Comparative Analysis on Polyphenolic Composition of Different Olive Mill Wastewater and Related Extra Virgin Olive Oil Extracts and Evaluation of Nutraceutical Properties by Cell-Based Studies

Abstract

This study reports a comparative analysis of the polyphenolic composition and nutraceutical properties of different olive mill wastewater (OMWW) and corresponding extra virgin olive oil (EVOO) extracts. Specifically, four OMWWs and corresponding EVOOs from cultivars Frantoio (A) and Leccino (B) obtained from different crushing seasons (early-stage (A1 and B1) and later-stage (A2 and B2)) were analyzed. Employing HPLC-DAD and LC-MS methods, the primary polyphenol content was identified and quantified. Overall, OMWW extracts showed a greater polyphenolic content compared to corresponding EVOO extracts, with OMWW B1 displaying the highest levels of polyphenols. The antiradical properties of extracts towards radical species (DPPH, ABTS, O₂^-, and HOCl^-) were demonstrated in vitro, revealing a correlation with polyphenolic content. In fact, OMWW B1 and B2 demonstrated the strongest antiradical activity. Exploring nutraceutical properties of OMWWs, the intestinal permeation of the main polyphenols in a co-culture model (Caco-2 and HT29-MTX cell lines) was assessed, with tyrosol achieving a permeation of almost 60%. Furthermore, the involvement in the inflammation process has been evaluated in cell studies on THP1-derived macrophages by immunocytochemistry, demonstrating that OMWW B1 may exert an anti-inflammatory effect by modulating specific phenotype expression on macrophages. In conclusion, this study provides evidence supporting the reuse of OMWWs as a source of polyphenols with nutraceutical properties.

Keywords: antiinflammatory; by-products; nutraceutical; oleacein; olive mill wastewater; polyphenols.

Figure 1

Total phenolic content (TPC, mg/kg) of EVOO and OMWW extracts. Values are expressed as mean ± standard deviation (n = 3). Different letters (a, b, c, d, e) in the same sample represent significant differences (p < 0.05) between different concentrations, according to Tukey’s HSD test.

Figure 2

Evaluation of maturation stage of olives during the different time collections. (A1). olive cultivar Frantoio early-stage. (B1). olive cultivar Leccino early-stage. (A2). olive cultivar Frantoio later-stage. (B2). olive cultivar Leccino later-stage.

Figure 3

Effects of OMWW extract exposure on the viability of HT29-MTX cell line at different concentrations, as measured by the MTT assay. Values are expressed as mean ± standard deviation (n = 3). Different letters (a, b, c) in the same sample represent significant differences (p < 0.05) between different concentrations, according to Tukey’s HSD test.

Figure 4

Effects of OMWW extract exposure on the viability of Caco-2 cell line at different concentrations, as measured by the MTT assay. Values are expressed as mean ± standard deviation (n = 3). Different letters (a, b, c, d) in the same sample represent significant differences (p < 0.05) between different concentrations, according to Tukey’s HSD test.

Figure 5

MRM chromatogram of detected polyphenols in a representative sample. For each analyte, multiple reaction monitoring (MRM) transitions/fragmentations were monitored: the one with the higher signal-to-noise ratio (S/N), usually corresponding to the most intense trace, was used for the quantification of the analyte (Q), while the other transitions, typically of lower intensity, were used to confirm that the peak is attributable to the analyte (q). Both transitions are necessary to ensure accurate quantification and detection of the compounds of interest. In this figure, two transitions per peak were extracted from the ion chromatogram (XIC) and associated with each specific target and its retention time. Chromatograms were obtained using Sciex Analyst^® software (version 1.7).

Figure 6

Permeation (%) of detected polyphenolic compounds from the B1 OMWW sample (500 µg/mL) at 30, 60, 120, 180, and 240 min through the intestinal model (n = 3). The bar graph uses different colors for each polyphenol to indicate the permeability rate achieved at different time points by the polyphenols detected within the OMWW sample dissolved and used for the permeation assay. The gray bar identifies the maximum achievable percentage permeability, represented by the apical side of the intestinal layer, from which the experiment begins.

Figure 7

In vitro effect of OMWW exposure on THP-1-derived macrophage proinflammatory polarization, assessed as variation of CD86 protein expression by ICC. (A). Confocal images of THP-1-derived macrophages treated for 24 with OMWWs stained with Hoechst (blue, nuclei) and anti-CD86 (red). (B). Quantification of CD86 protein expression reported as AU of fluorescence intensity per cell. Data are reported as mean ± S.E.M. of three different experiments, each completed in triplicate. * p < 0.05.

Figure 8

In vitro effect of OMWW exposure on THP-1-derived macrophage anti-inflammatory polarization, assessed as variation of CD163 protein expression by the ICC. (A). Confocal images of THP-1-derived macrophage treated for 24 with OMWWs stained with Hoechst (nuclei, blue) and anti-CD163 (red). (B). Quantification of CD163 protein expression was reported as AU of fluorescence intensity per cell. Data are reported as mean ± S.E.M. of three different experiments, each completed in triplicate. ** p < 0.01, *** p < 0.001, **** p < 0.001.