## 4 Discussion  
Seaweed phenolic compounds are considered to exert antioxidant and anti-inflammatory properties that mitigate oxidative stress and inflammation at the cellular levels [20,21]. Studies have shown that Gangneung-grown AC exerts antioxidant activity and inhibits the gene expression of pro-inflammatory cytokine such as IL-1/B, IL-6, and TNF\(\alpha\) in LPS-induced macrophages [15,16,22]. However, the effects of Busan-grown AC ethanol extract and its fractions on antioxidant and inflammation in LPS-induced macrophages were not confirmed. In this study, we found that Busan-grown ACE and its EtOAc fraction inhibited the mRNA expression of pro-inflammatory cytokines and antioxidant genes in LPS-stimulated RAW 264.7 macrophages accompanied by high TPC and total antioxidant activity. Phlorotannins are known to be the most representative phenolic compounds in edible brown seaweed extracts [23]. In the present study, we found that the EtOAc fraction of ACE exhibited the highest TPC among five fractions of ACE, and thus had the highest content of phlorotannins formed through the polymerization of phloroglucinol. In addition, ACE and its EtOAc fractions showed high DPPH and ABTS radical scavenging activity and FRAP activity. The antioxidant properties of ACE and its EtOAc fraction are partly attributed to a high phlorotannin content, which has a unique structure. The strong antioxidant capacity of phlorotannin is associated with the degree of polymerization, the phenol ring, and the phenolic hydroxyl group on the benzene ring skeleton [24]. The presence of phlorotannins in the ACE and EtOAc fractions may confer the intrinsic stability of the polyphenol structure and potent free-radical scavenging activity. Furthermore, studies have well supported that phlorotannins exert excellent antioxidant properties through their free-radical scavenging activity, inhibition of ROS production, and antioxidant defense mechanism [25]. Phlorotannin compounds showed a high scavenging ability on DPPH and superoxide anion radicals two-to-ten-folds higher than ascorbic acid, \(\alpha\)-tocopherol, and catechin, which are known as strong antioxidants [26]. Phlorotannin compound-isolated _Ishige okamurae_ significantly reduced intercellular ROS production in RAW 264.7 macrophages [27]. Furthermore, in zebrafish exposed to ethanol, AAPH, high glucose condition, and UV-B radiation, the phlorotannin compounds from _Eisenia cava_ inhibited intercellular ROS, DNA damage, and lipid peroxidation [28]. Thus, the study to find the phlorotannin compounds of the EtOAc fraction of ACE with potent total antioxidant activity by reducing free-radical scavenging activity is warranted to test this possibility. Figure 4: The effects of ACE and its EtOAc fraction on the expression of antioxidant genes in LPS-induced RAW 264.7 macrophages. Macrophages were treated with (**a**) ACE and (**b**) EtOAc fraction at 0, 25, 50, and 100 \(\upmu\)g/mL for 6 hr and then stimulated by LPS (100 ng/mL) for 3 hr in absence or presence of ACE and its EtOAc fraction for gene analysis by RT-PCR. GAPDH was employed as an internal control. Different letters in the same bar mean a significant difference (\(p<0.05\)). Data are presented as the means \(\pm\) SD. LPS, a major pathogenic infectious agent of Gram-negative bacteria, directly activates macrophages, which promotes the secretion of inflammation-inducible enzymes and pro-inflammatory cytokines [29]. In this study, ACE and its EtOAc fraction significantly suppressed the gene expression of IL-1B, TNF\(\alpha\), iNOS, and COX2 in LPS-induced macrophages. In addition, we found that ACE and its EtOAc fraction showed high TPC and total antioxidant capacity by reducing DPPH and ABTS radical scavenging activity and FRAP activity. Oxidative stress by increasing ROS production can lead to the oxidation of amino acids, lipid peroxidation, and oxidative DNA damage, triggering an inflammatory response [30].