基于PINK1/Parkin通路的醉鱼草果实抗帕金森病活性成分及作用机制研究

Study on the discovery activity constituents and mechanisms of anti-Parkinson's disease(PD) has been a research focus in medical field during recent years. Some triterpenoids isolated from the fruit of Buddleja lindleyana Fort. by our group, which could protect nerve cells and inhibit apoptosis, demonstrate good activity of anti-Parkinson's disease. These compounds were selected as candidate active constituents for anti-PD. On this basis, the fruit of Buddleja lindleyana Fort. was isolated by the method of gradient elution with macroporous resin to obtain individual compounds. And then we screen their activity in vitro by preventing the apoptotic detection of substantia nigra neuronal cells in 6-hydroxydopamine (6-OHDA)-induced PD model in order to get the active compounds. Taking the connection among PINK1/Parkin pathway, mitochondrial dysfunction and PD as the starting point, we research the effects and the molecular mechanisms of compounds' anti-PD activity through the ubiquitin-proteasome system, which could be described in the whole and on the cell level by using the Nematodes PD model and the α-Syn gene transfected PC12 cell model. And then modify the structure of compounds if possible. Such study could provide the experimental basis for the development and utilization about the fruit of B. lindleyana Fort. resources, and it can also offer a new source of anti-PD drug research. Furthermore, this botanicals' research mode, expressed as the link among activity screening, Mechanism and new drugs' developing, will be a good example which can provide new ideas for the modernization of traditional Chinese medicine research.

抗帕金森病的活性物质发现及机制研究一直是医药学界研究的热点，课题组前期从醉鱼草果实中分离出三萜类成分具有一定的神经细胞保护作用和较强的生物活性，显示出良好的抗PD作用，为抗PD候选活性物质。本课题在此基础之上，采用经典的6-羟多巴致多巴胺神经元损伤体外模型与大孔树脂梯度洗脱分离方法相结合，对醉鱼草果实成分进行活性筛选和追踪，分离纯化活性单体；分别采用线虫PD模型和转染α-Syn基因PC12细胞模型，以PINK1/Parkin通路-线粒体功能障碍-帕金森症密切相关联为切入点，在整体与细胞水平上，从泛素-蛋白酶体系统研究该化合物抗帕金森病的作用效果及分子机制，并可能对该活性结构进行优化修饰。本课题研究不仅为抗PD药物研究提供新来源、为中药抗PD机制研究提供新思路，而且为神经退行性疾病活性物质筛选提供评价平台，为中药物质基础研究的"活性追踪-机制研究-新药研发"的研究模式提供范例。

从中草药中发现具有抗帕金森病活性物质及机制研究，是研发新型抗帕金森病药物的有效途径。醉鱼草作为民间常用中草药，根据前期研究发现其具有较好的神经细胞保护活性，作为抗PD药物研发的候选活性物质。本课题通过对醉鱼草果实进行系统分离，共分离得到50余个化合物，运用现代波谱技术鉴定了其中45个，发现了5个新化合物，分别命名为buddlintriterpene A, buddlintriterpene B, buddlintriterpene C, Mimengoside J, Mimengoside K；累积了足量化合物用于成分分析、活性筛选和分子机制的探究；建立了HPLC法测定醉鱼草果实中木犀草素含量，UPLC法对醉鱼草药材茎、叶、花、果实中4种黄酮类成分含量，HPLC-ELSD法测定不同产地醉鱼草果实中代表性齐墩果烷型三萜皂苷的含量等一系列成分分析方法；基于PC12、SH-SY5Y细胞平台对醉鱼草果实成分进行活性筛选，利用秀丽隐杆线虫模型对醉鱼草果实成分进行活性评价及分子作用机制探究，证实了醉鱼草果实中多种三萜类成分具有抗PD活性，揭示了醉鱼草果实活性成分可通过降低α-突触共核蛋白(α-synuclein, α-syn)的聚集，发挥抗PD活性的潜在应用价值。为醉鱼草果实活性成分的有效评价和预测提供了科学依据，为醉鱼草药用资源的综合开发利用提供了基础研究资料。