## 3 Results  
### Inhibitory Effect of DM Extracts on Nitrite and PGE\({}_{2}\) Production  
Before the quantification of proinflammatory compounds, the cytotoxicity of RAW264.7 cells treated with various DM extracts was tested (Figure 2). When DM extracts were treated at concentrations of 50, 100, 250, and 500 \(\upmu\)g/mL for all extracts, cytotoxicity was not observed in 50 \(\upmu\)g/mL of all extracts. The DM extracts harvested in May showed approximately 98% cell viability for all extracts. In DM extract groups harvested in August, the cell viability was high, in the order of the 30% ethanol extract > hot water extract >60% ethanol extract. The DM extracts harvested in November showed a higher cell viability in hot water and the 60% ethanol extract than in the 30% ethanol extract. Therefore, all DM extracts of 50 \(\upmu\)g/mLwere considered non-toxic, like the control group. Based on the cytotoxicity test results, optimal and physiological attainable concentrations of DM extracts (12.5, 25, and 50 \(\upmu\)g/mL) were selected for further experiments. To confirm the anti-inflammatory effects, nitrite and PGE\({}_{2}\) levels in RAW264.7 cells treated with various DM extracts were quantified. The DM extracts expectedly decreased the nitrite level in 50 \(\upmu\)g/mL of extracts, regardless of solvents and seasons, except for hot water extracts obtained in May. Sometimes, a nitrite increase was observed in 12.5 \(\upmu\)g/mL of the hot water extract harvested in November compared to the LPS-treated group (Figure 3a). The 30% ethanol extract harvested in May and August and the 60% ethanol extract harvested in August and November displayed dose-dependent inhibitory effects. Similarly, the PGE\({}_{2}\) level also declined in all 50 \(\upmu\)g/mL extracts except for the 30% ethanol extract harvested in August. Sometimes, 12.5 or/and 25 \(\upmu\)g/mL of 30% ethanol extracts harvested in all seasons exhibited increased PGE\({}_{2}\) production compared to the LPS-treated group (Figure 3b). Of note, all extracts obtained with hot water and 60% ethanol harvested in all seasons significantly inhibited inflammatory responses. Compared to control cells, 60% ethanol extracts harvested in August and November decreased both nitrite and PGE\({}_{2}\) levels to normal control levels. When the concentration of the sample was set at 50 \(\upmu\)g/mL, 60% ethanol extracts harvested in August and November showed the most prominent inhibitory activity. To prove a decreasing trend for nitrite and PGE\({}_{2}\) levels, the mRNA expression of downstream elements was also quantified. When iNOS and COX-2 mRNA levels were analyzed in RAW264.7 cells treated with LPS and each extract at 50 \(\upmu\)g/mL, the treated cells displayed significantly decreased mRNA expression levels of iNOS and COX-2 (\(p<0.05\) for all treated samples; Figure 3c,d). In a group treated with extracts harvested in May, the mRNA expression levels of iNOS and COX-2 were lower in the 30% ethanol extract group than in the 60% ethanol extract group. All DM extracts inhibited the mRNA levels of iNOS and COX-2 compared with the LPS-treated group. Among various extracts, 60% ethanol extracts harvested in August and November decreased the values to those of normal control cells and showed the most prominent decreasing capability. Figure 2: Cell viability of RAW264.7 macrophage cells treated with _Dendropanax morphifera_ extracts. W100, hot water extracts; E30, 30% ethanol extracts; and E60, 60% ethanol extracts. Data are presented as the mean \(\pm\) SEs of triplicate determinations of independent wells. Figure 3: Effects on the production of nitrite (**a**), prostaglandin E\({}_{2}\) (**b**), mRNA expression levels of inducible nitric oxide synthase (**c**), and cyclooxygenase-2 (**d**) in the RAW264.7 cells stimulated with lipopolysaccharideacharide. W100, hot water extracts; E30, 30% ethanol extracts; and E60, 60% ethanol extracts. Data are presented as the mean \(\pm\) SEs of triplicate determinations of independent wells.