旱莲草凝血和止血活性成分及其作用机理研究

Eclipta alba, as one of the important medicinal plants, is commonly used to cure diseases of haemorrhage. However, the procoagulant and hemostatic compounds of E. alba and the action mechanisms of these active compounds are still unclear. Our previous study found that there were abundant procoagulant and hemostatic compounds with various action mechanisms in E. alba, and some of the compounds showed more efficiency in hemostasis and lower toxicity than medicines commonly used in clinic, suggesting that these compounds showed great potential in developing new hemostatic medicine with high efficiency and low toxicity. In this project, these active compounds would be tracked by the bioassay, and separated by using bioseparation technologies. The structures of these compounds would be elucidated using spectroscopic data. And then we will study the sampling time of E. alba, and confirm the growing period of high content of special procoagulant and hemostatic compounds, and analyze the content of specific active compounds in the root, stem and leaf of E. alba. The procoagulant and hemostatic efficiency of the active compounds, the synergistic effects of these compounds and the relationships between the structures and the activities of the analogues were measured through in vitro and in vivo tests. The influence of the active compounds on the contents of blood coagulation factors, such as serozyme, platelet, fibrinogen etc, would be studied. The time of haemorrhagia and cruor, the blood capillary shrinking or not, would be also involved in this study. Through these studies above, the main procoagulant and hemostatic compounds would be identified, along with the influences of these active compounds on the functions of the intrinsic and extrinsic coagulation pathways, and the action mechanisms of the active compounds would be illustrated. This study would provide the foundation for developing new hemostatics with high efficiency and low toxicity by using the abundant source of E. alba.

旱莲草是一种重要的药用植物，常用来治疗出血性病症，但其凝血和止血活性成分及作用机理尚不明确。我们研究发现，旱莲草的凝血和止血活性成分丰富，作用机理多样，部分物质止血效果远优于临床常用药物，且毒性更低，在研制高效低毒的新型止血药物方面潜力巨大。本项目拟在前期工作基础上，采用生物活性跟踪分离纯化技术，分离凝血和止血物质，利用波谱数据解析其结构；研究旱莲草采集时间，确定特定活性成分含量较高的生长期，分析其在根茎叶中的含量；采用体内和体外试验，研究活性成分的凝血和止血效率、协同作用、及结构类似物的构效关系；研究活性成分对凝血酶原、血小板和纤维蛋白原等凝血因子含量的影响，及对出血时间、凝血时间和毛细血管是否收缩等的影响。通过以上研究，明确旱莲草的主要凝血和止血活性成分、及其对内源性和外源性凝血途径功能的影响，初步阐明其作用机理，为利用丰富的旱莲草资源研发高效低毒的新型止血药物提供前期工作基础。

旱莲草是一种重要的药用植物，常用来治疗出血性病症，但其凝血活性成分及作用机理尚不明确，限制了其在止血药物研发中的应用。项目主要研究了旱莲草预处理、活性成分提取、分离纯化、结构鉴定、凝血效率、凝血机理、黄酮类化合物硫酸酯钾合成与凝血活性。研究发现，旱莲草采集后先在通风阴处晾2-3天，再晒干或烘干，会提高活性成分含量；用水、甲醇、乙醇和乙酸乙酯混合溶剂反复浸泡提取活性成分，除去其中有机溶剂后用大孔吸附树脂吸附，先用去离子水和25%甲醇水溶液洗脱，除去大极性无活性物质，再用70%水甲醇溶液洗脱得到活性组分，除去溶剂即得到黄褐色粉末，含量约为旱莲草干重的2-5%；该组分对红细胞具有很强的凝集活性，还可特异性地絮凝纤维蛋白原；碱性物质降低其凝血活性，酸性物质增强其凝血活性；加入0.56 mmol/l的酒石酸后，活性组分在0.05 mg/ml或0.2 mg/ml时，对去除血浆的红细胞或全血中的红细胞凝集率超过90%；无酸条件下，在1.3 mg/ml或1.9 mg/ml时，活性成分对2种血样的红细胞凝集率才能达到90%；加入0.56 mmol/l的酒石酸后，在0.2 mg/ml时，活性成分对人和牛的纤维蛋白原絮凝率分别为95.9±3.6%和101.9±4.1%，比无酸条件下分别增加14.6%和59.2%；木犀草素-7-硫酸酯是旱莲草主要凝血活性成分，占其干重0.4-1%；eclalbasaponin I占旱莲草干重约0.1%，具有凝血和溶血双重活性；黄酮类化合物硫酸酯钾在酸性条件下普遍具有促凝血活性，0.6-1 mmol/l的酒石酸处理后，部分化合物在0.2 mg/ml时对全血中的红细胞凝集率超过90%，在0.3-0.4 mg/ml时对纤维蛋白原絮凝率超过90%。项目研究结果对利用旱莲草及黄酮类化合物硫酸酯盐，研制新型创伤和胃出血治疗用药具有重要的意义，同时也为新型止血药物的研发提供了大量新材料。