潮间带大型绿藻中新颖结构抗凝血活性多糖及其作用机制研究

Marine green algae are important marine economic algae from China sea, and enriched in the polysaccharides. Marine green algae represent potential sources of sulfated polysaccharides with innovative structure and functional properties to be explored. In the present study, the anticoagulant-active sulfated polysaccharides with novel structure from the marine green algae in intertidal zone were investigated. Four types of marine green seaweeds from Yellow sea were chosen. On the basis of the anticoagulant activity assays, the anticoagulant-active sulfated polysaccharides were isolated using various effective chromatographic techniques, and their chemical characterizations were identified. Based on microwave-assistance treatment of the polysaccharides and effective isolation methods, the oligosaccharide fragments were obtained from the hydrolytic products. The structural characterizations of the polysaccharides and their oligosaccharides were investigated by a combination of chemical and spectroscopic methods. The fine structures of the sulfated polysaccharides were elucidated. The effects of the sulfated polysaccharides on coagulant factors were analyzed using human plasma coagulant factor system and chromogenic substrate methods. The anticoagulant properties and reactive target proteases of the sulfated polysaccharides were discovered. The mechanisms of anticoagulant activity mediated by the different sulfated polysaccharides were revealed. The desulfated and sulfated derivatives of the sulfated polysaccharides were prepared, and their anticoagulant activities were studied by the assays of the activated partial thromboplastin time, prothrombin time, thrombin time and inhibitions of coagulation factors. The relationships between the sulfation specific patterns of sulfated polysaccharides and the inhibitions of coagulant factors were elucidated. The investigation promotes the construction of the "marine polysaccharides database", and creates a good fundament for making full use of marine green seaweeds and further research of new marine drugs.

绿藻是我国重要的海洋经济海藻，蕴含着丰富的多糖物质，绿藻是寻找新颖活性多糖的潜在资源。本研究拟对海洋潮间带大型绿藻中新颖结构抗凝血多糖进行研究。从中国黄海海域潮间带大型绿藻中选择具有代表性的4种绿藻，以抗凝血活性为切入点，运用高效色谱分离方法，追踪分离绿藻中抗凝血活性多糖，探讨多糖的化学特征；通过微波辅助降解法和高效分离分析技术制备寡糖，采用现代波谱技术及化学方法的有机结合，进行多糖及其寡糖的结构研究，阐明多糖的细微结构；采用纯人缘凝血因子系统和酶学生色底物反应法，研究多糖对凝血因子的影响，探明不同结构多糖的抗凝特性和作用靶蛋白，揭示这些硫酸多糖的抗凝血作用机制，通过多糖脱硫和硫酸化衍生物及其抗凝血活性研究，阐释多糖硫酸化特定形式与抑制活化凝血因子活性之间的关系。该研究促进了“海洋糖库”的建设，对开发利用海洋大型绿藻资源和发展新型海洋药物，具有重要的借鉴价值和潜在的应用前景。

绿藻是我国重要的海洋经济海藻，蕴含着丰富的多糖物质，是备受关注的新颖活性多糖的潜在资源。本研究针对海洋潮间带大型绿藻中新颖结构抗凝血多糖进行研究，从中国黄海海域潮间带大型绿藻中选择具有代表性的4种绿藻，以抗凝血活性为切入点，运用高效色谱分离方法，追踪分离这些绿藻中抗凝血活性多糖，采用现代波谱技术及化学方法的有机结合，包括一维和二维核磁共振波谱、负离子模式下电喷雾电离质谱及碰撞诱导解离二级质谱、高效液相-质谱联用技术，对多糖的结构进行研究，探明多糖结构的新颖性，揭示多糖的细微结构。通过活化部分凝血活酶时间、凝血酶时间及凝血酶原时间对多糖的抗凝血活性进行研究，探明不同结构多糖的抗凝血活性特点，采用纯人缘凝血因子系统和酶学生色底物反应法，研究多糖对凝血因子的影响，揭示这些硫酸多糖的抗凝血作用机制，探讨分子量和硫酸基对抗凝血活性的影响。通过体内血浆纤维蛋白降解产物、D-二聚体，纤溶酶原激活物抑制剂1水平及体外血凝块溶解率，对部分多糖的纤溶和溶栓活性进行评价，并对部分多糖体内抗凝血活性进行研究。在本项目支持下，完成了4种海洋潮间带大型绿藻—袋礁膜、叉枝松藻、囊生刚毛藻和蛎菜中新颖的抗凝血活性多糖的研究，从中分离获得新颖的海洋多糖25个，在抗凝血活性方面表现出一定的潜在药用价值；发现多糖中包括了首次报道的新型鼠李糖型硫酸多糖、半乳糖型硫酸多糖、阿拉伯糖型硫酸多糖和葡萄糖型硫酸多糖。另外，获得了38个新颖的海洋寡糖。这些研究成果发表论文11篇，其中SCI收录论文8篇，包括Carbohydrate Polymers等本领域国际公认优秀期刊。申请发明专利1项。以上研究促进了我国“海洋糖库”的建设，对发现药物先导化合物，研制海洋创新药物和开发利用海洋大型绿藻资源奠定了基础。