Synergistic Mechanism Of Astragalus And Peony In The Regulation Of Cellular Antioxidant

In recent decades,ROS and free radicals have become the research frontier in modern life science.Evaluation and screening of natural antioxidants have increasingly been the trendency in the fields of biology,medicine and food science.China has abundant resources of traditional C hinese herbs,which have attracted wide attention in the world due to their being rich in various antioxidant components.Both Astragalus and Peony have strong antioxidant properties.It is evidenced that there is antioxidant synergism when they were used in combination.Previously we have investigated the synergistic antioxidant activity of Astragalus and Peony,and identified seven potential antioxidant components by 1,1-diphenyl-2-picrylhydrazine(DPPH)radical scavenging test followed by a high performance liquid chromatography separation combined with diode array detection and tandem mass spectrometry analysis(DPPH-HPLC-DAD-MS/MS).These seven components are:quercetin,calycosin-7-O-β-D-glucoside,formononetin-7-O-β-D-glucoside,9,10-dimethoxy sandalwood alkoxy-3-O-β-D-glucoside and 2’-hydroxy-3’,4’-dimethoxy-isoflavan-7-O-β-D-glucoside from Astragalus;oxypaeoniflorin and catechin from Peony.The main research contents of this study are as follow:1.The DPPH and ABTS radical sca venging activity of quercetin,catechin,calycosinglucoside,ononin,oxypaeoniflorin alone and their combinations were investigated.The results showed that both quercetin and catechin had the strong free radical scavenging ability.Calycosin-glucoside,ononin,oxypaeoniflorin had weak scavenging activity.The combination of quercetin and catechin(12.5μM:12.5μM)showed the strongest synergy.Other combinations,such as quercetin/calycosin-glucoside,quercetin/ononin,quercetin/oxypaeoniflorin,catechin/calycosin-glucoside,catechin/ononin,and catechin/oxypaeoniflorin showed significant antioxidant synergy.2.Cellular antioxidant activity of the selected components were assessed.MTT assay was performed to analyze the cytotoxicity of quercetin,catechin,calycosin,calycosinglucoside,and oxypaeoniflorin.Non-cytotoxic concentration were used for cell antioxidant activity assay.The combination of quercetin/catechin(12.5μM:12.5 μM)was chosen for further experiment based on the dose-effect,time-effect and the CI value.Verification experiment showed that quercetin and catechin were able to synergistically protect cells against oxidation in H2O2-induced HepG2 cells.3.Further analysis indicated that the protective effect of quercetin,catechin may be associated with their modulatory effect on intracellular antioxidant enzymes.Therefore,the activities of SOD,GPx,CAT and the MDA content were measured in ABAP and H2O2 induced cellular oxidative stress model.The results showed that quercetin and catechin were able to regulate the activity of SOD,GPx,CAT antioxidant enzymes.4.RNA-seq transcriptome sequencing was performed to further elucidate the mechanism of antioxidant synergy of quercetin and catechin in H2O2-induced Hep G2 cells.Significant difference analysis in gene expression showed that co-treatment of quercetin and catechin synergistically modulated the cellular antioxidant enzyme genes,including SOD1,GPX2,PRX1 and PRX7.Furthermore,seven antioxidant-associated transcription factors were identified as: HSF4,TWIST2,NFIC,TFAM,ATF3,AP-2α and SP1.These significant genes were substantially enriched in Alzheimer’s disease,Calcium signaling pathways,Oxidative phosphorylation,Huntington’s disease,and MAPK signaling pathways.