Pharmacodynamics And Pharmacokinetics Study Of Qingkailing Injection （Lyophilized） For CCl₄-induced Acute Hepatic Injury

Objective:（1）To study the protective effect of QKL injection on acute hepatic injury induced by CCl₄ in mice,and to explore them echanism of action.（2）To determine the baicalin,geniposide,cholicacid,hyodeoxycholic aicd and chlorgenic acid in CCl₄-induced acute hepatic injury models rat and normal rats plasma after i.p.of QKL injection,to clarify the regularity of blood drug concentration over time.Methods:（1）After administration of drug or solvent for seven days,the mice were intragastric administration with peanut oil of 0.2%（V/V）CCl₄ to establish the model of acute hepatic injury.The serum levels of AST and ALT were measured by biochemical method;the TNF-α,IL-1βand IL-6 were measured by enzyme-linked immunosorbent assay（ELISA）;the hepatic contents SOD、MDA、NO and NOS were measured by biochemical method.（2）The rats were sacrificed by i.p.of QKL injection,then the blood was collected at the corresponding time to determine by UPLC-MS/MS after treatment,Chromatographic separation was performed on a Waters ACQUITY UPLC?BEH C18 column（2.1 mm×100 mm,1.7μm）with a gradient mobile phase consisting of acetonitrile-water（containing 0.1%formic acid）.The compounds were ionized in the electrospray ionization ion source of the mass spectrometer and detected in MRM mode.The pharmacokinetics parameters were fitted and the differences of the disposal of several active ingredients in QKL injection in normal and CCl₄-induced acute hepatic injury rats were analyzed.Results:（1）Compared with the model group,the QKL injection could significantly decrease the serum activity of AST,ALT（P<0.01）,inhibit the expression of TNF-α,IL-1β,and IL-6 in serum（P<0.01 or P<0.001）,and enhance the activity of SOD,decrease the content of MDA,NO and NOS of l hepatic tissue（P<0.01 or P<0.001）.（2）The calibration curve of Baicalin,Geniposide,Cholic acid,Hyodeoxycholic acid,and Chlorogenic acid were linear in the range of 1.0650-260μg·mL^-1,0.1638-40μg·mL^-1,0.5734-140μg·mL^-1,0.9830-240μg·mL^-1,and 0.0819-20μg·mL^-1 in plasma（r≥0.99）.The recoveries obtained for five kinds of compounds were more than 70%,respectively.The validated method was successfully applied to the determination of content study of five kinds of compounds after i.p.to rats.The pharmacokinetic parameters in model group and normal group Rats were demonstrated as followed:the Tmax of Baicalin were 0.211±0.064 h and 0.145±0.035 h,the Cmax were96.938±8.68μg·mL^-1 and 100.773±13.422μg·mL^-1,the t_1/2 were over 4.0 h and 3.0 h,and the AUC were 178.417±32.261μg·h·mL^-1 and 124.54±19.094μg·h·mL^-1.The Tmax of Geniposide were 0.156±0.027 h and 0.159±0.056 h,the Cmax were11.477±2.194μg·mL^-1 and 12.237±2.31μg·mL^-1,the t_1/2 were 0.708±0.197 h and0.65±0.566 h,and AUC were 10.17±1.347μg·h·mL^-1 and 10.875±3.612μg·h·mL^-1.The Tmax of Cholic acid were 0.122±0.035 h and 0.078±0.017 h,the Cmax were105.243±10.519μg·mL^-1 and 86.909±8.386μg·mL^-1,the t_1/2 were 1.075±0.559 h and0.225±0.042 h,and AUC were 73.54±11.00μg·h·mL^-1 and 32.659±3.666μg·h·mL^-1.The Tmax of Hyodeoxycholic acid were 0.209±0.069 h and 0.17±0.048 h,the Cmax were 158.379±22.284μg·mL^-1 and 197.322±28.223μg·mL^-1,the t_1/2 were1.225±0.444 h and 0.187±0.057 h,and AUC were 108.836±14.641μg·h·mL^-1 and81.194±13.799μg·h·mL^-1.The Tmax of Chlorogenic acid were 0.167±0.092 h and0.181±0.034 h,the Cmax were 9.545±3.26μg·mL^-1 and 8.167±0.646μg·mL^-1,the t_1/2were 0.763±0.668 h and 0.497±0.221 h,and AUC were 8.077±4.691μg·h·mL^-1 and6.447±2.453μg·h·mL^-1.Conclusion:The QKL injection have a protective effect on CCl₄-induced acute hepatic injury in mice,and the mechanism of protecting hepatic function maybe related to anti-inflammation and anti-oxidation.Compared with the normal control group,the absorption of baicalin and cholic acid was delayed and the clearance was slowed down,the difference has statistical significance（P<0.05）,the absorption of hyodeoxycholic acid was attenuated,and the elimination was slowed down,the difference has statistical significance（P<0.01）,and there was no statistically significant difference in the pharmacokinetics of geniposide and chlorogenic acid in normal CCl₄ induced hepatic injury model rats.