Isolation of cytotoxic triterpenes from the mangrove plant, Scyphiphora hydrophyllacea CFGaertn (Rubiaceae)

Purpose: To isolate active cytotoxic compounds from the hexane and chloroform extracts of the leaves of the mangrove plant, Scyphiphora hydrophyllacea C.F. Gaertn (Rubiaceae), grown in Sri Lanka. Methods: Dried pulverized leaves of S. hydrophyllacea were extracted with hexane and chloroform. Vacuum liquid chromatography (VLC), column chromatography (size exclusion chromatography, Sephadex LH-20) and reversed phase preparative recycling high performance liquid chromatography (HPLC) techniques were used to isolate three compounds (compounds 1, 2 and 3). The structures of the isolated compounds were established with the aid of H-1, C-13 and two-dimensional nuclear magnetic resonance (2D-NMR) and electron ionization-mass spectrometry (EI-MS) techniques. 3-(4,5Dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to evaluate the cytotoxic effects of the compounds on oestrogen receptor positive breast (MCF-7) and non-small cell lung (NCIH- 292) cancer cells. Results: The isolated compounds were identified as oleanolic acid (1), ursolic acid (2) and eichlerianic acid (3). Ursolic acid and eichlerianic acid showed strong cytotoxic effects {IC50-ursolic acid: 8.47 mu g/mL (24 h, MCF-7), 7.78 mu g/mL (24 h, NCI-H292) and eichlerianic acid: 8.86 mu g/mL (24 h, MCF-7), 10.15 mu g/mL (24 h, NCI-H292)} in MCF-7 and NCI-H292 cancer cells at 24, 48 and 72 h post-incubation periods. Conclusion: Hexane and chloroform extracts of the leaves of S. hydrophyllacea yielded three compounds namely oleanolic acid, eichlerianic acid and ursolic acid. Ursolic acid and eichlerianic acid have been isolated for the first time from the leaves of S. hydrophyllacea grown in Sri Lanka and demonstrate in-vitro cytotoxic effects in oestrogen receptor positive (MCF-7) and non-small lung cancer (NCI-H-292) cells.