Planta Med 2001; 67(2): 146-149
DOI: 10.1055/s-2001-11494
Original Paper

© Georg Thieme Verlag Stuttgart · New York

In Vitro Propagation of Withania somnifera and Isolation of Withanolides with Immunosuppressive Activity

Mirosława Furmanowa1,*, Dorota Gajdzis-Kuls1 , Joanna Ruszkowska2 , Zbigniew Czarnocki2 , Grażyna Obidoska3 , Ałła Sadowska3 , R. Rani4 , Shakti N. Upadhyay4
  • 1 Department of Biology and Pharmaceutical Botany, The Medical University of Warsaw, Warsaw, Poland
  • 2 Laboratory of Natural Products, Faculty of Chemistry, The Warsaw University, Warsaw, Poland
  • 3 Department of Plant Genetics, Breeding and Biotechnology, Agricultural University, Warsaw, Poland
  • 4 National Institute of Immunology, Aruna Saaf Ali Marg., New Delhi, India
Further Information

Publication History

March 23, 2000

May 1, 2000

Publication Date:
31 December 2001 (online)

Abstract

Withania somnifera plantlets were produced in vitro from the shoot-tip of aseptically germinated seedlings. Culture conditions were optimized using different plant growth regulators which gave rise to 120 shoots from a single bud. The plantlets were then transferred to pots and maintained in greenhouse for 4 months. 90 % of these in vitro propagated plantlets survived and showed normal growth. Leaves from these plants were used for isolation of the withanolides. Methanolic extract of leaves from plantlets growing in tissue culture and those transferred to the greenhouse were evaluated for immunomodulatory activity. While the extract from greenhouse samples showed potent immunosuppressive activity, those from tissue cultures samples did not show any activity. Fractionation and characterization of withanolides, using HPLC, NMR, MS methods revealed the presence of withaferin A in the greenhouse samples. Our results indicate that Withania species may require longer time and better differentiation and also natural environment for the production of withaferin A.

Abbreviations

2,4-D:2,4-Dichlorophenoxyacetic acid

BA:6-Benzyladenine

IBA:Indole-3-butyric acid

IAA:Indole-3-acetic acid

K:Kinetin

AS:Adenine sulfate

References

  • 1 Proksa B, Uhrin D, Fuska J. Hydrolysis of withaferin A-4,27-diacetate.  Pharmazie.  1986;  41 282
  • 2 Bessalle R, Lavie D. Semi-quantitative reversed-phase high-performance liquid chromatographic analysis of the ecotypes of Withania somnifera chemotype III.  J. Chromatog.. 1987;  389 195-210
  • 3 Devi P U, Sharada A, Solomon F E, Kamath M. In vivo growth inhibitory effect of Withania somnifera (Ashwagandha) on a transplantable mouse tumor, Sarcoma 180.  Ind. J. Exp. Biol.. 1993;  30 169-72
  • 4 Shohat B, Kirson I, Lavie D. Immunosuppressive activity of two plant steroidal lactones: withaferin A and withanolide E.  Biomedicine. 1978;  28 18-24
  • 5 al-Hindawi M K, al-Khafaji S, Abdul-Nabi M. Anti-granuloma activity of iraqi Withania somnifera .  J. Ethnopharmacol.. 1992;  37 113-6
  • 6 Furmanowa M, Gajdzis-Kuls D, Starosciak B, Stefanska J. Antibacterial activity of Withania somnifera (L.) Dun. organs cultivated in vitro .  Herba Polonica. 1998;  44 265-9
  • 7 Bhattacharya S K, Satyan K S, Ghosal S. Antioxidant activity of glycowithanolides from Withania somnifera .  Indian J. Exp. Biol.. 1997;  35 236-9
  • 8 Fuska J, Fuskowa A, Rosazza J P, Nicholas A W. Novel cytotoxic and antitumor agents. IV. Withaferin A: relation of its structure to the in vitro cytotoxic effects on P-388 cells.  Neoplasma. 1984;  31 31-6
  • 9 Ganasoundari A, Zare S M, Devi P U. Modification of bone marrow radiosensitivity by medicinal plant extracts.  Br. J. Radiology. 1997;  70 599-602
  • 10 Bähr V, Hänsel R. Immunomodulating properties of 5,20α(R)-dihydroxy-6α,7α-epoxy-1-oxo-(5α)-witha-2,24-dianolide and solsodine.  Planta Medica. 1982;  44 32-3
  • 11 Ghosal S, Lal J, Srivastava R. Immunomodullatory and CNS effects of sitoinosides from Withania somnifera .  Phytotherapy Research. 1989;  3 201
  • 12 Habtemariam S. Cytotoxicity and immunosuppressive activity of withanolides from Discopodium penninervium .  Planta Medica. 1996;  63 15-7
  • 13 Upadhyay S N. Therapeutic potential of immunomodulatory agents from plant products. In: Shakti N, Upadhyay, ed Immunomodulation. New Delhi, India; Narosa Publishing House 1997: 149-54
  • 14 Heble M R. Multiple shoot cultures: a viable alternative in vitro system for the production of known and new biologically active plant constituents. In: Neumann HK, Barz W, Reinhard E, editors Primary and Secondary Metabolism of Plant Cell Cultures. Heidelberg, Germany; Springer Verlag 1985: 281-9
  • 15 Murashige T, Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures.  Physiol. Plant. 1962;  15 473-9
  • 16 Nitsch J P, Nitsch C. Haploid plants from pollen grains.  Science. 1969;  163 85-7
  • 17 Starck J R. Uprawa roli i nawożenie roslin ogrodniczych (Cultivation of soil and fertilization horticultural plants). Warsaw, Poland; PWRiL 1997: 202
  • 18 Olszowska O, Furmanowa M. Micropropagation of Coluria geoides by axillary shoots.  Planta Medica. 1986;  52 521
  • 19 Lavie D, Glotter E. Constituents of Withania somnifera Dun. III. The side chain of Withaferin A.  J. Org. Chem.. 1965;  30 1774-8
  • 20 Pelletier S, Moody N, Nowacki J, Bhattacharya J. Carbon-13 nuclear magnetic resonance spectral analysis of naturally occurring withanolides and their derivatives.  J. Nat. Prod.. 1979;  42 512-21

Prof. Dr. Mirosława Furmanowa

Department of Biology and Pharmaceutical Botany

The Medical University of Warsaw

Banacha Str. 1

02-097 Warsaw

Poland

Phone: Tel./Fax 00 48 22 8235984

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