Study On The Correlations Between Metabolism Process And Anti-influenza Effect Of Total Flavonoids In Mosla Chinensis Maxim

Objective A HPLC method was established to determine the determination of luteolin and apigenin in rat plasma,and the pharmacokinetic characteristics of total flavonoids from Mosla chinensis Maxim in rats were studied on the basis of the HPLC method.Chose the inhibition rate of influenza virus proliferation and IFN-β,IL-2 and SOD secretion in MDCK cell infected with influenza virus as the pharmacodynamic indices,furthor study the pharmacodynamics.With the combination of pharmacokinetic and pharmacodynamic characteristics,the changes of anti-influenza effect of total flavonoids of Mosla chinensis Maxim with concentration and time were studied,and the relationship between the metabolic process of total flavonoids and the anti-influenza effect of Mosla chinensis Maxim was clarified.Methods（1）A HPLC method was established to determine the concentration of luteolin and apigenin in plasma of total flavonoids from Mosla chinensis Maxim,and its methodology was investigated with reference to the technical guidelines for non-clinical pharmacokinetic study of chemical drugs.（2）The plasma concentration of Mosla chinensis Maxim at different time points after oral administration of total flavonoids was determined by the established HPLC method.The pharmacokinetic was analyzed by the PKsolver software,and obtained the pharmacokinetic parameters of luteolin and apigenin.（3）Study on serum containing drugs at different time points,and chose the inhibition rate of influenza virus proliferation,IFN-β,IL-2 and SOD secretion in MDCK cell infected with influenza virus as the pharmacodynamic indices.Pharmacokinetic study was carried out on the serum containing drugs at different time points,and the related effect-time curves were drawn,and the relevant pharmacokinetic parameters were obtained by PKsolver software.（4）According to the AUC ratio of luteolin and apigenin,the weight coefficients of each component in the integration concentration were calculated,the Cint（integrated plasma concentration）of luteolin and apigenin at different time points were calculated.The pharmacokinetic analysis of integrated blood concentration-time was carried out by PKsolver software,obtained the relevant parameters.Establishment of logarithmic linear model by divergence point correlation method,the fitting equation and R²were obtained,and the correlation between the metabolic process of total flavonoids of Mosla chinensis Maximin vivo and the anti-influenza effect was discussed.Results（1）A HPLC method to determine the concentration of luteolin and apigenin in rat plasma was established successfully.The analysis conditions were as follows:Hypersil BDS C₁₈column（250 mm×4.6 mm,5μm）,the column temperature was30℃,the detection wavelength was 350 nm,the mobile phase consisted of 0.2%aqueous solution of phosphoric acid（A）and methanol（B）,the flow rate was 0.8m L/min,the injection volume was 20μL,and the elution methods were as follows:0～20 min,54%～47%（A）,20～25 min,47%～40%（A）,25～30 min,40%～54%（A）.The established HPLC method for the determination of luteolin and apigenin had good specificity,sensitivity,accuracy,stability,and the precision,accuracy,stability,recovery and medium effects were in accordance with the technical guidelines for the study of non-clinical pharmacokinetics of chemical drugs.（2）The PKsolver software was used to fit the atrioventricular and calculate the pharmacokinetic parameters.The results showed that the metabolic processes of luteolin and apigenin in normal rats were in accordance with one-compartment model.The peak time of luteolin was 124.11 min,the peak concentration of luteolin was0.8205μg/m L,the area under the drug time curve was 271.20μg/m L·min,and the peak time of apigenin was 104.98 min,the peak concentration was 0.9790μg/m L,the area under the drug time curve was 480.11μg/m L·min.（3）The serum containing total flavonoids of Mosla chinensis Maxim has a good inhibitory effect on influenza virus,and can promote the secretion of IFN-β,IL-2 and SOD.The pharmacokinetic process of anti-influenza effect in vitro indices of total flavonoids of Mosla chinensis Maxim could be described by one-compartment model.The main pharmacokinetic parameters were as follows:T_{1/2 appear,E},T_{1/2 eliminate,E}and AUE of influenza virus proliferation inhibition rate were 22.56 min,383.88 min and29392.82%·min.T_{1/2 appear,E},T_{1/2 eliminate,E}and AUE of IFN-βwere 25.44 min,361.89min,13893.45 pg/m L·min.T_{1/2 appear,E},T_{1/2 eliminate,E}and AUE of IL-2 were 41.31 min,191.81 min,133568.21 pg/m L·min;T_{1/2 appear,E},T_{1/2 eliminate,E}and AUE of SOD were26.51 min,367.80 min,15058.28 pg/m L·min.（4）Integrated pharmacokinetics of luteolin and apigenin in plasma of total flavonoids in Mosla chinensis Maxim fitted to one-compartment model.The result of correlation analysis showed that the integrated correlation of luteolin and apigenin was higher than single component,and there was a good positive correlation between the effective value of anti-influenza indices（E）and the natural logarithm integrative concentration ln（Cint）.Among all the effect indices,the fitting equation of inhibition rate of influenza virus proliferation was Y=231.47 ln（x）-383.06,and the R²was0.9670,which have the best correlation.The others were the secretion of IFN-βin MDCK cells infected by inflenza virus[Y=111.24 ln（x）-184.46,R²=0.9668],the secretion of SOD[Y=89.283 ln（x）-148.71,R²=0.9321],the secretion of IL-2[Y=1724.6 ln（x）-2969.8,R²=0.8209].For individual effect indiex,according to the R²,luteolin had a better correlation with the secretion of IFN-βin MDCK cells infected by inflenza virus（R²was 0.9536）,apigenin had a better correlation with the inhibition rate of influenza virus proliferation and the secretion amount of SOD in MDCK cells infected by inflenza virus（R²were 0.9546 and 0.9204,respectively）,whereas the correlation of luteolin and apigenin with the secretion amount of IL-2 were not so good,and the R²were 0.7851 and 0.7968.Conclusion（1）A HPLC method to determine the concentration of luteolin and apigenin in rat plasma was established.The method was characterized by high specificity,high sensitivity and good stability,the precision,accuracy,recovery rate and the medium effect et al were in accordance with the technical guidelines for the study of non-clinical pharmacokinetics of chemical drugs,which was suitable for the determination of luteolin and apigenin in rat plasma and the subsequent pharmacokinetic study.（2）The pharmacokinetic characteristics of luteolin and apigenin from total flavonoids of Mosla chinensis Maxim in normal rats were obtained.The pharmacokinetics of luteolin and apigenin in normal rats could provide evidence for the development and utilization of the total flavonoids from Mosla chinensis Maxim and provide a reference for the pharmacokinetics of the compound or preparation containing Mosla chinensis Maxim.（3）In this study,the pharmacokinetics and anti-influenza pharmacokinetic equations of total flavonoids of Mosla chinensis Maxim in rats were established.According to the R²,there was a good positive correlation between the effective value of anti-influenza indices（E）and the natural logarithm integrative concentration of C_int,C_Lutand C_Api.It was found that total flavonoids of Mosla chinensis Maxim could significantly inhibite the proliferation of influenza virus,and the mechanism of anti-influenza effect of total flavonoids in the serum of Mosla chinensis Maxim is related to its promotion of IFN-β,IL-2 and SOD secretion in MDCK cell infected by inflenza virus.（4）In this study,by using the method of integrating pharmacokinetics of multi-effector components with anti-influenza pharmacokinetics in vitro,the relationship between the in vivo generation process of total flavonoids from Mosla chinensis Maxim and the anti-influenza effect was investigated.It reflected the metabolic characteristics of the pharmacodynamic materials in vivo,and the correlation between pharmacokinetics and pharmacodynamics of effecting materials was also considered.It provided a method for the study of the correlation between pharmacokinetics and pharmacodynamics of multi-effector components.