Hydroxytyrosol

Immunohistochemical analysis was performed as already described [54 (link)]. Sections were probed overnight with anti-zonula occludens (ZO) antibody (1:100; Millipore, Abingdon, UK) or anti-occludin antibody (1:100; Santa Cruz Biotechnology) or anti-intracellular adhesion molecule-1 (ICAM-1) (1:100; Santa Cruz Biotechnology) or anti-P-selectin (1:100; Santa Cruz Biotechnology). Sections were washed with phosphate-buffer saline (PBS) and incubated with peroxidase-conjugated bovine anti-mouse IgG, secondary antibody (1:2000 Jackson Immuno Research, WestGrove, Pennsylvania, USA). Specific labeling was provided with a biotin-conjugated goat anti-mouse IgG and avidin-biotin peroxidase complex (Vector Laboratories, Burlingame, CA, USA). Images were collected using a Leica DM6 (Milan, Italy) microscope. The histogram profile reports the positive pixel intensity value taken from a computer program.

Hydroxytyrosol and its metabolites
in plasma were determined by liquid–liquid extraction followed
by LC-MS/MS analysis as previously described.^{11 (link)} Briefly, 200 μL of plasma was mixed with 10 μL of freshly
prepared 10% ascorbic acid followed by 10 μL of 0.5% acetic
acid and 10 μL of 2-(3-hydroxyphenyl) ethanol (10 μM,
I.S.). Then, 2 mL of ethyl acetate was added to the samples that were
vigorously mixed in a vortex (5 min), placed into an ultrasonic bath
(10 min), and centrifuged at 1500g at 4 °C (15
min) (Centrifuge Megafuge 1.0R). A second extraction of the pellet
with 2 mL of ethyl acetate was carried out. Subsequently, the two
pooled supernatants were evaporated to dryness, reconstituted with
100 μL of 80% methanol, placed into amber vials, and immediately
analyzed by LC-MS/MS.
An Agilent 1260 liquid chromatograph (Agilent
Technologies, Santa Clara, USA) coupled to a QTRAP 4000 mass spectrometer
(AB Sciex, Toronto, Canada) equipped with a Turbo V electrospray ionization
(ESI) source was used for the determination of hydroxytyrosol and
its metabolites. Analyst software, version 1.6.2. (AB Sciex) operated
the instrument and was employed for data analysis. The equipment was
located at the Scientific and Technological Centre of the Universitat
de Barcelona (CCiTUB).
Injections of 2 μL of each sample
were performed by an automated
autosampler that maintained vials at 10 °C to avoid degradation.
Chromatographic separation of hydroxytyrosol and its metabolites was
performed in a Zorbax Eclipse XDB-C18 reversed-phase column (150
mm × 4.6 mm, 5 μm) protected with a guard cartridge of
the same material (Zorbax Eclipse XDB-C18, 12.5 mm × 4.6 mm,
5 μm) with the temperature set at 30 °C. The mobile phase
consisted of phase A formed by Milli-Q water with 0.025% acetic acid
and phase B containing acetonitrile with 5% acetone delivered at a
flow of 0.8 mL/min. The following gradient elution was used: 0 min,
95% A and 5% B; 1 min, 90% A and 10% B; 10 min, 35% A and 65% B; 10.5
min, 0% A and 100% B. Solvent B was maintained at 100% for 5 min to
prevent carryover prior to returning to initial conditions. A 6 min
delay was programmed before the next injection to ensure the equilibration
of the system. Moreover, the injector needle was washed with 1:1:1
(v/v) 2-propanol, tetrahydrofuran, and Milli-Q water to avoid further
carryover.
The ESI source, operating in negative mode, was set
as follows:
temperature, 600 °C; curtain gas (N₂), 25 arbitrary
units (au); ion source gas 1 (source heating gas, N₂);
50 au; ion source gas 2 (drying gas, N₂); 50 au, and ionization
spray voltage, −3500 V. The MS analysis was performed in multiple
reaction monitoring (MRM) mode and the specific parameters are shown
in Figure 1. Within
each analytical run, a full set of calibration standards was injected
including a reagent blank and blank plasma.
The plasmatic concentrations were calculated by
the interpolation
of the peak area ratio of hydroxytyrosol versus I.S. on a calibration
curve. Calibration standards were constructed with blank plasma obtained
by cardiac puncture from overnight fasted rats that had never received
either table olives or hydroxytyrosol. Then, 190 μL of blank
plasma was spiked with 10 μL of working standards at 0, 200,
500, 1000, 2000, 3000, and 5000 nmol/L to obtain the final concentrations
of 0, 10, 25, 50, 100, 150, and 250 nmol/L. Metabolites were identified
with the m/z indicated in Figure 1 and were assumed
to possess a LC-MS/MS response similar to that of hydroxytyrosol.
Hence, the concentrations of the sulfate and glucuronide derivatives
were quantified using the standard curve of the parent compound. Results
were expressed in nmol per liter of plasma (nmol/L). The method was
validated following the EMA guidelines³³ at six different concentrations ranging from 0 to 250 nmol/L analyzed
on three different days. Validation results indicated that the analytical
method is linear (R² > 0.998), precise
(CV < 15%), with satisfactory recovery (98.4 ± 1.64%), absence
of matrix effect (96.7 ± 2.75%), no carry-over, and adequate
sensibility with a limit of quantification (LOQ) of 0.2 nmol/L.

Bovine Serum Albumin, fluorescein isothiocyanate-dextran (wt 4,000), dimethyl-sulfoxide (DMSO), Bradford reagent, Cell Lytic-M, lipopolysaccharide from Escherichia coli, hydroxytyrosol, tyrosol, NaCl, NaF, K₂HPO₄, KH₂PO₄, MgSO₄x7H₂O, CaCl₂x6H₂O, NaHCO₃, Tween 80 and all solvents of analytical grade were purchased from Sigma Aldrich (Milano, Italy). tyrosol glucuronide, tyrosol sulfate sodium salt, 3′- hydroxytyrosol 3′-glucuronide, hydroxytyrosol 3-sulfate sodium salt, were obtained from LGC standards (Sesto San Giovanni, Italy). The Phosphatase and Protease Inhibitor Cocktail, nitrocellulose membranes, gels and all material for electrophoresis were purchased from ThermoFisher Scientific (Massachusetts, United States). ReagentPack Subculture Reagents with Trypsin/EDTA, TNS (Trypsin Neutralizer solution) and HEPES (2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid) solutions were obtained from Lonza (Basel, Switzerland). Transwell inserts were obtained from Corning Costar Corp. (New York, N.Y., United States).

The OMWW extract enriched in polyphenols (hydroxytyrosol and tyrosol) was provided by Stymon Natural Products P.C., Patras, Greece (www.stymon.com, accessed on 21 December 2023). This product derives from OMWW of the olive (Olea Europaea L.) variety Koroneiki and is produced based on a unique patent (Patent number 1,010,150 IOBE (INT.CL.2021.01) A23L 19/00 A23L 33/105, Stymonphen Liquid) using only green technologies. Its total polyphenol content was equal to 15,000 ± 592 mg/kg, according to the Folin–Ciocalteu method [66 (link)]; hydroxytyrosol and tyrosol were the main phenolic compounds (8784 mg/kg and 1638 mg/kg, respectively), detected by HPLC-DAD [67 ]. The stock solution was filtered, vortexed, and diluted in phosphate-buffered saline (PBS, Euroclone, Milan, Italy) to reach 1400 µg/mL; from this, different scalar concentrations of polyphenols (0.35; 0.7; 7; 14; 70; 140 µg) were obtained for the successive analyses by diluting them in complete culture medium.