基于氧化应激相关酶抑制活性的黄檀属植物抗缺血性心脏病新黄酮的发现及构效关系研究

Under the ischemic heart disease (coronary heart disease) , the myocardial cell injury caused mainly by restoration of oxygen and blood in the myocardium , during myocardial ischemia reperfusion. When oxidative stress reaction occurred during myocardial ischemia reperfusion, several enzymes , which could product free radicals, are activated. Then, The free radicals lead to occur imbalance of the anaerobic metabolism. Furthermore, myocardium is injured. Our previous research results is that as the characteristic components of Dalbergia L. f. (Leguminosae)， neoflavonoids have better protective effect on myocardium injury than other flavonoids in the Dalbergia odorifera . But, the free radical scavenging ability of neoflavonoids is weaker than the ability of other components in the plant. So, we guess that neolavonoids could have good protective effect on myocardium injury, because of the activity of several enzymes inhibited during oxidative stress reaction. We will choose several plants from Dalbergia L. f., as the research object. The neoflavonoids are prepared from these plants by the chemical method. Some enzymes are found by comparing neoflavonoids inhibiting the activity of several enzymes about oxidative stress reaction at the cell level and molecular level. On the base, we could build the structure-activity relationship between neoflavonoids and the enzyme about oxidative stress reaction ,by using "Molecular Docking combining with CoMFA and CoMSIA". Then, at the cell level and whole animal level, some neoflavonoids, which have good effect on inhibiting the activity of the enzyme about oxidative stress reaction, are verified.。At last, The mechanisms of the function of neoflavonoids are completely known, in order to provide evidence for the structure optimization of neoflavonoids.

心肌缺血/再灌注时血氧的恢复是引起缺血性心脏病（冠心病）心肌细胞损伤的主要原因。当心肌缺血/再灌注时，激活氧化应激相关酶，发生氧化应激反应，出现氧代谢失衡，导致大量自由基产生，进而损伤心肌。本课题组前期研究发现：降香黄檀中新黄酮类（黄檀属植物特有的成分）较其它黄酮类成分具有更好的抗心肌缺血损伤作用，但在清除自由基的能力上，却与其他黄酮类成分相近。表明新黄酮类可能还通过抑制氧化应激相关酶的活性而保护心肌。基于此，本课题拟选取多种黄檀属植物作为研究对象，提取分离一系列新黄酮类化合物。通过分子与细胞实验考察该类化合物对氧化应激相关酶的抑制作用，确定相关的氧化应激酶和有效成分。在此基础上，利用分子对接技术结合CoMFA模型和CoMSIA模型，构建基于氧化应激相关酶抑制活性的新黄酮类化合物的构效关系。再利用细胞和整体动物实验进行验证，从而阐明其作用机制，也为该类化学成分的结构优化提供依据。

心肌缺血/再灌注时血氧的恢复是引起缺血性心脏病（冠心病）心肌细胞损伤的主要原因。主要原因是激活氧化应激相关酶，发生氧化应激反应，出现氧代谢失衡，导致大量自由基产生，进而损伤心肌。本课题组前期研究发现，降香黄檀中新黄酮类可能可以通过抑制氧化应激相关酶的活性而保护心肌。因此分别对阔叶黄檀、交趾黄檀、东非黑黄檀进行了系统的化学成分分离，从中共分离得到172化学成分，其中新黄酮成分32个。黄嘌呤氧化酶和髓过氧化物酶是两种氧化应激相关酶，它们在心肌缺血/再灌注时具有重要作用。利用文献报道的黄酮类化合物对黄嘌呤氧化酶抑制活性数据，通过DS 4.5软件中分子对接和CoMFA等模块建立其3D-QSAR 模型，并利用此模型对新黄酮类成分抑制黄嘌呤氧化酶的活性进行预测。针对预测结果，利用H9c2心肌细胞缺氧复氧损伤模型对已经分离得到的32个新黄酮类化学成分进行全面的活性筛选验证。从中筛选出11个新黄酮类成分具有较好的活性，与预测结果基本一致，说明这3D-QSAR模型具有良好的预测能力。在此基础上，对 11个有活性的新黄酮类成分与髓过氧化物酶（MPO）进行分子对接，建立了新黄酮类化合物对MPO抑制活性的3D-QSAR构效关系，其非交叉验证系数r2达到了1.000，交叉验证系数q2也达到了0.982。H9c2细胞实验以及3D-QSAR构效关系研究均表明，主要新黄酮Latifolin是黄檀属植物抗缺血性心脏疾病的主要活性成分，因此在细胞及整体动物两个层面分别对其抗心肌缺血再灌注损伤进行系统的药效及机制研究。细胞实验结果表明，Latifolin可清除H9c2心肌细胞缺氧/复氧后所产生的ROS，缓解ROS对H9c2造成的氧化损伤，该作用可能与促进胞浆中Nrf2从Nrf2与Keap1的聚集体中解离并进入细胞核，促进Nrf2与抗氧化反应元件（ARE）的结合，从而促进了HO-1等抗氧化酶的产生有关。动物实验结果表明，latifolin对Pit及ISO诱导的大鼠急性心肌缺血均具有保护作用。其作用可能与其清除ROS，抑制Keap1的表达，促进Nrf2核转移，从而激活Nrf2信号通路有关。本课题通过对黄檀属植物主要新黄酮对氧化应激相关酶抑制活性构效关系研究，揭示了其主要药效物质基础，并阐明了其作用机制，为其综合开发利用提供了实验依据。