Virgin Olive Oil Extracts Reduce Oxidative Stress and Modulate Cholesterol Metabolism: Comparison between Oils Obtained with Traditional and Innovative Processes

Abstract

This study was aimed at demonstrating the substantial equivalence of two extra virgin olive oil samples extracted from the same batch of Coratina olives with (OMU) or without (OMN) using ultrasound technology, by performing chemical, biochemical, and cellular investigations. The volatile organic compounds compositions and phenolic profiles were very similar, showing that, while increasing the extraction yields, the innovative process does not change these features. The antioxidant and hypocholesterolemic activities of the extra virgin olive oil (EVOO) phenol extracts were also preserved, since OMU and OMN had equivalent abilities to scavenge the 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) radicals in vitro and to protect HepG2 cells from oxidative stress induced by H₂O₂, reducing intracellular reactive oxygen species (ROS) and lipid peroxidation levels. In addition, by inhibiting 3-hydroxy-3-methylglutarylcoenzyme a reductase, both samples modulated the low-density lipoprotein receptor (LDLR) pathway leading to increased LDLR protein levels and activity.

Keywords: EVOO extract; HepG2 cells; IOC methods; LDLR; PCSK9; antioxidant.

Figure 1

Analysis of secoiridoids. (A) Ratios of the integrals corresponding to the signals of secoiridoidic monoaldehydes in ¹H-NMR spectra of the dry extracts from OMN and OMU samples. Oleur. Ag, aglycone of oleuropein; Ligstr. Ag. aglycone of ligstroside. (B) Total tyrosol + hydroxytyrosol (after hydrolysis) and phenolic content (according to the IOC method) in the dry extracts from OMN and OMU oil; each data represents the mean of the analysis of three extracts of the same samples.

Figure 2

Antioxidant effects of OMN and OMU extracts. (A) In vitro radical scavenging activity of OMN and OMU phenol extracts by DPPH assay. (B) In vitro radical scavenging activity of OMN and OMU phenol extracts by ABTS assay. Data represent the mean ± s.d. of six determinations performed in triplicate. All the data sets have been analyzed by Two-way ANOVA. In particular, the reductions of DPPH (****) p < 0.0001 and ABTS (***) p < 0.001 radicals are significant as function of the concentrations, whereas no significant difference have been observed between OMN and OMU extracts.

Figure 3

MTT assay. Effect of OMU extract on the HepG2 cell viability. Data represent the mean ± s.d. of three independent experiments performed in triplicate. The statistical significance of C vs OMU 200 µg/mL was analyzed by t-student test. (****) p < 0.0001, C: control cells.

Figure 4

Antioxidant effects of OMN and OMU extracts on HepG2 cells. (A) OMN and (B) OMU reduce the H₂O₂ (1 mM)-induced ROS levels in HepG2 cells. (C) OMN and (D) OMU decrease the lipid peroxidation in the same cells after oxidative stress induction by H₂O₂. Data represent the mean ± s.d. of six independent experiments performed in triplicate. All the data sets were analyzed by One-way ANOVA; basal vs H₂O₂ samples were analyzed by t-student test, whereas H₂O₂ vs OMN/OMU + H₂O₂ samples by One-way ANOVA. (*) p < 0.5; (**) p < 0.01; (***) p < 0.001; (****) p < 0.0001.

Figure 5

Effect of OMN and OMU extracts on the in vitro activity of HMGCoAR. Data represent the mean ± s.d. of three determinations performed in triplicate. All the data sets were analyzed by two-way ANOVA. In particular, the reduction of enzyme activity is significant as function of the all the tested concentrations (**) p < 0.01, whereas no significant difference was observed between OMN and OMU extracts.

Figure 6

Modulation of cholesterol biosynthesis. (A) Western blot of SREBP-2 (precursor); (B) western blot of the LDLR; (C) western blot of HMGCoAR; (D) western blot of PCSK9; (E) western blot of HNF1- α. Data represent the mean ± s.d. of eight independent experiments performed in duplicate. All the data sets were analyzed by One-way ANOVA and OMN vs OMU by t-student test. (*) p < 0.5; (**) p < 0.01; (***) p < 0.001; ns: not significant. C: control sample.

Figure 7

Modulation of the LDLR on HepG2 cell surface and uptake of environmental LDL. (A) LDLR protein levels on HepG2 cell surface evaluated by in cell western. (B) Uptake of fluorescent LDL from the environment by HepG2 cells. Data represent the mean ± s.d. of five independent experiments performed in triplicate. All the data sets were analyzed by One-way ANOVA and OMN vs OMU by t-student test. (*) p < 0.5; (**) p < 0.001. ns: not significant, C: control sample.