Estimation of Antioxidant Potential of Individual Components Present in Complex Mixture of Withania somnifera (Ashwagandha) Root Fraction by Thin-Layer Chromatography—2,2-Diphenyl-1-Picrylhdrazyl Method

Summary

A thin-layer chromatography–2,2-diphenyl-1-picrylhdrazyl (TLC—DPPH) method has been developed for the quantification of withanolides and simultaneous estimation of antioxidant potential of each constituent present in a complex mixture. High-performance thin-layer chromatography (HPTLC) of withanolide-rich fraction of Withania somnifera root was performed on Si 60F₂₅₄ HPTLC plates with dichloromethane-toluene-methanol-acetone-diethyl ether (6.5:7:4:1.5:1, v/v) as a mobile phase for the separation of withanolides and on the basis of R_F values 0.07, 0.55, 0.61, 0.63, 0.66, and 0.77 for withanoside V, withaferine A, 1,2-deoxywithastramonolide, withanone, withanolide A, and withanolide B, respectively. The concentrations of withanoside V, withaferine A, 1,2-deoxywithastramonolide, withanone, withanolide A, and withanolide B were 0.86, 12.9, 1.92, 1.52, 5.24, and 4.52 mg g⁻¹ respectively. A TLC-DPPH rapid test was used to assess the free-radical scavenging activity of constituents in withanolide-rich fraction for the first time. In withanolides, withanoside V and withanolide B show the highest antioxidant potential. In unknown compounds, peak No. 17 at R_F 0.92 has the highest antioxidant potential and peak No. 16 at R_F 0.87 has recorded the lowest antioxidant potential. It is also found that some of the unidentified components had significant antioxidant potential. This technique has several advantages; therefore, this method will be useful for rapidly validating the Ayurvedic products containing W. somnifera.