基于与生物大分子相互作用探讨滇桂艾纳香有效成分及衍生物止血活性的分子机制

Because in normal third trimester of pregnancy, pregnant woman blood is in hypercoagulabale state, the reasons of pathologic haemorrhage in gynecology and obstetrics may be the hypofunction of coagulative fuction or the hyperfunction of anticoagulant function and fibrinolytic activity. Choosing the drugs that can active body coagulative fuction or deactive anti-coagulant function and fibrinolytic activity will be propitious to hemostasis. In our previous research, some components, in Blumea ripara (Bl.) DC., and their derivatives have a two-ways regulation of hemostasis and blood circulation promotion by removing blood stasis. However, no literature reports how the activity performs or the molecular mechanism of hemostasis. Meanwhile, the hemostasis and coagulation molecular mechanisms of other small organic compounds are not studied before. In this study, 50 components and their derivatives showing better hemostasis activities are selected. At the same time, up to three anticoagulant and coagulant enzymes are chosen according to previous experience, medical knowledge, and molecule docking technology. The interactions of enzymes and organic small molecules are investigated by biomolecular interaction analysis (BIA), isothermal titration calorimetry (ITC), circular dichroism (CD), molecular fluorescence spectrometry(MFS), and UV. With above results, ACL-TOP test, and the data of anticoagulant and coagulant, the effects of these compounds on anticoagulant and coagulant factors are analyzed and their molecular mechanisms are explored. We hope that this study can reveal the mechanism of the two-ways regulation, providing theory evidences to develop more effective hemostatic drugs used in obstetrics and gynecology.

正常妊娠晚期孕妇血液处于高凝状态,病理性妇产科出血，原因可能为体内凝血功能低下，或抗凝功能和纤溶活性亢进。选用激发体内凝血功能或抑制抗凝功能和纤溶活性的药物，有助止血。前研究发现壮族妇科药滇桂艾纳香有效成分及衍生物有止血而不瘀血的双向调节功能。其如何发挥药效、止血活性分子机制未见报道。也未见其它系列化合物止血、凝血分子机制研究报道。拟根据前期研究的结果、文献资料和分子对接等技术，筛选出50个以上止血活性良好的相关化合物和3个以上凝血、抗凝血因子；采用Biacore、ITC、 CD、MFS、UV等手段研究有效成分与相关的酶和蛋白质(生物大分子)相互作用的情况；并利用ACL-TOP测试得到的凝血、抗凝血各种数据，分析有效成分对抗凝血、凝血因子具有的激发或抑制作用，寻找靶蛋白，探讨止血分子机制。该研究有望揭秘滇桂艾纳香有效成分及衍生物的止血化瘀双向调节机理，为开发高效妇产科止血药物提供理论依据．

正常妊娠晚期孕妇血液处于高凝状态,病理性妇产科出血，原因可能为体内凝血功能低下，或抗凝功能和纤溶活性亢进。选用激发体内凝血功能或抑制抗凝功能和纤溶活性的药物，有助止血。之前的研究发现壮族妇科药滇桂艾纳香有效成分及衍生物有止血而不瘀血的双向调节功能。其如何发挥药效、止血活性分子机制未见报道。也未见其它系列化合物止血、凝血分子机制研究报道。本项目选择31种与凝血相关的酶、蛋白为靶点对拟合成的衍生物进行对接、筛选，预测可能具有止血、抗凝血活性的衍生物，根据预测的结果，设计并合成了酚酸类、黄酮类、氨基酸类衍生物259个，其中新化合物231个。进行了表面等离子共振法（SPR）测试衍生物与凝血酶和纤维蛋白原的相互作用、ACL-TOP测定凝血四项、血浆复钙（传统与改良的荧光/紫外动力学法）、血小板聚集测试、抑制凝血酶活性研究、人C型利钠肽测试、ITC/CD/UV和荧光光谱法测试衍生物与血清白蛋白/肝素的相互作用、抗氧化活性研究、抗菌活性研究和体外促软骨细胞增殖活性研究，共10种活性的测试；探讨了部分衍生物与纤维蛋白原、凝血酶、肝素、人血清白蛋白等生物大分子的相互作用情况。已发表研究论文26篇，其中SCI收录14篇，EI收录6篇，中文核心刊物6篇；获得授权中国发明专利4项。培养博后2人、博士3人、硕士10人。相关研究为开发新的止血药物提供一定的实验数据。