宽体金线蛭有效成分分子量分布和纯化工艺考察

Molecular Weight Distribution on Active Ingredients and Purification Process of Whitmania pigra Whitman

目的:采用不同方法对宽体金线蛭活性溶液进行不同分子量的含量测定和物质分离,获得最优条件。方法:采用不同分子通透性的透析膜对宽体金线蛭活性溶液进行分子量的含量测定。再分别采用大孔树脂、Q Sepharose FF、SP Sepharose FF对宽体金线蛭活性溶液进行纯化。通过考察不同纯化条件以达到分离有效物质的目的,并结合体外抗凝实验对最佳组分进行活性检测。结果:分别采用500 Da、1000 Da、3000 Da的透析膜对活性溶液进行分子量含量测定,得出<500 Da的物质占37.6%;500 Da^1000 Da的物质占到18.5%;1000 Da^3000 Da的物质占28.1%;>3000 Da物质占到15.7%。采用大孔树脂DA201-C处理得出50%乙醇洗脱液具有很好的活性。再采用Q Sepharose FF、SP Sepharose FF分别对宽体金线蛭活性溶液进行处理,采用不同洗脱剂进行洗脱然后进行活性验证得出1.0%Na Cl的p H值=8.5的Tris缓冲溶液洗脱组分活性比较强。结论:采用不同方法对宽体金线蛭活性溶液进行研究,获得较纯的活性组分,为后期的继续研究做好基础。

Objective：By using different methods to get determination of different molecular weight and material separation of Whitmania pigra Whitman active solution and obtain the optimal condition. Methods：We carried out the content determination of the molecular weight for Whitmania pigra Whitman macroscopic resin, Q Sepharose FF and SP Sepharose FF respectively. The ac- tive solution of Wh#rnania pigra Whitman was isolated by components and tested in vitro by anticoagulant assay. By examining the different conditions, we achieved the separation of the active substances. Results ： The molecular weight of active solution was deter- mined by dialysis membrane of 500 Da, 1000 Da and 3000 Da. The accounted substances of 500 Da was 37.6% ;that of 3000 Da was 28.1% ;that of more than 3000 Da was 15.7 %. The accounted material of 500 Da - 1000 Da was 18.6%. The results showed that the 50% ethanol solution with DA201 - C had good activity. And then Q Sepharose FF and SP Sepharose FF were used to treat the active solution and the solution was carried out with different solutions. The obtained activity of 1.0% NaC1 in Tris buffer solution of Q Sepharose FF was relatively strong. Conclusion： Whitmania pigra Whitman activate solution, studied by different methods, had purificated activated components, providing the basis for the later research.