| Diabetes has become one of the major chronic diseases that threaten human life and health in the 21st century.Porphyra yezoensis is rich in various active ingredients and has high health care value.The?-amylase inhibitor??-AI?extracted from Porphyra yezoensis is a natural product for preventing and administreting diabetes,which provided new ideas for the development of high-value products.This study aimed at the extraction,purification,degradation,characterization and postprandial glycemic regulation of?-AI in Porphyra yezoensis.Firstly,the polysaccharide of Porphyra yezoensis?-AI was prepared by hot water extraction,alcohol precipitation and protein removal.The molecular weight distribution of the polysaccharide was homogeneous by the high performance liquid phase?HPGFC?,which was mainly galactose?98.60%?and a small amount of glucose?1.40%?.The effects of heat,acid,alkali,protease,and simulated gastrointestinal environmental treatment on the activity of polysaccharide?-AI of Porphyra yezoensis were studied to reflect the stability of?-AI under the condition of 30.00 mg/m L concentration.The results showed that the polysaccharide?-AI has a wide range of acid-base tolerance?pH 210?,great thermal stability?3090??,and good stability in gastrointestinal digestive juice.Then,optimization parameters of the extraction of?-AI in Porphyra yezoensis showed that the addition of CaCl2 was 0.5%,the ratio of material to liquid was 1:20,the extraction temperature was ambient temperature,the extraction time was 1 h and the concentration of absolute ethanol was 60%.While the extraction rate of?-AI was 237.26 U/g under those optimized conditions.In addition,when the concentration of TCA was 2%and the action time was 50 min,the maximum protein clearance rate was 89.14%,and the polysaccharide loss rate was 23.72%.Thirdly,the component F1,F2,F3,F4 and F5 obtained by isolating and purificating from crude polysaccharide of Porphyra yezoensis was analyzed by high performance gel filtration chromatography?HPGFC?.According to the results,the F1 is not a single component and needed further purification.The relative molecular weights of F2F5 were 197.04 KDa,206.91 KDa,231.40 KDa,and 266.98 KDa,respectively.The extraction rates of?-AI from F1,F2,F3,F4 and F5 were 372.60 U/g,5906.25 U/g,3334.37 U/g,3467.85 U/g,and 3512.50U/g,respectively.According to the analysis of monosaccharide composition,the main component of the F1,F2,F3,F4 and F5 was galactose and a small amount of glucose.The characteristic absorption peak of polysaccharide of both the five components were identified by infrared spectrum analysis.Fourthly,the crude polysaccharide of Porphyra yezoensis was degraded by hydrogen peroxide at different concentrations?0%,1%,2%,3%,5%and 7%?,and the molecular weight distribution of degradation components?PD1,PD2,PD3,PD4,PD5 and PD6?was homogeneous,and the purity degree was high.The average molecular weights of PD1,PD2,PD3,PD4,PD5 and PD6 were 200.13 KDa,6.54 KDa,5.96 KDa,5.05 KDa,3.40 KDa and2.59 KDa,and the inhibition rates were 22.35%,23.56%,66.78%,100%,100%and 100%,respectively.The PD6-1 was purified by Sephadex G-25 gel chromatography and Sephadex G-10 gel chromatography from PD-6,while the IC50 of?-amylase was 12.72 mg/m L.The study on the characteristics of PD6-1 found that the inhibitory effect of PD6-1 on?-amylase was a reversible competitive inhibition.In addition,the?-amylase and PD6-1 conformation changes and the significant reduce of amylase activity were observed by CD chromatography and fluorescence spectroscopy.Finally,through the mice experiment,the postprandial blood glucose level of normal mice and db/db mice decreased significantly,while the level of reduce was similar to acarbose after PD6-1 treatment.However,there was no significant difference in the PD6-1 group and the acarbose dose group in reducing the fasting blood glucose concentration and weight in the short term. |
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