Pharmacokinetic Study In Rats And Determination Of Plasma Protein Binding Rate Of Robustaside B

This paper is mainly divided into three parts.The first part is about the extraction,separation,purification,and identification of robustaside B from Quezui tea.In second part,a method was established for the quantitative determination of robustaside B in rat plasma using liquid chromatography mass spectrometry,and the method was applied to the study on pharmacokinetics.In the third part,a method was established to quantitatively determine the concentration of robustaside B in the internal and external dialysate of rats.Using the principle of balanced dialysis,the plasma protein binding rate of robustaside B in normal and hyperuricemic rats was measured.Objectives:To establish an accurate,reliable,efficient,and convenient quantitative analysis method for determining the mass concentration of robustaside B in rat plasma,dialysate,and external dialysate,and further apply in the pharmacokinetic study and calculating pharmacokinetic parameters of robustaside B.Based on the principle of balanced dialysis,the plasma protein binding rates of normal and hyperuricemic rats were investigated in order to provide reference for the in-depth research and development of Quezui tea and its active components.Methods:1.The active ingredient robustaside B was extracted and separated from Quezui tea by a chromatography method.2.The method for the quantitative determination of robustaside B concentration in rat plasma,including the optimal internal standards selecting,the liquid phase and mass spectrometry conditions optimizaition,and the appropriate biological sample pretreatment methods,was established,and the methodological validation was also completed.3.SD male rats were administered 1200 mg/kg and 120 mg/kg of robustaside B by gavage and tail vein,respectively.The blood samples were collected at different time points,the plasma concentrations were measured,and the drug time curves were plotted.Using DAS analysis software,major pharmacokinetic parameters such as t_1/2,C_max,and AUC were obtained.4.The quantitative method for the robustaside B in the internal and external dialysate of rats was established,as well as the methodological study.5.Hyperuricemia rat model was induced by continuous intragastric administration of800 mg/kg potassium oxazinate combined with 100 mg/kg adenine in male SD rats for 7 days.The external dialysate consists different content of robustaside B,including 500,1000,1500 ng/m L,respectively.Plasma of normal and HUA rats were collected,respectively,and plasma protein binding rates of rats under the two conditions were determined by equilibrium dialysis method.Results:1.The purity of robustaside B from Quizui tea is 95%.2.The plasma was pretreated by methanolic protein precipitation,the quercetin was used as the internal standard,the methanol-0.1%formic acid was used as the mobile phase for gradient elution.Under these conditions,the linear relationship of robustaside B was good with the range of 50 to 3000 ng/m L,the results of accuracy and precision were88.8 to 112.2%,the range of RSD is from 1.1 to 9.6%,and the extraction recovery ranged from 74.6 to 94.3%.and the robustaside B was stable during the analysis,the analytical method met the requirements for quantitative determination.3.After intragastric and caudal intravenous injection of robustaside B,the main pharmacokinetic parameters were as follows:t_1/2was 0.81±0.24 h,0.20±0.02 h;C_maxwas 3639.67±262.24 ng/m L,56359.63±2216.17 ng/m L;V_zwas 2.02±0.56 L/kg,0.002 L/kg;MRT_（0-t）was 0.39±0.02 h,0.08±0.003 h;and F_abswas0.08%.4.In both the intra-and extra-dialysis fluid matrices,robustaside B was in the range of 50 to 2000 ng/m L,the linearity was good,and the accuracy and precision results were 95.5 to 106.5%,90.7 to 106.3%,RSD ranges from 3.3 to 8.9%,4.1 to7.2%,respectively,and the extraction recoveries were between 72.4 to 79.9%and79.3 to 88.2%,respectively.During the analysis process,robustaside B remained stable,and the analytical method met the requirements for quantitative determination.5.At low,medium,and high concentrations,the plasma protein binding rates of normal rats were 34.5±3.0%,48.0±1.5%,and 48.4±1.0%,respectively.The plasma protein binding rates of HUA rats were 47.1±1.6%,46.9±0.8%,and 48.1±0.8%,respectively.The plasma protein binding rate of normal rats in low concentration group was lower than that in medium and high concentration groups and HUA rats in low concentration group.Conclusion（s）:The established analytical method was able to accurately determine the mass concentration of robustaside B in rat plasma,dialysis internal fluid and dialysis external fluid.The pharmacokinetic results showed that robustaside B has the pharmacokinetic characteristics of rapid absorption,small distribution volume,rapid elimination and low bioavailability;The binding rate of plasma protein is about 50%,which belongs to moderate binding.In terms of drug interaction,it has good safety when used in combination with other drugs in clinic.