*4.3. Sample Preparation and HPLC-DAD Analysis for Phenols Quantification and Characterization*

The extract was dried under vacuum and resuspended in 9 mL of ethanol:water solution (75:25 *v/v* adjusted at pH 2 by formic acid addition) and partitioned with 5 mL of n-hexane in order to remove chlorophylls and other pigments, which could interfere in the analysis of phenolic compounds. The procedure was repeated three times. The last partition was carried out with chloroform instead of n-hexane. The polar phase was reduced to dryness, and the residue resuspended in 0.5 mL of methanol:water solution (50:50 *v/v* adjusted at pH 2.5 by formic acid addition).

Aliquots of the samples (15 μL) were injected into the Perkin® Elmer Flexar liquid chromatograph equipped with a quaternary 200Q/410 pump and an LC 200 diode array detector (DAD) (all from Perkin Elmer®, Bradford, CT, USA). The stationary phase was composed by a reverse-phase Agilent® Zorbax® SB-18 column (250 × 4.6 mm, 5 <sup>μ</sup>m) (Agilent Technologies Inc., Santa Clara, CA, USA) kept at 30 ◦C. A gradient solvent system of solvent A (acidified water, 0.1% formic acid) and solvent B (acetonitrile, 0.1% formic acid), over a 59 min run in a flow rate of 0.6 mL/ min was applied: 0–5 min (0% B), 5–8 min (0–3% B), 8–53 min (3–40% B), 53–58 min (40% B), 58–59 min (0% B).

The chromatograms were acquired at 280 and 350 nm, the most common wavelengths for the analysis of phenolic compounds. The putative identification of the phenolics detected was carried out based on the retention time, UV spectral characteristics, and comparison with standards, as well as based on literature data. A calibration curve of gallic acid (R2 = 0.99) was used to quantify the compounds and the result of the total phenolic content was given in mg GAE/g dry weight. The analysis was conducted in triplicate.

#### *4.4. Fucoxanthin Determination in the Methanolic Extract*

FX content of *T. lutea* F&M-M36 extract was carried out by chromatographic analysis according to a modification of the method by Kim et al. [8]. FX separation was achieved with an HPLC 1050 (Hewlett Packard, Palo Alto, CA, USA) equipped with a C30 reversephase column (YCM Carotenoid, 4.6 mm × 250 mm, 5 μm particle size) (Waters, MA, USA), and a UV photodiode array detector 1050 (Hewlett Packard, Palo Alto, CA, USA). A gradient method with two eluents were used; eluent A: 81% Methyl Tert-Butyl Ether (MTBE), 10% methanol, and 9% deionized water, and eluent B: 93% MTBE and 7% methanol. The injection volume was 20 μL with a constant flow rate of 1 mL/min, at 25 ◦C temperature. The detection was performed at 450 nm. The quantification was performed by internal standard calibration. Commercial FX (Sigma-Aldrich, Milan, Italy) standard solutions (20, 40, 60, 80, 100, 120 μg/mL in methanol/MTBE 4:1), with β-apo-carotenal (50 μg/mL) and Sudan Red (90 μg/mL) were prepared. The rate between the area under the peaks of FX standard solutions and the area under the internal standard peak was plotted against FX standard solution concentrations (μg/mL) to obtain a calibration curve adopted to quantify the concentration of FX in the *T. lutea* F&M-M36 extract.

### *4.5. In Vitro Model of Inflammation and Anti-Inflammatory Assay*

RAW 264.7 macrophages were purchased from the American Tissue Type Culture Collection (Manassas, VA, USA) and cultured in Dulbecco's modified Eagle's medium (Thermo Fisher Scientific, Milan, Italy) with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific) and 100 U/mL penicillin-streptomycin (Thermo Fisher Scientific), in 5% CO2 at 37 ◦C. The cytotoxicity of the extracts was first evaluated by MTS assay as previously described [18]. FX was dissolved in DMSO and diluted in a complete cell-culture medium in order to obtain the appropriate concentrations to be tested. The final concentrations of DMSO were below 0.1%, and the control cells were exposed only to DMSO 0.1%. The cultured cells were treated with lipopolysaccharide (LPS, 1 μg/mL Sigma-Aldrich, Milan, Italy) and with *T. lutea* F&M-M36 methanolic extract (1–100 μg/mL) or FX (4.7–470 ng/mL) (Sigma-Aldrich, Milan, Italy). After incubating for 18 h at 37 ◦C, the cells were harvested for RNA and protein extraction, and the cell medium was collected and stored at −20 ◦C for PGE2 determination [18,20].
