The Mechanism Exploration On The Activity Changes Of Antioxidant Peptides By PEF Technology Based On HepG2 Cell Experimental Methods

The study was granted by National 863 Program under Grant research and development on the stability and processing applicability of active protein of pine nut(Pinus koraiensis)(2013AA102206-5).This paper investigated the effects of pulsed electric field(PEF)treatment on antioxidant activity of two foodborne protein hydrolysates and the obtained antioxidant peptides SHECN,SHCMN,KDHCH,and QDHCH,using chemistry experiments in vitro and cell experiments.Moreover,to reveal the mechanism of activity improvement of antioxidant peptides by PEF,the reversed-phase high performance liquid chromatography(RP-HPLC),fourier transform infrared spectroscopy(MIR),circular dichroism spectrum(CD),FT-IR imaging,and 1H-NMR were used to study the effects of PEF treatment on the structure of antioxidant peptides and the state of antioxidant peptide solution system.This study has provided theoretical basis for further carrying out molecular simulation of PEF treatment on antioxidant peptides.The results of this study were as follows:(1)The improvement of antioxidant activity of two foodborne protein hydrolysates by PEF was studied.One-factor-at-a-time test and response surface methodology experiment were used to optimize PEF treatment conditions for 1-3 k Da soybean protein hydrolysates and 3-10 k Da pine nut protein hydrolysates.A quadric equation was obtained for antioxidant activity of 1-3 k Da soybean protein hydrolysates as Y= 83.994 + 2.240 X1 + 1.691 X2-2.240 X3-0.423 X1 X2-0.573 X1 X3 + 2.614 X2 X3-0.253 X12-3.022 X22-7.244 X32;A quadric equation was obtained for antioxidant activity of 3-10 k Da pine nut protein hydrolysates as Y= 83.494 + 0.355 X1 – 0.028 X2 – 1.301 X3 – 0.366 X1 X2 – 1.182 X1 X3 + 0.499 X2 X3 – 2.898 X12– 0.533 X22 – 3.508 X32.In the equations,Y indicates DPPH free radical scavenging,X1 indicates electric field intensity(k V/cm),X2 indicates pulse frequency(Hz)and X3 indicates flow velocity(m L/min).The DPPH free radical scavenging of 1-3 k Da soybean protein hydrolysates achieved the maximal value of 90.22 ± 0.90% at conditions of 15 k V/cm,1600 Hz and 2.93 m L/min,while 3-10 k Da pine nut protein hydrolysates achieved the maximal value of 85.12 ± 1.24% at conditions of 13 k V/cm,1500 Hz and 2.85 m L/min.(2)The chemical activity in vitro of antioxidant pentapeptides treated by PEF was studied.Four peptides SHECN,SHCMN,KDHCH and QDHCH were identified from 1-3 k Da soybean protein hydrolysates and 3-10 k Da pine nut protein hydrolysates using LC-MS/MS Q Trap technology.DPPH radical scavenging,ABTS radical scavenging and hydroxyl radical scavenging were used as evaluation index to evaluate the antioxidant activity of the four peptides.The optimal conditions for the SHECN was 1800 Hz and 15 k V/cm,for SHCMN was 2400 Hz and 5 k V/cm,for KDHCH was 1800 Hz and 15 k V/cm,for QDHCH was 2400 Hz and 15 k V/cm.(3)The protective effect improvement on oxidative Hep G2 cell model of peptides treated by PEF was studied.Firstly,effects of PEF-treated and untreated peptides SHECN,SHCMN,KDHCH and QDHCH on Hep G2 cell growth were investigated.The results indicated that PEF-treated and untreated peptides had no toxic effect or promoting proliferation on Hep G2 cells.Secondly,the CAA test was used to evaluate the effects of PEF treatment on intracellular antioxidant activity of peptides.The results indicated that the EC50 of peptides was decreased,while intracellular antioxidant activity was increased by PEF processing.Thirdly,the Hep G2 cell oxidative damage model was constructed with the indentified conditions as concentration 400 ?M and incubating time 6 h of H2O2,under these conditions the cell viability was 50.27 ± 2.36%.Finally,the protective effects of peptides on Hep G2 cell oxidative damage model were tested.The results indicated that the protective effects of peptides on oxidative damage cells were enhanced by PEF treatment and the protective effects of four antioxidant peptides was in order of KDHCH > SHCMN > QDHCH > SHECN.(4)The effects of pentapeptides treated by PEF on the antioxidant system of Hep G2 cells were studied.Firstly,KDHCH which has the best intracellular antioxidant activity was chosen as the research object.It was found that PEF treatment can significantly improve the ability of antioxidant peptide KDHCH to inhibit ROS production,thus antagonising the oxidative stress induced by H2O2.Secondly,the MDA level,LDH release rate and activity of antioxidant related enzymes were tested.The results indicated that the MDA level and LDH release rate of cells were decreased by KDHCH,meanwhile activity of CAT,SOD,GSH-PX and GSH-RX was increased.KDHCH has the effects of inhibiting cell lipid peroxidation,maintaining the integrity of cell membrane,improving the anti-oxidase defense system,and the PEF-treated KDHCH behaves better effects.Finally,the effects of KDHCH before and after PEF treatment on mitochondrial membrane potential(MMP)and caspase-3 level were investigated.Compared with untreated KDHCH,the PEF treated peptide can maintain the stability of MMP and reduce the activity of caspase-3 in cells,thus inhibiting the cell apoptosis induced by H2O2.(5)Based on the structural analysis method,the reason of antioxidant activity improvement of peptides by PEF treatment was studied.The basic structures,functional groups and secondary structures of peptides were analyzed using RP-HPLC,NMR and CD spectra technology.The RP-HPLC and NMR spectra showed that the basic structures of SHECN,SHCMN,KDHCH and QDHCH were not changed by PEF and there were no novel H protons produced in the antioxidant peptide system.This results indicated that the antioxidant activity improvement of peptides was not result from changing composition of antioxidant peptides or rupturing the peptide bonds.The MIR spectra showed that the functional groups of peptides were not changed by PEF,while the absorption strength of amide band was affected.The CD spectra showed that the content of ?-helix of peptides was reduced after PEF processing.This phenomenon indicated that PEF treatment promoted the transformation of secondary structure.The FT-IR imaging result showed that the spatial distribution state of KDHCH was changed by PEF and it was proved that the secondary structure of antioxidant peptides was changed and the spatial structure may be affected under the action of PEF.(6)The effects of spatial configuration changes on antioxidant activity of peptides after PEF processing were studied.The effects of PEF treatment on conductivity,zeta potential,average particle size and H-H cosy of peptides were analyzed.The conductivity of peptides was increased,it was indicated that PEF the effective ion concentration of antioxidant peptides.Zeta potential and average particle size of peptides were decreased by PEF treatment,this results showed that PEF treatment can change the charge distribution of peptide solution.After PEF processing,the antioxidant peptide complex was destroyed into smaller complexes inducing a change in spatial configuration.In addition,the chemical environment of active hydrogen proton of antioxidant peptide KDHCH was affected by PEF treatment.A chemical shift of 0.08 ppm was found in hydrogen protons of the-OH from 13-15 ppm.The TOCSY and NOESY spectra showed that with the space state of the antioxidant peptides solution system changing,the short-range or long-range connectivity between hydrogen protons of antioxidant peptide solution were changed by PEF.