In Vivo and In vitro Anti-Inflammatory Effect of the Chloroform Extract of Annona Muricata (soursop) leaves

Abstract

This study ascertained the anti-inflammatory effect and the mechanisms of action of the chloroform extract of Annona muricata leaves. The plant material was extracted using a mixture of chloroform and methanol in a ratio of 2:1. The chloroform extract was used to further investigate the in vivo effect on agar-induced rat paw oedema, and the in vitro activities of phospholipase A2 and prostaglandin synthase, membrane stabilization and platelet aggregatory response. The percentage yields of the chloroform and methanol extracts were 6.80 and 0.29% respectively. Phytochemical analyses of the extracts revealed the presence of phenols (26.290 ± 0.004 mg/g), flavonoids (24.123 ± 0.003 mg/g), terpenoids (19.040 ± 0.005 mg/g), alkaloids (15.691 ± 0.004 mg/g), tannins (5.446 ± 0.004 mg/g), reducing sugars (4.783 ± 0.004 mg/g), saponins (1.007 ± 0.005 mg/g), glycosides (0.576 ± 0.004 mg/g), steroids (0.236 ± 0.003 mg/g), hydrogen cyanides (0.147 ± 0.002 mg/g) and soluble carbohydrates (0.105 ± 0.003 mg/g) for the chloroform extract and phenols (18.790 ± 0.006 mg/g), alkaloids (10.382 ± 0.005 mg/g), reducing sugars (6.848 ± 0.003 mg/g), terpenoids (5.857 ± 0.004 mg/g), tannins (4.947 ± 0.003mg/g), flavonoids (4.163 ± 0.003 mg/g), saponins (0.736 ± 0.004 mg/g), hydrogen yanides (0.503 ± 0.002 mg/g), steroids (0.464 ± 0.003 mg/g), glycosides (0.448 ± 0.007 mg/g) and soluble carbohydrates (0.153 ± 0.017 mg/g) for the methanol extract respectively. Acute toxicity study on the chloroform extract showed it was toxic at 1000 mg/kg body weight. The LD50 was calculated as 316.23 mg/kg body weight. Agar-induced rat paw oedema test showed that at 50, 100 and 150 mg/kg body weight, the extract exhibited a significant (p < 0.05) reduction in paw volume from 1.5 to 5.5 hr compared to the control. The extract also significantly (p < 0.05) inhibited phospholipase A2 activity in a concentration-related manner compared to the control, with a range of 0.2 to 0.6 mg/ml inhibiting the enzyme activity by 23.91 to 43.48%. The effect of the extract on prostaglandin synthase activity showed a significant (p < 0.05) inhibition of enzyme activity at the concentrations 0.1, 0.5 and 1.0 mg/ml compared to the control. The highest percentage inhibition (87.46) attained at 0.5 mg/ml was comparable to that of 1.0 mg/ml indomethacin. At various concentrations (0.1 - 0.8 mg/ml), the chloroform extract also significantly (p < 0.05) inhibited heat and hypotonicity-induced haemolysis of human red blood cells (HRBCs) compared to the control. The highest percentage inhibition of heat-induced haemolysis (53.03) was obtained at 0.4 mg/ml of the extract while the highest percentage inhibition (77.91) of hypotonicity-induced haemolysis was obtained at 0.8 mg/ml. The extract also significantly (p < 0.05) and concentration-dependently inhibited platelet aggregatory response compared to the control. The anti-inflammatory effect exerted by the chloroform extract was found to be higher than that of the standard anti-inflammatory drugs, indomethacin and prednisolone. These results thus demonstrated that the chloroform extract of A. muricata leaves has potentials in curtailing inflammatory disorders through the inhibition of phospholipase A2 and prostaglandin synthase ctivities, membrane stabilization and inhibition of platelet aggregation.