灵芝-黄芪双向发酵菌质多糖的分离纯化及生物活性研究

Isolation,identification and immunological activity of polysaccharides from Ganoderma-Astragalus bidirectional fermentation

本文旨在探究灵芝-黄芪双向固体发酵体系对灵芝多糖生物活性的增效作用。分别以灵芝-黄芪双向发酵菌质和未添加黄芪的灵芝菌发酵菌质为原材料提取多糖,采用热水浸提和离子交换色谱对粗多糖进行分离,得到PG-1和PG-2,添加黄芪发酵的菌质多糖中的PG-1组分相较于未添加黄芪发酵的菌质多糖中的PG-2组分增加了325%的产量。对PG-1和PG-2进行初步的结构鉴定和免疫活性、抗肿瘤活性的研究。运用排阻色谱法测定其分子量,高效液相色谱、紫外光谱和红外光谱等方法分析其单糖组成、官能团、糖苷键构型以及糖分子链链型。PG-1和PG-2都由葡萄糖醛酸、葡萄糖、木糖和阿拉伯糖4种组成,单糖组分摩尔比分别为12∶58∶21∶9和16∶41∶32∶11。分子量分别为9.2×10^4和8.9×10^4。PG-1和PG-2均由β-糖苷键连接,二者糖链间有—O—连接。PG-1和PG-2均是具有三螺旋空间构象的分子量相对均一的多糖。体外细胞实验结果显示:加入PG-1和PG-2后,巨噬细胞的增殖作用、巨噬细胞对中性红的吞噬作用、巨噬细胞的保护作用和对肿瘤细胞增殖的抑制作用都显著地提高。在细胞实验中,PG-1显示出比PG-2更强的免疫增强活性和对肿瘤细胞增殖的抑制作用,说明添加黄芪促进了灵芝菌质多糖的分泌,使灵芝多糖的免疫增强活性和抗肿瘤活性增强。

The synergistic effect of Ganoderma-Astragalus bidirectional fermentation system on the biological activity of Ganoderma lucidum polysaccharides was investigated.The polysaccharides were extracted from Ganoderma-Astragalus bidirectional fermentation products and Ganoderma fermentation productsby hot-water.The polysaccharide components of PG-1and PG-2 were separated by ion exchange chromatography.The addition of Astragalus membranaceus promoted the secretion of Ganoderma lucidum polysaccharides,and the PG-1 yield from the Ganoderma-Astragalus bidirectional fermentation products was increased by 325% compared with PG-2 from the Ganoderma fermentation products.The primary structure identification,immune activity and antitumor activity of polysaccharides were determined.The relative molecular mass of PG-1 and PG-2 were 9.2×10^4 and 8.9×10^4 which were determined by size exclusion chromatography (SEC).The monosaccharide composition,functional group,glycosidic bond configuration,and sugar chain type were analyzed by high performance liquid chromatography,ultraviolet spectroscopy,and infrared spectroscopy.Both of PG-1 and PG-2 were composed of glucuronic acid,glucose,xylose and arabinose,and the monosaccharide component molar ratio of PG-1 and PG-2 was 12 ∶ 58 ∶ 21 ∶ 9 and 16 ∶ 41 ∶ 32 ∶ 11.Both of PG-1 and PG-2 are polysaccharides linked by β-glycosidiclinkages,and containing a three-helix spatial conformation and O-glycosedes.Cell experiment results showed that PG-1 and PG-2 have a significant role in macrophage proliferation,macrophage phagocytosis of neutral red,macrophage protection,and antitumor activity which indicated that PG-1 and PG-2 have a certain degree of immune activity and antitumor activity.PG-1 showed stronger immune-enhancing activity inhibition of cell proliferation than PG-2 in cell experiments.