Hollow Fibre Cell Fishing And Hollow Fibre Liquid Phase Microextraction With High Performance Liquid Chromatography For Studying Major Antitumor Components In Yinchenhao Decoction

Objective:Based on human hepatoma cell HepG-2 and human renal tubular cell ACHN,hollow fiber cell fishing?HFCF?and hollow fiber liquid phase microextraction?HFLPME?coupled with high performance liquid chromatography/ultraviolet detection?HPLC/UV?were developed and used for study of the major antitumor active components in Yinchenhao decoction formula?YCHD?and its constituent herbs,as well as their pharmacokinetics parameters.Methods:Before application,the various validations,e.g.,cell growth on the fiber inner wall,cell viability before and after screening,repeatability of the screened active components were investigated.The two hollow fibers separately seeded with HepG-2 and ACHN cells were sealed with cotton threads tied on their both ends and then inserted simultaneously into the sample vial to screening the major active components in herb or biological sample.Then we studied the major antitumor active components in YCHD,verified their synergetic or antagonistic effect,correlated their relationship between in vitro and in vivo and determined the major effective components.Furthermore,based on the optimization and methodological investigation of HFLPME-HPLC,the fiber filled with heptanol was sealed with cotton threads tied on its both ends and immersed in the sample vial containing herb or biological sample to extract the active components.Combined with HPLC,the distribution and content of active components from YCHD in vitro and in vivo were analyzed.Results:The HFCF-HPLC with HFLPME-HPLC showed that:?1?The major anticancer active components screened by HFCF-HPLC based on HepG-2 and ACHN cells were chlorogenic acid,geniposide,p-hydroxyacetophenone,crocin,and rhein,as well as their content in YCHD were 0.41±0.03,3.02±0.16,0.01±0.00,0.20±0.01 and 0.06±0.00 mg/g,respectively.?2?After intragastric administration of YCHD for 30 min,chlorogenic acid,geniposide and rhein could be absorbed into the blood and interacted with cancer cells;among them,geniposide and rhein could reach the liver and act on HepG-2 cells,chlorogenic acid and rhein could reach the kidney and act on ACHN cells.?3?The non-compartmental model pharmacokinetic parameters calculated by DAS 2.0 software of p-hydroxyacetophenone,crocin and rhein were as following:C_max is 0.35±0.06,0.78±0.07,2.95±0.76?g/m L;t_1/2 is 27.59±12.24,2.48±0.31,0.39±0.06 h;AUC_0-?is 11.09±3.41,2.90±0.26,2.24±0.58 h·?g/m L.Conclusion:?1?In vitro study found that the antitumor active components in YCHD were chlorogenic acid,geniposide,p-hydroxyacetophenone,crocin,and rhein.The types of active components in YCHD were more abundant than any its component herbs,and geniposide,crocin,and rhein exerted an obvious synergetic effect in YCHD compared with in the individual herbs.?2?In vivo study found that chlorogenic acid,geniposide and rhein could be absorbed into blood,reach the target organs and then act on HepG-2 or ACHN cells,suggesting that geniposide and rhein were the major effective components of anti-liver cancer,and chlorogenic acid and rhein were the major effective components of anti-kidney cancer.?3?The YCHD had multi-component and multi-target features,and the binding ability between the active components and the cells had nothing to do with theirIn summary,HCFC-HPLC is a simple,rapid,and reliable method that simultaneously screens active components from traditional Chinese medicine?TCM?.HFLPME-HPLC is a practical active components detection method with high purification ability and high sensitivity.HFCF-HPLC coupled with HFLPME-HPLC was successfully applied to screening and pharmacokinetic studying of the major antitumor active components of YCHD in vitro and in vivo.Our study provides a thought and valuable reference in probing the antitumor material basis of YCHD,as well as personally controlling its quality.