The Research On Pheretima Anticoagulant Active Components

Objective:Pheretima is also known as earthworm,it is the dry body of Megascolecidae animal Pheretima asperillum(E.Perrier),Pheretima vulgaris Chen,Pheretima guillelmi(Michaelsen)or Pheretima pectinifera Michaelsen.As one of Chinese traditional medicine,it is widely used in a variety of cardiovascular diseases.It is recorded by Compendium of Materia Medica that Pheretima has the effect of promoting blood circulation to remove blood,prevention and treatment of cardiovascular and cerebrovascular,etc.Modern research shows that Pheretima has the effect of anticoagulation and fibrinolysis and improving blood circulation.This paper studied on the extraction process and property of anticoagulant active components in two kinds of Pheretima,gel chromatography and anion exchange chromatography were applied to separate the the extracts of optimal extraction conditions of Pheretima asperillum.Methods:1 With the Whitmania pigra as positive control group,the 0.9% NaCl solution as a negative control group and Tris-HCl buffer solution(pH=7.40)as a blank control,six methods were applied to compare the difference of anticoagulant activity of extracts of two kinds of Pheretima by application of the thrombin titration method: the decocting method,the 0.9% NaCl solution decocting method and the 70% ethanol reflux extraction from Pheretima and petroleum ether skimmed Pheretima,respectively.2 The gel chromatography and anion exchange chromatography separation system combined with the thrombin titration method were applied to detect the anticoagulant activity of the 0.9% NaCl solution decocting methodpetroleum ether skimmed Pheretima.The chromatography separation system included Sephadex G50 separation,DEAE Cellulose-52 separation,Sephadex G50 desalination and Sephadex LH-20 separation,and used the ninhydrin reaction and the biuret reaction for characterization of the fractions.Result:1 With the Whitmania pigra as positive control group,after compared with blank and negative control group,under the six extraction methods,two kinds of Pheretima expressed anticoagulant activity.The different extraction pretreatment and different extraction methods expressed anticoagulant activity,and after petroleum ether skimmed the anticoagulant activity was significantly enhanced.Among these six methods,both the anticoagulant activity of the 0.9% NaCl solution decocting method from petroleum ether skimmed Pheretima was strongest.2 The extract of the 0.9% NaCl solution decocting method from petroleum ether skimmed Pheretima asperillum was separated by Sephadex G50 obtained the anticoagulant activity fraction I and II.Fraction I and II was separated respectively by DEAE Cellulose-52 obtained the anticoagulant activity fraction II-2,II-3,II-4.Fraction II-2,II-3,II-4was separated respectively by Sephadex G50 desalting obtained the anticoagulant activity II-2-1,II-3-1,II-4-2.Fraction II-2-1,II-3-1,II-4-2 was finally separated respectively by Sephadex LH-20 obtained the final anticoagulant activity fraction II-2-1-2,II-3-1-1,II-4-2-2.Both the ninhydrin reaction and the biuret reaction showed positive reaction.These showed that the anticoagulant activity fraction of the fractions may be proteins or peptides.Conclusion:1 The anticoagulant activity of extracts from different kinds of Pheretima are associated with raw treatment and extraction methods of materials.The different extraction pretreatment and different extraction methods of two kinds of Pheretima expressed different anticoagulant activity,and after petroleum ether skimmed the anticoagulant activity was significantly enhanced.After petroleum ether skimmed the extracts were easier for further separation,maybe the liposoluble impurities dissolved in water were removed,and the unit volume of water could dissolve more anticoagulant active components,at the same time,petroleum ether skimmed was benifit to extracts decolorization and removing the pigment interference,and the solution was clearer.2 The extract of the 0.9% NaCl solution decocting method from petroleum ether skimmed Pheretima asperillum was separated by gel chromatography and anion exchange chromatography separation system obtained three anticoagulant activity fraction II-2-1-2,II-3-1-1,II-4-2-2,both the ninhydrin reaction and the biuret reaction of three anticoagulant activity fractions showed positive reaction that the anticoagulant activity fractions may be proteins or peptides,laid the foundation for further research on novel Pheretima anticoagulant activity drugs.