黄连解毒汤自沉淀物质基础、形成机理及其神经保护活性研究

Compounds in the form of precipitation (CFP) are universally forming during the decocting of Chinese medicine prescription, such as Huanglian Jiedu decoction. CFP are abandoned with herb residue in the hospital decocting service or separation and extraction process in pharmaceutical companies. Systematic study on the pharmacological activity and formation mechanism of CFP will be certainly beneficial on evaluating its medicinal value and raising its utilization. According to reference, the CFP might derive from the interaction between acidic components and basic components of Huanglian Jiedu decoction. Moreover, acid−base complexation’s structure and relative stereochemistry were previously identified in our previous study. In addition, our former study proved that neuroprotective effect of CFP was better than the solution of Huanglian Jiedu decoction. .Components of the CFP will be analyzed by HPLC-MSn. Based on acid−base complexation theory and reductionism science, we will synthesize a series of acid−base complexation. Moreover, structural features of acid−base complexation will be elucidated by HRMS, NMR spectroscopy, X-ray, et al. Based on the thermodynamic parameters, generation mechanism of acid−base complexation will be analyzed by isothermal titration calorimeter (ITC), such as gibbs free energy (∆G), enthalpy (∆H) and entropy (∆S). .Furthermore, CFP neuroprotective effects will be evaluated on PC12 cells, CAM model and MCAO rats. Meanwhile, CFP neuroprotective mechanisms will be investigated by morphological examination, fluorescence staining, flow cytometric analysis, immunohistochemistry technology, et al. In conclusion, we are going to reveal the pharmacological activities, structure characteristic and formation mechanism of the CFP. Our study will provide experimental evidence for further exploring medicinal value of CFP and prove it to be a new effective fraction of Huanglian Jiedu decoction.

中药复方水煎过程自沉淀现象普遍存在。传统中医临床上自沉淀多与汤药同服，但现代临床服用汤药及企业提取生产时自沉淀常随药渣弃去。复方自沉淀的药用价值、结构特点及其形成机制均有待深入研究。课题组前期成功表征酸碱络合物的立体结构，已初步利用HPLC-MSn发现黄连解毒汤自沉淀主要由酸碱类化学成分组成，且自沉淀神经保护活性优于上清液。因此，本课题拟在前期研究基础上，以络合理论和还原论为指导，利用现代谱学技术和X-ray单晶衍射方法，对黄连解毒汤自沉淀进行结构表征；以等温滴定量热法解析自沉淀形成过程中的分子热力学特征，探讨自沉淀形成机制；通过PC12细胞、鸡胚CAM和大鼠MCAO模型，研究自沉淀的神经保护活性和作用机制。研究结果对黄连解毒汤的临床合理用药和二次开发具有理论价值和实际意义，为复方“自沉淀”成为创新中药“新源头”提供依据。

研究背景：复方水煎过程中产生自沉淀的现象十分普遍，如黄连解毒汤等。传统中医临床上自沉淀多与汤药同服，但医院代煎汤药时自沉淀常随药渣弃去。复方自沉淀为何产生，自沉淀的成分组成、结构特点、形成机制如何？是否具有药用价值等问题尚无深入研究。.研究内容：本课题以黄连解毒汤水煎自沉淀为例，开展研究：（1）自沉淀活性测定：评价黄连解毒汤水煎上清、自沉淀，黄芩-黄连合煎上清、沉淀和黄芩苷-小檗碱分子络合物的神经保护活性；（2）自沉淀成分分析：结合HPLC/MS和ICP-MS解析自沉淀中有机和无机成分的组成，测定代表性成分含量；（3）自沉淀形成机制：综合波谱学、材料化学表征自沉淀的结构特点；通过拆方研究和等温滴定量热法解析自沉淀形成过程中的分子热力学特征，探讨自沉淀形成机理；（4）受启发于自沉淀中酸碱络合物结构特征，开展黄芩苷化学修饰和神经保护先导化合物发现研究。 .重要结果：（1）黄连解毒汤自沉淀由黄芩苷、小檗碱等为代表的活性成分组成，具有较好的神经保护作用，相同剂量下强于上清液；（2）自沉淀主要源自黄芩-黄连药对，分子热力学研究表明自沉淀形成是有效分子间发生焓驱动的化学结合反应；（3）自沉淀形成本质是有效成分水煎煮过程中分子相互作用，自组装形成超分子，且超分子形成后活性发生变化，初步揭示超分子是中药复方物质基础重要组成；（4）受启发自沉淀结构特征：合成新型黄芩苷类衍生物15个，获得神经保护先导化合物1个。.关键数据：已发表ACS Nano（IF=13.9）、ACS Applied Materials & Interfaces（IF 8.4）、中国中药杂志等研究论文10篇（其中6篇SCI论文），申请国家发明专利4项，已培养研究生1名，在读2人，获得省部级人才项目1个。.科学意义：本研究阐明了中药复方水煎自沉淀形成机制，揭示自沉淀形成主要是有效成分间自组装形成超分子后聚集沉降。研究结果对揭示中药复方物质基础真实形态和为超分子作为中药复方物质基础的重要组成提供依据，相关研究结果入选2019年国家自然科学基金优秀资助成果。