基于体内存在形式和PI3K/Akt信号通路的黄芪黄酮类成分心肌保护作用研究

Cardiovascular disease is the primary reason for the death at present. Astragali Radix is a famous qi-tonifying drug and widely used for the treatment of cardiovascular disease. Flavonoids are the main bioactive constituents in Astragali Radix, and possess a wide range of pharmacological properties including cardioprotective activity. However, pharmacokinetic studies have indicated that these constituents have poor oral bioavailability due to its extensive metabolization in vivo. Therefore, the in vivo subsistent forms of Astragalus flavonoids were significant different from those in vitro. This study aims to screen the effective forms of Astragalus flavonoids with myocardial protective activity from the in vivo subsistent forms and explore their mechanism. To clarify the in vivo subsistent forms of Astragalus flavonoids, the metabolism of six main flavonoids (calycosin-7-O-β-D-glucoside, ononin, astrapterocarpan- 7-O-β-D-glucoside, astraisoflavan-7-O-β-D-glucoside, calycosin, and formononetin) was firstly studied by HPLC-ESI-IT-TOF-MSn. And then the metabolism of total Astragalus flavonoids was studied. The original constituents were detected in vivo can be acquired by separation of the total Astragalus flavonoids. The metabolites of Astragalus flavonoids were isolated from the biological samples and the rat liver S9 incubation samples. The protective effects of the in vivo subsistent forms of Astragalus flavonoids on H9c2 myocardial injury cells induced by hypoxic and hypoglycemia were studied. The mechanism was explored based on the PI3K/Akt signaling pathway. The results provide a foundation for screening the effective forms and exploring the mechanism of Astragalus flavonoids that possess the myocardial protective activity in vivo, and promote the further study on the prevention of cardiovascular diseases.

心血管病是目前我国人口死亡的首要原因，黄芪乃补气药之长，广泛用于心血管病防治。黄酮类成分是黄芪中一类主要的活性成分，然而口服生物利用度低，体内存在形式与体外化学组成形式差别显著。因此，本项目拟从单体与总体两个层面阐明黄芪黄酮类成分的体内存在形式，从中确定心肌保护作用的显效形式，并探讨其作用机制。首先采用HPLC-ESI-IT-TOF-MSn对6种主要黄酮类成分毛蕊异黄酮苷、芒柄花苷、黄芪紫檀烷苷、黄芪异黄烷苷、毛蕊异黄酮和芒柄花素分别进行体内代谢研究，然后对总黄酮进行体内代谢研究，阐明它们的体内存在形式。从总黄酮中分离体内原形成分，从生物样品和肝S9孵育体系中分离代谢产物。建立缺氧缺糖诱导的H9c2心肌细胞损伤模型，研究体内存在形式的心肌保护作用，以PI3K/Akt信号通路为切入点，探讨作用机制。该研究结果为揭示黄芪黄酮类成分心肌保护作用的科学内涵奠定基础，促进在心血管病防治方面的研究。

本项目从黄芪黄酮类化学成分体内存在形式入手，从中寻找在体内发挥心肌保护作用的“显效形式”。首先完成了黄芪中6种主要异黄酮类单体化学成分芒柄花苷、芒柄花素、毛蕊异黄酮葡萄糖苷、毛蕊异黄酮、黄芪紫檀烷苷和黄芪异黄烷苷的体内代谢研究。在此基础上，完成了黄芪总黄酮的体内代谢研究。结果，共鉴定黄芪黄酮类化学成分体内存在形式500多种，其中新化合物300多种。因此，基本阐明了黄芪黄酮成分口服以后在体内的具体存在形式。芒柄花苷与芒柄花素在体内代谢产生了42种相同的代谢产物；毛蕊异黄酮苷与毛蕊异黄酮在体内代谢产生了36种相同的代谢产物；芒柄花苷与毛蕊异黄酮苷在体内代谢产生了27种相同的代谢产物；黄芪紫檀烷苷和黄芪异黄烷苷在体内可相互转化；芒柄花素、毛蕊异黄酮、黄芪紫檀烷和黄芪异黄烷在体内均可代谢为活性更强的大豆苷元和雌马酚。在体内存在形式表征过程中，发现1种新代谢反应，即乙基取代反应，鉴定乙基取代产物14种。首次发现毛蕊异黄酮在体内可发生糖苷化代谢反应。首次报道2种硫酸酯化反应发生在糖基上的异黄酮类代谢产物。通过分离、委托合成和购买等方式获得了33种体内存在形式对照品，对15种开展了与心肌保护作用密切相关的小鼠子宫增重作用实验研究。结果，染料木素、雌马酚、鹰嘴豆芽素、大豆苷元和染料木苷被证明具有体内雌激素样作用，推测为体内发挥心肌保护作用的“显效形式”。该项目取得的科研成果，为黄芪黄酮类成分体内存在形式研究以及黄芪黄酮类成分在体内发挥心肌保护作用的药效物质基础研究奠定了基础。