Determination Of Multi-components And Attribution Of Fingerprint Peak From Shuanghuanglian Injection Based On "Prediction And Verification Of Active Components"

Shuanghuanglian injection,consisting of Lonicerae japonicae Flos,Forsythiae Fructus and Scutellariae Radix,is an injection prepared by modern technology.In this study,the active compounds in Shuanghuanglian injection were preliminarily screened based on network pharmacology;the antioxidant components in Shuanghuanglian injection were characterized by combining oline antioxidant activity screening with mass spectrometry analysis;twenty-two chemical components were quantified by UPLC chromatography;through the characterization and attribution for common chromatographic peaks of Shuanghuanglian injection using fingerprint method,the correlation among herbs,intermediates and shuanghuanglian injection was investigated.The main compounds in Lonicerae japonicae Flos,Forsythiae Fructus and Scutellariae Radix were searched in the traditional Chinese medicine database and some related literatures.With the help of TCMSP,the disease-drug targets were mapped,finally getting the potential targets of Shuanghuanglian injection for disease intervention.Through the integration of related information in TCMSP,Gene Cards,String,Uniprot and other databases,the relevant active ingredients in Shuanghuanglian injection were screened,and a “drug target-disease,active-chemical composition” network was simultaneously established.Furthermore,by binding the pharmacodynamic function with the ingredients,the active components associated with pharmacodynamic function in Shuanghuanglian injection were obtained.The antioxidant components in Shuanghuanglian injection were verified by online antioxidant activity analysis.DPPH·-Oline/UPLC-PDA method was established to screen antioxidant components in Shuanghuanglian injection by using UPLC-PDA multi-wavelength switching method.Relying on the changes of related chromatographic peak area before and after the reaction between Shuanghuanglian injection and DPPH·,the components with antioxidant activity were screened out quickly.Thirty-one compounds of Shuanghuanglian injection were preliminarily identified through liquid chromatography-mass spectrometry,among which 22 compounds were determined by comparison of reference substances.The components with antioxidant activity were forsythoside E,caffeic acid,isoforsythiaside,forsythoside A,isochlorogenic acid A,baicalein and wogonin,basically consistent with the results of the antioxidant components from virtual screening.Through UPLC-PDA multi-wavelength switching method coupled with proportional dilution method,22 chemical components in Shuanghuanglian injection were quantified,which realized the simultaneous determination of components varying significantly in contents in complex traditional Chinese medicine.Verified by systematic methodology,the analytical method had high precision,stability and feasibility,and was applied to the multi-component quantification for 20 batches of Shuanghuanglian injection.The results indicated that the 20 batches of Shuanghuanglian injection samples produced in 2018 and2019 were stable in contents of components,with perfect consistency between batches,and the contents of the 22 components in Shuanghuanglian injection accounted for 38.7% of the total solid content.In order to further explore the rule of chemical composition transfer in the manufacturing process of Shuanghuanglian injection,the correlation analysis of Shuanghuanglian injection,intermediate and raw materials was performed,and the fingerprint peaks of Shuanghuanglian injection and intermediate were characterized and assigned,through the fingerprint method analysis of several batches of Shuanghuanglian injection,honeysuckle and forsythia intermediate,scutellaria intermediate and raw materials(honeysuckle,forsythia and scutellaria).The results showed that neochlorogenic acid,chlorogenic acid,cryptochlorogenic acid,caffeic acid and isochlorogenic acids A and C in Shuanghuanglian injection and intermediate were derived from honeysuckle;caffeic acid,forsythiaside A and Isomer of forsythiaside A were derived from forsythia;oroxylin A-7-O-β-D-glucuronide and baicalin were from scutellaria.Aiming at the key problems in the quality research of traditional Chinese medicine injection,this study established the research method based on “active ingredient prediction-active ingredient verification-multicomponent quantification-fingerprint peak attribution and characterization”,improving the quality control method for Shuanghuanglian injection,and provided scientific evidence for the safe application of Shuanghuanglian injection clinically.