The Development Of New Methods In Quality Control, And New Pharmaceutical Application Research For Dextran

Dextran,a family of natural polysaccharide with line type structure,consisting of an α(1â†'6) linked-glucose main chain and a number of branches, Dextran is one of the most important polysaccharides used widely in industrial and medical areas and often produced as extra-polysaccharide by bacteria such as Lactobacillus species, Leuconostoc mesenteroides, and Weissella genera. Glucan produced by microbial fermentation hydrolyzed by acid to obtaine dextran products,with different molecular weight and size of the de-classification of ethanol..However, different microorganism fermentation will acquire kinds of dextrans with different Mw. Moreover, there are also significant differences in branched form and degree of branching. Also, The average molecular weight(Mw) of a dextran typically decides the applications. Low molecular weight dextran used in early clinical shock, larger molecular weight dextran can be absorbed into the body and then hydrolyzed to glucose as an energy supplement. In addition, the types and the quantity of branches are also important for dextran to play different roles, and there is a lack of effective way to determine the types and quantity of branches in dextran at present.Gas chromatography coupled with mass spectrometry(GC-MS) and nuclear magnetic resonance(NMR) are two major strategies to investigate the types and quantity of branches in dextran. However, multiple steps of chemical derivatization, such as methylation, hydrolyzation, acetylation, etc. are necessary before GC-MS analysis. Some structural information may lose during those processes of chemical derivatization and degradation.Furthermore, GC-MS method provides linkage information of branches rather than their accurate composition because of the different ionization of hydrolysates from derived polysaccharide in MS detection. NMR is an effective method to determine the structure and branching degree of dextran. However, NMR need a large amount of sample, and the measurements and interpretation of the results can be very demanding and time consuming.In this work, an ultra-performance hydrophilic interaction liquid chromatography(UP-HILIC)-Q/TOF-MS/MS method was developed to analyze digested products of dextran by a dextranase from Penicillium sp. The optimized chromatography provided effective separation of digested oligosaccharides directly not only by size but also by different linkage patterns. In addition, no any coexistence of α and β configurations was observed and affected separation in this new mothed without any derivatization. MS/MS was applied to sequence each oligosaccharide, including those with the same degree of polymerization(dp) but different linkage patterns.The linkage of enzyme-resistanceoligosaccharidescharacterize the types of branches asdextranase hydrolysisα-1, 6 connection specifically. However, the resolution of isomers with same dp but different linkages still has big room to improve, and quantitative analysis of dextran branches is still remaining.Anion exchange chromatography(HPAEC) with pulsed amperometric detection(PAD) offers an effective platform to separate and quantitate carbohydrates with high selectivity, sensitivity and accuracy. However, high concentration of nonvolatile salt used in HPAEC-PAD prevents it from the application to combine MS directly. An ion suppresser with the electrolysis-driven cation-exchange membranehad been install between HPAEC and MS that allow continuous online desalting and conversion of high salt eluents into MS compatible solvent. This online system, HPAEC coupled with PAD, and ESI- Q/TOF-MS in parallel, provides high resolution, quantitative data and structural information of digested dextran oligosaccharides. Based on the qualitative and quantitative analysis of dextran digested products, the types and quantity of the branches in dextran were determined.Simultaneously, polysaccharide carboxymethylation can improve the solubility and electronegativity, change the stretch direction and three-dimensional structure, which brings new biological activities to polysaccharides.In our researches, the protective effect on neurons with oxygen glucosedeprivation, the relationship between carboxymethy amounts or degree of substitute and anti-oxygen glucose deprivationactivities were investigated,based on carboxymethylated dextran oligosaccharides.