KYAMBOGO UNIVERSITY

FACULTY OF SCIENCE

DEPARTMENT OF CHEMISTRY

EVALUATION OF PHYTOCHEMICAL PROPERTIES AND

DYEING OF JUSTICIA BETONICA EXTRACT ON COTTON
FABRIC

BY

ANGUZU OLEGA BREVIS, 16/U/4817/CHD/PD

Project Report Submitted to the Department of Chemistry

in Partial Fulfilment of the Requirements for the Award of
Bachelor of Science in Chemical and Process Engineering
Degree of Kyambogo University

DECEMBER, 2020

i
DECLARATION
I, Mr. Anguzu Olega Brevis, 16/U/4817/CHD/PD, declare that the work contained in this project
report is my original work derived from experiments carried out under the guidance of my
supervisor. Where other people’s work has been used, this has been dully acknowledged in the
references.

Name: ……………………………

Reg. no: …………………………

Sign: ………………......................

Date: ………………………………

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APPROVAL
This is to certify that the project titled: “EVALUATION OF PHYTOCHEMICAL
PROPERTIES AND DYEING OF JUSTICIA BETONICA EXTRACT ON COTTON FABRIC”
has been done under my guidance and supervision.

Dr. Wacco Paul Alex

Supervisor

Sign: …………………………..

Date: …………………………..

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DEDICATION
I dedicate this report to my parents, Mrs. Draleru Francesca and Mr. Olega Christopher, my
siblings; Okudinia Bridget and Ayikoru Belinda Faith whose sacrifice towards my education has
exposed me to the world of Engineering

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ACKNOWLEDGEMENT
I would like to thank the Almighty God for His ever-present help in time of need, for the love,
wisdom, guidance, hope and strength upon successfully completing this thesis. I would also like
to express my earnest appreciation to my parents and siblings for their encouragement, prayers,
love and comfort throughout this academia experience.

Furthermore, I must express my heartfelt gratitude to my supervisor Dr. Wacco Paul Alex for his
sustained and appreciable guidance, diligent advice and knowledge imparted to me in fine tuning
this report until completion. Surely, the Lord who rewards may He make you flourish as He
grants you the desires of your hearts. Further gratitude goes to Mr. Wilber of Uganda Research
Institute for the assistance offered during the experimental processes of my study.

Finally, most sincere gratitude to my mother Draleru Francesca, my father Olega Christopher,
my sisters Okudinia Bridget and Ayikoru Belinda, my relatives, my colleagues, classmates,
friends and lecturers for their inspiring words of financial support, council, wisdom and
encouragement.

May the Good Lord bless and reward you abundantly.

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ABBREVIATIONS AND ACRONYMS
WHO World Health Organization

TCM Traditional and complementary medicines

GAE/gm Gallic Acid Equivalent per gram

mg milligram

UV Ultraviolet

ml milliliter

MLR Material to liquid ratio

w/w Weight/weight

o.w.f on weight of fabric

NMR Nuclear magnetic resonance

FT-IR Fourier transform-infra red

UV-Vis Ultraviolet visible

g, gm, gms grams

g/l grams per liter

HCL Hydrochloric acid

H2SO4 Sulphuric acid

CHCl3 Chloroform

v/v Volume/volume

FeCl3 Ferric chloride

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LIST OF TABLES

Table 1: Taxonomy tree for Justicia betonica L..............................................................................8

Table 2: optimized extraction conditions of some natural dyes....................................................25
Table 3: Phytochemical Screening of Various Extracts Justicia betonica.....................................47
Table 4: Reflectance at different wavelengths...............................................................................49
Table 5: K/S at different wavelengths...........................................................................................51

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LIST OF FIGURES
Figure 1:Habit of Justicia betonica L...............................................................................................9
Figure 2:Flowering twig of Justicia betonica L.............................................................................10
Figure 3: Basic structures of some pharmacologically important plant derived alkaloids............14
Figure 4: Basic structure of tannin.................................................................................................15
Figure 5: Basic structures of some pharmacologically important plant derived tannins...............15
Figure 6: Basic structure of saponin..............................................................................................16
Figure 7: Basic structure of terpenoid...........................................................................................17
Figure 8: Basic structure of anthraquinone....................................................................................17
Figure 9: Basic structures of some pharmacologically important plant derived anthraquinone. . .18
Figure 10: Basic structure of cardiac glycoside.............................................................................19
Figure 11: Basic structures of some pharmacologically important plant derived glycosides.......19
Figure 12: Basic structure of flavonoid.........................................................................................20
Figure 13: Basic structures of some pharmacologically important plant derived flavonoids.......20
Figure 14: Simple illustration of natural dye extraction process...................................................23
Figure 15: Graph of reflectance against wavelength.....................................................................36
Figure 16:Relationship between reflectance and colour and wavelength.....................................37
Figure 17:Reflection of light from multiple fibres........................................................................38
Figure 18: Kubelka-Munk function formular................................................................................39
Figure 19: Colour absorption value formular................................................................................39
Figure 20: Undyed cotton fabric....................................................................................................48
Figure 21: Dyed cotton fabric........................................................................................................49
Figure 22: Washed dyed fabric......................................................................................................49
Figure 23: A graph of reflectance against wavelength..................................................................50
Figure 24: A graph of K/S against wavelength.............................................................................51
Figure 25: Perkin Elmer UV-Vis Spectrophotometer...................................................................61
Figure 26: Dye extract...................................................................................................................61
Figure 27: Leaf extract...................................................................................................................62
Figure 28: Phytochemical screening test results............................................................................62

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TABLE OF CONTENTS
DECLARATION..............................................................................................................................i

APPROVAL....................................................................................................................................ii

DEDICATION...............................................................................................................................iii

ACKNOWLEDGEMENT..............................................................................................................iv

ABBREVIATIONS AND ACRONYMS........................................................................................v

LIST OF TABLES..........................................................................................................................vi

LIST OF FIGURES.......................................................................................................................vii

ABSTRACT....................................................................................................................................x

CHAPTER ONE: INTRODUCTION..............................................................................................1

1.1 BACKGROUND..............................................................................................................1

1.2 PROBLEM STATEMENT....................................................................................................4

1.3 OBJECTIVES........................................................................................................................4

1.3.1 General objective.............................................................................................................4

1.3.2 Specific objectives...........................................................................................................4

1.4 SCOPE OF THE STUDY......................................................................................................4

1.5 RESEARCH QUESTIONS....................................................................................................5

1.6 JUSTIFICATION OF THE STUDY.....................................................................................5

1.7 SIGNIFICANCE OF THE STUDY.......................................................................................5

CHAPTER TWO: LITERATURE REVIEW..................................................................................7

2.1 THE FAMILY ACANTHACEAE.........................................................................................7

2.2 GENUS JUSTICIA................................................................................................................7

2.2.1 Taxonomy tree for Justicia betonica L............................................................................8

2.3 NATIVITY AND DISTRIBUTION OF JUSTICIA BETONICA..........................................8

2.4 DESCRIPTION AND EMBRYOLOGY OF THE PLANT..................................................9

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 2.5 USES OF JUSTICIA BETONICA IN TRADITIONAL MEDICINES..............................11

2.6 PHYTOCHEMICAL INVESTIGATIONS CARRIED OUT ON J. BETONICA..............12

2.6.1 structure of phytochemicals...........................................................................................12

2.7 NATURAL DYES...............................................................................................................20

2.7.1 Advantages and disadvantages of natural dyes.............................................................21

2.7.2 Future use of dyes..........................................................................................................21

2.8 EXTRACTION METHODS OF NATURAL DYES..........................................................22

2.8.1 Extracting using aqueous solution.................................................................................23

2.8.2 Optimized aqueous extraction conditions.....................................................................26

2.8.3 Solvent extraction..........................................................................................................26

2.9 APPLICATION OF NATURAL DYE ON COTTON FABRICS......................................27

2.9.1 Mordanting of natural dyes...........................................................................................29

2.9.2 Methods of mordanting Used in Dyeing with Natural Dyes.........................................31

2.10 PURIFICATION AND CHARACTERIZATION OF NATURAL DYES.......................34

2.10.1 UV-Vis Spectrophotometry.........................................................................................34

CHAPTER THREE: METHODOLGY.........................................................................................41

3.1 MATERIALS AND REAGENTS.......................................................................................41

3.1.1 Materials........................................................................................................................41

3.1.2 Reagents........................................................................................................................41

3.2 METHOD.............................................................................................................................42

3.2.1 Sample preparation........................................................................................................42

3.2.2 Desizing of substrate.....................................................................................................43

3.2.3 Scouring of the Cotton Fiber.........................................................................................43

3.2.4 Extraction......................................................................................................................43

3.2.5 Phytochemical analysis of the dye extract....................................................................43

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 3.2.6 Dyeing of fabric.............................................................................................................45

3.2.7 Evaluation of Color Strength (K/S) of Wash-offs.........................................................45

CHAPTER FOUR: RESULTS AND DISCUSSION....................................................................47

4.1 EVALUATION OF NATURAL COLOURANT................................................................47

4.1.1 Analysis of Dye extract.................................................................................................47

4.1.2 Phytochemical analysis of Dye extract.........................................................................47

4.2 DYEING OF JUSTICIA BETONICA DYE EXTRACT ON FABRIC..............................48

4.2.1 Colour strength analysis................................................................................................49

CHAPTER FIVE: CONCLUSION AND RECOMMENDATIONS............................................53

5.1 CONCLUSION....................................................................................................................53

5.2 RECOMMENDATIONS.....................................................................................................53

5.3 DELIMITATION.................................................................................................................53

5.4 LIMITATIONS....................................................................................................................53

5.5 REFERENCES.....................................................................................................................54

5.6 APPENDIX..........................................................................................................................62

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ABSTRACT
Nature has gifted us more than 500 dye-yielding and medicinal plant species. Coloring agents of
these plants are derived from roots, leaves, barks, trunks or fruits. Justicia betonica L., family
Acanthaceae is a widely used traditional folk medicinal herb. It is a unique source of jusbetonin
and the leaves yield bluish purple dye. Natural dyes have better bio degradability and generally
have higher compatibility with the environment. They are non-toxic, non-allergic to skin, non-
carcinogenic, easily available and renewable. Color fastness is the resistance of a material to
change any of its color characteristics or extent of transfer of its colorants to adjacent white
materials in touch. The leaves of Justicia contains different phytochemicals which are
responsible for giving colour, antimicrobial and antioxidant properties to the fabric. In this study,
natural colourants were extracted using different solvents. The dye potential of the colourants
obtained from the Justicia betonica dye was evaluated by colouring cotton fabric as well as for
its phytochemical properties by qualitative laboratory tests. Simultaneous mordanting of cotton
fabric was done by using alum and the dyed fabric samples were allowed to age for a week and
then washed in standard industrial detergent solution. The wash offs from the samples were
assessed for dye bleeding using the conventional photo-spectrometric method. The absorbance
measurements were conducted using spectrophotometer at 100nm intervals between 400-700nm
wavelengths and the reflectance evaluated. The colour absorption values of the wash offs was
evalutated using the Kubelka-Munk equation. Various phytochemicals were found present in the
extract including flavonoids, terpenoids and others and spectral reflectance curves for the dyed
fabrics were plotted and indicated colour shades between 400 and 700 nm.

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CHAPTER ONE: INTRODUCTION

1.1 BACKGROUND

The World Health Organization (WHO) recognizes that traditional and complementary
medicines (TCM) are a vital part of the global health care system (Burton A, 2015). In Africa, it
is estimated that over 80% of the population continues to rely on medicinal plant species to meet
their basic health care needs (Ngbolua K.N, 2011). Justicia is the largest genus of Acanthaceae.
Its species are widespread in tropical regions of the world (Wasshausen D.C, 2004)and are
poorly represented in temperate regions (D.J, 1997). Justicia this genus of Acanthaceae, its
species are found in Asia, America and Africa. They are cultivated in Democratic Republic of
the Congo (DRC). Many parts of those plants are used in traditional medicine to treat several
diseases include, diabetes, cancers, respiratory and gastrointestinal diseases and as well as
inflammation, central nervous system as hallucinogens, somniferous agents, sedatives,
depressors and treatments for epilepsy and other mental disorders. Other species are used in the
treatment of headache and fever, sedative, analgesic, bronchitis, arthritis, vaginal discharges,
dyspepsia, and eye disease. Biological activities of different extracts of Justicia are well
documented and a considerable number of phytochemicals was isolate from different parts of
those plants (Wasshausen D.C, 2004).

In the past, this plant was grown in the local surroundings as an ornamental until locals
discovered its medicinal properties. The leaves of this plant were subsequently used to cure
various illnesses such as anemia or hypertension and are now widely used for many other
medicinal applications. The phytochemical composition of various cultivar leaf extracts revealed
the presence of polyphenols such as flavonoids, tannins, leuco-anthocyanins and anthocyanins)
(Mpiana P.T, 2010). Flavonoids were the largest class of phenolic compound found. Several
species of Justicia are widely used in folk medicine for the treatment of respiratory and
gastrointestinal diseases as well as inflammation. The plants are also utilized for their effects on
the central nervous system as hallucinogens, somniferous agents, sedatives, depressors, and
treatments for epilepsy and other mental disorders, with eleven occurrences. Other species are

1
popularly used in the treatment of headache fever, analgesic properties, cancer, diabetes, and
HIV. Whole plant and aerial parts are used in folk medicine. Extracts made from only the leaves
are the most used, followed by those extracts made from only the roots. (Adams M, 2007).

Nature in its occurrence is full of fascinating colors without which life would have been dull and
monotonous. Color is a property by which an object has the ability of producing different
sensations to the eye due to the way the object reflects or emits light. Dyeing is an ancient art
which really predates written records. Its practice can be traced back to the Bronze Age era in
Europe where primitive techniques of dyeing included sticking plants to fabric and rubbing
crushed pigments onto clothes (Ado A, 2014).

The first use of the blue dye, wood, beloved by the ancient Britons may have originated in pales
tine where it was found growing wild. The most famous and highly prized color through the ages
has been trian purple, noted in the Bible, a dye obtained from the spiny dye-murex shellfish. The
Phoenician prepared it until the seventh century, when Arab conquerors destroyed their dyeing
installations. All these produced high-quality dark colors. Until the mid-19th century all dyestuff
was made from natural materials mainly from vegetable and animal matter (Siva.R, 2007). This
form of coloring changed in 1856 when William Henry Perkin discovered the first synthetic dyes
called Mauvein. The latter being cheap and easier to apply than the natural dyes quickly replaced
natural dyes, and found favor in textile industries causing a cultural revolution which resulted
into the commercialization of synthetic dyes (Perkin, 1856).

Most textile dyers and manufacturers therefore shifted towards the use of synthetic colorants
because they were cost advantageous, easy to obtain in different grades, types and classes. As a
result, this considerably lowered the cost of natural dyes because the quality of dyeing needed
improvement (Taylor, 1989). For many decades, the practice and application of synthetic dyes
and colorants expanded as that of natural dyes tremendously declined since the existing natural
dyes could not satisfy the demands of the market. Despite their being threatened by the
application of synthetic dyes for over a century, the use of natural dyes never eroded away
completely.

However, the increasing application of synthetic dyes amidst worldwide conscience of

environmental pollution brought to the fore the threats and glitches associated with its production

2
and usage. Their attendant side effects on human health, has led to a regenerated overwhelming
international interest in natural dyes and natural fiber products dyed with eco-friendly natural
dyes containing organic value. Therefore, a significant revival of interest in natural colorants has
emerged.

Today, the use of natural dyes in textile dyeing has once again gained interest as an important
alternative to synthetic dyes because some of these (synthetic dyes) have been reported to have
carcinogenic effects besides causing environmental pollution (Paitoon A, 2002). Apart from
dyeing and printing, the demand of natural dyes for use in food manufacturing industries,
pharmaceuticals and cosmetic has greatly increased over the last decade. This is so because
natural dyes produce very extraordinary soothing, and soft shades that complement each other as
compared to synthetic dyes.

They have also gained economic advantage over synthetic dyes because some are antimicrobial,
non-toxic, non-carcinogenic and biodegradable. On the other hand, synthetic dyes which are
widely available at an economic price and produce a wide variety of colors, occasionally cause
skin allergy and other harmful effects to the human body as well as embodying undesirable
toxicity/chemical hazards accruing from their synthesis, application and life cycle. Aniline for
example which is used extensively in the production of certain types of dye releases toxic vapor
which is potentially carcinogenic (Sengupta S, 2015)

In Uganda, some research on extraction of natural dyes has been carried out and the natural
colorants obtained utilized as sources of dyes in the dyeing of mats, ropes and other home-based
craft materials (Wanyama P.A.G, 2011). The use of natural dyes can therefore be a substitute for
many hazardous synthetic dyes. Serious efforts are now being made to identify more raw
materials so as to boost, standardize the recipes and usage of natural dyes. In this research
Justicia betonica has been identified for study.

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1.2 PROBLEM STATEMENT
Today, most processes executed in the textile and clothing industry discharge atmospheric
emissions that are disturbing the eco-balance. Dyeing of fabrics which accounts for most of the
waste is a long process where at each stage some harmful chemicals used generate waste. This
waste poses a threat to the workers’ health and also to the general population through
environmental pollution from production of some synthetic dyes. Such environmental issues
associated with the production and application of synthetic dyes, coupled with the modern
dynamics of development, have once again revived consumer interest in natural dyes. Similarly,
the worldwide growing appreciation of the organic value of eco-friendly products, has focused
consumer interest towards use of textiles dyed with natural dyes. The current dyestuff
requirement for the textile industry is about 3 million tons (natural dyes taking only 10-25%) and
considering this fact, the use of natural dyes in mainstream textile processing is a big challenge.
Besides that, available scientific studies and systematic reports on dyeing of textiles with natural
dyes are still insufficient, yet there are plenty of natural products still untouched, hence the
relevance of this research project

1.3 OBJECTIVES
1.3.1 General objective
The general objective of this research work was to evaluate the phytochemical properties and
analyze the dyeing of cotton fabrics using Justicia betonica plant extract

1.3.2 Specific objectives

i. To extract natural coloring matter from the leaves of Justicia betonica plant and evaluate
its phytochemical properties
ii. To analyze the reflectance and color strength of wash-off of the dyed substrate

1.4 SCOPE OF THE STUDY

In this research, the coverage was; extraction of natural colorants from leaves of Justicia
betonica, evaluation of extract phytochemical properties, application of colorants on cotton
fabric using alum as a mordant, identification of the shades obtained, analysis of reflectance and
color strength of wash-off and comparison of results using statistical techniques

4
1.5 RESEARCH QUESTIONS
1. What are some of the natural plants from which dyes can be extracted?
2. How can these extracted dyes be used to dye textiles?
3. Are there any phytochemicals that are important in the plant?

1.6 JUSTIFICATION OF THE STUDY

Nature has gifted us with more than 500 dye-yielding and medicinal plant species, and a rich
biodiversity harboring a wealth of useful resources. Natural dyes are considered eco-friendly
because they are renewable, biodegradable, skin friendly safe and easy to handle (Gupta, 1998).
Though natural coloration is known from ancient time as artisanal practice for handicrafts,
painting and handloom textiles, the chemistry of interaction of such colorants with textile
materials is of relatively high interest in production of eco-friendly textiles. Therefore, there is
need to build a knowledge base and database with the production of appropriate shade cards for
different textiles using improved, biodegradable, environment friendly mordant extraction
methods. This will help popularize the use of natural dyes and solve some of its problems
relative to application methods, reproducibility and color fastness. Furthermore, there still exists
an enormous presence of unexploited plant resource base in the wild that is readily available and
eco-friendly, and guaranteeing safety of operation. Despite that, no synthetic dye has the luster,
under-glow of rich color, soft light and shadow that gives so much pleasure to the eye as the
natural dye. Moreover, presently it is more urgent to revitalize the sacrament of natural dye and
dyeing techniques as an alternative to the use of unsafe synthetic dyes

1.7 SIGNIFICANCE OF THE STUDY

Exploitation of the plant will offer not only a great potential of rich diverse source of dyes, but
also provide an alternative and sustainable income to farmers and local populations through
harvest and sale of plant dyes, thus enhancing and increasing national resource development of
domestic products. Furthermore, the natural dyes and colorants will not only be used for textile
dyeing but as alternative coloration of foods, medicines, hair dye, cosmetics, handicraft items
and toys, leather processing and other economic purposes. The exploitation of Justicia betonica
in this study will reinforce and enhance knowledge and database as research efforts in this area
have been negligible. Natural dyes are therefore an essential part of the world’s ecological and
cultural heritage as their selection and use to create colors is beneficial to all civilizations

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CHAPTER TWO: LITERATURE REVIEW

Synthetic dyes have been globally used in textile industries due to their availability. However,
the advantages and disadvantages associated with their production and usage have obliged dyers
to search for alternative sources, particularly natural dyes. For the last one decade or more,
research has been carried out to explore the concealed components found in plants, animals,
insects and other natural sources. This chapter therefore contains accounts of literature reviewed
on the extraction, application and analysis of natural dyes on textile materials. The literature
survey indicates that there is little work reported in the field of extraction and dyeing of cotton
fabrics with Justicia betonica and its phytochemical compounds.

2.1 THE FAMILY ACANTHACEAE

Acanthaceae family is comprised of herbs, shrubs and vines of 2500 species classified under 200
genera (Willis, 1966). Most of them are shrubs, tropical herbs or twining vines. The
characteristic features of Acanthaceae are opposite pairs of leaves, presence of calcium carbonate
crystals in vegetative parts, bisexual flowers, large and petaloid bracts, 4-5 petals and sepals, 2-4
stamens, staminodes, superior ovary with 2 fused ovules. The members of Acanthaceae are
horticulturally important and cultivated as ornamentals (A.J. Amirul-Aiman, 2013)

Presence of valuable secondary metabolites and biological activities of the family members are
well documented. The medicinal properties of various genera of the family are well explored by
folk, traditional and modern systems of medicines (M.T. Islam, 2012).

2.2 GENUS JUSTICIA

Genus Justicia occupies major place and represents as largest genus with approximately 700
species of the family Acanthaceae with many unresolved species (Daniel, 2011). The genus
Justicia possesses 1695 species with scientific names, out of which 376 are accepted species
names and 32 scientific names of intraspecific. Justicia species are reported to occur in tropical
to warm temperate regions of the Americas, India, Indonesia, Southeast Asia, Malaysia, Pakistan
and Africa (A. Perveen, 2010).

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Species of Justicia are widely explored in folk medicines and are used to cure respiratory,
gastrointestinal, inflammatory disorders, malaria, viral fever, epilepsy, rheumatism, headache,
cancer, diabetes, mental disorders, arthritis and HIV (G.M. Correa, 2012). The phytochemical
characterization of Justicia species shows the presence of quinazoline as major compound with
other important bioactive substances viz., Vasicine, Vasicinone and Vasicinol. These secondary
metabolites are well studied for their bronchodilator, hypotensive, abortifacient, anticancer,
cytoprotective, antimycobacterial, hepatoprotective, antidiabetic, antispasmodic, anti-bleeding,
male contraceptive and hallucinogenic properties (K. Kavitha, 2014).

2.2.1 Taxonomy tree for Justicia betonica L.

Table 1: Taxonomy tree for Justicia betonica L

Kingdom Plantae
Division Tracheophyte
Class Magnoliopsida
Order Lamiales
Family Acanthaceae
Sub-family Nelsonioideae
Tribe Ruellieae
Sub-tribe Justiciinae
Genus Justicia
Species Betonica L

2.3 NATIVITY AND DISTRIBUTION OF JUSTICIA BETONICA

The generic name ‘Justicia’ honors James Justice (1698–1763), a famous Scottish horticulturist
and the specific epithet ‘betonia’ mean betony-like, resembling Betonica, a plant genus which
come from a name Vettonica, medicinal plant from Vectones (Vettones), Spain (R.H.S, 2008).
Justicia betonica is reported to be native of eastern and southern Africa (i.e., Kenya,
Mozambique, Lesotho, Namibia, South Africa and Swaziland) and the Indian subcontinent (i.e.,
India and Sri Lanka) and widely naturalized in warmer coastal districts of eastern Australia and
Pacific islands.

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2.4 DESCRIPTION AND EMBRYOLOGY OF THE PLANT
Justicia betonica is a perennial shrub that grows up to the height of 2 m. Leaves are simple,
opposite-decussate, lanceolate to ovate, sometimes elliptic, measures approximately 6- 17.5 ×
2.2-4 cm, leaf base acute-attenuate, tapering into petiole (2 cm long) with entire to crenate
margin. Stem is terete, herbaceous, tumid and purple above the nodes. Inflorescence (6- 25 cm
long) is simple, rarely branched terminal spikes and peduncles long (3-8.5 cm). The bracts are 4-
ranked, imbricate (overlapping), white to pale green, broadly ovate, acute at apex, usually
scarious at margins, 3-nerved with purplish nerves and elliptic bracteoles.

Figure 1:Habit of Justicia betonica L

Flowers are hermaphrodite, 0.8-1.5 cm long, hypogynous, zygomorphic and usually on one side
of the spike. Coralla bilipped infundibular, white with 2 pinkish ridges or spots in the corolla
throat with upper lip hooded and limb pinkish mauve. The corolla tube is 5-8 mm, greenish
white, finely velvety with scattered stalked glands (Khare, 2007). There are two stamens,
epipetalous, posses 3 types of epidermal hairs, two types are restricted to anther and third type is
present on the filament. Anatomy of anther reveals the presence of outer wall layer epidermis,
fibrous endothecium, single ephemeral middle layer and glandular dimorphic tapetum. Distinct

8
variation reported in the cells of anther wall and connective in terms of size, shape and number
of nuclei. The microspore mother cells give rise to tetrahedral and decussate pollen tetrads. The
endothecium is absent, the pollen grains are dimorphic in size, tricolporate (6 38.1 ± 8.9 in polar
view and 23.1 ± 5.1 in equatorial view), possess pseudocolpi and sheds at 2-celled stage (K.
Rueangsawang, 2013) Gynoecium is bicarpellary, syncarpous, ana-campylotropous, unitegmic
and tenuinucellate. Four ovules are arranged in axial placentation on 2 longititudinal rows. A
single archesporial cell acts as megaspore mother cell, embryo sac development is Polygonum
type. The antipodals and synergids are ephemeral, funiculus with distinct micropyle. The
micropylar end has 4-nucleate stage (Narayanan, 1956). The fruits are pubescent capsules, 2
lobed, ovoidly clavate (10-20 x 5-6 mm) with a short solid base.

Flowering and fruiting reported throughout the year. Seeds 4-8 mm broad, are pale/dark brown
in color, orbicular (spinulose) to suborbicular in shape, sub-compressed, rugulose-tuberculate,
3.25–4.48 mm width × 3.32–4.52 mm length, endosperm is cellular, testa is densely tuberculate
and is made of loosely arranged cells and comparatively large among the Justicia species (K.
Rueangsawang, 2012).

Figure 2:Flowering twig of Justicia betonica L

9
2.5 USES OF JUSTICIA BETONICA IN TRADITIONAL MEDICINES
Entire plant of J. betonica is boiled with the stem or root of Plectranthus ternifolius, whole plant
parts of Barleria strigosa, Lepidagathis fasciculata and rhizome of Smilax glabra and taken
orally three times a day to treat insect bites in Thailand (W. Chuakul, 1996). It’s well
acknowledged for gastrointestinal complaints. The infusion made from the whole plant is used to
cure stomach ache by Lou tribe of Tanzania (J.O. Kokwaro, 1998).

In India, the inflorescence extract of this plant is given orally to treat vomiting and constipation
and used externally to wash hairs (D.M. Rao, 2006). Leaves crushed and applied as poultice on
abscesses to relieve pain and swelling (Khare, 2007). Nandi community of Kenya devours leaf
and flower ash internally for the treatment of cough, diarrhea and orchitis (P. Jeruto, 2008).
Decoction of J. betonica leaves were used by Kipsigis people in Kericho County of Kenya to
cure vomiting and headache due to plasmodial infection and malarial symptoms (B.C. Pacifica,
2018). Fresh roots are homogenized in water and given orally to aid snake bite in human and
cattle (T.H. Bekalo, 2009).

In Uganda the leaves of J. betonica are used against HIV/AIDS (M. Lamorde, 2008). J. betonica
leaves are boiled with the leaves of Cannabis sativa and Tetradenia riparia and used to control
helminthes in calves and cattles. The plants are grown with Banana plantation in Uganda to
extract bluish purple dye form the leaves and reported to possess more preponderance of dye
compared to stem and root (Wanyama P.A.G, 2011). Nyakayojo sub country in southwestern
Uganda uses J. betonica to treat malaria (T. Stangeland, 2011). J. betonica is administered to
lower cholesterol and used to treat paralysis, earaches, headaches, bruises, diarrhea, vomiting,
constipation, pain and inflammation and malaria in India (K. Gangabhavani, 2013).

Leaves are crushed, mixed with water and allowed to take bath to alleviate weakness during
pregnancy and malaria. Root infusion is taken orally to treat hernia. Root is pounded in water
and taken orally to cure worm infection (P. Tugume, 2016). Luhya community of Kakamega
East sub-County from Kenya pounds the aerial parts with cold water and boiled extract is
administered for malaria. Due to its bitter taste resembling their native anti-malarial plant Ajuga
integrifolia, J. betonica plants were introduced in the river beds of Kenya to control malaria (N.
Mukungu, 2016). Luhya community in Kakamega County of Western Kenya chews the whole
plant of J. betonica to treat toothache and malaria (E. Odongo, 2018).

10
2.6 PHYTOCHEMICAL INVESTIGATIONS CARRIED OUT ON J. BETONICA
Aqueous and methanolic extracts of various parts (leaf, stem and root) of J. betonica were
investigated for the presence of polyphenolic contents using Folin-Ciocalteu assay. The total
phenolics detected from aqueous extracts of leaf, stem and roots are 2.57, 2.11 and 9.31 mg
GAE/gm respectively (S. Sini, 2018). Simultaneously absolute methanolic extracts exhibited
2.22, 1.77 and 4.95 mg GAE/gm for leaf, stem and root of J. betonica. The standard
phytochemical procedures using aqueous and methanolic extracts reveal that all plant parts
possess alkaloids, flavonoids, glycosides, carbohydrates, gum and mucilage, proteins, fixed oils
and fat, phenolics and tannins (G. Bbosa, 2013).

Alkaloids, fixed oils and fat are absent in the roots and the whole plant is devoid of saponins and
sterols. Total phenolic content is reported to be least in J. betonica as compared with other
Justicia species such as J. adhatoda, J. beddomei, J. carnea, J. gendarussa, J. montana and J.
wayanadensis (S. Sini, 2018). Methanol extracts of leaf, stem and roots shows the presence of
total phenolics and flavonoid compounds. Leaves of J. betonica reported to possess high number
of flavonoids (2.86 mg QE) than J. adhatoda, J. beddomei, J. gendarussa, J. montana and J.
wynaadensis (B. John, 2013).

Qualitative phytochemical analysis using standard procedures reveals that the ether extracts of J.
betonica possess steroids and triterpenoids, alkaloids and saponins and these compounds are
reported to be active against malaria parasite (G. Bbosa, 2013).

2.6.1 structure of phytochemicals

Phyto-chemicals are nonnutritive plant chemicals that have protective or disease preventive
properties. Plant produces these chemicals to protect itself but research works demonstrates that
many phytochemicals can protect humans against diseases. The different qualitative chemical
tests were carried out on the aqueous extract using standard procedures to identify the
constituents as described. Phytochemical screening of Justicia betonica leaves were reported to
contain tannins, Flavonoids, Saponins, phenolics and traces steroids and alkaloids (B. John,
2013).
2.6.1.1 Alkaloids
These are the largest group of secondary chemical constituents made largely of ammonia
compounds comprising basically of nitrogen bases synthesized from amino acid building blocks

11
with various radicals replacing one or more of the hydrogen atoms in the peptide ring, most
containing oxygen. The compounds have basic properties and are alkaline in reaction, turning
red litmus paper blue. In fact, one or more nitrogen atoms that are present in an alkaloid,
typically as 1°, 2° or 3° amines, contribute to the basicity of the alkaloid. The degree of basicity
varies considerably, depending on the structure of the molecule, and presence and location of the
functional groups (Jones W.P, 2006).

Majority of alkaloids exist in solid such as atropine, some as liquids containing carbon,
hydrogen, and nitrogen. Most alkaloids are readily soluble in alcohol and though they are
sparingly soluble in water, their salts of are usually soluble. The solutions of alkaloids are
intensely bitter. These nitrogenous compounds function in the defense of plants against
herbivores and pathogens, and are widely exploited as pharmaceuticals, stimulants, narcotics,
and poisons due to their potent biological activities. In nature, the alkaloids exist in large
proportions in the seeds (Doughari, 2012).

Structure of alkaloids

12
Figure 3: Basic structures of some pharmacologically important plant derived alkaloids

2.6.1.2 Tannins
Tannins are phenolic-based natural products (Gandini, 1989). They are phenolic compounds of
high molecular weight. Tannins are soluble in water and alcohol and are found in the root, bark,
stem and outer layers of plant tissue. Tannins have a characteristic feature to tan, i.e., to convert
things into leather. They are acidic in reaction and the acidic reaction is attributed to the presence
of phenolics or carboxylic group. They form complexes with proteins, carbohydrates, gelatin and
alkaloids. Tannins are divided into hydrolysable tannins and condensed tannins. Hydrolysable
tannins, upon hydrolysis, produce gallic acid and ellagic acid and depending on the type of acid
produced, the hydrolysable tannins are called gallotannins or egallitannins. On heating, they
form pyrogallic acid. Tannins are used as antiseptic and this activity is due to presence of the
phenolic group. Common examples of hydrolysable tannins include theaflavins (from tea),
daidezein, genistein and glycitein. Tanninrich medicinal plants are used as healing agents in a
number of diseases. In Ayurveda, formulations based on tannin-rich plants have been used for
the treatment of diseases like leucorrhoea, rhinnorhoea and diarrhea (Doughari, 2012).

13
Chemical structure of tannin

Figure 4: Basic structure of tannin

Figure 5: Basic structures of some pharmacologically important plant derived tannins

2.6.1.3 Saponins
Saponins comprise a large family of structurally related compounds containing a steroid
(sapogenin) linked to one or more oligosaccharide moieties. They are characterized by their
hemolytic activity and foaming properties and are responsible for imparting a bitter taste to plant
materials containing a high concentration of saponins. Saponins are very poorly absorbed. Most
of the saponins form insoluble complexes with 3-b-hydroxysteroids and are known to interact
and form large mixed micelles with bile acids and cholesterol (V.R. Mohan, 2016). 

These are glycosides with a distinctive foaming characteristic. They consist of a polycyclic
aglycone that is either a choline steroid or triterpenoid attached via C3 and an ether bond to a
sugar side chain. The aglycone is referred to as the sapogenin and steroid saponins are called

14
saraponins. The saponins show the antimicrobial activity by inhibiting the growth of Gram
positive or Gram-negative microorganism. Some saponins are not effective against Gram
negative microorganisms because of the reason that they are not able to penetrate into the cell
membranes of the microorganisms. (Riguera, 1997)

Structure of saponin

Figure 6: Basic structure of saponin

2.6.1.4 Terpenoids
Terpenoids otherwise known as isoprenoids are a large and diverse class of naturally occurring
compounds derived from five carbon isoprene units. Terpenoids are differentiated from one
another by their basic skeleton and functional groups. Terpenoids are ubiquitous; thus, they are
found in almost all classes of living organisms. They contribute to the flavor, scents, and color of
plant's leaves, flowers, and fruits. Terpenoids are also essential for plant growth and
development. The terpenoids produced by plants do not only protect them against insects and
herbivores but also provide protection from fungal diseases and infestations, whereas in animals
terpenoids serve as the precursor of steroid and sterols (Beverly A.S. Reyes, 2018).

15
The structure of terpenoids

Figure 7: Basic structure of terpenoid

2.6.1.5 Anthraquinones
These are derivatives of phenolic and glycosidic compounds. They are solely derived from
anthracene giving variable oxidized derivatives such as anthrones and anthranols (Doughari,
2012). Other derivatives such as chrysophanol, aloe-emodin, rhein, salinos poramide, luteolin
and emodin have in common a double hydroxylation at positions C-1 and C-8. To test for free
anthraquinones, powdered plant material is mixed with organic solvent and filtered, and an
aqueous base, e.g., NaOH or NH4OH solution, is added to it. A pink or violet colour in the base
layer indicates the presence of anthraquinones in the plant sample (Jones W.P, 2006).

Structure of anthraquinone

Figure 8: Basic structure of anthraquinone

16
Figure 9: Basic structures of some pharmacologically important plant derived anthraquinone

2.6.1.6 Cardiac glycosides
Cardiac glycosides are plant-derived steroid-like compounds which have been used for the
treatment of congestive heart failure for many years (Rengul C.A, 2018). These are a class of
organic compounds that increase the output force of the heart and increase its rate of contractions
by acting on the cellular sodium-potassium ATPase pump. They are also beneficial for cardiac
arrhythmias; however, their relative toxicity prevents them from being widely used. Most
commonly found as secondary metabolites in several plants such as foxglove plants, these
compounds nevertheless have a diverse range of biochemical effects regarding cardiac cell
function and have also been suggested for use in cancer treatment (Patel, 2016).

Glycosides in general, are defined as the condensation products of sugars (including

polysaccharides) with a host of different varieties of organic hydroxy (occasionally thiol)
compounds (invariably monohydrate in character), in such a manner that the hemiacetal entity of
the carbohydrate must essentially take part in the condensation. Glycosides are colorless,
crystalline carbon, hydrogen and oxygen-containing (some contain nitrogen and sulfur) water-
soluble phytoconstituents, found in the cell sap. Chemically, glycosides contain a carbohydrate
(glucose) and a non-carbohydrate part (aglycone or genin). Alcohol, glycerol or phenol
represents aglycones. Glycosides are neutral in reaction and can be readily hydrolyzed into its
components with ferments or mineral acids. Glycosides are classified on the basis of type of
sugar component, chemical nature of aglycone or pharmacological action (Doughari, 2012).

17
Structure of cardiac glycoside

Figure 10: Basic structure of cardiac glycoside

Figure 11: Basic structures of some pharmacologically important plant derived glycosides

2.6.1.7 Flavonoids
Flavonoids are important group of polyphenols widely distributed among the plant flora.
Structurally, they are made of more than one benzene ring in its structure (a range of C15
aromatic compounds) and numerous reports support their use as antioxidants or free radical
scavengers. The compounds are derived from parent compounds known as flavans. Over four
thousand flavonoids are known to exist and some of them are pigments in higher plants.
Quercetin, kaempferol and quercitrin are common flavonoids present in nearly 70% of plants.

18
Other group of flavonoids include flavones, dihydroflavons, flavans, flavonols, anthocyanidins,
proanthocyanidins, calchones and catechin and leucoanthocyanidins (Doughari, 2012).

Structure of flavonoid

Figure 12: Basic structure of flavonoid

Figure 13: Basic structures of some pharmacologically important plant derived flavonoids

19
2.7 NATURAL DYES
The word ‘natural dye’ covers all the dyes or colorants derived from natural sources like
different parts of various tree species and plants, invertebrates and minerals (Konar A, 2011).
Scientifically, dyes are defined as chemical compounds that are attracted to substrates in a more
or less permanent state, and evoke the visual sensation of color. They can further be defined as
molecules which have the ability to absorb and reflect the visible part of light at specific
electromagnetic spectrum to give the human eye a sense of color (Technology, 2016). Such dyes
that are majorly from plant parts are extracted by either aqueous method, acids or basic solvent
method. For some plant parts like fruits, the dyes are extracted by pressing out the juice or drying
the fruit followed by crushing it (Antima S, 2012).

2.7.1 Advantages and disadvantages of natural dyes

The advantage of natural is that unlike synthetic dyes which are synthesized from petrochemical
processes, natural dyes are friendly to the environment. Some dyes like madder are considered as
host in tea gardens while indigo is renewable and biodegradable i.e., the waste residue after
extraction is used as fertilizer for crops thus eliminating disposal problems. Natural dyes can be
extracted from by- products of some industries, hence fit the zero-emission approach (Konar A,
2011).

Secondly, natural dyes are obtained from renewable sources that contain value-added benefits to
our health. Many natural dyes are UV protective (Zhang B, 2010), antimicrobial (Wangatia L.M,
2015) and deoxidizing agents (Lee, 2004).

Thirdly, natural dyes can produce special coloring effects that cannot be produced by synthetic
dyes. The shades produced are usually soft, lustrous, soothing to the human eye and are
polygenetic (Sanjeeda I, 2014). The drawbacks associated with usage of natural dyes is the low
to medium fastness properties especially poor light fastness. Almost all-natural dyes except
substantive dyes need fixation with mordants such as metal salts which are toxic and
environmentally unfriendly. Natural dyes also have poor reproducibility, particularly the plant
sources since they are affected by environment conditions like varying planting time, place and
species. Other disadvantages include: non-availability due to difficulty in collection, bulk
isolation of dye-stuff, standardization of dyeing procedure, color yield and complexity of the
dyeing process.

20
2.7.2 Future use of dyes
Natural dye itself is considered good to the environment. However, the extraction method,
mordanting and dyeing methods used may not be environmentally-friendly, hence the need for
developing dyes and techniques that are as green as possible. This includes extraction of natural
dyes from wastes of other industries; new methods of improving extraction/dyeing efficiency,
use of biodegradable mordants extracted from natural sources, environmentally green treatment
of fabrics to improve dye uptake, minimizing environmental pollution and energy consumption
and dyeing of synthetic fibers with natural dyes. In order to minimize pollution during
mordanting, natural mordants such as myrobolan, mango bark, tea leaves, pomegranate, cow
dung, baking powder, aloe Vera as well as lemon juice can be used. For this research, alum has
been chosen for use.

2.8 EXTRACTION METHODS OF NATURAL DYES

The control loop for modelling a dye bath first requires the monitoring of the bath, followed by
modelling the parameters of the bath, and lastly controlling the process to achieve the target
result (shade). In order to maximize the color yield for suitable application onto the textile
material, extraction of colorant from its source as a vital step, requires standardizing and
optimizing extraction process and variables. This is of commercial significance because it leads
to a reduction of the cost of extraction and dyeing process by limiting on wastage of samples.

Natural dyes as demonstrated in Figure 14 have been extracted using different methods such as;
aqueous method, enzyme assisted extraction, supercritical fluid extraction, alcoholic/organic
solvent extraction, and Soxhlet extraction method using either alcohol or benzene. Using these
methods, the mixture is filtered, evaporated and dried using ultra filtration equipment, centrifuge
rotary vacuum pump or extraction under reduced pressure, so as to obtain a solidified sample.

21
Figure 14: Simple illustration of natural dye extraction process

As a rule, vegetable dyes are extracted by pounding or cutting up the coloring material,
immersion in water, heating to just below boiling point, and simmering until the color has been
fully extracted. Extraction can therefore be defined as the separation of desired color components
by physical or chemical means, with the aid of a solvent or aqueous media (Konar A, 2011). The
common solvents used are water, hexane, ethyl acetate, ether, benzene, chloroform, methanol
and ethanol. These are commonly used in combination with other technologies such as
solidification/stabilization, precipitation and electro spinning (Bechtold T, 2006). Extraction of
bioactive compounds is influenced by various process parameters such as solvent composition,
pH, temperature, extraction time and solid to liquid ratio. However, optimum conditions of
extraction can be determined by varying extraction parameters of the colorant.

2.8.1 Extracting using aqueous solution

This type of extraction also known as the traditional/conventional method has been used to
extract natural dyes from diverse sources. The main medium of extraction is water with or
without addition of salt/acid/alcohol or alkaline. For optimal extraction conditions, the finely cut
material or plant part is dissolved in measured quantity of water and extraction carried out under
varying conditions such as extraction temperature, time, pH, material to liquid ratio,
concentration of salt and mordant.

22
Various scientists have carried out experiments on extraction of natural dyes using this method.
Grover (2011), extracted natural dye from the floral parts of Woodfordia fruticosa and applied it
on cotton fabrics. The collected floral parts were divided into three parts and natural dye
extracted in three different manners. The first method involved preparing an aqueous solution
(10g of flowers in 100mL distilled water) and extraction carried out at temperature ranges of 80-
850oC. In the second method, 10g of uncrushed flowers were placed in distilled water and the
pasty mass was for 10-15 days. After extraction, the extract was filtered and used for dyeing. In
method three, 10g of crushed flowers were placed in a pot, left undistorted for 20-25 days and
thereafter filtered through a piece of cloth and used for dyeing. The results established that the
second method which gave a dark yellow-brown was the paramount extraction method (Grover
N, 2011).

In another experiment, colorant was extracted from the leaves of eucalyptus hybrid (grewia
optiva) using aqueous method under varying conditions (Mongkholrattanasit R, 2011). Aqueous
filtrates have also been used to assess various activities such as antioxidation, UV protection and
antimicrobial activity.

The common procedure associated with aqueous extraction under normal conditions is boiling
either the dried or fresh samples from low temperatures to the boiling point of water, cooling the
filtrate, filtering and storing it for further analysis. However, to optimize the extraction method
of color components in aqueous media, the sample/material is dried, finely cut, ground to
powdered form, and finally extracted in water under a standard process. The extraction process
of dye liquor is then carried out under varying condition such as extraction time, extraction
temperature, pH of extraction liquor, concentration of color/source material and material to
liquor ratio (MLR). The continued usage of this technique has led to the optimization of
extraction conditions of some natural dyes such as pomegranate rind, babool and marigold as
illustrated in Table 2.

23
Table 2: optimized extraction conditions of some natural dyes

Name of dye Procedure Temperatur Time pH M:LR

e (ºC) (mins)
Pomegranate Pre-cut and dried rind is 90 45 11 1:20
Rind crushed to powder form.
Residue is filtered to
40% (w/w)

Babool Sun-dried pre-cut chips 100 120 11 1:20

of babool bark are
crushed to powder form
and extracted in water
Marigold Dried petals of marigold 80 45 11 1:20
(Genda) are crushed to powder
and extracted. Residue
is filtered
Catechu Pre-dried powder of 90 Not 12 1:20
(Khayer catechu is crushed to specified
powder and extracted in
aqueous medium.
Jack fruit Pre-cut and dried chips 100 30 11 1:10
wood of jack fruit wood are
crushed to powder form

Red sandal Dried pre-cut chips are 80 90 4.5 1:20

wood crushed to powder form

24
2.8.2 Optimized aqueous extraction conditions
After the extraction process shown in Table 2.3, the samples are filtered to obtain 40% (w/w) of
a clear aqueous solution. This method (aqueous method of extraction) has been reported to be
amongst the best extraction methods (Jones W.P, 2006). This is because with water, it’s assumed
that the inevitable solvent residue is not harmful. In contrast to this, in industrial solvents,
maximum residues are defined for a certain purpose. Water also preferentially extracts polar
compounds such as plant pigments and tannins since these need some special post treatment like
ion exchange and caustic wash (Bart, 2016). Therefore, for the extraction of natural colorant
from Justicia betonica, pre-cut and dried leaves will be crushed into powder form, and the
pigment extracted in aqueous solution under varying extraction conditions.

2.8.3 Solvent extraction

Flavonoids and anthocyanins are soluble in polar solvents with the glucosides and aglycones
more soluble in water and alcohols respectively. These phenolics are commonly extracted from
plant materials with water/alcohol or methanol/ethanol mixtures acidified with mineral acids
(Basak S, 2012) to prevent the degradation of the non-acylated anthocyanin pigments. This
method has been applied successfully in the extraction of a variety of organic compounds
ranging from herbs to other plant material, as well as natural colorant from natural source dye
materials. Literature relating to the extraction of natural dyes using solvent extraction technique
includes Manasa (2013) who used 50% ethanol and methanol for the extraction of natural dye
from onion skin powder. After preparation, the solutions were soaked for 1, 2 and 3 days
respectively, thereafter filtered using Whatman No. 1 filter paper and used for the estimation of
total physicochemical parameters present in the dye. The study established that for the extraction
of quercetin, rutin and total phenolic content, methanol gave the best results (Manasa M, 2013).

The extraction of anthocyanins can be done using one solvent with various solvent techniques as
demonstrated in the article (Sharif A, 2010), or a mixture of solvents with acid like Geetha
(2011) who treated 0.5gm of onion peel with 10ml of two different solvents (methanol and
acidified methanol). The solution was then centrifuged at 10,000 rpm for 10 minutes and the
supernatant analyzed. Methanol emerged as the better solvent followed by acidified methanol,
because it extracted more flavonoids (Geetha M, 2011). This method has been used to extract
other natural dyes.

25
Although the methods of extraction of natural dyes are vast, in this research, aqueous method
and solvent (using ethanol and methanol) extraction method was employed because of lack of
equipment. These methods have also proven that they yield good fastness properties (Rosa M. R,
2010).

2.9 APPLICATION OF NATURAL DYE ON COTTON FABRICS

Color obtained from multitudes of plants, animals and fungi has been used to color skin, hair,
food we eat, clothes and other different kinds of natural fibers that have been collected,
examined and analyzed as sources of raw materials for textile industries (Konar A, 2011). The
most common taxonomy of natural fibers used are those classified by botanical type. Using this
system, there are six basic types of fibers namely; Bast/core fibers such as jute, flax, hemp,
ramie, grass and kenaf; Leaf fibers such as banana, sisal, agave and pineapple; Seed fibers such
as coir, cotton and kapok and finally reed fibers such as wheat, corn and all other types such as
wood and roots (Broadbent, 2001).

Ancient artisans transformed these available fibers (mainly long fibers) into fabrics, at first by
hand and later using simple mechanical devices. The short fibers on the other hand were first
carded or combed, then drawn and finally spun by pulling with gradual twisting to produce yarn
which was interlaced to form fabric. Techniques of fabric production and their coloration has
subsequently developed over many hundreds of years with the most suitable fibers being natural
fibers. These have then become the basis of the textile industry in the world (Cordon, 2010).

Today, cotton, wool, jute, flax and silk are the most common important natural textile fibers
used, while cotton remains the most common conventional fiber preferred. This fiber has been
used in various textile applications such as dyeing. Dyeing occurs at any stage of textile
manufacture either on loose fiber or on the intermediate forms such as sliver or yarn, fabric or
towards the end of the manufacturing cycle including garments and finished articles. The art of
coloring with natural dyes which dates as far as the ancient civilization of Egypt, Samaria, Rome,
Greece, Mexico, India and China (Cordon, 2010) is where color is applied either on the fibers,
yarns (known as yarn dyeing), fabric or on finished textile products, by either printing or dyeing
(Saxena S, 2014).

26
Printing is the localized application of different dyes to different specific areas on one face of a
fabric according to a predetermined design, while the art of dyeing on the other hand is the
method that involves contact between an aqueous solution/ dispersion of dyes and the textile
material, under conditions that promote substantivity for the production of uniform coloration.
This can be attained through the following approaches;

a) Direct dyeing: where the dye in the aqueous solution in contact with the material is gradually
absorbed into the fibers due to its inherent substantivity.

b) Dyeing with a soluble precursor of the dye which forms an insoluble pigment deep within the
fiber on treatment after dyeing.

c) Direct dyeing: which follows a chemical reaction of the dye with appropriate groups in the
fiber.

d) Adhesion of the dye/pigment to the surface of the fibers using an appropriate binder.

Dyeing can be carried out in any medium say alkaline, acidic or in a neutral medium, using any
fibers such as natural/synthetic, fabrics like woven/knitted with either synthetic or natural dyes.
For uniform coloration during the dyeing process, factors such as the selection of proper dye
according to the textile material to be dyed, fiber characteristics like denier, staple length, luster,
cloth construction and selection of the method of dyeing the fiber, yarn or fabric should be
critically observed. The process of dyeing substrates may also necessitate additional treatment
while in the dyeing machine and upon finishing the dyeing process, the material is scoured to
remove adhering solution, unused and unfixed dyes. Different researchers have reported on the
available methods of dyeing fibers such as cellulosic, protein and synthetic with different
synthetic and natural dyes. For example, Prabhavathi (2014) improved the color fastness of
selected natural by optimizing the dyeing conditions of cotton fabrics. His study established that
the depth of shape increased with increase in mordant concentration. Very little attention has
been paid on improving color fastness with natural fixing agents as sources of dyes and
colorants. However, some studies have been made such as Prabhavathi (2014) who used five
fixing agents namely vinegar, lime juice, alum, ammonia, calcium chloride and myrobolan to
mordant fabrics using eucalyptus bark dye. The study concluded that all agents gave good
fastness results, vinegar giving the most excellent results (Prabhavathi P, 2014).

27
In another study, (Wangatia L.M, 2015) compared mango bark mordanted cotton samples with
those mordanted using alum. The investigation established that fabrics unmordanted and those
pre-mordanted with mango bark had the highest K/S values. However, in comparison with
natural fixing agents, the artificial coloring agents (synthetic mordants) show greater resilience
and stability when exposed to oxidation, changes in temperature, pH and other factors. Often,
these factors led to a shift in the resonance structures in the dye molecules while pH is known to
change the spectra of dyes significantly. This is because at higher pH values, protons can be
abstracted, which change the electronic configuration of the dye molecule. Since absorbance of
light is a factor of the electron configuration, any change in the configuration yields a change in
the spectrum.

2.9.1 Mordanting of natural dyes

2.9.1.1 Types of Mordants
Natural dyes are non-substantive to textiles thus should be applied with the help of metallic salts
or natural mordants that produce affinity for both the coloring matter and the fiber (Ferreira
E.SB, 2004). Transition metal ions such as stannous chloride, ferrous sulphate, alum, chrome and
copper sulphate have been used as mordants as they have strong coordinating power capable of
producing weak to medium attraction or interaction forces that act as bridging materials thereby
creating substantivity for the subsequent fiber. These salts combine with the fiber forming
insoluble precipitates (fixing both the dye and mordant into the fiber) and as a result form
reasonable fastness level (Konar A, 2011).

Many reviews have already been dedicated to developing mordanting agents which are nontoxic
and ecofriendly. Some of the approaches exploited include the use of metals which are
considered ecologically safe such as alum and iron sulphates; use of natural oil products, tannins
and other natural plant extracts (Saxena S, 2014).

A mordant can be defined as a chemical agent used to set/fix dyes on fabrics through the
formation of insoluble compounds with dyes i.e., facilitate the bonding of the dyestuff to the
fiber. Mordanting on the other hand is the treatment of textile fabrics with metallic salts or other
complex forming agents with the ability to bind the natural dyes onto textile substrates. This can
be achieved by either pre, simultaneous, or post mordanting, using different selective mordants
that result into the development of various shades, and increment in the dye uptake and

28
improvement of color fastness behavior of any natural dye. There are different types of mordants
but the major classifications are according to metal salts, tannins/tannic acid and natural oils
(Sengupta S, 2015).

Amongst all, mordanting with metal salts such as aluminum, chromium, copper, Iron and tin has
been the most exhausted method while less had been done concerning the use of natural
mordants like tannins. Tannins/tannic acid are bitter and stringent substances occurring as
excretions in plant parts such as barks, leaves, cutch, fustic, black oak, galls, pomegranate and
fruits. Tannic acid occurs in many types of vegetable substances but nut galls are the richest
source containing about 60-70% tannic acid (Saxena S, 2014) The main tannins used in place of
tannic acid on account of their low prices are natural tannins like myrobolan, mango bark and
leaves and tea. These mordants can be a clear solution (aqueous) or added into the dye bath with
the fiber (powder). Dried myrobolan (Terminalia chebula) fruits for example is a natural dye
with a have high tannin content used for producing bright yellow shades. The mordant has been
used to fix different natural dyes onto textile substrates majorly cotton and cellulosic fibers.
Besides application in textile industry, it has been established to have medical properties such as
antimicrobial and antifungal.

The main function of an oil mordant is to form a complex with alum when it is used as the main
mordant. This is because oil is insoluble in water and doesn’t have affinity for cotton hence
won’t easily be washed out from the treated fabric. Oil mordants contains fatty acids such as
palmitic, stearic, oleic, ricinoleic and glycerides. During mordanting, the -COOH group of the
fatty acids reacts with metal salt leading to the formation a COO-M, where M denotes the metal.
Among all the kinds of oil available, sulphonated oils are well known owing to their better metal
binding capacity than the natural oils. They are usually used in dyeing of madder to obtain
Turkey red color. All these mordants find application in the mordanting of numerous textiles
such as silk, cotton, sisal, blends of fabrics, synthetic fabric and dyeing madder to obtain Turkey
red color (Saxena S, 2014). However, mordanting with salts such as chrome, copper and tin
although they fix the dye well while providing an alternate palette, have been proven to cause
health hazards in the sense that they produce toxic waste which requires special disposal hence
not recommended for use. On the other hand, mordants such are alum, iron and tannins are safer

29
and can produce myriad colors when used in conjunction with the appropriate natural dye. This
is so because they are naturally present in the environment in large amounts (Saxena S, 2014).

Metals salts of iron, Aluminum, iron, chromium was used as mordants by traditional dyers, alum
however did not combine as readily with cellulosic fibers as it does with protein fibers.
Mordanting can be attained by either pre mordanting (before dyeing), simultaneous mordanting
(dyeing the fabric and mordanting at the same time) or post mordanting (after dyeing) using
different types of mordants. The selection of mordant and mordanting techniques depends on
conditions such as the nature of the substrate, its affinity for the substrate and type of dye to be
used. Extensive work has been reported for the dyeing of textiles with natural dyes while
adopting specific mordanting systems for a particular textile material such as (Saxena S, 2014),
(Prabhavathi P, 2014) and (Wangatia L.M, 2015).

2.9.2 Methods of mordanting Used in Dyeing with Natural Dyes

2.9.2.1 Pre-mordanting
Pre-mordanting is a technique which involves primarily mordanting the fabric before dyeing i.e.,
the mordant is applied to the fabric prior to dyeing. It’s the most common method of mordanting
practiced on cotton, cellulosic and some animal fibers such as cochineal because these often in
unmordanted form don’t have affinity for many natural dyes. The advantage of this method is
that the standing baths can be reused many times after replenishing with the mordants. This
makes the process economical as well as reducing the pollution load hence sufficient for large-
scale application (Saxena S, 2014). The procedures for mordanting are;

i). Dissolve the mordant and added to a dye bath containing ample amount of water.

ii). Add the substrate and bring the whole mixture to boil for a period of about ¾ of an hour

iii). Cool the solution

iv). Gently remove the substrate to ensure even take-up of the mordant.

v). Rinse the substrate to remove unfixed dyes, dry under room temperature and analyze. The
fabric can afterwards be dyed.

The disadvantage associated with this method is that there is a lot of fabric handling.

30
2.9.2.2 Simultaneous mordanting
As the name states, both the dyeing and mordanting process is carried out in the same dye bath
and at the same time. For cotton and other cellulosic, the mordant is usually added to the dye
bath at the start of dyeing so that both mordanting and dyeing processes take place concurrently
(Saxena S, 2014). The procedures for this method are;

i). First dissolve mordant in a separate bath.

ii). Transfer mordant solution to the dye bath and add the wetted fabric.

iii). Bring whole mixture to boil while stirring at intervals using a stirring rod.

iv). Simmering until maximum dye uptake.

v). Cool solution, remove fabric, rinse with mild detergent, dry and analyze.

Although this method is associated with less fabric handling, it gives colors that are not as
permanent as pre-mordanted fabrics because mordanting is dissolved at a separate stage and later
on introduced into the dye solution.

2.9.2.3 Post mordanting

In this method, the fabric after dyeing is treated with the mordant in a separate bath. The final
color is developed during the last phase. Iron salts are very often applied in this manner for
producing grey and black colors (Saxena S, 2014). The procedures are as follows;

i). Dissolve mordant in ample amount of water.

ii). During simmering (after dyeing), add solution to dye bath in the final five to ten minutes.

iii). Cool solution, remove substrate.

iv). Rinse with cold water to remove unfixed dyes, dry and analysis. Tin and iron are often used
in this way after pre-mordanting or simultaneous mordanting with another substance. This is
because tin has the effect of blooming i.e., it enhances and brightens whereas iron saddens or
dulls colors. This type of mordanting is quite satisfactory in the dyeing of several skeins with
different salts in the same dye bath, because once combined with the fibers, the metallic ions do
not react with one another. Many scientists have used these mordanting techniques in the dyeing

31
of various natural fibers like cotton, silk, wool and synthetics such as polyamide and polyester
(Kumaresan M, 2012). Some of the studies that have been conducted include; Prabhu (2011)
who used CuSO4 and FeSO4 to study the influence of concentrations of mordants on cotton
fabrics dyed with turmeric dyes. It was established that when fabrics were treated with tannic
acid and then dyed with metal salts, the depth and performance properties of the fabric such as
fastness to light, washing, rubbing (dry as well as wet) improved (Prabhu K.H, 2011).

Simultaneous mordanting is suitable when the dyer wants to achieve little or no loss of the dye
properties. The mordant chosen further has a marked effect on the final color. However, each dye
has different interactions with each mordant thus different shade can be obtained with the same
mordant using different methods of mordanting (Asif A, 2010).

Further collected works relating to the mordanting and dyeing of substrates with natural dyes
include Sundarajan (2011) who used tannin acid, cow dung, lemon juice and pomegranate rind as
natural mordants to standardize the dyeing effect of marigold and turmeric dyes on silk and
knitted cotton fabric (Sundarajan M, 2011). The study established that all the mordanted fabric
showed good dye uptake. Kumaresan (2012) dyed silk fabrics using extracts from the stems of
Achras sapota and flowers of Spathodea campanulata. Dyeing was performed under varying
conditions using pre-mordanting, simultaneous and post mordanting methods. The findings were
that the flowers of Spathodea campanulata with simultaneous method at three percent mordant
concentration gave the best results (Kumaresan M, 2012).

Under different dyeing conditions, different mordanting methods yield various results as most of
the mordants used generally affect the hue (shade) and purity of color. Ali (2011) who studied
the effect of pre-mordanting and post-mordanting of wool fibers with Aluminium sulfate, ferrous
sulfate under different conditions reported that during pre-mordanting and post-mordanting with
ferrous sulfate, there were huge changes in hue and a great deal of decrease in the chroma (purity
of color). The study further established that, alum and iron did not have any effect on fastness as
appreciable increase in fastness properties occurred (Ali N.F, 2011).

2.10 PURIFICATION AND CHARACTERIZATION OF NATURAL DYES

Colourants are characterised by their ability to absorb specific visible parts of the
electromagnetic spectrum in the range of 380 -780nm. For good colouring property, it has to

32
have high absorption coefficient of about 10,000 to 40,000 mol/cm. Furthermore, its properties
should enable it keep up with coloured material, stable in conditions such as phytochemical,
chemical and oxidation, and be harmless to human health. Studies on the analysis/ identification
of natural dyes started as early as nineteen hundred thirties where a French chemist performed
micro chemical analysis and achieved the result by colour reactions with different chemicals.
The identification of natural dyes advanced with time and since then till today, many workers
have used various methods and techniques such as thin layer chromatography, to identify natural
dyes in textiles (Samanta A.K, 2009).

Currently, there are numerous ways of analyzing the chemistry of natural colourants such as UV‐
Vis spectrometry, Chromatographic analysis, FT‐IR, NMR and Mass spectrometry. However, for
each method, the colourant is prepared differently. Ultraviolet/Visible absorption (UV/VIS)
spectroscopy is used as qualitative tools for identifying and characterizing molecular structures
and the characteristic absorption spectra that constitute the characteristic fingerprint of the
colourant (Espinosa-Morales Y, 2012).

2.10.1 UV-Vis Spectrophotometry

In this method, the hue and absorbance of the dye is determined using UV-Vis spectral scan of
aqueous or non-aqueous extract/solution of the purified natural dye whose UV-zone and visible
zone range from 190 to 700 nm or higher. The presence of dye along this range is indicated by
peaks and troughs in different wave length. Peaks and troughs in visible zone indicate the main
colour and absorption, while the UV-Zone with or without peaks, shows the property of the dye
under UV-light which may be correlated with its fastness behavior.

Various UV-Visible spectroscopic studies have been conducted by different scientists who
performed a number of UV-Vis spectral scan on a number of natural dyes namely madder,
cochineal, neem, beet sugar and indigo using different solvents for extraction. Neem bark
colourant showed two absorption maxima at 275 and 374 nm while beet sugar showed three
absorption bands at 220, 280 and 530 nm. In another experiment, Gomphrena globosa flower
colourant extracted in 2005 showed one major peak at 533 nm. The dye did not show much
difference in the visible spectrum at pH 4 and 7. Extractions, spectroscopic and colouring
potential of the dye from ginger rhizome were studied using UV- Vis Spectroscopy.

33
Reports indicate that the dye is soluble in hydroxyl organic solvents, giving one homogenous
component of Rf. value 0.86 on chromatographic separation with a wavelength of maximum
absorption at 420 nm. Furthermore, colour measurements and chemical analysis have been
performed on extracts from Possur plant using Video Spectral Comparator and UV-Vis
spectrophotometer respectively. Maximum absorbance of the crude dye extracted at different
temperatures was measured at a wavelength from 250-600nm. The results obtained were
compared with the traditional method of dye extraction and it was observed that at a maximum
wave length of 477nm, colourant extracted from PHWE was higher than the traditional method
(Razak N.A, 2011).

However, for this research, characterization of extracts from Justicia betonica plant will be
performed using UV Spectrophotometer. One percent aqueous dye solution will be prepared and
an aliquot solution subjected to a wave length scan in a micro-processor or computer attached to
a UV Vis absorbance Spectrophotometer at a wavelength ranging from 200-1000 nm.

2.10.1.1 The Measurement of Colour

The measurement of colour is known as colorimetry. A variety of instruments are used in this
field. The most sophisticated, the spectrophotometers, analyze light in terms of the amount of
energy present at each spectral wavelength and the reasons colour is measured in everyday life
with a scientific basis for

 colour communication
 colour matching
 colour difference

2.10.1.2 Relationship between reflectance and colour and wavelength

A spectrophotometer produces a reflectance curve that can indicate the color being measured.
White light contains all the colors of the rainbow in the visible spectrum. When white light falls
upon an opaque object, the object interacts with that light. If the object appears red it is because
the object absorbed most of the white light selectively, reflecting only the red portion, which is
observed by the eye.

34
Likewise, if an opaque object is green, it absorbs all of the white light except the green portion.
White objects produce a reflectance curve which is essentially flat at nearly 100 percent
(reflecting all wavelengths), and black objects produce a curve which is essentially flat at nearly
0 percent (absorbing all wavelengths). For transparent objects the process is similar, except the
white light is selectively absorbed, transmitting (rather than reflecting) only the color observed.
This interaction accounts for all the colors visible.

Many single pigments and pigment mixtures can be identified by their spectrophotometer curves.
Reflectance curves are like fingerprints. Each chemical variety of a pigment or dye has its own
specific spectrophotometric fingerprint (Sabic, 2017).

Figure 15: Graph of reflectance against wavelength

(Clarkvision, 2018)

Different surface features reflect and absorb the sun's electromagnetic radiation in different
ways. The reflectance properties of an object depend on the material and its physical and
chemical state, the surface roughness as well as the angle of the sunlight. The reflectance of a
material also varies with the wavelength of the electromagnetic energy. The amount of
reflectance from a surface can be measured as a function of wavelength, this is referred to

35
as Spectral Reflectance. Spectral Reflectance is a measure of how much energy (as a percent) a
surface reflects at a specific wavelength. Many surfaces reflect different amount of energy in
different portions of the spectrum. These differences in reflectance make it possible to identify
different earth surface features or materials by analyzing their spectral reflectance
signatures. Spectral reflectance curves graph the reflectance (in percent) of objects as a function
of wavelengths (Humboldt, 2020).

Figure 16:Relationship between reflectance and colour and wavelength

(Brady, 2017)

2.10.1.3 Scattering and absorption by a textile substrate

Figure 17 shows a simplified illustration of the different sources of scattering and reflectance in a
textile fabric. Johansson (2015) studied light scattering by paper and found out that the origin of
the light scattered from paper can either be the bulk of the paper or the surface of the paper. An
important component that is the pores of the paper also contribute significantly to the scattering.
Surface reflectance is light reflected directly at the interface between the surrounding medium
(air) and the substrate. Since the light has not interacted with the bulk, its spectral content

36
remains unaltered. Bulk reflectance consists of light that penetrates into the bulk of the paper,
and after multiple scattering events inside the material, is eventually reflected back through the
top surface. The last component is fluorescence, caused by the interaction of light and
fluorophores added to the paper (Johansson, 2015). In this study, this is the dye extract added to
the fabric.

2.10.1.4 Reflection from multiple fibres

Figure 17:Reflection of light from multiple fibres

2.10.1.5 The Beer-Lambert law

Dyes inside fibres behave as though they are dissolved in the fibre and their absorption
behaviour is similar to that of solutions. The absorption of light in dyed fibres is described by the
Beer-Lambert law:

I = I0 10-ecl

where I is the intensity of the transmitted radiation, I0 is the intensity of the incident radiation, e
is the molar extinction coefficient (l mol-1 cm-1), c is the concentration of the absorbing
substance (mol l-1) l is the path length through which the radiation passes (cm).

When the percentage of incident light transmitted (T) is measured, the law can be written:

A = log10 (100/T) = ecl

where A is the absorbance (Brady, 2017).

37
Color strength is a measure of the ability of a dye to impart color to materials. The color strength
is evaluated by light absorption in the visible region of the spectrum. Relative color strength can
be defined as the ratio of K/S values for samples as compared to a standard at same wavelength,
which expressed as percentage. ‘K’ and ‘S’ are absorption and scattering coefficients of dyed
sample. Relative color strength (%) is calculated from reflectance, R using Kubelka-Munk
equation as follows:

K/S = (1-R)2 / 2R

(Salima S.S, 2015)

The quantitative value for the color strength was obtained by measuring the percent value of
reflectance (% R) at the same wavelength and then converted to K/S value with K/S table
assistance by Kubelka-Munk theory. Reflectance value is the reflection of light amount reflected
by objects that contain color (F. Failisnur, 2018)

2.10.1.6 The Kubelka-Munk function

Figure 18: Kubelka-Munk function formular

where K is the absorption coefficient S is the scattering coefficient R ∞ is the reflectance of the
surface having such a thickness that there is no further change in the reflectance by increasing
the thickness

For dark shades, R∞ is replaced by a corrected value: R∞ = R - R0.

38
Figure 19: Colour absorption value formular

where R is the measured value of reflectance at a particular wavelength R 0 is the minimum value
of reflectance obtained with the deepest possible dyeing at the wavelength of maximum
absorption (Brady, 2017).

39
CHAPTER THREE: METHODOLGY

In this work a natural dye was extracted from Justicia betonica plant. Phytochemical analysis,
dyeing with and without mordant of the extract was carried out. The Justicia betonica leaves
were collected from central region in Wakiso district, Uganda and leaves of the plant were
collected and used for experimentation. The fabric was procured from a market.

3.1 MATERIALS AND REAGENTS

3.1.1 Materials
Electric blender

Perkin Elmer Lambda 25 UV-Vis spectrophotometer

Fabric type (cotton 100%)

Beakers

Conical flasks

Beakers

Test tubes

Water bath

Weighing scale

Filter paper

3.1.2 Reagents
Justicia betonica leaves dye powder

40
Sodium carbonate

Methanol

Distilled water

Drangendroff’s reagent

Sodium hydroxide

Benzene

Ammonia

Concentrated Sulphuric acid

Ferric chloride

Lead acetate

Concentrated Hydrochloric acid

Chloroform

Benedict’s reagent

Alum

Sodium sulphate

Acetic acid

3.2 METHOD
For standardization and optimization of a particular plant dye to maximize color yield, cost of
extraction as well as rate of dyeing, proper extraction conditions were chosen.

3.2.1 Sample preparation

The coloring substance that was used in this research work was extracted from Justicia betonica
plant. The leaves of the plant were thoroughly washed with freshly prepared sterile distilled
water and left to shade dry for 20 days. After drying, the samples were cut into small pieces,

41
grounded into powder using an electric blender, packed in clean sample containers and finally
stored for analysis

3.2.2 Desizing of substrate

White cotton fiber substrates were desized to 11cm by 9cm. The cut fibers were bleached and
scoured cotton fabrics were used for dyeing

3.2.3 Scouring of the Cotton Fiber

As preparatory processes for dyeing of the cotton fabrics, the desized cotton fabrics samples
were washed in a solution containing 0.5g/l of sodium carbonate and 2g/l of detergent at 50°C
for 25minutes. The scoured material was thoroughly rinsed with water and dried at room
temperature.

3.2.4 Extraction
3.2.4.1 Aqueous extraction
Aqueous extract was prepared by adding 5 g of Justicia betonica leaves dye powder to 100 ml
distilled water. The mixture was heated at 100ºC for 1 hour, allowed to stand for overnight and
then filtered. The extracts were sieved through Whatman No. 1 Filter paper. The filtrates were
concentrated by water evaporation. The filtrate was used for dyeing.

3.2.4.2 Methanolic extraction

5 g of Justicia betonica leaves dye powder was soaked in 100 ml methanol and kept in water
bath for period of 2 hours for complete dye extraction and then it was filtered.

3.2.5 Phytochemical analysis of the dye extract

3.2.5.1 Alkanoids
To 1ml of the filtrate, added 2ml of Drangendroff’s reagent. Turbid orange colour indicates the
presence of alkaloids (Harborne, 2001).

3.2.5.2 Flavonoids
To 1ml of filtrate, added 2ml of dilute NaOH. Development of golden yellow colour confirms
the presence of flavonoids (Parekh J, 2008).

42
3.2.5.3 Anthraquinones
To 1ml of the filtrate, added 10ml benzene, filter and now add 5ml of 10% (v/v) ammonia to the
filtrate and shake well. Development of pinkish coloured solution indicates the presence of
anthraquinones (Parekh J, 2008).

3.2.5.4 Cardiac glycosides

To 1ml of the filtrate, added 1ml of FeCl 3 reagent (mixture of 1 vol. of 5% FeCl 3 solution + 99
vol of glacial acetic acid) and a few drops of conc. H2SO4. Greenish blue colour appears within
few minutes (Arulpriya P, 2010).

3.2.5.5 Carbohydrates
Took 1ml of the filtrate with 5ml Benedict’s reagent and boiled for 5 minutes. Bluish green
colour indicates the presence of carbohydrates (Fernandez, 2001).

3.2.5.6 Phenolic flavonoids

To 1ml of filtrate, added 2ml of 10% lead acetate. Brown precipitate indicates presence of
phenolic flavonoids (I.J, 2008).

3.2.5.7 Tannins
To 1ml of filtrate, added 2ml of Ferric chloride. Dark green colour indicates presence of tannins
(Chopra, 2012).

3.2.5.8 Saponins
To 1ml of filtrate,added 2ml distilled water, shook vigorously and allowed it to stand for 10
minutes. Development of foam on the surface of the mixture, lasting for 10 minutes indicates the
Presence of saponins (Riguera, 1997).

3.2.5.9 Anthocyanides
To 1ml of filtrate, added 5ml of dilute HCl. Pale pink colour indicates presence of
anthrocyanides.

3.2.5.10 Terpenoids
Took 1ml of the filtrate and added 2ml CHCl3 and carefully added 3 drops of conc. H2SO4. An
interface with a reddish-brown coloration confirms the presence of terpenoids. (Rajic et al.,
2000)

43
3.2.5.11 Proteins
To 1ml of filtrate, added 6 drops of Millon’s reagent. Development of a white precipitate which
turned red on heating indicates presence of proteins (Fernandez, 2001).

3.2.6 Dyeing of fabric

Cotton fabrics were dyed by a simultaneous mordanting method as described by (P. A. G.
Wanyama, 2011). Fabrics (8 × 10 cm) weighing approximately 1.41 g were dyed in a beaker
(250 ml) containing 10% on weight of fabric (o.w.f) of alum (potassium aluminium sulphate)
mordant and 20% o.w.f of sodium sulphate. The pH of each dye solution was maintained
between 6.5 and 7.5 with 3% w/w of acetic acid (40% solution) monitored by test papers during
the dyeing process. A material to liquor ratio (MLR) of 1:200 was used in all dyeings.

3.2.7 Evaluation of Color Strength (K/S) of Wash-offs

After dyeing, the cotton samples were allowed to age for a week (seven days) before they were
washed. Each dyed sample was washed in three (3) separate detergent solution containing 250
ml of water and 2g of normal detergent powder. The wash-offs of each sample was analyzed
with the UV-visible spectrophotometer to assess the dye fastness and ultimately to determine the
degree of dye bleeding from the fabric. Reflectance values were measured at 100 nm intervals
between 400 and 700 nm, the range commonly covered in textile applications and from these
values, the corresponding K/S (colour concentration absorption) values were calculated. Plots of
reflectance values against wavelength were made for the coloured fabric samples resulting into
spectral reflectance curves, a fingerprint record of reflectance characteristics of the natural dyes
in this study (P. A. G. Wanyama B. T., 2011). The percentage of light reflected (R) by the wash-
offs sample was evaluated as a function of the percent absorbed (A) and percent transmitted (T)
by recourse to the standard procedure as presented in Equation below.

R = 100-(A+T)

The values of the three components of the incident light on the wash-offs could be used to
evaluate the concentration of the dye in the wash-offs, i.e., the degree of dye bleeding from the
fabric sample by adopting the conventional procedure of measuring liquid solution using the
well-known Kubelka – Munk theory. The theory illustrated in Equation 2 defines the relationship

44
between spectral reflectance (R) of the wash-off of the sample, its light absorption (K) and its
scattering characteristic (S).

K/S= (1- R) 2 /2R

Where, K and S are the absorption and scattering co-efficient of the sample. R is the absolute
(spectral) reflectance

The ratio K/S is the color strength of the wash-off, in other words, the higher the value of this
ratio the higher the degree of dye bleeding, or the less color fast the dye is to the fabric.

45
CHAPTER FOUR: RESULTS AND DISCUSSION

In view of the objectives of this study, a series of experiments were carried out in the extraction
of Justicia betonica dye from the leaves of the plant under already optimized conditions. The dye
was then characterized by examining the presence of different phytochemicals, dyeing of cotton
substrate and analysis of the dyed substrate.

4.1 EVALUATION OF NATURAL COLOURANT

4.1.1 Analysis of Dye extract
In this study, the dye extract obtained from Justicia betonica leaves from both aqueous and
methanol extract was a purple bluish crude dye extract in colour. This was in accordance with
(Wanyama P.A.G, 2011) whose research also obtained the same result, a purple bluish dye.

4.1.2 Phytochemical analysis of Dye extract

The phytochemical analysis of Justicia betonica dye extract obtained through extraction into
different medium i.e., aqueous and methanol. The results of phytochemical analysis of Justicia
betonica dye extracts are presented in the table below; -

Table 3: Phytochemical Screening of Various Extracts Justicia betonica

Chemical test Aqueous extract Methanol extract

Alkanoids + +
Flavonoids + +
Saponins - -
Carbohydrates + +
Tannins + +
Anthraquinones + +
Anthrocyanides + +
Proteins + +
Terpenoids - +

46
 Cardiac glycosides + +
Phenolic flavonoids + +

The extraction of phytochemicals in Justicia leaves was performed using water and methanol
and it was observed in Table that the extraction procedures affected the detection of chemicals.
Methanol and water extracts showed the presence of all phytochemicals in the plant leaves
except for saponins which were not present in both extracts. These results are in accordance with
(G. Bbosa, 2013) who established the presence of alkaloids, flavonoids, glycosides,
carbohydrates, gum and mucilage, proteins, fixed oils and fat, phenolics and tannins using the
standard phytochemical procedures using aqueous and methanolic extracts in Justicia betonica.
The research also established methanol as a better extracting solvent than water as the methanol
extract showed presence of terpenoids that were not present in the aqueous extract. These results
are in accordance with in (Jiang, 2016) from with terpenoids can be extracted by using nonpolar
organic solvents in this case methanol was used which agrees with the findings. The
anthrocyanins in the dye extract is responsible for the purple bluish colour of the dye. This is in
accordance with (Khoo, 2017) from their work on anthrocyanins.

4.2 DYEING OF JUSTICIA BETONICA DYE EXTRACT ON FABRIC

The aqueous extract of Justicia betonica leaves showed the satisfactory presence of
phytochemicals. Hence the aqueous dye extract of Justicia betonica was used for the dyeing of
cotton fabric. Dyeing of the fabric with the alum as a mordant using simultaneous mordanting
technique imparted a purple colour on the cotton fabric. In simultaneous mordanting, the dye and
mordant are added simultaneously as this method fixes the dye onto the fabric better than other
mordanting techniques leading to very little dye bleeding.

47
Figure 20: Undyed cotton fabric

Figure 21: Dyed cotton fabric

Figure 22: Washed dyed fabric

48
4.2.1 Colour strength analysis
4.2.1.1 Reflectance
Spectral reflectance curves for the dyed fabrics were indicative of colour shades between 400
and 700 nm. The results obtained from the spectrophotometer are in table 4 below; -

Table 4: Reflectance at different wavelengths

Wavelength (nm) Reflectance (%)

400 18
500 23
600 10
700 20

A plot of reflectance against wavelength

25

20
reflectance(%)

15

10

0
400 450 500 550 600 650 700 750
wavelength(nm)

Figure 23: A graph of reflectance against wavelength

Microsoft excel was used to plot the graph. There is an increase in reflectance from 400 nm to
500 nm then a gradual decline between 500 nm and 600 nm and finally an increase to 700 nm.

The initial increase in reflectance is due to the dye reflecting light from the violet end of the
visible spectrum absorbing all other colours of light as this is the colour of the crude dye extract.

49
The decline in reflectance between 500 nm and 600 nm is because most of the electromagnetic
radiation is absorbed with little reflected. The increase from 600 nm and 700 nm is because the
dye wash-off reflects light from the red region or infrared region of the visible spectrum. These
results don’t concur with (P. A. G. Wanyama, 2011)from their work on characterization of
colour from dye-yielding plants in Uganda from which their findings reveal the main spectral
colour for J. betonica occupied the wavelength bands between 450 for violet and 480 for blue
with values of reflectance increasing gradually between 400 nm and 700 nm.

4.2.1.2 Colour absorption value

Using the Kulbeka-Munk theory to calculate the colour absorption value (K/S) at each
wavelength and its variation with wavelength

K/S= (1- R) 2 /2R

Table 5: K/S at different wavelengths

Wavelength (nm) K/S

400 1.87
500 1.29
600 4.05
700 1.60

50
 A plot of K/S against wavelength
4.5

3.5

2.5
K/S

1.5

0.5

0
400 450 500 550 600 650 700 750
wavelength(nm)

Figure 24: A graph of K/S against wavelength

The graph was plotted using Microsoft excel. The colour absorption values (K/S) for the crude
dye extract showed a decline from 400 nm to 500 nm then an increase from 500 nm to 600 nm
and finally a slight decline from 600 nm to 700 nm.

This generally shows a gradual increase in the colour absorption value which is a negative result
or disagrees with results of (P. A. G. Wanyama, 2011)which showed a gradual decrease with
increase in wavelength from the violet to the red end (450 to 700 nm) of the visible spectrum
according to for the dye extract.

CHAPTER FIVE: CONCLUSION AND RECOMMENDATIONS

5.1 CONCLUSION
The purpose of this project has been to explore the natural reserves, identify plants from which
dyes can be extracted and applied to textiles. This task was accomplished by means of assisted
visits to various the location to identify, photograph and collect samples of the plants for the
experiments and to explore the dye yielding properties.

51
The above test results strongly indicate that the dye extract has a great potential in textile
coloration and can serve as a source of raw material for fabric dyeing in the future. Natural dyes
are not only having dyeing property but also having the wide range of medicinal properties due
to presence of phytochemicals with an increasing awareness among people towards natural dyes
and dye yielding plants

5.2 RECOMMENDATIONS
In view of the results of the study the following are recommended for consideration.

With more research on this plant and in the field of natural dyes in other colour fastness tests like
wash fastness, rub fastness, perspiration tests and light fastness of the dye would be better suited
for use in the textile industry if the results turn out to be suitable.

5.3 DELIMITATION
The study was limited to exploring a natural plant in this case Justicia betonica from which a dye
can be extracted and used as colourants for cotton as a textile fabric.

5.4 LIMITATIONS
The research was characterized by extensive traveling to enable the researcher collect and
ascertain data on the existence of some natural plant dye for use our local textile industry.
However, resource and funding constraints was a major drawback in gathering sample and data.

5.5 REFERENCES
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175-191.

A.J. Amirul-Aiman, T. N.-A. (2013). Petal anatomy of four Justicia (Acanthaceae) species. AIP Conference
Proceedings 1571, (pp. 368-371).

Adams M, G. F. (2007). Plants traditionally used in age related brain disorders – a survey of
ethnobotanical literature. J Ethnopharmacol. Plants traditionally used in age related brain
disorders – a survey of ethnobotanical literature. J Ethnopharmacol, 113 : 363-381.

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Ado A, Y. H. (2014). Dyeing of Textiles with Eco-Friendly Natural Dyes: A Review. International Journal of
Environmental Monitoring and Protection. 1(5), 76-81.

Ali N.F, E.-M. R. (2011). Eco-freindly and protective natural dye from red prickly pear (opuntia
Lasiacantha Pfeiffer) plant. Journal of Saudi Chemical Society, 15, 257-261.

Antima S, D. L. (2012). Dye yielding plants of Garhwal Himalya India. International Research Journal of
Biological Science, 1(4), 69-72.

Arulpriya P, L. P. (2010). In vitro antioxidant testing of the extracts of no Samanea saman (Jacq.) Merr.
Der Chemica Sinica. 73-79.

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5.6 APPENDIX

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Figure 25: Perkin Elmer UV-Vis Spectrophotometer

Figure 26: Dye extract

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Figure 27: Leaf extract

Figure 28: Phytochemical screening test results

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