## 4 Discussion  
Most chronic diseases are initiated by imbalances in major overarching processes, such as oxidative stress, metabolic stress, psychological stress, and inflammatory stress [22]. Plant food containing various secondary metabolites has been proposed to display anti-inflammatory responses [23]. Therefore, this study investigated the anti-inflammatory effect of various DM extracts according to seasonal variation and different ethanol concentrations as a solvent using the RAW264.7 murine macrophage cell line stimulated with LPS. DM leaves were harvested in May, August, and November, and each leaf sample was extracted using aqueous ethanol solutions (0, 30, and 60%). In the cytotoxicity assay, most extracts displayed cell viability close to that of the control group in 50 \(\upmu\)g/mL, except for a few extracts. The hot water and 30% ethanol extracts harvested in August and all extracts harvested in November showed a higher cell viability than the control group. Previous studies have reported that plant extracts can enhance cell proliferation and increase the cell viability [24]. Likewise, hot water and 30% ethanol extracts harvested in August and all extracts harvested in November could have potential which increases cells. Except for a few extracts, most DM extracts showed lower inflammatory responses in LPS-stimulated RAW264.7 cells relative to LPS-treated cells. Among the extracts, the 30% ethanol extract harvested in May and 60% ethanol extracts harvested in August and November exhibited prominent inhibitory activity. This inhibition was almost close to the normal control cell level. Sometimes, the inflammatory reaction increased at a low concentration (12.5 \(\upmu\)g/mL). Unlike human and in vivo studies, inflammation could be excessively induced in an in vitro study, as plants contain various phytochemicals and interact with each other [25; 26; 27]. Furthermore, this study was primarily based on cell viability data, and anti-inflammatory effects were revealed at a high concentration (50 \(\upmu\)g/mL). Although identifying the principal bioactive components is a theoretically simple process to design, the actual discovery of the active compound may be considerably delayed, given that multiple metabolic pathways are involved in the plant physiology [23]. Therefore, further research should be conducted to determine what causes increased inflammation in a low concentration sample group compared to an LPS-treated group. When inflammation is processed, NF-kB and MAPK pathways regulate the stimulation of macrophages as a trigger of events [17; 28]. LPS-treated macrophages transcribe the NF-kB protein, which acts as a transcription factor by promoting inflammation [29; 30]. In the cytoplasm, NK-kB, which is a heterodimer protein, maintains the complex inactivation state with the inhibitory protein IxB\(\alpha\). However, IxB\(\alpha\) is phosphorylated and ubiquitinated when extracellular stimulation occurs, and NF-kB is translocated into the nucleus [31]. Likewise, MAPKs are activated to phosphorylate downstream subjects, including protein kinases, and transcription factors can transcribe the MAPK-regulated gene [32]. JNK and p38, which are part of the MAPK family and known to be directly involved in the cellular inflammatory response, are also phosphorylated when activated [29; 33]. If proinflammatory cytokines are excessively produced, tissue damage and some proinflammatory responses occur, highlighting the significant role of proinflammatory cytokines as indicators in cell signaling and systematic inflammation [34]. The oxidation of L-arginine was catalyzed by nitric oxide synthase (NOS), releasing unstable and reactive NO. Among the isoforms of NOS, iNOS is activated by inflammatory stimulation and concerned with NO production [35]. A well-known inflammatory response in macrophages starts by combining LPS and TLR, which brings about a cascade of kinases [36]. COX has two isoenzyme forms: COX-1 and COX-2.