ANTIOXIDANT AND ANTICLASTOGENIC POTENTIALS OF METHANOLIC EXTRACT OF DEFATTED HOLARRHENA FLORIBUNDA LEAVES (G.DON)

Abstract

Holarrhena floribunda is a widely used plant in herbal formulations against number of disease in Nigeria. There is no sufficient information on the scientific basis for its use. Methanolic leaf extract of defatted H.floribunda was therefore investigated to determine its antioxidant, anticlastogenic and hepatoprotective effects against sodium arsenite (NaAsO3)-induced clastogenicity and hepatotoxicity. Antioxidant activity of H.floribunda leaves was examined by the in vitro scavenging abilitilities of the extract against 1,1 diphenyl picylhdrazyl (DPPH), hydroxyl radical (OH), nitric oxide radical (NO) and lipid peroxidation inhibition. Total antioxidant activity, reductive potential, total phenol, proximate and phytochemical constituents were also determined using standard procedures. Male wister rats were randomly alloted into six groups of five rats each and treated as follows; Group A (2.5 mg/kg NaAs0₃), Group B (100 mg/kg extract), Group C (100 mg/kg extract plus 2.5 mg/kg NaAs0₃) and Group D (200 mg/kg extract), Group E (200 mg/kg extract plus 2.5 mg/kg NaAs0₃) and Group F had distilled water. NaAs0₃ was given intraperitioneally once per week. The extract was administered through oral gavage for 28 days. Clastogenicity was assessed by micronuclei formation in Polychromatic Erythocytes Cells (PCEs) in bone marrow. Markers of liver function (plasma Gamma Glutamyl Transferase (yGT), Aspartate Amino Transferase (AST) and Alanine Transferase (ALT) were determined. Markers of oxidative stress (hepatic Reduced Glutathione (GSH), Superoxide Dismutase (SOD), Catalase (CAT) and Lipid perioxidation) were also estimated. Liver histopathological evaluation was also carried out. Regression analysis and ANOVA were used for statistical analysis. The extract exhibited scavenging activities with IC₅₀ values of 12.6ug/ml, 1,377.0 ug/ml, 244.0 ug/ml and 73.8 ug/ml for DPPH, OH, NO and lipid peroxidation respectively. Total antioxidant capacity equivalent of gallic acid and vitamin C were 195.6 ug/mg and 519.3 ug/mg of the extract respectively. Reductive potential increased with increase in concentration of the extract and the total phenol was 1427.9 ug/mg gallic acid equivalents. Proximate analysis revealed that the leaves contained 0.2% moisture, 12.8% ash, 9.6% crude fat, 23.3% crude fibre, 21.2% protein and 32.7% carbohydrate. The leaves also contain alkaloids, saponins, tannins and cardia glycosides. Furthermore, NaAs0₃-induced micronuclei formation in PCEs was reduced at 100 and 200 mg/kg of the extract by 7.7% and 38.5% respectively while elevated plasma yGT and ALT levels were significantly ameliorated (p<0.001). There were no significant differences (p<0.05) in plasma AST levels and hepatic SOD activities in all the treated groups when compared with the control. NaAsO3-induced reduction of GSH concentration was elevated by the extract at 100 and 200 mg/kg by 18.5% and 11.9% respectively. The reduction of CAT activity by NaAs0₃ was ameliorated only at 200 mg/kg by 23.3% . The extract at 100 mg/kg significantly reduced NaAs0₃-induced lipid peroxidation by 16.4% (p<0.05) while the reduction was not observed at 200 mg/kg. Histological examinations showed that the extract at 100 mg/kg only had hepatoprotective effect. The extract has antioxidant activity that ameliorated sodium arsenite-induced hepatotoxicity and clastogenicity and therefore could be used for prophylaxis in sodium arsenite-exposed populations.