Chemical and reagents
The chemicals used were analytical grade and include: methanol 99.8%, hexane 96.0% and ethyl acetate 99.5%. With the exception of anisaldehyde sulphuric acid which was a general purpose reagent, all other reagents used were laboratory reagents and include: galcia acetic acid, sulphuric acid, acetic acid, chloroform, bismuth subnitrate, distilled water, potassium iodide, alcoholic potassium hydroxide 20%, alcoholic aluminium chloride 2% and alcoholic ferric chloride 2%.
Plant collection and identification
The leaves of Azadirachta indica and Tamarindus indica; and stem-barks of Anogeissus leiocarpus, and Khaya senegalensis were collected in Area BZ of the main campus of Ahmadu Bello University (ABU), Zaria (11°9′48.21048″N 7°38′5.91828″E). The plants were identified at the Herbarium, Department of Botany, Faculty of Life Science, ABU, Zaria by Namadi Sunusi and the voucher number of each plant was obtained as follow: A. indica- 900,151, T. indica- 602, A. leiocarpus- 1738 and K. senegalensis- 900,181. The collected plant parts were separately dried in the laboratory to a constant weight.
Five hundred gram of each of the pulverized plants was extracted with 2.5 L of absolute methanol using Soxhlet apparatus. Each distilled extract was concentrated to dryness in vacuo using a rotary evaporator. The dried extracts were kept in labeled containers and stored at room temperature in the laboratory until required. The percentage yield of each extract was calculated.
Preliminary phytochemical screening of the extracts
Phytochemical screening of each extract for the presence of phenolic compounds, alkaloids, anthraquinones and steroids and triterpenes was done on thin-layer chromatography plates (TLC)- silica gel 60 TLC254 plate (Merck KGaA, Germany). The plates were sprayed with ferric chloride, Dragendoff’s, Bontragers and Liebermann-Burchard reagents for phenols, alkaloids, anthraquinones and steroids and triterpenes, respectively.
The Wistar rats used for the study were obtained from the Animal Room of the Department of Veterinary Pharmacology and Toxicology, A.B.U., Zaria. Outbred rats were used for the study. The rats were fed rat chow and water was provided ad libitum. The animals were kept in clean iron cages at room temperature throughout the study. The bedding was changed twice weekly throughout the duration of the experiment. Permission for the use of rats was given by the Ethical Committee on Animal Use and Care, Ahmadu Bello University, Zaria (reference number: ABUCAUC/2019/005) and the rats were handled according to its guideline, in line with international best practices for animal experimentation.
Determination of median lethal dose (LD50)
Limit dose of 5000 mg/kg  was used to determine the LD50 of each extract.
Acute, sub-acute, sub-chronic and chronic toxicities studies
Acute, sub-acute, sub-chronic and chronic toxicity studies  of the crude methanol extract of each plant were carried out. Twelve nulliparous rats were divided into four groups of 3 rats each. Single oral administration of 5000 mg/kg of the extract of A. leiocarpus was used for acute study (AS) while daily administration of 2000 mg/kg of the same extract for 2, 12 and 14 weeks was used for sub-acute study (SAS), sub-chronic study (SCS) and chronic study (CS), respectively. Rats in group IV were administered distilled water (negative control, NC). The same procedure was replicated for A. indica, K. senegalensis, and T. indica. All the rats were observed closely for any sign of toxicity within first 6 h of administration and twice daily: morning (7:00 am) and evening (7:00 pm). A thorough physical examination was carried out at the end of each experiment. Thereafter, the rats were euthanized by jugular venesection. Three milliliters of blood was collected from each rat into a plain sample bottle, allowed to stand on the laboratory bench in an inclined position for 15 min and then centrifuged at 3000 ×g for 10 min. The resulting serum was transferred into an appropriately labeled Eppendorf tube for serum biochemical evaluations. One milliliter of blood was also collected from each rat into an EDTA bottle for haematology.
Percentage bodyweight changes and organosomatic index (OSI) of liver and kidneys
The percentage bodyweight change of each rat was obtained by dividing the bodyweight of each rat at the end of the experiment with the bodyweight at the beginning of the experiment and multiplied by 100%. While OSI of liver and kidney of each rat was obtained by dividing the weight of the organ with the weight of that rat at the end of the experiment.
Packed cell volume was determined with hematocrit centrifugation at 3000 ×g for 15 min, total and differential white blood cells were determined by mechanical expansion, optical magnification and supravital cell staining .
Liver function test
Serum levels of transaminases, alkaline phosphatase, total protein, albumin and globulin were determined with AGAPPE® assay kits (Switzerland) according to the manufacturer’s instructions.
Kidney function test
Serum levels of urea, creatinine, Na+ and K+ were determined with AGAPPE® kits (Switzerland) according to the manufacturer’s instructions.
Histology liver and kidney
After euthanasia, the rats were placed on dorsal recumbency and a midline incision was made on the ventral aspect starting from the base of the neck down to the umbilicus. The liver and the kidneys were carefully exteriorized and cut off and immediately fixed in Bouin’s solution. Each sample was dehydrated through a graded series of ethanol and embedded in paraffin. Paraffin sections of the sample were sliced into 5 mm thickness and mounted onto coated slides. Before staining, sections were dewaxed in xylene and rehydrated through a decreasing series of ethanol. The sections were stained with hematoxylin and eosin.
Values obtained were expressed as mean ± standard error of the mean (±SEM), and subjected to two-way analysis of variance followed by Bonferoni post-test for multiple comparisons of the groups. Graph Pad Prism version 5.0 was used. Values of P < 0.05 were considered statistically significant.