Antioxidative Damage Effect And Active Ingredients Of Chrysanthemum Morifolium Ramat.,Chrysanthemum Indicum L. And Chrysanthemum Indicum Var. Aromaticum

Objective:The purpose of this study was to carry out the anti-oxid ative effects of Chrysanthemum Morifolium Ramat.,Chrysanthemum indi cum L.and Chrysanthemum indicum var.aromaticum,screen the effecti ve parts of antioxidation,identify the chemical components of the effect ive parts of antioxidation,and explore the biological mechanism of antio xidation,which is the rational application of Hangbaiju,wild chrysanthe mum and Shennongxiangju and provide the basis for finding natural anti oxidants.Methods:1.Antioxidative effect research and active site screening of Chrysanthemum Morifolium Ramat.（HBJ）,Chrysanthemum indicum L.（YJH）and Chrysanthemum indicum var.aromaticum（SNXJ）.Precisely weigh the medicinal materials,extract twice with 80%ethanol,combine the extracts,recover the ethanol under reduced pressure until there is no alcohol smell,and the residue is the total extract of each tested medicinal material;Add water to dissolve,adopt the system solvent extraction method,extract with petroleum ether,ethyl acetate,n-butanol,respectively,extract the solvent and the aqueous solution under reduced pressure to recover the solvent,dry,and the dried product is the corresponding extraction part of each test medicinal material;Appropriate amount of extraction parts of the tested medicinal materials were prepared into test solutions of different extraction parts of the tested medicinal materials;DPPH method,ABTS method and FRAP method were respectively used to determine the total extract of each tested medicinal material and the antioxidant effect of different extraction parts,and compare the different extracts.The antioxidant activity of medicinal materials was tested,and the antioxidant activity sites were screened.2.The protective effect of the active parts of HBJ,YJH and SNXJ on H₂O₂-induced oxidative damage in Hep G2 cells.Using the MTT method,Hep G2 cells were used as model cells,and the cell survival rate was used as an indicator to optimize the concentration and modeling time of the H₂O₂-induced cell oxidative damage model to form a stable oxidative damage cell model.（MDA,ROS）,antioxidant enzyme（CAT）and antioxidant（GSH）contents were used as indicators,and the cell survival rate was determined at the same time,and the protective effect of the active parts of the tested medicinal materials on the model of H₂O₂-induced cell oxidative damage was investigated and evaluated.The anti-oxidative damage effect of the active parts of the tested medicinal materials.3.Preliminary research on the material basis and mechanism of anti-oxidative damage of HBJ,YJH and SNXJ.UPLC-QTOF-MS/MS technology was used to determine the total ion chromatogram and mass spectrometry signals of the active site compounds of HBJ,YJH and SNXJ,and to identify the compounds contained in the active sites of the tested medicinal materials.The Target Prediction database predicts the potential targets of each compound,searches the Gene Cards database for oxidative stress-related proteins,comprehensively analyzes the potential targets and oxidative stress-related targets,and screens to determine the anti-oxidative stress targets of each compound;Perform GO and KEGG enrichment analysis on the identified antioxidant stress targets,and combine with Cytoscape software to construct a compound-target-pathway network map,and use topological analysis to select compounds and targets with high interaction degrees as core components and core targets;Compounds and targets with high degree of interaction were selected as core components and core targets;Using Autodock software,virtual verification of the combination of core components and core targets was carried out with binding energy as the index;using enzyme-linked immunosorbent assay（Elisa）assay,Hep G2 cell oxidative damage model induced by H₂O₂ was used as the subject.Subjects,using the core target protein expression as an indicator,analyze the effect of potential active compounds on the expression of the core target,and preliminarily clarify the anti-oxidative damage mechanism of each tested medicinal material.Results:Antioxidant experiment in vitro to detect the antioxidant capacity of different parts of HBJ,YJH and SNXJ.ethyl acetate fraction>n-butanol fraction>total extract>petroleum ether fraction≈water fraction.In vitro cell experiments:The cell viability was detected by MTT method,and it was found that the concentration of HBJ group,YJH group and SNXJ group had no effect on the activity of Hep G2 cells at 20μg/m L.Hep G2cells were treated with 800μmol/L H₂O₂ for 4 h as the best modeling condition.When the concentration of HBJ,YJH and SNXJ was 20μg/m L,it showed better protective effect in oxidative stress model.Further research found that pre-administration of HBJ group,YJH group and SNXJ group for24 h could effectively reduce the content of ROS and MDA in cells,and increase the activity of CAT and GSH content in cells.The HBJ group had the best effect,followed by the YJH group and the SNXJ group the worst.Using UPLC-QTOF-MS/MS technology,22 compounds were identified by comprehensive characterization of the ethyl acetate fractions of HBJ,YJH and SNXJ,including 16 flavonoids and their glycosides,and 6 phenylpropanoids.The corresponding target proteins of these 22 compounds were predicted using the Swiss Target Prediction database,and the oxidative stress-related proteins were intersected in the Gene Cards database,and a total of 47intersecting targets were obtained.The compound-target-pathway map was constructed by Cytoscape software,and then sorted according to the degree value.It was found that the flavonoids in the ethyl acetate parts of HBJ,YJH and SNXJ are important active components of anti-oxidative stress,among which the top The four compounds,Acacetin,Eupatilin,Kaempferol and Quercetin,have strong binding ability to core targets NOX4,XDH,EGFR and MMP2,respectively.The cell viability experiments showed that both Kaempferol and Quercetin could protect cells from oxidative damage,and the enzyme-linked immunosorbent assay（ELISA）showed that Kaempferol and Quercetin could effectively down-regulate the expression of MMP2 and NOX4 target proteins.Conclusion:The ethyl acetate parts of HBJ,YJH and SNXJ had the strongest antioxidant capacity,and had a certain protective effect on H₂O₂-induced oxidative damage in Hep G2 cells.HBJ,YJH and SNXJ may achieve protective effect by acting on MMP2 and NOX4,the core targets of anti-oxidative stress,through compounds such as Kaempferol and Quercetin.