Seperation,purification And Identification Of Free Phenolics In Adlay Bran Phenolic Extract And The Mechanisms Underlying Its Antioxidant Activities

Adlay is a nourishing food,and is also used in traditional Chinese medicine.Adlay bran is a by-product during the treating process of adlay and rich in polyphenols.How to use adlay by-products comprehensively has been an important problem for our country.There are quite abundant researches on the extraction methods of different parts of adlay and the officinal effects of coixenolide and coican,but little information about the types of adlay bran phenolics,their antioxidant and xanthine oxidase inhibitory activities and their mechanisms.The aim of this study is to evaluate the protective effects and involved mechanisms of adlay phenolic extracts against H₂O₂-induced oxidative damage in human erythrocytes,and seperate and identify the adlay bran free phenolics,and then evaluate their superoxide anion radical scavenging activities and xanthine oxidase inhibitory activities and correlation mechanisms.The findings provide important contributes to the critical estimation of adlay bran's use in food and pharmaceutical industries.?1?The intracellular antioxidant activities of millet,rice,wheat,brown rice,oat,buckwheat,corn and adlay phenolic extracts were evaluated in an ex vivo erythrocytes system.Buckwheat,corn,and brown adlay free phenolic extracts could effectively attenuate H₂O₂-induced erythrocyte hemolysis.The protective effects and involved mechanisms of polished adlay,brown adlay and adlay bran phenolic extracts were studied further.The intracellular antioxidant activities of adlay phenolic extracts decreased in the following order: adlay bran > brown adlay > polished adlay.Adlay bran free phenolic extract possessed stronger intracellular antioxidant activity than chlorogenic acid,p-coumaric acid and ferulic acid.The free phenolic extracts were more effective than bound phenolic extracts to suppress hemolysis and plasma oxidation through inhibition of MDA equivalents,MetHb and ROS generation and to restore the activities of ATPase and intracellular antioxidant enzymes to normal levels.?2?The adsorption and desorption characteristics of six macroporous resins including XAD-7HP,XAD-16,HP-20,HP-2MGL,SP-207 and SP-825 for enrichment of adlay bran free phenolics were studied.XAD-16,SP-207 and SP-825 were chosen for further studies due to their strong adsorption and desorption capacities.XAD-16,SP-207 and SP-825 had similar phenolics adsorption and desorption behaviors.Pseudo-second-order kinetics model and Freundlich isotherm model were suitable for describing the whole exothermic and physical adsorption processes of adlay bran free phenolics on XAD-16,SP-207 and SP-825 resins,which were affected by the mutual effects of boundary layer diffusion and intra-particle diffusion kinetics.The adsorption behaviors were of mono-molecule layer adsorption.After treatment with gradient elution on XAD-16 resin column,the free phenolics were mostly enriched in 30%,50% and 70% ethanol fractions.?3?The ethanol fractions with high phenolic contents and antioxidant activities were treated with gradient elution on C18 SPE column,then seperated and identified by UPLC-MS/MS.29 phenolics including protocathchuic acid,gallic acid,ferulic acid,chlorogenic acid,p-hydroxybenzoic acid,esculetin,vanillic acid,caffeic acid,octyl gallate,syringic acid,vanillin,perilla acid,p-hydrobenzaldehyde,sinapic acid,p-coumaric acid,coniferyl,umbelliferone,gingerol,?iso?swertisin,2'-hydroxy-3,7,8,4',5'-7-tetramethoxflavone,nobiletin,4-methoxy cinnamic acid,hesperidin,lupin isoflavone,myristicin,coniferyl alcohol,phloretin,kaempferol?luteolin?and chrysoeriol were identified.?4?The superoxide anion radical scavenging activities and xanthine oxidase inhibitory activities of sinapic acid,ferulic acid,syringic acid,p-coumaric acid,chlorogenic acid and caffeic acid were evaluated.Sinapic acid,ferulic acid and syringic acid decreased the amount of superoxide anion radical by inhibiting xanthine oxidase activities.p-Coumaric acid could scavenge superoxide anion radical and inhibit xanthine oxidase activities at the same time.Besides,chlorogenic acid and caffeic acid could decrease the amount of superoxide anion radical directly.The additive and synergistic inhibitory effects of sinapic acid,ferulic acid,syringic acid,p-coumaric acid and chlorogenic acid on xanthine oxidase were responsible for better antioxidant activities of adlay bran free phenolics.The Lineweaver-Burk and Dixon plots illustrated the inhibitory mechanisms underlying varied with type and concentration of substrate and inhibitors and they were all mix-inhibitors.Sinapic acid,ferulic acid,syringic acid,p-coumaric acid and chlorogenic acid could bind to xanthine oxidase with many binding sites and the micro-environments of xanthine oxidase were altered upon interaction with these phenolics.Sinapic acid and ferulic acid made the polarities around tryptophan and tyrosine residues increased and hydrophobicities decreased.For syringic acid and p-coumaric acid,the micro-environments around tyrosine residues displayed no discernable changes during the binding processes,but the polarity around tryptophan residues increased and hydrophobicity decreased.Chlorogenic acid could make the polarity around tryptophan residues decreased and hydrophobicity increased,and make the polarity around tyrosine residues increased and hydrophobicity decreased.