Essay: Influence of Elicitors on major Withanolide Content in Withania somnifera

REVIEW OF LITERATURE

Ever since ancient times, people looked for drugs in nature for rescue for their disease (Petrovska, 2012). Medicinal plant species constitute important alternatives to conventional medicine in a large number of developing countries. Gonzalez et al. (2013) reported that native medicinal plants of both the palaeotropics and neotropics have traditionally had a high value for indigenous communities because of their healing properties and also due to other uses.

Before the introduction of chemical medicines, man relied on the healing properties of medicinal plants. Plants are created to supply man with food, medical treatment, and other effects. It is thought that about 80% of the 5.2 billion people of the world live in the less developed countries and the World Health Organization estimates that about 80% of these people rely almost exclusively on traditional medicine for their primary healthcare needs. Medicinal plants are the “backbone” of traditional medicine, which means more than 3.3 billion people in the less developed countries utilize medicinal plants on a regular basis. There are nearly 2000 ethnic groups in the world, and almost every group has its own traditional medical knowledge and experiences( Ahvazi, 2012).

Plant derived secondary metabolites have played an essential role as medicine for thousands of years. Currently, secondary metabolites with bioactivity are being isolated and used either directly or after chemical modification. Their pharmacological value is increasing due to the constant discoveries of their potential roles in healthcare and as lead compounds for new drug development. (Shilpa et al., 2010)

The wide diversity of plant secondary metabolites is largely used for the production of various pharmaceutical compounds. In vitro cell tissue or organ culture has been employed as a possible alternative to produce such industrial compounds. Tissue culture techniques provide continuous, reliable, and renewable source of valuable plant pharmaceuticals and might be used for the large-scale culture of the plant cells from which these secondary metabolites can be extracted (Ahmad et al., 2013)

As the demand for medicinal plants is growing at a very fast pace, consequently some of them are increasingly being threatened even in their natural habitats (Kumar et al., 2004).Therefore, in search for alternatives to production of desirable medicinal compounds from plants, cell culture technologies were introduced as a possible tool for studying and producing plant secondary metabolites as in vitro regeneration holds tremendous potential for the production of high-quality plant-based medicines (Vanisree et al., 2004).

In ayurveda Withania somnifera is considered as Rasayana herb. The plant is well known for its roots rich in steroids and alkaloids and are a valuable constitute of traditional ayurvedic drug preparation against many diseases (Williamson,2002). The major biochemical constituents of this plant are a class of secondary metabolites known as withanolides. The biological activity of withanolides, especially of the dominant withanolide A and withaferin A have been studied extensively by various researchers.

The literature relevant to the present study entitled “ A Study on the Influence of Elicitors on major Withanolide Content in Withania somnifera JA 20” is reviewed in this chapter under the following headings:

2.1. Withania somnifera: Ashwagandha

2.2. Bioactive compounds present in W.somnifera

2.3. Pharmacological properties of W.somnifera

2.4. Studies on in vitro culture techniques

2.5. Elicitation

2.6. Analytical approaches to identify and quantify major withanolides

2.1. Withania somnifera: Ashwagandha

Withania somnifera Dunal (Solanaceae) popularly known as Ashwagandha or winter cherry has been an important herb in the ayurvedic and indigenous medical system for over 3000years and is one of the most valuable plants in the traditional Indian systems of medicine (Alam et al., 2011; Nathiya et al., 2012). Ashwagandha attains the special name as the root smells like horse (“Ashwa”) and it is believed that on guzzling it provides power of a horse. Different parts of Ashwagandha have significant therapeutic potency either as a whole plant extract or as separate constituents (Bhatt et al., 2006; Gupta and Rana, 2007).

The genus Withania belongs to the family Solanaceae and consists of 23

species. Of the 23 species, only two Withania somnifera and Withania coagulans(Linn.) Dunal have been reported from India. Withania Linn. genus is distributed in the east of the Mediterranean regions and South Asia. Withania somnifera is a native of drier part of India and Africa and old world. It is cultivated in large scale as commercial crop in Madhya Pradesh, Gujarat and some parts of Rajasthan. Withania coagulans is found as a commercial plant in the Punjab region. Withania somnifera is known as one of the most useful herbs in pacifying “vata” properties and the plant has been reported to have adaptogenic activity, anticancer, anticonversant, immunomodelatory, anti oxidative and neurological effects and also used in dietary purposes (Murthy and Sarala, 2010).

Ashwagandha posses several health benefits and is a well known herb in Indian Ayurveda and was used since centuries for its miraculous advantages (Mahima et al., 2012). Both the modern medical literature and traditional Ayurveda writings report many potential health benefits of the Ashwagandha herb (Wankhede et al., 2015).

2.1.1. Taxonomical Classification (Bano et al., 2015)

Kingdom: Plantae Order: Tubiflorae

Division:Angiosperms Family:Solanaceae

Class: Dicotiledoneae Genus: Withania

Species: somnifera

2.1.2. Botanical description of W.somnifera(Bhandari, 1995)

S.No Description Withania somnifera (L.) Dunal

1 Habit Undershrub

2 English Name Winter Cherry, Indian Ginseng

3 Vernacular Name Ashwagandha

4 Leaves Alternate, broadly ovate, sub-acute, entire margins

5 Inflorescence Axillary, umbellate cymes

6 Flowers Monoecious

7 Calyx Acorescent, gamosepalous with 5 sepals

8 Corolla Campanulate, greenish-yellow with 5 petals

9 Androecium Anthers 1.2 mm long, broadly ovate

10 Gynoecium Ovary ovoid/globose, glabrous

11 Style Filiform

12 Stigma Mushroom-shaped, 2-lamellate

13 Fruit (Berry) Globose, enclosed in the persistent calyx, seeds yellow, reniform

14 Seeds Globose, enclosed in the persistent calyx, yellow, reniform

15 Flowering Throughout the year

2.2. Bioactive compounds present in W.somnifera

The chemistry of Withania species has been extensively studied and several groups of chemical constituents such as steroidal lactones, alkaloids, flavonoids, tannin etc. have been identified, extracted, and isolated (Bandyopadhay et al., 2007). 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. Some of the known chemical constituents present in ashwagandha was reported by Christian (2013) which was given in table 2.1.

Table 2.1. Chemical constituents present in W.somnifera

Alkaloids Withanine, Withaninine, Somniferine, Tropeltigloate, Somniferinine, Somninine, Nicotine, Visamine, Withasomine

Salts Cuscohygrine, Anahygrine, Tropine, Pseudotropine, Anaferine

Steroidal Lactones Withaferin-A, Withanone, WS-1, Withanolide E C28H38O7, Withanolide F C28H38O6, Withanolide G C28H36O4, Withanolide H C28H36O5, Withanolide I C28H36O5, Withanolide J C28H36O5, Withanolide K C28H36O5, Withanolide L C28H36O5, Withanolide M C28H36O6

Nitrogen containing compounds Withanol C25H34O5, Somnisol C32H46O, Somnitol C33H46O7

Steroids Cholesterol, -sitosterol, Stigmasterol, Diosgenin, Stigmastadien, Sitoinosides VII, Sitoinosides VIII, Sitoinosides IX, Sitoinosides X

Flavonoids Kaempferol, Quercetin

Much of Ashwaganda’s pharmacological activity has been attributed to two main withanolides, withaferin A and withanolide A. The withanolides are a group of naturally occurring C28- steroidal lactones built on an intact or rearranged ergostane framework, in which C-22 and C-26 are appropriately oxidized to form a six-membered lactone ring. The basic structure (Figure 2.1) is designated as the withanolide skeleton.

Fig 2.1: Basic structure of Withanolide

The withanolide skeleton may be defined as a 22-hydroxyergostan-26-oic acid-26, 22-lactone. The characteristic feature of withanolides and ergosane-type steroids is one C8 or C9-side chain with a lactone or lactol ring but the lactone ring may be either six-membered or five membered and may be fused with the carbocyclic part of the molecule through a carbon-carbon bond or through an oxygen bridge. Appropriate oxygen substituents may lead to bond scission, formation of new bonds, aromatization of rings and many other kinds of rearrangements resulting in compounds with novel structures. Examination of W. somnifera roots has resulted in the isolation of a new dimeric thiowithanolide, named as ashwagandhanolide (Mirjalili et al., 2009).

Withaferin A (Fig.2.2) is the most important of the withanolides isolated from Withania somnifera to which the curative properties of the leaves are attributed. It has been receiving a good deal of attention because of its antibiotic and antitumour activities. Roots of Withania somnifera contains four steroidal lactones, called withanolides, viz withaferin A 5, 20a (R)-dihydroxy- 6a, 7a-epoxy-1 -oxo- (5a)-witha-2,24 – dienolide (m.p. 282-84 °C) and two minor withanolides, of which one is probably 5a, 17a-dihydroxy-1-oxo-6a, 7a-epoxy-22R-witha-2, 24-dienolide (the so-called withanone). The first two withanolides also occur in the roots (Supe et al.,2011).

Fig 2.2.Structure of major withanolides

A) WithaferinA B) Withanolide A

2.3. Pharmacological properties

Leaves and roots of this plant are abortifacient, aphrodisiac, diuretic, nervine tonic, alterative, narcotic, sedative, astringent, growth promoter and anthelmintic. It has antiarthritic, anti-bacterial, anti-dote for scorpion sting, anti-stress, anti-tumour and anti-cancer activities. It is used in toning of uterus, consumption, dropsy, leucoderma, impotence, rheumatism, debility from old age, ulcer, sexual and genital weakness, assumption, rheumatic swelling, loss of memory, loss of muscular energy, spermatorrhoea, syphilis, sterility of women, blood discharge, leucorrhoea, anemia with emaciation, nervous exhaustion, multiple sclerosis, neoplasia, cancer and fatigue. Fruits and seeds are diuretic and used in coagulation of milk (Christian, 2013).

2.3.1. Anticancer activity

Yadav et al.(2011) evaluated in vitro cytotoxicity in 50% ethanol extract of root, stem and leaves of Withania somnifera against five human cancer cell lines of four different tissues i.e. PC-3, DU-145 (prostrate), HCT-15 (colon), A-549 (lung) and IMR-32 (neuroblastoma) and concluded that root, stem and leaves extracts showed cytotoxicity activity ranging 0-98% depending on the cell lines but maximum activity was found in 50% ethanol extract of leaves of Withania somnifera. Methanolic extract of W. somnifera has been used in stem cell proliferation and also inhibited growth of breast, lung, central nervous system and colon cancer cell lines by decreasing their viability in doze dependent manner and therefore holds promise as a chemotherapeutic agent (Mir et al.,2012). W.somnifera enriched withaferin-A induces apoptosis through mechanisms (Fig. 2.3.) such as, inhibiting the activation of nuclear factor kappa-B (NF-κB) by preventing the TNF-induced activation of IκB kinase β via a thioalkylation sensitive redox mechanism (Oh and Kwon, 2009), activation of tumor suppressor proteins such as p53 and pRB (Wadhwa et al., 2013).

Fig.2.3. Withaferin-A induced cell apoptosis mechanisms

2.3.2. Antioxidant activity

The active principles of sitoindoside VII-X and withaferin A have antioxidant activity by enhancing the free radical scavenging enzymes such as superoxide dismutase, catalase, glutathione peroxidase(Singh et al., 2010). The study conducted by Maheswari and Manisha clearly suggests that Withania somnifera (L.) aqueous root extract has potent antiradical and antioxidant properties, attributed to the bioactive compounds like alkaloids and steroidal present in aqueous root fraction which is responsible for amelioration of toxin (CM) induced toxicity and oxidative stress in circulation(Maheshwari and Manisha,2015). Palash et al. (2010) observed strong antioxidant scavenging activities in mature root and young root bark portion of W.somnifera. Pal et al. (2011) reported antioxidant activity in methanolic extract of Ashwagandha root.

2.3.3. Antimicrobial activity

Leaf extracts at concentrations 6.25 mg/ml and 12.5 mg/ml inhibited the growth of five Gram-negative pathogenic bacteria (Escherichia coli, Salmonella typhi, Citrobacter freundii, Pseudomonas aeruginosa and Klebsiella pneumonia) (Alam et al.,2012). Isolated flavonoids and alkaloids from W. somnifera show growth inhibitory activity against Enterobacter aerogens, Proteus mirabilis, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis, Klebsiella pneumoniae, Raoultella planticola and Agrobacterium tumefaciens at concentration 0.039 mg/ml (Singh and Kumar, 2011; Singh and Kumar, 2012). In another study, crude extract of leaves of W. somnifera was tested against clinical pathogens Staphylococcus aureus, roteus mirabilis Streptococcus mutans, Streptococcus sobrinus and Salmonella paratyphi B, where it was found that 100 μl of extracts (100 mg/ml) was able to inhibit the growth of all the pathogenic bacteria (Al-Ani et al., 2013; Pandit et al., 2013). W. somnifera reported to exhibit antibacterial activities and it shows its activity against both Gram-positive and Gram-negative pathogenic bacteria (Singariya et al., 2012). In another study, it was argued that W. somnifera shows its bactericidal and fungicidal activity through mechanisms attributed to cytotoxicity, gene silencing and immune potentiation, where aerial extract at concentration 1.56 mg/ml shows good antimicrobial potency (Mwitari et al., 2013).

2.3.5. Antiepileptic activity

Studies with rodent models show that W. somnifera and its bioactive withanolides are effective in reducing seizures through Gama amino butyric acid (GABA)A receptor modulation mechanism in brain, where subeffective dose of W. somnifera (50 mg/kg), with a subprotective dose of either GABA (25 mg/kg) or Diazepm (0.5 mg/kg) increases the seizure threshold in brain (Kulkarni et al., 2008). In another study, it was demonstrated that W. somnifera root extract and withanolide-A were capable of restoring spatial memory deficit by inhibiting oxidative stress induced alteration in glutamergic neurotransmission, where W. somnifera reduces the expression of N-metyl-D-aspartate (NMDA) receptor, which is responsible for spatial memory loss in epileptic rats (Soman et al., 2012).

2.3.4. Neuroprotective activity

Preclinical research and clinical trials support the use of W. somnifera for the treatment of neurological conditions such as anxiety, depression, cognitive disorders, senile dementia and neurodegenerative disorders (Alzheimer’s and Parkinson’s diseases) (Kumar et al.,2015). Computational docking evidences reveal that withanolide-A inhibits brain acetyl cholinesterase, which could be a therapeutic alternative for the treatment of neurodegenerative Alzheimer’s disease (Grover et al., 2012). W. somnifera root extract significantly increases the levels of brain antioxidant enzymes (superoxide dismutase (SOD), chloramphenicol acetyltransferase, glutathione, and glutathione S-transferase) and total proteins to protect the brain when animal was exposed to lead nitrate (Sharma et al., 2011). W. somnifera and its bioactive withanolides are also effective in treating Parkinson’s disease, where it was revealed that its root extract enhances brain dopamine level in Parkinson’s animals and also improves physiological abnormalities seen in Parkinson’s disease (RajaSankar et al., 2009).

Fig.2.4. Neuroprotective mechanisms of W.somnifera(Kumar et al.,2015)

2.3.6. Anti-inflammatory activity

W. somnifera at dose levels 600 & 800 mg/kg significantly decreased the severity of arthritis by effectively suppressing the inflammatory mediators and improving the functional recovery of motor activity in experimental animals (Gupta and Singh, 2014). Ashwagandha acts as an anti-inflammatory agent through inhibition of complement, lymphocyte proliferation, and delayed-type hypersensitivity (Rasool & Varalakshmi, 2006). Roots of W.somnifera and withanolides are also effective in treating arthritic inflammation, inflammation in cystic fibrosis and irritable bowel syndrome, through various mechanisms such as inhibiting NF-κB activation, inhibition of COX-2 generation, inhibition of endothelial cell protein C receptor through antioxidant effect and cytokines release, thus in turn causes depletion of inflammatory mediators (Ku et al., 2014; Mulabagal et al., 2009; Oh and Kwon, 2009). W. somnifera and its bioactive withaferin-A down regulate the production of inflammatory mediators like prostaglandins, histamine, interleukins and cytokines (Gupta and Singh, 2014; Paval et al., 2009).

2.3.7. Hepatoprotective activity

Investigations have given numerous evidences, where W. somnifera at a dose 500 mg/kg significantly reduces the elevated biomarkers (aspartate aminotransferase, alanine transaminase, alkaline phosphatase, and Bilirubin) in experimental animals when exposed to hepatotoxic dose of paracetamol. It significantly reduces the lipid peroxidation, enhances glutathione content, catalase, glutathione reductase and glutathione peroxidase activity in liver (Malik et al., 2013; Sabina et al., 2013). W.somnifera extract at a dose of 100mg/kg for 7 days produced significant hepatoprotective effect by preventing the increase in weight of the liver, serum enzymes, bilirubin and lipid peroxidation while it significantly arrested the decrease in levels of GSH, SOD and CAT (Ganguly et al., 2009). Sultana et al. (2013) data suggests that higher doses of gentamicin produce hepatocellular damage which can be prevented by W.somnifera.

2.4. Studies on in vitro culture techniques

W.somnifera was collected from wild and used as raw material for bulk manufacturing in medicinal industries for its high medicinal value, leading to over exploitation and it has now become an endangered species (Patel and Murthy, 2013). The in vitro plant tissue culture provide an alternative to field grown plants harvested for the production of therapeutically valuable compounds (Jain et al.,2011).Production of secondary metabolites in tissue cultures is usually higher when plant cells are organized into tissues or organs (Abouzid et al.,2010). Hence, tissue culture plants through micropropagation are desirable. Micropropagation of Withania somnifera employs different explants such as shoot tips, auxiliary meristems, auxiliary leaves, auxiliary shoot and hypocotyl and root segments.

2.4.1. Micropropagation

In vitro micro propagation technology has sound and extensive potential for commercial rapid multiplication of plants because it is a quick method, allows round the year propagation of identical plants, and produces plants free from diseases (Kumar et al., 2013). Micropropagation is the process of vegetative growth and multiplication from tissues or seeds. It is carried out in aseptic and favourable conditions on growth media using various plant tissue culture techniques (Zhou and Wu, 2006). The advantages of in vitro micropropagation of medicinal plant are listed below:

 Higher rate of multiplication

 Environment can be controlled or altered to meet specific needs of the plant.

 Plants are available in all year (independent of regional or seasonal variations).

 Identification and production of clones with desired characteristics.

 Production of secondary metabolites.

 New and genetically engineered plants can be produced.

 Conservation of threatened plant species.

 Preservation of genetic material by cryopreservation.

Duhan et al., (2014) observed maximum number of shoots/explants in W.somnifera on MS medium supplemented with 1.5 mg-1 BAP and 0.5 mg-1 NAA after 45 days of culture. Sivanesan (2006) compared efficient culture media for growth of W.somnifera and found that MS medium supplemented with BAP + IAA (each at 2.0 mg/L) was optimal for induction of shoot buds whereas MS medium supplemented with 0.3 mg/L GA3 was the most suitable for shoot elongation. Dewir et al. (2010) used leaves as explants source and 2 mg/L BA and 0.5 mg/L IAA for indirect differentiation of shoots in W. somnifera.

2.4.2. Adventitious root culture

Adventitious root culture renders the secondary metabolites in a very large amount and it is the unique technique .In the field of medicine, agriculture, drug production, pigment production, dye production, and for many other field adventitious root culture fulfills the global demand. Root cultures can be used in various ways which also includes studies of carbohydrate metabolism, mineral nutrients requirements, essential need for of vitamins, growth regulators, differentiation of the root apex and gravitropism (Loyolo-Vargas & Ham, 1995). The advantages of using root cultures are as follows:

 The plants grow rapidly

 It is easy to prepare and maintain

 It show a low level of variability and

 It can be easily cloned to produce a large supply of experimental tissues (Pop et al., 2011).

Murthy et al (2008) found that mass cultivation of roots in vitro will be an efficient technique for the huge production of secondary metabolites because roots contain a number of therapeutically applicable withanolides. The main problem in analysis and isolation of these metabolites is that the structural diversity of withanolides present in Withania spp. In the United States the root extract of Withania species has freshly been accepted as an additional section of a dietary. There is a growing interest in root culture as an alternative source for this important metabolite because harvesting roots is ruinous for the plants (Murthy et al., 2008).

Thilip et al.,(2015) induced adventitious roots from cell suspension culture of W. somnifera for the production of withanolides and observed half strength MS liquid medium containing 0.5 mg/L IBA in combination with 0.25 mg/L IAA showed higher production of adventitious roots from cell suspension culture after 4 weeks. Sivanandhan et al. (2012) have reported that the brown, semi-friable callus (500 mg FW) derived from leaf explants produced higher number of primary adventitious roots (9 roots/callus) in half strength MS medium fortified with IBA (0.5 mg l−1) and NAA (0.1 mg l−1). Pradeepa et al. (2014) studied and found that in vitro adventitious root from the leaf explants of Withania

somnifera, was achieved on full strength MS medium supplemented with 45g/ L sucrose. She also used different concentration of auxin in her study and concluded that basal medium supplemented with 1mg/ L indole butyric acid (IBA) and 1mg/ L indole acetic acid (IAA) achieved the maximum number of roots with 100% response.

2.5. Elicitation

The qualitative and quantitative improvement of biologically active compounds by plant cell, tissue and organ culture have been used as an alternative source for the production of important bioactive secondary metabolites using different strategies of metabolic engineering. The biotechnological production of valuable secondary metabolites in plant cell or organ cultures is a good alternative to the extraction of whole plant material because many plants of high medicinal products are difficult to cultivate. The attractive concepts in the scaling up technology of natural products are enhancement of secondary metabolites and use of biological and chemical elicitors. It has been found that plants elicit the same response when come in contact with the compounds of the pathogen as attacked by the pathogen itself. These compounds are known as elicitors (Vakil and Mendhulkar, 2013). Elicitors are compounds triggering the formation of secondary metabolites (Karuppusamy, 2009). Elicitation is a good strategy to induce physiological changes and stimulate defense or stress-induced responses in plants. The elicitor treatments trigger the synthesis of phytochemical compounds in fruits, vegetables and herbs. Plants respond to these stressors by activating an array of mechanisms, similar to the defense responses to pathogen infections or environmental stimuli, affecting the plant metabolism and enhancing the synthesis of phytochemicals (Baenas et al., 2014) which was given in Fig.2.5..

Fig 2.5.Mode of action of elicitors

SAR -Systemic Acquired Response ISR-Induced Systemic Resistance

ROS -Reactive Oxygen Species RNS -Reactive Nitrogen Species

NADPH -Nicotinamide Adenine Dinucleotide Phosphate

SA- Salicylic acid JA -Jasmonic Acid

ET- Ethylene

Elicitors can be classified on the basis of their nature as abiotic elicitors or biotic elicitors, or on the basis their ‘origin’ like exogenous elicitors and endogenous elicitors.

2.5.1. Abiotic elicitors

Abiotic elicitors are the substances of non-biological origin, predominantly inorganic salts, and physical factors acting as elicitors like Cu and Cd ions, Ca2+ and high pH ((Namdeo, 2007) . SA is one of numerous phenolic compounds, defined as compounds containing an aromatic ring with a hydroxyl group or its derivative, found in plants which act as an elicitor. Exogenously supplied SA was shown to affect a large variety of processes in plants, including stomatal closure, seed germination, fruit yield and glycolysis.

Chaichana and Dheeranupattana,2012 found that SA elicitation in general has a negative effect on growth but acts as a key-signaling component involved in the signal transduction pathways of plant defense mechanisms thus enhancing secondary metabolite production .These workers finally concluded that SA slightly decreased the root growth in Stemona sp. in many concentrations and especially at 1.0 mM concentration and the production of Stemona alkaloids was found to increased after being cultured for 1 week, however the production after a 2-week culture period with decreased. 0.1 mM SA resulted in the

highest level of 1′, 2′-didehydrostemofoline and stemofoline production at 1.69 fold and 1.61 fold respectively.

2.5.2. Biotic elicitors

Biotic elicitors are substances with biological origin, they include polysaccharides derived from plant cell walls (pectin or cellulose) and micro-organisms (chitin or glucans) and glycoproteins or G-protein or intracellular proteins whose functions are coupled to receptors and act by activating or inactivating a number of enzymes or ion channels. Exogenous elicitors are substances originated outside the cell like polysaccharides, polyamines and fatty acids whereas ‘endogenous elicitors’ are substances originated inside the cell like galacturonide or hepta-β-glucosides etc (Namdeo, 2007). The use of cell wall components from fungi such as A. niger have been used in cell culture systems to elicit the synthesis of low molecular weight compounds. This effect has been studied in various plants, including Gymnema sylvestre, Psoralea corylifolia and Andrographis Paniculata. The fungal cell wall works as a polysaccharide elicitor that increases calcium concentrations in the cell and activates various defense responsive pathways. This, in turn, leads to the accumulation of phytoalexins and low molecular weight antimicrobial compounds (Vakil and Mendhulkar, 2013). The results obtained in the present study are in agreement with the findings of other researchers which suggest that the lower dose of A. niger extract is effective in eliciting the target compound (Ibrahim et al., 2007). It is reported that 2.5 ml of fungal extract prepared from fungal strain (F5) which was isolated from inner bark of 15-20-m high Taxus chinensis tree showed enhanced paclitaxel content on 14 days of treatment exposure in T. chinensis cell suspension culture (Zhang et al. 2000).

2.6. Analytical approaches to identify and quantify major withanolides

Chromatography is essentially a group of techniques used for separation of the constituents of mixture by continuous distribution or adsorption of analyte between two phases(Sripathi et al., 2011). The chromatographic techniques used in the isolation of various types of natural products can be broadly classified into two categories: classical or older and modern.

A) Classical or older chromatographic techniques

Thin-layer chromatography (TLC)

Preparative thin-layer chromatography (PTLC)

Open-column chromatography (CC)

Flash chromatography (FC)

B) Modern chromatographic techniques

High performance liquid chromatography(HPLC)

High performance thin layer chromatography(HPTLC)

Multiflash chromatography

Vacuum liquid chromatography

Droplet countercurrent chromatography

2.6.1. Column chromatography

In adsorption chromatography technique column chromatography is a basic technique. Based on the separation of components the extent of adsorption takes place in stationary phase. The solid material is packed in a vertical column. It may be a glass or metal (Davies et al., 2000). The individual components moves with a different rate compared to the mixture component. The lower affinity phase moves faster compared to the higher affinity. Disadvantages of column chromatography was it is complicated when comes to larger samples. It is not suitable to separate two very similar compounds. The most important drawback is it is time consuming and appropriate resins are used to prepare the column (Solomons and Fryhle, 2006).

2.6.2. Thin-layer chromatography

TLC is a simple, quick and inexpensive procedure that gives quick answer about components in a mixture. TLC is also used to support the identity of a compound in a mixture when the Rf of a compound is compared with the Rf of a known compound (Mohrig et al., 2010). But in TLC zone broadening is most noticeable for longer separation distances and at higher Rf values. This is a consequence of the use of capillary forces to promote and maintain the flow of mobile phase and is a considerable disadvantage for TLC compared with pneumatically regulated column systems. Hence to overcome these difficulties modern analytical tools such as HPLC and HPTLC can be used more precisely for quantification of compounds in plants (Sreekumar and Ravi, 2007).

2.5.3. High performance liquid chromatography

HPLC is a versatile, robust and widely used technique for the isolation of natural products. It is a chromatographic technique that can separate a mixture of compounds and is used in phytochemical and analytical chemistry to identify, quantify and purify the individual components of the mixture (Piana et al., 2013). Several authors describe the use of HPLC characterization and quantification of secondary metabolites in plant extracts, mainly phenol compounds, steroids, flavonoids, alkaloids (Boligon et al., 2012). Besides its advantages certain limitations have been observed in HPLC systems which includes,

 High pressure is required

 Sample clean up becomes mandatory

 High running cost

 High maintenance

 Long time is needed for column equilibration

 Limited choice of detector

2.5.4. High performance thin layer chromatography

Among various chromatographic analytical techniques, HPTLC has a firm place as a reliable method for analysing several samples of divergent nature and composition at the same time (Sripathi et al., 2011). High Performance Thin Layer Chromatography is one of the modern sophisticated techniques that can be used for wide diverse applications. It is a simple and powerful tool for high‐resolution chromatography and trace quantitative analysis is made possible. It is most widely used for quick and easy determination of quality, authenticity and purity of the crude drugs and market formulations (Mamatha, 2011). Quantification of withaferin-A was done on both chloroform and hydroalcoholic fraction along with methanolic extract by using solvent system toluene: ethyl acetate: formic acid in the ratio of (5: 5: 1) (Prasad et al., 2010). Shetty and Nareshchandra, (2012) have revealed that HPTLC fingerprints of mother plants and their regenerants produce variability in their chemical constituents. Jirge et al., 2011 have validated a High Performance Thin Layer Chromatography method for simultaneous estimation of two biomarkers present in Ashwagandha viz., withaferin A and beta‐sitosterol‐D‐glucoside. Patel etal., (2009) developed the fingerprint profile and analysed withaferin A in young and old root samples . Pankajavalli et al., (2014) quantified both withanolide A and Withaferin A present in in vitro cultures of W.somnifera using HPTLC and concluded that withanolide A to be the dominant metabolite in in vitro cultures. Alam et al., (2012) have concluded that the HPTLC method was found to be specific and accurate and can be ussed for qualitative estimation of crude extract of Withania and its polyherbal formulations.

2016-3-1-1456838938