*2.1. Effects of the Methanol Extract and Isolated Lignans on Cytotoxicity*

Cytotoxicity of the methanol extract and isolated lignans from *M. sirikitiae* leaves, including (−)-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**), 3 ,4-*O*-dimethylcedrusin (**6**) (Figure 1), was evaluated in RAW 264.7 cells using MTT colorimetric assay in order to assign the optimal concentrations of the samples before investigating anti-inflammatory activities in the cells.

**Figure 1.** Isolated lignans **1**–**6** from the methanol extract of *M. sirikitiae* leaves.

After treatment of the cells with various concentrations of the samples for 24 h, the number of viable cells was measured and calculated as the percentage of viable cells compared to a nontreated (control) group. As shown in Figure 2, the MTT results revealed that the number of survival cells more than 80% were found in the groups treated with 0.05–10 μg/mL of the methanol extract, lignans **1**, **2**, or **6**, and 0.05–5 μg/mL of lignans **3**, **4**, or **5**. In order to avoid the cytotoxic effect and allow at least 80% cell viability, the suitable

sample concentrations that were used for determining anti-inflammatory effects were 10 μg/mL for the methanol extract, lignans **1**, **2**, and **6**, and 5 μg/mL for lignans **3**, **4**, and **5**.

**Figure 2.** Cytotoxic effects of the methanol extract and isolated lignans **1**–**6** from *M. sirikitiae* leaves in RAW 264.7 cells; The percentage of cell viability of RAW 264.7 cells exposed to 0.05–50 μg/mL of methanol extract and 0.1–250 μg/mL of isolated lignans **1**–**6**. Data are shown as the mean ± SEM (*n* = 5).
