Pharmacodynamics Research Of Danshensu Isopropyl Ester

Free radicals are atomic or molecular species with unpaired electrons on an otherwise open shell configuration, such as superoxide anion radical (O₂^-·), hydroxyl radical (OH), Lipid alkoxyl radical (LO·). Under normal conditions, free radicals are continuously produced and eliminated at good balance and benefit the body; while, under pathological conditions, excessive free radicals can be accumulated in the body due to either increase in generation or decrease in elimination, which will cause damages to the body. Many studies showed that over 100 kinds of diseases are related to metabolic disturbance of free radical. For example, cancers, cardio-cerebrovascular diseases, gastrointestinal diseases and so on. Therefore, it is important to illuminate the physiological and pathologic effects of free radical in order to prevent and treat the diseases caused by free radicals.Compound danshen dropping prill (CDDP), a combination of Radix Salviae Miltiorrhizae, Radix Notoginseng and Borneolum Syntheticum, is a Chinese remedy, which has been reported has the effects of activating the blood and reducing phlegm. In the study of metabolins which are from CDDP in vivo, our cooperative team find that Danshensu isopropyl ister (DIE), a kind of active metabolin which is from CDDP in vivo, is a yellowish-brown liquid which dissolves easy in some dissolvent such as water, colonial spirit, alcohol and acetic ether. In the study to DIE, it was found that DIE can expand mesentery artery in rats and enhance ventricular contraction force. But nobody study the good positive effect, or other pharmacologic action and mechanism against free radical of DIE. In this project, we studied mainly the pharmacological action of DIE along the following three aspects. First, Model of D-galactose induced aged rats was established to observe how DIE effect SOD vigour, MDA density and the apoptotic index of purkinje fibers in cerebellum of D-galactose induced aged rat , and Vitamine E (VE) was used as positive control. Second, the model of myocardial ischemia/reperfusion injury was established in isolated rat heart perfused with Langendorff thechnique to observe how DIE effect the content of high energy phosphates and myocardial ultrastructure, and CDDP was used as positive control. Third, the model of cardiac arrhythmia was established to observe how DIE effect the arrhythmias induced by exogenous free radical, and verapamil (VPL) was used as positive control.Methods:In the first part, 80 healthy Wistar rats which were 6 months, were applied in this study and divided into 5 groups at random: normal group, aging model group, positive control (VE 100.0 mg/Kg) group, two DIE groups with the dose of 21.0 mg/Kg and 7.0 mg/Kg, and each group had 16 rats. The rats of normal group was treated with daily subcutaneous injection of 4.0 mL/kg saline (0.85%), The rats of model group was treated with daily subcutaneous injection of 2.0 mL/kg saline (0.85%) and 2.0 mL/kg D-gal (2.5%), The rats of VE group and two DIE groups with the dose of 21.0 mg/Kg and 7.0 mg/Kg was treated with daily subcutaneous injection of 2.0 mL/kg drug of corresponding concentration and 2.0 mL/kg D-gal (2.5%), for successive 8 weeks. In each group, the half of animal were used to detect SOD vigour and the content of MDA; the other half to detect the apoptotic index of purkinje fibers of cerebellum cortex of rats in each group by methy/green pyronin stain.In the second part, 80 healthy Wistar rats were applied in this study and divided into 8 groups at random: normal group, ischemia/reperfusion injury model group, CDDP prevention group, CDDP protection group, two DIE prevention groups with the dose of 4.00 mg/L and 0.04 mg/L, two DIE protection groups with the dose of 4.00 mg/L and 0.04 mg/L, and each group had 10 rats. A myocardial ischemical/ reperfusion injury model was established with the Langendroff method. Normal group: the hearts of rats were mouted on a Langendorff apparatus and perfused with modified Krebs-Hanseleit phosphate buffer for 75 min; I/R group: the hearts were perfused with modified Krebs-Hanseleit phosphate buffer for 15 min followed by global ischemia for 40 min, and then reperfused for 20 min; Each drug groups: the hearts were perfused or reperfused with modified Krebs-Hanseleit phosphate buffer which has drug of corresponding concentration, the concentration of drug is by turns 50.00 mg/L, 50.00 mg/L, 4.00 mg/L, 4.00 mg/L, 0.04 mg/L or 0.04 mg/L. In each group, High energy phosphates concentrations were determined by HPLC in myocardium of seven rats and the ultrastructure morphological changes of the other three rats'myocardial cell were observed by transmission electron microscope.In the third part, 50 healthy Wistar rats were applied in this study and divided into 5 groups at random: normal group, model group, Verapamil 1.00 mg/L group, two DIE groups with the dose of 4 mg/L and 0.04 mg/L, and each group had 10 rats. The arrhythmias in the isolated rat heart were induced by FeSO₄ perfusing (0.025 mmol/L)/Vitamine C (VC)(0.100 mmol/L) with the Langendroff method. normal group: the hearts of rats were mouted on a Langendorff apparatus and perfused with modified Krebs-Hanseleit phosphate buffer for 45 min; Model group: the hearts were perfused with modified Krebs-Hanseleit phosphate buffer which has FeSO₄ perfusing (0.025 mmol/L)/VC (0.100 mmol/L) for 15 min, and then with modified Krebs-Hanseleit phosphate buffer for 30 min; Each drug groups: the hearts were perfused with modified Krebs-Hanseleit phosphate buffer which has FeSO₄ perfusing (0.025 mmol/L)/VC (0.100 mmol/L) and drug of corresponding concentration for 15 min, and then with modified Krebs-Hanseleit phosphate buffer for 30 min. The ECG was recorded when the hearts were perfused.Results:In the first part, comparing the aging group with the normal group, there is significant decrease in SOD vigour, significant increase in the content of MDA, significant decrease in apoptotic index of purkinje fibers (P<0.01). comparing the effects between two DIE groups with the dose of 21.0 mg/Kg and 7.0 mg/Kg and VE 100.0 mg/Kg group with the aging group, there is significant increase in SOD vigour, significant decrease in the content of MDA, significant decrease in apoptotic index of purkinje fibers (P<0.01). The difference of the effects between DIE group with the dose of 21.0 mg/Kg and VE 100.0 mg/Kg group is insignificant (P>0.05). comparing DIE group with dose of 7.0 mg/Kg with the former two groups, there is significant decrease in SOD vigour, increase in the content of MDA, significant decrease in apoptotic index of purkinje fibers(P<0.05).In the second part, Energy-rich phosphate concentrations: the study showed that as compared with the normal control group, ATP, ADP, AMP and TAN levels were significantly decreased in the ischemia/reperfusion injury model group (P<0.01), as compared with the model group, ADP, AMP and TAN levels were significantly increased in the CDDP prevention group, the CDDP protection group, the DIE prevention groups with the dose of 4.00 mg/L, the DIE protection groups with the dose of 4.00 mg/L, the DIE prevention groups with the dose of 0.04 mg/L and the DIE protection groups with the dose of 0.04 mg/L (P<0.01) (except ADP levels in the DIE prevention groups with the dose of 0.04 mg/L); ADP levels was increased in the DIE prevention groups with the dose of 0.04 mg/L (P<0.05); ATP levels were significantly increased in the CDDP prevention group, the CDDP protection group, the DIE prevention groups with the dose of 4.00 mg/L and the DIE protection groups with the dose of 4.00 mg/L (P<0.05). The ultrastructure damages in myocardium tissue. In the normal control group, myoneme of myocardium cell was integral, sarcomere was regulation, light band and dark band was clear, Karyotheca of myocardium cell and mitochondrial membrane was integrity. In nucleus density of chromatin was proport, distribution of chromatin was uniformity, mitochondrial matrix was pycnotic. It was rich in the number of mitochondria and glycogen particle. The ultrastructure damages of myocardium tissue was very severe in the ischemia/reperfusion injury model group. sarcomere was arranged disorderly or disappeared, the dropsy of base material was very obvious in nucleus, mitochondria was swollen, mitochondrial cristae was fused or disappeared, Mitochondria and glycogen particle significantly diminished. The ultrastructure is damaged quite a little in the CDDP prevention group, the CDDP protection group, the DIE prevention groups with the dose of 4.00 mg/L, the DIE protection groups with the dose of 4.00 mg/L. In the DIE prevention groups with the dose of 0.04 mg/L and the DIE protection groups with the dose of 0.04 mg/L, the ultrastructure damages of myocardium tissue was lessened, as compared with the ischemia/reperfusion injury model group, but it was still severe.In the third part, normal group has no the arrhythmias; the incidence rate of arrhythmias induced by the exogenous free radical was 100%, it has chiefly conduction blockade and premature ventricualr contraction; the incidence rate of arrhythmias reduces significantly in VPL 1.00 mg/L group and two DIE groups with the dose of 4.00 mg/L and 0.04 mg/L, as compared with the model group.Conclusions:1. DIE has the anti-oxidative and anti-apoptotic effects, The anti-oxidative and anti-apoptotic effects of DIE is similar to VE.2. DIE can protect cardiomyocytes against ischemia/reperfusion injury, The protection of DIE is similar to CDDP.3. DIE has the preventing effect on the arrhythmias induced by the exogenous free radical, the effect of DIE is similar to VPL.