The 13 herbs that form GHJGS were purchased from Kwangmyungdang Medicinal Herbs (Ulsan, South Korea). The taxonomic classification of the 13 herbs was verified by Professor Je-Hyun Lee from Dongguk University (Gyeongju, South Korea). Voucher specimens (2012-KE32-1 to KE32-13) were deposited at the K-herb Research Center, Korea Institute of Oriental Medicine (Daejeon, South Korea).

Liquiritin, glycyrrhizin and 6-gingerol were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Hesperidin and rosmarinic acid were purchased from Acros Organics (Morris, NJ, USA) and Sigma-Aldrich (St. Louis, MO, USA), respectively. The purity of each component was determined to be ≥98% using high-performance liquid chromatography (HPLC) analysis. The chemical structures of the five marker compounds are presented in Fig. 1A. HPLC-grade reagents, methanol, acetonitrile and distilled water were obtained from J.T. Baker; Avanto Performance Materials (Phillipsburg, NJ, USA). Acetic acid was obtained from Merck KGaA (Darmstadt, Germany).

GHJGS was composed of 13 herbs (Table I; total weight, 5.0 kg, ~148.15 times the composition of a single dose) and extracted in distilled water at 100°C for 2 h under 98 kPa pressure using a COSMOS-660 electric extractor (KyungSeo Machine Co., Incheon, South Korea). The extracted solution was filtered using a standard sieve (no. 270; mesh size, 53 µm; Chung Gye Sang Gong Sa, Seoul, Korea) and freeze-dried. The yield of the extract was 12.89% (644.5 g). The lyophilized GHJGS extract (40 mg) was dissolved in 50% methanol (20 ml) and mixed for quantitative analysis. The solution was filtered through a 0.2-µm SmartPor GHP syringe filter (Woong Ki Science Co., Ltd., Seoul, South Korea) prior to being injected into a HPLC column.

The quantitative determination was performed using a Prominence LC-20A series HPLC system (Shimadzu Corporation, Kyoto, Japan) consisting of a solvent delivery unit (LC-20AT), online degasser (DGU-20A3), column oven (CTO-20A), auto sample injector (SIL-20AC), and photodiode array (PDA) detector (SPD-M20A). Data were collected and processed using LC solution software (version 1.24; Shimadzu Corporation). A Gemini C18 column (250 mm × 4.6 mm; particle size, 5 µm; Phenomenex, Inc., Torrance, CA, USA) was used for separation of the marker compounds and maintained at 40°C. The mobile phases consisted of 1.0% (v/v) acetic acid in distilled water (designated as A) and 1.0% (v/v) acetic acid in acetonitrile (designated as B). The gradient flow was as follows: 5–70% B for 0–40 min; 70–100% B for 40–50 min; 100% B for 50–55 min; and 100-5% B for 55–60 min. The analysis was conducted at 1.0 ml/min with PDA detection at 254 nm (glycyrrhizin), 280 nm (liquiritin, hesperidin and 6-gingerol), and 330 nm (rosmarinic acid). The sample injection volume was 10 µl.

The murine macrophage cell line, RAW 264.7, was obtained from the American Type Culture Collection (Rockville, MD, USA). The cells were cultured in Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 5.5% heat-inactivated fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.), penicillin (100 U/ml; HyClone Laboratories, Inc., Logan, UT, USA), and streptomycin (100 µg/ml; HyClone Laboratories, Inc.) in an incubator with 5% CO₂ at 37°C.

Cell viability assay was performed to determine the cytotoxicity of GHJGS using a Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan). Cells were plated onto a 96-well microplate at 3×10³ cells/well and treated with 0, 15.625, 31.25, 62.5, 125, 250, 500 or 1,000 µg/ml GHJGS for 24 h. Following incubation with CCK-8 reagent for 4 h, optical density (OD) at a wavelength of 450 nm was determined using a Benchmark plus microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Cell viability was calculated using the following equation: Cell viability (%) = mean OD_{GHGJS-treated cells} / mean OD_{untreated cells} ×100

Cells were pretreated with 0, 250, 500 or 1,000 µg/ml GHJGS for 4 h and stimulated with LPS (1 µg/ml) for an additional 20 h. Production of TNF-α, IL-6 and PGE₂ in the culture supernatants was measured using commercial ELISA kits from R&D Systems, Inc. (Minneapolis, MN, USA), BD Biosciences (San Jose, CA, USA) and Cayman Chemical Company (Ann Arbor, MI, USA), respectively. Indomethacin (2.5 ng/ml; Sigma-Aldrich) was used as a positive control.

Whole cell extract (WCE) was prepared by suspending cells using the Mammalian Cell Lysis kit (Sigma-Aldrich) containing protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN, USA). Nuclear extract (NE) was isolated using NE-PER^® Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, Inc., Rockford, IL, USA) according to the manufacturer's protocol. The protein concentration was determined using the Bio-Rad Protein Assay kit II (Bio-Rad Laboratories, Inc.). Equal quantities of cell extract (30 µg) were resolved by 4–20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Bio-Rad Laboratories, Inc.) at 100 v for 1 h and transferred to a polyvinylidene fluoride membrane (GE Healthcare Life Sciences, Piscataway, NJ, USA). The membrane was incubated with blocking solution [5% skimmed milk in Tris-buffered saline containing Tween-20 (TBST); DyneBio, Seongnam, Korea], followed by an overnight incubation at 4°C with the appropriate primary antibody, including rabbit polyclonal phosphorylated (p)-p38 MAPK (1:1,000 dilution; cat. no. 9211; Cell Signaling Technology, Inc., Danvers, MA, USA), rabbit polyclonal p-extracellular signal-regulated kinase (ERK; 1:1,000 dilution; cat. no. 9101; Cell Signaling Technology, Inc.), rabbit polyclonal p-c-Jun N-terminal kinase (JNK; 1:1,000 dilution; cat. no. 9251; Cell Signaling Technology, Inc.), mouse monoclonal HO-1 (1:1,000 dilution; cat. no. ab13248; Abcam, Boston, MA, USA), rabbit polyclonal NF-κB p65 (1:1,000; cat. no. sc-372; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and mouse monoclonal β-actin (1:5,000; cat. no. sc-47778; Santa Cruz Biotechnology, Inc.). The membranes were washed three times with TBST, and then incubated with polyclonal horseradish peroxidase-conjugated goat anti-mouse IgG (cat. no. 115-001-003; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA; 1:2,000 dilution) and goat anti-rabbit IgG (cat. no. 111-001-003; Jackson ImmunoResearch Laboratories, Inc.; 1:2,000 dilution) secondary antibodies for 1 h at room temperature. The membranes were washed three times with TBST, and developed using the SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Inc.). Image capture was performed using Chemi-Doc (Bio-Rad Laboratories, Inc.).

To examine the generation of ROS, the ROS-ID™ Total ROS Detection kit (Enzo Life Sciences, Inc., Plymouth Meeting, PA, USA) was used. The effect of GHJGS on ROS generation was examined by immunofluorescence staining. Cells were plated on µ-Dishes 35 mm (Ibidi, Aarhus, Denmark), treated with GHJGS (1,000 µg/ml) and LPS (1 µg/ml) for 30 min, and fixed in 4% paraformaldehyde (Sigma-Aldrich) and 100% acetone (Sigma-Aldrich). The ROS detection solution was loaded to the cells and incubated at room temperature for 1 h. Following the addition of a mounting medium (Vector Laboratories Inc., Burlingame, CA, USA), the stained cells were visualized using a FLUOVIEW FV10i confocal microscope (Olympus Corporation, Tokyo, Japan).

The data are expressed as the mean ± standard error of the mean. Data were analyzed using one-way analysis of variance and Dunnett's multiple comparisons test. P<0.05 was considered to indicate a statistically significant difference.

The novel HPLC-PDA method was applied for simultaneous quantification of the five marker compounds in GHJGS. The typical chromatogram patterns for standard compounds and the GHJGS decoction are presented in Fig. 1B and C. The retention times of the liquiritin, hesperidin, rosmarinic acid, glycyrrhizin and 6-gingerol were ~ 17.35, 19.35, 20.66, 33.39, and 33.88 min, respectively. The concentrations of the five components were between 1.18 and 3.16 mg/g (Table II).

Cytotoxicity of GHJGS was evaluated using RAW 264.7 cells. Cells were treated with serial dilutions of GHJGS for 24 h. Fig. 2 demonstrates that no cytotoxic effect was observed up to 1,000 µg/ml GHJGS treatment. For the subsequent assays, cell treatment with GHJGS was performed in the nontoxic concentration range (250-1,000 µg/ml).

To examine the anti-inflammatory effect of GHJGS, production of TNF-α and IL-6 was assessed in LPS-stimulated RAW 264.7 cells. LPS stimulation significantly increased levels of TNF-α and IL-6 in RAW 264.7 cells, compared with untreated controls. By contrast, GHJGS treatment significantly reduced LPS-induced production of TNF-α and IL-6 (P<0.01; Fig. 3A and B). The quantity of PGE₂ was also determined and indomethacin served as a positive control. The level of PGE₂ was significantly increased in cells treated with LPS alone. By contrast, GHJGS treatment significantly reduced PGE₂ production by LPS stimulation (P<0.01; Fig. 3C).

As demonstrated by Fig. 4A, LPS markedly enhanced phosphorylation of p38 MAPK, ERK and JNK in RAW 264.7 cells, compared with untreated controls. LPS-induced activation of MAPKs was inhibited when cells were pretreated with GHJGS. NF-κB p65 expression in the nucleus of RAW 264.7 cells was also analyzed. LPS increased the expression level of NF-κB p65, compared with that of untreated controls. When cells were exposed to LPS and GHJGS, the GHJGS exerted no influence on NF-κB p65 in the LPS-stimulated RAW 264.7 cells (Fig. 4B).

Cells distinctly stained with green fluorescence were observed in LPS-stimulated cells compared with the undifferentiated control (Fig. 5A). By contrast, GHJGS treatment blocked LPS-mediated ROS generation (Fig. 5A). Additionally, GHJGS induced HO-1 expression in a dose-dependent manner (Fig. 5B).