药用真菌马勃多糖的分离纯化及结构分析

Isolation,purification and characterization of polysaccharides from Calvatia gigantea

目的研究马勃多糖的分离纯化及基本性质。方法以马勃为材料，经热水浸提，用Sevag法脱蛋白，乙醇沉淀，经DEAE—sepharoseFF和Sephacryl S-300HR层析纯化制备多糖；采用Sephacryl S-300HR凝胶层析，200～360nm扫描和SDS—PAGE鉴定多糖的纯度，并测定多糖的表观分子质量；GC—MS分析单糖的组成和摩尔比；咔唑-硫酸法测定糖醛酸的含量；高碘酸氧化、Smith降解和红外光谱分析其结构。结果经分离纯化得到CGP—Ⅰ、CGP-Ⅱ、CGP-Ⅲ主要多糖组分，经凝胶层析检测显示单一对称峰；琼脂糖电泳显示单一多糖条带，未见蛋白染色条带，200～360nm扫描未见特征吸收峰。CGP—Ⅰ、CGP-Ⅱ和CGP-Ⅲ的表观分子质量分别为9．12×10^4、18．6×10^4、15．6×10^4。3种多糖均含有甘露糖、葡萄糖和半乳糖，CGP—HI还含有另外一种未知单糖，各自的摩尔比分别为1．22：11．28：3．92、1．74：26．45：0．36、1．06：9．9：3．29。CGP-Ⅱ和CGP-Ⅲ的糖醛酸含量分别为17．2％和20．5％，为酸性多糖，CGP—Ⅰ为中性多糖。3种多糖均为1→4连接，有少量的1→6连接。CGP— Ⅰ以α-和β-差向异构为主；CGP—Ⅱ、CGP—Ⅲ以β型差向异构为主，均主要以吡喃糖形式存在。结论对所纯化的马勃多糖进行了初步的性质研究与结构分析。

OBJECTIVE To explore the methods for purification of polysaccharides from Calvatia gigantea, and investigate the char- acteristics of the polysaccharides. METHODS Polysaccharides form Calvatia gigantea were separated and purified by hot water extraction, ethanol precipitation, and fractionation by ionexehange chromatography on DEAE - sepharose fast flow and Sephaeryl S - 300 HR chromatography. Sephacryl S - 300 HR chromatography, agarose gel eleetrophoresis, SDS - PAGE, UV spectrum were used to iden- tify the homogenicity of the polysaccharides. The apparent average molecular weight was identified by Sephacryl S - 300 HR chromatog- raphy. The component of the polysaccharides were identified by GC - MS. The content of uronic acid was identified by earbazole - sul- phuric acid analysis. The structural characterizations of the polysaccharides were confirmed by NalO4 oxidation, Smith degradation and IR. RESULTS Three water - soluble polysaccharides CGP - Ⅰ, CGP - Ⅱand CGP - Ⅲ were obtained from Calvatia gigantea. Three fractions were shown to be homogeneous by Sephacryl S -300 HR chromatography, agarose gel electrophoresis, SDS -PAGE, and UV spectrum. The apparent average molecular weight of CGP - Ⅰ,CGP - Ⅱ and CGP - Ⅲ were estimated to be 9.12 ×10^4 Da, 18.6 ×10^4 Da and 15.6 ×10^4 Da, respectively. The component of polysaccharides ：mannosc, glucose and galactose were found to be in the molar radio ofl.22 ： 11.28：3.92inCGP- Ⅰ ,1.74：26.45：0.36inCGP- 11,1.06：9.9：3.29inCGP-Ⅲ, and an unknownmon- osaccharide was found. The content of uronic acid in CGP - Ⅱ , CGP- Ⅲ was 17.2 % and 20.5% ,respectively, and there was no uronic acid in CGP - Ⅰ. The results of structural characterization showed that they were all branching chain polysaccharide with more 1→4 linking, fewer 1→6 linking. CGP - Ⅰ was neutral polysaecharide which was mainly composed of pyranose and had both β - and α - epimer, CGP - Ⅲ , CGP - Ⅲ were acidic polysaecharides which were composed of pyranose and had β - epimer. CONCLUSION The preliminary property and structural characterization of the polysaecharides purified from calvatia gigantea were investigated.