Collection and processing of plant materials
Fresh leaves of S. purpurea were collected from Area F, in Zaria. The Plant was identified at the Department of Plant Biology Federal University of Technology Minna and the voucher specimen number was FUT/PLB/AN/003. The leaves were rinsed under running tap water, air dried at ambient temperature and grinded to powder form and stored in an air tight container prior to use.
The powdered plant materials were extracted in two solvent such as water and absolute ethanol. Hundred (100) grams of the powdered dried leaves were soaked in 400 ml of solvent and stored at room temperature for 24 h. The extracted solution was filtered through muslin cloth and the filtrate was evaporated to dryness over a water bath at 50 °C.
Experimental animals
Female wistar rats weighing (121.5 ± 30.41 g) were obtained from the animal house, Faculty of Pharmaceutical science, Ahmadu Bello University Zaria. They were housed in cages and allowed to acclimatize at room temperature for 2 weeks with free access to feed and water ad-libitum [14].
Acute toxicity study
Acute toxicity study was carried out by method described by Lorke, 1983 [15]. In the first phase, rats were divided into 6 groups of 3 rats each and administered with aqueous and ethanol extracts of S. purpurea leaf by gavage at doses 10, 100 and 1000 mg/kg body weight. All animals were observed for 24 h. In the absence of death, the second phase was carried out. Rats were divided into 6 groups of one rat each and treated with the extracts at doses of 1600, 2900 and 5000 mg/kg body weight. The animals were observed for 24 h for signs of toxicity, including death.
Experimental design
The rats were weighed and randomly assigned into seven groups of four rats each. They were orally administered at doses of 500, 1000 and 1500 mg/kg of the extracts daily [16].
Group 1: served as normal control group and received 0.5 ml of normal saline.
Group 2: received 500 mg/kg body weight of the aqueous extract.
Group 3: received 1000 mg/kg body weight of the aqueous extract.
Group 4: received 1500 mg/kg body weight of the aqueous extract.
Group 5: received 500 mg/kg body weight of the ethanol extract.
Group 6: received 1000 mg/kg body weight of the ethanol extract.
Group 7: received 1500 mg/kg body weight of the ethanol extract.
The extracts were orally administered daily for a period of 14 days. Twenty-four hours after the last administration, the animals were anaesthetized with phenobarbital and blood samples were collected from orbital plexus by means of heparinized capillary tubes. The animals were sacrificed, quickly dissected, relevant organs collected and fixed in 10% formal saline for histopathological studies.
Determination of haematological parameters
Packed cell volume (PCV) was determined using micro-hematocrit method, White Blood Cell (WBC) by hemocytometer method. The hemoglobin concentrations were calculated from PCV values. Differential White blood cell (WBC) count was determined by Giemsa stain method.
Estimation of total plasma protein
Total protein concentration was determined according to the method of Lowry et al. [17]. In an alkaline medium, protein reacts with the copper in the Biuret reagent leading to an increase in absorbance due to formation of colored complex. Reagent (2.5 ml) and 0.05 ml serum sample were mixed. It was then incubated at room temperature for 10 min. The absorbance was read at 540 nm against reagent blank.
Histopathological studies
Histopathology slides were prepared by the method described by Aliyu et al., [18]. The extracted tissues (liver, kidney and spleen) were fixed in 10% normal saline for 72 h after which the tissues were sliced to a thickness of 2.1 mm each. These were dehydrated using alcohol of graded concentrations. They were further treated with paraffin wax and cast into blocks; sections of the tissues were cut on a microtome to 5 μm. these were later attached to a slide and dried. The samples slides were then stained in haematoxylin and eosin. The slides were then viewed on a photomicrograpic microscope to detect any damage.
Statistical analysis
The analysis was performed using the statistical package for social sciences (SPSS) for WINDOWS (version 21.0; SPSS Inc., Chicago). Results were subjected to analysis of variance (ANOVA) to determine their level of significance. Data were expressed as the mean ± standard deviation. Values were considered statistically significant at p > 0.05.