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DEVELOPMENT OF IN VITRO ROOT INDUCTION PROTOCOL AND HPTLC FINGERPRINT FOR

WITHANIA COAGULANS

ARCHANA .T.M

(Reg. No.10PB02)

A Thesis submitted to Avinashilingam Deemed University for Women, Coimbatore

In Partial fulfillment of the requirement for the Degree of

MASTER OF SCIENCE IN BIOCHEMISTRY

April, 2012

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CCEERRTTIIFFIICCAATTEE

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AACCKKNNOOWWLLEEDDGGEEMMEENNTT

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ACKNOWLEDGEMENT

“Peace on the outside comes from knowing God on the inside”. First and foremost I express

my heartfelt respect and thanks to God Almighty for all the blessings and strength he gave me in

all my endeavors.

“The best and the most beautiful things in this world can neither be seen nor be

touched, but can be felt with the heart”. Ability is of little account without opportunity. I

sincerely thank Ayya Avargal and Amma Avargal for creating a portal to exhibit our

abilities.

I owe my respectful gratitude and sincere thanks to Thiru. T.S.K. Meenakshi Sundaram

Chancellor, Avinashilingam University for Women, Coimbatore, for providing all the facilities to

conduct the project.

“Success is never found. Failure is never fatal. Courage is the only thing this is your

words” I record my heartfelt thanks to Dr. Sheela Ramachandran, Vice Chancellor, Avinashilingam

University for women, Coimbatore, for extending all possible help towards the completion of the

study.

I express my sincere thanks to Dr. Gowri Ramakrishnan, Registrar, Avinashilingam

University for Women, Coimbatore, for providing opportunity to carry out this piece of work.

I wish to express my profound gratitude to Hon. Colonel. Dr. Saroja Prabhakaran, Former

Vice Chancellor, Director of The Hall of Residence, Avinashilingam Deemed University for Women,

Coimbatore, for her constant support and encouragement during the period of study.

“To be simple is to be great” I am indebted to Dr. R. Parvatham, Dean, Faculty of Science,

Head of the Department of Biochemistry, Biotechnology and Bioinformatics, Avinashilingam

University for Women, Coimbatore, for her constant motivation, encouragement and support in

eliciting this project in a facile manner. I express my heartfelt thanks to my guide Dr. R.

Parvatham, Dean of Sciences, Head of the Department of Biochemistry, Biotechnology and

Bioinformatics, Avinashilingam Deemed University for Women, Coimbatore, for her guidance,

encouragement, amicable suggestions and help for the successful completion of this project.

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“The good teacher explains. The superior teacher demonstrates. The great teacher

inspires like you” I articulate my reverential gratitude to my teacher Dr. K. Kalaiselvi,

Assistant Professor, Department of Biochemistry, Biotechnology and Bioinformatics, for the

guidance rendered at every stage of the dissertation. Without her dynamic guidance, valuable

suggestions, untiring help, meticulous efforts and enduring support, this study would never have

seen the light of the day.

“Nothing that is worth knowing can be taught” I express my sincere thanks to Staff

members of the Department of Biochemistry, Biotechnology and Bioinformatics, Avinashilingam

University for Women, Coimbatore, for their help and cooperation.

"It’s great to work with somebody who wants to do things differently" A special word of

thanks to Prathipa. D, Pankajavalli. T, Nithya. K, Kalaiselvi. R, Preethi M.P and Rajalakshmi P.V

who timely helped and supported me throughout my project.

“A real friend is one who walks in when the rest of the world walks out” All glories of this

world are not worth a good friend. I deem it a great privilege to thank all my friends and well-

wishers for their immense help in times of dire need and for their constant support. A special word

of thanks to my friends Nafiya.P and Renugadevi.T for their timely help and support throughout my

project.

“Seek no praise, no reward for anything you do”. My heart has no bounds to thank my

parents, grandparents and brother who has sacrificed many things in their life for me, expecting

nothing in return since any great work can be done without sacrifice. Their mental and emotional

support, motivation, prayers and loving care provided has been the source of my strength.

ARCHANA.T.M

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CCOONNTTEENNTTSS

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CONTENTS

CHAPTER NO. TITLE PAGE

NO.

List of Tables

List of Figures

List of Plates

List of Appendices

1 Introduction 13

2 Review of Literature 17

3 Methodology 36

4 Results And Discussion 43

5 Summary And Conclusion 72

6 Bibliography 75

7 Appendices 89

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LIST OF TABLES

TABLE

NO. TITLE

PAGE

NO.

3.1 Hormone supplementation in MS media for root induction 38

4.1 Response of explants to variation in IBA concentration on Root

Induction

47

4.2 Response of explants to variation in IAA concentration on root

induction

52

4.3 Response of explants to variation in auxin concentration on root

induction

53

4.4 Growth index of roots in suspension culture 55

4.5 Quantitative phytochemical analysis of different in vivo and in

vitro roots of Withania coagulans and Withania somnifera45

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LIST OF FIGURES

FIGURE

NO. TITLE

PAGE

NO.

2.1 Withanolide 21

2.2 Withaferin A 22

2.3 Coagulin C 33

4.1 Response of Explants to Variation in IBA Concentration on

Root Induction

48

4.2 Response of Explants to Variation in IAA Concentration on

Root Induction

52

4.3 Response of explants to variation in auxins concentration on

root induction

54

4.4 Quantitative estimation of carbohydrates 61

4.5 Quantitative estimation of flavanoids 62

4.6 Quantitative estimation of proteins 63

4.7 Quantitative estimation of saponins 64

4.8 Quantitative estimation of steroids 65

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LIST OF PLATES

PLATE

NO. TITLE PAGE NO.

4.1 Influence of Auxins on root induction

45

4.2 Growth of roots in suspension 56

4.3 Mass production of roots in bioreactor 57

4.4 Standardization of solvent system for in vivo and in vitro roots of Withania coagulans and Withania somnifera

67 - 69

4.5 Comparative HPTLC finger print for in vitro and in vivo root of Withania somnifera (Ws) &Withania coagulans (Wc)

71

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IINNTTRROODDUUCCTTIIOONN

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1. INTRODUCTION

The traditional definition of medicinal plants is given in Ashtaanga Hrdaya (600

AD), Sutra sthana Chapter 9 verse 10 as “…Jagtyevam anoushadham na kinchit vidyate

dravyam,Vashaannaarthayogayoh” (There is nothing in this universe, which is non-

medicinal, which cannot be made use of for many purposes and by many modes). This

definition rightly suggests that in principle, all plants have a potential medicinal value

although 'in practice' a plant is referred to as medicinal when it is so used by sonic system

of medicine. The plant-based, traditional medicine systems continue to play an essential

role in health care, with about 80% of the world’s inhabitants relying mainly on traditional

medicines for their primary health care (Tripathy, 2004). India has several traditional

medical systems, such as Ayurveda, Siddha and Unani, which has survived through more

than 3000 years, mainly using plant-based drugs. Over the past decades, herbal medicine

has become a topic of global importance, making an impact on both world health and

International trade. Continuous usage of herbal medicine by a large proportion of the

population in the developing countries is largely due to the high cost of western

pharmaceuticals and healthcare. Thus, recognition and development of the medicinal and

economic benefits of these plants are on the increase in both developing and industrialized

nations (Dixit and Ali, 2010).

There are at least 121 chemical substances of known structure still extracted

from plants that are useful as drugs around the globe (Alothman et al., 2003).Rather than

using a whole plant as different types of organic extracts, pharmacologists identify, isolate,

extract, and synthesize individual components, thus capturing the active properties. There

are so many groups or families of phytochemicals that aid the human body in several ways

(Hossain et al.,2011).

Chemical constituents are non-nutritive plant bioactive chemicals that have

protective or disease preventive properties. Plant produces itself these bioactive chemicals

to protect itself but recent research demonstrates that many chemical constituents can

protect humans against diseases. There are so many groups of bioactive chemicals in fruits,

vegetables and herbs and each works differently (Hossain et al., 2011). The functional

bioactivity of a plant organic extract, in general, depends upon the presence of compounds

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such as polyphenols, carotenoids, terpenoids and chlorophyll (Negi et al., 2002). Plants also

can contribute in this area primarily due to the antioxidant activity of phenolic and

flavonoid compounds (Mhatreet al., 2009).

Plant tissue culture can be a potential source for important secondary metabolites

such as pharmaceuticals and food additives. This technology depends on using plant

cultures in a similar manner to microbial fermentation for factory-type production of target

metabolites (AbouZid et al., 2010). In vitro techniques have been found to be useful in the

propagation of a large number of threatened and endangered plants (Sarasan et al., 2006).

The technology bears many advantages over conventional agricultural methods: production

is independent of variation in crop quality or failure, yield of target compounds would be

constant and geared to demand, there is no difficulty in applying good manufacturing

practice to the early stages of production, production would be possible anywhere under

strictly controlled conditions, independency of environmental problems, free from risk of

contamination with pesticides, herbicides, agrochemicals or fertilizers and new methods of

production can be patented (AbouZid et al., 2010).

Production of secondary metabolites in tissue cultures is usually higher when plant

cells are organized into tissues/organs. The expression of secondary metabolic pathways in

organized cultures is not surprising because it mimics exactly what the plant does. Root

cultures are typical examples that can be used for production of phytochemicals. Root

cultures have been used as standard experimental system in studies of inorganic nutrition,

nitrogen metabolism, plant growth regulation, and root development (Loyola-Vargas &

Miranda-Ham, 1995).

Among the twenty-three known species of Withania, only two (Withania somnifera

(L.) Dunal and Withania coagulans Dunal) are economically significant and widely

cultivated (Mirjalili et al., 2009). Withania coagulans Dunal belonging to the family

Solanaceae is a small bush which is widely spread in south Asia .W. coagulans is

commercially important for its milk coagulating properties (Ali et al., 2009). It is well

known in the indigenous system of medicine for the treatment of ulcers, dyspepsia,

rheumatism, dropsy, consumption and sensile debility (Hemalatha et al., 2008). It has

received much attention in recent years due to the presence of a large number of steroidal

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alkaloids and lactones known as withanolides.Withanolides, chemically nomenclatured as

22- hydroxy ergostane-26-oic acid 26, 22-d-lactones, are C28-steroidal lactones based on

an intact or rearranged ergostane frame through appropriate oxidations at C-22 and C-26 to

form a d-lactone ring. Major Withanolides, like withaferin A and withanolide A of the plant

have been demonstrated to possess significant therapeutic actions (Kaileh et al. 2007).

Withania is distributed in the east of the Mediterranean region and South Asia (Negi et al.,

2006). It was abundant until a few decades ago, but ruthless collection for medicinal

purposes, habitat destruction and climate changes makes the species to become endangered

in their natural habitats. Jain et al. (2009) reported that overexploitation and the

reproductive failures forced the species W. coagulans towards the verge of extinction.

Therefore, it is important to propagate and conserve them to meet up with future demand.

The conventional propagation of this species is performed through seeds and cuttings of

stem since root is too slow and laborious. In vitro propagation technique may be the best

solution for its rapid multiplication and reestablishment in nature (Valizadeh and

Valizadeh, 2011). The in vitroshoot cultures could provide an alternative to field

plantharvesting for the production of therapeutically valuable compounds (Sangwan et al.

2007). Mirjalili et al (2009) reported that the withanolide contents of the hairy root cultures

of W. coagulans were higher than in the root of the plant. Therefore, there is a need to

develop an efficient protocol for the induction of in vitro adventitious roots and thereby

screen the accumulation of phytoconstituents in the roots of Withania coagulans.

With this information available, the present study was formulated with the

following objectives:

1. To identify the optimum concentration of growth hormones for root

induction in Withania coagulans and their mass culture in suspension.

2. To develop a HPTLC finger print for in vitro roots and compare it with in

vivo roots of Withania coagulans.

3. To perform quantitative estimation of selected phytochemicals present in

different in vivo roots collected from various regions of Iran and in vitro root

of Withania coagulans.

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RREEVVIIEEWW OOFF LLIITTEERRAATTUURREE

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2. Review of Literature

Since the beginning of human civilization, medicinal plants have been used by

mankind for its therapeutic value. Nature has been a source of medicinal agents for

thousands of years and an impressive number of modern drugs have been isolated from

natural sources. Many of these isolations were based on the uses of the agents in traditional

medicine. The term “herbal drug” determines the part/parts of a plant (leaves, flowers,

seeds, roots, barks, stems, etc.) used for preparing medicines As source of medicines, plants

have formed the basis for sophisticated traditional systems and continue providing mankind

with new remedies. It is a fact that the 25% of all medical prescriptions are based on

substances derived from plants or plant-derived synthetic analogues. The efficacy and

safety of herbal medicine have turned the major pharmaceutical population towards

medicinal plant’s research (Sara et al., 2009).

The Withania coagulans belonging to family Solanaceae is distributed from the

East of Mediterranean region, extending to South Asia. This plant is rich in withanolide.

Different parts of this plant have been reported to possess a variety of biological activities.

The fruit and berries are used commercially for milk coagulation (Sanjay et al., 2007).

This chapter focuses on a review on the various studies conducted using Withania

coagulans:

2.1 Withania coagulans

2.2 Secondary metabolites present in Withania coagulans

2.3 Pharmacological properties of Withania coagulans

2.4 Invitro culture studies onWithania coagulans

2.5 Methods employed for the study of secondary metabolites and their

purification

2.6 Properties of purified compounds from Withania coagulans

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2.1 Withania coagulans

Withania coagulans (L.) Dunal belonging to the family Solanaceae is commonly

known as “Indian cheese maker”. It is well known for its ethnopharmacological activities.

Withania coagulans Dunal distributed in the east of the Mediterranean region and extends

to South Asia. The plant is native of the Asia-temperate (Western Asia: Afghanistan) and

Asia-tropical (Indian Subcontinent: India, Nepal) regions. It shows the presence of esterase,

lignan, alkaloids, free amino acids, fatty oils, essential oils and withanolides. (Kiritikar,

1999). Withania coagulans (L.) Dunal is a small, evergreen shrub that is reputed to be used

as a remedy for dyspepsia, flatulent colic and other intestinal diseases. These activities have

been attributed to withanolides that are present in the plant (AbouZid et al., 2010, Rahman

et al., 2003). Antimicrobial, anti-inflammatory, antitumor, hepatoprotective,

antihyperglycemic, cardiovascular, immunosuppressive, free radical scavenging and central

nervous system depressant activities of the plant have also been demonstrated (Maurya et

al., 2010). The twigs are chewed for cleaning of teeth and the smoke of the plant is inhaled

for relief in toothache. The plant is known by different names in different local languages,

such as ‘Akri’ or ‘Puni-ke-bij’ in Hindi, ‘Tukhme- Kaknaje-hindi’ in Persian. Spicebajja in

Afghan, ‘Khamjira’ in Punjabi and ‘Punir band’ or ‘Punir-ja-fota’ in Sindhi (Mathur et al.,

2011). A survey of the literature has shown that in various traditional systems of medicine

the plant has been recommended for the treatment of various disorders (Maurya et al.,

2010).

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Taxonomical Classification

Kingdom :Plantae, Plants

Subkingdom : Tracheobionta, Vascular plants

Super division : Spermatophyte, Seeds plants

Division : Angiosperms

Class : Dicotyledons

Order : Tubiflorae

Family : Solanaceae

Genus : Withania

Species : Withania coagulans Dunal.

(Hemalatha et al., 2008)

Botanical Description

Withania coagulans Dunal is a rigid, grey under shrub, 60-120 cm high, occurring

in drier parts of the Punjab. The plant flowers during November-April and the berries ripen

during January-May. The natural regeneration is from the seed. The flowers are dioceous,

in auxiliary clusters; pedicles 0.6 mm long, Deflexed, slender. Calyx 6 mm long,

campanulate, clothed with fine stellate gray tomentum; teeth triangular, 2.5 mm long.

Corolla 8 mm long stellately mealy outside, divided about 1/3 the way down; lobes ovate

oblong, sub-acute. Male flowers stamens about level with the top of the corolla-tube;

filament 2 mm long, glabrous; anthers 3-4 mm long. Ovary ovoid, without style or

stigma.Female flowers stamens scarcely reaching 1/2 way up the corolla-tube; filaments

about 0.85 mm long; anther smaller than in the male flowers, sterile. Ovary is ovoid, style

glabrous; stigma mushroom-shaped, 2 lamellate. Berry 6-8 mm globose, smooth, closely

girt by the enlarged membranous calyx, which is scurfy-pubescent outside. Seeds are 2.5-

3.0 mm in diameter, somewhat ear shaped, glabrous. (Hemalatha et al., 2008)

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2.2 Secondary metabolites present in Withania coagulans

The pharmacological properties of Withania coagulans is diverse, including anti-

inflammatory, anti-tumor, and anti-stress, antioxidant, immunomodulatory, hemopoetic and

cardio-protective activities (Gupta et al., 2007). The major components responsible for

these biological activities are the withanolides (Fig 2.1); a group of naturally occurring C28

steroidal lactones built on an intact or rearranged ergostane framework, in which C-22 and

C-26 are appropriately oxidized to form a six-membered lactone ring. The basic structure is

designated as the withanolide skeleton. Withanolides are known as plant hormones, which

can be used instead of physiological human hormones. Withanolides are amphiphilic

compounds which are able to regulate activities and the physiological body hormones

processes. According to a theory, when these plant hormones enter the human body, they

occupy the active receptor of the cell wall, and don’t allow the animal hormones to get

binding to this site and express their true activities. (Alternative Medicine Review,

Monograph, 2004). At present, more than 12 alkaloids, 40 withanolides, and several

sitoindosides (a withanolide containing a glucose molecule at carbon 27) have been isolated

and reported from aerial parts, roots and berries of Withania species (Mirjalili et al., 2009, .

Anonymous, 2004). However, there is little information to date about the withanolide

contents of W. coagulans (Mirjalili et al., 2009, Rahman et al., 2003). One of the most

important withanolides isolated from Withania extracts is the anticancer compound

withaferin A (H. Yang et al., 2007).

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Withaferin A (Fig.2.2), the first member of this group, was isolated from Withania

somnifera in 1965 (AbouZid et al., 2010, Lavie et al., 1965). The root cultures of W.

coagulans synthesized withanolides of which withaferin A was the major compound

(AbouZid et al., 2010). The quantitative evaluation of Withaferin A in leaf and root of W.

coagulans is 2.299% and 0.076%, and that of W. somnifera is 1.13% and 0.044%,

respectively (Dalavayi et al., 2006).Several properties of Withaferin A have been reported:

antiangiogenesis through NF- кB inhibition (Yokota et al., 2006); cytoskeletal architecture

alteration by covalently binding annexin II (AbouZid et al., 2010, Falsey et al., 2006) and

apoptosis induction through the protein kinase C pathway in leishmanial cells (Sen et al.,

2007). The primary molecular target of withaferin A was shown to be the ß5 subunit of the

proteosome (Yang et al., 2007). It is well established that the various compounds of

Withania species, such as withaferin A from the leaves, are known to possess anti-cancer

Fig 2.1Withanolide

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properties (Jayaprakasam et al 2003). They have been reported to inhibit the cell growth of

various human cancer cell lines, including lung cancer (NCI-H460). Withaferin A showed

antiproliferative activity against head and neck squamous carcinoma, by reduced cell

viability in cell lines in vitro (Subramanian et al 1969).

The neuropharmacological properties of withanolide A have also recently attracted

interest, since it has been found to promote neurite outgrowth and synaptic reconstruction

(Kuboyama et al., 2005), and could thus be useful in treating neurological disorders such as

Alzheimer’s disease and Parkinson’s disease. The study on pattern of withanolide

accumulation in the hairy root cultures of W. coagulans showed that withanolide A was the

most abundant compound whereas only small quantities of withaferin A were detected and

quantified in the analyzed samples. However, the levels of withanolide A in W. somnifera

plants are usually very low and, contrary to the other withanolides, it occurs mainly in the

roots (Mirjalili et al., 2009).

W. coagulans was previously reported to contain withanolides and coagulin H, a

withanolide derivative isolated from this plant and reported to have a powerful inhibitory

Fig 2.2 Withaferin A

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effect on lymphocyte proliferation and Th-1 cytokine production (Mesaik et al., 2006,

Huanga et al., 2009).Coagulin-H was evaluated for its effect on various cellular functions

related to immune responses including lymphocyte proliferation, interleukin-2 (IL-2)

cytokine expression. These results were compared with prednisolone. Coagulin-H was

found to have a powerful inhibitory effect on lymphocyte proliferation and the Th-1

cytokine production. The inhibition of the phytohaemagglutinin (PHA) activated T-cell

proliferation by coagulin-H (Mesaik et al 2006).

The extracted coagulin L from W. coagulans fruits has antihyperglycemic activity in

rats. It showed significant drop of a fasting blood glucose profile and improved the glucose

tolerance of db/db mice. The extracted coagulin L from fruits of W. coagulans also has anti

dyslipidemic effect on mice (Maurya et al 2008).

2.3 Pharmacological properties of Withania coagulans

2.3.1 Anti-Inflammatory Effect

Inflammation is a complex process occurring through a variety of mechanisms,

leading to changes of local blood flow and the release of several mediators. Lalsare and

Chutervedi (2010) reported that various extracts of W. coagulans fruits have anti-

inflammatory activities. The same activity was produced by powdered roots of Withania

somnifera (Begum and Sadique., 1988).The alcoholic extract of W. coagulans showed

significant anti-inflammatory effects in acute inflammation induced with egg albumin

(Budhiraja et al 1984). 3β-Hydroxy-2, 3- dihydrowithanolide F exhibited a significant anti-

inflammatory activity at 10 mg/kg in sub-acute models of inflammation such as granuloma

formation and formalin-induced arthritis in rats. The effect was comparable with that

obtained with 50 mg/kg phenylbutazone and 10 mg/kg hydrocortisone. However, it did not

show any significant activity in acute models of inflammation (Budhiraja et al., 1987).

The rheumatoidrats given powdered root of Withania somnifera orally one hour

before being given injections of an inflammatory agent over a three day period showed that

Aswagandha produced anti-inflammatory responses compared to that of hydrocortisone

sodium succinate (Begum and Sadique., 1988). Administration of withaferin A to rats with

induced arthritis showed that the W. somnifera had a similar structure and function to

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glucocorticoids suggesting that W. somnifera has a complex influence on inflammation and

immune response (Davis and Kuttan, 2000).Bhattacharya et al., (2000) reported that a

liver synthesized plasma protein called alpha-2-macroglobulin greatly increases during the

inflammatory process. W. somnifera was found more effective at decreasing this protein

during inflammation than standard anti-inflammatory drugs.

2.3.2 Anti-hyperglycemic Activity

The drug W. coagulans exhibited hypoglycemic activity which is an effective and

safe alternative treatment for diabetes (Hemalatha et al 2004). Isolated alkaloids and

steroids from plant sources are responsible for hypoglycemic activity of those sources

(Adebajo et al 2006). Jaiswal et al (2009) reported that there was a significant

improvement in symptoms and signs and euglycemia was attained (diabetes mellitus type

2). Also the extracted coagulin L from W. coagulans fruits has antihyperglycemic activity

in rats (Maurya et al 2008). Lalsare and Chutervedi (2010) reported that various extracts of

W. coagulansfruits to have anthihyperlipidemic activity. The aqueous and chloroform

extracts of the fruits decreased the blood glucose (55%), also the fruits aqueous extract

decrease blood glucose by (52%), (Hoda et al 2010). Extracted coagulin L from fruits of W.

coagulans was determined about 25 mg/kg in streptozotocin-induced diabetic rats, which is

comparable to the standard antidiabetic drug metformin (Maurya et al 2008).Aswagandha

has been evaluated in clinical studies with human subjects for its hypoglycemic effects

(Andalluet al., 2000).Six type 2 diabetes mellitus subjects were treated with a powder

extract of the herb for 30 days. A decrease in blood glucose comparable to that which

would be caused by administration of a hypoglycemic drug was observed (Singh et al.,

2010).

2.3.3 Hypocholesterolemic Activity

The aqueous extract of W. coagulans fruits in high fat diet induced hyperlipidemic

rats, significantly reduced elevated serum cholesterol, triglycerides, lipoprotein and the

LPO levels. This drug also showed hypolipidemic activity in induced triton

hypercholesterolemia. The hypolipidemic effect of W. coagulans fruits were found to be

comparable with ayurvedic product containing Commiphora mukkul (Hemalatha et al

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2006). Hoda et al (2010) showed the aqueous and chloroform extracts of the fruits

decreased triglyceride, total cholesterol, LDL and VLDL increased the HDL levels .At the

same time Aswagandha has been evaluated in clinical studies with human subjects for its

hypocholesterolemic effects (Andallu et al., 2000).Six mildly hypercholesterolemic

subjects were treated with a powder extract of the herb for 30 days. A decrease in serum

cholesterol, triglycerides, and low density lipoprotein were seen(Singh et al., 2010).

2.3.4 Cardioprotective Activity

An isolated new withanolide with a special chemical structure that was similar to

the aglycones of the cardiac glycosides was examined for its cardiovascular effects of W.

coagulans fruits. The withanolide caused a moderate drop of blood pressure in dogs (34 +/-

2.1, mm Hg) which was blocked by atropine and not by mepyramine or propranolol

(Budhiraja et al 1983). Extracted coagulin L from W. coagulans fruits also showed

significant drop of a fasting blood glucose profile and improved the glucose tolerance of

db/db mice (Maurya et al 2008).In case of Withania somnifera, Mohantyet al (2004)

reported that Withania somnifera (25, 50 and 100 mg/kg) exerts a strong cardioprotective

effect in the experimental model of isoprenaline-induced myonecrosis in rats.

Augmentation of endogenous antioxidants, maintenance of the myocardial antioxidant

status and significant restoration of most of the altered haemodynamic parameters may

contribute to its cardioprotective effect. Among the different doses studied, Withania

somnifera at 50 mg/kg dose produced maximum cardioprotective effect.

2.3.5 Anti-Carcinogenic Activity

It is well established that the various compounds of Withania species, such as

withaferin A from the leaves of W. coagulans, are known to possess anti-cancer properties

(Jayaprakasam et al 2003). Aswagandha is reported to have anti-carcinogenic effects

(Ichikawa et al.,2006). But the mode of action varies between both. The extract of W.

coagulans showed remarkable DMSO (Dimethyl sulfoxide) inhibitory activity which was

induced to produce cytotoxicity and decreased the TNF-G production in chicken

Lymphocyte (Chattopadhyay et al 2007). Withaferin A has been reported to inhibit the cell

growth of various human cancer cell lines, including lung cancer (NCI-H460). Withaferin

26

A showed antiproliferative activity against head and neck squamous carcinoma, by reduced

cell viability in cell lines in vitro (Subramanian et al 1969). This mechanism of action is a

part of the result of G2/M cell cycle arrest and induction of apoptosis in HNSCC cells.

Withanolide A is well-known for its neuronal regenerating effect. Research on animal cell

cultures has shown that the herb Aswagandha decreased levels of the nuclear factor

kappaB, suppresses the intracellular necrosis factor, and potentiates apoptic signaling in

cancerous cell lines (Ichikawa et al., 2006).

2.3.6 Immunomodulating Effect

Coagulin H, a withanolide derivative isolated from W. coagulans exhibited effects

on the immune response, including an inhibitory effect on lymphocyte proliferation, and

expression of interleukin-2 (IL-2) cytokine. A complete suppression of

phytohaemagglutinin-activated T-cells was observed at ≥2.5 µg/ml coagulin H and this

suppression activity was similar to that of prednisolone, a commonly used immune

modulating drug. Coagulin H also significantly inhibited IL-2 production by 80%. Docking

studies predicted that coagulin H bound to the receptor binding site of IL-2 more effectively

than prednisolone. Based on the computational and the experimental results, coagulin H

was identified as a potential immunosuppressive candidate (Mesaik et al., 2006). The same

study using W. somnifera on animal models showed to have profound effects on healthy

production of white blood cells, which means it is an effective immunoregulator and

chemoprotective agent (Kuttan, 1996). In a study using mice, administration of powdered

root extract from Ashwagandha inhibited delayed-type hypersensitivity re actions and

enhanced phagocytic activity of macrophages when compared to a control group (Davis

and Kuttan., 2000).Research has also shown Ashwagandha to have stimulatory effects, both

in vitro and in vivo, on the generation of cytotoxic T lymphocytes, and demonstrated

potential to reduce tumor growth (Davis and Kuttan., 2002).

2.3.7 Anti-microbial Activity

Antifungal and antibacterial properties have been demonstrated in the withanolides

isolated from the ethanolic extract of the whole plant and leaves of Withania coagulans,

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respectively (Choudhary et al, 1995, Khan et al.1993). Withaferin A exhibited a significant

antibacterial activity against Gram-positive microorganisms at the concentrations 6–100

µg/ml, whereas it was inactive against Gram-negative bacteria or nonfilamentous fungi.

Methanolic, petroleum ether and Dichloromethane extract of Withania coagulans, at the

concentration of 25µg/ml were checked by using serial dilution tube method against seven

different fungal strains i.e. Trichoderma viridis, Aspergillus flavus, Fusarium laterifum,

Aspergillus fumigatus,Trichophyton mentogrophytes, Microsporum canis and Candida

albicans. The zones of inhibitions were measured and statistical analysis was applied on the

results of antifungal assay. The fungal strains were checked against the following standards

Ketoconazole, Econazole, Nystatin, Amphotericin, Clotrimazole and Miconazole as

positive control. The petroleum ether, methanolic and dichloromethane extract of Withania

coagulans showed highest activity against all the tested fungal strains Trichoderma viridis,

Aspergillus flavus, Fusariumlaterifum, Aspergillus fumigatus, Trichophyton

mentogrophytes, Microsporum canis and Candida albicans (Mughal et al., 2011).

In comparison, two new withanolides were found in a study done to test the antimicrobial

effect of W. somnifera. These were 4-deoxywithaperuvin (withanolide 1) and 17beta-

dihydroxywithanolide (withanolide 2) .They was tested against many different kinds of

bacteria, viruses and fungi. They were found to be effective against some bacteria

particularly Bacillus cereus, Streptomyces spp. and Pseudomonas flourescens. There was a

complete or partially complete inhibitory action on the fungi Aspergillus fumigatus, A.

terreus, Penicillum funiculosumand P. waksmani (Ahmad 2002).

2.4 Invitro culture studies on Withania coagulans

Adventitious root culture is the unique technique which renders the secondary metabolites

in huge amount and it fulfills the global demand in field of medicine, agriculture, drug

production, pigment production, dye production and so on. Root cultures can be used in

many ways including studies of carbohydrate metabolism, mineral nutrients requirements,

essential need for of vitamins, growth regulators, differentiation of the root apex and

gravitropism. The advantage of using root cultures is that they grow rapidly, relatively easy

28

to prepare and maintain, show a low level of variability and can be easily cloned to produce

a large supply of experimental tissues (Nagarajanet al., 2011). As roots contain a number of

therapeutically applicable withanolides, mass cultivation of roots in vitro will be an

effective technique for the large scale production of secondary metabolites (Murthy et

al.,2008). Structural diversity of withanolides present in Withania spp. is the main problem

in analysis and isolation of these metabolites. The root extract of withania species has

recently been accepted as a dietary supplement in the United States. Harvesting roots is

destructive for the plants and hence there is a growing interest in root culture as an

alternative source for this important metabolite.

Root cultures are typical examples that can be used for production of

phytochemicals. Root cultures have been used as standard experimental system in studies

of inorganic nutrition, nitrogen metabolism, plant growth regulation, and root development.

However, the relatively slow growth remains the main disadvantage of this system (Vargas

& Ham, 1995). More recently, Murthy et al. described the production of withanolide A,

which has also been reported in in vitro regenerated roots (Murthy et al., 2008). Hairy root

cultures can constitute a valuable tool for studies on the biosynthesis and biotechnological

production of secondary metabolites (Sabiret al., 2008).

Adventitious roots are natural, grow vigorously in phytohormone supplemented

medium and have shown tremendous potentialities of accumulation of valuable secondary

metabolites (Nagarajanet al., 2011). Among phytohormones, auxin plays an essential role

in regulating roots development and it has been shown to be intimately involved in the

process of adventitious rooting (Pop et al.,2011) Auxins are a group of tryptophan-derived

signals, which are involved in most aspects of plant development (Woodward and Bartel,

2005). Auxins plays a major role in controlling growth and development of plants, early

stages of embryogenesis, organization of apical meristem (phyllotaxy) and branching of the

plant aerial parts (apical dominance), formation of main root, lateral and adventitious root

initiation (Went and Thimann, 1937).Auxin and ethylene are often described as activators,

while cytokinins and gibberellins are seen as inhibitors of adventitious root formation, even

when some positive effects have been observed.

29

The physiological stages of rooting are correlated with changes in endogenous

auxin concentrations (Heloiret al., 1996). High endogenous auxin concentration is normally

associated with a high rooting rate at the beginning of the rooting process (Blažkováet al.,

1997; Caboniet al., 1997).When applying exogenous auxin on cuttings; the endogenous

auxin concentration reaches a peak after wounding (Gaspar et al., 1996; Gatineau et al.,

1997) coinciding with the initiation of the rooting process. Auxin enters cuttings mostly via

the cut surface (Kenney et al., 1969), even in microcuttings that are known to have a poorly

functioning epidermis (Guan and De Klerk, 2000) and is rapidly taken up in cells by pH

trapping (Rubery and Sheldrake, 1973) and by influx carriers (Delbarreet al., 1996).The

widely used sources of growth hormones for cuttings rooting are the IBA, NAA, IAA and

commercialization root promoters (root-growing powders). IAA was the first used to

stimulate rooting of cuttings (Cooper, 1935). It was discovered that a second, ‘synthetic’

auxin indole-3-butyric acid (IBA) also promoted rooting and was even more effective than

IAA (Zimmerman and Wilcoxon, 1935). Nowadays IBA is used commercially to root

microcuttings and is more efficient than IAA (Epstein and Ludwig-Müller, 1993). The

greater ability of IBA to promote adventitious root formation compared with IAA has been

attributed to the higher stability of IBA versus IAA both in solution and in plant tissue

(Nordstrom et al., 1991). The effective concentration of IBA in these kinds of studies was

also dependent on the pH of the medium. It was shown that, at lower pH values, lower IBA

Concentrations in the medium were sufficient to induce rooting of apple cuttings (Harbage

and Stimart, 1996).The performance of IBA versus IAA can be explained by several

possibilities: higher stability, differences in metabolism, differences in transport and IBA as

a slow release source of IAA. The conversion of IBA to IAA occurs in many plant species

(Ludwig-Muller et al., 2005). Several lines of evidence are now emerging which suggest

that part of the effects of IBA are the direct action of the auxin itself (Ludwig-Mu ller,

2000; Poupart and Waddell, 2000), although other functions may be modulated by the

conversion of IBA to IAA via β-oxidation (Zolman et al., 2000; Bartel et al., 2001).

Studies have shown that any small change in the growth supplement or culture

condition affected root growth in vitro and the accumulation of secondary metabolite

thereof (Praveen and Murthy, 2010). The development of a fast growing root culture

30

system would offer unique opportunities for producing root drugs in the laboratory without

having to depend on field cultivation. As roots contain a number of therapeutically

applicable withanolides, mass cultivation of roots in vitro will be an effective technique for

the large scale production of these secondary metabolites (Murthy et al., 2008). For

commercial withanolide production, roots from field grown plant material are generally

used. The quality of these products may be highly affected by different environmental

conditions, pollutants and fungi, bacteria, viruses and insects, which can result in heavy

loss in yield and alter the medicinal content of plant. Plant cell and organ cultures are

promising technologies to obtain a constant supply of standardized plant-specific valuable

metabolites (Verpoorte et al., 2002). Cell and organ cultures have a higher rate of

metabolism than field grown plants because the initiation of cell and organ growth in

culture leads to fast proliferation of cells/organs and to a condensed biosynthetic cycle.

Further, plant cell/organ cultures are not limited by environmental, ecological and climatic

conditions and cells/organs can thus proliferate at higher growth rates than whole plant in

cultivation (Rao and Ravishankar, 2002). Adventitious roots induced by in vitro methods

have been reported to show a high rate of proliferation and active secondary metabolite

accumulation (Hahn et al., 2003).

2.5 Methods employed for the study of secondary metabolites and their purification

Plant tissue culture can be a potential source for important secondary metabolites such as

pharmaceuticals and food additives. Since the early days of mankind, plants with secondary

metabolites have been used by humans to treat infections, health disorders and illness (Wyk

and Wink, 2004). Many higher plants are major sources of useful secondary metabolites

which are used in pharmaceutical, agrochemical, flavor and aroma industries.

Samples of powdered root of W. somnifera (20 g; Shimadzu Libror AEG-220 balance)

were extracted separately with ethyl acetate (50 ml × 4) under reflux. Extracts of the same

sample were combined and evaporated to dryness under vacuum. The residue (230 mg) was

dissolved using same solvent and used for HPTLC (Patel et al., 2009).The same procedure

was followed for Withania coagulans.

31

Phytochemical analysis reflects the presence of active chemical constituents like

steroid, flavonoids, and saponins etc. in the plant extract. Phytochemical screening of

methanolic and aqueous extracts of fruits of Withania coagulans showed the presence of

alkaloids, steroids, phenolic compounds, tannins, saponin, carbohydrates, proteins, amino

acids and organic acids (Mathur et al ., 2011) Phytochemical investigation of a given plant

will reveal only a very narrow spectrum of its constituents. Historically pharmacological

screening of compounds of natural or synthetic origin has been the source of innumerable

therapeutic agents. For the pharmacological as well as pathological discovery of novel

drugs, the essential information’s regarding the chemical constituents are generally

provided by the qualitative phytochemical screening of plant extracts (Talukdar et al.,

2010).

High Performance Thin Layer Chromatography is one of the modern sophisticated

techniques that can be used for wide diverse applications (Mamatha A., 2011). Due to

several advantages, such as the rapidity, the less amount of sample, and an extremely

limited solvent waste, HPTLC has gained widespread interest as a favorable technique for

the determination of pharmacologically interesting compounds in biological matrices, such

as plants, leaves, and flowers and herbal formulations (Bhandari et al.,2006). HPTLC

technique is precise, specific, accurate, and reproducible which is preferred, especially to

routine applications, to the comparatively more time-consuming and cost-intensive HPLC

(Bhandari et al., 2007). TLC profiling of the plant extracts gives an idea about the presence

of various phytochemicals. Different Rf (Retention factor) value of various phytochemicals

provide valuable clue regarding their polarity and selection of solvents for separation of

phytochemicals (Talukdar et al., 2010). The major advantage of HPTLC is that several

samples can be analyzed simultaneously using a small quantity of mobile phase (Ketanet

al., 2008). It is necessary to develop methods for rapid, precise and accurate identification

and estimation of active constituents or marker compound/s as the qualitative and

quantitative target to assess the authenticity and inherent quality ( Shrikumaret al., 2007).

Through various analytical techniques like TLC, HPLC and HPTLC we can ascertain the

presence of these compounds in plants and also quantify them. Densitometric HPTLC has

been widely used for the phytochemical evaluation of the herbal drugs (Rakeshet al., 2009).

32

2.6 Properties of purified compounds from Withania coagulans

The term “withanolide” is a structural term that has been used for “withan” from the

genus Withania, and “olide” is chemical term for a lactone. To this date, about 400

withanolides or closely related congeners have been discovered in altogether 58

solanaceous species belonging to 22 genera (Eich 2008). Different withanolides,

withacoagin and coagulan were reported from W. coagulans(Khare 2007). One new

withanolide, (17S,20S,22R)-14a,15a,17b,20b-tetrahydroxy-1-oxowitha-2,5,24-trienolide)

named coagulanolide along with four known withanolides, coagulin C, 17b-

hydroxywithanolide K, withanolide F isolated for the first time from this plant and coagulin

L(14R,17S,20S,22R)-14,17,20-trihydroxy-3b-(O-b-D-glucopyranosyl)-1-oxowitha-5,24-

dienolide have been isolated from Withania coagulans fruits and their structures were

elucidated by spectroscopic techniques.( Maurya et al.,2008).

Coagulin C was isolated as an optically active, colorless solid ([a]20D ¼þ5

(c¼0.09, CHCl3)). The molecular formula was determined as C28H36O5. The IR spectrum

displayed bands at 1712 and 1684 cm-1, indicating six membered cyclic ketone and α, β-

unsaturated lactone functionalities, respectively. The 1H- and 13C-NMR spectra of 1 were

characteristic for the steroidal structure of withanolides (Gottlieb and. Kirson, 1981).

Withacoagulin C had relatively good activities (IC50<20 mm) on the inhibition of both Con

A-induced T cell and LPS-induced B-cell proliferation. Withacoagulin C exhibited a

satisfactory SI value. Withanolides induces apoptosis in HL-60 leukemia cells via

mitochondria then the cytochrome C is released and caspase activation (Senthilet al 2007).

Coagulin C was evaluated for their antihyperglycaemic activity in the normoglycaemic rat

model (SLM) and in the streptozocin induced diabetic rat model (STZ). Exhibited

significant antihyperglycaemic activity,( 22.8%) in SLM and 16.9% in STZ, models,

respectively. Coagulin C (at 50 mg/kg body weight) in db/db mice for 10 consecutive days

significantly lowered the postprandial blood glucose level by 22.7% (P < 0.01), whereas

metformin decreased the postprandial blood glucose by 18.6% (P < 0.05) (Maurya et al.,

2008).

33

Coagulanolide is an amorphous powder. The FAB mass and HRESIMS spectra

showed peaks at m/z 509 [M+Na]+ and 486.2611 [M]+ respectively, corresponding to the

molecular formula C28H38O7. This conclusion was supported by the 13C NMR and DEPT

spectra. The 1H and 13C NMR of compound 4 showed that it had close resemblance in

substitution pattern of rings A, B and C with withanolide F, the difference being the

presence of a hydroxyl group at C-15. On the basis of these spectroscopic evidences led to

the structure (17S, 20S, 22R)-14a,15a,17b,20b-tetrahydroxy-1-oxowitha- 2,5,24-trienolide

(4) for this new withanolide, named Coagulanolide. The compound were evaluated for their

antihyperglycemic activity in normoglycemic rat model (SLM) and in streptozotocin

induced diabetic rat model (STZ) exhibited significant antihyperglycemic activity, 28.1% in

SLM and 19.3% in STZ, models (Maurya et al., 2008).

Withacoagulin A (¼(20S,22R)-17b,20b-Dihydroxy-1-oxowitha-3,5,14,24-

tetraenolide; Amorphous colorless powder. [a]20 D ¼þ5 (c¼0.09, CHCl3). IR (KBr): 3453,

2933, 1712, 1684, 1452, 1382, 1319, 1135, 597. 1H- and 13C-NMR: HR-ESI-MS (pos.):

475.2455 ([MþNa]þ , C28H36NaOþ5 ; calc. 475.2460). HR-ESI-MS (neg.): 497.2542

([MþCOOH]_, C29H37O7 ; calc. 497.2539).

Fig.2.3 Coagulin C

34

Withacoagulin D (¼(20S,22R)-14a,17b,20b,27-Tetrahydroxy-1-oxowitha-3,5,24-

trienolide; Amorphous colorless powder. [a]20 D ¼þ60 (c¼0.21, MeOH). IR: 3488, 3419,

2966, 1689, 1654, 1392, 1322, 1143, 1026, 1006, 810, 644. 1H- and 13C-NMRHR-ESI-MS

(pos.): 995.5131 ([2MþNa]þ, C56H76NaOþ 14 ; calc. 995.5132). HR-ESI-MS (neg.):

485.2540 ([M_H]_, C28H37O7 ; calc. 485.2539).

Withacoagulin E (¼(20R,22R)-14b,20b-Dihydroxy-1-oxowitha-2,5,24-trienolide; .

Amorphous colorless powder. [a]20 D ¼þ179 (c¼0.21, CHCl3). IR: 3415, 2941, 1689,

1384, 1319, 1124, 962, 761. 1H- and 13C-NMR: see Tables 2 and 3, resp. HR-ESI-MS

(pos.): 931.5331 ([2MþNa]þ, C56H76NaOþ 10 ; calc. 931.5336). HR-ESI-MS (neg.):

499.2683 ([MþCOOH]_, C29H39O7 ; calc. 499.2695)(Huang et al., 2009).

All 3 compounds, Withacoagulin A, Withacoagulin D and Withacoagulin E was

reported to possess anti-carcinogenic activity. The compounds had relatively good activities

(IC50<20 mm) on the inhibition of both Con A-induced T cell and LPS-induced B-cell

proliferation (Senthilet al 2007).

35

MMAATTEERRIIAALLSS AANNDD MMEETTHHOODDSS

36

3. MATERIALS AND METHODS

The various materials and experimental procedures employed in the study

“Development of in vitro root induction protocol and HPTLC fingerprint for Withania

coagulans” are described under the following headings.

3.1 Materials

3.1.1Plant material

3.1.2 Chemicals and Equipments

3.1.3 Media used

3.2 Methods

3.2.1 Inoculation of explants for root induction

3.2.2 Establishment of roots in suspension

3.2.3 Mass cultivation of roots in bioreactor

3.2.4 Extraction of secondary metabolites

3.3 Quantitative estimation of selected phytochemicals

3.4 High Performance Thin Layer chromatographic profiling of root extracts

3.5 Statistical analysis

37

3.1 Materials

3.1.1Plant material

Surface sterilized seeds of Withania coagulans were germinated in vitro and

seedlings were maintained on MS basal medium with regular sub culturing. The seeds were

obtained from Banaras Hindu University, Varanasi. Leaves excised from two months old

aseptic plantlets were used as explants.

3.1.2 Chemicals and Equipments

HIMEDIA chemicals and Elix-3 water were used for the entire study. HPTLC was

performed on precoated Silica gel aluminum60 F254 plates (E.MERCK, Germany) in a

semiautomatic CAMAG Linomat5 device.

3.1.3 Media used

MS basal medium was prepared essentially based on the procedure described by

Murashigae and Skoog (1962). The composition of the medium is given in Appendix 1. pH

of the media was adjusted to 5.6 - 5.8 using 0.1N NaOH or 0.1N HCl and the volume was

made up to one liter with Elix – 3 water. Solidifying agent (0.8% agar agar type I) was

added to the media and steamed to melt the agar. It was then dispensed in culture bottles

(30 ml/ bottle) and autoclaved at 15 lbs pressure at 121°C for 20 minutes.

For root induction, MS basal media with different combinations of indole butyric acid

(IBA) and indole acetic acid (IAA) and 3% sucrose were used. Hormone combinations are

given in Table 3.1.

For maintaining the in vitro induced roots in suspension, liquid basal MS media and that

supplemented with respective combinations of IAA and IBA and 3%sucrose were used.

It was then transferred to air-lift bioreactor provided with liquid basal MS media for mass

cultivation of roots.

38

Table 3.1.Hormone supplementation in MS media for root induction

S.No IBA

mg/L

IAA

mg/l

S.No IBA

mg/L

IAA

mg/l

T0 0 0 T13 2.0 1.0

T1 0.5 0 T14 4.0 1.0

T2 1.0 0 T15 0 2.0

T3 2.0 0 T76 0.5 2.0

T4 4.0 0 T17 1.0 2.0

T5 0 0.5 T18 2.0 2.0

T6 0.5 0.5 T19 4.0 2.0

T7 1.0 0.5 T20 0 4.0

T8 2.0 0.5 T21 0.5 4.0

T9 4.0 0.5 T22 1.0 4.0

T10 0 1.0 T23 2.0 4.0

T11 0.5 1.0 T24 4.0 4.0

T12 1.0 1.0

3.2 Methods

3.2.1 Inoculation of explants for root induction

The working table of the Laminar Air Flow chamber was first surface sterilized

with 70% ethanol. Sterile petridishes and tools (Forceps, scalpels, sterile cotton and paper

towels) that were used for inoculation were kept in the Laminar Air Flow chamber. The

ultra violet light was switched on for 20 minutes. Prior to inoculation, hands were

sterilized with ethanol. The forceps and scalpels used for inoculation were dipped in 70%

ethanol and flame sterilized. For root induction studies, the leaves were trimmed into

pieces of about 1cm2 and inoculated on to the medium. The inoculated explants were

39

cultured at 25ºC and observed regularly for contamination or for any other morphological

changes. Each experiment had 4 replicates with three explants in each. A photoperiod of

16 / 8 h light was maintained for all experiment.

3.2.2 Establishment of roots in suspension

After a period of 30 days, the root induced in various combinations was checked

and the combination with increased number of roots was identified as best. The root of that

particular combination was then cultured in liquid MS basal media (suspension)

supplemented with and without the respective hormone combinations. Thirty root tips or

branch (1.5 gm.) measuring in length 2-3 mm were cut under sterile conditions and

transferred to sterile conical flask containing 30 ml liquid MS with the respective media.

This study was taken to analyze the growth pattern of roots in suspension culture under the

influence of hormones. They were sub cultured regularly at 15 days intervals in culture

bottles.

3.2.3 Mass cultivation of roots in bioreactor

After 30 days of regular sub culturing, a part of the well grown roots were

transferred to air-lift bioreactor for mass cultivation of roots. The bioreactor was provided

with proper aeration supply and temperature was maintained at 22°C. The roots were

harvested and their wet and dry weight was noted. The increase in root mass was

calculated.

3.2.4 Extraction of secondary metabolites

3.2.4.1 Extraction from root sample

One gram of the in vitro and in vivo dried root sample of Withania coagulans was

extracted four times with 200ml of ethyl acetate (4 × 50ml). After each extraction, the

extract was filtered off using Whatmann No: 1 filter paper and the residue were allowed to

interact with another 50ml of ethyl acetate for overnight. The same procedure was followed

till the completion of fourth extraction. The entire extraction was carried out at 22ºC on a

shaker, maintained at 104 rpm. All the four extracts were combined and evaporated to

40

dryness under vacuum using flash evaporator, maintained at 45ºC and 150 rpm (Patel et

al., 2009). The residue was dissolved in HPLC grade methanol and the concentrated

extracts were used for HPTLC analysis.

3.3 Quantitative estimation of selected phytochemicals

3.3.1 Estimation of carbohydrates

The estimation procedure of Hedge and Hofreiter (1962) was followed for the

estimation of total carbohydrates present in 1g of Withania coagulans root sample

(Appendix 2).

3.3.2 Estimation of protein

The estimation procedure of Lowry et al., (1951) was followed for estimation of

proteins present in1g of Withania coagulans root. The optical density read at 660nm gave

the protein content of the sample (Appendix 3).

3.3.3 Estimation of flavonoids

To estimate the flavonoids present in 1g of Withania coagulans root samples, 0.1 ml

of extract was pipetted into test tube and evaporated to dryness. The procedure by Boham

and Kocipal-Abyazan, (1994) was followed for the estimation (Appendix 4).

3.3.4 Estimation of steroids

A modified procedure of Wall et al., (1952) was followed for the estimation of

steroids in1g of Withania coagulans root, where the green colour developed was observed

at 640nm and the total Steroid present in1 g of the sample was calculated (Appendix 5).

3.3.5 Estimation of saponins

The amount of saponins present in 1g of Withania coagulans root was calculated by

following the estimation procedure by Buccoet al.,1977 (Appendix 6).

41

3.4 High Performance Thin Layer chromatographic profiling of root extracts

The High Performance Thin Layer Chromatography analysis was carried out on

20cm 10cm precoated silica gel aluminum plate 60F254(E.MERCK, Germany). The plates

were pre-washed with methanol. The methanolic extract of samples were applied to the

plates as 8mm bands, under a stream of nitrogen, by means of a CAMAG (Switzerland)

Linomat V semiautomatic sample applicator fitted with a 100µl Hamilton HPTLC syringe.

Linear ascending development to a distance of 8cm was carried out on 20cm 20cm twin

trough chamber saturated with 11ml of the mobile phase, Toluene: Ethyl Acetate: Formic

acid (5: 5: 1). The optimized chamber saturation time for mobile phase was 30min at room

temperature (25ºC±2).Subsequent to scanning; TLC plates were dried in a current of air

with the help of an air dryer. The banding patterns were visualized in 254nm, 366nm and

white light and the Rf values were calculated Densitometric scanning was performed with

Camag TLC scanner III in the reflectance –absorbance mode at 540nm after spraying with

10% sulphuric acid and operated by Win CATS software (1.3.0 Camag) (Jirgeet al, 2011).

3.5 Statistical analysis

The data obtained from the various experiments were subjected to statistical

analysis by using the statistical software SIGMASTAT and AGRES, in completely

randomized design (CRD). Each experiment was repeated twice with a minimum of 3

replicates in each.

42

RREESSUULLTTSS AANNDD DDIISSCCUUSSSSIIOONN

43

4. RESULT AND DISCUSSION

This chapter deals with the results and discussion obtained during the course of the

present study entitled “Development of in vitro root induction protocol and HPTLC

fingerprint for Withania coagulans”, discussed under the following subheadings:

4.1 In vitro rhizogenesis in Withania coagulans.

4.2 Mass production of roots in suspension.

4.3 Mass production of roots in bioreactor

4.4 Quantitative estimation of selected phytochemicals

4.5 Comparative HPTLC fingerprint of in vitro and in vivo root extracts of

Withania coagulans and Withania somnifera.

4.1 In vitro rhizogenesis in Withania coagulans

Adventitious rooting is a complex process and a key step in the vegetative

propagation of economically important woody, horticultural and agricultural species,

playing an important role in the successful production of elite clones. The formation of

adventitious roots is a quantitative genetic trait regulated by both environmental and

endogenous factors (Pop et al., 2011). The formation of adventitious roots is a process

induced and regulated by environmental and endogenous factors, such as temperature, light,

hormones (especially auxin), sugars, mineral salts and other molecules. Phytohormones

have direct (involved in cell division or cell growth) or indirect (interacting with other

hormones or molecules) effects on plants (Jaillais and Chory, 2010).

While research to date has succeeded in producing a wide range of valuable

secondary phytochemicals in unorganized callus or suspension cultures, in other cases

production requires more differentiated micro plant or organ cultures (Dörnenberg and

Knorr, 1997). This situation often occurs when the metabolite of interest is only produced

in specialized plant tissues or glands in the parent plant. A prime example is ginseng

(Panax ginseng). Since saponin and other valuable metabolites are specifically produced in

44

ginseng roots, root culture is required in vitro. Pop et al., (2011) reported that several

factors such as concentration of rooting media, auxin type and concentration affect in vitro

rooting stage. Among phytohormones, auxin plays an essential role in regulating roots

development and it has been shown to be intimately involved in the process of adventitious

rooting (). Among auxins IAA was the first, used to stimulate rooting of cuttings (Cooper,

1935) and soon after another auxin which also promoted rooting; IBA was discovered and

was considered even more effective (Zimmerman and Wilcoxon, 1935). Wadegaonkar et

al. (2006) reported that combination of IAA and IBA was effective in the induction of

adventitious roots from leaf explants of W. somnifera. Thus, different combinations of IAA

and IBA were checked in case of Withania coagulans in the present study.

From the preliminary studies MS media supplemented with 1 mg/L IBA+0.25mg/L

IAA with 3% sucrose concentration in light was found to be the suitable media for highest

percentage of root induction (M.Sc thesis Uma maheswari, 2010).So the present study was

carried out to study the effect of IBA and IAA in 25 different combinations supplemented

with 3% sucrose concentration on root induction in Withania coagulans under 16 hrs

photoperiod. The results are presented in. (Plate 4.1). The different combinations of IAA

and IBA gave different responses. The results show that auxin plays an important role in

root induction.

45

46

4.1.1 Effect of IBA on root induction

Differences in the ability to form adventitious roots have been attributed to

differences in auxin metabolism (Epstein and Ludwig-Muller, 1993). The results of present

study showed that increasing concentration of IBA showed an increasing effect on root

induction (Table 4.1, Fig. 4.1). The effect followed an elevated pattern as the days

progressed. From Fig. 4.1it is obvious that, within 20 days of culture in media

supplemented with 4mg/L of IBA, maximum root induction occurred and on increasing the

culture period to 30 days, no significant increase was observed. This result is in accordance

with the observation of Muller et al., (2005) who reported that increasing the incubation

time of the second treatment on IBA resulted in more segments showing adventitious root

formation in Arabidopsis. When compared to the effect of IAA on root induction, IBA

shown to be more effective. Application of IBA to cuttings of many plant species results in

the induction of adventitious roots, in many cases more efficiently than IAA (Epstein and

Ludwig-Muller, 1993).The result showed 94.4% root induction in MS media supplemented

with 4mg/L of IBA. Several possibilities exist to explain the better performance of IBA

versus IAA: (i) higher stability, (ii) differences in metabolism, (iii) differences in transport,

and (iv) IBA is a slow release source of IAA. But the results suggested that IBA alone

could not bring 100% root induction, since a better result was obtained in the combination

of both IAA and IBA. This may be due to the fact that IBA may be a very simple

‘conjugate’ of IAA and must be converted to IAA by b-oxidation to have an auxin effect as

suggested by Muller et al., (2005) and that either IBA itself is active or that it modulates

the activity of IAA (van der Krieken et al., 1992, 1993).

47

Table 4.1 Response of Explants to Variation in IBA Concentration on Root Induction

* Data represents mean ± SE of 3 replications with 2 explants per replicate.

S.No

IBA

mg/L

IAA

mg/L MEAN ± SE PERCENTAGE OF

RESPONSE

PERCENTAGE OF

ROOT INDUCTION

After

10

days

After

20

days

After

30

days

After

10

days

After

20

days

After

30

days

After

10

days

After

20

days

After

30

days

T0 0 0 0±0

0.333±0.19 0.333±0.21 0 33.33 33.33 0 1.02 0.58

T1 0.5 0 1.0±0 6.833±1.33 10.167±0.79 100 100 100 10.71 20.81 17.68

T2 1.0 0 0.833±0.31 7.0±0.37 15.667±0.67 66.67 100 100 8.93 21.32 28.99

T3 2.0 0 1.0±0.37 8.667±0.56 18.667±0.49 66.67 100 100 10.71 26.9 32.46

T4 4.0 0 3.0±0.63 31.0±0.52 56.667±1.05 100 100 100 39.29 94.42 98.55

48

Fig 4.1 Response of Explants to Variation in IBA Concentration on Root Induction

10 days

20 days

30 days

2

49

4.1.2 Effect of IAA on root induction

It was clear from the results that increasing in concentration of IAA shown to have a

positive effect on root induction but a concentration higher than optimal showed a negative

impact(Table 4.2) (Fig. 4.2).Pilet and Saugy (1987)reported that the fast growing roots

were stimulated by IAA and ABA when applied at a low concentration (5.10-9 M), while at

a higher concentration (1.10-6 M) both hormones inhibited the growth rate. At initial

stages, there was a high percentage of root induction observed, but it gradually decreased as

the period of incubation progressed to 20 days. At the final stages i.e. by about 30 days the

percentage of root induction was appreciable. Divisions of the first root initial cells are

dependent on either endogenous or applied auxins (Hartman et al., 1997). An increase in

root induction at initial stage may be due to the endogenous IAA and that at final stage in

response to the exogenous IAA. This suggests that both endogenous and exogenous supply

of IAA had an effect on root induction as the days progressed. Pilet and Saugy (1987)

reported that endogenous IAA hormones are working in a smaller concentration spectrum

than exogenous hormones in the roots of maize. The exogenous supply of IAA had a

positive effect in the advancement of rooting in L. laxumgrown in a) peat/polystyrene and

b) bark/river sand/polystyrene mediums (Laubscher and Ndakidemi,2008). There is no

direct evidence that the synthetic auxin might substitute for a natural one in cells (Davis et

al., 1988), but they can reach the plant’s IAA-pool (Bartelet al., 2001).

4.1.3 Effect of combination of IAA and IBA on root induction

In the present study maximum root induction were observed in MS media

supplemented with 1mg/L IAA and 4mg/L IBA. 100% root induction was obtained in this

combination of IAA and IBA (Table 4.3) (Fig.4.3). Compared to that MS media

supplemented with IBA alone i.e. 4mg/ L showed 94.4% root induction, followed by that

provided with 2mg/L IAA and 4mg/ L IBA by 89.3%, during a period of 20 days. This

shows that percentage of root induction increases with increase in concentration of IBA and

IAA. It is suggested by Muller et al., (2005) that adventitious rooting in Arabidopsis stem

segments is due to an interaction between endogenous IAA and exogenous IBA.But in case

of IAA, after an optimal concentration (<1.0mg/L or >1.0mg/L) there was a decline in

50

number of roots. With IBA, it was observed that on increasing its concentration increased

root induction along with an increase in callus. In present study a combination of 6mg/L

IBA with 0.5, 1.0, 2.0, 4.0 mg/L of IAA were carried out. It was observed that a higher

percentage of roots were induced on increasing medium concentration of IBA to 6mg/L

compared to that supplemented with 4.0 mg/L IBA. But the roots mainly aroused from the

high amount of callus induced in response to the increased concentration of IBA. This

found to be insignificant and was dropped.

The results revealed that the percentage of explant response increased with increase

in concentration of IBA. Ali et al (2009) reported that IBA produced maximum number of

roots (5.03) per rooted explant at 1.5 mg/ L in Olive plants. Benelli et al., (2001) and

Tanimoto (2005) have proved that IBA is the most effective auxin in olive rhizogenesis as

compared to NAA. The percentage of response increased as the period of incubation

progressed. Callus induction was observed in some explants during the initial stages of the

culture. The callus was produced in response to IBA rather than IAA. The age, type and the

plant from which it was collected of determined the percentage response of the leaf explant.

At the same time, there was a difference in pattern of number of roots induced per

explant i.e. percentage of root induction in each combination. Though many of the explants

gave fast response in presence of varying proportions of auxin supplemented, the number of

roots induced varied. 100% of root induction was observed in the combination 1IAA and

4IBA, followed by 94.4% in 4 IBA and 89.3% in 2IAA and 4IBA.This shows that IBA play

a major role in root induction compared to IAA. The reason for these differences in root

inducing ability may be the slow and continuous release of IAA from IBA (Krieken et al.,

1993; Liu et al., 1998) and release of IBA through hydrolysis of conjugates (Epstein &

Muller, 1993).

51

Table 4.2 Response of Explants to Variation in IAA Concentration on Root Induction

* Data represents mean ± SE of 3 replications with 2 explants per replicate.

S.No

IBA

mg/L

IAA

mg/L MEAN ± SE PERCENTAGE OF

RESPONSE

PERCENTAGE OF

ROOT INDUCTION

After

10

days

After

20

days

After

30

days

After

10

days

After

20

days

After

30

days

After

10

days

After

20

days

After

30

days

T0 0 0 0±0

0.333±0.19 0.333±0.21 0 33.33 33.33 0 1.02 0.58

T1 0 0.5 0.667±0.42 1.167±0.31 7.167±0.54 33.33 83.33 100 7.14 3.55 12.46

T2 0 1.0 1.667±0.56 2.0±0.73 3.333±0.21 66.67 66.67 100 17.86 6.09 5.79

T3 0 2.0 0.667±0.24 0.667±0.42 2.167±1.38 83.33 33.33 33.33 8.93 2.03 3.77

T4 0 4.0 1.0±0.37 1.5±0.34 3.833±0.98 66.67 83.33 83.33 10.73 4.57 6.67

52

10 days

20 days

30 days

2

Fig4.2 Response of Explants to Variation in IAA Concentration on Root Induction

53

*Data represents mean ± SE of 3 replication with 2 explants per replicate

MEAN ± SE PERCENTAGE OF RESPONSE

PERCENTAGE OF ROOT

INDUCTION

S.No IBAmg/L IBAmg/L

After 10

days After 20 days After 30 days

After 10

days

After 20

days

After 30

days

After 10

days

After 20

days

After 30

days

T0 0 0 0±0

0.333±0.19 0.333±0.21 0 33.33 33.33 0 1.02 0.58

T1 0.5 0.5 2.333±0.21 9.167±0.70 18.0±0.58 100 100 100 25.0 27.92 31.3

T2 1.0 0.5 2.333±0.21 15.5±0.85 24.333±0.33 100 100 100 25.0 47.21 42.32

T3 2.0 0.5 2.833±0.54 22.667±0.67 28.333±0.67 100 100 100 30.36 69.04 49.28

T4 4.0 0.5 3.5±0.56 26.667±0.61 28.833±0.54 100 100 100 37.50 81.22 50.14

T5 0.5 1.0 2.667±0.60 7.333±0.84 9.333±0.76 83.33 100 100 23.21 22.34 16.23

T6 1.0 1.0 2.833±0.31 7.833±0.40 16.0±0.68 100 100 100 30.36 23.86 27.83

T7 2.0 1.0 2.0±0.63 19.833±0.34 22.333±4.51 66.67 83.33 83.33 21.14 60.41 38.84

T8 4.0 1.0 9.333±0.21 32.833±0.40 57.5±1.12 100 100 100 100 100 100

T9 0.5 2.0 0±0 0.833±0.40 2.333±0.56 0 50.0 83.33 0 2.54 4.06

T10 1.0 2.0 0.833±0.48 4.333±0.33 10.333±0.76 50 100 100 8.93 13.19 17.97

T11 2.0 2.0 1.5±0.34 9.167±0.65 16.333±0.50 83.33 100 100 16.07 27.92 28.40

T12 4.0 2.0 3.0±0.52 29.333±1.09 40.5±0.34 100 100 100 32.14 89.34 70.43

T13 0.5 4.0 0±0 0.833±0.40 9.0±2.9 0 50.0 66.67 0 2.54 15.65

T14 1.0 4.0 0.160.177± 1.167±0.31 11.167±0.6 16.67 66.67 100 1.79 3.55 19.42

T15 2.0 4.0 0.167±0.17 1.0±0.37 8.167±2.6 16.67 50.0 66.67 1.79 3.05 14.20

T16 4.0 4.0 0.833±0.48 5.833±0.48 12.833±0.75 50 100 100 8.93 17.77 22.32

Table 4.3 Response of explants to variation in auxins concentration

54

Table 4.3 Response of explants to variation in auxins concentration on root induction

(after 20 days)

55

4.2 Mass production of roots in suspension

For the establishment of roots in suspension, the roots obtained from MS media

supplemented with 1 mg/L IAA and 4mg/L IBA in 3% sucrose was taken, which was

chosen as the best auxin combination for root induction (Plate 4.2). After 30 days of root

cultures in suspension, the fresh weight of the root was noted and finally Growth index was

calculated (Wu et al., 2008) (Table 4.4). Media change was given after 15 days interval in

order to supply adequate nutrients for growth. At the end of 30th day the growth index was

found to be 17.0, which indicated an increase in root mass. The percent increase in root

mass was found to be 94%.The roots appeared healthy, thin and white in color.

The results suggest that adventitious root cultures of W. coagulans are promising for

large-scale biomass production in suspension cultures and that it can be successfully taken

to bioreactorsfor mass production. Similarly, adventitious root suspension cultures are

proved to be efficient for biomass accumulation in P. notoginseng (Gaoet al., 2005) and

Echinacea purpurea (Wu et al., 2008). Gamborg’s B5 medium was used for the production

of tropane alkaloids by adventitious root cultures of Scopoliaparviflora (Min et al., 2007).

Table 4.4 Growth index of roots in suspension culture

Media

Inoculated

fresh

weight(g)

Harvested

fresh

weight(g)

Growth index

1mg/L IAA and 4mg/L IBA 0.5 9.0 17.0 (After 30 days)

56

Plate 4.2 Growth of Roots in suspension

Initiation

After 30 days

57

Plate 4.3 Mass production of roots in bioreactor

Harvested root

58

4.3 Mass production of roots in bioreactor

Since the roots contain a number of therapeutically applicable withanolides, mass

cultivation of roots in vitro will be an effective technique for the large scale production of

these secondary metabolites. The development of a fast growing root culture system would

offer unique opportunities for producing root drugs in the laboratory without having to

depend on field cultivation (Murthy et al., 2008). The mass cultivation of in vitro roots was

performed on MS media without auxin supplements. The results showed an increase in root

mass by 39.4% after a period of 20 days (Plate 4.3). The roots then obtained appeared

slightly pale in color. This suggested that mass cultivation of roots of W. coagulans in vitro

could be successfully preceded and would offer unique opportunities for producing drugs

from roots. The withanolide contents of the hairy root cultures of W. coagulans were higher

than in the root of the plant. In the hairy root cultures all the withanolides were

accumulated in the root tissues and withaferin A or withanolide A were not detected in the

culture medium samples (Mirjalili et al., 2009).

4.4 Quantitative estimation of selected phytochemicals

Progress over the centuries towards a better understanding of plant derived

medicine has depended on 2 factors that have gone hand in hand. On has been the

development of increasing strict criteria of proof that a medicine really does what it is

claimed to do and other has been the identification by chemical analysis of the active

compound on the plant (Holiman, 1989). Knowledge of chemical constituents of plants is

desirable, not only for the discovery of therapeutic agents, but also because such

information may be of value in disclosing new sources of such economic materials as

tannins, oils, gums, precursors for the synthesis of complex chemical substances. In

addition, the knowledge of chemical constituents of plants would further be valuable in

discovering the actual value of folkloric remedies (Mojabet al., 2003).

All plant parts synthesize some chemical themselves to perform their physiological

activities. The medicinal value of these secondary metabolites is due to the presence of

chemical substances that produce a definite physiological action on human body

59

(Kubmarawa et al., 2008). The most important of these bioactive compounds are alkaloids,

flavonoids, tannins and phenolic compounds (Duraipandiyan et al., 2006). Thus to develop

a quantitative estimate of phytochemicals present in Withania coagulans was aimed at.

The quantitative estimation of selected compounds like carbohydrates (Fig. 4.4),

flavonoids (Fig. 4.5), proteins (Fig. 4.6), saponin (Fig. 4.7) and steroids (Fig. 4.8)present in

different in vivo roots and in vitro root of Withania coagulans were carried out. The result

was shown in (Table 4.5). The samples also included an in vivo Gujarat root and an in vitro

root of Withania somnifera, so that a comparative study between both the plants could be

done. The results showed that among the 15 samples evaluated, in vivo Gujarat root of

Withania somnifera contained the highest amount of carbohydrate (4.74±0.108 mg/g). The

flavonoid content was reported to be high in in vivo Withania coagulans root 019 (USB

WC 019) (4.43±0.123 mg/g) and that the protein content in in vitro roots of Withania

somnifera (25.30mg/g). The in vitro roots of Withania coagulans showed an increased

amount of saponin in it (14.40 mg/g). Steroids were reported to be high in in vivo Withania

coagulans root 008 (USB WC 008) (3.27 mg/g).The variation in amount of different

phytochemicals in different root samples may be due to the influence of the environmental

condition they were grown on. There is a wide variation in the amount and type of chemical

constituents in samples of different species, in samples that differ in method and time of

collection (ICH Harmonised Tripartite Guidelines, 1996).

60

Table 4.5 Quantitative Phytochemical Analysis of Different in vivo and in vitro roots of

Withania coagulans and Withania somnifera

*Data represents Mean± SE twice repeated, expressed in mg/g

S.No. CARBOHYDRATES FLAVANOIDS PROTEINS SAPONINS STEROIDS

WC001 0.72±0.21 1.04±0.06 7.16±0.32 3.57±0.10 2.1±0.05

WC 002 1.48±0.10 1.47±0.12 8.13±0 5.19±0.05 2.35±0

WC 003 2.79±0.10 1.10±0 5.70±0.16 5.03±0 2.15±0.10

WC 004 1.37±0.21 2.08±0.12 3.92±0.32 6.07±0 2.50±0.05

WC 005 3.22±0.10 3.19±0 7.48±0 8.05±0 2.71±0.05

WC 006 1.37±0 1.78±0.06 3.75±0.16 10.76±0.10 2.25±0

WC 007 2.02±0 1.84±0 7.16±0.32 7.89±0.05 2.86±0

WC 008 0.72±0 1.16±0.06 4.56±0.32 3.15±0 3.27±0

WC 010 0.72±0 2.70±0 5.54±0 9.09±0.10 3.06±0

WC 018 0.28±0 1.53±0.06 3.91±0 5.65±0.10 2.61±0.05

WC 019 0.28±0 4.43±0.12 6.51±0 10.45±0 2.50±0.05

WC 021 0.07±0 1.65±0.06 5.21±0 3.73±0.05 2.45±0

in vitro WC 3.01±0.10 2.76±0.06 22.06±0 14.40±0 2.76±0

in vitro WS 2.35±0.1 2.82±0 25.30±0 9.72±0 2.61±0.05

WS Gujarat 4.74±0.10 2.82±0.12 8.13±0 5.24±0 3.07±0.103

61

Fig 4.4 Quantitative estimation of Carbohydrate

62

Fig 4.5 Quantitative estimation of Flavanoids

63

Fig 4.6 Quantitative estimation of Proteins

64

Fig 4.7 Quantitative estimation of Saponin

65

Fig 4.8 Quantitative estimation of Steroids

66

4.5 Comparative HPTLC fingerprint of in vitro and in vivo root extracts of Withania

coagulans and Withania somnifera.

Withania coagulans is well known in the indigenous system ofmedicine for the

treatment of ulcers, dyspepsia,rheumatism, dropsy, consumption and sensile

debility(Hemalatha et al., 2008). According to Kubmarawa et al., (2008) the medicinal

value of the secondary metabolites is due to the presence of chemical substances that

produce a definite physiological action on human body. It is necessary to develop methods

for rapid, precise and accurate identification and estimation of active constituents or marker

compounds as the qualitative and quantitative target to assess the authenticity and inherent

quality (Jirge et al., 2011). The separation and purification of phytoconstituents of the

extract was mainly carried out using a combination of the chromatographic techniques. The

choice of technique depends largely upon the solubility properties and volatilities of

compound to be separated (Vinod et al., 2010).Through various analytical techniques like

TLC, HPLC and HPTLC we can ascertain the presence of these compounds in plants and

also quantify them. HPTLC offers many advantages over other chromatographic techniques

such as unsurpassed flexibility (esp. stationary and mobile phase), choice of detection, user

friendly, rapid and cost effective. Thus, HPTLC is most widely used at industrial level for

routine analysis of herbal medicines (Jirge et al., 2011).

The results of standardization of solvent system for root extracts of Withania

coagulans showed that the solvent system Toluene: Ethyl acetate: Formic acid in the ratio

of 5:5:1 to be the best (Plate 4.4). 10% Sulphuric acid was used as the derivatizing agent.

The other solvent systems employed include Chloroform: Ethyl acetate: Methanol: Toluene

(7.4: 0.4: 0.8: 3.0) and Chloroform: Methanol (9.0:1.0) and the derivatizing agent

Anisaldehyde sulphuric acid (Conc. Sulphuric acid: Glacial Acetic acid: Methanol:

Anisaldehyde in 5: 10: 85: 0.5). The Toluene: Ethyl acetate: Formic acid solvent system

showed a higher resolution and clear banding patterns, indicating a higher solubility of

compounds when compared to the other 2 solvent systems.

67

Plate 4.4 Standardization of solvent system for in vivo and in vitro roots of Withania coagulans and Withania somnifera

Solvent system- Chloroform: Ethyl acetate: Lane 1 – invitro Withania coagulans root

Methanol: Toluene (7.4:0.4:0.8:3.0) Lane 2 – invivo Withania coagulans 006root

Developing agent – a) 10% H2SO4 Lane 3 – invivo Withania coagulans 019 root

b) Anisaldehyde sulphuric acid Lane 4 - invivo Withania somnifera Maharashtra root

Lane 5 - Withaferin A

a) b)

1 2 3 4 5 1 2 3 4 5

68

69

70

An earlier study by Sharma et al., (2007) reported the use the same solvent system

in Withania somnifera as mobile phase. HPTLC of W.somnifera methanolic extract was

performed on Si 60 F254 (20 cm ×20 cm) plates with Toluene: Ethyl acetate: Formic acid

(5:5:1), as mobile phase (Sharma et al., 2007).The choice of solvent depends upon two

factors: (a) nature of substance to be separated, (b) material on which separation is to be

carried(Vinod et al., 2010). The spots appeared clearly on derivatizing with 10%sulphuric

acid rather than with anisaldehyde sulphuric acid.

The HPTLC analysis of the in vivo roots collected from different regions of Iran, in

vitro roots of Withania coagulans, in vivo roots Withania somnifera collected from Gujarat

and in vitro root of Withania somnifera were performed using the solvent system Toluene:

Ethyl acetate: Formic acid (5:5:1) in order to check the accumulation of various

phytoconstituents in it (Plate 4.5). The results revealed that among the in vivo roots of

Withania coagulans, USBWC 019 showed various spots indicating an increased number of

phytoconstituents in it (Fig. 4.5, Lane 14). The banding pattern of in vivo and in vitro root

extract of Withania coagulans to a greater extend showed to be similar but the

accumulation were found to be higher in in vitro roots. The in vitro roots of Withania

coagulans and Withania somnifera almost showed similar banding patterns, though the

accumulation of some compounds with Rf values 0.31, 0.50, 0.72 and 0.82 varied between

both. With reference to the standard withaferin A the Rf value being 0.39, the in vivo roots

of Withania coagulans (USB WC 019 and USBWC 010) and Withania somnifera (Gujarat)

showed a spot with similar Rf value, which indicates they contained withaferin. Though all

the samples showed the presence of withaferin, the accumulation varied significantly. The

qualitative evaluation of the plate was done by determining the migrating behavior of the

separated substances given in the form of Rf. The HPTLC of Dendrophthoefalcata

ethanolic extract showed eleven spots in UV, further resolving the separation of DFEE

done by TLC value (Vinod et al., 2010).

71

72

SSUUMMMMAARRYY AANNDDCCOONNCCLLUUSSIIOONN

73

5. SUMMARY AND CONCLUSION

The present study on “Development of in vitro root induction protocol and HPTLC

fingerprint for Withania coagulans” was carried out with an aim of mass culturing

Withania coagulans in bioreactors and to analyse the variability in phytochemical

composition in in vivo roots of Withania coagulans collected from different geographical

areas of Iran.

The available literature was surveyed and relevant information compiled. Materials

used in the study are of standard quality and the methods followed are established ones

reported in reputed journals.

The results of the study are summarized here under.

Among the 25 different combination of auxins (IAA and IBA) tested, MS media

supplemented with 1mg/L IAA and 4mg/L IBA and 3% sucrose was found to

be the best medium for adventitious root induction in Withania coagulans.

The roots were subjected to suspension culture and then to mass cultivation in

bioreactor, showed an increased mass and bioactive compound accumulation.

The quantitative phytochemical estimation of carbohydrates, flavonoids,

proteins, saponins and steroids on in vivo and in vitro roots of Withania

coagulans and Withania somnifera showed that in vivo Gujarat root of Withania

somnifera contained the highest amount of carbohydrate (4.74±0.108 mg/g).

The flavonoid content was reported to be high in in vivo Withania coagulans

root 019 (USB WC 019) (4.43±0.123 mg/g) and that the protein content in in

vitro roots of Withania somnifera (25.30mg/g). The in vitro roots of Withania

coagulans showed an increased amount of saponin in it (14.40 mg/g). Steroids

were reported to be high in in vivo Withania coagulans root 008 (USB WC 008)

(3.27 mg/g).

The HPTLC fingerprint of in vivo and in vitro roots of Withania coagulans

andWithania somnifera showed that the in vitro roots of Withania coagulans

andWithania somnifera had similar banding patterns but the accumulation of

compounds varied. The in vivo roots USBWC 010 and USBWC 019 of

74

Withania coagulans and in vivo roots Withania somnifera of from Gujarat

showed high withaferin content compared to other roots.

The in vitro roots contained a higher number and accumulation of secondary

metabolites compared to in vivo.

To conclude, as observed, there is a wide variation with the phytochemical contents

of in vivo roots collected from different location, and hence, culturing in vitro adventitious

roots in bioreactors could be used as a fast and efficient method of generating roots that

would offer unique opportunities for producing root drugs without having to depend on

field cultivation.

75

BBIIBBLLIIOOGGRRAAPPHHYY

76

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AAPPPPEENNDDIICCEESS

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APPENDIX-I

COMPOSITION OF MS MEDIUM

Ingredients Composition (mg/ L) Stock Solution (W/V) (g)

MS Macro I (10 X) 1000ml NH4NO3 1650 16.5 KNO3 1900 19 MgSO4.7H2O 370.6 3.7 KH2PO4 170 1.7 100 ml MS Macro II (10 X) 1000 ml CaCl2.2H2O 439.8 4.398 100 ml Fe-Na EDTA (1000 X) 100 ml Fe-Na EDTA 36.7 36.7 1 ml Micro Nutrients (1000 X) 100 ml NaMoO4.7H2O 0.25 0.025 CuSO4.5H20 0.025 0.0025 CoCl2.2H2O 0.025 0.0025

MnSO4.4 H2O 13.2 1.32

ZnSO4.4H2O 8.6 0.86 H3BO3 6.2 0.62 1 ml KI (1000X) 0.83 100ml

Myo-Inositol (100 X) 100 ml Myoinositol 100 1 10 ml

MS Vitamins (1000 X) 100 ml Nicotinic Acid 0.5 0.05 Pyridoxine HCl 0.5 0.05 Thiamine HCl 0.1 0.01 Glycine 2 0.2 1 ml

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APPENDIX 2

ESTIMATION OF TOTAL CARBOHYDRATE BY ANTHRONE METHOD

Hedge and Hofreiter (1962)

Carbohydrates are the important components of storage and structural materials in the plants. They exist as free sugars and Polysaccharides. The basic units of Carbohydrate are the Monosaccharide which cannot be split by hydrolysis into more simple sugars. The Carbohydrate content can be measured by hydrolyzing the Polysaccharides into simple sugars by Acid hydrolysis and estimating the resultant Monosaccharide.

PRINCIPLE

Carbohydrates are first hydrolyzed into simple sugars using dilute hydrochloric acid. In hot acidic medium glucose is dehydrated to hydroxymethyl furfural. This compound forms with Anthrone a green colored product with the absorption maximum at 630 nm.

MATERIALS

• 2.5 N HCl

• Anthrone Reagent: Dissolve 200 mg of Anthrone Reagent in 100 ml of 95% ice cold sulphuric acid. Prepare fresh before use.

• Standard Glucose

Stock standard: Dissolve 100 mg in 100 ml of water.

Working standard: 10 ml of stock diluted to 100 ml with distilled water and stored in refrigerator, after adding few drops of Toluene.

PROCEDURE

1. Take 0.1 of the sample into boiling test tubes.

2. Prepare the standards by taking 0, 0.2, 0.4, 0.6, 0.8 and 1ml of the working standard '0' serves as blank.

3. Hydrolyse by keeping it in a boiling water bath for three hours with 5 ml of 2.5 N HCl and cool to room temperature.

4. Neutralise it with solid sodium carbonate until the effervescence ceases.

91

5. Make up the volume to 1ml in all the tubes including the sample tubes by adding distilled water.

4. Then add 4 ml of Anthrone Reagent.

5. Heat for eight minutes in a boiling water bath.

6. Cool rapidly and read the green to dark green colour at 630 nm.

7. Draw a standard graph by plotting concentration of the standard on the X –axis versus absorbance on the Y – axis.

8. From the graph calculate the amount of Carbohydrate present in the sample tube.

CALCULATION

Amount of Carbohydrate present in 100 mg of the sample = (mg of glucose) (Volume of Test Sample) *100

92

APPENDIX – 3

ESTIMATION OF PROTEIN

Lowry et al. (1951)

Protein can be estimated by different methods as described by Lowry and also by estimating the total nitrogen content. No method is 100% sensitive. Hydrolyzing the protein and estimating the amino acids alone will give the exact quantification. The method developed by Lowry et al is sensitive enough to give a moderately constant value and hence largely followed. Protein content of enzyme extracts is usually determined by this method.

PRINCIPLE

The blue color developed by reduction of the Phosphomolybdic –Phosphotungstic components in the Folin – Ciocalteau reagent by the Amino acids Tyrosine and Tryptophan present in the Protein and also colour developed by the Biuret Reaction of the Protein with the alkaline cupric tartrate are measured in theLowry’s method.

MATERIALS

Reagent A: 2% Sodium Carbonate in 0.1N Sodium Hydroxide

Reagent B: 0.5% Copper Sulphate (CuSO4 .5 H2O) IN 1 % potassium sodium tartrate

Reagent C:

Alkaline Copper Solution: Mix 50 ml of A and 1 ml of B prior to use

Reagent D:

Folin –Ciocalteau Reagent: Reflux gently for 10 hours a mixture consisting of 100 g Sodium Tungstate (Na2WoO4.2H2O), 25 g Sodium Molybdate (Na2MoO4.2H2O), 700 ml water, 50ml of 85% Phosphoric acid, and 100 ml of concentrated Hydrochloric acid in a 1.5 L flask. Add 150 g Lithium Sulphate, 50 ml water and few drops of bromine Water. Boil the mixture for 15 minutes withoutcondenser to remove excess bromine. Cool, dilute to 1 litre and filter. The reagent should have no greenish tint. (Determine the acid concentration of the reagent by titration with 1 N NaOH to a Phenolphthalein end - point).

Stock Standard Solution:

Weigh accurately 50 mg of Bovine Serum Albumin and dissolve in distilled water and make up to 50 ml in a Standard flask.

Working Standard Solution

93

Dilute 10 ml of the Stock solution to 50 ml with distilled water in a Standard flask. One ml of this solution contains 200 µg.

PROCEDURE

1. Pipette out 0.2, 0.4, 0.6, 0.8 and 1 ml of working standard into a series of test tubes.

2. Pipette out 0.1ml of the sample extract in to the another test tubes.

3. Make up the volume to1 ml in all the test tubes. A tube with 1 ml of water serves as the blank.

4. Add 5 ml of Reagent C to each tube including the blank. Mix well and allowed to stand for 10 minutes.

5. Then add 0.5 ml of Reagent D, mix well and incubate at room temperature in the dark for 30 minutes. A blue colour is developed.

6. Take the readings at 660 nm.

7. Draw a Standard graph and calculate the amount of the sample present in the sample.

CALCULATION

Express the amount in mg / g or 100 g sample.

APPENDIX –4

ESTIMATION OF FLAVANOIDS

PRINCIPLE

A Portion of plant was weighed and carried out in two steps, firstly MeoH: H20 (9:1) and then MeoH: H2O (1:1) solvent added to make liquid slurry and mixture left to 12hrs .Filtration to separate the extract from plant material was / carried out rapidly for using glass wool or cotton wool plug in neck of filter funnel two extracts were combined andevaporated 1/13 original volume or most of MeoH had been removed. Resultant aqueous extract was cleared if low polarity contaminants such as Fats, Terpenes, Chloroform and Xanthophylls’ by extraction with hexane and or chloroform. This was repeated several times and extract combined. The solvent extracted aqueous layer containing bulk of Flavonoids was concentrated.

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MATERIALS

Vanillin reagent - 1%in 70%H2SO4

Catechin standard - 110 g/ml

PROCEDURE

1. Aliquot of extract was pipette into test tube and evaporated to dryness.

2. Then added 4ml of vanillin reagent.

3. A standard was also treated in the same manner.

4. Then equal amount of distilled water was added.

5. Kept in boiling water bath for 15 minutes.

6. Take the readings at 360 nm.

7. Draw a Standard graph and calculate the amount present in the sample.

CALCULATION

Express the amount in mg / g or 100 g sample.

APPENDIX –5

ESTIMATION OF STEROIDS

MATERIALS

LibermannBurchard Reagent (Acetic Anhydrate and Sulfuric acid)

Standard: 10mg Cholesterol dissolved in 10ml of chloroform.

LibermannBurchard reagent: 0.5ml sulfuric acid dissolved 10ml of a acetic anhydrates and kept in ice.

PROCEDURE

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1. Pipette out 0.5, 1, 1.5, 2, and 2.5 ml of Standard Cholesterol into a series of test tubes.

2. Pipette out 0.3ml of the sample extract into the another test tubes.

3. 2ml of LibermannBurchard reagent was added.

4. Then equal amount of chloroform was added.

5. Covered with carbon paper and then kept in dark place.

6. Incubated at room temperature in dark for 30 minutes. A green colour is developed.

7. Take the readings at 640 nm.

CALCULATION

Express the amount in mg / g or 100 g sample.

APPENDIX –6

ESTIMATION OF SAPONIN

MATERIALS

Standard: 0.1 g Diosgenin dissolved in 1 ml of HPLC grade methanol.

Reagent A: 0.5 ml anisaldehyde in 99.5 ml ethyl acetate.

Reagent B: 50ml con.H2SO4 in 50 ml ethyl acetate.

PROCEDURE

1. Pipette out 0.2, 0.4, 0.6, 0.8 and 1.0 ml of Standard into a series of test tubes.

2. Pipette out 0.3ml of the sample extract into the another test tubes.

3. Make up the volume to1 ml in all the test tubes. A tube with 1 ml of ethyl acetate serves as the blank.

4. 0.5ml of Reagent A was added.

5. Equal amount of Reagent B was added.

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6. Kept in boiling water bath maintained at 600C for 20 minutes. After cooling to room temperature.

7. The absorbance was measured at 430 nm.

CALCULATION

Express the amount in mg / g or 100 g sample.