Activity Determination Of CYP1A2,CYP2C9 And CYP2E1 And Effects Of Chinese Medicine Injection On Activities Of The Three Drug-metabolizing Enzymes In Human Liver Microsomes

Objective: To evaluate the influences of the Chinese Traditional Medicine injections including troxerutin and cerebroprotein hydrolysate injection,sofren injection,safflower yellow injection,and panax notoginseng saponins injection on CYP1A2,CYP2E1,and CYP2C9 activities.Experimental methods:(1)Phenacetin,diclofenac,and chlorzoxazoneas were selected as the probe drugs to determine CYP1A2,CYP2C9,and CYP2E1,respectively in vitro in the human liver microsomal incubation system.The influence of the metabolites of the probe drugs on the concentrations of acetaminophen,4-Hydroxydiclofenac and 6-Hydroxychlorzoxazone via calculating Km and Vmax by using HPLC were carried out.(2)Troxerutin-cerebroprotein hydrolysate injection in different concentration,sofren injection in different volume percentage concentrations,safflower yellow injection,and panax notoginseng saponins injection with different concentrations into the established system,measuring the concentrations of metabolites and calculating the half inhibitory concentrations(IC50).Results:(1)Phenacetin,diclofenac,and chlorzoxazone and their internal standards had standard peak types and were well separated from their metabolites.The linear concentration ranges of acetaminophen,4’-Hydroxydiclofenac,and 6-Hydroxychlorzoxazone were 1.00 ~ 50.0 μmol·L-1,0.50 ~ 30.0 μmol·L-1 and 0.50 ~ 30.0 μmol·L-1,respectively.Both intra-day and inter-day precision were less than 10% and the accuracy was 85% ~ 115%.Treatment recovery rate was higher than 85% and the stability was less than 5%.(2)Investigation on enzyme kinetics.The kinetic parameters Km and Vmax of acetophenetidin were 93.0 μmol·L-1 and 0.99 nmol·min-1·mg protein-1,respectively.The kinetic parameters Km and Vmax of diclofenac were 18.2 μmol·L-1 and 1.42 nmol·min-1·mg protein-1,respectively.The kinetic parameters Km and Vmax of chlorzoxazone were 45.7 μmol·L-1 and 0.65 nmol·min-1·mg protein-1,respectively.(3)IC50 determination.The IC50 value of sofren injection on CYP1A2 and CYP2C9 were 10.8% and 75.5%,respectively,demonstrating that the inhibition exists.No significant inhibitory effects on CYP2E1 were observed.The IC50 value of troxerutin cerebro-protein hydrolysate injection on CYP2E1 was 33.4%,implicating the existence of inhibition;no significant inhibitory effects on CYP1A2 and CYP2C9 were observed.The IC50 value of safflower yellow injection on CYP2C9 was 574.6 μmol·m L-1,indicating that the inhibition exists;no significant inhibitory effects on CYP1A2 and CYP2E1 were observed.The IC50 values of panax notoginseng saponins for injection on CYP1A2 and CYP2C9 were 68.6 μg·ml-1 and 9μg·ml-1,respectively,showing the existence of inhibition;no significant inhibitory effects on CYP2E1 were observed.Conclusions: The results indicated that under the therapeutic concentration,sofren injection exerted inhibitory effects on CYP1A2 and CYP2C9 but did not exert significant inhibitory effects on CYP2E1.The other three traditional Chinese medicine injection did not significantly influence the activities of CYP1A2,CYP2E1,and CYP2C9.