Kunming strain mice weighing 20-22 g, Grade II, Certificate SCXK (Lu) 20080006, were purchased from the Experimental Animal Center, Shandong University, China. The mice were maintained at room temperature under alternating natural light/dark photoperiod, and had access to standard laboratory food and fresh water ad libitum. This study was performed in accordance with the Guide for the Care and Use of Laboratory Animals. Care was taken to minimize discomfort, distress, and pain to the animals.
Alloxan and adrenaline were analytical grade. Alloxan was purchased from Sigma Co., Ltd and adrenaline was purchased from Tianjin Amino acid Co., Ltd. China. Xiaoke pills were purchased from Jilin Liuhe Pharmaceutic Factory, China. Xiaoke pill is a kind of Chinese medicine used in the treatment of diabetes. It is composed of glibenclamide and several traditional Chinese herbs, including Radix Puerariae, Radix Rehmannia, Radix Astragali, Radix Trichosanthis, Corn Stigma, Fructus Schisandrae and Rhizoma Dioscoreae.
Corn silk extract (CSE) preparation
Corn silk was obtained from local market. Sample was produced by the way introduced by Velazquez . Briefly, corn silk was dried at room temperature (24.2 ± 1.0°C) and an aqueous extraction was performed by adding 100 ml boiling water to 10 g corn silk, filtering after 20 min and then lyophilizing.
Blood samples from alloxan-induced hyperglycemic mice
One hundred mice were fasted for 12 h and then injected (iv) with alloxan (75 mg/kg) dissolved in sterile saline . Forty-eight hours later, blood samples were collected from the tail veins of the mice. The blood glucose was analyzed with a Glucometer-4 (Bayer). Sixty hyperglycemic mice (the blood glucose level greater than 11.1 mmol/L) were selected and randomly divided into 6 groups. From then on, the 6 groups of mice were administered orally saline (control), Xiaoke Pill and 0.5, 1.0, 2.0 or 4.0 g/kg body wt. of corn silk extract (CSE) dissolved in the same amount of saline (experimental). The body weights of the mice were measured on the 0th day, 5th day, 10th day, 15th day and the 20th day. At the same time, after fasting the mice for 12 h on the 20th day, blood samples were obtained from the tail veins to determine the blood glucose levels. On the 45th day, blood samples were collected from the orbital veins to measure the HbA1c with the HbA1c Apparatus (Variantα, Bio-Rad Laboratories) and insulin with an enzyme-linked immunosorbant assay (ELISA) kit (Biosource, Europe) respectively . Then, the mice were sacrificed. The pancreas was dissected out and placed in 10% buffered formalin and the liver was dissected out for the measurement of hepatic glycogen. Estimation of the damaged pancreatic β-cells
The pancreatic tissues were embedded in paraffin blocks after formalin fixation
Paraffin sections were cut at 4- μm thickness and were deparaffinized in xylene twice for 5 min and then were rehydrated with the graded ethanol. The sections were examined after hematoxylin and eosin (H&E) staining .
Estimation of hepatic glycogen
The liver was homogenized in ice-cold 0.6 M HClO4. The mixture was immediately centrifuged at 3000 g for 10 min at 4°C to obtain the supernatant. Free glucose in the tissue was measured with the glucose oxidase method. Amyloglucosidase solution (10 U/ml) in 0.2 M sodium acetate buffer (pH 4.8) was then mixed and incubated in the mixture at 40°C for 2 h. After incubation, pH of the mixture was adjusted to 7 and subjected to determination of total glucose. Free glucose was subtracted from total glucose to obtain glycogen content. The glycogen was expressed as mg/g wet tissue .
Estimation of gluconeogenesis
Thirty normal mice were selected and allocated equally into 3 groups: Xiaoke Pill-treated group, CSE-treated group and saline group used as the control group. From then on, these 3 groups of mice were administered orally with Xiaoke Pill, CSE (4.0 g/kg) and saline respectively. At the end of the experimental period (15 days later), animals were fasted 12 h. After administration 1 h later, the mice were injected (s.c.) with L-alanine. The blood samples from the tail vein of the mice were collected at the 0th min and 60th min to determine blood glucose level.
Blood samples from adrenaline -induced hyperglycemic mice
Sixty healthy mice were allocated equally into 6 groups. Then these 6 groups of mice were also orally administered with saline, Xiaoke Pill and CSE (0.5, 1.0, 20. and 4.0 g/kg), respectively. On the 14th day, they were fasted over night. After administration 1 h later, animals were injected (sc) with adrenaline. Blood samples from the tail vein of the mice were collected at the 0th min and 60th min to determine blood glucose level just as above.
All data were analyzed by a one-way analysis of variance, and the differences between means were established by Duncan's multiple-range test . The data represents means and standard deviations. The significant level of 5% (p <0.05) was used as the minimum acceptable probability for the difference between the means.