Chemicals and reagents
Dulbecco’s modified eagle’s medium (DMEM) was obtained from Welgene (Daegu, Korea). Fetal bovine serum (FBS) was obtained from Corning cellgro (Oneonta, New York, USA). Penicillin–streptomycin and HEPES were obtained from Gibco (Rockville, MD, USA). LPS from Escherichia coli serotype 055:B5 (L4391), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, sulfanilamide, phosphoric acid, naphthylethylenediamine, dihydrochloride, and fisetin were obtained from Sigma-Aldrich (St. Louis, MO, USA).
Preparation of standardized Rhus verniciflua stokes (RVS) extract
The RVS extract standardized with 3 % fustin was kindly provided by Lifetree Biotech Co., Ltd. (Suwon, Korea). Briefly, dried branches of RVS collected from Kangwon Province (Korea) were cut into chips with a chip maker. The chips were added to water and extracted at 90–110 °C for 4 h. The extract was concentrated and treated with alcohol. The alcohol-treated extract was then freeze-dried. Fustin and sulfuretin standards were also provided from Lifetree Biotech Co., Ltd.
High-performance liquid chromatography
Preliminary chemical analysis of RVS extract was carried out using high-performance liquid chromatography (HPLC). The HPLC system consisted of a 1260 infinity HPLC system with a 1260 quaternary pump (Agilent Technologies, Palo Alto, CA). The chromatographic separation of the compounds was achieved using a Capcell Pak C18 (4.6 mm I.D. × 150 mm, 3 µm, Shiseido, Tokyo, Japan) with the column oven temperature maintained at 30 °C. The mobile phase consisted of 0.1 % formic acid (solvent A) and 90 % acetonitrile containing 0.1 % formic acid (solvent B). The mobile phase flow rate was 1 mL/min with gradient elution: 0–1 min, 10 %; 1–15 min, 80 %; 15–16 min, 10 %; 16–25 min, 10 % of solvent B. The injection volume was 10 µL, and the UV detection wavelength was set at 260 nm.
Measurement of nitric oxide, pro-inflammatory cytokines, and prostaglandin E2 production
RAW264.7 cells (Korean Cell Line Bank, Seoul, Korea) maintained in DMEM medium containing 10 % FBS, 2 % penicillin–streptomycin, and 2 % HEPES at 37 °C in a humidified incubator (5 % CO2 and 95 % air). Cells were incubated with a pre-treated different sample for 24 h and then stimulated with LPS (2 µg/mL) for 48 h. Nitric oxide (NO) production was quantified using the Griess reagent (0.1 % naphthylenediamine and 1 % sulfanilamide in 5 % phosphoric acid). Interleukin-6 (IL-6, USCN Life Science Inc., Wuhan, Hubei, China) and prostaglandin E2 (PGE2, Abcam, Cambridge, Massachusetts, USA) were analyzed using ELISA Kits as described in the supplier manuals.
Measurement of intracellular reactive oxygen species (ROS)
After culture and stimulation, cells were washed with DPBS and incubated for 30 min with dichlorofluorescein diacetate (DCF-DA) dissolved in DMSO (final concentration at 50 µM). Fluorescence was measured using a fluorescent microscope (Motic, Richmond, Canada) and spectrophotometer (PerkinElmer Inc., Waltham, MA, USA) at 480 nm excitation and 530 nm emission.
Results were expressed as the mean ± standard deviation (SD). Comparisons between groups were performed using one-way analysis of variance (ANOVA), followed by Duncan’s multiple range test. A value of p < 0.05 was considered statistically significant.