Study On Anticoagulant Peptide Components Of Leech Hydrolysate And Anticoagulant Mechanism Of Leech

The paper roots in the project "Research on Anticoagulant Active Peptides and Mechanism of Whitmania Pigra Enzymatic Hydrolysate",which is supported by the National Natural Science Foundation of China.Based on the above-mentioned research,the author further explored the anticoagulant active ingredients of the enzymolysis product of Whitmaniapigra Whitman widely used in Traditional Chinese Medicine and widely cultivated in our country.Besides,the author studied the anticoagulant mechanism of different species of Leeches.The main experimental methods and results are as follows.Nol.Research on Anticoagulant Active Peptides of Whitmania Pigra Enzymatic Hydrolysate1.The separation and anticoagulant activity of different molecular fractions from enzymatic hydrolysis of Whitmania pigraThe ultrafiltration method were used to separate the enzymolysis product of Whitmania pigra into parts of different molecules,and Thrombin Time(TT)was chosen as an activity index to measure the anticoagulant activity of different fractions.In the meantime,the methodology of everted sacs was used to investigate the absorption characteristics of the fraction of MW>10KD and the part of MW<10KD.Results:Each fractions with different molecular weight isolated by ultrafiltration method has obvious anticoagulant activity,especially for the fraction of MW>10KD.However,compared with the components of MW<10KD,the former showed poor anticoagulant activity after intestinal absorption.Conclusion:The anticoagulant active peptides in enzymatic hydrolysis of Whitmania pigra were maybe some peptides with low molecular.2.The separation and anticoagulant activity of different polar fractions from enzymatic hydrolysis of Whitmania pigraThe reverse phase chromatography was used,and Thrombin Time(TT)was chosen as an activity index to measure the anticoagulant activity of different fractions.In the meantime,the methodology of everted sacs was used to investigate the absorption characteristics of every part.Result:Each fraction of different polarity has some anticoagulant activity.The fraction with strong polarity showed better anticoagulant activity before and after intestinal absorption than the weak polarity fraction.Conclusion:A active part named LFO-1 was separated from enzymatic hydrolysis of Whitmania pigra3.The analysis of peptides from the active fraction of Whitmania Pigra Enzymatic HydrolysateLTQ-Obitrap MS was used,and the processing of data was done by UNIPROT.Result:According to the result of LTQ-Obitrap MS analysis,there were 42 peptides and 26 proteins identified because of the lack of the known information of Leeches.Conclusion:The anticoagulant active peptides in enzymatic hydrolysis of Whitmania pigra were decentralized and sophisticated,and it need further study.4.The separation of direct thrombin inhibitor from Whitmania Pigra Enzymatic HydrolysateA kind of thrombin affinity chromatography was prepared by our team.So it was applied in the screening and purification of thrombin inhibitors of Whitmania Pigra Enzymatic Hydrolysate.The activity of thrombin and anticoagulant were tested.Result:the component separated by the thrombin affinity chromatography had no activity of thrombin and anticoagulant.Conclusion:Whitmania Pigra Enzymatic Hydrolysate maybe had no effect on thrombin.No.2 The effect of Leeches on coagulation pathwayAnti-thrombin titration were applied to study the antithrombin activities of Whitmania pigra Whitman and Hirudinaria manillensis;APTT,PT,TT were determined by a clotting assay to compare the effect of the two leeches for the pathway of blood clotting.Result:The anticoagulant activity of the two leeches measured by anti-thrombin titration were shown as follow.Living Hirudinaria manillensis>Dried Hirudinaria manillensis>>Dried Whitmania pigra Whitman.Each kind of leeches could prolong APTT?PT?TT.However,living Hirudinaria manillensis mainly affected TT,and dried Hirudinaria manillensis mainly affected APTT.Dried Whitmania pigra Whitman influenced dramatically APTT and TT.Conclusion:There was a big difference on coagulation pathway between Whitmania pigra Whitman and Hirudinaria manillensisNo.2 The effect of Leeches on last coagulation pathway1.Comparative studies on anti-thrombin among the LeechesFirstly,chromogenic substrate assay was applied to test the anti-thrombin activities of Whitmania Pigra Enzymatic Hydrolysate.Secondly,chromogenic substrate assay-extraction-HPLC was applied to study the anti-thrombin activities of Whitmania pigra Whitman and Hirudinaria manillensis.Result:The anti-thrombin activities of Whitmania Pigra Enzymatic Hydrolysate could not been test because of the trypsin.The result of chromogenic substrate assay-extraction-HPLC indicated that the low dose of aqueous extract of leech promoted the thrombin activity,high dose inhibit the thrombin activity.And Hirudinaria manillensis dramatically inhibited the activity of thrombin,while Whitmania pigra Whitman showed weak anti-thrombin activity only on a higher dose.Conclusion:this was first to confirm that low dose of aqueous extract of leech promoted the thrombin activity,while high dose inhibit the thrombin activity.2.Comparative studies on fibrinogen among the LeechesThe SDS-PAGE was used to study on the effect of Leech and fibrinogen.Result:Compared with Hirudinaria manillensis,Whitmania pigra Whitman could hydrolyze fibrinogen with its polypeptide chain a and ?.Conclusion:there was a big difference on fibrinogen between Whitmania pigra Whitman and Hirudinaria manillensis.