Skip to main content
SpringerLink
Log in

In vitro and in situ screening systems for morphological and phytochemical analysis of Withania somnifera germplasms

  • Original Paper
  • Published:
Plant Cell, Tissue and Organ Culture (PCTOC) Aims and scope Submit manuscript

Abstract

We report, for the first time for Withania somnifera, the use of a modified in vitro system for morphological and phytochemical screening of true to type plants as compared with those grown in a conventional in situ system. Eleven germplasms of cultivated W. somnifera from different regions of India were collected to examine chemotypic variation in withaferin A (WA). Methods were developed to optimize WA extraction. The maximum concentration of WA was extracted from manually ground leaf and root material to which 60 % methanol was added followed by sonication in a water bath sonicator. Variation in WA concentration in whole plants was observed amongst the different germplasms. In the in vitro system, the concentration of WA ranged between 0.27 and 7.64 mg/g dry weight (DW) and in the in situ system, the range in concentration was between 8.06 and 36.31 mg/g DW. The highest amount of WA found in leaves was 7.37 and 41.42 mg/g DW in the in vitro and the in situ systems respectively. In roots, the highest WA concentration was 0.27 mg/g DW in the in vitro and 0.60 mg/g DW in the in situ system. There are distinct advantages in using the in vitro grown plants rather than those grown in the in situ system including the simplicity of design, efficient use of space and nutrition and a system which is soil and contaminant free. The proposed in vitro system is therefore ideal for utilization in molecular, enzymatic and biochemical studies.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

Abbreviations

ESI:

Electron spray ionization

DW:

Dry weight

HPLC:

High performance liquid chromatography

HPLC–MS:

High performance liquid chromatography mass spectrometry

MS:

Murashige and Skoog

PDA:

Photo diode array

WA:

Withaferin A

References

  • Aalinkeel R, Hu Z, Nair BB, Sykes DE, Reynolds JL, Mahajan SD, Schwartz SA (2010) Genomic analysis highlights the role of the JAK-STAT signaling in the anti-proliferative effects of dietary flavonoid ‘ashwagandha’ in prostate cancer cells. Evid based Complement Altern Med 7:177–187

    Article  Google Scholar 

  • Anon (1976) The wealth of India, raw materials, vol. X: SpW. Publications and Information Directorate. CSIR, New Delhi, pp 581–585

    Google Scholar 

  • Atal CK (1975) Pharmacognosy and phytochemistry of Withania somnifera (Linn.) Dunal (ashwagandha). Central Council for Research in Indian Medicine and Homoeopathy publisher, New Delhi

  • Chatterjee S, Srivastava S, Khalid A, Singh N, Sangwan RS, Sidhu OP, Roy R, Khetrapal CL, Tuli R (2010) Comprehensive metabolic fingerprinting of Withania somnifera leaf and root extracts. Phytochem 71:1085–1094

    Article  CAS  Google Scholar 

  • Chaurasiya ND, Uniyal GC, Lal P, Misra L, Sangwan NS, Tuli R, Sangwan RS (2008) Analysis of withanolides in root and leaf of Withania somnifera by HPLC with photodiode array and evaporative light scattering detection. Phytochem Anal 19:148–154

    Article  PubMed  Google Scholar 

  • Ciddi V (2006) Withaferin A from cell cultures of Withania somnifera. Indian J Pharm Sci 68:490–492

    Article  CAS  Google Scholar 

  • Dewir YH, Chakrabarty D, Lee SH, Hahn EJ, Paek KY (2010) Indirect regeneration of Withania somnifera and comparative analysis of withanolides in in vitro and greenhouse grown plants. Biol Plant 54:357–360

    Article  Google Scholar 

  • Dhar RS, Verma V, Suri KA, Sangwan RS, Satti NK, Kumar A, Tuli R, Qazi GN (2006) Phytochemical and genetic analysis in selected chemotypes of Withania somnifera. Phytochem 67:2269–2276

    Article  CAS  Google Scholar 

  • Dhar RS, Lattoo SK, Kumar A, Qazi GN (2008) Use of ISSR and RAPD markers to facilitate molecular characterization of Withania germplasm—a repository of pharmaceutically active metabolite. J Plant Biol 35:107–113

    CAS  Google Scholar 

  • Doner LW, Becard G (1991) Solubilization of gellan gels by chelation of cations. Biotechnol Tech 5:25–28

    Article  CAS  Google Scholar 

  • Ganzera M, Choudhary MI, Khan IA (2003) Quantitative HPLC analysis of withanolides in Withania somnifera. Fitoter 74:68–76

    Article  CAS  Google Scholar 

  • Hoagland DR, Arnon DI (1950) The water-culture method for growing plants without soil. Calif Agric Exp Stn Circ 347:1–32

    Google Scholar 

  • Jain SK, Bordia PC, Joshi A (2007) Genetic diversity in ashwagandha. J Med Aromat Plant Sci 29:11–15

    Google Scholar 

  • Kaileh M, Berghe WV, Boone E, Essawi T, Haegeman G (2007) Screening of indigenous Palestinian medicinal plants for potential anti-inflammatory and cytotoxic activity. J Ethnopharmacol 113:510–516

    Article  PubMed  Google Scholar 

  • Khajuria RK, Suri KA, Gupta RK, Satti NK, Amina M, Suri OP, Qazi GN (2004) Separation, identification, and quantification of selected withanolides in plant extracts of Withania somnifera by HPLC-UV (DAD)—positive ion electrospray ionisation–mass spectrometry. J Sep Sci 27:541–546

    Article  CAS  PubMed  Google Scholar 

  • Kothari KS, Singh PC, Kumar VY, Singh K (2003) Morphology, yield and quality of ashwagandha (Withania somnifera L. Dunal) roots and its cultivation economics as influenced by tillage depth and plant population density. J Hortic Sci Biotechnol 78:422–425

    Google Scholar 

  • Kual KN (1957) The origin, distribution and cultivation of ashwagandha the so called Withania somnifera of Indian literature. Symposium on the utilization of Indian medicinal plants. New Delhi, pp 7–8

  • Kulkarni AA, Thengane SR, Krishnamurthy KV (2000) Direct shoot regeneration from node, internode, hypocotyl and embryo explants of Withania somnifera. Plant Cell Tissue Organ Cult 62:203–209

    Article  Google Scholar 

  • Kumar A, Kaul MK, Bhan MK, Khanna P, Suri KA (2007) Morphological and chemical variation in 25 collections of the Indian medicinal plant, Withania somnifera (L.) Dunal (Solanaceae). Genet Resour Crop Evol 54:655–660

    Article  CAS  Google Scholar 

  • Kumar A, Mir B, Sehgal D, Dar T, Koul S, Kaul M, Raina S, Qazi G (2011) Utility of a multidisciplinary approach for genome diagnostics of cultivated and wild germplasm resources of medicinal Withania somnifera, and the status of new species, W. ashwagandha, in the cultivated taxon. Plant Syst Evol 291:141–151

    Article  Google Scholar 

  • Kushwaha S, Roy S, Maity R, Mallick A, Soni VK, Singh PK, Chaurasiya ND, Sangwan RS, Misra-Bhattacharya S, Mandal C (2012) Chemotypical variations in Withania somnifera lead to differentially modulated immune response in BALB/c mice. Vaccine 30:1083–1093

    Article  CAS  PubMed  Google Scholar 

  • Lee J, Hahm ER, Singh SV (2010) WA inhibits activation of signal transducer and activator of transcription 3 in human breast cancer cells. Carcinogenesis 31:1991–1998

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Lee W, Kim TH, Ku SK, Min KJ, Lee HS, Kwon TK, Bae JS (2012) Barrier protective effects of Withaferin A in HMGB1-induced inflammatory responses in both cellular and animal models. Toxicol Appl Pharmacol 262:91–98

    Article  CAS  PubMed  Google Scholar 

  • Madina BR, Sharma LK, Chaturvedi P, Sangwan RS, Tuli R (2007) Purification and characterization of a novel glucosyltransferase specific to 27β-hydroxy steroidal lactones from Withania somnifera and its role in stress responses. Biochim Biophys Acta 1774:1199–1207

    Article  CAS  PubMed  Google Scholar 

  • Maitra R, Porter AM, Huang S, Gilmour PB (2009) Inhibition of NFκB by the natural product Withaferin A in cellular models of Cystic Fibrosis inflammation. J Inflamm 6:15

    Article  Google Scholar 

  • Manickam VS, Elango Mathavan R, Antonisamy R (2000) Regeneration of Indian ginseng plantlets from stem callus. Plant Cell Tissue Organ Cult 62:181–185

    Article  Google Scholar 

  • Mayola E, Gallerne C, Esposti DD, Martel C, Pervaiz S, Larue L, Debuire B, Lemoine A, Brenner C, Lemaire C (2011) Withaferin A induces apoptosis in human melanoma cells through generation of reactive oxygen species and down-regulation of Bcl-2. Apoptosis 16:1014–1027

    Article  CAS  PubMed  Google Scholar 

  • Min KJ, Choi K, Kwon TK (2011) Withaferin A down-regulates lipopolysaccharide-induced cyclooxygenase-2 expression and PGE2 production through the inhibition of STAT1/3 activation in microglial cells. Int Immunopharmacol 11:1137–1142

    Article  CAS  PubMed  Google Scholar 

  • Mirzajani F, Ghassempour A, Jalali-Heravi M, Mirjalili MH (2010) Optimisation of a microwave-assisted method for extracting Withaferin A from Withania somnifera Dunal. Using central composite design. Phytochem Anal 21:544–549

    Article  CAS  PubMed  Google Scholar 

  • Misra OH, Sharma RJ, Lal KR (1998) Genetic divergence in ashwagandha (Withania somnifera). J Med Aromat Plant Sci 20:1018–1021

    Google Scholar 

  • Mohan R, Hammers H, Bargagna-mohan P, Zhan X, Herbstritt C, Ruiz A, Zhang L, Hanson A, Conner B, Rougas J, Pribluda V (2004) Withaferin A is a potent inhibitor of angiogenesis. Angiogenesis 7:115–122

    Article  CAS  PubMed  Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497

    Article  CAS  Google Scholar 

  • Nagella P, Murthy HN (2010) Establishment of cell suspension cultures of Withania somnifera for the production of withanolide A. Bioresour Technol 101:6735–6739

    Article  CAS  PubMed  Google Scholar 

  • Nakajima H, Wakabayashi Y, Wakamatsu K, Imokawa G (2011) An extract of Withania somnifera attenuates endothelin-1-stimulated pigmentation in human epidermal equivalents through the interruption of PKC activity within melanocytes. Phytother Res 25:1398–1411

    CAS  PubMed  Google Scholar 

  • Negi MS, Singh A, Lakshmikumaran M (2000) Genetic variation and relationship among and within Withania species as revealed by AFLP markers. Genome 43:975–980

    Article  CAS  PubMed  Google Scholar 

  • Negi MS, Sabharwal V, Wilson N, Lakshmikumaran MS (2006) Comparative analysis of the efficiency of SAMPL and AFLP in assessing genetic relationships among Withania somnifera genotypes. Curr Sci 91:464–471

    CAS  Google Scholar 

  • Patra DD, Singh K, Misra HO, Gupta AK, Singh J, Singh SC, Khanuja SPS (2004) Agrotechnologies of ashwagandha Withania somnifera. J Med Aromat Plant Sci 26:332–335

    Google Scholar 

  • Ramesh Kumar R, Prasanna Anjaneya Reddy L, Niranjana Kumar A, Komaraiah K, Purnanand S, Sastry KP (2011) Root textural quality in ashwagandha (Withania somnifera) as influenced by crop growth periods and morphotypes. Ind Crops Prod 34:1231–1234

    Article  Google Scholar 

  • Ramesh Kumar R, Reddy LPA, Kumar JV, Komaraiah K, Purnanand S, Sastry KP (2012) Multivariate analysis and genetic diversity for morphometric and root textural quality traits in ashwagandha (Withania somnifera Dunal). Ind Crops Prod 35:199–202

    Article  Google Scholar 

  • Rani G, Grover IS (1999) In vitro callus induction and regeneration studies in Withania somnifera. Plant Cell Tissue Organ Cult 57:23–27

    Article  Google Scholar 

  • Roja G, Heble MR, Sipahimalani AT (1991) Tissue cultures of Withania somnifera: morphogenesis and withanolide synthesis. Phytother Res 5:185–187

    Article  CAS  Google Scholar 

  • Sangwan RS, Chaurasiya ND, Misra LN, Lal P, Uniyal GC, Sharma R, Sangwan NS, Suri KA, Qazi GN, Tuli R (2004) Phytochemical variability in commercial herbal products and preparations of Withania somnifera (Ashwagandha). Curr Sci 86:461–465

    CAS  Google Scholar 

  • Sangwan RS, Chaurasiya ND, Lal P, Misra L, Uniyal GC, Tuli R, Sangwan NS (2007) Withanolide A biogeneration in in vitro shoot cultures of ashwagandha (Withania somnifera Dunal), a main medicinal plant in ayurveda. Chem Pharm Bull 55:1371–1375

  • Scartezzini P, Antognoni F, Conte L, Maxia A, Troìa A, Poli F (2007) Genetic and phytochemical difference between some Indian and Italian plants of Withania somnifera (L.) Dunal. Nat Prod Res 21:923–932

    Article  CAS  PubMed  Google Scholar 

  • Sen J, Sharma AK (1991) Micropropagation of Withania somnifera from germinating seeds and shoot tips. Plant Cell Tissue Organ Cult 26:71–73

    Article  CAS  Google Scholar 

  • Senthil K, Wasnik N, Kim YJ, Yang DC (2010) Generation and analysis of expressed sequence tags from leaf and root of Withania somnifera (ashwgandha). Mol Biol Rep 37:893–902

    Article  CAS  PubMed  Google Scholar 

  • Sharma LK, Madina BR, Chaturvedi P, Sangwan RS, Tuli R (2007a) Molecular cloning and characterization of one member of 3β-hydroxy sterol glucosyltransferase gene family in Withania somnifera. Arch Biochem Biophys 460:48–55

    Article  CAS  PubMed  Google Scholar 

  • Sharma V, Gupta A, Bhandari P, Gupta R, Singh B (2007b) A validated and densitometric HPTLC method for the quantification of Withaferin-A and Withanolide-A in different plant parts of two morphotypes of Withania somnifera. Chromatographia 66:801–804

    Article  CAS  Google Scholar 

  • Sivanandhan G, Kapil Dev G, Jeyaraj M, Rajesh M, Muthuselvam M, Selvaraj N, Manickavasagam M, Ganapathi A (2013) A promising approach on biomass accumulation and withanolides production in cell suspension culture of Withania somnifera (L.) Dunal. Protoplasma 250:885–898

    Article  CAS  PubMed  Google Scholar 

  • Vaishnavi K, Saxena N, Shah N, Singh R, Manjunath K, Uthayakumar M, Kanaujia SP, Kaul SC, Sekar K, Wadhwa R (2012) Differential activities of the two closely related Withanolides, Withaferin A and Withanone: bioinformatics and experimental evidences. PLoS One 7(9):e44419

  • Voets L, Providencia I, Fernandez K, Ijdo M, Cranenbrouck S, Declerck S (2009) Extraradical mycelium network of arbuscular mycorrhizal fungi allows fast colonization of seedlings under in vitro conditions. Mycorrhiza 19:347–356

    Article  PubMed  Google Scholar 

  • Wadegaonkar PA, Bhagwat KA, Rai MK (2006) Direct rhizogenesis and establishment of fast growing normal root organ culture of Withania somnifera Dunal. Plant Cell Tissue Organ Cult 84:223–225

    Article  Google Scholar 

  • Yang ES, Choi MJ, Kim JH, Choi KS, Kwon TK (2011) Withaferin A enhances radiation-induced apoptosis in Caki cells through induction of reactive oxygen species, Bcl-2 downregulation and Akt inhibition. Chemico Biol Interact 190:9–15

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The authors acknowledge Deakin University, Australia and The Energy and Resources Institute for financial assistance and infrastructure support. Leena Johny was the recipient of a Deakin University postgraduate scholarship. We thank Dr Hashmath Inayath Hussain for his support during data analysis and Shailendra Kumar for assistance in the in situ experiments.

Author information

Authors and Affiliations

  1. Faculty of Science Engineering and Built Environment, School of Life and Environmental Sciences, Deakin University, Waurn Ponds, Geelong, Australia

    Leena Johny, Xavier Conlan & David Cahill

  2. TERI-Deakin Nanobiotechnology Centre, Biotechnology and Bioresources Division, The Energy and Resources Institute, New Delhi, India

    Leena Johny & Alok Adholeya

Authors
  1. Leena Johny
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Xavier Conlan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. David Cahill
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Alok Adholeya
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Alok Adholeya.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Johny, L., Conlan, X., Cahill, D. et al. In vitro and in situ screening systems for morphological and phytochemical analysis of Withania somnifera germplasms. Plant Cell Tiss Organ Cult 120, 1191–1202 (2015). https://doi.org/10.1007/s11240-014-0673-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11240-014-0673-3

Keywords

Search

Navigation