Research On PK And PD Of Chuan Xiong-tian Ma Anti-migraine With Hyperactivity Of Liver-yang 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".Pharmacodynamic differences of rhizoma ligustici wallichii-gastrodia elata curing migraine with hyperactivity of liver-yang induced by electrical stimulation of trigeminal ganglion and monkshood were compared,which was done in early.Next,used the HPLC method combined with blood microdialysis technology to analysis GAS and HBA content after oral administration of different ratios of rhizoma ligustici wallichii-gastrodia elata extracts.Reveal the rules of rhizoma ligustici wallichii-gastrodia elata from PK-PD,and provide references for clinical apply.2 Method2.1 Pharmacodynamics research on rhizoma ligustici wallichii-gastrodia elata curing migraine rats with hyperactivity of liver-yang.56 Rats are randomly divided into 7 groups,such as normal group(Z)、sham operation group(J)、model group(M),chuang xiong-tian ma(4:1,1:1,1:4)groups A、B、C,sumatriptan group(Y).Except group Z and J,each group was given aconitum carmichali debx water extracts for 21d by gavage.From 15thd on,gave drugs for 7 days,and 1h after the last time given drugs,stimulation of the Gasserian ganglion(-3.2,-2.8,-9.3-9.5mm;Stimulation parameters as follows:cycle 200 ms,wave width 5 ms,10 v)was performed for 10 min except group Z,J.But bipolar electrode was planted inton group J.After 30 min stimulation,collect serum and erebral homogenate to determine calcitonin gene related peptide(CGRP)、6-keto-prostaglandin Fla(6-Keto-PGF1α)、nitric oxide(NO)、endothelin(ET)、Thromboxane B2(TXB2).2.2 Pharmacokinetic research on rhizoma ligustici wallichii-gastrodia elata curing migraine rats with hyperactivity of liver-yang.2.2.1 Determination of GAS and HBA by HPLC Chromatographic column Kromasil C18(250 mm×4.6 mm,5μm);temperature 25℃,flow rate 1 ml.min-1,sample size 10μ1,mobile phase methol-0.05%phosphoric acid(10:90),isocratic elution,220nm.2.2.2 Pharmacodynaamic research of rhizoma ligustici wallichii-gastrodia elata curing migraine rats with hyperactivity of liver-yang.based on blood microdialysis combined with HPLCRats were randomly divided into tian ma group A,chuan xiong-tian ma(1:1,0.25:1)groups(6 each)B、C.each group was given aconitum carmichali debx water extracts for 21d(2 g · kg-1 · d-1)by gavage.At the last day,each group was induced migraine with nitroglycerin(10mg · kg-1)subcutaneous injection.30 min later,all groups were given GAS 183 mg`kg-1,HBA 24 mg · kg-1.Meanwhile,group B was given FA.TMP 63,5.25 mg · kg-1 respectively,and group C.was given FA.TMP 15.94,1.31 mg· kg-1 respectively.Use Ringer’s solution for perfusate,perfusion speed 1.5μ1 · min-1,collect sample every 20 min in jugular vein by microdialysis,toally 10 h.Determine the GAS and HBA in dialysate.The pharmacokinetic parameters were computed using PKsolver 2.0.3 Result3.1 Pharmacodynamics results of rhizoma ligustici wallichii-gastrodia elata curing migraine rats with hyperactivity of liver-yang.In serum:compared with group z,6 indexs have no significant differences in group J,CGRP、6-keto-PGFla、NOS increased,TXB2 decreased in group M,(P<0.05).Compared with Group M,CGRP、NOS decreased,TXB2、TXB2/6-keto-PGFl a increased in group Y.A.CGRP decreased in group B,CGRP、6-keto-PGF1α、NOS decreased,TXB2/6-keto-PGF1α increased in group C,(P<0.05～0.01).Compared with group B,TXB2 increased,NOS decreased in group A,6-keto-PGF1α、NOS decreased,TXB2/6-keto-PGFLα increased,(P<0.05～0.01)。In cerebral homogenate:compared with group Z,CGRP、6-keto-PGFla reduced,ET、TXB2/6-keto-PGFl α raised in group J;compared with group J,CGRP reduced,ET、NOS、6-keto-PGF1α、TXB2/6-keto-PGF1α raised in group M,(P<0.05～0.01).Compared with group M,6-keto-PGF1 α、.TXB2/6-keto-PGF1 α increased,NOS decreased in group Y,NOS decreased in group A,6-keto-PGF1α、TXB2/6-keto-PGFl α increased,ET decreased in group B,6-keto-PGF1 adecreased、TXB2/6-keto-PGF1 α increased in group C,(P<0.05～0.01)。Compared with group B,6-keto-PGF1 aincreased,NOS decreased in group A,6-keto-PGF1 a decreased、TXB2/6-keto-PGF1a increased in group C,(P<0.05～0.01)。3.2 Pharmacokinetic result of rhizoma ligustici wallichii-gastrodia elata curing migraine rats with hyperactivity of liver-yang.3.2.Result of the determination of GAS and HBA by HPLCThe linear range of GAS and HBA were 1.52～228.00μg · ml-1(r=0.9993),0.41～51.60μg· ml-1((r=0.9997)respectively.Both RSDs of within-day and between-day of GAS and HBA were lower than 10%.In linear range,the concentration made no differences on probe recovery,but with the flow rate increasing,probe recovery was decreased.The probe recovery was stable in 10 h.3.2.2 Pharmacokinetic result of blood microdialysis combined with HPLC3.2.2.1 The TMP and FA pharmacokinetic influences on GASThe pharmacokinetic parameters of GAS t1/2(min)、Tmax(min)、Cmax(μg/ml)、AUC 0-inf_obs(μg/ml*min)、MRT 0-inf_obs(min)of three groups:A group,65.32±8.16、86.67±10.32、70.31±7.36、15385±1205、182.29±5.05;B group,41.28±8.17、76.67 ±8.16、232.84±26.62、36325±5647、136.34±6.65,C group,55.44±15.27、40±0、34.17±4.55、5183±997、123.6±12、15。compared with group A,t1/2、MRT was shortened,Cmax、AUC increased in group B,t1/2、Tmax、Cmax、AUC、MRT dropped in group C,(p<0.01).Compared with group B,tl/2、Tmax、Cmax、AUC、MRT in group C all have significant differences(p<0.01),and t1/2 was shortened,Tmax、Cmax、AUC、MRT raised。3.2.2.2 The TMP and FA pharmacokinetic influences on HBAThe pharmacokinetic parameters of HBA,t1/2(min)、Tmax(min)、Cmax(μg/ml)、AUC 0-inf_obs(μg/ml*min)、MRT 0-inf_obs(min)of three groups:A group,115.83±8.44、120±48.99、4.65±0.19、1575.04±132、236.62±6.21;Bgroup,41.93±12.66、216.67±29.44、29.14±1.14、6368.14±557.59、237.16±9.27;C group,60.67±11.17、73.33±10.33、8.20±0.09、2206.57±74.95、186.98±5.80。compared with group A,t1/2 was shortened、Tmax、Cmax、AUC increased in group B(p<0.01);t1/2、Tmax、MRT was shortened,Cmax、AUC increased in group C,(p<0.01)。Compared with group B,t1/2 was lenthened,Tmax、Cmax、AUC、MRT diminished in group C,(p<0.01～0.05).4 Conclusion4.1 Chuan xiong-tian ma(4:1,1:1,1:4)were effective on migraine with hyperactivity liver-yang,and the(1:4)group was best.It was speculated that the treatment mechanism was related to vasoconstrictor and vasodilator like CGRP、NOS、6-keto-PGF1 a、TXB2.But it was not related to NO、ET according to the experiment.4.2 The established HPLC method was accurate,simple,specific,and could be used for the determination of GAS and HBA in blood dialyate.4.3 Chuan xiong could speed up the absorption and elimination rate of GAS in the blood.However,it’ s effect on bioavalability was associated with the amount of chuanxiong extracts.Chuan xiong could speed up the elimination of HBA and increase bioavailability,but it’ s effect on absorption was depended on dosage of chuan xiong exstrats.