| 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.
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