虾头类肝素的制备、理化性质及抗凝血活性评价

Preparation,Physicochemical Properties and Anticoagulant Activity of Heparinoid from Shrimp Head

本研究以虾头为原料,通过酶解、阴离子交换树脂、醇沉的方法提取并分离纯化了虾头中的类肝素;采用乙酸纤维素薄膜电泳初步鉴定了纯化后类肝素的种类,通过紫外吸收光谱、高效凝胶渗透色谱分析其纯度和分子质量,通过傅里叶变换红外光谱分析其官能团结构,采用柱前衍生化高效液相色谱分析其单糖组成;并通过体外凝血实验研究了其抗凝血活性。结果表明:组分F1.1和F1.5的类肝素质量分数均达83%,纯度较高,分子质量分别为7.8、19.8 kDa;F1.1的电泳迁移率与硫酸软骨素相似,F1.5接近肝素标准品的迁移速率;F1.1和F1.5均具有羧基、乙酰氨基、硫酸基团等吸收峰;单糖组成为不同比例的葡萄糖醛酸、艾杜糖醛酸、N-乙酰半乳糖胺、半乳糖和岩藻糖;F1.1和F1.5通过内源凝血途径和共同凝血途径发挥抗凝血作用,其中F1.5的抗凝血活性优于F1.1,可以作为潜在的抗凝血治疗剂。

In this study,heparinoid was separated and purified from an enzymatic hydrolysate of shrimp heads by anion exchange column chromatography and alcohol precipitation and was identified by cellulose acetate film electrophoresis.Its purity and molecular mass were analyzed by ultraviolet(UV)spectroscopy and high-performance gel permeation chromatography(HPGPC),its functional group structure by Fourier transform infrared spectroscopy,and its monosaccharide composition by high performance liquid chromatography(HPLC)after pre-column derivatization.Its anticoagulant activity was evaluated by in vitro coagulation test.The results showed that the heparinoid contents of fractions F1.1 and F1.5 were 83%,indicating their high purity,and their molecular masses were 7.8 and 19.8 kDa,respectively.The electrophoretic mobility of F1.1 was similar to that of chondroitin sulfate,and F1.5 was close to the heparin standard.Both had the absorption peaks of carboxyl,acetamido,and sulfuric acid groups.Monosaccharide composition analysis demonstrated that they were composed of glucuronic acid,iduronic acid,N-acetylgalactosamine,galactose and fucose in different proportions.F1.1 and F1.5 exerted a significant anticoagulant effect through the endogenous coagulation pathway and the common coagulation pathway,and F1.5 had better anticoagulant activity than F1.1 and could be used as a potential anticoagulant agent.