Elisa method

The experimental procedure was illustrated in Figure 2A. After a 1-week-adaptation period, mice were randomly divided into three groups: (1) distilled water (n = 8; Water); (2) low-dose water extract (n = 8; LWE; 350 mg/kg body weight) and (3) high-dose water extract (n = 8; HWE; 700 mg/kg body weight). The water extract of D. officinale or its vehicle (distilled water) was administered intragastrically once daily at 9:00 a.m. for 2 weeks. After that, mice were treated with multiple low-dose streptozocin (STZ, Sigma-Aldrich, St. Louis, MO USA) after overnight fasting. The STZ solution was prepared in citrate buffer (0.5%, pH = 4.3) and given by intraperitoneal injection at dosage 40 mg/kg of body weight for 5 consecutive days according to the protocol of STZ-induced diabetic mice [46 (link)]. At 0, 2 and 4 weeks, blood glucose level was measured from a tail nick by a portable glucose monitor (ACCU-CHEK Active, Mannheim, Germany). Meanwhile, body weight was recorded with a digital balance (JY, Shanghai Minqiao Precise Science Co. Ltd., Shanghai, China). At 4 weeks, fasting insulin level was measured in the serum using an ELISA method (Merck, Darmstadt, Germany). Moreover, oral glucose tolerance test was conducted by measuring blood glucose from a tail nick at 0, 15, 30, 60 and 120 min after oral feeding with 40 mg glucose.

Serum insulin was measured using an ELISA method (Merck, Darmstadt, Germany). Serum total cholesterol, triglycerides and LDL was measured using the Architect C16000 Clinical Chemistry Analyser (Abbott Laboratories, Ill, USA). Non-esterified fatty acids (NEFA) were measured using a WAKO kit (Osaka, Japan).

Serum insulin, GLP-1 and IL-6 levels were determined by using an ELISA method (Millipore, Bellerica, MA, USA). The assessment of insulin resistance, the homeostasis model assessment of insulin resistance (HOMA-IR), was calculated from the following formula: FBG (mmol/L) x fasting serum insulin (μIU/mL) / 22.5.