Study On Extraction,Decolorization And Degradation With Immune-stimulating Activities Of Sulfated Polysaccharide Ascophyllan Isolated From Brown Alga Ascophyllum Nodosum

The extraction,decolorization,purification,degradation,sugar composition,basic structural characteristics and immunostimulatory activity of ascophyllan from Ascophyllun nodosum were investigated in this stuty.To determine the optimal conditions for efficient yield of ascophyllan by enzyme-assisted extraction.Liquid-ratio,cellulase concentration,time and temperature were selected as influencing factors based on single-factor experiment.The simulated quadratic polynomial regression equation of prediction model was set up by Box-Benhnken central composite design and response surface analysis theory with the yield of ascophyllan as measure idicator.The optimum process parameters for the extraction of ascophyllan were obtained as follows:ratio of liquid to material 30:1,cellulase concentration 200 U/m L,time 2 h and temperature 50 ^oC.The tensile under the condition was 14.75%.The verified value was 14.65±0.21%.The relative error between them was 0.68%which was in close agreement with the predicted values.In this study,the efficiencies of various decolorization methods with different materials including activated carbon,organic solvent,macroporous resin,and hydrogen peroxide were compared and aimed to obtain the optimum decolorization method for removing the pigment in ascophyllan.To further determine the optimum decolorization technologic parameters of H₂O₂-decolorization method,the quantitative effects of time,temperature,H₂O₂ concentration,and p H value on the whiteness value and the retention rate of decolored ascophyllan were investigated using a single factor experiment and a L₉?3⁴?orthogonal design experiment.Our results showed that the optimum decolorization technologic parameters were as follows:the H ₂O₂ concentration 8.6%,the decoloring time4 h,the decoloring temperature 60 ^oC,and the p H 11.0.Under this condition,the whiteness value of ascophyllan was improved to 61.05±1.12%increased by 4.1 times,and its retention rate reached 90.02±0.03%.A chemical modification test using the sulphur trioxide-pyridine method for improving the immune function of NA,showed that the modified MA had no effect on the immunostimulatory activities.As the result,NA and MA were separated into six fractions by DEAE-52 anion-exchange column chromatography with different concentration of sodium chloride.We investigated the immunomodulatory effects of the six fractions on murine macrophage cells and results revealed that the six fractions markedly induced nitric oxide production.To further analyze this phenomenon,immunostimulatory activities of NA by the treatment with EDTA were investigated in vitro.As expected,EDTA-treatment resulted in significant increase the production of NO in macrophages,and the results revealed that metal ions such as Fe²⁺?Zn²⁺in the NA might be one of the inhibitors on its immunostimulating activities.To determine the optimum conditions for improving the degree of degradation on ascophyllan,the low-molecular weight ascophyllans were prepared by degradation with acid and free radical,respectively.The effects of concentration,time and temperature on degrading ascophyllans were investigated by athe single factor experiments.Our results show the optimum conditions of degradation with acid and degradation with a free radi cal to prepare the low-molecular weight ascophyllans.To further studying,the separation and purification of four fractions which the molecular weight range 1000 to 10000 Da were conducted by Sephadex chromatography.The immunostimulatory activities in vi tro and the basic structure on the four fractions of the research shows that the immunostimulatory activities of the four fractions are related to the conformation forms of monosaccharide,sulfate and uronic acid.