Study On The Chemical Constituents And Anti-MCAO Pharmacodynamic Mechanism Of Bu-yang Huan-wu Decoction Based On Nrf2/ARE Pathway

Bu-yang Huan-wu Decoction(BYHW) is a traditional Chinese prescription of Yiqi-Huoxue,derived from "Yilin Gaicuo" which is written by Qingren Wang in the Qing Dynasty. It contains seven herbals:Astragali radix 120g、Angelicae sinensis radix 6g、Paeoniae radix rubra 5g、Pheretima 3g、Chuanxiong rhizoma 3g、Persicae semen 3g、Carthami flos 3g. It is used widely to treat or prevent the deseases. For example,Transient Ischaemic Attack、 Coronary Atherosclerotic Heart Disease、Acute Myocardial Infarction and Hromboembolic Disorders. The main purpose of the project is to study the chemical constituents of Bu-yang Huan-wu Decoction, then to study the pharmacodynamic effects and mechanism for anti cerebral ischemia reperfusion injury of BYHW.By contrast the retention time tR of AstragalosideⅣ in the ELSD chromatogram of BYHW and AstragalosideⅣ, it is concluded that the AstragalosideⅣ is in BYHW. It is one of the main components of monarch drug Astragali radix. Then UFLC/DAD/ESI/MS is used to analyze Astragali radix and Angelicae sinensis radix of BYHW. In positive and negative ion modes, it is to make more compounds have a higher response by adjusting the capillary voltage, cone voltage and other parameters as far as possible.According to the retention time of chromatographic peaks, the mass spectrometric fragmentation information, m/z information to determine the initial 5 additional compounds. It is identified as Ferulic acid, Calycosin-7-glucoside, Ononin, Calycosin, Formononetincan, AstragalosideⅣ. The content of isoflavones and saponins are higher in BYHW, which come from monarch drug Astragali radix; the content of organic acids is relatively high,which is from the official medicine Angelicae sinensis radix. The result is the same as the status of the two medicine in BYHW.Using the suture method to make the model of Middle cerebral artery occlusion/ reperfusion, the result of Longa method which is for neural function score shows BYHW can improve the neural function after cerebral ischemia-reperfusion injury. After the score, the antioxidant enzyme in the brain tissue ischemia side should be detected the activity of SOD, GST, GSH-Px and the content of MDA. The result shows that the activity of antioxidant enzyme in model is greater than sham, BYHW is greater than model; the content of MDA in model is lower than sham, BYHW is further low than model. It suggests that cerebral ischemia after reperfusion for 24h,the defense system of organism response to radical compensatory activation, while giving BYHW can improve the body’s own defense system. Then it takes Nissl staining to observe brain tissue, the result further shows that BYHW can improve brain tissue injury caused by cerebral ischemia reperfusion.It is an important part in the treatment of ischemic stroke neuroprotective therapy sessions by taking the treatment of anti-oxidative and free radical damage. The Nrf2/ARE signaling pathway is the core pathway of endogenous oxidative stress. In order to explore the mechanism of BYHW on cerebral ischemia reperfusion injury, we choose the Nrf2/ARE pathway. Nrf2 and HO-1 are the indicators of detection in this pathway. Using immunohistochemical method、Western-blotting、RT-PCR to observe the activation of BYHW to MCAO rats of Nrf2 and HO-1.It shows that BYHW can activate the Nrf2 pathway,increase the expression of the transcription factor Nrf2 and its second enzyme HO-1. It shows BYHW plays one of its antioxidant effect through the Nrf2/ARE signal transduction pathway.Using Pearson correlation to analyze the study of correlation for BYHW chemical composition and anti MCAO efficacy. Then the result of Ferulic acid, Ononin, Calycosin, Formononetincan, AstragalosideⅣ is greater than 0.8, it can improve activity of SOD, GST, GSH-Px; the result of Ferulic acid, Calycosin-7-glucoside, Ononin, Calycosin, Formononetincan, AstragalosideⅣ is lower than -0.5, it tends to treduce the content of MDA, thereby to reduce oxidative stress injury that is caused by brain ischemia reperfusion. It provides a certain basis to explore the substance foundation of BYHW to anti MCAO.