羟基自由基降解琼胶及其产物的活性研究

琼胶是一种从石花菜等红藻中提取的，目前生产工艺和结构等方面研究比较成熟的海藻多糖，广泛应用于医药、仪器等行业。但是，海藻多糖因为具有分子量大，粘度大，溶解度较小的等特点，而使其应用范围受到限制。利用降解的手段对其进行修饰，降低分子量和粘度，改善溶解性，可以拓展其应用范围。并且根据文献报道，琼 胶寡糖具有一些特殊的生物活性，如抗氧化性，抗炎症等。因此，对琼胶降解的研究具有生要意义。本研究中，为了选择一种合适的降解方法，进行了几种水解方法的尝试，其中包括在不同湿度和酸度下盐酸水解，过氧化氢和醋酸催化水解，Fenton体系羟基自由基降解。对于酸水解和Fenton体系氧化还原降解方法，通过粘度法对反应的速度进行了比较，表明氧化还原降解反应中琼胶的粘度降低比较快，并且具有代表性和新意，确定为本实验的降解琼胶的方法并对氧化还原降解所得的产物进行了活性实验。通过模仿自然界普遍存在的氧化还原降解反应，利用Vc诱导的Fenton体系产生的羟基自由基氧化还原降解琼胶得到低分子量的琼胶。降解产物经过高速离心、60％乙醇沉淀，除去分子量比较大的降解产物和磷酸盐，得到可溶于60％乙醇的分子量估计小于3000的降争产物，其产率为85％。利用经Sephadex-G25凝胶色谱分离所香的不同分子量的级分进行分子量和α－葡萄糖苷酶抑制活性关系的实验。降解产物对α－葡萄糖苷酶的抑制率和各级分的浓度呈线性正相关，并且各级分的IC_(50)则随着分子量的降低而降低。另外，对所得的降解产物混合物进行了红外吸收光谱、质子去偶核磁共震碳谱和负离子基质辅助激光诱导－飞行时间质谱结构分析。结果表明，氧化还原降解反应的专一性差，在得到寡糖的同时，在光谱图中出现一些比较复杂的副产物的结构信息。最后，根据MTT法的原理，以有体皮肤成纤维细胞为材料，通过紫外线辐射产生自由基造成氧化损伤，研究降解产物对成纤维细胞的保护作用。当无紫外线辐射时，降解产物对成纤维细胞具有显著的促进生长增殖作用：当经UVa、UBb辐射时则可以显著地表现出对损伤的保护作用，并且这种促进生长和保护作用呈显著的量效关系，表明降解产物具有清除基自由基的作用。但是，因为氧化还原降解以应的机理尚不十分明的以及琼羟胶的特殊结构，使得反应的副产物很难预测，也就使得分离工作难以进行，所以，根据目前所得的信息，尚不能确定是降解产物的什么级分产生的以上两种生物活性。

Seaweed polysaccharides are important natural products from algae, widely used in food industry and medicine for years. The size or degree of polymerization of polysaccharide plays a critical role in determining their applications. Polysaccharides with large size tend to be insoluble or highly viscous in solution, which may have limited their applications. A decrease in molecular weight can substantially enhance their utility. Now they are showing increasingly great value for the deeper research and development of physiological and bioactive compounds. For example, some oligosaccharides produced by chemical or enzymatic degradation of seaweed polysaccharides showed some interesting biological activities, such as anti-inflammation, anti-tumor and antioxidant activities. It is critical to understand the precise roles they play in biological processes and elucidate the structure-bioactivity relationship for the deeper development of new medicine. Agar is a gelatinous polysaccharide extracted from certain species of the Rhodophyceae, the predominant structure was found to be a linear, alternating DL-galactan comprising of β-(1→4)- linked units of 3,6-anhydroα-D-galactopyranosyl-(1→3)- D-galactopyranose. This sequence was defined as 'agarose' and comprises approximately 70% of the agar polysaccharide. The remainder is agarose substituted with sulphate half-ester, methyl ether and pyruvic acid ketal. In our study, we used agar as the starting material and tried several degradation method to find a suitable method to prepare agaro-oligosaccharides, such as acid hydrolysis catalyzed by dilute hydrochloric acid, H_2O_2 and CH_3COOH, and oxidativ-ereductive depolymerization (ORD) via OH, using "Fenton" type reagents (hydrogen peroxide in conjunction with ferrous ions) as OH sources. The reaction processes of ORD and the acid hydrolysis by HCl have been monitored by viscosity measurement. The initial results indicated that the accelerated degradation of agar by Fenton type reagents to mimic naturally occuring ORD reaction has several advantages over methods like acid hydrolysis particularly in questions regarding the characteristic composition of agar, 3,6-AG and the degradation rate aspect. The low molecular weight agars (LMWA). produced by OH using the ascorbate induced Fenton type reagents as sources, have been characterized by IR spectra, ~(13)C NMR, Negative MALDI-TOF-MS and biological activities evaluation. The results of these instrumental analyses showed that the ORD is more representative but less specific than acid hydrolysis. In order to investigate the structure-activity relationship of the degradation products, we have prepared by size exclusion chromatography agar fractions of different molecular masses. Our experiments showed that the inhibitory activities of α-gluosidase are dependent on the molecular weight of the products. The reduction in molecular weight significantly enhanced the inhibitory capacity of the degradation products. In addition, according to the principle of MTT method, we designed to form OH by UVa and UVb radiating the fibroblast of human derma, and then evaluated the radical scavenging activity of the degradation products through the protective action of these injury fibroblast. The results showed that the protective effects under these two conditions were dose dependent upon the concentration of samples. What's more, the degradation products also markedly stimulated the fibroblast growth while no UV radiation, and this stimulative effect was dose dependent, too. Because of the complex byproduct produced by some unknown mechanisms of side reactions, the information available can not tell us what are the main components with these biological activities. But we can get a conclusion that the degaradation products of agar by OH may be available for developing physiologically functional foods endowed with antioxidative and hypoglycemic activity towards humans.