多糖蛋白复合物GM2的多层次结构与抗肿瘤活性研究

项目名称： 多糖蛋白复合物GM2的多层次结构与抗肿瘤活性研究

项目编号： No.30800193

项目类型： 青年科学基金项目

立项/批准年度： 2009

项目学科： 化学工业

项目作者： 彭燕飞

作者单位： 中国海洋大学

项目金额： 20万元

中文摘要： 多糖蛋白复合物是药用真菌中非常重要的抗肿瘤活性成分，但由于结构的复杂性，其构效关系研究并不完善。本项从紫芝发酵产物中分离纯化出一系列多糖及其蛋白复合物，并进行了化学结构表征和抗肿瘤活性筛选。研究结果表明：（1）90%饱和度的硫酸铵盐析可以去除胞外培养液和菌丝体PBS室温提取液中的绝大部分游离蛋白；菌丝体PBS的40度和80度提取物经DEAE–epharose柱层析后均获得四个组分；其中组分I和组分II为多糖组分，分子量均高达百万；组分III和组分IV分子量不到两万，分别含有0.6%和3.2%的结合蛋白。（2）羧基甲脂化是非常有效的通过MALDI-TOF MS检测含唾液酸聚糖的方法，通过一次衍生化即可同时在阳离子模式下检测所酶解下来的中性聚糖和含唾液酸聚糖，并且对聚糖中存在的微量去垢剂和盐等杂质不敏感。（3） PBS在40度和80度的粗提取物具有非常明显的体外抑制BEL-7402肝癌细胞生长的活性；80度粗提取物经柱层析纯化后的组分对BEL-7402和HL-60有抑制作用而对SGC-803没有效果；第二个和第三个组分在极低浓度下的有效性具有进一步的研究价值。

中文关键词： 紫芝；多糖蛋白复合物；质谱；化学结构；抗肿瘤

英文摘要： Fungal protein bound polysaccharides have been found to be important antitumoral substances, however the relationship between their structure and functions are imcompletely understood due to the limited information of their complex structure. In this project, series of polysaccharide and their protein conjugates were isolated and purified from Ganoderma Sinense (G. Sinense) obtained through submerged liquid fermentation, and their chemical structures were investigated and the antitumor activities screened out. Our findings included: (1) 90% saturated (NH4)2SO4 salting out could remove most of the unbounded protein in the extracellular cultural medium of G. Sinense and the isolates from G. Sinense mycelia at room temperature by phosphate buffer saline (PBS) extraction, and a few protein bound polysaccharides were removed simutaniously. After going through a DEAE-Sepharose CL-6B column, four fractions were obtained from isolates from G. Sinense mycelia at 40oC and 80oC by PBS extraction, respectively. Both Fraction I and Fraction II were pure polysaccharides with a molecular weight of 98x104 and 203x104. Fraction III and fraction IV were protein bound polysaccharide with a molecular weight of 1.8x104, and a protein content of 0.6% and 3.2%,respectively. (2) Methylation was an efficient way to detect sialyated glycans by MALDI-TOF MS. Both neutral and sialyated glycans enzymatically released could be identified in one manipunation in the positive mode. Methylation could work even with the trace coexistance of detergent and salts. (3) The crude isolates extracted from G. Sinense mycelia at 40oC and 80oC by PBS exhibited direct cytotoxity against human hepatoma cell line BEL-7402. The purified fractions from the isolate at 80oC showed similar inhibitional efficiency against BEL-7402, but no effect on SGC-803 tumor cell. It was notable that faction II and fraction III could inhibite efficiently the proliferation of HL-60 tumor cell even at a low concentration of 31 ug/mL, which was worthy of further investigation.

英文关键词： Ganoderma Sinense; Protein bound polysaccharide; Mass spectrometry; chemical structure; antitumor