【作者】 张旭；

【导师】 杨中铎；

【作者基本信息】 兰州理工大学 ， 微生物与生化药学， 2010， 硕士

【摘要】 阿尔茨海默病(Alzheimer’s disease,AD)是造成老年痴呆的最主要原因,是一种进行性和致命性疾病。迄今为止,AD的发病机制尚未明确,但是在众多假说中胆碱能学说和B-淀粉样蛋白学说得到了最广泛的支持。这两种学说都表明乙酰胆碱酯酶(acetylcholinesterase,AChE)和AD的形成密切相关,会从多个方面加重AD患者的症状。目前,抑制AChE活性从而间接增强胆碱能活性仍然是治疗AD最有效的方法。植物中含有许多活性好、毒性低的天然AChE抑制剂(acetylcholinesterase inhibitor,AChEI),为了准确快速的得到活性单体化合物,本课题对薄层色谱(thin layer chromatography,TLC)生物自显影方法在跟踪分离天然乙酰胆碱酯酶抑制剂方面的应用进行了研究。首先,对现有的TLC生物自显影方法进行了改进和优化,将所用酶溶液的浓度从原方法中的6.67U/mL降低到1.0U/mL,大大降低了实验所需费用。酶用量降低后TLC方法的检测限降低了两个数量级,这对发现植物中微量的AChEI非常有利。此外,对显色背景等进行了优化,使TLC生物自显影方法更适合于在活性成分跟踪分离过程中使用,并建立了排除假阳性结果的实验方法,进一步提高了方法的准确率和结果的可信度。然后,用改进的TLC生物自显影方法结合假阳性排除实验对31种植物提取物进行了抗AChE活性测试,发现荷叶、独活、五味子、徐长卿、香附、细辛、白芍很有可能含有天然AChEI。对荷叶总生物碱提取物进一步分离,得到了一种未被报道过的活性较好的天然AChEI,2-羟基-1-甲氧基阿朴啡,其IC50值为5.3±0.4μM。动力学研究表明,2-羟基-1-甲氧基阿朴啡是AChE的可逆非竞争性抑制剂。三维分子模拟实验表明,它和AChE通过两条氢键形成一个复合体,从而对AChE形成抑制。更多还原

【Abstract】 Alzheimer’s disease(AD)is the most common form of senile dementia, which is a progressive and fatal disease. The pathogeny of AD is undefined till now. However, cholinergic hypothesis and amyloid hypothesis which explain the cause of AD earn widspread respect. From the two hypothesies, we know that acetylcholinesterase (AChE)will aggravate AD in multiple ways. Therefore, inhibiting AChE is a therapy to AD.Natural AChE inhibitors(AChEI)are widly distributed in plants, of which many are with good activity but low toxicity. The research on separation of natural AChEIs with TLC bioautography for tracking was carried on in this paper.First, modifications to an existing TLC were carried on and then the concentration of enzyme was reduced from 6.67U/mL to 1.0U/mL. As a consequence, the expense of the assay was reduced remarkably and sensitivity was improved simultaneously, which was benefit to research groups pursuing for natural acetylcholinesterase inhibitors.To exculed false positive result due to inhibition of 1-naphthol reaction with Fast Blue B salt, a sample experiment was designed.Second, extracts of 31 plants were tested for AChE inhibiting activity by modified TLC bioautography method. We found that extracts of Folium Nelumbinis, Radix Paeoniae Alba,Radix Angelicae Pubescentis, Fructus Schisandrae Chinensis, Radix Cynanchi Paniculati, Rhizoma Cyperi and Radix et Rhizoma Asari showed AChE inhibiting activity. The further research on chemical composition of Folium Nelumbinis leaded to the isolation of 2-hydroxy-1-methoxy-aporphine, a new-found strong natural AChEI. The IC50 value of the compound was 5.3±0.4μM. The mode of AChE inhibition by 2-hydroxy-1-methoxy-aporphine was reversible and non-competitive.In addition, molecular modelling was performed to explore the binding mode of the compound with AChE. The result revealed that a complex was formed with two hydrogen bonds, which may explain why 2-hydroxy-1-methoxy-aporphine has inhibitory effect on AChE.更多还原