Chemical Reagents
N-haxane and carbon tetrachloride and were gotten from Sigma Aldrich. All other chemicals were obtained from standard chemical suppliers and were of analytical grade.
Animals
Male adult Wistar rats (100–120g) were used. The animals were used according to the standard guidelines of the Committee on Care and Use of Experimental Animal Resources.
Plant materials
Matured fruits of Tetracarpidium conophorum were collected from private farm land in Ondo Town, Ondo State, Nigeria. The fruits were authenticated by the Department of Plant Biotechnology, University of Medical Sciences, Ondo.
Preparation of Sample
The collected fruits were cleaned with a moist soft cotton wool and then the seeds were carefully separated from the fruits. Part of the separated nuts were immediately used for oil extraction, while the remaining part was dried at 650C for 4 hours in an oven, crushed with a laboratory mortar and pestle and kept in a well labelled air tight polythene bags.
Extraction of oil from Tetracarpidium conophorum seeds
The seeds of Tetracarpidium conophorum were removed from their shells, cut into small pieces, and air dried at room temperature for 2 weeks. The method of extraction used in this study was mirrored after the soxhlet extraction method reported by [18]. Using this method, the dried seeds of Tetracarpidium conophorum were grounded into powdered form using a blender and further air dried. The ground sample (50 g) was weighed and placed in the thimble. The thimble was placed in the extraction chamber of the soxhlet extractor. The solvent (n-hexane) was measured to 500ml and poured into separate round bottom flasks. The apparatus was then fitted with the help of clamps and stand to support the soxhlet extractor, round bottom flask, and condenser, all of which was placed on a heating mantle. The solvent was heated and extraction under reflux was carried out. At the end of extraction, the extract (oil) was collected in the round bottom flask and was placed in a rotary evaporator to evaporate the solvent leaving a concentrated form of the oil. The extraction was carried out in the laboratory of the Department of Biochemistry, University of Medical sciences, Ondo, Ondo state.
Experimental Design
In this study, the rats procured were divided into 4 groups, which are;
Group A: Rats were fed with rat pellet and water only (Control)
Group B: Rats were administered n-hexane extract of Tetracarpidium conophorum seed oil only
Group C: Rats were pre-treated with n-hexane extract of Tetracarpidium conophorum seed oil and administered carbon tetrachloride (200 mg/kg intraperitoneally).
Group D: Rats were administered toxicant only (Carbon tetrachloride) (200 mg/kg).
The Tetracarpidium conophorum seed oil was administered by mixing it with the rat pellets and the toxicant was administered intraperitoneally.
Preparation of homogenates
2 g of the liver tissue stored in normal saline was homogenized in 10 ml of Tris buffer. The homogenized samples were then centrifuged at 10000 g for about 10 minutes after which the supernatant was collected and used for subsequent analysis of antioxidant enzymes [13].
Biochemical Enzyme Analysis
The whole blood was collected from the hearts of the rats in plain sample bottles and allowed to clot by leaving it undisturbed at room temperature for 10 minutes after which the clots were removed by centrifuging at 1000g for 10 minutes, the resulting supernatant which is the serum collected was used to assay for the liver enzyme activity.
Estimation of Alanine Aminotransferase Activity
The Alanine transaminase activity was determined using commercial kits and carried out as described in the kit instruction leaflet (Product code: BXC0212).
Procedure
0.5 ml of R1 buffer containing phosphate buffer (100 mmol/L, pH7.4), L-alanine (200 mmol/L) and alpha-oxoglutarate (2.0 mmol/ L) was added to 0.1 ml of sample. The mixture was incubated for 30 minutes at 370C. 0.5 ml of R2 Dye Reagent containing 2,4-Dinitrophenyl Hydrazine (2.0 mmol/L) was added to the reaction mixture and allowed to stand for 20 minutes at 20-250C. 5 ml of 4.0 mol/L sodium hydroxide was then added and the absorbance was read against the sample blank after 5 minutes at 546nm.
Estimation of Aspartate Aminotransferase Activity
The Aspartate transaminase activity was carried out using commercial kit as described in the kit instruction leaflet (Product code: BXC0202).
0.5 ml of R1 AST buffer containing phosphate buffer (100 mmol/L, pH7.4), L-aspartate (200 mmol/L) and alpha-oxoglutarate (2.0 mmol/L) was added to 0.1 ml of sample. The mixture was incubated for 30 minutes at 370C. 0.5 ml of R2 Dye Reagent containing 2,4-dinitrophenyhydrazine (2.0 mmol/L) was added to the reaction mixture and allowed to stand for 20 minutes at 20-250C. 5 ml of sodium hydroxide (4.0 mol/L) was then added and the absorbance was read against the reagent blank after 5 minutes at 546nm.
Estimation of Reduced Glutathione (GSH) Level
The level of reduced glutathione (GSH) was estimated by using the method of [3]. In this method, thiol residues of GSH reduced Ellman’s reagent to 2-nitro-5-benzoic acid was read at 412 nm.
Assessment of Lipid Peroxidation
Lipid peroxidation was determined by measuring the formation of thiobarbituric acid reactive substances (TBARS) present in the test sample according to the method of Varshney and Kale (1990)[22]