Developed The New Thin-layer Chromatography Bioautography Method And Evaluated The Anti-acetylcholinesterase Ability Of Derivatives Of Nuciferine

Alzheimer's disease (AD), the most common in the elderly, is a progressive neurodegenerative disorder characterized by cognitive impairments and memory loss. To date, the cause of AD is mainly explained by cholinergic hypothesis and amyloid hypothesis. According to the theory, it is effective method to treatment of AD by inhibition of acetylcholinesterase activity, extension of present time of acetylcholine and increasing levels of acetylcholine in brain. So acetylcholinesterase inhibitors which were used as drugs for AD can get obvious effects. But now, most of the medicines can only delay the development of the state of AD, can not cure the disease in nature. These drugs also contain many faults, such as, having side-effects, low activity, low selectivity. In order to find new and more efficient inhibitors of acetylcholinesterase, the following two aspects have been carried out in this study.1. Established a new thin-layer chromatography (TLC) bioautographic method which is used for detecting of actylcholinesterase inhibitors from plant extracts To date, TLC bioautographic assay and Ellman's method are commonly used for detecting the AChEI. The former is advantageous for the screening of crude extracts of plants as it allows separation and detection of active compounds at the same time. However, the latter is more appropriate for the screening from libraries of synthetic or isolated natural compounds. But,the high consumption of enzyme (6 U/mL) and the high cost of test in current methods made the procedure expensive, which was an obstacle to scientific research centers lacking enough funds.Therefore,it is importantly to develop a new TLC bioautographic method which can reduce the consumption of enzyme and have the low of detection limits.Firstly, a total of 22 compounds, as the new substrates for AChE, were synthesized by acetylation. Secondly, their ability to be hydrolyzed by acetylcholinesterase was evaluated by the HPLC method. The results showed that five compounds could be easily hydrolyzed (hydrolytic yield >50%). Thirdly, Diazotization of benzidine, diazotization of 4-nitroaniline, potassium ferricyanide and iron chloride hexahydrate, hydroxylamine and iron chloride were used as chromogenic agents. The results show that only 4-Methoxyphenyl acetate (14) as substrate, with a mixture solution of K3(FeCN)6 and FeCl3?6H2O as chromogenic agent, a new TLC bioautographic method can be successfully established. In this method, the consumption of enzyme was reduced and the detection limit of two known acetylcholinesterase inhibitors, huperzine A(0.1ng) and physostigmine(1ng), was low.The advantage of the new method not only reduce the consumption of enzyme, but also contrast obviously between the background and the spots of inhibition and observe easily. It is more convenient to find new AChEIs.2. Synthesis and evaluation the anti-acetylcholinesterase ability of nuciferine derivativesIn our group, 2-hydroxy-1-methoxyaporphine(34) which was separated from Nelumbo nucifera, a new-found strong AChEI, have been reported at the previous study. In this study, we synthesized the compounds which have a higher inhibitory activity for AChE and study the structure-activity relationships.(1). the nuciferine, as raw materials, was modified by O-dealkylation, N-dealkylation and ring aromatization reactions. Eight derivatives were obtained, its was 1-Hydroxy-2-methoxyaporphine, 1,2-Dihydroxyaporphine, 6-Hydroxydehydron - uciferine, Nuciferine N-oxide, Dehydronuciferine, Nornuciferine, 1-Hydroxy-2-meth- oxynoraporphine and 1,2-Dihydroxynoraporphine.(2). all compounds were detected anti-acetylcholinesterase ability by Ellman's method at final concentration 0.1mg/mL. The results show that 1,2-dihydroxy aporphine(25) and dehydronuciferine(28) have moderate anticholinesterase activity with an IC50 value of 28μg/mL and 25μg/mL.(3). aromatization of C ring and the alkoxyl of C-1, the hydroxy at C-2 position and the substituent on N atom would have significant effect for the anti-acetylcholinester- ase activity.This work will help us further to synthesize the higher activity anti-acetylcholine sterase compounds...