Thesis (M.Sc. (Biochemistry) -- University of Limpopo, 2018 / Dicerocaryum senecioides is a plant widely used as a nutritional source. It is used also for treatment of measles, wounds and to facilitate birth in domestic animal and humans in many parts of southern Africa (Mampuru et al., 2012). Findings in our laboratory have shown that a dichloromethane fraction of D. senecioides possesses antiinflammatory properties in human t-lymphocytes (Madiga, 2009), while the methanol crude extract possesses anti-proliferative and proapoptotic properties against Jurkat T cancer cells (Mphahlele, 2008). In this study, the probable anti-cancer effect of D. senecioides crude methanol leaf extract was investigated on cervical HeLa cancer cells. Dried powdered leaves of D. senecioides were extracted with absolute methanol to obtain a crude extract. To assess the cytotoxicity effect of the extract, KMST-6 and HeLa cell cultures were exposed to various extract concentrations (0 to 600 µg/ml) for 24 and 48 hours and subjected to the MTT assay. The results showed the extract to have no significant increase in the viability inhibition of HeLa cells at all tested concentrations after 24 hours of treatment. However, treatment with 400, 500 and 600 µg/ml of the extract for 48 hours revealed significantly increased HeLa cell viability inhibition. Furthermore, the extract showed to have no effect on the viability of normal human fibroblast KMST-6 cells at concentrations below 600 µg/ml, after 24 and 48 hours of treatment, thus showing selective cytotoxicity of the extract. To determine the mode of cell death associated with the increase in HeLa cell viability inhibition, the Hoechst 33258 nuclear staining assay and inverted light microscopy were employed. The data proposed apoptosis as the mode of cell death associated with the inhibition of HeLa cell viability. This was evidenced by changes in cell morphology such as the loss of HeLa cell radial extensions, cell shrinkage, as well as nuclear morphological features such as chromatin condensation. Apoptosis induction was further confirmed by the annexin-V/PI and multicaspase assays, using flow cytometry. The results showed an increase in the percentage of cells stained with annexin-V/PI, as well as increased caspase activity in extract-treated HeLa cells. To elucidate proapoptotic mechanisms of the extract, Western blotting analysis as well as the human apoptosis antibody array kit were used. This was to measure the expression profile of a number of apoptosis regulatory proteins. The results demonstrated modulation of some anti- and pro-apoptotic proteins, as well as the release of mitochondrial proteins required xiii for initiation of apoptosis, in the cytoplasm. The D. senecioides extract showed to have no effect on the cell division cycle of HeLa cells as determined by the PI staining assay. In conclusion, D. senecioides crude methanol leaf extract induced some degree of apoptosis in cervical HeLa cancer cells via the intrinsic apoptosis pathway. This was by modulating some of the members of the Bcl-2 family of proteins, which, facilitated the release of cytochrome C and activation of a caspase cascade. / South African Medical Research Council (SAMRC)

Thesis (M.Sc. (Biochemistry)) --University of Limpopo, 2011 / Dicerocaryum senecioides is a crawling herb that is found growing mostly in sandy areas of southern and south-eastern Africa and its small, hairy leaves have been used over the years as food, shampoo, and for treatment of various ailments. In this study, the dichloromethane (D2) fraction was prepared from a crude methanol extract of D. senecoides leaves, and its effect on the proliferation of RAW 264.7 murine macrophages was investigated. Treatment of the macrophages with the extract resulted in a dose- and time-dependent decrease in cell viability as determined by the MTT assay and real time cell analysis. Cytotoxicity of the D2 fraction on the macrophages was demonstrated to be due to apoptosis by staining the cells with DAPI nucleic acid stain. Anti-inflammatory activity of D2 fraction on RAW cells was determined by evaluating intracellular ROS production by the DCFH-DA fluorescent assay. Cells treated with the D2 fraction and stimulated with PMA were found to have a lower fluorescence intensity compared to untreated, stimulated cells; thus mimicking the response observed in the resting cells. The percentage fluorescence in untreated, stimulated cells doubled, while no significant change was observed in the D2-treated cells. The effect of the D2 fraction on iNOS activity was also assessed. The fraction reduced the NO synthesised by iNOS in cells treated with the D2 fraction and stimulated with LPS dose-dependently. The D2 fraction was further fractionated by semi-preparative HPLC; and thin layer chromatography was used to analyse phytocompounds of the 96 HPLC sub-fractions as well as to screen these sub-fractions for anti-oxidative activity. Sub-fractions 1-7 and 33-39 showed an intensely pronounced DPPH-scavenging compound and this scavenging ability was confirmed by a quantitative DPPH assay that provided parallel results. The reducing potential of the sub-fractions was assessed by evaluating their Fe3+-reducing ability through the FRAP assay. Sub-fractions 1-7 and 33-39 displayed remarkable reducing potential. Taken together with the DPPH-scavenging activity, these findings suggest that HPLC sub-fractions 1-7 and 33-39 possess a compound(s) with impressive antioxidant activity. These findings merit the D2 fraction as an extract that can be used to control chronic inflammation as it does not only inhibit free radical production, but also scavenges excessive ROS and has the ability to induce apoptosis in the macrophages responsible for dysregulated production of the free radicals. The extract also has commendable chemoprotective and chemotherapeutic potential as it demonstrated pro-apoptotic activity along with prevention of excess free-radical production. / National Research Foundation and the University of Limpopo Research Office