Int J Pharm Pharm Sci, Vol 8, Issue 7, 17-26Review Article


IN VITRO AND OMICS TECHNOLOGIES OPENS A NEW AVENUE FOR DECIPHERING WITHANOLIDE METABOLISM IN WITHANIA SOMNIFERA

PANKAJAVALLI THIRUGNANASAMBANTHAM1, KALAISELVI SENTHIL1*

1Department of Biochemistry, Biotechnology and Bioinformatics, Avinashilingam Institute for Home Science and Higher Education for Women, Coimbatore 641043, Tamil Nadu, India
Email: kalaiselvi_bc@avinuty.ac.in      

Received: 16 Mar 2016 Revised and Accepted: 17 May 2016

ABSTRACT

Withania somnifera,commonly known as Ashwagandha or winter cherry, is a principal medicinal plant that has been used in Ayurvedic and native medicine. In view of its varied therapeutic prospective, it has also been the subject of considerable modern scientific attention. Ashwagandha roots are an integral of over 200 formulations in Ayurveda, Siddha and Unani medicine, which are used in the treatment of various physiological disorders. The major chemical constituents of this plant, the withanolides, are a group of naturally occurring C28-steroidal lactones. It has been extensively investigated in terms of chemistry and bioactivity profiling. However, there exists only very little fragmentary evidence about the dynamics of withanolide biosynthesis. This review examines different in vitro approaches that had been carried out over past decade of years and newly developed omics technologies for the large scale production of withanolides as well as for the analysis of genes associated with withanolide biosynthesis.

Keywords: Withania somnifera, Withanolides, in vitro cultures, Genomics, Transcriptomics, In silico, Proteomics, Metabolomics

© 2016 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)

INTRODUCTION

Withania somnifera (L.) Dunal commonly known as Ashwagandha/ Indian ginseng/winter cherry, is one of the most esteemed medicinal plants used in Indian Ayurveda for over 3000 y [1]. It is used as herbal medicine in various forms (decoctions, infusions, ointments, powder and syrup) in different parts of the world [2, 3] for all age groups of patients without any side effects even during pregnancy.

The medical importance of W. somnifera is mainly because of the presence of steroidal lactones namely “withanolides” [4-8]. The pharmaceutically important compounds are withanolide A, withanolide B, withaferin A, and withanone (major constituents) and 12-deoxy withastramonolide, withanoside IV and withanoside V (minor constituents). Each withanolide is having a wide array of therapeutic values.

The annual requirement of dried plant material for withanolide production in India has been estimated to about 9,127 tons as against the annual production of about 5,905 tons [9]. A major bottleneck in the biosynthesis of withanolides depends on the plant's tissue type and growth conditions in natural habitats as the commercial products are entirely derived from field-grown plants [10]. This ultimately leads to difficulties in the compositional standardization of Withania formulations and its commercial exploitation.

To meet the current Ashwagandha worldwide market requirement, in vitro cultures could provide an alternative to field-grown plant for the production of therapeutically valuable compounds and thereby suggests that in vitro cultures can be applied for secondary metabolite production in cell/organ culture system [7]. As roots contain a number of therapeutically applicable withanolides, mass cultivation of roots in vitro will be an effective technique for the production of these secondary metabolites on a trade scale. Therefore, the development of a fast-growing root system would offer unique opportunities for producing root drugs in tissue culture without depending on field cultivation, which is prone to show variables [11].

A few attempts have already been made for in vitro root culture of W. somnifera. Several authors [12-15] induced direct rooting from leaf explants. Even though works on producing adventitious root cultures of W. somnifera with higher content of withanolides using different strategies are available, until a date, not more than one report is available on its mass cultivation. Hence, it is essential to develop an efficient protocol for mass cultivation of adventitious roots in a superior variety with the potential to be utilized in the production of withanolides applicable for large-scale high-throughput processing in the industry.

The metabolic constituents, particularly secondary metabolites are reported to differ with the variety, tissue type and sometimes with growth conditions [16]. Such variations often lead to poor reproducibility of promoting properties of various commercial Withania preparations [17,18]. This causes difficulties in the compositional standardization of herbal formulations and the commercial exploitation of this plant. A recent review [19] narrates cases where multi-component W. somnifera extracts showed better medicinal efficiency than the purified compounds. Hence, a comprehensive phytochemical fingerprinting needs to be carried out on the plant material to be used for health benefits [20-22]. The comprehensive chemical analysis is required not only to establish a correlation between complex chemical mixtures and molecular pharmacology but also to understand complex cellular processes and biochemical pathways via metabolite-to-gene network [23].

Although this plant has been well characterized in terms of phytochemical profiles as well as pharmaceutical activities, only very little information about the genes responsible for biosynthesis of these compounds is available to date. Recently, various biochemical and molecular studies have been commenced to elucidate the biosynthetic pathway for various withanolides in W. somnifera [6]. Putatively, withanolides (C-30) are synthesized via both mevalonate (MVA) and non-mevalonate-1-deoxy-D-xylulose 5-phosphate/2-Cmethyl-D-erythritol 4-phosphate (DOXP) pathways through cyclization of 2,3-oxidosqualene to cycloartenol; wherein 24-methylene cholesterol is the first branching point towards the biosynthesis of various withanosteroids.

Production of withanolides includes a series of desaturation, hydroxylation, epoxidations, cyclization, chain elongation, and glycosylation steps. In plants, MVA is the general precursor of variously identified isoprenoids, such as sterols, plant growth regulators, and terpenoids [24, 25]. It has been recently reported that, in W. somnifera, both MVA and DOXP pathway participate in the biosynthesis of the withanolide [26].

An attempt to engineer the efficient production of secondary metabolites requires an understanding of their biosynthetic pathway(s), and our present knowledge of withanolide biosynthesis is limited to only a few genes involved in the pathway. Over the past decade, many attempts have been made in tissue culture field to manifest the tissue-specific accumulation of the legendary withanolides. Very recently, Senthil et al. [27] reported first large-scale transcriptome profiling of in vitro tissues for W. somnifera and provides a comparative expression profiling of pathway genes involved in withanolide biosynthesis and their potential biological activity. This panorama propels us to write this review that presents a characteristic overview of studies done related to in vitro and molecular ideas engaged in W. somnifera to understand the regulation of withanolide production.

Why withanolides

Much of ashwagandha's pharmacological activity has been attributed to two main withanolides, withaferin A and withanolide A [28] of which, withaferin being the dominant metabolite of leaf tissue and withanolide A, reported to be accumulated more in root tissues [29] (fig. 1). The withanolides serve as important hormone precursors that can convert into human physiologic hormones as needed. According to the literature, Ashwagandha is thought to be amphoteric; i.e., it can help regulate important physiologic processes. The principle behind this theory is that when there is an excess of a certain hormone, the plant-based hormone precursor occupies cell membrane receptor sites so the actual hormone cannot attach and exert its effect. If the hormone level is low, the plant-based hormone exerts a small effect [30]. Withanolides have been researched in a variety of clinical examinations for their numerous therapeutic activities including cancer and immune functioning [31]. Withanolide B, withaferin A and with anyone also have remarkable activities in physiological and metabolic restoration, anti-arthritic, anti-aging, anti-cancer, cognitive function improved in geriatric states and recovery from neurodegenerative disorders [32]. Withanolide A is considered as a good candidate for neurodegenerative diseases and potentiating humoral and cell-mediated Th1 immunity [33-35].

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Fig. 1: Dominant metabolites among different organs of Withania somnifera

Withaferin A is reported to induce apoptosis through Reactive Oxygen Spices (ROS) generation mediating modulation of both intrinsic and extrinsic apoptosis signaling cascades together with abrogation of NF-kB functions [34], as an anti-inflammatory agent, to protect cardiovascular systems, as an anti-cancer agent, antioxidant and antiplatelet activity [36].

The protective effect of W. somnifera in epilepsy is considered to be through GABAergic modulation. The profound anticonvulsant activity of W. somnifera root extract as reported in various animal models is hypothesized to be through GABAA receptors [37]. In another study, the antiparkinson effects of W. somnifera extract was evaluated using 6-hydroxy dopamine (6-OHDA)-induced Parkinson's-like effect in rats [38]. Meena and Lakshmi [39] demonstrated the antiepileptic activity of leaf extracts of Ashwagandha. Anti-aging property of W. somnifera was studied by [40] and [41], who proved that W. somnifera root powder at a dosage of 3 grams daily for one year, significantly improve hemoglobin, red blood cell count, hair melanin, and seated statue. In another study conducted by [42] provided evidence that root and leaf extracts of W. somnifera significantly reduces experimental and biochemical indicators of stress without adverse effects in humans. The available scientific data thus support the conclusion that Ashwagandha is a real potent regenerative tonic due to its multiple pharmacological actions like anti-stress, neuroprotective, antitumor, anti-arthritic, analgesic and anti-inflammatory, etc.

Biotechnological approaches for augmentation of withanolides in Withania somnifera

Inevitable In vitro cultures

For commercial withanolide production, field grown plant material has generally been used but as per the literature, the quality of these products may be highly affected by different environmental conditions, pollutants, and fungi, bacteria, viruses and insects, which can result in a heavy loss in yield and alter the medicinal content of the plant. Moreover, these methods are time-consuming, laborious, and they are not able to encounter the current Ashwagandha global market requirement [8]. To try to overcome these problems, many attempts were made during the last decades to evaluate the possibility of producing withanolides by in vitro plant cell and organ cultures [43, 44].

At the international level, there has been an ever-increasing demand for roots of W. somnifera in larger quantities [8]. Attempts to produce withanolides through tissue culture have been reported by many authors [45-50]. Rani et al. [51] were the first to report on somatic embryogenesis from calli obtained from axillary shoots, internodal segments, root and cotyledonary leaf segments. Sivanesan and Murugesan [52] developed an efficient protocol for high-frequency plant regeneration from leaf explants of W. somnifera on Murashige and Skoog (MS) medium supplemented with 6-Benzyl amino purine (BAP), Kinetin (Kn) and Naphthalene acetic acid (NAA). Synthetic seed production and subsequent conversion of encapsulated shoot tips into plantlets have been reported by [53]. Direct regeneration from apical bud explants [54], somatic embryogenesis and plantlet regeneration from leaf explants [55], direct shoot regeneration using petiole and leaf explants [56] and direct and indirect organogenesis from nodal explants [57] of Indian ginseng have also been reported.

Withanolide A was reported to be de novo synthesized within root tissues [58], hence, studies were focused on tissue specific synthesis of withanolides under in vitro conditions. Compared to cell cultures, adventitious roots were reported to show higher stability in their growing environment and synthesize cosmic amounts of secondary metabolites into their intercellular spaces, which can be more easily extracted, and can be grown in a phytohormone amended medium with low inoculum but a high growth rate [59]. Wasnik et al., [60] established a protocol for large-scale cultivation of in vitro adventitious root (fig.2) cultures of W. somnifera in a bubble column bioreactor.

Alteration in the media composition enhanced the accumulation of withanolides

Carbon source

Plant cell cultures are usually grown heterotrophically using simple sugars as a carbon source and inorganic supply of other nutrients. The concentration of sucrose has been shown to affect the productivity of secondary metabolite-accumulating cultures. Sucrose is the most common choice in tissue culture media as it is reported to be the main sugar that can translocate in the phloem of many plants. Doma and co-workers [61] analyzed the influence of different concentrations of carbon sources on hairy root cultures of W. somnifera on enhancing withanolide A and withaferin A accumulation.

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Fig. 2: Induction of in vitro adventitious root cultures of W. somnifera and its mass cultivation [60]. (a) Leaf explants inoculated in rooting medium. (b) Induction of adventitious roots from leaf explants. (c) Multiplication of in vitro roots in bubble column bioreactor. (d) Mass cultivation of in vitro roots

The authors concluded that 1733 µg/g dry weight of Withaferin A accumulated in MS basal medium supplemented with 4% sucrose and added that significantly higher amount of Withanolide A and withaferin A were accumulated (890 and 886 µg/g dry weight respectively) only in medium supplemented with 3% sucrose. Similarly, Murthy et al. [62] reported that only low amounts of Withanolide A (157.4 µg/g dry weight) were accumulated in hairy root cultures in MS basal medium supplemented with 4% sucrose concentration. Sivanandhan et al. [8] reported that the hairy roots of W. somnifera grown in half MS liquid medium supplemented with sucrose (4%) stimulated higher production of withaferin A (2.21 mg/g DW) and withanone (2.41 mg/g DW) on the 40th day of culture, followed by a combination of sucrose and glucose enriched medium.

Nitrate levels

Nitrogen concentration was reported to affect the level of proteinaceous or amino acid products in cell suspension cultures. The plant tissue culture medium such as MS, LS or B5 has both nitrate and ammonium as sources of nitrogen. However, the ratio of the ammonium/nitrate–nitrogen and overall concentration of total nitrogen have been shown markedly to influence the production of secondary plant products. Nagella and Murthy [63] observed that the nitrate and ammonium ions have different effects on primary and secondary metabolism in plant cell and tissue cultures and concluded that cell suspensions of W. somnifera have shown that both the biomass and secondary metabolite accumulation were influenced by the concentration and composition of macro elements and the ammonia–nitrate ratio. In their experiment, maximum biomass growth (110.45 g l-1 FW and 9.29 g l-1 DW) was achieved at in cell suspension medium supplemented with an NH4+/NO3-in the concentration of 7.19/18.80, while withanolide A production was greatest (3.96 mg g-1 DW) in medium supplemented with an NH4+/NO3-in the concentration of 14.38/37.60 mM.

Growth regulators

The concentration of growth regulator is often a crucial factor in secondary product accumulation [64, 65]. The type and concentration of auxin or cytokinin or the auxin/cytokinin ratio alter both the growth and the product formation in cultured plant cells [66] dramatically. Phytohormones, specifically auxin plays an essential role in regulating root development, and it has been shown to be intimately involved in the process of adventitious rooting. Auxin, Indoleacetic acid (IAA) was shown to be involved in the rooting process by Thimann and Went as far back as 1934 [67], and a second ‘synthetic’ auxin indole-3-butyric acid (IBA) also promoted rooting [68]. Adventitious root formation has many practical implications in horticulture and agronomy and there is a lot of commercial interest because of the many plant species that are difficult to root [69, 70].

Ray and Jha [50] showed the accumulation of withanolides such as withaferin A and withanolide D in micro shorts in MS liquid medium supplemented with BAP and coconut water. Ahuja et al. [71] studied the accumulation of glycol withanolides. Mir et al. [72] reported that Withaferin A was produced in relatively high amounts (1.30 % and 1.10 % DW) in shoots of W. somnifera cultured in half and full strength MS liquid media respectively enriched with 0.5 µM BAP as compared to natural field grown plants (0.85 % DW).

Sivanandhan et al. [7] recorded that the polyamines along with plant growth regulators enhanced the withanolides production in in vitro-raised plants when compared to field grown parent plants. Shukla and co-workers [73] concluded that half strength liquid MS medium enriched with IAA in both 100 and 200 ppm concentration significantly elevated total alkaloid and withanolide content in hairy root cultures of W. somnifera when compared with control untreated root cultures. Thirugnanasambantham and co-workers [74] reported that IBA is an effective inducer of lateral root formation when compared to IAA. Accumulation of withanolide A and the biomass increases as the concentration of IBA increased to 1 mg/l (2576±0.37 μg/g DW and 12.89±0.25 g/dL respectively) and added that IAA at lower concentration favors relatively high accumulation of withanolide A (1147±0.77 μg/g DW) in 30 d old in vitro adventitious root cultures of W. somnifera.

Elicitation

It is well established in recent years that application of elicitors in root cultures can upgrade the secondary metabolite production in plant cell/organ culture. Secondary pathways are triggered in response to stress. Elicitors can be abiotic or biotic and act individually or in combination to turn on the biosynthesis of molecules that may only be produced in small amounts or may even produce new compounds [75]. Abiotic elicitors such as methyl jasmonate (MJ) and salicylic acid (SA) are used in combination with the culture for the highest level of secondary metabolite production.

Recently, Sivanandhan et al. [76] reported that when hairy roots of W. somnifera with an initial inoculum mass of 5 g fresh weight elicited separately with methyl jasmonate (MJ) and salicylic acid (SA) at various concentrations for different exposure times after 30 d of culture lead to an enhanced production of biomass (32.68 g FW and 5.54 g DW; 1.23-fold higher), withanolide A (132.44 mg/g DW; 58-fold higher), withanone (84.35 mg/g DW; 46-fold higher), and withaferin A (70.72 mg/g DW; 42-fold higher) from 40 d-old culture. Production of secondary metabolites in plant tissue, cell cultures, and adventitious root cultures was reported to be enhanced through elicitation with SA as in Glycyrrhiza glabra and W. somnifera, respectively [77,8]. Ciddi [78] reported 50-fold enhancement of withaferin A production (25 mg/l) using salacin as an elicitor in cell suspension culture of W. somnifera.

In a study conducted by Sivanandhan et al. [8], exposure to 30-day-old adventitious root cultures to 150 μM SA for 4 h as elicitor resulted in the production of 64.65 mg g−l dry weight (DW) withanolide A (48-fold), 33.74 mg g−l DW withanolide B (29-fold), 17.47 mg g−l DW withaferin A (20-fold), 42.88 mg g−l DW withanone (37-fold), 5.34 mg g−l DW 12-deoxy withastramonolide (nine fold), 7.23 mg g−l DW withanoside V (sevenfold), and 9.45 mg g−l DW withanoside IV (nine-fold) after 10 d of elicitation (40th day of culture) when compared to untreated cultures [8].

UV-B irradiation

Kalidhasan et al. [79] reported that Ultraviolet (UV-B) enhanced radiation have triggered higher level of withaferin A synthesis than that of control root sample and suggested a possible enhancement of the enzymes necessary for the biosynthesis of withaferin A. Same team workers have concluded that along with withaferin A, some other UV absorbing compounds have also accumulated in higher concentration in the field grown root samples. These results suggested that W. somnifera could possess possibly more precursors for the formation of phenolic compounds or UV absorbing compounds.

Thus, the above-mentioned techniques and approaches carried out in in vitro cultures enhances the tissue-specific accumulation of therapeutically significant withanolides in W. somnifera.

Understanding withanolide biosynthetic pathway

Understanding the steps involved in withanolide biosynthesis is essential for metabolic engineering of this plant to increase withanolide production. Withanolides are biosynthesized through the isoprenoid pathway, probably via both the mevalonate and nonmevalonate pathways [80] (fig. 3). The head-to-tail condensation of isopentenyl pyrophosphate (IPP) leads to the formation of farnesyl diphosphate (FPP) which is the main precursor for triterpenoids [81]. A key intermediate compound, 24-methylenecholesterol is an immediate precursor for biosynthesis of different withanolides, 24-methylene cholesterol is the first branching point towards the biosynthesis of different withanolides through a series of desaturation, hydroxylation, epoxidation, cyclization, chain elongation, and glycosylation steps [82, 83].

In plants, MVA is the general precursor of variously identified isoprenoids, such as sterols, plant growth regulators, and terpenoids [24, 25]. It has been recently reported that, in W. somnifera, both MVA and DOXP pathway participate in the biosynthesis of the withanolide and plastidic activity regulate this cross-talk to a varying level [83].

Fig. 3: Putative biosynthetic pathway of withanolide. 1-Deoxy-D-xylulose-5-phosphate synthase DXPS; 1-Deoxy-D-xylulose-5-phosphate reductoisomerase DXPR; 2-C-Methyl-D-erythritol 4-phosphate cytidylyltransferase MEP-CT; 4-(Cytidine 5′-diphospho)-2-C-methyl-D-erythritol kinase CDP-MEK; 2-C-Methyl-D-erythritol 2,4-cyclodiphosphate synthase MECDPS; (E)-4-Hydroxy-3-methylbut-2-enyl-diphosphate synthase HMBPPS; 4-Hydroxy-3-methylbut-2-enyl diphosphate reductase HMBPPR; Isopentenyl-diphosphate delta-isomerase IDI; Acetyl-CoA C-acetyltransferase ACAT; Hydroxymethylglutaryl-CoA synthase HMGCS; Hydroxymethylglutaryl-CoA reductase HMGR; Hydroxymethylglutaryl-CoA reductase HMGR; Mevalonate kinase MK; Phosphomevalonate kinase PVMK; Diphosphomevalonate decarboxylase MVD; Geranyl diphosphate synthase GPPS; Farnesyl diphosphate synthase FPPS; Squalene synthase SS; Squalene monooxygenase SE; Cycloartenol synthase CAS; Sterol 24-C-methyltransferase SMT1; Methyl sterol monooxygenase/Sterol-4a-methyl oxidase 2 SMO1/SMO2; Cycloeucalenol cycloisomerase CEC1; Obtusifoliol 14-demethylase CPY51G; Delta 14-sterol reductase FK; C-7,8 Sterol isomerase HYD1; C-5 Sterol desaturase STE1; 7-Dehydro cholesterol reductase DWF5; Sterol glycosyltransferases SGT; Methyltransferases MT

Elucidation of genes involved in withanolide biosynthetic pathway

3-Hydroxy-3-methylgutary coenzyme A reductase (HMGR)

In MVA pathway, 3-Hydroxy-3-methylgutary coenzyme A reductase (HMGR, EC 1.1.1.34) catalyzes the NAD (P) H-dependent reduction of HMG-CoA to mevalonate, the first committed step in the isoprenoid pathway, which produces the largest group of contemporary natural products [84]. This enzyme is located in the endoplasmic reticulum. According to Hemmerlin et al. [84], plant HMGR is known to be controlled by a number of developmental and environmental signals like phytohormones, calcium, calmodulin, light, wounding, elicitor treatment and pathogen attack. Akhtar et al. [85] reported that the expression level of WsHMGR (WsHMGR1) is the maximum in the flower followed by root tissue in W. somnifera. Recently, Senthil et al. [27] analyzed the expression pattern of HMGR in in vitro root and leaf cultures of W. somnifera. The authors observed that HMGR expression was higher in 30-day-old in vitro adventitious root cultures, whereas, in leaf tissue, the expression of HMGR was observed to be much lower.

Farnesyl diphosphate synthase (FPPS)

In these pathways, farnesyl diphosphate (FPP), which is synthesized by catalytic action of the enzyme farnesyl diphosphate synthase (FPPS), serves as a substrate for first committed reaction of several branched pathways [86] leading to the synthesis of compounds that are essential for plant growth and development as well as of pharmaceutical interest [87]. FPPS is one of the key enzymes [86] for isoprenoid biosynthesis which synthesizes sesquiterpene precursors for several classes of essential metabolites, including sterols, dolichols, ubiquinones and carotenoids as well as substrates for farnesylation and geranylgeranylation of proteins. This also plays an important role in commencing steps of triterpenoid precursor synthesis catering to withanolide biosynthesis.

FPPS catalyzed reaction occurs in two consecutive steps; condensation of isopentenyl diphosphate (IPP) with dimethylallyl diphosphate (DMAPP) to form 10-C intermediate geranyl diphosphate (GPP) and condensation of GPP with another molecule of IPP which results into 15-C FPP [87]. This gene has been characterized from variety of plant species including Arabidopsis [88], Artemisia [89], Hevea [90], maize [91], rice [92] and Taxus media [93].

Gupta et al. [6] isolated and characterized the gene encoding FPPS from W. somnifera. The authors also analyzed the expression pattern of FPPS in field grown young leaf, mature leaf, flower, fruit (green berry), stem and root tissues of W. somnifera and concluded that WsFPPS transcripts levels were significantly higher in young leaf than in mature. The same team reported that higher level of WsFPPS in flowers. On the contrary, lower expression of WsFPPS was reported to be recorded in roots. Recently, Thirugnanasambantham et al. [74] analyzes the expression of FPPS in field grown root and leaf tissues at different developmental stages of W. somnifera and concluded that WsFPPS expression levels were higher in leaves than the root tissues.

Sabir et al. [80] compared the expression levels of FPPS between in vitro and field grown tissues and observed that WsFPPS slightly upregulated in in vitro shoots in comparison to in vitro roots.

Squalene synthase (SQS)

Squalene synthase (SQS: EC 2.5.1.21) is an important regulatory enzyme of cholesterol biosynthetic pathway. It is a bifunctional enzyme which catalyzes the condensation of two molecules of farnesyl pyrophosphate (FPP) in a head-to-head manner to form pre-squalene diphosphate (PSPP) and then converts the PSPP to squalene in the presence of NADPH and Mg2+. As studied in engineered yeasts [94, 95] down-regulation of the squalene synthase in the sterol biosynthetic pathway leads to the accumulation of FPP, which is redirected away from this pathway and toward the synthesis of other commercially important isoprenoids. Gupta et al. [6] analyzed the expression levels of WsSQS using Quantitative real time–polymerase chain reaction (qRT-PCR) and demonstrated that WsSQS has a tissue-specific expression with highest expression in leaves and lowest in roots. The similar results were found with the expression of some other genes of the same pathway [96, 6]. On the contrary, Bhat et al. [97] reported that expression of squalene synthase (WsSQS) was seen in all tested tissues, including roots, stem and leaves with the highest level of expression in leaves.

Squalene epoxidase (SE)

The biosynthesis of the withanolide pathway up to squalene (catalyzed by SS) is an anaerobic process [98]. Squalene epoxidase (SE) catalyzes first step of oxygenation in this pathway and steps after squalene epoxidation are shared by sterol/brassinosteroids biosynthetic pathway [58]. SE (EC. 1.14.99.7) is one of the rate-limiting enzymes in the biosynthesis of triterpenoids, catalyzing the stereospecific epoxidation of squalene to 2,3-oxidosqualene. It requires the participation of flavoprotein NADPH-cytochrome P-450 reductase (E. C. 1.6.2.4) [98] and functions as a rate-limiting step in the sterol and triterpenoids biosynthesis [99]. A perusal of literature reveals that SE is a microsomal protein and also present in lipid droplets, but only ER-associated protein has been found to be active [100]. In addition to 2, 3-oxidosqualene, SE activity can result in the formation of 6, 7-oxidosqualene, 10, 11-oxidosqualene and dioxide squalene [101]. Being a rate limiting enzyme [99], overexpression of SE may have an important role in the regulation of phytosterols and steroidal lactones in W. somnifera.

Senthil et al. [27] very recently analyzed the expression of important pathway genes involved in withanolide biosynthesis. The authors reported that among the selected pathway genes, an expression level of SE was significantly higher in in vitro root tissues of Withania somnifera. Sabir et al. [80] also reported that higher expression of SE transcripts in field grown root than shoot tissues of W. somnifera. Gupta et al. [102] made the first attempt of cloning and characterization of this gene from W. somnifera. Thirugnanasambantham et al. [74] also analyzed expression levels of SE in field grown leaf and root tissues at different developmental stages of W. somnifera and concluded that expression levels of SE were significantly higher in field grown root tissue at yellow berry stage than leaf tissue and concluded that expression levels of WsSE exhibit a direct correlation with that of withanolide biosynthesis.

Cycloartenol synthase (CAS)

Cycloartenol and lanosterol are important membrane constituents that can serve as precursors to steroid hormones. It is formed from (S)-squalene-2, 3-epoxide by a cyclization reaction catalyzed by cycloartenol synthase (EC 5.4.99.8.). CAS performs the important function of breaking 11 bonds and forming 11 new ones to transform 2, 3-epoxysqualene to the plant sterol precursor cycloartenol [103].

It is presumed that cycloartenol bifurcation takes place for the biogenesis of sterol and withanolides in W. somnifera. Probably because of this division of cycloartenol, Dhar et al. [104] reported that WsCAS expression was the maximum and on the rise with each advancing phenol phase to generate a reservoir of cycloartenol which may get channelized towards the two routes leading to the biosynthesis of phytosterols and withanolides. The same team of workers identified that WsCAS exist in a copy number of two, thereby indicating the separate role of each copy of WsCAS in sterol and withanolide biosynthesis. The duplicate copy number of WsCAS is reported to be plausibly a trigger for higher expression.

It has been well documented that CAS plays an essential role in the plant cell viability and in the regulation of triterpenoid biosynthesis [105]. The differences in the biosynthesis of sterols between higher plants and yeast/mammals are generally accepted to begin at the cyclization step of 2, 3-oxidosqualene, a common precursor. Phytosterols, such as campesterol and sitosterol, are biosynthesized via cycloartenol and catalyzed by cycloartenol synthase (CAS) in higher plants [106]. Senthil et al. [27] reported that CAS genes exhibited higher expression at 45 d of growth in both leaf and root tissue under in vitro condition. Thirugnanasambantham et al. [74] expression of gene encoding cycloartenol synthase exhibited higher levels in both field grown leaf and root tissues, throughout the different developmental stages of W. somnifera.

Glucosyltransferase (GT)

Glycosylation of secondary plant products, such as flavonoids, coumarins, terpenoids, and cyanohydrins, is generally catalyzed by Plant secondary product glycosyltransferase (PSPGs) [107], which belong to family-1 glycosyltransferases, catalyzing glycosyl transfer (GT) from nucleoside diphosphate-activated sugars (donor) to aglycon substrate (acceptor) molecules. The activated sugar form is typically uridine diphosphate (UDP)-glucose, but UDP-galactose, UDP-glucuronide, UDP-xylulose, and UDP-rhamnose are also reported. Glycosylation not only stabilizes the products but also modulates their physiological activities and governs intracellular distribution [108].

In plants, sterols are biosynthesized by mevalonate and non-mevalonate pathways. They occur in highly diversified skeletal and structural forms that are finally glycosylated. Some of these (e. g. sitosterol, stigmasterol, brassinosteroids) are ubiquitous in plants whilst others (e. g. withanolides, limonoids) are highly restricted in occurrence. Earlier reports from Myxoamoeba and human fibroblastoma cell lines showed the activation of glucosyltransferase and the production of sterol glucoside following heat stress. The glucosides have been reported to induce the signal transduction pathway, leading to the synthesis of heat shock proteins during heat stress [109,110] in animal cells.

Madina et al. [82] discussed that expression of sterol glucosyltransferases was enhanced in leaves of W. somnifera following the application of salicylic acid. GTs that use UDPactivated sugars as donors and various types of small molecules as acceptors are called UDP-glycosyltransferases (UGTs) and represent family 1GTs. Such UGTs are present commonly in plants and animals but have been reported in a few cases only in microorganisms. In higher plants, UGT catalyzed glycosylation constitutes a prominent terminal modification in the biosynthesis of secondary metabolites and generates diverse natural glycosides [111]

In higher plants, secondary metabolites are often converted to their glycoconjugates, which are then accumulated and compartmentalized in vacuoles. These glycosylation reactions are catalyzed by glycosyltransferases (GTases) [112]. In general, glycosylation is the last step in the biosynthesis of secondary metabolites [113]. A vast variety of glycosyltransferase genes have been identified thus far, which are currently classified on the basis of their phylogenetics into 70 families [111].

Cytochrome P450 reductase (CPR)

CPRs (EC 1.6.2.4) are membrane-bound proteins localized to the ER, contain an N-terminal positioned Flavin mononucleotide (FMN) binding domain linked to NADPH binding domain via Flavine Adenine Dinucleotide (FAD) domain [114]. CPR shuttles electrons derived from NADPH through FAD and FMN domains into the heme iron centre of the various P450 enzymes and thus confront the high demand of electron supply during biotic and abiotic stress or differential expression at various stages of plant development [114]. It has been earlier demonstrated that CPR1 and CPR2 from different plant species have different specific activities and most of them have been assayed using a microsomal fraction or truncated polypeptide (without membrane anchor) [115]. Rana et al. [116] suggested that Cytochrome P450 reductase is the most imperative redox partner of multiple P450s involved in primary and secondary metabolite biosynthesis. Dhar and co-workers [104] cloned and functionally characterized two paralogs of NADPH-cytochrome P450 reductase (WsCPR1 and WsCPR2) and studied the expression pattern of the same in tissues of W. somnifera cultures in the field during different developmental stages.

The authors concluded that WsCPR2 showed a slight increase along the developmental phases and added that this expression level might be possibly implicating its role in the biosynthesis of withanolides. Rana et al. [116] conducted Quantitative real-time PCR to analyze the expression of WsCPR1 and WsCPR2 in various tissues of W. somnifera and concluded that both genes were widely expressed in leaves, stalks, roots, flowers and berries with the relatively higher expression level of WsCPR2 in comparison to WsCPR1. 

Transcriptome analysis

Next-generation sequencing (NGS) technology for transcriptome (RNA-seq) dispenses a new resolution for both obtaining gene sequences and quantifying transcriptome of any organism. In recent years, the RNA-seq has been an influential method for distinguishing genes involved in important secondary metabolite pathways such as biosynthesis of ginsenosides in Panax ginseng [117,118], carotenoids in Momordica cochinchinensis [119], flavonoids, theanine and caffeine in tea (Camellia sinensis), Initial efforts have been made to generate expressed sequence tags (ESTs) from in vitro tissues of W. somnifera [120].

Complete transcriptome analysis of W. somnifera leaf and root tissue was performed using next-generation sequencing in order to gain insights into withanolide biosynthesis pathways and their regulations [102]. A total number of 47,885 and 54,123 unigenes generated from leaf and root tissues, respectively, have been annotated using TAIR10 protein database (http://www. arabidopsis. org; Tair10), NCBI protein database NR (http://www. ncbi. nlm. nih. gov). Based on the sequence homology, the unigenes were categorized into 45 functional groups. On the basis of the annotation, the genes encoding enzymes involved in the biosynthesis of triterpenoid backbone (including MVA and MEP pathways) were identified from both leaf and root libraries. Apart from these, a number of methyltransferases, cytochrome P450s, glycosyltransferase, and transcription factors have also been identified and reported by [102]

Dasgupta et al. [121] analyze the salicylic acid induced leaf transcriptome of W. somnifera using Illumina Genome Analyzer. A total of 45.6 million reads was generated, and the de novo assembly yielded 73,523 transcript contig with average transcript contig length of 1620 bp were reported. A total of 71,062 transcripts was annotated and 53,424 of them were assigned GO terms. Mapping of transcript contigs to biological pathways revealed the presence of 182 pathways. Seventeen genes representing 12 pathogenesis-related (PR) families were mined from the transcriptome data and their pattern of expression post 17 and 36 h of salicylic acid treatment were documented. The authors reported that significant up-regulation of all families of PR genes by 36 h post-treatment.

Very recently, Senthil et al. [27] had used RNA-seq for large-scale transcriptome profiling and generated a comprehensive Transcriptome for W. somnifera by assembling the transcriptomes of in vitro adventitious root and leaf tissues from the millions of short sequence reads generated by Illumina. The authors obtained a total of 177,156 assembled transcripts with an average unigene length of 1,033 bp. About 13% of the transcripts were reported to be unique to in vitro adventitious roots, but no such transcripts were observed in in vitro-grown leaves. Annotations including functional annotation, Gene encoding enzymes involved in withanolide biosynthesis were identified using Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways.

In silico analysis

Application of in silico tools can significantly improve the detection of genes and variation. In silico drug design or the computer-aided drug design (CADD) play a significant role in all stages of drug development from the preclinical discovery stage to late stage of clinical development. Recently, few in silico studies were carried out demonstrating the role of withanolides as a potent drug. High-throughput docking has become increasingly important in the context of compound drug identification [122-124].

Aishwarya and Santhi [125] described the docking of 26 withaferins and 14 withanolides from W. somnifera into the three-dimensional structure of PknG of M. tuberculosis using GLIDE. The authors concluded that among the withanolides, withanolide E, F and D and Withaferin-diacetate 2 phenoxy ethyl carbonate was identified as potential inhibitors of PknG. This data provides the molecular insights to the consideration of Withanolides as potential candidates against the PknG target in M. tuberculosis. In another study conducted by Santhi and Aishwarya [126] alkaloids namely withasomine, cuseohygrine and ana hygrine from W. somnifera were docked deeply within the binding pocket region forming interaction with binding site residues of both 5-LOX and COX (anti-inflammatory drug target proteins) of humans. The authors concluded among the alkaloids: withasomine, cuseohygrine and anahygrine showed better inhibitory activity to both 5-LOX and COX, whereas, the other three alkaloids showing anti-inflammatory activity are anahygrine, 3-tropyltgloate and tropinine.

Vaishnavi et al. [127] examined the docking efficacy of Withaferin A (WA) and Withanone against four target proteins, namely, mortalin, p53, p21 and Nrf2.  The authors demonstrated that Withaferin A that binds strongly to the selected targets acts as a strong cytotoxic agent both in normal and cancer cells. Withanone, on the other hand, has a weak binding to the targets; it showed milder cytotoxicity towards cancer cells and was safe for normal cells. This study revealed an important insight to the use of Withaferin A and Withanone for cancer treatment and development of new anti-cancer phytochemical cocktails.

Prabhakaran et al. [128] extensively used graphical software (MOLSOFT) for the identification of the binding energy of selected Withanolides like Withaferin-A, Withanolide-D from W. somnifera and to screen the phytoconstituents that will dock/bind to the active sites of COX-2 enzyme. The authors concluded that among the two constituents, withaferin A possesses great activity on COX-2 followed by withanolide-D. Also added that Withanolides may have a direct action on cox-2 enzyme by binding to the Cys-907, Cys-910, Cys-962 and Cys-964 residue.

Recently, Saha et al. [129] carried out molecular docking studies to find the potentiality of Withaferin A, a key metabolite of W. somnifera, as an inhibitor of vascular endothelial growth factor (VEGF). Molecular Docking calculations were carried out on Withaferin A/Bevacizumab-VEGF protein model. The authors concluded that Withaferin A showed favorable binding with VEGF, and the results were highly comparable with the commercially available drug Bevacizumab. Bikadi et al. [130] demonstrated direct covalent binding of Withaferin A to Cys303 of tubulin in MCF-7 cells. Further added that WA-binding pocket is located on the surface of tubulin and characterized by a hydrophobic floor, a hydrophobic wall, and a charge-balanced hydrophilic entrance.

Proteomics

To have a better understanding of the processes that occur in W. somnifera, proteome analyses were initiated on seeds and leaf tissues by Gupta and Co-workers [6], From these analyses, 70 individual proteins from seeds and 74 from leaves were identified by protein sequence database interrogation and were cataloged accordingly to different protein functions. Senthil and co-workers [131] investigated comparative protein changes between the root tissues cultivated in vitro and from the field. The authors recorded a similarity in protein spots in both in vitro and in vivo root samples and concluded that in vitro roots that are developed independently of shoot organs appear to have a similar developmental process as that of in vivo roots.

Metabolomics

The comprehensive chemical analysis is required not only to establish a correlation between complex chemical mixtures and molecular pharmacology but also to understand complex cellular processes and biochemical pathways via metabolite-to-gene network [23]. Chatterjee et al. [132] carried out metabolic profiling of crude extracts of leaf and root of Withania somnifera using Nuclear Magnetic Resonance (NMR) and chromatographic (High-Performance Liquid Chromatography (HPLC) and Gas Chromatography–Mass Spectrometry GC–MS)) techniques. A total of 62 major and minor primary and secondary metabolites from leaves and 48 from roots were unambiguously identified. Senthil et al. [133] compared the total metabolome profiling between in vitro and field grown root tissues of Withania somnifera using GC-MS technique. The authors reported that total of 29 metabolites was identified in in vitro cultured and field-grown roots by GC-MS analysis. The metabolites included alcohols, organic acids, purine, pyrimidine, sugars, and putrescine. In their study, vanillic acid was only observed in the in vitro cultured root samples. Very recently, Thirugnanasambantham et al. [134] compared the metabolite profiles between leaf tissues of W. somnifera cultured in in vitro and field grown conditions. The authors highlighted that in vitro leaf cultures able to accumulate metabolites in a similar fashion as that of field grown tissues and can be utilized as alternative resources to field-grown leaves for the production of useful metabolites such as γ-aminobutyric acid and putrescine. These results highlighted the potentiality of in vitro roots as an alternative to field-grown roots.

CONCLUSION

Until date only very less information is available on structural and functional aspects of enzymes involved in withanolide biosynthetic pathway of Withania somnifera. This review gives a clear understanding of various technical approaches carried out in W. somnifera and emphasized the worldwide achievements associated with understanding the synthesis and regulation of pharmaceutically important secondary metabolites. These new technologies will serve to extend and enhance the continued usefulness of in vitro cultured plants as renewable sources of medicinal compounds.

ACKNOWLEDGEMENT

This work was supported by WOS-A Program of the Department of Science and Technology [SR/WOS-A/lS-532/2011(G)], New Delhi.

CONFLICT OF INTERESTS

We declare that we have no conflicts of interest in the authorship or publication of this contribution.

REFERENCES

  1. Gupta GL, Rana AC. Withania somnifera (aswagandha): a review. Pharmacognosy 2007;1:129–36.
  2. Davis L, Kuttan G. Effect of Withania somnifera on DMBA induced carcinogenesis. J Ethanopharmacol 2001;75:165–8.
  3. Kumar A, Kaul MK, Bhan MK, Khanna PK, Suri KA. Withania somnifera (L.) Dunal (Solanaceae). Genet Resour Crop Evol2007;54:655–60.
  4. Asthana R, Raina MK. Pharmacology of Withania somnifera (L.) Dunal-a review. Indian Drugs 1989;26:199–205.
  5. Singh S, Kumar S. Withania somnifera:The Indian Ginseng Ashwagandha, Central Institute of Medicinal and Aromatic Plants, Lucknow, India; 1998.
  6. Gupta P, Akhtar N, Tewari SK, Sangwan RS, Trivedi PK. Differential expression of farnesyl diphosphate synthase gene from Withania somnifera in different chemotypes and in response to elicitors. Plant Growth Regul 2011;65:93–100.
  7. Sivanandhan G, Arun M, Mayavan S, Rajesh M, Jeyaraj M. Optimization of elicitation conditions with methyl jasmonate and salicylic acid to improve the productivity of withanolides in the adventitious root culture of Withania somnifera (L.) Dunal. Appl Biochem Biotechnol2011;168:681–96.
  8. Sivanandhan G, Arun M, Mayavan S, Rajesh M, Mariashibu TS. Chitosan enhances withanolides production in adventitious root cultures of Withania somnifera(L.) Dunal. Ind Crop Prod2012;37:124–9.
  9. Sharada M, Ahuja A, Suri KA, Vij SP, Khajuria RK, Verma V, et al. Withanolide production by in vitro cultures of Withania somnifera and its association with differentiation. Biol Plantarum 2007;51:161–4.
  10. Sangwan RS, Chaurasiya ND, Lal P, Misra L, Uniyal GC, Tuli R, et al. Withanolide A biogeneration in in vitroshoot cultures of Ashwagandha (Withania somnifera Dunal), a main medicinal plant in Ayurveda. Chem Pharm Bull2007;55:1371–5.
  11. Sudha CG, Seeni S. Establishment and analysis of fast-growing normal root culture of Decalepis arayalpathra, a rare endemic medicinal plant. Curr Sci 2001;81:371–4.
  12. Rani G, Arora S, Nagpal A. Direct rhizogenesis from in vitro leaves of Withania somnifera (L.) Dunal. J Herbs Spices Med Plants2003;10:47–54.
  13. Wadegaonkar PA, Bhagwat KA, Rai MK. Direct rhizogenesis and the establishment of fast-growing normal root organ culture of Withania somnifera Dunal. Plant Cell Tissue Organ Cult 2006;84:223–5.
  14. Praveen N, Murthy HN. Production of withanolide A from adventitious root cultures of Withania somnifera. Acta Physiol Plant 2010;5:1017–22.
  15. Pradeepa D, Kalaiselvi R, Pankajavalli T, Senthil K. Effect of sucrose and auxin concentration on induction of in vitro adventitious roots of Withania somnifera. Int J Pharma Bio Sci 2014;5:596–603.
  16. Abraham A, Kirson I, Glotter E, Lavie DA. Chemotaxonomic study of Withania somnifera (L.) dunal. Phytochemistry 1968;7:957–62.
  17. Dhar RS, Verma V, Suri KA, Sangwan RS, Satti NK, Kumar A, et al. Phytochemical and genetic analysis in selected chemotypes of Withania somnifera. Phytochemistry 2006;67:2269–76.
  18. Sangwan RS, Chaurasia ND, Mishra LN, Lal P, Uniyal GC, Sharma R, et al. Phytochemical variability in commercial herbal products and preparation of Withania somnifera (Ashwagandha). Curr Sci 2004;86:461–5.
  19. Deocaris CC, Widodo N, Wadhwa R, Kaul S. Merger of ayurveda and tissue culture based function genomics: inspiration from system biology. Rev J Trans Med 2008;6:1–8.
  20. Mohn T, Plitzko I, Hanburger MA. Comprehensive metabolite profiling of Isatis tinctoria leaf extracts. Phytochemistry 2009;70:924–34.
  21. Shyur LF, Yang NS. Metabolomics for phytomedicine research and drug development. Curr Opin Chem Biol 2008;12:66–71.
  22. Wang M, Robert JA, Lamers N, Kothout HAAJ, Nesselrooij JHJV, Witkamp RF, et al. Metabolomics in the context of system biology: bridging traditional Chinese medicine and molecular pharmacology. Phytother Res 2005;19:173–82.
  23. Nakabayashi R, Kusano M, Kobayashi M, Tohge T, Keiko YS, Kogure N, et al. Metabolomics oriented isolation and structure elucidation of 37 compounds including two anthocyanins from Arabidopsis thaliana. Phytochemistry 2009;70:1017–29.
  24. Wani MC, Taylor HL, Wall ME, Coggon P, McPhail AT. Plant antitumor agents: The isolation and structure of taxol, a novel antileukemic and antitumor agent from Taxus brevifolia. J Am Chem Soc 1971;93:2325–7.
  25. Schepmann HG, Pang J, Matsuda SP. Cloning and characterization of Ginkgo biloba Clevo pimaradiene synthase which catalyzes the first committed step in ginkgolide biosynthesis. Arch Biochem Biophys 2001;392:263–9.
  26. Chaurasiya ND, Uniyal GC, Lal P, Mishra L, Sangwan NS, Tuli R, Sangwan RS. Analysis of withanolides in roots and leaf of Withania somnifera by HPLC with photodiode array and evaporative light scattering detection. Phytochem Anal 2008;19:148–54.
  27. Senthil K, Jayakodi M, Pankajavalli T, Lee SC, Pradeepa D, Purushotham PM, et al. Transcriptome analysis reveals in vitro cultured Withania somnifera leaf and root tissues as a promising source for targeted withanolide biosynthesis. BMC Genomics 2015;16:14.
  28. Elsakka M, Grigorescu E, Stanescu U, Dorneanu V. New data referring to the chemistry of Withania somnifera species. Rev Med Chir Soc Med Nat Iasi 1990;94:385–7.
  29. Thirugnanasambantham P, Kalaiselvi R, Pradeepa D, Senthil K. Effect of exogenous indole-3-butyric acid and indole-3-acetic acid on biomass and legendary withanolides from in vitro root cultures of Withania somnifera–Jawahar 20 cultivar, Int J Pharma Bio Sci 2014;5:971–9.
  30. Bone K. Clinical applications of ayurvedic and Chinese herbs: monographs for the Western herbal practitioner. Phytotherapy Press: Queensland, Australia; 1996. p. 137–41.
  31. Grandhi A, Mujumdar AM, Patwardhan B. A comparative pharmacological investigation of ashwagandha and ginseng. J Ethnopharmacol 1994;44:131–5.
  32. Mishra LC, Singh BB, Dagenais S. Scientific basis for the therapeutic use of Withania somnifera (ashwagandha): a review. Altern Med Rev 2000;5:334–46.
  33. Kuboyama T, Tohda C, Komatsu K. Neuritic regeneration and synaptic reconstruction induced by withanolide-A. Br J Pharmacol 2005;144:961–71.
  34. Malik F, Singh J, Khajuria A, Suri KA, Satti NK. A standardized root extract of Withania somnifera and its major constituent withanolide-A solicit humoral and cell-mediated immune responses by up-regulation of Th1-dominant polarization in BALB/c mice. Life Sci 2007;80:1525–38.
  35. Khan NA, Nazar R, Anjum NA. Growth, photosynthesis and antioxidant metabolism in mustard (Brassica juncea L.) cultivars differing in ATP-sulfurylase activity under salinity stress. Sci Hort 2009;122:455–60.
  36. Ku SK, Bae JS. Antiplatelet, anticoagulant, and profibrinolytic activities of withaferin A. Vasc Pharmacol 2014;60:120–6.
  37. Mehta AK, Binkley P, Gandhi SS, Ticku MK. Pharmacological effects of Withania somnifera root extract on GABAA receptor complex. Indian J Med Res 1991;94:312–5.
  38. Ahmad M, Saleem S, Ahmad AS, Ansari MA, Yousuf S, Hoda MN, et al. Neuroprotective effects of Withania somnifera on 6-hydroxydopamine induced Parkinsonism in rats. Hum Exp Toxicol 2005;24:137–47.
  39. Meena M, Lakshmi T. Antiepileptic activity of indigenous herbal extracts a review. Asian J Pharm Clin Res 2013;6:12–4.
  40. Misra L, Lal P, Sangwan RS, Sangwan NS, Uniyal GC, Tuli R. Unusually sulfated and oxygenated steroids from Withania somnifera. Phytochemistry 2005;66:2702–7.
  41. Lal P, Misra L, Sangwan RS, Tuli R. New withanolides from fresh berries of Withania somnifera. Z Naturforsch B: J Chem Sci 2006;61:1143–7.
  42. Auddy B, Hazar J, Mitra A, Abedon B, Ghosal S. A standardized Withania Somnifera extract significantly reduced stress-related parameters in chronically stressed humans: a double-blind, randomised, placebo-controlled study. Cite Seer 2008;11:50–6.
  43. Berlin J, Rehm HJ, Reed G. editors. Secondary products from plant cell cultures. In: Biotechnology a comprehensive treatise. Weinheim; 1986;4:630–58.
  44. Alfermann AW, Petersen M. Natural product formation by plant cell biotechnology–results and perspectives. Plant Cell  Tissue Organ Cult 1995;43:199–205.
  45. Yu PLC, El Olemy MM, Stohs ST. A phytochemical investigation of Withania somnifera tissue cultures. J Nat Prod 1974;37:593–7.
  46. Heble MR, Neumann KH, Barz W, Reinhard E. editors. Multiple shoot cultures: a viable alternative in vitro system for the production of known and new biologically active plant constituents. In: Primary and Secondary Metabolism of Plant Cell Cultures, Springer-Verlag, Berlin; 1985:281–9.
  47. Roja G, Heble MR, Sipahimalini AT. Tissue cultures of Withania somnifera:morphogenesis and withanolide synthesis. Phytother Res 1991;5:185-7.
  48. Banerjee S, Naqvi AA, Mandal S, Ahuja PS. Transformation of Withania somnifera (L.) Dunal by Agrobacterium rhizogenes:Infectivity and phytochemical studies. Phytother Res 1994; 8:452–5.
  49. Vitali G, Conte L, Nicoletti M. Withanolide composition and in vitro culture of Italian Withania somnifera. Planta Med 1996;62:287–8.
  50. Ray S, Jha S. Production of withaferin A in shoot cultures of Withania somnifera. Planta Med 2001;67:432–6.
  51. Rani G, Virk GS, Nagpal A. Somatic embryogenesis in Withania somnifera (L.) dunal. J Plant Biotechnol 2004;6:113–8.
  52. Sivanesan I, Murugesan K. In vitro adventitious shoot formation from leaf explants of Withania somnifera dunal. Plant Cell Biotechnol Mol Biol 2005;6:163–6.
  53. Singh AK, Varshney R, Sharma M, Agarwal SS, Bansal KC. Regeneration of plants from alginate-encapsulated shoot tips of Withania somnifera (L.) Dunal, a medicinally important plant species. J Plant Physiol 2006;163:220–3.
  54. Sivanesan I. Direct regeneration from apical bud explants of Withania somnifera dunal. Indian J Biotechnol 2007;6:125–7.
  55. Sharma MM, Ali DJ, Batra A. Plant regeneration through in vitro somatic embryogenesis in ashwagandha (Withania somnifera L dunal). Researcher 2010;2:1–6.
  56. Ghimire BK, Seong ES, Kim EH, Lamsal K, Yu CY, Chung IM. Direct shoot organogenesis from petiole and leaf discs of Withania somnifera (L.) Dunal. Afr J Biotechnol 2010;9:7453–61.
  57. Rao S, Teesta VK, Bhattrai A, Khushi K, Bhat S. In vitro propagation of Withania somnifera and estimation of withanolides for neurological disorders. J Pharmacogn 2012;3:85–7.
  58. Sangwan RS, Chaurasiya ND, Sangwan PL, Misra LN, Tuli R, Sangwan NS. Withanolide A is inherently de novo biosynthesized in roots of the medicinal plant Ashwagandha (Withania somnifera). Physiol Plantarum 2008;133:278–87.
  59. Sivakumar G. Bioreactor technology: a novel industrial tool for the high-tech production of bioactive molecules and biopharmaceuticals from plant roots. Biotechnol J 2006;1: 1419–27.
  60. Wasnik NG, Muthusamy M, Chellappan S, Vaidhyanathan V, Pulla R, Senthil K. Establishment of in vitro root cultures and analysis of secondary metabolites in Indian Ginseng-Withania somnifera. Korean J Plant Res 2009;22:584–91.
  61. Doma M, Abhayankar G, Reddy VD, Kishor PB. Carbohydrate and elicitor are enhanced withanolide (withaferin A and withanolide A) accumulation in hairy root cultures of Withania somnifera (L.). Indian J Exp Biol 2012;50:484–90.
  62. Murthy HN, Dijkstra C, Anthony P, White DA, Davey MR, Power JB, et al. Establishment of Withania somnifera hairy root cultures for the production of Withanolide A. J Integr Plant Biol 2008;50:975–81.
  63. Nagella P, Murthy HN. Effects of macro elements and nitrogen source on biomass accumulation and withanolide-A production from cell suspension cultures of Withania somnifera (L.) Dunal. Plant Cell Tiss Org 2011;104:119–24.
  64. DiCosmo F, Towers GHN. Stress and secondary metabolism in cultured plant cells. In: Phytochemical adaptations to stress: Springer US; 1984. p. 97–175.
  65. Deus NBS, Zenk MH. Exploitation of plant cells for the production of alkaloids in Catharanthus roseus cell suspension cultures. Planta Med 1982;50:427–31.
  66. Mantell SH, Smith H. Cultural factors that influence secondary metabolite accumulations in plant cell and tissue cultures. In: Seminar series-Society for Experimental Biology; 1984. p. 75–108.
  67. Thimann KV, Went FW. On the chemical nature of the root forming hormone. Proc Kon Ned Acad Wetensch 1934;37:456–59.
  68. Zimmerman PW, Wilcoxon F. Several chemical growth substances which cause initiation of roots and other responses in plants. Contrib Boyce Thompson Inst 1935;7:209–29.
  69. Davies JR, Davis TD, Kester DE. The commercial importance of adventitious rooting to horticulture. In: Biology of adventitious root formation. Springer US; 1994. p. 53–9.
  70. Kovar JL, Kuchenbuch RO. The commercial importance of adventitious rooting to agronomy. In: Biology of adventitious root formation, Springer US; 1994. p. 25–34.
  71. Ahuja A, Kaur D, Sharada M, Kumar A, Suri KA, Dutt P. Glycowithanolides accumulation in in vitroshoot cultures of Indian ginseng (Withania somnifera Dunal). Nat Prod Commun 2009;4:479–82.
  72. Mir BA, Khazir J, Khalid R, Hakeem R, Koul S, Don A. Cowan enhanced production of withaferin-a in shoot cultures of Withania somnifera (L) Dunal. J Plant Biochem Biotechnol 2014;23:430–4.
  73. Shukla KK, Mahdi AA, Ahmad MK, Shankwar SN, Jaiswar SP, Tiwari SC. Mucana pruriens reduces stress and improves the quality of semen in infertile males. J Evidence-Based Complementary Altern Med 2012;7:137–40.
  74. Thirugnanasambantham P, Roy IM, Nancy S, Senthil K. Ontogenetic assessment of Withanolide biogenesis and expression of selected pathway genes in Withania somnifera, a traditional medicinal herb. J Pharm Res 2014;8:1344–51.
  75. Weathers PJ, Towler MJ, Xu J. Bench to batch: advances in plant cell culture for producing useful products. Appl Microbiol Biotechnol 2010;85:1339–51.
  76. Sivanandhan G, Kapil Dev G, Jeyaraj M, Rajesh M, Arjunan A, Muthuselvam M. et al. Increased production of withanolide A, withanone, and withaferin A in hairy root cultures of Withania somnifera (L.) Dunal elicited with jasmonate and salicylic acid. Plant Cell Tiss Organ Cult 2013;114:121–9.
  77. Shabani L, Ehsanpour AA, Asghari G, Emami J. Glycyrrhizin production by in vitro cultured Glycyrrhiza glabra elicited by methyl jasmonate and salicylic acid. Russ J Plant Physiol 2009;56:621–6.
  78. Ciddi V. Withaferin A from cell cultures of Withania somnifera. Indian J Pharm Sci 2006;68:490–2.
  79. Kalidhasan N, Boopala Bhagavan N, Kannan ND. Ultraviolet-B (280-320 nm) enhanced radiation-induced changes in secondary metabolites and photosystem-II of medicinal plant Withania somnifera Dunal. J Med Plant Res 2013;7:3112–20.
  80. Sabir F, Mishra S, Sangwan RS, Jadaun JS, Sangwan NS. Qualitative and quantitative variations in withanolides and expression of some pathway genes during different stages of morphogenesis in Withania somnifera Dunal. Protoplasma 2012;250:539–49.
  81. Kuzuyama T. Mevalonate and nonmevalonate pathways for the biosynthesis of isoprene units. Biosci Biotechnol Biochem 2002;66:1619–27.
  82. Madina BR, Sharma LK, Chaturvedi P, Sangwan RS, Tuli R. Purification and characterization of a novel glycosyltransferase specific to 27b-hydroxy steroidal lactones from Withania somnifera and its role in stress responses. Biochem Biophys Acta 2007;1774:1199–207.
  83. Chaurasia ND, Sangwan NS, Sabir F, Misra L, Sangwan RS. Withanolide biosynthesis recruits both mevalonate and DOXP pathways of isoprene genesis in Ashwagandha Withania somnifera L. (Dunal). Plant Cell Rep 2012;31:1889–97.
  84. Hemmerlin A, Harwood JL, Bach TJ. A raison d’être for two distinct pathways in the early steps of plant isoprenoid biosynthesis? Prog Lipid Res 2012;51:95–148.
  85. Akhtar N, Gupta P, Sangwan NS, Sangwan RS, Trivedi PK. Cloning and functional characterization of 3–hydroxyl–3 methyl glutaryl Withania somnifera:an important medicinal plant. Protoplasma 2013;250:613–22.
  86. Chappell J. The biochemistry and molecular biology of isoprenoid metabolism. Plant Physiol 1995;107:1–6.
  87. Mirjalili MH, Moyano E, Bonfil M, Cusido RM, Palazon J. Steroidal lactones from Withania somnifera, an ancient plant for novel medicine. Molecules 2009;14:2373–93.
  88. Closa M, Vranova E, Bortolotti C, Bigler L, Arro M, Ferrer A, et al. The Arabidopsis thaliana FPP synthase isozymes have overlapping and specific functions in isoprenoid biosynthesis, and complete loss of FPP synthase activity causes early developmental arrest. Plant J 2010;63:512–25.
  89. Matsushita Y, Kang W, Charlwood BV. Cloning and analysis of a cDNA encoding farnesyl diphosphate synthase from Artemisia annua. Gene 1996;172:207–9.
  90. Takaya A, Zhanga YW, Asawatreratanakul K, Wititsuwannakul D, Wititsuwannakul R, Takahashi S, et al. Cloning, expression, and characterization of a functional cDNA clone encoding GGPPS of Hevea brassiliensis. Biochem Biophy Acta 2003;1625:214–20.
  91. Cervantes MC, Gallagher CE, Zhu C, Wurtzel ET. Maize cDNAs expressed in endosperm encode functional farnesyl diphosphate synthase with geranylgeranyl diphosphate synthase. Plant Physiol 2006;141:220–31.
  92. Sanmiya K, Iwasaki T, Matsuoka M, Miyao M, Yamamoto N. Cloning of a cDNA that encodes farnesyl diphosphate synthase and the blue-light-induced expression of the corresponding gene in the leaves of rice plants. Biochim Biophys Acta 1997;1350:240–6.
  93. Liao ZH, Chen M, Gong YF, Zuo KJ, Wang P, Tan F, et al. A new farnesyl diphosphate synthase gene from Taxus media rehder: cloning, characterization, and functional complementation. J Integr Plant Biol 2006;48:692–9.
  94. Paradise EM, Kirby J, Chan R, Keasling JD. Redirection of flux through the FPP branch-point in Saccharomyces cerevisiae by down-regulating squalene synthase. Biotechnol Bioeng 2008;100:371–8.
  95. Shimada H, Kondo K, Fraser PD, Miura Y, Saito T, Misawa N. Increased carotenoid production by the food yeast Candida utilis through metabolic engineering of the isoprenoid pathway. Appl Environ Microbiol 1998;64:2676–80.
  96. Yin T, Liu J, Ye N, Chen Y, Yin T, Liu M, Hassani D. Transcriptome analysis of the differentially expressed genes in the male and female shrub willows (Salix suchowensis). PloS one 2013;8:e60181. Doi:10.1371/journal.pone.0060181. [Article in Press]
  97. Bhat WW, Lattoo SK, Razdan S, Dhar N, Rana S, Dhar RS, et al. Molecular cloning, bacterial expression and promoter analysis of squalene synthase from Withania somnifera (L.) Dunal. Gene 2012;499:25–36.
  98. Abe I, Abe T, Lou W, Masuoka T, Noguchi H. Site directed mutagenesis of conserved aromatic residues in squalene rat epoxidase. Biochem Biophys Res Commun 2007;352:259–63.
  99. Han JY, In JG, Kwon YS, Choi YE. Regulation of ginsenoside and phytosterol biosynthesis by RNA interferences of squalene epoxidase gene in Panax ginseng. Phytochemistry 2010;71:36–46.
  100. Leber R, Landl K, Zinser E, Ahorn H, Spok A, Kohlwein SD, et al. Dual localization of squalene epoxidase, Erg1p, in yeast reflects a relationship between the endoplasmic reticulum and lipid particles. Mol Biol Cell 1998;9:375–86.
  101. Bai M, Prestwich GD. Inhibition and activation of porcine squalene epoxidase. Arch Biochem Biophys 1992;293:305–13.
  102. Gupta P, Goel R, Pathak S, Srivastava A, Singh SP, Sangwan RS, et al. De novo assembly, functional annotation and comparative analysis of Withania somnifera leaf and root transcriptomes to identify putative genes involved in the withanolides biosynthesis. PLoS One 2013;8:e62714. Doi: 10.1371/ journal.pone.0062714. [Article in Press]
  103. Rees HH, Goad LJ, Goodwin TW. 2, 3-Oxidosqualene cycloartenol cyclase from Ochromonas malhamensis. Biochim Biophys Acta 1969;176:892–4.
  104. Dhar N, Rana S, Bhat WW, Razdan S, Pandith SA, Khan S. Dynamics of withanolide biosynthesis in relation to temporal expression pattern of metabolic genes in Withania somnifera (L.) Dunal: a comparative study in two morpho-chemovariants. Mol Biol Rep 2014;40:7007–116.
  105. Diarra S, He J, Wang J, Li J. Ethylene treatment improves diosgenin accumulation in in vitrocultures of Dioscorea zingiberensis via up-regulation of CAS and HMGR gene expression. Electron J Biotechnol 2013;16:6.
  106. Ohyama K, Suzuki M, Kikuchi J, Saito K, Muranaka T. Dual biosynthetic pathways to phytosterol via cycloartenol and lanosterol in Arabidopsis. Proc Natl Acad Sci 2008;106:725–30.
  107. Noguchi A, Saito A, Homma Y, Nakao M, Sasaki N, Nishino T. A UDP-glucose: isoflavone 7-O-glucosyltransferase from the roots of soybean (Glycine max) seedlings. Purification, gene cloning, phylogenetics, and implication for an alternative strategy of enzyme catalysis.J Biol Chem 2007;282:23581–90.
  108. Ullmann P, Ury A, Rimmele D, Benveniste P, Bouvier-Nave P. UDP-glucose sterol beta-D-glucosyltransferase, a plasma membrane-bound enzyme of plants: enzymatic properties and lipid dependence. Biochimie 1993;75:713–23.
  109. Murakami-Murofushi K, Nishikawa K, Hirakawa E, Murofushi H. Heat stress induces a glycosylation of membrane sterol in myxoamoebae of a true slime mold, Physarum polycephalum. J Biol Chem 1997;272:486–9.
  110. Kunimoto S, Murofushi W, Kai H, Ishida Y, Uchiyama A, Kobayashi T, et al. Steryl glucoside is a lipid mediator in stress-responsive signal transduction. Cell Struct Funct 2002;27:157–62.
  111. Sharma LK, Madina BR, Chaturvedi P, Sangwan RS, Tuli R. Molecular cloning and characterization of one member of a 3b-hydroxy sterol glucosyltransferase gene family in Withania somnifera. Arch Biochem Biophys 2007;460:48–55.
  112. Taguchi G, Yazawa T, Hayashida N, Okazaki M. Molecular cloning and heterologous expression of novel glycosyltransferases from tobacco cultured cells that have broad substrate specificity and are induced by salicylic acid and auxin. Eur J Biochem 2000;268:4086–94.
  113. Sun YG, Wang B, Jin SH, Qu XX, Li Y, BHou BK. Ectopic expression of Arabidopsis glycosyltransferase UGT85A5 enhances salt stress tolerance in tobacco. PLoS One 2013;8:e59924. Doi:10.1371/journal.pone.0059924. [Article in Press]
  114. Simmons DL, Lalley PA, Kasper CB. Chromosomal assignments of genes coding for components of the mixed-function oxidase system in mice. Genetic localization of the cytochrome P-450PCN and P-450PB gene families and the nadph-cytochrome P-450 oxidoreductase and epoxide hydratase genes. J Biol Chem 1985;260:515–21.
  115. Huang FC, Sung PH, Do YY, Huang PL. Differential expression and functional characterization of the NADPH cytochrome P450 reductase genes from Nothapodytes foetida. Plant Sci 2012;190:16–23.
  116. Rana S, Lattoo SK, Dhar N, Razdan S, Bhat WW, Dhar RS, et al. NADPH-cytochrome P450 reductase: molecular cloning and functional characterization of Two paralogs from Withania somnifera (L.) dunal. PLoS One 2013;8:e57068.  Doi:10.1371/journal.pone.0057068. [Article in Press]
  117. Luo H, Sun C, Sun Y, Wu Q, Li Y. Analysis of the transcriptome of Panax notoginseng root uncovers putative triterpene saponin-biosynthetic genes and genetic markers. BMC Genomics 2011;12:S5.
  118. Hyun TK, Rim Y, Jang HJ, Kim CH, Park J, Kumar R. De novo transcriptome sequencing of Momordica cochinchinensis to identify genes involved in the carotenoid biosynthesis. Plant Mol Biol 2012;79:413–27.
  119. Shi CY, Yang H, Wei CL, Yu O, Zhang ZZ, Jiang CJ. Deep sequencing of the Camellia sinensis transcriptome revealed candidate genes for major metabolic pathways of tea-specific compounds. BMC Genomics 2011;12:131–5.
  120. Senthil K, Wasnik NG, Kim YJ, Yang DC. Generation and analysis of expressed sequence tags from leaf and root of Withania somnifera (Ashwgandha). Mol Biol Rep 2010;37:893–902.
  121. Dasgupta MG, George BS, Bhatia A, Sidhu OP. Characterization of Withania somnifera leaf transcriptome and expression analysis of pathogenesis–related genes during salicylic acid signaling. PLoS One 2014;9.  Doi:10.1371/journal.pone.0094803. [Article in Press]
  122. Schneider G, Böhm HJ. Virtual screening and fast automated docking methods. Drug Discovery 2002;7:64–70.
  123. Waszkowycz B. Structure-based approaches to drug design and virtual screening. Curr Opin Drug Discovery 2002;5:407–13.
  124. Toledo-Sherman LM, Chen D. High-throughput virtual screening for drug discovery in parallel. Curr Opin Drug Discovery 2002;5:414–21.
  125. Aishwarya S, Santhi N. Insights from the molecular docking of withanolide derivatives to the target protein PknG from Mycobacterium tuberculosis. Bioinformation 2011;7:1–4.
  126. Santhi N, Aishwarya S. Identification of potential anti-inflammatory bioactive compounds from Withania somnifera (L.) Dunal: Molecular docking studies using Glide. Poster presented at The Eighth Asia Pacific Bioinformatics Conference; 2011.
  127. Vaishnavi K, Saxena N, Shah N, Singh R, Manjunath K, Uthayakumar M, et al. Differential activities of the two closely related withanolides, Withaferin A and Withanone: bioinformatics and experimental evidences. PloS One 2012;7:e44419. Doi:10.1371/journal.pone.0044419. [Article in Press]
  128. Prabhakaran Y, Dinakaran SK, Macharala SP, Ghosh S, Karanam SR, Kanthasamy N, et al. Molecular docking studies of withanolides against Cox-2 enzyme. Pak J Pharm Sci 2012;25:595–8.
  129. Saha S, Islam MK, Shilpi JA, Hasan S. Inhibition of VEGF: a novel mechanism to control angiogenesis by Withania somnifera’s key metabolite Withaferin A. In Silico Pharmacol 2013;1:11–5.
  130. Bikadi Z, Demko L, Hazai E. Functional and structural characterization of a protein based on analysis of its hydrogen bonding network by hydrogen bonding plot. Arch Biochem Biophys 2007;461:225–34.
  131. Senthil K, Karunanithi N, Kim GS, Nagappan A, Sundareswaran S, Natesan S, et al. Proteome analysis of in vitro and in vivo root tissue of Withania somnifera. Afr J Biotechnol 2013;74:16866–74.
  132. Chatterjee S, Srivastava S, Khalid A, Singh N, Sangwan RS. Comprehensive metabolic fingerprinting of Withania somnifera leaf and root extracts. Phytochemistry 2010;71:1085–94.
  133. Senthil K, Pankajavalli T, Oh TJ, Kim SH, Choi HK. Free radical scavenging activity and comparative metabolic profiling of in vitro cultured and field grown Withania somnifera roots. PLoS one2015;4:e0123360. Doi:10.1371/journal.pone.0123360. [Article in Press]
  134. Thirugnanasambantham P, Senthil K, Oh TJ, Coi HK. Comparative chemometric profiles between leaf tissues of Withania somnifera Cultured in vitro and field. Int J Pharm Pharm Sci 2015;7:66-71.