## 2 Materials and Methods
### Determination of Wedelolactone Contents in the Extracts  
To measure the contents of wedelolactone in the extracts from two different extraction processes, high performance liquid chromatography (HPLC, 1260 series, Agilent, Santa Clara, CA, USA) was used with a C\({}_{18}\) column (250 mm \(\times\) 4.6 mm, Jupiter 5u 3000A, Santa Clara, CA, USA) under the following isocratic conditions [18; 19]: The mobile phase was water:acetic acid:methanol (4.5:0.5:95) at a flow rate of 0.8 mL/min measured at a wavelength of 352 nm with the injection of 10 \(\upmu\)L of 1000 ppm of wedelolactone as the standard (Sigma-Aldrich, St. Louis, MO, USA).  
Estimation of the Production of Interleukin-6(IL-6), Tumor Necrosis Factor-\(\alpha\)(TNF-\(a\)), and Prostaglandin\(E_{2}\) (PGE\({}_{2}\))  
The amounts of two different cytokines, IL-6 and TNF-\(\alpha\), produced from a mouse macrophage (RAW 264.7m KCLB, Seoul, Korea) were measured with Quantikine ELISA Mouse IL-6 and TNF-\(\alpha\) kits (R&D Systems, Minneapolis, MN, USA) as follows [20]: 5.0 \(\times\) 10\({}^{5}\) cell/mL of RAW 264.7 cells were inoculated into a 96-well plate and cultured for one day at 37 \({}^{\circ}\)C with 5% CO\({}_{2}\). Then, various concentrations of the extracts, 1 \(\upmu\)M of vitamin D\({}_{3}\) (cholecalciferol) dissolved in 0.1% ethanol and 1.5 \(\upmu\)g/mL of wedelolactone as positive controls, respectively, were added with 25 \(\upmu\)g/mL of lipopolysaccharide for one more day. Thereafter, according to the manuals of the ELISA kits, 50 \(\upmu\)L of the assay diluent was put into the 96-well plate, which was appropriately shaken. The standard prepared in the kit and the sample that had been cultured thus far were then put into the 96-well plate, which was shaken for one week and left unattended for 2 h at room temperature while light was blocked. Thereafter, the plate was washed with a wash buffer, and 100 \(\upmu\)L of the substrate solution was added into the plate for 30 min at room temperature while light was blocked. After blocking the reaction by a stop solution, the concentrations in the plate were measured at a wavelength of 450 nm by an ELISA reader (Thermo Fisher Scientific, Waltham, MA, USA).  
The inhibition of the production of PGE\({}_{2}\) from human skin fibroblasts (CCD-986 sk, KCLB, Seoul, Korea) was measured by treating the extracts as follows [21]: 4.0 \(\times\) 10\({}^{5}\) cell/mL of human skin fibroblasts was inoculated into a 96-well plate for one day at 37 \({}^{\circ}\)C, treated with 150 \(\upmu\)L of each extract at various concentrations, 1 \(\upmu\)M of vitamin D\({}_{3}\) (cholecalciferol) dissolved in 0.1% ethanol and 1.5 \(\upmu\)g/mL of wedelolactone as positive controls, respectively, and again cultured for one more day. Thereafter, 50 \(\upmu\)L of the primary antibody solution was added into the plate, which was shaken at 100 rpm and left unattended for 1 h at room temperature. Then, the PGE\({}_{2}\) conjugate was added into the plate and left for 2 h at room temperature. The plate was washed four times using a wash buffer, and 200 \(\upmu\)L each of the substrate solutions was added to the plate for 30 min in a dark room. Thereafter, 100 \(\upmu\)L of a stop solution was added into the plate, and the amounts of PGE\({}_{2}\) were measured at a wavelength of 450 nm by comparing with the standard curve.