通俗环毛蚓、参环毛蚓、赤子爱胜蚓的分子鉴定及纤溶、抗凝活性对比  被引量:4

Molecular identification and comparison of anticoagulant activity and fibrinolytic activity of Pheretima vulgaris, Pheretima aspergillum and Eisenia foetida

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作  者:杨麒琳 马韫楠 杨万青[1] 汪文杰 钟宛凌 樊箫雨 杨天姿 杜守颖[1] 李鹏跃[1] YANG Qi-lin;MA Yun-nan;YANG Wan-qing;WANG Wen-jie;ZHONG Wan-ling;FAN Xiao-yu;YANG Tian-zi;DU Shou-ying;LI Peng-yue(School of Chinese Materia Medica,Beijing University of Chinese Medicine,Beijing 100029)

机构地区:[1]北京中医药大学中药学院,北京100029

出  处:《中南药学》2023年第2期427-432,共6页Central South Pharmacy

基  金:基于转录组学-蛋白组学-活性评价整合模式的地龙抗血栓有效部位研究(No.2020-JYB-ZDGG-032)。

摘  要:目的 鉴定参环毛蚓、通俗环毛蚓以及药典外研究较多的赤子爱胜蚓,再比较3种鲜体蚯蚓在两种不同提取方法下的纤溶和抗凝活性,并检测乙醇沉淀物的分子量。方法 采用DNA分子鉴定法鉴别3种蚯蚓。采用水提法提取鲜体蚯蚓蛋白,进一步采用乙醇沉淀法对水提物中的蛋白进行富集,利用纤维蛋白平板法和纤维蛋白原-凝血酶时间法(Fibg-TT法)测定纤溶活性和抗凝活性,利用SDS-聚丙烯酰胺凝胶电泳检测3种蚯蚓乙醇沉淀物的分子量。结果 以线粒体细胞色素c氧化酶亚基I(COI)基因片段为识别片段准确鉴定出了3种蚯蚓。比较3种蚯蚓水提液的抗血栓活性:参环毛蚓水提液的抗凝活性和纤溶活性最强,而赤子爱胜蚓水提液活性最弱;比较3种蚯蚓乙醇沉淀物的抗血栓活性:参环毛蚓乙醇沉淀物的纤溶活性最强,而通俗环毛蚓乙醇沉淀物的抗凝活性最强;赤子爱胜蚓的乙醇沉淀物纤溶和抗凝活性均最弱。参环毛蚓和通俗环毛蚓乙醇沉淀物的蛋白分子量主要分布在25~70 kDa,赤子爱胜蚓乙醇沉淀物的蛋白分子量主要分布在25~40 kDa。结论 药典地龙品种通俗环毛蚓和参环毛蚓的纤溶、抗凝活性远大于药典外的常用品种赤子爱胜蚓,3种蚯蚓中抗血栓活性蛋白有明显差异;乙醇沉淀法可有效富集抗血栓蛋白。Objective To indentify Pheretima aspergillum, Pheretima vulgaris and Eisenia foetida,compare the fibrinolytic and anticoagulant activities of 3 fresh earthworms by two extraction methods,and measure the molecular weights of ethanol precipitates. Methods DNA molecules were used to the 3 earthworms. Earthworm protein was extracted by water, and enriched by ethanol precipitation method.Fibrinolytic activity and anticoagulant activity were measured by fibrin plate method and fibrinogen thrombin time method. The molecular weights of ethanol precipitates from the earthworms were determined by SDS-PAGE. Results Mitochondrial COI gene fragment was used for the recognition,3 earthworms were accurately identified by molecular method. The antithrombotic activities of the 3 earthworm extracts were compared: the anticoagulant and fibrinolytic activities of Pheretima aspergillum were the strongest, while those of Eisenia foetida were the weakest;the antithrombotic activities of the 3 earthworm ethanol precipitates were compared. The fibrinolytic activity of Pheretima aspergillum was the strongest, the anticoagulant activity of Pheretima vulgaris was the strongest, and the fibrinolytic and anticoagulant activities of Eisenia foetida were the weakest. The protein molecular weight of ethanol precipitates of Pheretima aspergillum and Pheretima vulgaris was mainly 25 ~ 70 kDa, and that of ethanol precipitates of Eisenia foetida was mainly 25 ~ 40 kDa. Conclusion The fibrinolytic and anticoagulant effects of Pheretima aspergillum and Pheretima vulgaris in Chinese Pharmacopoeia are more active than Eisenia foetida outside Chineses Pharmacopoeia. The antithrombotic protein in the 3 earthworms is greatly different. The antithrombotic protein can be effectively enriched by ethanol precipitation.

关 键 词:通俗环毛蚓 参环毛蚓 赤子爱胜蚓 分子鉴定 抗凝活性 纤溶活性 

分 类 号:R96[医药卫生—药理学]

 

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