Ryu_2014_J.Agric.Food.Chem_62_1338

Enzyme binding affinity has been recently introduced as a selective screening method to identify bioactive substances within complex mixtures. We used an assay which identified small molecule binders of acetylcholinesterase (AChE) using the following series of steps: incubation of enzyme with extract; centrifugation and filtration; identification of small molecule content in the flow through. The crude extract contained 10 peaks in the UPLC chromatogram. However, after incubation the enzyme, six peaks were reduced, indicating these compounds bound AChE. All these isolated compounds (2, 3, and 5-8) significantly inhibited human AChE with IC(5)(0)s = 5.4-15.0 muM and butyrylcholinsterase (IC(5)(0)s = 0.7-11.0 muM). All compounds exhibited reversible mixed kinetics. Consistent with the binding screen and fluorescence quenching, gamma-mangostin 6 had a much higher affinity for AChE than 9-hydroxycalabaxanthone 9. This validates this screening protocol as a rapid method to identify inhibitors of AChE.