*2.2. Effects of the methanol Extract and Isolated Lignans on LPS-Induced PGE2 and TNF-α Secretion*

Lipopolysaccharide (LPS), known as a major factor involved in inflammation, is a component of the outer membrane of Gram-negative bacteria. Moreover, it is called endotoxin because of its endotoxic properties. LPS contributes to the pathogenicity of bacteria and can activate several immune cells, including macrophages [20]. In this study, LPS was used for inducing inflammation in RAW 264.7 macrophages. Incubation of the cells with LPS (5 μg/mL) for 24 h markedly increased PGE2 and TNF-α secretion (Figure 3). The methanol extract of *M. sirikitiae* leaves was first assessed for the inhibitory effects on LPS-induced PGE2 and TNF-α secretions. Treatment of the cells with the methanol extract of *M. sirikitiae* leaves resulted in a decrease in LPS-induced PGE2 secretion in a dosedependent manner, and the maximal inhibitory effect of the methanol extract was observed at a concentration of 10 μg/mL (Figure 3A). However, the methanol extract exhibited little effect on inhibition of LPS-induced TNF-α secretion at the same concentration and did not inhibit LPS-induced TNF-α secretion at a concentration of 1 μg/mL (Figure 3B). These results indicated that the compounds found in *M. sirikitiae* leaves might have antiinflammatory effects by reducing the secretion of PGE2 in RAW 264.7 macrophages.

**Figure 3.** Effects of the methanol extract of *M. sirikitiae* leaves on LPS-induced PGE2 and TNF-α secretion in RAW 264.7 cells; Serum-starved cells were pretreated with various concentrations of 0, 1, and 10 μg/mL of crude methanol extract for 3 h, and then stimulated with LPS (5 μg/mL) for 24 h at 37 ◦C. The levels of PGE2 and TNF-α secreted into the medium were assessed by ELISA assay. The PGE2 (**A**) and TNF-α (**B**) levels were quantified using a standard curve and expressed as the mean ± SEM (*n* = 3). \*, *p* < 0.05 vs. vehicle; #, *p* < 0.05 vs. LPS.

Lignans are polyphenols, and the main components of *M. sirikitiae* leaves. Our previous study has revealed that lignans (−)-epieudesmin (**1**), (−)-phylligenin (**2**), 2-(3,4 dimethoxyphenyl)-6-(3,5-dimethoxyphenyl)-3,7-dioxabicyclo[3.3.0]octane (**3**), magnone A (**4**), mitrephoran (**5**), and 3 ,4-*O*-dimethylcedrusin (**6**) are the main secondary metabolites in *M. sirikitiae* leaves [10]. As shown in Figure 4A, treatment of the cells with (−)-phylligenin (**2**) (10 μg/mL) significantly inhibited LPS-induced PGE2 secretion. However, (−)-epieudesmin (**1**), 2-(3,4-dimethoxyphenyl)-6-(3,5-dimethoxyphenyl)-3,7 dioxabicyclo[3.3.0]octane (**3**), magnone A (**4**), mitrephoran (**5**), and 3 ,4-*O*-dimethylcedrusin (**6**) had no effect on the inhibition of LPS-induced PGE2 secretion. These results are consistent with previous studies, which reported that phylligenin and koreanaside A, lignans isolated from *Forsythia koreana* fruits and flowers, could inhibit PGE2 secretion in LPS-stimulated RAW 264.7 cells in a dose-dependent manner [15,16]. Moreover, the antiinflammation of koreanaside A was also represented by the inhibitory effects on NO, TNF-α, and IL-6 productions [16].

**Figure 4.** Effects of the methanol extract and isolated lignans **1**–**6** from *M. sirikitiae* leaves on LPSinduced PGE2 and TNF-α secretion in RAW 264.7 cells; Serum-starved cells were pretreated with crude methanol extract (C) (10 μg/mL), lignans (5 or 10 μg/mL), or indomethacin (10 μM) for 3 h, and then stimulated with LPS (5 μg/mL) for 24 h at 37 ◦C. The levels of PGE2 and TNF-α secreted into the medium were assessed by ELISA assay. The relative PGE2 (**A**) and TNF-α (**B**) levels were quantified using a standard curve and expressed as the mean ± SEM (*n* = 3). \*, *p* < 0.05 vs. vehicle; #, *p* < 0.05 vs. LPS.

Interestingly, all lignans isolated from the caulis of *Urceola rosea* exhibited antiinflammatory activities by suppressing LPS-induced synthesis of NO, TNF-α, and/or IL-6 in RAW 264.7 cells. In addition, among those lignans, ecdysanol A and ecdysanol F exhibited potent anti-inflammatory activity against TNF-α secretion with IC50 values of 22.9 and 41.9 μM, respectively [17]. Consistent with this previous study, our results showed that among those six lignans isolated from *M. sirikitiae* leaves, 2-(3,4-dimethoxyphenyl)-6- (3,5-dimethoxyphenyl)-3,7-dioxabicyclo[3.3.0]octane (**3**) and mitrephoran (**5**) (5 μg/mL) significantly inhibited LPS-induced TNF-α secretion in RAW 264.7 cells (Figure 4B).
