Antithrombotic and anti-inflammatory activity of ethyl acetate extract of Protorhus longifolia stem bark
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Date
2019
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University of Zululand
Abstract
Blood clotting related disorders are the major underlying cause of cardiovascular diseases, which are the major cause of premature deaths globally. Despite great efforts made in the discovery of antithrombotic agents, ischemic events continue to claim more lives. Thus, there is a need to discover and develop new and more effective antithrombotic drugs, preferably from natural sources. The study is aimed at investigating the anti-thrombotic and anti-inflammatory activity of the ethyl acetate extract of Protorhus longifolia stem bark. The in vitro antioxidant activity of the crude ethyl acetate was evaluated against DPPH, ABTS and nitric oxide radicals. Its reducing power and metal ion chelating potential were also determined. A chromogenic substrate was used to determine the antithrombin activity of the extract. While the thrombolytic activity of the extract was determined in rat whole blood clot, the antiplatelet aggregation activity was evaluated on rat platelet rich plasma. The carrageenan induced rat paw edema and rat tail bleeding time models were used to investigate the anti-acute inflammatory and antithrombotic activity of the extract, respectively. The experimental rats were orally pre-treated with the extract at 100 and 350 mg/kg for 8 consecutive days. While the water displacement method was used to measure the rat paw edema, the time it took for the rat tail to stop bleeding following a 5 mm amputation was taken as the bleeding time of the rats. At the end of the experiments, all the rats were euthanized and blood and the hind paws of the rats were collected for biochemical analysis of some inflammation and oxidative stress biomarkers. The extract showed, to a varying degree of efficacy, concentration dependent scavenging activity on DPPH, ABTS and nitric oxide radicals. While the plant extract showed a strong reducing power, only a moderate metal ion (Fe2+) chelating activity was observed. The extract also exhibited concentration dependent antithrombin and antiplatelet aggregation activities. At the highest concentration of 10 mg/ml, the extract exhibited up to 35.7% thrombolytic activity. While a marked decrease in tail bleeding time (3.18 min) was observed in the untreated inflammation induced rats when compared to the normal control group, prior oral administration of the extract (at 100 and 350 mg/kg) to the rats seemed to prolong the bleeding time up to 8.8 min. The animals pre-treated with the extract also displayed anti-inflammatory activity as they effectively reduced the rat paw swelling. Even though no significant
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changes were observed in the tissue levels of IL-6 and TNF-α on the extract treated groups, a marked decrease in COX-2 and TGF- β1 was observed. Increased tissue levels of superoxide dismutase and total antioxidant status were also observed in the inflammation induced rats pre-treated with the extract at both 100 and 350 mg/kg in comparison to the untreated control group. Even though lower serum levels of some kidney and liver function biomarkers (urea, creatinine, AST, ALT and ALP) were observed in the group treated with the extract at 100 mg/kg, relatively higher serum levels of BUN and AST were observed in the rat group administered with the extract at a higher concentration of 350 mg/kg. The results obtained suggest that the ethyl acetate extract of P. longifolia stem bark possess antithrombotic and anti-inflammatory activities. The observed increases in serum levels of BUN and AST at the higher dosage of the extract indicate its potential nephro-and hepatotoxicity and that it be used as a treatment with caution.
Description
A thesis submitted in fulfillment of the academic requirements for the degree of Master if Science in the Department of Biochemistry and Microbiology in the Faculty of Science, Agriculture and Engineering, University of Zululand, 2019.
Keywords
antithrombotic agents, cardiovascular diseases, anti-inflammatory activity