Study On Pharmacodynamic Material Basis Of Danhong Injection On Myocardial Ischemia/reperfusion Injury In Rats By Differential Efficacy Of Serum Chromatographic Analysis

Background：Coronary heart disease is a major disease which threatens human health.Along with the wide application of coronary thrombolysis and coronary bypass surgery,the subsequent myocardial ischemia/reperfusion (MI/R) injury has drawn much moreattention among medical scientists. Traditional Chinese Medicine (TCM) plays animportant role in the treatment of MI/R injury. Especially, Danhong Injection (DHI) hasthe most excellent therapeutic effect. Although the bright prospects clinical application ofDHI, when given uncertainty of its pharmacodynamic material basis, its acceptance as avalid form of medical treatment around the world is quite difficult. Circumventing theseproblems involve the identification of the pharmacodynamic material basis of DHI. In thisstudy, we put forward a new research method, differential efficacy of serumchromatographic analysis (DESCA), to study the real pharmacodynamic material basis ofDHI. Through analysis standard combined with a HPLC-MS method to determine itsstructure, we find the pharmacodynamic material basis of Danhong Injection on myocardial ischemia/reperfusion injury in rats. It provides a new theory and idea for DHIto be made an international I chemical new drug and it provides a new idea for find theharmacodynamic material basis of TCMs anti-MI/R injury by this mechanism.Objectives：Through evaluating the expression of Akt and eNOS, the change of infarctsize, the changes of myocardial injury maker enzyme including creatine kinase isoenzyme(CK-MB), the changes of lactate dehydrogenase (LDH), cardiac troponin (cTnI), theinflammatory cytokines including: tumor necrosis factor-alpha (TNF-α), interleukin6(IL-6), interleukin10(IL-10), histopathological changes, we find the maximum andminimum pharmacodynamic time points when MI/R injury rats were treated with DHI.Combined the spectrum difference in serum between maximum and minimumpharmacodynamic time points when MI/R injury rats were treated with DHI, we find thepharmacodynamic material basis of DHI. Through analysis standard combined with aHPLC-MS method, we determine their structures. Finally, we feedback verify the abovepharmacodynamic material basis of DHI, and define they are really the pharmacodynamicmaterial basis of DHI. We also preliminary study their mechanism when MI/R injury ratswere treated with DHI.Methods：The study includes four parts:Part1Study on the ameliorated effects of DHI on MI/R injury rats on the expression ofAkt and eNOS, infarct size, myocardial injury maker enzyme including creatine kinaseisoenzyme (CK-MB), lactate dehydrogenase (LDH), cardiac troponin (cTnI), theinflammatory cytokines including: tumor necrosis factor-alpha (TNF-α), interleukin6(IL-6), interleukin10(IL-10), histopathological changes. Finally define the maximum andminimum pharmacodynamic time points when MI/R injury rats were treated with DHI.Part2Established the chemical fingerfrints:(1) fingerprint of blank serum;(2) fingerprintof danshen, honghua and danshen: honghua (3:1);(3) fingerprint of DHI;(4) fingerprint ofstandards;(5) fingerprint of serum at maximum and minimum pharmacodynamic timepoints.Part3Based on the above pharmacodynamic results, we analyzed the PMB of DHI usingDESCA. We compared the fingerprint of blank serum, DHI and fingerprint at the maximum (60min) and the minimum (10min) pharmacodynamic time point bychromatogram analytical method to find the possible pharmacodynamic time points ofDHI in rats with MI/R injury.Part4Further, we verify the above pharmacodynamic material basis of DHI, determinethe content of those pharmacodynamic material basis of DHI, made into injection A whichonly just include the pharmacodynamic material basis and injection to further verify thematerial basis for efficacy. Finally, we determine and evaluate the pharmacodynamicmaterial basis of DHI used to treat with MI/R injury rats, and clarify the important effectsof DESCA on studying pharmacodynamic material basis.Results：1. DHI can ameliorate on the expression of Akt and eNOS, infarct size, myocardial injurymaker enzyme including creatine kinase isoenzyme (CK-MB), lactate dehydrogenase(LDH), cardiac troponin (cTnI), the inflammatory cytokines including: tumor necrosisfactor-alpha (TNF-α), interleukin6(IL-6), interleukin10(IL-10), histopathologicalchanges and the hemorheological indictors on MI/R injury rats. The maximum andminimum pharmacodynamic time points when MI/R injury rats were treated with DHIare10min and60min.2. Through analysis the fingerprint of danshen, honghua and danshen:honghua (3:1), wecan find that there is no difference between Danshen and honghua extracted separatelyand together.3. By using DESCA, we preliminary find the possible pharmacodynamic time points ofDHI in rats with MI/R injury. Through compared with standard substances and theresult of HPLC-MS, we defined their structures, and respectively are danshensu,protocatechualdehyde, hydroxysafflor yellow A, rosmarinic acid and salvianolic acidB.4. We determined the content of those danshensu, protocatechualdehyde, hydroxysaffloryellow A, rosmarinic acid and salvianolic acid B in DHI, they are respectively1000.3μg/mL、88.2μg/mL、24.0μg/mL、205.2μg/mL、728.8μg/mL.5. Injection A can ameliorate on the expression of Akt and eNOS, infarct size, myocardial injury maker enzyme including creatine kinase isoenzyme (CK-MB), lactatedehydrogenase (LDH), cardiac troponin (cTnI), the inflammatory cytokines including:tumor necrosis factor-alpha (TNF-α), interleukin6(IL-6), interleukin10(IL-10) onMI/R injury rats, and the effects in equivalent to more than90%of DHI.Conclusions：1. Through study on myocardial injury, anti-inflammatory and related mechanismindicators, we find the maximum and minimum pharmacodynamic time points whenMI/R injury rats were treated with DHI are10min and60min.2. The possible pharmacodynamic material basis of DHI in rats with MI/R injury aredanshensu, protocatechualdehyde, hydroxysafflor yellow A, rosmarinic acid andsalvianolic acid B.3. DHI can protect MI/R injury rats by Akt-eNOS signaling pathway.