Research On Major Antitumor Active Components In Zi-Cao-Cheng-Qi Decoction Based On Hollow Fiber Cell Fishing And Oil-in-salt Hollow Fiber Liquid Phase Microextraction With High Performance Liquid Chromatography

Objective:Hollow fiber cell fishing(HFCF)based on hepatoma HepG-2 cells,human renal tubular ACHN cells or human cervical carcinoma He La cells,coupled with high-performance liquid chromatography(HPLC),was developed and employed to research the major active components in Zi-Cao-Cheng-Qi decoction(ZCCQD)both in vitro and in vivo.A novel hollow fiber liquid phase microextraction based on oil-in-salt was proposed and the mechanism of this method was analyzed and discussed.The method was used to determine the contents and total plasma protein binding rates of the major active components screened out in vivo and in vitro from ZCCQD.The research preliminarily seeks the truly antitumor active components in ZCCQD,elucidate the traditional Chinese medicine(TCM)characteristics of multiple components and multiple targets and lay a foundation for expounding the antitumor efficacy material basis in TCM.Methods:(1)To screen the active components from ZCCQD in vitro,the hollow fiber seeded hepatoma HepG-2 cells,human renal tubular ACHN cells,or human cervical carcinoma He La cells was inserted into ZCCQD extract and the single TCM included in the formula respectively and the HPLC was utilized to identify the components.Then the plasma,liver or kidney tissue of rats was collected after orally administered ZCCQD extract,the cancer cell-seeded fiber was used to screen the active components from them,identifing the majorcomponents entered into the body,comparing and discussing preliminarily the binding capacity between active components and different cancer cells;(2)using the hollow fiber covered a thin salt membrane on the fiber wall pore filling organic solvent as the extraction platform,oil-in-salt hollow fiber liquid phase microextraction(oil-in-salt HF-LPME)was proposed and developed based on the optimization of conditions and methodological research.Coupling with HPLC,the method was used to enrich and determine the active components of ZCCQD both in vitro and in vivo and their total plasma protein binding rates.At the same time,compared with the traditional HF-LPME method,and based on this,the extraction mechanism of oil-in-salt HF-LPME was analyzed and expounded.Through the qualitative and quantitative study on the main active compoonents of ZCCQD in vivo and in vitro,the research laid a foundation for expounding the antitumor efficacy material basis in ZCCQD.Results:In our study,it can be observed through the studying anticancer active components of ZCCQD by HFCF and oil-in-salt HF-LPME coupling with HPLC:(1)the active components screened out by HFCF,such as hesperidin(48.62 μg/g),magnolol(3.09 μg/g),honokiol(6.87 μg/g),shikonin(0.34 μg/g),emodin(1.28 μg/g)and β,β′-dimethylacrylshikonin(0.38 μg/g),all can be quantified;(2)the six active components screened out in vitro can be absorbed into blood and their plasma concentration determined by oil-in-salt HF-LPME were 0.45,0.40,0.74,0.48,0.11 and 1.11 μg/m L respectively.In plasma,hesperidin,emodin and β,β′-dimethylacrylshikonin acted on HepG-2,ACHN and He La cell,shikonin acted on HepG-2 and He La cell,whereas honokiol only acted on ACHN cell;(3)hesperidin and emodin reached the liver and acted on HepG-2 cell,hesperidin,shikonin and emodin reached the kidney and acted on ACHN cell;(4)the total plasma protein binding rates of the six active compounds were 42.0%(hesperidin),71.8%(honokiol),64.6%(shikonin),77.7%(magnolol),75.3%(emodin)and 75.7%(β,β’-dimethylacrylshikonin),respectively.(5)oil-in-salt HF-LPME showed a significantly positive effect on the target analytes′ EFs.This may be because salt is covered on solventsurface to form the oil-in-salt membrane in which the salt concentration around the extraction solvent is higher,the distance between the salt and solvent is closer,and the degree of hydration film around the target analytes is lower than the salt is added in sample phase.These can significantly improve the microenvironment of the target analytes enriched and concentrated Conclusion:HFCF and oil-in-salt HF-LPME coupled with HPLC was successfully applied to the screening,characterization and determination of anticancer components in ZCCQD.It has been concluded that ZCCQD can inhibit HepG-2,ACHN and He La cells.The active components screened in vitro can be absorbed into the blood and reach the corresponding target tissues.The distribution of active components in plasma is not related to their contents in ZCCQD.Oil-in-salt HF-LPME showed no significant difference in accuracy and precision compared with the conventional HF-LPME,but oil-in-salt HF-LPME had higher EFs and better extraction efficiency.In conclusion,HFCF and oil-in-salt HF-LPME coupled with HPLC can be used to study anticancer bioactive components in TCM and provide a new idea and new method for studying the material basis of anticancer activity of TCM.