桑葚多糖的分离纯化及组成分析

Separation,purification and composition analysis of polysaccharides from Mori fructus

目的探讨桑葚多糖(PMF)的分离纯化方法,研究纯化组分的组成及结构。方法首先采用DEAE-52纤维素离子交换柱进行分级洗脱纯化PMF,再用Sephadex G-100葡聚糖凝胶色谱柱进一步纯化得纯化多糖;用苯酚-硫酸法测定总糖的含量、考马斯亮蓝法测定蛋白质的含量、硫酸-间羟联苯法测定糖醛酸的含量、氯化钡-明胶比色法测定硫酸基的含量、糖腈乙酸酯衍生物GC法测定单糖的组成、分子排阻HPLC法测定纯度及分子量,采用IR法、高碘酸氧化、Smith降解分析其结构。结果经分离纯化得到PMF1、PMF2、PMF3等多糖组分;PMF1由木糖、半乳糖组成,其摩尔百分比分别为3.26%、96.74%;PMF2仅由葡萄糖组成;PMF3由鼠李糖、岩藻糖、木糖、甘露糖、葡萄糖、半乳糖组成,其摩尔百分比分别为9.31、1.76、13.09、14.18、25.49、36.17。PMF1、PMF2、PMF3的平均分子量分别为71.69、84.33、103.17 kDa,均为均一组分,具多糖的红外特征吸收峰,含糖醛酸和硫酸基,存在1→3、1→6或1→2键合吡喃己糖残基,PMF2、PMF3中存在α,β-糖苷键。结论对所纯化的桑葚多糖进行了初步的性质研究与结构分析。

OBJECTIVE To explore the methods for separation and purification of polysaccharides from Mori fructus （ PMF）, and investigate composition and structure of the polysaccharides. METHODS The crude PMF was purified by DEAE - 52 ion exchange column chromatography and further purified through a Sephadex G - 100 column to afford the purified polysaccharide. The content of total carbohydrate, protein, uronic acid and sulfuric radical were measured by the method of sulfuric acid - phenol coloration, coomass- ie brilliant blue coloration, sulfate - 3 - Phenylphenylol assay, gelatin - barium chloride assay, respectively. The composition of mono- saccharide was investigated by using the method of Sugar and acrylic acid ester derivatives of gas chromatography. The relative molecular weight was determined by HPLC - SEC. The structural characterizations of the polysaccharides were confirmed by NaIO4 oxidation, Smith degradation and IR. RESULTS Three polysaccharides of PMF1, PMF2 and PMF3 were obtained from crude polysaccharides. PMF1 was composed of xylose and galactose in a molar percent of 3.26 and 96.74, respectively, and PMF2 was only composed of glucose. PMF3 was composed of rhamnose,fucose,xylose, mannose, glucose and galactose with a molar percent of 9.31,1.76,13.09, 14.18,25.49,36.17 ,respectively. The relative molecular weight of PMFI, PMF2 and PMF3was 68.6 kDa, 80.6 kDa and 100.6 kDa, respectively. They were all homogenital components, infrared characteristic absorption peak of polysaccharides, uronic acid and sulfuric radical,branching chainpolysaccharide with 1→3,1→6 or 1→2 bonding pyran hexose residues. There was α,β -glycosidic bond in structural of PMF2 and PMF3. CONCLUSION It has been successfully carried out preliminary property and structural characterization of the polysaccharides purified from Mori fructus.