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1、重庆大学本科学生毕业设计（论文）五种抗糖尿病中药提取物的 PPARs 激动活 性研究学 生： 高 蝶学 号： 20087063指导教师： 夏之宁教授专 业： 药 学重庆大学化学化工学院二 O 一二年六月Graduation Design (Thesis) of Chongqing UniversityActivation on PPARs of extracts from five anti-diabetic traditional Chinese medicinesUndergraduate: Gao DieSupervisor: Prof.Xia ZhiningMajor: Pharmac
2、yCollege of Chemistry and Chemical EngineeringChongqing University June 2012重庆大学本科学生毕业设计（论文） 中文摘 要I摘 要本文通过研究具有抗糖尿病或利尿生津作用的中药金线莲、熟地黄、生地黄、地骨皮以及芦根的提取物对 PPARs 的两个亚型 PPAR 和 PPAR 的激动作用，探讨其抗糖尿病的作用是否可能是通过 PPARs 介导的。本文首先对五种中药进行了提取，得到其石油醚、乙酸乙酯、70%醇提、水提以及水提醇沉五个部分，然后配制不同浓度的各提取物，以 0.1% DMSO 及水作为对照组，每组作 3 个复孔的平行对
3、照，在 96 孔细胞培养板中，针对实验所用的 Hela 细胞进行了提取物细胞毒性实验，确定了五种中药各个提取物进行PPARs 研究实验的浓度范围,接着采用 Hela 细胞报告基因筛选模型，对中药的提取物进行 PPARs 的激动实验，将报告质粒和 PPAR 质粒（或 PPAR 质粒）共转染 Hela 细胞，得到大量表达 PPAR（或 PPAR）的 Hela 细胞，用不同浓度的提取物作用转染细胞，并分别用 0.1% DMSO 及水作为阴性对照组，以 0.5 g/mL的非诺贝特作 PPAR 组的阳性对照，以 0.5 g/mL 罗格列酮作 PPAR 组的阳性对照。最终进行荧光素酶活性检测，以药物作用组
4、与阴性对照组的比值为参考标准，筛选出对 PPAR（或 PPAR）有激动作用的提取物 对于 PPAR 有激动作用的包括金线莲（石油醚部分：200g/mL） 、熟地黄（石油醚部分：100g/mL）及芦根（乙酸乙酯部分：200g/mL） ，激活倍数分别为 4.93 倍、4.93 倍及 3.33 倍。五种中药对于 PPAR 均有一定激动作用，激活倍数分别为 3.69 倍、2.9 倍、2.95 倍、6.27 倍、7.48 倍（金线莲、熟地黄、生地黄、地骨皮、芦根） ；其中以芦根的乙酸乙酯部分的激动作用最为显著，激活倍数达7.48 倍。关键词：糖尿病，中药提取物，过氧化物酶体增殖物激活受体(PPARs)，
5、天然激动剂重庆大学本科学生毕业设计（论文） ABSTRACT IIABSTRACTIn the present study, the activities of the crude extracts of traditional Chinese medicines (TCMs) on PPAR and PPAR were investigated. The tested TCMs include Ancecotochilus roxburghii, Radix rehmanniae praeparata, Rehmannia glutinosa, Lycii cortex and Rhizom
6、es phragmitis, which have anti- diabetes or diursis nourish effects. Furthermore, the anti-diabetes mechanism of the tested TCMs, which may act on PPARs, is also discussed. Firstly, the five extracts of petroleum ether, ethyl acetate, 70% ethanol, water and water extraction and alcohol precipitation
7、 for each TCM were prepared. Then using a 96-well plate, the MTT toxic test was done on different concentrations, while 0.1% DMSO and water were used as control groups. Each sample repeat for three-cells and the range of preferable administration concentrations for the TCM extracts on the PPARs scre
8、ening model were chosen. At last, transfecting the report plasmid and the report plasmid with PPAR (or PPAR) expressed plasmid (screening cell model of PPAR or PPAR agonist) into Hela cells ( known as not expressed PPAR), in order to get a mass of Hela cells, which expressed PPAR plasmid (or PPAR).
9、The experiment was done with different concentrations of the TCM extracts, using 0.5 g/mL rosiglitazone and nuobei fidels as the positive drugs for PPAR and PPAR, respectively. And 0.1% DMSO and water were used as the negative control groups. Luciferase activity in the report plasmids was measured t
10、o evaluate the activation level. By compared with the result of negative control groups, the PPAR of PPAR active extracts of the tested TCMs can be found.In conclusion, Ancecotochilus roxburghiis petroleum ether extract (200 g/mL), Radix rehmanniae praeparatas petroleum ether extract (100 g/mL), Rhi
11、zoma phragmitiss ethyl acetate extract (200 g/mL) can activate PPAR with the activating multiple of 4.93 times, 4.93 times and 3.33 times, respectively. Furthermore, all the tested TCMs can activate PPAR with the activating multiple of 3.69 times, 2.9 times, 2.95 times, 6.27 times and 7.48 times for
12、 Ancecotochilus roxburghii, Radix rehmanniae praeparata, Rehmannia glutinosa, Lycii cortex and Rhizomes phragmitis extracts, repesctively. Rhizoma phragmitiss ethyl acetate extract had the strongest effect with activating multiple of 7.48 times.重庆大学本科学生毕业设计（论文） ABSTRACT IIIKey word: Diabetes; TCMs; Peroxisome proliferation activation receptors (PPARs); Natural agonists重庆大学本科学生毕业设计（论文） 目 录IV目 录中文摘要.I ABSTRACT.II 目 录.IV 1 绪论.11.1 五种中药的抗糖尿病作用研究概况.11.1.1 金线莲的抗糖尿病作用研究概况.11.1.2 熟地黄及生地黄的抗糖尿病作用研究概况.21.1.3 地骨皮的抗糖尿病作用研究概况.
