STZ and glibenclamide were purchased from Sigma Chemicals Co. (Saint Louis, Missouri, USA) and all other reagents and chemicals used in the present study were of analytical grade.
Collection and identification of plant material
The ripe fruits (835 g) of P. macrocarpa were collected and identified with Voucher specimen (no.: P-0230). The mesocarp and pericarp of the fruits were sliced and dried in a normal room temperature at 25 °C for 10 days, then it was pulverized to a powdered form (713 g) using Fritsch Universal Cutting Mill-Pulverisette 19, Germany, and kept at 4 °C until further use .
Preparation of ethanol extract
The sample (710 g) was extracted by cold maceration with ethanol (4:1, solvent: solid ratio) for 72 h at room temperature, filtered into a sterile round bottom flask using adsorbent cotton wool and filter paper (Whatman No. A-1). The extraction procedure with the solvent was repeated several times to ensure the highest percentage of the yield of ethanol-soluble compounds from the EEPM fruit powder. The ethanol extract was concentrated (temperature at 45 °C, 175 mbar, and rotation 80-85 rpm) by using a rotary vacuum evaporator (BUCHI R-205) to a final corrected volume of 500 ml. This was further frozen at −80 °C and shifted instantly to three week’s successive freeze drying at −50 °C using bench-top freeze dryer (ALPHA 1-4LD-2), to give an ultimate yield of 213 g.
Experimental procedure of this study was approved by the Institutional Animal Care and Use Committee/IACUC bearing an approval ID: /IACUC Approval/2014/(3) (8). It conformed to the International guidelines for the use and care of laboratory animals (OECD, 425). Efforts were made to minimize the number of animals and their discomfort in the laboratory uses. Healthy adult male Sprague-Dawley (SD) strain rats (8–10 weeks) weighing from 180 to 200 g were obtained from the Laboratory Animal Centre, Universiti Putra Malaysia (UPM), Serdang, Selangor, Malaysia. The current study was performed with a single-sex (male) to reduce variability in the test animals. A pair of animals were housed in a standard propylene cage, acclimatized for 7 days to the animal laboratory conditions at room temperature (22 ± 3 °C), relative humidity (RH) of 46–70%, and 12 h light/dark cycle , allowing adequate cross ventilation prior to the test. Moreover, the animals were fed with standard commercial dry pellet (Gold Coin Feed Mills Sdn. Bhd., Kuala Lumpur, Malaysia) diet containing 22% crude protein, 46% fat, 4% fiber, 7.6% ash, 12.0% moisture, 1.2% calcium, 0.73% phosphorus, and water ad libitum. Fasting animals were deprived of food for at least 16 h with free access to drinking water prior to the study .
Acute oral toxicity study
Acute oral toxicity of EEPM was performed on male Sprague-Dawley strain rats, according to the procedure described by the revised up-and-down method OECD (Organization of Economic Cooperation and Development) Guideline 425 . Two groups, with six rats each, were used for the study. Group I served as a control and received drinking water. Group II received a single oral dose of EEPM (5000 mg/kg). The rats underwent fasting (16 h) overnight and the body weight (g) of each rat was recorded prior to the test. The test substance was administered in a single dose by oral gavage using a stomach tube and a suitable intubation cannal and observed closely at 4 h initially, then every 6 h intervals for changes in skin, fur, eyes, mucous membranes, behavioral (alertness, restlessness, irritability, recumbence, vomiting, fearfulness), neurological (spontaneous activity, convulsion, gait, bleeding orifices, touch/pain response), autonomic (defecation, micturition) profiles, and/or mortality, and any significant morbidity or mortality within 24–168 h was recorded .
Induction of diabetes in experimental animals
Healthy male Sprague-Dawley rats were made to fast overnight (16 h) and their basal weight (g) and fasting blood sugar (mg/dl) were appropriately measured and recorded. Diabetes was induced under light ether anesthesia by a single i.p. injection of STZ (Sigma-Aldrich, USA) at 65 mg/kg, based on our result of the pilot study. STZ was first weighed individually for each animal according to the weight, solubilized with 0.1 ml of freshly prepared cold Na-citrate buffered (NaB- 0.1 M, pH 4.5), and administered within 5 min to prevent degradation. The injection of STZ volume was prepared to contain 1.0 ml/kg. Rats were supplied with 5% glucose solution for 48 h immediately after STZ injection to counteract severe acute hypoglycemic effect. Control rats received an equivalent volume of PBS without STZ interperitoneal . Diabetes induction was confirmed by determination (Life Scan One-touch Ultra Glucose Meter, USA) of high fasting blood glucose level alongside diagnostic triad (polyuria, polydipsia, weight loss) on the 3rd day after STZ administration. Only the rats that displayed the fasting blood sugar (FBS) level ≥ 300 mg/dl were selected for subsequent experiments .
The rats were made to fast (16 h) overnight and their baseline glucose level was measured and recorded. All the animals were given oral administration of glucose (2 g/kg) 30 min after dosing and their glycemic status was re-evaluated intermittently at 0, 30, 60, 90, and 120 min respectively. Fasting blood sugar levels ≥ 300 mg/dl further confirmed diabetes in these rats. Mild and sub diabetics rats after day 3 of STZ injection were further assessed by the oral glucose challenge test [23, 24].
Six groups of rats, six in each (6 normal; 30 diabetic, total 36) received the following treatment schedule.
Group I: Normal control (drinking water)
Group II: Diabetic control (STZ, 65 mg/kg + drinking water)
Group III: Drug control (STZ, 65 mg/kg + glibenclamide, 0.5 mg/kg)
Group III: Drug control (STZ, 65 mg/kg + EEPM, 50 mg/kg)
Group III: Drug control (STZ, 65 mg/kg + EEPM, 100 mg/kg)
Group III: Drug control (STZ, 65 mg/kg + EEPM, 200 mg/kg)
Blood glucose measurement
The fasting plasma glucose levels were recorded before the induction of STZ and observed very carefully for the first 24 h; 5% glucose solution was prepared in order to prevent any accidental loss of animal life due to hypoglycemia. Then they were kept for another 48 h in a diabetic state for the stability of physiological characteristics. Blood glucose was measured repeatedly in group-wise before starting the extraction treatment and was continued for 35 consecutive days using glucose-oxidase, peroxidase reactive strips, and a glucometer (One Tuch Ultra Life Scan Inc. USA) with a similar dose of 0.5 mg/kg (glibenclamide), 50, 100, 200 (EEPM) mg/kg/day orally using oral gavage.
Blood collection and preparation
At the end of the 7-day period of EEPM fruits oral administration, the rats were humanely sacrificed with an anesthetic agent (diethyl ether). Before the sacrifice, about 3 to 5 ml of blood was collected directly from the heart of each rat via intra cardiac puncture using 23 G syringe. The blood was collected in ethylene diamine tetraacetic acid (EDTA) tubes. Plasma was separated using refrigerated ultra-centrifuge (Allegra X-12R), 4 °C at 2000xg for 10 min. The plasma was stored in working aliquots at −80 °C for subsequent biochemical analysis.
The following parameters were determined calorimetrically by employing the standard ready-to-use kits (Human Gesellschaftfür Biochemica and Diagnostica MBH, Germany), creatinine (mg/dl), phosphorus (mg/dl), HDL cholesterol (mg/dl), LDL cholesterol (mg/dl), cholesterol (mg/dl), triglyceride (mg/dl), aspartate aminotransferase (AST-U/L), alanine aminotransferase (ALT- U/L), alkaline phosphatase (ALP-U/L), total bilirubin (T.BIL-mg/dL), albumin (ALB-g/dL), glucose (Glu-mg/dL), and total proteins (T.PROT-g/dL) (Cobas Mira, The automated Thermo Scientific Konelab 20 and 20XT analyzers). The manufacturer’s instructions for each biochemical parameter were strictly followed in the course of these investigations.
Data were expressed as a mean ± standard error mean (SEM) for six rats in each group, and analyzed using the SPSS software for Windows Version 21.0. Statistical differences were computed using one-way analysis of variance (ANOVA, SPSS, and Version-21), followed by Tukey’s honest significant difference (HSD) test. Data were considered statistically significant at *Ρ ˂ 0.05 and highly significant at **Ρ ˂ 0.01 and 95% confidence interval.