ROS Scavenging Activity And Regulation On The Expressions Of Antioxidant Genes Of Corn Antioxidant Peptides

This work was funded by the National Natural Science Foundation of China?NO.31471597?.Since 2012,the production of corn is over 200 million tons,which30%⁴0%of them are used in industrial processing in China.Corn gluten meal is the main by-product of the corn processing.It is known that corn gluten meal mainly contains zein but is lacked in essential amino acids such as lysine and tryptophan,limiting the use of corn gluten meal in food.It implies the importance of the deep-processing of corn gluten meal to the corn processing industry.In this study,corn gluten meal was used to prepare antioxidant peptides by enzymatic hydrolysis.The free radical scavenging activity,cellular reactive oxygen species?ROS?scavenging activity,antioxidant enzyme regulating capacity,and antioxidant gene expression regulating capacity of corn gluten hydrolysates were determined,as well as the hydrolysis and transepithelial transport of two novel corn gluten derived antioxidant peptides in human Caco-2 cell monolayers.The main research work and results were summarized as follows:?1?The corn gluten meal was hydrolyzed by Alcalase.Then the supernatant was fractionated using ultrafiltration and the fractions with molecular weights less than 1kDa?CGH1?and between 1 and 3 kDa?CGH2?were collected.Results showed that both of CGH1 and CGH2 exhibited good DPPH radical scavenging activities,hydroxyl radical scavenging activities,superoxide anion radical scavenging activities,ABTS radical scavenging activities and oxygen radical absorbance capacities?ORAC?.And CGH1 displayed higher free radical scavenging activities than CGH2.Both of CGH1 and CGH2 also showed Fe²⁺chelating activity and ferric-reducing antioxidant activity.In addition,amino acid composition analysis revealed that the total amino acids contents in CGH1 and CGH2 were 63.07 g amino acid/100 g peptide and 68.78g amino acid/100 g peptide,respectively.What should be noted was that the contents of some antioxidant amino acids,such as Ala,Val,Met,Ile,Leu,Phe,Pro,and Trp,in CGH1 and CGH2 were 28.79%and 28.24%,respectively.?2?A cellular antioxidant activity?CAA?assay was used to assess the cellular antioxidant activities of CGH1 and CGH2.Results showed that both of CGH1 and CGH2 exhibited high cellular antioxidant activities with EC₅₀ values of 12.40±2.01mg/mL and 13.86±0.78 mg/m L,respectively,in the presence of PBS wash.While in the absence of PBS wash,the EC₅₀ values were 2.85±0.19 mg/mL and 5.05±0.32mg/mL for CGH1 and CGH2,respectively.It was obvious that CGH1 had high cellular antioxidant activity than CGH2 in CAA assay without PBS wash protocol.Hydrogen peroxide was used to induce oxidative stress in HepG2 cells.Results showed that both of CGH1 and CGH2 also exhibited cytoprotective effects and intracellular ROS scavenging activities in H₂O₂-induced Hep G2 cells.In addition,both of CPF1 and CPF2 exhibited positive effects on the activities of the intracellular antioxidant enzymes SOD,CAT and GR,as well as on the total GSH levels in HepG2cells under conditions of oxidative stress.At the concentrations of 2.50 mg/m L,the CGH1 and CGH2 increased the activity levels of superoxide dismutase?SOD?,catalase?CAT?,and glutathione reductase?GR?,as well as the total glutathione?GSH?levels in oxidized HepG2 cells?from86.54%to 114.14%?CGH1?or 109.72%?CGH2?for SOD activity;from71.91%to 107.64%?CGH1?or 106.50%?CGH2?for CAT activity;from 70.52%to 103.01%?CGH1?or 104.10%?CGH2?for GR activity;and from 81.39%to 114.00%?CGH1?or 108.82%?CGH2?for total GSH levels?.?3?The effects of CGH1 and CGH2 on the expressions of 84 antioxidant defense and ROS metabolism relevant genes in HepG2 cells were determined using RT-PCR array.Results showed that the influences of CGH1 on the expressions of genes involving in antioxidant defense and ROS metabolism was more significant than that of CGH2.Moreover,genes such as GPX3,GPX5,SOD3,CYGB,SEPP1 and MT3that down-regulated in oxidative damaged HepG2 cells were,however,up-regulated in CGH1-pretreated cells,suggesting that CGH1 could improve the expressions of several antioxidant genes that were initially suppressed by oxidative stress.In addition,the results of GO analysis showed that the differentially expressed genes were highly concentrated at the terms of oxidative stress and ROS metabolism,and the biological functions of these genes and their products were mainly localized in the cell membrane.Furthermore,the KEGG analysis showed that the antioxidant action of CGH1 in cells was associated with arachidonic acid metabolism.CGH1 suppressed the expression of EPHX2,which may in turn increase cellular EETs and form EET-phospholipids that will help protect HepG2 cells against cell membrane damage induced by H₂O₂.?4?Tow novel antioxidant peptides of YFCLT and GLLLPH with ABTS radical scavenging EC₅₀ values of 37.63?M and 41.38?M respectively were identified from corn gluten meal using MALDI-TOF mass spectroscopy.The transepithelial transport of YFCLT and GLLLPH was determined using Caco-2 cell monolayers.Results showed that both of YFCLT and GLLLPH could transport in intact form across Caco-2 cell monolayers with apparent permeability coefficient(P_app)values of?1.10±0.16?×10^-7 cm/s and?1.98±0.23?×10^-7 cm/s,respectively.However,it was found that the two peptides were susceptible and easily hydrolyzed by brush border membrane peptidases.In the presence of diprotin A,an inhibitor of dipeptidyl peptidase IV?DPPIV?,the hydrolysis of YFCLT and GLLLPH decreased from 36%and 75%to 29%and 26%,respectively.Moreover,it was found that cytochalasin D,a tight junctions?TJs?disruptor,increased the permeability significantly.While wortmannin,a transcytosis inhibitor,and sodium azide,an ATP synthesis inhibitor,both decreased the permeability significantly.It indicated that the TJs-mediated paracellular pathway and energy-dependent transcytosis might be involved in the transport of YFCLT and GLLLPH across Caco-2 cell monolayers.