2.3. In Vitro Antioxidant Capacity Assessment

Cell culture: In this study, the Caco-2 human colon cell line was purchased from American Type Cell Culture (ATCC, catalog no. CCL-2102). The cells were cultured in DMEM culture medium supplemented with 10% fetal bovine serum in a humidified 5% CO₂ atmosphere at 37 °C.

Culture treatment protocol: When the cells reached 80% confluence, a 0.25% (w/v) Trypsin - 0.53 mM ethylenediamine tetraacetic acid (EDTA) solution (Sigma-Aldrich, St. Louis, MO, USA) was used to detach the cells. The enterocytes were counted and cultured at a density of 3 × 10⁴ cells/cm² into 24-well plates (for viability assay and intracellular reactive oxygen species (ROS) measurement) or in 25 cm² culture flasks (for glutathione content and lipid peroxidation analysis) and allowed to adhere overnight. Afterwards, Caco-2 cells were incubated with different concentrations (5, 25, 50, and 100 μg/mL) of MT for 12, 24, and 48 h (for MTT assay), whereas for oxidative stress evaluation, two doses (25 and 100 μg/mL) and two intervals of exposure (24 and 48 h) were selected. The oxidative stress was also tested, both in the presence and absence of the oxidizing agent (250 μM H₂O₂). For each experiment, untreated cells (untreated control) and cells treated with the oxidant (250 μM H₂O₂) were used.

Cell viability: It was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test [21]. This method consists of reducing the yellow MTT compound to purple formazan crystals by mitochondrial dehydrogenase-NAD(P)H (nicotinamide adenine dinucleotide phosphate reduced) dependent on the metabolically active cells. Formazan is then solubilized with 100% isopropanol and the concentration is determined spectrophotometrically at 595 nm. The culture medium was removed from each well and 0.5 mL of 1 mg/mL MTT was added. After 2 h of incubation at 37 °C, the MTT solution was removed and the formazan crystals from each well were solubilized with 0.5 mL of isopropanol. The absorbance of the samples was measured at 595 nm using a FlexStation 3 multi-mode microplate reader (Molecular Devices LLC, San Jose, CA, USA).

Preparation of cell lysates: Cells were collected from culture flasks, washed with PBS, and the cell lysates were obtained by sonication (30 s × 3 times) on ice with an ultrasonic processor (Hielscher UP50H, Teltow, Germany). The homogenate was centrifuged at 3000× g for 10 min at 4 °C and the total proteic extracts (EPT) represented by supernatants were collected for biochemical assays.

Determination of protein concentration by Bradford method: Protein concentrations of cell lysates were determined using the Bradford reagent (Sigma-Aldrich, St. Louis, MO, USA) and bovine serum albumin (BSA) as protein standard. Sample absorbance was determined at 595 nm using a FlexStation 3 multi-mode microplate reader (Molecular Devices LLC).

Malondialdehyde (MDA) level measurement: A precise and often used method for monitoring lipid peroxidation uses thiobarbituric acid (TBA) as a reactive substance. MDA–TBA adducts formed following the reaction between the MDA present in the biological sample and the TBA at 37 °C can be measured by the fluorimetric method. Briefly, 200 μL of cell lysate was mixed with 700 μL of 0.1 M HCl and incubated at room temperature for 20 min. Afterwards, 900 μL of 0.025 M TBA was added and incubated again for 65 min at 37 °C, at which time TBA–MDA products were formed. Finally, the relative fluorescence units (RFU) were recorded using a Jasco FP-750 fluorimeter (excitation wavelength = 520 nm and emission wavelength = 549 nm) (FP-750 Spectrofluorometer, Jasco, Tokyo, Japan) and converted to nmols of MDA using a standard curve of 1,1,3,3-tetramethoxypropane, with concentrations ranging 0–0.5 mM. Also, the MDA level in each sample was expressed as nmol of MDA/mg protein.

Glutathione (GSH) level measurement: Determination of reduced glutathione (GSH) concentration was performed with the Glutathione Assay Kit from Sigma. Therefore, cell lysates were deproteinized with 5% 5-sulfosalicylic acid (SSA) (in a ratio of 1:1) and centrifuged at 3000× g for 5 min at 4 °C to remove the precipitated proteins. Further, the samples were incubated with 5,5’-dithiobis-2-nitrobenzoic acid (DTNB) at room temperature for 5 min to allow the reduction of DTNB to 5-thio-2-nitrobenzoic acid (TNB) under GSH action. The optical density of the samples was measured at 412 nm using a microplate reader (TECAN GENios, Grödic, Germany). The GSH content was calculated by extrapolation on a GSH standard curve and expressed as nmols/mg protein.