IN VITRO AND OMICS TECHNOLOGIES OPENS A NEW AVENUE FOR DECIPHERING WITHANOLIDE METABOLISM IN WITHANIA SOMNIFERA
Keywords:Withania somnifera, Withanolides, in vitro cultures, Genomics, Transcriptomics, In silico, Proteomics, Metabolomics
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.
Gupta GL, Rana AC. Withania somnifera (aswagandha): a review. Pharmacognosy 2007;1:129â€“36.
Davis L, Kuttan G. Effect of Withania somnifera on DMBA induced carcinogenesis. J Ethanopharmacol 2001;75:165â€“8.
Kumar A, Kaul MK, Bhan MK, Khanna PK, Suri KA. Withania somnifera (L.) Dunal (Solanaceae). Genet Resour Crop Evol 2007;54:655â€“60.
Asthana R, Raina MK. Pharmacology of Withania somnifera (L.) Dunal-a review. Indian Drugs 1989;26:199â€“205.
Singh S, Kumar S. Withania somnifera: The Indian Ginseng Ashwagandha, Central Institute of Medicinal and Aromatic Plants, Lucknow, India; 1998.
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;65:93â€“100.
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 Biotechnol 2011;168:681â€“96.
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 Prod 2012;37:124â€“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;51:161â€“4.
Sangwan RS, Chaurasiya ND, Lal P, Misra L, Uniyal GC, Tuli R, et al. Withanolide A biogeneration in in vitro shoot cultures of Ashwagandha (Withania somnifera Dunal), a main medicinal plant in Ayurveda. Chem Pharm Bull 2007;55:1371â€“5.
Sudha CG, Seeni S. Establishment and analysis of fast-growing normal root culture of Decalepis arayalpathra, a rare endemic medicinal plant. Curr Sci 2001;81:371â€“4.
Rani G, Arora S, Nagpal A. Direct rhizogenesis from in vitro leaves of Withania somnifera (L.) Dunal. J Herbs Spices Med Plants 2003;10:47â€“54.
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;84:223â€“5.
Praveen N, Murthy HN. Production of withanolide A from adventitious root cultures of Withania somnifera. Acta Physiol Plant 2010;5:1017â€“22.
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;5:596â€“603.
Abraham A, Kirson I, Glotter E, Lavie DA. Chemotaxonomic study of Withania somnifera (L.) dunal. Phytochemistry 1968;7:957â€“62.
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;67:2269â€“76.
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;86:461â€“5.
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;6:1â€“8.
Mohn T, Plitzko I, Hanburger MA. Comprehensive metabolite profiling of Isatis tinctoria leaf extracts. Phytochemistry 2009;70:924â€“34.
Shyur LF, Yang NS. Metabolomics for phytomedicine research and drug development. Curr Opin Chem Biol 2008;12:66â€“71.
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;19:173â€“82.
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;70:1017â€“29.
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;93:2325â€“7.
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;392:263â€“9.
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;19:148â€“54.
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.
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;94:385â€“7.
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 somniferaâ€“Jawahar 20 cultivar, Int J Pharma Bio Sci 2014;5:971â€“9.
Bone K. Clinical applications of ayurvedic and Chinese herbs: monographs for the Western herbal practitioner. Phytotherapy Press: Queensland, Australia; 1996. p. 137â€“41.
Grandhi A, Mujumdar AM, Patwardhan B. A comparative pharmacological investigation of ashwagandha and ginseng. J Ethnopharmacol 1994;44:131â€“5.
Mishra LC, Singh BB, Dagenais S. Scientific basis for the therapeutic use of Withania somnifera (ashwagandha): a review. Altern Med Rev 2000;5:334â€“46.
Kuboyama T, Tohda C, Komatsu K. Neuritic regeneration and synaptic reconstruction induced by withanolide-A. Br J Pharmacol 2005;144:961â€“71.
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;80:1525â€“38.
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;122:455â€“60.
Ku SK, Bae JS. Antiplatelet, anticoagulant, and profibrinolytic activities of withaferin A. Vasc Pharmacol 2014;60:120â€“6.
Mehta AK, Binkley P, Gandhi SS, Ticku MK. Pharmacological effects of Withania somnifera root extract on GABAA receptor complex. Indian J Med Res 1991;94:312â€“5.
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;24:137â€“47.
Meena M, Lakshmi T. Antiepileptic activity of indigenous herbal extracts a review. Asian J Pharm Clin Res 2013;6:12â€“4.
Misra L, Lal P, Sangwan RS, Sangwan NS, Uniyal GC, Tuli R. Unusually sulfated and oxygenated steroids from Withania somnifera. Phytochemistry 2005;66:2702â€“7.
Lal P, Misra L, Sangwan RS, Tuli R. New withanolides from fresh berries of Withania somnifera. Z Naturforsch B: J Chem Sci 2006;61:1143â€“7.
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;11:50â€“6.
Berlin J, Rehm HJ, Reed G. editors. Secondary products from plant cell cultures. In: Biotechnology a comprehensive treatise. Weinheim; 1986;4:630â€“58.
Alfermann AW, Petersen M. Natural product formation by plant cell biotechnologyâ€“results and perspectives. Plant Cell Tissue Organ Cult 1995;43:199â€“205.
Yu PLC, El Olemy MM, Stohs ST. A phytochemical investigation of Withania somnifera tissue cultures. J Nat Prod 1974;37:593â€“7.
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; 1985:281â€“9.
Roja G, Heble MR, Sipahimalini AT. Tissue cultures of Withania somnifera: morphogenesis and withanolide synthesis. Phytother Res 1991;5:185-7.
Banerjee S, Naqvi AA, Mandal S, Ahuja PS. Transformation of Withania somnifera (L.) Dunal by Agrobacterium rhizogenes: Infectivity and phytochemical studies. Phytother Res 1994; 8:452â€“5.
Vitali G, Conte L, Nicoletti M. Withanolide composition and in vitro culture of Italian Withania somnifera. Planta Med 1996;62:287â€“8.
Ray S, Jha S. Production of withaferin A in shoot cultures of Withania somnifera. Planta Med 2001;67:432â€“6.
Rani G, Virk GS, Nagpal A. Somatic embryogenesis in Withania somnifera (L.) dunal. J Plant Biotechnol 2004;6:113â€“8.
Sivanesan I, Murugesan K. In vitro adventitious shoot formation from leaf explants of Withania somnifera dunal. Plant Cell Biotechnol Mol Biol 2005;6:163â€“6.
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;163:220â€“3.
Sivanesan I. Direct regeneration from apical bud explants of Withania somnifera dunal. Indian J Biotechnol 2007;6:125â€“7.
Sharma MM, Ali DJ, Batra A. Plant regeneration through in vitro somatic embryogenesis in ashwagandha (Withania somnifera L dunal). Researcher 2010;2:1â€“6.
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;9:7453â€“61.
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;3:85â€“7.
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;133:278â€“87.
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: 1419â€“27.
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;22:584â€“91.
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;50:484â€“90.
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;50:975â€“81.
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;104:119â€“24.
DiCosmo F, Towers GHN. Stress and secondary metabolism in cultured plant cells. In: Phytochemical adaptations to stress: Springer US; 1984. p. 97â€“175.
Deus NBS, Zenk MH. Exploitation of plant cells for the production of alkaloids in Catharanthus roseus cell suspension cultures. Planta Med 1982;50:427â€“31.
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. 75â€“108.
Thimann KV, Went FW. On the chemical nature of the root forming hormone. Proc Kon Ned Acad Wetensch 1934;37:456â€“59.
Zimmerman PW, Wilcoxon F. Several chemical growth substances which cause initiation of roots and other responses in plants. Contrib Boyce Thompson Inst 1935;7:209â€“29.
Davies JR, Davis TD, Kester DE. The commercial importance of adventitious rooting to horticulture. In: Biology of adventitious root formation. Springer US; 1994. p. 53â€“9.
Kovar JL, Kuchenbuch RO. The commercial importance of adventitious rooting to agronomy. In: Biology of adventitious root formation, Springer US; 1994. p. 25â€“34.
Ahuja A, Kaur D, Sharada M, Kumar A, Suri KA, Dutt P. Glycowithanolides accumulation in in vitro shoot cultures of Indian ginseng (Withania somnifera Dunal). Nat Prod Commun 2009;4:479â€“82.
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;23:430â€“4.
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;7:137â€“40.
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;8:1344â€“51.
Weathers PJ, Towler MJ, Xu J. Bench to batch: advances in plant cell culture for producing useful products. Appl Microbiol Biotechnol 2010;85:1339â€“51.
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;114:121â€“9.
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;56:621â€“6.
Ciddi V. Withaferin A from cell cultures of Withania somnifera. Indian J Pharm Sci 2006;68:490â€“2.
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;7:3112â€“20.
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;250:539â€“49.
Kuzuyama T. Mevalonate and nonmevalonate pathways for the biosynthesis of isoprene units. Biosci Biotechnol Biochem 2002;66:1619â€“27.
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;1774:1199â€“207.
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;31:1889â€“97.
Hemmerlin A, Harwood JL, Bach TJ. A raison dâ€™Ãªtre for two distinct pathways in the early steps of plant isoprenoid biosynthesis? Prog Lipid Res 2012;51:95â€“148.
Akhtar N, Gupta P, Sangwan NS, Sangwan RS, Trivedi PK. Cloning and functional characterization of 3â€“hydroxylâ€“3 methyl glutaryl Withania somnifera: an important medicinal plant. Protoplasma 2013;250:613â€“22.
Chappell J. The biochemistry and molecular biology of isoprenoid metabolism. Plant Physiol 1995;107:1â€“6.
Mirjalili MH, Moyano E, Bonfil M, Cusido RM, Palazon J. Steroidal lactones from Withania somnifera, an ancient plant for novel medicine. Molecules 2009;14:2373â€“93.
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;63:512â€“25.
Matsushita Y, Kang W, Charlwood BV. Cloning and analysis of a cDNA encoding farnesyl diphosphate synthase from Artemisia annua. Gene 1996;172:207â€“9.
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;1625:214â€“20.
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;141:220â€“31.
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;1350:240â€“6.
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;48:692â€“9.
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;100:371â€“8.
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;64:2676â€“80.
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]
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;499:25â€“36.
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;352:259â€“63.
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;71:36â€“46.
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;9:375â€“86.
Bai M, Prestwich GD. Inhibition and activation of porcine squalene epoxidase. Arch Biochem Biophys 1992;293:305â€“13.
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]
Rees HH, Goad LJ, Goodwin TW. 2, 3-Oxidosqualene cycloartenol cyclase from Ochromonas malhamensis. Biochim Biophys Acta 1969;176:892â€“4.
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;40:7007â€“116.
Diarra S, He J, Wang J, Li J. Ethylene treatment improves diosgenin accumulation in in vitro cultures of Dioscorea zingiberensis via up-regulation of CAS and HMGR gene expression. Electron J Biotechnol 2013;16:6.
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;106:725â€“30.
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;282:23581â€“90.
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;75:713â€“23.
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;272:486â€“9.
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;27:157â€“62.
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;460:48â€“55.
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;268:4086â€“94.
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]
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;260:515â€“21.
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;190:16â€“23.
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]
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.
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;79:413â€“27.
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;12:131â€“5.
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;37:893â€“902.
Dasgupta MG, George BS, Bhatia A, Sidhu OP. Characterization of Withania somnifera leaf transcriptome and expression analysis of pathogenesisâ€“related genes during salicylic acid signaling. PLoS One 2014;9. Doi:10.1371/journal.pone.0094803. [Article in Press]
Schneider G, BÃ¶hm HJ. Virtual screening and fast automated docking methods. Drug Discovery 2002;7:64â€“70.
Waszkowycz B. Structure-based approaches to drug design and virtual screening. Curr Opin Drug Discovery 2002;5:407â€“13.
Toledo-Sherman LM, Chen D. High-throughput virtual screening for drug discovery in parallel. Curr Opin Drug Discovery 2002;5:414â€“21.
Aishwarya S, Santhi N. Insights from the molecular docking of withanolide derivatives to the target protein PknG from Mycobacterium tuberculosis. Bioinformation 2011;7:1â€“4.
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.
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]
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;25:595â€“8.
Saha S, Islam MK, Shilpi JA, Hasan S. Inhibition of VEGF: a novel mechanism to control angiogenesis by Withania somniferaâ€™s key metabolite Withaferin A. In Silico Pharmacol 2013;1:11â€“5.
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;461:225â€“34.
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;74:16866â€“74.
Chatterjee S, Srivastava S, Khalid A, Singh N, Sangwan RS. Comprehensive metabolic fingerprinting of Withania somnifera leaf and root extracts. Phytochemistry 2010;71:1085â€“94.
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 one 2015;4:e0123360. Doi:10.1371/journal.pone.0123360. [Article in Press]
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.