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1
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
2
CCEERRTTIIFFIICCAATTEE
3
4
AACCKKNNOOWWLLEEDDGGEEMMEENNTT
5
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.
6
“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
7
CCOONNTTEENNTTSS
8
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
9
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
60
10
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
11
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
12
IINNTTRROODDUUCCTTIIOONN
13
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
14
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
15
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.
16
RREEVVIIEEWW OOFF LLIITTEERRAATTUURREE
17
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
18
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).
19
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)
20
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).
21
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
22
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
23
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
24
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
25
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,
27
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
90
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.