右旋糖酐质量控制新方法和新的药学应用研究

发布时间：2018-05-12 09:15

本文选题：右旋糖酐 + 亲水相互作用色谱　；参考：《苏州大学》2016年硕士论文

【摘要】：右旋糖酐(Dextran)是葡聚糖的一种,为一线型结构的中性多糖,广泛存在于微生物中,构成右旋糖酐主链的糖酐键为α-1,6糖酐键,并含有少量以α-1,3连接的支链结构。右旋糖酐是应用最广泛的葡聚糖,在医药、食品、化工等行业都有不同的用途。目前,右旋糖酐的生产是以蔗糖为底物,经链球菌、乳酸杆菌、明串珠菌等微生物发酵而生成的高分子葡聚糖,再经酸水解和乙醇分级得到具有不同相对分子质量大小的右旋糖酐产品。但不同种类微生物发酵产生的右旋糖酐分子量差异很大,且支链形式和支链度上也存在很大差异。右旋糖酐分子量大小决定了其应用范畴,如低分子右旋糖酐临床上应用于休克早期,中分子量的右旋糖酐其大小与人体血浆蛋白及球蛋白分子相近,可吸收进入体内然后水解为葡萄糖而作为能量补充剂。同时,右旋糖酐支链形式和支链度对右旋糖酐的实际应用影响重大,但目前对右旋糖酐支链形式和支链度的判定缺乏有效的方法。在以往的文献报导中,对右旋糖酐支链形式及支链度的研究主要有气质联用和核磁共振两种方式。气质联用方法分析的前提是对样品进行甲基化、水解、乙酰化等一系列衍生化,在此过程中无可避免地导致了样品部分结构信息的丢失。并且由于不同连接方式寡糖的离子化效率不同,气质联用方法不能准确判断支链度,只能判断支链形式。核磁共振(NMR)是判断支链形式和支链度的有效方式,但是NMR样品耗量较大,且样品的核磁谱信息会叠加在一起,费时费力很难解析。本文以右旋糖酐的右旋糖酐酶(dextranase)水解产物为研究对象,旨在建立超高效亲水相互作用色谱-飞行时间质谱联用(UP-HILIC-Q/TOF)方法,通过优化色谱方法对不同聚合度以及同一聚合度不同连接方式寡糖同分异构体进行分离,可以有效消除寡糖α,β异构影响且不需要衍生化。利用二级质谱MS/MS对酶解终产物进行结构解析,确证酶解终产物中寡糖聚合度及其连接方式。基于dextranase特异性水解葡聚糖α-1,6连接的作用,根据酶不识别片段的结构即可推断出右旋糖酐的支链形式。但同一聚合度不同连接方式的寡糖同分异构体在此液相方法上的分离有限,且不同结构的寡糖质谱响应各异,不能很好地进行定量分析。因此在此研究工作的基础上,鉴于离子色谱(HPAEC,简称IC)对糖类化合物很好的分离效果,以及安培脉冲检测器(PAD)对糖类化合物高灵敏度的特异性检测作用,我们改进仪器设置,在离子色谱和质谱间安装阳离子树脂交换抑制器(MSM),对离子色谱得流动想进行在线脱盐处理,克服了离子色谱高盐浓度洗脱剂无法直接进入质谱来分析的难点,使在线安培检测器和在线质谱检测平行存在成为可能。该方法成功搭建了HPAEC-PAD-QTOF联用平台,很好地结合了离子色谱和QTOF高选择性、高灵敏度、高准确度等特性,能很好地应用于右旋糖酐支链形式支链度的确定,该方法简便、高效、样品耗量少,是糖类化合物支链定性定量分析的有效方法。、基于多糖羧甲基化能提高多糖的溶解度、电负性,改变多糖分子伸展方向及立体结构,能让多糖具备新的生物活性,本文主要研究了羧甲基化右旋糖酐寡糖在氧糖剥夺损伤模型中对神经元细胞缺糖缺氧保护作用,并探索羧甲基数量、羧甲基取代度与抗氧糖剥夺活性的密切关系。
[Abstract]:Dextran (Dextran) is a kind of glucan. It is a linear structure of neutral polysaccharide, which is widely found in microbes. The sugar anhydride key of the main chain of dextran is a -1,6 sugar anhydride bond, and contains a small amount of branched chain structure connected by alpha -1,3. Dextran is the most widely used glucan and has different use in pharmaceutical, food, chemical and other industries. At present, the production of dextran is a high molecular weight glucan produced by fermentation of Streptococcus, Lactobacillus, and Streptococcus mutans with sucrose as the substrate. The dextran products with different molecular weight are obtained by acid hydrolysis and ethanol classification. However, the molecular weight difference of dextran produced by different kinds of microorganism fermentation The molecular weight of dextran determines its application, such as low molecular dextran in the early stage of shock, and the size of the middle molecular weight dextran is close to the human plasma protein and globulin molecule, and can be absorbed into the body and hydrolyzed into glucose. As an energy supplement. At the same time, the form of dextran branched chain and the degree of branch chain have great influence on the practical application of dextran, but there is no effective method to determine the form of dextran branched chain and the degree of branched chain. In the previous literature, the study of the form of dextran branched chain and the chain degree of the branched chain mainly include GC-MS and NMR The two methods. The precondition of GC-MS analysis is a series of derivatization, such as methylation, hydrolysis and acetylation of samples, which can not avoid the loss of some structural information in the sample. And because of the different ionization efficiency of the oligosaccharides in different connections, the GC-MS method can not accurately determine the degree of the branched chain. NMR (NMR) is an effective way to judge the form of the branched chain and the degree of branched chain. However, the consumption of NMR samples is larger, and the information of the samples will be superposed together. The time-consuming and hard work is difficult to analyze. This paper is aimed at establishing super efficient hydrophilic interaction with dextran dextran enzyme (dextranase) hydrolysate. Chromatography - time of flight mass spectrometry (UP-HILIC-Q/TOF) was used to separate the oligosaccharides and isomers of oligosaccharides with different polymerization degrees and the same degree of polymerization with the same degree of polymerization. The effects of oligosaccharide, beta isomerism and no derivatization were eliminated. Structure analysis of the final products of the enzyme hydrolysis was determined by two stage mass spectrometry MS/MS. The degree of oligosaccharide polymerization and its connection in the final product of enzymatic hydrolysis. Based on the role of dextranase specific hydrolysate glucan alpha -1,6 connection, the branch form of dextran can be deduced according to the structure of the unidentified fragment. However, the separation of oligosaccharide isomers from the same degree of polymerization with the same degree of polymerization is limited and different in this liquid phase method. The structure of the oligosaccharide mass spectrum response is different and can not be quantified well. Therefore, on the basis of this research work, in view of the good separation effect of HPAEC, IC, and the specific detection effect of the ampere pulse detector (PAD) on the high sensitivity of saccharides, we have improved the instrument setting. The cation resin exchange suppressor (MSM) is installed between the chromatograph and the mass spectrum. The flow of ion chromatography is to be desalted online, which overcomes the difficulty that the high salt concentration eluant can not enter the mass spectrometry directly. It is possible that the on-line ampere detector is parallel to the on-line mass spectrometry detection. The method has successfully built the HPAEC. -PAD-QTOF combined with high selectivity of ion chromatography and QTOF, high sensitivity and high accuracy can be used to determine the degree of branched chain in the form of dextran branch. This method is simple, efficient, and less sample consumption. It is an effective method for the quantitative analysis of carboxymethyl groups based on Carboxymethylation of polysaccharides. The solubility and electronegativity of high polysaccharide can change the stretching direction and stereostructure of polysaccharide molecules, which can make the polysaccharide have new biological activity. In this paper, the protective effect of carboxymethylation dextran oligosaccharide on oxygen glucose deprivation in the oxygen glucose deprivation model is mainly studied, and the number of carboxymethyl, the degree of carboxymethyl substitution and the stripping of oxygen glucose are explored. The close relationship between the activity and the activity.

【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R917

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