In vitro anti-platelet aggregation activity of the extracts of protorhus longifolia
Platelet aggregation beyond purpose of haemostasis is the underlying cause of blood-clotting related diseases. Concoctions of P. longifolia are used by Zulu traditional healers to manage such diseases. This work aimed at investigating the anti-platelet aggregation activity of the extracts of this plant and to identify and characterise the active components present and responsible for the anti-platelet aggregation activity. Phytochemical screening of the plant material revealed the presence of various secondary metabolites (tannins, flavonoids, alkaloids and terpenoids). Crude extracts (obtained by sequential extraction of plant material with hexane, chloroform, ethyl acetate, methanol and water) and two triterpenes (3-oxo- 5á-lanosta-8,24-dien-21-oic acid, and 3â-hydroxylanosta-9,24-dien-24-oic acid) isolated and characterized (using various chromatographic and spectrometric techniques-IR, MS, 1H-NMR, and 13C-NMR) from the crude chloroform extract were screened for antioxidant, anti-platelet aggregation, anti-inflammatory activities, and cytotoxicity. The antioxidant activity of the plant components was determined on DPPH and ABTS+ radicals. Their reduction potential and chelating activity on Fe2+ were also determined. Except the methanol extract (IC50 of 0.07 and 0.16 mg/ml), the crude extracts and the isolated compounds showed poor (< 50%) antioxidant activities as they weakly scavenged DPPH and ABTS+ radicals, exhibited low reduction potentials and poor Fe2+ chelating activities. The anti-platelet aggregation activity of both the crude extracts and isolated compounds was separately investigated on thrombin, ADP and epinephrine induced rat platelet aggregation. The extracts and the isolated triterpenes exhibited a concentration dependent anti-platelet aggregation activity induced by the three agonists. The highest activity by the hexane extract (IC50 of 0.59 mg/ml) was observed on the thrombin-induced platelet aggregation. In addition, the isolated compound also exhibited in vitro anticoagulant activity on the whole rats blood. The acute anti-inflammatory activity of the isolated triterpene was determined using the carrageenan- induced rat paw oedema model. The compound (500 mg/kg body weight) significantly (p<0.05) inhibited the acute inflammation of rat paw. The hexane and chloroform extracts showed weak cytotoxic effects on brine shrimps with LC50 39.6 and 54.7mg/ml respectively. Also the pure compound- 3â-hydroxylanosta-9,24-dien-24-oic acid exhibited weak cytotoxic effects on HEK293 and HEPG2 cell lines (IC50 8520 and 7960 ìg/ml respectively). These results support the use of P. longifolia in folk medicine in the management of blood-clotting related diseases.
Platelet aggregation, Protorhus longifolia
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