M. africana seeds were collected from the Samburu area of Kenya. Identification and authentication was done at the herbarium, School of Biological Sciences, University of Nairobi.
Extraction of seeds materials
The M. africana seeds were cleaned in tap water and rinsed in distilled water. After air-drying at room temperature (22-26 °C) to a constant weight, the seeds were ground to a uniform powder using an electric mill. The powder (100 g) was soaked in 1 L distilled water for 48 h. The mixtures was filtered through cotton wool and then with filter paper (125 mm) after which the filtrate was frozen at -20 °C for 24 h followed by freeze drying. The powdered extract was packed and sealed in air tight polythene bags, stored in the refrigerator at 4 °C and used within five days.
Laboratory animals
A total of 15 male Wistar albino rats (195 – 225 g) were obtained from the animal house of the department of Biochemistry, University of Nairobi. The animals were housed in the research room of this department and the temperatures were maintained at 27– 30 °C. The room was well ventilated and maintained on light for 12 hours and 12 h darkness. The rats were provided with the standard rat pellets and clean water ad libitum. The animal studies were in compliance with the ethical procedure for the care and use of laboratory animals approved by the “Animal care and use committee (ACUC)” of the Faculty of Veterinary Medicine University of Nairobi.
Experimental design for acute toxicity
The animals were randomly assigned into three groups of five rats each. They were kept overnight fasting prior to extract administration. Group 1 served as the control and the rats were administered with 2 ml distilled water once. Two concentrations of the aqueous extract, 1000 and 5000 mg/kg body weight were constituted each in 2 ml distilled water respectively. These extracts were orally given as a single dose and only once to groups 2 and 3 respectively using a gavage. Food was withheld for further 3 h post extract administration.
Animal bleeding, hematological and biochemical assays
Blood was collected at from each rat at 48 h and the 14th day post extract administration via the tail lateral vein using a 2 ml hypodermic syringe and needle when the animal was restrained. A blood aliquot (1.3 ml) was put into the EDTA tubes and thoroughly mixed for hematological analysis. The remaining 0.5 ml of the blood was put in the plain tube for biochemical analysis. Hemoglobin concentration (HB), packed cell volume (PCV), red blood cells (RBC), white blood cells (WBC), mean corpuscular hemoglobin (MCH), mean cell volume (MCV), mean corpuscular hemoglobin concentration (MCHC) and thrombocytes were analysed within six hours using “Melet Schoesing MS4” hematological analyzer. The blood in the plain tubes was immediately centrifuged at 3000 revolutions per minute for 10 min to extract serum which was stored at −20 °C and used within 12 h for biochemical assays. Aspartate aminotransferase (AST), alanine aminotransferase (ALAT), total proteins, creatinine, urea were assayed for each rat using commercial kits according to the manufacturer’s protocol using “UVmini-1240 UV–vis” spectrophotometer manufactured by “Shimadzu”.
Physical observation
Clinical observations were made after every 30 min post extract administration for the first four hours and latter once a day up to the 14th day. Mortality, moribund, ill health or reaction to treatment, such as changes in skin and fur, eyes and mucus membranes, behavior pattern, tremors, salivation, diarrhea, sleep and coma were observed. Weight recording was done before extract administration, at 48 h, day 7 and day 14.
Statistical analysis
The analysis was done using SPSS 17.0 and the results were expressed as mean ± standard deviation of the mean (SD). One-way analysis of variance (ANOVA) was employed for between and within group comparison. 95 % level of significance (p ≤ 0.05) was used for the statistical analysis.