4.1. Plant Materials and Sample Preparation
The leaves of the olive tree (Olea europea, cultivar Coratina) were collected in Roccelletta di Borgia, Calabria, Italy, latitude 20°25’47’’N and longitude 34°27’36’’E (February 2023, temperature 9,2° C. Several washes with MilliQ water were carried out to remove pollution or other contaminants from leaves. 100 g of dried and milled leaves and 800 ml of water were placed in a Pyrex round bottom flask equipped with a jacketed coiled condenser in a domestic microwave oven [23]. After extraction, performed by irradiation at 800 W for 10 min, leaves were filtered, and the obtained solution was dried under pressure. To eliminate the solid residue, the extract was filtered with acetone, and the solution was evaporated under pressure to obtain the crude extract (OE). Subsequently, OE was placed in dark bottles, and stored at 4° C before being used. To carry out the experiments, OE was dissolved in a hydroalcoholic solution composed of EtOH: water (70:30).
4.1.1 s-OE preparation
s-OE was obtained by dissolving the OE powder in a hydroalcoholic solution in which the ratio of EtOH to water was 50:50 and then subjected to a sonication treatment (frequency of 15 kHz, power 470 W for 10 min, Bandelin electronic 12207, Berlin, Germany) under constant stirring (150 rpm). s-OE was always kept in a container containing ice. s-OE was placed in dark bottles, and stored at 4° C before being used for experimentation. The choice of power to be used was justified by previous experiments in our laboratory.
4.2 UHPLC-UV-ESI-HRMS analysis
The quantification of hydroxytyrosol and oleuropein in Coratina leaves dry extract was performed by reverse-phase ultra-high-performance liquid chromatography followed by electrospray high-resolution mass spectrometry, with ionization in negative mode, as reported by Frisina et al. [24]. Chromatography separation was performed using Dionex Ultimate 3000 RS (Thermo Scientific - Rodano, MI, Italy), equipped with a Hypersil Gold C18 column (100 x 2.1 mm, 1.9 µm particle size, Thermo Scientific), which was thermostated at 24°C. The chromatographic column was equilibrated in 98% solvent A (Ultrapure water containing 0.1% of formic acid) and 2% solvent B (methanol). The flow rate on the column was maintained at 300 µl min-1 and the concentration of solvent B was linearly increased from 2% to 23% in 6 min, remaining in isocratic for 5 min, then linearly increased from 23% to 50% in 7 min, and from 50% to 98% in 5 min, remaining in isocratic for 6 min and finally returned to 2% in 6 min, remaining in isocratic for 3 min. The UV/VIS detector was set at 235, 254, 280, and 330 nm. Mass detection was performed by a high-resolution Q-Exactive orbitrap mass spectrometer (Thermo Scientific, Rodano, MI, Italy). In each scan, the negative exact mass [M-H]- of hydroxytyrosol (153.0557 m/z) and oleuropein precursors (539.1170 m/z) were selected in Parallel Reaction Monitoring. Hydroxytyrosol and oleuropein were characterized according to the corresponding HRMS spectra, accurate masses, characteristic fragmentations, and retention times. Xcalibur software (version 4.1) was used for instrument control, data acquisition, and data analysis.
4.3 Measurement of Polyphenols Content
The total polyphenols were quantified by the Folin–Ciocalteu assay using gallic acid 99% (Sigma-Aldrich, St. Louis, MO, USA) as the reference standard. The extracts OE or s-OE were prepared by adding 20 mL of ethanol/water 50:50 (w/w) to 1 g of powder to obtain absorbance values within the linearity range of the calibration curve. The mixture obtained was stirred for 24 h at 20° C. 400 µl of extract was added to 0.8 ml of Folin-Ciocalteu reagent diluted 10 times. After 3 minutes, 0.8 ml of sodium carbonate 7% (w/v) was added and the mixture was allowed to stand for 2 h with constant stirring. The absorbance was measured at 760 nm with a Prism V-1200 spectrophotometer. The total phenolic content was determined from the linear equation of a standard curve prepared with different concentrations of gallic acid and the results were expressed in mg of gallic acid equivalents per g of dry weight.
4.4 Measurement of Flavonoids Content
The flavonoid content of the extracts was measured by the colorimetric technique of aluminum chloride. Specifically, 1 mL of extract was mixed with 1 mL of 2% aluminum chloride in methanol. After 30 min, the absorbance at 510 nm was measured, and the rutin equivalents (mg RE/g extract) were used to represent the estimated content of flavonoids.
4.5 Free radical scavenging activity
The free radical scavenging activity of both extracts was determined using the stable radical 2,20 -diphenyl-1 picrylhydrazyl (DPPH) in which samples were tested at different concentrations (0,01-0.32 mg/ml) [25]. Experimentally, an aliquot (0,5 ml) of samples was added to 3 ml of DPPH solution prepared at the time of use, and keeping the mixture in the dark for 20 min. Subsequently, the absorbance was read by a UV-Vis spectrophotometer (Multiskan GO, Thermo Scientific, Denver, CO, USA) at 517 nm and at room temperature. Ascorbic acid was used as a reference. The results were expressed as a percentage of DPPH radical scavenging activity (%). The latter was defined by the formula [(A0 - Ac)/A0] x100, where A0 is the absorbance of the control and Ac is the absorbance in the presence of the sample or standard. The results are reported as an average percentage of radical scavenging activity (%) and were expressed also as % inhibition value and IC50.
4.6 Reducing power assay
This method is based on the principle that substances with a reducing potential react with potassium ferricyanide (Fe3+) to form potassium ferrocyanide (Fe2+). The reducing power of OE and s-OE extracts has been evaluated by spectrophotometric detection of the Fe3+-Fe2+ transformation method [26]. The samples were tested at different concentrations (0,01-0.32 mg/ml); different amounts of samples in 1 ml solvent were mixed with 2,5 ml phosphate buffer (0,2 M, pH 6,6) and 2,5 ml potassium ferricyanide at 1 % [K3Fe(CN)6]. The mixture was incubated at 50° C for 20 min. The solution was quickly cooled, mixed with 2,5 ml of 10 % trichloroacetic acid, and centrifuged at 3000 rpm for 10 min. The resulting supernatant was mixed with 2,5 ml of distilled water and 0,5 ml of 0,1% fresh ferric chloride (FeCl3) and the absorbance was measured at 700 nm after 10 min. Ascorbic acid was used as a reference. The reducing potential was expressed in ascorbic acid equivalent (ASE/ml).
4.7 Ferrous ions (Fe2+) chelating activity
The Fe2+ ions have pro-oxidant properties capable of determining lipid oxidation resulting in cellular alterations and chelating agents can reduce these damages. The chelating activity can be studied by spectrophotometric evaluation of the inhibition of the Fe2+-ferrozine complex in which the agents reduce the formation of this complex: the reduction of the color is proportional to the chelating activity produced [27]. Experimentally different concentrations of each sample in 1 ml solvent were mixed with 0.5 ml methanol and 0.05 ml of 2 mM FeCl2. The mixture obtained was vigorously mixed and maintained at room temperature for 10 min. The absorbance of the solution was measured spectrophotometrically at 562 nm. The percentage of the chelating effect was calculated using the following equation:
% Chelating effect = [1- (At/A0)] x 100
where A0 is the absorbance of the control and At is the absorbance of the tested compound. The results were expressed as IC50 concentration, where the inhibition of 50% of the Fe2+-ferrozine complex was obtained and compared with the standard solution of ethylenediaminetetraacetic acid (EDTA).
4.8 Cell cultures
A cell line of human neurons (SH-SY5Y) was acquired from the American Type Culture Collection (Sesto San Giovanni, Milan, Italy) and kept in Eagle’s minimum essential medium supplemented with nonessential amino acids, 10 % fetal bovine serum, penicillin (100 IU/ml) and streptomycin (100 µg/ml). The cell line was cultivated in a 5% humidified CO2 atmosphere at 37 ° C and the medium was changed every 2-3 days. To differentiate SH-SY5Y cells, the neuroblastoma line was treated with 10 µM of all-trans-retinoic acid for 5 days. When the cells reached 60% confluence, they were treated with OE or s-OE 25 μg/ml, for 24h. In neurotoxicity-induced studies, the cells were pre-treated with OE or s-OE, as described, and then exposed to Pb2+ for an additional 24h. Finally, in experiments conducted to study the antioxidant effect of NAC, the cells were treated with NAC, 2 mM for 6 hours, and successively exposed to Pb2+ for an additional 24h.
4.9 Measurement of cell proliferation through MTT test
3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) was used to evaluate cell proliferation in a colorimetric assay. 8 x 103 cells were grown in 96-well plates and, after 24 hours, the culture medium was replaced with fresh medium containing one of the treatments indicated at the appropriate concentrations. At the end of the required time, the medium was replaced with a phenol-free medium containing an MTT solution (0,5 mg/ml). Finally, after 4h incubation, 100 µl of 10% SDS was added to each well to solubilize formazan crystals, and the optical density was measured at wavelengths of 540 and 690 nm using a spectrophotometric reader (X MARK Microplate Bio-Rad Spectrophotometer).
4.10 Lipid quantification by flow cytometry
Nile Red (excitation/ emission ~552/636) is a dye that binds tightly to cellular lipids. At the end of the appropriate treatments, the neurons were collected and adequately incubated with 1 µg / ml of Nile Red dye for 15 min in the dark and protected from light. Subsequently, the cells were washed in PBS (pH = 7,4) to remove the excess dye and immediately read in the flow cytometer FACS Accury (Becton Dickinson, Italy).
4.11 Intracellular Calcium Measurements
The Rhodamine 2 fluorescent probe is frequently used to measure the intracellular calcium since it binds to this ion and, if subject to excitation (552 nm), emits fluorescence (581 nm). After being treated as indicated, the neurons were exposed to Rhod 2 for 1 h at 25 ° C and protected from light. At the end of the exposure time, they were washed in a calcium and magnesium-free solution, to prevent the entering of the calcium from the extracellular environment and subjected to flow cytometric analysis. It is important to note that the cells were also treated with decoupling of mitochondrial oxidative phosphorylation, carbonylcianuro-4-(trifluoromethoxy) phenylhydrazone (FCCP, 1 µM), and oligomycin (1 µg/ml), a mitochondrial ATPase inhibitor. Both treatments may exclude mitochondrial calcium involvement. Thus, any movement of calcium was related to endoplasmic reticulum calcium. A first cytofluorometer reading (FACS Accury, Becton Dickinson) provides the basal concentration of the calcium ion in cells. Then exposure to TAPSI (1 µM), leads to an increase in the concentration of cytosolic calcium that is released from the endoplasmic reticulum. Under these conditions, three new cytometric readings were carried out and finally, ethylene glycol-bis (β-aminoethyl ether)-N,N,N0,N0-tetraacetic acid (EGTA) 500 µM was added to chelate intracellular calcium. This scheme shows that the protocol is working. However, the interesting value is provided by the measurement of basal calcium that will be compared with that obtained following the different treatments.