Extraction And Purification Of The Anticoagulant From Whitmania Pigra Whitman

Whitmania pigra Whitman has used as a remedy for blood stasis and traumatic injury since hundreds and thousands years. Up to now, it is a main kind of medical herb used in Traditional Chinese Medicine for promoting blood circulation by removing blood stasis, and ofen, it is used after being processed. For the past few years, it more and more widely used at medical, especially the anticoagulants purified from leec. The main anticoagulant agent is hirudin.The Whitmania pigra Whitman is widely used in Traditional Chinese Medicine and widely cultivation at our country. But the active component of Whitmania pigra Whitman was few reported and the sequence of anticoagulant substance was not reported. So for further research of the element of anticoagulant, we take Whitmania pigra Whitman as the experiment subject, separation and purified the anticoagulant and make sequence analysis. The major findings of this study are as follows:First used Whitmania pigra Whitman as material. The result showed that: When used dried Whitmania pigra Whitman as material and acetone and trichloracetic acid for the method. We had acquired 0.394g crude product for per gram and the specific activity was 1.961IU/mg. Compared with vital Whitmania pigra Whitman, the method we recommended before is the best method.Then we compared different column. When use Sephadex G-50 as column packing, the total activity coefficient of recovery was 52.17%, and purification multiple was 18.39; when used Sephadex G-75 as column packing, the total activity coefficient of recovery was 59.63%, and purification multiple was 8.29. When its absorption peak and toatal activity coefficient is similar, compared to Sephadex G-75, purification multiple of Sephadex G-50 are more higher. So, we used Sephadex G-50 as column packing. When we use DEAE-sepharose CL-6B as anion exchange chromatography column packing, the total activity coefficient of recovery and purification multiple are more higher than DEAE-cellulose DE-52. At the same time, when used DEAE-cellulose DE-52 as column packing, SDS-PAGE analysis have three protein channel. But DEAE-sepharose CL 6B purification eluate only have two band after SDS-PAGE analysis. We use Sep-pak C18 to further separate and after SDS-PAGE analysis its only one band and the molecular weight is about 14.9KD. The result of Two Dimensional Gel Electrophoresis is only one band and the isoelectric point is about 5.9. And we make analysis on protein sequence of the extraction. According to isoelectric point and protein sequence, we deduction the anticoagulant we had acquired is a new protein.The stability of the anticoagulant result showed that: the anticoagulant we geted is a stable protein. It is stable under extreme pH(1-13), high temperat ure(100℃), and in the presence of some denaturants(1%SDS, 0.1mol/l DTT and 25% trifluoroacetic acid).In 0.1% DTT at 70℃for 1h, it is partially inactivated. One condition which rapidly inactivates hirudin is the combined effect of elevated temoerature and high pH. pH12 at 80℃for 15min,it is inactivated.This research provided some data for the clinical application and rationale for drug mechanism. And we first report the amino acid of anticoagulant substances from Whitmania pigra Whitman, this provide a background for further research.