## 4 Discussion
COX-1 appears in various cells and mediates physiological activity, whereas COX-2, known as an inducible enzyme, is found in immune cells treated with LPS and inflammatory cytokines [37]. A previous study described that COX-2 is accountable for the synthesis of prostanoids, which are relevant in critical and chronic inflammatory conditions [38]. Therefore, COX-2 hyperactivation can trigger PGE\({}_{2}\) production. Similarly, in this study, iNOS and COX-2 mRNA expression was upregulated in the LPS-treated group, and the resultant nitrite and PGE\({}_{2}\) production were confirmed to be increased. In comparison, all DM extract groups significantly inhibited iNOS and COX-2 expression compared to the LPS-treated group. In terms of the nitrite and PGE\({}_{2}\) level, among the extracts, the 60% ethanolextract harvested in August particularly suppressed both. Furthermore, DM aqueous ethanol extracts inhibited the phosphorylation of NF-kB, IKK\(\alpha\), JNK, and p38. Downregulation of these proteins was related to iNOS and COX-2 mRNA expression. Therefore, the downregulation of proinflammatory transcription factors reduced the production of proinflammatory cytokine (IL-6, IL-18, and TNF-\(\alpha\)) and cellular mediators (nitrite and PGE\({}_{2}\)). DM leaves contain several polyphenic compounds, such as chlorogenic acid, rutin, 4-methylcatenhol, and p-coumaric acid and terpenoids, including \(\alpha\)- and \(\beta\)-amyrin [12; 13; 19]. In the current study, these significant compounds were analyzed. Our results showed that terpenoids increased in the August and November extracts compared to extracts of May, and higher ethanol concentrations increased \(\alpha\)- and \(\beta\)-amyrin. However, except for rutin, the major polyphenic compounds were higher in the August extracts than in the May and November extracts. The rutin concentration was the highest in the May extract. Ethanol concentrations showed a similar pattern for extracting these active compounds. We identified the main DM active compounds through the literature review and analyzed them. However, given the preliminary nature of this study, we could not fully-characterize the active compounds from DM extracts. However, we estimated which components might contribute to suppressing inflammation by comparing the chief composition and anti-inflammatory activity. When the correlation between the concentrations of these compounds and anti-inflammatory activity was analyzed, \(\alpha\)- and \(\beta\)-amyrin showed significant correlations with iNOS and COX-2 inhibition. The 60% ethanol extracts harvested in August and November displayed the strongest inhibitory activity, and the concentrations of \(\alpha\)- and \(\beta\)-amyrin were the highest. Although the correlation between other compositions and inflammatory cytokines was also analyzed, there was no significant difference. This finding suggests a potential mechanism underlying the inhibition of inflammatory responses. Organic solvent extraction methods, such as ethanol and methanol, are conventional and have been applied to extract phenolic compounds from plants. In this study, overall, as the ethanol concentration increased, both \(\alpha\)- and \(\beta\)-amyrin in various extracts also increased. Among the well-known active chemicals in DM, \(\alpha\)- and \(\beta\)-amyrin were the most active compounds, as evidenced by correlation analysis. Amyrin is a pentacyclic triterpene that displays biological activities, such as anti-inflammatory, antinociceptive, hepatoprotective, antipruritic, gastroprotective, antitumor, and anxiolytic activity [39; 40]. Previous studies have reported that \(\alpha\)- and \(\beta\)-amyrin can prevent NF-kB activation and that the inhibition of NO plays a prominent role in an L-arginine-induced pancreatitis model [41]. Furthermore, the release of TNF-\(\alpha\) or its proinflammatory response was constrained by \(\alpha\)- and \(\beta\)-amyrin [42].