芋头多糖的分离纯化及对细胞免疫的调节作用

Isolation, Purification and Immunoregulatory Property of Polysaccharides from Taro(Colocasia esculenta)

对自提精制芋头多糖(TPS)进行分离纯化,研究各组分的化学特征及对小鼠体外免疫细胞功能的影响。通过水提醇沉,酶法辅助Sevag法去蛋白,透析,冷冻干燥得TPS,经DEAE-52离子交换柱和SephadexG-75凝胶色谱柱分离纯化得到3种多糖组分。紫外光谱扫描(UV)和高效液相色谱法(HPLC)鉴别多糖的纯度,气相色谱法(GC)分析单糖组成及物质的量比,小鼠体外MTT实验测定脾淋巴细胞增殖活性,吞噬中性红实验测定巨噬细胞吞噬功能。结果表明:TPS多糖含量为95.21%,分离纯化后得到TPS1p、TPS2p1和TPS3p2 3种主要组分,HPLC和UV分析结果表明TPS1p和TPS2p1是单一纯品多糖,而TPS3p2可能是一种糖蛋白;GC分析结果表明3种组分均是由阿拉伯糖、甘露糖、葡萄糖和半乳糖4种单糖组成;MTT实验表明25、50、100μg/mL 3种质量浓度的TPS1p和100μg/mL的TPS2p1、TPS3p2均可较强的协同ConA促进小鼠脾淋巴细胞的增殖;吞噬实验结果表明25、50、100μg/mL质量浓度的TPS1p和50μg/mL的TPS2p1和TPS3p2均可提高小鼠巨噬细胞的吞噬能力。

The present study aimed to isolate and purify polysaccharides （TPS） from taro （Colocasia esculenta）. The chemical characteristics and immunoregulatory property of purified fractions were explored. The crude TPS was extracted with hot water and then subjected to protein removal by the enzymatic-assisted Sevag method. TPS obtained after dialysis and freeze-drying underwent further purification by DEAE-52 and Sephadex G-75 column chromatography and three homogeneous components were obtained. The purity and chemical composition were determined by UV spectroscopy and HPLC and by gas chromatography （GC）, respectively. The viability of mouse spleen cells treated with TPS fraction was determined by MTT assay and the activation of peritoneal macrophages were analyzed by neutral red phagocytosis assay. The results showed that TPS contained 95.21% carbohydrate. Three fractions TPS1p, TPS2p1 and TPS3p2 were obtained after separation and purification. Data from UV spectroscopy and HPLC analysis revealed that TPS1p and TPS2p1 were two types of homogeneous polysaccharides while TPS3p2 might be a glycoprotein. All the three homogeneous polysaccharides were made of arabinose, mannose, glucose and galactose as demonstrated by GC analysis. The MTT assays showed that at the doses of 25, 50 μg/mL and 100 μg/mL, TPS1p could function synergistically with ConA to promote the cell proliferation of spleen lymphocytes in normal mice, while this synergy was also observed for TPS2p1 only at 100 μg/mL and TPS3p2 at 50 μg/mL and 100 μg/mL. Meanwhile, TPS1p at varying doses could markedly improve the phagocytotic capacity of macrophages in mice. At 50 μg/mL, TPS2p1 and TPS3p2 also showed this activity.