Study On The Difference In Chemical Composition And Spectrum-effect Relationship Of Epimedium From Different Origins

Background and Objective:Epimedium is a traditional tonic herb with a wide distribution and a long history of use in China.It is effective in tonifying kidney yang,strengthening tendons and bones,and dispelling wind and dampness,and is commonly used clinically to treat osteoporosis,rheumatism,hypertension and heart disease.The chemical composition of herbal medicines is the basis for their efficacy,while the genetic information and environmental factors of the primitive can affect the biosynthetic pathways and metabolic pathways of the herbs,thus leading to different chemical compositions in the herbs,which in turn affect the clinical efficacy.The main characteristic components of Epimedium are flavonoids,which have strong antioxidant effects and can scavenge free radicals in the body,reducing the damage of oxidative stress and reducing the development of diseases.Quality evaluation and control of Chinese medicine has been the focus and difficulty of Chinese medicine research.The current quality evaluation method of Epimedium in the 2020 version of pharmacopoeia is to determine the content of total flavonol glycosides by chromatography,the measured components are single and not associated with the efficacy of Epimedium,so it is not possible to comprehensively evaluate the quality of Epimedium.Therefore,it is important to screen the potential active components closely related to the efficacy of Epimedium for the quality evaluation of Epimedium.In this study,we investigated the differences in chemical composition of Epimedium from different basal sources,combined with UPLC-Q-TOF/MS technique to reveal the differences in chemical composition of Epimedium from different basal sources,and explored the correlation between the chemical composition of Epimedium and antioxidant and protective effects on chondrocytes under oxidative stress environment based on the spectrum-effect relationship,which provided the pharmacodynamic material basis for the further development and utilization of Epimedium.Methods:1.A chemical composition histological study of different basal Epimedium herbs:The chemical components of Epimedium were comprehensively analyzed by UPLC-Q-TOF/MS technique,and principal components analysis（PCA）,orthogonal partial Least Squares-Discrimination Analysis（OPLS-DA）were used to screen the potential differential metabolites of Epimedium.Squares Discrimination Analysis（OPLS-DA）were used to screen the potential differential chemical components of different basal herbs of Epimedium and provide scientific basis for the specificity of the material basis of different basal herbs of Epimedium.2.Study on the antioxidant effect of Epimedium officinale from different origins:The antioxidant effects were evaluated by three assays,DPPH,ABTS and hydroxyl radicals,and the antioxidant potency composite（APC）was used to comprehensively evaluate the antioxidant activities of different basal sources of Epimedium.3.Study on the protective effect of different basidiogenic Epimedium herbs against oxidative stress damage in chondrocytes:Hydrogen peroxide（H₂O₂）was used to induce oxidative stress injury model in C28/I2 cells,and cell viability was used as an indicator to evaluate the protective effect of different basidiogenic Epimedium on chondrocytes damaged by oxidative stress4.Modeling of antioxidant spectrum-effect relationship of Epimedium:The mass spectral data of chemical components of Epimedium were used as independent variables,APC composite index and chondrocyte survival rate were used as dependent variables,and Grey Relational Analysis（GRA）and Partial least square（PLS）were used to establish the spectro-effective relationship model between chemical components of Epimedium and antioxidant and protection of chondrocytes damaged by oxidative stress,and to screen the active components of Epimedium related to antioxidant and protection of chondrocytes damaged by oxidative stress.5.Validation of the efficacy of antioxidant-related ingredients:In vitro experimental validation studies were conducted on chemical components highly associated with antioxidant and chondrocyte-protective effects.Three assays,DPPH,ABTS and hydroxyl radical,were used to verify their antioxidant capacity and CCK-8method was used to detect their protective effects on chondrocytes to verify the reliability of the results screened by the above model.Results:1.Chemical composition study of Epimedium herbs from different base origins:3935 common chemical composition characteristic ions were identified from Epimedium herbs,among which a total of 53 chemical components were identified by database and literature comparison.PCA and OPLS-DA analyses proved that there were significant differences in the chemical composition of Epimedium from different basal origins.Initially,M1697,M1723,M1875,M2324,M2404,M2524,M2657,M3411,M3749,M3809,M3842,M3846,M3875,M3932 were identified as potentially different components of different basal epimediums.This section provides data support for the study of the material basis of different basal epimedium herbs.2.Study on the antioxidant effect of different basal herbs of Epimedium:different basal herbs of Epimedium have certain ability to scavenge DPPH,ABTS and hydroxyl radicals,and the APC composite index ranged from 38.31 to 80.60%,indicating that different basal herbs of Epimedium have different antioxidant ability.3.Study on the protective effect of different basidiogenic Epimedium herbs against oxidative stress damage in chondrocytes:the C28/I2 cell line identified by toluidine blue staining was chondrocytes.the H₂O₂ concentration of 1.1 m M/L was determined by CCK-8 method to construct the oxidative stress model of C28/I2 cells,and the duration of action was 24 h.The administration group of 100μg/m L of Epimedium had a significant protective effect on chondrocytes compared with the model group,except for S3,S4,S7,S10,S14,S16,S17,S18,S20,S25,S31,S36,S10,S14,S16,S17,S18,S20,S25,S31,and S36 had no significant protective effect on chondrocytes,except for S3,S4,S7,S10,S14,S16,S17,S18,S20,S25,S31,and S36,the remaining 24 batches of Icariin had significant protective effect on chondrocytes with statistical significance（P<0.05）.Among them,S30（Korean Epimedium）had the strongest protective effect on chondrocytes,which proved that there may be differences in the protective effect of different basal Epimedium on chondrocytes damaged by oxidative stress.4.Modeling of the spectrum-effect relationship of Epimedium:Combining the results of GRA and PLS models,it was predicted that the main components influencing the difference in antioxidant activity of different basal sources of Epimedium might be M3809,M1750,M3701,M3097,M1921;The main components predicted to influence the difference in the activity of different basidiogenic Epimedium on the protection of chondrocytes damaged by oxidative stress may be M3809,M3844,M3846,M736,M3412,M366,and M1232;among them,M3809（Epimedin B）was the main active ingredient in Epimedium that was associated with both antioxidant and protecting oxidative stress-injured chondrocytes.Therefore,patchouli B,which is related to both antioxidant and chondroprotective effects,was selected for the subsequent efficacy study.5.Validation of the efficacy of antioxidant-associated ingredients:patchouli B has the ability to scavenge DPPH,ABTS and hydroxyl radicals in a dose-dependent manner.15 and 20μM of Epimedin B acting on oxidative stress-injured chondrocytes can significantly increase cell viability（P<0.05）,indicating its protective effect on chondrocytes,which verifies that the above-mentioned models screened with antioxidant and protection of oxidative stress-injured chondrocytes are reliable,The results of the chemical components related to antioxidant and oxidative stress-injured chondrocytes protective activities screened by the above model were reliable.Conclusions:In this study,the chemical composition of Epimedium was comprehensively analyzed and characterized using non-targeted metabolomics techniques to reveal the differential composition of Epimedium from different base sources.Meanwhile,the chemical constituents of Epimedium were combined with pharmacological effects based on the spectral effect relationship to explore the chemical constituents associated with antioxidant and oxidative stress chondroprotection activities.This study provides a data reference for the identification of Epimedium from different base sources and also provides a methodological reference for the quality evaluation of Epimedium.