|Place of Conferral||北京|
|Keyword||复方金鹳草降糖有效部位 糖尿病 胰岛素抵抗 内质网应激|
复方金鹳草由丘陵老鹳草（Geranium collinum Stephan ex Willd）和糙枝金丝桃（Hypericum scabrum L.）组成，其医药用途在《中国药典》、《维吾尔药志》、《阿维森纳医典》均有记载，在民间应用中两种草药按7:3比例的混合具有治疗糖尿病的疗效，是临床疗效显著的经典验方。据查证，这两种药材按特定配比的提取物具有降糖活性，但其机理及其化学成分迄今未见详细报道。本论文通过响应面软件优化了该方的提取工艺，利用大孔树脂富集了其中的活性部位，建立了活性部位指纹图谱，测定了其活性部位主要成分的含量，通过分析谱效关系推测了其中主要降糖活性成分；观察了该复方对L6骨骼肌细胞、db/db糖尿病小鼠的降糖、改善胰岛素抵抗的作用，探索了该复方通过对内质网应激和胰岛素信号转导通路的影响，改善胰岛素抵抗作用的机制，其结果如下：1.以提取率、多酚含量、降糖活性（PTP-1B和α-葡萄糖苷酶）、抗氧化活性（ABTS和DPPH）为考察指标，利用响应面确定了制备复方提取物的最佳工艺：乙醇浓度为50%、提取温度为70 ℃、料液比为1:20 g/mL、提取时间为3 h、提取次数为3次，其模型预测值和实际得到的结果具有良好的一致性；利用大孔吸附树脂对降糖有效部位进行纯化，获得的最佳纯化工艺参数为：HPD-300型大孔吸附树脂，上样质量浓度为60 mg/mL，吸附速率为3 BV/h，上样量达到5 BV时树脂达到吸附饱和状态，洗脱时先用水洗除杂3 BV，再用30%乙醇洗脱 3 BV并收集药液，再用70%乙醇洗脱收集2 BV，合并两种不同乙醇浓度洗脱部位，得到复方金鹳草降糖有效部位（KWZ）。降糖活性检测表明，PTP-1B和α-葡萄糖苷酶的IC50分别为0.48±0.27 μg/mL和0.39±0.29 μg/mL。进行3次验证试验结果表明制备工艺可靠。2.通过标准品比对，确定了KWZ中主要的12个化合物，含量测定与谱效关系结果表明：KWZ中鉴定的12种化合物总含量达到10.67%。其中，儿茶素含量最高，达到3.12%，没食子酸含量最低，为0.12%。以药材7:3配比为参考，对其他8种不同药材配比PTP-1B降糖活性与指纹图谱中获得的25个共有峰进行灰度关联度分析，结果表明：其抑制活性与指纹图谱中获得的22个共有峰有关联，说明这22个峰在降糖作用中具有协同作用。3.利用L6骨骼肌细胞观察KWZ对其糖代谢及作用机制的影响时，结果显示：KWZ在3.125 μg/mL- 200 μg/mL浓度范围内对L6细胞无毒性作用，反而可促进细胞生长；通过对L6细胞的葡萄糖代谢实验发现，处理12 h时，6.25 μg/mL KWZ能够有效地促进L6细胞的葡萄糖消耗量；并发现它可能通过抑制GRP78蛋白的表达及激活PERK/eIF2α信号通路缓解内质网应激反应。另外，它还能显著的增加IRS-1、Akt、GSK-3β等蛋白的表达水平及可能通过AMPK途径改善糖原合成。因此，该部位可能有利于改善细胞内质网的稳定性，防止胰岛素β细胞的损伤和减轻胰岛素抵抗。4.经过4周的动物药效试验发现：KWZ显著地降低各组动物的血糖浓度和各组之间的每周体重增加；有效的改善了糖尿病db/db小鼠的OGTT和ITT的水平，增加了肝糖原的合成；同时可调节脂质代谢：有效地增加血清和肝脏中HDL-C的含量水平，降低LDL-C、TC和TG的含量水平，降低血清中游离脂肪酸和果糖的含量；它良好的抗氧化活性使它有效地改善了小鼠肝脏中抗氧化酶（SOD、CAT、MDA、GSH-PX）的异常表达状况；蛋白印记分析表明KWZ通过PERK/eIF2α与IRE1/XBP1途径能够缓解胰岛素抵抗，从而提高内质网的稳定性。本文复方金鹳草降糖有效部位的新药研发提供了支撑，为说明传统中医药在糖尿病治疗中应用的科学性提供依据。
Kursi Wufarikun Ziyabit (KWZ) was made of Geranium collinum Steph.ex Willd and Hypericum Scabrum Linn, as a classic traditional medicine (7:3 was the best proportion) used for the prevention and treatment of diabetes central Asia. It was mentioned in Avicenna’s “Canon of Medicine” and recorded in “Chinese pharmacopoeia”, “Kazakh medicine”, and widely used in folk tea drinking for its health care effect in treatment of T2DM. However, the underlying mechanism and effective substance basis of KWZ in T2DM has not been investigated extensively. In this study, the extraction conditions of effective part were optimized using response surface methodology (RSM), effective part was purificated using macroreticular resin and named KWZ, fingerprint of KWZ was established, main chemical compounds of it were identified by standard compounds, and the content of them were determined. Moreover, the hypoglycemic effect of KWZ was observed on L6 rat skeletal muscle cells and db/db T2DM mice, the effect of KWZ on the endoplasmic reticulum stress and insulin signaling pathway, which to improve the mechanism of insulin resistance were investigated. All results were showed below:1. By evluating indicated markers such as extraction yield, total polyphenolic content, antidiabetic activities (PTP-1B and α-glycosidase), and antioxidant activity (ABTS and DPPH), the extraction conditions of the prescription were optimized by RSM. The best extraction conditions for the prescription were: ethanol concentration 50 %, extraction temperature 70 ℃, solid-to-solvent ratio 1:20 g/mL, and extraction time 3 h; The best purification conditions were: macroporous resin HPD 300, sample concentration 60 mg/mL, flow rate on adsorption 3 BV/h, adsorption sample volume 5 BV, elution by 3 BV water to reduce the sugar negative reaction, elution solvent was 30 % and 70 %, 2 BV 30 % and 3 BV 70 % ethanol part were collected and combined, the purified effective part named as Kursi Wufarikun Ziyabit (KWZ). The IC50 of PTP1B and α-glycosidase were 0.48±0.27 μg/mL and 0.39±0.29 μg/mL, respectively. The results of verification test were shows that the preparation process is reliable.2. The quantitative analysis of 12 main components in the effective part of prescription was identified. The total content of the 12 components is 11.18 %, catechin is the highest content component in the sample and its content reached 3.63 %, the content of gallic acid is 0.15 %, and it is the lowest content component. The 25 common peaks were obtained from the chromatography fingerprint of 8 different proportion of the prescription through the similarity evaluation. The analysis showed that inhibition activity was related to 22 common peaks obtained in the fingerprint spectrum, indicating that these 22 peaks had synergistic effect with the hypoglycemic activity part.3. The pharmacodynamic mechanism of KWZ on L6 skeletal muscle cells was studied and the results showed that: No cytotoxicity of KWZ (3.125 μg/mL- 200 μg/mL) was found on L6 cell line by the MTT assay. The best effect of glucose consumption of cells was shown at 6.25 μg/mL after treatment for 12 hours. Expression of GRP78 protein was inhibited and PERK/eIF2α was activated by prescription KWZ in L6 myotubes. Moreover, KWZ significantly enhance the expression of IRS-1, Akt, GSK-3β, which suggest that KWZ possibly improve glycolysis through AMPK activation. Thus, KWZ might be beneficial for improving the stability of ER, preventing the damage of cells and alleviating insulin resistance.4. The results of pharmacological effects of KWZ in T2DM db/db mice showed that KWZ can significantly reduce the weekly body weight gain and decreased the fasting blood glucose, improved glucose tolerance and insulin sensitivity in db/db mice. KWZ treatment significantly decreased the serum/ liver lipid profiles such as LDL-C, TC, TG and serum free fatty acid/Fructosamine levels and increased the serum/ liver HDL-C levels. Moreover, the significantly improvement observed in glucose metabolism enzymes and antioxidant enzymes in experimental mice’s liver after KWZ treatment. The western blot result showed that KWZ significantly regulated the ER stress response through the PERK/eIF2α and IRE1/XBP1 signaling pathways, which involve in glucose metabolism and insulin resistance.This paper provides the support for the research and development of the new drug, which provides the basis for the application of traditional Chinese medicine in the treatment of diabetes.
|萨拉麦提·艾迪热斯. 复方金鹳草降糖有效部位的制备工艺及药理作用研究[D]. 北京. 中国科学院大学,2018.|
|Files in This Item:|
|复方金鹳草降糖有效部位的制备工艺及药理作（4606KB）||学位论文||开放获取||CC BY-NC-SA||Application Full Text|
|Recommend this item|
|Export to Endnote|
|Similar articles in Google Scholar|
|Similar articles in Baidu academic|
|Similar articles in Bing Scholar|
Items in the repository are protected by copyright, with all rights reserved, unless otherwise indicated.