薄层色谱生物自显影跟踪分离天然乙酰胆碱酯酶抑制剂的研究
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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。
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.
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。
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.
引文
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[2]王淑月.乙酰胆碱酯酶抑制剂治疗阿尔茨海默病的研究.中国临床康复,2004,8(25)：5336-5337
[3]吴思缈,周黎明.阿尔茨海默病的发病机制及药物治疗的进展.四川生理科学杂志,2009,31(1)：36-39
[4]邢宏义.Alzheimer病的胆碱能系统研究进展.国外医学神经病学神经外科学分册,2000,27(5)：262-264
[5]Parnetti L, Senin U,Mecocci P. Cognitive enhancement therapy for Alzheimer's disease:the way forward. Drugs,1997,53(5):752-768
[6]Corey-Bloom J,Tiraboschi P,Hansen LA, et al. E4 allele dosage does not predict cholinergic activity or synapse loss in Alzheimer's disease. Neurology,2000,54(2): 403-406
[7]王向阳.参芪六味散治疗阿尔茨海默病的实验研究：[内蒙古医学院硕士学位论文].内蒙古：内蒙古医学院,2009,24
[8]林逢春,闫也.阿尔茨海默病发病机制研究的现状与进展.辽宁医学院学报,2009,30(6)：558-560
[9]Smitha MA,Rottkampa CA,Nunomura A. Oxidative stress in Alzheimer's disease. Biochimica et Biophysica Acta-Molecular Basis of Disease,2000,1502(1):139-144
[10]Multhaup G,Ruppert T,Schlicksupp A,et al. Reactive oxygen species and Alzheimer's disease. Biochemical Pharmacology,1997,54(5):533-539
[11]Polidori MC. Oxidative stress and risk factors for Alzheimer's disease:clues to prevention and therapy. Journal of Alzheimer's Disease,2004,6(2):185-191
[12]Bleich S,Romer K,Wiltfang J,et al. Glutamate and the glutamate receptor system: a target for drug action. International Journal of Geriatric Psychiatry,2003,18(S1): S33-S40
[13]苏红丽.老年痴呆病的药物治疗进展.河南医药信息,2000,8(6)：64-65
[14]王斌.老年期痴呆动物模型及药物治疗的研究进展.中国药理学通报,1998,14(6)： 502-503
[15]Zandi PP,Anthony JC,Khachaturian AS. Reduced risk of Alzheimer disease in users of antioxidant vitamin supplements:the cache county study. Archives of Neurology,2004,61(1):82-88
[16]Zlokovic BV. Neurovascular mechanisms of Alzheimer's neurodegeneration. Trends in Neurosciences,2005,28(4):202-208
[17]张千.乙酰胆碱酯酶生物功能的研究进展及其应用.国外医学卫生学分册,2008,35(3)：143-147
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