Isolation and characterisation of potential anticancer compounds from medicinal plants
The research work presented in this thesis deals with the anticancer activity of four medicinal plants: 'Caralluma tuberculata' (Asclepiadaceae), 'Fagonia indica' (Zygophyllaceae), 'Solanum surattense' (Solanaceae) and 'Arisaema utile' (Araceae) that originate from the North West and Himalayan regions of Pakistan. Through a bioactivity-guided fractionation approach, the crude and resultant organic fractions were tested on cultured breast cancer cells (MCF-7 and MDA MB-468) and colorectal carcinoma cells (Caco-2) in vitro. Five new compounds out of seven in total were isolated from potent fractions of the new medicinal plants using repeated flash column chromatography. Structural elucidation was carried out through a series of spectroscopic experiments (1-D and 2-D NMR, GC-MS, LC-MS). SIngle crystal X-ray structure was determined using X-ray crystallography for the crystalline compounds, which showed a defraction pattern. The apprent IC[sub]50 for compounds (1-6) were estimated from serial dilutions of eight concentrations (0.78-100 [mu]M) of each compound, tested against breast and colon cancer cell lines, using two cell viability assays (MTT and neutral red uptake assays) for 24 h and 48 h treatments. Two new steroidal glycosides, acylated pregnane (1) and acylated androstane (2) glycosides, isolated from the ethyl acetate fraction of 'Caralluma tuberculata' showed highly significant (P<0.001) percentage growth inhibition in Caco-2 cells (IC[sub]50) 1.56-6.25 [mu]M) and MCF-7 cells (IC[sub]50 6.25 - 25 [mu]M), however, oestrogen independent cancer cells (MDA MB-468) were less responsive with IC[sub]50 25 - 50 [mu]M. These steroidal glycosides induced apoptosis in cancer cells as measures of cytoxic activity (NRU, PARP clevage, DNA ladder) on MCF-7, MDA MB-468 and Caco-2 cells were inhibited by pre-treatment with the pan-caspase inhibitor (Z-VAD-FMK). Another pregnane glycoside (3), isolated for the first time from 'Fagonia indica', was found to be more potent in suppressing cell growth (IC 6.25-25 [mu]M), in oestrogen negative breast cancer cells (MDA MB-468,) as compared to oestrogen positive cancer cells (MCF-7). Although a cleaved PARP (89kDa) was detected by Western blotting, cytomorphological alterations and in cells pre-treated with a pan-caspase inhibitor (NRU assay), indicated that the necrosis mode of cell death is more likely. Moreover, three esters: hexadecanoic acid ethyl ester (4), phtalic acid 1-(1, 1-dimethyl-pentyl) ester 2-(2-ethyl-dec-5-enyl) ester (5) from chloroform fraction of 'Solanum surattense', and 5-Oxo-19-propyl-docosanoic acd methyl ester (6) from 'Arisaema utile', showed a highly significant )p<0.001) decrease in cell numbers for MDA MB-468 and Caco-2 cells with apparent IC 6.25-12.5 [mu]M in cell viability assays (MTT and NRU) after 48 h treatment, while MCF-7 cells were less responsive (IC[sub]50 25 [mu]M). Compunds 5 and 6 (first report from a natural source) did not restrict the growth inhibition in MCF-7 and Caco-2 cells, pre-treated with Z-VAD-FMK, which indicated less involvement of Capase-dependent apoptosis, while DAPI staining and Western blots (cleaved-PARP) showed characteristics of apoptosis that suggested the possibility of aponecrosis phenomenon of cell death. In preliminary screening (Western blot and DNA ladder assays), compounds (1-6) were not toxic to normal human cells (HUVEC and U937) and indicated some selctivly between malignant and normal cells.