Chemicals and drugs
Streptozocin was a product of Sigma- Aldrich, USA. Metformin (glucophage 500 mg) was manufactured by Merck santé, France and lisiofil (lisinopril) was manufactured by Fourrts (India) Laboratories Pvt. Limited. All the reagent kits used for bioassay were sourced from RANDOX Laboratories Ltd., Crumlin, Co. Antrim, UK. All other chemicals used were of analytical grade and sourced from the Biochemistry laboratory, Kwara State University.
Plant collection, identification and crude extract preparation
The stem bark of E. chlorantha was obtained from Oja tuntun, Ilorin, Kwara State in June 2018. The plant sample was identified and authenticated at the department of Plant Biology, University of Ilorin, Kwara State, Nigeria. A voucher number UIH/001/1356 was assigned to the plant and a sample specimen was thereafter deposited in the Herbarium. The stem bark was cleaned to remove adhering dirt, air-dried for 2 weeks and ground into powder using an electric blender. Extraction was carried out by cold maceration of 800 g of the coarse powder with 5 L of 70% ethanol for 72 h, with constant shaking. The resultant mixture was filtered using Whatman filter paper (No.1) and the filtrate was concentrated using a rotary evaporator at 40 °C. Aliquot portions of the extract were weighed and the final yield was determined to be 12.5%. The extract was finally reconstituted in distilled water for use in the study.
Experimental protocols
Experimental animals
The sub-chronic toxicity study was carried out on healthy forty-nine (49) male Wistar rats weighing between 164 and 176 g while the acute toxicity studies was carried out on nine male Wistar rats weighing between 119 and 122 g. The rats were obtained from Can farm Ilorin and housed in cages at the animal house of the Department of Medical Biochemistry and Pharmacology, Kwara State University, Malete, Nigeria. The rats were acclimatized for 14 days and fed with commercial diets and water ad libitum. They were all maintained at 25 ± 2 °C light and dark cycle of 12/12 h respectively.
Acute toxicity test
The acute toxicity test was performed using the OECD guidelines 423 [20, 24]. Standard two-phase approach described by Lorke [17] was used. Nine rats of average weight 120.09 ± 2.51 g were used for this study. The rats were divided at random into three groups of three rats each in the first phase. The rats were first deprived of feed and water for 12 h. Groups I, II and III were orally treated with ethanolic stem bark extract of E. chlorantha at 10 mg/kg, 100 mg/kg and 1000 mg/kg body weight respectively. They were then observed for 48 h for signs of toxicity or mortality. In the second phase of the experiment, the remaining three rats were assigned into three groups (IV, V and VI) of one animal each and thereafter administered 1600 mg/kg, 2900 mg/kg and 5000 mg/kg body weight of the extract respectively. The rats were thereafter carefully observed individually after dosing. The observation was done once during the first 30 min, periodically during the first 24 h, with special attention given during the first 4 h and daily thereafter, for a total of 14 days. All the rats were then subjected to detailed gross necropsy by careful examination of the external surface of the body, all orifices and cranial, thoracic and abdominal cavities. Behavioral changes, lethargy, depression, salivation, diarrhea, muscular weakness, sedation and ailment signs were also observed. The LD50 was thereafter estimated based on the mortality observed in each group adopting the method of Ajani et al. [2].
$$ {\mathrm{LD}}_{50}\ge \mathrm{Maximum}\hbox{-} \mathrm{dose}\ \mathrm{Y}/\mathrm{number}\ \mathrm{of}\ \mathrm{rats}\ \mathrm{per}\ \mathrm{group} $$
$$ \mathrm{Where}\ \mathrm{Y}=\mathrm{Sum}\ \mathrm{of}\ \mathrm{mean}\ \mathrm{death} $$
Induction of diabetes
Type 2 diabetes was induced following the method of Wilson and Islam [34]. The rats were first fed 15% fructose solution (w/v) for 4 weeks, after which they were fasted overnight and thereafter administered streptozotocin (40 mg/kg i.p) freshly prepared in 0.1 M sodium citrate buffer. The diabetic state was confirmed 72 h after streptozotocin injection. All rats having fasting blood glucose levels greater than 200 mg/dl were considered diabetic.
Sub-acute oral toxicity study
Animal grouping/administration
Forty- two (42) diabetic rats were randomly assigned to 6 groups consisting of 7 rats each. The rats were labelled and treated as follow; Diabetic model group (DM); Diabetic treated groups (administered E. chlorantha [T1], Glucophage [T2], E. chlorantha+ Glucophage [T3], Lisinopril [T4], and lisinopril + Glucophage [T5]). Another 7 rats (non-diabetic) were kept as the control group (NC). The extract was administered at 200 mg/kg dose while glucophage and lisinopril were administered at 7.14 mg/kg and 0.14 mg/kg dose respectively. All administrations were carried out orally as a single dose daily by gavage for 4 weeks. The rats were housed in rat cages in the animal facility center at the department of Medical Biochemistry and Pharmacology, Kwara State University, Malete, Nigeria. The rats were maintained in accordance with the principles of laboratory animal care [22] guidelines. The experiment protocol was designed according to the Department Animal Ethics Committee guidelines. Twenty- four hours after the last administration and under mild diethylether anesthesia, the animals were sacrificed and blood was obtained from the jugular vein. Blood sample was transferred into plain centrifuge tube and allowed to clot at room temperature. It was then centrifuged within 1 h of collection at 4000x g for 10 min to obtain the serum.
Assay procedure
Biochemical and Haematological assay
Liver, renal and cardiac function parameters were evaluated according to manufacturer’s instructions on the assay kits. Serum Aspartate Aminotransferase (AST) and Alanine Aminotransferase (ALT) were assayed for by Reitman and Frankel [29] method. Alkaline Phosphatase (ALP) and Lactate Dehydrogenase (LDH) were assayed according to the recommendations of the Deutsche Gesellschaft fur Klinische Chemie [36]. γ-glutamyl transferase (GGT) was assayed for by Reitman and Frankel [29] method while total protein was assayed for by Peters [26] method. Total bilirubin was assayed for by Roma-Bikai et al. [30] method and creatine kinase (CK-MB) was assayed for using enzyme immunoassay technique [6]. Creatinine was assayed for by Tietz et al. [32] method. The plasma concentrations of sodium (Na+) and potassium ions (K+) were determined by Flame Photometry using Coming 410C Flame Photometer. Urea was assayed by Fawcett and Scott (1960) method. Automated hematological analyzer Sysmex, KX-21 (Japan) was used to analyzed haematological parameters.
Histopathological evaluation
The rats were quickly dissected and the liver and pancreas isolated, blotted with clean tissue paper and cleaned of fat. These were then used for histological examination. The method of Thapa and Anuj [31] was adapted for the histopathological examination of the harvested liver sections. Microscopic features of the cells of the treated rats were compared with both normal and the model groups.
Data analysis
All statistical analysis was performed with SPSS software version 16.0. Data are expressed as the means standard error of mean (S.E.M). Student’s t-test and analysis of variance (ANOVA) were employed in comparing means of continuous variables as appropriate. Differences were considered statistically significant if p < 0.05.
