Anti-atherothrombotic effects of Berberis vulgaris L., Teucrium polium L. and Orthosiphon stamineus Benth extracts in vitro and Berberis vulgaris L. extracts in vivo

Coronary artery disease is a group of diseases that includes stable angina, unstable angina, myocardial infarction and sudden cardiac death. Coronary artery disease is a leading cause of mortality and morbidity worldwide. The pathogenesis mainly due to atherosclerosis, plaque rupture and platelet thrombus formation. The main risk factors for coronary artery diseases are obesity, hypercholesterolemia, smoking, diabetes and high blood pressure. As part of disease management, anticoagulant and antiplatelet drugs are the options of treatment together with lipid-lowering medication. In combating this disease, medicinal plants comprised of anti-atherothrombotic effects can be options other than drug therapies that may be considered to have lesser adverse effects. Nevertheless, the effectiveness of medicinal plants in treating coronary atherothrombotic disease has yet to be fully explored. Therefore, the haematological, biochemical, gross and histological effect of Berberis vulgaris L, Teucrium polium Land Orthosipohon stamineus Benthextracts in preventing and treating coronary atherothrombotic disease were studied at the in vitro (phase I) and in vivo (phase II) level. Inphase I, three types of extraction including aqueous, methanol and polysaccharide of the B. vulgaris, T. polium and O. stamineus were studied for antiplatelet and anticoagulant effect using human whole blood. All extracts were subjected to the prothrombin time (PT) and activated partial thromboplastin time (APTT) test for anticoagulant activity and then investigated using an electrical impedance method for antiplatelet activity. B. vulgaris aqueous extract (BVAE), B. vulgaris polysaccharide extract (BVPE) T. polium aqueous extract (TPAE) and T. polium polysaccharide extract (TPPE) were found to significantly prolong the coagulation time in a concentration dependent manner (p<0.05). In addition, phytochemical screening revealed that aqueous extract of all three medicinal plants contained polysaccharides. Hence, it waS concluded that the present of polysaccharides in the medicinal plants, especially in B. vulgaris and T. polium but not in O. stamineus played a role in prolonging blood clotting in coagulation activity. However, this current study on also proved that not all polysaccharides gave equal effects on coagulation test. Though, for antiplatelet activity, the BVAE was the most effective sample against platelet aggregation caused by arachidonic acid (AA) and collagen. These effects are probably due to the present of berberine content in B. vulgaris and higher total phenolic compound thus inhibit platelet aggregation activity.In continuation of result in phase I, BVAE was proceeded for phase II (in vivo) due to its optimum effect of anticoagulation and antiplatelet activity. Phase II was divided into two parts, preventive and treatment studies. Atherosclerotic-induced male New Zealand white rabbits were divided into nine groups and antihyperlipidemic effect, anti-inflammatory effect, antiplatelet properties, anticoagulation properties, liver function test and renal profile were studied. After the treatment period for preventive study (10 weeks) and treatment study (12 weeks) were completed, the rabbits were sacrificed for gross and histological analyses (H&E, Masson trichrome and Modified Verhoeff stains), immunohistochemistry analyses (RAM 11) and biochemistry analyses were performed on extracts and control groups in both studies. All data were analysed using one-way ANOVA followed by LSD’s post-hoc test. The values were considered significant when p value is less than 0.05. The current study provided biochemical and histological evidences that BVAE possesses antihyperlipidemic effect through significant reduction on total cholesterol, triglyceride and low-density lipoproteins levels, and histologically for intima-media ratio and collagen score compared to control (p<0.05). For the anti-inflammatory effect, BVAE was shown to attenuate inflammatory cells biochemically including TNF-alpha and interleukin-6 levels, and significant reduction on macrophages cells number in concentration dependent manner (p<0.05) Toxicity analysis showed both concentrations of the extracts had no effect on the liver and renal function biochemically and histologically These finding provide an important evidence that BVAE is safe to use, and possible to proceed for further testing in a clinical setting. In conclusion, B. vulgaris had been proven to have anti-atherothrombotic properties and can be considered as a safe option for anti-atherothrombotic agent.

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