Application Of Novel Screening And Liquid Phase Microextraction Methods In The Study Of Flavonoids Anticancer Active Components

Objective:This paper proposed a solid membrane filtration three-way microfluidic cell screening method,using the human ovarian cancer SKOV3 cells hold in solid membrane to screen anti-cancer flavonoids from traditional Chinese medicines(TCMs),which may be related to human ovarian cancer,and according to the screening of flavonoids,a phosphatidylcholine supramolecular solvent dispersive liquid microextraction(DLPME)combined with high performance liquid chromatography(HPLC)method was established and applied to extraction,enrichment and quantitative determination of flavonoids in three TCMs(Ginkgobiloba,Platycladus orientalis,Scutellaria)and in biological samples(human plasma).Methods:(1)In the first part of this paper,using a three-way microfluidic cell screening device with human ovarian cancer SKOV3 cells as screening platform,a new screening method was established: solid membrane filtration three-way microfluidic cell screening technology,and it combined with HPLC was apply to screening of a variety of anti-cancer active ingredients from three kinds of TCMs.By comparing the chromatographic retention time with the referencesubstance,some of the six flavonoids(myricetin,quercetin,isorhamnetin,baicalein,wogonin,kaempferide)were screened and identified.Experiments on a variety of screening condition were optimized,including the type of solid membrane and its load function to SKOV3 cell,the influence of the methanol concentration for cell survival,cell mitochondria membrane potential changes,differences of cell survival ratio before and after screening experiment,negative and positive control experiment,screening of reproducibility,etc.,and cell fishing factor(CFF)of flavonoids were calculated to evaluate screening efficiency.(2)In the second part of this paper(I),a phosphatidylcholine supramolecular dispersive liquid microextraction(PCSS-DLPME)was developed to concentrate six flavonoids found in cell screening results.It used a supramolecular solvent formed by phosphatidylcholine/chloroform as the extraction solvent.A quantitative analysis method for the extraction and determination of the 6 flavonoids in TCMs was established combined with HPLC.The important parameters affecting the enrichment factors(EFs)of the six target analytes in the experiment,such as extraction conditions,were optimized,and the methodology was verified,and the extraction mechanism of this method was clarified.In the second part of this paper(II),six flavonoids in human plasma were extracted,concentrated,enriched and determined by phosphatidylcholine supramolecular solvent dispersive liquid phase microextraction combined with high performance liquid chromatography.Several key parameters affecting the EFs were optimized,and the methodological parameters were verified.The extraction results of six flavonoids in two different matrices were compared and the influence of matrix was discussed.In the second part of this paper(III),PCSS-DLPME was compared with other extraction methods,and its characteristics are analyzed.Results:(1)In the first part of the experiment: six flavonoids were preliminarily identified by comparing with the retention time of the reference substances,including myricetin,quercetin,isorhamnetin,baicalein,wogonin,and kaempferide.In optimizing the screening process,it was found that solid membrane in screening device had a greater influence on the the screening efficiency,among which the polycarbonate membrane had the least adsorption interference on active compounds,and it had a better load-effect on SKOV3 cells by the scanning electrostatic mirror,so it was used as a load-membrane.In negative and positive control experiments,the good reliability of the method was relized,and the CFFs of the six flavonoids were ranged from 2.2 to 12.5.(2)In the second part of the experiment(I): the EFs of six flavonoids in TCMs for PCSS-DLPME were between 107.6-205.3;The linear ranges were 0.002-0.8 μg/m L for myricetin and quercetin,0.001-0.7 μg/m L for isorhamnetin,baicalein,wogonin,and kaempferide;the LODs were between 0.15-0.5 ng/m L;The intra-day precisions(RSDs)and inter-day precisions(RSDs)were respectively: 1.8%-8.9%,1.7%-11.2%,and the average recoveries were between 87.6%-104.4%.In the second part of the experiment(II): the EFs of six flavonoids in human plasma for PCSS-DLPME were between 39.6-191.8;The linear range were 0.06-9 μg/m L for myricetin and quercetin isorhamnetin,0.005-8 μg/m L for baicalein,wogonin,and kaempferide;the LODs were 4-60 ng/m L;The intra-day precisions(RSDs)and inter-day precisions(RSDs)were respectively: 2.0%-9.8%,0.6%-7.2%;The average recoveries were between 87.0%-99.5%.In the second part of the experiment(III): compared with other methods,PCSS-DLPME has the advantages of simple operation,simple preparation of supramolecules,low detection limits,high sensitivity and short extraction time.Conclusion:In this paper,a solid membrane filtration three-way microfluidic cell screening method combined with high performance liquid chromatography was established to screen six flavonoids in TCMs.On this basis,a phosphatidylcholine supramolecular solvent dispersive liquid phase microextraction method combined with HPLC was established for the analysis and determination of 6 flavonoids active components screened,and their extraction behavior in different matrices,such as water matrix and plasma matrix was compared,and clarify the extraction mechanism.This method has the advantages of simple operation,rapid operation,low cost and easy availability of materials,high enrichment efficiency and high sensitivity.It has a broad application prospect in the screening and quantitative determination of trace active components of TCMs.