两色金鸡菊降血糖总黄酮有效部位及有效活性成分研究

The Coreopsis tinctoria Nutt., Asteraceae, is a medicinal plant in Xinjiang, and its flowering tops has been reported with a wide significant biological activities, especially in antihyperglycemic activity, regulating blood lipid, antihypertensive effect, and so on. However, the research on the C. tinctoria flowering tops (CTF) for antihyperglycemic activity are limited, such as the material basis of efficacy, active ingredients, and the targets for hypoglycemic effect. In order to clarify the chemical constituents for antihyperglycemic activity of effective parts or active ingredients from CTF, the objective of this study was to prepare the total flavonoids from CTF, and test its pharmacological activity in vivo and in vitro, and identify its active ingredients using column chromatographic method, HPLC-DAD-ESI/MSn, NMR, among others. Furthermore, the active ingredients and targets for hypoglycemic effect of total flavonoids were predicted using computer simulation and pattern recognition technology through the System Pharmacology research technology platform. The Systems Pharmacology technical parameters include pharmacokinetic parameters of active ingredients, namely Absorbtion(A), Distribution(D), Metabolism(M), Excretion (E), and Toxicity(T), as well as the targets of effects. In addition, the active ingredients that Systems Pharmacology selected, which were make further investigation for its antihyperglycaemic activity and confirmation the rationality of pharmacodynamic efficacy tested by experiments. Based on the findings of these studies, the antihyperglycaemic activity and the targets for hypoglycemic effect of active ingredients and effective material basis from CTF will be investigated. Therefore, the results of the present work will provide scientific support for the appropriate using of CTF for treatment of Diabetes mellitus. This work also provide a new research method and new idea for finding active ingredients and its targets from CTF for antihyperglycemic activity.

两色金鸡菊具有多样而显著的生物活性，特别是在降血糖、调血脂和降血压方面。但是，针对该植物能够有效降血糖的深层次药效物质基础、药效作用途径、有效活性成分及其作用靶标等方面的研究尚未深入进行，也不明确。本项目以两色金鸡菊总黄酮有效部位和有效活性成分为研究对象，制备具有降血糖功效的总黄酮有效部位（经体内和体外药效活性测试）；同时分离纯化和制备其中主要化学成分，并结合HPLC-PDA-ESI/MSn等检测手段对总黄酮有效部位化学成分进行分析和鉴定，获得化学成分信息；再通过系统药理学研究平台，从系统水平分析和筛选出有效活性成分，获得有效活性成分的药动学参数（吸收/分布/代谢/排泄和毒性）和作用靶标，并进行实验验证。通过本研究，对阐明两色金鸡菊中具有降血糖功效的药效物质基础、药效作用途径、有效活性成分和作用靶标等方面提供新的研究方法和研究思路；同时也为综合利用和开发新疆两色金鸡菊植物资源提供科学依据。

本课题以新疆两色金鸡菊为研究对象，对其降血糖总黄酮有效部位和有效活性成分进行了针对性研究。包括两色金鸡菊总黄酮有效部位的优化提取和制备、总黄酮中主要化学成分的定性和定量分析、总黄酮系列生物活性测试、总黄酮主要化学成分在肠道吸收特性，以及总黄酮主要化学成分对糖尿病关键酶的作用能力预测和实验验证等方面。结果表明，采用中心设计-响应面法优化提取两色金鸡菊黄酮类成分，再经大孔吸附树脂纯化和富集，可获得>50%的总黄酮有效部位。经多种分析检测手段定性和定量分析，总黄酮中含有矢车菊-3-O-β-D-吡喃葡萄糖苷、绿原酸、栎草亭-7-O-β-D-吡喃葡萄糖苷、芦丁、马里苷、异奥卡宁、奥卡宁、黄诺马苷、木犀草素、槲皮素和山萘素等化学成分；其中绿原酸、马里苷、异奥卡宁和奥卡宁等化学成分含量较高。构建糖尿病大鼠实验模型，结果表明总黄酮有效部位对糖尿病大鼠血糖和糖化血红蛋白值、胰岛素和胰高血糖素水平，肿瘤坏死因子TNF-α值，蛋白酪氨酸磷酸酶1B（PTP1B)）和二肽基肽酶IV（DPP-4）酶活性均有显著影响，说明总黄酮具有降血糖的药效作用。采用大鼠外翻肠囊模型考察了总黄酮主要化学成分在不同肠段的吸收特性。结果表明绿原酸、黄诺马苷、栎草亭-7-O-β-D-葡萄糖苷、异奥卡宁、3,5-O-二咖啡酰奎宁酸等化学成分在肠道内以被动扩散的方式进行吸收；矢车菊素-3-O-β-D-葡萄糖苷和马里苷在空肠和回肠中除了被动扩散外还存在主动运输过程。体外酶抑制能力结果显示总黄酮对α-糖苷酶、PTP1B 酶和DPP-4酶均有一定的抑制能力。利用分子对接技术，发现总黄酮中的绿原酸、栎草亭-7-O-β-D-吡喃葡萄糖苷、马里苷、异奥卡宁和奥卡宁均可与α-糖苷酶、PTP1B 酶和DPP-4酶有相互作用。经实验验证，上述5种化合物对α-糖苷酶、PTP1B和DPP-4酶均有不同程度的活性抑制作用。.基于以上研究内容和实验结果，本项目对揭示和阐明新疆两色金鸡菊中具有降血糖功效的药效物质基础、药效作用途径、有效活性成分和作用靶标等方面提供了研究基础，同时也为综合利用和开发新疆两色金鸡菊植物资源提供了科学依据。