竹节参多糖的结构表征及体外免疫活性研究

Structural Characterization and Immune Activity In Vitro of Polysaccharides from Panax japonicus

目的:从竹节参中分离纯化均一多糖,对其进行结构表征和体外免疫活性研究。方法:采用水提醇沉法制备粗多糖,经Sevag法脱蛋白得到精制的竹节参多糖(PJPS),通过乙醇分步沉淀法、DEAE-52和Sephacryl S-400柱色谱法分离纯化得到均一多糖PPS,采用高效凝胶渗透色谱HPGPC-RID测定PPS的相对分子量,1-苯基-3-甲基-5-吡唑啉酮(PMP)柱前衍生HPLC法测定单糖组成,GC-MS分析甲基化产物,IR、一维和二维核磁等方法对PPS进行结构表征,并采用小鼠单核巨噬细胞RAW 264.7细胞株,观察了PJPS和PPS对细胞活力、NO释放、肿瘤坏死因子-α(TNF-α)和白细胞介素-6(IL-6)表达的影响。结果:PPS是一种葡聚糖,相对分子质量为3.3 kDa,PPS的主链为α-D-(1→4)-连接的葡聚糖,每5个Glcp糖基的6位与α-t-Glcp连接形成侧链。PPS能促进RAW 264.7细胞增殖,PJPS和PPS能促进细胞分泌NO、TNF-α和IL-6。结论:PPS为从竹节参中分离得到的均一多糖,具有一定的免疫活性,对PPS结构和活性的研究可为竹节参的深入开发和利用提供参考。

Objective:To isolate and purify the homogeneous polysaccharide from Panax japonicus,and to study its structure characterization and immunoactivity in vitro.Methods:The crude polysaccharides of Panax japonicus were prepared using water extraction and alcohol precipitation method.After deproteinization with Sevag method,the refined polysaccharides of Panax japonicus(PJPS)were obtained.The ethanol fractional precipitation method,DEAE-52 and Sephacryl S-400 column chromatography were used to isolate and purify homogeneous polysaccharide PPS.The relative molecular weight and monosaccharide composition of PPS were determined by high performance gel permeation chromatography(HPGFC-RID)and 1-phenyl-3-methyl-5-pyrazolone(PMP)pre-column derivatization HPLC,respectively.The methylation products were analyzed by GC-MS.IR,1D-and 2D-NMR analyses were used for structural characterization of PPS.The effects of PJPS and PPS on cell viability,release of NO,expression of tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)were observed using RAW 264.7 cell line of mouse mononuclear macrophages.Results:PPS was a glucan with a relative molecular mass of 3.3 kDa.The backbone of PPS wasα-D-(1→4)-connection of glucoside,withα-t-Glcp attached to the six position of each of the five Glcp sugar groups.PPS could promote RAW 264.7 cell proliferation.PJPS and PPS could promote the secretion of NO,TNF-αand IL-6.Conclusion:PPS is a homogeneous polysaccharide isolated from Panax japonicus with certain immunological activity.The study of the structural characterization and immune activity of PPS can provide a reference for further development and utilization of Panax japonicus.