title: High-resolution α-amylase assay combined with high-performance liquid chromatography-solid-phase extraction-nuclear magnetic resonance spectroscopy for expedited identification of α-amylase inhibitors: proof of concept and α-amylase inhibitor in cinnamon.
abstract: Type 2 diabetes affects millions of people worldwide, and new improved drugs or functional foods containing selective α-amylase inhibitors are needed for improved management of blood glucose. In this article the development of a microplate-based high-resolution α-amylase inhibition assay with direct photometric measurement of α-amylase activity is described. The inhibition assay is based on porcine pancreatic α-amylase with 2-chloro-4-nitrophenyl-α-D-maltotriose as substrate, which this gives a stable, sensitive, and cheap inhibition assay as requested for high-resolution purposes. In combination with HPLC-HRMS-SPE-NMR, this provides an analytical platform that allows simultaneous chemical and biological profiling of α-amylase inhibitors in plant extracts. Proof-of-concept with an artificial mixture of six compounds-of which three are known α-amylase inhibitors-showed that the high-resolution α-amylase inhibition profiles allowed detection of sub-microgram amounts of the α-amylase inhibitors. Furthermore, the high-resolution α-amylase inhibition assay/HPLC-HRMS-SPE-NMR platform allowed identification of cinnamaldehyde as the α-amylase inhibitor in cinnamon (Cinnamomum verum Presl.).
authors.affiliation: Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen , 2100 Copenhagen, Denmark.
authors.forename: Leyla
authors.initials: L
authors.lastname: Okutan
authors.forename: Kenneth T
authors.initials: KT
authors.lastname: Kongstad
authors.forename: Anna K
authors.initials: AK
authors.lastname: Jäger
authors.forename: Dan
authors.initials: D
authors.lastname: Staerk
mesh_terms: D000171:Acrolein / Q000031:analogs & derivatives / Q000032:analysis; D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002935:Cinnamomum zeylanicum / Q000737:chemistry*; D004791:Enzyme Inhibitors / Q000032:analysis*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D055292:Pancreatic alpha-Amylases / Q000037:antagonists & inhibitors*; D010936:Plant Extracts / Q000737:chemistry; D015203:Reproducibility of Results; D052616:Solid Phase Extraction / Q000379:methods*; D013552:Swine; D014312:Trisaccharides / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D010936:Plant Extracts; D014312:Trisaccharides; C057413:2-chloro-4-nitrophenylmaltotrioside; D000171:Acrolein; D055292:Pancreatic alpha-Amylases; C012843:cinnamaldehyde
keywords: HPLC−HRMS−SPE−NMR; high-resolution α-amylase assay; microplate; type 2 diabetes