Cytotoxic withanolides from the leaves of Moroccan Withania frutescens

Withania species are a rich source of interesting phytochemical substances (withanolides) which have shown several biological properties.

To investigate the cytotoxic potential of Withania frutescens (L.) Pauquy (Solanaceae) leaf extracts and isolated active compounds against cultured tumor cell lines.

The crude methanol extract of W. frutescens leaves was partitioned with dichloromethane, ethyl acetate and n-butanol. MeOH extract and its fractions were tested for their cytotoxic activity against cancer cell lines (HepG2 and HT29) using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. Bioassay-guided fractionation was performed for the active CH₂Cl₂ fraction employing column chromatography and preparative high-performance liquid chromatography. Structural elucidation of the isolated active compounds was carried out mainly by 1D and 2D NMR and mass spectrometry. The compounds were then tested for their cytotoxic activity.

The CH₂Cl₂ fraction was the most active against HT29 cell line. The fractionation procedure resulted in the isolation of 4β,17α,27-trihydroxy-1-oxo-22-R-witha-2,5,24-trienolide (1), 5β,6β-epoxy-4β,17α,27-trihydroxy-1-oxowitha-2,24-dienolide (2) and 2,3-dihydroxywithaferin A-3β-O-sulfate (3). The latter exhibited the strongest cytotoxic activity against HT29 cancer cell lines (IC₅₀ of 1.78 ± 0.09 µM) which was comparable to that of 5-fluorouracil (5-FU) used as the positive antimitotic control.

Compounds 2 and 3 were isolated from W. frutescens for the first time. Data obtained suggest that the sulfated steroidal lactone (3) can be considered as a compound with potential application in the new anticancer drugs development field.