New withanolides from leaves along with alkyl glucoside from roots and stem as well as variations in withanolide contents in leaves of accessions of Withania somnifera, Ashwagandha- the Indian ginseng$.

Based on the major components in the leaves, the ashwagandha has been found to exist in several chemotypic forms in India. From the leaves of various accessions of Withania somnifera, which were maintained in our institute, three new steroids namely, 4-acetoxy-20ß-hydroxy-1-oxo-witha-2,5,24-trienolide (7), 24,25-dihydro-14α-hydroxy withanolide D (9), 5α,6ß,17α,27-tetrahydroxy-1-oxo-witha-2,24-dienolide (12) together with thirteen known withanolides were identified by spectroscopic methods. From the roots and stem of one accession and leaves of another, a new alkyl ester glucoside (4) has also been isolated. The new withanolides 7, 9 and 12 have been tentatively named as withanolide 135 A, withanolide 135B and withanolide 108, respectively.

Withanolide A is inherently de novo biosynthesized in roots of the medicinal plant Ashwagandha (Withania somnifera).

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 withanolides 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.

Selective reactivity of 2-mercaptoethanol with 5beta,6beta-epoxide in steroids from Withania somnifera.

2-Mercaptoethanol reacts selectively with the 5beta,6beta-epoxy steroids isolated from Withania somnifera substituting the epoxide by a six-membered oxyethylene-2'-thio ring whereas it failed to show such reactivity on 6alpha,7alpha-epoxy withasteroids. The structure of the product has been elucidated by spectroscopic methods, especially applying extensive 2D NMR methods. The anticancer activity of withaferin A was lost in the reaction product indicating that its activity is also linked to the free 5beta,6beta-epoxide functional group.

Analysis of withanolides in root and leaf of Withania somnifera by HPLC with photodiode array and evaporative light scattering detection.

A reversed-phase HPLC method for the simultaneous analysis of nine structurally similar withanolides, namely, 27-hydroxy withanone, 17-hydroxy withaferin A, 17-hydroxy-27-deoxy withaferin A, withaferin A, withanolide D, 27-hydroxy withanolide B, withanolide A, withanone and 27-deoxywithaferin A, has been developed using a linear binary gradient solvent system comprising methanol and water containing 0.1% acetic acid. Both photodiode array and evaporative light scattering detection were used to profile the extract compositions and to quantify the withanolides therein. Homogeneity and purity of each peak was ascertained by comparative evaluation of the on-line UV spectra of the eluted compounds with those of the reference compounds. The method has been validated with respect to various parameters of performance quality including computation regression analysis based on calibration curves, peak resolution factor, asymmetry factor, tailing factor, RSD (%) of retention time and peak area response, limit of quantivation, limit of detection, precision and recovery. The developed method has been applied to the analysis of leaf and root tissues of Withania somnifera for withanolide content.

Withanolide A biogeneration in in vitro shoot cultures of ashwagandha (Withania somnifera DUNAL), a main medicinal plant in Ayurveda.

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.

Unusually sulfated and oxygenated steroids from Withania somnifera.

Four (1, 8-10) and six known (2-7) withanolides were isolated from the leaves of Withania somnifera. Among the new compounds, 10 possessed the rare 3-O-sulfate group with the saturation in A ring and 9 contained unusual 1,4-dien-3-one group. Compound 8 did not have usual 2,3 unsaturation in A ring while 1 had the rare C-16 double bond. The structures of all the compounds were elucidated by spectroscopic methods and chemical transformation.