| Objective:In order to screen Q-markers in traditional Chinese medicine,this paper intended to propose a hollow fiber cell fishing method based on human breast cancer cell MCF-7,combined with high performance liquid chromatography-ultraviolet detection(HPLC-UVD)for the preliminary screening and identification of Q-markers in Scutellariae Barbatae Herba.The molecular mechanism of Scutellariae Barbatae Herba against breast cancer was preliminarily discussed by network pharmacology technology.To overcome the limitations of traditional extractants and improve the detection sensitivity,new extractant of reverse micelle was prepared.And the reverse micellar floating solidification liquid-phase microextraction method was proposed to be used for the simultaneous enrichment and purification of the screened Q-markers of flavonoids in Scutellariae Barbatae Herba.Methods:The hollow fibers with MCF-7 cells immobilized on the inner wall were placed in the sample solution prepared from the extract of Scutellariae Barbatae Herba,and stirred at37°C for screening;the factors affecting the screening were optimized,and the methodological investigation was carried out;under the best screening conditions,Q-markers in Scutellariae Barbatae Herba were preliminary screened and identified.In addition,relevant data of Scutellariae Barbatae Herba and breast cancer were collected through databases such as TCMSP,OMIM,Drugbank,and TTD,and the data were analyzed using platforms STRING and Metascape.Cytoscape software was used to construct a network of"active components of Scutellariae Barbatae Herba-targets of breast cancer-signal pathway-biological process",and the topological parameters of the nodes were used to preliminarily explore the molecular mechanism of Scutellariae Barbatae Herba’s anti-breast cancer effect.The reverse micelle of Aliquat 336/dodecanol as the extractant was injected into the sample phase(p H 4),and a cloudy solution was generated under magnetic stirring at 1000rpm and 40℃.After extraction,the reaggregated and floating reverse micelle above the sample phase were recovered through solidification for high performance liquid chromatography analysis.The synthesis of the reverse micellar extractant was investigated,and its microstructure was characterized by transmission electron microscope.The variables affecting the experimental procedure were optimized and methodology was investigated under the optimal conditions.The developed method was applied to analyze quantitatively the Q-marker of flavonoids in Scutellariae Barbatae Herba combined with HPLC-UVD.To further confirm the feasibility of the established method for the extraction of target compounds in complex samples,the results of the proposed method were compared with those of other reported methods.Results:Scutellariae Barbatae Herba has obvious inhibitory effect on the proliferation of MCF-7 cells.The best screening conditions for the hollow fiber cell fishing method were as follows:hollow fiber type,PP(0.6 mm,i.d.;0.2μm,p.s.);sample phase concentration,25μg/m L;screening time,1.5 h;cell density,5×10~6/m L;elution solvent,methanol;reconstitution solvent,methanol.The number and area of chromatographic peaks of active compounds screened by hollow fibers with fixed cells in the inner wall,were significantly larger than those of hollow fibers perfused with culture fluid.Under this screening and measurement condition(335 nm/277 nm),four known flavonoids of scutellarin(t _R 8.769),scutellarein(t _R 11.275),luteolin(t _R 13.354),apigenin(t _R 15.333),and four unknown components X1(t _R 10.439),X2(t _R 15.012),X3(t _R 16.255)and X4(t _R16.396).According to the analysis results of hollow fiber cell fishing and network pharmacology,we preliminarily identified scutellarin,baicalin,scutellarein,luteolin,apigenin and wogonin as potential Q-markers of Scutellaria barbata;and speculated that they may act on cancer pathways,breast cancer,cell cycle,PI3K-AKT,MAPK,proteoglycan,P53,HIF-1 and other signaling pathways by regulating core targets(such as AKT1,TP53,CCND1,EGFR,CDK2 and PI3KCG),and participate in the responses to xenogeneic stimuli,regulation of kinase activity,regulation of mitotic cell cycle regulation,growth regulation,protein phosphorylation,development of reproductive structures,negative regulation of cell population proliferation,cellular senescence and other biological processes,thereby exerting an anti-breast cancer effect.The optimal experimental conditions of the reverse micellar floating solidification liquid-phase microextraction method were as follows:extractant,40μL 5 mmo L/L of Aliquat 336-dodecanol reverse micelle;sample phase p H,4.0;extraction temperature,40°C;extraction time,10 min;stirring rate,1000 rpm.Under the optimum conditions,enrichment factors between 155 and 436 for the target analytes,satisfactory linear ranges(r≥0.9983),low detection limits(0.05～0.25 ng/m L),acceptable precisions(relative standard deviations of 0.2%～8.9%)and accuracies(recoveries of 87.2%～108.5%)were obtained.The average contents of scutellarin,scutellarein,luteolin and apigenin in Scutellariae Barbatae Herba were 6.99,0.92,0.16 and 0.19 mg/g,respectively.Conclusions:In this paper,a hollow fiber cell fishing method was established,which provided a fast,efficient and convenient method for the identification of Q-marker in traditional Chinese medicine.The molecular mechanism of Scutellariae Barbatae Herba against breast cancer was preliminarily explored by network pharmacology method,which provides theoretical basis for the clinical application of Scutellariae Barbatae Herba.The developed reverse micellar floating solidification liquid-phase microextraction approach was successfully applied for the concentration and purification of six Q-markers of flavonoids with different polarities from a traditional Chinese medicinal herb of Scutellariae Barbatae Herba. |
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