【作者】 李立；

【导师】 陈朝银；

【作者基本信息】 昆明理工大学 ， 生物化工， 2007， 硕士

【摘要】 多糖是天然存在的生物大分子,由于其广泛的生理活性特别是药理活性而倍受关注。鼠尾草属植物是传统的中药材,在中药中占有重要的地位。有关鼠尾草的研究很多,但关于鼠尾草中多糖的研究才刚起步。本文在前人对多糖的研究基础上,开展了鼠尾草多糖的提取、硫酸酯化、结构测定和构效关系的研究。主要研究结果如下:(1)研究了鼠尾草多糖的提取工艺。通过单因素实验和二次旋转正交组合实验设计分析,讨论了提取温度、提取时间、提取次数及水料比对多糖得率的影响,利用统计软件进行了数据处理。确立了最佳提取工艺条件,即提取时间106.37min,水料比148.68:1,提取次数3次。(2)对比了化学法和冻融法两种除蛋白工艺。结果表明:冻融法除蛋白效果好,且多糖损失较少,是用于粗多糖除蛋白的首选方法。(3)通过凝胶层析,将粗多糖进行了分离纯化,得到一个主要组分SP1。(4)研究了SP1的硫酸酯化工艺。通过单因素实验和二次旋转正交组合实验设计分析,讨论了反应温度、反应时间和磺化试剂的比例对产物硫含量的影响,利用统计软件进行了数据处理。建立了相应的数学模型,为优化提取工艺提供了依据。(5)建立了新的测定硫酸酯化多糖硫含量的方法,与目前常用的几种方法对比,认为新方法用于硫酸酯化多糖中硫含量的测定是可行的。(6)用化学分析和仪器分析的方法研究了SP1及其硫酸酯化产物的结构信息,结果表明:SP1的分子量为234kd,单糖组成为:鼠李糖:阿拉伯糖:葡萄糖=2:3:18,此外,还含有少量木糖和半乳糖。糖苷键的类型及比例为:1,4(2)-糖苷键:1,3-糖苷键:1,6-糖苷键(或非还原末端)=2:12:3。SP1经硫酸酯化修饰后,分子量增大,单糖的种类不变,但葡萄糖的含量明显减少,1,4(2)-糖苷键的比例也降低。SP1及SPS均不具有三股螺旋的空间结构。(7)SP1及SPS均能使BSA的荧光猝灭,猝灭类型为静态猝灭。其与BSA的结合位点分别为1.45和1.47,作用距离分别为3.65nm和3.33nm。SP1和SPS与BSA的作用均为以疏水作用力为主的自发过程。同步荧光研究发现,样品并不能使BSA的构象发生明显改变。(8)用细胞培养体外实验的方法研究了SP1及其硫酸酯化产物的抗HIV活性。通过分析结构与活性之间的关系,研究了SP1及其硫酸酯化产物抗HIV的构效关系。结果表明:为磺化后活性增强,磺化度适中的样品,活性较高,而磺化度过高活性反而降低;分子中1,2(4)-糖苷键比例较高,也即侧链较多的样品,活性较高。更多还原

【Abstract】 Polysaccharide was a kind of natural biomacromolecule.And it has been payed close attention to,because of the wide physiological activities,especially the pharmacal activities. Salvia was the orthodox Chinese crude drug,which possess a considerable position in the traditional Chinese drug.There were many researches about salvia,but only a few of them were about polysaccharides of Salvia.Through previous researches of polysaccharides,the extraction condition,sulfated condition,structure determination,and the structure-activity relationship were studied.The main results were got as follows.(1)The extraction condition of polysaccharide of Salvia was studied.The crude polysaccharides were extracted with hot water.Three factors in extraction were tested,such as extraction time,liquide ration:solid,extraction frequency.By means of response orthogonal test and statistic software STATGRAPHICS plus 5.1,the optimization extraction condition was established.The extraction time was 106.37min,the liquide ration: solid was 148.68:1,and extraction frequency was thrice.(2)The two methods of deproteinization were compared.The freeze thawing method was better than chemical method,and had a lower expense of the product,Freeze thawing method was the best selection to deproteinate in polysaccharide.(3)A principal component named SP1 was obtained through gel chromatography method.(4)By means of response orthogonal test and statistic software STATGRAPHICS plus 5.1,the molecule modification of SP1 was studied and the influence of three factors to the sulphur content of product was made clear,and the mathematical model has been established as well.(5)Compared with the general methods,a more feasible new method to measure the sulphur content of Sulfated polysaccharide was established.(6)The structure of SP1 and sulfated SP1 were confirmed by chemical and instrumental analysis methods.The results indicated:the molecular weight of SP1 was 234kd;SP1 was composed of six monosaccharides that is rhamnose,arabinose,glucose,and a few of rhaglucuronic acid,xylose and galactose,the proportion of the three main monosaccharides were 2:3:18;SP1 has three types of polysaccharide bond that is 1,4(2)-,1,3-,and 1,6-(or non-deoxidized terminal),and the proportion is 2:12:3;the molecular weight o f sulfated SP1 was heavier than SP1,and the variety of monosaccharides was same as SP1,but the content of glucose was decreased;the proportion of 1,4(2)- was also decreased.And the spatial structures of the both were not triple helix.(7)Both of SP1 and SPS can quench the fluorescence of BSA,and each of them was a single static quenching process.BSA was strongly binded to SP1 with the molar ratio of 1.45:1,and the ratio wasl.47:1 when binded to SPS.The binding distance between SP1 and BSA was 3.65nm,and between SP1 and BSA was 3.33nm.The hydrophobic force was the main binding force.The samples cannot make the conformational changes of BSA,studied by synchronization fluorescence.(8)With the cyto-active experiment,the activity of SP1 and sulfated SP1 were studied. The structure activity relationship of SP1 and sulfated SP1 were confirmed.The results show that the anti-HIV activities of products were related to their sulphur content,the activityof sulfated SP1 was higher than SP1.The sulfated SP1 reached higher activity when their degrees of sulfonation were suitable.And the sulfated SP1 which had a higher ratio of 1,2(4)-glucosidic bond reached higher activity.更多还原