基于分子自组装的甘草-芫花配伍禁忌物质基础及毒性机制研究

“Incompatible” is the focus of the “Eighteen Incompatible Medicaments” in Traditional Chinese Medicine. At present, most experts in traditional Chinese medicine supported that “Incompatible” is relative, that is, the conversion between “Incompatible” and appropriate compatibility can occur under certain conditions. Glycyrrhizae Radix et Rhizoma and Daphne genkwa are one of the representative of incompatible pairs in “Eighteen Incompatible Medicaments”, which had been well studied. Unfortunately, the rational application in clinic of them was not clear yet. 18β glycyrrhizic acid from Glycyrrhizae Radix et Rhizoma reacts with some flavonoid glycosides from Daphne genkwa to form supramolecular by molecular self-assembly in our previous studies, which showed some special physical and chemical properties. It was also found that 18β glycyrrhizic acid formed a colloid complex with yuanhuacine from Daphne genkwa. Combining with literature reports, glycyrrhizic acid may also react with diterpenes from Daphne genkwa to form complexes, which increased the diterpenes’ dissolution to prompted toxicity. In this study, the compounds in Daphne genkwa were rapidly, oriented and efficiently separated by “precipitation with 18β glycyrrhizic acid” and “HPLC-MS” technique, and then the target monomers of Daphne genkwa were obtained, and then self-assembled with 18β glycyrrhizic acid to obtain the target supramolecules. These target supramolecules were taking as the research object, multiple methods were used to characterize them structures and physicochemical properties. Meanwhile, the toxicity mechanism of them were studied at the cellular and animal levels to explore the nature of solubilization and toxicity enhancement, providing scientific basis for the rational application in clinic.

“反”是中药“十八反”概念的重心，目前中医药界专家的观点倾向于认为“反”是相对的，即在一定条件下可发生配伍禁忌和适宜配伍之间的转换。甘草-芫花反药组合是“十八反”的代表性组合之一，研究的也较为充分，但是其临床应用宜忌条件尚不清楚。我们前期研究发现甘草中的18β-甘草酸和芫花中的部分黄酮苷类成分通过分子自组装生成了超分子，同时初步发现18β-甘草酸与芫花中的二萜芫花酯甲形成胶体状复合物，结合文献报道甘草酸与芫花中的二萜可能形成复合物，并促使二萜溶出增加致毒性增加，本课题首先采用“以与18β-甘草酸生成沉淀”的方法和“液质联用”技术对芫花进行快速、定向、高效分离，获得芫花目标单体，进而与18β-甘草酸发生自组装，获得目标超分子。以目标超分子为研究对象，采用多种方法进行结构和物化属性表征，同时在细胞和动物水平进行毒性评价和毒性机制研究，探索其增溶增毒本质，为临床应用宜忌条件的确定提供科学依据。

本课题首先采用“以与18β-甘草酸生成沉淀”的方法和“液质联用”技术导向分离芫花花蕾95%乙醇提取物，主要获得量大的单体化合物14个，包括10个二萜和4个黄酮类化合物。并在超分子自组装技术的指导下，制备得到芫花乙酸乙酯部位-甘草酸超分子。采用色谱、场发射扫描电子显微镜、MTT 细胞毒性评价等方法研究芫花乙酸乙酯部位单煎及其与甘草酸共煎的主要化学成分、形貌学和毒性变化，结果显示芫花乙酸乙酯部位与甘草酸共煎煮后促进毒性二萜芫花酯甲的溶出, 并使该部位形成分布均一的纳米颗粒, 有利于毒性成分吸收, 从而致毒性增强。采用UHPLC-Q-Orbitrap HRMS技术对芫花乙酸乙酯部位-甘草酸超分子中化学成分进行定性分析，解析到58个化学成分，以解析到的芫花中主要毒性成分芫花酯甲与甘草酸进行超分子模拟，通过1D/2D-NMR技术确定其结合位点，并确定该超分子的组装机理是由疏水作用驱动的，甘草酸疏水性五环三萜和芫花酯甲向内，甘草酸亲水性葡萄糖向外。为了研究芫花-甘草配伍在临床可能的应用，我们选取了临床尚难解决的乳腺癌作为研究对象，小鼠体内抑瘤实验发现，芫花乙酸乙酯部位与甘草酸形成超分子后，抗肿瘤活性明显增强。芫花乙酸乙酯部位-甘草酸超分子不仅具有明显的抑制肿瘤生长的作用，还有抑制肺转移的效果。并继续从基因和蛋白两个层面证明芫花乙酸乙酯部位-甘草酸超分子是通过调控BCL2-CASP3线粒体凋亡通路抑制肿瘤生长；通过调控CXCL1/2-S100A8/9通路抑制肿瘤生长和肺转移。该研究不仅探索了芫花-甘草反药配伍的增溶增毒本质，还对实现其毒效转化应用，确定其临床宜忌条件提供了重要参考。