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

Abstract

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.

Withaferin A inhibits breast cancer invasion and metastasis at sub‐cytotoxic doses by inducing vimentin disassembly and serine 56 phosphorylation

“…21 The root extract of W. somnifera is composed of 14 compounds known as Withanolides, with Withaferin A (WFA) being the most abundant. 22,23 WFA directly binds vimentin and inhibits vimentin assembly through a covalent modification of cysteine 328 in the conserved vimentin a-helical coiled coil 2B domain. 24 At micromolar doses, WFA has vimentin-dependent proteasome inhibitory activity that leads to an anti-angiogenic effect.…”

Estimation of antioxidant potential of individual components present in complex mixture ofWithania somnifera(Ashwagandha) root fraction by thin-layer chromatography-2,2-diphenyl-1-picrylhdrazyl method

“…Withanolides are biosynthesized by de novo pathway in roots as well as in leaf [19][20][21]. Quantitative determination of withanolides has been carried out by high-performance liquid chromatography (HPLC) and high-performance thin-layer chromatography (HPTLC) [22][23][24][25]. Withanolides are steroidal lactones, and about 35 withanolides have been isolated from the roots of W. somnifera.…”

LC-MS/MS method for the quantification of withaferin-A in plant extracts ofWithaniaspp.

“…Thus, our limit of detection (LOD) in the standards was found to be 0.6 ng/mL (ppb), and applying the method to plant extracts, the LOD was approximately 10 times higher than in the standards, being 6 ng/g. Examples of reported detection limits for withaferin-A include 500 ng/mL using single-ion monitoring [10], 190 ng/mL using photodiode array [5] and 120 ng/mL using HPTLC [12], thus confirming that the LOD decreases in MS/MS compared to other methods. We required an analysis time of 10 min, which is less than in other reported studies on withanolides, in which total analysis times are 24 min (retention time for withaferin-A 14 min), 35 min (retention time for withaferin-A 21.35 min) and 50 min (retention time for withaferin-A 19.2 min) [4,9,10], respectively.…”

“…To extract the withaferin-A, we followed the method reported by Chaurasiya et al [5] with some modifications. Lyophilized and powdered plant material (0.5 g) was extracted with 25% MeOH (80 mL) using Soxhlet (8 h).…”

“…The most active withanolide found in Withania extracts and herbal products is the potent growth inhibitor [2] withaferin-A, which exhibits in vivo antitumor and antiangiogenesis activity. Several studies have identified the withanolide compounds of W. somnifera using HPLC coupled to a photodiode array detector (UV/DAD) [3,4] or PDS and evaporative light scattering detection [5]. However, since the withanolide UV absorption spectrum varies considerably in the number of permutations of chromophoric groups [6] and most withanolides show a low UV maximum, these compounds are in-creasingly being determined by HPLC/MS [7].…”