2种地龙饮片不同提取法体外抗凝活性对比研究

Comparative Study on Anticoagulant Activity in Vitro of Two Kinds of Pheretima by Different Extraction Methods

目的研究酒制对地龙体外抗凝活性的影响,为地龙临床应用提供科学依据。方法分别采用传统水提法和仿生酶解提取法对生品地龙、酒制地龙进行提取,以活化部分凝血活酶时间(activated partial thromboplastin time,APTT)、凝血酶原时间(prothrombin time,PT)、凝血酶时间(thrombintime,TT)和抗凝血酶活力为指标进行抗凝活性测定,并采用BCA蛋白测定法测定可溶性蛋白、多肽含量。结果采用水提取法时,PT和抗凝血酶滴定法均显示酒地龙与生地龙抗凝血活性相当,TT测定结果显示酒地龙抗凝血活性强于生地龙,APTT测定结果则显示地龙具有促凝血活性,且在一定范围内,浓度越高,促凝效果越强,蛋白、多肽含量为酒地龙大于生地龙;采用仿生酶解提取法时,APTT和PT测定结果显示酒地龙的抗凝血活性强于生地龙,并且蛋白、多肽含量测定结果显示酒地龙要多于生地龙,但TT和抗凝血酶滴定法并未比较出生品与酒制品之间的差异。结论与传统水提法相比,仿生酶解提取法能更多地提取出蛋白多肽类成分,仿生酶解提取液具有更强的抗凝血活性,2种提取法均显示地龙酒制有利于可溶性蛋白、多肽的溶出,能增强体外抗凝血活性。

OBJECTIVE To study the effect of wine processing on the anticoagulant activity of Pheretima in vitro,and to provide scientific basis for clinical application of Pheretima.METHODS The traditional water extraction method and bionic enzymatic extraction method were used to extract the raw and wine made Pheretima.Activated partial thromboplastin time(APTT),prothrombin time(PT),thrombin time(TT)and antithrombin activity were seleced as activity indexes to evaluate the anticoagulant activity of raw and wine made Pheretima.Then BCA method was applied to measure the content of soluble protein and polypeptide.RESULTS When water extraction was used,the results of PT and antithrombin titration showed that the anticoagulant activity of raw products and wine-processed products was the same.The results of TT showed that wine-processed products had stronger anticoagulant activity than raw products.The results of APTT showed that Pheretima had procoagulant activity,and in a certain range,the higher the concentration,the stronger the procoagulant effect.The content of protein and polypeptide in wine-processed products was higher than that in raw products.When biomimetic enzymolysis extraction method was used,the results of APTT and PT showed that the anticoagulant activity of wine-processed products was stronger than raw products.The results of protein and polypeptide content showed that the content of wine-processed products was higher than that of raw products.TT method and antithrombin titration method were not compared the difference between wine-processed products and raw products.CONCLUSION Compared with the traditional water extraction method,the bionic enzymatic extraction method can extract more protein and peptide components,and the bionic enzymatic hydrolysate has stronger anticoagulant activity.The results of the two extraction methods show that the wine making of Pheretima is beneficial to the dissolution of soluble protein and polypeptide,and could enhance the anticoagulant activity in vitro.