Study On Pharmacokinetics Of Rats With Blood-Stasis Migraine With Different Proportions Of Chuan Xiong And Tian Ma

Objective:This topic is part of the research project of the National Natural Science Foundation of China"Based on the Microdialysis Technology Chuanxiong-Tianma Lifting and Satisfactory Compatibility（NO.81274107）":pharmacokinetics research of Tianma Pills（Chuanxiong-Tianma 4:1）.In this paper,The method for the determination of Tetramethylpyrazine（TMP）and Ferulic Acid（FA）in Ringer’s solution and artificial cerebrospinal fluid had been established.The stability,matrix effects and other as-pects were investigated.At the same time,the in vitro recovery rate of blood,brain tissue microdialysis probes,and the stability in vivo for 10 h were examined.Based on the previous experiments,the pharmacokinetics of Chuanxiong and Tianma with different ratios in blood stasis migraine model rats were compared.The results were as follows:the pharmacokinetic parameters of ligustrazine and ferulic acid in blood and brain tissue dialysate were effected by Chuanxiong single herb,Chuanxiong-Tianma 1:0.25,Chuanxiong-Tianma 1:1 groups.This research aimed to explore the pharmacokinetics of TMP and FA in the blood and brain tissue of blood stasis migraine model rats.To clarify the rationality of the combination of Chuanxiong and Tianma in the treatment of blood stasis migraine which provides a reference for clinical rational drug use.Methods:1.Determination of tetramethylpyrazine,ferulic acid in the blood,brain dialysate determination by UPLC-MS/MSPrepared artificial cerebrospinal fluid and Ringer’s solution containing tetramethylpy-razine and ferulic acid respectively,and to build the method for the determination of ligustrazine and ferulic acid in blood and brain tissues dialysate.UPLC-MS/MS method was established and precision,Matrix effect,stability was inspected.2.Methods and inspection of Ligustrazine and ferulic acid in blood and brain microdialysatesTMP,FA in Vitro Recovery of Blood and Brain Tissue Microdialysis Probes was examined under different flow rates（0.5,1.0,1.5,2.0μL/min）、different tempera-tures（25°C,30°C,35°C,40°C）,different concentrations（tetramethylpyrazine/ferulic acid 30/80,140/300,460/800,1000/1800 ng/m L）,10 h In Vivo Stability and 6 con-secutive use in blank artificial cerebrospinal fluid,Ringer’s solution（incremental method）and drug-containing artificial cerebrospinal fluid,Ringer’s solution（decrease method）perfusion.3.Pharmacokinetics of Chuanxiong-Tianma with Different Proportions in Blood and Brain TissueCopying and evaluating the Blood stasis type migraine rat model,and rats were randomly divided to 4 groups,6 per group.On the established methodologies,the contents of Chuanxiong single herb,Chuanxiong-Tianma 1:0.25,Chuanxiong-Tianma1:1 groups of TMP and FA in blood and brain tissue were determined.Kinetic param-eters were analyzed to investigate the pharmacokinetics of Chuanxiong-Tianma in the blood stasis migraine rat model.Results:1.Determination of tetramethylpyrazine,ferulic acid in the blood,brain dialy-sate by UPLC-MS/MS1.1 Specificity:The retention time of TMP and FA in brain tissue and blood dialysate were 1.67 min and 2.03 min,respectively and the specificity was good.1.2 Linearity:The linear regression equations of TMP in artificial cerebrospinal fluid and Ringer’s solution are:Y=0.005957X+0.03816,R²=0.9989;Y=0.005901X+0.07584,R²=0.9980.The minimum detection limit was 3.21 ng/m L and the concen-tration range was 3.21～1444.5 ng/m L.The linear regression equations of ferulic acid in artificial cerebrospinal fluid and Ringer’s solution were:Y=0.00425X+0.02186,R²=0.9995;Y=0.004085X+0.07622,R²=0.9996.The minimum detection limit was 5.47 ng/m L and the concentration range was 5.47 to 2461.5 ng/m L.1.3 Precision:The RSD of the concentration of TMP in artificial cerebrospinal fluid at low,medium,and high（3.21,481.5,1444.5 ng/m L）concentrations was 4.35%,3.92%,and 2.60%,respectively;during the daytime in Ringer’s solution.The precision RSDs were 3.39%,5.85%,and 2.93%,respectively.The inter-day precision RSD of FA in artificial cerebrospinal fluid at low,medium,and high（5.47,820.5,2461.5 ng/m L）concentrations was 5.16%,2.35%,and 3.17%,respectively;daytime precision in Ringer’s solution.The RSDs were:4.53%,2.84%,and 3.49%.1.4 Matrix effects:The matrix effects of TMP at low,medium,and high concentra-tions of artificial cerebrospinal fluid were 93.40%,92.62%,and 91.29%,respectively;in Ringer’s solution,the matrix effects were:90.43%,90.29%,and 93.08%,respec-tively.The matrix effects of FA in artificial cerebrospinal fluid at low,medium and high concentrations were 94.61%,93.95%,and 93.81%,respectively;the matrix ef-fects in Ringer’s solution were 94.42%,96.45%,and 93.55%,respectively.1.5 Stability:The stability of TMP at low,medium and high concentrations showed that the samples remained stable in artificial cerebrospinal fluid and Ringer’s solution at room temperature for 6 h,4°C for 24 h,repeated freezing and thawing 3 times.The stability of ferulic acid at low,medium and high concentrations showed that the samples remained stable in artificial cerebrospinal fluid and Ringer’s solution at room temperature for 6 h,4°C for 24 h,repeated freezing and thawing 3 times.This method has a good linear relationship,precision,matrix effects,and stability are in line with relevant regulations,and can be used for the determination of content of TMP,FA in blood,and brain tissue dialysates.2.Method of TMP and FA in blood and brain microdialysates.2.1 Probe in vitro recovery rate:the results of different concentrations showed that different flow rates have influence on the recovery rate of TMP and FA in blood and brain probes.When the flow rate was 1.5μL/min and the sampling interval was 20min,the recovery rates of TMP in artificial cerebrospinal fluid and Ringer’s solution were 34.81±7.51%and 32.50±8.63%,respectively;FA in artificial cerebrospinal fluid The RR values in Ringer’s solution were 31.46±5.94%,33.19±9.10%,and the sample volume was 30μL.Take into account the sample size and the liquid injection requirements,determine the flow rate of 1.5μL/min.The results of different temperatures show that temperature changes have an im-pact on the recovery rates of TMP and FA.Therefore,the body temperature of rats should be kept constant during the microdialysis experiment.The results of different concentrations showed that the concentration had no sig-nificant effect on the recovery rate of TMP and FA.2.2 Probe Stability in vivoThe 10 h stability of blood and brain tissue microdialysis probes showed that the recovery rates of TMP,FA blood probes and brain tissue probes remained stable within 10 h and the fluctuation range was small,which could meet the requirements of subsequent microdialysis animals.3.Pharmacokinetics of Chuanxiong-Tianma with different Proportions in blood and brain tissueThe kinematical parameters of TMP and FA in blood and brain tissue showed that:（1）The pharmacokinetics of TMP:compared with the single herb group,Chuanxiong and Tianma 1:1 and 1:0.25 groups of blood and brain Response Half-time(T_1/2),Mean Retention Time（MRT）,Concentration_max(C_max),and Area Under Curve(AUC_0-∞)increase（large）.There was a significant difference（P<0.05～0.01）.Compared with the 1:1 group,the T_1/2,MRT,Cmax,and AUC_0-∞were in-creased（large）in the 1:0.25 group（P<0.05～0.01）.（2）Effect on pharmacokinetics of ferulic acid:compared with single-taste drug group,T_1/2,MRT,Cmax,and AUC_0-∞increase（large）in the blood of 1:1 and 1:0.25 groups of Chuanxiong and Tianma,and the brain MRT,Cmax,and AUC_0-∞increased（large）in the tissue（P<0.05～0.01）;compared with the 1:1 group,T_1/2,MRT,and AUC_0-∞increased（large）in the 1:0.25group.Significant（P<0.05～0.01）.The Drug Targeting Index（DTI）showed that the combination of Chuanxiong and Tianma could increase the brain targeting of Chuanxiong,with the highest brain target index of Chuanxiong-Tianma 1:0.25.Conclusion:1.This paper established a blood-brain simultaneous microdialysis technique com-bined with UPLC-MS/MS to study the method of the mobilization process of TMP and FA in blood and brain tissue.This method is reliable and stable,and can be used in pharmacokinetic experiments.It is of great significance for revealing the dynamic distribution of effect components of Chuanxiong and Tianma in blood and brain and explaining the compatibility mechanism of traditional Chinese medicine.2.Combination of different doses of Tianma can increase the absorption of TMP,FA in blood and brain tissue,slow down the elimination rate,prolong residence time,and improve brain targeting of TMP and FA,which Chuanxiong and Tianma 1:0.25was the best.