罗汉果根多糖的分离纯化及免疫活性研究

Isolation,purification and immunomodulatory activity of polysaccharides from roots of Siraitia grosuenorii

目的从罗汉果Siraitia grosuenorii根中分离纯化多糖组分,初步分析其结构特征并研究其免疫调节作用。方法干燥罗汉果根经95%乙醇脱脂,用70℃水提醇沉得到粗多糖,再经Cellulose DE-52纤维素阴离子交换柱、Sephadex LH-20凝胶柱分离纯化,得到均一多糖LGP-A。采用高效凝胶渗透色谱法(high performance gel permeation chromatography,HPGPC)与PMP柱前衍生化法测定其相对分子质量和单糖组成;通过傅里叶红外光谱(FT-IR)、核磁共振波谱(NMR)、刚果红实验、扫描电子显微镜(scanning electron microscope,SEM)等方法对LGP-A的初步结构进行分析。利用CCK法、ELISA试剂盒、中性红实验对RAW264.7细胞的增殖、一氧化氮(NO)、白介素-6(interleukin-6,IL-6)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)及吞噬能力进行检测,评价LGP-A的免疫活性。结果LGP-A的相对分子质量为1.83×106,主要由半乳糖(Galp,51.23%)和阿拉伯糖(Araf,44.68%)组成。红外光谱及核磁波共振波谱对其结构分析证明,LGP-A可能主要含有T-α-L-Araf(A)、→3)-α-L-Araf-(1→(B)、→5)-α-L-Araf-(1→(C)、→3,5)-α-L-Araf-(1→(D)、→3)-β-D-Galp-(1→(E)、→3,6)-β-D-Galp-(1→(F)、→6)-β-D-Galp-(1→(G)和→3)-α-D-Galp-(1→(H)片段。质量浓度在0.625~5.0μg/mL时,LGP-A能促进RAW264.7细胞的增殖,并能明显促进细胞分泌NO、IL-6和TNF-α,提高细胞吞噬率,表明LGP-A具有显著的免疫调节活性。结论LGP-A是从罗汉果根中得到的天然均一多糖,其初步结构和活性的研究为罗汉果根资源的开发提供了一定的依据。

Objective To isolate and purify the main polysaccharide components from roots of Siraitia grosuenorii and preliminarily analyze its structural characteristics and immunomodulatory effects Methods The crude polysaccharides of S.grosvenorii roots was degreased with 95%ethanol and extracted by hot water.The Cellulose DE-52 and Sephadex LH-20 columns were used to separate and purify homogeneous polysaccharides(LGP-A).The relative molecular mass and monosaccharide composition were analyzed by high-performance gel permeation chromatography(HPGPC)and PMP pre-column derivatization,and the structures of LGP-A were preliminarily identified by FT-IR,NMR,Congo red test and scanning electron microscope(SEM).The CCK-8,ELISA kit and neutral red colorimetric assay were utilized to determine the proliferation and phagocytic ability and the release levels of IL-6,NO,and TNF-αof RAW 264.7 cells,respectively to evaluate the immunomodulatory activity of LGP-A.Results LGP-A,with molecular weight of 1.83×106,was mainly composed of galactose(Galp,51.23%)and arabinose(Araf,44.68%).The structure analysis of LGP-A by IR and NMR verified that LGP-A might mainly contained T-α-L-Araf(A),→3)-α-L-Araf-(1→(B),→5)-α-L-Araf-(1→(C),→3,5)-α-L-Araf-(1→(D),→3)-β-D-Galp-(1→(E),→3,6)-β-D-Galp-(1→(F),→6)-β-D-Galp-(1→(G),and→3)-α-D-Galp-(1→(H).At the concentration of 0.625—5.0μg/mL,LGP-A promoted the proliferation of RAW 264.7 cells,significantly induced the secretion of NO,IL-6 and TNF-α,improved the phagocytic ability,thus exerting significant immunomodulatory effects.Conclusion LGP-A is a kind of natural homogeneous polysaccharides isolated from roots of S.grosuenorii.Structural characterization and immunomodulatory activity of LGP-A provided the significant reference for the further development of root resources of S.grosuenorii.