Purification of phenoloxidase from Haliotis discus hannai and its anti-inflammatory activity in vitro

Haliotis discus hannai, a food with a high protein content, is widely consumed in Asian countries. It is known to have antioxidant, anticancer, and antibacterial effects. Since the biological significance of H. discus hannai hemolymph has not been widely studied, the objective of the present study was to purify phenoloxidase (PO) and investigate its immunological effects on human colonic epithelial cells. PO was purified through ammonium sulfate precipitation and one step column chromatography. The molecular weight of the protein was about 270 kDa. When PO was mixed with Gram-negative bacteria-derived lipopolysaccharide (LPS) at various ratios (10:1-1:10, w/w), the amount of residual LPS was reduced. PO at concentrations up to 200 μg/mL was not cytotoxic to HT-29 cells. The inflammatory response induced by LPS in HT-29 cells was regulated when the concentration of PO was increased. With increasing concentration of PO, production levels of pro-inflammatory cytokines, cytokines associated with hyperimmune responses such as IL4, IL-5, and INF-γ, and prostaglandin 2 (PGE2) were regulated. It was thought that simultaneous treatment with PO and LPS anti-inflammatory effects in HT-29 cells showed by regulating the ERK1/2-mediated NF-κB pathway. Results of this study suggest that H. discus hannai hemolymph is involved in the regulation of Gram-negative bacteria-related inflammatory immune responses in human colonic epithelial cells.