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ACKNOWLEDGEMENT
ACKNOWLEDGEMENT
This is a golden opportunity for me to convey my sincere regards for all
those people who enabled me to accomplish my dissertation work successfully.
“Peace on the outside comes from knowing the God from 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”. I sincerely thank Ayya Avargal
and Amma Avargal for creating a portal to exhibit our abilities.
I express my immense gratitude to Thiru.T.K.Meenakshi Sundarum
B.A., B.L., Anna Avargal, Chancellor, Avinashilingam Institute for Home Science and
Higher Education for Women, Coimbatore, for providing me the opportunity to undertake
this project work.
I record my heartfelt thanks to Dr. (Mrs). Sheela Ramachandran M.Sc.,
PGDF &P., PhD., Vice Chancellor, Avinashilingam Institute for Home Science and
Higher Education for Women, Coimbatore, for providing all the amenities required and
immense support in the conduct of the study.
I express my sincere thanks to Dr. (Mrs.) Gowri Ramakrishnan,
Registrar, Avinashilingam Institute for Home Science and Higher Education for Women,
Coimbatore, for providing opportunity to carry out this piece of work.
It is my privilege to enunciate my heartfelt gratitude to Dr. Saroja
Prabakaran, Director, Hall of Residence and Former Vice-Chancellor, Avinashilingam
Institute for Home Science and Higher Education for Women, Coimbatore for rendering
benevolent support during the tenure of the 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 Institute for Home Science and Higher Education for
Women, Coimbatore, for her constant support in eliciting this project in a facile manner.
I am greatly indebted to my guide Dr. Mrs. Sivagami Srinivasan,
Professor, Department of Biochemistry, Biotechnology and Bioinformatics,
Avinashilingam Institute for Home Science and Higher Education for Women,
Coimbatore, who has always been beside me, extending love, strong guidance,
suggestions, and encouragement and for her unique route of attaining fruitfulness in my
present study. She has always been a source of moral support and strength. She is entirely
responsible for bringing out the best in me and to give me the confidence to perform to the
fullest. She has always encouraged thinking ahead and trying experimenting new ventures.
I am extremely grateful to God Almighty to have given me this experience which I will
cherish throughout my life.
I extend my heartfelt thanks to all the Staff members of the Department of
Biochemistry, Biotechnology and Bioinformatics, Avinashilingam University for Women,
Coimbatore, for their help and cooperation throughout the study.
“A Well-wisher sacrifices, works with dedication but never burdens
theirself”. They may lead but every moment sees that the individual concerned is in the
front. I would also like to convey my overwhelming gratitude to especially my senior
Pankajavalli. T who supporting me at every moments. And also I would like to thank lab
seniors Pratheepa. D, Kalaiselvi. R and Preethi M.P. to their selfless help and their
motivation in my all stages.
“A friend is one that knows you as you are, understands where you have
been, accepts what you have become, and still, gently allow you to grow”. Friends are
ones whom we know well and are fond of, intimate associate, close acquaintance, a
supporter and sympathizer, someone who always do good for us. A special word of thanks
to my friends Uheetha Banu.N and Sangeetha.U 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, Palaniappan and Mani who have sacrificed many things in
their life for me, expecting nothing in return since no great work can be done without
sacrifice.
I dedicate my work to my parents, sisters, brothers and my class friends.
Their mental and emotional support, motivation, prayers and loving care provided has
been the source of my strength.
SARANYA.P
CONTENTS
CONTENTS
CHAPTER
NO.
TITLE PAGE NO.
List of Tables
List of Plates
1 Introduction 1
2 Review of Literature 4
3 Methodology 20
4 Results And Discussion 25
5 Summary And Conclusion 42
6 Bibliography 43
.
LIST OF TABLES
TABLE
NO
TITLE PAGE
NO
1 Biological Importance of each part of Withania somnifera 8
2 Secondary metabolites accumulation of UV treated root and
plantlet
40
LIST OF PLATES
PLATE NO TITLE PAGE NO
1 Mass multiplication 27
2 UV Treated in vitro roots 27
3 Micropropagation and maintenance of aerial parts 28
4 UV treated in vitro plantlets 28
5 Screening to detect the presence of Withanolide A on in vitro UV-
treated roots 33
6 Peak area for control and UV treated in vitro root 33
7 Screening to detect the presence of Withanolide A on in vitro UV-
treated Plantlets 34
8 Peak area for control and UV treated in vitro plantlet 34
9 Regression graph for Withanolide A in UV treated in vitro root and
plantlet 35
10 Withanolide A accumulation on In vitro UV treated root and plantlet
of Withania somnifera 35
11 Screening to detect the presence of Withaferin A on in vitro UV-
treated roots 37
12 Peak area for control and UV treated in vitro root 37
13 Screening to detect the presence of Withaferin A on in vitro UV-
treated Plantlets 38
14 Peak area for control and UV treated in vitro plantlet 38
15 Regression graph for Withaferin A in UV treated in vitro root and
plantlet 39
16 Withaferin A accumulation on In vitro UV treated root and plantlet
of Withania somnifera 39
INTRODUCTION
1.0 INTRODUCTION
Ayurveda the 'science of longevity' stems philosophically from the
Sankhya and Vedanta. Ayurveda has been a lively system of health care in India with an
unbroken practice since 6000 years but growth as an industry has commenced only a few
years back (Supriya et al., 2011). Various compounds produced by plants can be broadly
grouped into two categories namely, “Primary Metabolites” and “Secondary Metabolites”.
The secondary metabolites are also referred to as “Natural Products” (Bowles et al., 1994).
Plant secondary metabolites are of tremendous importance, both for the plant itself (for
plant–environment interactions) and to humans, for their biological activities that can have
therapeutic value. Secondary metabolites have been used for centuries in traditional
medicine. Nowadays, they correspond to valuable compounds such as pharmaceuticals,
cosmetics, fine chemicals or more recently, nutraceutics (Jha et al., 2001).
Various technologies have been adopted for enhancing bioactive molecules in
medicinal pants. Large-scale use of plant tissue culture is found to be attractive alternative
approach to traditional methods of plantation as it offers a controlled supply of
biochemicals independent of plant availability (Khan et al., 2009). The use of plant cell
and tissue culture methodology as a means of producing medicinal metabolites has a long
history (Rout et al., 2000). Owing to its potential, researchers have endeavored to utilize
plant cell biosynthetic capabilities for obtaining useful products and for studying the
metabolism (Matkowski, 2008). The advantage of this method is that it can ultimately
provide a continuous, reliable source of natural products (secondary metabolites). The
major advantage of the cell cultures include synthesis of bioactive secondary metabolites,
running in controlled environment, independently from climate and soil conditions
(Karuppusamy, 2009).
Plants have been an important source of medicine for thousands of years. Even
today, the World Health Organization estimates that up to 80 per cent of people rely
mainly on traditional remedies such as herbs for their medicines. The definition of
Medicinal Plant has been formulated by WHO as "Any plant which, in one or more of its
organ, contains substance that can be used for therapeutic purpose or which is a precursor
for synthesis of useful drugs” (Sofowora, 1982). Withania somnifera Dunal (Solanaceae),
commonly called Ashwagandha, (Sanskrit) is one of an Ayurvedic medicinal plant
(Archana and Namasivayam, 1999). In Withania genus three species are found in India
namely, W.somnifera, W.coagulans and W.obtusifolia it is distributed in tropical and
subtropical region (Kumar et al., 2011). All major parts of Withania somnifera such as the
roots, fruits and leaves provide potential benefits for human health (Alam et al., 2011). It
has been recommended for the treatment of various ailments which include polyarthritis,
rheumatoid arthritis, lumbago, painful swellings, spermatorrhoea, asthma, leucoderma,
general debility, sexual debility, (Ali, 1997) scabies, ulcers marasmus and leucorrhoea
(Uddin et al., 2012) .
The biologically active chemical constituents are alkaloids (ashwagandhine,
cuswhygrine, anahygrine, tropine, etc), steroidal compounds, including ergostane type
steroidal lactones, withaferin A, withanolides A-Y, withasomniferin-A, withanone, etc
(Senthil et al., 2011). Withanolides are biologically active secondary metabolites present in
roots and leaves of W.somnifera. The biological activities of withanolides, especially of the
dominant Withanolide A and Withaferin A. They are having anti-cancerous activity
(Sumithradevi et al., 2011). This popular medicinal herb of Ayurveda is used for health,
vitality, longevity and rejuvenation properties (Ojha and Arya, 2009). It possesses
antioxidant, antitumor, antistress, anti-inflammatory, immunomodulatory, hematopoetic,
anti-ageing, anxiolytic, ant depressive rejuvenating properties and also influences various
neurotransmitter receptors in the central nervous system. In recent studies done on human
breast, lung and colon cancer cell lines (Sharma et al., 2011). Withania extract
administration was found to increase the haemoglobin level, RBCs and decrease serum
cholestrol, ESR etc (Davis and Kuttan, 1999).
Based on the importance of this plant; several studies have been reported on
the therapeutic application of Withanolide A and Withaferin A in Withania somnifera. To
till date not much information is available about the in vitro root cultures as potential
source of secondary metabolites from this plant. In an attempt to increase the secondary
metabolites accumulation on in vitro root and plantlet of Withania somnifera.
In the present study, with the above information discuss about changes of
secondary metabolites accumulation on in vitro adventitious root and plantlet of Withania
somnifera by UV radiation. Secondary metabolites accumulation was characterized and
quantified by HPTLC analysis.
REVIEW OF LITERATURE
2.0 REVIEW OF LITERATURE
Withania somnifera, commonly known as Ashwagandha, is a valued herb in
Ayurvedic medicine. W.somnifera mainly contains withanolides which are specific to the
Solanaceae family. Withanolides are biologically active secondary metabolites present in
roots and leaves of W.somnifera. The major withanolides, Withanolide A and Withaferin A
which are present on in vitro root and plantlet of Withania somnifera. In the present study,
we have entitled about “Effect of ultra violet radiation on in vitro roots and leaves of
Withania somnifera and its influence on major secondary metabolites accumulation”. This
study involves a detailed investigation about the accumulation of secondary metabolites in
the in vitro roots and plantlets of Withania somnifera quantitatively by HPTLC analysis.
Medicinal Plants
Medicinal plants are plants that have a recognized medical use. The medicinal
plants are widely used by the traditional medical practitioners for curing various diseases
in their day to day practice. In traditional systems of medicine, different parts (leaves,
stem, flower, root, seeds, bark and even whole plant) of Withania somnifera (Verma et al.,
2011). They range from those used in the production of mainstream pharmaceutical
products to plants used in herbal medicine preparations. Plants that have medical uses can
be found growing in many settings all over the world. Ashwagandha [Withania
somnifera L. Dunal] (Solanaceae) is an important medicinal plant, commonly-used as a
domestic remedy for several diseases in India as well as other parts of the world .Withania
somnifera is a medicinal plant extends over a large area, from the Atlantic ocean to South
East Asia and from the Mediterranean region to South Africa .
Withania somnifera is mentioned in Indian systems of medicine as a herbal
tonic and health food. It is an official drug and is mentioned in the Indian
pharmacopoeia. Medicinal plants have been explored for treatment of numerous health
problems ( Kulkarni et al., 2008). It has been used as a tonic and antistress supplement.
Pharmacological activities include physiologic and metabolic restoration, antiarthritic,
antiaging, cognitive function improvement in geriatric states, and recovery from
neurodegenerative disorders (Bhattacharaya et al., 2002).
It is a popular medicinal herb of Ayurveda is used for health, vitality,
longevity and rejuvenation properties. It is a common ingredient of polyherbal or
herbomineral formulations and used for preventive or therapeutic polypharmaceutical use.
Kumar and Arya, (2009) investigated the pharmacological properties and its use in
cardiovascular diseases.
Various alkaloids, withanolides and sitoindosides have been isolated from this
plant. Among the various withanolides reported withaferin A and withanone are customary
major withanolides of the plant whereas the amount of withanolide A is usually very low
(Zhao et al., 2002). Recently, withanolide A has attracted interest due to its strong
neuropharmacological properties of promoting out growth and synaptic reconstruction
(Kuboyama et al., 2005). Withanolide A is an important candidate for the therapeutic
treatment of neurodegenerative diseases, like Alzheimer‟s disease, Parkinson‟s disease,
convulsions, cognitive function impairment, as it is able to reconstruct neural networks
(Tohda et al., 2005).
The chemistry of Withania somnifera has been extensively studied and over
35 chemical constituents have been identified, extracted, and isolated. The biologically
active chemical constituents are alkaloids (isopelletierine, anaferine), steroidal lactones
(withanolides, withaferins), saponins containing an additional acyl group (sitoindoside VII
and VIII), and withanolides with a glucose at carbon 27 (sitoindoside IX and X). Withania
somnifera is also rich in iron (Mishra et al., 2000).
The topic “Effect of ultra violet radiation on in vitro roots and leaves of
Withania somnifera and its influence on major secondary metabolites accumulation”
has been reviewed under following headings
2.1Withania somnifera – secondary metabolites
2.2 Importance of plant tissue culture
2.3 Micropropagation and In vitro root induction
2.4 Physical stressors
2.5. Chromatographic analysis
2.1. Withania somnifera – secondary metabolites
Withania somnifera, popularly known as Ashwagandha, Indian ginseng, or winter
cherry has been used for millennia in Ayurveda, Indian system of traditional medicine
Ojha and Arya, (2009). It is a small, woody shrub in the Solanaceae family that grows
about two feet in height. It can be found growing in Africa, the Mediterranean, and India.
An erect, evergreen, tomentose shrub, 30-150 cm high, found throughout the drier parts of
India in waste places and on bunds. Roots are stout fleshy, whitish brown; leaves simple
ovate, glabrous, those in the floral region smaller and opposite; flowers inconspicuous,
greenish or lubrid-yellow, in axillary, umbellate cymes; berries small, globose, orange-red
when mature, enclosed in the persistent calyx; seeds yellow, reniform.The roots are the
main portions of the plant used therapeutically. The bright red fruit is harvested in the late
fall and seeds are dried for planting in the following spring. Whole plant, roots, leaves,
stem, green berries, fruits, seeds, bark are used (Gupta et al., 2007). Different parts of
Withania somnifera have shown a significant importance since long time, as a medicinal
remedy for many diseases. Jamal et al., (1995) have revealed that Withania somnifera is a
rich source of interesting new withanolides with potentially useful biological activities.
Alam et al., (2011) revealed a great deal of evidence indicating that excessive free
radical production and lipid peroxidations are actively-involved in the pathogenesis of a
wide number of chronic diseases, including atherosclerosis, cardiac and cerebral ischemia,
neurodegenerative disorders , carcinogenesis , diabetes and rheumatic disorders and
contributes a major role in the ageing process. Plant-derived antioxidants such as vitamin
E, vitamin C, and polyphenols including phenolic acids, phenolic diterpenes, flavonoids,
catechins, procyanidins and anthocyanins are becoming increasingly important as dietary
factors. Plant acids are known to have anticarcinogenic activity and phenolic compounds
are believed to be an important part of the general defense mechanism of many plants
against infections. Therefore, it is useful to measure the presence of phenolic compounds
in natural substances. Plant products are frequently considered to be less toxic and more
free from side effects than synthetic ones (Singh et al., 2010).
Taxonomical Classification of Withania somnifera
Kingdom: Plantae
Order: Solanales
Family: Solanaceae
Genus: Withania
Species: somnifera
Binomial name : Withania somnifera
Vernacular Names
Arabic : Kaknaj-e-Hindi
Bengali : Ashvaganda, Asvagandha
English : Winter cherry
Gujarati : Asan, Asana, Asoda, Asundha,
Ghodaasoda
Hindi : Asgandh, Punir
Malayalam : Amukkiram, Pevetti
Marathi : Askandha, Kanchuki, Tilli
Odiya : Asugandha
Persian : Kaknaj-e-Hindi, Asgand Nagaori
Sanskrit : Ashvagandha,Ashvakandika,
Gandhapatri, Palashaparni
Tamil : Amukkira, Asubam, Asuvagandi
Telugu : Asvagandhi, Penneru, Pennerugadda,
Dommadolu
Urdu : Asgand, Asgand Nagori(Anonymous, 2007).
The main constituents of Ashwagandha are alkaloids and steroidal lactones
which are commonly called withanolides. The withanolides have C28 steroidal nucleus
with C9 side chain having six membered lactone ring (Padmavathi et al., 2005). Among
the various alkaloids, withanine is the main constituent. The other alkaloids are
somniferine, somnine, somniferinine, withananine, pseudo-withanine, tropine,
pseudotropine, cuscohygrine, anferine and anhydrine.
The roots of the plant are categorized as rasayanas, which are reputed to promote
health and longevity by augmenting defense against disease, arresting the ageing process,
revitalizing the body in debilitated conditions, increasing the capability of the individual to
resist adverse environmental factors and by creating a sense of mental well being. The
biological activities of withanolides, especially of the dominant withanolide A and
withaferin A, have been studied extensively and, more recently, have been shown to have
anti-cancerous activity. (Nadia et al., 2011). High performance thin layer chromatography
(HPTLC) analysis of secondary metabolites viz. withanolides, withaferin-A and total
alkaloids of the diseased leaves vis-à-vis control revealed reduction in withaferin-A and
withanolides contents by 15.4% and 76.3% respectively (Pati et al., 2008).
Table 1. Biological Importance of each part of Withania somnifera
S.No Part of the
Plant
Biological Activity Reported
1 Leave Anti-inflammatory, anti-helminthic, antibiotic, anti-pyretic,
protective against hepatotoxicity and syphilitic sores.
2 Fruit Diuretic, anti-tumor, anti-inflammatory and used for the
treatment of carbuncle, tubercular glands and ulcers.
3 Seeds Hypnotic, coagulant, diuretic and maslicatory.
4 Tuber Anti-bronchitic, anti-psoriatic, anti-ulcer, anti-
inflammatory, anti-scabielic and anti-helminthic.
5 Root Anti-rheumatic , nutritive, health restorative protective
against cold, chill , loss of memory, dyspepsia, nervous
exhaustion and hypertension.
(Jamal et al., 1995).
Plants synthesize a bewildering array of chemical compounds with a variety of
physiological roles, starting from air, water, minerals and sunlight as the energy source.
Various compounds produced by plants can be broadly grouped into two categories
namely, “Primary Metabolites” and “Secondary Metabolites”. The secondary metabolites
are also referred to as “Natural Products”. It is believed that more than 100,000 different
structures of secondary metabolites maybe synthesized by organisms, to a tune of 109 tons
per year (Bowles and Leyser , 1994). Out of these, more than 80% are found in plants
(Harborne, 1993). They are used either as medicines/pharmaceuticals, foods,
neutraceuticals (foods as well as medicines used for preventive and curative treatments),
flavors, colors, spices or fragrances by humans while in plants they constitute a chemical
response to pollinators and distributors, to competitors and herbivores, to symbionts and
pathogens and to stress.
Plant cell and organ cultures are promising technologies to obtain 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 (Ramachandra et al., 2002). Recently, Sharadha et al., (2002) have reported the
presence of withaferin A in the shoots and withanolide D in the roots of tissue culture
regenerated plants.
Structures of the main active compounds isolated from the roots and leaves of
Withania somnifera (Rekha et al., 2006).
The root extract of Withania somnifera has been shown to have health promoting
effects such as anti-stress, anti-arthritic, anti-inflammatory, analgesic, anti-pyretic, anti-
oxidant and immunomodulatory properties (Agarwal et al., 1998). Methanolic root extracts
of Withania somnifera includes a variety of withanolides and was shown to induce nitric
oxide synthase expression that could account, at least in part, for its immunostimulant
properties (Iuvone et al., 2003). Similar extracts protected rats against hepatic, renal and
skin pathology induced by fungicide (carbendazim) and DMBA (dimethyl benzanthracene)
(Akbarsha et al., 2000). They are also found to induce growth of human neuronal cells in
culture (Kuboyama et al., 2002 2005). Only a few studies have investigated the effects of
leaf extract of Withania somnifera. Similar to the root extracts, major components of the
methanol extract of leaf are withanolides. These are structurally diverse steroidal lactones
and are suggested to have anti-cancer, anti-oxidative and anti-mutagenic properties (Devi
et al.,1996).
2.1.2 Biosynthetic pathway of secondary metabolites (withanolide A and withaferin
A) (Sangwan et al., 2008).
Withanolide A is an important secondary metabolite in Withania somnifera,
which is having a high medicinal value and possesses potent anti-tumor and antioxidant
properties. Distribution of withanolide A in various organs of Withania somnifera was
investigated. The content of withanolide A gradually decreased from aerial parts i.e., from
young leaves to the root. In root, the root tip accumulated higher concentration when
compared to middle and basal portion (Praveen et al., 2010).
A large number of withanolides have been isolated from different species of
Withania Physalis/and Datura. Some of these were found to have antitumor, cytotoxic and
antimicrobial activities (Kaur et al., 2004).
Withanolide A
Tohda et al., (2005) concluded Withanolide A is important candidate for the
therapeutic treatment of neurodegenerative diseases, like Alzheimer‟s disease, Parkinson‟s
disease, convulsions, cognitive function impairment, as it is able to reconstruct neural
networks.
Withaferin A
Kaur et al., (2004) reported the leaf extracts of Ashwagandha (Lash) for
anti-genotoxicity in Allium cepa root tip cells and found that it confers substantial
protection against the MNNG-induced genotoxicity.The withanolides are steroidal
lactones, one of which, withaferin-A, was isolated for the first time from the leaves of
Withania somnifera in 1956.
Mirjalili et al., (2009) elucidated the structure of withaferin A in leaves of this
plant, which is mainly valued for its anti-cancerous properties. The yields of withaferin A
from intact plants of Withania somnifera are 0.2-0.3% of DW of leaves and withaferin A
is totally absent in roots, stems, seeds and persistent calyx of fruits of intact plants but
present in leaves (1.6%).
2.2. Importance of plant tissue culture
Environmental stimuli are used as cues to rapidly modify and
influence developmental programming to provide flexibility in response throughout the
growth cycle of plant. The entire technology of “Plant Tissue Culture” is based on this
ability of plant cells to be influenced by their surroundings and differentiate to give rise to
a range of organs dependent on the culture conditions (Bowles and Leyser, 1994). “Plant
Tissue Culture” is the maintenance and propagation of plant parts, as small as a single cell,
in axenic culture under controlled environmental conditions (Pauls, 2000). Plant tissue
culture techniques are extensively used for mass production of elite plants as well as to
study the basic aspects of primary and secondary metabolism, morphogenesis and genetic
engineering (George and Sherrington, 1984). Plant tissue culture work was initiated with
the aim to micro propagate selected superior chemo types from various explants and to
assess for the possible presence of Withaferin -A in tissue culture raised plants, callus
cultures and hairy root cultures (Kulkarni et al., 2000). Tissue culture methods offer an
alternative means of plant vegetative propagation. Clonal propagation through tissue
culture popularly called micro propagation can be achieved in a short time and space. Use
of plant tissue culture for micro propagation was initiated by and found commercially
viable approach for orchid propagation (Morrel, 1960). Nagella and Murthy, (2010)
established that cell suspension cultures of Withania somnifera for the production of
withanolide A. The productivity of withanolide A was found to be dependent on type and
concentration of culture medium, growth regulators and carbohydrate sources.
Suspension cultures of Withania somnifera cells were established and
shown to produce Withaferin A. Banerjee et al. (1994) reported the hairy root cultures of
Withania somnifera able to produce Withaferin A.
2.3.Micropropagation and Adventitious root induction
Tissue culture, also known as microprpropagation, is a propagation method used to
produce plants under sterile condition. Micropropagation is the practice of rapidly
multiplying stock plant material to produce a large number of progeny plants, using
modern plant tissue culture methods. Micropropagation is the growing of plants from
meristematic tissue or somatic cells of superior plants on nutrient suitable media under
controlled aseptic physical conditions.
It is used to multiply novel plants, such as those that have been genetically
modified or bred through conventional plant breeding methods. It is also used to provide a
sufficient number of plantlets for planting from a stock plant. Within a production period
less than 4 months, a commercial laboratory can obtain as many as one million plantlets of
boston fern per year starting from a single explant.
An efficient method of in vitro shoot propagation of six elite accessions of
Withania somnifera collected. Maximum numbers of shoots in all accessions were
achieved from axillary explant on Murashige and Skoog (MS) medium supplemented with
1 mgL−1
BAP and 1 mgL−1
kinetin. The highest number of shoots (60 ± 1.82 and 60.05 ±
2.03) was observed. Stable production of withanolides from in vitro regenerated shoots
was comparable to the yields from field grown mother plants, indicating the in vitro
methodology could be used successfully for the true-to-type plant regeneration of Withania
somnifera accessions (Sabir et al., 2008).
Tissue cultures were established from axillary meristems of the plant Withania
somnifera. Growth hormones influenced the morphogenetic responses of the cultures. The
explants gave multiple shoots in Murashige and Skoog‟s (MS) medium supplemented with
benzyladenine (BA) (Roja and Heble, 1991).
Sterile plants were achieved in vitro from a shoot-tip of aseptically germinated
seedlings using MS medium. Existing meristems (shoot-tips or axillary buds) were used
for multiple shoot development and rooting on Murashige and Skoog-MS (15) and Nitsch
and Nitsch-NN media. Different plant growth regulators such as 2,4-D + BA, IAA + BA,
IBA + BA were used. Light regime was: 12 h light and 12 h dark (Furmanowa et al.,
2000).
Dewir et al., (2010) introduced a protocol for indirect shoot organogenesis and
plantlets regeneration of Withania somnifera. Leaf explants were cultured on Murashige
and Skoog (MS) medium supplemented with different concentrations and combinations of
6-benzylaminopurine (BAP) and indole-3-acetic acid (IAA). The highest callus induction
rate (89.5 %) and shoot regeneration rate (92 %) were obtained when 2 mg dm-3 BAP was
combined with 0.5 mg dm-3 IAA. Three major withanolides (withaferine A, 12-
deoxywithastramonolide and withanolide A) were investigated. . Leaves contained higher
contents of withanolides and phenolics than roots or stems.
Adventitious root are roots in an unusual place, that originates from stem or leaf
tissue rather than from another root, often where a branch or other part contacts soil or
damp material. Adventitious roots are not ordinarily expected, and often they are the result
of stress or injury. A plant's normal growth comes from meristematic tissue, but
adventitious growth comes from non-meristematic tissue. Adventitious roots are indeed
very common in vascular plants.
Thomas et al., (2006) reported that, for root induction, excised shoots with three or
more leaves were transferred to MS basal medium fortified with IAA (0.2 - 0.6 mg/l) and
IBA (0.2 - 0.6 mg/l).
Root growth does not always occur in the earlier stages in plant cell culture, and is
of course a requirement for successful plant growth after the micro propagation. It is
performed in vitro by transferring the plantlets to a growth medium containing auxin(s).
Various concentrations and combinations of IAA, IBA and NAA were added in the
medium. The pH of the medium adjusted to 5.6 to 5.8 before sterilization. It is well
established that root development is controlled by hormonal signals, especially auxins
(Wadegaonkar et al., 2006).
Praveen and Murthy, (2010) reported that Withanolides are biologically active
secondary metabolites present in roots and leaves of Withania somnifera. Adventitious
roots were induced directly from leaf segments of Withania somnifera on half strength
Murashige and Skoog (MS) semisolid medium (0.8% agar) with 0.5 mg l-1 indole-3-
butyric acid (IBA) and 30 g l-1 sucrose. Adventitious roots cultured in flasks using half
strength MS liquid medium with 0.5 mg l-1 IBA and 30 g l-1 showed higher accumulation
of biomass (108.48 g l-1FW and 10.76 g l-1 DW) and withanolide-A content (8.8 ± 0.20
mg g-1 DW) within five weeks.
The healthy leaf explants from the plantlets maintained in MS basal medium were
inoculated in adventitious root induction medium (MS basal medium supplemented with
0.5 mg/l IAA and 2.0 mg/l IBA) under 16hr photoperiod (Neha et al., 2009).
2.4. Effect of ultraviolet radiations
Plants use sunlight for photosynthesis and, as a consequence, are exposed to the
ultraviolet (UV) radiation that is present in sunlight. UV radiation is generally divided into
three classes: UV-C, UV-B, and UV-A (Ann and Stapleton, 1992).UV-B radiation can
activate the self-protective secondary metabolism system. (Gu et al., 2010) found the
method to induce bioactive secondary metabolites from mulberry leaves (Morus alba L.)
by UV-B radiation in vitro.
Ktitorova et al., (2005) investigated that in 95% of irradiated roots, the length
increment was between 0.3 and 1.7 mm by UV radiation.
Diagram showing UV-B-induced changes in leaf and plant morphology. Part (a) is the
control; part (b) is a plant exposed to supplementary UV-B (Marcel et al., 1998).
Sarghein et al., (2011) investigated about UV-A,B and C radiation in pepper plants
(Capsicum longum A.DC.). It was exposed to UV radiation under greenhouse conditions.
Leaf area also decreased in UV-R-exposed plants and decreased significantly in UV-C-
exposed plants. Root thickness was not affected by UV treatment, but stem and leaf
thickness significantly increased in response to UV-A and UV-C treatment. It was different
to my study with in vivo growth study.
2.5. HPTLC analysis
A simple, sensitive and accurate high performance thin layer chromatographic
(HPTLC) method has been developed for the estimation of withaferin-A and withanolide-
A in different plant parts such as, leaf, root, stem and fruit of two morphotypes of Withania
somnifera. HPTLC of Withania 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. Quantitative evaluation of the plate was performed in the absorption-reflection
mode at 530 nm. The method was validated for precision, repeatability, and accuracy. The
average recovery of withaferin-A and withanolide-A in two levels were 96.0 and 96.7%,
showing the excellent reproducibility of the method (Sharma et al., 2007).
Praveen et al., (2010) reported that a new method of identification of withaferin A,
using a destructive reagent, and an HPTLC method for quantification of the compound.
Chromatography on silica with toluene–ethyl acetate–acetone 2:3:3 as mobile phase
enabled good resolution of withaferin A without interference from other compounds
present withania somnifera. After spraying with anisaldehyde–sulfuric acid reagent and
heating for 15 min at 105°C, characteristic orange fluorescence was observed for
withaferin A only among all the spots resolved.
Jigar et al., (2009) reported a new method of identification of withaferin A, using a
destructive reagent, and an HPTLC method for quantification of the compound.
Chromatography on silica with toluene–ethyl acetate–acetone 2:3:3 as mobile phase
enabled good resolution of withaferin A without interference from other compounds
present in Withania somnifera. After spraying with anisaldehyde–sulfuric acid reagent and
heating for 15 min at 105°C, characteristic orange fluorescence was observed for
withaferin A only among all the spots resolved. When scanned at 214 nm the RF of
withaferin A was 0.62. Interestingly, old root did not contain withaferin A whereas young
root contained a large amount. The method was validated for accuracy, precision,
specificity, linearity, and limits of detection and quantification.
MATERIALS AND METHODS
3.0 MATERIALS AND METHODS
The study discussed about “Effect of ultra violet radiation on in vitro roots
and leaves of Withania somnifera and its influence on major secondary metabolites
accumulation” has various materials and methods which are described under the following
headings
3.1. Material
3.1.1. Plant material
3.1.2. Chemicals
3.2. Methods
3.2.1 In vitro culture of Withania somnifera plantlet
a. Induction of adventitious roots
b. Mass multiplication
c. Micropropagation and maintenance of aerial parts
d. UV- treatment for roots and plantlets
3.2.2. Extraction of sample
3.2.3. HPTLC analysis
3.1.1. Plant Material
One month old in vitro plantlets maintained in MS basal medium and one month
old in vitro adventitious roots were used for this study.
3.1.2. Chemicals
a) HIMEDIA chemicals were used for this study.
b) Toluene (LR), Ethyl acetate (LR), Formic acid (LR) and Hexane (LR)-
solvents were used.
c) Withania Standards - Withanolide A, Withaferin A from (Chromodex,
USA).
d) Pre-coated silica gel plates 60 F254 for TLC (Merck, Germany).
3.2. METHODS
3.2.1 In vitro culture of Withania somnifera plantlet
a. Induction of adventitious roots
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)
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 were dipped in 70% ethanol and flamed, cooled and used
for inoculation. For root induction studies, the leaves from MS0 grown plantlets were used
and inoculated on medium containing 1 mg/L IBA and 0.25 mg/L IAA. The inoculated
explants were incubated at 25 0C for 16hrs photoperiod for one month.
b. Mass multiplication in suspension
The root tips and branches from in vitro induced adventitious roots were cultured
in liquid MS basal media (suspension). Media change was given once every 15 days. After
30 days of regular sub culturing, the well grown root was subjected to treatment.
c. Micropropagation and maintenance of aerial parts
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) 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 were dipped in 70% ethanol and flamed,
cooled and used for inoculation. For micropropagation studies the shoot tips (or shoots,
meristem) of Withania somnifera were inoculated on the MS media. By this method plant
yield was more.
d. UV- treatment for roots and plants
3g of one month old in vitro root was used for the treatment. 100ml of suspension
medium was added into each sterilized conical flask. Consequently, 3gms of roots were
inoculated into the media aseptically; these were performed under aseptic condition in a
Laminar Air Flow chamber. After inoculation, root was exposed to UV at 1.35µE/(m²/s) in
different time intervals- 4hrs, 8hrs, 12hrs, 24hrs & 48hrs. After exposure, the treated roots
were sub cultured into 100ml MS suspension media. Control roots were not exposed to
UV. After one week control and treated roots were harvested. The wet weight was noted
and allowed to dry completely, dry weight was also noted.
For UV treatment on leaves, instead of root, the whole MSO grown plantlet was
exposed to UV. The time interval was same as root. The control plant was not exposed to
UV. After exposure, the treated plantlets were sub cultured in MS0 basal medium. After
one week, control and treated plantlets were harvested. Fresh weight was calculated and
allowed to dry, dry weight was also noted.
3.2.2 Extraction of secondary metabolites
The treated and control roots of Withania somnifera (Ashwagandha) were taken for
extraction. These roots were ground to powder. 0.3ml of ammonia was added to 0.3g of the
powder. It gives basicity to the root followed by sonication for 20 minutes to disperse the
cells. Root was extracted four times with 60ml of ethyl acetate (4 × 15ml) (Jigar et al.,
2009). After each extraction, the extract was filtered off using Whatmann No: 1 filter paper
and the residue were allowed to interact with another 15ml of ethyl acetate for overnight.
The same procedure was followed till the completion of fourth extraction. The entire
extraction was carried out in shaker, maintained in 220C. All the four extracts were
combined and evaporated to dryness under vacuum using flash evaporator. The residue
was dissolved in 1.8ml of HPLC grade methanol concentrated extracts were used for
HPTLC analysis.
For leaf extraction, treated and control leaves were taken. The leaves were
ground to powder. Chlorophyll content was removed from the leaf by using hexane (Gupta
and Rana, 2007). After adding hexane, it was kept for overnight in shaker. The next day it
was filtered using wattmanno.1 filter paper. The final residue was allowed to dry. This dry
powder was taken for extraction. Ammonia was added to the leaf powder. It gives basisity
to leaf followed by sonication for 20 minutes to disperse the leaf cells. Extraction
procedure was carried out similar that of root extraction procedure.
3.2.2 Extraction of secondary metabolites from in vitro roots and plantlets
Kept in overnight in
shaker and filtered
Sonication for 20minutes, kept 2hrs in shaker
.
Followed by filtration, repeated 4times with
ethyl acetate
Residue was dissolved in HPLC grade
methanol
In vitro UV treated root
powder
In vitro UV treated plantlet
powder
Chlorophyll was removed
by hexane
Filtered residue was taken
To 0.3g of the powder
300µl of ammonia was
added
Extracted with 15ml of
ethyl acetate
Four filtered extracts were pooled and
flash evaporated
Incubated in shaker for 2hrs
Concentrated extracts were used for
HPTLC analysis
3.2.3 HPTLC analysis
HPTLC analysis was carried out in order to view the secondary metabolite
accumulation in the in vivo root and in vitro root. This was done on precoated silica gel
aluminum plate 60F254. The UV treated and control methanolic root extracts were applied
to the plates as 8mm bands, under a stream of nitrogen, by means of a CAMAG Linomat V
semiautomatic sample applicator fitted with a 100µl Hamilton HPTLC syringe. Linear
ascending development to a distance of 9 cm was carried out on twin trough chamber
saturated the mobile phase, Toluene: Ethyl Acetate: Formic acid (5: 5: 1). After run, the
plates were removed from the chamber and air dried and visualized at 254 and 366 nm.
The plate was then subjected to scanning. It was performed with Camag TLC scanner III
in the reflectance – absorbance mode at 223 (or) 234nm [withaferin (or) withanolide]
according to the presence of secondary metabolite. Then, the plates were derivatized using
concentrated sulfuric acid: methanol: glacial acetic acid: anisaldehyde in the ratio of
5:85:10:0.5 and kept in hot-air oven for 10 minutes at 110°C, for the development of spots.
The plate was visualized at 254 nm, 366 nm and white using CAMAG TLC visualiser and
each compound were recorded (Jirge et al., 2011).
RESULTS AND DISCUSSION
4.0 RESULTS AND DISCUSSION
The results of the present study entitled “Effect of ultra violet radiation on
in vitro roots and leaves of Withania somnifera and its influence on major secondary
metabolites accumulation” are presented and discussed in this chapter.
4.1 Induction of adventitious root
4.2 Mass multiplication
4.3 Micropropagation and maintenance of Arial parts
4.4 UV-treatment for in vitro roots and plantlets
4.5 Extraction of secondary metabolites
4.6 HPTLC analysis
4.1 Induction of adventitious root
Adventitious roots were induced directly from healthy leaf segments of Withania
somnifera on Murashige and Skoog (MS) semisolid medium (0.8% agar) with 1.0mg/l of
Indole-3-butyric acid (IBA), 0.25mg/l of Indole acetic acid (IAA) and 3% sucrose under
16hr photoperiod for 1month. After one month the adventitious roots were developed from
leaves. These roots were inoculated into hormone free MS suspension medium with
3%sucrose. The cultures were maintained at 22℃ at 80-90 rpm. My study also similar to
(M.Sc thesis Uma maheswari, 2010) study. In her study, earlier MS media supplemented
with 1mg/l IBA, 0.25mg/l IAA and 3% sucrose was standardized for root induction (M.Sc
thesis Uma maheswari, 2010). This hormone combination was suitable media for highest
percentage of root induction. So roots grown on this combination were taken for my study.
Neha et al., (2009) established root induction in the Jawahar variety of Withania
somnifera using MS media supplemented with 0.5(mg/l) IAA and 2 (mg/l) IBA.
Thomas et al., (2006) reported that, for root induction, excised shoots with three or
more leaves were transferred to MS basal medium fortified with IAA (0.2 - 0.6 mg/l) and
IBA (0.2 - 0.6 mg/l).
Wadegaonkar et al. ,(2006) has reported that direct rooting from leaf explants of
Withania somnifera on half strength Murashige and Skoog‟s medium supplemented with
15 gl-1
sucrose with 2.85 µM indoleacetic acid and 9.85 µM indolebutyric acid achieved
maximum number of roots with 100% response.
Earlier in our laboratory adventitious root induction studies was standardized by
inoculating the leaf explants in MS medium supplemented with 1mg/l IBA and 0.25mg/l
IAA along with 3% sucrose. The same procedure was followed for the present study. The
established roots were subjected to suspension culture (Full strength MS medium with 3%
sucrose) for the mass multiplication. Nagella and Murthy, (2010) reported root induction
by inoculating the leaf explants on full strength MS medium supplemented with 2,4-D and
KN and 3% (w/v) sucrose. 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. Similar to his report, our results also suggested that maximum root induction
can be obtained by using the combination of IAA and IBA.
Vanisree et al., (2004) revealed that cell suspension culture is preferred for large
scale production due to its rapid growth cycles. Thus cell suspensions are used for large
amount of roots transferred from MS medium with growth responses and metabolism of
novel chemicals.
4.2 Mass multiplication
Plate 1.Withania somnifera In vitro Withania somnifera In vivo
Plate 2. UV Treated in vitro roots
UV-4hrs UV-8hrs UV-12hrs
UV-24hrs UV-48hrs
4.3 Micropropagation and maintenance of aerial parts
Plate 3. Withania somnifera In vitro Withania somnifera In vivo
Plate4.UV
Treated in vitro plantlets
UV-4hrs UV-8hrs UV-12hrs
UV-24hrs UV-48hrs
Micropropagation is the practice of rapidly multiplying stock plant
material to produce a large number of progeny plants, using modern plant tissue
culture methods. A tissue culture technique for plant propagation in which offspring are
cloned from tissue taken from a single plant.
The healthy shoot tips were inoculated in MS medium (hormone free media) under
sterile condition. Shoots with 2-3nodes were inoculated in MS media supplemented with 1
mg/l BAP and 0.5mg/l Kinetin under sterile condition for elongation of shoots and for
maintenance of aerial parts. This combination provides maximum shoots within short
period. The well grown plants from MS0 media was taken for the study. Siddique et al.,
(2004) reported the combination of BAP and kinetin provides maximum shoot
proliferation. Similar to their study maximum shoot proliferation was obtained.
Sabir et al., (2008) achieved highest number of shoots (60 ± 1.82 and 60.05 ± 2.03)
from axillary explant on Murashige and Skoog (MS) medium supplemented with 1 mgL−1
BAP and 1 mgL−1
kinetin. Supe et al., (2006) revealed that Withania somnifera seed was
inoculated in MS supplemented with BAP at 0.6 mg/l with 0.4 mg/l IAA was found to be
most effective in initiating multiple shoots at the rate of ten per explant.
4.4 UV-treatment for in vitro roots and plantlets
One month old in vitro adventitious roots were taken for the study. 1.0g of root was
inoculated into 100ml of suspension media. The roots were exposed to UV at
1.35µE/(m²/s) in different time intervals 4hrs, 8hrs, 12hrs, 24hrs and 48hrs. This was done
in triplicates. The control root was not exposed to UV.
The well grown in vitro plantlet was used for the study. The well grown plant on
MSO media was taken for UV treatment. Similar to in vitro root, in vitro plantlets were
also exposed to UV treatment.
Ann and Stapleton, (1992) reported that like all living organisms, plants sense and
respond to UV radiation, both the wavelengths present in sunlight (UV-A and UV-B) and
the wavelengths below 280 nm (UV-C). Gu et al., (2010) suggested UV-B radiation can
activate the self-protective secondary metabolic system. It is one of the method to induce
bioactive secondary metabolites by UV-B irradiation in vitro. Ehsanpour et al., (2005)
revealed UV-C radiation is used to induce physiological and genetic changes in plant.
Calluses of peanuts, an easily obtainable source, as the material to induce
piceatannol production under controlled conditions. To induce resveratrol and piceatannol,
calluses were exposed to the ultraviolet (UV) irradiation. Significant quantities of
resveratrol and piceatannol were produced by calluses upon UV irradiation in suspension
culture. The amounts of piceatannol and resveratrol produced in 1g of calluses ranged from
2.17 to 5.31 µg and from 0.25 to 11.97 µg, respectively, in static culture. The quantities of
induced piceatannol and resveratrol reached a maximum at 18 h after UV irradiation
treatment in static culture (Ku et al., 2001). Soy- bean (Glycine max) leaves that had higher
concentrations of screening compounds, and lower epidermal transmittance, experienced
less DNA damage when challenged with a short exposure to solar UV-B (Mazza et al.,
2000).
4.5 Extraction of secondary metabolites
UV treated and control roots were powdered. This powder was taken for extraction.
Secondary metabolites were extracted with ethyl acetate and concentrated in flash
evaporator (Patel et al., 2009). This extracted root was dissolved in HPLC grade methanol
based on the dry weight of the root powder (0.3g in 1.8ml of methanol).The precipitate
was filtered using syringe filter. The major bioactive secondary metabolites present in the
sample was quantified by HPTLC analysis.
UV treated and control plantlets were powdered and taken for extraction.
Chlorophyll was removed from the leaves using hexane. Hexane was added to leaf powder,
kept in shaker for overnight and filtered. This residue was taken for the extraction. Using
this residue the extraction was carried out similar as root extraction. The extracted sample
containing secondary metabolites were used for HPTLC analysis.
Alam et al., (2011) described a procedure for sample extraction of Withania
somnifera root. In her study, ground dry root (500 mg) was weighed into a test tube
followed by the addition of a total of 10 ml of 80% aqueous methanol. The tubes were
sonicated for 5 min and centrifugated for another 10 min at 1500 rpm and the resulting
supernatants were collected and evaporated to a volume of approximately 1 ml.
Sharada et al., (2002) isolated withanolides from 5 g of air-dried,
lyophilized plant parts and in vitro cultured tissues of Withania somnifera. The samples
were percolated four times with ethanol: water (1:1) at 25 ± 2 °C. The aqueous ethanolic
extracts were concentrated by evaporation at reduced pressure and temperature (50 ± 5 ºC).
Singh et al., (2001) suggested the detailed extraction procedure for
an active aqueous sub fraction. Shade dried coarsely powdered roots (13 kg) of Withania
somnifera were extracted with 70% alcohol. The extract so obtained was dissolved in
water and extracted to obtain an aqueous fraction.
Ravi et al., (2004) extracted secondary metabolites from Withania
somnifera root powder. The powdered roots of Withania somnifera were percolated four
times with ethanol: water (1:1) at room temperature. The four extracts were combined,
centrifuged, and finally concentrated to 1/8 of the original volume under reduced pressure
at 55°C.
Jyothi et al., (2010) performed soxlet extraction using classical sox
let apparatus with accurately weighed 10g leaf powder for 14hr. Extraction was performed
with 500ml methanol as the extracting solvent. Finally extracts were evaporated to dryness
under vacuum.
4.6 Quantification of secondary metabolites present in roots and leaves of Withania
somnifera
The treated root and leaf samples were subjected to quantification using HPTLC
technique. The samples on silica gel plate by means of a CAMAG Linomat V
semiautomatic sample applicator. After loading, it was kept inside the chamber containing
mobile phase. After it reached 8cm, the plate was removed from the solvent. Allowed to
air dry and visualized at254nm and 366nm. Scanned with Camag TLC scanner III in the
reflectance – absorbance mode at 223 (or) 234nm [withaferin (or) withanolide]
respectively. The peek area was observed and Rf value was calculated. Then the plate was
derivatised using Anisaldehyde sulphuric acid. After derivatisation it was kept in oven at
110º C. Then the plate was visualized CAMAG TLC visualiser and each compound was
recorded at 254nm, 366nm and White R. It was observed that UV-treated 8hrs root
samples contained more amount of withanolide A compared with withaferin. Withaferin A
accumulation was high in UV treated 8hrs leaves than roots. It was similar to Sharma et
al., (2007) study.
Sharma et al., (2007) established HPTLC method for quantification of the
compound. Chromatography on silica with toluene: ethyl acetate: formic acid (5:5:1)as
mobile phase enabled good resolution of Withanolide A and Withaferin A without
interference from other compounds present in Withania somnifera. After spraying with
anisaldehyde–sulfuric acid reagent and heating for 15 min at 105°C, characteristic orange
fluorescence was observed for withaferin A only among all the spots resolved. When
scanned at 214 nm the Rf value was calculated.
Plate 5. SCREENING TO DETECT THE PRESENCE OF
WITHANOLIDE A ON IN VITRO UV TREATED ROOTS
Derivatised plate White R
Control root 8hrs UV treated root
Plate 6. Peak area for control and UV treated in vitro roots
Standard
Withanolide A
Plate 7. SCREENING TO DETECT THE PRESENCE OF WITHANOLIDE A ON
IN VITRO UV TREATED PLANTLETS
Derivatised plate at White R
a. Control Plantlet b. 4hrs UV treated Plantlet
Plate 8. Peak area for control and UV treated in vitro plantlet
Standard
Withanolide A
Plate 9. Regression graph for Withanolide A in UV treated in vitro root and plantlet
Withanolide A in UV treated Root Withanolide A in UV treated Plantlet
Plate 10. Withanolide A accumulation on In vitro UV treated root and plantlet of
Withania somnifera
HPTLC analysis showed secondary metabolite (Withanolide A)
accumulation was higher in 8hrs UV treated in vitro roots. The accumulation started to
increase from 4hrs treatment. Optimum concentration occurs at 8hrs UV- treatment.
Accumulation of Withanolide A started to decline after 8hrs UV-treatment.
Withanolide A concentration was not significant in UV treated in vitro plantlets
compared with untreated (control) plantlets. After UV treatment Withanolide A
accumulation was started to decline.
Plate 11. SCREENING TO DETECT THE PRESENCE OF
WITHAFERIN A ON IN VITRO UV TREATED ROOTS
Derivatised plate at White R
Control Root 12hrs UV treated Root
Plate 12. Peak area for control and UV treated in vitro root
Standard
Withaferin A
Plate 13. SCREENING TO DETECT THE PRESENCE OF WITHAFERIN A ON
IN VITRO UV TREATED PLANTLETS
Derivatised plate at White R
Control Root 8hrs UV treated Root
Plate 14. Peak area for control and UV treated in vitro plantlet
Standard
Withaferin A
A
Plate15. Regression graph for Withaferin A in UV treated in vitro root and
Withaferin A in UV treated root UV treated Plantlets tre
Plate 16. Withaferin A accumulation on In vitro UV treated root and plantlet of
Withania somnifera
Withaferin A concentration was high in untreated (control) in vitro root
compared with UV treated in vitro roots. Its concentration was started to increase from
4hrs UV treated in vitro roots.It reaches high in 12hrs UV treated in vitro roots compared
with other UV treated in vitro roots.
Withaferin A concentration was higher on in vitro leaves compared in vitro to root.
Withaferin A accumulation was started to increase from 4hrs UV treated in vitro plantlets.
In 8hrs UV-treated in vitro plantlet contained high concentration of Withaferin A. Then it
started to decline after 8hrs UV treatment. Optimum concentration was observed at 8hrs
UV treated in vitro leaves.
Table 2. Secondary metabolites accumulation of UV treated root and plantlet
S.No
Explants
Withanolide A Withaferin A
Root
(mg)
Leaf (mg) Root (mg) Leaf (mg)
1 Control 0.44575 0.36000 0.09981 0.26677
2 UV-4hrs 0.44617 0.31239 0.07654 0.33000
3 UV-8hrs 0.49872 0.18345 0.07870 0.71100
4. UV-12hrs 0.47400 0.15289 0.12939 0.5478
5. UV-24hrs 0.47000 0.11520 0.04485 0.5200
The table shown that major secondary metabolites withanolide A and
withaferin A were present in 8hrs UV-treated in vitro root and leaf of Withania somnifera
compared to other period. In root, higher concentration of Withanolide A has been
obtained at 4hrs UV treatment. But in leaves, significant amount of Withaferin A was
obtained at 12hrs UV treatment compared with other UV treated leaves. P- Value was
Significantly varied in secondary metabolites accumulation between the all samples
(control and UV treated root and plantlet).
Ktitorova et al., (2005) reported UV-B radiation of Barley root. In 8 h, root
diameter in the sub apical zone increased root length increased by only 0.5 mm for the
period from 6 to 24 h after irradiation. Some irradiated roots did not grow at all, whereas in
95% of irradiated roots, the length increment was between 0.3 and 1.7 mm. Similar to my
study, but instead of secondary metabolites accumulation he proven plant length was
increased.
Similar to my study, Gu et al., (2010) suggested that UV-B radiation method could
induce chalcomoracin and moracin N in mulberry leaves in vitro.
Ehsanpour and Razavizadeh, (2005) revealed that, UV-C radiation increases fresh
weight of callus culture of Alfaifa (Medicago sativa). In that within 15, 30 and 60 minutes
fresh weight of callus increased 10% , 20% and 37.96% respectively.
Ktitorova et al., (2006) reported that UV-B irradiation of barley (Hordeum vulgare L.)
roots (1 W/m2, 15 min) or leaves (3 W/m
2, 3.3 h) and also one-day-long root incubation in
the Knop solution supplemented with 1–4 μM ABA, 1 mM salicylic acid, 16 μM
ionomycin, or 0.1 mM colchicine induced growth retardation and subapical root swelling.
During the first hour after unilateral root UV-B irradiation, their growth sharply retarded
and hydraulic conductivity of membranes in the rhizodermis of growth zone rose 1.5-fold.
In 2.5 h, root tips bent toward the source of irradiation. In 4.5 h, the ratio of longitudinal to
transverse root extensibility in the root growth zone reduced twofold. In 8 h, root diameter
in the subapical zone increased and root hairs appeared in this zone and attained 300 μm in
length.
The result shown that withanolide A accumulation was high in root during 8hrs UV-
treatment. Similar to the root, UV treated plantlet contained more accumulation of
withaferin A at 8hrs UV treatment.
SUMMARY AND CONCLUSION
5.0 SUMMARY AND CONCLUSION
The results of the present study entitled “Effect of ultra violet radiation on
in vitro roots and leaves of Withania somnifera and its influence on major secondary
metabolites accumulation” are summarized as follows.
Induction of in vitro adventitious roots, mass cultivation and Micropropagation were
carried out using standard tissue culture practices. One month in vitro tissues were exposed
to UV radiation at different time intervals (4hrs, 8hrs, 12hrs, 24hrs and 48hrs).
Withanolide A accumulation increased in 8hrs UV treated in vitro root
(0.49872g) and 4hrs UV treated in vitro plantlets (0.31239g). Withaferin A concentration
was high in 12hrs UV treated root (0.12939g) and 8hrs UV treated in vitro plantlets
(0.71100g).
Secondary metabolites Withanolide A and Withaferin A accumulation high in 8hrs
UV treated in vitro Root and Plantlet.
To conclude, the present study can be accounted for validating the use UV stress
treatment to in vitro tissues as alternative source to secondary metabolites accumulation
increment.
BIBLIOGRAPHY
5.0 BIBLIOGRAPHY
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immunomodulatory activity of Withania somnifera (Ashwagandha) extracts
in experimental immune inflammation, Journal of Ethnopharmacology., 67,
27-35.
Akbarsha,M.A., Vijendrakumar,S., Kadalmani,B., Girija,R., Faridha,A. (2000),
Curative property of Withania somnifera Dunal root in the context of
carbendazim-induced histopathological changes in the liver and kidney of
rat, Phytomedicine., 7, 499–507.
Alam,N., Hossain,M., Khalil,I., Moniruzzaman,M., Sulaiman.S.A., and Gan,S.H.
(2011), High catechin concentrations detected in Withania somnifera
(ashwagandha) by High Performance Liquid Chromatography analysis,
BMC Complementary and Alternative Medicine., 11(65), 1-8.
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