Physiologia Plantarum 133: 278–287. 2008 Copyright ª Physiologia Plantarum 2008, ISSN 0031-9317 Withanolide A is inherently de novo biosynthesized in roots of the medicinal plant Ashwagandha (Withania somnifera) Rajender Singh Sangwan a, *, Narayan Das Chaurasiya a , Payare Lal a , Laxminarain Misra a , Rakesh Tuli b and Neelam Singh Sangwan a a Central Institute of Medicinal and Aromatic Plants (CSIR), PO CIMAP, Lucknow 226015, India b National Botanical Research Institute (CSIR), Lucknow 226 001, India Correspondence *Corresponding author, e-mail: sangwan.lab@gmail.com; rs.sangwan@cimap.res.in Received 26 June 2007; revised 12 January 2008 doi: 10.1111/j.1399-3054.2008.01076.x Ashwagandha (Withania somnifera Dunal., Solanaceae) is one of the most reputed medicinal plants of Ayurveda, the traditional medical system. Several of its traditionally proclaimed medicinal properties have been corroborated by recent molecular pharmacological investigations and have been shown to be associated with its specific secondary metabolites known as withanolides, the novel group of ergostane skeletal phytosteroids named after the plant. Withanolides are structurally distinct from tropane/nortropane alkaloids (usually found in Solanaceae plants) and are produced only by a few genera within Solanaceae. W. somnifera contains many structurally diverse with- anolides in its leaves as well as roots. To date, there has been little biosynthetic or metabolism-related research on withanolides. It is thought that withanolides are synthesized in leaves and transported to roots like the tropane alkaloids, a group of bioactive secondary metabolites in Solanaceae members known to be synthesized in roots and transported to leaves for storage. To examine this, we have studied incorporation of 14 C from [2- 14 C]-acetate and [U- 14 C]- glucose into withanolide A in the in vitro cultured normal roots as well as native/orphan roots of W. somnifera. Analysis of products by thin layer chromatography revealed that these primary metabolites were incorporated into withanolide A, demonstrating that root-contained withanolide A is de novo synthesized within roots from primary isoprenogenic precursors. Therefore, withanolides are synthesized in different parts of the plant (through operation of the complete metabolic pathway) rather than imported. Introduction Ayurveda and traditional Chinese medicines are the key knowledge resources driving phytomolecule-based drug development programs world over. Ashwagandha (With- ania somnifera Dunal., Solanaceae) is one of the most reputed medicinal plant of Ayurveda. The herb forms essential constituent or whole of more than 100 tra- ditional medicines. The well-descript pharmacologi- cal activities of the plant include physiological and metabolic restoration, anti-arthritic, anti-aging, cognitive function improvement in geriatric states and recovery from neurodegenerative disorders (Lal et al. 2006, Misra et al. 2005, Sangwan et al. 2004a). In vitro and in vivo molecular pharmacological investigations have eluci- dated association of these activities of the herb with its specific secondary metabolites known as withanolides Abbreviations – FAB, fast atom bombardment; FW, fresh weight; MS, Murashige and Skoog; Radio-TLC, radioactivity scanned TLC; TCS, three cycle subcultured. 278 Physiol. Plant. 133, 2008