Basic Research On Composition Rules Of Herba Ephedrae Couplet Medicine—changes In Plasma Pharmacokinetics,Tissue Distribution And Excretion Of Epherae Herba-gypsum Fibrosum Herb Pair

BackgroundIngredients of Chinese medicine are complex,one prescription or one drug is difficult to obtain a regular understanding.Although the composition of Herba couplet is simple,it represents core features of series derived prescription and contains a wealth of objective rule.Research on Herba couplets mostly based on traditional Chinese medicine theory,from aspects in vitro analysis,pharmacological effects,pharmacokinetics and metabolism by modern techniques in order to explain the empirical clinical medication.This topic is from the basic research of Herba Ephedrae couplets(the Key Project of Natural Science Foundation of China),and we take Epherae Herba-Gypsum Fibrosum as research object.They have contrary taste,one warm one cold,one ascent one descent,one releasing one clearing.They seem contrary but the real is complementary in eleven "Zhongjing prescriptions".In classic prescription of"Maxing Shigan decoction",Epherae Herba-Gypsum Fibrosum(1:2)was good at treating lung heat,asthmatic cough and body heat.Preliminary pharmacological and metabolomic study showed:Epherae Herba-Gypsum Fibrosum(1:2)had a better effect on antifebrile and antiasthmatic than Epherae Herba-Gypsum Fibrosum(1:1)and Epherae Herba-Gypsum Fibrosum(1:4).Epherae Herba and Gypsum Fibrosum combined use or alone could improve the fever rat’s metabolic disorder which induced by yeast.Especially combination adjusted the biomarkers call-back maximum.Nowadays,researches on Epherae Herba and Gypsum Fibrosum are focus on pharmacodynamics and content changes of chemical component in aqueous extract.Pharmacokinetics of this Herba couplet are rarely reported,even though Herba couplets are the major predecessor of herbal medicinal compound prescribes.For this reason,combined use of Epherae Herba-Gypsum Fibrosum of"Increasing efficacy and Reducing toxicity" may relation to "dynamic changes" of effect or toxic ingredients in our body.We analyze blood concentration,tissue distribution,excretion behavior of index component in rats after oral administration aqueous extract of Epherae Herba-Gypsum Fibrosum,in order to explain the rationality and scientificity of Epherae Herba-Gypsum Fibrosum matched in different dose.AimThis study is the extend of chemical composition in vitro,pharmacodynamics,metabolomics of Epherae Herba-Gypsum Fibrosum herb pair.We take effect or toxic ingredients of Epherae Herba-Gypsum Fibrosum herb pair as a core to carry on our pharmacokinetic research.In order to find the intrinsic relationship between dose-time-toxicity-effect,to deepen the rich compatibility meaning of Epherae Herba-Gypsum Fibrosum.1.To explore the influence on pharmacokinetic parameters of five ephedrine(NME,NMP,E,PE,ME)after Epherae Herba-Gypsum Fibrosum matched in different ratio(1:0,1:1,1:2,1:4).Five ephedrine were detected with UPLC-MS/MS.2.To compare the changes of tissue distribution,excretion condition of ephedra alkaloids after Epherae Herba-Gypsum Fibrosum matched.3.To inspect the influence of Epherae Herba-Gypsum Fibrosum different proportions(1:0,0:2;2:2,1:2,0.5:2)to pharmacodynamics of three elements(Ca,Na,Mg)in serum.4.To establish the inner relationship among chemical composition,pharmacological effect and toxicological effect after having a comprehensive analysis of past researches.MethodsMaxing Shigan decoction first appeared in "Treatise on Fevers",said:"Ephedrae four tael(removed head);Semen Armeniacae fifty,peeled tip;Glycyrrhiza two tael,processed;Gypsum Fibrosum half a kilo." According to preliminary pharmacology and toxicology experiment,We selected Epherae Herba-Gypsum Fibrosum as the classical herb pair to research,and designed the dose ratio of Epherae Herba-Gypsum Fibrosum(1:0,1:1,1:2,1:4,0:1).1.To explore the influence on ephedra alkaloids plasma pharmacokinetics after Epherae Herba-Gypsum Fibrosum matched in different dose ratio.Firstly we established UPLC-MS/MS method to detect the content of NME,NMP,E,PE and ME in rat plasma simultaneously.SD rats were randomly divided into four groups and orally administered with different-dose Epherae Herba-Gypsum Fibrosum decoction(1:0,1:1,1:2,1:4).Their blood were collected at specific time points,the main content of five ephedra alkaloids were detected.The concentration-time curve and main pharmacokinetic parameters were calculated.To explore the influence on pharmacological effect or toxicologic ingredient’s pharmacokinetic behavior after Ephedra matched different proportions of Gypsum;Analyze the line to effect changes after Epherae Herba-Gypsum Fibrosum matched.2.We researched the tissue distribution after Epherae Herba-Gypsum Fibrosum matched.After UPLC-MS/MS method detecting the content of NME,NMP,E,PE and ME in tissue was established,SD rats were randomly divided into two groups and orally administered with Epherae Herba(1:0)and Epherae Herba-Gypsum Fibrosum decoction(1:2).Their heart,liver,spleen,lung,kidney and brain were collected at specific time points.Then we detected five ephedra alkaloids in their organs and compared the tissue distribution and accumulation of pharmacological effect or toxicologic ingredient between Epherae Herba-Gypsum Fibrosum matched and alone.In order to research the correlation to effect or poison intensity change after Epherae Herba-Gypsum Fibrosum matched.3.To study the excrete characteristics of ephedra alkaloids in urine and feces after Epherae Herba-Gypsum Fibrosum matched.After UPLC-MS/MS method detecting the content of NME,NMP,E,PE and ME in urine and feces was established.SD rats were randomly divided into two groups and orally administered with Epherae Herba(1:0)and Epherae Herba-Gypsum Fibrosum decoction(1:2).Their urine and feces were collected at specific time points.Then we detected five ephedra alkaloids in their excreta and analyzed its excretion preliminarily.4.We analyzed the change of Ca,Na and Mg level in serum after Epherae Herba-Gypsum Fibrosum matched in different proportions.SD rats were randomly divided into five groups and orally administered with different dose Epherae Herba-Gypsum Fibrosum decoction(1:0,0:2,2:2,1:2,0.5:2).Their blood were collected at specific time points.After detecting the content of Ca,Na and Mg by Roche biochemical analyzer,we obtained concentration-time curve and associated pharmacokinetic parameters.Then we analyzed the change of Ca,Na and Mg level after matched different doses of Ephedra,so explored relationship of antipyretic effect enhanced but not sweating after Epherae Herba-Gypsum Fibrosum combine used.5.Data analysis.DAS 3.2.2 software is used to calculate pharmacokinetic parameters and SPSS 13.0 software is used to analyze statistic.Using One-way ANOVA to compare pharmacokinetic parameters among groups,while LSD method(homogeneity of variance)and the Games-Howell(missing variance)for multiple comparisons.Using independent samples T test for mean parameters between two groups.And repeating measurement data analysis was for tissue distribution data analysis(p<0.05,mean statistically significant).Results1.The quantitative analysis method of NME,NMP,E,PE,ME in rat biological samples was established successfully.Specificity,linearity,limit of quantification,precision,accuracy,extraction recoveries,matrix effects and dilution factor study of this method are in line with the requirements of the relevant analysis in vivo.2.Study on pharmacokinetic behavior of ephedra alkaloids in rat plasma after Epherae Herba-Gypsum Fibrosum matched in different dose ratio.Absorptive trend(AUC0-t/Dose)was NME≈NMP>E≈PE≈ME in all groups.There were significant differences in AUC0-t and tmax of four groups,as Cmax had no difference(P>0.05);compared with Epherae Herba group,Epherae Herba-Gypsum Fibrosum 1:1 and 1:2 decreased the AUC0-t of NME,NMP and E,while Epherae Herba-Gypsum Fibrosum 1:2 and 1:4 shorten their tmax;however PE and ME had same trend but no difference(P>0.05).In three Epherae Herba-Gypsum Fibrosum groups,as dose of Gypsum Fibrosum increased,the change of five ephedrine alkaloids were similar shown t1/2z,MRT and Vz/F increased yet tmax and CLz/F decreased.3.The tissue distribution of ephedra alkaloids after Epherae Herba-Gypsum Fibrosum matched.Time factors and position factors all could affect alkaloids distribution in vivo and there was a significant interaction between time and organization.The same ingredients had different trends in different tissues and the distribution amount of lung,kidney,spleen were in the majority.Compared with the Epherae Herba group,the absorption of E,PE and ME in lung of Epherae Herba-Gypsum Fibrosum 1:2 group accelerated(tmax shortening)and equilibrium phase extended.E and PE in kidney also had the same changes even bimodal phenomenon.While equilibrium phase narrowed of E in heart and brain organ,and the occupied share of E smaller than PE(AUC0-t(E)/AUC0-t(PE)).4.Research on urine and feces excrete characteristics of ephedra alkaloids after Epherae Herba-Gypsum Fibrosum matched.Among five alkaloids of accumulative urine excrete rate,NME and NMP were at most(up to 80%-98%),then E and PE,ME was the least(just 4%-6%).The Rmax of Epherae Herba group was detected in 0-2 h,while Epherae Herba-Gypsum Fibrosum 1:2 group was 2-6 h.There was no different of excretion rate,t1/2,Rmax and Ke value between two groups,except accumlative excretion of ME in Epherae Herba-Gypsum Fibrosum 1:2 group more than Epherae Herba group(P<0.05).Meanwhile feces excretion was less,Accumulative excrete were less than 1%.As the same the kinetic parameter had no difference between two groups.5.The Ca,Na and Mg level in serum after Epherae Herba-Gypsum Fibrosum in different proportions matched.There were significant differences of pharmacodynamic parameters in Ca,Na/Ca and Mg among five groups,as Na had no difference(P>0.05).Compared with the Epherae Herba group,Epherae Herba-Gypsum Fibrosum 1:2 group could extend mean residence time(MRT),increased distribution volume(Vz/F)of Ca,and increased peak concentration(Cmax),extended MRT of Na/Ca and Mg.Gypsum Fibrosum group had no significant difference between Epherae Herba group(P>0.05)except Vz/F(Ca,Mg).In three Epherae Herba-Gypsum Fibrosum combined groups(2:2,1:2,0.5:2),the ratio of 1:2 showed climax or trough effect,because of the distribution volume of Ca maximum,peak concentration and AUC0-t of Mg maximum,as peak concentration of Na/Ca minimum.Conclusion1.The method of UPLC-MS/MS to detect the content of NME,NMP,E,PE and ME in biological sample(blood,tissue,feces,etc.)was more sensitive and rapid the past way.This method was applied to monitor drug content in vivo which guiding clinical medication safely.2.Gypsum Fibrosum in different dose would regularly effect the pharmacokinetics of active or toxic ingredients of Epherae Herba.While Epherae Herba-Gypsum Fibrosum 1:2 and 1:4,E and PE absorption rate accelerated,residence time extended,distribution volume and absorption degree increased,bioavailability enhanced,which linked up with when the amount of Epherae Herba less than Gypsum Fibrosum antipyretic and asthma effect better.But clinical proportion was 1:2,the reason possibly was NME and E in Epherae Herba-Gypsum Fibrosum 1:4 group the pharmacokinetics were similar to Epherae Herba,so the incidence of adverse reaction might enhance.As Epherae Herba-Gypsum Fibrosum 1:1,the ratio of AUC0-t、Vz/F between E and PE were less than Epherae Herba-Gypsum Fibrosum 1:2.The former occupied the share of ascension,as fluctuant process antiasthmatic effect reduced while diuretic effect enhance,in order to achieve the aim of diuretic effect better.In addition,After Epherae Herba-Gypsum Fibrosum combine used AUC0-t and Cmax of E and ME decreased,elimination rate accelerated,in order to decreased toxic and side efFect.This study confirmed the science and rationality of Epherae Herba-Gypsum Fibrosum in clinical medication from plasma pharmacokinetic view.3.Whether Epherae Herba or Epherae Herba-Gypsum Fibrosum compatibility group,the distribution of five alkaloids tended to lung,kidney and spleen organ.It is agree with the tropism of Epherae Herba and Gypsum Fibrosum.Gypsum Fibrosum could selectively affect the alkaloids include in the same target position(lung,kindey,heart and brain).achieving to adjust efficacy/toxicity ingredients proportion,in order to obtain the goal of effect-enhancing and toxicity-reducing of combine used.4.Five ephedra alkaloids mainly excreted in urine.The excretion rate of NME,NMP which were at the end of the transform chain were up to 80%～98%,as ME was just 4%～6%which at the top site.Furthermore the proportion of five ingredient in urine was different from that in aqueous extract.This terminals of the drug metabolism information proofed that ephedra alkaloids metabolism in vivo follow direction ME→E→NME,PE→NMP.Meanwhile the reason for alkaloids Rmax delay mightly were absorption faster and distribution equilibrium phase extended.5.Compared with Epherae Herba,Gypsum Fibrosum,Epherae Herba-Gypsum Fibrosum(2:2)group and Epherae Herba-Gypsum Fibrosum(0.5:2)group,Ephedra-Plaster(1:2)group had the best intervention effect of Ca(raised),Na/Ca(down)and Mg(raised)in serum.From the perspective of serum element,we explained compatibility connotation that Epherae Herba-Gypsum Fibrosum(1:2)antipyretic made cool down but not sweating,so no injury of body fluid.