Cellular Antioxidant Effect of an Aronia Extract and Its Polyphenolic Fractions Enriched in Proanthocyanidins, Phenolic Acids, and Anthocyanins

Abstract

Oxidative stress and chronic inflammation contribute to some chronic diseases. Aronia berries are rich in polyphenols. The aim of the present study was to characterize the cellular antioxidant effect of an aronia extract to reflect the potential physiological in vivo effect. Cellular in vitro assays in three cell lines (Caco-2, HepG2, and SH-SY5Y) were used to measure the antioxidant effect of AE, in three enriched polyphenolic fractions (A1: anthocyanins and phenolic acids; A2: oligomeric proanthocyanidins; A3: polymeric proanthocyanidins), pure polyphenols and microbial metabolites. Both direct (intracellular and membrane radical scavenging, catalase-like effect) and indirect (NRF2/ARE) antioxidant effects were assessed. AE exerted an intracellular free radical scavenging activity in the three cell lines, and A2 and A3 fractions showed a higher effect in HepG2 and Caco-2 cells. AE also exhibited a catalase-like activity, with the A3 fraction having a significant higher activity. Only A1 fraction activated the NRF2/ARE pathway. Quercetin and caffeic acid are the most potent antioxidant polyphenols, whereas cyanidin and 5-(3',4'-dihydroxyphenyl)-γ-valerolactone showed the highest antioxidant effect among polyphenol metabolites. AE rich in polyphenols possesses broad cellular antioxidant effects, and proanthocyanidins are major contributors. Polyphenol metabolites may contribute to the overall antioxidant effect of such extract in vivo.

Keywords: (poly)phenols; NRF2; anthocyanins; antioxidant cell bioassays; aronia; chokeberry; oxidative stress; phenolic acids; proanthocyanidins.

Figure 1

Polyphenolic characterization of AE and the three polyphenolic fractions (A1, A2, and A3). Values are expressed in milligrams of polyphenols per gram of extract or fraction.

Figure 2

Dose–response curves of AE assessed with the AOP1 assay on different cell lines. HepG2 (panel A), SH-SY5Y (panel B) and Caco-2 cells (panel C) were treated for 4 h with AE in dose–response mode. Left column: kinetic profile of raw data. The raw data (RFUs, relative fluorescence units) were plotted in a kinetics-like mode showing the effect of light flashes on the fluorescence level. Data points, mean RFUs of triplicates; bars, SD. Central column: same data after normalization. Antioxidant indices correspond to control AUC minus data AUCs. Right column: antioxidant-index-based dose–response and sigmoid fit curve used for log EC₅₀ estimations (µg/mL). Data points correspond to measured antioxidant indexes. EC₅₀, efficacy concentration which leads to a half-maximal response; R², coefficient of determination obtained by non-linear regression model fit.

Figure 3

NRF2/ARE-mediated pathway activation by the fraction A1 in stable ARE-driven luciferase expression HepG2 cells. Cells were treated for 17 h with increasing doses of AE and fractions A1, A2, and A3. Only the A1 fraction showed a dose–response effect. On the left side, results are presented in gene expression fold increase compared with the control (cell culture medium only, gene expression = 1). Values under 1 indicate cytotoxicity effect. The bars are SD for mean relative luminescence units of duplicate. On the right side, the same results are presented as a dose–response curve (EC₅₀: 742 µg/mL, 95% CI: [582.5, 944.9]). Data points correspond to the fold increase for each concentration and bars are SD. EC₅₀ is Efficacy Concentration which leads to half-maximal response. R² is the coefficient of determination obtained by non-linear regression model fit.