Leech Anticoagulant Active Ingredients

Whitmania pigra Whitman, as a popularly used traditional Chinese medicine for removing blood stasis, has a great effect of anticoagulant,antiplatelet, antithrombus, the main active elements of Whitman are protein and enzyme. In this article, we tried several means, such as normal saline extracting, salting out method, gel chromatography and so on, to extract and purify the element from Whitmania pigra Whitman.Section one is about extracting methods of Whitman. Firstly, normal saline extracting methods is decided through comparing the effect anticoagulant of the 3 kinds of Whitman extracting solution.Secondly, Decide use all body of Whitman by comparing head side and tail side of its anticoagulant effect, two of them having no apparent difference.After that, the best extracting condition is decided by orthogonal test. They are 5 multiple solution, extracting 2 hours and 3 times.At last, salting out condition is decided by comparing the Whitman' s anticoagulant of extracting solutions, which come from different saturation of (NH4)2SO4.Section two is about isolating methods of Whitman's anticoagulant. Firstly, Whitman' s anticoagulant was isolated by DEAE sephadex A~50 column under the condition of 0-0. 5mol/L and pH=8. Secondly also try isolating Whitman' s anticoagulant through CM Sepharose F. F. column.Section three is the purification of the Whitman' s anticoagulant. Try several methods, such as Sepharose 6 Fast Flow column and Sephacryl S-200 High Resolution column, to purificate Whitman' s anticoagulant.Section four is about qualitative determination .Through above methods we received a high purification of Whitman' s anticoagulant.