Theses (MSc. Molecular Medicine)

Moringa oleifera L (Family: Moringacea) is a type of perennial angiosperm tree that consists of a few plant species. Moringa is a common vegetable used by inhabitants of tropical and sub-tropical countries. At present, M. oleifera is noted to possess a wide range of biological roles including anti-cancer, neuroprotective, anti-inflammatory and hepato-protective activities. In addition, there are numerous studies that have reported many other therapeutic effects of M. oleifera, which include anti-rheumatoid arthritis, anti-infertility, diuretic, thyroid regulation, anti-depression, anti-diabetes, pain relief and atherosclerosis. Over the last decade, the bioactivity of M. oleifera has achieved vast attention due to these bio-activities. The anti-cancer activities could be attributed to the presence of several bioactive compounds such as β-sitosterol-3-O-β-D-glucopyranoside, 4-(α-L-rhamnosyloxy) benzyl isothiocyanate and niazimicin in the Moringa oleifera leaves extracts. It has been noted that Moringa oleifera leaf extracts have anticancer effects, which is reported to take place through induction of apoptosis. To date, the ability of extracts from M. oleifera leaf to induce apoptosis in breast cancer cells has not been fully elucidated. This study involves evaluating the ability of extracts of Moringa oleifera, from various solvents, to modulate apoptosis. Cell based assays using two types of human breast cancer cell lines (MCF-7 and MDA MB-231) as well as a normal breast epithelial cell line (MCF-10A) were used in the experiments. Expression of several genes (human Caspase9, Bcl2, Ccnd1 and Brca2 genes) in the cells when treated with the extracts were also investigated. A commercial source of dried Moringa oleifera leaf powder was subjected to sequential extraction using n-hexane, followed by ethyl acetate, methanol and water-based extraction. The effect of these extracts (0, 10, 20, 40, 60, 80, 100 μg/mL) on cell proliferation was evaluated using the MTT cell proliferation assay. Data analysis was carried out on the MTT assay results. The half maximal inhibitory concentration (IC50) of M. oleifera extracts following 72 hours of exposure was found to be: (a) MCF-7 [IC50 for ethyl acetate (hex-EA): 95 g/mL; water: 100 g/mL and quercetin: 90 g/mL] and MCF-10A [IC50 for ethyl acetate (hex-EA): 52 g/mL]. The IC50 values for MDA MB-231 cells could not be attained in the studied concentrations range. Difficulty in obtaining the IC50 values for MDA MB-231 cells might be due to as this cell line is a triple-negative breast cancer [lack ER, PR and human epidermal growth factor receptor 2 (HER2) amplification)], which means it is an aggressive kind of breast cancer with limited therapeutic approach. The RNA from MCF-7 and MCF-10A cells treated with various extracts for 72 hrs of exposure was extracted for gene expression studies. The genes studied were [human Caspase9, Bcl2, Ccnd1 and Brca2 genes]; all of which are reported to be involved in the regulation of apoptosis. Following this, the purified RNA was analysed for gene expression using the real-time PCR approach. Gene expression studies showed that Caspase9, Bcl2, Ccnd1 and Brca2 genes were upregulated in MCF-7 cells treated with water extract of Moringa oleifera leaf powder. In the MCF-10A cells, the expression of the Bcl2 gene was downregulated in cells treated with hexane extract whilst the expression of the Ccnd1 gene was downregulated in cells treated with ethyl acetate (hex-EA) and water extracts. The findings from this study suggest that Moringa oleifera extracts can differentially regulate genes related to apoptosis in MCF-7 cells. More studies need to be conducted to elucidate the molecular signalling pathways.

Introduction: Saffold virus (SAFV), of the Picornaviridae family is currently associated with unknown pathogenicity although the virus had been detected in infants with neurological disorders, including meningitis and cerebellitis. Exposure to SAFV infection mouse models showed that the virus is able to induce apoptosis of neuronal and glial cells. Orientin and madecassoside are potential compounds having an effect against SAFV infection due to their anti-viral, anti-inflammatory, antioxidative and anti-apoptotic abilities. Therefore, the aim of this study is to investigate if orientin and madecassoside provide preventive, direct inhibitory or a rescue effect on BV2 microglia cells and NE-4C neural stem cells infected with SAFV. Methods: The effects of orientin and madecassoside on BV2 and NE-4C cells infected with SAFV were examined at three different time-points: prevention (cells were treated with the two compounds respectively first, followed by SAFV infection), direct inhibition (the two compounds respectively were administered onto SAFV first and then added to the two cell lines) and rescue (cells were first infected with SAFV followed by administration of the two compounds respectively). The IC50 dose of orientin and madecassoside, and the CCID50 of SAFV on BV2 and NE-4C cells were determined via MTT assays. Subsequently, the two compounds were administered on the BV2 and NE-4C cells infected with SAFV at the three aforementioned time-points. The cells’ viability was measured via the MTT assay while the virus titer was determined by real-time PCR. Effectiveness of the compounds was determined based on improvements in cell viability and reductions in virus titer compared to the negative control. Results: Results from the efficacy experiments indicated that orientin was more effective than madecassoside in inhibiting SAFV as shown in the direct inhibition and rescue modes. However, both compounds were ineffective when administered in a preventive manner. Direct inhibition was selected as the optimum method as it provided more consistent results. Orientin significantly improved cell viability in BV2 cells and NE-4C cells by 94% and 90% respectively and had significantly lower virus titer when administered via direct inhibitory mode at a dose of 1μg. Whereas, madecassoside was only effective in NE-4C cells during direct inhibition with a significant improvement in cell viability of 87% and significantly lower virus titer compared to the negative control at a dose of 1μg. Conclusion: This study enabled the development of a cell-based assay with suitable methodology and criteria for analysis of results for drug testing against SAFV. Positive results were obtained for both rescue and direct inhibition modes but were more consistent for latter mode of action. Hence, direct inhibition of SAFV was the preferred mode of action and results indicated that orientin was superior to madecassoside in improving cell viability and reducing SAFV virus titer.