2.5.1. Cellular Antioxidant Activity

Cellular antioxidant assay (CAA) was performed using HepG2 cells (ATCC, USA) maintained in Minimum Essential Medium α (MEMα), supplemented with 10% (*v*/*v*) heat-inactivated fetal bovine serum, penicillin (50 units/mL), and streptomycin (50 μg/mL). Cells were incubated at 37 ◦C in a fully humidified environment under 5% CO2, and HepG2 cells at passage 80–100 were used for the experiments. Cells were subcultured at 7 days intervals before reaching 90% confluence.

CAA assay was performed on spray dried extracts using HepG2 cells at a density of 6 × 10<sup>4</sup> cells/well seeded black 96-well plates (BD Falcon™) in 100 μL growth medium/well according to Wolfe and Liu (2007) [27] and Samaranayaka et al. (2010) [28] with minor modifications. Briefly, 24 hours after cell seeding, 100 μL of DCFH-DA probe (1 μM in HBSS) was added to the cells and incubated at 37 ◦C in the dark for 30 min. Cells were then treated with different concentrations of extract and incubated for 1 h at 37 ◦C. Subsequently, after removal of the antioxidant-tested compounds, 100 μL of peroxyl radical initiator AAPH (750 μM in HBSS) were added to the cultured cells. Fluorescence readings (λexcitation = 493 nm, λemission = 527 nm) were recorded using a POLARstar OPTIMA (BMG Labtech) every 10 min for 90 min after addition of AAPH. Each plate included four replicates of both blank and controls: the blank consisted of cells exposed to only the DCFH-DA probe, and the control consisted of cells with the DCFH-DA probe and the AAPH added, but in the absence of test compounds.

#### 2.5.2. Cell Protective Effects against Induced Oxidative Stress

HepG2 cells were seeded in culture T75 flasks (16.9 × 10<sup>4</sup> cells/cm2), and after 24 h, they were incubated with 62.5 μg/mL seaweed extract (batch B200314) for 48 h. Finally, 200 μM of tert-butyl hydroperoxide (TBUT) (Sigma-Aldrich, Missouri, USA) was added and incubated for 3 h. Experiments included untreated cells that were not exposed to seaweed and/or TBUT. Cells were harvested using trypsin/EDTA and centrifuged at 1000 × g for 5 min. The supernatant was removed and the cell pellets were subjected to enzymatic assays and glutathione extraction with 10% metaphosphoric acid.

To this aim, cells were homogenized on ice in H2O, and the supernatant was assayed for protein content according to Lowry method (1951) [29] and used to perform enzyme assays as previously described [30].

Briefly, catalase activity was assayed by measuring the consumption of H2O2 at 240 nm for 1 min at 30 ◦C according to Aebi (1984) [31]. The incubation mixture included: 50 μL H2O2 200 mM, 50–100 μg of proteins of sample, and Na-phosphate buffer (50 mM pH 7.0) to reach a final volume of 1 mL. One unit of catalase activity is defined as amount of enzyme required to catalyze the decomposition of 1 μmol H2O2 min−1.

The glutathione reductase (GR) activity was assayed according to Pinto et al. (1984) [32]. Briefly, GSSG reduction and NADPH consumption were recorded at 340 nm. The incubation mixture included: 20 μL GSSG 125 mM, 11 μL NADPH 11 mM, 50–100 μg of proteins of sample, and K-phosphate buffer (100 mM pH 7.0) to 1 mL final volume.

The activity of selenium-dependent glutathione peroxidase (GPx) was assayed according to Prohaska and Ganther (1976) [33] by following the decrease in the absorbance at 340 nm for 5 min, which corresponds to the rate of GSH oxidation to GSSG in the presence of NADPH and GR. The incubation mixture included: 20 μL GSH 100 mM, 10 μL NADPH 11 mM, GR 1 Unit, 10 μL TBUT 20 mM, 50–100 μg of proteins of sample, and EDTA-K phosphate buffer to 1 mL final volume. One unit of GR or GPx activity is defined as the amount of enzyme required to catalyze the oxidation of 1 mmol NADPH min−1.

Total glutathione content was assayed according to Griffith (1985) [34] with slight modifications. Briefly, the sulfhydryl group of GSH, also generated from GSSG by adding GR, reacts with DTNB (5,50-dithio-bis-2-nitrobenzoic acid) and produces a yellow-colored 5-thio-2-nitrobenzoic acid (TNB). The rate of TNB production is directly proportional to the concentration of GSH in the sample. Measurement of the absorbance of TNB at 412 nm provides an accurate estimation of the GSH level present in the sample.

The end product of lipid peroxidation, malonyldialdehyde (MDA) was measured in cell extracts and quantified using an HPLC-UV system (Jasco, Japan) as previously described [35]. The MDA standard and sample preparation was carried out according to Karatas et al. (2002) [36]. To prepare MDA standards, 10 μL of 1,1,3,3-tetraethoxypropane (TEP) were accurately diluted to 10 mL with 0.1 M HCl in a screw-capped test tube and placed in a boiling water bath for 5 min and then rapidly cooled on ice, producing hydrolyzed acetal. A working stock solution of MDA was prepared by adding 1 mL of the hydrolyzed acetal to 99 mL of water; the working stock solution was 40 μM MDA. The stock solution was further diluted and used to construct the calibration curve. MDA was determined by HPLC (Jasco, Japan) equipped with a UV detector. A C18 column (Waters, 1.7 μm, 50 × 2.1 mm) was used at room temperature. Samples were suspended in water and HClO4 0.1 M, centrifuged at 4500× *g* for 5 min and supernatants were used for HPLC analysis. The mobile phase was KH2PO4/CH3OH/acetonitrile (72/18/11; *v*/*v*/*v*) and the flow rate was 1.0 mL/min. Chromatograms were monitored at 254 nm and injection was 20 μl (0.5 × 10<sup>6</sup> cells). The retention time of MDA was 2.5–3 min.
