Thesis submitted in fulfilment of the requirements for the degree of Master of Technology: Biomedical Technology In the Faculty of Health and Wellness Sciences At the Cape Peninsula University of Technology Supervisors: Prof. Adriaan J Esterhuyse Dr Dirk J Bester Bellville January 2014 / Background: Oxidative stress and chronic inflammation contributes significantly to the pathogenesis of several ischaemic heart diseases, including atherosclerotic plaque rupture and myocardial infarction. It is widely demonstrated that ischaemia, followed by reperfusion, results in alterations of the mitochondrial and endothelial function through uncontrolled cascades of events characterized by free radical release and inflammation. Recent experimental evidence shows that modulation of inflammatory and antioxidant signaling mediators may determine the host outcome following myocardial ischaemia-reperfusion injury. Investigations from the past decade indicate that food supplements may play an important role in the prevention and management of chronic inflammatory diseases. Garcinia kola seeds are flavonoid rich nut from a tropical flowering, non-timber plant of the Guttiferae family. This plant is highly valued in several African cultures for its use in herbal medicine. Recently, the majority of experimental research has linked phytochemicals found in Garcinia kola nut, to its proposed beneficial effects in treatment and management of oxidative stress related-chronic diseases. Research performed in our laboratory demonstrated that kolaviron, a prominent Garcinia kola flavonoid extract, reduces myocardial apoptosis during ischaemia-reperfusion injury. Therefore, the aim of our current study was to determine the effects of Garcinia kola supplementation on cardiac inflammatory and antioxidant signaling pathways during ischaemia-reperfusion using a Wistar rat heart model. Materials and Methods: Male wistar rats were randomly divided into two groups: a control group receiving 2ml/kg corn oil and the experimental group receiving 100mg/kg Garcinia kola dissolved in corn oil, daily for 4 weeks. After the feeding period, blood samples were collected and lymphocytic DNA damage was analyzed using the alkaline comet assay. Furthermore, rat hearts were isolated and perfused with Krebs-Henseleit buffer on a working heart perfusion apparatus to measure myocardial functional parameters. Myocardial functional recovery was measured after 15 minutes global ischaemia followed by 25 minutes reperfusion. Hearts were freeze clamped at three different time points for myocardial cytokine concentration determinations using multiplex electrochemilunescent immunoassay. Nuclear factor kappa beta (NF- kβ), p38 mitogen activated protein kinases (p38 MAPK), protein kinase B/Akt (PKB/Akt), nitro-tyrosine, inducible nitric oxide (iNOS), cyclooxygenase-2 (COX-2), poly (adenosine-di-phosphate) ribose polymerase-1 (PARP-1) and caspase-3 expression and their phosphorylated forms (where applicable) were analyzed using the Western blot technique. Results: Dietary Garcinia kola supplementation significantly improved functional recovery when compared to the control group as reflected by the improved aortic output recovery (68.47 ± 6.16% versus 44.96 ± 7.00%; p<0.05). Our biochemical results supports the hypothesis that, dietary Garcinia kola supplementation modulates different cardiac proteins in terms of expression and activation at different time points when compared to the control group. We show that, before induction of ischaemia, Garcinia kola supplementation attenuates expression of inflammatory mediators and pro-apoptotic proteins when compared to the control group. The improved functional recovery was associated with a prompt inflammatory response, activation of PKB/Akt and attenuation of protein nitrosylation after 10 minutes of reperfusion. Modulation of NF-kβ and the p38 MAPK family proteins expression could have also played a significant role in myocardial functional recovery. Conclusion: We have shown that a 4 week period of dietary Garcinia kola supplementation at 100mg/kg daily improves cardiac functional recovery following ischaemia-reperfusion injury. We propose that dietary Garcinia kola supplementation protects cardiac myocytes from ischaemia-reperfusion induced oxidative stress through the induction of a prompt inflammatory response and controlled expression and/or activation of the, NF-kβ, PKB/Akt and p38 MAPK protein signaling pathways PARP-1 and caspase. Finally, we demonstrated that dietary Garcinia kola supplementation did not induce rat lymphocytic DNA damage when compared to the control group.

Thesis submitted in fulfillment of the requirements for the Doctor of Technology: Biomedical Technology In the Faculty of Health and Wellness At the CAPE PENINSULA UNIVERSITY OF TECHNOLOGY 2014 / Diabetes mellitus (DM) results in severe metabolic imbalances and pathological changes in many tissues. Chronic inflammation and oxidative stress have been implicated in the pathophysiology of diabetes mellitus. Garcinia kola (Family: Guttiferae) is a plant well known for its ample medicinal values. The seed of the plant also known as ‘bitter kola’ due to its bitter taste is used as a masticatory agent in traditional hospitality, cultural and social ceremonies in Africa. Kolaviron (KV) is a defatted ethanol extract from the seeds of Garcinia kola (GK). Kolaviron has been shown in experimental models of diseases to have numerous beneficial effects due to the presence of flavonoids (mainly Garcinia biflavonoid (GB)-1, GB-2 and kolaflavanone). However, there is paucity of information regarding the possible effect of kolaviron on inflammatory mediators and oxidative stress in diabetes mellitus. Therefore, this study was carried out to investigate the potential beneficial effects of kolaviron on antioxidant status, inflammatory mediators and apoptosis. Other biochemical and histological alterations in the blood, liver and kidney of streptozotocin-induced diabetic rats were also evaluated. A single intraperitoneal injection of freshly prepared solution of streptozotocin (50 mg/kg.b.wt.) in citrate buffer (0.1M, pH 4.5) was administered to overnight fasted rats for diabetes induction. Diabetes was confirmed by stable hyperglycemia (>18 mmol/l) in the tail blood glucose after 5 days of streptozotocin injection. Kolaviron (100 mg/kg b.wt.) was administered to diabetic rats (by gastric gavage) on the 6th day after the induction of diabetes and treatment continued for 6 weeks (5 times weekly). The effects on blood glucose, body weight, organ (liver and kidney) weight, serum biochemical parameters, oxidative status, inflammatory mediators and histology of the liver, kidney and pancreas were assessed. Kolaviron (KV) treatment lowered blood glucose in diabetic and normoglycemic rats and reduced glycated haemoglobin [HbA1C (%)]. Plasma insulin level was raised in diabetic rats treated with KV. Histomorphometric analysis of the pancreas revealed increased β-cell area of pancreatic islets of kolaviron-treated diabetic group. The indices of organ (liver and kidney) damage were increased in diabetic rats. However, KV treatment protected against liver and kidney damage. The characteristic features of diabetic dyslipidemia such as elevated serum triglyceride and cholesterol concentration which are major risk factors for cardiovascular disease were also significantly reduced in KV-treated diabetic rats. Alteration in antioxidant enzymes status was observed in the liver, kidney and blood (erythrocyte, plasma and serum) of diabetic rats. Lowered catalase (CAT) activity was observed in the liver and kidney of diabetic rats while KV treatment significantly (p < 0.05) elevated catalase activity in the liver and kidney. There was no significant change (p > 0.05) in erythrocyte catalase activity among all treatment groups. Erythrocyte of diabetic rats showed a marked reduction in the activity of superoxide dismutase (SOD) with no significant changes in liver and kidney SOD activity of diabetic rats compared to control whereas KV administration to rats markedly increased SOD activity. Glutathione peroxidase (GPX) activity was elevated in the erythrocyte and kidney of STZ-induced diabetic rats with no significant effect on liver GPX activity. KV treatment reversed the alteration in GPX activity in the kidney and erythrocyte. Level of reduced glutathione (GSH), a non-enzymatic antioxidant was decreased in the both liver and kidney of diabetic rats and treatment of diabetic rats with KV elevated GSH concentration in both tissues. Also, malondialdehyde (MDA), a marker of lipid peroxidation was elevated in the liver, kidney and plasma of diabetic rats and significantly (p < 0.05) lowered following KV treatment. Diabetes induction reduced the capacity of liver and kidney to absorb oxygen radicals as demonstrated by lowered oxygen radical absorbance capacity (ORAC) values. KV administration to normal and diabetic rats significantly increased ORAC values. Increased rate of apoptosis, a major cellular response to high glucose induced stress was observed in the renal and hepatic tissues of diabetic control rats. Kolaviron treatment of diabetic rats protected the liver and kidney against hyperglycemia-induced apoptosis and decreased the number of TUNEL positive cells A significant (p < 0.05) elevation of pro-inflammatory cytokines; monocyte chemoattractant protein (MCP-1), Interleukin-1β (IL-1β), IL-6 and tumor necrosis factor (TNF)-𝛂 was observed in the liver of diabetes rats. KV treatment lowered these inflammatory biomarkers. On the other hand, the kidney of diabetic rats showed elevated concentration of pro-inflammatory IL-1β with no significant effect on kidney TNF-𝛂. An increase in the serum concentration of MCP-1 and IL-1β was observed in the untreated diabetic rats while kolaviron treatment normalized the alteration in serum concentration of MCP-1, IL-1β and vascular endothelial growth factor (VEGF). In conclusion, persistent and chronic hyperglycemia promotes the generation of free radicals and inflammatory molecules which contributes to progressive development of micro- and macro vascular complications and multi-organ damage. Kolaviron demonstrated beneficial effects on markers of oxidative stress and inflammation in the diabetic rats and also promoted the survival and functional integrity of the liver and kidney.