Extraction, Isolation, and Characterization of Bioactive Anticancer Compounds from Withania somnifera

In recent years, an increasing attention has been focused on the identification of new anticancer agents of natural origin that are safer and more effective. Bioactive constituents were isolated, characterised and identified from roots of Withania somnifera in the present investigation. Serial solvent extraction of DAE was done with hexane, chloroform, ethyl acetate, methanol and water, and bioassay-guided fractionation. The methanol fraction showed the most potent cytotoxic activity among the extracts with IC50 values from 36.2 to 44.6 µg/mL on MCF-7, HeLa and A549 cancer cells. Subsequent chromatographic isolation of this fraction led to isolation of two major active compounds which were characterized as Withaferin A and Quercetin. Characterization was performed by UV–Vis, FTIR, ¹H, ¹³C NMR, LC-MS/MS analyses for their identification. In-vitro cytotoxic activity of Withaferin A was found to be highly potent with IC₅₀ ranges from 1.8 -2.5 µM when compared to reference standard (IC₅₀ ≥) against cancer cells (doxorubicin). Quercetin demonstrated modest cytotoxicity (IC₅₀ 12.8-16.2 µM). Dose response analysis revealed that Withaferin A induced a rapid decrease in cell viability while gradual dose dependent decrease was observed for Quercetin. These results implied that Withaferin A might be a potential lead compound for anticancer drug, and Quercetin might exert synergism or supportive action for the crude extracts. In general, this study has characterized W. somnifera as a potent repository of natural anticancer agents and laid a platform for further mechanistic, in vivo, and formulation studies to promote these phytoconstituents to therapeutic regime.