Methodological Study On Rapid Evaluation Of Flavonoid Content In Epimedii Folium Germplasm Resources

Epimedium Folium was first written in the Shen Nong’s Classic of the Materia Medica（Shén Nóng B?n C?o Jīng）,which traditionally has the functions of tonifying kidney and strengthening yang,dispelling wind and dampness,strengthening muscles and bones.Modern studies have shown that Epimedium has many pharmacological effects in the treatment of hypertension,coronary heart disease,osteoporosis,climacteric syndrome,impotence,hemiplegia,limb numbness and so on.In the 2015 edition of Chinese Pharmacopoeia,there are five sources for the use of Epimedium L.-Epimedium brevicornu Maxim.,E.sagittatum（Sieb.et Zucc.）Maxim.,E.pubescens Maxim.,E.koreanum Nakai and E.wushanense Ying.Epimedium is rich in chemical components,mainly flavonoids,and flavonol glycosides with isopentenyl structure in flavonoids are the main active components of Epimedium.Before the 2015 edition of Chinese Pharmacopoeia,epimedium C and icariin were defined as the quality control index of Epimedium.In the publication of the 2020 edition of Chinese Pharmacopoeia,it is proposed that the total contents of Epimedium A,B,C and Icariin are used as the quality control standard of Epimedium.Objective:At present,there are many methods to determine the content of flavonoids in Epimedium.In Chinese Pharmacopoeia,the content of total flavonoids in Epimedium was determined by UV method,and several monomer compounds were determined by HPLC method.HPLC and UV methods are mature and accurate in content determination,but the experimental period is long and the consumption of reagents and medicinal materials is large.Therefore,based on the two mature methods of HPLC（UPLC）and UV,a rapid evaluation method for the content of flavonoids in Epimedium was established by Multiplex Research（MX）method or FTIR method.It is used for the selection of germplasm resources and the determination of a large number of samples in variety selection,in order to establish a technical method for the medicinal production and application of Epimedii Folium.Method:In this study,E.sagittatum（Sieb.et Zucc.）Maxim.of Hunan provenance（HNJY）and Guizhou provenance（GZJY）,E.pseudowushanense B.L.Guo（NWS）and E.pubescens Maxim.（RM）were selected as experimental subjects.Firstly,the main components were qualitatively analyzed by UPLC-Q-TOF MS,and the main components were determined for follow-up quantitative analysis.MX method:the flavonoid content of 63 HNJY fresh leaves of Epimedium was determined by MX’s flavonoid index（FLAV）.The total flavonoid content of leaves was determined by the sum of front and back FLAV index values,and the total flavonoid content determined by UV and HPLC were analyzed by linear regression analysis.FTIR method:the leaf powder of Epimedium sample（HNJY-71,GZJY-40,NWS-19）was scanned by FTIR method,and the content of Epimedium was determined by UPLC method.Three different quantitative models were established by PLS stoichiometry.60 calibration set samples and 11 verification set samples of HNJY were selected to establish the quantitative model of the sum of four components（ABCI%）and the sum of six components of rhamnose icariinⅡand icariinⅡ（TF%）.And select 90 correction sets of HNJY,GZJY and NWS and 20 samples of verification set to study the quantitative model of ABCI%,select the best fitting model and calculate the prediction accuracy.Results:Qualitative analysis of HNJY,GZJY,RM and NWS Epimedii Folium samples by UPLC Q-TOF MS showed that 31 components were identified,including 29 flavonoids（including 24flavonoids with isopentenyl structure）,1 alkaloid and 1 organic acid.The main components of each species were Epimedium A,B,C,Icariin,rhamnosideⅡand IcariinⅡ.Among them,GZJY increased the content of Epimedium A₁and NWS decreased IcariinⅡ.In the experimental results of rapid determination of flavonoids in Epimedium by MX,the correlation between MX&HPLC,MX&UV was less than 40%,and the prediction accuracy was low.Three quantitative models constructed by FTIR method for rapid determination of flavonoid content in Epimedium germplasm resources,quantitative model 1 of ABCI%percentage content of HNJY Epimedium samples,obtained the correction model R²=0.997,SEE=0.116 and SEP=0.135,the linear equation was Y₁=0.9973X₁+0.0231,and the average prediction accuracy of correction set and verification set samples were 98.985%and 96.087%,respectively;The second model is the TF%quantitative model of HNJY Epimedium samples.The correction model R²=0.997,SEE=0.119,SEP=0.137,the linear equation Y₂=0.997X₂+0.0245,the average prediction accuracy of correction set and verification set samples are 98.998%and 94.771%,respectively.The third model is the ABCI%quantitative model of HNJY,GZJY and NWS Epimedium samples.The calibration model R²=0.997,SEE=0.166 and SEP=0.173.The linear equation is Y₃=0.9970X₃+0.0223.The average prediction accuracy of calibration set and verification set is97.471%and 92.36%,respectively.Conclusion:In this study,the correlation between the determination of flavonoids by MX and HPLC,MX and UV did not get the expected results.The possible reasons are as follows:（1）the composition of secondary components in the leaves of Epimedii Folium is complex and contains a large number of other components that can be absorbed under 365nm;（2）Icariin flavonoids may also exist in mesophyll,which is not suitable for the determination of epidermis by MX method.3.There are differences in the composition of fresh leaves determined by MX and dried leaves determined by HPLC and other methods.FTIR method combined with UPLC method to explore the flavonoid content of Epimedium germplasm resources and achieved good results.The average prediction accuracy of the predicted value and actual value of the three quantitative models were all more than 97%.The average prediction accuracy of the predicted value and the actual value of the verification set is more than 94%for the models based on the same species,and more than 92%for the models based on different species.For the verification samples of Epimedium kweichuensis,the accuracy is lower,the average is 82%,while the other two species are 94%.The reason for the analysis is that the chemical composition is different,so the production model is used.The accuracy of prediction can be improved by using the same or similar groups as far as possible.