Research On PK Of Chuan Xiong-tian Ma Anti-migraine With Hyperactivity Of Liver-yang Rats Based On Microdialysis

1 ObjectiveThis research was based on the National Science Foundation”the study on ascending and descending compatibility of rhizoma ligustici wallichii?gastrodia elata based on brain microdialysis technology".Harmacodynamic differences of rhizoma ligustici wallichii-gastrodia elata in different doses water detection,alcohol extract curing migraine with hyperactivity of liver-yang induced by nitroglycerin and radix aconiti carmichaeli+electrical stimulation of trigeminal ganglion and monkshood were done,Pharmacokinetic differences of gastrodin(GAS)and 4-hydroxybenzyl alcohol(HBA)content after oral administration of different ratios of rhizoma ligustici wallichii-gastrodia elata extracts was studied by combining with blood microdialysis technology and high performance liquid chromatography(HPLC)method which was done in early.Next,used the HPLC method combined with blood-brain microdialysis technology to analysis GAS and HBA content,studied pharmacokinetic differences of different proportions of different ratios of rhizoma ligustici wallichii-gastrodia elata,and provide references for clinical apply.2 Method2.1The HPLC establishment of GAS and HBAChromatographic conditions:chromatographic column for Kromasil C18 column(250 mm×4.6mm,5?m);Column temperature:250C,the flow rate:lml/min,sample quantity:10ul,mobile phase:methanol:0.05%phosphoric acid(10:90),wavelength:220nm.Methodology examination:take blank artificial cerebrospinal fluid,GAS/HBA standard solution and artificial cerebrospinal fluid after the treatment then test respectively according to the 2.1 conditions.Linear and detection limit:precision weighing the GAS and HBA reference substance,use artificial cerebrospinal fluid volume,dilute the mother liquor concentration to get seven samples,and record peak area,get the regression equation and the standard curve.Precision of the experiment:prepared respectively the hybrid reference substance solution of GAS/HBA concentration of high,medium and low,according to the chromatographic conditions under 2.1,continuous sample introduction 5 times,continuous determination for 3d,record peak area and RSD value.2.2 The HPLC establishment of brain micro dialysis and the investigate of influence factor2.2.1 The rate influence on GAS/HBA relative recoveries and relative loss effect in vitroBrain probe were immersed in the mixed GAS/HBA reference substance solution which concentration for 103.23/51.48 1 g/ml.With artificial cerebrospinal fluid under 1.0?1.5?2.0?2.5 ?1/min flow perfusion,collected 4 dialysate samples each concentration,30ml/each sample,need to be balanced for 30min when switch to a new concentration,HPLC determination,according to the formula of RR=Cdialysis/Cperfusate×100%to calculate the relative recovery(RR).Brain probe were immersed in the artificial cerebrospinal fluid solution then perfused use the mixed GAS/HBA reference substance solution which concentration for 103.23/51.48 ?g/ml in the rate of 1.0?1.5?2.0,2.5 u 1/min.Collected 4 dialysate samples each concentration,30ml for each sample,need to be balanced for 30min when switch to a new concentration,and then HPLC determination,according to the formula of RL=(Cperfusate?Cdialysis)/CperfusateX100%to calculate the relative loss(RL).2.2.2 The effect of drug concentration to the GAS/HBA relative recoveries and relative lossBrain probe were immersed in the mixed GAS/HBA reference substance solution which concentrations for 103.23/51.48?57.35/28.6?34.41/17.16?11.47/5.72 ?g/ml.With artificial cerebrospinal fluid under 2ml/min flow perfusion,collected 4 dialysate samples each concentration,30ml for each sample,need to be balanced for 30min when switch to a new concentration,then HPLC determination,according to the formula of RR=Cdialysis/Cperfusate X 100%to calculate the relative recovery(RR).Brain probe were immersed in the artificial cerebrospinal fluid solution then perfused use the mixed GAS/HBA reference substance solution which concentrations for 103.23/51.48?57.35/28.6?34.41/17.16?11.47/5.72 ?g/ml in the rate of 2.0 u 1/min.Collected 4 dialysate samples each concentration,30ml for each sample,need to be balanced for 30min when switch to a new concentration,and then HPLC determination,according to the formula of RL=(Cperfusate?Cdialysis)/Cperfusate X 100%to calculate the relative loss(RL).2.2.3 RL stability test of GAS/HBA in vivoAnaesthesia rat with 20%urethane solution(0.8ml/100g),fastened the rat to the brain stereotaxic instrument,implanted brain probe(coordinates:AP+0.2mm,ML+0.5mm,DV-3.5mm),balance for 0.5h,with 2ul/min flow perfusion,every 30 min to collect a dialysate,collect 8 hours continuously.Determination of GAS and HBA,calculate the relative loss.2.3 PK study on rhizoma ligustici wallichii-gastrodia elata in different doses in migraine with hyperactivity of liver-yang rats which based on the blood-brain micro dialysisThe preparation of aconite root extract:weighing the coarse powder of aconite root,with 10 times of water immersion30 min and heating reflux extraction for 2 times,each time 1h,filtered and merged the filtrate,stress concentration of crude drugs containing 2mg/ml solution.According to the rats weight given the aconite root extract(2g/kg/d),lavage 21 days in a row,copying the hyperactivity of liver-yang model.Model rats were randomly divided into group A(GAS/HBA:183/24 mg/kg),group B(GAS/HBA/FA/TMP:183/24/63/5.25mg/kg)and group C(GAS/HBA/FA/TMP:183/24/15.94/1.31mg/kg),six rats in each group.The method of blood-brain probe implantation:anesthesia rats with 2%pentobarbital sodium(0.2ml/100g).Shaving the hair between ears,fastened the rats to the brain stereotaxic instrument,brain probe casing were buried(coordinates:AP+0.2mm,ML+3.5 mm,DV-3.5mm),fixed brain probe casing,and removed the rats,waiting for its awake and free activities,implanted vascular probe in the second day,and put in brain probe.When complete blood-brain probe implantation then subcutaneous injected the nitroglycerin(l0mg/kg)immediately to the right shoulder of rats for duplicate the migraine model,intragastric administration according to each set of corresponding dose after 30min,collect dialysates for 8h immediately.2.4 PK study on rhizoma ligustici wallichii-gastrodia elata in different doses in model and normal rats based on the blood-brain micro dialysisCopy the model rats according to the method of "2.3",and the model rats and normal rats were randomly divided into group A(GAS/HBA:915/120 mg/kg)and group B(GAS/HBA/FA/TMP:915/120/79.7/6.55mg/kg)and group C(GAS/HBA/FA/TMP:915/120/315/26.25mg/kg),3 rats for each group.According to the“2.3”operations implanted blood-brain probe,and testing samples.3 Result3.1 GAS/HBA in the medicated artificial cerebrospinal fluid had the same peak time with GAS/HBA in standard solution,blank artificial cerebrospinal fluid had no interference,show that the established test method was a good specificity.GAS standard curve:y=23660x+8574.8,R2=0.9998;HBA tandard curve:y=45886x+10014,R2=0.9991;GAS/HBA in the artificial cerebrospinal fluid had good linear relationship within the scope of 0.28675-103.23/0.143-51.48?g/ml.GAS/HBA of high,medium and low concentration precision in one day RSD were 0.058%/0.110%?0.075%/0.467%?0.61%/0.933%;Daytime precision RSD were 0.764%/0.754%?1.517%/1.105%?2.691%/3.125%,show that instrument precision is good.3.2 With the increase of flow,GAS and HBA relative recoveries and relative loss rate had a decreasing trend;GAS in the range of 11.47~103.23 ? g/ml,and HBA in the range of 5.72~51.48 ?g/ml that probe relative recovery had nothing to do with the concentration.Relative Loss rate of GAS and HBA was stable within 8h.3.3 Blood GAS in the pharmacokinetic results:compared with group A,group B Tmax had difference(P<0.05);Group C of the peak concentration(Cmax)increased,there is a very significant difference(P<0.01),AUC increased,there is significant difference(P<0.01).Compared with group C:Cmax and AUC in B group was reduce,there was a difference(P<0.01).Blood HBA pharmacokinetic results:compared with group A,Cmax had increased in the group B,there was significant difference(P<0.01),but also AUC,greatly enlarged,have difference(P<0.05),the average residence time(the MRT)decreased significantly,there were significant differences(P<0.01),T1/2 significantly shortened(P<0.05).Group C Tmax,Cmax,AUC,MRT,T1/2 were very significant differences(P<0.01).Compared with group C,group B Tmax significantly shortened(P<0.01).The pharmacokinetic results of GAS in the brain:compared with group A,AUC in group B was obviously increased(P<0.05),the MRT also increased(P<0.05);Cmax increase compared with group C,group B(P<0.05),AUC also significantly increased(P<0.01),the MRT increased,there was significant difference(P<0.05).Different proportion set HBA in concentration in the brain was not a complete pharmacokinetic parameters.3.4 Blood GAS results:in model group,compared with group A,group B and group C Tmax and Cmax were increased,the T1/2 were reduced,the MRT of group B was reduced,the group C of the MRT increases;Group B compared with group C,group C Tmax,Cmax is reduced,AUC,the MRT,T1/2 are increased.In the normal group,compared with group A,group B and group C,Tmax,Cmax,AUC and the MRT were reduced,join after rhizoma ligustici wallichii,less GAS absorption,eliminate the slow down.HBA in the blood results:in model group,compared with group A,group B and group C Tmax,Cmax,AUC were increased,the MRT,T1/2 B group were decreased,while the MRT,T1/2 C group were increased.Group B compared with group C,Tmax,Cmax,AUC was reduced,the MRT,T1/2 were increasedIn the normal group,compared with group A,group B Tmax,AUC,the MRT,T1/2 were increasing,Cmax was reduced,Tmax,Cmax in C group were increased,contrary to group B,group C AUC,the MRT,T1/2 are decreased;Group B compared with group C,Tmax,AUC,the MRT,T1/2 are reduced.Brain GAS results:in model group,compared with group A,group B and group C Tmax,Cmax,AUC were increasing,T1/2,the MRT were reduced;Group B compared with group C,group C increase Tmax,Cmax,AUC were reduced,the MRT,T1/2 basically remain unchanged,In the normal group,compared with group A,group B and group C Tmax,Cmax,AUC and the MRT were reduced,the T1/2 was increasing;Group B compared with group C,Tmax,AUC,the MRT were reduced,the T1/2 basically remain unchanged,Brain HBA results:in model group,compared with group A,group B and group C Tmax and Cmax all increase,group B AUC is reduced,group C AUC increased.In the normal group,compared with group A,group B and group C Tmax,Cmax,AUC,the MRT,T1/2 are reduced.4 Conclusion4.1 HPLC instrument precision was good,for GAS,HBA detection specificity was strong,can be used in the next HPLC combined micro dialysis technology methodology.4.2.In fixed perfusion speed,stable drug concentration range RR,RL stability was good,in the body of this detection method can be used as rhizoma ligustici wallichii,gastrodia elata with different in vivo pharmacokinetic study of test methods.4.3 After joining rhizoma ligustici wallichii can promote the absorption of GAS;Rhizoma ligustici wallichii,gastrodia elata 0.25:1 set of GAS through the blood brain barrier ability optimal;After adding rhizoma ligustici wallichii,promote the absorption and elimination of the HBA.4.4 The pharmacokinetic results in blood:you can see in the model group,rhizoma ligustici wallichii accelerate the absorption of GAS,HBA,rhizoma ligustici wallichii and two-way adjustment function for the GAS and the absorption of the HBA.For normal rats,adding rhizoma ligustici wallichii is not conducive to the GAS absorption.The pharmacokinetic results in brain:In the model group,increase the amount of rhizoma ligustici wallichii conducive to GAS through the blood brain barrier.For normal rats,rhizoma ligustici wallichii against GAS,HBA through the blood brain barrier.