【作者】 廖洪梅；

【导师】 戴玲；

【作者基本信息】 安徽大学 ， 生物化学与分子生物学， 2007， 硕士

【摘要】 从丹皮中分离水溶性多糖，所得粗多糖经反复冻融、高速离心、DEAE-52离子交换和分子筛分离等方法，得级分PSM-2bA与PSM-2bB，经高压液相色谱方法证明为均一组分，经GC-MS分析，PSM-2bA的单糖组成为Rha、Ara、Xyl、Man、Glc、Gal，摩尔比依次为3.55：7.06：2.11：1.00：5.60：12.91；PSM-2bB的单糖组成为Rha、Ara、Xyl、Man、Glc、Gal，摩尔比依次为3.43：8.05：1.00：1.00：24.94：9.01。苯酚硫酸法测定了两组分的多糖含量分别为91.8％、82.6％；间羟联苯法测定糖醛酸含量分别为4.2％、15.2％；考马斯亮兰法测定蛋白质含量分别为2.7％、1.3％。以上结果表明，PSM-2bA与PSM-2bB为酸性杂多糖。对PSM-2bA和PSM-2bB的结构研究采取了¹³C-NMR分析，部分酸水解，高碘酸氧化和Simth降解，IR分析，GC-MS分析，甲基化分析等方法，甲基化产物经水解、乙酰化后用GC-MS进行测定。结果分析表明：PSM-2bA与PSM-2bB均为多分枝结构；PSM-2bA为具有多分支点的主链连接短支链的结构；其主链的重复结构可能为：PSM-2bB则为长支链的结构。其主体结构由Glc（1→3），Glc（1→2），Gal（1→2，4），Rha（1→3）等单元组成，其主链的重复结构可能为：通过刚果红试验，透射电镜及环境扫描电镜研究和观察了PSM-2bA的显微结构，推导出PSM-2bA的显微结构为由亚基组成的螺旋结构相互缔和，形成了网状高级结构，在天然状态下，PSM-2bA为条带状形态。基于乙酰化效果是影响GC或GC-MS分析结果的关键因素，本文对单糖乙酰化条件也进行了研究。利用气相色谱—质谱联用技术（GC-MS）检测五种常见单糖（木糖、阿拉伯糖、甘露糖、葡萄糖、半乳糖）的乙酰化产物含量，采用正交试验综合考察了还原时间、还原温度、乙酰化温度、乙酰化时间等因素对单糖回收率的影响及规律。结果表明，还原温度和乙酰化时间的影响最为显著。总结出了单糖衍生化最佳的反应条件为还原温度50℃、还原时间2.5h；乙酰化温度90℃、乙酰化时间2.5h。更多还原

【Abstract】 A water soluble polysaccharide has been isolated from cortex of PaeoniaOstill（Moutan）. The crude polysaccharide was deproteinized by freezed-meltedmethod repeatedly, and fractionated by DEAE-52 and molecular sieve, giving twopolysaccharide fractions termed PSM-2bA and PSM-2bB respectively.Homogeneity of them were proved by HPLC. GC-MS show that PSM-2bA consistsof Rha, Ara, Xyl, Man, Glc and Gal with the molarity rate of 3.55: 7.06: 2.11:1.00: 5.60: 12.91 in turn and PSM-2bB consists of Rha, Ara, Xyl, Man, Glc andGal with the molarity rate of 3.43: 8.05: 1.00: 1.00: 24.94: 9.01 in turn.The polysaccharide contents of PSM-2bA and PSM-2bB measued by phenol-sulphuric acid method were 91.8%、82.6%, respectively, and protein contents were2.7%、1.3% measured by coomassie brilliant blue method and uronic acids were4.2%、15.2% measured by sulfate-3-phenylphenylol method. These indicated thatPSM-2bA and PSM-2bB were a kind of acidic heteropolysaccharides. In the structureanalysis of PSM-2bA and PSM-2bB, ¹³C-NMR, partial hydrolysis with acid, periodateoxidation, Smith degradation, IR, GC-MS and methylation etc. were the principalmethods used. After acetylation, the methylated productions were analyzed by GC-MS. The results revealed that PSM-2bA and PSM-2bB both were a kind of highbranched structure. Meanwhile PSM-2bA was some kind of structure with lots ofdivarication at main-chain and small branch; The possible main chain unit of PSM-2bA was The microstructure of PSM-2bA was studied by Congo Red, TEM and ESEM.And the conclusion was deduced that the microstructure of PSM-2bA was somethinglike network which formed by helical structure composed by sub-particle. The naturestate of PSM-2bA was band shape.The thesis also investigated the reduction-acetylation conditions ofmonosaccharides basing on it’s importance to the analysis by GC or GC-MS. Thederivatives of 5 kinds of monosaccharides Xyl, Ara, Man, Glc, Gal were detected anddetermined respectively by GC-MS. The influences of several factors, such asreductive time, reductive temperature, acetylating temperature and acetylating time,on recovery of monosaccharides were studied by orthogonal experiments. The resultsshowed that the reductive temp. and the acetylating time influences derivatizationsignificantly. The optimal derivatization condition is reducting reaction 2.5h at 50℃followed by acetylating 2.5h at 90℃.更多还原