Samples of E. abyssinica were collected from their natural habitats in Siaya and Kisumu counties, Western Kenya during the month of January 2020 with the help of a plant taxonomist. The stem barks of the plant were carefully harvested from mature healthy plants with minimal injury to the plant. Leaves, branches and fruits were used to prepare a voucher specimen (OSB/01/2020/001) which was deposited at the University of Eldoret Herbarium, Botany department for correct botanical authentication and reference purposes. The harvested stem barks were packed in sacks and transported to the Chemistry laboratory at Moi University for drying and pulverization. The samples were chopped into small pieces and air-dried under shade at room temperature (25.0 ± 2.0°C) for 4 weeks until a constant mass was obtained. The samples were then pulverized using an electric grinder (NutriBullet^® 600 Series), packed and stored in clean labelled paper envelopes at room temperature until extraction.

Methanol of analytical grade was purchased from Merck-Sigma Aldrich and used to extract phytochemicals from the samples. A sample (300 g of powder) was macerated in 1,000 ml of methanol for 3 days with occasional shaking. The mixture was first filtered using cotton wool and then through Whatman’s filter paper No. 1 (pore size 11 µm) to obtain the crude extract. The extract was concentrated to a minimum volume using a rotary evaporator (Hahnvapor HS-2005S) at 40°C and reduced pressure. The concentrated crude extracts were dried in a desiccator over anhydrous copper (II) sulphate to constant weight at room temperature. The concentrated crude extract was stored in clean labeled bottles at 4°C in a refrigerator until further use.

Mature healthy inbred Wistar albino rats (100–200 g) about 8–10 weeks old were purchased from the animal facility at the College of veterinary medicine, Animal Resources and Biosecurity, Makerere University, Kampala, Uganda. Three animals of the same sex were housed in standard wooden cages (15 × 21 × 29 cm) bedded with wood chips and equipped with continuous flow nipple watering devices. The animals were fed ad libitum on standard feeds, allowed free access to water and cage beddings changed every 2 days. The animals were maintained in clean animal facility at 23–27°C, with a 12-h light and darkness cycle. The animals were acclimatized to the housing conditions for 2 weeks prior to commencement of the study and were handled in conformity with guidelines for handling laboratory animals (Kilkenny et al., 2010). Animals which died before the end of the experiment and those sacrificed were pooled in a bio-hazard container and stored at −20°C (for approximately 24 h) before being incinerated. All animals at the end of the experiment were sacrificed under general anesthesia using an overdose of pentobarbitone sodium solution.

The concentrated extract (1000 mg) were dissolved in 10 ml of 1% tween 80 in distilled water at room temperature (25.0 ± 2.0°C) to make an extract suspension of 100 mg/ml. The suspension was vortexed (Analog Vortex mixer OHAUS) for 20 min and after digitally shaken (VWR–digital shaker) for 2 h to allow maximum dissolution. The prepared extract solution was then poured in clean labelled flask for administration to the animals. All the extract solutions were freshly prepared every day for use.

The OECD 407 guidelines on oral repeated toxicity testing of chemicals in rodents were adopted (Olayode et al., 2019). Wistar Albino rats (24) of both sexes were randomized basing on their body weight into four groups (A–D). Each group consisted of six rats (three males and three females) which were housed in different cages. The administered doses (150, 300, 600 mg/kg) were determined based on the median lethal dose (4183 mg/kg) that was predetermined using the Lorke’s method. Three groups (A, B and C) were orally administered different doses of the extract (150, 300, 600 mg/kg) respectively while group D received 1% tween 80 in distilled water (control) depending on their body weight daily for 28 days. The volume of the extract administered was calculated using the equation below. Volume given to each animal  ( ml ) = Body weight of the animal  ( kg ) × Dose  ( mg/kg ) Concentration of the extract  ( mg/mL ) (1)

Observations of any toxic symptoms including death manifested were noted and recorded systematically daily up to the end of the experiment. Body weights of the rats were taken on day 0, 7, 14, 21, and 28. On the 29th day, the surviving rats were sacrificed under general anesthesia using anesthetic ether solution. The rats were dissected to obtain blood and vital organs (liver, kidney, heart, and spleen) for biochemical, hematological and histopathological analyses. The weights of the vital organs were measured using a digital analytical balance.

Blood (2 ml) was collected by cardiac puncture from each rat into non-heparinized vacutainers using syringes. Blood was centrifuged at 3,000 rpm for 5 min to obtain serum which was assayed using an automated chemistry analyzer (HumaStar 200) for levels of different biochemical parameters. Test kits for measurement of different parameters were purchased from Sigma-Aldrich and used according to the manufacturer’s instructions. The parameters assayed included creatinine, urea, uric acid, aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP), serum proteins, bilirubin, triglycerides, total cholesterol, Low density lipoproteins (LDL), high density lipoprotein (HDL) and serum electrolytes (Na⁺, K⁺, Ca²⁺, Cl⁻, H⁺).

Blood (2 ml) was collected by cardiac puncture from each rat into heparinized vacutainers using syringes and analyzed using an analyzer (Sysmex 1000i) for hematological counts of different parameters. These included White blood cell (WBC), neutrophils (NEUT), lymphocytes (LYMP), monocytes (MONO), eosinophil (EO), basophils (BASO), red blood cells (RBC), hematocrit (HCT), hemoglobin (Hb), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC) and platelet count (PCH).

The obtained vital organs (liver, heart, spleen, and kidney) were grossly examined for the observable histomorphological changes and the weight of each organ measured using a digital weighing scale. The isolated organs were fixed in 10% (v/v) buffered formalin labeled bottles for 72 h. After fixation, the tissues were trimmed and loaded in cassettes for processing using an automated tissue processer (Leica 40). They were first dehydrated by placing them in tissue cassettes with graded alcohol concentration (70, 80, 90, and 96%, v/v) and then removed and placed into xylene solution baths to clear off the alcohol. They were then impregnated with molten wax and allowed to dry. The tissues were then sectioned by use of Rotary microtome (at 5 µm thickness), and then stained with hematoxylin and eosin (H & E). Slides were prepared and then examined using a research light microscope connected to computerized camera (Lieca LB₂–image analyzer). Photomicrographs were captured and then examined for histopathological changes by two independent pathologists who were not aware of the biochemical and hematological data.

Quantitative data was entered in Microsoft excel version 2013 and its means and standard error of mean calculated. The results were presented as means ± standard error of mean. Statistically significant differences were determined using one-way analysis of variance (ANOVA) and/or Student’s t-test followed by Dunnett’s post hoc test using Graph Pad Prism version 5.01 (Graph Pad software, San Diego, California, United States). Differences were considered statistically significant at p < 0.05.

The stud was approved and registered by the Scientific and Ethics Research Unit of Kenya Medical Research Institute (KEMR/SERU/CTMDR/CSCP085/4067).

All the animals in the treatment and control groups increased in body weight over the 28-day period except for those administered with the highest dose (600 mg/kg). Over the 28-day period, the increase in body weight was only significant (p < 0.05) for animals dosed with 300 mg/kg of the extract. There was no statistically significant differences (p > 0.05) in the body weight between animals in the treatment and control group (Table 1).

Majority of the biochemical parameters were not significantly altered by administration of the extracts except for serum albumin, AST, Uric acid, LDL and HDL (Table 2). All doses of the extract significantly increased the uric acid levels. The extracts (at 300 and 600 mg/kg) significantly increased (p < 0.05) the level of AST but significantly decreased serum albumin (p > 0.05). At doses of 150 and 300 mg/kg, the extract significantly decreased the level of HDL. At 150 mg/kg of the extract, the extract significantly increased the level of LDL. All these differences are in comparison with the control (1% tween 80 in distilled water).

Extract administration at all doses did not significantly alter the concentration of sodium, potassium, chloride, and calcium ions except at 300 mg/kg where it elevated the potassium ions (Table 3). On the other hand, all doses significantly elevated the hydrogen ion concentration (lowered the pH).

The extract did not have a significant effect (p > 0.05) on the white blood cells and its differentials at all doses (Table 4). However, it significantly (p < 0.05) altered some red blood differentials (HCT, MCV, MCHC, and RDW-CV) and Platelet differentials (MPV and PCT). At all doses the extract significantly reduced the MCV and HCT while increased the MCHC. MPV was significantly reduced at 150 and 300 mg/kg of extract while PCT at 150 mg/kg only.

The extract did not significantly alter the weight of the various organs as compared to the control group except for the liver and kidney which were significantly reduced (p < 0.05) at 600 mg/kg dose of the extract (Table 5).

No significant organ lesions were associated with administration of the extract in the heart, liver, kidney and spleen (Figures 1A–D). However, at the 600 mg/kg dose, the extract caused mild to moderate multifocal parenchymal hepatocytes necrosis (Figure 1A), periportal mononuclear inflammatory cell infiltration and focal interstitial nephritis (Figure 1C). In all the treatment and control groups, the spleen exhibited nonspecific immunostimulation as indicated by mild to moderate diffuse lymphoid hyperplasia within the white pulp (Figure 1D). A summary of the organ specific findings is indicated in Table 6.