Biotechnological interventions in Withania somnifera (L.) Dunal.

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Biotechnological interventions in Withania somnifera (L.) Dunal a

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Pritika Singh , Rupam Guleri , Varinder Singh , Gurpreet Kaur , b

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Hardeep Kataria , Baldev Singh , Gurcharan Kaur , Sunil C. Kaul , c

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Renu Wadhwa & Pratap Kumar Pati a

Department of Biotechnology, Guru Nanak Dev University, Amritsar, Punjab, India b

Zentrum für Molekulare Neurobiologie, Universitätsklinikum Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany

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Cell Proliferation Research Group and DBT-AIST International Laboratory for Advanced Biomedicine, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305 8562, Japan Published online: 19 Mar 2015.

To cite this article: Pritika Singh, Rupam Guleri, Varinder Singh, Gurpreet Kaur, Hardeep Kataria, Baldev Singh, Gurcharan Kaur, Sunil C. Kaul, Renu Wadhwa & Pratap Kumar Pati (2015): Biotechnological interventions in Withania somnifera (L.) Dunal, Biotechnology and Genetic Engineering Reviews, DOI: 10.1080/02648725.2015.1020467 To link to this article: http://dx.doi.org/10.1080/02648725.2015.1020467

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Biotechnology and Genetic Engineering Reviews, 2015 http://dx.doi.org/10.1080/02648725.2015.1020467

Biotechnological interventions in Withania somnifera (L.) Dunal Pritika Singha, Rupam Guleria, Varinder Singha, Gurpreet Kaura, Hardeep Katariab Baldev Singha, Gurcharan Kaura, Sunil C. Kaulc, Renu Wadhwac and Pratap Kumar Patia*

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Department of Biotechnology, Guru Nanak Dev University, Amritsar, Punjab, India; bZentrum für Molekulare Neurobiologie, Universitätsklinikum Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany; cCell Proliferation Research Group and DBT-AIST International Laboratory for Advanced Biomedicine, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305 8562, Japan (Received 9 December 2014; accepted 10 February 2015) Withania somnifera is one of the most valued plants and is extensively used in Indian, Unani, and African systems of traditional medicine. It possess a wide array of therapeutic properties including anti-arthritic, anti-aging, anti-cancer, anti-inflammatory, immunoregulatory, chemoprotective, cardioprotective, and recovery from neurodegenerative disorders. With the growing realization of benefits and associated challenges in the improvement of W. somnifera, studies on exploration of genetic and chemotypic variations, identification and characterization of important genes, and understanding the secondary metabolites production and their modulation has gained significant momentum. In recent years, several in vitro and in vivo preclinical studies have facilitated the validation of therapeutic potential of the phytochemicals derived from W. somnifera and have provided necessary impetus for gaining deeper insight into the mechanistic aspects involved in the mode of action of these important pharmaceutically active constituents. The present review highlights some of the current developments and future prospects of biotechnological intervention in this important medicinal plant. Keywords: Withania somnifera; secondary metabolites; withanolide biosynthesis; anti-cancer; neuroprotective

Introduction Withania somnifera (L.) Dunal is an important medicinal plant of family Solanaceae. It is commonly called as Ashwagandha, Asgandh, or Indian ginseng and has been extensively used in Indian, Unani, and African traditional medicine (Mishra, Singh, & Dagenais, 2000). W. somnifera also appears in World Health Organization (WHO) monographs on selected medicinal plants (Mirjalili, Moyano, Bonfill, Cusido, & Palazón, 2009). Besides India, it is widely spread in Asia, Africa, Mediterranean region, and Middle East. W. somnifera is mainly propagated through seeds and the plant grows well in red soil (slightly basic) receiving about 500–750 mm rainfall and the optimal temperature range is 20–32 °C. The crop is harvested in about 180–210 days (Rajeswara, Rajput, Nagaraju, & Adinarayana, 2012) and different parts of the plant are

*Corresponding author. Email: [email protected] © 2015 Taylor & Francis


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used for commercial purposes (Sharada, Ahuja, & Vij, 2008; Yadav, Bajaj, Saxena, & Saxena, 2010). The other cultivated species of Withania is W. coagulans, which is generally found in Afghanistan, India, Iran, and Pakistan (Ahmad, 2007; Mirjalili et al., 2009). W. somnifera possesses immense therapeutic potential and is known for its immunomodulatory (Malik et al., 2009; Rasool & Varalakshmi, 2006), anti-stress (Archana & Namasivayan, 1998), cardioprotective (Mohanty et al., 2004), anti-aging (Singh, Narsimhamurthy, & Singh, 2008), antioxidant, anti-inflammatory (Mishra et al., 2000), anti-tumor (Wadhwa et al., 2013; Widodo et al., 2007, 2008), neuroprotective, and anti-brain cancer activities (Kataria, Shah, Kaul, Wadhwa, & Kaur, 2011; Kataria, Wadhwa, Kaul, & Kaur, 2012, 2013). The medicinal properties of W. somnifera are attributed to the presence of a wide array of secondary metabolites, including alkaloids [tropine, pseudotropine, hygrine, 3-trigloyloxytropine, cuscohygrine, choline, dl-isopelletierine, anaferine, anahygrine, and withanosomine (Schröter, Neumann, Katritzky, & Swinbourne, 1966; Schwarting et al., 1963)], flavanol glycosides [6,8-dihydroxykaempferol 3-rutinoside, quercetin and its 3-O-rutinoside and 3-rutinoside-7-glucoside (Kandil, El Sayed, Abou-Douh, Ishak, & Mabry, 1994)], glycowithanolides [sitoindoside VII to X (Bhattacharya, Satyan, & Ghosal, 1997)], steroidal lactones [withanolide A, withanolide D, withanone, withaferin A], sterols, and phenolics (Chatterjee et al., 2010; Chaurasiya, Sangwan, Misra, Tuli, & Sangwan, 2009; Chaurasiya, Sangwan, Sabir, Misra, & Sangwan, 2012; Ghosal, Kaur, & Shrivastava, 1988; Sangwan et al., 2008; Xu, Gao, Bunting, & Gunatilaka, 2011). In traditional home medicine, W. somnifera leaves (Jayaprakasam, Zhang, Seeram, & Nair, 2003) and roots (Kumar et al., 2011) are mainly used for herbal formulations. However, bark, seeds (Kulmi & Tiwari, 2005), and fruits are used only rarely (Bolleddula, Fitch, Vareed, & Nair, 2012; Lal, Misra, Sangwan, & Tuli, 2006). Recently, a total of 62 major and minor primary and secondary metabolites from leaves and 48 from roots have been identified, out of which 29 are common to both (Figure 1). It is also reported that the distribution of secondary metabolites varies significantly with

Figure 1.

Metabolic profile of W. somnifera.


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respect to different tissues, developmental stages, and chemotypes (Chatterjee et al., 2010; Dhar et al., 2013). Withaferin A and withanone are major metabolites present in leaves, whereas withanolide A is principle metabolite found in roots (Figure 2). Further, NMR and HR-MAS NMR studies on different chemotypes of W. somnifera show a clear distinction in the metabolome of different organs (Bharti, Bhatia, Tewari, Sidhu, & Roy, 2011; Namdeo et al., 2011). NMR spectroscopy reveals that the leaves of W.somnifera have the most wide array of metabolites which includes amino acids, flavonoids, lipids, sugars, organic acids, withanolides, trigonelline, ferulic acid, tryptamine, and kaempferol glycosides (Namdeo et al., 2011). The NMR spectra also reveal the presence of two types of withanolides: 4-OH and 5,6-epoxy withanolides (withaferin A-like steroids) and 5-OH and 6,7-epoxy withanolides (withanolides A-like steroids). It was further observed that ratio of these two withanolides was a major discriminating feature of W. somnifera leaf samples from different origins (Namdeo et al., 2011). Likewise, NMR technique has also been employed for qualitative and quantitative analysis of metabolites in W. somnifera fruits in different chemotypes (Bhatia, Bharti, Tewari, Sidhu, & Roy, 2013) and during different stages of development (Sidhu et al., 2011). It was observed that the early stages of fruit development had relatively higher concentrations of withanolides, alanine, aspartate, choline, phosphocholine, sucrose, and caffeic acid, whereas higher accumulation of citrate and withanamides was observed during maturation phase. Hence, identifying particular stage when fruits can be harvested for obtaining significant amount of bioactive ingredients is critical. Emerging challenges for W. somnifera Recently, there has been a global shift of preference toward natural chemicals and herbs for medicines and health supplements (Briskin, 2000). Herbal medicines are being widely accepted for their safety, efficacy, cultural acceptability, better compatibility with the human body, and lesser side effects. WHO estimates that more than 80% of population in developing countries still relies primarily on traditional remedies such as herbs for their medicines (Haq, 2004). Moreover, approximately one-quarter of prescribed drugs contain plant extracts or active ingredients obtained from plant source. With such

Figure 2. Distribution of pharmaceutically important secondary metabolites among different organs of W. somnifera.

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worldwide shift in consumer choice and preferences for herbal medicines, intensive efforts are being made toward identification of pharmaceutically important secondary metabolites and their experimental validation using a variety of model systems as well as understanding their biosynthesis, transport, accumulation, and modulation. Biosynthesis and transport of withanolides Withanolides are the signature metabolites of W. somnifera synthesized only in a few genera within Solanaceae family. Withanolides are ergostane skeleton-based C28-steroidal lactones derived from triterpenoids. De novo biogenesis and accumulation of withanolides were found to be most active in young leaves. Reports suggest that the process of withanolide biogenesis begins as early as in primordial (very young) stage but is maximal in the young leaves and starts to decline as leaves reach maturation stage (Chaurasiya, Gupta, & Sangwan, 2007). Further, it has been observed that a particular organ has an inherent capacity to biogenerate specific metabolites and their production is tightly associated with the morphogenetic differentiation of the tissue. Moreover, each developmental stage is characterized by differential accumulation of withanolides. Withanone and withanolide A accumulate considerably during vegetative stage whereas maximum withaferin A content is recorded at fruit set stage in W. somnifera (Dhar et al., 2013). Reports suggest that 24-methylene cholesterol, which has a metabolic origin from isoprenoid pathway, serves as the precursor for withanolide biosynthesis (Sangwan et al., 2008). Recent reports further suggest that withanolides may be synthesized from 24-methylene cholesterol via stigmasterol route (Singh et al., 2014). The isoprenoid biosynthesis in plants occurs via two independent pathways: cytosolic mevalonate (MVA) pathway and plastid localized 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway. Isoprenoids synthesized from these pathways are channelized into various metabolic pathways that produce an array of specialized metabolites involved in a number of cellular and regulatory processes (Hunter, 2007). The classical mevalonate pathway begins with activation of acetyl moieties to form acetoacetyl CoA. Acetyl CoA molecule further condenses with acetoacetyl CoA to form 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) in a reaction catalyzed by HMG-CoA synthase. 3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) then catalyzes irreversible conversion of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) into mevalonic acid. In the subsequent steps, mevalonic acid undergoes two phosphorylation reactions catalyzed by mevalonate kinase and phosphomevalonate kinase to form 5-phosphomevalonate and 5-pyrophosphomevalonate, respectively. Mevalonate-5-pyrophosphate decarboxylase then catalyzes the formation of 3-isopentenyl pyrophosphate (IPP) from 5-pyrophosphomevalonate. The molecules of IPP condense in a head-to-tail manner with its isomer DMAPP to give geranyl pyrophosphate (GPP), a reaction catalyzed by farnesyl diphosphate synthase (FPPS). FPPS also catalyzes condensation reaction of trans-geranyl pyrophosphate with another molecule of IPP to yield farnesyl pyrophosphate (FPP). Squalene synthase (SQS) then catalyzes condensation of two molecules of farnesyl diphosphate to form a linear 30-carbon compound squalene. Squalene is converted to 2,3-oxidosqualene by the enzyme squalene epoxidase (SE). The latter undergoes ring closure to form lanosterol which is then converted into a variety of different steroidal triterpenoidal skeletons (Figure 3) (Mirjalili et al., 2009). Apart from mevalonate pathway, molecules of IPP are also generated from MEP pathway that operates independently in plastids. Recently, significant involvement (about



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Figure 3. Withanolide biosynthesis in W. somnifera. 3-hydroxy-3-methylglutaryl-CoA (HMGCoA); HMG-CoA synthase (HMGS); 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR); 3-isopentenyl pyrophosphate (IPP); Dimethylallyl pyrophosphate (DMAPP); Isopentenyl pyrophosphate isomerise (IPPI); Geranyl pyrophosphate (GPP); Farnesyl diphosphate synthase (FPPS); Farnesyl pyrophosphate (FPP); Squalene synthase (SQS); Squalene epoxidase (SE). Pyruvate (Pyr); D-glyceraldehyde-3-phosphate (GP3); 1-deoxy-D-xylulose-5-phosphate (DXP); DXP synthase (DXS); 2-methyl D-erythritol 4-phosphate (MEP); DXP reductoisomerase (DXR); 4-diphospho-cytidyl-2-methyl-D-erythritol (CDP-ME); 4-(cytidine-5-diphospho)-2-C-tmethyl-Derythritol synthase (CMS); 4-(cytidine-5-diphospho)-2-C-methyl-D-erythritol kinase (CMK); 2-Cmethyl-D-erythritol-2-phosphate (CDP-MEP); 2-C-methyl-D-erythritol-2,4-cyclodiphosphate (MEcPP); 2-C-methyl-D-erythritol-2,4-cyclodiphosphate synthase (MCS); Hydroxy methyl butenyl 4diphosphate synthase (HDS); Hydroxy methyl butenyl 4-diphosphate (HMBPP); Hydroxy methyl butenyl 4-diphosphate reductase (HDR).

25%) of MEP pathway in withanolides production has been established (Chaurasiya et al., 2012). In MEP pathway, the first step is condensation of pyruvate with D-glyceraldehyde-3-phosphate to form 1-deoxy-D-xylulose-5-phosphate (DXP), a reaction catalyzed by DXP synthase (DXS). In the subsequent step, DXP is converted into 2-methyl D-erythritol 4-phosphate (MEP) by DXP reductoisomerase (DXR) in presence of NADPH. Further, MEP is converted into 4-diphospho-cytidyl-2-methyl-D-erythritol (CDP-ME) in a CTP-dependent reaction catalyzed by 4-(cytidine-5-diphospho)-2-C-tmethyl-D-erythritol synthase (CMS). CDP-ME undergoes phosphorylation by 4-(cytidine-5-diphospho)-2-Cmethyl-D-erythritol kinase (CMK) to yield 2-C-methyl-D-erythritol-2-phosphate (CDPMEP). CDP-MEP is then converted into 2-C-methyl-D-erythritol-2,4-cyclodiphosphate (ME-cPP) by the enzyme 2-C-methyl-D-erythritol-2,4-cyclodiphosphate synthase (MCS).

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In the final steps of pathway, hydroxy methyl butenyl 4-diphosphate synthase (HDS) catalyzes the formation of hydroxy methyl butenyl 4-diphosphate (HMBPP) from ME-cPP, which is directly converted into a mixture of IPP and DMAPP (5:1) by the enzyme hydroxy methyl butenyl 4-diphosphate reductase (HDR) (Figure 3) (RodriguezConcepcion & Boronat, 2002; Wanke, Skorupinska-Tudek, & Swiezewska, 2001).


Transport of withanolides Plant secondary metabolites that perform various biological activities are produced in different parts of the plant. It was observed that these secondary metabolites are often synthesized in the source tissue and are transported to the sink tissue where they get accumulated. Transport of each metabolite is presumed to be very specific and highly regulated. In case of W. somnifera, leaves and roots accumulate withanolides differentially. Withaferin A and withanone are predominantly present in leaves, while withanolide A and withanolide D are present in substantial amount in roots. Hence, it was hypothesized that withanolides might be synthesized in leaves and transported to roots like other tropane alkaloids (Ray & Jha, 2001). Studies have shown that both shoots and roots of W. somnifera contain withaferin A and withanolide D but in excised nontransformed as well as transformed root cultures only withanolide D could be detected (Ray, Ghosh, Sen, & Jha, 1996). This suggested that withanolide D is probably synthesized in roots and withaferin A is transported from leaves to roots (Ray & Jha, 2001). It was also observed that withanolide A, that was not present in the aerial parts of fieldgrown plants, accumulated significantly in the in vitro shoot cultures (Sangwan et al., 2007). Moreover, significant similarity in the characteristics of withanolides from leaf and root tissues and occurrence of concentration gradient between the tissues (higher in leaves and lower in roots) provides evidence in support of transport of withanolides from leaves to roots. However, radiotracer studies suggested that the roots might have a complete and independent system for biosynthesis of withanolides. Radio-labeled precursors [2-14C]-acetate and [U-14C] glucose were fed to root cultures of W. somnifera. Analysis of metabolites by thin-layer chromatography revealed that these primary metabolites were incorporated into withanolide A, demonstrating that withanolide A is de novo synthesized within roots from primary isoprenogenic precursors (Sangwan et al., 2008). Recently, EST and transcriptome databases generated from the root tissue of W. somnifera also revealed presence of biosynthethic genes in roots (Gupta, Goel, et al., 2013; Senthil, Wasnik, Kim, & Yang, 2010). Expression analysis of some of the key biosynthetic genes in root tissue further showed their active role in biosynthesis of withanolides (Akhtar, Gupta, Sangwan, Sangwan, & Trivedi, 2013; Bhat et al., 2012; Gupta, Agarwal, et al., 2013; Gupta, Sharma, Kumar, Vishwakarma, & Khan, 2012). In this background, more studies are warranted to get deeper insights into identifying the genes involved in the biosynthesis and transport of secondary metabolites for systematic metabolic engineering. Identification and characterization of pathway genes Identification and overexpression of key genes of withanolide biosynthesis pathway would be instrumental in understanding their synthesis and would also provide wealth information at molecular level for metabolic engineering in this medicinal plant. Several workers have reported cloning and characterization of biosynthetic pathway genes from W. somnifera such as: 3-Hydroxy 3-methylglutaryl CoA Reductase (HMGR) (Akhtar



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et al., 2013), farnesyl diphosphate synthase (FPPS) (Gupta, Akhtar, Tewari, Sangwan, & Trivedi, 2011), squalene synthase (SQS) (Bhat et al., 2012), squalene epoxidase (SE) (Razdan, Bhat, Rana, Dhar, & Lattoo, 2013), 1-deoxy-D-xylulose-5-phosphate synthase (DXS), 1-deoxy-D-xylulose-5-phosphate reductase (DXR) (Gupta, Agarwal, et al., 2013), cytochrome P450 reductase (Cyt P450) (Rana et al., 2013), glycosyltransferase (Singh et al., 2013), a member of 3β-hydroxysterol glucosyltransferase gene family (SGTL1) (Sharma, Madina, Chaturvedi, Sangwan, & Tuli, 2007), members of oxidosqualene cyclase (OSC) super-family: ß-amyrin synthase (OSC/BS), lupeol synthase (OSC/LS), and cycloartenol synthase (OSC/CS) (Dhar et al., 2014) (Table 1). The expression analysis of some of the key biosynthetic genes such as HMGR, FPPS, SQS, DXR, and DXS suggests that these genes are differentially expressed in different chemotypes, tissues, and in response to elicitors such as salicyclic acid (SA), methyl jasmonate (MeJ), as well as mechanical injury (Akhtar et al., 2013; Bhat et al., 2012; Gupta et al., 2012; Gupta, Agarwal, et al., 2013). HMGR, FPPS, SQS, DXS, and DXR showed higher expression in young leaves as compared to mature leaves and roots (Gupta et al., 2011; Gupta, Agarwal, et al., 2013), which may be due to higher rate of withanolide biosynthesis in young leaves as compared to mature leaves (Chaurasiya et al., 2007). Further, the observed lower expression of DXS and DXR in roots indicates that both the enzymes are plastid localized. These observations suggested that roots and leaves may possess independent system for withanolide synthesis but leaves may be the prime site for withanolide biosynthesis (Sangwan et al., 2008). Similar to the varying content of specific withanolides, different chemotypes show differential expression of key biosynthetic genes suggesting that the different chemotypes may have differential activities of enzymes involved in secondary conversion of withanolides. Recently, three OSCs (OSC/LS, OSC/BS, and OSC/CS) have been characterized in W. somnifera that utilize the common substrate pool of 2,3-oxidosqualene and lead to the formation of various triterpenoids, phytosterols, and withanolides which serves both primary and secondary functions for the plant (Dhar et al., 2014). Such branch point genes act as favorable candidate for metabolic engineering by redirecting the metabolic Table 1.

Withanolide biosynthetic genes cloned from W. somnifera.

Gene 3-hydroxy-3-methylglutaryl coenzyme A reductase Farnesyl pyrophosphate synthase Squalene synthase Squalene epoxidase 1-deoxy-D-xylulose-5-phosphate reductase 1-deoxy-D-xylulose-5-phosphate synthase Glycosyltransferase Cytochrome P450 reductase Sterol glucosyltransferase Flavonoid glycosyltransferase ß-amyrin synthase Lupeol synthase Cycloartenol synthase

Accession no.

Gene size

Reference

HQ293119.1

2021

Akhtar et al. (2013)

HM855234.1 1253 GU181386.1 1560 GU574803.1 JQ710679.1

1829 1653

Gupta et al. (2011) Gupta, Sharma, Santosh Kumar, Vishwakarma, and Khan (2012) Bhat et al. (2012) Gupta, Agarwal, et al. (2013)

JQ710678.1

4162

Gupta, Agarwal, et al. (2013)

FJ560880.2 GU808569.1 DQ356887.1 FJ654696.1 JQ728553.1 JQ728552.1 HM037907.1

1575 2297 2532 1413 2289 2268 2277

Kumar et al. (2013) Bhat et al. (2012) Sharma et al. (2007) Singh et al. (2013) Dhar et al. (2014) Dhar et al. (2014) Dhar et al. (2014)


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flux toward the production of desired secondary metabolites, thereby decreasing the flux through other pathways. Further, gene expression profile of these three OSC genes in different organs of W. somnifera shows that OSC/LS is highly expressed in roots followed by berries, stalk, and leaves. OSC/BS is expressed highly in both roots and berries followed by stalk and leaves. However, in case of OSC/CS, maximum expression was found in leaves followed by stalk and berries and minimum in roots. This expression pattern of OSC/CS is in correlation with the higher amounts of withanolides in the leaves (Dhar et al., 2014). As discussed above, expression of upstream genes like SQS, SQE, and cytochrome P450 reductase 2 (CPR2) was also found to be higher in leaves as compared to other tissues (Bhat et al., 2012; Rana et al., 2013; Razdan et al., 2013). Additionally, enhanced expression of obtusifoliol-14-demthylase (CYP51) and sterol methyl transferase (SMT-1) that constitute important part of downstream withanolide biosynthetic pathway has also been reported in leaves (Pal et al., 2011). Higher expression of these putative biosynthetic genes in leaves indicates that these genes could serve as favorable candidates in future endeavors aimed at enhancing withanolide production. Recently, transgenic cell lines of W. somnifera overexpressing a key regulatory gene of withanolide biosynthetic pathway (squalene synthase) were produced using agrobacterium-mediated transformation (Grover, Samuel, Bisaria, & Sundar, 2013). Transformed cell lines showed a significant 4-fold and 2.5-fold increment in squalene synthase activity and withanolide content, respectively, as compared to the nontransformed cell line. Further, Withaferin A was also produced by transgenic cell lines, which was completely absent in non-transformed cells (Grover et al., 2013). Semi-RT analysis of key biosynthethic pathway genes reveals that their expression is closely related with the developmental phases in W. somnifera. SQS, SE, and cycloartenol synthase (CAS) along with Cyt P450 transcripts exhibited increased expression with each progressive developmental stage (Dhar et al., 2013). However, transcripts of CAS gene were found to be most abundant in vegetative as well as reproductive phases. In another study, higher expression of HMGR, FPPS, SQS, SE, and glycosyl transferase (GT) was observed in in vitro shoots as compared to in vitro roots. HMGR was highly expressed in field grown shoots as compared to root tissues. The expression of FPPS and GT was upregulated, while that of SQS and SE was downregulated in field grown shoots in comparison to roots. However, expression of CAS was more or less similar in field grown shoot and root tissues (Sabir, Mishra, Sangwan, Jadaun, & Sangwan, 2013). Elicitors such as MeJ and SA act as key signaling molecules that regulate a network of interconnecting signal transduction pathways responsible for induced plant defense against biotic and abiotic stresses (Shah, 2009). Exogenous addition of elicitors is considered to be one of the most promising strategies for enhancing the production of secondary metabolites. Increase in the gene expression patterns of FPPS, DXS, and DXR was observed in response to different elicitors suggesting that the expression of these genes is tightly regulated with the different defense signaling pathways. In another study, differential transcript and translational profiles of OSC/LS, OSC/BS, OSC/CS were observed in response to elicitors [MeJ, Gibberllic acid (GA) and yeast extract] along with corresponding changes in withanolide content. Treatment with MeJ and GA, led to enhanced expression of OSC/BS while that of OSC/LS was downregulated (Dhar et al., 2014). However, OSC/CS transcript level remained relatively constant. At protein level, the three OSCs displayed no variation in MeJ-treated plants whereas in GA-treated plants, OSC/LS protein expression remained constant while OSC/CS and OSC/BS showed a gradual decrease (Dhar et al., 2014). Further, increased production of withaferin A in response to MeJ may be attributed to the ability of OSC/CS to utilize


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2,3-oxidosqualene from increased pool for synthesis of withanolides. These results suggest that by suppressing competitive branch pathways of OSCs like OSC/BS and OSC/LS could possibly lead to diversion of substrate pool toward OSC/CS for increased withanolide production (Dhar et al., 2014). Promoter analysis of biosynthetic pathway genes Promoter analysis is essential to understand the regulation of biosynthetic gene. Promoter analysis of squalene synthase gene isolated from W. somnifera provides the information about several cis-elements. Myb and Myc recognition sites were identified which have been shown to play important role in plant response to pathogens, low temperature, and drought. Apart from this, various phytohormone-induced cis-elements like TGA element (auxin-responsive element), GARE motif (gibberellins-responsive element), CGTCA-motif (MeJ-responsive element), ABRERATCAL (Abscisic acidresponsive element), and W-Box were also found on the squalene synthase promoter region. WRKY transcription factor involved in various physiological processes binds specifically to the W-Box. Moreover, low-temperature responsiveness and heat stressresponsive elements were also identified. Light-responsive cis-elements, 3-AF binding, GT1CONSENSUS, and GATA box were also present in the promoter (Bhat et al., 2012). Promoter analysis of another key regulatory gene squalene epoxidase indicates presence of calcium-responsive cis-element ABRERATCAL and some important phytohormone-induced regulatory elements like ABREOSRAB21 (Abscisic acid-responsive element) and ARFAT (auxin-responsive factor). WRKY71OS, a transcriptional repressor of gibberellin signaling pathway and ASF1MOTIFCAM element, involved in transcriptional activation of several auxin and salicylic genes, were also identified (Razdan et al., 2013). Promoter analysis of three OSCs genes resulted in identification of several important cis-acting elements like light-responsive, hormone-responsive, and various other stressrelated elements (Dhar et al., 2014). These include bZIP protein-binding motifs TGACG/CGTCA (MeJ-responsive element), GARE-motif (TCTGTTG) (GA-responsive element), and Box-W1 of consensus sequence TTGACC (fungal elicitor-responsive element). ESTs and transcriptomics studies EST database has been used to identify genes involved in specific plant metabolic pathways. Study of secondary metabolite genes that are differentially expressed in the EST library would be instrumental in increasing our understanding of regulation of secondary metabolism. In an effort to gain information on the genes involved in withanolide biosynthesis, ESTs from cDNA libraries of W. somnifera leaf and root tissue were isolated (Senthil et al., 2010). EST database analysis showed that the most abundant functional genes were those involved in cellular processes, membrane, and catalytic activities. ESTs assigned to metabolic processes were highly abundant in leaf (14.80%) and roots (19.43%). Presence of defense and secondary metabolism-related genes suggested that these genes are involved in maintaining a normal physiological state in plants in response to a variety of stresses. ESTs involved in MVA and MEP pathway of withanolide biosynthesis were also identified on the basis of amino acid sequence homology. The root library contained a gene CDP-ME kinase from MEP pathway, HMG CoA synthase and squalene epoxidase from MVA pathway. Presence of these enzymes in root


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EST library suggested that roots possess an independent system for production of withanolide biosynthesis. Cytochrome P-450 and glycosyltransferases genes were also found. These enzymes are known to participate in a variety of biochemical reactions, including triterpenoid and sterol metabolism. Glycosyltransferase is involved in glycosylation and is often involved in secondary metabolite synthesis. EST database also provides information about the enzymes involved in pathogenesis in W. somnifera (catalase and cytochrome P450) and were found to be expressed in both leaf (10.41%) and root (14.25%) tissues. However, genes involved in reproduction and embryonic development appear in low proportions in EST library (Senthil et al., 2010). Recently, 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 (Gupta, Goel, et al., 2013). 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), tomato (http://solgenomics. net/organism/Solanum lycopersicum/genome; version ITAG2.3), and potato (http://sol genomics.net/organism /Solanum_tuberosum/genome; v3.4) databases. 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 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. Earlier reports suggest that leaves of W. somnifera are rich in withaferin A, whereas withanolide A is majorly present in roots. Such tissue-specific withanolide accumulation can be attributed to the differential expression of cytochrome P450, methyltransferase, and glycosyltransferase which have been implicated in conversion of 24-methylene cholesterol to withanolides. Further, a considerable number of unigenes from these families showed unique as well as differential expression in leaves and roots. This suggests that these unigenes might be involved in synthesis of tissue-specific withanolides (Gupta, Goel, et al., 2013). In addition to gene identification, large number of EST-SSRs markers have also been identified which would facilitate marker-assisted breeding and would assist in future genetic studies especially in relation to chemotyping. Thus, the novel unigenes identified from this study would be valuable for elucidating the withanolide biosynthesis pathway and to understand the molecular mechanism underlying the biosynthesis of specific withanolides in root and leaf tissues. Proteomics studies in W. somnifera Proteomic studies offer a new approach to identify a broad spectrum of genes that are expressed in living system. One of the primary advantages of proteomics research based on two dimensional electrophoresis (2-DE) along with mass spectroscopy (MS) is the ability to investigate hundreds of proteins simultaneously (Jacobs, Van der Heijden, & Verpoorte, 2000). The feasibility of proteomic approach for identifying known and unknown genes responsible for biosynthesis of active compounds has also been elucidated. The knowledge gained from proteomic analysis would make a significant contribution to our future endeavor of characterization of proteins. Also, it would help to understand the physiology during development and the associated complex metabolic networks in W. somnifera.



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Total proteome analysis of leaf and seed tissues of W. somnifera distinguished proteins on the basis of stored and differentially expressed, their count, and function. The proteome analysis of seeds and leaves of W. somnifera has identified and characterized tissue-specific proteins (Dhar et al., 2012). A comparative analysis of the two tissues indicates that some enzymes/proteins involved in housekeeping pathways were common to both, whereas some others were tissue specific with specialized metabolic complement. Further, proteomic approach undertaken in Indian ginseng has given us an insight on its self-defense mechanism by identifying certain reference proteins such as antioxidant enzymes that can be used as markers for monitoring the defense response of the plant. Further, to understand the proteins and enzymes involved in withanolide biosynthetic pathway, detailed 2-DE and MS analysis of in vitro grown adventitious roots and in vivo root samples of W. somnifera was conducted (Senthil et al., 2011). A high level of similarity in protein spots in both in vitro and in vivo root samples was observed. The data suggested that though in vitro roots are developed independent of shoot organs, they might have a similar developmental process as that of in vivo roots. Also, out of 55 total proteins resolved in 2-DE gels, only 26 were identified and found in available protein databases. Among these proteins, only one was found to differentially expressed in in vitro root tissue. In vitro and in vivo preclinical studies and emerging concepts The mainstream pharmaceutical research is on its way toward veering from monomolecular or single target approach to combinations and multiple target strategies (Wermuth, 2004). Perhaps, multisite mechanisms of action of herbal preparations from the crude extracts may offer greater chances for success where conventional single-site agents have been disappointing. Auspiciously, many of these traditional herbal medicines are now increasingly being appreciated with western models of integrative health sciences and evidence-based approach both in research and practice. Several bioactive compounds have emerged from research in herbal medicine. These include Rauwolfia alkaloids for hypertension, psoralens for vitiligo, Holarrhena alkaloids in amoebiasis, guggulsterones from Commiphora as hypolipidemic agents, Mucuna pruriens for Parkinson’s disease, bacosides from Bacopa monnieri, antivirals from phyllanthins, withanolides, and many other steroidal lactones and their glycosides as immunomodulators (Mishra et al., 2000). Limited in vitro studies are available on the role and action of W. somnifera in central nervous system (CNS)-related tumors. Several withanolides from the leaves of W. somnifera have been tested on SF-268 CNS cell line along with other cell lines from different origins and it was shown that withanolides inhibit the cell proliferation in dosage-dependent manner (Jayaprakasam et al., 2003). Ashwagandhanolide a dimeric thiowithanolide isolated from the roots of W. somnifera displayed growth inhibitory effect on CNS cell line SF-268 along with colon (HCT-116), lung (NCI H460), human gastric (AGS), breast (MCF-7) cell lines, with IC50 values in the range 0.43–1.48 μg/ml (Subbaraju et al., 2006). Gliomas and neuroblastomas are the most common primary brain tumors with only limited options for treatment. Majority of these tumors develop into malignancy and remain incurable in spite of the therapies like external beam radiation, surgery, and chemotherapy and hence, call for the development of novel therapeutic approaches. W. somnifera induced upregulation of neuronal marker proteins has been correlated with the induction


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of differentiation in these cells (Kataria et al., 2011, 2013; Kuboyama, Tohda, & Komatsu, 2005; Kuboyama et al., 2002; Shah et al., 2009). The upregulation of glial fibrillary acidic protein (GFAP) in glioma, neurofilament protein (NF200) in neuroblastoma, and mortalin expression suggested the induction of differentiation in these cells. The upregulation of neural cell adhesion molecule (NCAM) and downregulation of its polysialylated form (PSA-NCAM) and matrix metalloproteases (MMPs) may explain the anti-migratory and differentiation inducing properties of W. somnifera leave extract in the glioblastoma and neuroblastoma cells (Kataria et al., 2011, 2013). Further, W. somnifera treatment led to cell cycle arrest at G0/G1 phase and increase in early apoptotic population along with modulation of cell cycle marker Cyclin D1, anti-apoptotic marker bcl-xl, and Akt-P (Kataria et al., 2013; Widodo et al., 2007). These studies provide evidence that W. somnifera appears to affect multiple pathways for its anti-cancer and differentiation-inducing role in glioma and neuroblastoma cells instead of targeting a single protein or pathway. Further using the combinational approach, it was found that the combinations of withaferin A, withanone, and withanolide A were effective to induce differentiation compared to individual component, as these combinations caused stronger growth inhibition and differentiation (Shah et al., 2009). Thus, W. somnifera may have the potential to be suitable as adjunct therapy by its differentiation inducing activity in brain tumors. The potential of W. somnifera for neural regeneration has been explored in some of the in vitro as well as in vivo studies. It is well known that neurite outgrowth may compensate for and repair damaged neuronal circuits in the dementia brain. W. somnifera root extract significantly increased the percentage of cells with neurites in human neuroblastoma SK-N-SH cells in dose and time-dependent manner which was associated with the increase in expression of dendritic markers MAP2 and PSD-95 (Tohda, Kuboyama, & Komatsu, 2000). The methnaolic extract of W. somnifera has been characterized to contain withanolides such as withanolide A, withanoside IV, and withanoside VI, which induce neurite outgrowth in human neuroblastoma SHSY5Y. Withanolide A, withanoside IV, withanoside VI, and coagulin Q have been shown to possess significant neurite outgrowth activity on a human neuroblastoma SH-SY5Y cell line (Zhao et al., 2002). In another study, using methanolic extract of W. somnifera, withanolide A, withanoside IV, and withanoside VI showed neuritic regeneration and synaptic reconstruction in Aβ(25– 35)-induced damaged cortical neurons (Tohda, Kuboyama, & Komatsu, 2005). Owing to W. somnifera’s well-known neuromodulatory properties, our lab explored its role in glutamate-induced neuroexcitotoxicity. Pre-exposure of retinoic acid (RA) differentiated neuroblastoma and glioma cell line, leads to significant increase in their viability against glutamate-mediated excitotoxicity. W. somnifera treatment rescued the glial and neuronal cells from glutamate-induced cytotoxicity by upregulation of plasticity marker proteins such as heat shock protein 70 (HSP70), NCAM, and PSA-NCAM suggesting its role in protection against the neurodegeneration associated with glutamate-induced excitotoxicty (Kataria et al., 2012). It has also been shown that withanolides cause oxidative stress to the cancer cells resulting in their selective death either by growth arrest or apoptosis. An interesting observation of some recent studies has been that whereas withaferin A caused cytotoxicity to cancer as well as normal cells, withanone was shown to be selectively toxic to cancer cells (Widodo et al., 2007, 2008; Widodo, Priyandoko, Shah, Wadhwa, & Kaul, 2010). Furthermore, low doses of withanone were shown to possess anti-aging activity for normal cells in culture in assays that involved premature induction of senescence either by oxidative or chemical stress or by inhibition of proteasomal activity (Priyandoko, Ishii, Kaul, & Wadhwa, 2011; Widodo et al., 2010). Similarly, it was



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shown that low doses of withanone could effectively protect brain-derived cells against amnesia and glutamate stress and could enhance their neuroplastic characteristics (Gautam, Wadhwa, & Thakur, 2013; Konar et al., 2011). These were shown to be mediated by upregulation of neurotropic factors BDNF and ARC (Gautam et al., 2013; Konar et al., 2011). In mice model studies, it was shown that mice pretreated with W. somnifera extracts were more tolerant to the stress conditions (Gautam et al., 2013; Konar et al., 2011) suggesting that W. somnifera leaves and the phytochemicals therein could be used for protection against stress, age-associated neurodegenerative diseases, and cancer. While further studies are warranted to dissect the molecular signaling responsible for these effects, it was suggested that W. somnifera leaf-derived withanolides may serve as economic, safe, and effective agents for healthy aging. Recently, we have identified triethylene glycol (TEG) in the water extracts of W. somnifera leaves to possess cytotoxicity to human cancer cells that was mediated by activation of pRB tumor suppressor protein (Wadhwa et al., 2013) (Figure 4) providing evidence to the occurrence of bioactive compounds, other than withanolides, in W. somnifera leaves. There is need for use of natural products as resources for drug discovery due to safety issues and failure of existing regimens. Moreover, as the recent approaches take advantage of biomimicry (mimicry of biological system) for the drug development, there arises the potential need of natural products as potential therapeutic agents that are safe and can be administered as dietary supplements. W. somnifera seems to be one of the most promising plant in this category; thus, evaluation and characterization of the bioactive components of W. somnifera extracts is highly warranted. Some novel molecules

Figure 4. Schematic representation of tumor management using water and alcoholic extract of W. somnifera.

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and/or a formulation with neuroprotective properties could serve as a valuable adjunct therapeutic agent(s) with existing conventional chemo- and radiotherapeutic modalities, as well as neurodegenerative diseases. In silico studies and their implications Recently, few in silico studies have advanced our knowledge regarding the role of withanolides, using an array of computational techniques. The concepts that have emerged from these studies are highlighted in this review.


Structural, docking, and simulation studies Significant information has been obtained from sequence and structure analysis of squalene epoxidase (WsSQE) and squalene synthase (WsSQS) genes involved in withanolides biosynthesis. The deduced WsSQE protein sequence revealed maximum homology with Dathura innoxia SQE (95%). WsSQE consists of many domains: DAO (D-amino acid oxidase), lycopene cyclase, SE, FAD binding-2 component of Mem1, and FAD binding-3. Secondary structure analysis predicted 36.35% alpha helices, 19.02% extended strands, 38.79% random coil, and 5.84% beta turns, and molecular modeling revealed binding sites for the FAD ligand in the three-dimensional structure of WsSQE. One transmembrane helix containing signal peptide toward the N-terminal of the sequence was found by hydrophobicity analysis (Razdan et al., 2013). Analysis of another gene, squalene synthase shows that the secondary structure consists of 65.69% α-helixes, 4.38% ß-turns, 8.03% extended strands, and 21.90% random coils. The threedimensional structural model is similar to the available crystal structures of several class I isoprenoid biosynthetic enzymes, where the conserved feature is an α-helical core surrounding a central active site cavity (Bhat et al., 2012). Anti-tumor properties of withaferin A have been established via structural studies, computational electron density analysis and in silico docking approaches. These techniques have shown C1 and C24 of withaferin A as the critical sites which inhibit the proteasomal chymotrypsin subunit (β5) by attacking the hydroxyl group of its N-terminal threonine, in human prostate cancer cells and tumors (Yang, Shi, & Dou, 2007). In addition to this, molecular dynamics simulations have been employed to further explore the other pharmacological activities of withaferin A against its various targets such as suppression of nuclear factor kappa B (NF-κB; a transcription factor involved in the immune response, differentiation, cell proliferation, and apoptosis) activity (Grover, Shandilya, Punetha, Bisaria, & Sundar, 2010), inhibition of human and bovine proteasomes (Grover, Shandilya, Bisaria, & Sundar, 2010), anti-herpetic action against Herpes simplex virus DNA polymerase (Grover, Agrawal, Shandilya, Bisaria, & Sundar, 2011), restraining Hsp90/Cdc37 interactions (Heat-shock protein 90 kDa/ Cell division cycle protein 37) (Grover, Shandilya, et al., 2011), and Leishmanial protein kinase C (LPKC) in Leishmaniasis (Grover, Katiyar, Jeyakanthan, Dubey, & Sundar, 2012). Withanone and withaferin A have recently been shown to bind mortalin (a member of Hsp70 family of proteins) via docking and simulation studies, thereby interfering with the interaction between mortalin and p53 tumor suppressor protein (Grover, Priyandoko, et al., 2012). Abrogation of mortalin-p53 interaction and reactivation of p53 leading to upregulation of its downstream effector p21WAF1 and growth arrest was shown in human cancer cells treated with withanone (Grover, Priyandoko, et al., 2012; Vaishnavi et al., 2012; Widodo et al., 2007). Another mechanism for the anticancer



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activity of withaferin A and withanone was shown through its interaction with TPX2Aurora A complex (TPX2 is a targeting protein for Xenopus kinesin-like protein 2 and Aurora A is a Ser/Thr kinase involved in mitosis and cytokinesis) that plays a key role in continued proliferation of cancer cells (Grover, Singh, et al., 2012). Further, this natural compound also shows antileishmanial action against Leishmanial protein kinase C in Leishmaniasis (Grover, Katiyar, et al., 2012). A recent study from our group reveals that withaferin A and withanone have differential activities in normal and cancer cells. Further, docking competency of these two structurally similar withanolides with four targets: mortalin, p53, p21, and Nrf2 was analyzed and it was observed that both withanolides exhibited differential binding characteristics with each of the selected targets. Withaferin A showed strong binding with all the targets, whereas withanone interacted weakly (Vaishnavi et al., 2012). W. somnifera transcriptome database and pathway analysis Recent transcriptome sequencing from W. somnifera root and leaf has led to the development of its transcriptome database. This has further helped to identify biological pathways operating in these tissues using Kyoto Encyclopedia of Genes and Genomes (KEGG) (Gupta, Goel, et al., 2013). Similar approach will be helpful to explore the different biosynthetic pathways in other parts of the plant and hence, the biosynthesis of various other natural products from W. somnifera and other medicinal plants. An effort has been made to identify key gene targets for W. somnifera crude extract and its two anti-tumor components (withaferin A and withanone), using a combined approach of siRNA and ribozyme library screening. Ingenuity Pathway Analysis software (http:// www.ingenuity.com/products/pathways_analysis.html) was used for network construction to explore the interaction between these gene targets as well as difference in their bioactivities (Deocaris, Widodo, Wadhwa, & Kaul, 2008). Conclusion A global shift of preference for herbal medicines for general health and various ailments is evident by their increasing consumption. It is of great irony that the therapeutic potential of the medicinal plants has lead to its harvesting from the wild, causing loss of genetic diversity, and habitat destruction. Hence, at this particular juncture, it is important to identify some high-value medicinal plants, propagate and bring them to the existing frame work of rational scientific use. The recent upsurge in Withania research is an indication in this direction. The present review has highlighted the world-wide efforts associated with understanding synthesis and regulation of pharmaceutically important secondary metabolites and lays the foundation for metabolic engineering in W. somnifera. The significant progress in clinical research using this plant material has undoubtedly provided a unique status to this plant among medicinal plants. Availability of new experimental tools, biological resources, and modern experimental approaches will further provide an impetus to Withania research and pave the way for it becoming a model medicinal plant in future.

Disclosure statement No potential conflict of interest was reported by the authors.

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Funding This work was supported by Department of Biotechnology, Government of India under DBTAIST, Japan collaboration program, CSIR, New Delhi and University Grants commission (UGC), Government of India, New Delhi, India.

ORCID Hardeep Kataria

http://orcid.org/0000-0001-8413-0600


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Effect of Withania somnifera (L.) Dunal on Sex Hormone and Gonadotropin Levels in Addicted Male Rats.

Microbial secondary metabolites ameliorate growth, in planta contents and lignification in Withania somnifera (L.) Dunal.

Germination behaviour of seeds of Withania somnifera (L.) Dunal: a high value medicinal plant.

Changes in the leaf proteome profile of Withania somnifera (L.) Dunal in response to Alternaria alternata infection.

Improved Agrobacterium rhizogenes-mediated hairy root culture system of Withania somnifera (L.) Dunal using sonication and heat treatment.

Validation of dbEST-SSRs and transferability of some other solanaceous species SSR in ashwagandha [Withania Somnifera (L.) Dunal].

Metabolic shift from withasteroid formation to phenylpropanoid accumulation in cryptogein-cotransformed hairy roots of Withania somnifera (L.) Dunal.

Evaluation and selection of candidate reference genes for normalization of quantitative RT-PCR in Withania somnifera (L.) Dunal.

Influence of plant growth regulators on callus mediated regeneration and secondary metabolites synthesis in Withania somnifera (L.) Dunal.

Stability indicating studies on NMITLI 118RT+ (standardized extract of withania somnifera dunal).

Sonication, Vacuum Infiltration and Thiol Compounds Enhance the Agrobacterium-Mediated Transformation Frequency of Withania somnifera (L.) Dunal.

Withania Coagulans and Somnifera.

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.

A Decade of Molecular Understanding of Withanolide Biosynthesis and In vitro Studies in Withania somnifera (L.) Dunal: Prospects and Perspectives for Pathway Engineering.

Enhanced biosynthesis of withanolides by elicitation and precursor feeding in cell suspension culture of Withania somnifera (L.) Dunal in shake-flask culture and bioreactor.

5,6-de-epoxy-5-en-7-one-17-hydroxy withaferin A, a new cytotoxic steroid from Withania somnifera L. Dunal leaves.

Anti-granuloma activity of Iraqi Withania somnifera.

Hypoglycemic activity of withanolides and elicitated Withania somnifera.

Withania somnifera: From prevention to treatment of cancer.

Withania somnifera root extract extends lifespan of Caenorhabditis elegans.

Withania somnifera Root Extract Enhances Chemotherapy through 'Priming'.

Neuro-protective potential of a vesicular system of a standardized extract of a new chemotype of Withania somnifera Dunal (NMITLI118RT+) against cerebral stroke in rats.

Functional and Morphological Correlates in the Drosophila LRRK2 loss-of-function Model of Parkinson's Disease: Drug Effects of Withania somnifera (Dunal) Administration.

Effect of cadmium stress on inductive enzymatic and nonenzymatic responses of ROS and sugar metabolism in multiple shoot cultures of Ashwagandha (Withania somnifera Dunal).