Study On The Sabia Parviflora Absorbed Into Plasma And Pharmacokinetics Of H4

Sabia parviflora is a common medicinal plant,the main medicinal parts are stems and leaves,most of which grow in southwest China,and are known as"small yellow medicine"and so on.Sabia parviflora is widely used in clinic and has remarkable pharmacological activity,but the material basis of its role in clinical efficacy has not been clarified.To analyze the blood components of Sabia parviflora extract in rats by UHPLC-Q-TOF-MS/MS.Liquid phase analysis method:the mobile phase was 0.1%formic acid water（A）-acetonitrile（B）,gradient elution,and the elution conditions were 0.1～2 min,5%B;2～20 min,5%～21%B;20～35 min,21%～95%B;35～37 min,95%B;37～37.1 min,95%～5%B;37.1～40 min,5%B,the injection volume 5μL,and the flow rate 0.3 m L/min.Mass spectrometry analysis method:electrospray ion source（ESI）,scanning in both positive and negative ion modes,scanning range m/z 50～1250,data acquisition time40min.Through the analysis and identification of plasma samples of blood ingredient,a total of 68 compounds were identified,including isocitrate,Azelaic acid,Oleamide,Liriodenine,1-[2-（dimethylamino）ethyl]-3,4-phenanthrenediol-4-O-β-D-glucoside,as well as 34 metabolites such as Protocatechuic acid,Tuduranine,Isoboldine,Syringic acid and Vanillic acid.The reaction pathway involves methylation,glucuronidation and sulfation.In this study,a UPLC-MS/MS quantitative analysis method was established to detect the content of H4 in rat plasma,with magnolia as the internal standard compound.The established method was investigated in all aspects,including specificity,minimum quantitative limit,accuracy,extraction recovery,matrix effect and stability.Liquid phase analysis method:the mobile phase was 0.1%formic acid water（A）-acetonitrile（B）,gradient elution,and the elution condition was 0～2.0 min,5%～25%B;2.0～3.0 min,25%～95%B;3.0～3.01 min,95%～5%B;3.01～6.0 min,5%B.the injection volume was 1μL,and the flow rate was 0.4 m L/min.Mass spectrometry analysis method:electrospray ion source（ESI）was used for scanning in both positive and negative ion modes,and the data acquisition time was 6 min.The mass spectrometric parameters of H4 are DP:131.3V;CE:39.1Ev,CXP:6.4V,DP:103.0V;CE:25.0e V,CXP:8.5V,and the ion pairs selected for monitoring are H4 m/z444.2→237.0 and Magnoflorine m/z 343.1→298.0,respectively.H4 in the range of 5～500 ng/m L showed good linearity at different concentrations,the standard curve was Y=0.0241X+0.0454,and the regress-ion coefficient（r）was 0.9985.The LLOQ was5.0ng/m L and the lower limit of quantification RE was 101.40%,The RE range of intra-day and inter-day RE of three groups of H4 plasma samples with different concentrations of quality control samples ranged from 91.2%to 103.3%,and the RSD was all less than 3.98%.The extraction recoveries were 93.95%±6.28%,102.85%±7.81%,90.25%±4.11%;the H4 standard plasma sample is stable in many cases.The experimental results show that the method has good linearity,specificity and sensitivity,and is suitable for the study of pharmacokinetics of H4.The drug concentration was determined by UPLC-MS/MS method.After oral administration of 2.1 mg/kg,7.0 mg/kg,21.0 mg/kg H4 and intravenous administration of 3.0 mg/kg H4,the results showed that the C_max of low,middle and high doses were12.48±8.25 ng/m L,29.46±3.14 ng/m L,180.50±69.40 ng/m L,respectively.AUC was 18.91±4.29μg·h/L,30.80±17.67μg·h/L,158.26±75.15μg·h/L,respectively.After oral administration of three dose levels,C_max and AUC were regressed to the dose respectively.The results showed that there was a linear relationship between C_max,AUC_（0-t）,AUC_（0-）∞and H4 dose curve,and the curve equations were as follows:Y=9.31X-19.25,Y=7.76X-8.49,Y=7.74X-7.28,the correlation coefficients（r）were 0.974,0.970 and 0.971,respectively,indicating that there was a linear relationship between Cmax and AUC at the dose of 2.1～21 mg/kg.In the low,middle and high dose groups,C_max and AUC_（0-t）showed obvious dose dependence,showing dose-dependent pharmacokinetics.The T_max was 0.18～0.23 h,and the T_1/2 was 1.88～5.56h.The CL was 112.13～271.76 L/h/kg.There was no significant difference in T_max,T_1/2 and CL at different doses,indicating that H4 conformed to the first-order kinetic process in rats.The AUC_（0-）∞was obtained after oral administration of 2.1 mg/kg H4and caudal vein injection of 3.0 mg/kg H4,and the absolute bioavailability of rats was3.23%.Finally,the metabolism of H4 was studied in vitro by rat liver microsome experiment.after administration of H4 rat liver microsomes,seven metabolites were identified,including methylation,hydroxylation,glucuronidation,sulfonation,deglucose metabolites and so on.It lays a foundation for the further study of the pharmacological activity and metabolic mechanism of H4 in the later stage.