Introduction:
The ability of flavonoids to bind to and inhibit the digestive enzyme α-amylase has been reported. Because of this ability, they may serve as an adjunctive therapy for the treatment and prevention of diabetes. We studied this interaction using the fluorescence polarization analysis (FPA) method on the example of the most common flavonoid quercetin. The method is based on the comparison of rotation speed of fluorescently labeled hapten (fast) and the complex of protein and labeled hapten (slow). The higher the molecular mass of the complex, the slower the rotation and the higher the polarization value (mP). Monoclonal antibodies are usually used for this method, but they have a high price. The use of available amylase as a high molecular weight component that binds to quercetin may reduce the cost of the assay. Also, the FPA method takes minutes and does not require multi-step sample preparation, does not use toxic reagents.

Methods:
Measurements were performed using Sentry 200 from Ellie (USA). Quercetin monohydrate was obtained from Acros Organics (Belgium). Tracer was synthesized by activation of the keto group followed by addition of ethylenediaminfluorescein (EDF). The statistical parameters of the method were determined: IC₅₀, limit of detection, limit of quantification, and detection range. Cross-reactivity was performed with dihydroquercetin.

Results:
EDF-labelled quercetin tracer was synthesized, purified and characterized. The method for the quantification of quercetin was based on the competitive interaction of quercetin and tracer with amylase. The IC₅₀ was 77 μg/mL, detection limit 25.5 μg/mL, determination range 12.3–37.5 μg/mL (p<0.05). No cross-reactivity with dihydroquercetin was observed.

Conclusions:
A quantification method based on the binding of quercetin and amylase using FPA was developed. Tracer quercetin-EDF was obtained. The basic statistical performance of the method is described.

Conflicts of interest:
The authors declare that they have no conflict of interest in the publication of this article. The authors have no conflicts of interest to report in this work. Abstract was not submitted elsewhere and was first published here.

Funding:
This research received no external funding from any funding agency in the public, commercial, or not-for-profit sectors.