Mechanism Of Antioxidant Interactions Of Lycopene,lutein,Chlorogenic Acid And Delphinidin By Nrf2 Pathway

Phytochemicals have the ability to neutralize reactive oxygen species(ROS)and are closely related to the pathogenesis of cardiovascular disease(CVD).However,there are few studies on the interaction between different phytochemicals in food and their mechanisms.Lycopene(LY),Lutein(LU),Chlorogenic acid(CA)and Delphinidin(DP)are abundant and widely distributed antioxidants in food,which have a variety of biological activities such as antioxidant,anti-inflammatory,and reducing the incidence of cardiovascular diseases.This paper use the above four kinds of phytochemicals as the research object,using DPPH and ABTS two chemical methods in vitro,analysing four kinds of phytochemicals interaction in vitro antioxidant properties,using H2O2 induced H9c2 rat myocardial cells oxidative damage model,The interactive antioxidant effects of binary combinations of four kinds of phytochemicals in different ratios were studied in protective effects of oxidative damage.On this basis,the intracellular absorption and degradation of lycopene and lutein were studied,and the mechanism of antioxidant interaction of phytochemicals was analyzed based on the Nrf2 pathway,providing theoretical basis for elucidating the mechanism of antioxidant interaction of phytochemicals and developing corresponding functional foods.Main research contents and results are as follows:1.The four natural phytochemicals(lycopene,lutein,chlorogenic acid and delphinidin)were combined in pairs with different molar ratios(1:9,3:7,1:1,7:3,9:1),and the antioxidant activities of ABTS,DPPH and isoradiation analysis were used to investigate the in vitro antioxidant activity and their interactions of the four phytochemicals.The results showed that the antioxidant activities of the water-soluble chlorogenic acid and delphinium ABTS and DPPH were stronger than that of the fat-soluble lycopene and lutein.According to the isometric analysis of ABTS,lycopene has a strong synergistic antioxidant activity with other three phytochemicals.In DPPH free radical scavenging experiments,most of the lipid-soluble lycopene and lutein complexes had antagonistic effects,while most of the water-soluble chlorogenic acid and delphinium complexes had synergistic effects.Different proportions of the two substances show different antioxidant interactions.In addition,different antioxidant interactions were found in different experimental methods when the two substances were mixed in the same proportion.2.Establish H9c2 cell model of oxidative damage to H2O2,and determine the effect of lycopene,lutein,chlorogenic acid and delphinium alone on ROS level.In combination with CAA and combined index method,6 groups of significant synergistic and antagonistic antioxidation were selected.The ROS levels of the significantly synergistic and antagonistic groups were determined by flow cytometry,and the activities of its antioxidant enzymes(SOD,GSH-Px and CAT)were studied.The results showed that,except delphinidin,with the increase of concentration,the ability of lycopene,lutein and chlorogenic acid to scavenge H2O2-induced ROS was enhanced,but began to decrease when reaching a certain concentration.Therefore,high concentration of phytochemicals may cause the pro-oxidation effect of H9c2 cells.The antioxidant interactions of four natural phytochemicals were determined by CAA method and combined index method.Six groups of significant synergistic and antagonistic combinations were selected as synergistic groups:CA-LU=3:7,DP-C A=7:3,DP-LY=1:1 and antagonistic group:LY-LU=1:1,CA-LU=9:1,DP-LY=7:3.Four natural phytochemicals play an antioxidant role by directly reducing ROS levels in H9c2 cells.In different enzyme systems,the activity of antioxidant enzymes in the significantly synergistic and antagonistic groups was different from the expected synergistic and antagonistic actions.For example,DP-CA=7:3 and DP-LY=1:1 in the significantly synergistic group showed significant synergistic effect on the expression of SOD activity,while there was no significant synergistic effect on antioxidant activity of GSH-Px.Due to the specific interaction between phytochemicals and endogenous antioxidant enzymes,the expected significant synergistic and antagonistic antioxidant enzyme activities were affected.May not be sensitive to the changes in antioxidant activity caused by interaction.3.Western blot was used to determine the expression of Nrf2 pathway related proteins,and the effects of four phytochemicals on Nrf2 nuclear translocation and mitochondrial membrane potential of H9c2 rat cardiomyocytes were investigated by immunofluorescence staining and JC-1 staining.The results showed that the selected combinations of lycopene,lutein,chlorogenic acid and delphinium had significant synergistic and antagonistic effects on the expression of HO-1 and GCLC proteins,respectively.The synergistic effect of the four phytochemicals was mainly played by the accumulation of Nrf2 in the nucleus,while the Nrf2 inhibitor did not affect the protein expression of the antagonist group,so the antagonist group did not target the Nrf2 pathway to play the antagonistic effect.Mitochondria were important targets of phytochemical interactions,and phytochemical combinations interact by regulating mitochondrial membrane potential.4.The stability of lycopene and lutein in DMEM culture was determined by high liquid chromatography,and the effect of another component in the phytochemical combination on lycopene and lutein was investigated.The results showed that lutein was more stable in DMEM medium,while lycopene was less stable in DMEM medium.In a certain concentration range,the greater the initial concentration of phytochemicals added to the medium,the greater the cell absorption rate.In the phytochemical combination,both chlorogenic acid and lycopene promoted the absorption of lutein,and the group with the strongest promotion was 2.85 times that of the control group.In the significantly synergistic group,delphinone promoted lycopene uptake(increased by 31.1%),while in the antagonistic group,lutein and delphinone inhibited lycopene uptake(decreased by 43.3%and 37.7%).Therefore,the antioxidant interactions of phytochemical combinations are also affected by the cell absorption rate.