title: The quenching effect of flavonoids on 4-methylumbelliferone, a potential pitfall in fluorimetric neuraminidase inhibition assays.
abstract: Many assays aimed to test the inhibitory effects of synthetic molecules, and naturally occurring products on the neuraminidase activity exploit the hydrolysis of 2'-O-(4-methylumbelliferyl)-N-acetylneuraminic acid (4-MUNANA). The amount of the released product, 4-methylumbelliferone (4-MU), is then measured fluorimetrically. The authors attempted an analysis of the inhibitory properties of 35 naturally occurring flavonoids on neuraminidase N3, where only 29 of them were sufficiently soluble in the assay medium. During the analysis, the authors noticed a strong quenching effect due to the test compounds on the fluorescence of 4-MU. The quenching constants for the flavonoids were determined according to the Stern-Volmer approach. The extent of fluorescence reduction due to quenching and the magnitude of the fluorescence reduction measured in the inhibition assays were comparable: for 11 of 29 compounds, the two values were found to be coincident within the experimental uncertainty. These data were statistically analyzed for correlation by calculating the pertinent Pearson correlation coefficient. Inhibition and quenching were found to be positively correlated (r = 0.71, p(uncorr) = 1.5 × 10(-5)), and the correlation was maintained for the whole set of tested compounds. Altogether, the collected data imply that all of the tested flavonoids could produce false-positive results in the neuraminidase inhibition assay using 4-MUNANA as a substrate.
authors.affiliation: Department of Pharmacognosy, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand.
authors.forename: Jarinrat
authors.initials: J
authors.lastname: Kongkamnerd
authors.forename: Adelaide
authors.initials: A
authors.lastname: Milani
authors.forename: Giovanni
authors.initials: G
authors.lastname: Cattoli
authors.forename: Calogero
authors.initials: C
authors.lastname: Terregino
authors.forename: Ilaria
authors.initials: I
authors.lastname: Capua
authors.forename: Luca
authors.initials: L
authors.lastname: Beneduce
authors.forename: Andrea
authors.initials: A
authors.lastname: Gallotta
authors.forename: Paolo
authors.initials: P
authors.lastname: Pengo
authors.forename: Giorgio
authors.initials: G
authors.lastname: Fassina
authors.forename: Orawan
authors.initials: O
authors.lastname: Monthakantirat
authors.forename: Kaoru
authors.initials: K
authors.lastname: Umehara
authors.forename: Wanchai
authors.initials: W
authors.lastname: De-Eknamkul
authors.forename: Stanislav
authors.initials: S
authors.lastname: Miertus
mesh_terms: D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D005419:Flavonoids / Q000737:chemistry / Q000494:pharmacology*; D006868:Hydrolysis / Q000187:drug effects; D006923:Hymecromone / Q000031:analogs & derivatives* / Q000032:analysis; D009439:Neuraminidase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D013050:Spectrometry, Fluorescence
chemical_list: D004791:Enzyme Inhibitors; D005419:Flavonoids; D006923:Hymecromone; D009439:Neuraminidase