Glucose induced toxicity to cells: protective effects of momordica charantia

Aljohi, Ali Omar (2014) Glucose induced toxicity to cells: protective effects of momordica charantia. Doctoral thesis (PhD), Manchester Metropolitan University.

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Abstract

Increased advanced glycation endproducts (AGEs) formation and oxidative stress are believed to underlie the pathogenesis of diabetic vascular complications including the impairment of the wound healing. In most developing countries, diabetic treatment is expensive and plants provide a cheap potential natural source of anti-diabetic remedies. Several studies have examined the beneficial effects of using Momordica charantia (MC) because of its hypoglycaemic properties in diabetic subjects. Here, for the first time, the anti-glycation and antioxidant properties of aqueous extracts of Momordica charantia pulp (MCP), flesh (MCF) and charantin were assessed in vitro. Since wound-healing is one of the most costly complications and affecting 15% of diabetic patients, the potential angiogenic activities of MCP, MCF and charantin in the presence or absence of AGEs were investigated. Lysozyme was glycated using either glucose or methylglyoxal in the presence or absence of 5 to 15 mg/ml of Momordica charantia extracts in 0.1 M sodium phosphate buffer (pH 7.4) at 37°C for three days. The formation of glucose or methylglyoxal-derived AGE crosslinks was assessed using sodium dodecyl sulphate-polyacrylamide gel electrophoresis followed by Coomassie blue staining. A non-competitive ELISA method was used to investigate the effect of Momordica charantia extracts on carboxymethyllysine (CML) concentrations. Antioxidant activities of all extracts of Momordica charantia were evaluated using the stable free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) and hydroxyl radical-scavenging activity, metal chelating activity and reducing power. The phenolic, flavonols and flavonoid contents of all extracts were also measured. In vitro angiogenic assays including cell proliferation, migration and endothelial tube formation in Matrigel™ were used to assess the potential angiogenic effects of the natural extracts. By Western blotting, the angiogenic signalling pathways induced by AGEs and potentially modulated by MCP, MCF and charantin were also investigated. Furthermore, the neutralization of the receptor for AGEs (RAGE) was performed using a monoclonal anti-RAGE antibody to highlight the role of RAGE in the modulation of AGE-induced signalling pathways followed addition of MCP, MCF and charantin. All extracts inhibited the formation of MG-derived AGEs in a dose-dependent manner and the MCF extract showed the most potent inhibitory effect on both AGE and CML formation. Antioxidant capacity of MCF was significantly higher than MCP based upon the DPPH and hydroxyl radical-scavenging activity (p < 0.005); however, MCP shows higher metal-chelating activity in comparison to other extracts. The content of phenolic compounds was expressed in gallic acid equivalents (GAE), whereas flavonols and flavonoid contents were expressed in rutin equivalents (RE). In addition, all Momordica charantia extracts increased bovine aortic endothelial cell (BAEC) proliferation, migration and tube formation with induction of p-ERK1/2 expression through RAGE. Moreover, these natural extracts decreased the anti-angiogenic effects of high concentration of BSA-AGEs. Momordica charantia does not only have established hypoglycaemic effects but this study shows that crude extracts are capable of preventing MG-derived cross-linked AGEs and Glyoxalic acid-derived CML at least in vitro. This anti-glycation activity might be due to their antioxidant properties from their phenolic content. Furthermore, because of its pro-angiogenic effects and its ability to reduce the AGEs-induced anti-angiogenic effect, Momordica charantia presents a promising natural product for the development of a new strategy to accelerate wound-healing especially in diabetic foot. Thus, use of Momordica charantia deserves more attention in particular its ability to reduce AGE formation and oxidative stress in diabetic subjects and as a pro-angiogenic therapy.