Application Of Novel Screening And Microextraction Techniques In The Study Of Anti-cancer Active Components Of Flavonoids In Huangqisan

Objective:In this paper,a multichannel solid film microperfusion（MCSFMP）method was established based on human hepatoma Hep G2 cells,and the anti-cancer active components in the ancient Huangqisan prescription（Puerariae lobatae radix,Astragali radix and Mori cortex）were screened.And the screened active ingredients were concentrate,enrich and determine by PEG-NL hollow fiber liquid-phase microextraction（PEG-NL-HF-LPME）method combined with high performance liquid chromatography（HPLC）.Methods:（1）The first part of the paper:Using the polypropylene solid membrane with carrying Hep G2 cells as the screening platform,using automated multichannel microperfusion system to perfuse the cells for activity screening,the multichannel solid film microperfusion（MCSFMP）technique was established to realize multicomponents screening.The factors affecting activity screening were investigated and optimized in the experiment.The methodological investigation was carried out under the optimum screening conditions,and the anticancer active components in Huangqisan were analyzed and determined by high performance liquid chromatography.（2）The second part of the paper:on the basis of hollow fiber liquid phase microextraction（HF-LPME）,polyethylene glycol（PEG）modified nano-liposomes were prepared and filled into the wall holes of hollow fibers as extractant.A PEG-NL hollow fiber liquid phase microextraction（PEG-NL-HF-LPME）method was established,and it combined with high performance liquid chromatography technology to enrich,concentrate and determine the active components in Huangqisan.Results:（1）The optimal experimental conditions for MCSFMP:the polypropylene filter membrane acts as a solid film for carrying cells,7 m L Chinese herbal extract（3μg/m L）was used as the perfusion phase,and the screening time was 30 min.By comparing the chromatographic retention time and peak area of the screened active ingredients with the reference substance,8 anticancer active ingredients were preliminarily identified in Huangqisan,which were puerarin,calycosin glycoside,ononin,morin,calycosin,genistein,isorhamnetin and formononetin.The CFF_{Hep G2}of these active ingredients ranged from 1.1 to 19.8.（2）The optimal experimental conditions of PEG-NL-HF-LPME:the nanoliposomes were modified with PEG and loaded on polyvinylidene fluoride hollow fibers（pore size:0.2μm,inner diameter:0.7 mm）.The volume,p H and salt concentration of sample phase were 7.0 m L,3.0 and 20%,respectively.The extraction was performe by magnetic stirring at 1500 rpm for 2 h.The eluent volume was 80μL.Under these conditions,the EFs of the eight flavonoids in Huangqisan were between 4 and 106,the linear ranges were 0.002-4.444μg/m L with the good linear correlation coefficient（r≥0.9956）;the detection limits and quantification limits were respectively in between 0.0001-0.0025μg/m L and 0.0005-0.0083μg/m L;the RSDs values of intra and inter-day precision were 0.1%-8.8%and 0%-9.5%;the average recoveries ranged from 88.2%to 96.0%.Conclusions:In this paper,a multichannel solid film microperfusion technology combined with high performance liquid chromatography was established to screen the anticancer active components in Huangqisan.On this basis,the PEG-NL hollow fiber liquid phase microextraction method was used to enrich,concentrate and determine the content of the screened active ingredients.This study provides a new idea and method for the screening and analysis of complex matrices of traditional Chinese medicine,and has broad prospects for development.