基于血中移行成分“谱-效”相关联的养阴通脑颗粒多组分配伍抗脑缺血再灌注损伤的药效物质基础研究

Compound prescription has good curative effect and unique advantages in the treatment of ischemic cerebrovascular disease. However, the ingredients of the compatible compound are complex, the material basis of its pharmacodynamics is not clear, and how it works is still unclear. Its pharmacodynamics may be related to the dynamic changes of fingerprint (In other words, it's related to the ingredients in the blood). Based on the preparation of multi-components of Yangyin Tongnao Granules and pharmacokinetics and pharmacodynamics of its main components in vivo, the related theories of fingerprint and pharmacodynamics will be deeply studied in this paper. Response surface method will be used to compose the Yangyin Tongnao Granules compatible prescription. The compatible formula will be used as a tool drug to treat the model of cerebral ischemia-reperfusion injury in rats. The fingerprints in vivo and in vitro of the compounding prescription will be determined. The oxidation (GSH-Px, etc.) and inflammation index (TLR-4, etc.) were measured simultaneously. By using data mining and other biological information processing technologies, the correlation between spectrum and pharmacodynamics of Yangyin Tongnao Granules compatible prescription will be studied. The optimum compatibility prescription will be obtained. The main chromatographic peaks that have strong correlation with pharmacodynamics will be obtained by separation and analysis. Finally, the pharmacodynamics of main chromatographic peaks was verified by feedback. It is conducive to clarify the principle of prescription compatibility, the optimization of prescription compatibility and the material basis of prescription pharmacodynamics from a new point of view, and to explore the mechanism and law of prescription compatibility. It also will be provided a new scientific basis for the research and development of multi-component compatibility of Yangyin Tongnao Granules.

配伍复方在治疗缺血性脑血管病上具有良好的疗效及独特的优势。然而配伍复方成分复杂、药效物质基础不明，且其如何发挥作用等目前尚不清楚。其药效的发挥可能与指纹图谱动态变化(即入血成分)有关。本项目在已完成养阴通脑颗粒多组分制备和体内主要组分药动学、药效学研究的基础上，从指纹图谱和药效等相关理论深入研究，采用响应面法配伍养阴通脑颗粒多组分，以配伍组方为工具药，制作大鼠脑缺血再灌注损伤模型，测定配伍组方体内外指纹图谱，并同步测定氧化(GSH-Px等)、炎症指标(TLR-4等)等。运用数据挖掘等多种生物信息处理技术，研究养阴通脑颗粒多组分配伍谱效间的相关性，并寻优得到得到最佳配伍组方；尝试分离分析得到与药效相关联较强的主要色谱峰，并对其药效学进行反馈验证。实现从新的角度阐明方剂配伍原理、组方配伍优化、方剂药效物质基础等，深入探讨方剂配伍作用机制及规律，为养阴通脑颗粒多组分配伍新药研发提供新的科学依据。

中药复方在治疗脑卒中上具有良好疗效及独特优势。然而配伍复方成分复杂、药效物质基础不明，且其如何发挥作用等目前尚不清楚。其药效的发挥可能与指纹图谱动态变化(即入血成分)有关。本项目通过研究养阴益气活血方药(养阴通脑颗粒)提取工艺，建立GNN数学模型，得到最佳提取工艺参数，为多目标情况下的实验和未来工业提取提供了参考和理论依据。通过研究养阴通脑方（YYTN）的指纹图谱及体外抗氧化作用，成功建立了YYTN的谱效关系，并识别得到5个活性成分密切相关峰。利用ThermoFisher Q-Exactive高分辨质谱系统，从养阴通脑颗粒(YYTNG)中共检测出黄芪甲苷、葛根素、没食子酸等240个化合物（负离子模式），其中黄酮类（37种）、酚酸类（53种）、有机酸类（57种）和氨基酸类（35种）等。通过建立大鼠大脑中动脉闭塞再灌注（MCAO/R）损伤模型，灌胃给予YYTNG，构建不同数学模型，研究不同时间点血浆的指纹图谱、抗炎水平变化情况及谱效相关性，发现色谱峰与炎症指数相关性如下：P2 >P6 >P5 >P1 >P3 >P4。灌胃给予MCAO大鼠不同配伍组方后，测定不同时间点血浆的指纹图谱及其药效指标（TNF-α、Cyt-C、T-SOD），并使用遗传神经网络进行数据模型，建立配伍组方指纹图谱与体内药效指标的谱效关系，得到各个色谱峰的贡献度。对YYTNG中的药效物质基础--芒柄花素进行大鼠MCAO/R损伤作用机制进行药理研究，发现其显著减轻神经功能缺损和脑组织病理状态，降低大鼠脑梗死体积、血浆IL-18和TNF-α炎症因子水平、IL-6和IL-1βmRNA水平，以及脑组织中p-JAK2、p-STAT3、NLRP3、ASC、cl-Caspase-1和cl-IL-1β的蛋白质水平。本项目的开展，将为YYTNG多组分配伍新药研发提供实验依据，也为阐明方剂配伍原理、组方配伍优化、方剂药效物质基础等提供思路与方法。