Study On Extraction,Purification,Structure And Antioxidant Activity Of Non-Starch Polysaccharides From Red Rice Bran

Red rice is the largest yield colored rice in China,with biological activities such as antioxidant and hypoglycemic properties.The research and exploitation of bioactive ingredients mainly focus on polyphenols of red rice.Polysaccharides are also a class of important bioactive ingredients,among which the structure and biological activity of non-starch polysaccharides from red rice bran（RRBP）have not been researched deeply and their resources have not been utilized fully.In this paper,non-starch polysaccharides were extracted and purified from red rice bran,their structural characterization and antioxidant activity were investigated to provide basis for fully utilizing functional features of red rice.The main results are as follows:1.Extraction and impurity removal of RRBPThe effects of decoloring and deproteinizing were analyzed by comparing pigments concentration,protein color and UV spectrogram of RRBP.In order to maintain original structure and biological activity of RRBP,the molecular weight of polysaccharides under different extraction conditions was compared,and the extraction condition with the highest molecular weight of polysaccharides was selected as optimal extraction condition.Finally,the extraction was carried out in a water bath at 60℃for 5 h,followed by decoloring,deproteinizing,alcohol precipitation and dialysis.RRBP was prepared through microporous absorption resin AB-8 decolorization,Sevag protein removal,alcohol precipitation and dialysis.The content of neutral polysaccharides,uronic acid and protein of RRBP were analyzed using phenol-sulfuric acid method,sulfuric acid-carbazole and Braford method,respectively.The results showed that there were more ethanol-soluble and water-soluble pigments in polysaccharides solution and some of pigments were combined with protein.Extraction red rice bran twice with ethanol can remove ethanol-soluble pigments,then the effect of complete decolorization can be achieved by microporous absorption resin AB-8.There were no protein and pigment absorption peaks found in UV spectrogram of RRBP after extraction,decolorization,protein removal,alcohol precipitation and dialysis.The yield of RRBP was（1.32±0.04）%,the content of neutral polysaccharides and uronic acid was（52.67±0.4）%and（7.24±0.84）%,total content of them was（59.91±1.24）%.2.Purification and structure characterization of RRBPRRBP1,RRBP2,RRBP3,RRBP4 and RRBP5 were isolated by DEAE-52 Cellulose,with the largest two proportion being the RRBP4 and RRBP5 component,and the proportion of RRBP4 and RRBP5 was 46.07%and 24.32%.Therefore,RRBP4 and RRBP5 were selected for Sephadex G-200 isolation,purification,structural characterization and antioxidant activity analysis.The structures of RRBP4 and RRBP5 were characterized using HPLC,FT-IR,complete acid hydrolysis,partial acid hydrolysis,β-elimination and amino acid analysis,I₂-KI experiment,particle size analysis,Congo red test methods,thermogravimetric analysis and scanning electron microscope.The result of HPLC showed that RRBP4 and RRBP5 were composed of mannose（1.95%,3.12%）,ribose（12.63%,15.11%）,rhamnose（2.34%,3.34%）,glucuronic acid（9.22%,8.93%）,galacturonic acid（3.26%,6.28%）,glucose（6.53%,10.77%）,galactose（36.45%,27.17%）,xylose（10.90%,18.19%）,arabinose（16.72%,7.08%）,the two components were acidic heteroglycan dominated by galactose.The results of partial acid hydrolysis and I₂-KI experiment showed that RRBP4 and RRBP5 were highly branched polysaccharides or with long side chain.RRBP4 and RRBP5 were polysaccharides withα-glycosidic bonds and intramolecular and intermolecular hydrogen bonds.Based on HPGPC,the molecular weight of RRBP4 and RRBP5 were 1.65×10⁵ Da和2.56×10⁴ Da.β-elimination showed that RRBP4 and RRBP5 were linked to the amino acid by an O-glycopeptide bond.The Congo red test showed that RRBP4 and RRBP5 had triple helix structure.Particle size distribution of RRBP4 and RRBP5 were 164 nm～459 nm and 142 nm～396 nm,peak particle size were 295 nm and 220 nm respectively.Scanning electron microscopy showed that lyophilized samples of RRBP4 and RRBP5 had smooth surface,which was similar as that of polysaccharides extracted by hot water.Thermogravimetric analysis showed that RRBP4 and RRBP5 were disintegrated at 257.8℃～475.2℃and 268.4℃～498.7℃.3.Antioxidant activity of RRBPIdentified the scavenging capacity of DPPH radicals,superoxide anionic radicals,hydroxyl radicals and ABTS radicals of RRBP,RRBP4 and RRBP5,and analyzed antioxidant activity of them.The results showed that the scavenging capacity of DPPH radicals,superoxide anionic radicals were relatively good.The IC₅₀ for RRBP,RRBP4 and RRBP5 to scavenge DPPH radicals were 0.43 mg/m L,0.40 mg/m L,0.43 mg/m L respectively,the IC₅₀ for RRBP,RRBP4 and RRBP5 to scavenge superoxide anionic radicals were 0.38 mg/m L,0.29 mg/m L,0.52 mg/m L,respectively.At the concentration of 5mg/m L,the hydroxyl radical scavenging rates of RRBP,RRBP4 and RRBP5 were 25.58%,14.11%and 26.85%respectively,and the ABTS radicals scavenging rates of RRBP,RRBP4 and RRBP5 were 10.79%,9.14%and 14.11%,respectively.