P a g e
and a few of them are in the stage of clinical trials (Duffy et al., 2012). The major drawback of
artemisinin is its poor bioavailability (short half life), which leads to less absorption in the cells
causing reduced efficacy with regard to the antimalarial activity (Parshikov et al., 2012).

The lower yield (< 1 % Dry wt.) and limited availability (A. annua) of artemisinin have steered the
research focus towards the development of different biotechnological approaches for the
continuous and consistent supply of this pharmaceutically important molecule (Weathers et al.,
2004, 1997). Although chemical synthesis of artemisinin is possible, but lack of economic feasibility
of this approach (Wang et al., 2008, Sivakumar et al., 2010) has led to the exploration of innovative
sources for its sustainable production. The high commercial demand of these metabolites have
necessitated implementation of various biotechnological approaches amongst which the “hairy
root” (HR) culture technique has evolved as the most efficient production alternative which
overcomes the constraints of geographical, seasonal / environmental factors (Pandey et al. 2014).
Although initially it was believed that hairy root can synthesized only the root specific metabolites of
the parent plant, but the hairy root culture of A.annua has successfully demonstrated its
biosynthetic potential in term of the leaf specific metabolite – artemisinin.

The application of HR culture technique has therefore been explored extensively in A. annua, not
only to obtain a sustainable production source for artemisinin and/ or its precursors / derivatives,
but also, to utilize it as a suitable model system for pathway engineering through either
overexpression or heterologous expression of the targeted genes for enhanced production of
artemisinin (Arsenault et al., 2008). The genetic / biochemical stability, ease of scalability and growth
potential in hormone-free media as well as long term production consistency enhanced the
credentials of HR cultures in A. annua with regard to the consistent production of the desired
secondary metabolites. The present review summarizes the overall reported progress made in the
area of hairy root mediated production of artemisinin or its derivatives in Artemisia species in
general and A. annua in particular coupled with the evaluations of the effects of bacterial strains,
medium composition and elicitors on the production efficiency of the HR cultures.

Major adopted approaches

A detailed literature survey revealed that the overall yield of artemisinin in the hairy root cultures
remained quite low during initial attempt, which required further modifications for increasing the
final productivity. The most widely adopted approaches can be categorized under the following
class:

i. Screening and selection of elite hairy root clone and media optimization:

Published literature has revealed that bacterial strain specificity and inter-clonal variability played a
crucial role in deciding upon the maximum productivity of artemisinin. With regard to the utilization

32 | P a g e
of A. rhizogenes strains for the induction of HR clones in A. annua, the LBA 9402 strain was
predominantly used followed by the 1601> YUT-16> ATCC 15834> LBA 8196> A4> 9365> 9340 (Giri
et al., 2001, Ahlawat et al., 2014, Kiani et al., 2012). The most frequently and successfully used
nutrient medium was found to be the full strength Murashige and Skoog (MS) (Giri et al., 2001; Patra
et al., 2013) medium followed by the full strength Gamborg’s medium (B5) (Ahlawat et al., 2014),
whereas half strength MS medium revealed lesser contribution towards the growth & maintenance
of A.annua having hairy root cultures.

ii. Efforts for increasing the content of metabolite through other means:

Several strategic attempts have been carried out by several researchers around the globe with
regard to the increasing the content of this therapeutically important drug molecule- artemisinin in
the HR culture of A.annua which are as follows:

a. Elicitation

The hairy root cultures of A. annua have been treated quite often with both biotic & abiotic elicitors
for improving the artemisinin content. The most widely used abiotic elicitors include methyl
jasmonate, chitosan, casein hydrolysate, oligosaccharide, cerebroside, gibberellic acid, which
represents about 64 % of the total reports (Putalun et al., 2007; Patra et al., 2013; Ahlawat et al.,
2014). On the other hand, the biotic elicitors (fungal and yeast) constitutes only 12 % of the reported
efforts (Wang et al., 2001; Ahlawat et al., 2014). Treatment with inorganic salts such as sodium
nitroprusside, potassium nitrate and sodium phosphate, sodium acetate had also be reported, which
revealed slight improvement in the artemisinin synthesis by the hairy root culture (Wang et al.,
2001; Liu et al., 2002; Zheng et al., 2001; Wang et al., 2009; Patra et al., 2013; Ahlawat et al., 2014).
The maximum content of artemisinin reported from the A. annua HR culture was found to be 0.7
mg/gm DW by using oligosaccharide elicitors (Zheng et al., 2008).

b. Polyploidization:

Ploidy status of HR cultures had been found to play a major role in the growth and secondary
metabolite production potentials in A. annua. The diploid HR cultures of A. annua has been
genetically manipulated by using mitotic inhibitor colchicines to produce tetraploid clones which
resulted six times more artemisinin content as compared to the parent diploid culture (Gonzalez
and Weathers, 2003 ).

c. Scalability in bioreactors:

Several reports have revealed that HR cultures have been up scaled in different types of bioreactors
for the enhancement of the productivity of artemisinin. The types of bioreactors used are diverse,
such as Mist Bioreactor, Bubble column, Stirred tank, Airlift bioreactor (Wang et al., 1998; Weathers

33 | P a g e
et al., 2000). Amongst them, the mist bioreactor revealed the maximum potential for the up-scaling
of the A. annua HR cultures followed by the bubble column reactor (Sivakumar et al., 2010; Kim et
al., 2002, 2003; Patra et al., 2014).

iii. Utilization of HR culture of A. annua for the production of transgenic plants:

The HR culture of A.annua has been reported to induce spontaneous regeneration of transgenic
plants which demonstrated the potentials to synthesize artemisinic acid and arteannuin B. The
transformed plant revealed higher amount of both the metabolites as compared to their respective
HR clones (Banerjee et al., 1997). The need for the discovery of innovative sources of not only
artemisinin, but also, other potential molecules to combat the problem of resistance development
towards the existing molecules can be met through such approach.

iv. Potentials of the hairy roots of other Artemisia species:

The hairy root culture of A. annua has gained maximum attention in terms of serving as a feasible
alternate production source of artemisinin or it derivatives. However, there are several reports that
have explored the potentials of HR cultures of other species of Artemisia as well in terms of
artemisinin synthesis. Hairy root cultures of A. dubia (Mannan et al., 2008), A. indica (Kiani et al.,
2012) and A absinthium (Kiani et al., 2012) have been reported to produce artemisinin, but that of
A.annua superseded the rest. The HR cultures of A. annua were found to be the most efficient for
the synthesis/ accumulation of artemisinin followed that of A. dubia, A. indica and A. absinthium in
reducing order (Mannan et al. 2008). There are certain different species of Artemisia such as, A.
bushriences and A. dracunculus, having the potentials of producing traces of artemisinin (Omar et al.,
2012). Apart from the known artemisinin, artediffusin, a sesquiterpene lactone from Artemisia
diffusa has also found to have antimalarial activity (Rustaiyan et al., 2011).

CONCLUSION

The HR culture of A. annua can be a plausible alternative production source of artemisinin or its
derivatives. Further, elicitation through diverse biotic/ abiotic elicitors and up-scaling in bioreactor
enhanced the production of the targeted metabolites. The modulation of the metabolic pathway by
over expression of single/ multiple genes in the HR cultures may lead to the higher production of
therapeutically important antimalarial drug- artemisinin.

REFERENCES

34 | P a g e
Ahlawat S, Saxena P, Alam P, Wajid S, Abdin MZ (2014). Modulation of artemisinin biosynthesis by
elicitors, inhibitor and precursor in hairy root cultures of Artemisia annua L., Journal of Plant
Interactions 9: 811-824.

Ahlawat S, Saxena P, Ram M, Alam P, Nafis T, Mohd A, Abdin MZ (2012). Influence of Agrobacterium
rhizogenes on induction of hairy roots for enhanced production of artemisinin in Artemisia
annua L. plants. African Journal of Biotechnology 11(35): 8684-8691.

Arsenault PR, Wobbe KK, Weathers PJ (2008). Recent advances in artemisinin production through
heterologous expression. Curr Med Chem; 15(27): 2886-2898.

Banerjee S, Zehra M, Gupta M M, Kumar S (1997). Agrobacterium rhizogenes-mediated

transformation of Artemisia annua: Production of Transgenic Plants, Planta Medica63: 467-
469.

Duffy R, Wade C, Chang R. Discovery of anticancer drugs from antimalarial natural products: a
Medline literature review. Drug Discovery Today 2012, 17: 942-953.

Giri A, Ravindra ST, Dhingra V, Narasu ML (2001). Influence of different strains of Agrobacterium
rhizogenes on induction of hairy roots and artemisinin production in Artemisia annua. Current
Science 81:378-382.

Gonzalez LDJ, Weathers PJ (2003) Tetraploid Artemisia annua hairy roots produce more artemisinin
than diploids. Plant Cell Rep. 21:809–81.

Kiani BH, Safdar N, Mannan A, Mirza B (2012). Comparative artemisinin analysis in Artemisia dubia
transformed with two different Agrobacteria harbouring rol ABC genes, POJ 5(4):386-391.

Kim Y, Wyslouzil BE, Weathers PJ (2002). Invited review: Secondary metabolism of hairy root
cultures in bioreactors. In Vitro Cell. Dev. Biol.-Plant 38:1–10.

Kim YJ, Weather PJ, Wyslouzil BE (2003). Growth dynamics of Artemisia annua hairy roots in three
culture systems. Biotechnology and Bioengineering. 83:428-443.

Kim YJ, Weathers PJ, Wyslouzil BE (2002). Growth of Artemisia annua hairy roots in liquid- and gas-
phase reactors. Biotechnology and Bioengineering 80: 454-464.

Liu C, Wang Y, Guo C, Ouyang F, Ye H, Li G (1998). Enhanced production of artemisinin by Artemisia

annua L hairy root cultures in a modified inner-loop airlift bioreactor. Bioprocess Engineering
19:389-392.

Liu CZ, Guo C, Wang YC, Ouyang F (2002). Effect of light irradiation on hairy root growth and
artemisinin biosynthesis of Artemisia annua L. Process Biochemistry 38:581-585.

Mannan A, Shaheen N, Arshad W, Qureshi RA, Zia M, Mirza B (2008) Hairy roots induction and
artemisinin analysis in Artemisia dubia and Artemisia indica. African Journal of Biotechnology
7(18): 3288-3292.

Nguyen KT, Arsenault PR, Weathers PJ (2011). Tricomes + roots+ ROS= artemisinin: regulating
artemisinin biosynthesis in Artemisia annua L.,In vitro cell. Dev. Biol-Plant 47:329-338.

35 | P a g e
Omar MN, Khan NT, Hasali NHM, Moin SF, Alfarra HY (2012). Microbial transformation of artemisinin
–antimalaria Drug, Adv. in Bioresearch 3:27–31.

Pandey P, Kaur R, Singh S, Chattopadhyay SK, Srivastava SK, Banerjee S (2014). Long-term stability in
biomass and production of terpene indole alkaloids by hairy root culture of Rauvolfia
serpentina and cost approximation to endorse commercial realism, Biotechnol. Lett. 36:1523–
1528.

Parshikov IA, Netrusov AI, Sutherland JB (2012). Microbial transformation of antimalarial terpenoids.
Biotechnology Advances, 30:1516–1523.

Patra N, Srivastava AK (2014) Enhanced production of artemisinin by hairy root cultivation of

Artemisia annua in a modified stirred tank reactor. App Biochem Biotechnol 174:2209-2222.

Patra N, Srivastava AK, Sharma (2013). Study of various factors for enhancement of artemisinin in
Artemisia annua hairy roots. International Journal of Chemical Engineering and Applications
4:157-160.

Putalun W, Luealon W, De-Eknamkul W, Tanaka H, Shoyama Y (2007). Improvement of artemisinin

production by chitosan in hairy root cultures of Artemisia annua L. Biotechnol Lett, 29: 1143–
1146.

Rustaiyana A, Masoudib S (2011). Chemical constituents and biological activities of Iranian Artemisia
species. Photochemistry Letter, 4:440–447.

Sivakumar G, Liu C, Towler MJ, Weathers PJ (2010). Biomass production of hairy roots of Artemisia
annua and Arachis hypogaea in a scaled-up mist bioreactor. Biotechnol Bioeng. 107(5): 802–
813.

Smith TC, Weathers PJ, Cheetham RD (1997) Effects of gibberellic acid on hairy root cultures of
Artemisia annua: growth and artemisinin production. In Vitro Cell. Dev. Biol. 33:75-79.

Souret FF, Kim Y, Wyslouzil BE, Wobbe KK, Weathers PJ (2003). Scale-up of Artemisia annua L. hairy
root cultures produces complex patterns of terpenoid gene expression. Biotechnology and
Bioengineering, 83:6,653-667.

Wang JW, Tan RX (2002). Artemisinin production in Artemisia annua hairy root cultures with
improved growth by altering the nitrogen source in the medium. Biotechnology Letters 24:
1153–1156.

Wang JW, Zhang Z, Tan RX (2001) Stimulation of artemisinin production in Artemisia annua hairy
roots by the elicitor from the endophytic Colletotrichum sp. Biotechnology Letters 23: 857–
860.

Wang JW, Zheng LP, Zhang B, Zou T (2009). Stimulation of artemisinin synthesis by combined
cerebroside and nitric oxide elicitation in Artemisia annua hairy roots. Appl. Microbiol.
Biotechnol. 85: 285–292.

36 | P a g e
Wang Y, Zhang H, Zhao B, Yuan X (2001). Improved growth of Artemisia annua L hairy roots and
artemisinin production under red light conditions. Biotechnology Letters 23:1971–1973.

Weathers PJ, Gonzalez LD, Kim YJ, Souret FF, Towler MJ (2004). Alteration of biomass and
artemisinin production in Artemisia annua hairy roots by media sterilization method and
sugars. Plant Cell Rep 23:414–418.

Weathers PJ, Hemmavanh DD, Walcerz DB, Cheetham RD, Smith TC (1997). Interactive effects of
nitrate and phosphate salts, sucrose, and inoculum culture age on growth and sesquiterpene
production in Artemisia annua hairy root cultures. In Vitro Cell. Dev. Biol. Plant 33:306-312.

Wyslouzil BE, Waterbury RG, Weathers PJ (2000). The growth of single roots of Artemisia annua in
nutrient mist reactors. Biotechnology and Bioengineering, 70:144-150.

Zheng LP, Guo YT, Wang JW, Tan RX (2008). Nitric oxide potentiates oligosaccharide-induced
artemisinin production in Artemisia annua hairy roots. Journal of Integrative Plant Biology 50
(1): 49–55.

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 In vitro production of Artemisinin acid and their derivatives

Anand Mishra, Pankhuri Gupta, Dr Sunita Singh Dhawan

Department of Plant Biotechnology, Central Institute of Medicinal and Aromatic Plants (CSIR-
CIMAP), PO CIMAP, Kukrail Picnic Spot Road, Lucknow 226015, India
Email: mishra.anand653@gmail.com

ABSTRACT

Globally, malaria causes death to 1 million people annually. The malaria parasite,
Plasmodium falciparum, has multidrug resistance which causes difficulty in developing
vaccines. Artemisinin is a valuable drug for the cure of malaria obtained from the plant,
Artemisia annua. Having wide spectrum use as anti- parasitic and anti- viral, the demand of
this chemical is on the rise worldwide. Due to its low production, there is needed to develop
certain novel methods to synthesize artemisinin. This review summarizes the research work
done for in vitro artemisinin production. .

INTRODUCTION

Artemisia annua is an annual green herb of family Asteraceae, a native of China, where it is
known as qinghao and used for over 2,000 years to treat symptoms like fever and malaria. In
United States, it is called as sweet Annie, annual or sweet wormwood (Ferreira et al., 1997).
Chinese researchers were first to find out antimalarial activity of diethyl ether extract of A.
annua followed by the isolation of artemisinin. A. annua is main source of artemisinin among
approx.. 400 species of Artemisia (Namdeo et al 2006). Artemisinin is a sesquiterpene lactone
with an unusual endoperoxide structure. Artemisinin inhibits the sarco/endoplasmic reticulum
Ca2+ ATPase (SERCA) of P. falciparum after activation by iron ions. Artemisinin and its
derivatives are also known to have anti-schistosomal, anti-viral and anti-cancer activities.
(Xiao et al., 2005; Marin et al., 2006; Efferth et al., 2006)

Artemisnin biosynthesis
The biosynthesis pathway of artemisinin has been studied from last many years, but the
whole process is not completely understood. The first committed step in the biosynthesis of
artemisinin is the cyclization of farnesyl diphosphate (FPP) by amorphadiene synthase (ADS)
to amorphadiene (Spamela et al., 2006). The precursor, IPP, originates from two independent
pathways: the mevalonate pathway (MVA) originating from acetyl CoA, and the mevalonate
independent pathway (MEP) stemming from pyruvate. Sugars that are produced in
chloroplast by photosynthesis are converted to acetyl-CoA in cytosol in plants. Acetyl-CoA is
feeder for cytosolic mevalonate pathway, which produces farnesyl diphosphate (FPP), further
cyclised to amorphadiene and subsequently enzymatically oxidized to artemisinic acid or
dihydroartemisinic acid, which may be accumulated to different levels according to genotype.
Dihydroartemisinic acid is the precursor of artemisinin which is thought to be non-enzymatic
photo-catalytic process.

Production of Artemisinin in vitro.

38 | P a g e
A. annua plant can be easily grown in synthetic medium (Ferreira et al., 2009) without
any hormone addition. Shoot proliferation can be stimulated by the addition of
cytokinins in the media, but decreases rooting with simultaneous increase in
vitrification. Artemisinin is produced in shoots in vitro and is enhanced by the presence
of roots (Ferreira et al., 1996). Most attempts reported for stimulating artemisinin
production, however, have not been encouraging (Patrick et al., 2008). None or trace
levels of artemisinin were found in roots, callus, cells, or cell free medium. Rao et al.,
(1998) showed that transformed root cultures produced artemisinin when transferred
from dark to light conditions, and concluded that photosynthesis and chloroplasts are
involved in the biosynthesis of artemisinin.

Heterologous expression
Different groups tried to reconstitute the steps of biosynthetic pathway of artemisinin in E
coli. Basically, two enzymes specific to artemisinin biosynthesis, ADS and CYP71AV1,
have been targeted for heterologous expression and genetic engineering mainly in microbes.
In E. coli, only MEP pathway is found which produces FPP and its derivatives and the
optimization of this pathway is a viable means to increase FPP availability (Keasling JD,
2006, 2007). Martin et al., 2003 engineered mevalonate pathway for IPP production by codon
optimization from yeast into E. coli for 10–36 fold yield and improvement in amorphadiene
production compared to wild type ADS mRNA sequences. Yeast is an eukaryotic oraganism
having mevalonate pathway for the biosynthesis of isoprenoids and sterols (Paltauf et al
1992). The cytosolic acetoacetyl‑CoA converts to IPP by five enzymatic steps via the
intermediate mevalonate (MEV). IPP is isomerized to DMAPP by IPP isomerase, and the two
molecules are combined to form larger isoprenoids, such as farnesyl diphosphate (FPP).
Keasling et al., 2006 reported S. cerevisiae can produce higher amount of artemisinic acid (up
to 100 mg/per litre) by an engineered mevalonate pathway, amorphadiene synthase, and a
novel cytochrome P450 monooxygenase (CYP71AV1) from A. annua that performs a three-
step oxidation of amorphadiene to artemisinic acid. Synthetic artemisinic acid was seen to be
transported out of cell and retained in medium which could be easily purified and used to
obtain the desired product. These processes need further optimisation and could be scaled up
to produce artemisinin commercially.

CONCLUSION

In present scenario, there is an urgent need for antimalarial drugs that are affordable and
effective against multi-drug resistant malaria, and that have low toxicity. Different groups are
working globally to increase production of artemisinin and related drugs. Breeders and
researchers are trying to produce new varieties and transgenics which produce more drug
molecules in comparison to traditional one. Heterologous systems have made good
contribution for production of artemisinin to meet the world’s demand but there is a need of
further optimisation. There is a need to fill the gaps in the biosynthetic pathway and its
associated steps that can provide new opportunities. Synthetic chemistry could also prove to
be beneficial if applicable in a cost effective manner.

REFERENCES

39 | P a g e
Rao, K.V., Venkanna, N., Narasu, M.L. 1998. Agrobacterium rhizogenes mediated
transformation of Artemisia annua. Journal of Scientific and Industrial Research 57:773-
776
Paltauf F., Kohlwein, SD. & Henry, SA. 1992. The Molecular and Cellular Biology of the Yeast
Saccharomyces 425–500 Cold Spring Harbor Laboratory Press,
Ro DK,. Paradise E. M, Ouellet M,. FisherKJ, NewmanK, NdunguJ, Ho KA, Eachus RA, Ham
TS, KirbyT, Chang M,. WithersS, Shiba Y, Sarpong R & Keasling JD 2006 Production of
the antimalarial drug precursor artemisinic acid in engineered yeast Nature 440, 940-943
Efferth T. Molecular pharmacology and pharmacogenomics of artemisinin and its derivatives in
cancer cells. Curr Drug Targets. 2006; 7:407–21.
Ferreira J & Janick J 2009 Annual Wormwood (Artemisia annuaL.) New Crop factsheet
Ferreira JS, Simon JE, Janick, J. 1997. Artemisia annua: botany, horticulture, pharmacology (A
Review). Horticultural Reviews 19:319–371.
Ferreira, J.F.S. and J. Janick. 1996. Roots as an enhancing factor for the production of artemisinin
in shoot cultures of Artemisia annua. Plant Cell Tissue Organ Cult. 44:211-217.

Martin VJ, Pitera DJ, Withers ST, Newman JD, Keasling JD. 2003 Engineering a mevalonate
pathway in Escherichia coli for production of terpenoids. Nat Biotechnol 2003;21:796–802.

Namdeo AG, Mahadik KR., and. Kadam SS, 2006.Antimalarial drug-Artemisia

annua,Pharmacognosy Magazine, vol. 2, no. 6, pp. 106–111,
Patrick R. Arsenault, Kristin K. Wobbe 2 , and Pamela J. 2008 Weathers Recent Advances in
Artemisinin Production Through Heterologous Expression Curr Med Chem. 2008; 15(27):
2886.
Pitera D, Newman KL, Keasling JD ,2006 : High-level production of amorpha-4,11-diene in a
two-phase partitioning bioreactor of metabolically engineered Escherichia coli. Biotechnol
Bioeng, 95:684-691
Pitera DJ, Paddon CJ, Newman JD, Keasling JD, 2007: Balancing a heterologous mevalonate
pathway for improved isoprenoid production inEscherichia coli. Metab Eng, 11:193-207.
28. Newman JD, Marshall J, Chang M, Nowroozi F, Paradise E,
Romero MR, Serrano MA, Vallejo M, Efferth T, Alvarez M, Marin JJ. Antiviral effect of
artemisinin from Artemisia annua against a model member of the Flaviviridae family, the
bovine viral diarrhea virus (BVDV) Planta Med. 2006; 72:1169–74.
Spamela J. Weathers, Shereen Elkholy1 , Andkristin K. Wobbe, 2006 Artemisinin: The
Biosynthetic Pathway And Its Regulation Inartemisia Annua, A Terpenoid-Rich Specie In
Vitro Cell. Dev. Biol.—Plant 42:309
Xiao SH. Development of antischistosomal drugs in China, with particular consideration to
Praziquantel and the Artemisinins. Acta Trop. 2005; 96:153–67.

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 Metabolic engineering approaches for enhanced artemisinin production
Sunita Jindal, Bendangchuchang Longchar and Vikrant Gupta*
Biotechnology Division, CSIR-Central Institute of Medicinal and Aromatic Plants, P.O.
CIMAP, Lucknow 226 015
Email: sunitajindal20@gmail.com

INTRODUCTION

In order to defend against various stresses like herbivory, fungal and bacterial infections,
plants synthesize a wide array of secondary metabolites. Most of these compounds have
immense pharmaceutical importance to humans. Such plants are subject to in-depth studies
and exploration ever since the beginning of human civilization. Artemisia annua L. (Family:
Asteraceae), known for the world’s most prominent anti-malarial drug ‘artemisinin’, is one of
such plants. A. annua is a native of China where it is known as ‘quinghao’. Its extract has
long been used in Chinese herbal preparations to treat many diseases such as fever,
tuberculosis, illness caused by worms, parasites and common cold. Its major recommended
use is against malaria which is one of the most devastating global health problems. The
World Health Organization (WHO) has recommended the use of Artemisinin-based
Combination Therapies (ACTs) to treat malaria caused by Plasmodium falciparum and P.
vivax, including multi-drug-resistant strains. However, the amount in which artemisinin is
naturally produced by A. annua (~0.3-0.8% DW) is too short to meet the needs of over 100
million courses of ACTs per year world-wide (Mutabingwa 2005). Therefore, enormous
research efforts have been put to enhance the yield of this vital compound and understanding
of the biosynthetic pathway leading to the formation of artemisinin serves as the fundamental
part of these research studies.

Artemisinin biosynthesis
Artemisinin is biosynthesized in the glandular trichomes present on the aerial surfaces of the
plant. The pathway of artemisinin biosynthesis includes complex multi-step enzymatic
reactions which have been investigated for many years because of so far non-replaceable
medicinal importance of artemisinin. The precursors of artemisinin are isopentenyl
diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMADP) (Croteau et al. 2000).
In plastids, IPP and DMADP are synthesized by condensation of pyruvate with D-
glyceraldehyde-3-phosphate. This condensation is catalyzed by deoxy-D-xylulose-5-
phosphate synthase (DXPS) and 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXPR)
sequentially leading to the formation of IPP and DMADP. IPP is synthesized in cytosol also
through a different pathway using acetyl-coA. The enzymes employed in the synthesis of
cytosolic IPP include 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS) and 3-hydroxy-3-
methylglutaryl-CoA reductase (HMGR). The condensation of IPP and DMADP in plastid
leads to the formation of linear molecule geranyl pyrophosphate (GPP) by the action of GPP
synthase (GPPS) (Schramek et al. 2010). The GPP is transferred into the cytosol where it
elongates further using IPP to form farnesyl pyrophosphate (FPP) in FPP synthase (FPPS)
dependent manner. FPP is further channelized into two competitive processes of making
either sesquiterpenes or sterols. The subsequent steps leading to synthesis of artemisinin, a
sesquiterpene, take place in glandular trichomes where the genes encoding the necessary
enzymes are expressed. The first committed step towards artemisinin biosynthesis is the
conversion of FPP into amorpha-4,11-diene which is catalyzed by amorpha-4,11-diene
synthase enzyme, encoded by ADS gene. The next three enzymatic steps after the formation
of amorpha-4,11-diene are catalyzed by a cytochrome P450 (CYP/P450) enzyme which is

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encoded by CYP71av1 in A. annua (Teoh et al. 2006). It shows both dehydrogenase and
reductase activities and catalyzes the oxidation of the amorpha-4,11-diene, artemisinic
alcohol and artemisinic aldehyde. Cytochrome P450 reductase (CPR) works synergistically
with CYP71av1. At some point, the double bond of amorpha-4,11-diene is reduced by
artemisinic aldehyde Δ11(13) reductase enzyme (encoded by DBR2). The double bond is
thought to occur first in artemisinin aldehyde but when it is expressed in yeast alongwith
other artemisinin biosynthetic pathway genes, dihydroartemisinic acid is accumulated (Zhang
et al. 2008). Further, aldehyde dehydrogenase enzyme encoded by ALDH1 converts
artemisinic aldehyde into artemisinic acid, an activity also ascribed to CYP. ALDH1 also
converts dihydroartemisinic aldehyde into artemisinic acid and dihydro-artemisinic acid. The
subsequent steps of conversion of artemisinic acid into artemisinin are not yet known.
According to Brown et al. (2007), artemisinic acid is converted non-enzymatically into many
molecules including artennuin B. The trichome-specific enzymatic steps leading to
artemisinin are depicted in Figure 1.

Cytosol FPP

ADS
DBR2
Amorpha-4,11 - Artemisinic Artemisinic Dihydroartemisinic
diene alcohol aldehyde aldehyde
CYP71av1/CPR CYP71av1/CPR
CYP71av1 ALDH1
ALDH1
Secretory cells Artemisinic acid
Dihyroartemisinic
acid
Trichome sunlight sunlight

Subcuticular space Arteannuin B Artemisinin

Figure 1: Artemisinin biosynthetic pathway subsequent to FPP

Genetic modulations in homologous or heterologous systems

The understanding of the biosynthetic pathway has facilitated metabolic engineering in
heterologous and homologous systems. The in planta approaches to improve artemisinin
content include the improvement of A. annua plant by overexpressing the pathway genes,
suppressing the squalene synthase gene, and, through heterologous/homologous gene
expression in A. annua and tobacco. The overexpression of Gossypium arboreum farnesyl
diphosphate synthase (FPPS) gene in A. annua increased the artemisinin content by 2- to 3-
folds in transgenic plants (Chen et al. 2000). The overexpression of Catharanthus roseus
HMGR in A. annua led to the production of 22.5% more artemisinin as compared to wild type
(Aquil et al. 2009). Similarly, the overexpression of A. annua CYP71av1 and its redox partner
CPR led to 38% higher artemisinin content in transgenic plants (Shen et al. 2012). Chen et al.
(2012) showed that co-overexpressing FPS, CYP71av1 and CPR genes lead to even higher
amounts of artemisinin content (3.6-fold) than that of control plant. Since sterol and
artemisinin biosyntheses are competitive pathways, efforts were invested to suppress the
squalene synthase (SQS), a key gene of sterol biosynthetic pathway. It was observed that in
RNAi lines of A. annua, artemisinin content got increased by 3.14-fold as compared to
untransformed plants (Zhang et al. 2009). In order to utilize heterologous plant system for
artemisinin production, the ADS and CYP71av1 genes from A. annua were expressed in
tobacco which led to the accumulation of amorphadiene and artemisinic alcohol. The
expression of DBR2 led to additional accumulation of dihydroartemisinic alcohol. The
artemisinic acid however, could not be produced because of the reduction favoring

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biochemical environment in tobacco cells (Zhang et al. 2010). Such approaches serve as
model for metabolic engineering of the plant for improvement of desired secondary
metabolite. Apart from the in planta metabolic engineering approaches, transferring
metabolic pathways in genetically modifiable industrial biological hosts (E. coli and yeast)
and developing heterologous system has emerged as much more attractive alternative to
produce important secondary metabolites in huge amounts. For the same reason, the Semi-
synthetic Artemisinin Project (Zhang 2005) was started to metabolically engineer a
microorganism which can produce a late stage precursor of artemisinin (artemisinic alcohol),
followed by chemical (in vitro) conversion into artemisinin. One of the earliest attempts of
synthetic biology application for artemisinin production was made by engineering the
expression of a synthetic ADS gene and the mevalonate isoprenoid pathway from S.
cerevisiae in E. coli (Martin et al. 2003). The ADS gene was codon optimized for expression
in E. coli. The mevalonate pathway was carried by E. coli on two plasmids encoding the
mevT operon and mevB operon. The mevT operon comprises three genes viz. atoB, ERG13
and tHMG1 which are required for the conversion of acetyl-CoA to mevalonate while the
mevB operon consists of idi, ispA, MVD1, ERG8 and ERG12 genes which are needed for the
conversion of mevalonate to FPP. The production of amorphadiene reached to 24 μg
caryophyllene equivalent/ml or 112 mg/L in the E. coli culture. By utilizing the volatility of
amorphadiene and trapping it in off-gas by using a two-phase partitioning bioreactor, the
amorphadiene production was further enhanced to 0.5 g/L (Newman et al. 2006). Steps
subsequent to amorphadiene were difficult to recapitulate in E. coli, and therefore it was
decided to switch to S. cerevisiae to produce artemisinic acid. Westfall et al. (2012)
overexpressed all the genes of mevalonate pathway including ERG20 in S. cerevisiae strain
CEN.PK2, which upon the development of fermentation process led to the production of >40
g/L amorphadiene. In the engineered CEN.PK2 strain, ADS, CYP72AV1 and CPR1 were
expressed through a single plasmid, which converted amorphadiene into artemisinic alcohol.
Two more A. annua genes ADH1 and ALDH1 which are required for conversion to
artemisinic acid were introduced into yeast chromosome. All A. annua genes were synthetic
and codon optimized for expression in yeast. The final step of artemisinic acid to artemisinin
required chemical conversion for which a novel chemical process was developed which
yielded 25 g/L of artemisinic acid of reasonable purity (Paddon et al. 2013). All these
approaches of metabolic engineering of plants and microorganisms have been demonstrated
to be used as efficient tools and can be adopted as model techniques to bring about the
desirable outcomes.

REFERENCES

Aquil S, Husaini AM, Abdin MZ and Rather GM (2009) Overexpression of the HMG-CoA reductase
gene leads to enhanced artemisinin biosynthesis in transgenic Artemisia annua Plants. Planta
Med 75:1453-1458.
Brown GD and Sy LK (2004) In vivo transformations of dihydroartemisinic acid in Artemisia annua
plants. Tetrahedron 60:1139-1159.
Chen D, Ye H and Li G (2000) Expression of a chimeric farnesyl diphosphate synthase gene in
Artemisia annua L. transgenic plants via Agrobacterium tumefaciens-mediated transformation.
Plant Sci 155:179-185.
Chen Y, Shen Q, Wang Y, Wang T, Wu S, Zhang L, Lu X, Zhang F,Jiang W, Qiu B, Gao E, Sun X
and Tang K (2012) The stacked over-expression of FPS, CYP71AV1 and CPR genes leads to
the increase of artemisinin level in Artemisia annua L. PlantBiotechnol Rep 7:287-295.
Croteau R, Kutchan TM and Lewis NG (2000) Natural products (secondary metabolites). In:
Biochemistry and Molecular Biology of Plants. Eds. Buchanan B, Gruissem W and Jones RL.
pp 1250-1318. American Society of Plant Physiologists, Rockville, MD.

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Martin VJ, Pitera DJ, Withers ST, Newman JD and Keasling JD (2003) Engineering a mevalonate
pathway in Escherichia coli for production of terpenoids. Nat Biotechnol 21:796-802.
Mutabingwa TK (2005) Artemisinin-based combination therapies (ACTs): best hope for malaria
treatment but inaccessible to the needy! Acta Tropica 95:305-315.
Newman JD, Marshall J, Chang M, Nowroozi F, Paradise E, Pitera D, Newman KL and Keasling JD
(2006). High-level production of amorpha-4,11-diene in a two-phase partitioning bioreactor of
metabolically engineered Escherichia coli. Biotechnol Bioeng 95:684-691.
Paddon CJ et al (2013). High-level semi-synthetic production of the potent antimalarial artemisinin.
Nature 496:528-532.
Schramek N, Wang HH, Romisch-Margl W, Keil B, Radykewicz T,Winzenhorlein B, Beerhues L,
Bacher A, Rohdich F, Gershen-zon J, Liu BY and Eisenreich W (2010) Artemisinin
biosynthesis in growing plants of Artemisia annua. A (CO2)-C-13 study. Phytochemistry
71:179-187.
Shen Q, Chen YF, Wang T, Wu SY, Lu X, Zhang L, Zhang FY, Jiang WM, Wang GF and Tang KX
(2012) Overexpression of the cytochrome P450 monooxygenase (cyp71av1) and cytochrome
P450 reductase (cpr) genes increased artemisinin content in Artemisia annua (Asteraceae).
Genet Mol Res 11:3298-3309
Teoh KH, Polichuk DR, Reed DW, Nowak G and Covello PS (2006) Artemisia annua L. (Asteraceae)
trichome-specific cDNAs reveal CYP71AV1, a cytochrome P450 with a key role in the
biosynthesis of the antimalarial sesquiterpene lactone artemisinin. FEBS Lett 580:1411-1416
Zhang JF (2005) A detailed chronological record of project 523 and the discovery and development of
qinghaosu (artemisinin). Yang Cheng Evening News Publishing Company, China.
Zhang Y, Teoh KH, Reed DW, Maes L, Goossens A, Olson DJ, Ross AR and Covello PS (2008) The
molecular cloning of artemisinic aldehyde ∆11(13) reductase and its role in glandular trichome-
dependent biosynthesis of artemisinin in Artemisia annua. J Biol Chem 283:21501-21508.
Zhang L, Jing F, Li F, Li M, Wang Y, Wang G, Sun X and Tang K (2009) Development of
transgenic Artemisia annua (Chinese wormwood) plants with an enhanced content of
artemisinin, an effective anti-malarial drug, by hairpin-RNA-mediated gene silencing.
Biotechnol Appl Biochem 52:199-207.
Zhang Y, Nowak G, Reed DW and Covello PS (2010) The production of artemisinin precursors in
tobacco. Plant Biotechnol J 9:445-454.

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 Molecular marker study in Artemisia annua for high yielding artemisinin content

Susheel Kumar Singh, Sunita Singh Dhawan

Plant Biotechnology Division, CSIR-CIMAP, Kukrail Picnic spot road, P.O. CIMAP,
Lucknow.
Email: susheelbt05@gmail.com

ABSTRACT

Artemisia, one of the larger genera in the family Asteraceae and the largest genus in the tribe
Anthemideae, comprises from 200 to more than 500 taxa at the specific or subspecific level.
It is widely used as an antimalarial agent. RAPD-PCR could be very useful in the
development of SCAR (Sequence Characterized Amplified Region) marker. SCAR markers
could be very specific, which allows detection and authentication of specific plants species.
The MAP-12 marker was found to be linked with the higher yield of artemisinin in Artemisia
annua var. CIM-Arogya. The developed SCAR marker was able to detect more than 0.4 %
artemisinin.

INTRODUCTION

Artemisia annua L. is a source of artemisinin, an antimalarial agent (Klayman et al., 1985).

Artemisia herbs are used for various purposes, such as medicine, food, spices and
ornamentation purposes (Lee et al., 2006). The relatively low content of artemisinin in
cultivated types of A. annua has been a limiting factor for its commercialization. Enhanced
production of artemisinin either in cell/tissue culture or in the whole plant of A. annua is
therefore highly desirable. To study the genomic constitution of plants and applying genetic
engineering methodology to develop new varieties, concept of molecular markers came into
existence (Bennetzen et al., 2000).

Artemisinin occurs in the plant in very small quantities. Therefore, economic and practical
considerations dictate that plants with maximum content of artemisinin found to increase
their artemisinin content be sought. The key to selection and breeding is to have a
comprehension of chemical and genetic variability in the available populations and stocks
and suitable selection(s) of elite clones for cultivation per se or to employ them as parental
types for scoring the promising plant in the progeny or its further pedigreed population.
Availability of biochemical and/or genetic markers related to artemisinin as well as essential
oil content will also be of great help in the genetic improvement programs (Sangwan et al.,
1999).

Molecular Marker

Polymorphism involves the existence of different forms (alleles) of the same gene in a plant
or a population of plants. These differences are tracked as molecular markers to identify
desired genes and the resulting trait. Most organisms are diploid, means they have two copies
of each gene, one from each parent. One gene usually dominates the other thus determining
the inherited trait. Randomly Amplified Polymorphic DNA (RAPD) is easy to develop and
simple molecular marker, but lack of reproducibility makes it less reliable for authentication
of herbal drugs. Besides RAPD, other popular PCR and non-PCR based markers like AFLP,
ISSR, SSR and RFLP are also used for authentication. However, these also have
disadvantages like use of radioactive isotopes, high cost and absolute requirement of

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sequence information. Therefore, it is a better option to improve the reproducibility of RAPD
by converting RAPD amplicons into Sequence Characterized Amplified Region (SCAR)
markers. A SCAR represents a specific region of the genome that is amplified by PCR using
a pair of specific oligonucleotide primers. These are identified as distinct single band in
agarose gel electrophoresis. SCAR markers are easy, reliable and reproducible, thus, have an
advantage over RAPD markers for authentication of medicinal herbs used in the preparation
of traditional medicines. These markers however, have been developed for only few
medicinal herbs.

Utility of SCAR marker in Artemisia

The role of SCAR markers in authentication of medicinal herbs used in traditional

formulations (Kiran et al., 2010). Based on nucleotide sequence of the primer 329 product,
designed a Fb (5-CAT CAA CCA TGG CTT ATC CT-3) and R7 (5-GCG AAC CTC CCC
ATT CCA-3) primer-set to amplify a 254 bp sized SCAR (sequence characterized amplified
regions) marker, through which A. princeps and A. argyi can be efficiently discriminated
from other Artemisia herbs, particularly, A. capillaris and A. iwayomogi (Lee et al., 2006).

SCAR marker for high yielding artemisinin

The development of SCAR marker for high artemisinin content was possible through RAPD
profile with MAP-12 primer at CSIR-CIMAP. The 850 bp sequence generated by of MAP-12
primer was very specific to the artemisinin content, which consistently showed its presence in
the genotypes containing more than 0.4% artemisinin and absent in the genotypes with trace
or no artemisinin. The certainly the DNA band of about 850 base pair size could be correlated
with the biosynthesis of more than 0.4% artemisinin in Artemisia annua var. CIM-Arogya
(Khanuja et al.,2005).

The genetic marker for artemisinin content was screened using random amplified
polymorphic DNA (RAPD) and sequence characterized amplified region (SCAR) techniques.
The random primer OPA15 (5'-TTCCGAACCC-3') could amplify a specific band of
approximately 1000 bp that was present in all high-artemisinin yielding strains, but absent in
all low-yielding strains in three independent replications. This specific band was cloned and
its sequence was analyzed. This RAPD marker was converted into a SCAR marker to obtain
a more stable marker (Lee et al., 2006).

CONCLUSION

The potential use of SCAR markers were in the identification of correct genotype of
Artemisia species. The developed SCAR markers for artemisinin content were highly specific
and accurate. The SCAR marker can be developed for other important agronomic traits.

REFERENCE

Bennetzen, J.L., (2000) Comparative sequence analysis of plant nuclear genomes:

microcolinearity and its many exceptions. Plant Cell 12:1021-9.
Khanuja, S.P.S., Paul, S., Shasany,A.K., Gupta, A.K., Darokar, M.P., Gupta, M.M., Verma, R.K.,
Ram,G., Kumar, A., Lal, R.K., Bansal ,RP, Singh AK,Bhakuni RS, Tandon S (2005)
High Artemisinin yielding plant genotype CIM-Arogya’ Pub. N0. : US 2005/0223447P1.

46 | P a g e
 Kiran, U., Khan, S., Mirza, K.J., Ram, M., Abdin, M.Z., (2010) SCAR markers: A
potential tool for authentication of herbal drugs. Fitoterapia 969-976
Klayman, D.L., (1985) Qinghaosu (artemisinin): an antimalarial drug from China. Science 1049-
1055.
Lee, M.Y., Doh, E.J., Park, C.H., Kim, Y.H., Kim, E.S., Ko, B.S. and Oh, S.E., (2006).
Development of SCAR marker for discrimination of Artemisia princes and A. argyi from
other Artemisia herbs. Biol. Pharm. Bull. 629-633.
Sangwan, R. S., Sangwan, N. S., Jain, D. C., and Ranade S. A., (1999) RAPD profile based
genetic characterization of chemotypic variants of Artemisia annua. Bioch. and Mol. Bio.
Inter. 935-944
Zhang, L., Ye, H.C., and Li, G.F., (2006) Effect of development stage on the artemisinin content
and the sequence characterized amplified region (SCAR) marker of high-artemisinin
yielding strains of Artemisia annua L. J. of Int. Pl. Bio 1054−1062

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 OMICS approaches for Withania somnifera: A need for exploration

Sanchita, Ashok Sharma*

Biotechnology Divison, CSIR-Central Institute of Medicinal and Aromatic Plants, Lucknow,
India
Email: 0804sanchita@gmail.com

ABSTRACT

This mini review represents the biological information available till date in one of the
important medicinal plant, Withania somnifera. Various researches covered in omics studies
have been explained to make the wide usefulness of available biological data. More
exploration of this plant is the requirement of the time for the best use of secondary metabolic
compounds.

W. somnifera is one of the important medicinal plants of Solanaceae family. This plant is also
known as Ashwagandha, winter cherry and Indian ginseng. It forms an essential constituent
of over 100 traditional medicinal formulations for different pharmacological activities [1].
The medicinal importance of W. somnifera is due to presence of diverse secondary
metabolites. The biologically active chemical constituents of W. somnifera include alkaloids
(isopelletierine, anaferine, cuseohygrine, anahygrine, etc.), steroidal lactones (withanolides,
withaferins) and saponins [2]. These metabolites are synthesized in almost every tissue of the
plant. The omics study is introduced in the biological sciences to better understand and
explore the available biological information. High-throughput 'omics' technologies (such as
genomics, transcriptomics and proteomics) are enabling detailed studies of its molecular
changes and revealing information about various biological pathways [3].

Although W. somnifera is very well characterized in terms of phytochemical profiles and

pharmaceutical activities. The gene and proteins involved in the synthesis of these
pharmaceutically active constituents are very limited. Although the genomic information of
W. somnifera is being continuously expanding, the basic steps of withanolide biosynthetic
pathway are still unknown. The precursor for withanolides is isoprenoids, they are
synthesized from classical cytosolic mevalonate (MVA), and plastid localized MEP pathway
leading to synthesis of IPP. The cloning and functional characterization of genes involved in
the different steps of withanolide biosynthesis such as 3-hydroxy-3-methylglutaryl coenzyme
A reductase (HMGR) [4], 1-deoxy-D-xylulose-5-phosphate synthase (DXS) and 1-deoxy-D-
xylulose-5-phosphate reductase (DXR) [5], farnesyl pyrophosphate synthase (FPPS) [6],
squalene synthase (SQS) [7, 8], squalene epoxidase (SQE) [9] and sterol methyl transferase
(SMT1) and obtusifoliol-14 -demethylase (ODM) [10]. HMGR is the first rate-limiting
enzyme that catalyzes the key regulatory step of MVA pathway affecting the biosynthesis of
withanolides. DXS and DXR genes are involved in MEP pathway for isoprenoid synthesis.
The FPPS gene acts in the first committed reaction of several branched pathways from
isoprenoids. SQS and SQE are involved in sesquirerpenoid and tritepenoid pathway and
divert the carbon flux from the terpenoid backbone pathway towards sterol biosynthesis.
SMT1 and ODM are involved in steroid biosynthesis. All the mentioned genes lie in the
upstream of withanogenesis. Although they show significance in withanolide biosynthesis but
there is no report available showing substrate and product relationship. Functional genomics
is related to the study of expression analysis of genes. GEO database of NCBI have only one
data on gene expression on W. somnifera analyzed from microarray technique [11].

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Transcriptomics is used to analyze the coding and non-coding RNAs and their expression
profiles and for W. somnifera two transcriptome sequence data are available in SRA database
of NCBI. The data were from leaf and root tissues using 454-sequencing platform [12] and
leaf transciptome treated with salicylic acid [13]. Few putative genes such as cytochome
P450s, glycosyltransferase (GTs) and methyltransferases are reported to be involved in
biosynthesis from 24-methylene cholesterol. 24-methylene cholesterol is considered to be the
prcursor for different withalide biosynthesis.

In terms of proteomic analysis, the prediction of three-dimensional structure of various

known proteins of W. somnifera has been analyzed. Various enzymes involved in withanolide
biosynthesis were studied for deducing the stuctural informaion from the sequence. HMGR,
DXS, DXR, FPPS, SQS, SQE, and CAS have been reported for their predicted structures
[14]. The structural information of SQE is also mentioned in another report [9]. The role of
squalene synthase enzyme is important which acts as a key regulator in flow of carbon flux
from main isoprenoid pathway towards sterol biosynthesis. The structural motif analysis have
also been analyzed showing binding site for the substrate, FPP, NADP(H) and Mg ++ [15].

The complete biosynthetic pathways of withanolides are still unclear and it is supposed to be
derived from cholesterol [16]. On the other hand, many biosynthetic pathways are linked
together to form a precursor that initiates the synthesis of secondary metabolites in Withania
somnifera. The synthesis starts from terpenoid backbone biosynthesis resulting in the
formation of farnesyl diphosphate (FPP). FPP is taken as a precursor for sesquiterpenoid and
triterpenoid biosynthesis that forms squalene 2, 3 epoxidase as the end product. The steroid
biosynthesis initiates with 2, 3 squalene epoxidase and results in formation of 24-methylene
cholesterol. 24-methylene cholesterol plays important role in the initiation steps of
withanolides biosynthesis. As the synthesis of withanolides occur in different tissues of the
plant rather than its transportation from one tissue to another. Therefore, it is necessary to
understand the complete metabolic pathway in every aspect of omics. The characterization of
all genes as well as proteins for deciphering complete biosynthetic pathway is required for
better understanding of withanolide biosynthesis in W. somnifera. The research in its
genomic, transcriptomic and proteomic area is necessary to facilitate the understanding about
secondary metabolism.

REFERENCES

[1] M. Kaileh, W. Vanden Berghe, E. Boone, T. Essawi, G. Haegeman, Screening of indigenous

Palestinian medicinal plants for potential anti-inflammatory and cytotoxic activity, J
Ethnopharmacol, 113 (2007) 510-516.
[2] N. Singh, M. Bhalla, P. de Jager, M. Gilca, An overview on ashwagandha: a Rasayana
(rejuvenator) of Ayurveda, Afr J Tradit Complement Altern Med, 8 (2011) 208-213.
[3] A.M. Valdes, D. Glass, T.D. Spector, Omics technologies and the study of human ageing, Nat
Rev Genet, 14 (2013) 601-607.
[4] N. Akhtar, P. Gupta, N.S. Sangwan, R.S. Sangwan, P.K. Trivedi, Cloning and functional
characterization of 3-hydroxy-3-methylglutaryl coenzyme A reductase gene from Withania
somnifera: an important medicinal plant, Protoplasma, 250 (2013) 613-622.
[5] P. Gupta, A.V. Agarwal, N. Akhtar, R.S. Sangwan, S.P. Singh, P.K. Trivedi, Cloning and
characterization of 2-C-methyl-D-erythritol-4-phosphate pathway genes for isoprenoid
biosynthesis from Indian ginseng, Withania somnifera, Protoplasma, 250 (2013) 285-295.
[6] P. Gupta, Akhtar, N., Tewari, S.K., Sangwan, R.S., Trivedi, P.K., Differential expression of
farnesyl diphosphate synthase gene from Withania somnifera in different chemotypes and in
response to elicitors, Plant Growth Regul, 65 (2011) 93-100.

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[7] W.W. Bhat, S.K. Lattoo, S. Razdan, N. Dhar, S. Rana, R.S. Dhar, S. Khan, R.A. Vishwakarma,
Molecular cloning, bacterial expression and promoter analysis of squalene synthase from
Withania somnifera (L.) Dunal, Gene, 499 (2012) 25-36.
[8] N. Gupta, P. Sharma, R.J.S. Kumar, R.K. Vishwakarma, B.M. Khan, Functional
characterization and differential expression studies of squalene synthase from Withania
somnifera, Mol Biol Rep, 39 (2012) 8803-8812.
[9] S. Razdan, W.W. Bhat, S. Rana, N. Dhar, S.K. Lattoo, R.S. Dhar, R.A. Vishwakarma,
Molecular characterization and promoter analysis of squalene epoxidase gene from Withania
somnifera (L.) Dunal, Mol Biol Rep, 40 (2013) 905-916.
[10] S. Pal, Singh, S., Shukla, A.K., Gupta, M.M., Khanuja, S.P.S., Shasany, A.K., Comparative
withanolide profiles, gene isolation, and differential gene expression in the leaves and roots of
Withania somnifera, Journal of Horticulture Science & Biotechnology, 86 (2011) 391-397.
[11] N. Mantri, Olarte, A., Li, C.G., Xue, C., Pang, E.C.K., Fingerprinting the asterid species using
subtracted diversity array reveals novel species-specific sequences, PloS one, 7 (2012) e34873.
[12] P. Gupta, R. Goel, S. Pathak, A. Srivastava, S.P. Singh, R.S. Sangwan, M.H. Asif, P.K. Trivedi,
De novo assembly, functional annotation and comparative analysis of Withania somnifera leaf
and root transcriptomes to identify putative genes involved in the withanolides biosynthesis,
PloS one, 8 (2013) e62714.
[13] M. Ghosh Dasgupta, B.S. George, A. Bhatia, O.P. Sidhu, Characterization of Withania
somnifera leaf transcriptome and expression analysis of pathogenesis-related genes during
salicylic acid signaling, PloS one, 9 (2014) e94803.
[14] Sanchita, S. Singh, A. Sharma, Bioinformatics approaches for structural and functional analysis
of proteins in secondary metabolism in Withania somnifera, Mol Biol Rep, 41 (2014) 7323-
7330.
[15] Sanchita, G. Singh, A. Sharma, In silico study of binding motifs in squalene synthase enzyme
of secondary metabolic pathway solanaceae family, Mol Biol Rep, 41 (2014) 7201-7208.
[16] N. Gupta, P. Sharma, R.J. Santosh Kumar, R.K. Vishwakarma, B.M. Khan, Functional
characterization and differential expression studies of squalene synthase from Withania
somnifera, Mol Biol Rep, 39 (2012) 8803-8812.

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 Stumbling blocks and their prospective solutions in research on
Catharanthus roseus

Maneesha Mall, Ashutosh K Shukla*

CSIR-Central Institute of Medicinal and Aromatic Plants, Lucknow 226015, UP, INDIA
Email: scholarmani@gmail.com

ABSTRACT

Several research groups are working worldwide on various aspects of terpenoid indole
alkaloid (TIA) biosynthesis in Catharanthus roseus. Two of the bisindole alkaloids -
vincristine (VCR) and vinblastine (VLB), are highly potent antineoplastic agents and they
occur in extremely low quantities in planta due to their highly cytotoxic nature, which poses
the problem of cellular containment. Major research effort on the plant aims at maximizing
its bisindole biosynthetic capability as the plant remains the most important source for these
alkaloids in spite of reports on total synthesis of vincristine and vinblastine. However there
are so many challenges in C. roseus research, for example, its recalcitrance to genetic
transformation, the complexity of the TIA structures, and the subcellular
compartmentalization of TIA biosynthesis. The present review has been compiled to
understand the major problems with the C. roseus research and the published successful
attempts to overcome it.

INTRODUCTION

Catharanthus is a promising medicinal plant with a number of gaps in its research, which
need to be filled. The genus Catharanthus comprises eight species, which are C. roseus, C.
ovalis, C. trichophyllus, C. longifolius, C. coriaceus, C. lanceus, C. scitulus and C. pusillus.
The recent research is focussed on C. roseus due to its pharmaceutically important terpenoid
indole alkaloids (TIAs), notably the anticancer bisindole alkaloids, vincristine (VCR) and
vinblastine (VLB). It is necessary to explore the therapeutic utility of the other species of
Catharanthus but lack of literatures and the availability of plants are major problems. Even
research on Catharanthus roseus itself faces many hurdles, which need to be tackled head-on.

Plant growth environment

C. roseus plants are erect, evergreen, everblooming perennial herbs or small shrubs. The
plant grows almost in all variety of soil and climate but the growth is checked in low
temperature condition (as in winter). The highly alkaline and waterlogged soils are also
unsuitable (Mishra and Kumar 2000; Virmani et al. 1978). The next problem is to control the
pests and diseases although the C. roseus plants are hardy and fairly resistant to it. Several
pathogens like virus, bacteria, fungi and mycoplasma infect the plant and affect its
functionality. The spike disease of Santalum album (Virmani et al. 1978) characterised by a
bushy appearance of plants is also seen in C. roseus plants. Green rosette disease (Garga
1958) is another one in which the leaf and flower size is reduced. Some other fungal
pathogens are Phytophthora nicotianae, Pythium debaryanum, P. butleri, Colletotrichum
dermatium causing twig blight/top-rot or die back. The plants are also susceptible to viruses
(Mishra and Kumar 2000; Samad et al. 2008). To avoid the detrimental effects of the
pathogens regular sprays of oxytetracycline, gibberellic acid (GA), tetracycline and 0.5%
aqueous solution of nicotine sulphate may be a useful approach. Foliar spray of the fungicide

51 | P a g e
Captafol Foltaf 80 WP, Carbendazim Bavistin 50 WP and Benomyl 50 WP may cure some of
fungal diseases (Kalra et al. 1991).

Alkaloid extraction and detection

Extraction and purification of specific alkaloid from the alkaloidal mixture is a tedious task.
Detection of different alkaloids with approximate same retention time needs advanced
detection techniques. So many factors have to be taken into consideration to extract a fair
amount of alkaloid like temperature, pH, solvent system, and chromatographic column, etc.
The methods, which are used till date are TLC (Fransworth et al. 1964), GC-MS (Ylinen et
al. 1990), LC-MS (Auriola et al. 1990), flow-injection electrospray ionisation mass
spectrometry (Favretto et al. 2001), HPLC (Naaranlahti et al. 1987; Volkov and Grodnitskaya
1994; Singh et al. 2000; Tikhomiroff and Jolicoeur 2002), specific radioimmunoassay (RIAs)
(Hirata et al. 1989; Huhtikangas et al. 1987; Westekemper et al. 1980; Deus-Neumann et al.
1987) and enzyme-linked immunosorbent assay (ELISA) (Cibotti et al. 1990). Some
advanced techniques like capillary electrophoresis-mass spectrometry (CE-MS) (Chen et al.
2011) and an ultraperformance liquid chromatography/mass spectrometry method (He et al.
2011) have also been developed. However HPLC is the most convenient method for the C.
roseus alkaloids and symmetry C18 column is a better choice (Uniyal et al. 2001).

Biosynthetic pathway gene prospecting

The first two published cDNA libraries of C. roseus provide an initial view of the gene sets
expressed in leaves or roots of the plant. However, due to low expression levels of TIA-
related genes and by dilution of the transcripts from the specialized cells in those whole organ
libraries, it was difficult to identify the new TIA biosynthetic candidate genes (Murata et al.,
2006; Shukla et al., 2006). However some advances have been made for the selection of
candidate genes like exploiting the epidermome resource, high throughput transcriptomics
(orthology comparisons, co-regulated gene clustering). The epidermome library produced by
the De Luca group is the first successful attempt for gene selection (Murata et al., 2008).
Another strategy is the comparison of transcriptomic resources of plants that produce, and
plants that do not produce specific compounds by the De Luca group. It resulted in the
identification of an unknown enzyme step in the late stage of secologanin biosynthesis (Salim
et al., 2013). Another approach is based on the postulate that genes of a biosynthetic pathway
are expectedly co-regulated. The first successful example of C. roseus gene clustering came
from the O’Connor group, to identify P450 encoding genes involved in the oxygenation of
tabersonine (Giddings et al., 2011).

Improving key TIA biosynthesis

VCR and VLB are the least abundant and the most expensive TIAs (De Bernonville et al.
2014). Therefore, several attempts were made to understand the biochemistry and cell
biology of C. roseus alkaloid biosynthesis and way to the alternative production platforms
(cell and root cultures) (St-Pierre et al., 2013; Shukla and Khanuja, 2013). VCR and VLB are
formed in planta by the condensation of monomeric TIAs, vindoline and catharanthine.
However, unlike the vindoline biosynthetic pathway, the catharanthine pathway is not
elucidated in literature. Besides, vindoline biosynthesis represents a bottleneck as it requires
differentiated cells having well-formed chloroplast structures. Till date large-scale production
of bisindole alkaloids in undifferentiated suspension-cultured cells (bioreactors) has been
unsuccessful due to non-expression of the vindoline biosynthetic pathway (which operates
only in the differentiated green aerial parts of the plant). Since catharanthine is easily
produced in suspension cultures, sourcing vindoline, either from alkaloid-rich field-grown
plants or alternatively, through different means, is the main issue. As C. roseus is the only

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source of key alkaloids (like vindoline, which is the major bottleneck in bisindole alkaloid
semi-synthesis), the need is to identify its genotypes that accumulate vindoline and factors
that promote its biosynthesis and accumulation in planta. The leads in this direction are the
elucidation of the effect of several biotic and abiotic factors on TIA accumulation. The
example of biotic factors are the effect of fungal homogenate (Godoy-Hernandez and Loyola-
Vargas 1991), endophyte treatment (Tang et al. 2011; Tiwari et al. 2013) etc. It is evident that
C. roseus leaves infected by phytoplasmas contain less fatty components and higher
vindoline compared to healthy leaves (Choi et al., 2004). Some of the abiotic factors are
osmotic stress (Godoy-Hernandez and Loyola-Vargas 1991), heavy metals (Srivastava NK
and Srivastava AK 2010), exposure to UV-B light (Binder et al. 2009; Ramani et al. 2008),
oxidative stress (Matsuura et al. 2014) etc.

Plant genetic transformation

Stable genetic transformation of the recalcitrant C. roseus plant is another major challenge.
Some successful attempts have been made towards this end - a protocol for genetic
transformation of C. roseus by Agrobacterium rhizogenes A4 (Zhou et al. 2012); A.
tumefaciens-mediated transgenic plant production via direct shoot bud organogenesis from
pre-plasmolyzed leaf explants of C. roseus (Verma and Mathur 2011); development of
efficient C. roseus regeneration and transformation system using A. tumefaciens and
hypocotyls as explants (Wang et al. 2012). Guirimand et al. (2009) have optimized the
transient transformation of C. roseus cells by particle bombardment. However, achieving
stable transformants of C. roseus plants in a robust manner is still a challenge. To improve
the content of key TIAs, it is required to isolate and characterize all the genes involved in the
pathway. Recently a virus induced gene silencing (VIGS) method has been developed, which
is a fast but transient method for knocking down expression of endogenous genes in plants
(Liscombe and O’Connor, 2011).

CONCLUSIONS AND FUTURE PROSPECTS

Although among the various species of Catharanthus, the C. roseus enjoys the major
research share, the other species also need to be researched upon. There are many gaps in
understanding of the TIA pathway in C. roseus. The recent reviews (De Bernonville et al.,
2014; Shukla and Khanuja, 2013) have elaborated the gaps to be filled in this area. For
example, the catharanthine pathway is yet to be elucidated, as well as the other unknown
regulatory genes of the TIA pathway. The initial steps in the conversion of the strictosidine
aglycone into tabersonine have not been elucidated. The effect of various biotic and abiotic
factors on TIA biosynthesis and accumulation in the plant must also be studied. The
elucidation of functions of novel genes / ESTs is another area, where significant research
effort is required through use of newly developed protocols like VIGS. This would also be
facilitated through development of yet unavailable efficient transformation systems for the
recalcitrant medicinal plant species. In future, alkaloid biotechnology research will focus on
heterologous production systems for therapeutically useful alkaloids and muta-synthesis of
novel alkaloid analogs.

REFERENCES

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Auriola, S., Naaranlahti, T., Kostiainen, R., Lapinjoki, S.P. 1990. Identification of indole alkaloids of
Catharanthus roseus with liquid chromatography/mass spectrometry using collision-induced
dissociation with the thermospray ion repeller. Biomed. Environ. Mass Spectrom. 19:400-4.
Binder, B.Y., Peebles, C.A., Shanks, J.V., San, K.Y. 2009. The effects of UV-B stress on the
production of terpenoid indole alkaloids in Catharanthus roseus hairy roots. Biotechnol Prog.
25:861-865.
Chen, Q., Li, N., Zhang, W., Chen, J., Chen, Z. 2011. Simultaneous determination of vinblastine and
its monomeric precursors vindoline and catharanthine in Catharanthus roseus by capillary
electrophoresis-mass spectrometry. J. Sep. Sci. 34:2885-2892.
Choi, Y.H., Tapias, E.C., Kim, H.K., Lefeber, A.W.M., Erkelens, C., Verhoeven, J.T.J., Brzin, J., Zel,
J., Verpoorte, R., 2004. Metabolic discrimination of Catharanthus roseus leaves infected by
phytoplasma using 1H-NMR spectroscopy and multivariate data analysis. Plant Physiology
135:2398–2410.
Cibotti, M.C., Freier, C., Andrieux, J., Plat, M., Cosson, L., Bohuon, C. 1990. Monoclonal antibodies
to bis-indole alkaloids of Catharanthus roseus and their use in enzyme-linked immuno-sorbent-
assays. Phytochemistry 29:2109-2114.
De Bernonville, T.D., Clastre, M., Besseau, S., Oudin, A., Burlat, V., Glevarec, G., Lanoue, A.,
Papon, N., Giglioli-Guivarc’h, N., St-Pierre, B., Courdavault, V. 2014. Phytochemical
genomics of the Madagascar periwinkle: Unravelling the last twists of the alkaloid engine.
Phytochemistry. [doi: 10.1016/j.phytochem.2014.07.023]
Deus-Neumann, B., Stockigt, J., Zenk, M.H. 1987. Radioimmunoassay for the quantitative
determination of catharanthine. Planta Med. 53:184-188.
Farnsworth, N.R., Blomster, R.N., Damratoski, D., Meer, W.A., Cammarato, L.V. 1964. Studies on
Catharanthus alkaloids. VI. Evaluation by means of thin layer chromatography and ceric
ammonium sulphate spray reagent. Lloydia 27:302-314.
Favretto, D., Piovan, A., Filippini, R., Caniato, R. 2001. Monitoring the production yields of
vincristine and vinblastine in Catharanthus roseus from somatic embryogenesis.
Semiquantitative determination by flow-injection electrospray ionization mass spectrometry.
Rapid Commun. Mass Spectrom. 15:364–369.
Garga, R.P. 1958. Studies on virus diseases of plants in Madhya Pradesh. I. Green rosette of Vinca.
Curr. Sci. 27:493-494.
Giddings, L.A., Liscombe, D.K., Hamilton, J.P., Childs, K.L., DellaPenna, D., Buell, C.R., O’Connor,
S.E., 2011. A stereoselective hydroxylation step of alkaloid biosynthesis by a unique
cytochrome P450 in Catharanthus roseus. J. Biol. Chem. 286:16751–16757.
Godoy-Hernandez, G., Loyola-Vargas, V.M. 1991. Effect of fungal homogenate, enzyme inhibitors
and osmotic stress on alkaloid content of Catharanthus roseus cell suspension cultures. Plant
Cell Rep 10:537-540.
Guirimand, G., Burlat, V., Oudin, A., Lanoue, A., St-Pierre, B., Courdavault, V. 2009. Optimization
of the transient transformation of Catharanthus roseus cells by particle bombardment and its
application to the subcellular localization of hydroxymethylbutenyl 4-diphosphate synthase and
geraniol 10-hydroxylase. Plant Cell Rep. 28:1215-1234.
He, L., Yang, L., Xiong, A., Zhao, S., Wang, Z., Hu, Z. 2011. Simultaneous quantification of four
indole alkaloids in Catharanthus roseus cell line C20hi by UPLC-MS. Anal. Sci. 27:433-438.
Hirata, K., Yamanaka, A., Kurano, N., Miyamoto, K., Miura, Y. 1987. Production of indole alkaloids
in multiple shoot culture of Catharanthus roseus (L.) G. Don. Agric. Biol. Chem., 51:1311-
1317.
Huhtikangas, A., Lehtola, T., Lapinjoki, S., Lounasmaa, M. 1987. Specific radioimmunoassay for
vincristine. Planta Med. 53:85-87.
Kalra, A., Ravindra, N.S., Chandrashekhar, R.S. 1991. Influence of foliar application of fungicides on
dieback disease caused by Pythium aphanidermatum and alkaloid yield of periwinkle
(Catharanthus roseus). Ind. J. Agric. Sci. 61:949-951.
Liscombe, D.K., O’Connor, S.E. 2011. A virus-induced gene silencing approach to understanding
alkaloid metabolism in Catharanthus roseus. Phytochemistry 72:1969-1977.
Matsuura, H.N., Rau, M.R., Fett-Neto, A.G. 2013. Oxidative stress and production of bioactive
monoterpene indole alkaloids: biotechnological implications. Biotechnol Lett. 36:191-200.

54 | P a g e
Mishra, P., Kumar, S. 2000. Emergence of Periwinkle Catharanthus roseus as amodel system for
molecular biology of alkaloids: Phytochemistry, pharmacology, plant biology and in vivo and
in vitro cultivation. J. Med. Arom. Plant Sci. 22:306-337.
Mishra, P., Kumar, S. 2000. Emergence of Perwinkle Catharanthus roseus as a model system for
molecular biology of alkaloids: Phytochemistry, pharmacology, plant biology and in vivo and
in vitro cultivation. J. Med. Arom. Plant Sci. 22:306-337.
Murata, J., Bienzle, D., Brandle, J.E., Sensen, C.W., De Luca, V., 2006. Expressed sequence tags
from Madagascar periwinkle (Catharanthus roseus). FEBS Lett. 580:4501–4507.
Naaranlahti, T., Nordstrom, M., Huhtikangas, A., Lounasmaa, M. 1987. Determination of
Catharanthus alkaloids by reversed-phase high-performance liquid chromatography. J.
Chromatogr. 410:488-493.
Ramani, S., Jayabaskaran, C. 2008. Enhanced catharathine and vindoline production in suspension
cultures of Catharanthus roseus by ultraviolet-B light. J Mol Signal. 3:9–14.
Salim, V., Yu, F., Altarejos, J., De Luca, V., 2013. Virus-induced gene silencing identifies
Catharanthus roseus 7-deoxyloganic acid-7-hydroxylase, a step in iridoid and monoterpene
indole alkaloid biosynthesis. Plant J. 76:754–765.
Samad, A., Ajaykumar, P.V., Gupta, M.K., Shukla, A.K., Darokar, M.P., Somkuwar, B., Alam, M.
2008. Natural infection of periwinkle (Catharanthus roseus) with Cucumber mosaic virus,
subgroup IB. Australas. Plant Dis. Notes 3:30-34.
Shukla, A.K., Khanuja, S.P., 2013. Catharanthus roseus: The metabolome that represents a unique
reservoir of medicinally important alkaloids under precise genomic regulation, OMICS
Applications in Crop Science. CRC Press, pp. 325–384.
Shukla, A.K., Shasany, A.K., Gupta, M.M., Khanuja, S.P.S., 2006. Transcriptome analysis in
Catharanthus roseus leaves and roots for comparative terpenoid indole alkaloid profiles. J.
Exp. Bot. 57:3921–3932.
Singh, D.V., Maithy, A., Verma, R.K., Gupta, M.M., Kumar, S. 2000. Simultaneous determination of
Catharanthus alkaloids using reversed phase high performance liquid chromatography. J. Liq.
Chrom. Rel. Technol. 23:601-607.
Srivastava, N.K., Srivastava, A.K. 2010. Influence of some heavy metals on growth, alkaloid content
and composition in Catharanthus roseus L. Indian J Pharm Sci. 72:775-778.
St-Pierre, B., Besseau, S., Clastre, M., Courdavault, V., Courtois, M., Crèche, J., Ducos, E., de
Bernonville, T.D., Dutilleul, C., Glévarec, G., Imbault, N., Lanoue, A., Oudin, A., Papon, N.,
Pichon, O., Giglioli-Guivarc’h, N., 2013. New light on alkaloid biosynthesis and future
prospects. Adv. Bot. Res. 73–109.
Tang, Z., Rao, L., Peng, G., Zhou, M., Shi, G., Liang, Y. 2011. Effects of endophytic fungus and its
elicitors on cell status and alkaloid synthesis in cell suspension cultures of Catharanthus
roseus. J. Medicinal Plants Research 5:2192-2200.
Tikhomiroff, C., Jolicoeur, M. 2002. Screening of Catharanthus roseus secondary metabolites by
high-performance liquid chromatography. J. Chromatogr. A 955:87-93.
Tiwari, R., Awasthi, A., Mall, M., Shukla, A.K., Srinivas, K.S., Syamasundar, K.V., Kalra, A. 2013.
Bacterial endophyte-mediated enhancement of in planta content of key terpenoid indole
alkaloids and growth parameters of C. roseus. Ind Crops and Prod. 43:306-310.
Uniyal, G.C., Bala, S., Mathur, A.K., Kulkarni, R.N. 2001. Symmetry C18 Column: a Better Choice
for the analysis of indole alkaloids of Catharanthus roseus. Phytochem. Anal. 12:206–210.
Verma, P., Mathur, A.K. 2011. Agrobacterium tumefaciens-mediated transgenic plant production via
direct shoot bud organogenesis from pre-plasmolyzed leaf explants of Catharanthus roseus.
Biotechnol. Lett. 33:1053–1060.
Virmani, O.P., Srivastava, G.N., Singh, P. 1978. Catharanthus roseus: The tropical periwinkle. Indian
Drugs 15:231-252.
Virmani, O.P., Srivastava, G.N., Singh, P. 1978. Catharanthus roseus: The tropical periwinkle. Indian
Drugs 15:231-252.
Volkov, S.K., Grodnitskaya, E.I. 1994. Application of high-performance liquid chromatography to the
determination of vinblastine in Catharanthus roseus. J. Chromatogr. B. Biomed. Appl.
660:405-408.

55 | P a g e
Wang, Q., Xing, S., Pan, Q., Yuan, F., Zhao, J., Tian, Y., Chen, Y., Wang, G., Tang, K. 2012.
Development of efficient Catharanthus roseus regeneration and transformation system using
Agrobacterium tumefaciens and hypocotyls as explants. BMC Biotechnol. 12:34.
Westekemper, P., Wieczorek, U., Gueritte, F., Langlois, N., Langlois, Y., Potier, P., Zenk, M.H. 1980.
Radioimmunoassay for the determination of the indole alkaloid vindoline in Catharanthus.
Planta Med. 39:24-37.
Ylinen, M., Suhonen, P., Naaranlahti, T., Lapinjoki, S.P., Huhtikangas, A. 1990. Gas
chromatographic- mass spectrometric analysis of major indole alkaloids of Catharanthus
roseus. J. Chromatogr. 505:429-434.
Zhou, M.L., Zhu, X.M., Shao, J.R., Wu, Y.M., Tang, Y.X. 2012. An protocol for genetic
transformation of Catharanthus roseus by Agrobacterium rhizogenes A4. Appl. Biochem.
Biotechnol. 166:1674-1684.

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 Trans-acting regulators of gene expression in Catharanthus roseus
Pravin Prakash, Dolly Ghosliya and Vikrant Gupta
Biotechnology Division, CSIR-Central Institute of Medicinal and Aromatic Plants, P.O.
CIMAP, Lucknow 226 015

ABSTRACT

The terpenoid indole alkaloid and phenylpropanoids pathways participate in biosynthesis of a

number of commercially important secondary metabolites in Catharanthus roseus.
MicroRNAs and transcription factors are potent trans-acting regulators of gene expression
and recently they have been shown to regulate plant secondary metabolism. Certain trans-
acting regulators represent themselves as potential candidates for metabolic engineering in C.
roseus. In-depth knowledge related to the metabolic pathways and their regulatory
mechanisms are essential for the application of biotechnological approach to enhance the
production of commercially important secondary metabolites in medicinal plants. The
identification and characterization of secondary metabolism-related trans-acting regulators
from C. roseus would unravel the still unknown complex gene regulatory networks operating
in this medicinally important plant and provide biotechnological resource for future use.

Keywords: Catharanthus roseus, trans-acting regulators, microRNAs, transcription factors,

metabolic engineering

INTRODUCTION

Trans-acting regulators are important regulatory molecules which control gene expression at
transcriptional and post-transcriptional levels. A trans-acting component is basically a DNA
sequence that harbors a gene which encode for a regulatory protein or microRNA, mediating
the regulation of its target gene. Transcription factors (TFs) and microRNAs (miRNAs) are
considered as potential trans-acting regulators of gene expression (Hobert, 2004).
Transcription factors are proteins that ensure coordinated and effective regulation of gene
expression. The promoter regions of certain genes contain sequence-specific cis-motifs where
specific transcription factors bind to regulate their expression (Gill, 2001; Latchman, 1997).
On the other hand, microRNAs are ~19-25 nucleotide long non-coding RNAs that are
involved in the regulation of gene expression at post transcriptional level (Axtell and Bartel,
2005; Bartel, 2004; Lai, 2003). Plant miRNAs exhibit complementarity to target mRNA
transcript(s), thus either cleave or repress them and ultimately regulate the expression of the
concerned gene (Reinhart et al., 2002). The microRNAs and transcription factors generally
exert their regulatory potential by mutual interaction. More than half of the targets predicted
for known miRNAs are transcription factors (Axtell and Bartel, 2005; Bartel, 2004).
Transcription factors and miRNAs have emerged as important players in gene regulation and
are being targeted for metabolic engineering in plants.
Catharanthus roseus (Madagascar periwinkle) is an important medicinal plant belonging to
the Apocynaceae family and well known for biosynthesis of valuable alkaloids having
anticancer properties. Important alkaloids produced by C. roseus include anti-neoplastic
vinblastine and vincristine, the anti-hypertensives reserpine and ajmalicine, and the anti-
arrhythmic ajmaline. Phenolic compounds produced include anthocyanins, flavonoids, 2, 3-
dihydroxybenzoic acid, and phenylpropanoids such as cinnamic acid derivatives (Memelink
and Gantet, 2007; Murata et al., 2008; Mustafa and Verpoorte, 2007). The plant produces a
wide array of terpenoid indole alkaloids and phenolic compounds via terpenoid indole

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alkaloid (TIA) and phenylpropanoids pathways, respectively (Mustafa and Verpoorte, 2007;
Rischer et al., 2006). The studies focused on trans-acting regulators-mediated gene
expression have potential to reveal the unexplored possibilities of genetic manipulation of
secondary metabolite biosynthesis in C. roseus.

Transcription factor-based metabolic engineering in C. roseus

Terpenoid indole alkaloid (TIA) pathway in C. roseus is tightly regulated at the
transcriptional level. The major transcription factors that are characterized and known to
regulate terpenoid indole alkaloid biosynthesis include Octadecanoid-responsive
Catharanthus AP2-domain ORCA2 and ORCA3 (Menke et al., 1999; van der Fits and
Memelink, 2000), Box P-binding factor BPF1 (van der Fits et al., 2000), G-box binding
factors GBF1 and GBF2 (Sibéril et al., 2001), zinc finger Catharanthus transcription factors
ZCT1, ZCT2 and ZCT3 (Pauw et al., 2004), basic helix-loop-helix protein CrMYC2 (Zhang
et al., 2011) and WRKY transcription factor CrWRKY1 (Suttipanta et al., 2011).
Overexpression of a jasmonate-responsive APETALA2 (AP2)-domain transcription factor
ORCA3 in C. roseus led to an enhanced expression of several key TIA pathway genes and
resulted in increased amounts of terpenoid indole alkaloids (Memelink et al., 2001; van der
Fits and Memelink, 2000). The ORCA3 transcription factor acts as a master regulator of
primary and secondary metabolism-related genes in C. roseus. WRKY family transcription
factors have also been reported to be involved in the regulation of secondary metabolites
biosynthesis (Kato et al., 2007). Transgenic hairy roots overexpressing CrWRKY1 showed the
accumulation of higher levels of serpentine suggesting root-specific abundance in C. roseus
due to root-specific expression of CrWRKY1 (Suttipanta et al., 2011). The C. roseus G-box
binding factors (CrGBF1 and CrGBF2) were suggested to be involved in coordinated
regulation of several TIA pathway genes. Upon transient expression, CrGBF1 and CrGBF2
repressed the expression of a key gene strictosidine synthase (Sibéril et al., 2001).
Transcription factor-based metabolic engineering in Catharanthus cell cultures has been
successfully employed for enhanced biosynthesis of alkaloids. A significant enhancement in
the production of ajmalicine was noted upon expression of Arabidopsis thaliana root-specific
MADS-box transcription factor agamous-like-12 (Montiel et al., 2007).

MicroRNAs: huge potential of tiny RNA molecules for metabolic engineering in C.

roseus
In plants, miRNAs play important roles in the regulation of diverse biological processes such
as flower and root morphogenesis (Aukerman and Sakai, 2003; Wang et al., 2005), anther,
leaf and vascular development (Millar and Gubler 2005; Palatnik et al. 2003; Kim et al.,
2005). Besides these, miRNA-like molecules targeting secondary metabolism-related genes
have also been reported recently from plants like Catharanthus roseus (Prakash et al., 2014),
Panax ginseng (Li et al., 2013), Populus trichocarpa (Lu et al., 2013) and Taxus chinensis
(Qiu et al., 2009). These secondary metabolism-related miRNAs identified from medicinal
plants have immense biotechnological applications as they can be utilized for genetic
manipulation for modulation of metabolite content in medicinally important plants. In
Populus trichocarpa, secondary metabolism-related laccase genes were predicted to be
targeted by ptr-miR397a. Overexpression of ptr-miR397a led to downregulation of 17
laccases, and reduced lignin content was observed in transgenic lines (Lu et al., 2013). A
total of 14 tch-miRNAs were assumed to be involved in taxoid biosynthesis by altering plant
metabolic differentiation upon treatment of Taxus chinensis with taxoid elicitor methyl
jasmonate (Qiu et al., 2009). Over-expression of miR393 increased glucosinolate levels and
reduced the levels of camalexin in Arabidopsis (Robert-Seilaniantz et al., 2011). The miR858
targets MYBs of diverse biological processes related to cell wall formation, lignification, and

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anthocyanin biosynthesis in apple (Xia et al., 2012). The above examples clearly indicate the
potential of miRNAs as biotechnological tools.

CONCLUSIONS

Commercial level production of minute amount of pharmaceutically important secondary

metabolites of medicinal plants is a major objective of medicinal plant research. C. roseus
being an anti-cancer medicinal plant has drawn attention towards its extremely valuable
compounds like vincristine and vinblastine. Large scale production of vinblastine and
vincristine is still a challenge in C. roseus research. Successful genetic manipulation of
secondary metabolic pathways in this important medicinal plant depends upon proper
understanding of still unexplored enzymatic and regulatory steps involved in the biosynthesis
of secondary metabolites of interest. The use of transcription factors as a tool has been proven
to be promising for manipulation of secondary metabolic pathways and to increase the
biosynthesis of secondary metabolites. Successful manipulation of secondary metabolic
pathways could also be achieved by identification of genes and transcription factors which
are targeted by miRNAs. MiRNA-based vector constructs for miRNA overexpression or
silencing could also be exploited for the genetic manipulation and metabolic engineering in
medicinal plants. Further investigations would be helpful to exploit the available trans-acting
regulators for metabolic engineering in C. roseus to enhance the yield of certain
pharmaceutically important secondary metabolites through biotechnological methods.

REFERENCES
Aukerman MJ, Sakai H (2003) Regulation of flowering time and floral organ identity by a microRNA
and its APETALA2-like target genes. Plant Cell 15: 2730–2741.
Axtell MJ, Bartel DP (2005) Antiquity of microRNAs and their targets in land plants. Plant Cell 17:
1658–1673.
Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116: 281–297.
Gill G (2001) Regulation of the initiation of eukaryotic transcription. Essays Biochem 37: 33–43.
Hobert O (2004) Common logic of transcription factor and microRNA action. Trends in Biochem Sci
29: 462–468.
Kato N, Dubouzet E, Kokabu Y, Yoshida S, Taniguchi Y, Dubouzet JG, Yazaki K, Sato F (2007)
Identification of a WRKY protein as a transcriptional regulator of benzylisoquinoline alkaloid
biosynthesis in Coptis japonica. Plant Cell Physiol 48: 8–18.
Kim J, Jung JH, Reyes JL, Kim YS, Kim SY, Chung KS, Kim JA, Lee M, Lee Y, Kim VN, Chua
NH, Park CM (2005) MicroRNA-directed cleavage of ATHB15 mRNA regulates vascular
development in Arabidopsis inflorescence stems. Plant J 42: 84–94.
Lai EC (2003) MicroRNAs: runts of the genome assert themselves. Curr Biol 13: R925–R936.
Latchman DS (1997) Transcription factors: an overview. Int J Biochem Cell Biol 29: 1305–1312.
Li C, Zhu Y, Guo X, Sun C, Luo H, Song J, Li Y, Wang L, Qian J, Chen S (2013) Transcriptome
analysis reveals ginsenosides biosynthetic genes, microRNAs and simple sequence repeats in
Panax ginseng C. A. Meyer. BMC Genomics 14: 245.
Lu SF, Sun YH, Shi R, Clark C, Li LG, Chiang VL (2005) Novel and mechanical stress-responsive
microRNAs in Populus trichocarpa that are absent from Arabidopsis. Plant Cell 17: 2186–
2203.
Memelink J, Gantet P (2007) Transcription factors involved in terpenoid indole alkaloid biosynthesis
in Catharanthus roseus. Phytochem Rev 6: 353–362.
Memelink J, Verpoorte R, Kijne JW (2001) ORCAnization of jasmonate-responsive gene expression
in alkaloid metabolism. Trends Plant Sci 6: 212–219.
Menke FL, Champion A, Kijne JW, Memelink J (1999) A novel jasmonate- and elicitor-responsive
element in the periwinkle secondary metabolite biosynthetic gene Str interacts with a
jasmonate- and elicitor-inducible AP2-domain transcription factor, ORCA2. EMBO J 18:
4455–4463.

59 | P a g e
Millar A, Gubler F (2005) The Arabidopsis GAMYB-like genes, MYB33 and MYB65, are microRNA-
regulated genes that redundantly facilitate anther development. Cell 17: 705–721.
Montiel G, Breton C, Thiersault M, Burlat V, Jay-Allemand C, Gantet P (2007) Transcription factor
agamous-like 12 from Arabidopsis promotes tissue-like organization and alkaloid biosynthesis
in Catharanthus roseus suspension cells. Metab Eng 9: 125–132
Murata J, Roepke J, Gordon H, Luca VD (2008) The leaf epidermome of Catharanthus roseus reveals
its biochemical specialization. Plant Cell 20: 524–542.
Mustafa NR, Verpoorte R (2007) Phenolic compounds in Catharanthus roseus. Phytochem Rev 6:
243–258.
Palatnik JF, Allen E, Wu XL, Schommer C, Schwab R, Carrington JC, Weigel D (2003) Control of
leaf morphogenesis by microRNAs. Nature 425: 257–263.
Pauw B, Hilliou FA, Martin VS, Chatel G, de Wolf CJ, Champion A, Pré M, van Duijn B, Kijne JW,
van der Fits L, Memelink J (2004) Zinc finger proteins act as transcriptional repressors of
alkaloid biosynthesis gene in Catharanthus roseus. J Biol Chem 279: 52940–52948.
Prakash P, Ghosliya D, Gupta V (2015) Identification of conserved and novel microRNAs in
Catharanthus roseus by deep sequencing and computational prediction of their potential
targets. Gene 554: 181–195.
Qiu D, Pan X, Wilson I, Li F, Liu M, Teng W, Zhang B (2009) High throughput sequencing
technology reveals that the taxoid elicitor methyl jasmonate regulates microRNA expression in
Chinese yew (Taxus chinensis). Gene 436: 37–44.
Reinhart BJ, Weinstein EG, Rhoades MW, Bartel B, Bartel DP (2002) MicroRNAs in plants. Genes
Dev 16: 1616–1626.
Rischer H, Oresic M, Seppänen-Laakso T, Katajamaa M, Lammertyn F, Ardiles-Diaz W, Van
Montagu MC, Inzé D, Oksman-Caldentey KM, Goossens A (2006) Gene-to-metabolite
networks for terpenoid indole alkaloid biosynthesis in Catharanthus roseus cells. Proc Natl
Acad Sci USA 103: 5614–5619.
Robert-Seilaniantz A, MacLean D, Jikumaru Y, Hill L, Yamaguchi S, Kamiya Y, Jones JD (2011)
The microRNA miR393 re-directs secondary metabolite biosynthesis away from camalexin and
towards glucosinolates. Plant J 67: 218–231.
Sibéril Y, Benhamron S, Memelink J, Giglioli-Guivarc'h N, Thiersault M, Boisson B, Doireau P,
Gantet P (2001) Catharanthus roseus G-box binding factors 1 and 2 act as repressors of
strictosidine synthase gene expression in cell cultures. Plant Mol Biol 45: 477–488.
Suttipanta N, Pattanaik S, Kulshrestha M, Patra B, Singh SK, Yuan L (2011) The transcription factor
CrWRKY1 positively regulates the terpenoid indole alkaloid biosynthesis in Catharanthus
roseus. Plant Physiol 157: 2081–2093.
van der Fits L, Memelink J (2000) ORCA3, a jasmonate-responsive transcriptional regulator of plant
primary and secondary metabolism. Science 289: 295–297.
van der Fits L, Zhang H, Menke FL, Deneka M, Memelink J (2000) A Catharanthus roseus BPF-1
homologue interacts with an elicitor-responsive region of the secondary metabolite biosynthetic
gene Str and is induced by elicitor via a JA-independent signal transduction pathway. Plant Mol
Biol 44: 675–685.
Wang JW, Wang LJ, Mao YB, Cai WJ, Xue HW, Chen XY (2005) Control of root cap formation by
microRNA-targeted auxin response factors in Arabidopsis. Plant Cell 17: 2204–2216.
Xia R, Zhu H, An YQ, Beers EP, Liu Z (2012) Apple miRNAs and tasiRNAs with novel regulatory
networks. Genome Biol 13: R47.
Zhang H, Hedhili S, Montiel G, Zhang Y, Chatel G, Pre M, Gantet P, Memelink J (2011) The basic
helix-loop-helix transcription factor CrMYC2 controls the jasmonate-responsive expression of
the ORCA genes that regulate alkaloid biosynthesis in Catharanthus roseus. Plant J 67: 61–71.

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 Transcriptome Analysis of Indian ginseng Withania somnifera
(Ashwgandha)

Swati Upadhyay
Plant Biotechnology Division, Central Institute of Medicinal and Aromatic Plants
Email: upadhyayswati4s2010@gmail.com

INTRODUCTION

Withania somnifera, (a member of Solanaceae family) is commonly known as “Ashwagandha”,

“Indian Winter cherry” or “Indian Ginseng”. It is one of the most important herb of Ayurveda
(the traditional system of medicine in India) used for millennia as a Rasayana for its wide ranging
health benefits (Singh et al., 2011). It has a number of pharmaceutical applications which makes
it a valuable medicinal plant. The roots of W. somnifera commonly known as Asgand are mainly
used for the medicinal properties. However, leaves of the plant are also reported to be used
medicinally. The fresh roots are collected during January to March and dried under shade for
several days. The drug retains its therapeutic efficacy for less than 2 years. It is prone to
decomposition and looses its potentials within 2 years. So the fresh dried roots are preferred for
medicinal uses (Uddin et al., 2012).

Uses
W. somnifera benefits the entire body and mind and its range of use is very broad. It protects the
body from various diseases, helps to prevent cancer and it also possesses aphoridisiac virtues,
acting as a powerful regulator. This plant effectively combats all forms of stress and promotes
sleep. It also promotes healthy metabolic functioning and protects various organs.

EST analysis
Expressed sequence tag (EST) analysis is an inexpensive, easy and less time consuming method
as compared to traditional gene cloning and whole genome sequencing methods. It quickly gives
the gene index of an organism and is used to identify the genes involved in specific plant
metabolic pathways. It is a rapid and cost effective approach to elucidate the transcriptome of an
organism. Successful examples of EST based gene discovery include the isolation of genes
encoding enzymes involved in the secondary metabolites production, manipulation and
elucidation of biosynthetic pathways. Due to its immense medicinal and pharmaceutical
advantages, molecular studies regarding the production of valuable secondary metabolite and for
deep understanding of signaling mechanism for overproduction of useful secondary metabolite in
vitro and in vivo, EST analysis was carried out. The first effort to identify the genes potentially
involved in the production of withanolides that will be beneficial to human and responsible for
other important cellular functions is the construction of cDNA library of withania leaf and root to
isolate ESTs and to find out tissue specific sequences. From withania ESTs, it was possible to
identify several candidate genes involved in withanolide biosynthetic pathway like HMG CoA
synthase, squalene epoxidase, sesquiterpene cyclase in MVA pathway and CDP-ME kinase from
MEP pathway. The later enzymes of withanolide biosynthesis are cytochrome p-450s and
glycosyltransferases (Senthil et al., 2010). This EST approach enhances our knowledge related to
secondary metabolite biosynthesis in withania and its regulation in some extent.

Trancriptome Analysis
After the EST studies it was found that the transcriptomic and genomic data of W. somnifera are
very limited and only 742 ESTs are available in the National Center for Biotechnology
Information (NCBI) database. The in-depth generation of EST databases of Withania may
provide information about all the expressed regions of genome and can be used to characterize
patterns of gene expression in specific tissues but after the advancement of next generation

61 | P a g e
sequencing approach the transcriptomic analysis of leaf and root tissues was performed to
increase our understanding related to withanolide (major and important secondary metabolite in
withania genus) biosynthesis. These transcriptomic data showed the expression profiling of
differentially expressed methyltransferases, cytochrome P450s, glycosyltransferase and
transcription factors which may be involved in withanolide biosynthesis in leaf and root tissues.
This information will help us in developing strategies of metabolic engineering and to increase
the production of specific withanolides. In addition to gene identification for pathway elucidation,
molecular markers were also identified using transcriptome data from leaf and root that will be
useful for marker assisted selection and breeding programme (Gupta et al., 2013).

In addition to the above mentioned leaf versus root transcriptome analysis to identify the gene
involved in secondary metabolite biosynthesis in W. somnifera the leaf transcriptome of W.
somnifera after exogenous application of SA was also done to understand the signaling
mechanism involved in the production of secondary metabolites. SA is the key hormone during
biotic defense response and levels of SA and its glycosylated conjugate (SAG) in tissues are
known to drastically accumulate both locally and systemically after pathogen infection.
Exogenously application of SA was reported to enhance disease resistance and induce
pathogenesis related (PR) gene expression in a wide variety of plant species. So this
transcriptomic study targeted the analysis of transcripts expressed in salicylic acid treated leaf
tissues. The production of secondary metabolites under in vitro condition are reported to be
enhanced by exogenous application of elicitors (biotic and abiotic) in culture media and methyl
jasmonate and salicylic acid are widely reported to induce production of secondary metabolites
under culture conditions. The transcriptomic study recorded the increase in production of three
major metabolites of W. somnifera including withanoside V, withaferin A and withanolide A in
leaf tissues, subsequent to exogenous application of SA (Ghosh et al., 2014).

The development of high throughput sequencing techniques and their analysis not only broadens
our knowledge related to Plant molecular and biochemical studies and metabolic engineering but
also valuable for drug development and breeding programme. In plants, this approach has
accelerated the understanding of complex transcriptional patterns and has provided measurements
of gene expression in different tissues or at different stages of development.

REFERENCES

Ghosh Dasgupta M, George BS, Bhatia A, Sidhu OP (2014). Characterization of Withania somnifera
leaf transcriptome and expression analysis of pathogenesis-related genes during salicylic acid
signaling. PLoS One, 9(4):e94803.
Gupta P, Goel R, Pathak S, Srivastava A, Singh SP, Sangwan RS, Asif MH, Trivedi PK (2013). De
novo assembly, functional annotation and comparative analysis of Withania somnifera leaf and
root transcriptomes to identify putative genes involved in the withanolides biosynthesis. PLoS
One, 8(5):e62714.
Senthil K, Wasnik NG, Kim YJ, Yang DC (2010). Generation and analysis of expressed sequence
tags from leaf and root of Withania somnifera (Ashwgandha). Mol Biol Rep, 37(2):893-902.
Singh N, Bhalla M, de Jager P, Gilca M (2011). An overview on ashwagandha: a Rasayana
(rejuvenator) of Ayurveda. Afr J Tradit Complement Altern Med, 8(5):208-13.
Uddin Q, Samiulla L, Singh VK, Jamil SS (2012). Phytochemical and Pharmacological Profile of
Withania somnifera Dunal: A Review. Journal of applied pharmaceutical science, 2 (1): 170-
175.

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 Vinca alkaloids: tubulin binding and cancer treatment

Shubhandra Tripathi, Ashok Sharma

Biotechnology Division, CSIR-Central Institute of Medicinal & Aromatic Plants, P.O:
CIMAP, Lucknow-226015, U.P., India
Email: shub.bioinfo@gmail.com

ABSTRACT

Vinca alkaloids, well known for antimitotic and antitubulin activity, are extracted from the
periwinkle plant Cataranthus roseus. There are four major type of vinca alkaloids (Maryam
et al., 2013, Bhanot et al., 2011) i.e. vincristine (VCR), vinblastine (VBL), vinorelbine
(VRL) and vindesine (VDS). Vinca alkaloids and there analogues are well known in cancer
treatment. Vinblastine is the first vinca alkaloid approved to be used as anticancer drug in
1961 (Duffin et al., 2000). Various semisynthetic derivatives of vinca alkaloids are nowadays
used in cancer therapy.

Microtubules, polymeric proteins forming the cytoskeleton of the cell, are made up of
heterodimers of α and β tubulin subunits. Microtubulin proteins filament produces motility in
cytoskeleton network by assembling and disassembling multi-subunit polymers (Amos et al.,
2011). During cell division, polymerization and depolymerization of microtubules occurs
normally. Any perturbing microtubules function leads to disruption of tubulin-cytoskeleton
network causing mitotic arrest (or mitotic block), resulting in cell death by apoptosis (Jordan
et al., 1998, Honore et al., 2005, Hadfield et al., 2003). Compounds interrupting tubulin
function are known as tubulin binding agents (TBA) (Dumontet et al., 2010), TBA are mainly
of two types; tubulin stabilizing agents (TSA) (Field et al., 2013) and tubulin destabilizing
agents (TDA). On the basis of tubulin binding site, TSA are broadly of two classes;
compounds binding mainly at paclitaxel binding site (Field et al., 2013) and compounds
binding at colchicine binding site, while tubulin destabilizing agents binds at the binding site
of vinca alkaloids (Dumontet et al., 2010).

The vinca alkaloids, have significant role in mitotic arrest, cytotoxicity occurs due to its
binding to the tubulin cytoskeleton proteins (αβ-tubulin dimers) (Owellen et al., 1976). The
crystal structure of tubulin-bound conformations of vinblastine, was available from crystal
structure (PDB ID: 1Z2B)11,12 of tubulin in complex with stathmin, colchicine and
vinblastine13. The vinca alkaloids binds have a destabilizing mechanism, thus preventing the
polymerization of the tubulin subunits. The mode of binding of vinca alkaloids is at the
interface of the β1-α2 tubulin heterodimer (Dumontet et al., 2010).

The binding of the vinca alkaloids to the α-β dimer of the tubulin, provides the destability that
is responsible for the mitotic arrest of the tumor cell. Vinblastine and vincristine are natural
compounds approved for various lymphomas and solid tumors. While, vinorelbine is a
semisynthetic compound used in breast cancer and NSCLC cancer. Vindesine is
semisynthetic compound for treatment all lymphomas and lung cancer. Vinflunine is also a
semsynthetic compound approved for bladder cancer (Bellmunt et al., 2009) and in clinical

63 | P a g e
trials for breast cancer in combination with trastuzumab
(http://www.accessdata.fda.gov/scripts/cder/drugsatfda).

In the area of cancer treatment, vinca alkaloids have provided promising opportunities, they
are used for the disrupting cytoskeleton network and causing cell death. Vinca alkaloids are
used in various types of cancer, in combinatorial chemotherapy for cancer treatment. In
recent studies, various vinca derivatives like 16-desmethoxyvinblastine, 4-desacetoxy-16-
desmethoxyvinblastine and a series of analogues having side chain modifications are
developed (Ishikawa et al., 2009) using in-silico approaches. In an advanced research, a
series of C20’ urea derivatives of vinblastine are synethesised and some of these chemically
modified compounds have shown good potency in cell growth inhibition assays (Silvestri et
al., 2013).

CONCLUSION

In summary, these studies demonstrates key role of vinca alkaloids in cancer therapy. Vinca
alkaloids have various derivative series having cell growth inhibitory properties. Using
various in silico approaches, more potent derivatives of vinca alkaloids are needed to be
developed. These finding opens new prospects of vinca alkaloids as anticancer agents.

Keywords: Tubulin, cancer, vinca alkaloids

REFERENCES

Maryam, M. & Go, R. Vinak alkaloids. Inter. Jour. Of. Prev. Med. 4, 1231-1235 (2013).

Bhanot, A. & Sharma, R. Natural sources as potential anticancerous agents: a review. Inter. Jour.
Of Phytomed. 3, 09-26 (2011).

Duffin, J. Poisoning the spindle: serendipity and discovery of the anti-tumor properties of the
Vinca alkaloids. Can. Bull. Med. Hist. Bull. Can. Hist. Médecine 17, 155–192 (2000).

Amos, L. A. What tubulin drugs tell us about microtubule structure and dynamics. Semin. Cell
Dev. Biol. 22, 916–926 (2011).

Jordan, A., Hadfield, J. A., Lawrence, N. J. & McGown, A. T. Tubulin as a target for anticancer
drugs: agents which interact with the mitotic spindle. Med. Res. Rev. 18, 259–296 (1998).

Honore, S., Pasquier, E. & Braguer, D. Understanding microtubule dynamics for improved cancer
therapy. Cell. Mol. Life Sci. CMLS 62, 3039–3056 (2005).

Hadfield, J. A., Ducki, S., Hirst, N. & McGown, A. T. Tubulin and microtubules as targets for
anticancer drugs. Prog. Cell Cycle Res. 5, 309–325 (2003).

Dumontet, C. & Jordan, M. A. Microtubule-binding agents: a dynamic field of cancer

therapeutics. Nat. Rev. Drug Discov. 9, 790–803 (2010).

Field, J. J., Díaz, J. F. & Miller, J. H. The Binding Sites of Microtubule-Stabilizing Agents.
Chem. Biol. 20, 301–315 (2013).

Owellen, R. J., Hartke, C. A., Dickerson, R. M. & Hains, F. O. Inhibition of tubulin-microtubule

polymerization by drugs of the Vinca alkaloid class. Cancer Res. 36, 1499–1502 (1976).

64 | P a g e
Ravelli, R. B. G. et al. Insight into tubulin regulation from a complex with colchicine and a
stathmin-like domain. Nature 428, 198–202 (2004).

Mitra, A. & Sept, D. Localization of the antimitotic peptide and depsipeptide binding site on beta-
tubulin. Biochemistry (Mosc.) 43, 13955–13962 (2004).

Cormier, A., Knossow, M., Wang, C. & Gigant, B. The binding of vinca domain agents to
tubulin: structural and biochemical studies. Methods Cell Biol. 95, 373–390 (2010).

Bellmunt, J. et al. Phase III trial of vinflunine plus best supportive care compared with best
supportive care alone after a platinum-containing regimen in patients with advanced
transitional cell carcinoma of the urothelial tract. J. Clin. Oncol. Off. J. Am. Soc. Clin.
Oncol. 27, 4454–4461 (2009).

Ishikawa, H. et al. Total Synthesis of Vinblastine, Vincristine, Related Natural Products, and Key
Structural Analogues. J. Am. Chem. Soc. 131, 4904–4916 (2009).

Silvestri, R. New Prospects for Vinblastine Analogues as Anticancer Agents. J. Med. Chem. 56,
625–627 (2013).

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PLANT CHEMISTRY
 A review of the extraction technologies for artemisinin ex Artemisia annua

Karishma Agarwal , Abhishek Nagar and Sudeep Tandon

Process Chemistry and Technology Department, CSIR-Central institute of Medicinal and
Aromatic Plants, Lucknow-226015.

ABSTRACT

Artemisia annua is a well known medicinal plant due the presence of artemisinin which has a
potent antimalarial activity. The content of artemisinin itself is very small at around 0.01 to 1.0%
of dry weight of plant. The present review is an attempt to highlight the various extraction
techniques being used for the isolation of Artemisinin. Extraction using volatile organic solvents,
ionic liquids, hydrofluorocarbons HFC-134a, and supercritical carbon dioxide are compared with
more sophisticated technologies such as microwave and ultrasonic assisted extraction, so that the
economic viabilities of the herb increases and new approaches to improve the existing
technologies can be developed.

INTRODUCTION
Artemisia annua L. (Asteraceae) is a medicinal plant originated from Asia and Eastern Europe. It
is an important medicinal plant due to the presence of artemisinin its major constituent which has
proven anti malarial activity. Artemisinin is a sesquiterpene endo peroxide found in aerial parts
of this plant. It has received a world wide exposure for the treatment of cerebral malaria caused
by the plasmodium species which is becoming resistant to the known antimalarial drugs.
Artemisia annua grown worldwide is traditionally processed through solvent extraction using
hexane and petroleum ether as extracting solvents. Although, both the solvents are cheap but are
hazardous and can be harmful to the environment. Several alternative solvents & techniques
have been studied for the extraction of artemisinin from the herb like using ionic solvents, super
critical fluid extraction and microwave assisted extraction and other technologies which claims
to have greater efficiencies and are safer to environment. The objective of this review is to
examine the technologies developed for extraction of Artemisinin.

Extraction using volatile solvents

Due to the physiochemical properties of Artemisinin i.e. low thermal and chemical stability and
low polarity hence poor solubility in water the miscibility in organic solvents enhances the use of
hexane, acetonitrile, ethyl acetate and methanol for extraction of the drug. Several studies have
been reported on the extraction efficiency using different combination of solvents and
methodology of extraction.

Hexane
In this method reported in a study by El-Sohly et al and Haynes et al the dried crush leaves of
Artemisia is soaked in hexane in ratio solvent: plant material 4:1, and each extraction cycle takes
10 to 40 hours. However the extraction efficiencies of artemisinin through this process is very
low. In order to improve the efficiencies of extraction sometimes a small amount of polar solvent
like ethyl acetate is added to increase the solubility of artemisinin by about two orders of
magnitude. The obtained crude extract is then evaporated to ten percent of its initial volume and

66 | P a g e
the remaining liquor is left to stand at ambient temperature for about 48 hours to crystallize crude
artemisinin. Further purification of artemisinin is achieved by chromatography. 1

Hexane and Acetonitrile

In this pilot scale experiment carried out at CIMAP by Tandon et al the dried herb of Artemisia
annua is extracted with a hydrocarbon solvent like hexane in a solvent extraction plant in which
four to five percolations are given for complete extraction of herb. The crude extract obtained
from non-polar solvent is further subjected to liquid-liquid partitioning using a polar modified
solvent like acetonitrile for removing of unwanted fats and other chemical entities. The polar
extract obtained is further purified using column chromatography for the isolation of artemisinin.
The extraction efficiency of extraction using this technology was 80-85 % with purity of
artemisinin upto 90%.2

Methanol, hexane and ethyl acetate

In another study by Dahnum Deliana et al solvent extraction of the herb is done using methanol,
followed by partition using hexane and column chromatographic separation process with ethyl
acetate / hexane as eluent. Based on the research done, artemisinin compound was obtained 2.0
mg (0.016% w / w) by methanol extraction. 3

Chloroform
In this study reported by Bioniqs limited the leaf material of Artemisia was extracted with
chloroform using percolation. The solvent was then removed in vacuum to yield 11.6 gms of
greenish semicrystalline solid residue. The extract was then redissolved in 50:50 diethyl ether ;
and 40:60 petroleum ether and analysed by HPLC. HPLC Chromatogram of primary Artemisia
extract show the presence of artemisinin along with 6 major co-extracted impurities.

Ethanol
Ethanol is potentially attractive solvent due to it’s local availability and lower price. According
to the study carried out by Rodrigues R.A.F et al Quantitative extraction of artemisinin with
ethanol is possible compared to 60% efficiency in hexane. 3 In case of ethanol no losses in
artemisinin content has been found in the liquid extract upto 6 months. However, in case of
ethanol extract contain higher amount of polar impurities mainly sugars which inhibit the
crystallization of artemisinin. So, a rectified study was been done for the extraction by Ethanol
aqueous azeotrope at room temperature claimed high efficiency of artemisinin. 4 However,
flammability, high toxicity and its high rish risk of use restricts the use of ethanol.

Extraction with Ionic liquids

Organic ionic liquids are a new class of solvents having negligible vapor pressure and are
inflammable. Also due to their property of bio-degradability and non-toxic nature they are often
considered as green alternatives. They are capable of being sourced and recycled. They are being
selective for artemisinin in presence of flavanoids and terpenoids.The various ionic solvents
which has been used are N,N-dimethylethanol ammonium octanoate (DMEA oct) , bis( 2-
methoxy ethyl) ammonium bis (trifluromethyl sulfonyl) imide (BMOEA bst) , N,N-dimethyl-(2-
methoxyethyl)amine pro. The review is based on the preliminary study undertaken by Bioniqs ltd
(UK).

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 Volume of Water g IL per g artemisinin
1. 0.5 62-85
2. 1 40-67
3. 1.5 36-58
4. 2.0 30-50
5. 5 15-35
6. 10 8-27
7. 25 4.5-18
8. 50 1.0-7
9. 75 0.6-1.5
10. 100 0.2-0.8

N,N-dimethylethanol ammonium octanoate (DMEA oct)

Extraction process with DMEA oct was similar to liquid-solid extraction and was performed at
25ºC.The observed concentration of artemisinin was considered to be similar to hexane but the
rate of extraction was more.

Extraction of Artemisia annua with the DMEA oct solvent was done using solvent: leaf ratio 2:1.
Further the extract was analysed with HPLC and the artemisinin was present at the concentration
of 0.36% of dried weight of plant. Further when the ratio of solvent: leaf was increased to about
7:1 the amount of artemisinin was increased to 0.45-0.47% of dried weight of plant. In addition
to artemisinin only three major impurities were extracted as compared to six impurities in
chloroform. Further the recovery of the precipitate of artemisinin was done using DMEA oct by
dividing primary extract into ten parts 5 ml each and adding a define volume of water varying
from 0.5 to 100 ml.

Bis( 2-methoxy ethyl) ammonium bis (trifluromethyl sulfonyl) imide (BMOEA bst)
Further to increase the yield of artemisinin 2 bis( 2-methoxy ethyl ) ammonium bis (trifluoro
methyl sulfonyl) imide has been used. In this case extraction rate was considerably slower than
DMEA oct. However, extraction efficiency was higher by 23% thus in comparison with volatile
solvent this gives higher extraction efficiency at the same rate.

N,N-dimethyl-(2-methoxyethyl)amine pro
DMMOEA pro was prepared by the neutralization of N,N-dimethyl-(2-methoxyethyl)amine with
the propionic acid. The main purpose for the use of this solvent is to minimize the amount the
amount of impurities observed in DMEA oct .

The extraction profile of DMMOEA pro indicate the absence of impurities found in DMEA oct
but substantially many other impurities of terpenoid class were extracted in addition to
artemisinin.

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So, in nut shell DMEA oct possess large number of desirable properties to extract artemisinin in
the absence of large amount of terpenoids but due to its poor reproducibility of precipitation
from solution of artemisinin by addition of water it is not considered for its in- field application.
So, DMMO pro was optimized for in field application in spite of its co- extraction of terpenoids.

Solvent Abs. saturation Optimal Primary Precipitation Purity after

concentration of extraction yield vs yield first
artemisinin (g/L) time (mins) chloroform recrystalization
@25 0C
DMEA oct 110 30-60 32% 82% >97%
DMMOEA 68 10-45 41% 88% >97%
pro

Extraction using supercritical CO2

In a paper published by Rodrigues R.A.F et al, it was observed that compared to the extraction
with hydrocarbon solvents, the extraction of artemisinin by sub-critical liquid CO2 is more
quantitative, rapid and with higher selectivity. There is a wide variability lies in extraction
efficiencies using supercritical CO2 due to the parameters such as scale of extraction, use of co-
solvents, temperature and pressure of extraction and superficial velocity of the solvent in the
extractor. Efficiency of extraction was reported to be lower in the absence of hydrophilic co-
solvent. The overall extraction efficiency including purification by crystallization was reported
about 80%.5

Extraction using hydrofluorocarbons HFC-134a (1,1,1,2-tetrafluoroethane)

Hydrofluorocarbons are gases under normal condition and liquefied at relatively low pressures.
Therefore, these solvents are ideally suited for the continuous extraction processes. It is
considered as non-flammable and has zero ozone depleting potential. It is used as a solvent in the
food flavourings in Japan, Europe and USA. 6

High global warming potency factor i.e. 1300 times larger than that of carbon dioxide is one
drawback. It is the reason due to which the complete recycling and capture of the solvent within
a process is of significant importance.

Extraction of other natural compounds by HFC-134a and iodotrifluoromethane (ITFM) have

been reported but not specifically for Artemisinin. 6 Ineos Fluor Ltd. Provided the data which are
reported in this study.

Microwave assisted extraction of Artemisinin

A new method has been evolved for the extraction of Artemisinin through microwave assisted
extraction using different solvents such as ethanol, trichloromethane, cyclohexane, n-hexane,
petroleum ether 30-60 0C and petroleum ether 60-900C,No.120 solvent oil and no.6 extraction
solvent oil.7 In this method , ten gram of Artemisia Annua was put in flask followed by addition
of proper volume of solvent. The flask was then exposed for 60 seconds in microwave extractor.

69 | P a g e
Then the flask was taken out, cool to room temperature and the above process was repeated up to
designed value.

Ultrasonic assisted extraction of artemisinin by response surface methodology

According to the method reported by Haihui Zhang et al extraction of artemisinin was done
using ultrasound-assisted extraction (UAE). In this study, response surface methodology (RSM)
was used to optimize UAE conditions for obtaining the maximum yield of artemisinin. The
optimal extraction conditions were as follows: 42.71 mL/g ratio of solvent to material, 41.86°C
extraction temperature and 120 W ultrasonic power. The predicted yield of artemisinin by model
was 0.7848%, whereas the actual yield in the extracts was 0.7826% ± 0.0790% in adjusted
optimal conditions, with a relative error of 0.28%8. The results undoubtedly demonstrated that
RSM could be used to explore the optimum conditions of artemisinin extraction.

Comparison of extraction efficiency

Based on the available data for each extraction process the efficiency of extraction related to
artemisinin content has been studied and reported by Alexei lapkin et al. Among these processes
microwave assisted extraction, HFC 134a, ionic liquid based extraction may offer the most
feasible methods for high artemisinin content. However, amongst the solvents studied for
extraction of artemisinin, the extraction efficiency was observed to be best using combination of
hexane and acetonitrile.

S.No. Extraction methods Duration of Extraction efficiency (in %)

Extraction cycle

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 1. Hexane 8-10 hrs 60%
2. Ethanol 7 hrs 73%
3. Ionic liquids 2.5-6 hrs 79%
4. sc CO2 3-6 hrs 82%
5. HFC-134a 6 hrs >62%
6. Microwave assisted 6 min 82%
7. Hexane & 4-6 hrs > 85%
Acetonitrile

REFERENCES

1 EI-Sohly, H. N.; Jr, E. M. C.; El-Feraly, F. S.; EI-Sherei, M. M. J. Nat. Products, 1990, 53,
1560-1564.
2 S Tandon ; A.Kahol ; DC Jain ; RS Bhakuni ; Sushil Kumar , Journal of Scientific and
Industrial Research , 2003 , 62 , 457-461.
3 Dahnum , D.; Abimanyu, H.; Senjaya , A.; International Journal of Basic & Applied
Sciences IJBAS-IJENS , 12 , 90-95.
4 Kohler, M.; Haerdi, W.; Christen, P.; Veuthey, J.-L. J. Chromatography A., 1997, 785,
353-360;
5 McCulloch, A.; Lindley, A. A. Int. J. Refrigeration 2003, 26, 865-872.
6 Wilde, P. F. US Patent 5,512,285, 1996;
7 Jin-yu Hao ; Wei Han ; Shun-de-Huang ; Bo-young Xue ; Xieu Deng , Separation and
Purification Technology , 2002 , 28 , 191-196.
8 Haihui, Z.; Li, Z.; Xigui, H.; Yonghong, Z.; Chunbang, D.; Ruiwu, Y.; Xiaoli,
W.; Dengchao, L.; Separation Science and Technology , 2014, 49(5).

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 A wonder plant Withania: Pharmacological and Chemical review

Surjeet Verma, Santosh Kumar Srivastava

Medicinal Chemistry Department,CSIR-Central Institute of Medicinal and Aromatic Plants,
Kukrail Picnic Spot Road, Lucknow-226015, India.
Email: surjeetjayram@gmail.com

ABSTRACT

The purpose of this review is to introduce the readers about the pharmacological and chemical
aspects of the genus Withania. Present review has a brief description about almost all the
pharmacological and chemical studies made on the six different species of this genus and tells
about the future prospectus of these plants in the field of drug discovery and health care. It also
provides key information about the structure activity relationship in withanolides which are the
major bioactive constituents of these plants.

INTRODUCTION

Withania is a well known genus of the flowering plants family Solanaceae with 23 species,
distributed over the parts of North Africa, the Middle East, the Mediterranean, and the Canary
Islands (Mirjalili, et al., 2009). Out of 23 species, Withania somnifera (Ashwagandha) also
known as Indian ginseng and Withania coagulans (Ashutosh booti) are well explored and
economically important species, cultivated in several regions for their medicinal uses (Mirjalili,
et al., 2009). W. somnifera and W. coagualans have shown diverse biological activities viz.,
cytotoxic, anti-inflammatory, anti-stress, antioxidant, anti-aging, diuretic, hypothyroid,
immunomodulatory, anti-alzheimer’s disease, antihyperglycemic, anti-hypercholesterolemic,
antimicrobial, anti-feedent, cardiovascular, cell-differentiation-inducing activity,
immunosuppressive and radio sensitizing activity, etc. (Maurya et al., 2010, Singh et al., 2010,
Chen et al., 2011, Khodaei et al., 2012, Kaur et al., 2013). Detailed bioassay guided
phytochemical investigations have revealed that almost all these biological activities are due to
the withanolides. Chemically, withanolides are steroidal lactones. The term withanolides is the
composition of the “withan” from the genus Withania, and “olide” a chemical term for the
lactone moiety. Withaferin A was the first withanolide isolated by Lavie in 1965 (Lavie et al.,
1965) from W. somnifera. Since then, more than 400 withanolides have been isolated from 58
solanaceous species belonging to 22 genera (Kirson and Glotter, 1981, Glotter, 1991, Chen et al.,
2011, Khodaei et al., 2012).

72 | P a g e
DISCUSSION

Beside these two species (W. somnifera and W. coagualans), four other species viz., W.
adpressa, W. aristata, W. frutescens and W. obtusifolia have also been investigated for the study
of their phytochemical and biological aspects. But still these species are not well explored. For
example, there are only two reports one on each of the antifungal (Askarne et al., 2012) and
cytotoxic (Abdeljebbar et al., 2009) activity of W. adpressa. However, this antifungal activity is
reported for the leaf of this plant but other parts have to be investigated. Further, no chemical
constituents have been identified which are responsible for this antifungal activity. Similarly,
three cytotoxic withanolides, 14α, 15α, 17β, 20β-tetrahydroxy-1-oxo-(22R)-witha-2,5,24-
trienolide (1), withanolide F (2) and withanolide J (3) are isolated from the dichloromethane
(CH2Cl2) fraction of the methanolic extract of the W. adpressa leaf. But no work has been carried
out on the phytochemical investigation of the other parts of this plant as well as on other
fractions of the methanolic extract of the leaves. Hence, the other parts viz., stem, root, fruits and
flower and other fractions viz., n-hexane, ethylacetate, n-butanol of the methanolic extract of the
leaf, require chemical investigation of the compounds responsible for the cytotoxicity and other
biological activities.

Figure-1. Cytotoxic withanolides isolated from W. adpressa.

Certainly, it will be a great effort in the area of drug discovery from the plants, which will further
improve the economical significance of W. adpressa as well as of the genus Withania.
Similarly, several withanolides have been isolated from the dichloromethane extract W. aristata
leaves too. These withanolides have shown significant antimicrobial (Araujo et al., 2008),
diuretic (Martın-Herrera et al., 2008; Benjumea et al., 2009) and anti-proliferative cytotoxic
(Llanos et al., 2008, 2010, 2012) activities. Similar to W. adpressa, other parts and different
extracts of W. aristata leaves also require chemical and biological investigations for other
biological activities and the novel withanolides responsible for these bioactivities.

73 | P a g e
74 | P a g e
 Figure-2. Cytotoxic withanolides from W. aristata.

Figure-3. Diuretic withanolides from W. aristata.

Withania frutescens (L.) Pauquy is another species of this genus. The leaf extract of this plant
showed significant protective and curative action against CCl4-induced hepatotoxicity (Montilla
et al., 1990), but so for no hepatoprotective chemical constituent has been isolated from this
extract. Another reports demonstrated the isolation of some cytotoxic withanolides from the
dichloromethane extract of W. frutescens leaves (Bouzidi et al., 2013). However, other plant
parts require further chemical and biological investigations for the above and new biological
activities as well.

75 | P a g e
 Figure-4. Cytotoxic withanolides from Withania frutescens.

Occurrence of withanolides has also been reported in Withania obtusifolia Dun by Modawi et al.,
in 1986. But surprisingly no chemical and biological investigations have been carried out so for
on this plant. Hence, it will be worth exploring this plant for the discovery of other withanolides
and their biological activities.

Withania somnifera is a very well phytochemically explored plant and a number of withanolides
have been isolated from the different part of this plant. The soil-less aeroponically grown W.
somnifera leaves and twigs afforded some unusual withanolides viz., 3α-(uracil-1-yl)-2,3-
dihydrowithaferin A (42), 3b-(adenin-9-yl)-2,3-dihydrowithaferin A (43) and 2,3-
dihydrowithaferin A-3β-O-sulfate (44) (Xu et al., 2009, 2011). The purpose of this study was to
maximize the production and the structural diversity of the plant metabolites and studying the
effect of W. somnifera grown under soil-less aeroponic conditions and its ability to produce
withaferin A and other withanolides. The sulphated withanolides have also been reported from
the normal withania somnifera (Misra et al., 2005). Further, 2,3-dihydrowithaferin A-3β-O-
sulfate was also isolated from W. frutescens with undefined stereochemistry at C-3 position
(Figure-4). These three novel withanolides possess significant cytotoxicity (0.54, 0.61 and 5.25
µM respectively) against human Ewing’s sarcoma cell line CHP-100 (Wijeratne et al., 2014).
Hence, the present report provides a new idea to phytochemists in search of the new withanolides
from W. somnifera. This study can further extend to other species and other parts of Withania
somnifera.

76 | P a g e
 Figure-5. Unusual withanolides from aeroponically grown W. somnifera.

Structure-activity relationship (SAR)

Withanolides isolated from W. somnifera are potent Cyclooxygenase-2 (COX-2) inhibitor
(Jayprakasham and Nair, 2003) and it has been observed that the presences of double bond in
α,β-unsaturated δ-lactone moiety is essential for the COX-2 inhibitory activity. Further, it has
been observed that epoxide ring at 5,6- position is important for cholinesterase inhibition
(Chaudhary et al., 2004, 2005). Ring A enone and epoxide ring at 5,6- position are also essential
for the cytotoxicity of the withanolides (Llanos et al., 2012, Joshi et al., 2014, Wijeratne et al.,
2014). Conversion of the epoxide ring into thioepoxide or cleavage of the epoxide bonds to
produce hydroxyl and thiol group resulted 10 to 25 fold reduction in the cytotoxicity of
withaferin A and other withanolides against different tested cancer cell lines (Joshi et al., 2014).
Further, acetylation of 4 and 27 OH groups also resulted in significant improvement in
cytotoxicity whereas introduction of the β-OH at C-12 position reduces the cytotoxicity of the
withanolides (Llanos et al., 2010, 2012, Wijeratne et al., 2014). Acylation of 4-hydroxy group
with 4-bromobenzoyl group in (4S,20S,22R)-4,27-Dihydroxy-1-oxo-witha-2,5,16,24-
tetraenolide isolated from W. aristata enhanced the cytotoxicity up to 3-7 fold (Llanos et al.,
2010). Hence, the above concept can be applied in the semi-synthesis of the other withanolide
derivatives with improved biological activities. Protection of the 27-OH group with
tertiarybutyldimethylsilyl (TBDMS) group also enhanced cytotoxicity by 3-10 folds against
various tested cancer lines. Hence, the above observation suggested that increasing the
lipophilicity of withanolides leads to the increase in their cytotoxicity.

77 | P a g e
 Figure-6. Structure-cytotoxicity relationship in different types of withanolides.

Structure-activity relationship for different biological activities has been summarized in Figure-
7. The functional groups within the circle are responsible for that particular activity.

Figure-7. Necessary units for different biological activities of withanolides.

CONCLUSION

78 | P a g e
Withania is the pharmacologically very important genus of the flowering plants and well known
for its diverse biological activities. Withanolides are the major bioactive constituents in all the
species of this genus. These withanolides possesses significant cytotoxicity against various
cancer cell lines and it has been investigated that increasing the lipophilicity of these molecules
enhances the cytotoxicity and vice-versa.

REFERENCES

Askarne, L.; Talibi, I.; Boubaker, H.; Boudyach, E. H.; Msanda, F.; Saadi, B.; Serghini, M. A.; Ait
Ben Aoumar, A. In vitro and in vivo antifungal activity of several Moroccan plants against
Penicillium italicum, the causal agent of citrus blue mold. Crop Protection 2012, 40, 53-58.
Abdeljebbar, L. H.; Benjouad, A.; Morjani, H.; Merghoub, N.; Haddar, S. E.; Humam, M.; Christen,
P.; Hostettmann, K.; Bekkouche, K.; Amzazi, S. Antiproliferative Effects of Withanolides
from Withania adpressa. Therapie 2009, 64 (2), 121–127.
Araujo, L.; Padilla, N.; Llanos, G. G.; Bazzocchi, I. L.; Moujir, L. Antimicrobial activity of
withanolides from the leaves of Withania aristata. Planta Med. 2008, 74 - PB70.
Benjumea, D.; Martin-Herrera, D.; Abdala, S.; Gutierrez-Luis, J.; Quinones, W. Cardona, D.; Torres,
F.; Echeverri, F. Withanolides from Whitania aristata and their diuretic activity. J.
Ethnopharmacol. 2009, 123, 351–355.
Bouzidi, L. E.; Mahiou-Leddet, V.; Bun, S. S; Larhsini, M.; Abbad, A., Markouk, M.; Fathi, M.;
Boudon, M. l.; Ollivier, E.; Bekkouche, K.. Cytotoxic withanolides from the leaves of
Moroccan Withania frutescens. Pharmaceut. Biol. 2013, Early Online, 1–7. DOI:
10.3109/13880209.2013.775162.
Chen, L. X.; He, H.; Qiu, F. Natural withanolides: an overview. Nat. Prod. Rep. 2011, 28, 705–740.
Choudhary, M. I.; Yousuf, S.; Nawaz, S. A.; Ahmed, S.; Rahman, A. U. Cholinesterase Inhibiting
Withanolides from Withania somnifera. Chem. Pharm. Bull. 2004, 52(11), 1358-1361.
Choudhary, M. I.; Nawaz, S. A.; Haq, Z.; Lodhi, M. A.; Ghayur, M. N.; Jalil, S.; Riaz, N.; Yousuf,
S.; Malik, A.; Gilani, A. H.; Rahman, A. Withanolides, a new class of natural cholinesterase
inhibitors with calcium antagonistic properties. Bioch. Biophys. Res. Commun. 2005, 334, 276–
287
Glotter, E. Withanolides and related ergostane-type steroids. Nat. Prod. Rep., 1991,8, 415-440.
Jayaprakasam, B.; Nair, M. G. Cyclooxygenase-2 enzyme inhibitory withanolides from Withania
somnifera leaves. Tetrahedron 2003, 59, 841–849.
Joshi, P.; Misra, L.; Siddique, A. A.; Srivastava, M.; Kumar, S.; Darokar, M. P. Epoxide group
relationship with cytotoxicity in withanolide derivatives from Withania somnifera. Steroids
2014, 79, 19–27.
Kaur, N.; Niazi, J.; Bains, R. A Review on Pharmacological Profile of Withania somnifera
(Ashwagandha). Res. Rev. J. Botan. Sci. 2013, 2(4), 6-14.
Khodaei, M.; Jafari, M.; Noori, M. Remedial Use of Withanolides from Withania Coagolans
(Stocks) Dunal. Advances in Life Sci. 2012, 2(1), 6-19.
Kirson, I. , Glotter, E. Recent Developments in Naturally Occurring Ergostane-Type Steroids. A
Review. J. Nat. Prod., 1981, 44(6), 633–647.
Lavie, D.; Glotter, E.; Shvo, Y. Constituents of Withania somnifera Dun. III. The Side Chain of
Withaferin A. J. Org. Chem., 1965, 30 (6), 1774–1778.
Llanos, G. G.; Araujo, L. B.; Jimenez, I. A.; Moujir, L.; Bazzocchi, I. L. New cytotoxic withanolides
from Withania aristata. Planta Med. 2008, 74 - PB131.
Llanosa, G. G.; Araujob, L. M.; Jimeneza, I. A.; Moujirb, L. M.; Vazqueza, J. T.; Bazzocchia, I. L.
Withanolides from Withania aristata and their cytotoxic activity. Steroids 2010, 75, 974–981.

79 | P a g e
Llanos, G. G.; Araujo, L. M.; Jimenez, I. A.; Moujir, L. M.; Bazzocchi, I. L. Withaferin A-related
steroids from Withania aristata exhibit potent antiproliferative activity by inducing apoptosis in
human tumor cells. Eur. J. Med. Chem. 2012, 54, 499-511.
Martin-Herreraa, D.; Abdalaa, S.; Benjumeaa, D.; Gutierrez-Luis, J. Diuretic activity of some
Withania aristata Ait. Fractions. J. Ethnopharmacol. 2008, 117, 496–499.
Maurya, R.; Akanksha; Jayendra. Chemistry and pharmacology of Withania coagulans: an Ayurvedic
remedy. J. Pharm. Pharmacol. 2010, 62, 153–160.
Mirjalili, M. H.; Moyano, E.; Bonfill, M.; Cusido, R. M.; Palazón, J. "Steroidal Lactones from
Withania somnifera, an Ancient Plant for Novel Medicine". Molecules 2009, 14 (7), 2373–
2393.
Misra, L.; Lal, P.; Sangwan, R. S.; Sangwan, N. S.; Uniyal, G. C.; Tuli, R. Unusually sulfated and
oxygenated steroids from Withania somnifera. Phytochem. 2005, 66, 2702–2707.
Modawi, B. M.; Iskander, G.M.; Karim, M. A.; Fair, C. K.; Schlemper, E. O. Crystal and molecular
structure of a new withanolide. Isomeric with withaferin a isolated from withania obtusifolia
dun. Journal für Praktische Chemie 1986, 328 (2), 291–294.
Montilla, M. P.; Cabo, J.; Navarro, M. C.; Risco, S.; Jimenez, J.; Aneiros, J. The protective and
curative action of Withania frutescens leaf extract against CCl4-induced hepatotoxicity.
Phytother. Res. 1990, 4(6), 212-215.
Singh, G.; Sharma, P. K.; Dudhe, R.; Singh, S. Biological activities of Withania somnifera. Annals of
Biol. Res. 2010, 1(3), 56-63.
Wijeratne, E. M. K.; Xu, Y.; Scherz-Shouval, R.; Marron, M. T.; Rocha, D. D.; Liu, M. X.; Costa-
Lotufo, L. V.; Santagata, S.; Lindquist, S.; Whitesell, L.; Gunatilaka, A. A. L.
Structure−Activity Relationships for Withanolides as Inducers of the Cellular Heat-Shock
Response. J. Med. Chem. 2014, 57, 2851−2863.
Xu, Y.; Marron, M. T.; Seddon, E.; McLaughlin, S. P.; Ray, D. T.; Whitesell, L.; Gunatilaka, A. A.
L. 2,3-Dihydrowithaferin A-3β-Osulfate, a new potential prodrug of withaferin A from
aeroponically grown Withania somnifera. Bioorg. Med. Chem. 2009, 17, 2210−2214.
Xu, Y.; Gao, S.; Bunting, D. P.; Gunatilaka, A. A. L. Unusual withanolides from aeroponically
grown Withania somnifera. Phytochem. 2011, 72, 518–522.

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 Anti-cancer properties of artemisinin and its derivatives

Rashmi Gaur*, Rajendra Singh Bhakuni

Medicinal Chemistry Division, CSIR- Central Institute of Medicinal and Aromatic Plants,
Lucknow-226015, India

ABSTRACT

A number of natural products, with diverse chemical structures, have been isolated as anticancer
agents. Several potential lead molecules are vincristine, vinblastine, camptothecin,
podophyllotoxin, taxol, combretastatins, artemisinin etc. In this tutorial review, the authors have
focused on the recent developments on various kinds of artemisinin derivatives including
artemisinin dimers, trimers and tetramers has been made and their efficacy towards different
cancer cell lines was compared with that of artemisinins, and various other anti-cancer drugs. It
is expected that this review will provide first-hand information on artemisinin chemistry wto
organic/medicinal chemists, and pharmacologists working on anticancer drug development.

INTRODUCTION

Cancer is a growing public problem its estimated worldwide new incidence is about 6 million
cases per year. The search for natural products as potential anticancer agents dates back, atleast,
to the Ebers papyrus in 1550 BC, but the scientific period of this search is more recent,
beginning with the investigations by Hartwell and co-workers in late 1960s on the application of
podophyllotoxin and its derivatives as anticancer agents.

Owing to the remarkable success of artemisinin in the treatment of malaria, there is a growing
interest in exploring the other therapeutic potentials of artemisinin. At the beginning of 1990s, it
was found that artemisinin endoperoxides such as artemisinin (1), artemether, arteether,
artesunate exhibited cytotoxicity to Ehrlich ascites tumour cells with IC50 values ranging from
12.2 to 19.9 μM [Woerdenbag, et al. 1993]. This study opened up a new research direction in
exploring the anticancer activities of artemisinin.

Discovery of artemisinin
14
2 10 O
3 9 O O O
15 O 1 O
O
O O
O
NC
4 O O O
O 6 7 8 O O
5 11 NC O
O O R SO2
O 12
13 O
O R R= H, F, Cl, Br, NO2 etc
Artemisinin

Artemisinin (1) is found in the leaves and flowers of Artemisia annua in 0.01– 0.8% of
dry weight [Van, et al. 1997]. The peroxide moiety of artemisinin (1) is crucial for its
antimalarial activity as deoxyartemisinin without the peroxide moiety is completely inactive
[Klayman, 1993].

The Cytotoxic Artemisinin Derivatives

81 | P a g e
At the beginning of 1990s, it was found that artemisinin endoperoxides such as artemisinin (1),
artemether, arteether, artesunate exhibited cytotoxicity to Ehrlich ascites tumour cells with IC 50
values ranging from 12.2 to 19.9 μM [Woerdenbag, et al. 1993]. This study opened up a new
research direction in exploring the anticancer activities of artemisinin.

Monomeric Cytotoxic Artemisinin Derivatives

It is generally accepted that ferrous iron (in the form of heme) reduces the crucial endoperoxide
moiety of artemisinin and triggers the formation of a series of reactive intermediates including
carbon-centered free radicals and reactive oxygen species (ROS), one or more of them kills the
parasites in malaria-infected human red blood cells. It is proposed that the anticancer activity of
artemisinin may share the mechanism of action similar to that of antimalarial activities because
of the higher concentration of Fe(II) in cancer cells [Karin, et al. 1981; May, et al. 1985]. This
hypothesis, coupled with preliminary experimental results, prompted the researchers to study the
cytotoxicity of simple artemisinin analogues such as artemisinin (1), DHA, artesunate against a
panel of cancer cell lines such as breast cancer (HTP27), small-cell lung cancer SCLC (H69VP),
human leukemia (Molt-4), cervical cancer (HeLa), uterus chorion cancer (JAR), embryo
transversal cancer (RD) and ovarian cancer (HO-8910).

Recently, a new class of thioacetal-containing artemisinins that showed antiangiogenic activity

was developed. Some of the analogues exhibited strong growth inhibition activity against
HUVEC proliferation with IC50 values ranging from 0.93 to 18.2 μM [Oh, et al. 2003; Oh, et al.
2004].

Artemisinin dimers, trimers and tetramers: novel leads in drug discovery

It has been established through the structure–activity relationship (SAR) studies of artemisinin
and its various kinds of C-12/C-13 ether/ester derivatives that only peroxide-linkage affects the
anti-cancer and anti-malarial activity. Furthermore, several drawbacks associated with these
compounds viz solubility, thermal and bioavailability, hydrolytic stability, and short half life etc,
have led to development of second generation C-12/C-13 trioxane-derivatives.

Artemisinin dimers reported till now, have displayed structural diversity, separated through
artemisinin monomer units with or without linkers of various length and flexibility with diverse
stereochemistry. Several of these C-12/C-13 carbon artemisinin dimers have shown outstanding
anti-cancer and anti-malarial activity and are better than C-12 ether/ester dimers. Artemisinin
trimers and tetramers of C-12/C-13 non-acetal derivatives have also been reported in recent
years, in which artemisinin units are connected through linkers of various kinds with diverse
length and stereochemistry. However, the number of artemisinin dimers synthesized so far are
far-ahead of the number of artemisinin trimers and tetramers. Many of these dimers, trimers and
tetramers have shown outstanding anti-cancer and anti-malarial activities compared to
artemisinin and related compounds, and are in various phases of clinical trials. These compounds
may become future potential leads in anti-cancer and anti-malarial chemotherapy.

I. Artemisinin dimers
Based on the C-12/C-13 linkage between artemisinin units through the linker, acetal or non-
acetal, artemisinin dimers have been classified as follows:

82 | P a g e
(a) C-12 oxa dimers: Here two artemisinin monomers units are linked through the C-12 acetal
(i.e. ether–ester linkage) linker.
O
O
O
O

OMe

OMe
m-dimer, 74%
O O BF3.OEt2

O O
H O H H H
O
O O O O O O O
O O O
O O O O O
O

O
O
O
O
O furan-dimer, 56%
BF3.OEt2

F Me2Al
H H O
O H H
O O AlMe2
O O O O O
O O O O
O
p-dimer, 27%

BF3.OEt2 m-dimer, 19%

(b) C-12 carba dimers: Here two artemisinin monomers units are linked through the C-12 non-
acetal (i.e. carbon–carbon linkage) linker. Although C-12 oxa dimers have displayed high
antimalarial, antiproliferative and anti-tumor activities in vitro, often they are hydrolytically
unstable. In order to enhance the hydrolytic stability, researchers have directed their efforts to
synthesize a variety of C-12 olefinic carba dimers.

O
O O
O O O O
O O O O O O O O
O O O O O
O O O O O O R
O O O O O O O
O O O O O HN
O O O O
O O O O O
O NH
O O NH
( O )n ( )n NH ( )n S S
( )n ( )n
S n=1, 2
O O O
F O n=0
n=0, 2 O F F
F (R=t-Bu)

The in vitro cytotoxicity of artemisinin and related dimers against murine and human cancer
cells. Sulfide dimer (IC50 = 0.40 µg mL-1) is active comparable to aldriamycin (IC50 = 0.39 µg
mL-1) and four times more active than mitomycin (IC50 = 1.50 µg mL-1) against-mouse fibroblast
leukemia (P388). Sulfone-linked dimer (IC50=1.04 µg mL-1) is active comparable to mitomycin
(IC50=0.85 µg mL-1) and six times more active than adriamycin (IC50 = 6.24 µg mL-1) and taxol
(IC50 = 7.39 µg mL-1) against human placental choricarcinoma cells (Bewo).

Later on, Posner’s group has synthesized [Posner, et al. 2003] a series of artemisinin dimers
starting from dihydroartemisinin acetate. Anti-cancer growth inhibitory activities of these dimers
were measured in vitro using a diverse panel of human cancer cell lines, indicates that alcohol
and diol dimers are strongly
O O
O O O O
O O O
O O
O
O O Art Art
O O

Art Art
72a, ortho
O O
72b, meta
O O Art Art COOH
72c, para
O 74 77
O 74a, ortho + -
72
RuCl3.NaIO4 O N O
74b, meta EDC, DMAP
74c, para NaBO3 78
O 58
O
O
Art Art
O O O + -
O O O BH3.SiMe2 HOOC N O
O O O
O O
O
O O
O O
P
O
OH
OR 75 ( )n
75a, R=Me O 59
O
75b, R=Ph 76a, n=2 O

76b, n=3
76 Scheme. 15

83 | P a g e
growth inhibitory but not cytotoxic towards several human cancer cell lines. These semi-
synthetic new chemical entities, and especially the easily synthesized dimer carboxylic acids,
therefore, deserve further preclinical evaluation as potential drug candidates for chemotherapy of
malaria and cancer.
Later on, Posner and O’Neill’s group synthesized [Jeyadevan, et al. 2004] a new series of
artemisinin dimers starting from their previously prepared key intermediate. All of these dimers
prepared displayed potent low nanomolar anti-malarial activity vs. the K1 and HB3 strains of P.
falciparum. The most potent compound assayed was phosphate dimer (IC50 = 0.2 nM), which
was greater than 50 times more potent than the parent drug artemisinin 1 (IC50 = 12.3 nM), and
about 15 times more potent than the clinically used acetal artemether. All of the dimers
expressed poor anticancer activity. Furthermore, detailed studies on these dimers in vitro in
HL60 cells demonstrate that both phosphate ester dimers (IC50 = 0.14 µM) and (IC50 = 0.24 µM)
are more potent than the anti-cancer agent doxorubicin (IC50 = 0.51 µM).

Posner’s group has further synthesized [Posner, et al. 2004] isobutyric acid dimer and
isonicotinate N-oxide dimer starting from alcohol dimer. Both alcohol dimer and N-oxide dimer,
but not carboxylic acid dimer, very strongly inhibit the growth of prostate cancer cells.

Recently, Posner et al. have also reported [Paik, et al. 2006; Mott, et al. 2013] the synthesis of
another new series of artemisinin bis-benzylic dimers starting from conjugated dimer, obtained
directly from dihydro-artemisinin acetate.

Art Art Art Art

O
O O Art Art
O O
O O
O O
O
O
(EtO) 2(O)PO OP(O)(OEt) 2 O
82
HOOC COOH O O
P O
Art Art O O
Art Art O OPh O O
O
O
O HN O
Art Art
NH
NH
HO OH
MeOOC COOMe 81
80 O
O

Preliminary growth inhibitory activities at nanomolar to micromolar concentrations were

measured in vitro using a diverse panel of 60 human cancer cell lines. It has been realized that
trioxane phthalate dimer (IC50 = 500 nM) is approximately 10-20 times more potent than
trioxane-monomer DHA and that trioxane diol dimer (IC50 = 46.5 nM) is approximately 110-220
times more potent than DHA, without being toxic to primary normal cervical cells.
(c) C-13 carba dimers: Here two artemisinin monomers units are linked through the C-13 non-
acetal (i.e. carbon–carbon linkage) linker. In order to search for more hydrolytically stable and
potent compounds, researchers became interested to synthesize the C-13 dimers of artemisinin 1,
starting from the key intermediate artemistine. Ekthawatchai et al. have reported [Ekthawatchai,
et al. 2001] synthesis of C-13 carba-dimers starting from the key intermediate artemisitene.
Anticancer activities of artemisinin dimers show high potency against vitro cells only.

Recently, Grellepois et al. have reported [Grellepois, et al. 2005] synthesis of C-13 carba dimer
starting from C-13 allylic ether compound using Grubbs metathesis reaction. Preliminary
growth-inhibitory activity of these dimers was evaluated in vitro using a diverse panel of 60
human cancer cell lines.

84 | P a g e
 86a, n=2

O 86b, n=3 O CF 3
O Nu O
O O
O O
86c, n=4 O
O O
O
O 86d, n=5 O O O O
O S S
O
O ( )n O O
O 86 O
O O O CF 3 O
88 O 92
O
O O
O
O O O HO CF 3
O O
O O O
O S S O
( )n O O
- + O O O O
OM S S O
O 89 89a, n=2 O
( )n O
89a, n=2 OM
- +
CF 3 OH O
O 90 90a, n=2 O
90a, n=2 94

Particularly, TGI data shows the selectivity and potency of dimer against a few cancer cell lines
(e.g. leukemia HL-60, non-small cell lung cancer NCI-H-226, colon cancer COLO 205, and KM-
12, CNS cancer SF-295). In order to search for more potent, more bioavailable, hydrolytically
stable, and less toxic compounds of artemisinin derivatives, researchers have directed their
efforts towards the synthesis of trimer and tetramer derivatives of artemisinin.

Later on, Jung et al. synthesized [Jung et al. 2003] another artemisinin trimer through the
coupling of their previously synthesized key intermediates. The in vitro cytotoxicity of this
trimer was tested against murine and human cancer cells. The trimer (IC 50 = 0.12 µg mL-1) is
three times more active than adriamycin (IC50 = 0.39 µg mL-1), 12 times more active than
mitomycin ((IC50 = 1.50 µg mL-1), and 20 times more active than taxol (IC50=2.27 µg mL-1)
against P388. Furthermore, it has been realized by these researchers that this trimer 109 is most
potent in almost all the human cancer cell lines tested, and should receive more attention as a
possible anti-cancer drug candidate.

Mode of anti-cancer activity

(a) Endosome Fe(III)-Art Lysosomal damage ROS

O .
(c) ER stress
O Fe(II)
O Fe(II) HO (d) DNA damage
FPFe(II) O
O
O
O
O

Mitochondria Heme-Art Electron transport chain ROM

Cytochrome c release

Caspase activation3/9

Apoptosis

Figure 2: Postulated anticancer mechanisms of action of artemisinins.

CONCLUSIONS

In this review, we have attempted to give a comprehensive overview on the recent

developments of artemisinin and its derivatives as potential anti-cancer agents. Comparative
anticancer activities of these derivatives were discussed and a study on the efforts towards the

85 | P a g e
development of various artemisinin dimers, trimers and tetramers as potential ‘leads’ for
anticancer drugs have been carried out, where the activities have been correlated with artemisinin
and various other standard anti-cancer drugs. From the current degree of development, it is
understandable that several new potent dimers and trimer ‘lead’ molecules have been discovered
in recent years which are in the various phases of clinical trials. In view of development of new
‘lead’ compounds, it is hoped that interest in this rapidly growing area will continue further to
yield exciting results in the coming years.

REFERENCES

Beekman, A. C., A. R. W. Barentsen, H. J. Woerdenbag, Planta Med, 1998, 64(7), 615-619.

Efferth, T., Benakis, A., Romero, M. R. et al., Free Rad. Biol. Med, 2004, 37(7), 998–1009.
Ekthawatchai, S., Kamchonwongpaisan, S., Kongsaeree, P., Tarnchomopoo, B., Thebtaranonth, Y.
and Yuthavong, Y. J. Med. Chem., 2001, 44, 4688.
Grellepois, F., Crousse, B., Delphon, D. B. and Begue, J. P., Org. Lett., 2005, 7, 5219.
Jung, M., Lee, K., Kim, H. and Park, M., Curr. Med. Chem., 2004, 11, 1265.
Jung, M., Lee, S., Ham, J., Lee, K., Kim, H. and Kim, S. K., J. Med. Chem., 2003, 46, 987.
Jung, M., Tak, J., Chung, W. Y. and Park, K. K., Bioorg. Med. Chem. Lett., 2006, 16, 1227.
Kain, K. C. Curr. Opin. Infect. Dis. 1993, 6, 803–811.
Karin, M.; Mintz, B. J. Biol. Chem. 1981, 256, 3245.
Klayman, D. L., ACS Symp.Ser. 1993, 534, 242–255.
Luo, X. D., Shen, C. C. Med. Res. Rev. 1987, 7, 29–52.
May, W. S., Cuatrecasas, P. J. Membr. Biol. 1985, 88, 205.
Mott B T., He R, Chen X, Fox J M., Civin C I., Arav-Boger R, Posner G H. Bioorg. & Med. Chem.
2013, 21, 3702–3707.
Oh, S., Jeong, I. W., Ahn, C. M., Shin, W. S., Lee, S. Synthesis and antiangiogenic activity of exo-
olefinated deoxoartemisinin derivatives. Bioorg. Med. Chem. 2004, 12, 3783–3790.
Oh, S., Jeong, I. W., Shin, W. S., Lee, S. Bioorg. Med. Chem. Lett. 2003, 13, 3665–3668.
Oh, S., Shin, W S., Ham, J., Lee, S., Bioorg. & Med. Chem. Lett. 2010, 20, 4112–4115.
Posner, G. H., and O’Neill, P. M., Acc. Chem. Res., 2004, 37, 397.
Posner, G. H., McRiner, A. J., Paik, I. H., Sur, S., Borstink, K., Xie, S., Shapiro, T. A., Alagbala, A.
and Foster, B. J. Med. Chem. 2004, 47, 1299.
Posner, G. H., Northrop, J., Paik, I. H., Borstink, K., Dolan, P., Kensler, T. W., Xie, S. Shapiro, T.
A., Bioorg. Med. Chem., 2002,10, 227.
Posner, G. H., Paik, I. H., Sur, S., McRiner, A. J., Borstnik, K., Xie, S. and Shapiro, T. A., J. Med.
Chem. 2003, 46, 1060.
Posner, G. H., Ploypradith, P., Hapangama, W., Wang, D., Cumming, J. N., Dolan, P., Posner, G. H.,
Ploypradith, P., Parker, M. H., Dowd, H. O., Woo, S. H., Northrop, J., Krasavin, M., Dolan, P.,
Kensler, T. W., Xie, S. and Shapiro, T. A., J. Med. Chem., 1999, 42, 4275.
Van Geldre, E.; Vergauwe, A.; Van den Eeckhout, E. Plant Mol. Biol. 1997, 33, 199–209.
Woerdenbag, H. J., Moskal, T. A., Pras, N., Maringle, T. M., ElFeraly, F. S., Kampinga, H. H.,
Konings, A. W. T. J. Nat. Prod. 1993, 56, 849–856.

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 Artemisinin and its derivatives as potent antimalarial agents
Deepak Singh Kapkoti‫٭‬, Dr R.S.Bhakuni
Medicinal Chemistry Department
Central Institute of Medicinal and Aromatic Plants,
CSIR, Lucknow-226015, India
singhdeepak98@gmail.com

ABSTRACT
Artemisinin and its derivatives are the well known antimalarial agents. Some of its derivatives
are in clinical use. In this minireview, we have tried to give an overview on recent development
of artemisinin and its derivatives as potential antimalarial agents.

INTRODUCTION
Artemisinin (1) is a sesqueterpene lactone found in non polar (hexane) extract of the palnt
Artemisia annua (Asteraceae)( Lin, et al. 1979). A. annua is a traditional Chinese medicinal plant
which was used for the treatment of fever from the ancient time. The plant is also known as
qinghao and its active ingredient artemisinin is also known as qinghaosu in Chinese. Artemisinin
has an endoperoxide linkage which is essential for its antimalarial activity. Artemisinin is active
against both uncomplicated and severe forms of malaria. Unlike the conventional antimalarial
agents like chloroquine, quinine etc artemisinin lack nitrogen containing heterocyclic system.
CH3
H
O
H3C O
H
O

O
CH3

O
1

Discovery of artemisinin
During 1960s there was a demand of new antimalarial agent because in most of the part of the
world there was emergence of parasites resistant to most of the existing antimalarial drugs of
that time such as chloroquine. In 1967 Chinese government began a program to screen the
traditional Chinese medicinal plants to find an adequate treatment for malaria. In 1971 scientists
of the Pharmaceutical Institute of the Academy of Traditional Chinese Medicine showed that the
extracts of Artemisia was able to kill Plasmodium berghei in mice and in 1972 they had
identified the active molecule from plant – artemisinin(Klayman, et al. 1985). In 2011, Tu
YouYou was awarded by the prestigious Lasker-DeBakey Clinical Medical Research award for
her role in the discovery and development of artemisinin.

Artemisinin derivatives

C-12 ethers/esters derivatives

Artemisinin is active against both the chloroquine sensitive and chloroquine resistant strains of
P.falciparum but it has some drawback including poor water and oil solubility and not well

87 | P a g e
absorbed by gastro-intestinal tract. To overcome this problem some ether and ester derivatives
of dihydroartemisinin (DHA, 2) were made such as artemether(3), arteether (4), artesunate (5)
,artelinate (6) etc.( Li, et al. 1981, Brossi, et al. 1988, Binns, et al. 1989, Dutta, et al. 1987, Liu,
et al. 1987)
Artemether and arteether are oil soluble derivatives. Among them methyl ether (β-epimer) is
most active (Gu, et al. 1980). Artesunate, the sodium salt of artesunic acid is the water soluble
derivative which can be given by the oral, intravenous, intramuscular and even intrarectal route,
but it has the problem of stability in aqueos solution due to rapid hydrolysis and very short
plasma half life. All these clinically useful artemisinins are finally metabolized to DHA which
is associated with short half life and some toxicity.

CH3 CH3
CH3 H
H H
O O
O O
O H3C O ether/esterification H3C H
H3C H Reduction H
O O
O

O O
O
CH3 CH3
CH3
OH OR
O
1 2
3 R =Me
4 R=Et
5 R=COCH2CH2COONa

6 R=CH2C6H4COONa

O’ Neill et al synthesized a series of the hydrolytically stable C-12 phenoxy derivatives. Among
them trifluoromethyl derivative (R=CF3 , IC50=3.90nm) (7) showed better in vivo activity
(ED50=2.12 mg kg -1) than artemether (ED=6 mg kg -1) (O’Neill, et al. 2001).
CH3
CH3 H
H CH3
H O
H3C O
O H
H3C O O
H O
H3C O
H 9a R=H,3'OH, 4'CH2NR2
O
O O
O CH3 9b R=H,3'OH, 4'CH2-morpholine
CH3 O R1
CH3 O
O NR 1R2
O
OH
R=CF3 n n=2,3
8
CH 2NR 2
R 9
7

Li et al reported the synthesis of 30 amino group containing derivatives of DHA. Among them
compound 8 showed 4-5 folds higher activity against P.berghei infected mice than artesunic acid
(Li, at el. 2000).

88 | P a g e
Yang et al have synthesized mannich base group containing derivatives of artemisinin and
screened them against P.berghei and P.falciparum in k1 and NF54 cells. Among them compound
9a (IC50=0.18 and 0.36ng ml -1) and 9b (IC50=0.25 and 0.17ng ml -1) were found more active than
artesunate ((IC50=1.20 and 1.20 ng ml -1) (Li, et al. 2003).
Recently Singh et al have synthesized new series of ether and ester derivatives. In the ether series
α biphenyl ether derivatives 10a and 10b are the most active than β arteether. In ester series most
of the compounds were found to be more active than β arteether (Singh, et al. 2006; Singh, et al
2008).
CH3 CH3
H H

O O
O H3C O
H3C H H

O 10aR=4-biphenylmethyl O

O 10b R=2-biphenlyethyl O
CH3 CH3

OR O R

10 O
11
R=substituted admantane&biphenyl

C-12 carbon analogues

Due to the presence of acetal group in oxo derivatives of artemisinin, they shows poor
bioavailability and rapid clearance. C-12 carbon analogues have carbon in palce of oxygen at C-
12 position and they have longer half life and lower toxicity. Example of some C-12 carbon
derivatives which were showing good antimalarial activity given below (Fig. 1)
CH3 CH3
H H
CH3
O O H
H3C O H3C O CH3
H H
O H
O O H3C O CH3
H
O H
O O H3C O
O H
CH3 O
CH3 O O
O H3C H
O O CH3
O O
O CH3
O
OH CH3

COOEt O
F/CF 3 F/CF 3 CF 3

Ma, et al. 2000 R

O' Neil, et al. 1999)
R=Me, Et, iPr, tBu

Figure 1

Artemisinin dimers, trimers and tetramers

It was thought that the extent of antimalarial activity of artemisinins depends upon the extent of
the number of peroxide units. Therefore, numbers of artemisinins dimmers trimers and tetramers
were synthesized and these compounds showed outstanding antimalarial activity.

89 | P a g e
Mechanism of Action
The Plasmodium parasite enter the human body through the bite of a female mosquito (genus
Anopheles). They enter into the blood stream as sporozites and then migrate to the liver where
they infect liver cells (hepatocytes) and replicate there into merozoites for two weeks. Then the
merozoites invade red blood cells, develop ring forms, trophozoites and schizonts that in turn
further produce new merozoites. Most of the merozoites continue this cycle but some of them
differentiate into male and female gametocytes which are taken up by the female mosquite and
continue the life cycle.
CH3
H CH3 (III)FeO H
(III)FeO H .
. H3C HO
H3C O H
H
1,5 H shift O
O TS1
CH3 O
H O CH3
CH3 Pathway 1
O 2+ O
O Fe O
H3C H
O
2+ CH3
Fe CH3 H
O . .
O H CH 3
CH3
(III)FeO
O H
H3C (III)FeO
H
1 C-C cleavage A cO
O TS2
O
O Pathway 2 CH3
CH3
O
O
Figure 2

The artemisinin compounds mainly act on the blood stage of the parasite life cycle . The
endoperoxide linkage of artemisinin and heme iron (Fe2+) play critical roles in their mechanism
of action. It was considered that Fe2+ iron is found in the food vacuole of parasite where
digestion of haemoglobin takes place and free iron is detoxified and deposited in haemozoin
which is an insoluble dimer of haem which form hydrogen bond aggregates.
The mechanism of action comprises two step (Posner,et al. 2004) (Fig. 2). In the first activation
step, heme iron attacks the endoperoxide linkage and produces an oxy free radical which after
rearrangement gives a carbon free radical. In the second step this carbon free radical will alkylate
specific malarial proteins and kill the malarial parasites.
There are two possible pathways in activation step. In the first pathway, heme iron attack at the
O2 position and gives free radical at O1 position then there is an intramolecular 1,5-H shift takes
place and gives C4 free radical. In the second pathway heme iron attack at the O 2 position and
gives free radical at O1 position. Then homolytic cleavage of C3-C4 bond takes place resulting
into the C4 free radical. This C4 free radical is very crucial for antimalarial activity of
artemisinins.

Conclusion
In this mini reveiw we have try to cover some important development of artemsinin and
its derivatives as a potential antimalarial agents. Comparative antimalarial activities of these
derivatives were discussed and a study on the efforts towards the development of various
artemisinin derivatives as potential ‘leads’ for antimalarial drugs have been carried out where

90 | P a g e
the activities have been correlated with artemisinin. From the current degree of development, it is
understandable that several new potent artemisinin derivatives have been discovered in recent
years which are in the various phases of clinical trials. In view of development of new ‘lead’
compounds, it is hoped that interest in this rapidly growing area will continue further to yield
exciting results in the coming years.

References-

Binns. F and Wallace. T. W, Tetrahedron let, 1989, 30, 1125

Brossi. A, Venugopalan. B, Gerpe. L. D, Yeh. J. C, Flippen-anderson. J. L, Buchs. P, Luo. X. D.,
Milhous. W, Peters. W, J. Med. Chem, 1988, 31, 645
China Co-operative research group on Qinghouso and its derivatives as anti-malarials, J.
Tradit. Clin. Med, 1982, 2, p. 9
Dutta. G. P, Bajpal. R, Vishwakarma. R. A, Indian J. Parasitol, 1987, 11, 253
Gu. H, Lu. B, and Qu. Z , Acta Pharmacol. Sinica, 1980, 1, 48
Klayman. D. L, Qinghaosu (artemisinin): an antimalarial drug from China. Science
1985,228, 1049–1055.
Li. Y, Yu. P, Chen. Y, Li. L, Gai. Y, Wang. D, Zheng. Y, Acta Pharmaceut. Sinica, 1981, 16,
430.
Li. Y, Zhu. Y. M, Jiang. H. J, Pan. J. P, Wu. G. S, Wu. J. M., Shi. Y. L, Yang. J. D,
Wu. B. A, J. Med. Chem, 2000, 43, 1635.
Li. Y, Yang. Z. S, Zhang. H, Cao. B. J, Wang. F. D, Zhang. Y, Shi. Y. L, Yang J. D,
Wu. B. A, Bioorg. Med. Chem, 2003, 11, 4363
Lin. J. M, Ni. M.Y, Tou. Y. Y, Wa. Z. H, Wu. Y. L, Chou. W. S , Acta Chim. Sinica,
1979, 37, 129.
Liu. X, Patent CN 85, 100, 781, 20 Aug 1986 (Chem. Abstr, 1987,107, 78111).
Ma. J, Katz. E, Kyle D. E, Ziffer. H, J. Med. Chem, 2000, 43, 4228
O’Neill. P. M, Searle. N. L, Kan. K. W, Storr. R. L, Maggs. J. L, Ward. S. A, Raynes
K, Park. B. K, J. Med. Chem, 1999, 42, 5487
O’Neill. P. M , Miller A, Bishop. L. P. D , Stephen H , Maggs. J. L, Ward. S. A,
Roberts. S. M, Scheinmann. F, Stachulskii. A. V, Posner G. H, Park B. K, J. Med.
Chem, 2001, 44, 58.
Posner. G. H, O’Neill. P. M , Acc. Chem. Res, 2004, 37, 397
Singh. C, Chaudhary. S, Puri. S. K, J. Med. Chem, 2006, 49, 7227.
Singh. C, Chaudhary. S, Puri. S. K, Bioorg. Med. Chem. Lett, 2008, 18, 1436

91 | P a g e
 Semi-synthetic approach of withaferin A: Related steroidal lactones as a
potent anti proliferative agents

Imran Ahmad
Medicinal Chemistry Department, CSIR-Central Institute of Medicinal and aromatic Plants, PO.
CIMAP, Kukrail Road, Lucknow-226015, India,
Email: imranlcc@gmail.com

ABSTRACT

Cancer is hyper proliferative disorder of normal cells that involves transformation, dysregulation
of apoptosis, proliferation, invasion, angiogenesis and metastasis. Millions of people die every
year with different types of cancer.

In the last century, great discoveries have been made in modern medical system to cure and
prevent the cancer. However, success rates are very low. For decades, Withania somnifera is a
medicinal plant that has been used as traditional medicine for the treatment of various diseases
including cancer. The different parts of Withania somnifera and its constituents are effective in
prevention and treatment of different kinds of cancer like colon cancer, lung cancer, blood
cancer, skin cancer, breast cancer, renal cancer, fibrosarcoma, prostate cancer, pancreatic cancer.
The major biochemical constituents of Withania somnifera root are steroidal alkaloids and
steroidal lactones in a class of constituents called withanolides. This mini review focuses on
anticancer activity especially on antiproliferative activity of some steroidal compounds present in
Withania somnifera and their structure activity relationship (SAR).

Keywords: Withania somnifera, withanolides, withaferin A, anti-proliferative activity, SAR

INTRODUCTION

Cancer is the second major cause of death worldwide. There are about 100 different types of
cancers that affect humans having no curative therapy; therefore, there is an urgent need to
discover novel leads for chemotherapy. Natural products represent a rich source of drug leads [1]
and they have played a significant role in the discovery and development of new anticancer
agents.

According to estimates from the International Agency for Research on Cancer (IARC), there
were 12.7 million new cancer cases in 2008 worldwide, of which 5.6 million occurred in
economically developed countries and 7.1 million in economically developing countries. The
corresponding estimates for total cancer deaths in 2008 were 7.6 million (about 21,000 cancer
deaths a day), 2.8 million in economically developed countries and 4.8 million in economically
developing countries. By 2030, the global burden is expected to grow to 21.4 million new cancer
cases and 13.2 million cancer deaths simply due to the growth and aging of the population, as
well as reductions in childhood mortality and deaths from infectious diseases in developing
countries [2].

92 | P a g e
Withanolides are a group of naturally occurring chemical compounds, consisting a steroidal
backbone attached to a lactone or one of its derivatives [3]. This class of steroids has attracted
much interest due to their complex structural features, but also their broad range of biological
activities [4]. Withaferin A, isolated from Winter cherry (Withania somnifera) a prototype of the
withanolides class of natural products, responsible for anticancer effect, in a variety of human
cancer cells, including prostate, colon, breast, leukemia, pancreatic, renal, head and neck [5],
among others. It was shown that withaferin A actives p38 MAP kinase, inhibits the Notch
signaling pathway and nuclear factor-lB activation [6,7], induces Akt inactivation, death receptor
5 (DR5) up-regulation and down-regulation of c-FLIP [8,9] as well as induces apoptosis through
its ability to generate reactive oxygen species [10,11].

The present work provides a novel class of withanolides that have been isolated from Withania
somnifera under aeroponic conditions or produced semi-synthetically from withanolide natural
products. They are also the part of various pharmaceutical compositions and methods for using
the same in proliferative diseases [12].

WITHAFERIN A AND ITS SEMISYNTHTIC ANALOGS

Chemical constituents
W. somnifera has diversified class of compounds which are biologically active. The
representative structures of the main chemical constituents present in W. somnifera are as
follows:

Figure 1 some important compounds present in W. somnifera

The roots of the medicinal plant Withania somnifera have been used from ancient times in the
Ayurvedic tradition of India for various diseases [5].

Withania somnifera has been shown to possess anti-inflammatory, immunomodulatory, cardio

protective and anti-proliferative activities. The primary bioactive constituents of ashwagandha

93 | P a g e
are known as withanolides, are structurally diverse C28-steroidal compounds with an ergosterol
skeleton in which C-22 and C-26 are oxidized to form a δ-lactone ring on the nine carbon side
chain [19]. W somnifera provide bulk quantities of biomass under conditions that allow
improved yield and consistency of desired secondary metabolite production. Certain inventive
compounds, such as certain compounds derived from aeroponically cultured biomass, have been
found to inhibit cell proliferation/ survival in MCF-7 breast cancer cells (FIG. 1) [12]. Possible
biological targets of WithaferinA and related Withanolides are the actin bundling protein annexin
II[13], the 20S proteasome[14], the intermediate filament protein vimentin [15], the transcription
factor NFKB[16], protein kinase C[17], and the Par-4-dependent apoptosis path way [18]

Figure 2 structure of natural withanolides and derivative

The above mentioned withinolides are isolated and/or semi synthetically derived from
Withania somnifera and tested for their anti cancer activity.

Anti-Proliferative Activity of Withanolides:

94 | P a g e
 A B

C D

E F

Figure 3 Some activity graph of withanolides

A sulphated withinolides (Fig.2) isolated from aerial tissue of Withania somnifera, 2.3-
dihydrowithaferin A-3β-O-sulphate, shows concentration and time dependent inhibition of the
proliferation/survival of MCF-7 breast cancer cells (A). Withaferin A inhibits the growth of
cancerous cells at an early stage, but 2.3-dihydrowithaferin A-3β-O-sulphate is equipotent after
ca.72 hours. The same pattern was observed in two other cancer cell lines (NCI-H460 and PC-
3M).This maybe because of the conversion of dihydrowithaferin A-3β-O-sulphate to withaferin
A in the presence of cells, so that it may be acting as a prodrug of Withaferin A (B) [12].

95 | P a g e
Withaferin A also inhibits tumor cell migration and invasion in prostate cancer cells and Ewing ,s
sarcoma cells (C) and inhibit tumor growth in Ewing ,s sarcoma (D). Withaferin A disrupts the
endothelial cell network (E) and inhibit tumor vascularization (F) [12].

SAR Analysis

The effect of the substituents on the cytotoxic activity of the withanolides has the following
trends in the structure activity relationship.

The influence of substituents on the A and B rings was evident. Simple modification of the
double bond at C-2 or epoxy group at C-5 produced a considerable decrease in the activity [19].
Moreover, the elimination of those both essential groups caused the abolition of cytotoxicity
[20].

Furthermore, the most striking result was the replacement of a hydroxyl by a carbonyl group at
C-4 which results in a considerable increase of selectivity [19].

Substituents on the five member ring play an important role, a detrimental effect was observed
with the presence of a hydroxyl group [19].

Regarding the substituents on the lactone ring, the substitution of a C-27 methyl by a
hydroxymethyl group increases considerably the activity.

These trends, based upon substitution patterns provide valuable information on the
pharmacophore for withanolide-type compounds and will be helpful for the rational design of
more potent and selective anticancer drugs.

CONCLUSION

So far, much advancement has been done in modern medicine for anti cancer leads, but still there
is need to discover novel leads. Withania somnifera shows great potential as a safe and effective
in anti proliferation. Therefore, SAR guided modification is needed to improve its anti
proliferative action and increase effectiveness with devoid of side effects of conventional
treatments with the use of Withania somnifera.

Acknowledgements

I.A. acknowledges CSIR, New Delhi, India for financial support as SRF. Support received from
Director, CSIR-CIMAP is dully acknowledged.

REFERENCES

1. Bhuwan, B.M.; Vinod K.T. Eur. J. Med. Chem. 2011, 46, 4769-4807.
2. http://www.cancer.org/research/cancerfactsstatistics/global
3. Glotter, E, Nat. Prod. Rep. 1991 8 415-440.

96 | P a g e
4. Chen, L.-X.; Qiu, H. He. F. Nat. Prod. Rep. 2011, 28 ,705-740.
5. Verma,S.K.; Kumar, A. Asian J Pharmaceut Res Health Care .2011, 4.
6. Koduru, S; Kumar, R.; Srinivasan, S.; Evers M. B.; Damodaran ,C. Mol. Cancer Ther. 2010, 9,
202-210.
7. Malara, N.; Focá, D.; Casadonte, F.; Sesto, M. F.; Macrina, L.; Santoro, L.; Scaramuzzino, M.;
Terracciano, R.; Savino, R. Cell Cycle 2008, 7, 3235-3245.
8. Lee, T. J.; Um, H. J.; Min, D. S.; Park, J. W.; Choid, K. S.; Kwon, T. K. Free Radic. Biol.Med.
2009, 46, 1639-1649.
9. Oh, J. H.; Lee, T. J.; Kim, S. H.; Choi, Y. H.; Lee, S. H.; Lee, J. M.; Kim, Y. H.; Park, J. W.;
Kwon, T. K. Apoptosis 2008, 13, 1494-1504.
10. Hahm, E. R.; Moura, M. B.; Kelley, E. E.; Houten, B. V.; Shiva, S.; Singh, S. V. PloS ONE 2011,
6.
11. Mayola, E.; Gallerne, C.; Esposti, D. D.; Martel, C.; Pervaiz, S.; Larue, L.; Debuire, B.; Lemine,
A.; Brenner, C.; Lemaire, C. Apoptosis .2011, 16, 1014-1027.
12. Pub. No.: US 2011/0230551 A1
13. Falsey, R.R.; Marron, M. T.; Gunaherath, G. M. B.; Shirahatti, N.; Mahadevan, D.; Gunatilaka, A.
A. L.; Whitesell, L. Nat. Chem. Biol., 2006, 2, 33-38.
14. Yang, H.; Shi, G.; Ping Dou, Q. Mol. Pharmacol., 2007, 71, 426-437.
15. Bargagna-Mohan, P.; Hamza, A.; Kim, Y.; Khuan (Abby) Ho, Y.; Mor-Vaknin, N.; Wendschlag,
N.; Liu, J.; Evans, R. M.; Markovitz, D. M.; Zhan, C. G.; Kim, K. B.; Mohan, R. Chem. Biol.2007,
14, 603-728.
16. Srinivasan, S.; Ranga, R. S.; Burikhanov, R. Han, S.; Chendil, D. Cancer. Res. 2007, 67, 246-253.
17. Sen, N.; Banerjee, B.; Das, B. B.; Ganguly, A.; Sen, T.; Pramanik, S.; Mukhopadhyay, S.;
Majumder, H. K. Cell Death Differ 2006, 14, 358-367.
18. Kaileh, M.; Vanden, B. W.; Heyerick, A.; Horion, J.; Piette, J.; Libert, C.; Keukeleire, D. D ;
Essawi, T.; Haegeman, G. J. Biol. Chem. 2007, 282, 4253-4264.
19. LLanos, G. G.; Araujo, L. M.; Jiménez, I. A.; Moujir, L. M.; Bazzocchi, I, L. Eur. J. Med. Chem.
2012, 54, 499-511.
20. Xu ,Y. M.; Marron, M.T.; Seddon, E.; McLaughlin, S.P.; Ray, D.T.; Whitesell, L.; Gunatilaka, A.
A. L., Bioorg. Med. Chem. 2009, 17, 2210-2214.

97 | P a g e
 Substantiation of withanolides as anticancer agents

Pallavi Joshi*, Rashi Tewari, Amreen A. Siddique, Furkan Ahmad, Madhuri Gupta and
Jyotsna Bharadwaj
Chemical Science Division, CSIR- Central Institute of Medicinal and Aromatic Plants, P.O.
CIMAP, Lucknow 226015, India
*Email: pallavijs.joshi@gmail.com

ABSTRACT

Withanolides isolated from Withania somnifera possess good anticancer activity against different
types of cancer including breast, colon, prostate, lung and liver cancer cell lines. To substantiate
this, researchers have performed several experimentations and prepared derivatives to enhance
the anticancer activity of withanolides. The findings till date reveal that a small alternation in the
skeleton of withanolide can cause changes in its anticancer activity, also the activity is functional
group specific, as some results show increase but some show a decreased cytotoxicity. Overall
the withanolides as such in their natural chemical composition as well as their derivatives have
proved to be of great value in anticancer therapy.

INTRODUCTION

Medicinal plants have been acknowledged and accepted for therapeutic usage since ancient
times, primarily due to their being safe, readily available, feasible and effective. Among the
several plants known for medicinal value, Withania somnifera L. Dunal (Solanaceae) also known
as Ashwagandha, is one of the major herbal components, renowned for its therapeutic potential
and has been an important herb in the Ayurveda for over 30 centuries . It is an annual herb
growing in dry and arid soil as a wild plant. Its popularity as health promoting and therapeutic
medicinal plant in traditional systems of medicine in India for Ayurveda, Siddha and Unani has
been very well documented (Tuli et al, 2009). The traditional use of Withania somnifera was to
increase energy, health, strength, increase vital fluids, muscle fat, blood, lymph, semen, and cell
production (Jayaprakasam et al., 2003).

From the chemical perspective, Withania somnifera contains a special class of biologically active
alkaloids (withanine, withasomnin) and steroidal lactones and glycosides also called as
withanolides and sitoindosides, and possess significant biological activities including, anticancer
(Siddique et al., 2014), CVS and CNS activity. It is well known for anti-ageing, aphrodisiac,
improving vigor, etc. Valerian root is reported with validation to be useful in the treatment of
insomnia. The extract of Withania somnifera exhibits analgesic, anabolic, antibiotic,
immunomodulatory, antitumor, anticonvulsant, anti-oxidant, anti-inflammatory, hepatoprotective
and mildly sedative activities (Mishra et al., 2000).

Withanolides are very effective in anticancer therapy. Several withanolides have been isolated
which showed potent anticancer activity. Withanolides are C28 ergostane based steroidal
skeleton.

Significance of withanolides as anticancer

98 | P a g e
Withanolides as well as its derivatives have shown great antitumor activity. Withaferin A,
withanolide D & E exhibited significant antitumor activity in vitro. The alcoholic extract of the
dried roots of the plant as well as the active component withaferin A isolated from the extract
showed significant antitumor and radio-sensitizing effects in experimental tumours in vivo,
without any noticeable systemic toxicity. Some derivatives of isolated withanolides, viz.
withaferin A, 27-deoxywithaferin A, 17-hydoxywithaferin A, 17-hydroxy-27-deoxywithaferin A,
withanone, 27-hydroxywithanone and withanolide A have shown great antiproliferative activity.
The anticancer activity has direct relationship with the α,β-unsaturated ketone in ring A and the
epoxide moiety in ring B in withanolides. The chemical composition of various parts of Withania
somnifera has yielded a variety of chemical structures belonging to withasteroids (Mishra et al.,
2008, Sabir et al., 2011). The structure activity relationship in withasteroids has recently been
proved by us that the epoxide functional group in B ring has its positive relationship with its
anticancer activity (Mishra et al., 2008). In continuation of our interest on the structure activity
relationship in withasteroids from W. somnifera, we have further isolated several withasteroids
from its leaves and applied simple reactions to convert the epoxide of withanolides into various
functional groups and tested their anticancer activity.

Some withanolides which have been isolated from Withania somnifera are as such very active
against different types of cancer cell lines. The chlorinated and diepoxy withanolides isolated
from Withania sominfera showed effective anticancer activity. The 27-acetoxy-4b,6a-dihydroxy-
5b-chloro-1-oxowitha-2,24-dienolide and 5b,6b,14a,15a-diepoxy-4b,27-dihydroxy-1-oxowitha-
2,24-dienolide possess good anticancer activity against lung cancer cell lines (Choudhary et al.,
2010).

A new withasteroid named 5,6-de-epoxy-5-en-7-one-17-hydroxy withaferin A was isolated from

leaves of Withania somnifera, and showed cytotoxic activity against breast, liver, prostate and
colon cancer cell lines; among these, it shows strong activity against liver and breast cell lines
and moderate activity against colon and prostate cancer (Siddique et al., 2014).

Effects of alternation in the withanolide skeleton on its anticancer activity

Several researchers have prepared derivatives which have shown to enhance and even inhibit
anticancer activity as comparison to the parent withanolides.
Azide derivatives prepared by the researchers showed better activity than the parent withanolide
i.e. Withanolide A. Withanolide A in methanol was treated with basic solution of TMSN 3 at
room temperature (Scheme 1) (Yousufa et al., 2011).

99 | P a g e
 27
24 OH

27
22
O 24 OH
21 O
18 20
22
H O
12
O 17 21 O
20
9 14 16 TMSN3 18
2 H
12
H MeOH, rt O 17
7 16
9 14
2
O H
OH 7
Withaferin A N3
O
OH
3-Beta substituted

Scheme 1
Withaferin A showed activity against five cancer types viz., breast, colon, ovary, pancreatic and
prostate cancer while the 5,6-de-epoxy-5,6-(oxyethylene-2_-thio) withaferin A lost cytotoxic
active against all types of cell lines. For this, withaferin A was taken in ethanol and to it 2-
mercaptoethanol was added and the reaction was conducted at 37 OC temperature for 3 h which
lead to the formation of 5,6-de-epoxy-5,6-(oxyethylene-2_-thio) withaferin A (Scheme 2). Then
the comparison between the withaferin A and 5,6-de-epoxy-5,6-(oxyethylene-2_-thio) withaferin
A was made on the basis of activity results which revealed that withaferin A is more active
against cancer (Misra et al., 2008).
27
24 OH 27
24 OH
22
O
21 O 22
O
18 20 21 O
18 20
H
12 H
O 17 12
9 14 16 H+ -H2O O 17
9 14 16
2
2
H OH
7 HS 7 H

O O
OH OH S
Withaferin A
5,6-De-epoxy-5beta,6alpha-(oxyethylene-2'-thio) withaferin A

Scheme 2

Chemical transformations at the epoxide ring and α,β-unsaturated ketone show a considerable
loss in biological activity suggesting the importance of these functional groups in withasteroids.
Replacement of oxygen atom in epoxide with sulfur atom results in an appreciable decrease in
the cytotoxicity of these thiirane derivatives. Some literature revealed that epoxide derivative of
27-deoxywithaferin A, 17-hydoxywithaferin A, 17-hydroxy-27-deoxywithaferin A, and
withanone are more active than their thiirane derivatives. To confirm the significance of epoxide
derivatives over thiirane derivative of withanolides, several withanolides analogs were

100 | P a g e
synthesized. For synthesis, reactant was treated with ammonium thiocyanate, SbCl 3 and
acetonitrile (Scheme 3). Then their activity results were compared which lead to the conclusion
that epoxide derivatives possess more anticancer activity as comparison to the thiiranes (Joshi et
al., 2014). Biological activity testing of epoxide ring is often essential in withanolides for their
cytotoxicity because it mostly decreased in analogs which have no epoxide functional group.
28

27
24
23
25
R
21
20
22 (i)
26
18 O O
12
17 R1
11
O 19 13
16
1 9
14 15
2
10 8

3 5
4 7
6
O
OH
R

O O
R1
(ii) O

S
OH

Scheme 3. Reagents and conditions: (i) Ammonium thiocyanate, SbCl3, Acetonitrile, 3 h;

(ii) Ammonium thiocyanate, Cyanuric Chloride, THF, overnight.

Besides this, the reduction of 27-deoxywithaferin A , 17-hydoxywithaferin A, 17-hydroxy-27-

deoxywithaferin A, and withanone to diols enhanced their cytotoxic activity. Reduction to diol
using polymethyl hydrogen siloxane gave positive results across all 4 cell lines. The derivatives
were active, in particular, across prostate and liver cell lines, even at low micro molar
concentrations. For synthesis, 17-hydoxywithaferin A, 17-hydroxy-27-deoxywithaferin A, and
withanone were reacted separately with polymethyl hydrogen siloxane and iodine for 1.5 hours
(Scheme 4) (Joshi et al., 2014).

101 | P a g e
 R R

O O
O O R1
R1 O
O
PMHS
I2 1.5hrs
OH
O OH
OH
Scheme 4

CONCLUSION

In a nutshell, the literature reviews and also the experimental researches in our laboratory
substantiate that withanolides possess good anticancer activity. Besides this, changes in the
withanolide skeleton by preparing its derivation can either enhance or decrease its
antiproliferative activity. The thiirane derivatives were found to possess less anticancer activity
as compared to the parent withanolides, as they showed moderate reduction in the activities
along the prostate and liver cell lines as compared to the original values for reactant
withanolides. Similarly the epoxide derivatives were found to have better anticancer activity than
the parent withanolides.

Overall, Withania somnifera can be called the Indian ginseng or a wonder drug because the
withanolides isolated from this plant possess great pharmaceutical or medicinal values like
antioxidant, anti-stress, immunomodulatory, apoptosis etc., among which anticancer properties
are of great importance. The various experimentation and derivatization of withanolides
substantiates that they possess better anticancer activity.

REFERENCES

Choudhary MI , Hussain S , Yousuf S , Dar A , Mudassar , Chlorinated and diepoxy withanolides

from Withania somnifera and theircytotoxic effects against human lung cancer cell line.
Phytochemistry, 71, 2010 , 2205–2209
Jayaprakasam B, Nair MG. Cyclooxygenase-2 enzyme inhibitory withanolides from Withania
somnifera leaves. Tetrahedron 2003, 59, 841-9
Joshi P , Misra LN , Siddique AA, Srivastava M , Kumar S ,Darokar MP. Epoxide group
relationship with cytotoxicity in withanolide derivatives from Withania somnifera Steroids 79
2014 19–27
Misra LN, Lal P, Chaurasia ND, Sangwan RS, Sinha S, Tuli R. Selective reactivity of
2mercaptoethanol with 5,6--epoxide in steroids from Withania somnifera. Steroids2008,73,
245

102 | P a g e
Misra LN, Lal P, Sangwana RS, Sinha S, Tuli R .Selective reactivity of 2-mercaptoethanol with 5
,6-epoxide in steroids from Withania somnifera, Steroids 73 2008 245–25
Mishra LC, Singh BB, Dagenais S. Scientific basis for the therapeutic use of Withania somnifera
(ashwagandha): A review. Altern Med Rev. 2000, 5, 334-346
Sabir F, Sangwan RS, Singh J, Misra LN, Pathak N, Sangwan NS. Biotransformation of withanolides
from cell suspension cultures of Withania somnifera (Dunal).Biotechnology Letters 2011,
5,127-34
Siddique AA, Joshi P, Misra LN, Sangwan NS, & Darokar MP. 5,6-De-epoxy-5-en-7-one-17
hydroxy withaferin A, a new cytotoxic steroid from Withania somnifera L. Dunal leaves.
Natural Product Research, 2014, 28, 392-398
Yousufa SK, Majeed R,Ahmad M, Lal P Sangwan NS, A structural modified derivatives of
withaferin A and the evaluation of their cytotoxic potential, Steroids ,76, 2011, 1213-1222
Tuli R, Sangwan RS, Kumar S, Bhattacharya S, Misra LN, et al. Ashwagandha Withania somnifera
A model Indian medicinal plant. CSIR, New Delhi; 2009

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 Terpenoid Diversity in Artemisia annua

VS Pragadheesha,b, Ranjana Mauryab, Anju Yadavc, CS Chanotiyaa,b,c*

* a) Academy of Scientific and Innovative Research
b
) Laboratory of Aromatic Plants and Chiral Separation,
c) Central Instrument Facility,
CSIR-Central Institute of Medicinal and Aromatic Plants,
Lucknow - 226 015, India
Emails: cs.chanotiya@cimap.res.in

ABSTRACT

Artemisia annua L. (family Asteraceae ) is well known as a medicinal plant and source for the
potent anti-malarial drug artemisinin. Terpenoids are plant secondary metabolite derived from
isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). Terpenoids are
classified as monoterpenoid (C10), sesquiterpenoids (C15), diterpenoids (C20) and so on. Various
monoterpenoids and sesquiterpenoids were reported from essential oils and solvent extracts of
different parts of the plant. Since, artemisinin and its derivatives have been reviewed extensively
in many previous studies. Hence, this review deals with the glimpse of diversity of mono- and
sesquiterpenoids in aerial parts including seeds of Artemisia annua.

1. Diversity of mono- and sesquiterpenoids

In addition to artemisinin, the plant has been a rich source for several other terpenoids
constituents. Therefore, a need based investigations has been carried out time to time to explore
this diversity. The chemical diversity has been discussed in the following headings:

1.1 Chemistry of aerial parts of Artemisia annua

To investigate the composition of volatiles, the A. annua plant mainly aerial parts have been
hydro-distilled in Clevenger-type apparatus. The essential oil contained artemisia ketone (1),
(1R)-(+)-camphor (2), (1S)-(-)-camphor (3) and 1,8-cineole (4) etc. as markers among
oxygenated monoterpenoids constituents (Figure 1). Besides, several bornane derivatives have
also been reported. Among identified sesquiterpenoids, β-caryophyllene (5), germacrene D (6),
(E)-β-farnesene (7) and 1-epi-cubenol (8) have also contributed slight proportion to essential oil
[Lopes-Lutz et al., 2008; Padalia et al., 2011; Cavar et al., 2012].

Geographical environment has been proven to affect the terpenoid synthesis, which leads to
chemical diversity within the species. For example, oil extracted from Artemisia grown in USA
contained sesquiterpenoids rich composition, whereas the oil of France and Brazil origin
possessed camphor rich composition [Tellez et al., 1999; Juteau et al., 2002; Perazzo et al.,
2003]. Similarly, borneol (15.9%) and (Z)-β-farnesene (12.9%) have been identified as major
compounds of Chinese origin.

Oil sample of Iranian origin revealed trans-3(10)-caren-4-ol (22.3%), artemisia ketone (18.6%),
1,8-cineole (14.9%), δ-selinene (13.0%) and α-pinene (8.2%) in its essential oil [Habibi et al.,
2013]. In another work, plants from different origins have been grown and studied in Finland.
The results showed significant variations in proportions of mono- and sesquiterpenoid

104 | P a g e
compounds [Holm et al., 1997]. Similarly, bisnorcadinanes class of compounds has also been
characterized in hexane extract [Misra et al., 1993].

1.1.1 Enantiomeric composition

The enantiomeric composition of monoterpenoids of A. annua has not been much studied so far.
However, Holm et al., has reported chiral discrimination of camphene, α-pinene and β-pinene in
A. annua cultivated in Finland. The paper does not report enantiomeric differentiation of
camphor enantiomers. The results revealed that the (-)-enantiomers predominated over (+)-
enantiomers along with non-consistent ratios of (-)-camphene/ (+)-camphene and (-)-α-pinene in
entire growing period [Holm et al., 1997].

O
O O

2 3
1 4
OH

H
H

8
5 6 7
Figure 1. Terpenoids reported from essential oil of Artemisia annua.

Essential oil extracted from Artemisia annua grown in CSIR-CIMAP, Lucknow has been studied
for its enantiomeric composition. The composition has been expressed as enantiomeric excess
(ee) with respect to predominant enantiomers. Our results revealed (+)-camphene (ee >96%) and
(1R)-(+)-camphor (ee 88.2%; 2) as characteristic feature of essential oil of Indian origin
[Pragadheesh et al., 2014, unpublished data]. Therefore, we conclude that the plant mainly
synthesized (+)-enantiomers over corresponding (-)-enantiomers. Furthermore, the enantiomeric
results may be used as origin indicator for A. annua essential oil.

1.2 Chemistry of Artemisia annua seeds

Artemisia annua seeds have not been studied much for its chemical composition. In recent time,
Brown et al., have successfully characterised many amorphane and cadinane type sesquiterpenes
(9 - 35) from A. Annua seed extract. The characteristic feature of A. annua sesquiterpenoids is
that their amorphane and cadinane compounds occur as 11,13-dihydro and 11,13-dehydro forms
[Brown et al., 2003]. Figure 2 depicts the structural diversity of compounds in A. annua seeds.

CONCLUSION

105 | P a g e
Keeping the structural diversity in view, the plant may be further explored for the search of novel
chemicals in the interest of mankind.

106 | P a g e
 H OH OH OH

O O
H H OOH
H OH
O
10 11 12 13
9
OH H H

H O OH O OH
OH
HOOC HOH2C

14 15 16 17 18
O O
O H O
H H
H H

O
O O
O
O H
HOOC HOOC
HCOOH2C HOOC HOOC

19 20 21 22
23
H O
H H H OH
HO H
O

O H H O HOOC
O COOH H
O OH
HOOC
O
24 26 27 28
25

OH O
O
OH
HO
OH
OH OH
H 32
31
30
29

OH OH
OH 34 OOH
33
H

O
O
O
O
O

35
Figure 2. Terpenoids reported from the seeds of Artemisia annua.

107 | P a g e
REFERENCES

Brown, G.D., Liang, G-Y., Sy, L-K., (2003) Terpenoids from the seeds of Artemisia annua,
Phytochemistry, 64, 303–323
Cavar, S., Maksimovic, M., Vidic, D., Paric, A., (2012). Chemical composition and antioxidant and
antimicrobial activity of essential oil of Artemisia annua L. from Bosnia. Industrial Crops
and Products, 37, 479 – 485.
Habibi, Z., Ghanian, S., Ghasemi S., Yousefi, M., (2013). Chemical composition and antibacterial
activity of the volatile oil from seeds of Artemisia annua L. from Iran. Natural Product
Research, 27 (2), 198 – 200.
Holm, Y., Laakso, I., Hiltunen, R., Galambosi, B., (1997) Variation in the essential oil composition
of Artemisia annua L. of different origin cultivated in Finland. Flavour and Fragrance
Journal, 12, 241–246.
Juteau, F., Masotti, V., Bessiere, J.M., Dherbomez, M., Viano, J., (2002). Antibacterial and
antioxidant activities of Artemisia annua essential oil. Fitoterapia, 73, 532–535.
Lopes-Lutz, D., Alviano, D.S., Alviano, C.S., Kolodziejczyk, P.P., (2008). Screening of chemical
composition, antimicrobial and antioxidant activities of Artemisia essential oils.
Phytochemistry, 69, 1732–1738.
Misra, L.N., Ahmad, A., Thakur, R.S., Jakupovic, J., (1993). Bisnorcadinanes from Artemisia annua
and definitive 13C NMR assignments of β-arteether. Phytochemistry 33, 1461–1464.
Padalia, R.C., Verma, R.S., Chauhan, A., Chanotiya, C.S., Yadav, A., (2011). Variation in the
volatile constituents of Artemisia annua var. CIM-Arogya during plant ontogeny. Natural
Product Communications, 6(2), 239 – 242.
Perazzo, F.F., Carvalho, J.C.T., Carvalho, J.E., Rehder, V.L.G., (2003). Central properties of the
essential oil and the crude ethanol extract from aerial parts of Artemisia annua L.
Pharmacological Research, 48, 497–502.
Pragadheesh, V.S., Chanotiya, C.S., Yadav, A., (2014). Enantiomeric composition of chiral
terpenoids in Artemisia annua essential oil. unpublished data.
Tellez, M.R., Canel, C., Rimando, A.M., Duke, S.O. (1999). Differential accumulation of isoprenoids
in glanded and glandless Artemisia annua L., Phytochemistry, 52, 1035–1040.

108 | P a g e
 Vinblastine, Vincristine and their derivatives as anti-cancer agents

Yashveer Gautam, B. Sathish Kumar, Aastha Singh, Tanu Kaushal, Ankita Srivastava,
Ravi Kusumoori , Amit kumar Verma, Sadiya Khwaja

Medicinal Chemistry Department,

CSIR-Central Institute of Medicinal and Aromatic Plants, Lucknow-226015, India
Email: yashdynamic@yahoo.in

ABSTRACT

Vinblastine and vincristine are vinca alkaloids; a family of indole indoline dimeric compounds
isolated from the various medicinal plants belonging to Apocynaceae family, and represents one
of the most important classes of the antitumor drug. Vinblastine and Vincristine are more
clinically useful and its derivatives such as vindesine, vinorelbine and very recently vinfluinin
have been developed after intensive synthetic and structure activity relationship studies. Some
other analogous of vinblastine and vincristine also shows good binding affinity and less toxicity
towards tubulin, this review present discovery of vinblastine and vincristine and its synthetic
derivatives and its application.

INTRODUCTION

Vinblastine 1 and vincristine 2 is chemotherapeutic drugs which is most widely recognized

members of the class of bisindole alkaloids. Originally they were isolated in trace quantities
(0.00025% of dry leaf weight for vinblastine) from the leaves of Catharanthus roseus (L.) G.
Don, 2 and their biological properties were among the first to be shown to arise from inhibition
of microtubule formation and mitosis that today is still regarded as one of the most successful
targets for cancer treatment.1, 2, 14 They were the first natural products whose structures were
determined by X-ray crystallography4, they were also first for which X-ray analysis of a heavy
atom derivative was used to establish their absolute configuration. Structure of both vinblastine
and vincristine possess the similar having Catharanthus upper subunit and second is vindoline-
derived lower subunits .Both are differing only in the dihydroindole N-substituent. Beside this
small structural difference, vinblastine and vincristine differ in their antitumor properties and
dose-limiting toxicities. Two additional semisynthetic vinca alkaloids, vindesine and vinorelbine
have been developed as antitumor drugs and a third, vinfluinin is in late-stage clinical trials in
Europe. Vincristine is used in combination therapy to treat acute leukemia and lymphomas and
also treating childhood leukemia. Vinblastine is often used in combination to treat bladder and
breast cancers and Hodgkin's disease. Vinorelbine was approved for use in Europe (1991) and
the US (1995) for the treatment of non small-cell lung cancer and vindesine has been approved
for the treatment of melanoma. Neurotoxicity (vincristine) or myelosuppression (vinblastine) are
the main side effects of administration and neutropenia is the principal dose-limiting toxicity of
the vinca alkaloids, but recovery occurs following treatment. However, the major limitation to
the continued use of the vinca alkaloids is the emergence of drug resistance derived principally
from overexpression of phosphoglycoprotein (Pgp), an efflux pump that transports many of the
major drugs out of the cell. In fact, vinblastine represents one of the most studied prototypical
substrates for Pgp efflux responsible for multidrug resistance (MDR). Thus, in addition to

109 | P a g e
identifying vinblastine and vincristine analogues that may address the current dose-limiting
toxicities, the development of a modified vinca alkaloid that is not a substrate for Pgp efflux and
is efficacious against MDR tumors would constitute a major advance. Additionally, the emerging
evidence that the vinca alkaloids also possess anti-antigenic activity that may contribute to their
in vivo antitumor activity, especially in combination with other drugs, may provide additional
future clinical applications. Due to the pharmaceutical importance and low natural abundance of
vinblastine and vincristine, C. roseus has become one of the most extensively studied medicinal
plants. Their biosynthesis involves the participation of at least 35 intermediates, 30 enzymes, 30
biosynthetic and 2 regulatory genes and 7 intra- and intercellular compartments. Presently, the
clinical supplies of vinblastine and related drugs are derived from natural sources. The doses are
so small that the production amounts are manageable even with the trace natural abundance of
Vinblastine (0.01%) or vincristine (0.0003%) in the source plants. Development of an effective
coupling protocol starting with the more abundant naturally occurring (+)-catharanthine and (−)-
vindoline. Interestingly, only C. roseus produces catharanthine and does so with an absolute
configuration enantiomeric with structurally related alkaloids also found in C. roseus and related
alkaloids found in nature. More significantly, an effective synthetic approach would provide
access to analogues that incorporate deep-seated structural changes that have not yet been
explored. Typically, it has been semisynthetic derivatives of the natural products that have been
examined and improve on the biological properties of vinblastine or vincristin.14
HO

N 4'
N
3'

N
18'
N CH3
H 5
H
15 CH3
H3COOC H
1 3
H3CO
17
N OCOCH3 H3CO N OCOCH3
R1 CO2CH3 H
HO
H3C COOCH3
HO
1-R1=CH3 - Vinblastin
2-R1= CHO -Vincristin 3-vindoline

5' HO 19'
21'
4'
N 20' 18'
9' 6'
3'
15' 5
7' 3
14' 4
6 N 14
1' 16'
12' N 15 18
H 20
CH3
N
10 9 8 7
H3COOC H 17 19
1 2
11 13 16
H3CO N OCOCH3
12
R1 CO2CH3
HO N
biogenetic numbering H
R1= CHO -Vincristin COOCH3
R1=CH3 - Vinblastin 4-catharanthine

Mechanism of Action

110 | P a g e
Both were found to be cell cycle dependent antimitotic agent that interacts with tubulin. Both
vincristine and vinblastine agents induce the destabilization of polymerized tubulin, by binding
to a site recently localized on b-tubulin, and both have a high affinity for the protein. It has been
reported that this destabilizing effect results from the stoichiometric endwise poisoning of the
tubulin heterodimer, presumably preventing polymerization from occurring by blocking the
region involved in heterodimer attachment. As the microtubule is a dynamic protein, constantly
polymerizing and depolymerizing, vinca alkaloid poisoned dimers could easily be incorporated
into the microtubule polymer, preventing further growth (Figure-1). The incorporation of the
vinca alkaloids onto the heterodimer is rapidly reversible, and appears to occur at two sites per
tubulin dimer. At higher concentrations of drug, 13 microtubule crystals are formed, consisting of
two intertwined helices of tubulin. In addition to the two natural compounds a third effective
alkaloid, vindesine has been produced by functional group transformation, and appears to work
by the same mechanism11-12

Figure-1
Synthesis and derivatives

Vinblastine and vincristine isolated very minute quantities from the leaves of C. roseus. For that
reason total synthesis and semi-synthesis of these binary alkaloids has been the subject of a
number of studies, for the clinical trial and biological activity purpose.

The complex structure and stereochemistry of vinblastine and vincristine are established by
Neuss et al. and Moncrief and Lipscomb. Both are dimeric compound possessing an indole
moiety (velbanamine upper part) and vindoline dihydroindole nucleus. They differ only by the
substituent attached to the nitrogen of the dihydroindole part, vinblastine bearing an N-methyl
while vincristine bearing N–formyl functional group, both are strongly differ in their antitumor
properties and clinical toxicities.so for three other modified vinca alkaloids developed as a
antitumor compound, Vindesine, Vinorelbine and more recently a fluorinated structure analogue,
Vinfluinin.

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In this review only one synthesis we are discussing of vinblastine, Boger’s group developed a
recently coupling between catharanthine and vindoline moiety which is good natural abundance
in nature to give vinblastine and after few step vincristine. This is one pot method combine
FeCl3-promoted vindoline/catharanthine with a subsequent Fe2 (ox) 3-NaBH4/air concomitant
oxidation of the C3’=C4’ double bond and reduction of the imine intermediate (Scheme-1).14

HO
N
N N
+ . N
OCOCH3 1) FeCl3,25 C,2hr
HH
3CO2C
N CO2CH3 N H CO2CH3 2)Fe2(ox)3,NaBH4,air.O.C OCOCH3
H H3CO H3CO N H CO
CH3 62%C4' alcohaols 2CH3
CH3OH

Scheme-1 Direct coupling of catharantine and vindoline to vinblastin 41% beta -OH
21% alpha-OH

Structure activity relationship

All most 50 years, several hundred derivatives have been synthesized and evaluated for their
antitumor activities. Most of these products have been obtained by modification. 12 of the
vindoline “lower part” bearing several reactive functions. Structure changes in the upper part are
much less Trivial and have shown that even slight change can dramatically modify biological
activities.

112 | P a g e
 Catharanthine moiety
Important confugration for activity

HO

N
N
H
CH3
H3COOC H

H3CO N OR3
Important confugration for activity H
R1 COR2
HO

R1= CHO,CH3
Vindoline moiety

show activity

Figure-2

Modification on vindoline
some important derivatives have been synthesized and evaluated by Eli Lilly groups on vindoline
moiety, like Vinglycinate, vindesine, and vinzolidine.3
Vinglycinate is first vinblastine analog which inter phase I clinical trial in 1967 which is
substituted by a glycine residue at the vindoline C-4 position.

113 | P a g e
 F
HO F

N
N
H
CH3
H3COOC H

H3CO N OCOCH3
H
5-Viflunin R1 COOCH3
HO

N
N
H
CH3
H3COOC H

H3CO N OCOCH3
H
H3C CO2CH3
HO
6-Vinorelbin

HO
HO

N
N

N N
N N
H H
CH3 CH3
H3COOC H H3COOC H
O

H3CO N H3CO N O N
OH
H H
8-Vinzolidine O
CONH2 H3C O
H3C HO
N
7-Vindesine O Cl

HO

HO N

N
N
N H
CH3
N H3COOC H
H
CH3
H3COOC H H3CO N OH
O H
C O
H3CO N O N 10-Infosiltine H3C HO O P
H HN OEt
CO2CH3 OEt
9-VinglycinateH3C HO

114 | P a g e
Vindesine4 is considered as a chemical precursor of vinzolidine and vintriptol which is
substituted by C-3 position lead to the amido-derivative vindesine.5
Vinepidine, which was also developed by the Lilly group corresponding to 4’-epi-4’-
deoxyvincristine, for its increased tubulin affinity relative to vinblastine.

None of these semisynthetic compound exerted marked benefits in clinical evaluation relative to
vinblastine and vincristine. Due to presence of unusual high potency of Aminophasphate
derivatives vinfosilitin for clinical trial with clinical efficacy due to both in vitro and in vivo
compared with the vinblastine and vincristine. So many modifications performed at C-3 position
of anhydrous vinblastine with the formation of numerous amide, ester, ether, and carbamate
derivatives and their in vitro and vivo activities were evaluated, showing that a carbonyl group at
that position is important for activity. Out of these one more compound which is tenfold less
toxic than vinca alkaloids in vitro but presented, in vivo, superior antitumor activity to
vinorelbine against sarcoma 180 in mice as well as better pharmacokinetic profile its activity was
compared with that of vinfluinin that is (3-decarbomethoxy-acetyloxymethyl-anhydrovinblastin).

Modification of catharanthine portion

Upper moiety of vinblastine has been less studied due to lack of functionalities, it is still
considered to be a very important region in term of the potency and novelty of analogus. 7, 8
Stereo chemical configuration at C-16’-C14’ in the velbanamine portion are very important.
Inversion of this configuration at C-16’-C14’ configuration leads to loss of activity. The C16’
carbomethoxy group of velbanamine portion is also important, since the decarboxylated dimer is
inactive.

Structural modification at C15’-C20’ of the velbanamine portion is well tolerated. Leurosidine

(the C20’epimer), leurosine (the epoxide), the C20’-deoxy derivative, the C15’-C20’dehydro-
derivative, and C20’-desethyl derivatives, 3 all exhibit different inhibition activities for
microtubule assembly. Several N’-oxides have been prepared by oxidation of the parents
compounds with peroxide. they reduced antitumor as well as antitubulin binding affinity .some
other modification like D ring cleavage like Vinepidine also known as catharanthine which
nearer complete loss of activity.one of the most important derivatives of upper portion is
vinorelbine which retains excellent activity but is less neurotoxic than vincristine. Vinorelbine is
currently available worldwide for the treatment of non-small cell lung cancer and of breast
cancer.

Vinfluinin the difluoro-derivatives9-10 of vinorelbine this compound has been improved

antitumor activity over vinorelbine against B16 melanoma and a panel of human tumor
xenografts.it was discovered that fluorination at C19’ position of vinorelbine dramatically
increases the in vivo activity. The hydroxylation at the same position results in the total loss of
activity.

No effect on activity due to presence mono halogenation at C19’ but dihalogen significantly
decrease the activity. De acetylation at the C-17 position of dihydro vinorelbine and vinfluinin
resulted in increase of both in vitro cytotoxicity and in vivo activity. In velbanamine moiety
bisindole alkaloids is important for maintaining their potency. Only one of the tetrahydro-

115 | P a g e
isoquinoline derivatives are exerted marginal activity on the tubulin polymerization inhibition
test. other derivatives lacking the C5’-C6’ bond in the C-ring were found to be inactive.

Erick K. Leggans et al. synthesized urea derivatives at C20’ which exceed the potency of
vinblastine in functional cell-based growth inhibition assays. In addition to defining structural
features of the urea required for or potentiating their activity that is directly related to their
relative tubulin binding affinity.

CONCLUSION

Due to their potent anticancer activity, vinblastine and vincristine are being used for last 40
years. Both are isolated from the leaves of Catharanthus roseus in very low yields. To overcome
the problem of poor yield and toxicity, many synthetic methods have been developed and many
potent derivatives have been derived. In future we can also develop new method which will give
us high yield of vinblastine and vincristine and can also derived them for better activities

REFERENCES

1. Brossi, A., Suffness, M., Eds. Antitumor Bisindole Alkaloids from Catharanthus Roseus (L.).
In The Alkaloids; Academic Press Inc.: New York, NY, USA, 1990; Volume 37, pp. 1–240.
2. Bölcskei, H.; Szabó, L.; Szántay, C. Synthesis of vinblastine derivatives. Front. Nat. Prod.
Chem. 2005, 1, 43–49.
3. Eli Lilly Company, Neue Amin derivate von Vinblastine, Leurosidine und Leurocristin und
Verfahren zu ihrer Herstellung. DE Patent 22415980, 1974; [Chem. Abstr. 1974, 82,
579967b].
4. Barnett, C.J.; Cullinan, G.J.; Gerzon, K.; Hoying, R.C.; Jones, W.E.; Newlon, W.M.; Poore,
G.A.; Robison, R.L.; Sweeney, M.J.; Todd, G.C. Structure-activity relationships of dimeric
Catharanthus alkaloids. 1. Deacetyl vinblastine amide (vindesine) sulfate. J. Med. Chem. 1978,
21, 88–96.
5. Rao, K.S.P.B.; Collard, M.-P.M.; Dejonghe, J.P.C.; Atassi, G.; Hannart, J.A.; Trouet, A.
Vinblastin-23-oyl amino acid derivatives: Chemistry, physicochemical data, toxicity, and
antitumor activities against P388 and L1210 leukemia. J. Med. Chem. 1985, 28, 1079–1088
6. Brady, S.F.; Pawluczyk, J.M.; Lumma, P.K.; Feng, D.-M.; Wai, J.M.; Jones, R.; DeFeo-Jones,
D.;Wong, B.K.; Miller-Stein, C.; Lin, J.H.; et al. Design and synthesis of a pro-drug of
vinblastine targeted at treatment of prostate cancer with enhanced efficacy and reduced
systemic toxicity. J. Med. Chem. 2002, 45, 4706–4715.
7. Scott, I.L.; Ralph, J.M.; Voss, M.E. Vinca derivatives. WO Patent 2005/55939, 2005.
8. Voss, M.E.; Ralph, J.M.; Xie, D.; Manning, D.D.; Chen, X.; Frank, A.J.; Leyhane, A.J.; Liu,
L.; Stevens, J.M.; Budde, C.; et al. Synthesis and SAR of Vinca alkaloid analogues. Bioorg.
Med.Chem. Lett. 2009, 19, 1245–1249.
9. Lafitte, C.; Jouannetaud, M.-P.; Jacquesy, J.-C.; Fahy, J.; Duflos, A. Stereoselective ionic
hydrogenation of Vinca alkaloids and vinorelbine in Superacids: An access to 4′R-reduced
analogs. Tetrahedron Lett. 1998, 39, 8281–8282.
10. Fahy, J.; Duflos, A.; Ribet, J.-P.; Jacquesy, J.-C.; Berrier, C.; Jouannetaud, M.-P.; Zunino, F.
Vinca alkaloids in superacid media: A method for creating a new family of antitumor
derivatives. J. Am. Chem. Soc. 1997, 119, 8576–8577.
11. Kingston, David G. I. (2009). "Tubulin-Interactive Natural Products as Anticancer
Agents(1)". Journal of Natural Products 72 (3): 507–15. .

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12. Lixin Zhang, Arnold L. Demain (2005), Natural products: drug discovery and therapeutic
medicine.Natural products: drug discovery and therapeutic medicin Molecules 2012, 17 5912
13. Ivachtchenko, Alexandre; Kiselyov, Alex; Tkachenko, Sergey; Ivanenkov, Yan; Balakin,
Konstantin (2007). "Novel Mitotic Targets and Their Small-Molecule Inhibitors". Current
Cancer Drug Targets 7 (8): 766–84.
14. Hayato Ishikawa, David A. Colby, Shigeki Seto, Porino Va, Annie Tam, Hiroyuki Kakei,
Thomas J. Rayl, Inkyu Hwang, and Dale L. Boger*Total Synthesis of Vinblastine, Vincristine,
Related Natural Products, and Key Structural Analogues J Am Chem. Soc. 2009 April 8;
131(13): 4904–4916.
15. Thimmaiah, K.N.; Lloyd, W.D.; Sethi, V.S. A simple method for the chemical modification of
antitumor Catharanthus Vinca alkaloids. Ind. J. Chem. Sect. B: Org. Chem. Med. Chem. 1990,
29, 678–680.

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 WITHANIA COAGULANS PHARMACOLOGICAL AND PHYTOCHEMICAL
INVESTIGATION

Shalini Dixit*, Deepti Yadav, Madhumita Srivastava, Priyanka Maurya, M.M Gupta
CSIR- Central Institute of Medicinal and Aromatic Plants, P.O. CIMAP, Lucknow 226015, India
*Email: shasddixit@gmail.com

ABSTRACT

A number of health problems are attributed to the modern life style which may further lead to
fatal conditions. Due to many adverse effects of modern drugs people used to prefer herbal
drugs. It is the need of the day to extensively study the medicinal plants as it has been also
proved that human physiology are adaptable to digesting and utilizing plant-based remedies.
In this context Withania coagulans commonly known as "paneer ke phool”, is a promising plant
due to its various therapeutical effects.

The use of Withania coagulans has been highlighted in Ayurveda and has shown to exert
hepatoprotective, anti-inflammatory, antihyperglycaemic, hypolipidaemic, free radical
scavenging, antimicrobial, cardiovascular, central nervous system depressant,
immunomodulating, antitumour and cytotoxic activities [Maurya et al., 2010]. The present
review emphasizes the various chemical constituents and major biological activity of isolates.

INTRODUCTION

Withania coagulans commonly known as “Paneer ke phool”in Hindi, Desi Asgandh in Arbi &
vegetable rennet in English has been used since time immemorial in Indian Ayurvedic
medicines. It is a rigid gray-whitish small shrub, 30-90 cm tall, leaves 2.5-7.5 cm by 1.5 cm.
Flowers are 7-12 mm across, yellow dioeciously and polygamous. It is cultivated in drier parts of
Iran, Pakistan, Afghanistan, and India (Punjab, Rajasthan, Simla, Kumaun and Garhwal).

Traditionally all the parts of the plant has been used as indigenous medicine for the treatment of
various ailments and physiological disorder. Dry fruits soaked in water for overnight are
effective in diabetes mellitus and in blood pressure control as well. The twigs are chewed for
cleaning of teeth and the smoke of the plant is inhaled for relief in toothache [Gupta et al., 2013].

At present, more than 12 alkaloids, 40 withanolides, and several sitoindosides (a withanolide

containing a glucose molecule at carbon 27) have been isolated and reported from aerial parts,
roots and berries of Withania species. Isolated alkaloids and steroids from plant sources are
responsible for hypoglycemic activity [Adebajo et al., 2006].

The major chemical constituents of these plants, withanolides, are mainly localized in leaves, and
their concentration usually ranges from 0.001 to 0.5% dry weight [Anonymous, 2004., Atal et al,
1975., Kapoor, 2001]. The withanolides are a group of naturally occurring C28- steroidal
lactones built on an intact or rearranged ergostane framework, in which C-22 and C-26 are
appropriately oxidized to form a six-membered lactone ring [Khodaei et al., 2012].

118 | P a g e
Encouraging with the fact that W. coagulans have already been reported for different medicinal
aspects, they may provide leads for developing ethical drugs or vital drink. This is particularly
important in light of the fact that this plant species is having better chances to act at multiple
targets.

REFERENCES

Adebajo A, Ayoola O, Iwadewa E, Akindahunsi A, Omisore N, Adewunmi C, Adenowo T Antitrichomonal,

bio-chemical and toxicological activities of methanolic extract and some carbajole alkaloids
isolated from the leaves of Murraya koenigii growing in Nigeria. Phytomedicine, 2006; 13(4) 246-
54.
Anonymous, Monograph: Withania somnifera. Altern. Med. Rev. 2004, 9, 211-214.
Atal, C.K., Gupta, O.P., Ranghunathan, K., Dhar, K.L. Central Council for Research in Indian Medicine and
Homeopathy, 1975.
Gupta V, Bihari B, Keshari withania coagulans dunal. (paneer doda): a review, International journal of
ayurvedic & herbal medicine 2013; 1330-1336.
Kapoor, L.D. Handbook of Ayurvedic Medicinal Plants. 2001; 337-338.
Khodaei M, Jafari M, Noori M. Remedial Use of Withanolides from Withania coagulans (Stocks) Dunal.
Advances in Life Sciences 2012; 6-19.
Maurya R, Akanksha and Jayendra Chemistry and pharmacology of Withania coagulans: an Ayurvedic
remedy, Journal of Pharmacy and Pharmacology 2010; 62: 153–160

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BIOCHEMISTRY AND

PLANT PHYSIOLOGY
 Dynamics of Secondary Metabolite Biosynthesis in Withania somnifera

Jyoti Singh Jadaun and Rajender Singh Sangwan

CSIR-Central Institute of Medicinal and Aromatic Plants, Lucknow 226015, UP, INDIA
Email: jyotisingh.jadaun@gmail.com

Withania somnifera, an Indian perennial herb, is known as Indian ginseng or Indian winter
cherry. Since ancient time, it is used as an important ingredient in traditional systems of
medicines like Ayurveda, Siddha, and Unani (Sangwan et al., 2004). It is pronounced as a
‘Rasayana’ due to its use for increasing the longevity and vitality of human life (Singh et al.,
2001). Population of developing countries still depends on the use of traditional medicines to
fulfill their requirement and Withania genus is one of such demanding medicinal plants.
Recently extract of W. somnifera (WS) is used in preparation of herbal tea, powders and in
many skin ointments. All parts of WS have varied medicinal properties such as anti-
inflammatory, antitumor, immune-modulatory, antioxidant, antistress, anticonvulsant, anti-
sedative and hepatoprotective (Ghosal et al., 1989; Bhattacharya et al., 1996; Kumar and
Kulkarni., 2006; Oberholzer et al., 2008). This plant is adored due to presence of
pharmaceutically relevant compounds, and such novel compounds isolated from Withania
collectively nominated as withanolides. It is used for the treatment of many diseases such as
leprosy, insomnia, respiratory disorders, asthma, epilepsy, diabetes and hormonal imbalance.
Phytochemistry of WS entails about the presence of an array of economically important
molecules which may lead to the direction for the development of new drugs against some
incurative diseases like tumor and neurodegenerative disorders like Parkinson’s disease (Nair
and Jayprakasham., 2007). Withanolides are C28 steroidal lactones built on an intact or
rearranged ergostane skelton ring. Comprehensive metabolic profiling has been done in
leaves and root of WS by using the recent analytical techniques like HPLC, NMR and GC-
MS (Chatterjee et al., 2010). In comparative analysis of leaf and root it was found that some
compounds are common but some are restricted to particular tissue. Main chemical
constituents of WS are withaferin A, withasomniferin A, 5-dehydroxywithanolide,
withanosides I-VII, withanone, withanolide A, withanolide D, oxygenated and sulfated
withanolide, somniferin, somniferinin, tropine and pseudotropine (Mishra et al., 2000;
Jayaprakasham et al., 2004; Mishra et al 2005; Miralili et al., 2009, Kushwaha et al., 2013).
These compounds are chemically classified as terpenes and this has been reported through
radioactive assays that both, mevalonic and methylerythritol phosphate pathways are
involved in biosynthesis of these compounds (Chourasyia et al., 2012). Tissue cultured
multiple shoots and induced root also have the ability to synthesize these molecules.
(Sangwan et al., 2007; Sabir et al., 2013) Terpenes are formed as a consequence of
polymerization reaction of isoprenes, these are low molecular weight five carbon molecule.
Mevalonic acid pathway (MVA) is operated in cytosol while methyl erythritol phosphate
pathway (MEP) or 1-deoxy D-xylulose phosphate pathway (DOXP) is channelized in
chloroplast. Isopentenyl pyrophosphate (IPP) synthesized via both pathways participates in
synthesis of terpenes. Relative contribution of MEP and MVA pathway in withanolide
biosynthesis is approximately 25:75 respectively (Chourasiya et al., 2012). Further molecular
studies of genes involved in withanolide biosynthesis pathway also support the existence of
both pathways (Gupta et al., 2012). 3-Hydroxy 3-methyl glutaryl Co A reductase (HMGR)
gene, the first gene of MVA pathway was isolated and cloned from leaves of WS and after
that 1-deox D-yxylulose 5-phosphate synthase (DXS), first gene of DOXP pathway was also
identified and characterized (Akhtar et al., 2012; Gupta et al., 2013). Gap of knowledge in
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molecular studies of WS may be completed by the transcriptome data of leaf and root (Gupta
et al., 2013). We have explored so much about its biologically active phyto-constituents and
their biosynthetic pathways but there is also a requirement to generate more data about the
operation and regulation of withanolide biosynthesis mechanism.

REFERENCES

Akhtar N, Gupta P, Sangwan NS, Sangwan RS, Trivedi PK (2012) Cloning and functional
characterization of 3-hydroxy-3-methylglutaryl coenzyme A reductase gene from
Withania somnifera: an important medicinal plant. Protoplasma 250:613-622
Bhattacharya SK, Satyan KS, Ghosal S (1997) Antioxidant activity of glycowithanolides
from Withania somnifera. Indian J. Exp. Biol. 35:236–239
Chatterjee S, Srivastava S, Khalid A, Singh N, Sangwan RS, Sidhu OP, Roy R, Khetrapal
CL, Tuli R (2010) A Comprehensive metabolic fingerprinting of Withania somnifera
leaf and root extracts. Phytochemistry 71:1085–109
Chaurasiya ND, Uniyal GC, Lal P, Misra L, Sangwan NS, Tuli R, Sangwan RS (2008)
Analysis of withanolides in root and leaf of Withania somnifera by HPLC with
photodiode array and evaporative light scattering detection. Phytochem. Ana.
19(2):148-54
Chaurasiya ND, Sangwan NS, Sabir F, Misra L, Sangwan RS (2012) Withanolide
biosynthesis recruits both mevalonate and DOXP pathway of isoprenogenesis in
Ashwagandha, Withania somnifera Dunal. Plant Cell Reports 31(10):1889-1897
Gupta P, Agarwal AV, Akhtar N, Sangwan RS, Singh SP, Trivedi PK (2012) Cloning and
characterization of 2-C-methyl-D-erythritol-4-phosphate pathway genes for isoprenoid
biosynthesis from Indian ginseng, Withania somnifera. Protoplasma 250(1):285-95
Kushwaha AK, Sangwan NS, Trivedi PK, Negi AS, Misra LN, Sangwan RS (2013) Tropine
forming tropinone reductase gene from Withania somnifera (Ashwagandha):
Biochemical characteristics of the recombinant enzyme and novel physiological
overtones of tissue-wide gene expression patterns. PLOS ONE 2013 DOI:
10.1371/journal.pone.0074777
Mirjalili MH, Moyano E, Bonfil M, Cusido RM, Palazon J (2009). Steroidal lactones from
Withania somnifera, an ancient plant for novel medicine. Molecules 14: 2373-2393
Mishra LC, Singh BB and Dagenais S (2000). Scientific basis for the therapeutic use of
Withania somnifera (Ashwagandha): A Review. Altern. Med. Rev. 5(4):334-346
Nair MG, Jayaprakasam B (2007) Cyclooxyganase-2 inhibitory withanolide composition and
methods. United States Patent US 7195,784 B2
Oberholzer HM, Pretorius E, Smit E, Ekpo OE, Humphries P, Auer RE, Bester MJ (2008)
Investigating the effect of Withania somnifera, selenium and hydrocortisone on blood
count and bronchial lavage of experimental asthmatic BALB/c Mice. Scand. J. Lab.
Anim. Sci. 35: 239-248
Sabir F, Mishra S, Sangwan RS, Jadaun JS, Sangwan NS (2012b) Qualitative and
quantitative variations in withanolides and expression of some pathway genes during
different stages of morphogenesis in Withania somnifera Dunal. Protoplasma 250:539-
549
Sangwan RS, Chaurasiya ND, Misra LN, Lal P, Uniyal GC, Sharma R, Sangwan NS, Suri
KA, Qazi GN, Tuli R (2004) Phytochemical variability in commercial herbal products
and preparations of Withania somnifera (Ashwagandha). Curr. Sci. 86:461-465

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Singh B, Saxena AK, Chandan BK, Gupta DK, Bhutani KK, Anand KK (2001) Adaptogenic
activity of a novel, withanolide free aqueous fraction from the roots of Withania
somnifera. Phytother. Res. 15:311-318

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 Hairy Root Induction via Agrobacterium rhizogenes in Withania somnifera

Shilpi Bansal and Neelam S. Sangwan*

Metabolic and Structural Biology, CSIR-Central Institute of Medicinal and Aromatic Plants,
Lucknow 226015, UP, INDIA
Email: shilpi.success@gmail.com

INTRODUCTION

Withania somnifera is a traditional Indian medicinal plant known for its pharmacological and
therapeutic properties. These properties are attributed due to the presence of secondary
metabolites, called as withanolides synthesized via mevalonic and non-mevalonic pathway
with a contribution of 3:1 (Chaurasiya et al, 2012). These metabolites are synthesized in the
roots and aerial part of the plant (Sangwan et al, 2008; Anonymous, 2004). However the
concentration of these withanolides in plants grown in in vivo conditions is very less as
compared to the demand required to carry out various experimental studies. This problem call
for a need to develop a system which is fast, economical and also provide stable
accumulation of the compounds. Transformation via Agrobacterium is a tool which gives an
opportunity to enhance the content of metabolites.

A. rhizogenes is a gram negative soil bacterium infection by which leads to the induction of
hairy roots. The bacterium consists of a Ri plasmid which includes T-DNA region covered by
25 bp left border and right border boundaries, group of virulence (vir) genes and opine
synthesis genes. For a successful transformation event the T-DNA region is transferred to the
host plant with right border playing a major role in establishing the polarity and serving as the
attachment site for virD proteins. It is the activation of vir gene cluster which mediate transfer
of T-DNA. First in response to signal molecules like phenolics, virA and virG is activated
which turns on the cascade of activation of vir gene signals involving virD, virB complex and
virE2 (Gelvin, 2003).

W. somnifera and hairy roots

Enormous work has been done to induce hairy roots in W. somnifera, so as to improve growth
index, quality and quantity of withanolides. Correct choice of bacterial strain is a pre-
requisite for successful and effective transformation. Sivanandan et al, (2014) reported 88%
root induction by leaf explants and 64% by cotyledons when infected with R1000 strain while
A4 strain gave 79% and 38% on using leaf and cotyledons respectively. However
Bandopadhyay et al (2007) reported 85% infection, in both case of A4 and LBA4404 with the
occurrence of three different morphological types; hairy root, rooty callus and callus.
Accumulation of Withaferin A (0.44% on dry weight basis) along with Withanolide D
(0.23% on dry weight basis) was observed in transformed roots. It was the first report to
determine the presence of Withaferin A in transgenic root where preliminary hypothesis
regarding the accumulation of withanolides states that Withanolide D and Withanolide A are
mainly accumulated in roots while Withaferin A in aerial parts (Sangwan et al, 2008;
Anonymous, 2004). A number of explants can be used as a starting material for the induction
of hairy roots however its efficiency varies depending on the plant used and also on the stage
of the explants used, environmental conditions, hormones etc. (Hu and Du, 2006; Nin et al,
1997). Among various explants used, leaf produced maximum hairy roots followed by shoot
tips, cotyledons, hypocotyls etc. Though most reports state leaves as best explants an
123 | P a g e
exception to this was petiole which gave 64% hairy roots as reported by Saravanakumar et al,
(2012). Certain factors like the carbon source and its concentration, the ionic concentration of
the medium, pH, light, phytohormones, temperature, and inoculum are critical factors to
affect growth and secondary metabolism (Giri and Narasu, 2000). Optimization of the
medium composition for hairy root cultures is important for the increased biomass and also
for high production of secondary metabolites. Among different media such as MS, B5, NS
(Murashige and Skoog, 1962; Linsmeir and Skoog, 1965; Chu et al, 1975; Schenk and
Hildebrandt, 1972) the formulation most suitable for the plant was found to be MS (Murthy et
al, 2008). Sucrose is the major source of carbon which at a concentration of 3% alone or in
combination is most effective. Slight variation in the pH of the media from 5.8 to 6.0 resulted
in 13.84 mg/g DW Withanolide A production while sucrose at 4% favored the production of
13.28 mg/g DW Withanolide A (Nagella and Murthey, 2012). Changing the concentration of
the macroelements specifically of nitrogen can significantly upregulate the withanolide A
content. Application of certain biotic and abioiltic factors such as elicitors may further
augment the secondary metabolite content. Methyl jasmonate, salicyclic acid, fungal
pathogens are some of the common elicitors known for their ability to elevate the expression
of genes involved in secondary metabolite biosynthetic pathway and hence improve the
content of withanolides (Doma et al, 2005; Chaudhuri et al 2009).

Secondary metabolites produced in in vivo grown plants or roots attached to them or in

adventitious roots are very less. Also in vivo grown plants are dependent on various
environmental and seasonal factors with a prolonged cultivation period thereby further
making it difficult to isolate the compounds. Hairy root system negates such disadvantages by
providing a fast, rapid and a stable system for the increased production of secondary
metabolites. Random integration of T-DNA further provides chances of occurrence of certain
new metabolites. Apart from increasing the Withanolide content, A. rhizogenes mediated
transformation can be used as a system to determine the functional aspect of genes and
biosynthetic pathways. Despite being of immense importance hairy root system is yet not
been utilized to its fullest. Efforts need to be taken to commercialize it and to establish it up
to large scale bioreactors so as to meet the increasing demand of phytomolecules in industries
and therapeutics.

REFERENCES

Anonymous. Monograph: Withania somnifera. Altern Med Rev 2004; 9:211–214

Bandyopadhyay M, Jha S, Tepfer D. Changes in morphological phenotypes and withanolide
composition of Ri-transformed roots of Withania somnifera. Plant Cell Rep 2007; 26: 599-609
Chaudhuri K, Das S, Bandyopadhyay M et al. Transgenic mimicry of pathogen attack stimulates
growth and secondary metabolite accumulation. Transgenic Res 2009; 18: 121-134
Chaurasiya ND, Sangwan NS, Sabir F, Misra L, Sangwan RS. Withanolide biosynthesis recruits both
mevalonate and DOXP pathways of isoprenogenesis in Ashwagandha Withania somnifera L.
(Dunal). Plant Cell Rep 2012; 10: 1889-1897
Chu CC, Wang CC, Sun CS et al. Establishment of an efficient medium for anther culture of rice
through comparative experiments on the nitrogen sources. Sci Sin 1975; 18: 659-668
Doma M, Abhayanakar G, Reddy VD, Kavi Kishor PB. Carbohydrate and elicitor enhanced
withanolide (withaferin A and withanolide A) accumulation in hairy root cultures of Withania
somnifera (L.). Indian Journal of Experimental Biology 2012; 50:484-490
Gelvin SB. Agrobacterium-mediated plant transformation: the Biology behind the ''Gene-Jockeying''
Tool. Microbiol. Mol. Biol. Rev. 2003, 67(1):16. DOI: 10.1128/MMBR.67.1.16-37.2003

124 | P a g e
Giri A, Narasu ML. Transgenic hairy roots: Recent trends and applications. Biotechnol. Adv. 2000;
18:1-22
Hu ZB, Du M. Hairy root and its application in plant genetic engineering. Journal of Integrative Plant
Biology 2006; 48(2):121-127
Linsmaier EM, Skoog F. Organic growth factor requirements of tobacco tissue cultures. Physiol Plant
1965; 18: 100-127
Murashige T, Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue
cultures. Physiol Plant 1962; 15: 473-497
Murthy HN, Dijkstra C, Anthony P et al. Establishment of Withania somnifera hairy root cultures for
the production of withanolide A. J Integr Plant Biol 2008; 50: 975-981
Nagella P, Murthy HN. Synthesis of withanolide A depends on carbon source and medium pH in
hairy root cultures of Withania somnifera. Ind Crop Prod 2012; 35: 241-243.
Nin S, Bennici A, Roselli G, Mariotti D, Sehiff S, Magherini R (1997). Agrobacterium mediated
transformation of Artemisia absinthium L. (wormwood) and production of secondary
metabolites. Plant Cell Rep. 1997; 16:725-730.
Sangwan RS, Chaurasiya ND, Lal P, Misra L, Tuli R, Sangwan NS. Withanolide A is inherently de
novo biosynthesized in roots of the medicinal plant Ashwagandha (Withania somnifera).
Physiol Plant 2008; 133: 278-287.
Saravanakumar A, Aslam A, Shajahan A. Development and optimization of hairy root culture systems
in Withania somnifera (L.) Dunal for withaferin-A production. African Journal of
Biotechnology 2012; 11(98):16412-16420
Schenk RU, Hildebrandt AC. Medium and techniques for induction and growth of monocotyledonous
and dicotyledonous plant cell cultures. Can J Bot 1972; 50: 199-204.
Sivanandan G, Selvaraj N, Ganapathi A, Manickavasagam M. An efficient hairy root culture system
for Withania somnifera (L.) Dunal. African journal of biotechnology 2014; 13(43):4141-4147.

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 Micropropagation of Withania somnifera Dunal
Shiwani Maurya and Neelam S. Sangwan

Metabolic and Structural Biology, CSIR-Central Institute of Medicinal and Aromatic Plants,
Lucknow 226015, UP, INDIA
Email: shiwani.m.1907@gmail.com

INTRODUCTION

Withania Somnifera, commonly known as Ashwagandha is a perennial shrub from the Solanaceae
or Nightshade family. It is cultivated in many of the drier regions of India, such as Mandsaur
district of Madhya Pradesh, Punjab, Sindh, Rajasthan etc., and grown as late rainy-season
(kharif) crop. Withania Somnifera is used as herb in Ayurvedic medicine. Various parts of this
plant (berries, leaves and roots) are used as folk remedies. The herbal root extract has been
traditionally used as a tonic and as a sedative and recent research shows that the leaf extract
contains Withanolides which have been found to have regenerative properties on brain-cell
synapses in mice and in human cell lines in laboratory studies.

Plant tissue culture has emerged as a potential tool and forms the backbone of plant
biotechnology. Tissue culture techniques are widely applied for the improvement of field crops,
forests, horticulture and plantation crops for increased agricultural and forestry production. This
technique has been commercialized globally and contributed significantly towards the enhanced
production of high quality planting material. Micropropagation is a complex multistep process
and it is in effect the miniature version of conventional propagation which is carried out under
aseptic conditions. The ease by which plants can be micropropagated, varies from species to
species. Mostly seeds, seedlings and juvenile plant parts are used as starting materials since they
are easier to propagate. Tissue culture technique can play an important role in clonal propagation
and qualitative improvement of this medicinally important plant. Direct regeneration of
Ashwagandha plants from shoot buds and apical buds explants to regenerate W. Somnifera plants
have been explored.

The first step in any successful tissue culture programme is the selection of suitable explant
followed by complete disinfecting. Disinfecting the surface generally involves sterilization with
one or more disinfectants followed by washing. Withania somnifera nodal segments were
collected and washed with teepol detergent for 15 minutes at slow speed on a magnetic stirrer and
later washed thoroughly under running water for 2 hours. Surface sterilization was carried out
with 0.1 % HgCl2 for 10 minutes followed by washing thrice with double distilled water to
remove the traces of HgCl2. Sterilized explants were transferred aseptically to sterilized glass
plate under laminar flow hood. The deduced method for multiplication of shoot induction was
tried in five different chemotypes of Withania somnifera. These culture plates were finally kept in
the culture growth room with temperature conditions 25± 1 °C, with a photoperiod of 16 hrs
daylight and 8 hrs night break under the cool white fluorescent lightIn Withania, several
procedures were available for inducing in vitro response using leaf explants (Baburaj et al.,
1995). However, Furmanowa (2001) produced in vitro Withania somnifera plantlets from the
shoot-tip of aseptically germinated seedlings. We used MS medium supplemented with different
combinations of growth regulators for growth of Withania chemotypes. The highest percentage of
plant regeneration was found in BAP supplemented medium. Ray and Jha (2001) grew shoot tips
on MS media supplemented with BA (1.0 mg/l). Shoot induction was found to be 10.0 micro
shoots per explant. The shoot bud regeneration frequency gradually increased up to (0.6 mg/l) of
BAP; with further increase in hormone concentration there was a sharp reduction in the number
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of shoots. Another protocol was developed for large scale propagation of Withania somnifera
using seed as explants. The best callus production was observed in Murashige and Skoog (MS)
medium supplemented with 1.0 μM Kn, 4.5 μM BAP, and 1.5 μM NAA within a 14 day dark
period. Shoot initiation was observed in calli produced from shoot tips and nodal segments. The
highest shoot multiplication was observed in calli from nodal segments cultured in the presence
of 9.0 μM BAP and 1.0 μM IAA.

Propagation of Withania somnifera is primarily via seeds. However, conventional propagation of

this plant by seeds is not reliable and is inadequate to meet commercial demands because of the
low viability of the stored seeds and low seed germination rates (Sabir et al. 2008). Therefore, a
study was done to develop a high frequency direct regeneration system for Withania somnifera
using leaf and explants for direct shoot regeneration. Direct regeneration of shoot buds was
observed in MS basal medium supplemented with various concentrations of either benzyladenine
(BA) or thidiazouron (TDZ) depending on the explant. Nodal explants formed multiple shoots
both from pre-existing and de novo buds on Murashige and Skoog's medium (MS) containing
0.1–5.0 mg/l BA and a ring of de novo shoot buds on MS medium containing 0.2 and 0.3 mg/l
TDZ. Internodal explants generates shoot buds on MS with 1.0 and 5.0 mg/l BA while the
hypocotyl explants gave rise to multiple shoots only on MS with 0.5 mg/l BA. Callus cultures
were initiated from nodal segments on Murashige and Skoog medium supplemented with 2, 4-D,
BAP and Kinetin.

CONCLUSION

An efficient method of in vitro shoot propagation of six elite accessions of Withania somnifera
collected throughout India was developed. Maximum numbers of shoots in all accessions were
achieved from axillary explant on Murashige and Skoog (MS) medium supplemented with 1 mg/l
BAP and 1 mg/l kn (Sabir et al., 2008)

REFERENCES

Baburaj S, Gunakeswaran K, (1995). In vitro differentiation of shoots from leaf callus culture of
Withania somnifera (L.) Dunal. J. Indian Bot. Soc. 74:323–324.
Furmanowa M, Gajdzis-Kuls D, Ruszkowska J, Czarnocki Z, Obidoska G, Sadowska A, Rani R,
Upadhyay SN, (2001). In vitro propagation of Withania somnifera and isolation of withanolides
with immunosuppressive activity. Planta Med. 67:146–149.
Ray S, S Jha, (2001). Production of withaferin A in shoot culture of Withania somnifera. Planta Med.
67:432–436
Sabir F, Sangwan NS, Chaurasiya ND, Misra LN, Tuli R, Sangwan RS, (2008). Rapid
Micropropagation of Withania somnifera L. Accession form axillary meristems. Journal of
Herbs,Spices& medicinal plants13(4): 123-133
Sabir F, Sangwan NS, Chaurasiya ND, Mishra SK, Lal P, Mishra LN, Uniyal GC, Sangwan RS,
(2005). Biochemical and phytochemical investigation on shoot differentiation Induced by
growth regulator kinetin and BAP, in Withania somnifera (L.) Dunal. In
Sangwan NS, Chaurasiya ND, Misra S, Pandey S, Tiwari SP, Uniyal GC, Lal P, Misra LN, Tuli R,
Sangwan RS (2004). Phytochemical and molecular differentiation of Withania somnifera
genotypes, Second Global Summit on Medicinal &Aromatic Plants, 2004, New Delhi.
Sangwan RS, Chaurasiya ND, Misra LN, Lal P, Uniyal GC, Sharma R, Sangwan NS, Suri KA, Qazi
GN, Tuli R, (2004). Phytochemical variability in commercial herbal products and preparations
of Withania somnifera (Ashwagandha). Current Science 86:461–465.
Sangwan RS, Chaurasiya ND, Misra LN, Lal P, Uniyal GC, Sharma R, Sangwan NS, Suri KA, Qazi
GN, Tuli R, (2005). Process for isolation of withaferin-A from plant materials and products
therefrom. US Patent (AppFT) 20050226950
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 Mining of BAHD superfamily alcohol acyl transferases from Artemisia
annua trichome transcriptome

Lokesh K. Narnoliya, Neelam S. Sangwan and Rajender S. Sangwan

Department of Metabolic and Structural Biology, CSIR-Central Institute of Aromatic and
Medicinal Plants (CSIR), P.O. CIMAP, Lucknow-226015, India
Email: lokesh.narnoliya@gmail.com

Plants are well known to synthesize secondary metabolites such as terpene, phenolic, amines,
steroids, saponins, flavonoids, polyketides, lignin, fatty-acid-derived, alkaloids etc but
surprisingly only few pathways involve in biosynthesis of these compounds (Srivastava et al.,
2012, Sangwan et al., 2007). The diversity of these metabolites is achieved by secondary
transformation steps of the basic skeletons such as hydroxylation, decarboxylation,
oxidation/reduction, glycosylation, methylation and acylation. Out of these, acylation is the
most common type of modification of secondary metabolites and it is catalyzed by
acyltransferases enzymes. In acyltransferases enzymatic reaction, acyl group from acyl CoA
is transfer to OH group containing compound and esterified product produced. In this type of
reaction, activated acyl donors sources are acyl-sugars, acylated acyl carrier proteins or acyl-
activated coenzyme A thioesters and substrates can be terpenoids, alkaloids, fatty acids,
anthocyanin, volatile compound, alcoholic compound and other OH group containing
metabolites (D’ Auria, 2006). These acyltransferases enzymes belong to BAHD supper
family and the name BAHD stand for its first four biochemically characterized
acyltransferases enzymes. These enzymes are benzyl alcohol O-acetyltransferase (BEAT)
from the Clarkia breweri, which produces floral volatile benzyl acetate; deacetylvindoline 4-
O-acetyltransferase (DAT) from Catharanthus roseus, involve in alkaloid vindoline
biosynthesis; anthranilate N-hydroxycinnamoyl/benzoyltransferase (HCBT) from Dianthus
caryophyllus, produces a phytoalexins, anthramides; and anthocyanin O-
hydroxycinnamoyltransferase (AHCT) from Gentiana triflora producing acylated
anthocyanins (D’ Auria, 2006, Srivastava et al., 2012, Sharma et al., 2005 & 2013).

These alcohol acyl transferases (AAT) possess two most conserved amino acid sequence;
HXXXD motif located in middle of enzyme and DFGWG motif near C-terminal regain. Site
directed mutagenesis studies reveals that mutagen in one or both motif reduced enzyme
activity. The first crystal structure of vinorine synthase, an acetyltransferase enzyme from
Rauvolfia serpentina shows that histidine residue of HXXXD motif play key role in acyl
transferases activity (D’ Auria 2006, Srivastava et al., 2012) while Asp residue of HXXXD is
considered to be involved in its conformational suitability (St-Pierre and De Luca 2000;,
Suzuki et al. 2003;, Bayer et al. 2004). The actual role of DFGWG motif in enzyme catalysis
is not known but it might be participating in catalysis and acyl CoA binding process (D’
Auria ,2006; Ma et al., 2005). The next generation sequencing (NGS) technology provide
large amount of data (genome, transcriptome and libraries data) which would be mined into
discrete genes, and biochemically analyzed (Sangwan at el., 2013, Narnoliya et al., 2014,
Rajakani et al., 2014). More than 40 acyltransferases enzymes were characterized which were
involved in anthocyanin biosynthesis, alkaloids biosynthesis, volatile ester biosynthesis etc.
Arabidopsis thaliana genome data contained approximately 64 BAHD member and Oryza
sativa possess 119 BAHD family genes. There are many transcriptome and genome data
available in public databases which have to be analyzed to identify BAHD super family
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genes. Recently Artemisia annua transcriptome was analyzed for acyltransferases member
identification. (Luo et al. 2007, Srivastava et al 2012)

Artemisia annua produces various metabolites such as monoterpenes, sesquiterpenes and

their esters, including artemisinin which is well known for its antimalarial activity (Sangwan
et al., 1998 and 2001; Sangwan et al., 1993 and 1999). Due to such enormous importance,
various researches has been conducted with respect to its molecular biology, biochemistry
and phytochemistry. Recently A. annua glandular trichome transcriptome were published
(Wang et al. 2009). The assembly of ESTs, their alignment and other bioinformatics analysis
identified total 19 discrete non redundant BAHD acyltransferases cDNAs from Artemisia
annua trichome transcriptome which possess more than 50% similarity with other plants
acyltransferases. Out of these, 11 contigs showed similarity (50-84%) with
benzoyltransferase group, five contigs shows similarity (>98%) with anthranilate-N-hydroxy-
cinnamoyl/benzoyl transferases (HCBTs) group and remaining three shows similarity (45-
60%) with salutaridinol 7-O-acetyltransferase and deacetylvindoline 4-O-acetyltransferase.
All the BAHD members of Artemisia annua plant belong to all the four groups of BAHDs
super family (BEAT, HCBT, ACHT and DAT) (Srivastava et al,. 2012).

These contigs were further extended to obtain 3’ and 5’ region by in silico approach using
overlapping regain. By this approach three cDNA were assembled as full length namely
AAT-RS1, AAT-RS2 and AAT-RS3. Blastx and ORF finder provide appropriate coding
region containing start and stop codon. These three AATs contain both signature motifs
(HXXXD and DFGWG) and average molecular mass (48 – 55 kDa) which was in the range
of other known BAHD family acyltransferases. The alignment results of these AATs with
other plants AATs indicate that 16 residues are universally conserved and 8 – 10 amino acids
were around 90% conserved. These AATs shows low similarity around 24% at protein level
among themselves (Srivastava et al,. 2012).

Expression pattern of these 19 discrete AATs were analyzed by semiquantitative PCR in leaf,
stem and root. The results of expression analysis revealed that all the contigs show their
maximum expression in leaf tissue and almost all expressed in stem but none of them show
expression in root tissue. These results speculated that both stem and leaf tissue contains
identical glandular trichomes that’s why it shows expression but roots completely lack these
trichomes so expression not observe. This expression profile is ecologically relevant for plant
defense system because leaf and stem are directly exposed to herbivory, infections, insects
etc. but roots are relatively less exposed. Various metabolites are biosynthesized in both leaf
and stem tissue such as (Z)-α–trans bergamotal acetate, (Z)-3-hexenyl isovalerate, linyl
acetate, benzyl isovalorate etc which could be involved in plant defense system (Srivastava et
al., 2012; Goel et al., 2007).

Such type of studies could be performed for other plants also whose genome or transcriptome
was submitted to public databases such as Withania somnifera, Catharanthus roseus,
Azadirachta indica, Ocimum basilicum, Ocimum sanctum, Centella asiatica, Asparagus
racemosus etc.

REFERENCES

Bayer A, Ma X, Stockigt J (2004) Acetyltransfer in natural product biosynthesis - functional cloning

and molecular analysis of vinorine synthase. Bioorg Med Chem 12:2787 – 2795

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D’ Auria JC (2006) Acyltransferases in plants: a good time to be BAHD. Curr Opin Plant Biol 9:331
– 340
Goel DV, Singh M, Ali G, Mallavarupu KS (2007) Essential oils of petal, leaf and stem of the
antimalarial plant Artemisia annua. J Nat Med 61:187 – 191
Luo J, Nishiyama Y, Fuell C, Taguchi G, Elliott K, Hill L, Tanaka Y, Kitayama M, Yamazaki M,
Bailey P, Parr A, Michael AJ, Saito K, Martin C (2007) Convergent evolution in the BAHD
family of acyl transferases: identification and characterization of anthocyanin acyl transferases
from Arabidopsis thaliana. Plant J 50(4):678 – 695
Ma X, Koepke J, Panjikar S, Fritzsch G, Stockigt J (2005) Crystal structure of vinorine synthase, the
first representative of the BAHD superfamily. J Biol Chem 280:13576 – 13583.
Narnoliya LK, Rajakani R, Sangwan NS, Gupta V, Sangwan RS (2014) Comparative transcripts
profiling of fruit mesocarp and endocarp relevant to secondary metabolism by suppression
subtractive hybridization in Azadirachta indica (neem). Mol Biol Rep 41(5): 3147-3162.
Rajakani R, Narnoliya L, Sangwan NS, Sangwan RS, Gupta V (2014). Subtractive transcriptomes of
fruit and leaf reveal differential representation of transcripts in Azadirachta indica. Tree
Genetics & Genomes 1-21.
Sangwan NS, Farooqi AHA, Shabih F, Sangwan RS (2001) Regulation of essential oil production in
plants. Plant Growth Regul 34:3 – 21
Sangwan NS, Kumar S, Sangwan RS (1998) Isolation of genomic DNA from antimalarial plant
Artemisia annua L. Plant Mol Biol Rep 16:365
Sangwan NS, Sharma PK, Sangwan RS (2007) Geranyl acetate esterase is commonly present but
linalyl acetate esterase occurrence is highly limited in plants. Flav Frag J 22:173 – 177
Sangwan RS, Agarwal K, Luthra R, Thakur RS, Sangwan NS (1993) Biotransformation of arteannuic
acid into arteannuin-B and artemisinin in Artemisia annua L. Phytochemistry 34:1301 – 1302
Sangwan RS, Sangwan NS, Jain DC, Kumar S, Ranade S (1999) RAPD profile based analysis of
genetic characterization of chemotypic variants of Artemisia annua L. Biochem Mol Biol Int
47:933 – 944
Sangwan RS, Tripathi S, Singh J, Narnoliya LK, Sangwan NS (2013) De novo sequencing and
assembly of Centella asiatica leaf transcriptome for mapping of structural, functional and
regulatory genes with special reference to secondary metabolism. Gene 525(1):58-76.
Sharma PK, Sangwan NS, Sangwan RS (2005) TCA facilitated coenzyme A-SH stabilization based
end point spectrophotometric DTNB-assay for plant terpene alcohol acetyl transferases (AATs)
activity. Anal Biochem 346:176 – 178
Sharma, PK, Sangwan, NS, Bose, SK, Sangwan, RS (2013). Biochemical characteristics of a novel
vegetative tissue geraniol acetyltransferase from a monoterpene oil grass (Palmarosa,
Cymbopogon martinii var. Motia) leaf. Plant Science 203: 63-73.
Srivastava S, Sangwan, RS (2012) Analysis of Artemisia annua transcriptome for BAHD alcohol
acyltransferase genes: Identification and diversity of expression in leaf, stem and root. J Plant
Biochem Biotechnol 21(1): 108-118
St-Pierre B, De Luca V (2000) Evolution of metabolic pathways. Recent Adv Phytochemistry 34:285
– 315
Suzuki H, Nakayama T, Nishino T (2003) Proposed mechanism and functional amino acid residues of
Malonyl-CoA: Anthocyanin 5- O-Glucoside-6 ‴ -O-Malonyltransferase from flowers of Salvia
splendens, a member of the versatile plant acyltransferase family. Biochemistry 42:1764 – 1771
Wang W, Wang Y, Zhang Q, Qi Y, Guo D (2009) Global characterization of Artemisia annua
glandular trichome transcriptome using 454 pyrosequencing. BMC Genom 10:465

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 Modelling and Characterization of Tropine forming Tropinone reductase
gene in Withania.

Muktesh Chandra, Neelam S. Sangwan

Metabolic and Structural biology Division, CSIR-Central Institute of Medicinal and Aromatic
Plants, Lucknow.
Email: mchandra33@gmail.com

ABSTRACT

Withania somnifera is also known as Ashwagandha and Indian gensing, is a well versed plant
with medicinal properties and synthesis of secondary metabolites as withanolides, alkaloids
etc. In this review, we focus on enzymes involved in formation of tropane alcohols, viz.
tropinone reductases a class of enzyme possessing distinct sequence motifs and form a well-
established enzyme family of oxidoreductases distinct from functionally related enzyme
families such as medium-chain dehydrogenases/reductases or aldo-keto reductases i.e. SDR
(short chain dehydrogenase/reductase). Tropinone reductases (TRs) basically are NADPH-
dependant enzymes, TR-I and TR-II involved in conversion of Tropinone into tropine and
pseudotropine respectively. This review will give close insight about the TRs, and can serve
as informative for further characterization and deep study of particular enzyme in different
flora.

INTRODUCTION

Withania somnifera is reputed medicinal plant of the Indian systems of medicine and forms
essential constituent of >100 herbal and nutraceutical formulations [1]. The herb possesses
pharmacological activities like anti-arthritic, cognitive function improvement in geriatric
states and recovery from neurodegenerative disorders [2][3]. Also, Phytochemically, the plant
is unique in producing several types of secondary metabolites such as withanolides,
withanamides, and alkaloids [5][6][7][8][9]. In this review, we focus on enzymes involved in
formation of tropane alcohols, viz. tropinone reductases a class of enzyme possesing distinct
sequence motifs and form a well-established enzyme family of oxidoreductases distinct from
functionally related enzyme families such as aldo-keto reductases or medium-chain
dehydrogenases/reductases i.e. SDR (short chain dehydrogenase/reductase) [10]. At present,
about 3000 primary structures (including species variants) of the SDR family are annotated in
sequence databases. The enzymes included in the SDR family span several EC classes, from
oxidoreductases and lyses to isomerases, with oxidoreductases forming the majority [10]. In
this class, many enzymes of different substrate specificities are found acting on steroids,
prostaglandins, aliphatic alcohols and xenobiotics.

Tropinone reductases (TRs) basically, TR-I and TR-II involved in conversion of Tropinone
into tropine and pseudotropine respectively [10][11]. Tropinone reductases (TRs) are
NADPH-dependant enzymes belonging to protein family of SDRs (short-chain
dehydrogenases/ reductases) [13]. TRs catalyse reduction of tropinone into tropine and
thereby enabling biosynthesis of tropane and pseudotropane alkaloids. Two classes of TRs
are reported that catalyzes stereo-specific conversion of tropinone into tropine (TR-I) and
pseudotropine (TR-II) [14], [15]. Both, TR-I and TR-II, are present together in any given
tropane alkaloid-producing species and varying expression levels of the two TRs in the
tropane alkaloid-producing species, may govern the precursor metabolite flux distribution at
131 | P a g e
 the branch point across the two metabolic streams [14], since no inter-conversion between
tropine and pseudotropine has been observed in vivo [15].

CONCLUSION:

Tropine reductase of withania somnifera showed a high similarity to the TRs of other
members of SDR (short chain dehydrogenases/reductases) family of enzymes.
The characteristic motifs of the SDRs were also evident in TRs sequence, for example,
NADPH binding TGXXXGXG motif [12]. This motif was present at 27–34 position in TR-I.
Similarly, NNAG motif of SDRs was located at position 105–108 in Withania TR-I protein.
The tyrosine residue of catalytic sequence motif YXXXK of SDRs [12] was observed at
position 171 of TR-I. The tyrosine residue of the later motif is considered essential for the
catalytic activity of TRs, as discerned from the crystal structures and site directed
mutagenesis studies. The catalytically active enzyme was 60 kDa homodimeric protein. The
enzyme had catalytic characteristic and substrate specificity similar to other reported TR-Is.
Interestingly, the gene was considerably expressed in most of the plant parts rather than
remaining only restricted to roots reported for other species. Some of the observed novelties
in this study call for a new scope of fundamentals of alkaloids for this medicinal plant that is
gaining growing validations for its traditional pharmacological activities.

REFERENCE:

1. Sangwan RS, Chaurasiya ND, Misra LN, Lal P, Uniyal GC, et al Phytochemical variability in
commercial herbal products and preparations of Ashwagandha (Withania somnifera). Curr
Sci. 86: 461–465.(2004)
2. Tuli R, Sangwan RS (2010) Ashwagandha (Withania somnifera) – a model Indian
medicinal plant. Council of Scientific and Industrial Research (CSIR), New
Delhi, ISBN 978-93-80235-29-5.
3. Mirjalili MH, Moyano E, Bonfill M, Cusido RM, Palazo´n J (2010) Steroidal
lactones from Withania somnifera, An ancient plant for novel medicine. Molecules
14: 2373–2393.
4. Udo Oppermanna, Charlotta Fillinga, Malin Hulta, Naeem Shafqata, Xiaoqiu Wua, Monica
Lindha, Jawed Shafqata, Erik Nordlinga, b, Yvonne Kallberga, b, Bengt Perssona, b, Hans
Jörnvalla Short-chain dehydrogenases/reductases (SDR): the 2002 update
Chemico-Biological Interactions doi:10.1016/S0009-2797(02)00164-3(2003).
5. Chatterjee S, Srivastava S, Khalid A, Singh N, Sangwan RS, et al. (2010)
Comprehensive metabolic fingerprinting of Withania somnifera leaf and root
extracts. Phytochemistry 71: 1085–1094.
6. Jayaprakasam B, Strasburg GA, Nair MG (2004) Potent lipid peroxidation
inhibitors from Withania somnifera fruits. Tetrahedron 60: 3109–3121.
7. Misra LN, Misra P, Pandey A, Sangwan RS, Sangwan NS (2012) 1,4 dioxane
and ergosterol derivatives from Withania somnifera roots. J Asian Nat Prod Res 14:
39–45.
8. Chen LX, He H, Qui F (2011) Natural withanolides: an overview. Nat Prod Rep
28: 705–740.
9. Gupta P, Goel R, Pathak S, Srivastava A, Singh SP, et al. (2013) De novo
assembly, functional annotation and comparative analysis of Withania somnifera
leaf and root transcriptomes to identify putative genes involved in the
withanolides biosynthesis. PLoS ONE 8(5): e62714.
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10. Amit K. Kushwaha, Neelam S. Sangwan, Sandhya Tripathi, Rajendra S. Sangwan.Molecular
cloning and catalytic characterization of a recombinant tropine biosynthetic tropinone
reductase from Withania coagulans leaf.
Gene doi 516(2013) 238-247
11. Kushwaha AK, Sangwan NS, Trivedi PK, Negi AS, Misra L, et al. (2013) Tropine
Forming Tropinone Reductase Gene from Withania somnifera (Ashwagandha): Biochemical
Characteristics of the Recombinant Enzyme and Novel Physiological Overtones of Tissue-
Wide Gene Expression Patterns. PLoS ONE 8(9): e74777. doi:10.1371/journal.pone.0074777
12. Oppermann U, Filling C, Hult M, Shafqat N, Wua X, et al. (2003). Short-chain
dehydrogenases/reductases (SDR): the 2002 update. Chem Biol Interact 143–
144: 247–253.
13. Nakajima K, Yamashita A, Akama H, Nakatsu T, Kato H, et al. (1998) Crystal
structures of two tropinone reductases: different reaction stereospecificities in the
same protein fold. Proc Nat Acad Sci 95: 4876–81.
14. Hashimoto T, Nakajima K, Ongena G, Yamada Y (1992) Two tropinone
reductases with distinct stereospecificities from cultured roots of Hyoscyamus niger.
Plant Physiol 100: 836–845.
15. Yamada Y and Hashimoto T (1990) Possibilities for improving yields of
secondary metabolites in plant cell cultures. Curr Plant Sci Biotech Agric 9: 547–
556.

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 ROS Homeostasis of Withania somnifera against Cadmium Stress

Bhawana Mishra, Neelam S. Sangwan*

Metabolic and Structural Biology, CSIR-Central Institute of Medicinal and Aromatic Plants,
Lucknow 226015, UP, INDIA
Email: bhawna.mishra07@gmail.com

ABSTRACT

Withania somnifera (member of Solanaceae family) very important medical herb has been
recommended for more than 100 formulations in Unani, Ayurveda, and Siddha. In vitro
shoots cultures were treated with various concentration of Cadmium (Cd). Cadmium possess
toxic effect to Withania at higher concentration. Withania evolved enzymatic and non-
enzymatic antioxidant mechanism against Cd to minimize its toxic effect and maintain the
ROS homeostasis by balancing between ROS generation and scavenging.

INTRODUCTION

Medicinal plants gaining lots of attention as the important source of modern and traditional
medicine in the world, having significant therapeutic value in the human life. The therapeutic
properties of these plants are mainly due to abundance of various active constituent like
various secondary metabolites and essential oils (Lovkova et al. 2001). Withania somnifera
(Ashwagandha) is one of the most valued medicinal plant, considered as a Rasayana and
important medicinal herb, which is widely used in traditional health care systems, Ayurvedic
and traditional systems of medicine since over 3000 years. (Sangwan et al. 2003; Chaurasiya
et al. 2012) Ashwagandha is commonly known as “Indian Winter Cherry” or “Indian
Ginseng”. This herb is also commonly used in herbal formulation for its wide range of health
benefits. Ashwagandha is mainly cultivated in many drier regions of India such as Rajasthan,
Sind, Mansa, Neemuch, Punjab and Jawad tehsils of the Mandsaur district of Madhya
Pradesh. Due to the presence of unique steroidal lactone called as withanolides,
Ashwagandha has various range of pharmacological properties such as anti-apoptotic, anti-
stress, anti-tumor, anti-inflammatory, immunomodulatory, antioxidative property etc.
(Sangwan et al. 2004, and 2007, 2008 Mishra et al. 2014)

Antioxidant plays an important role in maintaining the ROS (reactive oxygen species)
homeostasis. Under metal stress, Plant produces excessive amount of ROS, which is toxic to
plant. Plants are continuously exposed to different biotic and abiotic stresses such as salinity,
UV- radiation, heat, draught, and heavy metal stress. Heavy metals are toxic to plants as well
as human when its concentration is higher in soil by affecting the growth and productivity.
Cadmium is one of the most toxic heavy metal, it possess deleterious effect to plant such as
stunted growth, alterations in membrane permeability and biochemical and physiological
processes like photosynthesis, respiration, protein metabolism, inhibition of certain enzymes
activities, inhibition of uptake of many essential elements, nutrient and their transport (Gill et
al. 2011; Sabir et al. 2012; Mishra et al. 2014). To minimize these effects plants have
developed antioxidative mechanism including both non-enzymatic and enzymatic
antioxidants. These antioxidants are low molecular weight substrate, which neutralize the
ROS production and their scavenging mechanism. Plants have enzymatic antioxidants such as
superoxide dismutase (SOD), catalase (CAT), peroxidase (POX), guaiacol peroxidase (g-
POD) and ascorbate peroxidase (APx), glutathione peroxidase (GPX), glutathione reductase
134 | P a g e
(GR) and non-enzymatic antioxidants such as total phenolics, sugars, ascorbate, carotenoid,
glutathione, and flavonoids etc. (Sabir et al. 2012; Mishra et al. 2014).

Multiple shoots cultures of Withania somnifera were treated with various concentration of Cd
and its effect on physiological and on antioxidative mechanism were examined. This study
suggested that at higher concentration, Cd has adverse effect on Withania multiple shoot
cultures such as stunted growth and chlorosis. Enhanced activities of antioxidative enzyme
such as CAT, APX, POX, and G-POD (except SOD) proved their active involvement in ROS
scavenging and Cd detoxification in multiple shoot cultures of Withania somnifera under Cd
stress. In case of non-enzymatic antioxidant, total proline, phenolics, carotenoids, and
ascorbate level were increased in Cd treated shoots suggesting their important role in
maintain the ROS homeostasis and Cd tolerance mechanism in Withania somnifera (Mishra
et al. 2014).

CONCLUSION

Antioxidative mechanism activate upon salt and Cu exposure in Withania somnifera to cope
the adverse conditions (Sabir et al. 2014, Khatun et al. 2008). Enzymatic antioxidants such as
SOD, CAT, APX, POX, and G-POD and non-enzymatic antioxidants like proline, phenolic,
and carotenoids were increased with increasing Cd concentration. These results reveal the
active involvement of these enzymatic and nonenzymatic antioxidative responses to minimize
the oxidative stress induced by scavenging the excess amount of ROS generated via Cd
exposure.

REFERENCES

Chaurasiya ND, Sangwan NS, Sabir F, Misra L, Sangwan RS (2012) Withanolide biosynthesis
recruits both mevalonate and DOXP pathways of isoprenogenesis in Ashwagandha Withania
somnifera L. (Dunal). Plant Cell Reports 31:1889-97
Gill SS, Tuteja N (2011) Cd stress tolerance in crop plants probing the role of sulfur. Plant Signal
Behav 6:215–222
Khatun S, AliMB,Hahn EJ, PaekKY (2008) Copper toxicity in Withania somnifera: growth and
antioxidant enzymes response of in vitro grown plants. Env Exp Bot 64:279–285
Lovkova MY, Buzuk GN, Sokolova SM, Kliment’eva NI (2001) Chemical Features of Medicinal
Plants (Review). Applied Biochemistry and Microbiology 37: 229–237
Mishra B, Sangwan RS, Mishra S, Jadaun JS, Sabir F, Sangwan NS (2014) Effect of cadmium stress
on inductive enzymatic and nonenzymatic responses of ROS and sugar metabolism in multiple
shoot cultures of Ashwagandha (Withania somnifera Dunal). Protoplasma 251:1031-45
Sabir F, Sangwan RS, Kumar R, Sangwan NS (2012) Salt stress induced responses in growth and
metabolism in callus cultures and differentiating in vitro shoots of Indian ginseng (Withania
somnifera Dunal). Journal of Plant Growth Regulation 31:537-548
Sangwan NS, Yadav U, Sangwan RS (2003) Genetic diversity among elite varieties of the aromatic
grasses, Cymbopogon martini. Euphytica 130:117–130
Sangwan RS, Chaurasiya ND, Mishra LN, Lal P, Uniyal GC, Sharma R, Sangwan NS, Suri KA, Qazi
GN, Tuli R (2004) Phytochemical variability in commercial herbal products and preparations of
Withania somnifera (ashwagandha). Curr Sci 86:461–465
Sangwan RS, Chaurasiya ND, Lal P, Misra L, Tuli R, Sangwan NS (2008) Withanolide A is
inherently de novo biosynthesized in roots of the medicinal plant Ashwagandha (Withania
somnifera). Physiologia Plantarum 133:278-287

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Sangwan RS, Chaurasiya ND, Lal P, Misra L, Uniyal GC, Tuli R, Sangwan NS (2007) Withanolide A
biogeneration in in vitro shoot cultures of ashwagandha (Withania somnifera DUNAL), a main
medicinal plant in Ayurveda. Chemical & Pharmaceutical Bulletin 55:1371-5.
Withania somnifera Reverses Alzheimer’s Disease by Degrading-Amyloid β
Peptides in Brain

Archana and Prema G. Vasudev

Department of Metabolic and Structural Biology, CSIR-Central Institute of Medicinal and
Aromatic Plants, Lucknow 226015 (U.P)
Email: archanaa843@gmail.com

ABSTRACT

Alzheimer's disease (AD) is commonly characterized by destruction of neural network,

resulting in accumulation of amyloid β peptides and neurofibrillary tangles in the brain.
Roots of Withania somnifera is used as nerve tonic and memory enhancer. Chemical
constituents of Withania somnifera and their derivatives act as neuroprotective agents by
enhancing the expression of LRP (Low density lipoprotein receptor – related protein 1) in
brain which result in degradation of amyloid β peptide. In this review, we summarize effect
of Withania somnifera on Alzheimer's disease.

Key Words: Alzheimer's disease, Withania somnifera, Amyloid β peptides, Tau protein, LRP
(Low density lipoprotein receptor – related protein 1).

INTRODUCTION

Alzheimer’s disease (AD) is a neurodegenerative disease characterized mainly by destruction

of neural network. In AD, neural network is not spontaneous leading to loss of neuron and
synapse in cerebral cortex and in subcortical region. The distinct causes of AD are plaques
and tangles in brain or atrophy by amyloid β, degeneration of temporal lobe and parietal lobe,
frontal cortex and cingulated gyrus, lobus coeruleus.(1,2,3) Several-drugs used for the
treatment of AD such as cholinesterase inhibitors, N-methyl–D-aspartate receptor antagonist,
secretase inhibitor etc, provide only symptomatic relief but they do not cure it.

Withania somnifera is also known as Ashwagandha, used for treatment of neurodegenerative

disease. The roots of Withania somnifera (WS) serves as nerve tonic and memory enhancer
and is shown to possess anti-stresss, immunomodulatory, antioxidant and
antineuropsychiatric effects.(4,5,6) In addition, WS has neuroprotectiv-effect against amyloid
β. The methanol:chloroform (1:3) extract of root of WS contain mainly Withanolide A and
Withanolide IV, which promote neurite outgrowth. In this review, we describe effect of
Withanolides and its derivatives, in the context of Alzheimer's disease.

Alzheimer's Disease

AD is autosomal dominant disease, triggered either by a mutation in one of the genes which
encode for APP ( Amyloid Precursor Protein), Presenilin 1 and 3 or by inheritance of E4
allele of apolipoprotein E. Mutation of any of the three genes results in the accumulation
amyloid β peptide, mainly Aβ42 (main component of the plaque).(7) Inheritance of E4 allele

136 | P a g e
of apolipoprotein E (APOE) is called sporadic AD (8, 9) Mutation in the TREM2 gene has
been associated with 3 to 5 times higher risk of development of AD.(10,11)

AD is characterized by β amyloid plaque formation or by neurofibrillary tangles in which

twisted protein threads are found inside the nerve cell. Amyloid β peptide, an extracellular
protein, is 40-42(amino acid) in length and is derived from β amyloid precursor protein
(APP). These are encoded by chromosome 21. Patients having trisomy have extra copy of
APP gene, leading to overproduction of Aβ and therefore they are more prone to AD. Aβ
protein also induces lipid peroxidation and generate free radical species. Brain is highly
vulnerable to ROS because of it high oxygen consumption. (19, 20) Neurofibrillary tangles
are mainly composed of a protein called tau. (12, 13) Tau protein has a certain number of
phosphate attached to it which binds to the microtubules and appears to stabilize the neuron.
(14) In AD, hyperphosphorylation of tau occurs and as a result the tau proteins detach from
microtubules and form a structure called paired helical filament. They appear as tangles
within the cell and damage the ability of neuron to communicate with each other. (15, 16) AD
can also damage neuron to the point they cannot function properly and die. (17, 18) Low
activation of one of the three neurotransmitters, viz, N – methyl D-aspartate glutamate
receptor, is also observed in AD. The low activation levels of this receptor triggers
continuous influx of calcium in the neuron causing an increase in APP and Aβ production
and phosphorylation of tau protein.(21, 22, 23, 24) The overall result of this is an increase in
the production of amyloid β and decrease in cholesterol synthesis in brain.

Active Constituents of Withania somnifera:

Withanolide A, Withanoside IV and Withanoside VI are active constituents of Withania

somnifera. All three compounds induce axonal growth even in the presence of amyloid β 25-
35 (active partial fragment of Aβ) in axonal atrophy model. (25, 26, 27) It has been found
that consecutive administration of three compound increases the densities of axon, synapsis
in parietal cortex and improve memory. Withanoside IV is glycosylated at C-3 position. After
oral administration of withanoside IV, deglycolsylation takes place by the intestinal bacteria,
which is then absorbed in blood. (28, 29) Withanoside IV is metabolized to active sominone,
which induces axonal regeneration, recovery of synapses and memory in Aβ 25-35 treated
mice. Single intraperitoneal administration of sominone increased axonal density in brain and
enhance memory. (30) Sominone activates rearranged during transfection (RET, proto-
oncogene), (part of receptor complex for glial cell line derived from neurotrophic factor) by
phosphorylation which induces axonal growth leading to memory enhancement.

Withanolide A (C28H38O6), is a steroidal lactone that can induce nerve development and
improve the function of nervous system. Axons are extended by Withanolide A and dendrites
are extended by Withanosides IV and VI. (31) Effect of Withanolide A on Alzheimer’s
disease in experimental model showed that Withanolide A decreases the level of β secretase
(enzyme that cleave the APP) and increases the expression of disintergin and
metalloproteinase domain – containing protein 10 (ADAM10 known as α secretase)(32).
Amyloid β is produced by APP in the presence of β secretase and γ secretase. APP in the
presence of α secretase form APPα which is non-toxic to the brain. Increase in the APPα
results in increase in insulin degrading enzyme (TDE), major enzyme that involve in the Aβ
degradation. Withanolide A treatment also increases the expression of PPARγ (peroxisome
proliferating activated receptor γ) directly correlates with neuroprotective effect. Withanolide
A causes up-regulation of liver LRP (Low density lipoprotein receptor – related protein 1)
137 | P a g e
and sLRP1 in plasma result in increase of amyloid β, then protease neprilysin causes the
degradation of amyloid β in plasma. Experimental study has shown that free Aβ re-enter in
brain by RAGE (Receptor of advanced glycation; product; blood brain barrier), leading to the
formation of neurotoxic oligomer.

LRPIV, recombinant wild type culture bind multiligand. (33) To enhance the LRPIV binding,
generate a library of LRPIV fragment. In this LPRIV in which Gly is replaced by Asp at
calcium binding sites. Therefore this mutant is known as LRPIV 36754G, this have 1.6 and
2.7 binding for Aβ40 and Aβ42. In vitro LRPIV contain 11 complement type repeat (CRs)
(CR21-CR31), they bind LRP1 ligand such as factor IX a receptor associated protein (RAP),
activated α2 macroglobulin. (34, 35) Each CR has 40 amino acid and single calciumbinding
domain for proper folding and structural integrity. LRPIV –D3674G mutant has highest in
vitro binding affinity for Aβ these help in clearance of Aβ from brain, thus improves the
blood flow response. Treatment with LRPIV-D3674G in AD mice result in increase in
cerebral blood fluid (CBF), lead to reduction of Aβ level in brain. CR24-CR28 repeat of
LRPIV most efficiently bind RAP, α2 macroglobulin, factor VIII light chain and factor IXa.
(36) CR24-CR26 involved in high affinity α2M, factor VIII light chain.(37) By the site
directed mutagenesis it is confirmed that CR26 has higher affinity for Aβ 42 than Aβ40.
Mutation in CR22 and CR26 reduced affinity for both Aβ 42 than Aβ40. Therefore LRPIV-
D3674G could be developed as potential therapeutic agent as monotheraphy.

Aqueous extract of Withania somnifera could inhibit acetylcholinesterase (AChE) activity in

Scopolamine induced Alzheimer’s rat. In case of AD there is increase in AChE and decrease
in acetylcholine. Acetylcholine is hydrolyzed into acetate and choline at central pheripheray
cholinergic synapse. AchE play a major role in acetycholine mediated neurotransmission
(36). In AD, AChE activity increases within and around amyloid plaque and promote
assembly of β peptide into fibrils leading to cytotoxicity. Aβ enhances acetylcholinesterase
activity by increasing calcium entry through L-type voltage dependent calcium channals. (37)
Withania somnifera reverse actylcholinesterase activity to modulate cholinergic function
clearing of Aβ by protease.

CONCLUSION

Withania somnifera and its constituents showed various activities against Alzheimer's
disease. Withanolide A is neuroprotective against Aβ induced cytotoxicity. Withania
Somnifera causes increase in the LRP in liver, a recombinant varient of LRP is LRPIV-
D3674G which could be developed as potential therapeutic agent as monotheraphy. Withania
somnifera extract and related compounds are novel for the treatment of Alzheimer's disease;
for instance, it enhances the memory, maintain the connectivity between the neurons, clear
Aβ in the brain. However, direct target molecule from Withania somnifera has not been
identified yet. Future treatment strategies focus on multiple mechanism to prevent the
Alzheimer's disease.

REFERENCES

1) Elise Caccappolo-Van Vliet, et al. The neuropsychological profiles of mild Alzheimer’s disease
and questionable dementia as compared to age-related cognitive decline, International Journal
of Neuropsychological Society, 2003, 75(9), 720-732.

138 | P a g e
2) Harkany T, Abraham I, Timmerman W, et al. Β amyloid neurotoxicity is mediated by a
glutamate-triggered excitotoxic cascade in rat nucleus basalis. European Journal of
Neuroscience, 2000, 12, 2735-2745.
3) Creeley CE, Wozniak DF, Nardi A, et al. Donepezil markedly potentiates memantine
neurotoxicity in the adult rat brain. Neurobiology of Aging, 2008, 29, 153-167.
4) Kulkarni SK, Dhir A Withania somnifera: An Indian ginseng. Progress in
Neuropsychopharmacology and Biological Psychiatry 2008, 32,1093–1105
5) Mishra LC, Singh BB, Dagenais S. Scientific basis for the therapeutic use of Withania
somnifera (ashwagandha): a review. Altern. Med. Rev., 2000, 5, 334–346
6) Waring SC, Rosenberg RN. Genome-wide association studies in Alzheimer disease. Archives
of Neurology, 2008, 65(3), 329–34.
7) Strittmatter WJ et.al. Apolipoprotein E: high-avidity binding to beta-amyloid and increased
frequency of type 4 allele in late-onset familial Alzheimer disease. Proceedings of the National
Academy of Sciences of the United States of America. 1993, 90(5), 1977–81.
8) Mahley RW, Weisgraber KH, Huang Y. Apolipoprotein E4: a causative factor and therapeutic
target in neuropathology, including Alzheimer's disease. Proceedings of the National Academy
of Sciences of the United States of America. 2006, 103(15), 5644–51.
9) Jonsson T, Stefansson H, Steinberg S et.al. Variant of TREM2 associated with the risk of
Alzheimer's disease. The New England Journal of Medicine. 2012, 368(2),107–16.
10) Guerreiro R, Wojtas A, Bras J et.al. TREM2 variants in Alzheimer's disease. The New England
Journal of Medicine. 2012, 368(2),117–27.
11) Alzheimer’s Disease Education and Referral Center Website. Alzheimer’s disease – unravelling
the mystery. Accessed 2005. Available at: http://www.alzheimers.org/unraveling/01.htm.
12) McPhee S, and Hammer G. "Alzheimer's disease" in Pathophysiology of disease an
introduction to clinical medicine. Lange, 6th edition, 2010, 172-174.
13) Iqbal K. Tau Pathology in Alzheimer disease and other tauopathies. Biochimca et Biophysica
Acta, 2005, 1739(2-3), 198-210.
14) Mudher A, Lovestone S. Alzheimer's disease-do tauists and baptists finally shake hands?
Trends in Neurosciences, 2002, 25(1), 22-26.
15) Goedert M, Spillantini MG, Crowther RA. Tau proteins and neurofibrillary degeneration. brain
pathology, 1991, 1(4), 279-8
16) Selkoe DJ. Alzheimer’s disease is a synaptic failure. Science. 2002, 298, 789-791.
17) Chun W, Johnson GV. The role of Tau phosphorylation and cleavage in neuronal cell Death.
Frontiers in Bioscience, 2007, 12, 733-56.
18) Butterfield DA, Griffin S, Munch G, Pasinetti GM. Amyloid beta-peptide and amyloid
pathology are central to the oxidative stress and inflammatory cascades under which
Alzheimer’s disease brain exists. Journal of Alzheimer’s disease, 2002, 4, 193-201.
19) Zhu X, Raina AK, Smith MA. Cell cycle events in neurons. Proliferation or death? American
Journal of Pathology, 1999, 155, 327-32
20) Parsons CG, Danysz W, Quack G. Glutamate in CNS disorders as a target for drug
development: an update. Drug News and Perspectives, 1998, 11, 523-569.
21) Abramov AY, Canevari L, Duchen MR. Calcium signals induced by amyloid beta peptide and
their consequences in neurons and astrocytes in culture. Biochimica et Biophysica Acta, 2004,
1742, 81-87.
22) Gordon-Krajcer W, Gajkowska B. Excitotoxicity-induced expression of amyloid precursor
protein (beta-APP) in the hippocampus and cortex of rat brain: an electron microscopy and
biochemical study. Folia Neuropathologica, 2001, 39, 163-173.
23) Harkany T, Abraham I, Timmerman W, et al. Β amyloid neurotoxicity is mediated by a
glutamate-triggered excitotoxic cascade in rat nucleus basalis. European Journal of
Neuroscience, 2000, 12, 2735-2745.
24) Asma Shaheda Shaik, A. Elaya Raja, M. Vijyalakshmi and G. Devalarao. Alzheimer’s disease
– pathophysiology and treatment. International Journal of Pharma and Bio Sciences, 2010, 1(2).

139 | P a g e
25) Alzheimer's Association. "While scientists know Alzheimer's disease involves progressive
brain cell failure, the reason cells fail isn't clear." 2011.
26) Hardy J, Allsop D. Amyloid deposition as the central event in the aetiology of Alzheimer's
disease. Trends in Pharmacological Sciences, 1991, 12(10), 383-88.
27) Akao T, Hayashi T, Kobashi K, Kanaoka M, Kato H, Kobayashi M, Takeda S, Oyama T.
Intestinal bacterial hydrolysis is indispensable to absorption of 18 beta-glycyrrhetic acid after
oral administration of glycyrrhizin in rats. J. Pharm. Pharmacol., 1994, 46, 135–137
28) Tawab MA, Bahr U, Karas M, Wurglics M, Schubert-Zsilavecz M. Degradation of
ginsenosides in humans after oral administration. Drug Metab. Dispos, 2003, 31, 1065–1071.
29) Tohda C, Joyashiki E. Sominone enhances neurite outgrowth and spatial memory mediated by
the neurotrophic factor receptor, RET. Br. J. Pharmacol., 2009, 157, 1427–1440.
30) Williams & Wikins LIPPINCOTT, 2002.
31) Iqbal K. Tau pathology in Alzheimer disease and other tauopathies. Biochimca et Biophysica
Acta, 2005, 1739(2-3), 198-210.
32) Abhay P. Sagare.et.al. A lipoprotein receptor cluster IV mutant preferentially binds amyloid-_
and regulates its clearance from the mouse brain. J Biol Chem. 2013, 288, 21- 24.
34) Meijer AB., Rohlena J, et al. Functional duplication of ligand-binding domains within low-
density lipoprotein receptor-related protein for interaction with receptor associated protein, 2-
macroglobulin, factor IXa and factor VIII. Biochim. Biophys. Acta, 2007, 1774, 714–722.
33) Obermoeller, L. M., Warshawsky, I., Wardell, M. R., and Bu, G. Differential functions of
triplicated repeats suggest two independent roles for the receptor-associated protein as a
molecular chaperone. J. Biol. Chem. 1997, 272, 10761–10768.
34) Benilova, I., Karran, E., and De Strooper, B. The toxic A_ oligomer and Alzheimer’s disease.
An emperor in need of clothes. Nat. Neurosci. 2012, 15, 349–357.
35) Meijer, A. B., Rohlena, J., et.al Functional duplication of ligand-binding domains within low-
density lipoprotein receptor-related protein for interaction with receptor associated protein, 2-
macroglobulin, factor IXa and factor VIII. Biochim. Biophys. Acta, 2007, 1774, 714–722.
36) Brookmeyer R, Gray S, Kawas C. Projections of Alzheimer's disease in the united states and
the public health impact of delaying disease onset. American Journal of Public Health, 1998,
88(9), 1337-42.
37) Melo JB, Agostinho P, Oliveira Involvement of oxidative stress in the enhancement of
acetylcholinesterase activity induced by amyloid beta-peptide. Neuroscience Research CR,
2003, 45: 117–127.

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 Withanolides: The Steroidal Lactones Biosynthesis
Yashdeep Srivastava and Neelam S. Sangwan

Email: yashiids@gmail.com

Total twenty-three Withania species are widely distributed, among them only two species, W. somnifera
and W. coagulans, are economically and medicinally important (Kulkarni et.al 1996). Withania somnifera,
also known as Ashwagandha, is an erect, evergreen, perennial shrub mainly cultivated in India and North
America, whose roots have been used for thousands of years by Ayurvedic practitioners for over 3,000
years (RajaSankar et.al 2009). In Ayurveda, Withania is widely claimed to have potent aphrodisiac,
sedative, anti-inflammatory, anti-tumor, anti-stress, antioxidant, mind-boosting, immune-enhancing and
rejuvenative properties. It is also used as a general energy-enhancing tonic known as “Medharasayana”
means ‘which promotes learning and memory’ and in geriatric problems (Sangwan et.al 2008, Rastogi et.al
1998).

Laboratory analysis has revealed over 35 chemical constituents contained in the roots of Withania
somnifera (Lal et.al 2006). The biologically active chemical constituents are alkaloids (isopellertierine,
anferine), steroidal lactones (withanolides, withaferins), saponins containing an additional acyl group
(sitoindoside VII and VIII), and withanoloides with a glucose at carbon 27 (sitonidoside XI and X).
Withania somnifera plant also possess iron element (Singh et.al 2010). The roots of Withania somnifera
consist primarily of compounds known as withanolides, which are believed to account for its extraordinary
medicinal properties. Withanolides are steroidal and bear a resemblance, both in their action and
appearance, to the active constituents of Asian ginseng (Panax ginseng) known as ginsenosides. Very less
information is available on structural and functional aspects of enzymes involved in withanolides
biosynthetic pathways of Withania somnifera.

Studies suggest that precursor molecules for withanolide biosynthesis are isoprenoids. These isoprenoids
are synthesized via classical cytosolic mevalonate (MVA) pathway and plastid localized 2-C methyl- D-
erythritol-4-phosphate (MEP) pathway, which leading to biosynthesis of 24-methylene cholesterol (C30
terpenoid) (Sangwan et.al 2008, Chaurasia et.al 2012). This central molecule through various biochemical
reactions including hydroxylation and glycosylation leads to the production of various withanolides (Gupta
et.al 2013).

Researchers still trying to explore withanolide biosynthesis pathway and try to characterize the enzymes
involved in this pathway. Based on the leaf and root transcriptomic data available in public database, it
could be possible to identify pathway related gene/enzymes, much more easily efficiently. The CYP 450 and
glucosyltransferases involved in the final steps of withanolide biosynthesis are still to be characterized.
The putative biosynthetic pathway of withanolide biosynthesis is summarized here.

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In Cytosol In Plastid

Acetyl Co-A Glyceralde-3-phosphate +

Acetoacetyl Co-A transferase 1-deoxy-D-xylulose-5-phosphate synthase
Pyruvate

Acetoacetyl Co-A 1-Deoxy-D-xylulose-5-phosphate (DOXP) +

HMG Co-A –synthase
NADPH DOXP reductoisomerase

HMG Co-A –reductase CDP - ME Synthase

HMG Co-A 2C-Methyl-D-erythritol-4-phosphate
(MEP) Mevalonate kinase CDP - ME Kinase

Mevalonic Acid
Phospho mevalonate kinase 4-Diphosphocytidyl -2C-methyl-D-erythritol (CDP-
Methylerythritol cyclodiphosphate (MEcPP) synthase
ME)
Mevalonate-5-pyrophosphate Decarboxylase
Hydroxymethylbutenyl 4-diphosphate (HMBPP) synthase
Mevalonate-5-Phosphate CDP-ME
IPP Isomerase
phosphate HMBPP reductase

Geranyl Diphosphate Synthase

Mevalonate -5-pyro phosphate Methylerythritol cyclodiphosphate
(MEcPP)
Farnesyl Diphosphate Synthase

Isopentenyl -5-pyrophosphate (IPP) Hydroxymethylbutenyl 4-diphosphate

Squalene Synthase
(HMBPP) IPP Isomerase
Cycloartenol Synthase
Dimethyl allyl pyrpphosphate (DMAPP) Isopentenyl -5-pyrophosphate
(IPP) Cycloartenol C-24 methyltransferase

GeranylSterol-4a-methyl
pyrophosphate oxidase 1

Farnesyl pyrophosphate
Cycloeucalenol + NADPH
cycloisomerase

Squalene
obtusifoliol 14-demethylase

D14-sterol reductase
Cycloartenol

24-Methylene Cycloartenol
C-7,8 sterol isomerase

Sterol-4a-methyl oxidase 2
Cycloeucalenol

C-5 sterol desaturase

Obtusifoliol

Delta- 8, 14-Sterol
Sterol D7 reductase

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 4 Alpha-methyl fecosterol

24-Methylenelophenol WITHANOLIDES

Episterol Withaferin-A

5-Dehydroepisterol Sitoindoside VII

24-Methylenecholestrol Withaferin E

Figure: The Putative pathway for withanolide biosynthesis in Withania somnifera

References

1. Kulkarni AA, Thengane SR, Krishnamurthy KV (1996). Direct in vitro regeneration of leaf
explants of Withania somnifera (L.) Dunal. Plant Sci (Limerick) 119:163–168.
2. RajaSankar S, Manivasagam T, Surendran S (2009). Ashwagandha leaf extract: A potential
agent in treating oxidative damage and physiological abnormalities seen in a mouse model of
Parkinson’s disease. Neurosci Lett. 454:11–15.
3. Sangwan RS, Chaurasiya ND, Lal P, Misra L, Tuli R, Sangwan NS (2008). Withanolide A is
inherently de novo biosynthesized in roots of the medicinal plant Ashwagandha (Withania
somnifera). Physiol Plant. 133(2):278-87.
4. Lal P, Misra L, Sangwan RS, Tuli R (2006). New withanolides from fresh berries of Withania
somnifera. Z Naturforsch 61b:1143–1147.
5. Rastogi RP, Mehrotra BN (1998). Compendium of Indian Medicinal Plants, Vol. 6. Central Drug
Research Institute, New Delhi.
6. Singh G, Sharma PK, Dudhe R, Singh S (2010). Biological activities of Withania somnifera.
Annals of Biological Research 1(3): 56-63.
7. Chaurasia ND, Sangwan NS, Sabir F, Misra L, Sangwan RS (2012) Withanolide biosynthesis
recruits both mevalonate and DOXP pathways of isoprenogenesis in Ashwagandha Withania
somnifera L. (Dunal). Plant Cell Rep 31: 1889–1897.
8. Gupta P, Agarwal AV, Akhtar N, Sangwan RS, Singh SP, et al. (2013) Cloning and
characterization of 2-C-methyl-D-erythritol-4-phosphate pathway genes for isoprenoid
biosynthesis from Indian Ginseng, Withania somnifera. Protoplasma 250: 285–295.

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 PLANT

SYSTEMATICS,

CONSERVATION

AND PROSPECTION
 A tale of three medicinal plants - Catharanthus roseus L., Artemesia annua
L. and Withania somnifera (L.) Dunal

Amit Kumar*, Priyanka Mishraa, Ashutosh K. Shukla b, Velusamy Sundaresan a

a
Department of Plant Biology and Systematics, CSIR - Central Institute of Medicinal and
Aromatic Plants, Research Centre, Bangalore 560065, India
b
Biotechnology Division, CSIR - Central Institute of Medicinal and Aromatic Plants, P.O.
CIMAP, Lucknow 226015, India
Email: amitsingh.twins@gmail.com

ABSTRACT

The use of plant-derived products in disease management is an important breakthrough in the

history of humankind. The plants are used to treat various ailments since an early era by the
different communities and are mentioned in various systems of medicine such as Ayurvedic,
Chinese, Unani, Sidhha, etc. In the present review, the medicinal uses of Catharanthus
roseus, Artemesia annua and Withania somnifera are discussed due to their importance in
various systems of medicine. These plants are used to treat various ailments by the different
indigenous communities from various parts of the world.

INTRODUCTION

Man is known to have utilized plants as source of drugs for millennia. A large number of
plants are used in different systems of medicinal practices such as Chinese, Indian, Unani,
Siddha, etc., for the treatment of various ailments. The widespread use of herbal remedies and
healthcare preparations, as those described in ancient texts such as the Vedas and the Bible,
and obtained from commonly used traditional herbs and medicinal plants, has been traced to
the occurrence of natural products with medicinal properties. The practice of traditional
medicine is widespread in China, India, Japan, Pakistan, Sri Lanka and Thailand. In China,
about 40% of the total medicinal consumption is attributed to traditional tribal medicines.
According to the World Health Organization (WHO), approximately 80% of the people in
developing countries (Farnsworth NR and Soejarto DD, 1985) and about 40% of the world
populations rely chiefly on traditional medicines for their primary health care needs (WHO,
2003). Furthermore, an increasing reliance on the use of medicinal plants in the industrialized
societies has been traced to the extraction and development of several drugs and
chemotherapeutics from these plants as well as from traditionally used rural herbal remedies
(UNESCO, 1998). Recent estimates suggests the over 9,000 plants have known medicinal
applications in various cultures and countries, and this is without having conducted
comprehensive research amongst several indigenous and other communities (Farnsworth NR
and Soejarto DD, 1991).

Medicinal Plants
Catharanthus roseus L.
Catharanthus roseus L. belongs to the family Apocyanaceae is the native to Indian Ocean
Island of Madagascar. There are about two common cultivars of C. roseus which are named
on the basis of their colour of the flower that is pink and white. The pink one is “rosea” and
the white flower is “alba”. Ethno-botanically, this plant is used to treat various ailments such
as leaves are useful in treating oliguria, haematuria, diabetic mellitus and menstrual disorders.
The roots and the leaves in the form of decoction or extract are active on hypertension. It is
also used in cancer, diabeties, swell body (Jain 1991). The extracts from the dried or wet
144 | P a g e
flowers and leaves of the plants are applied as a paste on wounds by some rural communities.
The fresh juice from the flowers made into a tea has been used by Ayurvedic physicians in
India for external use to treat skin problems, dermatitis, eczema and acne.

This plant has more than 130 known alkaloids, some of which are approved as antineoplastic
agents to treat leukemia, Hodgkin's disease, malignant lymphomas, neuroblastoma,
rhabdomyosarcoma, Wilms’ tumor, and other cancers. The alkaloids like vinblastine and
vincristine are mainly present in aerial parts of C. roseus, which are used in the treatment of
human cancers, so it is considered as magic plant for cancer chemotherapy (Shukla and
Khanuja, 2013). Pharmocolgically, the extracts of C. roseus have demonstrated significant
anticancer activity against numerous cell types (El-Sayed and Cordell 1981). This plant has
also seen in the treatment of wound healing activities (Nayak and Pereira, 2006).

Artemesia annua L.
Artemesia annua L., an annual herb belongs to the family Asteraceae having fern-like leaves,
bright yellow flowers and a camphor-like scent native to China, commonly found in the
northern parts of the Chahar and Suiyan provinces as part of the natural vegetation. However,
the plant now grows in several countries, including Argentina, Australia, Bulgaria, France,
Hungary, Italy, Spain, and the United States (Dhingra et al. 2000). Glandular structures
(trichomes) producing a wide range of bioactive compounds (mostly terpenoids) can be found
on the surface of leaves, stems and flowers. The herb A. annua has been used medicinally to
treat fevers for more than 2,000 years and to treat malaria for more than 1,000 years in China.
The current edition of the pharmacopoeia of China documents the therapeutic use of A. annua
for treating fevers and malaria. The herb is prepared with hot water according to traditional
Chinese medicine. In 1971, scientists demonstrated that the plant extracts had antimalarial
properties in primate models (van Agtmael, 1999). In 2010 it was discovered that A. annua
has already been cited in the earliest Chinese medical prescriptions, the Mawagndui tomb
texts dating back to 168 B.C. There, it is prescribed for female haemorrhoids and as a sexual
tonic, being mixed with other herbs, including cinnamon and ginger, and administered in
boiled urine (McGovern, 2010).

Biological activities reported for the compounds isolated from A. annua are antimalarial,
antibacterial, anti-inflammatory, angiotensin converting enzyme inhibitory, plant growth
regulatory, cytokinin-like and antitumour (Bhakuni et al. 2001).

Withania somnifera (L.) Dunal

Withania somnifera (L.) Dunal, a xerophytic shrub belongs to the family Solanaceae found in
the drier parts of India, Sri Lanka, Afghanistan, Baluchistan and Sind. It grows wildly
throughout India particularly in hotter parts, on waste places and on road sides. It is also
cultivated for medicinal purposes in fields and open grounds throughout India. In Unani
system of medicine, roots of W. somnifera commonly known as Asgand are known for their
medicinal properties. However, leaves of the plant are also reported to be used medicinally
(Anonymous, 1982). The roots are alternative aphrodisiac, tonic, deobstruent, diuretic,
narcotic hypnotic, sedative, restorative. These are used in rheumatic, cough, and debility from
old age, dropsy, emaciation of children, consumption and general weakness. It is valued as a
potent tonic, delays ageing and physical and mental strength. The paste of the roots and
leaves are applied to carbuncles, ulcers and painful swellings. The tuberous roots are
astringent, bitter, somniferous, stimulant, aphrodisiac, diuretic and tonic. Roots are used in
asthma, bronchitis, cough, dropsy, dyspepsis, seeds are used in chest complaints and
inflorescence in boils, swelling on hands and feet (Jain, 1999). Asgand (W. somnifera) has

145 | P a g e
also been recommended for the treatment of polyarthritis, rheumatoid arthritis, lumbago,
painful swellings, spermatorrhoea, asthma, leucoderma, general debility, sexual debility,
amnesia, anxiety neurosis, and leucorrhoea (Anonymous, 2007, Khare, 2007, Ghani, 1920).

Many biochemically heterogeneous alkaloids have been reported in the roots of W.

somnifera. Basic alkaloids include cuscohygrine, anahygrine, tropine, pseudotropine,
anaferine, isopelletierine, withananine, withananinine, pseudo-withanine, somnine,
somniferine, somniferinine. Neutral alkaloids include 3-tropyltigloate and an unidentified
alkaloid. Other alkaloids include withanine, withasomnine, and visamine. Withaferin A, a
steroidal lactone is the most important withanolide isolated from the extract of the leaves and
dried roots of W. somnifera exhibits fairly potent anti-arthritic and anti-inflammatory
activities (Khare, 2007).

REFERENCES

Anonymous 1982. The Wealth of India. Vol. X (Sp-W), Publications and Information
Directorate, Council of Scientific and Industrial Research (CSIR), New Delhi, India: pp.
580-585.
Anonymous 2007. The Unani Pharmacopoeia of India. Part I, Vol. I, Department of AYUSH,
Ministry of Health & Family Welfare, Govt. of India, New Delhi, India: pp. 7-8.
Bhakuni RS, Jain DC, Kumar S. 2001. Secondary metabolites of Artemisia annua and their
biological activity. Current Science 80(1): 35-48.
Dhingra V, Vishweshwar RK, Lakshmi NM. 2000 . Current status of artemisinin and its
derivatives as antimalarial drugs. Life Sci 66: 279-300.
El-Sayed A, Cordell GA. 1981. Catharanthamine, a new antitumor bisindole alkaloid from
Catharanthus roseus. J Nat Prod 44(3): 289-293.
Farnsworth NR, Soejarto DD. 1985. Potential consequence of plant extinction in the United
States on the current and future availability of prescription drugs. Economic Botany 39:
231-240.
Farnsworth NR, Soejarto DD. 1991. Global Importance of Medicinal Plants. In: Akerle O,
Heywood V, Synge H (eds.) Conservation of Medicinal Plants. Cambridge University
Press, Cambridge.
Ghani N. Khazainul Adviyah.1920. Vol. I, Munshi Nawal Kishore, Lucknow, India: pp. 230-231.
Jain 1991. Dictionary of Indian folk medicine and ethnobotany. Deep publication, India.
Khare CP. 2007. Indian Medicinal Plants–An Illustrated Dictionary. First Indian Reprint,
Springer (India) Pvt. Ltd., New Delhi, India: pp. 717-718.
McGovern PE, Christofidou-Solomidou M, Wang W, Dukes F, Davidson T, El-Deiry WS. 2010.
Anticancer activity of botanical compounds in ancient fermented beverages (review). Int J
Oncol 37(1): 5-14.
Nayak BS, Pereira LMP. 2006. Catharanthus roseus flower extract has wound-healing activity in
Sprague Dawley rats. BMC Complementary and Alternative Medicine 6: 41.
Shukla AK, Khanuja SPS. 2013. Catharanthus roseus: The metabolome that represents a unique
reservoir of medicinally important alkaloids under precise genomic regulation. In:
Debmalya Barh (Ed.) OMICS Applications in Crop Science, CRC Press (Taylor & Francis
Group), Boca Raton pp 325–384. (doi: 10.1201/b16352-11)
UNESCO. 1998. FIT/504-RAF-48 Terminal Report: Promotion of Ethnobotany and the
Sustainable Use of Plant Resources in Africa, Paris: pp. 60.
Van Agtmael MA, Eggelte TA, van Boxtel CJ. 1999. Artemisinin drugs in the treatment of
malaria: from medicinal herb to registered medication. Trends Pharmacol Sci 20(5): 199-
205.
World Health Organization. 2003. Guidelines for the Assessment of Herbal Medicine Programme
on Traditional Medicine.Doc. WHO/TRM/91.4.WHO, Geneva.

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 Anti-mitotic Property of Natural Dimers from Catharanthus roseus

Vijaya Dubey, Kaneez Fatima

Molecular Bioprospection Department of Biotechnology,
CSIR-Central Institute of Medicinal and Aromatic Plants, Lucknow-226015
Email: dubeyvijaya01@gmail.com

ABSTRACT

Medicinal plants are being in use for centuries to treat a number of diseases and ailments.

Catharanthus roseus is one such plant, recognized for its medicinal importance due to the

presence of alkaloids possessing strong antimitotic property. This mini review spotlight the

antimitotic property of the two most important alkaloids of Sadabahar namely ‘Vincristine’

and ‘Vinblastine’ that created history in the field of chemotherapeutic agents of natural

origin.

INTRODUCTION

Catharanthus roseus, Madagascar periwinkle ‘Sadaphuli’, Vinca rosea or Rosy periwinkle is

a tropical and subtropical plant belonging to the family Apocynaceae (Van Bergen et al.,

1996). It is known for its medicinal properties for centuries and Ayurvedic system of

medicine also acknowledged its anti-diabetic, anti-bacterial, anti-oxidant, anti-ulcer and anti-

diarrheal properties (Verpoorte et al., 2002, Nagle et al., 2006, Jayomthi et al., 2009,

Kyakulaga et al., 2011, Prajakta et al., 2010, Bhutkar et al., 2011). It is also good for patients

of asthma, tuberculosis and rheumatism (Sain et al., 2013). The plant extract and its

phytoconstituents both are essentially valuable for medication. Females find relieve from pain

in menstruation on using the plant extract. The leaves and seeds extract of the plant also

possesses antibacterial activities. The flower petals and seeds extract possess antioxidant

activity (Jayomthi et al., 2009, Kyakulaga et al., 2011, Prajakta et al., 2010, Bhutkar et al.,

2011). It was observed that the patient’s blood glucose level was lowered on consuming

extracts from this plant. The whole plant including roots, leaves, flowers and stalks is used

147 | P a g e
for the extraction of a number of alkaloids. Because of its vasodilating and blood thinning

properties, vinca alkaloids are good for increasing the oxygen, blood and glucose supply to

the brain and increase the level of serotonin neurotransmitter in brain. The clinical success of

vinblastine and vincristine came to the drug researchers at Eli Lilly in 1960s, interested in

finding hypoglycemic activity; by chance found its anticancer activity which happens to be a

milestone discovery in the field of clinical oncology (Jordan et al. 1991). Subsequently,

elucidation of their mechanism of action on cellular microtubules, have facilitated the

development of several semi-synthetic derivatives notably vindesine, vinorelbine and

vinflunine, which are now being used in the clinic for the treatment of leukemias,

lymphomas, and some solid malignancies. The plant is nowadays categorized in the

endangered plant species; still an important model for research in West and thus its

conservation is a matter of great concern.

Structure of Vinca Alkaloids

Vincristine and Vinblastine are bis-indole alkaloids, and natural dimer formed by the fusion

of two monomeric units Catharanthine and Vindoline. The difference in the structure of these

two bis-indole alkaloids is due to the presence of different functional moieties on the indole

nitrogen of the vindoline skeleton. Vinblastine has a methyl group while Vincristine has the

formyl group at this position. The chemists and biologists are very much interested in solving

the structure and biological activity of this natural dimer due to its marvelous biological

impact on cancer cell and its involvement in combination chemotherapy. Vinblastine can be

derived from vinblastine through chemical synthesis. Based on this structural core of this

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dimer new modified structures have been synthesized and tested for their medicinal

properties and safety profile (Tiong et al., 2013).

Isolation and Synthesis of Vinca Alkaloids

The vinca alkaloids, vinblastine and vincristine, were initially isolated from the leaves of the

Madagascar periwinkle. The amount of vinca alkaloids present in the leaves of periwinkle is

very less. Also they are very complex structural moieties to be synthesized in laboratory. So,

being present naturally in very minute quantities, researchers are utilizing cell tissue culture

studies to increase its yield by incorporating newer biosynthetic pathways (Tiong et al.,

2013).

Why Microtubules are Targeted in Cancer?

Microtubules are bestowed with some fundamental properties in a normal living cell like

maintaining the cell shape, cell motility, cell signaling cascades, intracellular transport of

biomolecules and muscular contractions. They govern the cellular dynamics and cell to cell

networking. They are solely responsible for separating the DNA in mitosis phase of cell

cycle, resulting into two daughter cells. So, because of this unique role they have been the

centre of attraction of chemotherapeutic research and became an important target. Vinca

alkaloids are the first anticancer agents of natural origin that affect the tubulin polymerization

followed by the taxanes in 1979. These compounds disturb the dynamic property of

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microtubule results in tubulin depolymerization, growth arrest in M phase of the cell cycle

and thus inhibit cell proliferation. These ‘antimitotics’ and their derivatives have transformed

cancer treatment and inspired the discovery of a number of other classes of tubulin-binding

compounds (Zhou J. and Giannakakou P, 2005).

How Do the Antimitotic Agents Work?

Vinca alkaloids interact with β-tubulin at a region adjacent to the GTP-binding site known as

vinca domain and causes conformational change (Rai and Wolff, 1996). Thus, formation of

spindle microtubules is prevented and cell is no longer able to mechanistically separate the

chromosomes (Rai and Wolff, 1996; Jordan and Wilson, 2004). Dynamic disturbance in

microtubules leads to the death of cancer cell. These agents inhibit the DNA repair and the

RNA synthesis processes. Binding is reversible, temperature independent and rapid. The

decay is first order with a half time of 3·5 h and is unusual in that it affects the affinity as

well as the number of sites available. At a lower concentration vinca alkaloids inhibit

microtubule dynamics only and at higher concentrations, they induce microtubule

depolymerization. In both situations, mitotic spindle formation is prevented, and cells can’t

complete a normal mitosis (Jordan et al., 1991).

Antimitosis Activity of Vinca Alkaloid and Its Impact in Clinical Oncology

Since 1960s, vinblastine has been used successfully in combination with other chemotherapy

drugs against lymphomas as well as testicular, ovarian, breast, bladder and lung cancers.

Vincristine is routinely used in combination regimes against childhood acute lymphoblastic

leukemia and non-Hodgkin lymphomas.

Negative Side of Antimitotic Drugs

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Neurological and hematological toxicities of these drugs are dangerous issue. Drug resistance
due to p glycoprotein is another issue which is undesirable while using these MTAs in cancer
chemotherapy. Although some researchers claim that the presence of some synergetic or
additive constituents in this plant can overcome the resistance towards these MTAs.

Other Molecules in the List

Vinorelbine, vinflunine that destabilize or depolymerize microtubules, and the taxanes

(paclitaxel, docetaxel) and epothilones (ixabepilone) that polymerize microtubules, thus
causing mitotic arrest.

Global Economic Profit

Global drug market has earned enormous profit from these antimitotic based drug products.
The sale of these agents around the globe is ~75 million dollar in a year. But sadly this huge
profit is not delivered to Madagascar directly or indirectly, the chief producer of this plant.
Pharmaceutical companies are now trying to find the ways in which the companies as well as
the local producers of this drug can equally share the benefit from this.

CONCLUSION: DIMER VS DIMER

This review highlights the antimitotic property of two natural dimers present in Catharanthus
plant emphasizing the mechanism through which they affect the microtubules, an interesting
polymer formed from heterodimeric tubulin.

REFERENCES

Alba Bhutkar M.A., Bhise S.B. Comparative study on antioxidant properties of Catharanthus
roseus and Catharanthus alba. Int. J. Pharmaceutical Tech., 3(3): 1551-1556 (2011).
Jayomthi M., Sow Balal N., Raja Lakshmi G., Siva Kumar V. Study of anti hyperglycemic
effect of Catharanthus roseus. In alloxan induced diabetic rats, Int. J. Pharmacy and
Phar. Sci., 4: 19-25 (2009).
Jordan M. A. and Wilson L. Microtubules as a target for anticancer drugs. Nat.Rev. Cancer,
4: 253-265 (2004).
Jordan M. A., Thrower D. and Wilson L. Mechanism of inhibition of cell proliferation by
Vinca alkaloids. Cancer Res., 51: 2212-2222 (1991).
Kyakulaga A., Hassan Alinda T., In vivo antidirraheal activity of the ethanolic leaf extract of
Catharanthus roseus L (Apocynaceae) in wistar rats. African J. Pharmacy and
Pharmacology, 15: 1797-1800 (2011).
Lobert S. and Correia J. J. Energetics of vinca alkaloid interactions with tubulin. Meth.
Enzymol, 323: 77–103 (2000).
Noble R.L., The discovery of the vinca alkaloids chemotherapeutic agent against cancer.
Biochemistry and Cell Biology, 689: 1344-1351 (1990).

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Prajakta J., Patil Jai S., Ghosh. Antimicrobial activity of Catharanthus roseus- A detailed
study. British J. Pharmacology and Toxicology, 1(1): 40-44 (2010).
Rai S. S. and Wolff J. Localization of the vinblastine-binding site on beta-tubulin. J. Biol.
Chem., 271: 14707-14711 (1996).
Sain M., Sharma V. Catharanthus roseus (An anti-cancerous drug yielding plant) - A Review
of Potential Therapeutic Properties. Int. J. Pure App. Biosci., 1 (6): 139-142 (2013).
Tiong S.H., Looi C.H., Hazni H. Antidiabetic and Antioxidant Properties of Alkaloids from
Catharanthus roseus (L.) G. Don Molecules, 18: 9770-9784 (2013).
Van Bergen M.A., Snoeijer W. Revision of Catharanthus G. Don. Series of Revisions of
Apocyanceae XLI; Backhuys Publisers: Leiden, The Netherlands, 32-35 (1996).
Verpoorte R., Contin A., Memelink. Biotechnology for the production of plant secondary
metabolites. Phytochemical Review, 1: 13-25 (2002).
Zhou J. and Giannakakou P. Targeting Microtubules for Cancer Chemotherapy. Curr. Med.
Chem. - Anti-Cancer Agents, 5: 1-7 (2005)

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 Biological activities of artemisinin and its derivatives
Himanshu Tripathi, Aparna Shukla and Feroz Khan*
Metabolic & Structural Biology Department, CSIR-Central Institute of Medicinal and Aromatic
Plants, Lucknow-226015 (U.P.), India
Corresponding Author: f.khan@cimap.res.in

ABSTRACT

The annual herb Artemisia annua, originally used to cure fever and malaria in Chinese traditional
medicine, is the chief source of its active constituent artemisinin, a sesquiterpene lactone
containing a peroxide bridge. Besides as an antimalarial drug, artemisinin and its derivatives have
shown a range of diverse biological activities. The present review encompasses brief account of
these activities along with proposed mechanism of actions.

Keywords: anticancer, antifungal, anti-inflammatory, antischistosomal, anti-psoriasis, herbicidal.

INTRODUCTION

A. annua, commonly known as sweet wormwood, is an aromatic herb native to temperate Asia,
now naturalized across the globe due to its therapeutic uses. Several flavonoids, monoterpenoids,
sesquiterpenoids, triterpenoids, phenolics, coumarins, steroids, and aliphatic compounds have
been isolated from different parts of the plant, A. annua (Bhakuni et al., 2001). Among these
phytomolecules artemisinin is of particular importance because, though its chemical synthesis is
achieved but large scale production is not cost effective, A. annua is the sole source for its large
scale production (Zhu and Cook, 2012). Artemisinin itself has high in vitro antiplasmodial
activity comparable to chloroquine, but low bioavalibalty limits it effectiveness; thus
semisynthetic derivatives viz., artesunate, artemether, dihydroartemisnin etc. is largely used for
disease therapy. A brief account of biological activities of artemisinin and its derivatives are
given below.

Antipalsmodial activity
Initially, artemisinin derivatives were largely used for the treatment of uncomplicated as well as
severe malaria patients due to rapid parasitemia clearance (Klayman, 1985). In addition, these
drugs were equally effective against chloroquine- susceptible and resistant malaria. Mechanism
of action of these drugs is largely debated with manifestation of multi-faceted response: heme-
peroxide bridge mediated Reactive oxygen species (ROS)/Reactive carbon species (RCS)
generation; heme-artemisinin adducts formation; protein alkylation; iron dependent inhibition of
PfATP6; parasite membrane disruption; drug induced ROS generation from parasite
mitochondria (Meshnick, 2002; Wang et al., 2010; Fugi et al., 2010; O’Neill et al., 2010). To
cope with resistance and high rate of recrudescence, WHO recommended artemisinin
combination therapies (ACT) for the treatment of P. falciparum malaria as first-line therapy
worldwide (WHO, 2006). Since last few years early sign of artemisinin/derivatives resistance has
been seen in many sites in Burma, Combodia, Vietnam, and Thailand (Dondrop et al., 2009;
Noedl et., 2010; Fairhurst et al., 2012; Tulloch et al., 2013). Recently, artemisinin resistance
against P. falciparum has been confirmed in Cambodia and the border areas of Thailand (Na-
Bangchang and Karbwang, 2013). Quantitative structure activity relationship (QSAR) studies on
artemisinin/derivatives have been also reported for antiplamodial activities (Ji-an and Ru-yun,
1982; Cheng et al., 2002; Qidwai et al., 2012).

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Anticancer activity
Artemisinin and its derivatives have been tested on many human cancer cell lines with IC 50 in
range of 10-20 µM (Lai et al., 2012). Exact mode of action is still not clear, but its selectivity of
cancer cells is supposed due to presence of free iron in cancer cells, resulting heme-mdiated
response like in plasmodium. Reports suggest that artemisinin/derivatives cause inhibition of NF-
κB and decrease in VEGF, and also affect EGF, NOXA, COX, HIFα, survivin, Wnt/β-catenin, c-
MYC oncoprotein, MAPK, and TNFα (Firestone and Sundar, 2009; Slade et al., 2009; Lai et al.,
2012). Recently, QSAR studies have been done on artemisinin/derivatives for anticancer activity
(Vieira et al., 2014; Wang et al., 2014).

Antimicrobial activity
Artemisinin and its derivatives have shown potent antifungal activity against Cryptococcus
neoformans as by amphotericin but fail to inhibit Candida albicans in the same range (Galal et
al., 2005; Appalasamy et al., 2014). Artemisinin have slightly less antibacterial potential against
both, gram- negative and positive (~100 µg/ml) bacteria (Appalasamy et al., 2014). However,
artemisinin/derivatives selectively inhibit Helicobacter pylori but most of the derivatives (except
artemisinic acid) fail to inhibit most common bacterial and fungal pathogens (Goswami et al.,
2012). A classical report suggests that artemisinin and its deoxy derivatives have no activity
against aerobic, anaerobic, facultative anaerobic and microphilic bacteria; only methyl diperoxy
derivatives had activity against all tested anaerobes (highest MIC 9.4 µg/ml) (Shoeb et al., 1990).
These drugs have also displayed inhibitory effect against phytopathogenic fungi (Algamal et al.,
2013).

Anti-inflammatory activity
In many studies, artemisinin/derivatives have reflected their potentials to inhibit pro-
inflammatory cytokine productions, through acting on NF-κB, Rig-G/JAB1, and MAPK
signaling pathways (Wang et al., 2007, 2009, 2011; Zhu et al., 2012).

Antischistosomal activity
Artemisinin/derivatives have been reported to have significant antischistosomal activity, but in
combination with praziquantel, an antischistosomal drug, have enhanced effect than the drugs
used alone (Utzinger et al., 2003; Liu et al., 2011; Villar et al., 2012). Like praziquantel, they
have broad spectrum of activity but act against different parasite stages (Schistosomula), and
cause synergetic effect (Utzinger et al., 2003).

Anti-psoriasis activity
Anti-psoriasis activities of artemisinin/derivatives have been unveiled too. Since these drugs have
inhibitory effect on (Th)1/Th17, TNF-α, IL-33, IL-17, IL-8, IL-6, VEGF expression and
receptors, and inducing apoptosis, potent anti-psoriasis activities cannot be ruled out; further
studies needed in this perspective (Thornfeldt, 1991; Feily, 2014).

Herbicidal activity
Artemisinin and its derivatives are phytotoxic (e.g. arteether is phytotoxic at 1 ppm for other
plants and for itself 33 ppm), and displayed inhibition of seed germination and seedling growth
(Chen and Leather, 1990; Hoagland, 2000). Recently, in an in vitro and in vivo study mode of
action is reported at chloroplast (Bharati et al., 2012). Saad et al., 2013 have reported that
artemisinin/derivatives have herbicidal potential in three common weeds, namely Panicum repens
(complete seed germination inhibition at 100 µM by dihydroartemisnin and artemether; root
growth inhibition EC50 0.39 µM by artesunate), Phalaris minor (complete seed germination
inhibition at 50 µM and root growth inhibition EC50 1.2 µM by artemether), and Silybum
marianum (root growth inhibition EC50 10 µM by artesunate). Apart from this, artesunate can
completely suppress Agrobacterium tumefaciens-induced crown gall tumor and wound

154 | P a g e
callus cells in the model plant Ricinus communis at 10 µM concentrations (Ullrich et al.,
2009).

CONCLUSION

Although artemisinin and its derivatives are largely used medication for cerebral malaria due to
its rapid clearance of pathogen, but it has some implications that must be topic of further research
in order to combat pathogen like P. falciparum. First, there is no established idea about
mechanism of actions. Second, these class of drug have very short life span in circulatory system
and 80% may be excreted within 24 hours, a major reason for high recrudescence. Therefore, by
utilising vast amount of knowledge in public domain as well as trans-disciplinary approaches
novel artemisinin derivatives/anlogs can be semi-synthesized with retained potency and enhanced
drug half-life. Mechanism of actions may also be explored that will help in designing leads and it
may also help in understating resistance. Besides, other therapeutic potentials of
artemisinin/derivatives must be explored. Particularly, as an anticancer agent, because apart from
anticancer activity these drugs also have anti-inflammatory, antiangiogenesis, and
antiproliferatiion potential that make them unique and beneficial over traditional anticancer
drugs. In addition, encouraging antifungal, anti-inflammatory, antischistosomal, anti-psoriasis
activities of artemisinin/derivatives also offer ample opportunities to develop new drugs for the
treatment of such complexities. Since, endoperoxide bridge in the oxepane ring of artemisinin is
the essential component for activity, many more derivatives or molecules of other classes with
this can be studied for the potential therapeutic applications.

REFERENCES

Appalasamy S, Lo KY, Ch'ng SJ, Nornadia K, Othman AS, Chan LK, Antimicrobial activity of
artemisinin and precursor derived from in vitro plantlets of Artemisia annua L, Biomed Res
Int. 2014;2014:215872.
Bharati A, Kar M, Sabat SC, Artemisinin inhibits chloroplast electron transport activity: mode of
action, PLoS One. 2012;7(6):e38942.
Chen PK, Leather GR, Plant growth regulatory activities of artemisinin and its related
compounds, J Chem Ecol. 1990 Jun;16(6):1867-76.
Cheng F at. al., Molecular docking and 3-D-QSAR studies on the possible antimalarial
mechanism of artemisinin analogues,Bioorg Med Chem. 2002 Sep;10(9):2883-91.
Dondorp AM et al, Artemisinin resistance in Plasmodium falciparum malaria,.N Engl J Med.
2009 Jul 30;361(5):455-67.
Fairhurst RM at al. Artemisinin-resistant malaria: research challenges, opportunities, and public
health implications, Am J Trop Med Hyg. 2012 Aug;87 (2):231-41.
Feily A. One friend among foes; anti-malarial drug aretesunate as a novel addition to anti-
psoriasis weaponry. Eur Rev Med Pharmacol Sci. 2014 Sep;18(17):2401-2.
Firestone GL, Sundar SN.Anticancer activities of artemisinin and its bioactive derivatives, Expert
Rev Mol Med. 2009 Oct 30; 11:e32.
Fügi MA, Wittlin S, Dong Y, Vennerstrom JL, Probing the antimalarial mechanism of
artemisinin and OZ277 (arterolane) with nonperoxidic isosteres and nitroxyl radicals,
Antimicrob Agents Chemother. 2010 Mar;54 (3):1042-6.
Galal AM, Ross SA, Jacob M, ElSohly MA, Antifungal activity of artemisinin derivatives, J Nat
Prod. 2005 Aug; 68(8):1274-6.
Goswami S, Bhakuni RS, Chinniah A, Pal A, Kar SK, Das PK, Anti-Helicobacter pylori potential
of artemisinin and its derivatives, Antimicrob Agents Chemother. 2012 Sep;56 (9):4594-
607.
Guidelines for the Treatment of Malaria. Geneva: World Health Organization. 2006. ISBN 92-4-
154694-8

155 | P a g e
Hoagland RE. Bioherbicides: Phytotoxic Natural Products. Agrochemical Discovery Chapter 7,
pp 72–90. ISBN13: 9780841237247
Klayman DL. Qinghaosu (artemisinin): an antimalarial drug from China. Science. 1985 May
31;228 (4703):1049-55.
Lai HC, Singh NP, Sasaki T, Development of artemisinin compounds for cancer treatment, Invest
New Drugs. 2013 Feb;31 (1):230-46.
Liu R, Dong HF, Guo Y, Zhao QP, Jiang MS, Efficacy of praziquantel and artemisinin
derivatives for the treatment and prevention of human schistosomiasis: a systematic review
and meta-analysis, Parasit Vectors. 2011 Oct 17;4:201.
Meshnick SR. Artemisinin: mechanisms of action, resistance and toxicity, Int J Parasitol. 2002
Dec 4;32 (13):1655-60.
Na-Bangchang K, Karbwang J. Emerging artemisinin resistance in the border areas of Thailand.
Expert Rev Clin Pharmacol. 2013 May;6(3):307-22.
Noed H et al., Artemisinin resistance in Cambodia: a clinical trial designed to address an
emerging problem in Southeast Asia, Clin Infect Dis. 2010 Dec 1;51 (11):e82-9.
O'Neill PM, Barton VE, Ward SA, The molecular mechanism of action of artemisinin--the debate
continues, Molecules. 2010 Mar 12;15 (3):1705-21.
Pérez del Villar L, Burguillo FJ, López-Abán J, Muro A, Systematic review and meta-analysis of
artemisinin based therapies for the treatment and prevention of schistosomiasis, PLoS One.
2012;7(9):e45867.
Qidwai T, Yadav DK, Khan F, Dhawan S, Bhakuni RS. QSAR, docking and ADMET studies of
artemisinin derivatives for antimalarial activity targeting plasmepsin II, a hemoglobin-
degrading enzyme from P. falciparum, Curr Pharm Des. 2012;18 (37):6133-54.
Saad MMG, Algamal MA, Abdelgaleil SAM. Effect of Artemisinin and Its Derivatives on
Germination and Seedling Growth of Three Weed Species. Alex. J. Agric. Res. Vol. 58,
No.3, pp.287‐294, 2013.
Shoeb HA, Tawfik AF, Shibl AM, el-Feraly FS, Antimicrobial activity of artemisinin and its
derivatives against anaerobic bacteria, J Chemother. 1990 Dec;2 (6):362-7.
Slade D et al., Antiprotozoal, anticancer and antimicrobial activities of dihydroartemisinin acetal
dimers and monomers, Bioorg Med Chem. 2009 Dec 1;17 (23):7949-57.
Thornfeldt CR. Treatment of skin diseases with artemisinin and derivatives. Patent no.
EP0428773 A1, 1991.
Tulloch J, David B, Newman RD, Meek S. Artemisinin-resistant malaria in the Asia-Pacific
region. Lancet. 2013 Jun 1;381(9881):e16-7.
Ullrich CI, Eitzen-Ritter M von, Jockel A, Efferth T, Prevention of plant crown gall tumor
development by the anti-malarial artesunate of Artemisia annua, Journal für Kulturpflanzen
2009 Vol. 61 No. 1 pp. 31-36
Utzinger J, Keiser J, Shuhua X, Tanner M, Singer BH, Combination chemotherapy of
schistosomiasis in laboratory studies and clinical trials, Antimicrob Agents Chemother.
2003 May;47(5):1487-95.
Vieira JB, A QSAR, pharmacokinetic and toxicological study of new artemisinin compounds
with anticancer activity, Molecules. 2014 Jul 24;19 (8):10670-97.
Wang J, Huang L, Li J, Fan Q, Long Y, Li Y, Zhou B, Artemisinin directly targets malarial
mitochondria through its specific mitochondrial activation, PLoS One. 2010 Mar 8;5
(3):e9582.
Wang JX1, Hou LF, Yang Y, Tang W, Li Y, Zuo JP, SM905, an artemisinin derivative, inhibited
NO and pro-inflammatory cytokine production by suppressing MAPK and NF-kappaB
pathways in RAW 264.7 macrophages Acta Pharmacol Sin. 2009 Oct;30(10):1428-35.
Wang L et al., Design, synthesis, and biological evaluation of artemisinin-indoloquinoline
hybrids as potent antiproliferative agents, Molecules. 2014 Nov 18;19 (11):19021-35.

156 | P a g e
Wang Y, Huang Z, Wang L, Meng S, Fan Y, Chen T, Cao J, Jiang R, Wang C, The anti-malarial
artemisinin inhibits pro-inflammatory cytokines via the NF-κB canonical signaling
pathway in PMA-induced THP-1 monocytes, Int J Mol Med. 2011 Feb;27(2):233-41.
Wang Z, Qiu J, Guo TB, Liu A, Wang Y, Li Y, Zhang JZ, Anti-inflammatory properties and
regulatory mechanism of a novel derivative of artemisinin in experimental autoimmune
encephalomyelitis, J Immunol. 2007 Nov 1;179 (9):5958-65.
Wu JA, Ji RY. A quantitative structure-activity study on artemisinine analogues, Zhongguo Yao
Li Xue Bao. 1982 Mar;3 (1):55-60.
Zhu C, Xiong Z, Chen X, Peng F, Hu X, Chen Y, Wang Q, Artemisinin attenuates
lipopolysaccharide-stimulated proinflammatory responses by inhibiting NF-κB pathway in
microglia cells, PLoS One. 2012;7 (4):e35125.

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 Elucidating Anticancer Activity of Withaferin A, Major Constituent of
Withania somnifera Through Suppression of NF-кB, a Prominent Marker
in Cancer
Lubna Siddiqui, Shilpi Singh
Molecular Bioprospection Department of Biotechnology Division, CSIR-Central Institute of
Medicinal and Aromatic Plants, Lucкnow-226015
Email: lubna2660@gmail.com

ABSTRACT

Cancer is a mounting health crisis around the world, treatment of which through radiotherapy and
chemotherapy with synthetic drugs evoke stern side effects. Thus, from therapeutic point of view,
it seems perceptible to overcome the above inadequacy with the use of natural products especially
from plants. Withaferin A, a foremost constituent of Withania somnifera has been found effective
in preventing cancer by targeting NFκB, a major target involved in biochemical pathways of
cancer. Activation of NFκB regulates genes that promote cellular proliferation, carcinogenesis,
metastasis, and inflammation. The effect of Withafarin A on NFκB and NFκB regulated gene
expression activated by various carcinogens is being reviewed. Withaferin A suppresses NFκB
activation through inhibition of IКК activation, IkBα phosphorylation, IkBα degradation, p65
phosphorylation, p65 nuclear translocation, and NFκB-dependent reporter gene expression
activation.

INTRODUCTION

Cancer is a major health problem around the world and the treatment of this dreaded disease
through conventional radiotherapy and chemotherapy with synthetic drugs evoke stern side
effects including severe immune suppression, suppression of bone marrow, nausea, vomiting, and
high toxicity in majority of cancer patient (Modha et al., 2007). To overcome the problem of side
effect and resistance, it seems apparent to use the natural products especially from plants. Plants
are known to possess high chemical diversity, structural complexity, easy accessibility, and are
compatible with the human body, showing lesser side effects (Cragg et al., 1997). One of the
plants, Withania somnifera has been used as traditional medicine for the treatment and prevention
of different kinds of cancer including colon, skin, breast and renal cancer. W. somnifera
commonly known as ‘Ashwagandha’, belongs to Solanaceae family and have a reputation of
being called as ‘Indian ginseng.’ It is widely cultivated in India and throughout the Middle East,
Eastern Africa. The plant is an erect, greyish, slightly hairy evergreen shrub with fairly long
tuberous roots. The flowers are small and greenish, single or in small clusters in the leaf axils.
The fruit is smooth, round, fleshy, and has many seeds, orange-red when ripe, enclosed in a
membranous covering (Кhare et al., 2007).

Chemical Constituents of Withania somnifera

The biologically active chemical constituents are alkaloids (isopellertierine, anferine), steroidal
lactones (withanolides, withaferins), saponins containing an additional acyl group (sitoindoside
VII and VIII) and withanoloides with a glucose at carbon 27 (sitonidoside XI and X) (Mishra et
al., 2000). The major chemical constituents of this plant, withanolides, are mainly localized in the
leaves and roots and their concentration usually ranges from 0.001 to 0.5% dry weight (Kapoor.,
2001). They are a group of C28-steroidal lactones built on an ergostane structure in which C-22
and C-26 are oxidized to form a six-membered lactone ring. Withaferin A, the first withanolide
isolated, is a C5, C6 epoxy compound with hydroxyl groups on C4 and C27. The compound has
been reported for its anti-tumorigenic activity against various cancer cells (Mayola et al., 2011).

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 CH3

OH
CH3
CH3 O O

O
CH3

A B

O
OH
Structure of Withaferin A

Anticancer Efficacy of Withania somnifera

W. somnifera have healing potential due to the presence of natural antioxidants functioning as
reducing agents, free radical scavengers and quenchers of singlet oxygen. The anticancer effect of
the plant has been reviewed earlier (Devi et al., 1996, Davis and Kuttan, 2000, Prakash et al.,
2002) and it was revealed that it prevent cancer through its ability to reduce the tumor size
(Prakash J et al., 2002, Jayapraкasam B et al., 2003). Anticancer activity of root, stem and leaves
of W. somnifera was first evaluated by Yadav et al., 2010 against various human cancer cell lines
depicting that root, stem and leaves extracts showed cytotoxic activity from 0-98% varies with
the type of cancer cell lines. It was also revealed that maximum activity was found in 50%
ethanolic leaf extract and withanolides containing an α, β-unsaturated ketone in ring A, a
5β,6β‐epoxy group in ring B, and a nine‐carbon side chain with an α,β‐unsaturated δ‐lactone
group exhibits most promising activity. The typical withanolide, Withaferin A contains these
three moieties and has been shown in vitro and in vivo to suppress the growth of an array of
tumor cells by inducing apoptosis as mention in Table 1 (Jeffrey et al., 2012).

Table 1: Biological activities of Withaferin A in different types of cancer

Component Type Assay Biological activities Reference

Withaferin A Colon cancer In vivo Decreased the activities of TCA Muralikrishnan
cycle key enzymes such as et al., 2010.
isocitrate dehydrogenase (ICDH),
succinate dehydrogenase (SDH),
malate dehydrogenase (MDH),
and alpha-keto glutarate
dehydrogenase (alpha-KGDH)
Skin cancer In vivo Decrease in incidence and average Mathur et al.,
number of skin lesions 2004
Breast cancer In vitro Vimentin cytoskeleton inhibitor Thaiparambi,
MCF-7 et al.,2011
cells

Blood cancer In vitro Withaferin A induced early ROS Malik et al .,

HL-60 cells generation and mitochondrial 2007
dysfunction in cancer cells trigger
events responsible for
mitochondrial-dependent and
independent apoptosis pathways

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Nuclear Factor-kappa B (NFκB) and Withanolides
NFκB was identified by David Baltimore in 1986 as a factor in the nucleus that binds to the
promoter of the kappa chain of immunoglobulins in β cells (Aggarwal et al., 2004). Five different
mammalian NFκB family members have been identified: NFкB1 (p50/p105), NFкB2 (p52/p100),
Rel A (p65), RelB, and c-Rel. All family members share a highly conserved Rel homology
domain (RHD; ~300 aa) responsible for DNA binding, a dimerization domain, and the ability to
interact with IкBs, the intracellular inhibitor for NFκB. In resting cells, NFκB consisting of p50
and Rel A is sequestered in the cytoplasm in an inactive form through its association with one of
several inhibitory molecules including IkBα, IкBβ, IкBγ, p105, and p100, among which IкBa is
the most abundant. Activation of NFкB regulates about 400 genes that promote cellular
proliferation, carcinogenesis, metastasis, and inflammation. These include inflammatory
cytokines (TNF, IL-1, IL-6, and chemokines), adhesion molecules, inflammatory enzymes (COX-
2, 5-LOX), viral proteins, telomerase, angiogenesis proteins (VEGF), anti- apoptotic proteins, and
cell cycle regulatory genes (Kumar et al., 2004). The effect of Withafarin A on NFкB and NFкB-
regulated gene expression activated by various carcinogens was examined and it was found that
Withafarin A suppressed NFкB activation.

Signaling Pathways Activating NFκB

In unstimulated cells, NFκB dimers usually exist as latent complexes with the IкB proteins in the
cytoplasm. There are two major mechanisms leading to NFκB activation: the canonical and non-
canonical NFκB pathways, which are based on the inducible degradation of IкBα and processing
of p100 to generate p52 (selective degradation of the C-terminal IкB like sequence of p100).

Canonical NFκB Signaling

In the canonical NFκB signaling pathway lipopolysaccharides (LPS), tumor necrosis factor α
(TNFα) or interleukin-1 (IL-1) activate Toll-like receptors (TLRs), tumor necrosis factor receptor
(TNFR) and interleukin-1 receptor (IL-1R) respectively (Perkins et al., 2006,2007). Through a
variety of adapter proteins and signaling kinases this leads to an activation of IkB in the IКК
complex, which can then phosphorylate IkBα on serine residues S32 and S36. This
phosphorylation is a prerequisite for its subsequent polyubiquitination, which in turn results in
proteasomal degradation of IkBα. NFκB homo- or heterodimers can then translocate to nucleus
and activate target gene transcription.

Non-canonical NFκB Signaling

In the non-canonical NFκB signaling pathway, activation of β-cell activation factor (BAFFR),
CD40, receptor activator for nuclear factor kappa B (RANК) or lymphtoxin β-receptor (LTβR),
leads to activation of IККα by the NFκB-inducing kinase (NIК), (Sun, 2010). IККα can then
phosphorylate p100 on serine residues S866 and S870. This phosphorylation leads to
polyubiquitination of p100 and its subsequent proteasomal processing to p52 (Xiao et al., 2001).
p52-RelB heterodimers can then activate transcription of target genes.

Mechanism and Gene Regulation

Withafarin A suppress both inducible and constitutive NFκB activation (Haruyo et al., 2006). It
was found that Withafarin A blocked the activation of NFκB without directly interfering with the
DNA binding of NFκB. This inhibition was mediated through inhibition of IКК by Withafarin A,
which led to the suppression of phosphorylation and degradation of IкBa. Withafarin A also
inhibits the TNF-induced phosphorylation of p65, nuclear p65 translocation, and NFкB
dependent reporter gene activity. Various in vitro kinase assay results show that with Withafarin
A is not a direct inhibitor of IКК it seems that this agent blocks the activation of IКК by
interfering with some upstream regulatory kinases like AKT, NIК, mitogen-activated protein
Kinase 1, and atypical protein kinase C (Aggarwal et al., 2004). Withafarin A also down regulate
the expression of genes regulated by NFкB which are involved in the proliferation and metastasis

160 | P a g e
of cancer are such as the expression of inhibitor of apoptosis protein 1, Bfl-1/A1, and c-FADD-
like IL-1h converting enzyme inhibitory protein as their overexpression in cancer has leads to
tumor survival and chemoresistance (Figure 1).

Figure 1. Anticipated mechanism of Withaferin A suppressing NFκB activation.

CONCLUSION

From the literature information available, it may be concluded that Withafarin A, a major
constituent of Withania somnifera has been found effective in preventing cancer by targeting
NFкB, a major target involved in biochemical pathways of cancer. Activation of NFкB regulates
genes that promote cellular proliferation, carcinogenesis, metastasis, and inflammation. The
effect of Withafarin A on NFкB and NFкB regulated gene expression activated by various
carcinogens was examined. It was found that Withafarin A suppressed NFкB activation through
inhibition of IКК activation, IkBα phosphorylation, IkBα degradation, p65 phosphorylation, p65
nuclear translocation, and NFкB-dependent reporter gene expression activation. However further
studies are needed to validate its effectiveness.

REFERENCES

Aggarwal, BB. Nuclear factor-kappa B: the enemy within. Cancer Cell 6: 203–208(2004.).
Aube J, Timmermann B, Cohen M, Samadi A. Novel Derivatives of Withanolides as Anticancer
Agents US 0196815(2012).
Cragg GM, Newman DJ, Snader КM. Natural products in drug discovery and development. J Nat
Prod 60:52–60(1997).
Davis L, Кuttan G. Effect of Withania somnifera on 20-methylcholanthrene induced fibrosarcoma. J
Exp Clin Cancer Res 19:165-167(2000).
Devi PU. Withania somnifera Dunal (Ashwagandha): potential plant source of a promising drug for
cancer chemotherapy and radiosensitization. Indian J. Exp. Biol 34:927-932(1996).
Ichiкawa H, Taкada Y, Shishodia S, Jayapraкasam B, Nair MG, and Bharat B. Aggarwal:
Withanolides potentiate apoptosis, inhibit invasion, and abolish osteoclastogenesis through
suppression of nuclear factor-КB (NF-КB) activation and NF-КB–regulated gene expression
Mol Cancer Ther 5:1434-1445(2006).

161 | P a g e
Jayapraкasam B, Zhang Y, Seeram NP, et al. Growth inhibition of human tumor cell lines by
withanolides from Withania somnifera leaves. Life Sci 74:125–32(2003).
Maliк F, Кumar A, Bhushan S, Кhan S, Bhatia A, Suri КA, Qazi GN, Singh J. Reactive oxygen
species generation and mitochondrialdysfunction in the apoptotic cell death of human myeloid
leuкemia HL-60 cells by a dietary compound withaferin A with concomitant protection by N-
acetyl cysteine. Apoptosis 12(11):2115-2133(2007).
Mathur S, Кaur P, Sharma M, Кatyal A, Singh B, Tiwari M, Chandra R. The treatment of sкin
carcinoma, induced by UV B radiation, using 1-oxo-5beta, 6beta-epoxy-witha-2-enolide,
isolated from the roots of Withania somnifera, in a rat model. Phytomedicine 11(5):452-
460(2004)
Mayola E, Gallerne C, Esposti DD, Martel C, Pervaiz S, Larue L, Debuire B, Lemoine A, Brenner C,
Lemaire C. Withaferin A induces apoptosis in human melanoma cells through generation of
reactive oxygen species and down-regulation of Bcl-2. Apoptosis 16(10):1014-1027(2011).
Mishra LC, Singh BB, Dagenais S. Scientific basis for the therapeutic use of Withania somnifera.
(Ashwagandha): A review. Alternative Medicine Reviews 5: 334-46(2000).
Modha J, Modha N. International Centre for Ayurveda Studies. Jamnagar, Gujarat, India: Gujarat
Ayurveda University Role of Ayurveda in the Management of Cancer(2007).
Muraliкrishnan G, Amanullah S, Basha MI, Dinda AК, Shaкeel F. Modulating effect of Withania
somnifera on TCA cycle enzymes and electron transport chain in azoxymethane-inducedcolon
cancer in mice. Immunopharmacol Immunotoxicol 32(3):523-527(2010).
Perкins ND, Gilmore TD: Good cop, bad cop: the different faces of NF-кappaB. Cell Death Differ
13:759–772(2006).
Perкins ND: Integrating cell-signalling pathways with NF-кappaB and IКК function. Nat Rev Mol
Cell Biol 8:49–62(2007)
Praкash J, Gupta SК, Dinda AК. Withania somnifera root extract prevents DMBA-induced squamous
cell carcinoma of sкin in Swiss albino mice. Nutr. Cancer 42:91-97(2002).
Sun S-C: Non-canonical NF-κB signaling pathway. Cell Res 21:71–85(2010).
Thaiparambil JT, Bender L, Ganesh T, Кline E, Patel P, Liu Y, Tighiouart M, Vertino PM, Harvey
RD, Garcia A, Marcus AI. Withaferin A inhibits breast cancer invasion and metastasis at
subcytotoxic doses by inducing vimentin disassembly and serine56 phosphorylation. Int J
Cancer (2011).
Xiao G, Harhaj EW, Sun SC: NF-кappaB-inducing кinase regulates the processing of NF-кappaB2
p100. Mol Cell 7:401–409(2001).
Yadav B. Bajaj A, Saxena M, Saxena К. In Vitro Anticancer Activity of the Root, Stem and Leaves of
Withania Somnifera against Various Human Cancer Cell Lines. Indian J Pharm SA 72:652-
63(2010).
Кapoor LD. Handbooк of Ayurvedic medicinal plants. CRC Press (2001).
Кhare CP. Indian Medicinal Plants: an IllustratedmDictionary, Heidelberg Springer, Berlin:717(2007)
Кumar A, Y Taкada, AM Borieк BB, Aggarwal L. Nuclear factor-кappaB:its role in health and
disease. J. Mol. Med. 82: 434–448(2004).

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 Role of Artemisinin and related compounds in cancer prevention and
treatment

Ravinder Naik Dharavath

Molecular Bioprospection Department of Biotechnology Division,
CSIR-Central Institute of Medicinal and Aromatic Plants, Lucknow-226015.
Email: naik.pharmacology@gmail.com

ABSTRACT

Artemisinin is the major constituent extracted from Artemisia annua. It has been used in
Chinese Traditional Medicine for years and approved by USFDA for the treatment of
malaria. The necessity to develop novel anticancer drugs with unaccountable adverse effects
unlike existing chemotherapeutic agents has motivated researches to look into naturally
occurring compounds for their anticancer activities. The expanding list of such anticancer
phytochemicals includes artemisinin, indole-3-carbinol, 3,3′-diindolylmethane, resveratrol,
genistein, kaempferol, curcumin, and epigallocatechin gallate. Artemisinin and its derivatives
have been evaluated for their anticancer activities and found to be very potent. Artemisinin
and its related compounds inhibit the tumour progression by interacting with various cellular
pathways involved in proliferation, metastasis, differentiation and apoptosis.

INTRODUCTION

Artemisinin, a sesquiterpene lactone, is extracted from the Chinese plant Artemisia annua. It
is commonly known as ‘Qinghaosu’ or ‘Sweet wormwood’ used for at least 2000 years to
treat fever and malaria [1]. Artemisinin and its bioactive derivatives, such as artesunate,
arteether, artemether, artemisone and dihydroartemisinin has been reported to meritoriously
treat different forms of malarial parasites including multidrug-resistant strains [2]. Due to
severe adverse effects of emerging chemotherapeutic drugs, natural compounds have been
brought on the stage for treating cancers because of their negligible adverse effects and
abundant availability in nature. The expanding list of such anticancer phytochemicals
includes artemisinin, indole-3-carbinol, 3,3′-diindolylmethane, resveratrol, genistein,
kaempferol, curcumin, and epigallocatechin gallate [3]. The results of recent studies on the
use of artemisinin in cancer treatment have made researchers to excavate deep into the
mechanism for their anticancer properties.

The important structural feature in artemisinin and its derivatives that mediates their
antimalarial activity is an endoperoxide bridge, which is cleaved in the presence of
intracellular iron (Fe+2) leading to generation of carbon centred reactive oxygen species
(ROS). It has been proposed that a similar mechanism is involved in both of the artemisinin
exerted antimalarial and anticancer activities [4,5]. Artemisinin has also been witnessed to
mitigate multidrug resistance in chemotherapy, activate various cellular pathways involved in
anti-proliferation (cell cycle arrest), disruption of mitochondria (initiation of apoptosis),
inhibition of tumour cell angiogenesis and metastasis. It has also been reported that
simultaneous activation of caspase-3 and/or caspase-9 by artemisinin and its derivatives is
responsible for their apoptotic responses [6,7].

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In a study done by the Developmental Therapeutics Program of the National Cancer Institute
(NCI), USA, artemisinin and its related compounds have been screened against 55 human
cancer cell lines and found that artesunate, the semisynthetic derivative is very potent against
leukaemia and colon cancer cell lines, and has intermediate effects on melanomas, breast,
ovarian, prostate, renal cancer cell lines and Central Nervous System [8]. Dihydroartemisinin
also display an outstanding anticancer activity against pancreatic, leukemic, osteosarcoma,
and lung cancer cells [7]. Among all the artemisinin derived compounds artesunate and
dihydroartemisinin are very potent cancer cell growth inhibitors. Results from most of the
studies points out dihydroartemisinin as the most potent anticancer artemisinin-like
compound and the order of activity was found to be dihydroartemisinin > artesunate >
arteeter > artemether [9]. It has recently been observed that artemisinin and its derivatives
can be used as an adjuvant with other anticanceragents due to its synergistic interactions.

Table: Effect of artemisinin and derived compounds in various cancer cell lines

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 Figure 1. Cellular pathways interrupted by artemisinin and derivatives

CONCLUSIONS

Till now, a lot of research has been carried out regarding the use of artemisinin and its
derivatives for their role in cancer prevention and treatment suggesting that they could be
used along with existing anticancer drugs as adjuvants. Though artemisinin and its
derivatives are showing significant anticancer activity, but are not potent enough to use them
alone in cancer treatment. Also, artemisinin has low water solubility and less oral
bioavailability, so there is an urgent need to get the synthetic artemisinins on to the stage to
address above limitations.

REFERENCES

[1]. Duffy PE, Mutabingwa TK. 2004. Drug combinations for malaria: time to ACT? The Lancet
363: 3-4.
[2]. Meshnick SR. 2002. Artemisinin: mechanisms of action, resistance and toxicity. International
journal for parasitology 32: 1655-1660.
[3]. Firestone GL, Sundar SN. 2009. Anticancer activities of artemisinin and its bioactive
derivatives. Expert reviews in molecular medicine 11: e32.
[4]. Rosenthal PJ, Meshnick SR. 1996. Hemoglobin catabolism and iron utilization by malaria
parasites. Molecular and biochemical parasitology 83: 131-139.
[5]. Paik IH, Xie S, Shapiro TA, Labonte T, Narducci Sarjeant AA, Baege AC, Posner GH. 2006.
Second generation, orally active, antimalarial, artemisinin-derived trioxane dimers with high
stability, efficacy, and anticancer activity. Journal of medicinal chemistry 49: 2731-2734.
[6]. Mukanganyama S, Widersten M, Naik YS, Mannervik B, Hasler JA. 2002. Inhibition of
glutathione S‐transferases by antimalarial drugs possible implications for circumventing
anticancer drug resistance. International journal of cancer 97: 700-705.

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[7]. Lu YY, Chen TS, Qu JL, Pan WL, Sun L, Wei XB. 2009. Dihydroartemisinin (DHA) induces
caspase-3-dependent apoptosis in human lung adenocarcinoma ASTC-a-1 cells. J Biomed Sci
16: 16.
[8]. Efferth T, Sauerbrey A, Olbrich A, Gebhart E, Rauch P, Weber HO, Hengstler JG, Halatsch M-
E, Volm M, Tew KD. 2003. Molecular modes of action of artesunate in tumor cell lines.
Molecular pharmacology 64: 382-394.
[9]. Jefford CW. 2007. New developments in synthetic peroxidic drugs as artemisinin mimics. Drug
Discovery Today 12: 487-495.
[10]. Weifeng T, Feng S, Xiangji L, Changqing S, Zhiquan Q, Huazhong Z, Peining Y, Yong Y,
Mengchao W, Xiaoqing J. 2011. Artemisinin inhibits in vitro and in vivo invasion and
metastasis of human hepatocellular carcinoma cells. Phytomedicine 18: 158-162.
[11]. Wu J, Hu D, Yang G, Zhou J, Yang C, Gao Y, Zhu Z. 2011. Down‐regulation of BMI‐1
cooperates with artemisinin on growth inhibition of nasopharyngeal carcinoma cells. Journal of
cellular biochemistry 112: 1938-1948.
[12]. Buommino E, Baroni A, Canozo N, Petrazzuolo M, Nicoletti R, Vozza A, Tufano MA. 2009.
Artemisinin reduces human melanoma cell migration by down-regulating αvβ3 integrin and
reducing metalloproteinase 2 production. Investigational new drugs 27: 412-418.
[13]. Disbrow GL, Baege AC, Kierpiec KA, Yuan H, Centeno JA, Thibodeaux CA, Hartmann D,
Schlegel R. 2005. Dihydroartemisinin is cytotoxic to papillomavirus-expressing epithelial cells
in vitro and in vivo. Cancer research 65: 10854-10861.
[14]. Efferth T, Sauerbrey A, Olbrich A, Gebhart E, Rauch P, Weber HO, Hengstler JG, Halatsch M-
E, Volm M, Tew KD. 2003. Molecular modes of action of artesunate in tumor cell lines.
Molecular pharmacology 64: 382-394.
[15]. W. Lijuan, “Effect of artesunate on human endometrial carcinoma,” Journal ofMedical
Colleges of PLA, vol. 25, no. 3, pp.143–151, 2010.
[16]. Wang J, Guo Y, Zhang B-C, Chen ZT, Gao JF. 2007. Induction of apoptosis and inhibition of
cell migration and tube-like formation by dihydroartemisinin in murine lymphatic endothelial
cells. Pharmacology 80: 207-218.
[17]. Cabello CM, Lamore SD, Bair III WB, Qiao S, Azimian S, Lesson JL, Wondrak GT. 2012. The
redox antimalarial dihydroartemisinin targets human metastatic melanoma cells but not primary
melanocytes with induction of NOXA-dependent apoptosis. Investigational new drugs 30:
1289-1301.
[18]. Lu YY, Chen TS, Wang XP, Li L. 2010. Single-cell analysis of dihydroartemisinin–induced
apoptosis through reactive oxygen species–mediated caspase-8 activation and mitochondrial
pathway in ASTC-a-1 cells using fluorescence imaging techniques. Journal of biomedical
optics 15: 046028-046028-046016.
[19]. Michaelis M, Kleinschmidt MC, Barth S, Rothweiler F, Geiler J, Breitling R, Mayer B,
Deubzer H, Witt O, Kreuter J. 2010. Anti-cancer effects of artesunate in a panel of
chemoresistant neuroblastoma cell lines. Biochemical pharmacology 79: 130-136.
[20]. Jiao Y, TONG J. 2007. Dihydroartemisinin is an inhibitor of ovarian cancer cell growth1. Acta
Pharmacologica Sinica 28: 1045-1056.
[21]. Hou J, Wang D, Zhang R, Wang H. 2008. Experimental therapy of hepatoma with artemisinin
and its derivatives: in vitro and in vivo activity, chemosensitization, and mechanisms of action.
Clinical cancer research 14: 5519-5530.
[22]. Singh NP, Panwar VK. 2006. Case report of a pituitary macroadenoma treated with artemether.
Integrative cancer therapies 5: 391-394.
[23]. Wang JX, Tang W, Shi LP, Wan J, Zhou R, Ni J, Fu YF, Yang YF, Li Y, Zuo JP. 2007.
Investigation of the immunosuppressive activity of artemether on T‐cell activation and
proliferation. British journal of pharmacology 150: 652-661.
[24]. Gravett AM, Liu WM, Krishna S, Chan WC, Haynes RK, Wilson NL, Dalgleish AG. 2011. In
vitro study of the anti-cancer effects of artemisone alone or in combination with other
chemotherapeutic agents. Cancer chemotherapy and pharmacology 67: 569-577.

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 Role of Withania somnifera (L.) in management of Alzheimer disease

Dhananjay Kumar Singh

Molecular Bioprospection Department, CSIR-Central Institute of Medicinal and Aromatic
Plants, Lucknow-226015.
Email: dhananjaypcology@gmail.com

Alzheimer (AD) is a progressive, degenerative disorder of brain characterized by the loss of

memory, judgment, verbal communication skills and activities. Among old age people, AD is
the common cause of dementia or the loss of logical power. Pathological features include
senile plaques, dystrophic neurites, gliosis, neuroinflammation and neurofibrillary tangles.
Current chemotherapy of AD includes cholinesterase inhibitors (Donepezil) and the NMDA
antagonist (Memantine) which provides only symptomatic aid but do not cure the disease (De
Strooper, 2010). Dietary and ethnobotanical therapies consist of Ginkgo, Panax ginseng,
Withania somnifera, huperzine A, polyphenols, phosphatidylserine, alpha-lipoic acid, omega-
3 fatty acids, acetyl L-carnitine, coenzyme Q10, some vitamins and minerals, and melatonin
(Wollen, 2010). Stimulatory therapies defined are physical exercise, cognitive training,
music, and socialization. There is no remedial treatment available till, therefore most of the
research and study are focused on development of drugs to slow disease advancement or
prophylaxis. Traditional systems of medicine such as Ayurveda, Unani, Sidha and Chinese
recommend many plant based therapy based on which we can develop novel therapeutic
strategies. Withania somnifera, commonly known as Ashwagandha, Indian ginseng, poison
gooseberry, or winter cherry, belongs to family Solanaceae, is a nootropic agent that enhance
learning and memory. Withanolide A and withanoside IV from its roots help to promote
neurite outgrowth in cultured neurons and in rodents injected with Aβ 25-35 (Dasilva et al.,
2010). Root of W. somnifera has been used for several purposes it is mainly considered a
nervine tonic in traditional Ayurvedic medicine. In a study by Tohda (2008) on components
from root, withanolide A, withanoside IV, and withanoside VI were reported as important
natural molecules for the treatment of neurodegenerative diseases, they also demonstrated
that the phytoconstituents such as withanolide A, withanoside IV, and withanoside VI,
renovated presynapses and postsynapses, after Aβ (25-35) induced injury in mice on oral
administration of withanolide A, withanoside IV, and withanoside VI (10 µM/kg/day for 12
days) specially, withanoside IV was found to direct neurons as well as glial cells for
reconstruction neuronal networks. Withanamides A and C were found to shield the PC-12
cells (neuronal cells) from β-amyloid induced cell damage. The cytotoxicity due to β-amyloid
was annulled by withanamide treatment, it was further evidenced by in silico studies which
exhibited that withanamides A and C exclusively bind to the β-amyloid (25-35) it also
suggested that withanamides might prevent the fibril formation (Jayaprakasam et al., 2010).
Acetylcholinesterase is a well-known clinically valuable approach for the treatment of AD.
Withanolide A, from this plant has revealed considerable neuro-protective capability. This
investigation endow with evidence for consideration of withanolide A as small ligand
molecule in treatment and prevention of AD (Grover, 2012). Semipurified extract of the root
of W. somnifera consisting mainly of withanolides and withanosides repealed behavioral
deficits, plaque pathology, accumulation of β-amyloid peptides (Aβ) and oligomers in the
brain of middle-aged and old Alzheimer's disease transgenic mice on 30 day oral
administration, notable therapeutic effect was mediated through upregulation of liver LRP
indicates that targeting the periphery offers a unique mechanism for Aβ clearance and
reverses the behavioural deficits and pathology seen in Alzheimer's disease models (Sehgal,
2012). Vareed and his co-worker (2014) reported that, when mice were administered with
250 mg/kg of W. somnifera extract i.p., and the blood and brain samples analyzed by Q-

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TOF/MS detection, four major withanamides were detected in brain and blood of mice they
concluded that withanamides crossed the blood-brain barrier, which may help to develop W.
somnifera fruit extract as a preventive or therapeutic botanical drug for stress-induced
neurological disorders. In conclusion, these major findings suggest that W. somnifera extracts
and its component could be taken up for further drug discovery process, it will also help to
bring some hope for the treatment of AD which otherwise seems incurable.

REFERENCES

Dasilva KA, Shaw JE, McLaurin J (2010). Amyloid-beta fibrillogenesis: Structural insight and
therapeutic intervention. Exp Neurol 223, 311–321.
De Strooper B, Vassar R, Golde T (2010). The secretases: Enzymes with therapeutic potential in
Alzheimer disease. Nat Rev Neurol 6:,99–107.
Grover A, Shandilya A, Agrawal V, Bisaria VS, Sundar D, (2012). Computational evidence to
inhibition of human acetyl cholinesterase by withanolide a for Alzheimer treatment. J Biomol
Struct Dyn 29, 651-62.
Haass C, Selkoe DJ (2007). Soluble protein oligomers in neurodegeneration: Lessons from the
Alzheimer’s amyloid beta-peptide. Nat Rev Mol Cell Biol 8,101–112.
Jayaprakasam B, Padmanabhan K, Nair MG. (2010). Withanamides in Withania somnifera fruit
protect PC-12 cells from β-amyloid responsible for Alzheimer's disease. Phytotherapy
Research 24, 859-63.
Sehgal N, Gupta A, Valli RK, Joshi SD, Mills JT, Hamel E, Khanna P, Jain SC, Thakur SS,
Ravindranath V. (2012) Withania somnifera reverses Alzheimer's disease pathology by
enhancing low-density lipoprotein receptor-related protein in liver. Proc Natl Acad Sci U S A
28, 3510-5.
Tohda C. (2008). Overcoming several neurodegenerative diseases by traditional medicines: the
development of therapeutic medicines and unraveling pathophysiological mechanisms.
Yakugaku Zasshi 128, 1159-67.
Vareed SK, Bauer AK, Nair KM, Liu Y, Jayaprakasam B, Nair MG.(2014). Blood-brain barrier
permeability of bioactive withanamides present in Withania somnifera fruit extract. Phytother
Res 28, 1260-1264.
Wollen KA. (2010). Alzheimer's disease: the pros and cons of pharmaceutical, nutritional, botanical,
and stimulatory therapies, with a discussion of treatment strategies from the perspective of
patients and practitioners. Altern Med Rev 15, 223-44.

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 The Time Travel of Artemisia: An Ethno-Botanical Review

Tanya Biswas, Archana Prasad and Archana Mathur

Plant Biotechnology Division, CSIR-Central Institute of Medicinal and Aromatic Plants,
Lucknow-226022

Disease cures and the search for drugs from nature has been an eternal quest for mankind
since times immemorial. Indeed, the oldest written evidence has been found on a Sumerian
clay tablet in Nagpur, aged 5000 years. Engraved on it, were 12 recipes from around 250
plants. Plant derived drug use, mostly instinctive in its origins, gradually developed through
experience and formed a treasure house of knowledge related to varied uses of a particular
medicinal plant and also in combinations with others. This knowledge repository is largely
culture-influenced with different plants introduced to the medicinal arena by different world
ethnicities. The Chinese, renewed for their TCM (Traditional Chinese Medicine; a current
favorite of Alternative Therapists), are known to promote uses of Podophyllum, ginseng,
jimson weed and Ephedra. Indians largely added to the database by introducing nutmeg,
pepper, cloves from the “Vedas”. The Arabs brought with them their traditional usage of
Aloe, Coffee, Saffron, Cinnamon and so forth (Petrovska, 2012). What adds to the intriguing
picture is the fact, that these traditional medicinal plants and their forms and mode of usage
are well differentiated by geographical and cultural barriers with one plant being renowed for
a particular ailment in a one culture group and a completely unrelated use in another.

Man and Artemisia are known to have joined hands since a long time. The genus belongs to
the family, Asterasceae (Compositae) which is one of the most largest and complicated taxa
to understand. Deriving its nomenclature from Greek mythology, “Artemis”: The Greek god
of the Wilderness and the Hunt is the major influence who was believed to have kickstarted
medicinal preparations from this “bitter” plant. One of the most prominent writers on plant
drugs, “Dioscorides” (The Father of Pharmacognosy; Author of “De Materia Medica”)
advocated the use of this plant for expelling intestinal worms, hence its common name of
“Wormwood”. More than 400 species of Artemisias exist and their uses range from culinary
to medicinal to even decorative.

Medicine
It is known to have been medicinally used as far back as 3500 years ago. Europeans and
Asians have been known to use wormwood (A. cina, A.pauciflora) for expelling intestinal
worms and relief from digestive complications. Native American tribes are known to use
wormwoods extensively for body cleansing in purification rites, basically by burning incense
sticks of white sage (A.tridentata). The Chinese are known to use rolled up wormwood leaves
to stop nosebleeds (Current practice prevails).

Two most prominent Artemisia species; A. annua (sweet annie) and A. vulgaris (Mugwort)
deserve special mention when medicinal properties are to be discussed. The Chinese are
credited with the isolation of artemisnin from A.annua (Quing-Hao for the Orients, Hsu,
2006, 2010) known to be effective against malaria and in dispelling fever chills. TCM
advocated soaking of A. annua leaves in urine (maximum retrieval of artemisnin) and
subsequent ingestion after proper crushing of leaves in the urine emulsion. Nowadays,
however a “tea” Infusion is preffered (Kooy and Sullivan, 2013). A. vulgaris is often
associated with a curious practice of pressing and burning dried mugwort onto acupuncture
points (Moxibustion) for curing “deficiency conditions” and also interestingly to stimulate
fetal movements to facilitate turning of breech babies. Traditional use of Mugwort in Japan is

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quite fascinating, with undertones of demonic protection. Its leaves are use to treat
psychogenic disturbances. Constant slapping with Mugwort brooms are believed to have been
effective in chasing away devils and subsequent body purification. Japanese people are
known to carry Mugwort leaves in scarves and also in shoes while long journeys on foot
(Kinoshita and Takemura, 1993). Additionally, they were used to treat venereal diseases such
as syphilis and also to alleviate dental problems. In India, Mugworts (known as Nagdauna,
Matajari etc) are used as tonics and antihelminthics. Hindu Vaidyas also advocate its use in
cases of obstructed menses (Wormwood species are well known to increase uterine
movements, increase in scanty menses, however with abortive tendencies in case of
pregnancies) and hysteria (Shah, 2014). Certain species are also religious offerings (A.
nilgarica; South India).Our close neighbours, Nepal and Pakistan also revel in its many uses.
Mugworts are used to cure cuts, offer protection from leeches and are used for insect
repelling in Nepal. It is also a popular antihelmintic, antipyretic and are useful in treating
gynecological problems in Pakistani culture (Nadeem et al, 2013).

Food/Cuisine/Beverages
The brightest star in the Artemisia galaxy, in terms of cuisine, is undoubtedly, A.dracunculus,
also known as the French Tarragon, valued for its sweet-anise: licorice flavor, when used
fresh. An integral part of French cuisine in the forms of hollandaise sauces, vinaigerettes, it is
an essential poultry stuffing, with none of the bitterness associated with the other Artemisias.
Mugwort are also sometimes used in poultry preparations which is supposed to prep the
gastrointestinal activites to aid in their easy digestion and dodging indigestion and bloating,
common with consumption of rich poultry. Japanese employ “yomogi” (A. princeps) as an
additive to their glutinous rice dumplings, apart from their use in soups and salads.

Among beverages, one of the very popular alcoholic drinks, “absinthe” (Popular Renaissance
artist Van Gogh, was thought to have painted some of his masterpieces under the
hallucinogenic effect of absinthe), is flavoured with A.abscinthium and A.pontica, right back
from 1200-1500 BC, prevalent in Europe, China and even India. Indeed “Absinthe” is the
French word for wormwood. However, its production was halted due to the presence of a
much debated and controversial compound “thujone”, a hallucinatory agent. However, this
ban was lifted due to inconclusive scientific evidence related to thujone induced brain
activities.

Aroma
Since Artemisias are essential oil producers, certain species are extremely deserving towards
their exploitation in the perfumery business. A.abronatum (southern wood) has a fruity odor,
while citrus southernwood has lemon undertones. Camphor southernwood (A. camphorata)
has a pleasant camphor smell. Closer to home turf, South India harbors a particular A. pallens
, which is renowed for its “ Davana” oil, used in high end perfumery.

Landscaping
The Greens, Greys and the Silver color palettes of the different Artemisias, offer a pleasant
visual addition to any landscapes. Some well known examples commonly are the “Silver
King”, “Silver Queen” and the much famed “Powis castle”. Silver King (A. ludoviciana) and
Silver Queen form luxuriant silver patches. Powis castle was awarded the “Award of Garden
Merit” by the Royal Horicultural Society in 1993, basically because of its outstanding colour
and shape. Another wormwood species, A. arborescens, deserves special mention due to its
stately mini silver-grey canopy.

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With its varied uses over centuries, across time and populations, Artemisias are surely plants
that deserve special scientific attention to unearth the plethora of its “magical” benefits.
Current Scientific attention is largely focused on A. annua, for its artemisnin molecule. This
molecule has shown extremely promising anti parasitic activity against malaria, even with
chloroquine resistant strains of Plasmodium falciparum. However different scientific groups
differ with their mode of artemisnin uptake i.e, whether the pure molecule is responsible for
its activity or is the activity due to synergism between artemisnin and other flavonoids that
are co-extracted when tea infusions are ingested (Lube et al, 2012). Detailed studies
regarding intestinal absorption and blood sera levels (bioavailability) following consumption
are carried out as a pharmacokinetic study. The balance slightly tips towards the use of Tea
infusions which demonstrate better antimalarial activity, suggesting possible synergism.
CSIR-CIMAP has long been a crucial piece of the Jigsaw puzzle related to the medicinal
Artemisia species in India. Since the early 1980’s, CIMAP has successfully carried out
demographic distribution studies of A. annua, throughout India. Certain new species were
also reported such as A. filiformiloburata and A.astro-himalayana from the Niti Border
(Chamoli, India; Shah, 2014). Over the years CIMAP has been actively involved in
developing new varieties of A. annua, with better artemisinin yields. Focus currently
currently lies on CIM- Arogya variety, developed by breeders through mass selection. The
corresponding technology has successfully been handed down to the IPCA, Indore Industries.
Many impact making patents are related to Artemisia cultivation method, CIM-Arogya herb
processing for artemisinin , artemisinin extraction process and CIM-Arogya - genetically
tagged high yielding variety of Artemisia annua and its cultivation technology
The current global focus now lies on A. annua extracts and new compounds which are being
actively investigated for their possible roles against HIV and cancer, currently prescribed by
NGO’s as ANAMED. A. annua has truly risen to the status of a global pharmaceutically
priceless medicinal plant.

REFERENCES

Kinoshita Y, Takimura H.1993. Studies on diseases and the medical treatments of Ainu
people. Takeuchi Publishing Company, Inc. SapporO
Kooy F, Sullivan SE. 2013. The complexity of medicinal plants: The traditional Artemisia
annua formulations, current status and future perspectives. Journal of
Ethnopharmacology: 150:1-13
Nadeem M, Shinwari ZK, Qaiser M. 2013. Screening of folk remedies by Genus Artemisia
based on Ethnomedicinal surveys and traditional knowledge of native communities of
Pakistan. Pakistani Journal of Botany: 45: 111-117
Petrovska BB. 2012. Historical review of medicinal plant usage. Pharmacognosy Reviews:
6:1-5
Shah NC. 2014. The Economic and Medicinal Artemisia Species in India. The Scitech
Journal:1: 29-37
Wormwoods-Herbal Encyclopedia
Praising the Artemisias; Richters.com
Herb of the Year: Artemisia, www.iherb.org (International Herb association)

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 Therapeutic Potential of Artemisia against Malaria
Archana Saxena and Dnyaneshwar U. Bawankule.
Molecular Bioprospection Division, CSIR-Central Institute of Medicinal and Aromatic Plants
Lucknow, India-226015
Email: archanamsubt@gmail.com

ABSTRACT

Malaria is one of the world's leading killer infectious diseases with high incidence and morbidity.
Artemisinin is the active antimalarial constituent of A. annua. The development of resistance to
front-line antimalarial drugs such as chloroquine is the greatest challenges facing malaria control
today. Artemisia annua commonly known as sweet wormwood is a Chinese antimalarial herb that
has been used for more than 2000 years. Herbs of artemisia have been used as tonics,
antimalarials, antihelmintics and antidiabetics, and in treating wounds, bronchitis, ulcers, and
tuberculosis in traditional system of medicine. Parasite resistance to artemisinins has been already
detected in 4 countries (Cambodia, Thailand, Myanmar, and Vietnam) in the Greater Mekong
subregion in Southeast Asia. To obviate emergence of drug resistance artemisinin-based
combination therapies (ACTs) have been recommended by the WHO for the treatment of
uncomplicated malaria. Artesunate, artemether, dihydroartemisinin (DHA) and arteether are the
semisynthetic derivatives of artemisnin, commonly used in combination therapy of malaria
treatment.

Keywords: Artemisia annua , malaria, Artemisinin- combination therapy (ACT).

INTRODUCTION:

Medicinal Plants (MPs) are being used for thousands of years in traditional system of medicine
for the cure of many ailments as for the safe and effective therapeutic strategy, demand of herbal
medicines is increasing both in the developing and developed countries (Ganesan and Bhatt,
2008). Generally Herbal drugs have no side effects (Bodhisattwa et al., 2011).

World Health Organization (WHO) reported that 80% of the world populations currently use
herbal drugs for healthcare. Malaria is a major parasitic infection caused by different species of
the genus Plasmodium in the developing world. World Health Organization (WHO) reported that
215 million cases of malaria occurred, with 4655,000 deaths; half the world's population is at risk
of contracting the disease (WHO, 2012).

Artemisia annua belongs to the family Asteraceae, is an aromatic herb with small, yellow flower
heads. This plant showing rich phytochemical diversity, is used in traditional medicine as well as
in modern pharmacology. In China A. annua plant extracts have been used to treat malaria and
fever for many centuries (Klayman, 1985). A. annua possesses antibacterial, anti-inflammatory,
and cytotoxicity (antitumour) activities in addition to its antimalarial activity (Hasheminia et al.,
2011; Efferth et al., 2011). Artemisinin, sesquiterpene lactone, is the active antimalarial
constituent of A. annua (Klayman, 1985). Aerial parts of A. annua are used to make anti-malarial
drugs. Artemisinin has been found in only two other species of genus Artemisia, Artemisia
apiacea and Artemisia lancea (Tan et al., 1998). This medicinal plant is very useful against
malaria so researchers try to prepare new and effective antimalarial drugs using this plant in
combination with other medicinal plant.

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Malaria treatment and artemisia
Malaria treatment is become more difficult due to the development of resistance in parasites, and
in vector against most of the available drugs. Chloroquine was recomended as safe and affordable
antimalarial drug, but now, it is not very useful because of resistance, and the reported
antifertility effect (Ebong et al., 1999). Currently, artemisinin is most effective antimalarial drug.
Artemisnin monotherapy is difficult to use against malaria treatment, due to its poor
pharmacokinetic properties and risk of resistance development. Artemisnin resistance parasite
was detected in Pailin, Cambodia, near to the Thai Cambodian border (WHO, 2010). To avoid all
these complications, WHO has been recommended artemisinin-based combination therapies
(ACTs) for the treatment of uncomplicated malaria (WHO, 2010). Artemisinin-based
antimalarials have several advantages over the quinines because have faster parasitemia clearance
(Barnes and White, 2005). Low yield in the plant and the cost of secondary anti-malarials in the
ACT, make artemisinin costly for the developing world. Artemisinin derivatives, such as
artesunate, artemether, dihydroartemisinin (DHA) and arteether were made in such a manner that
these having enhanced antimalarial activity with an unique structure and mode of action. (Li et
al., 2003). Development of resistance to artemisinin and their associate drugs limit the utility of
ACT in future. There is an urgent need for the discovery and development of new
chemotherapeutic compounds to meet with the challenges of drug resistance (Bathurst and
Hentschel, 2006). Natural plant products and their derivatives have traditionally been a common
source of drugs and are the safest and effective sources of malaria treatment.

CONCLUSION

This minireview has attempted to highlight the use of Chinese medicinal plant Artemisia annua in
the treatment of malaria like fevers. Further treatment of malaria by using artemisin in
combination with other antimlarials is required to pave the way of drug resistance. The
development of novel and safe antimalarial agents is an urgent priority for malaria control
especially in developing nations.

REFERENCES

Barnes, K.I., White, N.J. 2005. Population biology and antimalarial resistance: The Transmission
of antimalarial drug resistance in Plasmodium falciparum. Acta Trop 94: 230–240.
Bathurst, I., Hentschel, C. 2006. Medicines for Malaria Venture sustaining antimalarial drug
development. Trends Parasitol 7:301-307.
Bodhisattwa, M., Nagori, B.P., Rambir, S., Kumar, P., Upadhyay, N. 2011. Recent trends in
herbal drugs: a review. Int J Drug Res Tech 1: 17-25.
Ebong, P.E., Eyong, E.U., Eteng, M.U., Ukwe, C.N. 1999. Influence of chronic administration of
chloroquin on leydig cell integrity and testosterone profile of albino Wistar rats. Afr J
Reprod Health 3: 97–100.
Efferth, T., Herrmann, F., Tahrani, A.,Wink, M. 2011. Cytotoxic activity of
secondarymetabolites derived from Artemisia annua L. towards cancer cells in comparison
to its designated active constituent artemisinin. Phytomed 18: 59–969.
Ganesan, S., Bhalt, R.Y. 2008. Qualitative Nature of Some Traditional Crude Drugs Available
in Commercial Markets of Mumbai, Maharashtra, India. Ethnobotanical leaflets 12: 348-
360.
Hasheminia, S.M., Sendi, J.J., Jahromi, K.T., Moharramipour, S. 2011. The effects of Artemisia
annua L. and Achillea millefolium L. crude leaf extracts on the toxicity, development,
feeding efficiency and chemical activities of small cabbage Pieris rapae L. (Lepidoptera:
Pieridae). Pest Biochem Physiol 99: 244–249.
Klayman, D.L. 1985. Qinghaosu (artemisinin) an antimalarial drug from China. Sci 223: 1049-
1055.

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Li, Y., Wu, Y.L. 2003. An over four millennium story behind qinghaosu (artemisinin) – a
fantastic antimalarial drug from a traditional Chinese herb. Curr Med Chem 10:2197–2230.
Tan, R.X., Zheng, W.F., and Tang, H.Q. 1998. Biologically active substances from the genus
Artemisia. Planta Med 64:295–302.
WHO, 2010. Global Report on Antimalarial Drug Efficacy and Drug Resistance: 2000–2010.
Geneva: World Health Organization.
WHO, 2012. 10 Facts on Malaria.

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 Toxicological safety trends of Withania somnifera

Pone Kamdem Boniface, Sonam Khare and Anirban Pal

CSIR-Central Institute of Medicinal and Aromatic Plants Lucknow, India-226015
Email: ponekamdemboniface@gmail.com

ABSTRACT

Objective: The present study aim to review the literature pertaining to the toxicity profile of
Withania somnifera (ashwagandha, Ws), a common herb used in Ayurvedic medicine.

Methodology: This review is in a narrative format and consists of the data relevant to the
safety profiling of ashwagandha that were identified through a systematic search of major
computerized databases including www.scifinder.com, www.pubmed.com and
www.sciencedirect.com.

Results: Studies indicated that ashwagandha exhibit no adverse effects at acute and sub-acute
levels. Genotoxicity study revealed that Ws is safe during pregnancy at the acute level. Ws is
cytotoxic towards cancer cells.

Conclusion: Preliminary toxicity studies revealed that ashwagandha is safe at the acute and
subactue levels. However, sub-chronic and chronic toxicity, mutagenicity and teratogenicity
studies should be carried out in order to ascertain Ws safety limits.

Key words: Withania somnifera, toxicity, cell damage, mice, rats.

INTRODUCTION

Withania somnifera (Ws), also known as Ashwagandha, Indian ginseng, or winter cherry, has
been an important herb in the Ayurvedic system for over 3000 years (Sandhu et al., 2010).
Several other species in the genus Withania including Withania frutescens, Withania
coagulans and Withania adpressa are morphologically similar. In Ayurveda, the berries and
leaves are applied externally to tumors, tubercular glands, carbuncles, and ulcers (Mirjalili et
al., 2009). Ws was reported to possess a wide range of pharmacological properties including
antimalarial, anti-oxidant, anti-inflammatory, immunomodulatory and antiproliferative
activity (Mishra et al., 2000). The phytochemistry of Ws has also been investigated and
several pharmacologically active compounds including Withaferin and withanolides have
been identified. The chemistry and biological studies of Ws impelled many researchers to
focus their surveys on its toxicity profile. In the present review, we report the research work
done so far in conjunction with the safety profile of Ws in vitro and in vivo.

Methodology

The data on the toxicity studies of Ws were collected through an internet search in
www.scifinder.com, www.sciencedirect.com and www.pubmed.com.

Studies reported so far on acute, sub-acute and genotoxicity of Ws

Acute oral toxicity study of Ws at 2000 mg/kg did not reveal any mortality and morbidity in
rats after 14 days. 28 days oral administration of Ws at the doses of 500, 1000 and 2000
mg/kg did not modify physical, haematological, biochemical and histological parameters in

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Wistar rats (Prabu et al., 2013). Hence, the no observed adverse effect level (NOAEL) of Ws
was reported as 2000mg/kg. Additionally, 90 days administration of Ws alongside Panax
gensing did not reveal morbidity or mortality in rats while histological, haematological and
biochemical parameters remained unchanged (Aphale et al., 1998). Likewise, the oral
administration of Ws in pregnant Wistar rats (Days 5 to 19 gestation) at 500, 1000 and
2000mg/kg did not reveal any sign of toxicity. The NOAEL was concluded to be at least
2000 mg/kg (Prabu and Panchapakesan, 2014).

Cytotoxicity of Ws

Ws and its active constituents have been reported for their cytotoxicity activities to a wide
range of cancer cells. Ws was active against prostate, colon, lung, breast, and glioma cancer
cells (Joshi et al., 2014; Sumantran et al., 2007; Szarc vel Szic et al., Al-Fatimi et al., 2005).
Ws was also active against neuroblastoma and murine fibrosarcoma (Kataria et al., 2011).
Likewise, a new cytotoxic agent namely 5,6-de-epoxy-5-en-7-one-17-hydroxy withaferin A
from Ws leaves, has also been reported (Siddique et al., 2014). Ws extract inhibited cancer
metastasis, while L-asparaginase from Ws was cytotoxic against acute lymphoblastic
leukemia (Yang et al., 2013; Oza et al., 2010).

Toxicity of Ws to plants: Herbicidal effects of Ws

The herbicidal effect of aqueous, methanol and n-hexane extracts from Ws against Phalaris
minor was reported. 2 and 3% concentrations of Ws significantly reduced the shoot and root
dry biomass, while higher concentrations of 4 and 5% of residue incorporation completely
arrested, the germination of one-week Phalaris minor (Javaid et al., 2010).

Ameliorative effects of Ws towards cell damage

Besides the toxicity studies of Ws, the scientific validation of Ws in cell damage repair has
been the focus of many researchers. Ashwagandha reversed toxicity induced by lead, iron,
cadmium and silver nanoparticles (Kumar et al., 2014; Bhattacharya et al., 2000; Bharavi et
al., 2010; Anwar et al., 2014). Furthermore, Ws attenuated the effects of toxicity induced by
paclitaxel, scopolamine and 6-hydroxydopamine (Gupta et al., 2001; Konar et al., 2011;
Ahmad et al., 2005). Ws inhibited hyaluronidase activity of snake venoms (Machiah, 2006).
Moreover, withanone extracted from ashwagandha leaves decelerated the senescence of
human fibroblast cells (Widodo et al., 2009)

DISCUSSION AND CONCLUSION

As outlined above, results from previous studies indicate that ashwagandha exhibit no side
effects up to 2000 mg/kg attesting the wide safety margin of Ws when given orally for a short
term period. In addition, Ws is non selective to normal cells but cytotoxic to cancer cells.
More interestingly, Ws ameliorates the toxicological condition caused by some chemical
compounds (Paclitaxel, scopolamine, 6-hydroxydopamine and cyclophosphamide) and heavy
metals (Iron, lead, cadmium and silver) rather than inducing the toxicity. Although the results
from this review are quite promising for the toxicity profile of ashwagandha, several
limitations exist in the current literature. While ashwagandha has been used successfully in
Ayurvedic medicine for centuries, preclinical studies directed towards sub-chronic and
chronic toxicity, mutagenicity and teratogenicity studies of Ws should be conducted to
support its toxicological safety.

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REFERENCES

Ahmad, M., Saleem, S., Ahmad, A.S., Ansari, M.A., Yousuf, S., Hoda, M.N., Islam, F., 2005.
Neuroprotective effects of Withania somnifera on 6-hydroxydopamine induced
Parkinsonism in rats. Human and Experimental Toxicology, 24(3), 137-147.
Al-Fatimi, M., Friedrich, U., Jenett-Siems, K., 2005. Cytotoxicity of plants used in traditional
medicine in Yemen. Fitoterapia, 76(3-4), 355-358.
Anwar, M.F., Yadav, D., Rastogi, S., Arora, I., Khar, R.K., Chander, J., Samim, M., 2014.
Modulation of liver and kidney toxicity by herb Withania somnifera for silver
nanoparticles: a novel approach for harmonizing between safety and use of nanoparticles.
Protoplasma, 2014 Sep 24. [Epub ahead of print].
Aphale, A.A., Chhibba, A.D., Kumbhakarna, N.R., Mateenuddin, M., Dahat, S.H., 1998.
Subacute toxicity study of the combination of ginseng (Panax ginseng) and ashwagandha
(Withania somnifera) in rats: a safety assessment. Indian Journal of Physiology and
Pharmacology, 42(2), 299-302.
Bharavi, K., Reddy, A.G., Rao, G.S., Reddy, A.R., Rao, S.V., 2010. Reversal of Cadmium-
induced Oxidative Stress in Chicken by Herbal Adaptogens Withania Somnifera and
Ocimum Sanctum. Toxicology International, 17(2), 59-63.
Bhattacharya, A., Ramanathan, M., Ghosal, S., Bhattacharya, S.K., 2000. Effect of Withania
somnifera glycowithanolides on iron-induced hepatotoxicity in rats. Phytotherapy
Research, 14(7), 568-570.
Gupta, Y.K., Sharma, S.S., Rai, K., Katiyar, C.K., 2001. Reversal of paclitaxel induced
neutropenia by Withania somnifera in mice. Indian Journal of Physiology and
Pharmacology, 45(2), 253-257.
Javaid, A., Shafique, S., Shafique, S., 2010. Herbicidal activity of Withania somnifera against
Phalaris minor. Natural Product Research, 24(15), 1457-1468.
Joshi, P., Misra, L., Siddique, A.A., Srivastava, M., Kumar, S., Darokar, M.P., 2014. Epoxide
group relationship with cytotoxicity in withanolide derivatives from Withania somnifera.
Steroids, 79, 19-27.
Kataria, H., Shah, N., Kaul, S.C., Wadhwa, R., Kaur, G., 2011. Water extract of ashwagandha
leaves limits proliferation and migration, and induces differentiation in glioma cells.
Evidence-Based Complementary and Alternative Medicine, 2011, 267614.
Konar, A., Shah, N., Singh, R., Saxena, N., Kaul, S.C., Wadhwa, R., Thakur, M.K., Protective
role of Ashwagandha leaf extract and its component withanone on scopolamine-induced
changes in the brain and brain-derived cells. PLoS One, 6(11), e27265.
Kumar, P., Singh, R., Nazmi, A., Lakhanpal, D., Kataria, H., Kaur, G., 2014. Glioprotective
effects of Ashwagandha leaf extract against lead induced toxicity. Biomed Research
International, 2014, 182029.
Machiah, D.K., Girish, K.S., Gowda, T.V., 2006. A glycoprotein from a folk medicinal plant,
Withania somnifera, inhibits hyaluronidase activity of snake venoms. Comparative
Biochemistry and Physiology Part C: Toxicology & Pharmacology, 143(2):158-161.
Mirjalili, M.H., Moyano, E., Bonfill, M., Cusido, R.M., Palazón, J., 2009. Steroidal lactones from
Withania somnifera, an ancient plant for novel medicine. Molecules, 14 (7), 2373-2393.
Mishra, L.C., Singh, B.B., Dagenais, S., 2000. Scientific basis for the therapeutic use of Ws
(ashwagandha): a review. Alternative Medicine Review, 5:334-346.
Oza, V.P., Parmar, P.P., Kumar, S., Subramanian, R.B., 2010. Anticancer properties of highly
purified L-asparaginase from Withania somnifera L. against acute lymphoblastic leukemia.
Applied Biochemistry and Biotechnology, 160(6), 1833-1840.
Prabu, P.C., Panchapakesan, S., 2014. Prenatal developmental toxicity evaluation of Withania
somnifera root extract in Wistar rats. Drug and Chemical Toxicology, 2014 Mar 20. [Epub
ahead of print].

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Prabu, P.C., Panchapakesan, S., Raj, C.D., 2013. Acute and sub-acute oral toxicity assessment of
the hydroalcoholic extract of Withania somnifera roots in Wistar rats. Phytotherapy
Research. 27(8), 1169-1178.
Sandhu, J.S., Shah, B., Shenoy, S., Chauhan, S., Lavekar, G.S., Padhi, M.M., 2010. Effects of
Withania somnifera (Ashwagandha) and Terminalia arjuna (Arjuna) on physical
performance and cardiorespiratory endurance in healthy young adults. International
Journal of Ayurveda Research, 1(3), 144-149.
Siddique, A.A., Joshi, P., Misra, L., Sangwan, N.S., Darokar, M.P., 2014. 5,6-de-epoxy-5-en-7-
one-17-hydroxy withaferin A, a new cytotoxic steroid from Withania somnifera L. Dunal
leaves. Natural Product Research, 28(6), 392-398.
Sumantran, V.N., Boddul, S., Koppikar, S.J., Dalvi, M., Wele, A., Gaire, V., Wagh, U.V., 2007.
Differential growth inhibitory effects of W. somnifera root and E. officinalis fruits on CHO
cells. Phytotherapy Research 21(5), 496-499.
Szarc vel Szic, K., Op de Beeck, K., Ratman, D., Wouters, A., Beck, I.M., Declerck, K.,
Heyninck, K., Fransen, E., Bracke, M., De Bosscher, K., Lardon, F., Van Camp, G.,
Vanden Berghe, W., 2014. Pharmacological levels of Withaferin A (Withania somnifera)
trigger clinically relevant anticancer effects specific to triple negative breast cancer cells.
PLoS One, 9(2), e87850.
Widodo, N., Shah, N., Priyandoko, D., Ishii, T., Kaul, S.C., Wadhwa, R., 2009. Deceleration of
senescence in normal human fibroblasts by withanone extracted from ashwagandha leaves.
Journals of Gerontology Series A: Biological Sciences and Medical Sciences, 64(10),
1031-1038.
Yang, Z., Garcia, A., Xu, S., Powell, D.R., Vertino, P.M., Singh, S., Marcus, A.I., 2013. Withania
somnifera root extract inhibits mammary cancer metastasis and epithelial to mesenchymal
transition. PLoS One, 8(9), e75069.

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CROP PRODUCTION

AND

IMPROVEMENT
 Artemisia annua, artemisinin and stresses

Pragya Trivedi
Division of Agronomy and Soil Science, CSIR- CIMAP,
Lucknow
Email: pragyatrivedi89@gmail.com

INTRODUCTION

Artemisia annua, a traditional medicinal herb of Asteraceae family, is native to China and widely
cultivated in Asia, America and Europe1. It is commonly known as quinghao, sweet annie, sweet
sagewood, sweet wormwood, or annual wormwood. Plant shows luxurious growth, erect stem
having bright green foliage and inflorescence of loose panicles. In 1596, Chinese naturalist Li
Shi-Zhen described the use of A. annua for the treatment of malaria and other disease in his book
“Compendium of Materia Medica”. The essential oil extracted from A. annua aerial parts is also
reported to have antimicrobial and antioxidant activity2. Leaves and inflorescence of A. annua
consists of glandular trichomes which accumulate substantial quantities of unique sesquiterpenoid
artemisinin and other phytomolecules, and the characteristic essential oil3. Artemisinin, the most
important constituent of A. annua has antimalarial properties against chloroquine resistant strains
of Plasmodium falciparum4. The de novo synthesis is multi-step process with low yield and this
makes the plant the only feasible source of artemisinin.

Artemisia has ability to grow in different soil and environmental conditions and can withstand to
various environmental stresses such as water stress, salinity, physical stress, oxidative stress etc.
Therefore, it is important to review the available information to know the facts and exploit this
unique quality of Artemisia for remediation of various environmental hazards and artemisinin
production. Here I will discuss about tolerance of A. annua to various stresses and artemisinin
production. in following subheadings.

Drought
Drought is one of the limiting factors of plant growth. Water deficit can trigger secondary
metabolite accumulation depending upon growth stage of plant and intensity of stress. A study
conducted by Marchese et al.5 suggested that moderate water deficit induced artemisinin
accumulation and reduced the time required for drying, which increases crop profit margins.
Artemisinin production might be part of A. annua chemical defense system against water deficit.
Interestingly, some of minor monoterpenes, all sesquiterpenes and other low molecular weight
volatiles of essential oil components were induced by water deficit treatment.

Another study, revealed that artemisinin, arteannuin-B, artemisinic acid, dihydroartemisinic acid
and essential oil content were positively controlled by the growth and development however
negatively modulated by prolonged water deficit stress. Water deficit stress induces a decrease in
glandular trichome density and size as well3.

Heavy metals and salt stress

High salinity causes hyperionic as well as hyper-osmotic stress which are lethal to plant tissues. Both
heavy metals and salinity can cause oxidative stress in plant leaves in which active oxygen species
(AOS) are generated which may damage the photosynthetic apparatus resulting in a loss of
chlorophyll content and a decline in photosynthetic rate, biomass accumulation and artemisinin
production. NaCl and lead acetate treated soil enhanced lipid peroxidation at all stages of plant
growth and increased the concentration and yield of artemisinin, while other parameters declined at all
growth stages. The magnitude of changes was greater in lead acetate treatment than in salt-treated
plants. Both treatments induced oxidative stress 6.

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In another experiment conducted by Prasad et al.7 plant height decreased with the increase in salinity
and leaf stem ratio and vegetative yield increased while artimisinin was not influenced upto certain
limit.

Rai et al.8 explored physiological, biochemical and molecular changes in A. annua under arsenic (As)
stress. They found enhanced artemisinin production under As stress and upregulation of transcripts of
the genes involved in artemisinin biosynthesis. This suggests that A. annua has considerable As
tolerance, and thus can be successfully cultivated in As contaminated fields.

These studies thus support that AOS are generated through exposure of plants to heavy metals and salt
stress, and might be responsible for conversion of immediate precursors viz. dihydroartemisinic acid,
artemisinic acid and arteannuin B into artemisinin during early growth stages of the plant, when these
precursors are available in adequate amounts.The low level of artemisinin at later stages of plant
growth could be either due to its low synthesis because of impaired photosynthesis, its enhanced
degradation, or both.

Physical stress
One of the physical stress treatments i.e. sandblasting of the plants resulted in a significant
enhancement in the essential oil content as compared to non-treated plant 9.

Oxidative stress
Boron induced oxidative stress results in decrease in stem height, fresh weight, dry weight, net
photosynthetic rate, stomatal conductance, internal CO2 and total chlorophyll content and increase in
antioxidant enzymes like peroxidase, catalase and super oxide dismutase of A. annua. There is a
significant increase in H2O2 and artemisinin up to 1.00 mm concentration of boron compared to
control, and on applying higher doses artemisinin content was reduced 10. Therefore, a mild stress of
boron can be utilized for higher artemisinin production during the early phases of plant growth. This
may be due to a sudden conversion of its precursors (e.g. artemisinic acid/ dihydroartemisinic acid) to
artemisinin by activated oxygen species under oxidative stress. Asper suggestion given by Wallaart
et al. (1999), dihydroartemisinic acid might act as a scavenger of ROS (H2O2 in this case)
that are released in plant cells due to various reasons. During the biochemical reaction,
dihydroartemisinic acid hydroperoxide (DHAA-OOH) is generated that gets converted to
artemisinin.

Irradiation
Different studies are available showing effects of irradiation on A. annua artemisinin content. Aftab et
al.11 used sodium alginate irradiated by Co-60 rays together with various nitrogen doses, (N80
Kg N ha−1 together with Irradiated-SA 80 mg L−1) and found an increase in artemisinin content and
yield). Sodium alginate (SA) is a natural polysaccharide, derived from brown algae, available in
large quantities in nature. n Catharanthus and Papaver, respectively. It appears that ISA
promoted the artemisinin synthesis by recuperating the level of H2O2, which converts
dihydroartemisinic acid into artemisinin duringartemisinin biosynthesis 12,13.

In another experiment, A. annua was treated with artificial UV-B and UV-C, and it was observed that
radiation not only altered the growth responses, biomass production, pigment content and antioxidant
enzyme activity but also enhanced the secondary metabolites (including artemisinin and flavonoid)
content at all developmental stages as compared to non-irradiated plants14.

CONCLUSION
From the preceding review of literature it is concluded that yield of artemisinin of A. annua showed
various trends. In all the experiments there was increase in artemisinin content to a specific limit and
beyond that decrease was observed. This plant has ability to withstand various environmental stresses
like drought, oxidative stress, physical stress and irradiations as well as in various climatic and
edaphic conditions.

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REREFERENCES

1. Ozguven, M., Sener, B., Orhan, I., Sekeroglu, N., Kirpik, M., Kartal, M., Pesin, I.,
Kaya, Z., 2008. Effects of varying nitrogen doses on yield, yield compo-nents and
artemisinin content of Artemisia annua L. Ind. Crops Prod. 27 (1),60–64
2. Juteau, F., Masotti, V., Bessiere, J.M., Michel Dherbomez, Josette Vianoca., 2002.Antibacterial
and antioxidant activities of Artemisia annua essential oil. Fitoterapia 73 532–535.
3. Yadav, R.K., Sangwan, R.S., Sabir, F., Srivastava, A.K., Sangwan, N.S., 2014. Effect of
prolonged water stress on specialized secondary metabolites, peltate glandular trichomes, and
pathway gene expression in Artemisia annua L, Plant Physiology and Biochemistry 74, 70-83.
4. Klayman, D.L., 1985. Qinghaosu (artemisinin) an antimalarical drug from China. Science
223, 1049–1055.
5. Marchese J.A., Ferreira, J.f.s., Rehder, V.I.G., Rodrigues, O., 2010. Braz Water deficit effect on
the accumulation of biomass and artemisinin in annual wormwood (Artemisia annua L.,
Asteraceae). J. Plant Physiol., 22, 1-9.
6. Qureshi, MI., Israr, M., Abdin, M., Iqbal, M., 2005. Responses of Artemisia annua L. to lead
and salt-induced oxidative stress. Environmental and Experimental Botany 53, 185–193
7. Arun Prasad , Dinesh Kumar , M. Anwar , D. V. Singh & D. C. Jain (1998) Response of
Artemisia annua L. to Soil Salinity, Journal of Herbs, Spices & Medicinal Plants, 5:2, 49-55,
DOI: 10.1300/J044v05n02_07
8. Rai, R., Pandey, S., Rai, S.P., 2011. Arsenic-induced changes in morphological, physiological,
and biochemical attributes and artemisinin biosynthesis in Artemisia annua, an antimalarial
plant . Ecotoxicology, 20, 1900-1913.
9. Ivarsen, E., Kjær, A., Jensen, M., Grevsen, K., Christensen, L.P., Frett., X., 2013. Effect of
Chemical and Physical Stress Conditions on the Concentration and Composition of Essential
Oil Components in Leaves of Full-Grown A. annua L.. J Agro Crop Sci, ISSN 0931- 2250.
10. Aftab, T., Khan, M. M. A., Idrees, M ., Naeem, M., Ram, M., 2010. Boron Induced Oxidative
Stress, Antioxidant Defence Response and Changes in Artemisinin Content in Artemisia annua
L. J. Agronomy & Crop Science ISSN 0931-2250.
11. Aftab, T., Naeem, M., Idrees, M., Masroor, M., Moinuddin, A.K., Varshney, L., 2013.
Cumulative role of irradiated sodium alginate and nitrogen fertilizer on growth,
biochemical processes and artemisinin production in Artemisia annua . Industrial Crops
and Products 50, 874– 881.
12. Guo, X.X., Yang, X.Q., Yang, R.Y., Zeng, Q.P., 2010. Salicylic acid and methyl
jasmonate but not rose bengal enhance artemisinin production through invoking burst of
endogenous singlet oxygen. Plant Sci. 178, 390–397.
13. Pu, G.B., Ma, D.M., Chen, J.L., Ma, L.Q., Wang, H., Li, G.F., Ye, H.C., Liu, B.Y.,
2009.Salicylic acid activates artemisinin biosynthesis in Artemisia annua L. Plant Cell
Rep. 28, 1127–1135.
14. Rai, R., Meena, RP., Smita, S.S., Shukla, A., Rai, S.K., Rai, S.P., 2011. UV-B and UV-C pre-
treatments induce physiological changes and artemisinin biosynthesis in Artemisia annua L. –
An antimalarial plant, Journal of Photochemistry and Photobiology B: Biology 105, 216–225.

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Genetic Improvement for high yielding varieties in (Withania somnifera L.)

Rashmi Lahiri*, Sougata Sarkar, Smita Singh, Renu Yadav, R. K. Lal

Genetics & Plant Breeding Division, CSIR- Central Institute Of Medicinal and Aromatic
Plant, Lucknow (U.P.)
*Email id: rashmilahirigzb@gmail.com

INTRODUCTION

Ashwagandha (Withania somnifera), also known as Indian ginseng, is an important medicinal

plant, which is being cultivated for centuries in India. It is one of the most precious shrub and
hold important place in Indian traditional systems of medicine, Ayurveda and Unani. Root
and herb of ashwagandha are used as tonic, hypnotonic, sedative and diuretic (Jain and
Defillip, 1991), has anticancerous (Leyon and Kuttan,2004), anti-stress, anti-inflammatory,
anti-tumor (Devi et al, 1992) anti-oxidative properties and used to treat various diseases
associated with nerve tissue damage, heart disease, atherosclerosis, cancer, Aids and aging
because of its strong anti-oxidative property. It is an ingredient in many formulations
prescribed for a variety of musculoskeletal conditions (e.g., arthritis, rheumatism), and as a
general tonic to increase energy, improve overall health and longevity, and prevent disease in
athletes (Bhosale and More 2014).

Distribution
Ashwagandha belongs to family Solanaceae, is erect, evergreen, branched shrub, attains a
height of 30-60 cm. It grows well in dry and arid soil (Patra et al, 2004) with preference to
acid soil (Obidoska and Sadowska, 2003) in subtropicals and semiarid regions. In India it
grows in Uttar Pradesh, Maharastra, Madhya Pradesh, Haryana, Rajasthan and few regions of
Himanchal Pradesh.

Active Constitents
The biologically active chemical constituents are alkaloids (isopellertierine, anferine),
steroidal lactones (withanolides, withaferins), saponins containing an additional acyl group
(sitoindoside VII and VIII), and withanoloides with a glucose at carbon 27 (sitonidoside XI
and X). Withania somnifera is also rich in iron. There are presence of various chemical
constituents in the different parts of the plant which are as follows:

Roots
Roots contain alkaloids, amino acids, steroids, volatile oil, starch, reducing sugars,
glycosides, hentriacontane, dulcitol, withaniol. The total alkaloidal content of the Indian roots
has been reported to vary between 0.13 and 0.31 percent, though much higher yields (up to
4.3%) have been recorded elsewhere (Anonymous, 1982, Anonymous, 2007). Basic alkaloids
include cuscohygrine, anahygrine, tropine, pseudotropine, anaferine, isopelletierine,
withananine, withananinine, pseudo-withanine, somnine, somniferine, somniferinine. Other
alkaloids include withanine, withasomnine, and visamine .The free amino acids identified in
the root include aspartic acid, glycine, tyrosine, alanine, proline, tryptophan, glutamic acid,
and cystine (Khare, 2007).

Leaf
The leaves of the plant (Indian chemotype) are reported to contain 12 withanolides, 5
unidentified alkaloids (yield, 0.09%), many free amino acids, chlorogenic acid, glycosides,
glucose, condensed tannins, and flavonoids (Khare, 2007). Withaferin A, a steroidal lactone

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is the most important withanolide isolated from the extract of the leaves and dried roots of
Withania somnifera.

Fruit
The green berries contain amino acids, a proteolytic enzyme, condensed tannins, and
flavonoids. They contain a high proportion of free amino acids which include proline, valine,
tyrosine, alanine, glycine, hydroxyproline, aspartic acid, glutamic acid, cystine and cysteine.
The presence of a proteolytic enzyme, chamase, in the berries may be responsible for the high
content of the amino acid.

Other Parts
The tender shoots are rich in crude protein, calcium and phosphorous, and are not fibrous.
They are reported to contain scopoletin. The stem of the plant contains condensed tannins and
flavonoids. The bark contains a number of free amino acids (Anonymous, 1982).

Varieties released from CSIR-CIMAP

CHETAK (Naguri withania variety)

Variety CIMAP Chetak is a semi vigorous, medium green small leaves size and whitish green
stem, was found to be highly promising for high dry root yield (11.77 ql/ha v/s check 5.45
ql/ha) with high total Withanolide content (0.40 v/s 0.20% in check). The fresh and dry leaf
yield was also high (1.722 and 0.453 ql/ha v/s 0.872 and 0.147 ql/ha in check) with high
withaferine content 1.223 v/s 0.788 % in the check.

PRATAP
Variety CIMAP Pratap is a highly vigorous, dark green medium size leaves and dark green
stem; highly promising for high dry root yield (34.95 ql/ha v/s Poshita 21.99 ql/ha) with high
total Withanolide content (0.31 v/s 0.25% in Poshita). The fresh and dry leaf yield was also
high (5.39 and 0.87 ql/ha v/s 2.83 and 0.50 ql/ha in Poshita) with high witheferin contentin
dry leaves 0.720 v/s 0.528 % in the check variety Poshita.

POSHITA
Poshita is medium tall, semi broad, medium dark colour leaf with red coloured berries. The
estimated dry root yield was 14q/ha v/s check which is 8q/ha. Total withanolides (kg/h) is
0.25. This variety is very popular in South India specially Anantnag.

NMITLI 118
New chemotypes identified under a NMITLI project effort was made to collect the
Ashwagandha germplasm, which resulted into collection of 150 independent accessions from
various geographical locations, with many of them having contrasting chemotypes. Efforts
are underway to explore the pharmacological activities of selected chemotypes and individual
molecule to identify the best chemotype for adaptogenic activity.
Varieties developed by other universities and institutes are Jawahar, WS 20 and tall
Ashwagandha.

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REFERENCE

Anonymous. The Unani Pharmacopoeia of India. Part I, Vol. I, Depart. of AYUSH, Ministry of
Health & Family Welfare, Govt. of India, New Delhi (2007) 7-8.
Anonymous. The Wealth of India. Vol. X (Sp-W), Publications and Information Directorate,
Council of Scientific and Industrial Research (CSIR), New Delhi (1982) 580-585.
Bhosale RS and More AD (2014), Effect of Gamma Radiation on seed germination, seedling
height and seedling injury in withania somnifera, (L) Dunal.
Devi PU, Sharada AC, Solomon FE, Kamath MS. In vivo growth inhibitory effect of Withania
somnifera (Ashwagandha) on a transplantable mouse tumor, Sarcoma 180. Indian J Exp
Biol 1992;30:169-172.
Jain SK, DeFillips RA (1991). Medical plants of India Refrence Publications, Inc Algonac,
Michigan, USA.
Khare CP. Indian Medicinal Plants–An Illustrated Dictionary. First Indian Reprint, Springer
(India) Pvt. Ltd., New Delhi (2007) 717- 718.
Leyon PV, and G. Kuttan (2004). Effect of Withania somnifera on B16F-10 melanoma induced
metastasis in mice. Phytother, Res., 18:118-122.
Obidoska, G. and A. Sadowska (2003). Effect of pH and nitrogen content of the substrate on the
yield of Withania somnifera (L.) Dun. Biulentyn Instytutu Hodowli Aklimatyzacji Roslin,
288:373-381.
Patra, DD, K. Singh, H.O. Mishra, A.K.Gupta, J. Singh, S.C. Singh and S.P.S. Khanuja (2004).
Agrotechnologies of Ashwagandha, J.Med. Aro. Plant Sci., 26:332-335.
Qamar Uddin, Samiulla L., Singh V. K. and Jamil S. S., Phytochemical and Pharmacological
Profile of Withania somnifera Dunal: A Review, Journal of Applied Pharmaceutical
Science 02 (01); 2012: 170-175.
Rastogi RP, Mehrotra BN. Compendium of Indian Medicinal Plants, Vol.6. Central Drug
Research Institute, New Delhi, 1998.

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 Development of in-situ moisture conservation practices for enhancing
productivity of the crops under moisture stress conditions: A potential R &
D area for Ashwagandha (Withania somnifera L. Dunal)

Nilofer, Rupal Singh, Anil Kumar Singh and Saudan Singh

Department of Agronomy, CSIR-Central Institute of Medicinal and Aromatic Plants,
Lucknow
Email id: absar.nilofer74@gmail.com

Withania somnifera L. Dunal, known as ‘Indian Ginseng’ (Singh and Kumar, 1998), is an
important medicinal plant used in the Indian traditional system of medicines, like Ayurveda
and Unani since ancient times. Its roots are used as a medicine. It is commercially grown in
the semi arid parts of tropical and sub-tropical zones as rainfed crop where there is
availability of proper moisture to complete its life cycle. Production of high quality roots is
the observed limiting factor in the plant. Hence, there is a need to find out suitable moisture
conservation practices to enhance its productivity, water use efficiency and economics. Under
rainfed conditions, availability of moisture to the crops can be enhanced through different in-
situ and ex-situ moisture conservation practices such as retention ditches, contour farming,
water harvesting by external catchment, contour furrows, stone lines, grass stripes, planting
pits, semi-circular bunds, earth basins, cover crop ,conservation tillage, organic mulch,
inorganic mulch, use of antitranspirant etc. Out of these, use of different types of mulches are
the cheap and simple options for in-situ soil moisture conservation and enhancing water use
efficiency in dry land farming. Therefore, in the present review, efforts have been made to
assess the effect of different types of mulches along with different levels of available soil
moisture, on soil environment, root productivity and water use efficiency of Ashwagandha.
Compiled information revealed that different types of mulches are found to be beneficial for
enhancing productivity under moisture stress/ limited water supply. However, no effort has
been made to find out suitable mulch for Ashwagandha grown under rainfed/moisture stress
condition.

Allolli et al. (2008) found that different moisture conservation practices viz. ridges and
furrows along with mulch, enhanced the vigor and also helped to promote the productivity of
cluster bean and chillies. Mulching is one of the important practices which are used to
conserve soil moisture and improve crop productivity. Mulch is usually but not exclusively
organic in nature and it is incorporated naturally into the soil by the activity of worms and
other organisms. The process is used in commercial crop production and in gardening and
when applied correctly, can dramatically improve soil productivity (Jumps up, 2008).
Mulching is beneficial in conserving the soil moisture, suppressing the weeds, improve the
soil fertility and modify the soil physical environment (Yoo- Jeong et al., 2003). Organic
mulches have been used for different medicinal and aromatic crops viz. Citronella java
(Singh et al, 2010), Basil (Palada et al 2000), Mulberry (Shashidhar et al, 2009) and
Geranium (Ram et al., 2003). Singh et al (1999) reported that productivity and WUE
improved significantly with dust mulch in Mentha arvensis. Kumar et al. (2014) reported that
mulching significantly increased organic carbon, available nitrogen, phosphorus, potassium,
bacterial and fungal population compared to unmulched plots and consequently growth of
Stevia. Fontana (2006) found that mulching technique favored growth of rosemary, thyme
and lavendor. Different types of mulches have been used to obtain good crop growth and
yield in crops like banana (Ssali et al, 2003), vegetables (Richards et al 2002), tomato

185 | P a g e
(Rahman et al 2006), pepper (Aliyelaabe and Fawusi, 1986) and strawberry (Kumar and Dey,
2011). Mulching also helps in faster plant emergence, early canopy development and higher
tuber yield in potato (Mohammad et al, 2002). Zamir (2013) also reported significant effect
of wheat straw mulch together with zero tillage on autumn planted maize. Arora and Bhatt
(2008) reported that mulch spread on the whole plot increased the grain and straw yield of
maize by 58.5 and 35.0 % as compared to unmulched control.

Similarly as reported by several workers, it may be observed that for crops described above,
different types of mulch may act as good moisture conservation practices in Ashwagandha,
grown either as rainfed crop or under limited supply of irrigation water. These practices may
bring significant improvement in the yield and quality of the roots of the Ashwagandha.
Singh et al. (2013) reported that mulching either by spreading of organic waste between rows
and plants or by creating dust mulch after every rain/irrigation, improved the dry root and
withanolide yield significantly only under rain fed condition and irrigation at 20% ASM, over
no mulches. Further, a study is being conducted at experimental farm of CSIR- Central
Institute of Medicinal and Aromatic Plants, Lucknow to evaluate performance of Withania
somnifera L. Dunal under different mulching practices like dust mulch, distillation waste &
control with different levels of available soil moisture (ASM) i.e. rainfed, 10% ASM, 20%
ASM, 30% ASM, 40% ASM, 50% ASM and control. The experiment is in progress.

From the review it may be concluded that organic as well as dust mulch is an important
aspect on which lot of research work has been done to improve productivity of a number of
traditional food and agricultural crops. However work on medicinal and aromatic crops is
meager and only few attempts has been made on Ashwagandha, which holds promising scope
for an Institute like ours.

REFERENCES

Aiyelaagbe, I.O.O., Fawusi, M.O.A., 1986, Growth and Yield response of pepper to mulching,
Biotronics 15, 25-29
Allolli, T.B., Hulihalli, U.K., Athani, S.I., 2008, Influence of in situ Moisture Conservation
Practices on the Performance of Dryland chilli. Karnataka J.Agric.Sci., 21(2): 253-255
Allolli, T.B., Hulihalli, U.K., Athani, S.I., 2008, Influence of in situ Moisture Conservation
Practices on the Performance of Dryland Cluster Bean. Karnataka J.Agric.Sci., 21(2):
250- 252
Arora, S. and Bhatt, R., http://Tucson.ars.ag.gov/isco/isco15/pdf/Arora%20S
Impact%20of%20improved%20Soil.pdf.
Fontana, E., Hoeberechts, S. and Nicola, S., 2006, Effects of mulching on medicinal and aromatic
plants in organic farm guest. ISHS, Acta Horticulture.723: 405-410
Jump up RHS A-Z, encyclopedia of garden plants. United Kingdom: Dorling Kindersley, 2008
pp: 1136
Kumar, R., Sood, S., Sharma, S., Kasana, R.C., Pathania, V.L., Singh, B., Singh, R.D., 2014,
Effect of plant spacing and organic mulches on growth, yield and quality of natural
sweetner plant Stevia and soil fertility in Western Himalayas. Int. J. of Plant Production
8(3) July 2014, pp: 311-332
Kumar.,S., Dey, P.,2011. Effect of different mulches and irrigation methods on root growth,
nutrient uptake, water use efficiency and yield of strawberry. Sci.Hortic. 127: 318-324
Mohammad, MM, Farooq, K., Hussain, A. and Sher, R., 2002. Effect of mulching on growth and
yield of potato crop. Asian J. Plant Sci.2: 132-133

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Palada, M.C., Crossman, S.M.A., Kolualski, J.A.,Collingwood, C.D.,2000. Evaluation of organic
and synthetic mulches for basil production under drip irrigation. J. Herbs Spices Med.
Plants. 6, 39-48
Rahman, M.I., Uddin, M.S.,Bagum, S,A.,Mondol, a.T.M.A.I.,Zaman. M.M., 2006. Effect of
mulches on the growth and yield of tomato in the coastal area of Bangladesh under
rainfed condition. Int. J. Sustain Crop prod. 1,pp: 6-10
Ram, M., Ram, D., Roy, S.K., 2003.Influence of an organic mulching on fertilizer nitrogen use
efficiency and herb and essential oil yield in Geranium. (Pelargonium graviolens).
Bioresource Technol. 87, pp: 273-278
Richard, P.L., Bracy, R.P., Rosendale, R.M., 2002, Use of plastic mulch for successive crops.
Vegetable crop prod. 8, 63-70
Shashidhar, K.R., Bhaskar, R.N., Priyadarshini, P.,Chandra kumar, H.L., 2009. Effect of different
organic mulches on pH, organic carbon content and microbial status of soil and its
influence on leaf yield of M5 mulberry (Morus indica L.) under rainfed conditions.
Current Biotica, 2, 405-413
Singh, A., Singh, M., Singh, S., 2010. Effective utilization of distillation waste as organic mulch
for weed management in the aromatic grasses, Citronells java. Int. J. Pest Manage. 47,
253-257
Singh, S., Singh, A. K., Lal, R.K., Gangwar, S.P., Sangwan, N. S.and Patra, D. D.(2013). Agro-
technology for enhancing productivity of dry roots and withanolides in Ashwagandha
(Withania somnifera Dunal) under sub-tropical climate of North India. Paper presented in
the International conference on Biologically Active Substances and Materials held at
Novy Svet, AR Crimea, Ukraine during May 27-June 1, 2013. Published in the
proceedings of the Conference Vol. 1 pp 241
Singh, S., Singh, A., and Singh, V.P., 1999. Use of dust mulch and antitranspirant for improving
water use efficiency of menthol mint (Mentha arvensis). J. of Med. And Arom. Plant
Sciences, 21: 29-33
Singh. S., and Kumar, S., 1998. Withania somnifera, The Indian Ginseng Ashwagandha.CSIR-
Central institute of Medicinal and Aromatic Plants, Lucknow, pp: 293
Ssali, H., McIntyre, B.D.,Gold, C.S.,kashaija, I.N., Kizito, F.2003. Effects of mulch and mineral
fertilizer on crop, weevil and soil quality parameters in highland banana. Nutr. Cycl.
Agroecosys. 65, 141-150
Yoo-Jeong, Y., Dungam, R.S., Ibekwe, A.M., Valenzuela-Solano, C., Crohn, D.M.,Crowley,
D.E.,2003. Effect of organic mulches of soil bacterial communities one year after
application. Biol. Fertil. Soils. 38, 273-281
Zamir, M.S.I., Javeed, H.M.R., Ahmed, W., Ahmed, A.U.H., Sarwar, N., Shehzad, M., Sarwar,
M.A., Iqbal, S., 2013. Effect of tillage and organic mulches on growth,yield and quality
of autumn planted maize (Zea mays L.) and soil physical properties. Ceretari Agronomice
in Maldova, 2(154): 17-27

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 Optimization of plant population in Ashwagandha (Withania somnifera L.
Dunal) under rainfed conditions

Rupal Singh, Nilofer, Anil Kumar Singh and Saudan Singh

Department of Agronomy, CSIR-Central Institute of Medicinal and Aromatic Plants,
Lucknow
Email: rupalsingh90@yahoo.in

ABSTRACT

A plant would perform better only when it is provided with optimum environmental
conditions. The time and method of sowing, plant population/spacing, weed management,
harvesting schedule and post harvest practices are the major environmental/agronomic
parameters for the optimization of growth, yield and quality of a crop. Among these
environmental parameters the establishment of adequate plant population per unit area is
most important to realize the full yield potential of a crop. Plant population refers to the
number of plants per unit area of land eg. 40,000 plants ha -1 or 4 plants sq m-1. Inter and intra
row spacing on the other hand refers to the arrangement of plants on the area planted e.g.,
varying plant spacing such as 100 cm × 10 cm, 50 cm × 20 cm and 40 cm × 25 cm; all give a
plant population of 10 plants sq m-1. Variation in plant population has been found to affect
growth and dry matter accumulation due to variability in the availability of above and
underground resources such as light, CO2, O2, moisture and nutrients. Higher plant densities
restrict the growth of branches and number of reproductive parts per plant but yield may be
compensated by increased population densities. Hence, efforts have been made to review the
information available on the effect of plant population on growth and yield of Ashwagandha
which has been compiled and analyzed in this paper. Further, it is found that this aspect has
important role in enhancing productivity of Ashwagandha. Several workers have worked out
optimum plant population for different agro-climatic conditions. However, no attempt has
been made to workout optimum plant population for the sub-tropical climate of central Uttar
Pradesh.

The optimization of plant population in Withania somnifera L. Dunal has demonstrated its
positive effect on the productivity of Ashwagandha under different agro-climatic conditions.
Pakkiyanathan et al., (2004) reported that maximum plant height, root length, number of root
fresh weight of shoot, was recorded at 30 cm × 10 cm spacing in Ashwagandha under sub-
tropical climate of Hyderabad, where as maximum number of leaves, fresh and dry weight of
root was recorded under 30 cm × 15cm spacing. The optimum spacing requirement for
Ashwagandha, where roots are the economic parts was studied by Agarwal et al., (2004) at
Jobner on loamy sand soil. They reported the longest roots at closer spacing (20 cm x 5 cm)
compared to wider spacing (25 cm x 7.5 cm). In an experiment conducted on clayey alkaline
soils. Nigam et al., (1984) observed significant increase in root yield of Ashwagandha at
higher plant density of 6.6 lakh ha -1 (30 cm x 10 cm) as compared to 4.4 lakh ha -1 (45 cm x
5 cm) and 2.2 lakh ha -1 (45 cm x 10 cm). Nigam and Kandalkar, (1985) found that the
population of 8.0 lakh ha-1 (30 cm x 5 cm) was optimum for higher root yield of
Ashwagandha on medium black soils of Mandsaur, as compared to wider spacing.

In an experiment conducted on sandy loam and light red soils at University of Agricultural
Sciences, Bangalore, Farooqui and Sreenivas, (2001) reported that the optimum plant
population was 20,000 to 25,000 ha-1 for harvesting higher root yield of Ashwagandha. In a
study conducted by Kubsad et al., (2009) at Annigeriin (Karnataka) during rabi 2004-05 and
188 | P a g e
2005-06, they found that at 15 x 10 cm spacing, the dry root yield increase was 9.9, 34.9 and
× 53.7% more than 15 cm x 5 cm, 30 cm x 10 cm and 45 cm x 10 cm respectively in
Ashwagandha. The maximum dry root yield was mainly related to better performance of
yield attributes due to more plants per hectare.

As different plant population was advocated for different agro-climatic condition, the field
studies were conducted by Singh et al., 2012-2013 (unpublished) to workout optimum plant
population for Ashwagandha under sub- tropical climate of central Uttar Pradesh. The crop
was grown as rainfed during September- March in 2012-13 at the experimental farm of
Central Institute of Medicinal and Aromatic plants, Lucknow. The results revealed that dry
root yield increased with increase in the plant population upto 5.00 lakh plants ha -1, however,
significant increase was recorded only upto 4.00 lakh plants ha -1; Further, the increase in the
plant population beyond this plant population resulted in decrease dry root yield. The
opposite trend was observed in dry shoot yield as it was highest at the lowest plant population
and lowest at the highest plant population. The quality and nature of the roots was accessed to
be comparatively less fiberous and more powdery which was found to be enhanced with
enhancing plant population. Thus, sowing Ashwagandha at the time of recession of monsoon
with plant population of 4.0 lakh plants ha -1at a spacing of 25 cm × 10 cm is recommended
for obtaining 10.2 q ha-1 root yield under rainfed conditions of Central Uttar Pradesh.

It is concluded from present review that optimization of plant population for Ashwagandha
under different agro-climatic conditions, and varying levels of input supply is an important
area for R & D work and it should be undertaken for enhancing productivity, quality in a cost
effective manner for this valued crop.

REFERENCES

Agarwal, M., Singh, P., Agarwal, M.K., 2004. Effect of sowing dates and spacing on yield
attributes and root yield of Ashwagandha. J Med Pl Sci. 26: 473- 474.
Farooqui, A.A., Sreenivas, B.S., 2001. Arom Med Pl, IBH Publications, New Delhi, pp, 27-34.
Kubsad, V.S., Palled, Y.B., Mansur, C.P. 2009. Productivity, quality and economics of rainfed
Ashwagandha as influenced by spacing and fertilizer levels. Indian J. Agron. 54: 449-
453.
Nigam, K.B., Kandalkar, V.S., 1985. Evaluation of genetic variability in Asgandh (W. somnifera
L. Dunal). Proceedings of vi All India workshop for Med Arom Pl., Bangalore, 22-25
December.
Nigam, K.B., Rawat, G.S., BhagawatPrasad, 1984. Effect of methods of sowing, plant density
and fertility levels on Ashwagandha .South India Hort32: 356-359.
Pakkiyanathan, K., Pasha, Y.N., Narayan Reddy, Y.,ArunSathe, 2004. Effect of spacing and
phosphorus levels on growth and root yield of Ashwagandha (withania somnifera
Dunal.), Indian J. of Hort. 61(2), 195-197.
Patel, K., Kushwaha, N.K., 2013. Studies on influence of Species, nitrogen and spacing on
parameters of plant growth at various stages of Basil. Intl. J. of Pharm. and Life Sci.
(IJPLS), vol.4(10), 3028-3034.
Shahjahan, M., Solaiman, A.H.M., Sultana, N., Kabir, K., 2013. Effect of organic fertilizers and
spacing on Growth and Yield of Kalmegh (Andrographis paniculata Nees). Intl J Agri
Crop Sci. Vol., 6(11), 769-775.
Shaw, S., van de Westelaken, T., Sorrenson, I., Searle, B., Hederley, D., 2008. Effect of plant
population and planting date on growth and development of kumara cultivar Owairaka
Red (Sweet potato). Agronomy New Zealand 38.

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Singh, A., Singh, M., Singh, H.P., Singh, S., Singh, K., 2005. Improvement in productivity and
weed competition in Palmarosa through differential spacing and nitrogen application.
Indian perfumer 49(2):201-209.
Singh, M., Singh, V.P., Singh, S., Saini,P., 2003. Optimum planting time and row spacing for
bergamot mint (Mentha citrata Ehrh.) var. ‘Kiran’ under sub-tropical plains of Central
Uttar Pradesh. Journal of Spices and Aromatic Crops Vol.12(2):135-138.
Singh, M., Tripathi R.S., Singh, S., Yaseen, M., 2008. Influence of row spacing and nitrogen
levels on herb and essential oil production and oil quality of Tagetes minuta L. Journal of
Spices and Aromatic Crops Vol. 17(3):251-254
Singh, S., Kumar, S., 1998, Withania somnifera “The Indian Ginseng Ashwagandha”. CSIR-
Central Institute of Medicinal and Aromatic Plants (CIMAP), Lucknow, pp.293
Singh, S., Singh, A.K., Nilofer, 2012-2013, Optimization of plant population in Ashwagandha
(Withania somnifera L. Dunal.,) under rainfed conditions of Central Uttar-Pradesh
(unpublished).

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 Scientific Cultivation of Ashwagandha (Withania somnifera)

Prawal Pratap Singh Verma, Anand Singh

CSIR-Central Institute of Medicinal and Aromatic Plants, Research Centre Purara-
Bageshwer- 263688 (Uttrakhand)
Email: prawal.psv@cimap.res.in

ABSTRACT

Ashwagandha (Withania somnifera Dunal.) belongs to the family solanaceae having

chromosome number 2n = 48. It is one of the commercial medicinal crops under rainfed
condition. The dried roots are rich source of ‘withanine’ and ‘somniferine’ (Thakur et al.,
1989). It is also known as Asgandh in Hindi and commonly known as Winter Cherry in
English. It is mentioned as an important drug in ancient Ayurvedic literature. It is cultivated
over an area of 10,780 ha with a production of 8429 tonnes in India. While the annual
demand increased from 7028 tonnes (2001-02) to 9127 tonnes (2004-05) necessitating the
increase in its cultivation and higher production (http://nmpb.nic.in). Ashwagandha mainly
found in Rajasthan (Nagour), Punjab, Haryana, Uttar Pradesh, Gujarat, Maharastra and
Madhya Pradesh. Ashwagandha is an important cultivated medicinal crop of India. It is also
well known in the traditional system of medicines of several countries for its sedative,
hypnotic and antiseptic properties and occasionally the leaves and seeds are also used for
medicinal purpose, root is economic part of the plant. The roots are used for curing
rheumatism, dyspepsia, skin diseases, bronchitis, ulcers, sexual debility and snakebite. W.
somnifera grows well in sandy loam or light red soil, having pH 7.5-8.0 with good drainage.
It can be cultivated between 600-1200 m altitudes. The semi-tropical areas receiving 500-750
mm rainfall are suitable for cultivation of this rained crop. The crop requires dry season
during its growing period. Temperature between 200C to 350C is most suitable for
cultivation. Late winter rains are conducive for the proper development of the plant roots.
The crop can be sown either by broad casting or in lines. Live to line method is preferred as it
increases root production and also helps in performing intercultural practices properly. The
seeds are usually sown about 1-3 cm deep in June- July in nursery. A light shower after
shower after sowing ensures good germination. About 500-750 gm seeds are sufficient for 1
ha field. Seeds can be treated, with Thiram or Indofil or Dithane medicinal plants - 45 (@ 3
gm/kg seed), before sowing to protect seedlings from seed borne diseases (FChandra et al,
2011). The seedling after 25-35 days after sowing can be transplanted in the field marinating
60 x 60 cm. Spacing between the plants & the rows. It may be noted that since
'Ashwagandha' is a rainy season Kharif crop, the time of sowing is decided by date of arrival
of monsoon in that area. The seeds sown by broadcasting or in the line in furrows should be
thinned out by hand at 25-30 days after sowing to maintain a plant population of about 30-60
plants per square meter (about 3.5 to 6 lakh plants/hectare). The plant density to be used may
depend on the nature and fertility of the soil. On the marginal land the population is kept
high. The medicinal plants have to be grown without chemical fertilizers and use of
pesticides. Organic manures like, Farm Yard Manure (FYM), Vermi-Compost and Green
Manure etc may be used as per requirement of the species (Raja et al., 2013). Light shower
after transplantation ensures establishment of seedlings. There is no need of irrigation if
rainfall is at regular intervals. Excessive rainfall/water is harmful to the crop. Life saving
irrigations may be applied, if required. The plants start flowering and bearing fruits from
December onwards. The crop is ready for harvest in January-March at 150 to 180 days after
sowing. The maturity of crop is judged by drying out of leaves and yellow red berries. The
entire plant is uprooted for roots which are separated from aerial parts by cutting the stem 1-2

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cm above the crown. The roots are then either cut transversely into small pieces (7 to 10 cm)
or dried as it is in the sun. About 650-800 kg roots can be obtained from 1 ha on drying it
comes to 350-435 kg (Shrivastava et al., 2013). Berries are hand plucked separately. They are
dried and crushed to take out the seeds. The dried roots, entire or transversely cut into smaller
pieces, have to be further cleaned, trimmed and graded. The roots are beaten with a club
which removes adhering soil and breaks off the thin, brittle lateral rootlets. Lateral branches,
root crown and stem remains on roots are carefully trimmed with the help of knife. On an
average yield from one hectare land under commercial cultivation is approx 3-5 quintals of
dried roots and 50-75 kg seeds.

REFERENCES

Chandra, V. and Pandey, M.C., 2011. Jadi butiyon ki kheti, Indian Agriculture Research
Institute, New delhi, 132-136.
http://nmpb.nic.in/WriteReadData/links/7624249622.
Raja, G. and Veerakumari, L., 2013. Influence of vermicomposts on the yield and alkaloid
content of withania somnifera. International Journal of advanced biological research,
vol. 3 (2): 223-226.
Shrivastava, A.K. and Sahu, P.K., 2013.Yield and quality parameter of alkaloids of Withania
somnifera (L) Danal. International Journal of Agronomy and Plant Production. Vol., 4
(12), 3246-3254.
Thakur, R.S., Puri, H.S. and Husain, A., 1989.Major medicinal plants of India, CIMAP,
Lucknow, 531-535.

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 Unravelling the Periwinkle genetics and breeding efforts in last five decades

Pranshu1, T.Jhang1 and RN Kulkarni2*

Department of Genetics and Plant Breeding,
1
CSIR-CIMAP, Lucknow, 2CSIR-CIMAP Resource Center , Allasandra, Bangalore
*Correspondence: rn.kulkarni@cimap.res.in

ABSTRACT

Madagascar Periwinkle [Catharanthus roseus (L) G. Don], is one of the most extensively
investigated medicinal plant due to its monoterpene indole alkaloid (MTIAs) which occurs
as hetero-dimers (vinblastine, vincristine) and monomers (Vindoline, catharanthine,
ajmalicine) with antitumoral and hypertensive properties. The dimeric alkaloid are present in
very low levels in the plant as its high amount is cytotoxic to the plant and restricted to
specific leaf cell types. Although remarkable efforts by chemists have yielded the total
synthesis of dimeric alkaloids they or their monomeric precursors, catharanthine and
vindoline, are still harvested and purified from periwinkle plants and the process of alkaloid
extraction and purification produces low yields at very high cost. Catharanthus roseus plant
is the only viable economic and commercial source of the vindoline and catharanthine, and,
despite significant efforts, cell cultures are not yet a valid alternative for alkaloid production.
Since plant in vivo is the only viable option till date, breeding for higher content of
monomers MTIAs vindoline and catharenthine in Catharanthus roseus is one of the major
challenge to meet its growing demand at domestic and international level. Here we have tried
to compile the work carried out on various aspects of genetics and breeding to help
understanding Catharanthus roseus plant better with the aim that the generated information
will help to overcome the major bottlenecks for Catharanthus breeding programme.
Mutations, both natural and artificially induced, provide a valuable resource in Catharanthus
breeding and in genomics research. Since most of the MTIAs pathway in Catharanthus is
now known, allelic polymorphisms survey in Catharanthus roseus large germplasm can be
tapped and can serve as a valuable tool for mining for SNPs in germplasm, assessing
heterozygosity, uncovering variants for MTIA content by detecting natural variants for MTIA
targeted breeding.

Vinca or Catharanthus?
The Madagascar periwinkle [Catharanthus roseus (L.) G. Don], the model medicinal plant
for alkaloid secondary metabolism, is widely adaptable, ever-blooming perennial herb or
sub-shrub. It has been variously designated as Vinca rosea L., Lochnera rosea Reichb. and
Ammocallis rosea Small. Carl Linnaeus in 1753 established the genus Vinca with two species
V. minor and V. major. In 1759, tropical V. rosea was added into the temperate genus Vinca
although it did not fit into its generic description with regard to stamens. Reichenbach was
the first to recognize V. rosea as generically different from Vinca (Stearn, 1975). George Don
while describing this genera in 1835, retained the name Vinca for the genus containing Vinca
minor, Vinca major and Vinca herbaceae and established the genus Catharanthus typified by
Vinca rosea further described as Catharanthus roseus. The genus Catharanthus consists of
seven species (including C. roseus) indigenous to Madagascar and one species C. pusillus
(Murr.) G. Don endemic to India. It was introduced into Paris in 1757 and from there to India
by 1794 (Morton, 1977). Native to Madagascar, the Madagascar periwinkle has spread
worldwide as a cultivated ornamental. It may now be found naturalized in almost every
tropical and subtropical region of the world, occurring on every continent except Antarctica

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Literature update
Catharanthus importance as a medicinal plant was recognized with the discovery of anti-
cancer alkaloids, vinblastine (VLB) and vincristine (VCR) in its leaves by Nobel et al.
(1958). This stimulated intensive research on this plant. A critical survey of literature
published on periwinkle during last four decades revealed that initial interest in this plant
were primarily due to its medicinal properties and till 1968 research was focused only on its
phytochemistry and pharmacological value. With established medicinal and economical value
research focused to enhance its alkaloids production through cell, tissue and hairy root
cultures. Era 1980 decade was dedicated to understand the Catharanthus plant physiology
and alkaloid biochemistry. From 1994 workers started working out the molecular biology of
the plant to overcome the bottleneck of alkaloid production. About 2941 scientific
publications have appeared on this plant from 1963- November,2014, with an average of
about 69 publications per year .The impetus comes from 1990 onwards with the discoveries
of genomic tools adding about 1721 more publications with an average of 86 publications per
year indicating most productive era of prime work. Thus, it is evident that the interest in this
plant has not declined but continued to increase. The low content of medicinally important
alkaloids (VLB and VCR) in this plant has led to considerable research mostly on the
production of these alkaloids through synthesis, semi-synthesis and cell and tissue cultures
(Moreno et al., 1995). An overall overview of number of publications indicates large work
have appeared in the area of physiology and biochemistry, cell and tissue cultures followed
by molecular biology and genetic engineering and phyto-chemistry , only 5.42 per cent of
publications are reported in agronomy and 5.44% in genetics and breeding.

Genetics and breeding efforts in last five decades

Periwinkle is a diploid plant species with a chromosome number of 2n = 16 (Furusato, 1940;
Simmonds, 1960; Dnyansagar and Sudhakaran, 1966; De Padua et al., 1992). Recently Hong
et al 2009 reconfirmed that karyotype formula in C. roseus is 2n=2x=16=2m=12sm=2T.
Although a number of publications have reported experiments upon artificially produced
autotetraploids in cultivation, it appears that wild populations are, so far as is known,
exclusively diploid. There are numerous reports on induced autotetraploidy in periwinkle
(Furusato, 1940; Schnell, 1941; Eigsti and Schnell, 1943; Eigsti and Tenny, 1943; Janaki
Ammal and Bezbaruah, 1963; Dnyansagar and Sudhakaran, 1977; Gogitidze and Laptev,
1981; Mohan Kumar, 1980; Kulkarni, 1984 Kulkarni et al., 1984; 1987; Krishnan et al.,
1985; Handique and Sharma, 1990; Chauhan and Raghuvanshi, 1993). Autotetraploids were
found to be more vigorous in growth with broader leaves, larger stomata, flowers, pollen
grains and embryos but had low pollen fertility, poor fruit set and low seed production as
compared with diploids (Janaki Ammal and Bezbaruah, 1963; Gogitidze and Laptev, 1981;
Mohan Kumar, 1980). Landraces and wild species of Catharanthus (possess an underused
source of novel alleles that have great potential for crop improvement of cultivated
Catharanthus species (C.roseus) since they possess new genes that could be exploited for
yield increases and for developing resistance to biotic stresses and tolerance of abiotic
stresses. Natural hybridization between periwinkle species has been observed in Madagascar
(Veyret, 1974; Markgraf, 1976) and most of these hybridizations were between C. longifolius
and C. roseus (Sevestre-Rigouzzo et al., 1993). Artificial hybridization and selfing
techniques have been employed for carrying out genetic studies (Flory, 1944; Simmonds,
1960; Rajasekaran et al., 1981; Levy et al., 1983; Milo et al., 1985 Sevestre-Rigouzzo et al.,
1993; Kulkarni et al., 2001). Here we have compiled the efforts carried out on different
aspects such as genetic diversity and gene surveys in parental material,interspecific
hybridization and exploiting wild species,self pollination or cross pollination,functional male
sterility,exploitation of heterosis for alkaloid content,genetics of flower colour and

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pubescence,genetics of plant height,mutation breeding,disease resistance and catharanthus
molecular breeding(Kumari et al 2013,Kulkarni and Bhaskaran 2013 and Kulkarni and Jhang
2014 per communications).

Future Perspectives
Growing knowledge from all prospective disciplines of Catharanthus study has enabled a
better understanding of biosynthetic pathway genes. Development of distinct and
characterized gentic resources indicated the pleiotropic action of a wide array of genes
involved in metabolic differentiation , physiology and development .Perception of effect of
various elicitors tried for enhancing the expression of key MTIA genes lead to the suggestion
that a central regulation by transcription factors and other regulatory genes may be involved
.Bottlenecks from the self produced toxic secondary metabolites can be overcome by utilizing
recombinatorial approaches of homologous and heterologous expression by genetic
transformation. Wide hybridization with C. pussilus should be undertaken for molecular
characterization to understand failure of accumulation of MTIA. Breeding for higher content
of vindoline and catherenthine in Catharanthus roseus is one of the major challenges to meet
its growing demands at domestic and international level.

REFERENCES

Kulkarni RN and Jhang T.2014 .Unravelling the Periwinkle genetics and breeding efforts in
last five decades. Plant Genetic Resources and Utilization ( In communication).
Kulkarni RN, Baskaran K. 2013 From herkogamy to cleistogamy--development of
cleistogamy in periwinkle. J Hered. 2013 Jan-Feb;104(1):140-8.
Kumari R, Yadav G, Sharma V, Sharma V, Kumar S.2013 Cytosine hypomethylation at CHG
and CHH sites in the pleiotropic mutants of Mendelian inheritance in Catharanthus
roseus. J Genet. 2013 Dec;92(3):499-511

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 MICROBIAL

TECHNOLOGY AND

CROP PROTECTION
 A plethora of endophytic microorganisms interacting with Catharanthus
roseus

Sucheta Singh, Shiv Shanker Pandey, Deepamala Maji, Pooja Mishra, Samvedna
Shukla, Alok Kalra
Microbial Technology Department, CSIR-Central Institute of Medicinal and Aromatic Plants,
Lucknow, India-226015
Email: suchetasingh05@gmail.com

ABSTRACT

Plants may be considered complex microecosystems where different niches are exploited by a
number of organisms. Varieties of organisms reside not only on the external surfaces of
plants but also in internal tissues. Such organisms that inhabit internal tissues without
apparent harm to the host are called as endophytic organisms (Tiwari et al., 2010). These
organisms mainly endophytic microorganisms are involved in multitrophic interactions with
plant and diversely localized having remarkable functional lifestyles (Ryan et al., 2007).

Majority of biologically active molecules (>1 million) have been discovered from plants, but
microorganisms are also a rich source of more than 20,000 biologically active compounds.
Some common examples like gibberellins derived from Fusarium fujikuro, Taxol from Taxus
brevifolia associated fungi (Kharwar et al., 2008) and many other compounds from
Streptomyces genus.

Endophytic microorganisms may originate from rhizospheric soil microorganisms and being
adapted to the specific plant environment. Sometimes they contribute to the production of
metabolic compounds of host plant by following ways: (1) Some metabolites are produced
by the combined association of microorganisms (endophytes) and plants, for example flavour
of strawberries, where furanoids are responsible for the typical fragrant are produced by plant
associated methylobacteria (Koutsompogeras et al., 2007). (2) Some endophytes may
strongly influence the performance, growth and stress tolerance of the plants and may even
enhance the secondary metabolites e.g In Artemisia annua, actinobacterium,
Pseudonorcardia sp. is able to enhance the production of the antimalarial compound
artemisinin (Li et al., 2012). (3) Some other endophytes themselves may be the source of
bioactives like Fusarium proliferatum, a fungus isolated from plume poppy or Bocconia
cordata plant produces antibacterial, anthelmintic, anti-inflammatory compound sanguinarine
(Wang et.al, 2014) and production of vinblastine and vincristine from endophytic Fusarium
oxysporum fungus (Kumar et.al, 2013) isolated from Catharanthus roseus plant.

Catharanthus roseus, belonging to Apocyanaceae family is ornamental and medicinal plant

of enormous pharmaceutical interest because it is nothing less than a chemical factory
producing more than 130 terpenoid indole alkaloids (TIAs) (Shukla et al., 2012). The alkaloid
content is highest at the flowering stage. The principle alkaloids present in the plant are
vindoline, vincristine, vinblastine, vincarodine, vincoline, leurocolombine, vinamidine,
vincathicine, vincubine, isosotsirikine, vincolidine, lochrovicine, catharanthine, leurosine,
lochnerine, tetrahydroalstonine, vindolinine, ajmalicine, serpentine, reserpine,
coronaridine,11-methoxy tabersonine, tetrahydroalstonine, ajmalicine, vindorosidine but only
five alkaloids vinblastine, vincristine, 3,4-anhydrovinblastine, serpentine and ajmalicine are
being marketed (Hisiger, et. al., 2007).

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Endophytic microorganisms have been proved to be a rich source of structurally novel and
biologically active secondary metabolites and therefore, have become an attractive resource
for new compounds ever since the discovery of penicillin in 1929. Certain endophytes are
capable of producing secondary metabolites similar to those produced by the host plants.
Concisely, the utilization of the endophytic microorganisms could be harnessed as microbial
cell factory for obtaining non-plant-based bioactive compounds having broader therapeutic
applications.

Vincristine and vinblastine and their precursor vindoline accumulates in C. roseus leaves at
very low amounts (0.0003 to 0.01 %) (Rai et al., 2013). These compounds are currently being
obtained from natural plant sources which limits their supply. Since the chemical synthesis of
these alkaloids has proved to be inopportune and not economically viable, they are
exclusively isolated from plants to cater to the need of the pharmaceutical industry. A
suitable alternative source could be helpful to meet the demand of these compounds.
Microbial production can provide not only an inexpensive, streamlined method of production
but also new analogues with differential drug potential (Kusari et al., 2014). For this reason,
isolation and characterization of endophytic microorganisms are being considered as an
alternative solution. Fusarium oxysporum, endophytic fungus isolated from C. roseus can
produce the anticancer drugs vinblastine and vincristine to an extent of 76µg/liter and
67µg/liter concentration respectively with properties similar to the plant derived vinblastine
and vincristine (Kumar et.al, 2013).

Kharwar et al., (2008) isolated 183 endophytic fungi representing 13 fungal taxa from leaf,
stem and root tissues of C.roseus. They observed that root tissues were heavily colonized by
genera such as Alternaria, Cladosporium and Aspergillus. However, leaf tissues showed a
greater diversity of endophytes and Drechslera, Curvularia, Bipolaris, Alternaria and
Aspergillus spp. and a unidentified fungus (not having fruiting structure) were the dominant
fungi isolated. The root tissues had maximum colonization. It decreases from stem to leaf and
seeds had lowest colonization. Endophytic colonization or existence was proved by gfp
tagged strain in C. roseus (Torres et al., 2013).

Tiwari et al., (2012) isolated Staphylococcus sciuri and Micrococcus sp., from C. roseus.
When inoculated these endophytes, performed very well substantially improving plant
growth parameters, host defence response and enhanced alkaloids yields in inoculated plants
compared to control plant of C. roseus. Micrococcus sp. was outperformed Staphylococcus
sciuri and significantly increasing the root weight (65.81%), shoot weight (25.39%), leaf
weight (35.51%) besides, significant enhancement of vindoline content (68.51%) in leaves,
serpentine (54.74%) and ajmalicine content (46.34%) in roots of field grown-plants. But
endophytes could not significantly enhance the content of vincristine probably because of its
cytotoxicity and storage problems in plants. This experiment clearly established that
endophytes can enhance the alkaloids yield directly through increasing in planta content and
indirectly through enhancement in plant biomass. Besides, multifarious endophytes should be
tested as consortia as they will give tangible benefits in plants. In a recent study, culture
filtrate of Trichoderma harzianum, a fungal endophyte boosted the biomass accumulation and
alkaloid content in Vinca minor hairy roots and cell suspension grown in bioreactor (Verma
et al., 2014).

The in planta content of the terpenoid indole alkaloids is low and their total scaffold
generating chemical synthesis, although feasible, is not commercially appropriate (Tiwari et
al., 2012]. Thus presently, in spite of synthetic chemistry and biology efforts, the plant is still

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the more pertinent source for these alkaloids. For the commercial requirement of the
bisindole alkaloids, the best option lies in a semi-synthetic or synthetic biology approach
from their precursors, catharanthine and vindoline and to predict the remaining essential
component in the search for new medicines. Though catharanthine is available from number
of sources but vindoline is accessible only from the plant source (Shukla et al., 2010).
Vindoline is formed in green plant in the presence of natural light source.

Endophytic microorganisms produce variety of secondary metabolites themselves and when

inoculated on medicinal plant can promote abundant production of these life-saving drugs, in
an eco-friendly, non-toxic manner which will be easily available at reduced price. Thus, these
natural products from microbes can promote “effective healing & wellness practices that do
no harm to people and the global environment” in comparison to synthetic drugs causing
various harmful side effects to human beings. The potential of endophytic organisms, having
desirable functional traits will be a part of establishment of red biotechnology (Kusari et al.,
2014). In future, these discoveries will be used into industrial bioprocesses scale for
commercial production of biopharmaceuticals. However, these sustainable strategies can only
be build through optimizing conditions for their growth and secondary metabolites
production that can lead to harnessing of endophyte mediated pharmaceutical products.

REFERENCES

Hisiger S, Jolocoeur M, (2007) Analysis of Catharanthus roseus alkaloids by HPLC. Phytochem

Rev 6: 207-234
Kharwar RN, Verma VC, Strobel G, Ezra D (2008). The endophytic fungal complex of
Catharanthus roseus (L.) G.Don. Curr. Sci. 95(2): 228
Koutsompogeras P, Kyriacou A, Zabetakis I (2007). The formation of 2,5-dimethyl-4-hydroxy-
2H-furan-3-one by cell-free extracts of Methylobacterium extorquens and strawberry
(Fragaria X ananassa cv. Elsanta). Food Chem. 104 :1654–1661.
Kumar A, Patil D, Rajamohanan P R, Ahmad A, (2013) Isolation, Purification and
Characterization of Vinblastine and Vincristine from Endophytic Fungus Fusarium
oxysporum Isolated from Catharanthus roseus. PLos ONE 8(9): e71805.doi
:10.1371/journal.pone.0071805
Kusari S, Singh S, Jayabaskaran C (2014). Biotechnological potential of plant-associated
endophytic fungi: hope versus hype., Trends Biotechnol. S0167-7799(14)00059-6
1
Li J , Zhao GZ, Varma A, Qin S, Xiong Z, Huang HY, Zhu WY, Zhao LX, Xu LH, Zhang S, Li
WJ (2012). An Endophytic Pseudonocardia Species Induces the Production of Artemisinin
in Artemisia annua. PLoS ONE . 7(12):e51410.
Rai A, Smita SS, Singh AK, Shanker K, Nagegowda AD (2013). Heteromeric and Homomeric
Geranyl Diphosphate Synthases from Catharanthus roseus and Their Role in Monoterpene
Indole Alkaloid Biosynthesis. Mol Plant.1531-49
Ryan R, Germaine K, Frank A, Ryan D, Dowling D (2007) Bacterial endophytes: recent
developments and applications. DOI:10.1111/j.1574-6968.2007.00918.x
Shukla AK, Shasany AK, Khanuja SPS (2012). cDNA-AFLP-based numerical comparison of leaf
and root organ cDNAs in Catharanthus roseus. OMICS 16: 397–401.
Shukla AK, Shasany AK, Verma RK, Gupta MM, Mathur AK, Khanuja SPS (2010). Influence of
cellular differentiation and elicitation on intermediate and late steps of terpenoid indole
alkaloid biosynthesis in Catharanthus roseus. Protoplasma 242: 35–47.
Tiwari R, Awasthi A, Mall M, Shukla AK, Srinivas KVNS, Syamaundar KV, Kalra A (2012)
Bacterial endophyte-mediated enhancement of in planta content of key terpenoid indole
alkaloids and growth parameters of Catharanthus roseus. Indu. Crops and Prod. 43 (2013)
306-310

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Tiwari R, Kalra A, Darokar MP, Chandra M, Aggarwal N, Singh AK, Khanuja SPS (2010).
Endophytic bacteria from Ocimum sanctum and their yield enhancing capabilities. Curr.
Microbiol. 60:167e171.
Torres AR, Araujo WL, Cursino L, Rossetto PB, Mondin M, Hungria M, Azevedo JL (2013)
Colonization of Madagascar periwinkle (Catharanthus roseus ) by endophytes encoding
gfp marker. Arch Microbiol 195:483-489
Verma P, Khan S, Mathur A K, Shanker K, Kalra A, (2014) Fungal endophytes enhanced the
growth and the production kinetics of Vinca minor hairy roots and cell suspensions grown
in bioreactor. Plant Cell Tiss Organ Cult. 118:257-268
Wang XJ, Min CL, Ge M, Zuo RH (2014). An Endophytic Sanguinarine-Producing Fungus from
Macleaya cordata, Fusarium proliferatum BLH51.Curr. Microbiol. 68:336–341

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 Viruses: Invaders of a high-valued alkaloid crop (Catharanthus roseus)

Asifa Khan1, Seema Dharni2, Sana T. Saeed1, Abdul Samad1

1
Plant Pathology Department, 2Agronomy and Soil Science, CSIR-Central Institute of
Medicinal and Aromatic Plants, Lucknow – 226015.
Email:asifakhan101@gmail.com

INTRODUCTION

Catharanthus roseus (family Apocyanaceae), commonly known as periwinkle, is a perennial

herb. It is cosmopolitan in distribution, mostly grown in tropical countries and cultivated in
Madagascar, India, Israel and USA (Stearn, 1975). It is famous for its medicinal properties
that are derived from its various alkaloids (Svoboda and Blake, 1975). The major alkaloids
vinblastine and vincristine derived from the C. roseus are well known for its anticancerous
properties. The alkaloids derived from this plant have also been used in the treatment of
various diseases, such as diabetes, high blood pressure and some of them also act as powerful
tranquilizers (Favali et. al., 2004).

Viruses are microscopic pathogens that consist of small nucleic acid molecule surrounded by
a protein coat. The viral genome carries the information of a few proteins and depends on the
cellular machinery of host for their reproduction and protein synthesis. Different types of
viruses infect almost all the types of living organisms including animals, plants, fungi, and
bacteria. Plant viruses affect many economically and commercially important plants and are
responsible for huge losses in crop production and quality worldwide. The virus infected
plants show typical symptoms that include leaf yellowing (either whole leaf or in a pattern of
stripes or blotches), leaf distortion or curling stunting, abnormalities in flower or fruit
formation and overall retarded plant growth.

Thus it is important to study the various viruses that are affecting the growth and cultivation
of a plant with such important pharmaceutical properties. In the present review emphasis has
been given on the viruses which cause fatal diseases in C. roseus in recent years.

2. Cucumber mosaic virus (CMV)

Cucumber mosaic virus belongs to the genus Cucumovirus of Bromoviridae family with a
vast range of hosts that include the agricultural, ornamental and commercially important
medicinal and aromatic crops (Roossinck, 1999). It is transmissible mechanically and by
aphids in a non-persistent manner (Palukaitis et. al., 1992). Its symptoms include leaf mosaic,
ringspots, yellowing, stunting of the stem, and distortion of leaves, flowers and fruits.

CMV was first reported in Cucumis sativus from the USA (Prince, 1934). In India, CMV has
been reported to infect several important hosts such as Egyptian henbane (Samad et al. 2000),
Gladiolus (Raj et al. 2002), Lycopersicum esculentum (Sudhakar et al. 2006), Banana
(Aglave et. al., 2007), Rauvolfia serpentina and Jatropha curcas (Raj et. al., 2007, 2008) and
C. roseus in India (Samad et.al., 2008). Recently, it has been confirmed to have spread even
in Korea (Choi et. al., 2014).

Under transmission electron microscope it showed the presence of spherical viral particles of
~28 nm in diameter and serological method such as DAS-ELISA is used to detect its coat
potein from the sap of infected plant samples. The presence of CMV was also confirmed by
reverse transcription (RT)-PCR using CMV CP specific primers, which amplified the CP

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gene (657 bp). The CIMAP-India C18 isolate (EU310928) identified as strains of Cucumber
mosaic virus (CMV) and revealed the closest identity (98%) with Rauvolfia serpentina isolate
of CMV which belongs to subgroup IB.

3. Potyvirus
Potyviruses (family Potyviridae), a ssRNA virus, is named after Potato virus Y. More than
200 species of aphids belong to subfamily Aphidinae (genera Macrosiphum and Myzus) have
been reported as vector. It is also mechanically transmissible. The virus particles are
filamentous in shape with 680- 900 nm in length and 11-20 nm in diameter. The nucleocapsid
is made up of about 2000 copies of capsid protein. These viruses have a wide host range, out
of which two have been reported to be infecting C. roseus which are Catharanthus mosaic
virus and Lettuce mosaic virus.

3.1 Catharanthus mosaic virus (CatMV)

Maciel et. al. (2011) reported a new potyvirus causing mosaic and flower variegation in C.
roseus and named Catharanthus mosaic virus (CatMV). Mandevilla, an ornamental tropical
vine popular for its bright and attractive flowers has been infected by CatMV (Mollov et. al.,
2014). Filamentous virus particles with modal lengths 700-900 nm were observed by TEM in
partially purified preparations from symptomatic leaves. It was transmitted by vectors Aphis
gossypii and Myzus nicotianae. It was mechanical transmissible in C. roseus and N.
benthamiana and developed systemic mosaic, whereas C. amaranticolor and C. quinoa
exhibited chlorotic local lesions.

3.2 Lettuce mosaic virus (LMV)

A new isolate of Lettuce mosaic virus (LMV- Cr) reported from C. roseus, showed no close
affinities to previously known isolates of LMV. It therefore formed a novel clade of LMV
(Dumas et. al., 2014).

4. Geminivirus
Geminiviruses are very important plant pathogens and are characterized by twinned
icosahedral particles of ~18 x 30 nm size, containing circular single-stranded DNA genomes
of 2.5–3.0 kb length (Hanley-Bowdoin et. al., 2000). These viruses infect most of the
agriculturally important crops and cause significant yield loss throughout the world.
Begomovirus is the largest genus of the family geminiviridae, transmitted by
whitefly (Bemisia tabaci) and cause severe disease in dicot plants such as tomato, cotton,
chilli, soybeen etc. including some important medicinal and aromatic plants.

4.1 Catharanthus yellow mosaic virus (CYMV)

The first begomovirus infection has been reported on Catharanthus roseus in 2013, as
irregular yellow mosaic symptoms with severe curling and leaf distortion. The causal virus
was characterized as Catharanthus yellow mosaic virus (Ilyas et. al., 2013). Recently, we
have also reported the infection of the same virus on Andrographis paniculata plants (Khan
et.al., 2014). The infected plants initially showed symptoms like chlorotic streaks and later on
complete yellowing of the leaves, ultimately lead to premature plant death. Based on the
symptomology, heavy infestation of whiteflies (Bemisia tabaci) in the infected fields and lack
of mechanical transmission, the association of a begomovirus was suspected which was
confirmed via molecular detection methods. This study highlighted the spread of CYMV
from its preliminary to other new host (A. paniculata), across the south Asian countries.

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CONCLUSION

Plant viruses are very smart and devastative pathogen that cannot be directly controlled but
preventive measures as controlling of transmitting vectors, developing and promoting the
resistant varieties, using virus-free planting materials for vegetative propagation could be
helpful in the management of pathogen/diseases. More focused research is required for the
development of transgenic and or disease resistance varieties. Moreover, the recent advances
in detection methods, it is now possible to quickly identify and characterize viruses present in
single or mixed infections in plants. Several certified programs for many crops are used
which are based on these newly developed methods. The significant rise in the number of
viruses infecting periwinkle has been observed in the last few years and it may be time to
take up appropriate control measures.

REFERENCES

Aglave B.A., Krishnareddy M., Patil F.S., Andhale M.S. (2007). Molecular Identification of a
Virus Causing Banana Chlorosis Disease from Marathwada Region. International Journal
of Biotechnology and Biochemistry 3, 13-23.
Choi S.K., Cho I.S. and Choi G.S. (2014). First Report of Cucumber mosaic virus in
Catharanthus roseus in Korea. Plant Disease, 98(9), 1283.
Dumas L.S., Verdin E., Faure C., German-Retana S., Gognalons P., Danet J.L., Marais A. and
Candresse T. (2014). Adaptation of Lettuce mosaic virus to Catharanthus roseus Involves
Mutations in the Central Domain of the VPg. Molecular Plant Microbe Interactions,
27(5), 491-497.
Favali M.A., Musetti R., Benvenuti S., Bianchi A. and Pressacco L. (2004). Catharanthus roseus
L. plants and explants infected with phytoplasmas: alkaloid production and structural
observations. Protoplasma, 223: 45-51.
Hanley-Bowdoin L., Settlage S.B., Orozco B.M., Nagar S. and Robertson D. (2000).
Geminiviruses: models for plant DNA replication, transcription, and cell cycle regulation.
Critical Reviews in Biochemistry and Molecular Biology 35, 105–140.
Ilyas M., Nawaz K., Shafiq M., Haider M.S. and Shahid A.A. (2013). Complete nucleotide
sequences of two begomoviruses infecting Madagascar periwinkle (Catharanthus roseus)
from Pakistan. Archives of Virology, 158, 505–510.
Khan A., Saeed S.T. and Samad A. (2014). New Record of Catharanthus Yellow Mosaic Virus
and a Betasatellite Associated with Lethal Leaf Yellowing of Kalmegh (Andrographis
paniculata) in Northern India. Accepted for publication. Plant Disease,
http://dx.doi.org/10.1094/PDIS-08-14-0862-PDN.
Maciel S.C., da Silva R.F., Reis M.S., Jadão A.S., Rosa D.D., Giampan J.S., Kitajima E.W.,
Rezende J.A.M. and Camargo L.E.A. (2011). Characterization of a new potyvirus causing
mosaic and flower variegation in Catharanthus roseus in Brazil. Scientia Agricola, 68(6),
687-690.
Mollov D., Guaragna M.A., Lockhart B., Rezende J.A.M. and Jordan R. (2014). First report of
Catharanthus mosaic virus in Mandevilla in the United States. Accepted for publication.
Plant Disease. doi.org/10.1094/PDIS-09-14-0913-PDN.
Palukaitis P., Roossinck M.J., Dietzgen R.G., Francki R.I.B. (1992). Cucumber mosaic virus.
Advances in Virus Research, 41, 281-348.
Prince W.C. (1934). Isolation and study of some yellow strains of Cucumber mosaic virus.
Phytopathology 24, 743-761.
Raj S.K., Kumar S., Pratap D., Vishnoi R., Snehi S.K. (2007). Natural Occurrence of Cucumber
mosaic virus on Rauvolfia serpentina, a new record. Plant Disease, 91, 322.

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Raj S.K., Kumar S., Snehi S.K. (2008). First Report of Cucumber mosaic virus on Jatropha
curcas in India. Plant Disease, 92, 171.
Raj S.K., Srivastava A., Chandra G., Singh B.P. (2002). Characterisation of Cucumber mosaic
virus isolate infecting Gladiolus cultivars and comparative analysis of serological and
molecular methods for sensitive diagnosis. Current Science 83, 1132-1136.
Roossinck M.J. (1999). Cucumoviruses (Bromoviridae) - general features. In ‘Encyclopedia of
Virology’. 2nd edn (Eds L Granoof, RG Webster) Academic Press: San Diego. pp. 315-
320.
Samad A., Ajayakumar P. V., Gupta M. K., Shukla A. K., Darokar M. P., Somkuwar B. and
Alam M. (2008). Natural infection of periwinkle (Catharanthus roseus) with Cucumber
mosaic virus, subgroup IB. Australasian Plant Disease Notes, 3, 30-34.
Samad A., Raj S.K., Srivastava A., Chandra G., Ajayakumar P.V., Zaim M., Singh B.P. (2000).
Characterization of a Cucumber mosaic virus isolates infecting Egyptian henbane
(Hyoscyamus muticus L.) in India. Acta Virologica, English Ed. 44, 131-136.
Stearn W.T. (1975). A synopsis of the genus Catharanthus (Apocynaceae). In ‘Catharanthus
alkaloids’. (Eds WL Taylor, NR Fransworth) Marcel Dekker: New York. pp. 9-44.
Sudhakar N., Nagendra-Prasad D., Mohan N., Murugesan K. (2006). First Report of Cucumber
mosaic virus Subgroup II Infecting Lycopersicon esculentum in India. Plant Disease, 90,
1457.
Svoboda G.H., Blake D.A. (1975). The phytochemistry and pharmacology of Catharanthus
roseus. In ‘Catharanthus alkaloids’. (Eds WL Taylor, NR Fransworth). Marcel Dekker,
New York. pp. 45-124.

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 Current scenario of fungal diseases in Withania somnifera and its
sustainable management
Arvind Saroj,*Abdul Samad
Department of Plant Pathology, CSIR-CIMAP, Luknow- 226015
*Email: amicro27@gmail.com

ABSTRACT

Attention in traditional medicines and, in particular herbal medicines, has increased substantially
in both developed and developing countries over the past two decades. Global and national
markets for medicinal herbs have been increasing significantly. On the parameter of, the safety
and quality herbal medicines have become important concerns for health authorities and the
public. The safety and quality of raw medicinal plant materials and finished products depend on
factors that may be classified as intrinsic (genetic) or extrinsic (environment, collection methods,
cultivation, harvest, post-harvest processing, transport and storage practices) (Essono et al.,
2007). Inadvertent contamination by microbial or chemical agents during any of the production
stages can also lead to deterioration in safety and quality. Withania somnifera (Ashwagandha) is
extensively utilized in preparation of medicines in Ayurvadic system. W. somnifera is native to
India and commercially cultivated in different parts of the country. It promotes strength and vigor
as an aphrodisiac and rejuvenator, in treatment of rheumatism, inflammation of joints and certain
paralytic conditions (Basu and Kirtikar, 1991). Fungal plant pathogens are among the most
important factors that cause serious losses to agricultural products every year. Monitoring of
health and detection of diseases in plants and trees is critical for sustainable agriculture. Four
fungal diseases have been reported on W. somnifera plant since 2007, Pithomyces chartarum
causing leaf blight (Verma et al. 2007), Alternaria dianthicola causing leaf spot (Maiti et al.
2007), Choanephora cucurbitarum causing stem wet rot (Saroj et al. 2012) and
Pseudocercospora fuligena causing black leaf spot (Saroj et al. 2014). Interestingly all above
reported diseases affect foliar part of the plant and resulting in decrease of productivity of the
crop.

The application of pesticides for effective pest’s prevention and control of various diseases are
most popular and beneficial method applied in agriculture. At present application of synthetic
fungicides such as Blitox 50, Bavistin etc. are used for the treatment/ management of plant foliar
diseases. However, the consistent use of fungicides could be a risk to the environment,
particularly if residues endure in the soil or percolate off-site and enter waterways (e.g. due to
spray drift, run-off) (Kibria et al., 2010; Komarek et al., 2010). Regular use of synthetic
fungicides, may also lead to the development of resistant phytopathogens (Johnson et al., 1994;
Ishii, 2006) in future. Since last many decades, synthetic fungicides are used to protect plants
from fungal diseases and now their fatal/ hazardous effect to ecosystem has been realized. After
application, pesticide enters into ground water, lakes and marine water by various environmental
processes. Moreover, some of the recent studies revealed that the aquatic organisms are being
adversely affected by increased use of pesticides.

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Therefore, biological and plant products were thoroughly investigated for their antifungal
activity. Bio-control of plant diseases including fungal pathogens has been considered a viable
alternative method to chemical control. In plant pathology, the term biocontrol applies to the use
of microbial antagonists to suppress diseases. But it is also having some limitation for
commercial use. Mainly bio-control showed variable performances under different environmental
conditions in the field. Strategy to overcome this problem it is important to develop new
formulations and application method of biocontrol microorganisms with advanced level of
stability and survival.

Keeping in view the current scenario, it is essential to adopt safety concerns over the synthetic
antimicrobial chemicals in plant protection. We have focused to explore the potential applications
of essential oils as an alternate to chemical control measures. Essential oils have a broad spectrum
of anti-fungal properties (Kalemba and Kunicka, 2003; Sokovi´c and vanGriensven, 2006; Carmo
et al., 2008) and they are eco-friendly (biodegradable, do not leave toxic residues or by-products
to contaminate the environment) (Abdel-Kader et al., 2011) and have been used for the
management of several diseases and results were highly encouraging (Sharif et al. 2010).
Essential oil isolated from Cinnamomum camphora and Ocimum spp and its components were
investigated for antifungal activity against Withania wet rot pathogen (Pragadheesh et al. 2013a,
2013b) while other essential oils such as citronella oil (Seenivasan et al. 2006), lemon, oregano
(Vitoratos et al. 2013) were also studied against other plant pathogens. In vitro antifungal assay
showed many potent compounds of essential oil. Here, I would like to mention that we should
plan our research in such a way that we could be able to prepare sustainable fungicides for the
management of Ashwagandha diseases.

REFERENCES

Abdel-Kader, M., El-Mougy, N., Lashin, S., 2011.J. Plant Prot. Res. 51, 306-313.
Basu B. D., Kirtikar K. R., 1991. Indian Medicinal Plants: Plates, vol. 1-4. Bishen Singh
Mahendra Pal Singh, Dehradun, India.
Carmo E.S., de Oliveira Lima E., de Souza E.L., 2008. Braz. J. Microbiol. 39, 362-367.
chemicals. Environmental and biological aspects. New India Publishing Agency, Pitam
Essono G., Ayodele M., Akoa A., Foko J., Olembo S., Gockowski J., 2007. Afr. J. Microbiol.
Res. 1, 01-08.
Ishii H., 2006. Jpn. Agric. Res. Q. 40, 205–211.
Johnson K.B., Sawyer T.L., Powelson M.L., 1994. Plant Dis. 78, 572-577.
Kalemba D., Kunicka A., 2003. Curr. Med. Chem. 10, 813-829.
Kibria G., Yousuf Haroon, A.K., Nugegoda D., Rose G., 2010. Climate change and
Komarek M., Cadkova E., Chrastny V., Bordas F., Bollinger J.C., 2010. Enviro. International. 36;
138 – 151.
Maiti C.K. , Sen S., Paul A.K., Acharya K., 2007. Plant Dis. 91, 467.
Pragadheesh V.S., Saroj A., Yadav A., Chanotiya C.S., Alam M., Samad A., 2013a. Ind. Crop.
Prod. 49, 628-633.
Pragadheesh V.S., Saroj A., Yadav A., Samad A., Chanotiya C.S., 2013b. Ind. Crop. Prod. 50,
333-337.
Pura, New Delhi.
Saroj A., Kumar A., Srivastava A., Khaliq A., Absar N., Alam M., Samad A., 2014. Plant Dis.
98, 1275.

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Saroj A., Kumar A., Qamar N., Alam M., Singh H.N., Khaliq A., 2012. Plant Dis. 96, 293.
Seenivasan P., Manickkam J., Savarimuthu I., 2006. BMC Complementary and Alternative
Medicine 6, 39.
Sharif M. A., Atiqur R., Yunus A., Sun C.K., 2010. Pestic Biochem Phys. 96, 86–92
Sokovi´c M., van Griensven J.L.D., 2006. Eur. J. Plant Pathol. 116, 211-224.
Verma O.P, R.B.L. Gupta R.B.L., Shivpuri A., 2007. New Dis. Rep. 15, 47.
Vitoratos A., Bilalis D., Karkanis A., Aspasia Efthimiadou A., 2013. Not Bot Horti Agrobo. 41,
86-92

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Endophytes and PGPRs in artemisinin research: status and potential

Vikas Kumar Patel*, Deepamala Maji, Deepti Barnawal, Alok Kalra

Microbial Technology Department, CSIR-Central Institute of Medicinal and Aromatic Plants,

Lucknow (226015), India
*Email: vikaspatel15@gmail.com

ABSTRACT

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The last decade has seen an emergence of anti-malarial plant Artemisia annua owing to
presence of anti-malarial compound, artemisinin, in its leaves. As the production of
artemisinin from A. annua is insufficient to meet the global demand, there had been an
increasing trend towards discovery of new methods and techniques for enhanced production
of A. annua biomass and processing of the same for artemisinin. Because of this reality,
artemisinin focused research and development programmes to enhance the artemisinin
production need to be undertaken. Artemisinin based drugs against cerebral malaria currently
dominate the market, leading to concerns over sustainable supply of the required amounts.
Today’s microbiology and plant molecular biology methods present the potential to address
the short falls of A. annua feedstocks. Specifically, PGPRs (plant growth promoting
rhizobacteria) and endophytes may offer increased productivity through sequestering
nutrients from soil, providing tolerance against biotic and abiotic stress, and resistance
against pests and pathogens thus enhancing the A. annua biomass and content of artemisinin
as well as other target secondary compounds. However, PGPRs and endophytes based
technologies largely remain unexplored because of several limitations, including the lack of
competitive background and strains that complement of suitable traits within a single species
of PGPR or endophyte. The present review demonstrates that in spite of their potential,
mandates and funding support for artemisinin feedstock are largely overlooked globally, a
factor that may severely limit future artemisinin research and the development of
technologies to combat future anti-malarial drug scarcity.

Keywords: Artemisia annua, artemisinin PGPRs, endophytes, technology.

INTRODUCTION

Discrete microbial communities, their assemblages on and inside numerous plants and their
tissues have been reported, yet extent to which plant determine the community structure
inside its plant parts has been generally overlooked for important medicinal crops. The
microbial communities chiefly consist of actinobacteria, bacteroidetes, firmicutes,
proteobacteria, and some fungi (Bulgarelli et al., 2013), which may change the face of
modern plant life and metabolism. Presence of these PGPRs around the rhizosphere and
assemblages of root as well as shoot endophytes changes with host plant genotype.
Consequently, fine-tuning by these microbes drives the selection in establishment of
phyllosphere communities at leaf surface. Biochemicals attributes of these root and shoot
microbiota provide protection against pathogens and some insects, tolerance to biotic as well
as abiotic stress (drought, salt, temperature and water logging), by regulating plant metabolic
pathways. Additionally, PGPRs and other root microbiota colonizing the rhizosphere have
been reported for additional host functions through the acquisition of nutrients from soil. Not
all, but few members of these microbial communities in rhizosphere as well as endophytic
assemblages in plant tissues work as parasite (Ganley et al., 2004); eliminating these negative
regulators from host may largely improve the plant growth and secondary compounds. Here,
we emphasize the critical roles of PGPRs and endophyte research of A. annua with special
focus on bacterial communities up-regulating (Rosenblueth et al., 2006) host metabolism for
growth, tolerance to abiotic as well as biotic stressors and enhanced artemisinin (Duke,1987)
through enriching the host seedlings and plantlets with selected PGPRs and endophytes.

W.H.O has developed a number of artemisinin-based therapeutics to cure the severe ailment
‘cerebral malaria’ caused by Plasmodium falciperum and spreading into host through female
anopheles mosquito bite. Nevertheless, due to its lower yield and increased cost of labors the

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efforts are becoming uneconomical and efforts are continuing to increase artemisinin content
in Artemisia, but a very little success has been achieved.

Since artemisinin yield from plant A. annua is presently based on available arable land, the
utilization of marginal and costal area must be explored for growing A. annua and the growth
promoting role of microbiota must be looked into for growing the crop under biotic and
abiotic stress conditions. Several questions remain to be answer such as (a) Can PGPRs and
endophytes, enrichment and technology advances further increase A. annua growth and
artemisinin yields! if yes, then to what extent? (b) Could A. annua be grown under abiotic
stress conditions with selected bacterial communities to meet the current and future
artemisinin demand globally? (c)Whether the content of artemisinin could be regulated
through microbial (PGPRs, endophytes) inoculations?

Plant growth and metabolite production is fine-tuned by PGPRs and endophytes

Endophytes (Li et al., 2012) and rhizobacteria enhance (Compant et al., 2010) the Artemisia
annua growth and its artemisinin yields through a number of activities including mobilisation
of nutrients from soil, stimulation of root growth and shoot branching, defence against soil-
borne plant pathogens, and producing stress ameliorating enzymes. Metabolic potential of
these endophytes may differ in plants from their soil counterparts (Zhang et al., 2006).
Although, most of the endophytes facilitate plant growth by providing them phosphate,
nitrogen, iron (Barry et al., 2009), auxins (e.g. indole acetic acid-IAA), and alleviating stress
causing metabolites and hormones through their ACC deaminase activity (Bulgarelli et al.,
2013).

Enterobactin (Escherichia coli), Ferrichrome (Ustilago sphaerogena), enterobactin and

bacillibactin (Bacillus subtilis), mycobactin-(Mycobacterium), ferrioxamine B-(Streptomyces
pilosus), yersinia bactin (Yersinia pestis), fusarinine C (Fusarium roseum), azotobactin
(Azotobacter vinelandii), vibriobactin (Vibrio cholerae), pseudobactin-(Pseudomonas) ,
erythrobactin (Saccharopolyspora erythraea ) and ornibactin-(Burkholderia cepacia) are
known iron chelating compounds “ siderophores” from microbiota residing in rhizosphere
and plant tissues (Wandersman and Delepelaire.,2004). Recently we isolated a Burkholderia
cepacia strain ART7 from seeds of Artemisia annua L with same potential. Chelated iron
from mineral soil move to plant cells via active transport. Plants through their nitrate
reducing / denitrification feature also provide nitrogen, a major limiting element in plants.
Most of the reported PGPRs and endophytes reduce NO3 to nitrite (NO2), which further
converted to oxides of nitrogen (N2O and NO) or NH3. Nitric oxides may modulate the auxin
biosynthesis, altering root growth and branching in plants (Molina et al., 2008). Second
limiting nutrient, phosphorus is also facilitated by these endophytes (only during root
colonization and before entry in root cells), and rhizobia through their phosphate solubilising
trait. They secrete some organic acids and acid phosphatases leading to the microbial cell
acidification and their surroundings causing mineralization of organic phosphorus in soil
(Mehta and Nautiyal., 2001). Gluconic acids, 2-ketogluconic acid seems to be recurrent agent
of mineral phosphate solubilisation. Strains of Pseudomonas, Bacillus and Rhizobium
produced a mixture of isovaleric, lactic, isobutyric, acetic acids, oxalic, glycolic, succinic and
malonic acid in majority of agricultural crops (Mehta and Nautiyal., 2001). The levels of
secondary metabolites, extenuating shoot branching and root growth, are highly modulated
by PGPB (plant growth promoting bacteria). Among auxins; Indole Acetic Acid (IAA) is a
known hormone produced via bacteria; modulates the agricultural productivity in various
crops (Dobbelaere et al., 1999). ACC (1-aminocyclopropane-1-carboxylic acid) deaminase
enzyme reduces the levels of ethylene under stress conditions by attenuating the levels of 1-

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aminocyclopropane-1-carboxylic acid (precursors for ethylene) (Argueso et al., 2007,
Lizada., 1979), through converting it into NH3 and α ketobutyrate. Due to reduced levels of
ACC by activity of these bacteria, ethylene levels reduce (Glick et al., 2007).

Recently we isolated an endophyte Burkholderia cepacia strain ART7 from Artemisia annua
L seeds in our laboratory (Microbial Technology Department) having all of above described
plant growth promoting as well as stress ameliorating traits. Furthermore, this organism was
evaluated for salt and drought stress to see its efficacy to enhance the tolerance of Artemisia
under both abiotic stresses through a statistical method Response Surface Methodology
(RSM). Inoculation of A. annua plants with bacteria protected Artemisia upto 200 mM salt
and 66% drought stresses, resulting in increased herb as well as artemisinin yields in case
plants are exposed to both the stresses.

Bioactive metabolites secreted by endophytes of Artemisia

The fungal endophyte Colletotrichum sp., isolated from the stem of A. annua, secrete a
number of metabolites including a number of ergosterols, a phyto-hormone indole-3-acetic
acid (IAA) and three new antimicrobials (Lu et al., 2000). The cytotoxic compounds 10,13-
cyclotricho thecane derived macrolides, myrothecines A–C were identified from two
endophytes Myrothecium roridum IFB-E009 and IFB-E012; isolated from A. annua by Shen
et al(2006). Furthermore, Ge et al. (2006) identified another xanthene derivative metabolite,
paranolin 137, from Paraphaeosphaeria nolinae IFB-E011 endophyte of A. annua. These
endophytes also have the potential to drive the expression of regulatory genes against
pathogen defence and artemisinin increase in A. annua. Losgen et al., (2008) reported a
chromone-3-oxepine-polyketides; ‘Isofusidienol’ A-D from fungal endophyte of Artemisia
vulgaris. ‘Leptosphaeric acid’ from Leptosphaeria sp. IV403; an endophyte of A. annua (Liu
et al., 2003) and ‘Colletotric acid’ from endophyte Colletotrichum gloeosporioids of A.
mangolica, displaying allelopathy against bacteria and fungus Helminthsporium sativum (Zou
et al., 2000). Research group of Aftab et al. (2010) made efforts to enhance the artemisinin
content of A. annua. through chemical priming by applying triacontanol and gibberellic acid.
Yet in another study, salicylic acid promoted the salt stress acceptance of Artemisia annua L.
(Aftab et al., 2011). Although chemical priming enhanced the artemisinin and stress tolerance
but instead of applying these chemicals externally, native endophytes and PGPRs producing
such chemicals would be useful alternates protecting Artemisia from pathogen as well as
enhancing tolerance against abiotic stress and producing higher herb and enhanced
artemisinin yields.

Endophytes and PGPRs modulate the gene expression in Artemisia and enhance the
artemisinin yields
PGPB including rhizobia and endophytes have been investigated for their metabolic attributes
to promote plant growth as well as secondary metabolites in a number of medicinal crops, but
in Artemisia annua, till date, not much information exists in details, especially their role in
cumulative community functions in improving herbage yields and metabolite production
(Kloepper., 1996). A number of efforts are ongoing to enhance the artemisinin yields in
Artemisia. Wang (2001) could enhance the artemisinin content in A. annua hairy roots from
0.8 mg g-1 to 1 mg g-1 in dried biomass through priming by elicitor of fungal extracts from
endophyte Colletotrichum sp. Furthermore, inoculation of endophyte Pseudonocardia sp.
strain YIM 63111 in A. annua enhanced the plant growth and artemisinin yields through up
regulating the expression of CYP71AV1 and CPR genes (Li et al., 2012). Olofsson et al.,
(2011) enumerated the up as well as down regulated genes in A.rtemisia annua L. It was
clarified that 3-Hydroxy-3-methyl-glutaryl coenzyme A reductase was negatively regulated

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in older leaves of Artemisia than other plant tissues. Consequently, they observed that
Farnesyl diphosphate synthase (FDS), squalene synthase (SQS) and 1-deoxy-D-xylulose-5-
phosphate reducto isomerase (DXR) moderately regulate the artemisinin yields throughout
the plant life. Zhang et al., (2013) reported enhanced artemisinin yields through over-
expressing the AaPYL9; a ortholog of ABA receptor. Although, these methods have largely
contributed in artemisinin research yet some issues on other hand have to be looked into that
how to imply and provide these technologies to farmers and make it sustainable at global
scale. Employing endophytes for the same may largely contribute acceptability and ease of
use to farmers. However, before using these endophytes, these must be evaluated for their
metabolites influencing flora and fauna of the same field and reveal the potential role of their
metabolites in regulating the genes of artemisinin biosynthesis pathway.

A single endophyte or PGPR may not be able to provide all the suitable complement/s to the
plants to thrive against a number of stressors, and to produce enhanced target metabolites of
medicinal importance. Awasthi et al., (2011) investigated the synergistic effects of
mychorrhiza Glomus mosseae and a nitrogen fixing Bacillus subtilis strain Daz26 on content
of artemisinin in A. annua L and found that presence of both the organism can enhance the
herb yields and content of artemisinin. Some microbial strains from other crops, having
almost all plant growth promoting traits, such as production of IAA (Chaudhary et al. 2008,
Weathers et al., 2005, Lin et al., 2013), ACC deaminase (Glick et al., 2007), siderophores
(Wandersman and Delepelaire., 2004) and their ability towards P-solubilisation and nitrate
reduction (Molina et al., 2008) may also be considered for improving A. annua growth.
However, its efficacy could be decided only after experimentation (Bharti et al 2013, Singh et
al. 2013, Tan and Zou., 2001). Not all, but some individuals of endophytic community may
affect the plant metabolism negatively whilst others may protect the plant against pathogens
(Chung et al., 2008, Li et al., 2012). Huang et al., 2009 isolated some fungal endophytes from
three species of Artemisia with biocontrol activities.

CONCLUSIONS

The endophytes of A. annua and as well as of other medicinal crops may thrive and colonize
within stems, roots, leaves and reproductive parts of plants, and their colonization may be
observed at different stages of plant growth, as plant develops from seed to mature flowering
stages. Individually these organisms may be good for plant growth promotion and secondary
metabolites enhancements, but when they reside as plant microbiota assemblages in root,
stem, leaves or other plant tissues, their functions may be altered due to the metabolic
activities of individuals in different communities (Bulgarelli et al., 2013). Thus, selecting the
beneficial individuals PGPR/endophytes from plant microbiomes, and establishing their in
vivo stable functional communities, may largely contribute in enhancing the plant growth and
artemisinin yields in A. annua. Alternatively, the rhizobacteria around the rhizosphere of A.
annua may also increase the herbage yields of plant by altering the growth attributes and
metabolite production of endophytes. Zhao et al., (2011, 2011a, 2011b, 2011c, 2011d, 2011e,
2012) identified as well as characterized a number of endophytes including; Pseudonocardia
artemisiae, Pseudonocardia xishanensis sp. Nov, Pseudonocardia antimicrobica sp. Nov.,
Phytomonospora endophytica gen. nov., sp. nov, Nonomuraea endophytica sp. nov.,
Pseudonocardia kunmingensis sp. nov., Rhodococcus artemisiae sp. nov., Nocardia
artemisiae sp. nov., from A. annua for their different functional roles with special emphasis
towards secondary metabolites. Hong et al (2000) also reported new bioactive metabolites
from a fungus Colletotrichum sp. from A. annua. Metabolites from these endophytes may

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suppress the growth of other individuals of community through their metabolic potentials and
then regulate the A. annua growth.

We at CSIR-Central Institute of Medicinal and Aromatic Plants have isolated and identified
12 bacterial endophytes from A. annua L with different plant growth promoting as well as
stress ameliorating traits. Presently we are trying to engineer a suitable community of A.
annua endophytes to promote its growth and herb yields in abiotic stress conditions. The
developed community could be implied in combination with vermicompost to Artemisia
seedlings as well as plantlets, which could be later transferred to field.

Thus, In vitro engineering an endophytic community for enhanced artemisinin yields should
be emphasised. The individuals within community should have all the required features for
growth promotion, protection against pathogens, and tolerance to biotic as well as abiotic
stresses. Considering this fact, two types of communities one including only endophytes and
other having both rhizobacteria and endophytes could be considered. Engineered
communities for Artemisia herbage and artemisinin yields should be elucidated both under
biotic as well as abiotic stress environments. It will pave way for more sustained production
of A. annua herb and artemisinin yields and utilization of waste and marginal lands for A.
annua production.

REFERENCES

Aftab T., Khan M. M A., Idrees M., Naeem M., Singh M., Ram M. (2010) Stimulation of crop
productivity, photosynthesis and artemisinin production in Artemisia annua L. by
triacontanol and gibberellic acid application. J Plant Inter. 5: 273-281.
Aftab T., Khan M. M A., Teixeira d S J A., Idrees M. M., Moinuddin N. (2011). Role of Salicylic
Acid in Promoting Salt Stress Tolerance and Enhanced Artemisinin Production in
Artemisia annua L. J Plant Growth Regul 30:425–435
Argueso CT., Hansen M., Kieber JJ. (2007). Regulation of ethylene biosynthesis. J. Plant Growth
Regul. 26:92–105
Awasthi A., Bharti N., Nair P., Singh R., Shukla A.K., Gupta M.M., Darokar M.P., Kalra A.
(2011) Synergistic effect of Glomus mosseae and nitrogen fixing Bacillus subtilis strain
Daz26 on artemisinin content in Artemisia annua L. App. Soil Ecol. 49: 125-130
Barry S.M., Challis G.L. (2009) Recent advances in siderophore biosynthesis. Curr Opin Chem
Biol. 13: 205–215.
Bharti N., Yadav D., Barnawal D., Maji D., Kalra A. (2013) Exiguobacterium oxidotolerans, a
halotolerant plant growthpromoting rhizobacteria, improves yield and content of
secondarymetabolites in Bacopa monnieri (L.) Pennell under primary and secondary salt
stress. World J Microbiol Biotechnol. 29:379–387
Bulgarelli D., Schlaeppi K., Spaepen S., Themaat E V L V., Schulze-Lefert P.(2013) Structure
and Functions of the Bacterial Microbiota of Plants. Annu. Rev. Plant Biol. 64:807–38
Chaudhary V., Kapoor R., Bhatnagar AK (2008) Effectiveness of two arbuscular mycorrhizal
fungi on concentrations of essential oil and artemisinin in three accessions of Artemisia
annua L. Appl Soil Ecol 40:174–181
Chung B S., Aslam Z., Kim S W., Kim G G., Kang H S., Ahn J W., Chung Y R. (2008). A
Bacterial Endophyte, Pseudomonas brassicacearum YC5480, Isolated from the Root of
Artemisia sp. Producing Antifungal and Phytotoxic Compounds. Plant Pathol J.24 (4).
Compant S., Clement C., Sessitsch A. (2010) Plant growth-promoting bacteria in the rhizo- and
endosphere of plants: their role, colonization, mechanisms involved and prospects for
utilization. Soil Biol Biochem. 42:669–678.

212 | P a g e
Dobbelaere S., Croonenborghs A., Thys A., Vande Broek A., Vanderleyden J. (1999).
Phytostimulatory effect of Azospirillum brasilense wild type and mutant strains altered in
IAA production on wheat. Plant Soil 212:155–64
Duke SO., Vaughn KC., Croom EM., Elsohly HN (1987) Artemisinin, a constituent of annual
wormwood (Artemisia annua), is a selective pytotoxin. Weed Sci 35:499–505
Ganley R J., Brunsfeld S J., Combe G N. (2004) A community of unknown, endophytic fungi in
western white pine.. 101 (27):10107–10112
Ge HM., Song YC., Chen JR., Hu S., Wu JY., Tan RX., (2006) Paranolin: a New Xanthene-
Based Metabolite from Paraphaeosphaeria nolinae Helv.Chim. Acta, 89: 502.
Glick BR., Todorovic B., Czarny J., Cheng ZY., Duan J., McConkey B. (2007). Promotion of
plant growth by bacterial ACC deaminase. Crit. Rev. Plant Sci. 26:227–42
Huang WY., Cai YZ., Surveswaran S., Hyde KD., Corke H., Sun M.( 2009) Molecular
phylogenetic identification of endophytic fungi isolated from three Artemisia species.
Fungal Div. 36:69–88.
Kloepper JW (1996) Host specificity in microbe-microbe interactions. Bioscience 46:406–409
Li J., Zhao G.Z., Varma A., Qin S., Xiong Z., Huang HY., Zhu WY., Zhao LX., Xu LH., Zhang
S., Li WJ., (2012). An endophytic Pseudonocardia species induces the production of
artemisinin in Artemisia annua. PLoS ONE.;7:e51410.
Li J., Zhao GZ., Huang HY., Qin S., Zhu WY., Zhao LX., Xu LH., Zhang S., Li WJ., Strobel G.
(2012) Isolation and characterization of culturable endophytic actinobacteria associated
with Artemisia annua L. Antonie Van Leeuwenhoek. 101(3):515-27
Li J., Zhao GZ., Zhu WY., Huang HY., Xu LH., Zhang S., Li WJ (2011b) Phytomonospora
endophytica gen. nov., sp. nov., a novel actinomycete of the family Micromonosporaceae
isolated from the roots of Artemisia annua L. Int J Syst Evol Microbiol. doi:
10.1099/ijs.0.030205-0
Lin L, Xu X. (2013) Indole-3-acetic acid production by endophytic Streptomyces sp. En-1
isolated from medicinal plants. Curr Microbiol. 67:209-17
Liu JY, Liu C H, Zou W X, Tan R X (2003) Leptosphaeric Acid, a Metabolite with a Novel
Carbon Skeleton from Leptosphaeria sp. IV403, an Endophytic Fungus in Artemisia annua.
Helvetica Chimica Acta 86:657-660.
Lizada M.C.C., Yang S.F., (1979). A simple and sensitive assay for 1-aminocyclopropane-1-
carboxylic acid Anal. Biochem., 100:142–147
Losgen S., Magull J., Schulz B., Draeger S., Zeeck A.(2008). Isofusidienols: novel chromone-3-
oxepines produced by the endophytic fungus Chalara sp. European J. Org.Chem. 698–703.
Lu, H; Zou, WX; Meng, JC; Hu, J; Tan, RX; (2000) New bioactive metabolites produced by
Colletotrichum sp., an endophytic fungus in Artemisia annua. Plant Science 151: 67-73
Mehta S., Nautiyal C.S., (2001), An efficient method for qualitative screening of phosphate
solubilizing bacteria. Curr. Microbiol., 43 :57–58
Molina FC., Creus CM., Simontacchi M., Puntarulo S., Lamattina L. (2008). Aerobic nitric oxide
production by Azospirillum brasilense Sp245 and its influence on root architecture in
tomato. Mol. Plant-Microbe Interact. 21:1001–9.
Olofsson L., Engstrom A., Lundgren A., Brodelius P E (2011) Relative expression of genes of
terpene metabolism in different tissues of Artemisia annua L. Plant Biology 11:45.
Rosenblueth M., Martinez R E. (2006) Bacterial endophytes and their interactions with hosts.
Mol Plant-Microbe Interact. 19:827–837.
Singh R., Soni S K., Kalra A. (2013) Synergy between Glomus fasciculatum and a beneficial
Pseudomonas in reducing root diseases and improving yield and forskolin content in
Coleus forskohlii Briq. under organic field conditions. Mycorrhiza, 23:35–44.
Tan R. X., Zou W. X. (2001) Endophytes: a rich source of functional metabolites. Nat. Prod.
Rep., 18:448-459.
Wandersman C., Delepelaire P. (2004) Bacterial iron sources: from siderophores to hemophores.
Annu Rev Microbiol. 58:611-47.

213 | P a g e
Wang JW., Zhang Z., Tan RX. (2001)Stimulation of artemisinin production in Artemisia annua
hairy roots by the elicitor from the endophytic Colletotrichum sp. Biotech. Letters 23:857 –
860
Weathers PJ., Bunk G., NcCoy MC (2005). The effect of phytohormones on growth and
artemisinin production in Artemisia annua hairy roots. In Vitro Cellular and Developmental
Biology - Plant 41:47-53.
Zhang F., Lu X., Lv Z., Zhang L., Zhu M., Jiang W., Wang G., Sun X., Tang K. (2013)
Overexpression of the Artemisia Orthologue of ABA Receptor, AaPYL9, Enhances ABA
Sensitivity and Improves Artemisinin Content in Artemisia annua L PLoS ONE8:e56697.
doi:10.1371/journal.pone.0056697.
Zhang H W., Song Y C., Tan R X. (2006) Biology and chemistry of endophytes. Nat. Prod. Rep.,
18: 448
Zhao G Z., Li J., Huang H Y., Zhu W Y., Zhao L X., Tang S K., Xu L H., Li W J.(2011),
Pseudonocardia artemisiae sp. nov., isolated from surface-sterilized Artemisia annua L.
Inter J Syst Evol Microbiol 61:1061–1065
Zhao G Z., Li J., Qin Y L., Miao C P., Wei D Q., Zhang S., Xu L H., Li WJ. (2012)
Pseudonocardia antimicrobica sp. nov., a novel endophytic actinomycete associated with
Artemisia annua L. (sweet wormwood). J Antibiot 65: 469-472
Zhao G Z., Li J., Zhu W Y., Wei D Q., Zhang J L., Xu L H., Li W J. (2012).Pseudonocardia
xishanensis sp. nov., an endophytic actinomycete isolated from the roots of Artemisia
annua L. Inter J Syst Evol Microbiol 62: 2395–2399.
Zhao GZ., Huang HY., Zhu WY., Lee JC., Xu LH., Kim CJ., Li WJ (2011a) Nonomuraea
endophytica sp. nov., an endo- phytic actinomycete isolated from Artemisia annua L. Int J
Syst Evol Microbiol 61:757–761
Zhao GZ., Li J., Huang HY., Zhu WY., Park DJ., Kim CJ., Xu LH., Li WJ (2011b)
Pseudonocardia kunmingensis sp. nov., a novel actinobacterium isolated from surface-
sterilized roots of Artemisia annua L. Int J Syst Evol Microbiol 61:2292–2297
Zhao GZ., Li J., Zhu WY., Klenk HP., Xu LH., Li WJ (2011e) Nocardia artemisiae sp. nov., an
endophytic actinobacterium isolated from surface-sterilized stem of Artemisia annua L. Int
J Syst Evol Microbiol. doi: 10.1099/ ijs.0.029306-0
Zhao GZ., Li J., Zhu WY., Tian SZ., Zhao LX., Yang LL., Xu LH., Li WJ (2011d) Rhodococcus
artemisiae sp. nov., a new endophytic actinobacterium isolated from pharmaceutical plant
Artemisia annua L. Int J Syst Evol Microbiol. doi:10.1099/ijs.0.031930-0
Zou WX., Meng JC., Lu H., Chen GX., Shi GX., Zhang TY., Tan RX. (2000) Metabolites of
Colletotrichum gloeosporioids an endophyte fungus in Artemisia mangolica. J.Nat.Prod.
63:1529-1530.

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 Microbes mediated stress tolerance and plant growth promotion in
Withania somnifera
Tania Ray, Shivangi Mishra, Alok Kalra
Microbial Technology and Crop Protection, CSIR- CIMAP, Lucknow-226015
*Email: tani318ray@gmail.com

INTRODUCTION

Withania somnifera, also known as “Ashwagandha”, is an important medicinal plant, which is

used in Ayurveda, Siddha, Unani and indigenous systems of medicines with over 200
formulations. The plant is being extensively used in traditional system of medicine for treating
many ailments of animals and humans (Rajakumar et al. 2012). The roots contain several
alkaloids; withasomnine and steroids, lactones, withaferin A and withanolides (Arun et al. 1996).
The plant is used as a sedative, tonic, stimulant, aphrodisiac, diuretic, antipyretic and anthelmintic
(Gupta et al. 1993) and is known for anticancer (Devi et al. 1996), antiarthritic properties.

W. somnifera is prone to various pathogens and stresses and faces biodetoriation, therefore, being
one of the health heritage, it needs to be safeguarded. There are several means through which this
can be accomplished. One of the important techniques i.e. use of Plant growth promoting
rhizobacteria (PGPRs) has gained popularity for stress management in W. somnifera because of
restriction of use of chemicals especially in medicinal plants.

Role of PGPRs in W.somnifera

Plant growth promoting rhizobacteria (PGPRs) are root colonizing bacteria. Some form symbiotic
relationship with the plants and some are free living in soil and roots, but all influence plant
growth by various mechanisms. PGPRs are able to improve plant growth by increasing the rate of
seed germination and seedling emergence, improving nutritional uptake, minimizing the adverse
effects of external stress factors and protecting plants from soil-borne pests and diseases. Various
strains such as, Azospirillum, Azotobacter, Arthrobacter, Bacillus and Pseudomonas (Attia et al.
2001) have been earlier reported to possess PGPR activity. Rhizobium is also considered as a soil
bacteria with PGPR activity.

In a study, it was found that microbes can tolerate a variety of toxic metals in the environment
enriched with such metals (Martin-Laurent F et al. 2004) and can be useful in promoting plant
growth under such conditions. Tolerance to heavy metals was found more predominant among
rhizobacteria from Ni-Cd treated soil as compared to normal. Isolation of both nickel and
cadmium tolerant PGPRs communities (like, Bacillus, Pseudomonas and Azotobacter) from the
rhizosphere of W. somnifera was carried and tested for plant growth promoting activities and
heavy metal tolerance. The test rhizobacterium P-35 both metal tolerant with multiple PGP
activities like IAA, ammonia, HCN, catalase etc. was subjected to seed germination test. It was
shown that seed inoculation with test bacterium significantly enhanced seed germination, root and
shoot growth in W. somnifera (Rathaur et al.2012).

In another study, six bacterial isolates found to be efficient in producing PGP compounds like
IAA, ammonia, HCN, catalase and siderophore were further characterized and tested for their
growth promoting capabilities in W. somnifera seeds after treating with PGPRs. This seed
inoculation significantly enhanced seed germination and seedling vigour of W. somnifera. This
effect was also observed under UV-B stress. PGPR strains significantly enhanced plant growth as
compare to control. It has been reported that PGPRs might enhance plant height and productivity
by synthesizing phytohormones, increasing the local availability of nutrients, facilitating the
uptake of nutrients by the plants decreasing heavy metal toxicity and antagonizing plant

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pathogens. It was observed that plant height increased by 37%, 35% and 33% by isolates PG10,
PG9 and PG7 respectively (Rathaur et al. 2012).

Many microbes are adapted to peculiar soil environment and can be commercially and effectively
used for enhancing growth of medicinal plants like W. somnifera in soils comtaminated with
hydrocarbons. In a study it was found that such strains helped in plant growth promotion of W.
somnifera in the soil contaminated with petrol oil while surfactant property of the strains helped
in lowering the toxicity of hydrocarbons to the plant. Pseudomonas strains (RK 4 and RK 3)
isolated from contaminated soil were found to have PGPR as well as biosurfactants
(rhamnolipids) properties. Inoculating the strains and consortium of both the strains in petrol
hydrocarbon contaminated soil and its interaction with W. somnifera in presence of petrol oil
revealed that rhamnolipids property of the strains helped in lowering the impediment effect of
petrol engine oil hydrocarbons and the growth promotory activities enhanced the growth and
antioxidant activity of W. somnifera. Consortium of both the strains showed better results as
compared to the individual strain and the interaction were found to be beneficial (Kumar et
al.2014).

Diseases of Withania somnifera and their management by PGPRs

Several diseases affect W. somnifera, both under wild and cultivated conditions. But the crop is
mainly attacked by fungal pathogens under field conditions, although deterioration of these crops
due to other diseases has also been reported. Leaf spot is the most prevalent disease of W.
somnifera caused by a fungus Alternaria alternate (Pati et al. 2008). The plant has also been
reported to be affected by many other foliar diseases. It has been observed that Alternaria
alternata causes severe leaf spot disease, while Myrothecium roridum and Fusarium oxysporum
caused minor diseases and losses (Shivanna et al. 2013). Another disease of W. somnifera is wet
rot. The causal organism was identified as Choanephora sp., a fungus which produces white
aerial mycelia that later turned pale yellow (Saroj et al. 2012). In root rot and wilt disease the
plants in the nurseries show symptoms of yellowing, drooping and decay at seedling stage leading
to 30-50% mortality. The causal organism has been reported as Fusarium solani (Gupta et al.
2004). The typical symptoms of another disease i.e. Witches broom disease consist of little leaf,
shortening of internodes, excessive branching giving witches-broom appearance and premature
drying and death of infected twigs and leaves. Phytoplasma was found to be associated with the
disease on the basis of symptomatology (Bharti et al. 2012).

Various PGPRs Bacillus cereus and Pseudomonas fluorescence, have been investigated for
having a role in reducing the severity of root rot in ashwagandha (Liu et al.1995). Some
siderophore producing strains have been recognized as biocontrol agents against certain soil-
borne plant pathogens. They chelate iron and deprive pathogens of the iron required for their
growth and pathogenesis and hence reduce the incidence and severity of plant diseases caused by
Fusarium (Scher et al.1980).

Induction of resistance possible role of PGPRs

Many microbes especially PGPR have also been found to induce systemic resistance. In the
beginning of the past century, it was realised that upon infection by a pathogen, plants develop an
enhanced resistance to further pathogen attack (Beauverie et al. 1901). The analysis of the
priming phenomenon in intact plants for the first time was reported by (Mur et al. 1996. The role
of PGPR in enhancing resistance of the plants to various plant pathogens has been reported.

The root-endophytic basidiomycete Piriformospora indica confers systemic resistance to fungal

diseases and salt stress (Waller et al. 2005). P. indica was found to increase growth and yield of
the two medicinal plants Spilanthes and W. somnifera in the field. Significant higher growth and
yield of ashwagandha was recorded relative to uninoculated controls. A considerable increase in

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shoot and root length, biomass, basal stem, leaf area, overall size, number of inflorescences and
flowers and seed production were observed in the presence of the fungus. This was attributed to
greater root length and biomass because of extensive colonisation of roots by the fungus, leading
to greater absorption of mineral nutrients and water resulting in increased drought tolerance in
plants (Rai et al. 2005). With reference to an earlier report, it is also assumed that increase in
fresh and dry weight may be related to increased P- uptake (Sudha et al. 1998). Furthermore,
enhanced pathogen resistance is reported from plants whose roots have been colonized by
mycorrhizal symbionts (Pozo et al. 2005).

Bharti et al. (2012) found that both chemicals and PGPR can reduce the incidence of root rot.
Although both chemicals and PGPR could reduce the severity individually, combination of both
was found to be more effective. Three different resistant inducers viz. thiosalicylic acid (TSA),
O-acetyl salicylic acid (O-ASA), DL-2 amino butyric acid ( DL-2ABA) and two efficient PGPRs
viz. siderophores producing bacteria Bacillus cereus(Sd23) & Pseudomonas fluorescens (DPF)
were evaluated individually as well as in combinations. Of these thiosalicylic acid with Bacillus
cereus and O-Acetylsalicylic acid with Pseudomonas sfluorescens were found to be highly
effective in reducing severity of root rot (reduced by 85% and 88%) and root yield could be
enhanced by 358% and 419% respectively, against Fusarium solani induced root rot disease in
Withania somnifera (Bharti et al.2012).

Several enzymes like, peroxidase, polyphenol oxidase and phenyl ammonium lyase were found to
have an important role in reduction of disease severity. It has been found that increase in
peroxidase activity in O-salicylic acid treated plants could be directly correlated with the
reduction in disease and increase in yields in O-acetyl salicylic acid treated pots. Suppression of
disease in Bacillus cereus pots was also correlated with increase in peroxidase activity (Bharti et
al.2 012). Induction of peroxidases and other defence related enzymes through the use of
bioinoculants have been reported earlier (Karthikeyan et al. 2010). Although peroxidases have
not been shown to have direct antifungal properties unlike chitinases and β-1,3 gluconases but
may play a functional role in disease resistance indirectly by enhancing the production of
fungitoxic compounds and lignification.

With increased awareness towards naturals and harmful effects of chemicals used as form inputs.
The PGPR could be safe and cheap alternative to the agro-chemical inputs.

REFRENCES

A. Saroj, A. Kumar, N. Qamar, M. Alam, H. N.Simgh, and A. Khaliq.(2012) First Report of Wet
Rot of Withania somnifera caused by Choanephora cucurbitarum in India. Plant Disease.
Arun K, Tripati, Shukla YN, Kumar Sushil. 1996. Ashwagandha[Withania somnifera Dunal
(Solanaceae)]: A status report. J Med Arom Sci 18: 46–62
Attia, F.A. and Saad, O.A.O. (2001). Biofertilizers as potential alternative of chemical fertilizer
for Catharanthus roseus G. Don. Journal of Agricultural Science, 26(11): 7208–7193.
Beauverie J (1901) Essais d’immunisation des végétaux con- tre des maladies cryptogamique. C
R Acad Sci Ser III 133: 107–110
Bishal,(2009).Reduction of Fusarium root rot of ashwagandha with chemical resistance inducers
and siderophores producing strains of rhizobacteria. University of Lucknow.
Devi PU. 1996. Withania somnifera Dunal (Ashwagandha):Potential plant source of a
promising drug for cancer chemotherapy and radio sensitization. Indian J Exp Biol
34:927–932
Gupta S, Kumar A, Thakur RN. 1993. Some problems incultivation of Withania somnifera (L)
Dunel (Aswagandha) in Jammu region of India. J Res Edu Ind Med 12: 23–27

217 | P a g e
Gupta, M. L.; Misra, H. O.; Kalra, A.; Khanuja, S. P. S. (2004) Journal of Medicinal and
Aromatic Plant Sciences;Vol. 26 No. 2 pp. 285-287
Karthikeyan, B., Joe, M.M., Jaleel, C.A. and Deiveekasundaram, M. (2010). Effect of root
inoculation with plant growth promoting rhizobacteria (PGPR) on plant growth, alkaloid
content and nutrient control of Catharanthus roseus (L.) G. Don. Natura Croatica, 1: 205–
212
Liu L., Kloepper J.W. and Tuzan S. (1995) .Induction of systemic resistance in cucumber against
bacterial angular leaf spot by plant growth promoting rhizobacteria. Phytopathol 85:843-
847
M. B. Shivanna, T.R.Parashurama, K.G. Somashekhara Achar, & M. M. Vasanthakumari.(2013)
Fungal foliar diseases in Withania somnifera and its effect on secondary metabolites. Plant
biosystems
M. Zaim; A. Samad; (1995). Association of phytoplasmas with a witches-broom disease of
Withania somnifera (L) Dunal in India; Plant Science 109 (1995) 225-229
Martin-Laurent F., L. Ranjard, J. Lopez- Gutierrez and L. Philppot et al., 2004. Estimation of
atrazine degrading genetic potential and activity in three French agricultural soil. FEMS
Microbiol. Ecol., 48:425-435.
Mur,L.A.J.,Naylor,G.,Warner,S.A.J.,Sugars,J.M.,White,R.F., & Draper, J. (1996). Salicylic acid
potentiates defence gene expression in tissue exhibiting acquired resistance to patho- gen
attack. Plant Journal, 9, 559 –571
N. Bharti, P. Agrawal, B. Misra, A. Tripathi, R. Singh, Deepmala Maji. (2012) Efficacy of
combined applications of antagonist bacteria and chemical resistance inducers for the
management of Fusarium solani causing root rot in Withania somnifera. Biocontrol
Science and Technology.
N. Bharti, P. Agrawal, B. Misra, A. Tripathi, R. Singh, Deepmala Maji. (2012) Efficacy of
combined applications of antagonist bacteria and chemical resistance inducers for the
management of Fusarium solani causing root rot in Withania somnifera. Biocontrol
Science and Technology.
P. Rathaur, P. Wasudeo Ramteke, W. Raja and S. Ashish John 2012. Isolation and
Characterization of Nickel and Cadmium Tolerant Plant Growth Promoting Rhizobacteria
from Rhizosphere of Withania somnifera . J. BIOL. ENVIRON. SCI. 6(18), 253-261
P. Rathaur, W. Raja, P W Ramteke, S A John 2012. Effect of UV-B Tolerant Plant Growth
Promoting Rhizobacteria (PGPR) on Seed Germination and Growth of Withania somnifera
. Pelagia Research Library Advances in Applied Science Research, 2012, 3 (3):1399-1404
Pozo MJ, Van Loon LC, PitersevCMJ (2005) Jasmonates-Signals in plant-microbe interactions. J
Plant Growth Ragul 23:211-222
Pratap Kumar Pati · Monica Sharma · Raj Kumar Salar · Ashutosh Sharma · A. P. Gupta B.
Singh.(2008) Studies on leaf spot disease of Withania somnifera and its impact on
secondary metabolites. Indian journal of microbiology.
R. Kumar, A. Jyoti Das, Asha ,A Juwarkar 2014. Restoration of Petrol Contaminated Soil by
PGPR Consortium Producing Rhamnolipids and Enhancement of Growth and Antioxidant
activity of Withania somnifera . Petroleum & Environmental Biotechnology 55
Rai, M., Acharya, D., Singh, A., & Varma, A. (2001). Positive growth responses of the medicinal
plants Spilanthes calva and Withania somnifera to inoculation by Piriformospora indica in
a field trial. Mycorrhiza, 11, 123–128
Rajakumar N, Shivanna MB. 2012. Traditional veterinaryhealthcare practices in Shimoga
districts of Karnataka, India.Indian J Trad Knowledge 11(2): 283–287
Scher F.M. and Baker R. (1980). Mechanism of biological control in a Fusarium- suppressive
soil. Phytopathol 70: 412-417
Sudha, Hurek T, Varma A (1998) Active translocation of phos- phate (P32) to rice and carrot by
Piriformospora indica. In: Ahonen-Jonnarth U, Danell E, Fransson P, Karen O, Lindahl B,

218 | P a g e
 Rangel I, Finlay R (eds) Second International Congsssress on Mycorrhiza, Uppsala,
Sweden, July 5–10
Waller, F., Ahatz, B., Baltruschat, H., Fodor, J., Becker, K., Fischer, M., et al. (2005). The
endophytic fungus Piriformospora indica reprograms barley to salt-stress tolerance, disease
resistance, and higher yield. Proceedings of the National Academy of Sciences of the USA,
102, 13386–13391

219 | P a g e

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