Withanolide A Biogeneration in in Vitro Shoot Cultures of Ashwagandha (Withania somnifera DUNAL), a Main Medicinal Plant in Ayurveda

Abstract

Multiple shoot cultures of two experimental lines of Withania somnifera plants (RS-Selection-1 and RS-Selection-2) were established using nodal segments as explants. The hormonal combinations of benzyl adenine and kinetin not only influenced their morphogenetic response but also differentially modulated the level of biogeneration of withanolide A in the in vitro shoots of the two lines. Interestingly, withanolide-A, that was hardly detectable in the aerial parts of field-grown Withania somnifera (explant source), accumulated considerably in the in vitro shoot cultures of the plant. The productivity of withanolide A in the cultures varied considerably (ca. 10-fold, 0.014 to 0.14 mg per gram fresh weight) with the change in the hormone composition of the culture media as well as genotype used as source of the explant. The shoot culture of RS-Selection-1 raised at 1.00 ppm of BAP and 0.50 ppm of kinetin displayed the highest concentration of withanolide A in the green shoots of 0.238 g per 100 g dry weight tissue. This was a more analytical concentration keeping in view the isolation yields so far reported from the dried roots of the field-grown plant (ca. 0.015 g per 100 g dry weight), even if isolation losses are considered during purification. The enhanced de novo biogenesis of withanolide A in shoot cultures was corroborated with radiolabel incorporation studies using [2-(14)C] acetate as a precursor. Production of withaferin A was also found in the in vitro shoot cultures. As this compound is a predominant withanolide of native shoots as well and has been already reported to be accumulated in in vitro shoot cultures, its biogeneration observed in these shoot cultures is not discussed in detail.

Withanolides and Related Steroids

“…Incubation of withaferin A (1) with Cunninghamella echinulata gave 12b-hydroxywithaferin A (476) and 15b-hydroxywithaferin A (477) (194); the same hydroxylated products have been obtained upon incubation with Cunninghamella elegans (4). Several reports also describe the production of withanolides (mostly withaferin A) by shoot cultures of Withania somnifera (195)(196)(197)(198).…”

“…Accordingly, these bioactivity results should be considered with caution. Among the Physalis withanolides with an unmodified skeleton, potent cytotoxic activity has been reported for withangulatins A (511), B (94), and I (102), physangulin B (512), phyperunolide A (101), withaphysacarpin (195), philadelphicalactones A (197) and B (198), 18-hydroxywithanolide D (128), and withanone (513) (50,55,56,58,104,247).…”

“…LC-MS analysis of an ethyl acetate extract of the plant suggested that 196 may occur naturally although in minor amounts, being generated to a larger extent as a result of the extraction procedure. From leaves and stems of P. philadelphica, Kinghorn and coworkers isolated philadelphicalactones A and B (197,198) and compound 199, all of them with a saturated d-lactone side chain (104,105).…”

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

“…It appears to be relevant to adopt in vitro system which may serve as an alternative source of withanolides and thus may be exploited for efficient biogeneration of such substances throughout the year, which are pharmacologically promising but are severely limited in production. Production of withanolide D, withanolide A, withaferin A, withanone have been reported in organogenetic cultures including hairy roots (Banerjee et al 1994;Furmanowa et al 2001;Murthy et al 2008;Jha 1999, 2001;Ray et al 1996;Roja and Heble 1991;Sangwan et al 2007;Vitali et al 1996). Recently Nagella and Murthy (2010) reported production of withanolide A in the suspension cultures of Withania somnifera.…”

“…There are reports on in vitro culture of Withania somnifera using different explants (Sen and Sharma 1991;Kulkarni et al 2000;Manickam et al 2000;Sivanesan and Murugesan 2005;Sabir et al 2007;Sivanesan 2007) as well as generation of withanolides in vitro (Roja and Heble 1991;Furmanowa et al 2001;Ray and Jha 2001;Sangwan et al 2005;Sangwan et al 2007). There is also a report on the production of withanolide A in cell-suspension cultures of W. somnifera (Nagella and Murthy 2010).…”