2.7.1. Total Phenolic Content (TPC) by Folin–Ciocalteu (FC) Assay

Total Phenolic Content was determined spectrophotometrically by using the Folin–Ciocalteu method, with some modifications [31]. Briefly, the working reagent (WR) was prepared daily by diluting one part of commercially available Folin–Ciocalteau reagent (2N with respect to the acid, Sigma-Aldrich, St. Louis, USA) in 14 parts of ultrapure water. TPC was determined by pipetting 10 µL of each standard/sample’s concentration per well or 10 µL of DMSO as control (eight wells/condition). In four of the eight wells, 200 µL of WR was added, and in the other four, 200 µL of 0.1 M HCl was added. The plate was incubated in the dark for 10 min, after which 40 µL of a solution of Na₂CO₃ 20% (w/v) was added to every well. Afterward, the plate was incubated for 45 min at room temperature, after which its absorbance at 765 nm was read (EPOCH 2 microplate reader, BioTek^® Instruments, Winooski, VT, USA). The TPC was expressed as milligrams of phloroglucinol equivalents (PGE) per milligram of seaweed extract (mg PGE·mg⁻¹ extract). The assay was performed in triplicate.