| Objective:Based on the Q-marker theory of traditional Chinese medicine,this paper intends to comprehensively identify the potential Q-marker in Scutellariae Radix from the aspects of screening the active ingredients and its pharmacological effects.Afterwards,it is proposed to establish a sample pretreatment method based on a new green solvent for quantitative analysis of some preliminarily identified Q-marker,which is to further improve the quality control system of Scutellariae Radix.Methods:With human breast cancer MCF-7 cells and prostate cancer DU145 cells as target cells,a hollow fiber cell fishing（HFCF）method was established to screen the bioactive components in Scutellariae Radix.Some important factors affecting the screening results were optimized by using a one-variable-at-a-time method,and the methodology evaluation was conducted under the optimal conditions,the potential Q-marker of Scutellariae Radix was preliminarily identified by HPLC analysis.The molecular mechanism of the anti-tumor effect of Scutellariae Radix was preliminarily explored by using the network pharmacology and molecular docking:the relevant data of Scutellariae Radix,breast cancer and prostate cancer were searched from databases such as TCMSP,Uniprot and OMIM,and analyzed by STRING and Metascape platforms.The Cytoscape 3.7.2 software was used to construct relevant networks and analyze their topological parameters.And the SYBYL-X2.0 software was used for molecular docking verification.The above results combined with HFCF experiment results could further identify the potential Q-marker in Scutellariae Radix.The switchable deep eutectic solvent prepared from alkanolamine and fatty acid was used as the extractant,the variables affecting the extraction efficiency were optimized and the methodology was investigated under the best conditions,then a switchable deep eutectic solvent-homogeneous liquid-liquid microextraction（s DES-HLLME）was developed to quantitatively analyze the identified potential Q-marker combined with HPLC.Fourier transform infrared spectroscopy,proton nuclear magnetic resonance spectroscopy and differential scanning calorimetry were applied to characterize the extraction phase,and the microextraction mechanism was analyzed according to the characterization results.Results:The optimal screening conditions of the HFCF experiment were as follows:polypropylene hollow fiber（inner diameter:0.6 mm,pore diameter:0.2μm）;sample phase concentration,50 mg/m L（MCF-7 cells）and 25 mg/m L（DU145 cells）;screening time,2.0h（MCF-7 and DU145 cells）;cell density,5×106 cells/m L（MCF-7 cells）and 2.7×106cells/m L（DU145 cells）;elution solvent,methanol（MCF-7 and DU145 cells）and reconstitution solvent,methanol（MCF-7 cells）and 70%ethanol（DU145 cells）.Both two target cells captured five potentially active compounds from all batches of Scutellariae Radix:scutellarin（t R=14.045）,baicalin（t R=16.038）,wogonoside（t R=18.302）,baicalein（t R=21.156）and wogonin（t R=23.105）;and scutellarein（t R=16.708）could be captured from the first batch of Scutellariae Radix.The results of network pharmacology and molecular docking showed that all the six active compounds screened by HFCF method had anti-breast and prostate cancer activities,which were preliminarily considered as potential Q-marker of Scutellariae Radix.It was concluded that the above compounds could regulate core targets such as PTGS2,HSP90AB1,AKT1 and AR,act on PI3K-Akt,p53,IL-17,MAPK and other signal pathways,participate in the positive regulation of cell death,response to hormone and other biological processes,thereby achieving the anti-breast cancer and prostate cancer effect.The optimal extraction conditions of s DES-HLLME were as follows:the switchable DES extraction phase,90μL of DMEA-heptanoic acid（1:1,n:n）;phase-switching trigger,100μL of 5.0 mol/L HCl;10%（w/v）of salt concentration in sample phase;extraction time,0.3 min.Under these conditions,the enrichment factors for six target analytes were between0.4 and 104.The calibration curves were linear（r≥0.9866）in the range of 0.033-8.65 mg/L for scutellarin,0.022-5.77 mg/L for baicalin,0.0033-0.865 mg/L for scutellarein and wogonoside,and 0.0022-0.577 mg/L for baicalein and wogonin,respectively.Low detection limits(≤8.0×10-3 mg/L)and quantification limits(≤2.4×10-2 mg/L)as well as good precisions（relative standard deviations lower than 9.2%）and acceptable accuracies（spiked recoveries 89.3%-114.4%）were also obtained.The average contents of scutellarin,baicalin,scutellarein,wogonoside,baicalein and wogonin in Scutellariae Radix were 7.7、278.1、0.12、36.5、1.5 and 1.0 mg/g,respectively.Conclusion:In this paper,the established HFCF,network pharmacology and molecular docking methods were successfully applied to preliminary screen and identify the Q-marker closely related to the pharmacological effect of Scutellariae Radix,and to reveal the molecular mechanism of multi-component,multi-target and multi-channel synergy of it,which provided ideas for the improvement and innovation of the quality control system of Scutellariae Radix.The proposed s DES-HLLME method was also successfully applied to the enrichment,purification and determination of the identified Q-marker,which has a good application prospect in the pretreatment of trace analytes in complex matrix samples. |
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