Flavescens, Astragalus Drug Compatibility On The Therapeutic Effect And Mechanism Of Viral Myocarditis

Objection:1.To determine the proportion of the optimal compatibility and the optimal extracting methods of Sophorae Flavescentis Radix and Astragalus membranaceus couplet medicines through studying the enhancing immune function of mice.2.To compare the rats'plasma levels of Oxymatrine and Oxysophorcarpine after administering the extracting components of Sophorae Flavescentis Radix and Astragalus membranaceus couplet medicines with those only administering Sophorae Flavescentis Radix .3. To study of Antivirus effects against coxsackie virus B₃ of the extracting components of Sophorae Flavescentis Radix and Astragalus membranaceus couplet medicines and its protective effects on myocardial cells infected by Coxsackie virus B₃ in vitro.4. To investigate the curing effects of the extracting components of Sophorae Flavescentis Radix and Astragalus membranaceus couplet medicines on experimental coxsackievirus B₃ myocarditis in vivo.5. To study the underlying molecular mechanisms of the extracting components of Sophorae Flavescentis Radix and Astragalus membranaceus couplet medicines on murine virus myocarditis model induced by Coxsackievirus B₃. Material and method:1. Four methods of extracting were used: Water-extracting respectively and then combined the extracting-liquid together, and then fried; combined the two medicines together and then Water-extracting and then fried; ethanol-extracting respectively and then combined the extracting - liquid together, and then fried; combined the two medicines together and then ethanol-extracting and then fried. The dose ratio of the two herbs are 1:1,1:2,1:3,1:4,1:5,2:1,3:1,4:1,5:1. The contents of the total alkaloids of the extracts have been analyzed by ultraviolet spectrophotometry and the contents of Sophocarpin,Matrine,Oxysophoridine,Sophoridine,Oxymatrine and Astragalosideâ…£were analyzed by high performance liquid chromatography. The mice immune functions were studied after administered the components extracted from Sophorae Flavescentis Radix and Astragalus membranaceus couplet medicines. Using orthogonal design method, the optimal extraction process of the two herbs was determined.2. To investigate and compare with the Oxymatrine and Oxysophorcarpine pharmacokinetic parameters after giving the extracting components of Sophorae Flavescentis Radix and Astragalus membranaceus couplet medicines with those only giving the extracting components of Sophorae Flavescentis Radix using the internal standard method. There are two groups of mice, each six animals: one group administered the extracting components of Sophorae Flavescentis Radix and Astragalus membranaceus couplet medicines intragastrically, the other administered the extracting components of Sophorae Flavescentis Radix intragastrically. At 0.5 hours before the administration and at 0.5,1,2,3,4,6,8,10,12,24 h after the administration,0.5ml of the vein blood was sampled to observe the contents of Oxymatrine and Oxysophorcarpine in the blood serum. 3. Construct VMC model in vitro by infecting myocardiocytes of newborn Wistar rat. Groups: negative control(NC),virus group(VK),Ribavirin (RBV); Sophorae Flavescentis Radix high,medium,lower does treatment, Astragalus membranaceus high, medium,lower does treatment, the extracting components of Sophorae Flavescentis Radix and Astragalus membranaceus couplet medicines high,medium,lower does treatment, At 2,5 d after administering medicine, respectively, the cell beating rate of each hole are determined under the inverted microscope. The morphological changes of the cells were also observed under the inverted microscope. Cell survival rate was measured by MTT, and observe the protection from cytopathic effects (CPE),percentage of beat cells(PBC)and creatine kinase isoenzyme MB(CK-MB)4. Experimental research of the treatment of the extracting components of Sophorae Flavescentis Radix and Astragalus membranaceus couplet medicines on VMC model infected by CVB₃. Animals: pure male Balb/C mice, 4 weeks of age (weight about 12g). Groups: the extracting components of Sophorae Flavescentis Radix and Astragalus membranaceus couplet medicines high, medium and low dose group, control group, virus control group, Yu Dan Rong Xin Pill positive control group, 20 mice each group. Each group was divided into two sub-groups: one group sacrificed at 7d, the other at 14d. The survival rates, TCID50, histopathology changes were detected and compared.5. Using immunohistochemistry, in situ hybridization and in situ end labeling, Granzyme B protein, BCL-2 gene expression, Caspase3 protein, gene expression, and myocardial apoptosis were observed of each group sacrificed at 14d.Results:1. The optimal compatibility ratio of the Sophorae Flavescentis Radix and Astragalus membranaceus is 2:1. The optimal method of extracting is combining the two medicines together and then ethanol-extracting. The contents of the total alkaloids of the extracts have been analyzed using high performance liquid chromatography. The five highest contents of alkaloids were sophocarpine, matrine, oxysophocarpine, sophoridine and oxymatrine, And their contents 1.0128, 2.9458, 1.0803, 0.71790,3.0960 mg·g^-1 respectively. The astragaloside is 0.1192 mg·g^-1, the total alkaloid content 3.188 mg·g^-1 by spectrophotometry.However when the ratio of the two herbs 4:1 , using the method combining the two medicines together and then ethanol–extracting, the five highest content of alkaloids were 3.9020, 10.470, 2.4293, 2.2400, 6.9530 mg·g^-1, respectively. The astragaloside was up to 0.1306mg·g^-1 the total alkaloid content was 3.474 mg·g^-1.Through the study of the effects on immune function research, we found the method combining the two medicines (Sophorae Flavescentis Radix and Astragalus membranaceus ratio 2:1) together and then ethanol–extracting was better than other extraction methods on enhancing the immune function of mice. When the Chinese herbal medicines were used, we should not only consider the dose of the single herbs, but also pay attention to the ratio between the amounts of herbs. By compatibility, not only the role of the original herbs, but also their co-effect should be considered. Side effects and therapy effects play the same functions to the strengths of drug compatibility, so that the characteristics of the drugs with the formation of a new organic whole, to meet the needs of complex treatment of disease. In this experiment, with the guidance of the basic theory of Chinese medicine based on the results with pharmacological effect is mainly based on secondary indicators of active ingredient content, finalize flavescens, Astragalus dosage according to the compatibility of Health and the optimum ratio of 2:1.2. The pharmacokinetic results of Oxymatrine, oxysophocarpine after administered the extracting components of Sophorae Flavescentis Radix and Astragalus membranaceus couplet medicines and the extracting components of Sophorae Flavescentis Radix intragastrically : After administered the extracting components of Sophorae Flavescentis Radix and Astragalus membranaceus couplet medicines, the greatest concentration of Oxymatrine in rats'plasma is 2.01μg·mL^-1 and the peak time was at 6 hour.The peak time was at 12.91 hours after administering. The mean residence time of drug molecules staying in the body at 0-24 hours 22.61mg/L·h hours, The area under the curve of 0-24 hours was 43.31mg / L·h. After administered the extracting components of Sophorae Flavescentis Radix intragastrically, the greatest concentration of Oxymatrine in rats plasma 6.526μg·mL^-1, and the peak time was at 4 hours, The mean residence time of drug molecules staying in the body at 0-24 hours was 15.70 hours, The area under the curve of 0-24 hours was46.50mg/L*h. After administered the extracting components of Sophorae Flavescentis Radix and Astragalus membranaceus couplet medicines, the greatest concentration of Oxysophocarpine in rats'plasma is 3.954μg·mL^-1.The peak time was at 6 hours after administering. The mean residence time of drug molecules staying in the body at 0-24 hours 8.679 hours, The area under the curve of 0-24 hours was 43.31mg / L * h. After administered the extracting components of Sophorae Flavescentis Radix intragastrically, the greatest concentration of oxysophocarpine in rats plasma 2.202μg·mL^-1 , and the peak time was at 4 hours, The mean residence time of drug molecules staying in the body at 0-24 hours was 9.385 hours, The area under the curve of 0-24 hours was 26.11mg / L * h. According to the results the plasma Sophors peak time at 4 hours, after administering the extracting components of couplet medicines, compared with administered the extracting components of Sophorae Flavescentis Radix intragastrically,at 6 hours. The plasma peak time of extracting components of couplet medicines is significantly later than that of the extracting components of Sophorae Flavescentis Radix. The maximum concentration of oxysophocarpine in plasma administering the extracting components of couplet medicines was 2.202μg·mL^-1, while 3.954μg·mL^-1after the administration of the extracting components of Sophorae Flavescentis Radix. These meaned that Oxymatrine and oxysophocarpine was to be more easily absorbed in rats.3. The replication of virus be reduced after administer the the extracting component of Sophorae Flavescentis Radix, Astragalus membranaceus, the extracting components of couplet medicines in vitro and they can increase PBC, reduce CPE and CK-MB of myocardiocytes. At 2 days, there were significant differences in decreaseing the virus titer (P <0.05),between the high dose group Sophorae Flavescentis Radix, the extracting components of couplet medicines high, middle dose group and virus control group. There was no significant difference (P> 0.05) between these three groups respectively. On day 5, There are significant difference (P <0.05), compared Sophorae Flavescentis Radix high,medium does group and high dose group,couplet medicines high, medium and low dose group with virus control group. And there were significant difference (P <0.05) compare the couplet medicines high, medium group with Sophorae Flavescentis Radix high,medium does group. There was significant inhibition of viral replication in the groups of couplet medicines. The extracting components of Sophorae Flavescentis Radix and Astragalus membranaceus couplet medicines can reduce the cytopathic effect, raise the percentage of cultured myocardial cells and reduce the enzyme CK-MB, can have a protective effect myocardial cells against CVB₃ infection.4. Experimental results of the extracting components of couplet medicines t on CVB₃ drugs on infection of Balb / C mice with myocarditis: At 7d, only the extracting components of couplet medicines high dose group, can reduce mortality, and protect myocardial function against virus, according to mortality statistics, virus titration analysis, histopathology changes. At the time 14d, in the extracting components of couplet medicines high, medium and low dose group, TCID50 and histopathology changes in VMC mice myocardium were reduced significantly compared with other groups. The extracting components of couplet medicines can protect the myocardial function of VMC Balb / C mice and have a good dose-effect relationship. Yu Jung Dan Pill could also increase the survival rate of sick mice, significantly reduced inflammatory cell infiltration and myocardial necrosis.5. The result of Investigating the underlying mechanism: at 14d after administration by immunohistochemistry, in situ hybridization and in situ end labeling for apoptotic morphological analysis of the organization, the extracting components of couplet medicines can reduce the Granzyme B protein, BCL-2 gene expression, Caspase3 protein, gene expression, and myocardial expression of apoptosis, and the extracting components of couplet medicines were significantly lower on gene expression levels.Conclusion:1. The optimal compatibility ratio of the Sophorae Flavescentis Radix and Astragalus membranaceus is 2:1. The optimal method of extracting is combining the two medicines together and then ethanol-extracting2. The concentrtions of Oxymatrine and Oxysophorcarpine in the blood serum after administering the extracting components of couplet medicines intragastrically was higher than those of administering of Sophorae Flavescentis Radix intragastrically.3. The extracting components of couplet medicines can antiviral CVB₃ infection of myocardial cells and protection is better than single Chinese medicine Sophorae Flavescentis Radix and matrine, astragalus.4. The extracting components of couplet medicines has therapeutic effects. CVB₃ infection in Balb / C mice with myocarditis. 5. The extracting components of couplet medicines may be through the reduction of Granzyme B protein, Caspase3 protein and its gene expression, BCL-2 gene expression up-regulation, and myocardial expression of apoptosis play against some viral treatment.