## 4 Discussion
Similar to reported findings, this study also confirmed the anti-inflammatory activity of amyrin in DM extracts by suppressing the cellular regulators iNOS and COX-2 and proinflammatory cytokines by preventing activation of the NF-kB and MAPK pathways in the 30 and 60% ethanol extract-treated groups. Although precise underlying mechanisms were not revealed in this study, the anti-inflammatory effects of DM extracts varied by season and extraction solvent used. Among the extracts, the 30% ethanol extract harvested in May and 60% ethanol extracts harvested in August and November exhibited the highest anti-inflammatory effects in the RAW264.7 murine macrophage cell line stimulated with LPS. Unlike other seasons, in May, the 30% ethanol extract showed high anti-inflammatory activity. Therefore, we need to conduct further studies to identify unknown active components in the May harvest. To the best of our knowledge, this is the first study to show that DM alleviates inflammatory responses through its active components of \(\alpha\)- and \(\beta\)-amyrin. In this study, we found the optimal conditions, including the season and amount of ethanol for DM extracts, for maximizing the anti-inflammatory activity. We suggest that DM extracts could be developed as functional food ingredients for preventing inflammation-related diseases. This is because DM extracts significantly inhibited cellular mediators (nitrite and PGE\({}_{2}\)); the mRNA expression of iNOS and COX-2; and inflammatory cytokines, including IL-6, IL-18, and TNF-\(\alpha\). In addition to this, the NF-kB/MAPK signaling pathway was downregulated by DM extracts. Nevertheless, although amyrins could be beneficial in standard compositions, this study has major limitations. As a preliminary screening study, we only analyzed the major compounds in extracts and investigated the correlation between the flavonoids and inflammatory cytokines. Although it was found that \(\alpha\)-amyrin and \(\beta\)-amyrin were the first major starting points in anti-inflammatory effects, the roles of the active compounds and detailed mechanisms were not revealed in the current study. Therefore, additional studies on the profiling of various flavonoids and the physiological relevance of DM extracts using in vivo and human interventions should be conducted. This might produce the most proof for discovering detailed mechanisms underlying the anti-inflammatory activity of multi-compounds derived from DM extracts. The following are available online at [[http://www.mdpi.com/2076-3417/10/23/8756/s1](http://www.mdpi.com/2076-3417/10/23/8756/s1)]([http://www.mdpi.com/2076-3417/10/23/8756/s1](http://www.mdpi.com/2076-3417/10/23/8756/s1)), Figure S1: Relationship between \(\alpha\)- (a) or \(\beta\)-amyrin (b) and PGE\({}_{2}\) or proinflammatory cytokines. Correlations are presented as [PERSON]'s correlation coefficients (r2); Figure S2: Relationship between chlorogenic acid and inflammatory mediators (a) or proinflammatory cytokines (b). Correlations are presented as [PERSON]'s correlation coefficients (r2); Figure S3: Relationship between 4-methylcleatech and inflammatory mediators (a) or proinflammatory cytokines (b). Correlations are presented as [PERSON]'s correlation coefficients (r2); Figure S4: Relationship between p-coumaric acid and inflammatory mediators (a) or proinflammatory cytokines (b). Correlations are presented as [PERSON]'s correlation coefficients (r2); Figure S5: Relationship between Rutin and inflammatory mediators (a) or proinflammatory cytokines (b). Correlations are presented as [PERSON]'s correlation coefficients (r2); Table S1: The correlation r2 and p-values between flavonoid compounds and proinflammatory mediators or cytokines. Conceptualization, J.Y.K. ; investigation, K.J.K., J.S.Y., and Y.-J.K.; data curation, K.J.K., J.S.Y., and Y.-J.K.; writing--original draft preparation, K.J.K. and J.Y.K. ; writing--review and editing, K.J.K. and J.Y.K. ; supervision, J.Y.K. ; project administration, J.Y.K. ; funding acquisition, J.Y.K.