**4. Materials and Methods**

#### *4.1. Preparation of YA Peptide*

Crassostrea gigas specimens (length, 5.8 ± 0.4 cm; height, 3.2 ± 0.4 cm; body weight (BW), 9.8 ± 2.1 g) were harvested from a fish farm in Tongyeong (South Korea) in 2018–2019, frozen, and preserved for 1–2 years. The preparation of the oyster hydrolysate (OH) and YA was conducted according to a previous protocol [25]. The amino acid sequence of the purified peptide fragment in OH is determined using LC/MS/MS. The sequenced peptide YA was synthesized with a purity of 95% or higher to test their function. In addition, we validated histological changes in the liver and changes in liver enzymes (alanine aminotransferase, ALT; aspartate aminotransferase, AST) using synthetic YA purchased from Sigma-Aldrich (St. Louis, MI, USA).

#### *4.2. Measurement of Cyclooxygenase-2 (COX-2) and 5-Lipoxygenase (5-LO) Inhibition Activity*

The percentages of COX-2 inhibition and 5-LO inhibition of YA were measured according to the previous protocols [24]. Briefly, the assay mixture for COX-2 contained 450 μL of Tris-HCl buffer (pH 8.0, 100 mM), 100 μL of hematin (150 mM), 100 μL of ethylene-diaminetetraacetic acid (EDTA, 30 μM), 200 μL of COX-2 (40 U/mL), and 100 μL of YA. The mixture was incubated for 15 min at room temperature. The reaction was initiated by adding 20 μL of arachidonic acid (20 mM) and 25 μL of N,N,N',N'-tetramethyl-ρ-phenylenediamine (TMPD, 10 mM) and evaluated after 5 min at 590 nm. To measure 5-LO activity, 200 μL of the enzyme solution (160 U/mL) were prepared in a 0.2 M boric acid buffer (pH 9.0), mixed with 50 μL of YA (1, 3, 5, and 100 mg/mL in boric acid buffer), and then incubated at room temperature for 3 min. The reaction was initiated by adding 250 μL of the substrate solution (100 μM of linoleic acid) and evaluated for 2 min at 234 nm using the VERSAmax microplate reader (Molecular Devices, San Jose, CA, USA).
