2.4. Nicotinamide/streptozotocin-induced mice with type 2 diabetes mellitus

The use of nicotinamide/STZ to induce T2DM-ICR mice was performed as per previous reports with modifications.^21,22 After a 2-week acclimation period, the mice were randomly divided into two groups: blank (N = 6) and T2DM-induced (N = 18 for three subgroups: control, sitagliptin-treated, and vitisin B-treated). After fasting for approximately 16 h, blood was drawn from the tail vein of each ICR mouse to determine the basal BG level, and hyperglycemia was induced by nicotinamide/STZ for 2 weeks. For mice in the T2DM-induced group in the first stage (day 0–14), nicotinamide (120 mg/kg in 0.9% sodium chloride solution) was injected intraperitoneally on day 14, followed by the freshly prepared STZ (100 mg/kg in the 50 mM sodium citrate buffer, pH 4.5) in an ice bath 15 min later. On day −12, the same injection protocol was used to induce T2DM in each ICR mouse. For mice in the blank group, 0.9% sodium chloride solution was administered instead of nicotinamide solution. The 50 mM sodium citrate buffer (pH 4.5) was administered instead of STZ in the first induction stage. Each mouse with a BG level >230 mg/dL was identified as having T2DM successful induced. In the second stage, T2DM-induced ICR mice were randomly categorized into control, sitagliptin-treated (positive control), and vitisin B-treated. Each sample was sonicated in distilled water, as previously reported.^14,15,17 The control group received a single administration of distilled water daily by oral gavage using a feeding tube. However, the two intervention groups received either 10 mg/kg sitagliptin (sitagliptin-treated group) or 50 mg/kg vitisin B (vitisin B-treated group) for additional 28 days (days 0–28) to evaluate hypoglycemic activities. In the second stage, mice in the blank group were administered an equal volume of distilled water once daily via oral gavage using a feeding tube. Each mouse was weighed weekly, and the accumulated feed, water intake, and fasting BG levels from the tail vein were monitored every 7 days until the end of the experiments (days 14–28).