Application Of Hollow Fiber Cell Fishing And Supramolecular Solvent-based Liquid Phase Microextraction For Studying Q-markers Of Forsythiae Fructus

Objective:Based on the relevance between the ingredients of traditional Chinese medicine and its effectiveness,this paper seted to preliminarily screen and identify of Q-markers in Forsythiae Fructus,and quantitatively analyze them,in order to establish a scientific and reasonable quality control system for Forsythiae Fructus.Methods:Based on the TCMSP platform,Uniprot and CTD databases,the potential active components and targets of Forsythiae Fructus were collected and the types of disease treatment were predicted.The STRING and Metascape platforms were used to analyze the intersection targets.The relevant networks were constructed by Cytascape,and the potential Q-markers of Forsythiae Fructus were preliminarily predicted.The cancer cells were fixed on the inner wall of the hollow fiber,and placed in the sample phase solution prepared with the extract of Forsythiae Fructus,which was stirred for screening of potential Q-markers in Forsythiae Fructus.The primary factors affecting the screening results of hollow fiber cell fishing method were optimized and the methodology was investigated under the best screening conditions.Combined with HPLC,the potential Q-marker in Forsythiae Fructus was identified.A supramolecular solvent-based dispersive liquid-liquid microextraction was proposed for the extraction and determination of lignans in Forsythiae Fructus combined with HPLC.The extraction variables affecting the extraction results were optimized and the methodology was investigated under optimal conditions.Results:The TCMSP platform and Uniprot gene library were used to screen the potential active ingredients and targets of Forsythiae Fructus,and the CTD database was used to predict the potential therapeutic advantages of Forsythiae Fructus in the treatment of lung cancer and breast cancer.The“ingredients-targets-pathways-biological processes”network was constructed.It was preliminarily predicted that the potential Q-marker of Forsythiae Fructus were phillyrin,phillygenin,arctiin,quercetin,luteolin,wogonin and kaempferol.The best test conditions of HFCF method are as follows:concentration of Forsythiae Fructus extract,45 mg/m L;cell density,1.25×10~6/m L(MCF-7 cells),1.7×10~6/m L(A-549cells);screening time,1.5 h;elution solvent,acetonitrile;reconstitution solvent,methanol(30μL).Under this screening conditions,three lignans and six unknown components were fished by MCF-7 cells,and two lignans and two unknown components were fished by A-549 cells.According to the analysis results of HFCF and network pharmacology,we preliminarily established that phillyrin,phillyrin,arctiin and arctigenin are the potential lignans Q-markers of Forsythiae Fructus.It is speculated that it may play its role by interacting with core targets such as AKT1,TNF,TP53,BCL2,CCND1 and CDKN1A,regulating signal pathways such as cancer pathway,PI3K-Akt,proteoglycan in cancer,NF-kappa B and TGF-beta,and participating in biological processes such as hormone response,regulation of cell proliferation,regulation of apoptosis signal pathway,positive regulation of cell apoptosis and response to xenobiotic stimulation.The optimal conditions of SUPRAS-DLLME were as follows:extractant,5 mmo L/L of TBAB-hexanol supramolecular solvent,70μL;p H of sample phase,4.0;salt concentration,20%;extraction time,30 s;volume of sample phase,7 m L.Under the optimal conditions,the analytes’EF were between 6 and 170.Satisfactory linear ranges(r≥0.99),limits of detection(0.025-0.4 ng/m L),limits of quantitation(0.075-1.2 ng/m L)and precisions(<9.2%).The average recovery rate is between 96.5%and 104.8%.The average content of phillyrin,arctiin and philligenin in Forsythiae Fructus was 6.02,3.63and 0.081 mg/g,respectively.The arctigenin was not detected.Conclusions:Based on the core concept of Q-marker,and taking the relationship between the components and the effectiveness of Forsythiae Fructus as the research idea,this paper preliminarily predicted the Q-marker of Forsythiae Fructus through network pharmacology and hollow fiber cell fishing method,and revealed the mechanism of action of Forsythiae Fructus with multiple components and multiple targets,which provided a scientific basis for the establishment of its quality standard.The supermolecular solvent based dispersion liquid-liquid microextraction method was applied to the enrichment and determination of potential Q-marker in Forsythiae Fructus.