Study On Pharmacokinetics And Pharmacodynamics Of The Main Effective Constituents Of Danhong Injection After Compatibility

Objective Building a stable,feasible,successful and reproducible rat disease model of focal cerebral ischemia-reperfusion(I/R)injury and a HPLC-DAD method for the simultaneous quantitation of danshensu,protocatechuic acid,vanillic acid,salvianolic acid B and hydroxysafflor yellow A(HYSA)in cerebral I/R injury rat plasma to evaluate the influence of the main effective constituents in Danhong Injection on the pharmacokinetic characteristics after orthogonal design.Moreover,to establish PK-PD modelling of the main effective constituents in Danhong Injection based on the anti-inflammatory effects,and got the concentration-response equations about every index component,which could help forecast the change rules between plasma concentrations and effects with timeMethods(1)MCAO models:Using modified middle cerebral artery occlusion(MCAO)method for establishment of cerebral I/R model in SD rats by insertion of suture-embolus into the common carotid artery(CCA)without the pterygopalatine artery(PPA)ligation.The suture-embolus was removed for reperfusion at 1 h after occlusion.After 24 h of reperfusion,the neurological scores,cerebral infarction and brain histopathology were evaluated.(2)HPLC-DAD method:The MCAO model was established,in which all of the rats were intravenously injected of effective constituents of Danhong Injection simultaneously(danshensu,protocatechuic aldehyde,salvianolic acid B and HYSA).The analysis of the measured components’plasma concentration was carried at 30℃ on the reversed-phase column of Agilent Eclipse XDB-C18(4.6 mm×150 mm,5 μm)and eluted with acetonitrile and water containing 0.4%phosphate acid as mobile phase in gradient mode.Detection wavelengths were 280 nm for danshensu,protocatechuic acid,vanillic acid,salvianolic acid B and 403 nm for HYSA.The propyl p-hydroxybenzoate was used as the internal substance(3)Pharmacokinetic studies:According to the orthogonal design L9(34)composed of the main effective constituents(danshensu,protocatechuic aldehyde,salvianolic acid B and HYSA)of Danhong Injection.9 different dosage ratios combinations were formed,which were used for intravenous administration in MCAO rats to study the pharmacokinetics of effective constituents after compatibility.Using non-compartmental model of DAS 3.2.6 software to fit the pharmacokinetic parameters and using total quantum statistical moment analysis method to evaluate the total pharmacokinetic characteristics(4)PK-PD studies:Selecting the six combination to explore the relationships between pharmacokinetics and pharmacodynamics for effective constituents in MCAO rats with the decreases of TNF-α and IL-1β levels as PD indicators.Using DAS 3.2.6 software to fit the PK-PD modelling and get the quantitative equations between drug concentration and efficacy.Results(1)Successfully established the rat focal cerebral ischemia reperfusion model:After MCAO for 1 h followed by 24 h reperfusion,rats appeared apparent symptoms of neurological deficit and the average neurological score was 2.83±0.75.TTC staining showed that brain tissues stained uniform red in the sham group,whereas areas of infarction in the right cerebral hemisphere were white in the model group.HE staining showed that no histopathological abnormalities were observed and no inflammatory cells infiltrated in the sham group.But in the model group,most neurons in the ischemic zone appeared shrunken with eosinophilic cytoplasm and triangulated pyknotic nuclei,edematous glial cells,a fuzzy cell outline,loose cytoplasm,condensed nuclei,swelling,severe cell deformation and necrosis were observed(2)Successfully established the HPLC-DAD method:Protocatechuic aldehyde rapidly metabolized in MCAO rat,but its metabolites protocatechuic acid and vanillic acid were easily detected.The linear ranges of danshensu,protocatechuic acid vanillic acid,salvianolic acid B and HYSA were 0.35-140 mg·L-1(R2=0.999 8),0.15-60 mg·L-1(R2=0.999 0),0.05-20 mg·L-1(R2=0.998 8),0.25-100 mg·L-1(R2=0.999 6)and 0.075-30 mg·L-1(R2=0.998 5)respectively.The mean recoveries were between 85%and 115%and the RSD of intra-day and inter-day were less than 10%.The results of stability meet the requirements for biopharmaceutical analysis.(3)The pharmacokinetic results:There were different effects of the effective constituents with orthogonal compatibility on the on the phannacokinetic characteristics and the total quantum statistical moment parameters.Danshensu had the strongest effect on the total AUC and had a significant effect(P<0.05).A3B3C2D1 was the preferred dose combination for AUCt.HYSA had the strongest effect on the total MRT and its optimal ingredient composition was A1B3C1D1.(4)The PK-PD results:The effective constituents of Danhong Injection could inhibit the rise of inflammatory factors such as TNF-α and IL-1β in rats at the early stage of focal cerebral I/R,and the time delay phenomena(hysteresis phenomena)between the drug plasma concentrations and the anti-inflammatory effects were obvious.The best models for pharmacodynamics were Sigmoid Emax models.Fitting results are in good correlation and all P values were less than 0.05.Conclusion(1)The modified MCAO method for establishment of cerebral I/R model is relatively simple,controllable and higher successful ratio,which can provide stable and reliable animal disease model for the later experiments in regard to pharmacokinetic and pharmacodynamic studies(2)The HPLC-DAD method was simple,rapid,precise and stable,which can be used to the pharmacokinetics studies on the compatibilities of danshensu,protocatechuic aldehyde,salvianolic acid B and HYSA in MCAO rats(3)Compatibility of the main effective constituents in Danhong Injection had a certain influence on their pharmacokinetic behaviors in MCAO rats.The total quantum statistical moment analysis could combine the multi-component pharmacokinetic parameters and the total pharmacokinetic behaviors.The total quantum statistical moment analysis method can be used to study the pharmacokinetics of multi-component traditional Chinese medicine(TCM)compound prescription(4)Danhong Injection against cerebral I/R injury may be related to the anti-inflammatory properties of the effective constituents.PK-PD modelling can be used to predict and evaluate the correlation between pharmacokinetics and pharmacodynamics of TCM.