Chemicals. Streptozotocin (Merck Germany), Ethanol 70% (Caledon Canada), Serum Normal Saline and Distilled Water, Sodium Citrate Dihydrate Buffer (Merck Germany), Citric Acid Monohydrate (Merck Germany), STAR Glucometer for Blood Glucose (Made in Taiwan), Insulin Kit (manufactured by Shibayagi Company), and Malone Dealdehyde Kit (Jiancheng Institute of Bioengineering Company, Nanjing, China).
Preparation of samples and plant powder. E. billardieri plant was collected in August 2016 from Urmia region, Iran. After identification, its herbarium specimen with number 1249 was kept in the herbarium of Tabriz University of Medical Sciences. At first, the aerial parts of the mentioned plant were dried naturally in the open air, and turned into a very fine powder. The powder obtained from this step was very light with a low density.
Extraction of the Plant Material. Extraction was performed by maceration method; first, pour 200 g of plant powder into a wide-mouthed Erlenmeyer flask and add 70% ethanol ratio. Then, the Erlenmeyer was placed in a quiet place away from sunlight and shook the Erlenmeyer flask once a day to mix its contents. We did this until the color of the solvent did not change. The supernatant is passed through a filter and the solvent of the extract is dried via an evaporator at low pressure and temperature. The dried extract was placed in a sealed container and stored at 4°C for further procedure.
Experimental Animals. This study was conducted in four stages including streptozotocin injection, injection and gavage of the extract, collection of blood and tissue samples, measurement of serum insulin and malondialdehyde. Forty eight male NMRI mice race purchased from Pasteur Institute of Iran with an average age of 12 weeks and a weight range of 32 g in standard clear polyethylene cages under standard conditions of 21°C, and humidity ± 12 hours light-dark, and in normal temperature of laboratory (10 ± 60%) were maintained. They had free access to sufficient water and food until the experiment. In general, mice were randomly divided into 8 groups (n = 6): Healthy control group, diabetic control group (receiving STZ), healthy extract control group (gavage dose of 300 mg/kg extract), diabetic group (receiving 100 mg/kg extract from the third day as IP), diabetic group (receiving 300 mg/kg extract from the third day as IP), diabetic group (gavage 100 mg/kg extract from the third day), diabetic group (gavage 300 mg/kg extract from the third day), STZ receiving group and dose 300 mg/kg of extract (Oral) for 5 days.
Streptozotocin preparation. Since STZ is a powder and cannot be injected directly into the peritoneum, the powder was dissolved in a 0.1 M sodium citrate dihydrate buffer. Due to the sensitivity of STZ to light, all steps of STZ preparation were performed in the dark [13, 14].
Induction of diabetes in male mice by streptozotocin. First, the weight and blood sugar of each mouse was measured and recorded. Blood was taken from the tail vein of a mice to measure blood sugar; in this way, first the mice tail was disinfected with alcohol and then the tail picking method was used for blood sampling. The dose of streptozotocin (150 mg/kg) for each mice was calculated based on mouse weight [13, 14]. The injection site was disinfected with 70% ethanol and then streptozotocin was injected intraperitoneally. The mice were monitored for three days, during which time they had access to water and food. After three days, the rats' weight and blood sugar were measured again. Mice with blood glucose above 200 mg/dl were selected as diabetic groups and this procedure was repeated every three days.
Receiving the extract by diabetic mice as IP and Oral and their surgery. From the third day, diabetic mice (groups 4, 5, 6 and 7) received the extract for 18 days. After 18 days of receiving the extract, mice were anesthetized by intraperitoneal injection of ketamine (100–100 mg/kg) and xylazine (3–10 mg/kg) [15–17]. After deep anesthesia, cervical dislocation was performed to ensure the cessation of vital signs in mice. Blood samples were centrifuged at 4500 rpm and 4 ° C for 30 minutes and then isolated serum samples were stored at -70 ° C.
OGTT test. Three days before surgery, mice were given 2 g/kg glucose by gavage after 12 hours of fasting. At times, zero (before glucose consumption), 30, 60, 90, and 120 minutes after glucose consumption, blood samples were collected from the venous vein to measure blood glucose levels [18].
Measurement of serum insulin levels. Determination of insulin levels in serum samples was performed by ELISA method. For this purpose the company kit Shibayagi was used. 10 µl of serum samples were incubated in wells coated with biotin conjugated to insulin antimonoclonal for 2 hours at room temperature. After incubation, the wash buffer was added to the wells and incubated with streptavidin-conjugated HRP (Horseradish Peroxidase) for 30 minutes at room temperature. In the next step, the streptavidin-conjugated HRP residue reacted with the chromogenic reagent at room temperature for 30 minutes. The reaction was stopped by adding the solution. The absorbance was read with a microplate reader (Bio-Rad, Hercules, CA, USA) at 450 nm [19].
Measuring the amount of malondialdehyde (MDA). The amount of malondialdehyde was measured using colorimetry method and thiobarbituric acid reagent (TBARS) according to the instructions of the manufacturer (Jiancheng Institute of Bioengineering Company, Nanjing, China) and its absorption was determined at 500 nm [20].
Statistical analysis of data. Data were represented as Mean ± SEM. One-way ANOVA and post-hoc Tukey test were used as statistical methods. P < 0.05 is considered as a significant difference.