| Germinated brown rice contains a large number of physiologically active ingredients such as GABA,dietary fiber,?-oryzanol,etc.It has antioxidant,blood pressure lowering,blood lipid lowering,hypoglycemic and other active functions.It is a food that can prevent diabetes and other complications..In this study,germinated brown rice was used as raw material to extract polysaccharides from germinated brown rice to determine the optimum extraction process.The components of germinated brown rice polysaccharide were isolated by decolorization and deproteinization and purified by chromatography to identify the structure of each component.And the inhibition of ?-glucosidase was studied,and the components with the strongest inhibitory effect were screened to investigate its effect on insulin resistance in HepG2 cells.The main research contents and results are as follows:(1)Extraction,purification,and separation of polysaccharides from germinated brown rice:Extraction of polysaccharides from germinated brown rice using complex enzymatic hydrolysis and ultrasonic-assisted extraction.The optimum conditions are:enzymolysis temperature 50?,enzymolysis time 2.5 h,and enzyme complexes.(Papain:cellulase:pectinase)The amount of addition was 1.5%and the pH of the enzymolysis was 6.The average extraction rate of the germinated brown rice polysaccharide was 37.58%.Three protein removal methods(Sevag method,trichloroacetic acid method,enzyme combined with Sevag method)were used to analyze the protein removal effect of germinated brown rice polysaccharides and three methods of decolorization(activated carbon adsorption,hydrogen peroxide oxidation,macroporous resin).Adsorption method)Decolorization of germinated brown rice polysaccharide.The macroporous resin AB-8 had the best decolorization effect on germinated brown rice polysaccharides,with a decolorization rate of 86.57%and a polysaccharide loss rate of 28.96%.The enzyme-Sevag method has better deproteinization effect and low polysaccharide loss rate.The deproteinization rate is 74.36%,and the polysaccharide loss rate is 14.09%.Compared with DEAE-Sepharose CL-6B,DEAE Fast Flow and DEAE-Cellulose 52,the chromatographic purification effect of brown rice polysaccharides was compared.The optimal filler was selected as DEAE-Sepharose CL-6B with deionized water,The gradient elution of 0.1 mol/L,0.2 mol/L,0.3 mol/L,and 2 mol/L NaCl solution eluted with deionized water to elute a constituent polysaccharide with a content of 32.82%,eluted with different concentrations of NaCl gradient.Three component polysaccharides were obtained with contents of 9.91%,8.15%,and 7.42%,respectively.Sepharose CL-6B column chromatography was used to elute with 0.1 mol/L NaCl.The polysaccharide was washed with water to obtain a fraction of WPS with a content of 24.16%.The salt gradient eluted polysaccharide to obtain three components of SPS-1 and SPS-2.The contents of SPS-3 and SPS-3 were 8.28%,7.09%,and 5.94%,respectively.(2)Characterization of polysaccharides from germinated brown rice:Ultraviolet absorption spectroscopic analysis of polysaccharides from germinated brown rice showed that each polysaccharide component contained no protein or nucleic acid.High performance liquid chromatographic analysis showed that the water-washed polysaccharide WPS consisted of rhamnose,xylose,arabinose,mannose,and glucose in a molar ratio of 7.8:9.1:6.3:3.9:4.4;the salt-washed polysaccharide SPS-1 was composed of mannose.,galactose and glucose composition,molar ratio 8.3:4.5:10.1;salt washed polysaccharide SPS-2 from rhamnose,arabinose,mannose and glucose,molar ratio 10.3:7.9:6.7:4.1;salt washed polysaccharide SPS-3 consists of rhamnose,arabinose,and glucose in a molar ratio of 4.8:2.2:6.5.Infrared spectroscopy analysis showed that each polysaccharide contained characteristic absorption peaks of polysaccharides,while the washed polysaccharides mainly consisted of pyranoid rings.and the salt-washed polysaccharides contained pyran and furanose rings.Nuclear magnetic resonance 1H-NMR analysis showed that the water-washed polysaccharide component mainly contains the ?-configuration of the pyran ring,and the salt-scrubbed polysaccharide contains the furan and pyranose rings of ? and ?-configuration.(3)Study on the hypoglycemic mechanism of germinated brown rice polysaccharides germinated brown rice polysaccharides can inhibit ?-glucosidase activity better,and the inhibitory effect of 0.6 mg/mL germinated brown rice polysaccharide is the best,and it is non-competitive inhibition type..And it was found that the content of ?-glucosidase inhibitor in SPS-1 polysaccharide fraction eluted with 0.1 mol/L NaCl salt solution was the highest,the highest ?-glucosidase inhibitory rate and the SPS-3 polysaccharide fraction.The content of the enzyme inhibitor substance is the least.The germinated brown rice polysaccharide component having the activity of inhibiting ?-glucosidase can increase the insulin resistance of HepG2,make HepG2 cells absorb glucose more,and germinate brown rice polysaccharide with inhibition of ?-glucosidase activity regardless of the presence or absence of insulin.Both components can significantly promote glucose consumption in HepG2 cells.Therefore,germinating brown rice polysaccharide can reduce blood glucose by inhibiting ?-glucosidase activity and promoting glucose consumption of cells.The germinated brown rice polysaccharide component with inhibition of ?-glucosidase activity can also rapidly and significantly increase the enzymatic activity of hexokinase and pyruvate kinase in insulin resistant HepG2 cells.The pyruvate kinase enzyme activity was detected at the 12h and 24h time points.Increased 18.21%and 17.35%,respectively,hexokinase activity increased by 11.35%and 10.47%;glycogen content in the addition of polysaccharide components make insulin resistant HepG2 cell glycogen content increased significantly,the role of insulin resistance mitigation Significantly improved glycogen content in insulin resistance. |
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