Phytochemical Analyses of Bioactive Compounds in the Roots of Cassia Alata Linn and the Anti-Angiogenic Evaluation of Rhein as a Therapeutic Agent against Breast Cancer Cells

The research presented in this dissertation includes the novel phytochemical analysis of root extracts of Cassia alata L. and the anti-angiogenic evaluation of rhein in the treatment of breast cancer. To conduct these studies a combination of analytical techniques were employed, namely, chromatographic, spectroscopic, and bioanalytical. The first part of this research is focused on the phytochemical analysis of C. alata root extracts. A high performance liquid chromatography method was developed and validated for the determination of six phenolic compounds in the root extracts. The investigated compounds were identified by their ultraviolet spectra. Additionally, the presence of these phenolics was confirmed by on-line mass spectrometric detection using atmospheric pressure chemical ionization. Subsequently, the primary anthraquinone, rhein, was isolated by use of flash chromatography and purified using preparative thin layer chromatography. Lastly, characterization studies were performed by utilizing mass spectrometry, nuclear magnetic resonance, ultraviolet, and infrared spectroscopy. The final part of this study is focused on assessing the effectiveness of rhein as an anti-angiogenic agent in the treatment of breast cancer. The in vitro anti-angiogenic activity of rhein was evaluated using human umbilical vein endothelial cells, non-invasive (MCF-7) and invasive (MDA-MB-435s) breast cancer cells. Rhein’s mechanism of action was investigated by use of in vitro angiogenesis assays, enzyme-linked immunosorbent-assays, and Western Blotting. Rhein inhibited endothelial cell tube formation, MCF-7 and MDA-MB-435s cell viability, as well as MDA-MB-435s cell migration and invasion under normoxic and hypoxic conditions. In addition, rhein significantly reduced vascular endothelial growth factor levels in supernatant fractions and decreased hypoxia inducible factor-1 activities in nuclear extracts of both cell lines. These results indicate the novel use of rhein as an anti-angiogenic agent, which could have a vital impact in the prevention and treatment of breast cancer