Dimethyl sulfoxide (DMSO), RIPA buffer, and phosphate-buffered saline (PBS) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were obtained from Invitrogen-Gibco (Grand Island, NY, USA). ELISA kits for PGE₂, IL-1β, and IL-6 were purchased from R&D Systems, Inc. (St. Louis, MO, USA) and BD Biosciences (San Diego, CA, USA). Anti-phosphorylated IκB-α (anti-p-IκB-α), anti-NF-κB, anti-JNK, anti-phosphorylated JNK (anti-p-JNK), anti-ERK1/2, anti-phosphorylated ERK1/2 (anti-p-ERK1/2), anti-p38, and anti-phosphorylated p38 (anti-p-p38) mouse or rabbit antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). All other reagents were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA).

T. tetragonoides were collected from Jeju Island in the Republic of Korea. The samples were washed with water to remove salt and sand, rinsed carefully in fresh water, and then dried at room temperature for 1 week. Dried T. tetragonoides (2 kg) were soaked in water (80 L) and then distilled at 121 °C for 4 h using a high-speed, low-temperature vacuum extractor (DM-3000, Daehan median co., LTD, Seoul, Korea). The primary oil decanted from distillate (hydrosols) was filtered to remove particles and stored in a sealed vial at 4 °C until further testing.

Composition analysis of volatile oil was performed by gas chromatography-mass spectrometry (GC-MS) using Shimadzu GCMS-TQ8040 (Shimadzu Corporation, Japan) on a Rxi-5MS capillary column (30 m × 0.25 mm id, film thickness 0.25 μm; Shimadzu, Japan). The carrier gas was helium (flow rate of 1.36 ml/min). GC-MS was carried out using the following temperature program: initial temperature was set at 180 °C and held for 3 min, followed by 6 °C/min ramp to 240 °C and hold for 3 min, followed by 3 °C/min ramp to 275 °C and hold for 3 min, followed by 5 °C/min ramp to 300 °C and hold for 3 min, followed by 5 °C/min ramp to a final temperature of 330 °C and hold for 5 min. The injection temperature was set at 290 °C and the injection volume was 1 μl (splitless mode). Detector parameters used for GC-MS analyses were as follows: interface temperature, 320 °C; ion source temperature, 300 °C; mass spectrometry was performed using Q3 scan with an m/z 45-500 scanning range. Chromatograms and mass spectra were evaluated using the GCMS solution software (Shimadzu Corporation, Japan).

Raw 264.7 murine macrophage cells were purchased from the Korean Cell Line Bank (KCLB; Seoul, Korea). The cells were cultured in DMEM supplemented with antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin) and 10 % FBS at 37 °C in a 5 % CO₂ atmosphere.

RAW 264.7 cells (2 × 10⁵ Cells/well) were plated in 96-well plates and incubated overnight, and subsequently treated with LPS (1 μg/ml) in the absence or presence of various concentrations (0.5 %-5 %) of TTH at 37 °C for 24 h. After incubation, MTT solution (5 mg/ml) was added to each well and plates were incubated for 4 h in a CO₂ incubator at 37 °C. Subsequently, supernatant was removed and DMSO was added to dissolve formazan crystals. The absorbance was measured at 540 nm.

RAW 264.7 cells (2 × 10⁵ Cells/well) were plated in 24-well plates and subsequently treated with LPS (1 μg/ml) in the absence or presence of various concentrations (0.5 %-5 %) of TTH at 37 °C for 24 h. Culture supernatants were mixed with Griess reagent (1:1 mixture of 1 % sulfanilamide and 0.1 % naphthylethylenediamine dihydrochloride in 2.5 % phosphoric acid) and incubated at room temperature for 10 min. Subsequently, the NO concentration in supernatant was determined by measuring absorbance at 540 nm.

RAW 264.7 cells (2 × 10⁵ Cells/well) were plate in 24-well plates and subsequently treated with LPS (1 μg/ml) in the absence or presence of various concentrations (0.5 %-5 %) of TTH at 37 °C for 24 h. Culture supernatants were collected for determination of IL-1β, IL-6, and PGE₂ concentrations by ELISA according to the manufacturer's instructions.

RAW 264.7 macrophages were cultured at 2 × 10⁵ cells per 35 mm dish and incubated at 37 °C for 24 h. The cells were then treated with different concentrations of TTH and incubated for 24 h. The cells were lysed using RIPA lysis buffer. Protein concentrations were determined using a Bio-Rad protein assay kit; bovine serum albumin (BSA) was used as the calibration standard. Cell lysates were electrophoresed on SDS polyacrylamide gels (8-12 %) and the separated proteins were transferred to PVDF membranes. The membranes were incubated in the blocking solution (Tris-buffer /Tween 20, TBST) containing 3 % BSA (w/v) for 2 h under gentle shaking at room temperature. Subsequently, the membranes were incubated with primary antibodies (iNOS, COX-2, p-p65, p-p105, p-IκB-α, p65, p105, IκB, p-ERK, ERK, p-JNK and JNK diluted 1:1000 in 3 % BSA in TBST) for 24 h at 4 °C. After incubation, the membranes were washed three times with TBST buffer at room temperature, and then incubated with secondary antibodies (1:3000) in 3 % BSA in TBST for 2 h at room temperature. Signals were developed using ECL Western blotting detection kit and visualized on a Bio-Rad ChemiDoc system. The density of the band was quantified using ImageJ software (National Institutes of Health).

Cells were fixed in 4 % formaldehyde for 15 min at room temperature and permeated with methanol at −20 °C for 10 min. Cells were then blocked for 1 h with 1 % BSA in PBS and permeabilized by incubation with 0.4 % Triton X-100 for 30 min. Subsequently, cells were washed with PBS and incubated with anti-p65 primary antibody (1:100) at 4 °C overnight. After washing with PBS, cells were incubated with a secondary antibody (1:100) at 4 °C for 1.5 h. Cells were then washed with PBS and incubated with Alexa Fluor 488 goat anti-rabbit antibody (1:800) for 1.5 h at RT in the dark. After washing with PBS, slides were mounted and counter-stained using Vectashield mounting medium containing DAPI (Vector Laboratories, Burlingame, CA, USA). Slides were visualized under a laser scanning microscope (LSM 700; ZEISS, Jena, Germany).

All data are expressed as means ± S.D. Significant differences among the groups were determined using the unpaired Student's t-test. A value of *p < 0.05 was considered to be statistically significant.

The chemical profile, percentage content, and retention indices of the constituents of TTH are summarized in Table 1(Tab. 1). The following six compounds were identified in TTH, representing 86.4 % of hydrosols: tetratetracontane (29.5 %, nonacosane (27.6 %), oleamide (17.1 %), octacosane (7.6 %), 2,6,10,15-tetramethyl-heptadecane (2.6 %), and 2-methyleicosane (2.0 %).

We first treated the cells with TTH and measured the cytotoxicity using MTT assay to determine the non-cytotoxic concentration (Figure 1A(Fig. 1)). In all subsequent experiments, TTH was used at concentrations ranging from 0.5 % to 5 %. To determine whether TTH inhibited the production of LPS-induced NO and PGE₂, which play a central role in the inflammatory response, RAW 264.7 cells were pretreated with TTH for 1 h and then stimulated with LPS. Compared to that in unstimulated cells, NO and PGE₂ production was markedly induced in LPS-stimulated cells. TTH significantly inhibited the production of LPS-induced NO and PGE₂ in a concentration-dependent manner (Figure 1(Fig. 1)).

RAW 264.7 cells were treated with TTH to study the expression of inflammation-related proteins induced by LPS treatment. The LPS-stimulated RAW 264.7 cells showed significant iNOS and COX-2 protein expression. However, treatment with TTH dose-dependently inhibited iNOS and COX-2 protein expression in LPS-stimulated RAW 264.7 cells (Figure 2(Fig. 2)).

To further investigate the anti-inflammatory effect of TTH on LPS-stimulated macrophages, the production of pro-inflammatory cytokines was evaluated by ELISA. The treatment of RAW 264.7 cells with LPS significantly increased IL-6 and IL-1β production compared to that in the untreated (control) cells. However, TTH treatment significantly and dose-dependently decreased the levels of IL-1β and IL-6 compared to that reported with the LPS treatment (Figure 3(Fig. 3)).

Nuclear translocation of NF-κB is directly linked to IκB-α degradation and phosphorylation and is initiated by the phosphorylation, ubiquitination, and proteolytic degradation of IκB-α (Ghosh and Karin, 2002[11]). Therefore, the levels of phosphorylated IκB-α were examined by Western blot analysis. As shown in Figure 4A(Fig. 4), unstimuated RAW 264.7 cells did not show detectable form of phosphorylated IκB-α, whereas addition of LPS resulted in phosphorylation and degradation of IκB-α. However, pre-treatment with TTH markedly inhibited the LPS-induced phosphorylation and degradation of IκB-α. For further confirmation, the nuclear translocation of NF-κB/p65 was visualized under a laser confocal microscope. The LPS-stimulated RAW 264.7 cells showed a dramatic increase in the translocation of NF-κB into the nucleus. The LPS-stimulated NF-κB nuclear translocation was markedly suppressed after TTH pre-treatment (Figure 4B(Fig. 4)). These results suggest that TTH inhibits IκB-α degradation and prevents NF-κB translocation into the nucleus.

MAPKs are associated with the production of inflammatory mediators in marcrophages (Uto et al., 2005[29]). Therefore, western blot analysis was performed to determine whether TTH mediates its effect through the MAPK pathway. The LPS-induced phosphorylation of p38, ERK1/2, and JNK was evaluated in RAW 264.7 cells. As shown in Figure 4C(Fig. 4), LPS stimulation significantly increased the phosphorylation of MAPK, whereas TTH treatment markedly inhibited the expression of phosphorylated MAPK in LPS-stimulated cells (Figure 4C(Fig. 4)).