| Ling-Gui-Zhu-Gan decoction was originally described in "Treatise on Febrile Diseases".It is composed of four crude drugs:namely Poria cocos(Fuling),Ramulus cinnamomi(Guizhi),Atractylodis macrocephalae Rhizoma(Baizhu)and Radix glycyrrhizae(Gancao).It is mainly used in the treatment of acute myocardial ischemia in the clinic.In this study,using Ling-Gui-Zhu-Gan decoction as the research object,based on the characteristics of the integrity of traditional Chinese medicine,using serum spectroscopy and combination of pharmacokinetics methods,expounded the pharmacodynamic material basis and compatibility rules of Ling-Gui-Zhu-Gan decoction.Acute myocardial ischemic injury is one of the major causes of sudden death in the world.It has become a major disease that threatens human health and should arouse widespread concern.In this study,an acute myocardial ischemia model was established by intraperitoneal injection of isoproterenol hydrochloride 2 mg/kg in two consecutive days.The changes of creatine kinase,aspartate aminotransferase,lactate dehydrogenase and malondialdehyde and superoxide dismutase in the heart of model animals were determined in model rats.The results indicated that the model established successfully and screening the efficacy index changed significantly.The pharmacological index of creatine kinase is used as a pharmacodynamic evaluation indicator.UPLC-MS/MS-based metabonomics method was used to evaluate the stability of rat model of acute myocardial ischemia reproduced by the above method.The results showed that the model has good time continuity,and differences in urine metabolic profile after modeling can be effectively maintained for more than 3 days.This suggests that when using this model for related research,the evaluation time for therapeutic administration should not exceed 3 days.UPLC-Q-TOF-MS/MS method was used to collect the serum of rats after intragastric administration of Ling-Gui-Zhu-Gan decoction in both positive and negative ion mode.The transitional components in rats after administration were analyzed.By comparison with the references and blank plasma chromatograms,and according to the compound excimer ion peaks and secondary fragment ion peaks,and referring to the relevant literature,a total of 46 transitional components in plasma were identified,of which 39 were prototype components and 7 were metabolites.A simple and rapid UHPLC-MS/MS method was developed for the simultaneous determination of cinnamaldehyde,cinnamic acid,and 2-methoxy cinnamic acid in rat whole blood.Chromatographic separation was performed on a Thermo Scientific C18 column.Using acetaminophen as internal standards,samples were processed by a one-step protein precipitation with acetonitrile-37%formaldehyde(90:10,v:v)and eluted with a 0.005%formic acid water-acetonitrile gradient system.All three components were effectively separated,and endogenous components did not interfere with the assay.The method meets the validation requirements for the analysis of biological samples in terms of specificity,accuracy,precision,extraction recovery and matrix effects.A comparative pharmacokinetic study was conducted on the rats after intragastric administration of Fuling single decoction and a combination of Fuling-Baizhu herb pair decoction.The experimental results showed that the Cmax and AUC of the biologically active substances tumulosic acid,dehydroturmoroic acid and polyporenic acid C were significantly increased in the Fuling-Baizhu herb pair decoction group,indicating that the presence of Baizhu could significantly increase the effect of Fuling.In accordance with the compatibility theory of Baizhu can increase the efficacy of Fuling in Ling-Gui-Zhu-Gan Decoction At the same time,the differences in pharmacokinetics of cinnamic acid and 2-methoxycinnamic acid in plasma after administration of Guizhi single decoction and a combination of Guizhi-gancao herb pair decoction in healthy rats were compared.Both Cmax and AUC were significant increased in Guizhi-gancao herb pair decoction.The phenomenon further confirms the rationality of the compatibility of Ling-Gui-Zhu-Gan Decoction,and provides the necessary basis for the study of the efficacy-time relationship in the later period of the project.Using serum creatine kinase as a pharmacodynamic indicator,blood samples were collected at 9 different time points after oral administration of Ling-Gui-Zhu-Gan Decoction to the acute myocardial ischemia rats,and an effect-time relationship study was conducted.One hundred and eight rats were randomly divided into 9 groups according to their body weight and the time of blood collected after dosing.Each group consisted of 12 rats and were divided into 1 h group,1.5 h group,2 h group,2.5 h group,3 h group,4 h group,6 h group,8 h and 12 h groups,the acute myocardial ischemia model was replicated by subcutaneous injection of ISO.Two hours after modeling injection,each group of rats was divided into two parts by average body weight.One part was given physiological saline solution by gavage as a model group;the other part was given Decoction as a drug administration group.Blood was collected from the retro-orbital venous plexus and serum creatine kinase was measured.In order to eliminatee possible systematic errors and more objectively reflect the therapeutic effects of drugs on rats with acute myocardial ischemia,the present study calculated and compared the values of creatine kinase indexes at various time points based on Percent decrease as an effect against myocardial ischemia.With time as the horizontal axis,the effect indicator is plotted on the vertical axis,and the effect-time curve of the index is obtained.The experimental results showed that the maximum percentage of creatine kinase decreased in serum occurred at 4.0 h after administration.In addition,the percentage decrease of creatine kinase in plasma fluctuates at 5.0 h after administration.It is speculated that the reason for this phenomenon may be caused by comprehensive adjustment of multi-pathways and multi-targets of Ling-Gui-Zhu-Gan Decoction.The ultra-HPLC-MS/MS technique was used to detect the concentration of 11 indicators(tumoric acid,dehydroturmoroic acid,polyporenic acid C,cinnamic acid,atractylenolide Ⅰ,atractylenolide Ⅱ,atractylenolide Ⅲ,glycyrrhizic acid,glycyrrhetinic acid,glycyrrhizin and isoliquiritigen)in rats with acute myocardial ischemia at different time points.Schisandrin was used as the internal standard and plasma samples were precipitated with acetonitrile.After sample processed,the gradient elution was carried out using 0.1%formic acid water-acetonitrile as the mobile phase,and the MS data was obtained using both positive and negative in MRM ion mode.The method meets the requirements of the validation of biological sample analysis methodology in terms of specificity,accuracy,precision,extraction recovery rate and matrix effect,and provides accurate and reliable blood drug concentration data for the fitting of drug efficacy and blood drug concentration.In the time interval(at each time point)when the pharmacodynamic detection index changes,the concentration data(taking the natural logarithm value)of each index component in the plasma and the pharmacodynamic index data are linearly fitted.The correlation coefficient and the size of significance were used as the evaluation indexes of the correlation;the slope of the curve was used as the evaluation index of its contribution.Among the 11 indicators monitored,the components with significant changes in serum creatine kinase levels(significance<0.05)were in the following order:tumoric acid,dehydroturmoroic acid,polyporenic acid C,cinnamic acid,atractylenolide Ⅰ,atractylenolide Ⅱ,atractylenolide Ⅲ,glycyrrhizic acid,glycyrrhetinic acid,glycyrrhizin and isoliquiritigen.It is speculated that the combination of each herb in Ling-Gui-Zhu-Gan Decoction causes acute myocardium.The pharmacodynamic material basis for the change of creatine kinase in the serum of acute myocardial ischemia rat model may be the above index components.The slope(k)of the PK-PD correlation curve in the time interval where the pharmacodynamic index changes was used as a weight,and the weighted combination of the concentration of each index component(natural logarithm)in rat plasma at different time points was calculated by the formula as the overall "apparent effect concentration"(C’),draw the apparent effect concentration-time curve,calculate the "combination pharmacokinetics"(CPK)parameters,and use "apparent effect concentration" and pharmacodynamic indicators relative value(percentage of the pre-dose index value as a base)to analyze the CPK-PD linear model.Via "apparent effect concentration" calculation and CPK-PD fitting,results showed that there is a overall correlation between "apparent-effect concentration"-time curve and pharmacological effect-time curve of each effect component of Ling-Gui-Zhu-Gan Decoction which obviously affected the content of creatine kinase in serum.Results indicated that after CPK-PD fitting,using the correlation coefficient(R)and significance(Sig)as the pharmacodynamic material basis indicators,curve slope(k)as its contribution to the efficacy(including synergy and antagonism)evaluation indicators are feasible.Among the eleven detectable,transitional components in Ling-Gui-Zhu-Gan Decoction,all were "effect components" associated with serum creatine kinase,including 4 main effect components,3 synergistic components and 4 antagonistic components.Tumoric acid,polyporenic acid C,cinnamic acid,glycyrrhetinic acid are the main substances for decreasing the concentration of serum creatine kinase;atractylenolide Ⅰ,atractylactone Ⅲ,liquiritigenin and isoliquiritigenin negatively related to the decrease of creatine kinase in serum(with antagonistic effect);dehytumoric acid,atractylenolide Ⅱ,glycyrrhizic acid and other three components have a certain correlation(a synergistic effect)with the decrease of creatine kinase in serum.Baizhu(assistant herb)can significantly enhance the bioavailability of Fuling(monarch herb),which has a significant effect of enhancing the effect of Monarch herb,while Gancao can also significantly increase the bioavailability of Guizhi,and it has the effect of enhancing the minister effect.In this study,serum pharmacochemistry,serum pharmacology,and combined pharmacokinetics were used to construct a "combination plasma concentration"pharmacodynamic model" to study the pharmacodynamics basis of Ling-Gui-Zhu-Gan Decoction in the treatment of acute myocardial ischemia.Comparative pharmacokinetics method was used to illustrate the monarch,minister,assistant and guide compatibility rulers of Ling-Gui-Zhu-Gan Decoction in order to establish a new theoretical system for the systematic study of the material basis of traditional Chinese medicine and its compound,and to solve the bottleneck problem in the basic research of pharmacodynamic substances. |
PDF Full Text Request