| Haimin Chen (Marine biology) Directed by Professor Xiaojun YanAgaro-oligosaccharides are linear molecules compose of 1,4-linked 3,6-anhydro-a-L-galactose alternating with 1,3-linked p-D-galactopyranose, including neoagaro series and agaro series. In recent years, bioactivity studies demonstrated that neoagaro-oligosaccharides showed almost no physiological functions. In contrast, the agaro-oligosaccharides exhibited a variety of physiological activities, and the activities are correlated to the degree of polymerization (DP). The agaro-oligosaccharides with DP 2-4 are able to suppress the production of the pro-inflammatory cytokine TNF-a and the expression of iNOS. The oligosaccharides with DP 6-8 can elicit physiological response in algae. Therefore, due to their pharmaceutical and agrochemical importance, the research on agaro-oligosaccharides will be of potential value for the development of drugs and functional ingredient.In order to clarify the relationship between DP and bioactivities, each component of oligosaccharide with DP from 2 to 10 were firstly prepared in our study by treated agar with solid acid and isolated with Sephadex G25 and G10. The structure of purified agarobiose. agarotetraose. agarohexaose, agarooctaose and agarodecaose was determined by using TLC. GC-MS. FT-IR, NMR spectroscopy and MALDI-TOF-MS. Consequently, their physiological activities including inhibitory activity towards glycosidase, antioxidant activity, cytotoxicity and anti-tumor ability were studied. The results indicated that agarobiose showed highest ct-glucosidase inhibitory activity in the five oligosaccharides. and its IC50 was 1.39 mg/ml. No significant inhibition was observed when the DP of oligosaccharides are larger than 8. The extracellular and intracellular antioxidant activity was examined by using DPPH and DCFH-DA probe, of which, agarohexaose showed highest scavenging DPPH activity, while agarodecaose exhibited obvious activity against hydroxyl radicals. In liver cell4Abstractexperiments, agarohexaose could inhibit the oxidation of DCFH into DCF significantly, and it also exhibited protective effect towards antimycinA which induced apoptosis of Liver cell. Agaro-oligosaccharides showed selective cytotoxic activity towards different tumor cells. All of them possessed no cytotoxicity against normal liver cell, but exhibited significant activity against BGC-823 and Hela cells. In them, agrarotetraose expressed the excellent anti-tumor property. Its apoptosis activities towards BGC cell were also examined by TUNEL detection, DNA Ladder analysis and flow cytometric assays.In order to tackle the hydrolysis optimization problem, a quantitative method to monitor the agarobiose oligomers is in much needed. Therefore, in this research, by using HPLC technique, a sensitive precolumn labeling HPLC method was established. Based on this technique, agaro-oligosaccharides were hydrolytically obtained from agar using three acid forms including hydrochloric acid, citric acid, and solid acid. Orthogonal matrix method was applied to optimize the preparation conditions by analyzing the agaro-oligosaccharides yield and agarobiose ratio. The best way was achieved as 1 % agar in 20 % cationic exchange resin for 8 h at 90 ?C, which could give a high yield and avoid solution neutralization process. Agarobiose of high purity was consequently eluted from activated carbon column chromatography in the fractions of 5 % and 8 % ethanol, agarotetraose and agarohexaose in 10 % to 15 % ethanol. as well as high-DP oligosaccharides in 25 % ethanol.The activities including inhibiting glucosidase capability, antioxidant ability and cytotoxicity were examined again and the results were almost consistent with those obtained from the purified agaro-oligosaccharides. The antioxidant effect of the agaro-oligosaccharides on tissue peroxidative damage induced by carbon tetrachloride was also investigated in rat model. The result indicated that agaro-oligosaccharides could elevate the activity of SOD, GSH-Px and decrease the level of MDA, GPT, GOT...
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