Materials
All materials and solutions were purchased from Fisher Scientific (Mumbai, India) unless otherwise stated.
Garlic extract
Black garlic was extracted by refluxing water twice at 80 ° C (yield 12.8%). The resulting solution was evaporated, then lyophilized and stored at 4 ° C until use. Then it was fermented in edible Saccharomyces cerevisiae (KCTC 7910) by two-step culture. In the first stage culture, the microorganisms were cultured in a medium containing 3% (w / v) malt extract at 28 ° C for 36 hours to promote cell proliferation. Cell clusters were collected by centrifugation and recultured in a medium containing 5% (wt / vol) of garlic extract under the same conditions to increase the concentration of bioactive substances such as polyphenols and alicysteine. After culturing, the cells were removed by extracting the culture broth by filtering and heating. The solution was then lyophilized after evaporation and kept at 4 ° C until use. After that, it was dissolved directly in distilled water and orally administered at 100 milligrams / kilograms one time daily for eight weeks [19,21].
Melatonin
Melatonin from (Thermofisher, Mumbai, India) ten milligrams per kilograms every day /kg mixed in 0.05% ethanol then given to the mice in the water they drink [22].
Glibenclamide
Glibenclamide (GLC; Julphar Pharmaceuticals, Ras Al Khaimah, United Arab Emirates)
Mice
All Mice care and treatment procedures have been approved by the Institutional Review Board for Biomedicine and Research (HAPO-02-K-012-2021-10-789) at the University of Umm Alqura. A total of 48 male C57BL / 6J (B6) male mice were purchased from Harlan (Charles River Laboratories, Wilmington, Massachusetts, USA) for this study. Mice were housed in a temperature-controlled room (23 ± 1 ° C) under a 12-to-12-hour light-dark cycle. Mice were individually housed in standard cages with free access to water and standard solid diets (CRM pellets, SDS diet, USA).
Measurements were started at ten weeks of age and performed over eight weeks. Weight was monitored three times a week (Monday, Wednesday, and Friday) just before the lights were turned off throughout the experimental protocol. Eight weeks later, mice were fasted overnight, euthanized with CO2, and blood samples were taken by heart puncture.
Diabetes Induction of mice
The combination of STZ and nicotinamide [NA] can prepare a method for inducing type 2 diabetes with hyperglycemia and relatively low insulin levels. Mice were injected intraperitoneally (i.p.) with STZ (50 mg / kg body weight) in 0.1 M citrate buffer (pH 4.2) for 2 consecutive days. NA (120 mg / kg body weight) in physiological saline was administered intraperitoneally. Injections were made thirty minutes before the STZ injection on the first day after an overnight fast. Mice with an eight-hour fasting blood glucose level of 200 mg / dL, injected 7 days after the second injection, were recognized as hyperglycemia. Other mice that showed fasting blood glucose levels less than FBG 200 mg / dl, were infused with more ATZ and monitored until the FBG level reached 200 mg / dl. Fasting blood glucose was monitored with a glucose meter (Free style, USA). [22, 23]
Experimental design
The experimental design of our study used mice that were chosen randomly and then we divided them into six groups with eight mice in each group. We placed a group for control non-diabetic group and five groups were injected with NA/STZ to induce diabetes, the four groups received drug treatments daily for eight consecutive weeks:
• 1(C); is the control non-diabetic group that was treated with saline orally and citrate buffer.
• 2 (D); is the non-treated induced diabetic group.
• 3(D+GLC); is the induced diabetic group that was treated with the standard antidiabetic drug, glibenclamide (5 mg/kg/day) [24]
• 4 (D+M); is the induced diabetic group that is treated with melatonin (10 mg/kg/day in drinking water) [22]
• 5 (D+G); is the induced diabetic group that is treated with prepared garlic extract (100 mg/kg/day) [19]
• 6 (D+M+G);is the induced diabetic group that is treated with melatonin and garlic extract.
We choose the doses of melatonin and garlic because of previous research studies [25,26, 27]
At 2nd and 8th weeks, the levels of FBG and fasting insulin levels were examined. After 8 weeks; oral glucose tolerance test, plasma lipids and inflammatory cytokines (IL-6 and TNF-α Levels) were examined.
Assessment
Body weight
Changes in body weight and food intake were recorded every week in the studied groups throughout the experiment.
Fasting blood glucose and fasting insulin measurements
Mice were fasted overnight at week two and eight then blood were taken out from their tails to measure their fasting blood glucose and insulin levels. Fasting blood glucose was measured with a One Touch II glucose meter (Free style, USA). Fasting insulin were examined by using the enzyme-linked immunosorbent assay (insulin ELISA, # nr 10-1247-01, Mercodia, Sweden) and the spectrophotometric plate reader (Synergy HT Multi-Mode Microplate Reader, BioTek, USA).
Oral glucose tolerance test (OGTT)
One day before the end of experiment, all rats were fasted overnight and infused intragastrical with 2 g glucose per kilogram of body weight. Rat tail blood samples were taken at 0 min, 30 min, 60 min and 120 min to evaluate FBG, 30 min, 1 h postprandial blood glucose (PBG1) and 2 h postprandial blood glucose (PBG2) respectively [28].
Plasma lipids and Inflammatory Cytokines (IL-6 and TNF-α)
Blood samples that were taken from mice used in these measurements following manufacturer’s guidelines. IL-6 and TNF-α were determined from a standard curve and their levels were expressed in pg/ml [29].
Statistical Analysis
All results are expressed as group means ± standard deviation. Results were analyzed using one-way analysis of variance ANOVA, followed by Tukey’s post-hoc test to assess significance. Any P value of less than 0.05 was considered significant. We used GraphPad Prism version 6 (GraphPad Software Inc., California, USA).