title: Improved thin-layer chromatography bioautographic assay for the detection of actylcholinesterase inhibitors in plants.
abstract: BACKGROUND
  Thin-layer chromatography (TLC) bioautographic method is a simple and rapid method to screen acetylcholinesterase inhibitors from plant extracts. However, the high consumption of enzyme (6 U/mL) in current methods makes the procedure expensive, which is an obstacle to scientific research centers lacking funding.
  
  
  OBJECTIVE
  To develop a new low-cost TLC bioautographic method.
  
  
  METHODS
  A series of compounds, as substrates, were synthesised and their ability to be hydrolysed by acetylcholinesterase was evaluated by the HPLC method.
  
  
  RESULTS
  4-Methoxyphenyl acetate (14) was proved to be an appropriate substrate for TLC bioautographic assay. Therefore a new and cheap TLC bioautographic assay was set up. The mechanism of this new method is that the enzyme converts 4-methoxylphenyl acetate into 4-methoxyphenol, which reacts with a solution of potassium ferricyanide ([K₃(FeCN)₆]) and iron chloride hexahydrate (FeCl₃·6H₂O) to make an aquamarine blue coloured background on the TLC plates. Regions of the TLC plate which contain acetylcholinesterase inhibitors show up as light yellow spots against the background. The consumption of enzyme (1 U/mL) in the new method is low and the detection limit of two known acetylcholinesterase inhibitors, huperzine A (0.0001 μg) and physostigmine (0.001 μg), for this assay are close to published values.
  
  
  CONCLUSIONS
  A low-cost TLC bioautographic method was developed, which will benefit research groups pursuing natural acetylcholinesterase inhibitors.
authors: School of Life Science and Engineering, Lanzhou University of Technology, Lanzhou 730050, People's Republic of China.
authors: Zhong-Duo
authors: ZD
authors: Yang
authors: Zhu-Wen
authors: ZW
authors: Song
authors: Jin
authors: J
authors: Ren
authors: Ming-Jun
authors: MJ
authors: Yang
authors: Shuo
authors: S
authors: Li
mesh_terms: D000107:Acetylation; D000470:Alkaloids / Q000737:chemistry; D000840:Anisoles / Q000737:chemistry; D002712:Chlorides / Q000737:chemistry; D002800:Cholinesterase Inhibitors / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer / Q000379:methods*; D002863:Chromogenic Compounds / Q000737:chemistry; D003116:Color; D057075:Enzyme Assays / Q000379:methods*; D005290:Ferric Compounds / Q000737:chemistry; D005292:Ferricyanides / Q000737:chemistry; D006868:Hydrolysis; D057230:Limit of Detection; D015394:Molecular Structure; D032102:Phellodendron / Q000737:chemistry; D010648:Phenylacetates / Q000138:chemical synthesis / Q000737:chemistry*; D010830:Physostigmine / Q000737:chemistry; D024301:Plant Bark / Q000737:chemistry; D010936:Plant Extracts / Q000737:chemistry; D012717:Sesquiterpenes / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity
chemical_list: D000470:Alkaloids; D000840:Anisoles; D002712:Chlorides; D002800:Cholinesterase Inhibitors; D002863:Chromogenic Compounds; D005290:Ferric Compounds; D005292:Ferricyanides; D010648:Phenylacetates; D010936:Plant Extracts; D012717:Sesquiterpenes; C050426:huperzine A; C009760:mequinol; D010830:Physostigmine; C024555:ferric chloride; C028033:potassium ferricyanide