201012-1335嗯嗯呃(2)

嗯嗯呃

- 2 - 中国科技论文在线

1 Materials and methods

1.1 Reagents The commercial kits of blood glucose (Beijing zhongsheng Biotech Institute, Beijing, China), total cholesterol (TC), triglyceride (TG), low density lipoprotein-cholesterol (LDL-C) and high density lipoprotein-cholesterol (HDL-C), superoxide dismutase (SOD), maleic dialdehyde (MDA) were purchased from the Nanjing Jiancheng Bioengineering Institute (Jiangsu, China). Alloxan, streptozotocin (Sigma Chemical Co., USA). All other chemicals were of analytical grade.

1.2 Plant material

The entire parts of Potentilla chinesis Ser. were collected in Jixian county, Tianjin City, P.R. China and identified by Prof. Ye Zhou, Department of Botany, School of Pharmaceutical Sciences, Tianjin Medical University. A voucher specimen (No. D2*******) was deposited at the School of Pharmaceutical Sciences, Tianjin Medical University.

1.3 Isolation and identification

Air-dried and powdered plants of Potentilla chinesis (2.7 kg) were extracted three times with 95% ethanol (27L each) for 6h at reflux. The ethanol extract (335 g) was suspended in H 2O, and then successively partitioned with petroleum ether, EtOAc, and n-BuOH, to obtain petroleum ether fraction (38 g), EtOAc fraction (54 g), and n-BuOH fraction (55 g). Based on the results of previous screening of the three extracts, the anti-diabetic active EtOAc extract was chromatographed on a silica gel column, eluted with solvents of increasing polarity [petroleum ether-EtOAc (3:1, 1:1, 1:3), EtOAc, EtOAc-MeOH (19:1, 10:1, 5:1)] to give 14 fractions (1-14). Fraction 14 was chromatographed on an RP-18 column (H 2O/MeOH gradient) to give Fraction 14.1-14.5. Fraction 14.2 was purified on a Sephadex LH-20 column (MeOH-H 2O, 1:1) to give trans-tiliroside (84.7 mg). Its structure was identified by 1H, 13C NMR, and HRMS analysis . The spectroscopic and other physical data are in full agreement with those of the reported compound in literature [9].

1.4 Animals

Male Kunming mice (20±2 g) and albino male Sprague Dawley rats (180±20 g) were purchased from the China BK Experimental Animal Center. All the animals were maintained under laboratory conditions of temperature (22 ± 2 °C), relative humidity (50 ± 5%) and a 12 h day: 12 h night cycle and were allowed food (standard pellet diet) and water ad libitum. The experimental procedures are in compliance with the National Institutes of Health Guide for Care and Use of Laboratory Animals.

1.5 Preparation of diabetic animals and treatments

1.5.1 Alloxan -induced diabetic mice

Experimental diabetes were induced by the single intervenous injection of alloxan mono- hydrate (70 mg/kg bw) after overnight fasting for 12 h. Alloxan was first weighed individually in Eppendorf’s tube for each animal according to the weight and then solubilized with saline just prior to injection. The mice of control group were injected with normal saline only. 72 hours after alloxan injection, blood was collected from the eyes (venous pool). Blood glucose levels were measured using GOD-POD glucose estimation kit and animals with blood glucose greater than 11.1 mmol/L were used for the experiment. Treatment with tiliroside was started 72 h after alloxan