*3.6. Measurement of Nitric Oxide Production*

Nitrate and nitrite concentrations were assayed by Griess reagent using a nitric oxide assay kit as described previously [31]. RAW 264.7 cells (2 × <sup>10</sup><sup>5</sup> cells/well) were plated in 6-well culture plates overnight. Under the serum-free condition, cells were pretreated with the methanol extract (1, 10 μg/mL), or isolated lignans (5, 10 μg/mL) for 3 h and subsequently treated with LPS (5 μg/mL) for 24 h. The absorbance was measured at a wavelength of 540 nm using the Infinite M200 microplate reader (TECAN). Nitrate and nitrite concentrations in the samples were calculated from the standard curve following the manufacturer's instructions. The amounts of nitrate and nitrite accurately reflect the NO production in RAW 264.7 cells.

#### *3.7. Measurement of mRNA Expression of Inflammatory Biomarkers*

The effects of the methanol extract and the isolated lignans on the synthesis of inflammatory biomarkers were determined by measurement of mRNA expression. Under serumfree condition, RAW 264.7 cells (2 × <sup>10</sup><sup>5</sup> cells/well) were treated with the methanol extract (1, 10 μg/mL), or isolated lignans (5, 10 μg/mL) for 6 h. The total mRNA of RAW 264.7 cells was isolated with RNeasy kit (Qiagen). The mRNA expression of inflammatory biomarkers was determined by using the KAPA SYBR FAST One-step RT-qPCR kit with gene-specific primer sequences (Table 1) and Mx 3005p Real-Time PCR system (Stratagene) as previously described [32]. The mRNA expression level of inflammatory biomarkers was calculated by the comparative cycle threshold (CT) method. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the housekeeping gene.


**Table 1.** The gene-specific primers for RT-qPCR (mouse).
