Virgin Olive Oil By-Products: Biological Activity of Phenolic Extract of Pâté on AGS Gastric Cells

Abstract

Pâté is a by-product of olive oil production which represents an abundant source of phenolic compounds and can be used for food formulation, reducing its environmental impact and promoting a circular economy. In this context, the effects of a hydroalcoholic extract of pâté were evaluated for the first time in an AGS human cell line commonly used as model of gastric mucosa. Pâté was obtained from Tuscan olives; the total phenolic content was 16.6 mg/g dried extract, with verbascoside and secoiridoid derivatives as the most abundant phenols. The phenolic pâté extract did not alter viability, distribution of cell cycle phases or proliferation and migration of AGS cells at the tested concentrations. Seven enzymes were chosen to investigate the metabolic effect of the pâté extract in the context of oxidative stress. Pâté produced a statistically significant increase in the activity of key enzymes of some metabolic pathways: Lactate dehydrogenase, Enolase, Pyruvate kinase, Glucose 6-phosphate dehydrogenase, Citrate synthase, 3-Hydroxyacyl-CoA dehydrogenase and Hexokinase. Pre-treatments with the extract of pâté at 100 µg/mL or 200 µg/mL, as observed through PCA analysis, appeared able to counteract the enzymatic activity alterations due to oxidative stress induced by H₂O₂ 1 mM and 2 mM. The results indicate that dried pâté, due to its phenolic components, can be proposed as a new functional food ingredient.

Keywords: AGS cells; antioxidant action; bioactive compound; enzymatic activities; extra virgin olive oil; hydroxytyrosol; metabolism; olive oil by-product; polyphenols.

Figure 1

(a) Chromatographic profile of the hydroalcoholic extract of freeze-dried pâté: 1, hydroxytyrosol glucoside; 2, hydroxytyrosol; 3, tyrosol glucoside; 4, tyrosol; 5, chlorogenic acid; 6, β-OH-acteoside isomer 1; 7, β-OH-acteoside isomer 2; 8, rutin; 9, luteolin-7-O-glucoside; 10, verbascoside; 11, cafselogoside; 12, secoiridoid derivatives; 13, comselogoside; 14, luteolin. (b) Amount of each phenolic compound in the hydroalcoholic extract of freeze-dried pâté expressed as mg/g dried extract; (c) total content of tyrosol and hydroxytyrosol free and linked forms determined after acidic hydrolysis and expressed as mg/g DE.

Figure 2

Cell viability after treatment with the hydroalcoholic extract of pâté at 50, 100 and 200 µg/mL for 2, 24 and 48 h by MTT assay. *** indicates statistically significant difference respect to the control at the same time (Student’s t-test p < 0.001).

Figure 3

Percentage distribution in the different phases of cell cycle of samples treated with 50, 100 and 200 µg/mL of the extract of pâté 2 h, 24 h and 48 h.

Figure 4

Scratch closure curves (coloured circles, with standard deviation error bars) and relative best-fit linear models (continuous lines).

Figure 5

ROS assessment by the DCF-DA test in cells treated with the extract of pâté for 24 h and then incubated with two different concentrations of H₂O₂, 1 mM or 2 mM for 1 h. ** or *** indicates a statistically significant difference compared to the control sample (Student’s t-test ** p < 0.01, *** p < 0.001).

Figure 6

Specific activities of (from left to right and from top to bottom of each panel) LDH, G6PDH, ENO, PK, CS, HACoADH and HK for the two investigated concentrations of the extract of pâté: (top panel) with 100 µg/mL and (bottom panel) with 200 µg/mL. For each enzyme the activities were measured for control cells, cells treated with H₂O₂ 1 mM and 2 mM, 100 µg/mL or 200 µg/mL of the extract of pâté and cells treated with both the two concentrations of H₂O₂ and of the pâté, as specified in the legend. Error bars represent the standard deviation of the three replicates of the assay. Samples significantly different from control are marked with * (Student t-test p < 0.05), ** (p < 0.01) or *** (p < 0.001).

Figure 7

Biplots resulting from the PCA analysis for the two investigated concentrations of the extract of pâté. (Top panel): 100 µg/mL. (Bottom panel): 200 µg/mL. Coloured circles represent the various samples as specified in the legend (control cells, cells treated with H₂O₂ 1 mM and 2 mM, cells treated with 100 µg/mL or 200 µg/mL of the extract and cells treated with both the two concentrations of H₂O₂ and of the extract of pâté). The dashed lines of the same colour represent the envelope of each cluster of points to help the reader to view them. The red lines marked with the various enzymes names represent the projections of the relative enzymatic activities on the PCA1 and PCA2 coordinates.