Intracellular Redox Behavior of Quercetin and Resveratrol Singly and in Mixtures

Abstract

Polyphenols have attracted great interest as potent antioxidant compounds and nutraceuticals; however, their antioxidant properties represent a multifaceted phenomenon, including pro-oxidant effects under particular conditions and complex behavior when multiple polyphenols are simultaneously present. Moreover, their intracellular behavior cannot always be predicted from their ability to counteract the production of ROS in acellular assays. The present work aimed to study the direct intracellular redox activity of two polyphenols, resveratrol and quercetin, singly and in mixture in a cellular short-term bioassay under both basal and pro-oxidant conditions. The study was carried out by spectrofluorimetric assessment of the intracellular fluorescence of CM-H₂DCFDA-charged HeLa cells under either basal conditions, due to the reactive species associated with the normal cellular oxidative metabolism, or pro-oxidant conditions induced by H₂O₂ exposure. Under basal conditions, the obtained results showed a significant antioxidant effect of quercetin and a weaker antioxidant effect of resveratrol when used singly, while antagonism of their effect was detected in their equimolar mixtures at all the concentrations used. Under exposure of the cells to H₂O₂, quercetin exhibited a dose-dependent intracellular antioxidant activity whereas resveratrol manifested a pro-oxidant intracellular activity, while their equimolar mixtures showed an intracellular interaction between the 2 polyphenols, with additive effects at 5 µM and synergic at 25 µM and 50 µM. Thus, the results clarified the direct intracellular antioxidant/pro-oxidant activity of quercetin and resveratrol alone and in their equimolar mixtures in the cell model HeLa cells and highlighted that the antioxidant properties of polyphenols in mixtures at the cellular level depend not only on the nature of the compounds themselves but also on the type of interactions in the cellular system, which in turn are influenced by the concentration and the oxidative status of the cell.

Keywords: CM-H2DCFDA; antioxidant; mixture; pro-oxidant; quercetin; resveratrol.

Figure 1

Structural formula of quercetin and resveratrol.

Figure 2

(A–C) Effect of increasing concentrations of quercetin, resveratrol, and equimolar mixtures of the two compounds on the basal fluorescence of HeLa cells loaded with the CM-H₂DCFDA probe. The effect was measured spectrofluorimetrically after 1 h incubation and is reported as a % variation in the CM-H₂DCFDA calculated with respect to the control (unexposed cells). The statistical significance of the data was analyzed by one-way ANOVA and Tukey’s multiple comparison test. Data are expressed as mean ± SEM. ** p < 0.01; * p < 0.05.

Figure 3

(A,B) Effect of increasing concentrations of H₂O₂ on the fluorescence of HeLa cells loaded with the CM-H₂DCFDA probe. The effect was measured spectrofluorimetrically over 1 h. Control refers to cells loaded with the probe but not exposed to the peroxide. Data are expressed as mean ± SEM. (B) Representative confocal images of HeLa cells charged with the probe before and after the exposure to 300 µM H₂O₂.

Figure 4

(A–C) Effect of 1 h preincubation with increasing concentrations of quercetin (A), resveratrol (B), and an equimolar mixture of the 2 polyphenols on the fluorescence variation time course of HeLa cells loaded with the CM-H₂DCFDA probe and exposed to an oxidative challenge (H₂O₂ 300 µM). The effect was measured spectrofluorimetrically over 1 h. Data are expressed as mean ± SEM.

Figure 5

(A,B) Comparison between the activity (expressed as percentage variation of the control) of equimolar mixtures of resveratrol and quercetin measured experimentally in basal (A) and pro-oxidant conditions (B) with the corresponding theoretical values calculated by adding up the effect of the individual components at the same concentration analyzed separately. Data are expressed as mean ± SEM. * p < 0.05; *** p < 0.001.