Reagent and chemicals
Methanol, concentrated H2SO4, glacial acetic, ferric chloride, acetic anhydride, ammonia, chloroform, hydrochloric acid, mercuric chloride, potassium iodide, iodine, glucose and formalin were obtained from Sigma Chemical Company, St. Lious, Mo, U.S.A., and British Drug House (BDH) chemical Ltd., Poole, England. Metformin was purchased from Teva Pharmaceuticals, UK. Insulin (HumulinR) was purchased from Eli Lilly, USA. The diagnostic kits were obtained from Randox Laboratories Ltd., Crumlin, Co. Antrim, U.K. All reagents and chemicals used were of analytical grade.
A total of forty-nine albino rats were used for the study. The animals were purchased from College of Health Sciences, Osun State University animal house where they were kept in well ventilated cages. The cages were lined with wood husks, renewed every 24 h under a 12:12 h light/dark cycle at room temperature. The rats had free access to water and commercial standard pellets and were acclimatized for two weeks. Experimental animals were used according to the Department of Biochemistry, LAUTECH Ethics Committee Guidelines on the use of vertebrate animals for experiments, and the approval was deemed unnecessary having conformed to the National regulations and International guidelines of National Institute of Health (NIH publication 85-23, 1985) for laboratory animal care and use.
Collection and preparation of Eucalyptus globulus leaf
Eucalyptus globulus leaf was collected from the Osun State University botanical garden, identified and authenticated for this study by Mr. G.A. Ademoriyo at Ife Herbarium, Obafemi Awolowo University, Ile-Ife, Nigeria. The approval number allotted was 17,938. The leaf was shade-dried for 2 weeks and then pulverized into fine powder using electrical grinder.
Extraction of Eucalyptus globulus leaf
Dried powder of Eucalyptus globulus leaf (200 g) was subjected to cold maceration with frequent agitation in 2 L of 100% methanol for 72 h at room temperature . The filtrate was concentrated using standard procedure. Eucalyptus globulus leaf extract (EGLEX) was stored in the fridge until used.
Phytochemical screening of Eucalyptus globulus leaf extract
The Phytochemical tests were carried out on EGLEX to determine qualitatively the presence of cardiac glycosides, steroids, flavonoids, alkaloids, saponins, tannins, terpenoids and phlobatannins using standard procedures as described by Harborne and Turner .
GC–MS analysis of Eucalyptus globulus leaf extract
GC–MS was utilized to identify compounds in the methanol extract of the plant leaf according to the method described by Santos et al. .
Determination of mean lethal dose
Mean lethal dose (LD50) of Eucalyptus globulus leaf extract (EGLEX) was determined using protocol of Organization for Economic Cooperation and Development (OECD) guidelines, starting from a fixed dose of 50 mg/kg body weight. Female rats (3) of the same age group and weight were dosed up to 2000 mg/kg body weight through oral administration. The animals were observed for signs of toxicity for the first 1 h, hourly for 4 h and finally every 24 h for 14 days. Ten percent of LD50 was considered to be safe dose.
Sub-acute toxicity test
Sub-acute toxicity study was conducted according to OECD guidelines using twenty albino rats randomly divided into four groups (n = 5) as follows:
Group 1: control treated orally with distilled water
Group 2: treated orally with 50 mg/kg bwt EGLEX
Group 3: treated orally with 300 mg/kg bwt EGLEX
Group 4: treated orally with 2000 mg/kg bwt EGLEX
All rats were treated daily for 14 days. Prior to sacrifice, blood samples were collected via ocular puncture and serum separated for biochemical studies.
Oral glucose tolerance test
Monitored oral glucose tolerance test (OGTT) was performed on twenty rats fasted overnight and randomly divided (n = 5) following pre-treatment as follows:
Group 1: Normal control (administered distilled water only)
Group 2: 200 mg/kg bwt EGLEX + 2 g/kg bwt of 20% glucose
Group 3: 7 mg/kg bwt metformin + 2 g/kg bwt of 20% glucose
Group 4: 0.4 mg /kg bwt insulin + 2 g/kg bwt of 20% glucose
Baseline blood glucose was measured prior administration of single dose of 2 g/kg bwt of 20% glucose solution. Blood glucose level was measured by glucometer (Accuchek Adventage II; Roche, Germany) in each group at 0, 30, 60 and 120 min after glucose load.
Assay for urea and creatinine concentrations
Serum urea concentration was determined by urease-berthelot method as described by Fawcett and Scott  using RANDOX diagnostic kit while serum creatinine was determined by alkaline picrate method .
Assay for aspartate transaminase and alanine transaminase activities
Aspartate transaminase and alanine transaminase activities in the serum were determined according to the methods described by Reitman and Frankel  using Randox diagnostic kit.
The animals were sacrificed by cervical dislocation and dissected. The liver and kidney tissues were harvested, immediately fixed in 10% formalin and used for histomorphological study.
Data obtained were subjected to statistical analysis using ANOVA of SPSS (version 20.0) statistical package. Tests of homogeneity of variance was conducted using Levene statistic. Tukey’s test was used for multiple comparisons and homogenous subsets. A p-value of less than 0.05 was considered statistically significant.