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

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.

HepG2 cells, purchased from ATCC (HB-8065, ATCC from LGC Standards, Milan, Italy), were cultured in DMEM high glucose with stable L-glutamine, supplemented with 10% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin (complete growth medium) with incubation at 37 °C under 5% CO2 atmosphere. HepG2 cells were used for no more than 20 passages after thawing, because the increase in number of passages may change the cell characteristics and impair assay results.
OMN and OMU extracts were tested separately. Briefly, each EVOO extract was dissolved in DMSO in order to prepare stock solutions (50.0 mg/mL), which were diluted in order to reach the final concentration of 25.0 µg/mL in complete growth DMEM. The final 0.05% concentration of DMSO was kept constant either in treated or control cells. The growth medium of adherent HepG2 cells was discarded and each diluted EVOO extract in complete DMEM was replaced and incubated for the desirable incubation time based on the experiments.

HMGCoAR activity assay
The assay buffer, NADPH, substrate solution, and HMGCoAR were provided in the HMGCoAR Assay Kit (Sigma). The experiments were carried out following the manufacturer's instructions and conditions previously optimized [28] at 37 °C. Each reaction (200 µL) was prepared adding the reagents in the following order: 1 X assay buffer, pravastatin (1.0 µM) or OMN and OMU EVOO extracts (10-250 μg/mL) or vehicle (C), the NADPH (4 µL), the substrate solution (12 µL), and finally the HMGCoAR (catalytic domain) (2 µL). Subsequently, the samples were mixed and the absorbance at 340 nm read by a microplate reader Synergy H1 from Biotek at time 0 and 10 min. The HMGCoAR-dependent oxidation of NADPH and the inhibition properties of EVOO extracts were measured by absorbance reduction, which is directly proportional to enzyme activity

Western blot analysis
Briefly, HepG2 cells/well (1.5 × 10 5 cells/wel 24-well plate) were treated with OMN and OMU samples (25.0 μg/mL) for 24 h. After each treatment, cells were scraped in 30 μL ice-cold lysis buffer [RIPA buffer + inhibitor cocktail + 1:100 PMSF + 1:100 Na-orthovanadate] and transferred in an ice-cold microcentrifuge tube. After centrifugation at 13,300g for 15 min at 4 °C, the supernatant was recovered and transferred into a new ice-cold tube. Total proteins were quantified by Bradford method and 50 μg of total proteins loaded on a precast 7.5% Sodium Dodecyl Sulfate -Polyacrylamide (SDS-PAGE) gel at 130 V for 45 min. Subsequently, the gel was pre-equilibrated with 0.04% SDS in H2O for 15 min at RT and transferred to a nitrocellulose membrane (Mini nitrocellulose Transfer Packs,) using a trans-Blot Turbo at 1.3 A, 25 V for 7 min. Target proteins, on milk or BSA blocked membrane, were detected by primary antibodies as follows: anti-SREBP2, anti-LDLR, anti-HMGCoAR, anti-PCSK9, anti-HNF1-α, and anti-β-actin. Secondary antibodies conjugated with HRP and a chemiluminescent reagent were used to visualize target proteins and their signal was quantified using the Image Lab Software (Biorad). The internal control β-actin was used to normalize loading variations.

In-Cell Western (ICW) assay
A total of 3 × 10 4 HepG2 cells/well were seeded in 96-well plate and, the following day, they were treated with 25 μg/mL of OMN and OMU EVOO extracts in complete growth medium for 24 h. Subsequently, they were fixed in 4% paraformaldehyde for 20 min at RT. Cells were washed 5 times with 100 µL of PBS/well (each wash was for 5 min at RT) and the endogenous peroxides activity quenched adding 3% H2O2 for 20 min at RT. Non-specific sites were blocked with 100 µL/well of 5% bovine serum albumin (BSA, Sigma) in PBS for 1.5 h at RT. LDLR primary antibody solution (Abcam) (1:3000 in 5% BSA in PBS, 25 µL/well) was incubated O/N at +4 °C. Subsequently, the primary antibody solution was discarded, and each sample was washed 5 times with 100 µL/well of PBS (each wash was for 5 min at RT). Goat anti-rabbit Ig-HRP secondary antibody (Santa Cruz) solution (1:6000 in 5% BSA in PBS, 50µL/well) was added and incubated 1 h at RT. The secondary antibody solution was washed 5 times with 100 µL/well of PBS (each wash for 5 min at RT). Fresh prepared TMB Substrate (Pierce, 100 µL/well) was added and the plate was incubated at RT until the desired color was developed. The reaction was stopped with 2 M H2SO4 and then the absorbance at 450 nm was measured using a microplate reader Synergy H1 from Biotek. Cells were stained by adding 1 × Janus green stain, incubating for 5 min at RT. The dye was removed, and the sample washed 5 times with water. Afterward, 0.1 mL 0.5 M HCl per well were added and incubated for 10 min. After 10 s shaking, the OD at 595 nm was measured using the Synergy H1 fluorescent plate reader from Biotek.

Assay for the evaluation of fluorescent LDL uptake by HepG2 cells
Experiments have been performed using conditions previously optimized.
[31] Briefly, a total of 3 × 10 4 HepG2 cells/well were seeded in 96-well plates and kept in complete growth medium for 2 days before treatment. On the third day, cells were treated with 25 μg/mL of OMN and OMU extracts or vehicle (H2O) for 24 h. At the end of the treatment period, the culture medium was replaced with 50 μL/well LDL-DyLight™ 550 working solution. The cells were additionally incubated for 2 h at 37 °C and then the culture medium was aspirated and replaced with PBS (100 μL/well). The degree of LDL uptake was measured using the Synergy H1 fluorescent plate reader from Biotek (excitation and emission wavelengths 540 and 570 nm, respectively). Each compound was separately tested in the range of concentration 10-250 µM. The results clearly suggested that only HT and Ole but not Tyr are able to scavenge the DPPH radical ( Figure 1S)

In vitro Effect of HT, Ole and Tyr on the HMGCoAR activity.
Each compound was assessed in order to evaluate their effect on the HMGCoAR activity. Results suggest that only HT and Ole are slightly active at 15.4 and 54 µg/mL, which correspond to 100 µM, respectively, whereas Tyr is totally ineffective at all the tested concentrations. In particular, HT reduces the enzyme activity by 25±4.5% and Ole 14.3±7.1% at 100 µM, respectively. Figure S3. Effect of HT, Ole, and Tyr on the in vitro HMGCoaR activity.