Study On Mechanisms Of Drug Metabolism Of Smilax Glabrae Roxb

Two cytochrome enzymes,CYP3A4 and CYP2D6,which are closely related to the metabolism of human drugs,were used as targets respectively.Testosterone and dextromethorphan were used as probe substrates.The interaction between epicatechin,neoastilbin,astilbin,isoastilbin,engeletin,resveratrol in Smilacis glabrae Roxb（S.glabrae）and two enzymes was studied by ultra performance liquid chromatography and triple quadrupole mass spectrometry for the first time.When pH value of phosphate buffer solution was 7.4,incubation time was 120min,incubation temperature was 30℃,substrate concentration was 50μM,K_m was18.9 and 36.4μM,V_maxax was 0.02 and 0.20μM·min^-1for CYP3A4 and CYP2D6,respectively in vitro.Resveratrol showed the strongest inhibition of CYP3A4,followed by engeletin,astilbin,isoastilbin,epicatechin,neoastilbin and with IC₅₀values of 0.44,1.32,2.63,3.03,6.13,6.51μM.Neoastilbin showed the strongest inhibition of CYP2D6,followed by engeletin,epicatechin,resveratrol,isoastilbin,astilbin,with IC₅₀0 values of 1.48,2.87,5.58,6.71,11.87,14.16μM,respectively.Resveratrol had an irreversible inhibition on CYP3A4,but a reversible inhibition on CYP2D6.With the increase of incubation time and inhibitor concentration,resveratrol showed mechanical inhibition on CYP3A4.It was found that the other five components showed reversible inhibition on both enzymes.Epicatechin was a mixture and anticompetitive inhibitor of CYP3A4 and CYP2D6,neoastilbin and astilbin were noncompetitive inhibitors of CYP3A4 and CYP2D6,isoastilbin was a mixture and anticompetitive inhibitor of CYP3A4 and CYP2D6,engeletin was a mixture and competitive inhibitor of CYP3A4 and CYP2D6,respectively.The effects of two enzymes on the metabolic elimination rate of six active components in S.glabrae were further studied in this study.The results indicated that engeletin was the best substrate for CYP3A4 with K_m value at 0.79μM,followed by neoastilbin,epicatechin,isoastilbin,resveratrol and astilbin,K_m was 1.38,1.39,1.59,2.01 and 2.37μM,respectively.K_m at 1.20μM was obtained for epicatechin indicated that it was the best substrate for CYP2D6,followed by resveratrol,engeletin,neoastilbin,isoastilbin and astilbin with K_m value at 1.30,1.86,2.14,2.84,3.84μM,respectively.The metabolic elimination rate（Clint）of engeletin was the highest among 6 components in CYP3A4,Clint value was 1.42 min^-1.10^-2,followed by epicatechin,isoastilbin,resveratrol,neoastilbin and astilbin,Clint was 1.14,0.99,0.97,0.96,0.90 min^-1.10^-2,respectively.Clint of engeletin was the highest in CYP2D6,Clint value was 1.07 min^-1.10^-2,followed by isoastilbin,neoastilbin,epicatechin,astilbin and resveratrol,Clint was 1.05,0.93,0.83,0.78,0.76 min^-1.10^-2.An accurate and reliable method of ultra performance liquid chromatography coupled with a triple-quadrupole tandem mass spectrometer（UPLC-MS/MS）was firstly developed and fully validated for the simultaneous determination of epicatechin,neoastilbin,astilbin,isoastilbin,engeletin and resveratrol in rat plasma after administration of S.glabrae extract（1g/kg）.Naringenin was used as an internal standard（IS）.The method was validated in terms of selectivity,linearity,precision,accuracy,extraction recovery,matrix effect,and stability.The developed method was successfully applied to determine the main pharmacokinetic parameters of six components in rat plasma,such as C_max,T_max,T_1/2/2 and K_e,etc.