Evaluation Of Effects Of Selected Medicinal Plants On Angiogenesis By Determining Their Inhibitory Effects On Cox-2, Nos And Lox Enzymes

Abstract

Introduction: Angiogenesis is the process of forming new capillaries from existing ones. This process is important for tumor growth and metastasis. It is also important during wound healing. During tumor metastasis this process is amplified, as new capillaries are needed for cancer to spread, but in ulcerations, when this process does not occur, ulceration does not heal. Cyclooxygenase-2 (COX-2), nitric oxide synthase (NOS) and lipoxygenase (LOX) are some of the enzymes that are highly expressed during angiogenesis and therefore provide a useful way to detect angiogenesis. Some medicinal plants have been shown to possess angiogenetic modulating properties, which has led to the development of anti-angiogenic drugs. Euphorbia tirucalli, Spirostachys Africana sond, and Synadenium cupulare (Boiss.) L.C. have traditionally been used for treatment of open wounds, ulcers and cancer. Their mechanism(s) of action is, however, still not clear. These plants have therefore been selected to determine their effect on angiogenesis, as angiogenesis is a denominator for cancer and tissue regeneration, which is critical for wound healing and healing of ulcers.

Aim: The study aims to evaluate the effects of selected medicinal plants for their angiogenesis modulating effects by determining their inhibitory effects on COX-2, LOX and NOS enzymes, as well as their growth inhibitory effects on MCF-7 cells.

Methods: The plants were collected from their natural habitat in Pretoria and the Kruger National Park, Limpopo. Plants were dried and extractions were made with water and sequentially with hexane, dichloromethane, methanol and ethyl acetate. Phytochemical analysis was performed to determine the classes of compounds present in the plants. The MCF-7 cells were cultured in DMEM and 10% fetal bovine serum under normal culture conditions. The cells were treated with different concentrations of the plant extracts. After exposure, the cell viability was measured using MTT assay. The cell lysate was then evaluated for enzymatic activity of COX-2 and LOX. Pure enzymes obtained commercially were used as positive controls, while commercial substrates were used for standard curves using enzyme. The culture media were tested for the amount of nitric oxide produced by cells after incubation with extracts.

Results and discussion: The plant ingredients were extracted successfully and the yields were recorded. Phytochemical analysis revealed the presence of tannins, saponins and glycosides on all plants. Euphorbia tirucalli was one of the plants that had the lowest number of phytochemicals, namely saponins, glycosides, tannins and triterpenoids. This plant also had the lowest cell growth inhibitory activity around 10 μg/ml or higher and the lowest enzyme inhibitory activity. Spirostachys africana had most phytochemicals and the highest growth inhibition of MCF-7 cells with IC50 around 1 μg/ml and selectivity for cancer cells over MCF-10A and CHO cells. E. cupularis had the second highest number of phytochemicals and also exhibited good cell growth inhibition, ranging around 5 to 1 μg/ml, and good selectivity for cancer cells, especially the nonpolar extracts. The extracts induced NOS, which is indicative of apoptosis or which could imply induction of apoptosis by activating inducible NOS. Selective extracts of E. cupularis and S. africana might inhibit angiogenesis through cyclooxygenase inhibition. LOX inhibitory activity was concentration-dependent. The highest extract concentration had the highest inhibitory activity.

Conclusion: From our findings, we suggest that non-polar extracts of S. africana and E. cupularis have anti-cancer activity and anti-angiogenic activity as well. Our results were also contradictory to the traditional use of the plants.