【作者】 吴环；

【导师】 沈业寿；

【作者基本信息】 安徽大学 ， 生物化学与分子生物学， 2006， 硕士

【摘要】 本文以AMP-2为对象，研究确定其分离纯化的最佳方法及条件、化学组成、降血糖活性的鉴定及有效成份的确定。 针对AMP-2的主要成份为多糖和黄酮类，分别采用了分子筛分离法和大孔树脂分离法。研究两种分离方法的最适洗脱参数。结果表明分子筛法分离AMP-2的最佳参数条件为水洗、0.32mL／min、1管／12分钟；而大孔树脂分离法的最佳参数条件为径高比1：10、溶剂浓度60％、洗脱体积200mL。分子筛法分离AMP-2得到两个组份AMP-2-1和AMP-2-2，经圆盘状聚丙烯酰胺凝胶电泳和FPLC实验证实是均一组份；大孔树脂法在最佳分离条件下得到水洗部分和60％乙醇洗脱两个组份，水洗组份经过Sephadex G-75验证就是AMP-2-1，醇洗获得组份命名为AMP-2-2′，凝胶电泳法和FPLC法证实其为非均一组份。因此比较确定采用分子筛法分离AMP-2。 用HPLC法测得AMP-2-1、AMP-2-2的分子量分别为4.2万和0.8万。紫外光谱、颜色及荧光鉴定反应均证实AMP-2主要由多糖和黄酮苷组成。定量测得AMP-2的多糖含量为11.04％、黄酮含量为3.6％、蛋白质含量为2.1％、不含糖醛酸；AMP-2-1多糖含量为58.5％，不含蛋白质、黄酮和糖醛酸；AMP-2-2多糖含量为15.44％、黄酮含量为4.5％，无蛋白质和糖醛酸。同时检测AMP-2内的水和水不溶成份的含量，分别为12.43％和12.5％。 Gc结果表明AMP-2-1的单糖组成为Rha、Fuc、Ara、Xyl、Man、Glc、Gal，各单糖的摩尔比为Rha：Fuc：Ara：Xyl：Man：Glc：Gal为6.6：1：9.2：1：5.3：120：12.1；AMP-2-2中单糖组成则为Axa、Xyl、Man、Glc和Gal，各单糖摩尔比为Ara：Xyl：Man：Glc：Gal=1：14：7.9：41：6.7。其中葡萄糖含量与最低含量单糖的比值AMP-2-1中高达120：AMP-2-2中为41。由此可见葡萄糖是AMP-2-1和AMP-2-2糖链中的主要组成部分。 降血糖试验结果证明AMP-2能使腹腔注射葡萄糖的受试小鼠血糖值在2h内降至正常值；AMP-2对四氧嘧啶所致的糖尿病小鼠有显著的降血糖作用。更多还原

【Abstract】 This article use AMP-2( Amillaria mellea fruiting body is fragmented and then be hotted by water of 80℃ to extract carbohydrate) .And it invesgates the methods of isolated and purified AMP-2, these involve the main chemical component、decrease the blood sugar and the active site.On the basis of two majior chemical component flavone and carbohydrate of AMP-2, we can use methods of one of component.So I choose to use foramen magnum resin and Sephadex to separate the components.On the same time I can find the best parameter.The result of our experiments indicate the best paramete is water washing、0.32mL/min and one tube /12 min; And the best way for foramen magnum resin is the ratio of diameter and the length of the pivor bur、 the best dentisy of solvent is 60% alcohol 、 best washing volum is 200mL.Using the substance from isolation to make a purity experiment and a experiment of decreasing blood sugar .The round polyacrylamide gel electrophoresis and the result of FPLC experiment indicate AMP-2-1, AMP-2-2 are both uniform component.While the result of foramen magnum resin indicats the water washing component and the 60% alcohol washing are not uniform.And Sephadex experiment indicates the AMP-2-1 and the water washing component are the same, and I call the 60% alcohol washing AMP-2-2’. As these we can see the Sephadex is the best way to separate AMP-2.The HPLC experiment make we know the molecular weight is that AMP-2-1 be 42 thousand and AMP-2-2 be 8 thousand. The main chemical component can be deduced by the infrared light spectrum and the ultraviolet spectrum . There are carbohydrate and some small aglycone . Polysaccharide content can be detected about 11.04% by the way of Phenol and sulfuric acid. Flavone content can be deteced 4.5% ultraviolet spectrophometer. In addition we also measure the water ratio of AMP-2 is 12.43%. And the GC indicate that the corformation monosaccharide of AMP-2-1 is Rha、 Fuc、 Ara、 Xyl、 Man、 Glc, the corformation更多还原