Pharmacodymaics And Pharmacokinetics Study Of Chuan Xiong-Tian Ma Anti-migraine With Blood Stasis By Microdialysis

ObjectiveThis topic for the NSFC is a part of the research content which "based on brain micro dialysis technology of rhizoma ligustici wallichii,gastrodia elata lift assist pharmaceutical sexual compatibility research(NO.81274107)".Research advance of rhizoma ligustici wallichii-day anesthetic on the main effective components are studied,found that the content of effective components from the different ratio of drug to have obvious difference,and have a test showed that rhizoma ligustici wallichii-day anesthetic on rat liver Yang type of blood stasis syndrone and migraine has good efficacy,the ratio of different medicine on pharmacodynamics index has obvious differences.This study on the basis of the premise to study further the ice water bath combined injection of adrenal combined electrical stimulation of trigeminal ganglion and blood stasis type method research the pharmacodynamics of migraines model,and USES the micro dialysis technology joint UPLC-ms method in blood stasis type of migraine model rats in vivo pharmacokinetic study,respectively from the pharmacodynamic and pharmacokinetic aspects discusses different ratio of rhizoma ligustici wallichii-the feasibility of gastrodia elata in treating blood stasis type of migraine and difference.Method1 Pharmacodynamic research of Different matching of Rhizoma Ligusticum Chuanxiong and Gastrodia Elata in Migraine with Blood Stasis1.1 The Establishment of migraine with Blood Stasis modelStudied with migraine with Blood Stasis model caused by Subcutaneous injection Adrenaline,a total of two times,two times the interval of 4 h,And 2 h after the first injection put rats in 5-8 ? water for 5 min,after the rats with 10%chloral hydrate anesthesia,fixed on the stereotaxic instrument,with dental drill socket after positioning,and then insert the electrode(to dura mater is a depth of 9.3 mm)in trigeminal ganglia,postoperative continuous anesthesia.Stimulate the 10 min after debugging electrical stimulation parameters.2.2 ELISA detection of NO and NOS in blood and brain homogenate,CGRP and ET,TXB2,6-Keto-PGFIaDetermine the content of CGRP?NO?ET?TXB2 and 6-Keto-PGF1? in serum by ELISA,experimental data is expressed in(x ±s),analyzed by SPSS20.02 Established the method and methodology of determination of ligustrazine and ferulic acid in blood dialysis fluid at the same time2.1 Mass spectrometer conditionsLiquid conditions:The analysis was achieved with a Kinetex C18(100×2.1mm,2.6um)column by gradient elution of acetonitrile(A)-0.005%methanoic acid(B)at indoor temperature((0?5 min,20%A,80%B;5?10 min,20%?90%A,80%?10%B;5.1?10min,20%A,80B).The flow rate was 2ml/min and the sample volume was 10 ?L.The pallet temperature was 4?.Mass spectrometer conditions:The ion source was atmospheric pressure electrospray ion source at 350?.The detection mode was simple reaction monitoring with sheath gas(Arb):33,auxiliary gas(Arb):3.All of gas was nitrogen and the spray voltage was 3500V.2.2 Methodological evaluationStandard curve and the quantitative limit:dissolve 6.06 mg of ligustrazine hydrochloride standard(equivalent to 5.08 mg TMP)and 4.11 mg of ferulic acid in the ringer's solution as the standard of mother liquor.Eight concentration gradient were getted by diluting mother solution,and measured,recorded the peak area,obtained the regression equation and the standard curve.Matrix effect:Taking ringer's solution to preparate the FA/TMP high,medium and low three levels including matrix sample,and get the corresponding peak area.The methanol instead of ringer's solution,with the same method of operation,the preparation of three reference samples were analyzed,and the mass concentration of received the matrix of the peak area(average)for the determination of 6 times.Concentrated two different processing method of peak area ratio for calculate matrix effect.Precision inspection:mixturing respectively quantitative limit of ferulic acid and ligustrazine(LLOQ)and get low,medium and high three QC sample.Each mass concentration contained 6 samples along with standard curve,for continuous determination of 3 d,the calculation is based on the standard curve of the the LLOQ and QC sample concentration.Concentrating with standard solution,and getted ferulic acid,ligustrazine QC samples limit of detection in the batch,batch precision(RSD%).Stability test:collecting a about 100 ul dialysis fluid samples after drug administration.The samples were stored in refrigerator at 0 to 4 ? and measured the peak areas every two hour.3 The research on the method of micro blood dialysis and influencing factors3.1 The establishment of the method of rat micro dialysis.The way to implant probes:Firstly,anesthetize rats by 20%urethane,immobilize rats' all fours and make it in supine position,shave off fur on neck,disinfect skin around the left side jugular vein by tincture of iodine,striping jugular vein passively,ligaturing the port which beyond heart.Then,clamping the proximal part of jugular vein in one hand,the other hand inserting a probe on the tuberosity of jugular vein,and keep balance with the bleeding location in order to lift up the probe,and immobilize the probe by filament.Finally,to assure the flow velocity to perfuse after the completion of probe inserting.After 1 hour in balance,dialysate will be collected per 30 min,until to the 8th hour.3.2 The ratio of recovery on micro dialysis in vitro and the stability in vivo of ligustrazine and ferulic acid.By investigating the effect of probe recovery due to concentration:put the probe into pastille Ringer' s solution whose concentration known as FA/TMP.Then perfuse in a regular flow velocity by margin Ringer's solution,collect 5 samples in each concentration of Ringer' s solution after 30 min balance,and determine samples and the concentration of pastille Ringer's solutions by UPLC-MS.By investigating the stability in vivo:after inserting the probe,perfuse the pastille Ringer's solution whose concentration known as FA/TMP in a certain flow velocity,and collect dialysate per 20 min,determine samples and the concentration of pastille Ringer' s solutions by UPLC-MS 8 hours later.3.3 Comparison with 5 probe recoveriesFetch 5 SD rats,after inserting probe,perfuse the pastille Ringer' s solution whose concentration known as FA/TMP in a certain flow velocity,5 samples in each rat after 1 hour balance,determine samples and the concentration of pastille Ringer' s solutions by UPLC-MS.4 The in vivo Pharmacokinetic study research on different proportion of Ligusticum wallichii-gastrodia elata in blood stasis model migraine.Put rats into 3 groups randomly:single Ligusticum wallichii medication administration team,Ligusticum wallichii-gastrodia elata(1:1)medication administration team,Ligusticum wallichii-gastrodia elata(4:1)medication administration team.After inserting probes,firstly exclude bubbles in pipeline by flow velocity of 3-5ul/min,then perfuse margin Ringer's solutions in a regular flow velocity,balancing 1 hour to stabilize system.2 min later,when dose is completed(wipe off margin Ringer' s solutions in pipeline),start to collect dialysate.Then adopt blood dialysis method to sampling,determine the content of FA/TMP by UPLC-MS,calculate pharmacokinetic parameter by pharmacokinetic software-PKSolver.Results1.The rats masticatory muscle contraction,nose and mouth secretions increase with stimulation.Moreover,we choose the laser doppler blood flow meter detecting data to evaluated the blood stasis type migraine.The LDF value of model group decreased significantly compared with the normal control group with significant difference(P<0.05).It shows the success of the experimental model in rats with acute blood stasis syndrome according from blood flow quantity.2.SPSS20.0 statistical software analysis.we choose single factor analysis of variance between groups(One-way ANOVA),significant difference was recognized with P<0.05 and P<0.01.The NO,NOS,CGRP,6-Keto-PGF1?level in serum of model group increased and the NO,NOS,CGRP,6-Keto-PGF1?,ET level in brain homogenate rised compared with normal group.3.Establish UPLC-ms method for detect FA/TMP.The regression equation were Y=478.01+48.848,r=0.9967,Y-8352.8 X-2151.9,r=0.9932,respectively.The concentration of FA in 2.045?1023 ng·ml-1 shows excellent liner realationship,The concentration of TMP in 2.54 1270 ng/ml shows excellent liner realationship,The precision of the batch within/between(RSD%)of ferulic acid were 3.03/2.22,1.94/2.06,1.94/2.06,in high,medium and low concentrations levels respectively.The precision of the batch within/between(RSD%)of ligustrazine were 4.71/1.48?2.81/1.78?2.51/3.46,in high,medium and low concentrations levels,respectively.The matrix effect of FA in high,medium and low concentration levels were 96.07%(RSD = 2.73%),96.84%(RSD = 0.91%),94.41%(RSD = 1.18%),respectively.The matrix effect of TMP in high,medium and low concentration levels were 101.27%(RSD = 0.38%),101.78%(RSD = 1.64%),100.66%(RSD = 3.33%),respectively.All the RSD were below 15%,which meet the specification.The experiment samples were stored at 0 to 4 ? refrigerator and were added every 2 h.The stability of quality control sample(RSD%)of ferulic acid of high,medium and low concentration level were 8.46,8.80,5.74,respectively.The stability of quality control sample(RSD%)of ligustrazine of high,medium and low concentration level were were 3.32,1.19,2,59,respectively.4.The results showed that the recovery rate of FA and TMP decrease with the increase of flow velocity.The RR and RL under various flow velocity were basically the same.Put the probe into four medicated ringer's solution with the known FA/TMP concentrations.with 1.5 uL/min flow rate of ringer's solution perfusion blank,collect 5 samples after balance 30 min in each concentrations level.The results show that the recovery rate of FA and TMP has no relationship with the concentration and dialysis method in the certain concentration range.When the velocity is constant,RL of FA and TMP remain relatively stable within about 8 h,the average recovery rate of probe FA and TMP in RL were 65.20%,40.97%,RSD were 5.83%,9.07%,respectively,with good stability in the body.5.The TMP drug concentration level were high in all drug groups.This showed that TMP could be absorbed quickly in animal groups.The Cmax level of the 1:0.25 group,1:1 group were significantly increased by about 1.3 times,1.5 times compared with single drug group with significant difference(P<0.05,P<0.01).The T1/2 of the 1:0.25 group,1:1 group were significantly increased by about 1.4 times,1.2 times compared with single drug group with significant difference(P<0.05,P<0.01),and the time of the MRT is prolonged significantly.The T1/2 and MRT of the 1:0.25 group increase significantly compared with 1:1 group with significant difference(P<0.01).After analysis of AUCO-t?AUCO-? of each group,we found the AUC of the 1:0.25group,1:1 group were significantly increased by about 1.5 times,1.3 times compared with single drug group with significant difference(P<0.01).Pharmacokinetic parameters between groups in the FA showed that the Cmax of the 1:0.25 group,1:1 group were significantly increased by about 1.3 times,1.5 times compared with single drug group with significant difference(P<0.01).The T1/2 and MRT of 4:1 group and 1:1 group increased compared with single drug group.The T1/2 and residence time of 1:0.25 group increased about 2.5 times,1.3 times compared with single drug group,respectively,with significant difference(P<0.05,P<0.01).The MRT of 1:0.25group increased about 1.5 times compared with single drug group,and the residence time increased,too,But there were no significant difference.Conclusion1.Tn pharmacodynamics experiment part,the observation indexes of model group and normal group shoued significant difference,to research the scientific of drug compatibility of rhizoma ligustici wallichii-Gastrodia elata,further research showed that NOS?CGRP?6-Keto-PGF1?level decreased in serum and brain homogenate of all the drug groups and model group.The research preliminarily proves that the drug compatibility of rhizoma ligustici wallichii-gastrodia elata has certain curative effect on treating blood stasis type migraine,and 4:1 group showed significant better result compared with 1:4 group.2.Establish one UPLC-MS method to determination micro dialysis ligustrazine and ferulic acid content in the samples simultaneous.This method has high specificity and impurities without interference,quantitative precision lower limit,within-day precision and stability all meet the requirements of biological sample analysis.Therefore,this method could be used for ligustrazine and ferulic acid in rats in vivo pharmacokinetic study.3.We studied comprehensive inspection of Micro dialysis recovery in vivo and in vitro.The experimental results show that using micro dialysis technology study of ligustrazine and ferulic acid in migraine rats in vivo pharmacokinetic study was practical and feasible.4.The pharmacokinetic parameters all drug groups showed significant differences.The AUC data analysis showed that the 1:0.25 group increased significantly compared with single drug group and and 1:1 group,suggesting that Gastrodia elata improve the FA in vivo absorption and slows down the metabolism.In 1:0.25 group,the metabolism is slowest,the residence time is longest and the absorption degree is maximum.These illustrates that the scientific nature and rationality of 1:0.25 group for treating blood stasis type of migraine.