悬钩子木多糖的分离纯化及免疫调节活性研究

Separation, purification and immunomodulatory activity of polysaccharides from Rubus sachalinensis

目的从悬钩子木中分离纯化多糖,初步分析其结构特征并进行免疫调节活性研究。方法采用热水浸提法提取悬钩子木粗多糖(Rubus sachalinensis polysaccharides,RSP),再经Cellulose DE-52纤维素离子交换柱、Sephadex G-100凝胶柱色谱进行分离纯化;利用高效凝胶渗透色谱法(HPGPC)鉴定纯度并测定相对分子质量;采用气相色谱分析其单糖组成;采用红外光谱和核磁共振波谱对其结构进行初步分析;采用CCK-8法检测其对小鼠脾淋巴细胞增殖的影响;利用ELISA试剂盒测定其对白细胞介素-2(IL-2)、γ-干扰素(IFN-γ)和肿瘤坏死因子-α(TNF-α)释放能力的影响。结果从悬钩子木中分离纯化得到RSP1-1和RSP1-22种多糖,相对分子质量分别为13227(多分散指数1.15)和9343(多分散指数1.08);2种多糖均主要含阿拉伯糖、甘露糖、葡萄糖和半乳糖,其物质的量比分别是RSP1-1(9.5∶7.0∶10.3∶18.6)和RSP1-2(5.7∶11.1∶10.3∶14.2);红外光谱及核磁波共振波谱对其结构分析证明RSP1-1可能主要含有α-1,3-Ara、β-1,4-Gal、β-1,3-Glc、β-1,3-Man片段,RSP1-2可能主要含有β-1,4-Gal、β-1,3-Glc、β-1,3-Man片段。质量浓度在5~200μg/mL时,RSP1-1、RSP1-2和RSP2均可刺激小鼠脾淋巴细胞的增殖(P<0.05),并且明显促进淋巴细胞分泌IFN-γ和TNF-α(P<0.05),质量浓度为5μg/mL时能促进淋巴细胞分泌IL-2(P<0.05)。结论RSP1-1和RSP1-2均为首次从悬钩子木中分离得到的天然来源的均一多糖。红外光谱及核磁共振波谱进一步表征了其纯度和结构特征。RSP1-1、RSP1-2和RSP2均能不同程度促进脾淋巴细胞的增殖,促进淋巴细胞分泌IL-2、IFN-γ和TNF-α,呈现一定的免疫调节活性。

Objective To extract,separate and purify homogeneous polysaccharides from Rubus sachalinensis and study on monosaccharide component and immunomodulatory activity.Methods The crude polysaccharides of Rubus sachalinensis(RSP)were extracted by hot water.The Cellulose DE-52 and Sephadex G-100 columns were used to separate and purify homogeneous polysaccharides.The relative molecular mass was analyzed by high-performance gel permeation chromatography,and the monosaccharide composition and structure were preliminarily identified by GC,IR and NMR.The effects on proliferation function of mice spleen lymphocyte proliferation were determined by CCK-8,and the effects on the release capacity of IL-2,IFN-γand TNF-αwere determined by the ELISA kit.Results Two homogeneous polysaccharides,RSP1-1 and RSP1-2,were separated and purified,with molecular weights of 13227 and 9343 determined by HPGPC.They mainly contained arabinose,mannose,glucose and galactose,with the mole ratios at 9.5∶7.0∶10.3∶18.6 and 5.7∶11.1∶10.3∶14.2,respectively.The structure of RSP1-1and RSP1-2 was analyzed by IR and NMR,and RSP1-1 might mainly containα-1,3-Ara,β-1,4Gal,β-1,6-Glc,β-1,3-Man,and RSP1-2 might mainly containβ-1,4-Gal,β-1,6-Glc,β-1,3-Man.At 5—200μg/mL,RSP1-1and RSP1-2 stimulated proliferation of spleen lymphocytes(P<0.05)and promoted lymphocytes to secrete IFN-γand TNF-α.At 5μg/mL,RSP1-1and RSP1-2 promoted lymphocytes to secrete IL-2.Conclusion RSP1-1and RSP1-2 are natural homogeneous polysaccharides that are obtained from this plants for the first time.Its purity and structure were further characterized by IR and NMR.These two homogeneous polysaccharides promoted the proliferation of splenic lymphocyte in different degrees and promoted lymphocytes to secrete IL-2,IFN-γand TNF-αthat all possessing immunomodulating activity.