The dried fruit of T. terrestris (Fructus Tribuli) was purchased from the Gyeongdong oriental Herbal Store, Seoul, Korea, in March 2012 and was formally identified by Professor Joa Sub Oh (College of Pharmacy, Dankook University, Cheonan, Korea). A voucher specimen (G46) was deposited at the Natural Products Research Laboratory, Gyeonggi Institute of Science and Technology Promotion, Suwon, Korea. The air-dried, crushed fruits of T. terrestris (10 kg) were pulverized and the extract was removed with 80% ethanol (EtOH; 3×18 liters) at room temperature (twice each day for 2 days).

The 80% EtOH extract was filtered and concentrated in vacuo at 40°C to yield 673.5 g of residue, and the residue was then suspended in water and partitioned with hexane (3×1.5 liters) to produce a hexane-soluble layer (40 g). The aqueous layer was partitioned with CHCl₃ to provide a CHCl₃-soluble residue (8.1 g). The CHCl₃ layer was subjected to liquid chromatography [glass column (7×20 cm) packed with silica gel (230–400 mesh)] using CHCl₃:MeOH (100:0, 99:1, 98:2, 97:3, 96:4, 94:6, 92:8, 90:10, 80:20, 70:30, 60:40, 50:50; v/v) gradient mixtures as eluents. The eluent fractions G46-51-(1–13) were obtained from this initial liquid chromatographic separation. The fractions F001-F011 were subjected to an in vitro bioassay to evaluate their NO inhibitory activity. The fraction G46-51-7 exhibited promising inhibitory activity against NO production and was thus selected for further analysis. Column chromatography of the CHCl₃-soluble layer (8.1 g) on a silica gel using MeOH, with increasing polarity, yielded 13 fractions, G46-51-(1–13). Fraction G46-51-7 (2.71 g) was further applied to flash column chromatography on a sephadex LH-20 column using CHCl₃:MeOH (1:1), and 21 fractions were noted: G46-52-(1–21). Of these 21 fractions, CT (97.5 mg) was isolated from fraction G46-52-12, which was precipitated with CHCl₃. ¹H- and ¹³C-NMR spectra were recorded on a Bruker Ascend 700 MHz spectrometer (Bruker, Billerica, MA, USA) using CDCl3 as a solvent. Electrospray ionization (ESI) mass spectra were obtained on an LTQ Orbitrap XL (Thermo Scientific, Bremen, Germany) mass spectrometer.

Amorphous powder; ¹H-NMR (CD₃OD, 700 MHz) δ: 7.40 (1H, d, J=15.4 Hz, H-7′), 7.07 (2H, d, J=8.4 Hz, H-2, 6), 7.01 (1H, d, J=1.4 Hz, H-2′), 6.92 (1H, dd, J=8.4, 2.1 Hz, H-6′), 6.78 (1H, d, J=8.4 Hz, H-5′), 6.74 (2H, d, J=8.4 Hz, H-3, 5), 6.35 (1H, d, J=15.4 Hz, H-8′), 3.47 (1H, t, J=7.0 Hz, H-7), 2.77 (1H, t, J=7.0 Hz, H-8); ¹³C-NMR (CD₃OD, 175 MHz) δ 167.9 (C-9′), 155.5 (C-4), 147.3 (C-4′), 145.3 (C-3′), 140.8 (C-7′), 129.9 (C-1′), 129.3 (C-2, 6), 126.9 (C-1), 120.7 (C-6′), 117.0 (C-8′), 115.0 (C-5′), 114.8 (C-3, 5), 113.6 (C-2′), 41.1 (C-8), 34.4 (C-7); ESI mass spectrometry (ESIMS; negative) m/z 298 [M-H]⁻ (18). The structure of CT is presented in Fig. 1A.

The following pharmacological agents and antibodies were purchased from commercial sources: LPS from Escherichia coli serotype 0111:B4, celecoxib, N^G-monom ethyl-l-arginine (L-NMMA) and dexamethasone (all from Sigma-Aldrich, St. Louis, MO, USA); anti-COX-2 (M-19; sc-1747), anti-β-actin (13E5) and anti-GAPDH antibodies, and goat and mouse IgG-horseradish peroxidase conjugates (all from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA); anti-c-Jun N-terminal protein kinase (JNK; #9251) and anti-phospho-JNK (Thr183/Tyr185) antibodies (both from Cell Signaling Technology, Beverly, MA, USA).

RAW 264.7 murine macrophages (TIB-71) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS; both from Gibco^® Life Technologies, Inc., Grand Island, NY, USA), 100 U/ml penicillin and 0.1 mg/ml streptomycin (both from Gibco^® Life Technologies, Inc.) in a humidified atmosphere of 95% air with 5% CO₂ at 37°C. On day 0, the cells were seeded in 96 well plates. After 24 h, the cells were stimulated with medium (0.5 μg/ml LPS in 10% FBS-DMEM) for 2 h, and then this medium was replaced with maintenance medium (10% FBS-DMEM). The cells were treated with various concentrations of CT (0–50 μM) for 24 h. We then measured the levels of nitrite, a stable metabolite of NO, using Griess reagent (1% sulfanilamide and 0.1% N-(1-naphthyl) ethylenediamine dihydrochloride in 2.5% phosphoric acid; Sigma-Aldrich). Subsequently, the mixture was incubated at room temperature for 10 min, and the absorbance was measured at 540 nm. The quantity of nitrite was determined from a standard curve for sodium nitrite (Sigma-Aldrich).

The 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich) assay was used for the determination of cell viability in vitro in the RAW 264.7 cells. The cells were plated at a density of 4×10⁴ cells/well in 100 μl culture medium. One day after plating, a time zero control plate was made. Following stimulation of the cells with LPS for 2 h, CT was applied directly, and the cells were incubated for 24 h in a humidified atmosphere with 5% CO₂ at 37°C. Cell culture was then performed. MTT (5 mg/ml in PBS) was added to each well, followed by incubation for 90 min. The medium was removed from the wells by aspiration; subsequently, 0.1 ml of buffered dimethyl sulfoxide (DMSO; Sigma-Aldrich) was added to each well, and the plates were shaken. The absorbance was measured on a microtiter plate reader at 540 nm.

ELISA was performed for the determination of the levels of cytokines in vitro in the RAW 264.7 cells. The cells were plated at a density of 4×10⁴ cells/well in 100 μl culture medium. One day after plating, a time zero control plate was made. Following stimulation of the cells with LPS for 2 h, CT was applied directly and the cells were incubated for 24 h in a humidified atmosphere with 5% CO₂ at 37°C. Cell culture was then performed. The supernatants were harvested and assayed for cytokines by ELISA. The concentrations of interleukin (IL)-6, IL-10 and TNF-α in the culture medium were quantified using a platinum ELISA kit (eBioscience, San Diego, CA, USA), and the concentration of PGE₂ in the culture medium was quantified using a competitive enzyme ELISA kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions, respectively.

Total RNA was extracted using a total RNA extraction kit (Ambion, Carlsbad, CA, USA). Five micrograms of RNA were used as a template for each RT-PCR reaction using the SuperScript™ III One-Step RT-PCR system (Invitrogen, Carlsbad, CA, USA). Newly synthesized cDNA from the RAW 264.7 control cells and CT-treated cells was amplified using specific primers and the Accupower^® Pfu PCR PreMix (Bioneer, Daejeon, Korea). The sequences of the primers used for RT-PCR are shown in Table I.

The cells were harvested and washed with PBS and then collected by centrifugation at 13,000 rpm for 1 min at 4°C. To obtain the cell lysate, the cells were lysed on ice for 30 min in RIPA buffer [50 mM Tris-HCl, pH 7.5, 0.15 M NaCl, 1% NP-40, 0.1% sodium dodecyl sulfate (SDS), 1 mM dithiothreitol (DTT) and 1 mM phenylmethanesulfonyl fluoride (PMSF)], which contained protease inhibitors (Roche, Mannheim, Germany). Insoluble materials were removed by centrifugation at 13,000 rpm for 10 min at 4°C. A total of 50 mg of the supernatants was separated using a 10% polyacrylamide gel containing 10% SDS, 1.5 M Tris-HCl, 0.035% N,N,N′,N′-tetramethylenediamine and 7 mg ammonium persulfate. The separated proteins were electrically transferred onto a nitrocellulose membrane (Whatman, Dassel, Germany) at 36 mA in a transfer buffer containing 39 mM glycine, 48 mM Tris base, 0.037% SDS and 20% MeOH. All western blot analyses were performed at least in triplicate, and representative blots are shown.

Data are expressed as the means ± SD. The statistical significance of the experimental results was analyzed (Student's t-test and one-way ANOVA with a subsequent Dunnett's multiple-range test). P-values <0.05 were considered to indicate statistically significant differences.

The chemical structure of CT is illustrated in Fig. 1A. To examine the effects of CT on the inflammatory response, we measured the levels of NO production following treatment of the LPS (0.5 μg/ml)- stimulated RAW 264.7 cells with CT (0, 5, 25 or 50 μM) for 24 h. Treatment with CT induced a marked decrease in NO levels in the LPS-stimulated cells in a dose-dependent manner. Treatment with 50 μM CT induced an 84.07% decrease in NO production. We also confirmed that this result was similar to that achieved by treatment with 100 μM L-NMMA (Fig. 1B), as also previously demonstrated (19). To evaluate the cytotox-icity of CT, we conducted an MTT assay. Treatment with 5, 25 or 50 μM CT did not have a marked cytotoxic effect on the LPS-stimulated RAW 264.7 cells (Fig. 1C).

We investigated the effects of CT on the expression of TNF-α, IL-6 and IL-10, which are pro-inflammatory cytokines, in the LPS-stimulated RAW 264.7 cells. Firstly, we measured the mRNA expression levels of TNF-α, IL-6 and IL-10 by RT-PCR following treatment with 5, 25 or 50 μM CT. We observed that treastment with CT suppressed the mRNA levels of TNF-α, IL-6 and IL-10 in a dose-dependent manner (Fig. 2A). Treatment with dexamethasone (25 μM), which is a potent synthetic member of the glucocorticoid class of steroid drugs, also inhibited the mRNA expression of TNF-α, IL-6 and IL-10 (Fig. 2A). We then confirmed the effects of CT on TNF-α, IL-6 and IL-10 at the protein level by ELISA. The protein levels of TNF-α, IL-6 and IL-10 in the conditioned medium were decreased following treatment with 5, 25 or 50 μM CT. In particular, treatment with 50 μM CT significantly inhibited the release of TNF-α, IL-6 and IL-10 by up to 44.13, 18.38 and 84.99%, respectively (Fig. 2B–D).

To determine the effects of CT on COX-2 expression, we examined whether the expression of COX-2 is reduced at both the mRNA and protein level in LPS-stimulated RAW 264.7 cells following treatment with 5, 25 or 50 μM of CT. As shown in Fig. 3A, CT significantly inhibited COX-2 mRNA expression in a dose-dependent manner. Treatment with 5 μM of celecoxib, a well-known COX-2 inhibitor, significantly inhibited COX-2 expression at the mRNA level. In addition, treatment with 5, 25 or 50 μM CT also resulted in the suppression of COX-2 expression at the protein level in a dose-dependent manner, as evidenced by western blot analysis. Treatment with celecoxib also significantly inhibited COX-2 protein expression (Fig. 3B). Studies have demonstrated that the LPS-induced phosphorylation of MAPKs leads to the production of inflammatory cytokines (20,21). Thus, to determine whether the activation of the MAPK pathway is regulated by CT, we measured the phosphorylation levels of JNK. Treatment with CT (particularly with 50 μM CT) significantly inhibited the LPS-induced phosphorylation of JNK, but did not affect the expression of JNK (Fig. 3C).

To confirm the effects of CT on PGE₂, one of the mediators produced by COX-2, we measured the secretion levels of PGE₂ following treatment of the LPS-stimulated RAW 264.7 cells with CT (5, 25 or 50 μM) and celecoxib (5 μM). The conditioned media were collected and the PGE₂ content was measured by ELISA. As shown in Fig. 4, the levels of PGE₂ in the conditioned media were significantly decreased following treatment with CT (50 μM) and celecoxib (5 μM).