Extra Virgin Olive Oil Phenolic Extract on Human Hepatic HepG2 and Intestinal Caco-2 Cells: Assessment of the Antioxidant Activity and Intestinal Trans-Epithelial Transport

Abstract

In the framework of research aimed at promoting the nutraceutical properties of the phenolic extract (BUO) obtained from an extra virgin olive oil of the Frantoio cultivar cultivated in Tuscany (Italy), with a high total phenols content, this study provides a comprehensive characterization of its antioxidant properties, both in vitro by Trolox equivalent antioxidant capacity, oxygen radical absorbance capacity, ferric reducing antioxidant power, and 2,2-diphenyl-1-picrylhydrazyl assays, and at the cellular level in human hepatic HepG2 and human intestinal Caco-2 cells. Notably, in both cell systems, after H₂O₂ induced oxidative stress, the BUO extract reduced reactive oxygen species, lipid peroxidation, and NO overproduction via modulation of inducible nitric oxide synthase protein levels. In parallel, the intestinal transport of the different phenolic components of the BUO phytocomplex was assayed on differentiated Caco-2 cells, a well-established model of mature enterocytes. The novelty of our study lies in having investigated the antioxidant effects of a complex pool of phenolic compounds in an extra virgin olive oil (EVOO) extract, using either in vitro assays or liver and intestinal cell models, rather than the effects of single phenols, such as hydroxytyrosol or oleuropein. Finally, the selective trans-epithelial transport of some oleuropein derivatives was observed for the first time in differentiated Caco-2 cells.

Keywords: ROS; hydroxytyrosol; lipid peroxidation; oleuropein aglycone; phenolic phytocomplex; secoiridoids; trans-epithelial transport.

Figure 1

In vitro antioxidant power evaluation of the phenolic extract (BUO) extract by 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) (A), Oxygen Radical Absorbance Capacity (ORAC) (B), ferric reducing antioxidant power (FRAP) (C), and 2,2-diphenyl-1-picrylhydrazyl (DPPH) (D) assays. The data points represent the averages ± SD of four independent experiments performed in duplicate. All data sets were analyzed by One-way ANOVA followed by Tukey’s post-hoc test. ns: not significant and C: control sample (H₂O). (*) p < 0.5; (**) p < 0.01; (****) p < 0.0001.

Figure 2

Antioxidant capacity of the BUO extract assayed at the cellular level, by ferric reducing antioxidant power (FRAP) assay. On (A) HepG2 and (B) Caco-2 cells, the BUO extract reverted the oxidative stress induced by 1 mM H₂O₂. Data represent the mean ± SD. of three independent experiments performed in duplicate. All data sets were analyzed by One-way ANOVA followed by Tukey’s post-hoc test. ns: not significant; (*) p < 0.5; (**) p < 0.01. Basal: untreated cells.

Figure 3

Evaluation of the effects of the BUO extracts on H₂O₂-induced reactive oxygen species (ROS) production levels in human hepatic HepG2 (A) and intestinal Caco-2 (B) cells. The data points represent the averages ± SD of six independent experiments in duplicate. Basal vs. H₂O₂ samples were analyzed by t-student test, whereas All data sets were analyzed by One-way ANOVA followed by Tukey’s post-hoc test. ns: not significant; (*) p < 0.5; (**) p < 0.01; (***) p < 0.001; (****) p < 0.0001. Basal: untreated cells.

Figure 4

Evaluation of the effects of the BUO extract on H₂O₂-induced lipid peroxidation levels in human hepatic HepG2 cells (A) and intestinal Caco-2 cells (B) as assessed by intracellular MDA levels. The data points represent the averages ± SD of six independent experiments in duplicate. All data sets were analyzed by One-way ANOVA followed by Tukey’s post-hoc test. ns: not significant; (*) p < 0.5, (**) p < 0.01, (***) p < 0.001, (****) p < 0.0001. Basal: untreated cells.

Figure 5

Effect of the BUO extract on the H₂O₂ (1 mM)-induced NO levels (A,B) and inducible nitric oxide synthase (iNOS) protein levels (C–F) in human hepatic HepG2 cells (A,C,E) and intestinal Caco-2 cells (B,D,F). The data points represent the averages ± SD of six independent experiments in duplicate. All data sets were analyzed by One-way ANOVA followed by Tukey’s post-hoc test. ns: not significant; (*) p < 0.5, (**) p < 0.01, (***) p < 0.001, (****) p < 0.0001. Basal: untreated cells.

Figure 6

Chromatographic profiles at 280 nm showing OH-Tyr, Tyr, and the main secoiridoidic compounds in BUO and AP200 (AP side of the cells treated at 200 µg/mL) samples after 2 h incubation in the AP compartment.

Figure 7

Total ion current (TIC) and extract ion current (IC) profiles of the BUO extract (top) and AP200 sample after incubation (bottom); in both the samples isobars of oleuropein aglycone at 377 m/z, isobars of oleacin at 319 m/z, and isobars of ligstroside aglycones at 361 m/z.

Figure 7

Total ion current (TIC) and extract ion current (IC) profiles of the BUO extract (top) and AP200 sample after incubation (bottom); in both the samples isobars of oleuropein aglycone at 377 m/z, isobars of oleacin at 319 m/z, and isobars of ligstroside aglycones at 361 m/z.

Figure 8

Specific ratios involving OH-Tyr, Tyr, Ole-aglyc, (rt 41.6 min) and ligstroside aglycone (Ligst-aglyc, rt 45.8 min), evaluated as area values at 280 nm for BUO extract and AP sample (AP 200 µg/mL) after 2 h incubation with 200 µg/mL BUO. (****, p < 0.0001). ns: not significant. Bars were analyzed by Two-Way ANOVA followed by Tukey’s test.

Figure 9

Chromatographic profiles of the BL sample after incubation for 2 h of 200 µg/mL BUO extract in the AP side of Caco-2 cells with, from top to bottom: TIC, EIC at 377 m/z, EIC at 319 m/z, and EIC at 393 m/z.

Figure 10

Main phenols in the BL samples were evaluated after incubation with the BUO extract at 100 and 200 µg/mL for 2 h, respectively. The data are the mean of triplicate experiments; the Ole aglycone isobar had rt 41.6 min, its oxidated derivative rt 36.2 and oleacin isobar rt 34.4 min (**, p < 0.01; ***, p < 0.001; ****, p < 0.0001). Bars were analyzed by Two-Way ANOVA followed by Tukey’s test.