基于体内动态效应的中药苦参抑制血管新生组分间相互作用研究

Modern research suggests that different chemical component categories in Traditional Chinese medicine play a role in formation of clinical comprehensive effect through the best compatibility proportion.To clarify the interaction of pharmacokinetics and pharmacodynamics between different chemical categories of their compatibility change (including changes in the composition and proportion) is the key to reveal Chinese medicine overall function.Our preliminary study suggests that the flavonoids from Sophora flavescens are as same as its alkaloids have angiogenesis inhibition effect, and the combination of two types components does exist influence each other.Based on entirety theory of Chinese medical science, this investigation explore the compatibility rule of Sophora flavescens by taking the pharmacodynamics experiments on zebrafish in the screening of the best combination between different categories components.The efficacy substances are determined through the spectrum-effect relationship which was built by support vector machine (SVM) and LC-MS/MS.The compatibility mechanism of different categories components is clarified by compare the changes of pharmacokinetic parameters of the efficacy substances in alkaloids, flavones and the best combination.The PK-PD modeling will be built by total amount statistical moment and artificial neural network method to further clarify the scientific connotation of compatibility rule.The implement of this subject will provide the basis for comprehensive quality evaluation, new drug research and development of Sophora flavescens,and provide new research methods and ideas for the interaction of different chemical categories in Traditional Chinese medical.

现代研究认为，中药药效的发挥是多种有效组分按照最佳比例组合的结果。阐明组分间组合、量化后药效、药代的相互影响，是揭示中药整体作用的关键。我们前期的工作表明苦参黄酮成分同其生物碱一样具有血管新生抑制作用，且两类组分配伍后药效确实存在相互影响。基于中医药整体观，本项目以苦参两类有效组分为研究对象，结合整体动物药效学实验，筛选抑制血管新生最佳配伍组合，探索配伍规律；建立各配伍组指纹图谱及支持向量机（SVM）模型进行谱效关系拟合，明确药效物质及作用强弱；通过比较两类有效组分及最佳配伍中主要药效物质药动学参数变化，揭示组分间的互作机制；采用权重总量统计矩法整合药代动力学参数，人工神经网络法建立多成分整合药代动力学-药效学模型，进一步从药物与机体、药物与药效角度阐明中药苦参抑制血管新生物质基础和组分间相互作用的科学内涵，为全面评价苦参质量、研发组分新药提供依据，为中药组分间相互作用研究提供方法和思路。

基于中医药整体观，本项目以苦参两类有效组分为研究对象，从药物与机体、药物与药效角度阐明中药苦参抑制血管新生物质基础和组分间相互作用的科学内涵，主要包括如下几个方面： 一、建立了苦参生物碱、黄酮两类有效组分提取工艺、纯化工艺；并建立了溴甲酚绿苦参总生物碱及盐酸-镁粉苦参总黄酮含量测定方法；采用“一测多评”法对苦参药材、饮片、部位生物碱指标性成分含量进行了质控；采用LC-MS高分辨质谱仪，建立了苦参药材、饮片及提取部位中14种含量较大及新黄酮化合物质控方法。同时对苦参黄酮单体成分进行了研究，总计分离鉴定出化合物52个，其中黄酮类化合物40个，其中1个新化合物，5个为首次从该属或该植物中分离得到。二、建立了野生和fli1-eGFP转基因斑马鱼模型，分别对苦参生物碱、黄酮有效组分斑马鱼胚胎毒性进行了研究并确定了毒性剂量范围；考察了对斑马鱼胚胎体毛细血管形成的影响，结果显示苦参生物碱、黄酮组分均可抑制斑马鱼胚胎的血管生成。三、进行了组分药效学实验，考察了不同组对斑马鱼胚胎的影响，发现苦参生物碱、黄酮组分组合时抑制斑马鱼血管新生作用增强。四、鉴于研究三的结果，增加了对生物碱、黄酮单体成分的药效学研究，采用谱-效关系、分子对接虚拟筛选及药理实验相结合的办法开展，有效避免盲筛、假阳性和组合结果显著性不强带来的影响，同时继续在多个模型上进一步确认药效作用，开展分子生物学层面的机制探讨并准备专利申报等工作。五、增加了对苦参中结构比较有代表性的10余个化合物的体内代谢研究，总结了规律；同时采用LC-MS/MS方法对苦参组合大鼠血浆中苦参酮、降苦参酮、苦参碱、氧化苦参酮的药代动力学进行了研究。六、采用UPLC-MS的方法测定大鼠不同时间点的血浆中各药效成分浓度，根据谱效关系中单体成分对整体药效的贡献率确定权重系数，应用人工神经网络对各成分的药动学数据进行了整合，并尝试与药效学模型进行结合。