Plant material
The fresh stem bark of P. macrophylla was collected from Kufena village, Zaria, Kaduna State, Nigeria. Botanical authentication of the plant parts was carried out at the herbarium unit of Biological Sciences Department, Ahmadu Bello University, Zaria, Kaduna State, Nigeria. It was identified with a voucher specimen no.: 012341 and deposited at the herbarium unit.
Preparation of plant extracts
The fresh stem bark of P. macrophylla was air-dried and made into powder using pestle and mortar. The air-dried powdered plants (800 g) materials were extracted with water using soxhlets apparatus. The solvent was removed by vacuum evaporation using a rotary evaporator. A dark brown residue weighing 63.9 g was obtained and kept in a sealed container and stored in a refrigerator at about 4 °C until use.
Experimental animals
Thirty-two apparently healthy male Wistar strain albino rats (8–10 weeks old) weighing 100–150 g were obtained from Animal House, Department of Pharmacology and Clinical Pharmacy, Faculty of Pharmaceutical Sciences, Ahmadu Bello University, Zaria, Kaduna. The animals were kept in well-aerated laboratory cages and were allowed to adjust to the laboratory conditions for a period of 2 weeks before the commencement of the experiment. They were fed with grower and starter mash from Vital Feeds Company and water was provided ad libitum during the stabilization period. The principles governing the use and care of laboratory animals, and the experimental protocol were strictly followed in accordance with the National Research Council, Guide on the Care and Use of Laboratory Animals [22]. The study was approved by the Research Ethics Committee of Nigerian Defence Academy, Kaduna, Kaduna State, Nigeria (Ref: NDA/PGS/FS/M/1826/14).
Phytochemical screening
The preliminary phytochemical screening of the crude extracts of P. macrophylla was conducted at Pharmacognosy Department, Faculty of Pharmaceutical Sciences, Ahmadu Bello University, Zaria, Kaduna to ascertain the presence of their constituents by utilizing standard conventional protocols [23]. The extracts were screened for alkaloids using Mayer’s test and Wagner’s test, saponins using Frothing test, tannins using Ferichloride test, anthracene derivatives using Bontrager's test, cardiac glycosides using Keller-Killiani test and NaOH test, flavonoids using Shinoda test and NaOH test, carbohydrates using Mollish test, and steroids and triterpenes using Liebermann-Burchard test. These tests were carried following the standard protocols reported in the literature [24,25,26].
Acute toxicity studies
The median lethal dose (LD50) of the plant extract was determined by the modified method of Lorke [27] using 12 rats. In the first phase, rats were divided into 3 groups (3 rats per group) and were treated with the extract at doses of 10, 100, and 1000 mg/kg body weight orally. They were observed for 24 h for signs of toxicity. In the second phase, 3 rats were divided into 3 groups (1 rat per group) and were also treated with the same extract but at doses of 1600, 2900, and 5000 mg/kg body weight orally. The median lethal dose (LD50) was obtained using the second phase.
Induction of experimental diabetes mellitus
Twenty rats were used for the experiment. The rats fasted for 24 h but they were allowed water ad libitum. Diabetes was induced in 15 of the 20 rats using a single intra-peritoneal injection of alloxan monohydrate at the dose of 150 mg/kg [28]. Because alloxan has the potential to produce fatal, hypoglycemia as a result of massive pancreatic release of insulin, the rats were kept for the next 24 h on 5% glucose solution bottles in their cages to prevent hypoglycemia [29]. After 72 h of alloxan administration, the blood glucose levels (BGLs) of the rats were determined. The rats with BGL > 200 mg/dl were considered diabetic [30]. The body weight of the animals was monitored daily for 2 weeks.
Experimental design
The diabetic rats were randomly divided into three groups (B–D) comprising five rats per group (n = 5). Diabetes was not induced in group A, which represents the normal control group, but received 10 ml/kg of sterile distilled water orally. Group B which represents the diabetic control group received a standard oral hypoglycemic agent: 10 mg/kg of glibenclamide. Groups C and D (experimental groups) received oral dose of 1000 mg/kg and 2000 mg/kg body weight of aqueous stem bark extract of P. macrophylla respectively. All treatments were administered on daily basis for 2 weeks.
Determination of blood glucose levels
All blood samples were collected from the tail vein of the rats. BGL was determined at an interval of week 0 (0 day), week 1 (7th day), and week 2 (14th day). The determination of the BGLs was done by the glucose-oxidase method [31] using the digital glucometer (Accu-Check advantage, Roche Diagnostic, Germany), and results were reported in mg/dl.
Histological investigation of the pancreas
The animals in the normal control and the two experimental groups were sacrificed after the 2 weeks of treatment using chloroform inhalation as anesthesia. The rats were individually placed in an airtight plastic box containing drops of chloroform (37%) in cotton wool until they become unconscious (maximum time: 2 minutes). An incision was made in the abdomen and sections of their pancreatic tissue were removed and processed for histological examinations at the Gross Anatomy Research Laboratory, Department of Human Anatomy, Faculty of Medical Sciences, Ahmadu Bello University, Zaria, Kaduna State.
Statistical analysis
Data collected were summarized using descriptive statistics of mean and standard error of measurement (mean ± SEM). One-way analysis of variance (ANOVA) was used to assess difference between the groups in BGLs and body weight. Where ANOVA showed a significant difference, a post-hoc test with Turkey HSD was applied for multiple pairwise comparisons. All statistical analyses were performed using SPSS version 23.0 (IBM Co., Armonk, NY, USA) with the level of significance set at P < 0.05.