Preparation of plant material
Fresh leaves of Jatropha tanjorensis were collected from Nsukka in Enugu State of Nigeria and identified by a taxonomist at the Bioresource Development and Conservation Programme (BDCP), Nsukka, Enugu State, Nigeria. The leaf samples were deposited in their herbarium with voucher number InterCEDD/16045. The leaves were then air dried and pulverized, and 350 g of the powdered leaves was extracted by maceration in 1.4 L of 98% methanol for 48 h. This was followed by filtration, first with a mesh and then with Whatman No. 1 filter paper and the filtrate concentrated using rotary evaporator at 40 °C to obtain the crude methanol extract (JTME).
The methods of [12, 13] were adopted for the preliminary phytochemical analyses of the extract. The extract was screened for the presence of flavonoids, alkaloids, tannins, terpenoids, carbohydrates, and steroids.
Eighteen male albino mice of average body weight of 25 g and 24 male albino rats of the Wistar strain (6–8 weeks old and 85–150 g average weight) were used respectively for the acute and sub-acute toxicity study. They were kept in clean animal cages with proper ventilation and acclimatized to laboratory environment for 7 days while being maintained on standard animal feed (Vital feeds) and water ad libitum. Animal cages were cleaned every 3 days throughout the experimental period. The animals were handled in accordance with the relevant ethical guidelines compliant with the International Standard for the use of laboratory animals.
For the acute toxicity study, the protocol of  was adopted. The mice were administered, by oral intubation, doses of 10, 100, and 1000 mg/kg b.wt of the extract in the first phase and monitored for 24 h for any signs of toxicity. Due to the absence of any toxic sign or mortality, the doses were increased to 1600, 2900, and 5000 mg/kg b.wt in the second phase and administered to another group.
For the sub-acute toxicity studies, the method described by the Organization for Economic Cooperation and Development (OECD)  was adopted. The OECD guideline for sub-acute toxicity studies requires oral administration of graduated doses of a test substance to experimental animals daily for a period of 28 days using a minimum of three test groups along with a control group. In this study, 24 rats were distributed to four groups (n = 6) with group 1 (control group) administered 1 ml distilled water while groups 2–4 (test groups) were administered, by oral gavage, 100 mg/kg, 200 mg/kg, and 400 mg/kg body weight of the extract respectively for the duration of the experiment. Extract administration lasted for 28 days while the average body weight for each group was taken weekly and recorded. On the 29th day, the animals were weighed and then euthanized under mild chloroform anesthesia. Blood samples were collected from the orbital sinus into ethylene diamine tetra-acetic acid (EDTA)-lined sample bottles for hematological analysis and plain sample bottles for biochemical analysis. Blood samples for the biochemical analyses was allowed to clot and centrifuged at 4000 rpm for 30 min to obtain the serum which was stored at 4 °C in a refrigerator until use.
Estimation of hematological and biochemical parameters
Hematological parameters (RBC, PCV, Hb, PLT, and WBC) were estimated using the methods described by [16, 17]. Biochemical parameters were determined using commercially available kits. Total bilirubin concentration was determined by colorimetric method described by . Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were assayed following the protocol of . Alkaline phosphatase (ALP) activity was assayed by the method of . Lipid peroxidation was determined by the method described by . Total protein in whole blood was determined according to the method of . Serum urea and creatinine concentrations were determined using the method of .
Tissue samples from the liver and kidneys were also collected after dissecting the rats, fixed in 10% phosphate-buffered formalin, and prepared for histopathological examination. Hematoxylin- and eosin-stained tissues on different slides were examined using a compound light microscope with × 4, × 10, and × 40 objective lenses. The photomicrographs were taken using Motic© 9-megapixel microscope camera at magnifications × 100 and × 400.
Statistical analysis of obtained data was carried out using the Statistical Product and Service Solution (SPSS, version 21.0). All data were expressed as mean ± standard deviation (SD) and statistical different between means were determined by one-way analysis of variance (ANOVA).