基于电化学模拟技术的大川芎方治疗偏头痛的药效物质基础及作用机制研究

Migraine is a common chronic neurovascular disease that has seriously harm human health. The famous prescription Dachuanxiong formula (DCXF) plays an important role on the treatment of migraine in clinic. However, its efficacy substance is not yet clear. The preliminary works of applicant have shown that DCXF can significantly improve the effect of migraine model rats induced by nitroglycerin. Benzyl alcohol esters would convert to gastrodin and citric acid in vivo, and alkyl phthalides would convert to hydroxyl phthalides in vivo. What’s more, there are some differences in the amount and content of ingredients in the extract, blood plasma, and brain tissues of DCXF, which are manifested as multi-source normalization. In addition, the applicant have successfully simulate the metabolism of ligustilide and other components and isolated the product by using an electrochemical system. Therefore, this study will continue to identify drug-derived ingredients (prototypes and metabolites) in vivo, and study the regularity of metabolism, transmission and change between “in vitro-in vivo” components. Then, the metabolism and metabolites of DCXF will be simulated and separated by using the electrochemical system, and the prototype components will be prepared by modern separation techniques directly. Moreover, the mechanism and activity evaluation of prototypes and metabolites will be clarified clearly. The “component identification-isolation and identification-activity evaluation” strategy will be established. This study will clarify effective substance and its mechanism of DCXF for treating of migraine, which will lay foundation and scientific basis for discovering effective substances in vivo and developing new drugs.

偏头痛是常见的慢性神经血管性疾病，已严重危害人类健康。经典名方大川芎方在临床上治疗偏头痛效果显著，但其药效物质尚不明确。申请者前期工作显示，大川芎方能明显改善硝酸甘油致偏头痛模型大鼠作用，其苄醇酯类在体内转化为天麻素和柠檬酸，其烷基苯酞类在体内转化为羟基苯酞类，其药材、血浆、脑组织中的成分在数量及含量上存在差异，表现为多源归一性。此外，申请者成功利用电化学系统模拟藁本内酯等成分的代谢，并分离制备得到产物。本项目将继续从体内药源性成分（原型成分和代谢产物）识别入手，研究“体外-体内”成分之间的代谢、传递和变化规律，并利用电化学系统模拟其代谢及分离制备代谢产物，结合现代分离制备技术定向高效制备原型成分，在此基础上，展开活性评价和作用机制研究，建立“成分识别-分离鉴定-活性评价-作用机制”策略，阐明大川芎方治疗偏头痛的药效物质基础及作用机制，为发现体内药效物质及研发新药奠定基础和提供科学依据。

大川芎方治疗偏头痛效果显著，辨识真实存在于体内血浆和靶器官中的药源性成分（原型成分及代谢产物）以及突破药源性成分制备，对阐明其药效物质及作用机制具有理论和应用价值。本项目进展如下：①利用代谢组学差异识别大川芎方入血药源性成分有179个（原型成分42个、代谢产物137个），而脑组织中仅发现29个（原型成分12个，代谢产物17个），主要为酚酸类、羟基苯酞类及代谢产物。②通过HPLC-QQQ-MS方法定量测定体内血浆中原型成分暴露量，明确了阿魏酸、洋A、藁本内酯、正丁基苯酞、洋I暴露量较高，基于半定量法发现羟基苯酞类（如洋川芎内酯F、B和洋川芎内酯I/J的硫酸化、N-乙酰化半胱氨酸、谷胱甘肽结合物等）、酚酸类（如阿魏酸、香草酸及硫酸化结合物）暴露量较高，而体外制剂中含量较高的成分依次为天麻素、洋川芎内酯I、阿魏酸、巴利森苷、洋川芎内酯A等。③通过“体外-体内”成分之间的代谢、传递和变化规律研究发现烷基苯酞类成分进入体内易发生氧化、还原等一相代谢转化为羟基苯酞类，进而发生半胱氨酸、谷胱甘肽结合等二相代谢，酚酸类及巴利森苷类易发生酸、碱、酶水解，且基于峰面积进一步探讨了主要药源性成分血药浓度随时间变化规律，其中羟基苯酞类化合物进入血浆后呈双峰，分别在0.25/0.75, 1/1.5h达峰，而其谷胱甘肽结合物迅速在0.25h达峰；二聚体苯酞类化合物在1.5h达峰；大部分酚酸类部分化合物进入血浆后在5min迅速达峰后迅速代谢，在12h内基本消除。④利用电化学模拟系统等方法分离制备得到38个原型成分及12个代谢产物，并实验验证了天麻素及其代谢产物、藁本内酯、欧当归内酯A等对神经和内皮细胞具有保护作用活性，且在一定程度上天麻素的氧化代谢产物活性相较天麻素高。⑤通过分子对接筛选发现大川芎方中入脑的大部分药源性成分均与抗偏头痛靶蛋白结合，为潜在效应成分，其中羟基苯酞类成分的半胱氨酸和谷胱甘肽类代谢物与靶蛋白的结合作用最强；通过网络药理学研究发现大川芎方治疗偏头痛的作用机制有神经调节、血管作用、抗炎、氨基酸代谢，涉及GABA能突触、VEGF、IL-17和TNF等14个作用通路。本项目共发表文章7篇，2篇为SCI杂志收录论文，5篇为中文核心期刊论文，协助培养硕士2名。