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黑木耳多糖AAP-Ⅱa级分的制备及其生物活性的研究
Studies on Preparation and Bioactivities of the AAP-Ⅱa Fraction of Polysaccharides from Auricularia Auricula
【作者】 樊黎生；
【导师】 张声华；
【作者基本信息】 华中农业大学 ， 食品科学， 2006， 博士
【摘要】 黑木耳(Auricularia auricula)是我国重要的食用菌作物之一。黑木耳中含有大量的活性成分——黑木耳多糖。近代，由于黑木耳多糖具有重大保健价值而倍受学术界重视，本文在前人的工作基础上开展了对黑木耳多糖AAP-Ⅱa级分的特性、结构以及生物活性，包括抗菌作用、抑制肿瘤作用、抗衰老抗心肌缺血及其分子作用机理等的系统研究。主要研究结果如下： 1 黑木耳多糖AAP-Ⅱa级分的化学特性表征 将市售黑木耳干品去杂、粉碎、脱脂、浸提(超声波协同酶法)、离心或过滤、浓缩，醇沉、酶与Sevage复合法脱除蛋白质，经冷冻干燥后，即得黑木耳多糖。 黑木耳多糖的平均提取率为8.58％。采用DEAE Sephadex A-25色谱柱对脱除蛋白质后的黑木耳多糖进行分级，并用Sephadex G-200凝胶色谱柱纯化，得到黑木耳多糖AAP-Ⅱa级分，该级分得率为4.59％。并对AAP-Ⅱa级分的纯度和理化特性进行了鉴定。经过液相色谱，纸层析分析说明，AAP-Ⅱa级分是由葡萄糖、岩藻糖、甘露糖、葡萄糖醛酸和鼠李糖组成的杂多糖，摩尔比例为11.52：1.27：1.00：0.61：0.23。凝胶柱层析法测得其平均分子量为165000。AAP-Ⅱa级分中中性糖含量为58.93％，葡萄糖醛酸的含量为33.82％；蛋白质含量为1.90％；钙、镁、硒、铁元素含量分别为：3.01mg／g、2.23mg／g、23.51μg／g和1.04mg／g。采用原子力显微镜和扫描电镜观察了多糖分子的形貌。推测糖分子在固态时是产生大的岛屿状的宏观聚集，分子间分子内结合致密。 2 黑木耳多糖AAP-Ⅱa级分的抗菌效果研究 结果表明，AAP-Ⅱa级分对大多数革兰氏阳性细菌和革兰氏阴性细菌均有较强的抑菌作用，对部分酵母菌也有良好的抑制生长作用，且抑菌所需浓度低，抑菌pH值作用范围广、热稳定性强，其抗菌效果略低于苯甲酸钠，对霉菌没有明显的抑制作用。 3 黑木耳多糖AAP-Ⅱa级分对肝癌H22细胞的生长抑制作用 通过体内给药，结果显示AAP-Ⅱa级分能显著抑制肝癌H22细胞的生长，同时明显提高了荷瘤小鼠的免疫器官重量，促进小鼠淋巴细胞的增殖作用和迟发型超敏反应强度，增强免疫作用等，其作用效果与剂量呈正相关。上述结果提示，AAP-Ⅱa级分的体内抗肿瘤活性是通过增强机体的免疫活性来实现的。另外它可使荷瘤小鼠血清的SOD含量升高，MDA值降低。说明其抗肿瘤活性还与其抗氧化活性有关。
【Abstract】 The fruit bodies of Auricularia auricula is one of the most important edible fungi in our country. Auricularia auricula is rich of polysaccharides that have multiple pharmacological activities and bioactivities. Recently, Auricularia auricula polysaccharide (AAP) is being payed great attention by scientists because of its significant health protection functions. In this study, the characteristics, structure and bioactivities of Auricularia auricula polysaccharides- II a fraction (AAP- II a fraction) were investigated. Bioactivities of AAP-Ⅱ a fraction involved the effects on inhibiting microbe, tumor growth, anti-aging, acute myocardial ischemia and its molecular mechanisms. The main results are shown as follows: 1 The preparation and chemical structure analyses of AAP-Ⅱ a fractionThe fruit bodies of Auricularia auricula were broken and its powder was sieved. Firstly the lipids in it was extracted off by solvents, then the residue was extracted by water (ultrasonic wave combined with enzyme method), centrifugalized or filtered, enriched and precipitated by alcohol, the protein in it was taken off by Sevag and enzyme, and then was freeze-dried. Auricularia auricula polysaccharides (AAP) was gained.The average extraction rate of AAP was 8.58%。AAP was separated by DEAE Sephadex A-25 column. The second fraction of AAP separated by DEAE Sephadex A-25 column was purified by Sephadex G-200 column, and the AAP-Ⅱ a fraction was gained, its yield was 4.59%, and its purity and characteristics were identified.The monosaccharides in AAP-Ⅱ a fraction were identified by HPLC and paper chromatography, they are glucose, fucose, mannose, glucuronic acid and rhamnose with the molar ratio of 11.52: 1.27: 1.00: 0.61: 0.23, its molecular weight was 165000 tested by Sephadex G-200 column. The content of neutral polysaccharide in it is 58.93%, glucuronic acid is 33.82%; protein 1.90%; The content of calcium, magnesium, selenium and iron in AAP- Ⅱ a fraction are 2.23mg/g、23.51 μg/g、and 1.04mg/g, repectively.