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红花降糖有效部位的谱效关系及其作用机理研究
武鹤婷
学位类型硕士
导师信学雷
2016-06-03
学位授予单位中国科学院大学
学位授予地点北京
学位专业有机化学
关键词糖尿病 胰岛素抵抗 内质网应激 谱效关系 红花
摘要

糖尿病是一类严重威胁人类生命健康的慢性、非传染性疾病。它以胰岛素抵抗为特征,并伴有高血糖、高血脂、高血压、动脉粥样硬化等症状。降糖、降脂、抗氧化是改善胰岛素抵抗的重要手段。红花的干燥花作为一种传统的中药材,自古以来在活血化瘀、治疗心脑血管疾病及妇科疾病中有广泛应用。在新疆它不仅重要的经济作物资源,而且也是维吾尔医药常用的药食同源植物品种,对妇科病有治疗作用,也被发现具有止痛、抗炎及治疗糖尿病的作用。本文旨在研究红花水提物的高效液相色谱指纹图谱与降糖作用药效的相关性,初步阐明其药效的物质基础;观察了红花水提物对 3T3L-1脂肪细胞、2 型糖尿病模型大鼠的降糖、改善胰岛素抵抗的作用,以及对胰岛素信号转导通路、内质网应激和 SIRT1/PGC-1α 通路各因子的影响,初步揭示红花水提物改善胰岛素抵抗的作用靶点,其结果如下:1. 初步揭示红花水提物的高效液相色谱指纹图谱与降糖作用药效的相关性红花降糖有效部位是通过对红花加热回流提取,大孔树脂分离纯化获得的,经初步的活性筛选试验证明:具有蛋白酪氨酸磷酸酶1B(PTP1B)抑制作用。为解释其药效基础,我们建立了有效部位的指纹图谱,进行谱效关系分析。灰度关联度分析表明:其PTP1B抑制活性与指纹图谱中获得的7个共有峰有关联,说明这7个峰在降糖作用中具有协同作用,关联度从大到小,顺序为P5>P6>P3>P1>P7>P4>P8。其中关联度和含量都比较高的是羟基红花黄色素A(6 号峰)和6-羟基山奈酚-3, 6-双-O-葡萄糖-7-O-葡萄糖苷(3号峰),山奈素含量也与PTP1B抑制活力相关。含量测定结果表明,新疆红花中 HSYA及山奈素含量远高于药典中的含量要求。2. 确定了红花水提物改善胰岛素抵抗的作用,并处阐明其作用机制经体外降糖抑制剂筛选模型,发现红花水提物对 PTP1B 和α-GAA 酶活具有较强的抑制作用,其 IC50 分别为 4.43 和 389.80 μg/mL。红花水提物具有协同胰岛素促进小鼠3T3-L1前脂肪细胞葡萄糖消耗的作用。急毒试验证明:红花水提物对昆明小鼠最大药量为 26.4 g/kg,药后小鼠未见明显异常。通过高糖高脂饲料喂养,辅以低剂量腹腔注射链脲佐菌(STZ)的方法,建立糖尿病大鼠模型,经高脂饲料红花水提物灌胃 4 周,发现红花水提物能够降低血糖及肝脏中游离脂肪酸的含量,提高糖尿病大鼠肝脏中己糖激酶的活力,同时促进肝糖元和肌糖原的合成。进一步的机制探索研究表明:红花水提物作用于糖尿病大鼠肝脏,通过上调胰岛素信号传导通路标志性蛋白 IR、IRS、AKT、GSK3β、Glut4的表达水平;刺激了胰岛素信号转导同时抑制了糖尿病引起的内质网应激下调了该通路中 PERK、eIF2α、IRE1等蛋白的表达,减少了 ERS而引起的胰岛β细胞凋亡;也能增加了细胞线粒体生物合成及氧化代谢的主要调节者PGC-1α 的蛋白表达水平,改善肝脏组织线粒体功能损伤,对糖尿病大鼠肝脏起到保护作用。综上所述,红花水提物具有改善糖尿病大鼠胰岛素抵抗作用,其作用机制可能是通过激活胰岛素信号转导通路,抑制肝脏内质网应激,提高肝脏中己糖激酶和线粒体生物合成,保护肝脏细胞,从而起到改善胰岛素抵抗作用。 关键词:糖尿病,胰岛素抵抗,内质网应激,谱效关系,红花。

其他摘要

Diabetes is an endocrine metabolic disease that threatens human’s health and even life,which was characterized by insulin resistance and accompanied by systemic low-grade inflammation and hyperglycemia, hyperlipidemia, hypertension, atherosclerosis and other symptoms, and mainly caused by obesity. However, anti-inflammatory, hypoglycemic, lipid-lowering and antioxidant are important means of improving insulin resistance.Carthamus tinctorius L. is widely distributed in Xinjiang China. The crop has been well-known as Uighur’s traditional food preservative as well as a remedy for the treatment of gynecological disease. It is also demonstrated that safflower exerts analgesic, anti-inflammatory and antidiabetic activities. The dried flower of the Carthamus tinctorius L. gets its extensive application in traditional Chinese medicine owning to the therapeutic effect on activating blood circulation, eliminating blood stasis and treatment for gynecological disease and cardio-cerebrovascular diseases such as coronary heart disease and hypertension. To research for the water extract of Carthamus tinctorius L. as potent PTP1B inhibitors, the correlation between chromatography fingerprint of Carthamus tinctorius L. and the PTP1B inhibition activity was elaborated and the most effective substances were indicated by this way. The purpose of our research is to observe the effect on the treatment of water extract of Carthamus tinctorius L. to T2DM, and to find the mechanism of water extract of Carthamus tinctorius L..For purpose of further explain the antidiabetic spectrum-effect relationship of the water extract of Xinjiang safflower, the inhibition on PTP1B was tested in vitro, and it was found as an potent PTP1B inhibitors with IC50 in 8.38μg/mL, which could ameliorating the insulin-dependent signaling pathway are potential candidates for the treatment and prevention of diabetes. The correlation between chromatography fingerprint of 10 different Carthamus tinctorius L. and the PTP1B inhibition activity was evaluated to found the most effective compounds in the water extraction, and the content of two marker compounds, which collected in Chinese Pharmacopoeia in Carthamus tinctorius L., were quantitative analysis by HPLC. The efficacy of PTP1B inhibition of the water extract part of Carthamus tinctorius L. resulted in its chemical compositions together. The contribution order for PTP1B inhibition effect was (number of peaks): P5>P6>P3>P1>P7>P4>P8>P2, in which the number of peak 3,6 were identified as 3-O-caffeoylquinic acid,and hydroxysafflor yellow A.In vitro hypoglycemic screening model, the water extract fraction of Carthamus tinctorius L. has strong inhibition effect on PTP1B and α-GAA with IC50 of 2.67 and 365.24 μg/mL, respectively. The water extract part of Carthamus tinctorius L. promoted glucose consumption in 3T3-L1 cells, glucose consumption reached highest in cells when incubating in 25 μg/mL for 15 h. Fed with maximum dose of 26.4 g/kg, no obvious abnormity in Kunming mice, then the water extract part of Carthamus tinctorius L. lavage 4 weeks in diabetes rats induced by feeding high sugar, high fat, diet and injection of low dose streptozotocin (STZ). The water extract part of Carthamus tinctorius L. significantly reduced the blood sugar. It promote the synthesis of glycogen in liver by improving hexokinase activity in liver, and glycogen content increased in liver and muscle of diabetic rats. In addition, the water extract part of Carthamus tinctorius L. lowered the content of free fatty acids in the liver. That means the water extract part of Carthamus tinctorius L. reduced blood sugar, and improve the oxidation activity meantime it can protect the liver from oxidative damage of diabetic rats.Collectively, the water extract part of Carthamus tinctorius L. has obvious hypoglycemic effect in diabetic rats. The possible of mechanism of hypoglycemic could be by inhibit the expression of PTP1B and ERS pathway, improve the liver hexokinase and antioxidant enzymes activity.Key words: diabetes mellitus, insulin resistance, endoplasmic reticulum stress, spectrum-effect relationship, Carthamus tinctorius L..

文献类型学位论文
条目标识符http://ir.xjipc.cas.cn/handle/365002/4561
专题资源化学研究室
作者单位中国科学院新疆理化技术研究所
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武鹤婷. 红花降糖有效部位的谱效关系及其作用机理研究[D]. 北京. 中国科学院大学,2016.
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