Objective:Hollow fiber cell fishing with high performance liquid chromatography(HFCF-HPLC)was utilised to screen and identify the anticancer coumarins in vitro(Radix angelicae dahuricae extraction)and in vivo after the oral administration of the herb.Moreover,hollow fiber liquid phase microextraction with HPLC(HFLPME-HPLC)was developed and applied to detect and quantify the coumarins screened by HFCF in vitro and in vivo and their pharmacokinetics.Combine anticancer compounds screening in vivo and in vitro,content determination and multi-compounds pharmacokinetic parameters of Radix angelicae dahuricae to correlate the relationship between in vitro and in vivo results,seek the truly effective anticancer coumarins,lay a foundation for expounding the anticancer efficacy material basis in Radix angelicae dahuricae.Methods:(1)Various operation parameters of HFCF-HPLC,sucn as cell growth states and cell survival rates of human ovarian cancer SKOV3 cells,human renal tubular ACHN cells,human hepatoma HepG-2 cells on the inner walls of fibres,effect of ethanol concentration on Hep G-2 cells survival rate,binding between fibre active centres and coumarins and repeatability were investigated.Under the optimized conditions,the coumarins of psoralen,bergapten,oxypeucedanin,imperatorin and isoimperatorin were selected as model compounds,hollow fibre seeded with SKOV-3,ACHN,HepG-2 cells and Hep G-2 cell membranes,cell organelles was inserted into the Radix angelicae dahuricae alcohol extraction,water decoction and the plasma,liver and kidney tissue homogenates after the oral administration of the herb to screen the anticancer coumarins and preliminary clear the binding ablility and binding site of the coumarins with the three kinds of cancer cells.(2)Various operation parameters of HFLPME-HPLC,such as extraction solvent,sample phase pH,extraction time and stirring rate were optimised.Linear ranges,limits of detection,limits of quantitation,precisions and recoveries of the method were invest igated.Under the optimized conditions,the coumarins of psoralen,bergapten,oxypeucedanin,imperatorin and isoimperatorin were selected as model compounds,HFLPME-HPLC was developed and applied to detect and quantify the trace levels of coumarins screened by HFCF in vitro and in vivo and their multi-compounds pharmacokinetics.Base on the screening of active compounds in vivo and in vitro,content determination and multi-compounds pharmacokinetic parameters,this study research the anticancer coumarins and anticancer efficacy material basis in Radix angelicae dahuricae.Results:HFCF and HFLPME with HPLC were used to investigate the bioactive anticancer coumarins in RAD.The results showed that:(1)xanthotoxin,xanthotoxol,psoralen,bergapten,oxypeucedanin,imperatorin and isoimperatorin in Radix angelicae dahuricae were screened by the three kinds of cancer cells;the average contents of the five coumarins known reference substance in Radix angelicae dahuricae extract were 1.36 ?g/g(psoralen),3.05 ?g/g(bergapten),22.30 ?g/g(oxypeucedanin),7.78 ?g/g(imperatorin),2.83 ?g/g(isoimperatorin),respectively;(2)all seven active coumarins in vitro were absorbed into blood;their mean peak concentrations after oral administration of Radix angelicae dahuricae were 0.36 ?g/mL(psoralen),0.23 ?g/mL(bergapten),0.08 ?g/mL(oxypeucedanin),1.20 ?g/mL(imperatorin),0.47 ?g/mL(isoimperatorin),respectively;among them,xanthotoxol,xanthotoxin,bergapten and imperatorin were fished by the two cancer cells;(3)in kindey,xanthotoxol,xanthotoxin,bergapten and imperatorin were screened by ACHN cells;xanthotoxol,xanthotoxin,psoralen(0.72 ?g/g),bergapten(0.46 ?g/g),imperatorin(0.22 ?g/g),isoimperatorin(0.62 ?g/g)were detected and quantified;(4)in liver,only xanthotoxol and xanthotoxin were screened by Hep G-2 cells;xanthotoxol,xanthotoxin,oxypeucedanin(1.03 ?g/g),imperatorin(0.41 ?g/g)and isoimperatorin(0.10 ?g/g)were detected and quantified;(5)HFLPME-HPLC was introduced for simultaneously researching the pharmacokinetics and the pharmacokinetic parameters of five coumarins,i.e.psoralen,bergapten,oxypeucedanin,imperatorin and isoimperatorin,in rat after oral administration of Radix angelicae dahuricae extract.Conclusion:HFCF-HPLC,coupled with HFLPME-HPLC has applied successfully to research the anticancer compounds screening,content determination and multi-compounds pharmacokinetics of the trace levels of coumarins in Radix angelicae dahuricae.Under this experimental condition,xanthotoxin,xanthotoxol,psoralen,bergapten,oxypeucedanin,imperatorin and isoimperatorin were the active anticancer coumarins screened in vitro,and their binding ability with the cancer cells had nothing to do with their content in Radix angelicae dahuricae;the screening results in vitro and the results in vivo had certain differences;xanthotoxin,xanthotoxol,bergapten and imperatorin were screened by the two kinds of cell from drug-containing plasma;in kindey,xanthotoxol,xanthotoxin,bergapten and imperatorin were screened by AC HN cells;in liver,only xanthotoxol and xanthotoxin were screened by HepG-2 cells;and those active coumarins keep effective concentration in target tissues;the results showed that the trace levels of coumarins in Radix angelicae dahuricae had dramatic cancer sites,anti-ACHN,and anti-Hep G effect;but the time to peak of the five coumarins were similar,this phenomenon may result in the accumulation effect of active compounds.In addition,HFCF-HPLC,coupled with HFLPME-HPLC can be used to screen and find the active compounds(especially the trace levels of active compounds)in vivo and in vitro.The combination of the two methods provides us novel ideas and novel methods of anticancer efficacy material base. |