The prescription of DGHT was based on Donguibogam [2] and is shown in Table 1. The materials of the prescription used in this study were purchased from Omniherb Company (Gyeong-buk, Korea), and the hot aqueous extraction method is as follow. The materials of DGHT 192g ware boiled in 2L of distilled water for 4 hours. The extract was filtered and centrifuged (Hanil, Korea) at 3,000 RPM for 3 minutes. The supernatant was secondary filtered with a 0.03 mm filter (Nalgene, New York, USA) and concentrated to 100mL using a rotary evaporator (Korprotech, Korea) and frozen at -80°C. The concentrated extract was lyophilized for 7 days using a freeze dryer system (Labconco, USA) to obtain 38.42g of powdered DGHT extract (Fig. 1).

RAW 264.7 macrophages were obtained from the Korean Cell Line Bank (Seoul, Korea) and used for experiments. The RAW 264.7 macrophage cell line was cultured in DMEM containing 10,000 units/mL penicillin and 10,000 μg/mL streptomycin in a 37°C incubator (Sanyo, Japan). 1μg of LPS was dissolved in 1mL of 1 x PBS 1mL and filtered for treatment. In each experiment, the cells were cultured for 24 hours, and incubated for 1 hour, and then treated with LPS at a concentration of 1μg/mL.

Cell viability was evaluated using the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The stabilized cells were treated with 10, 50, 100, 200 and 400 μg/mL of DGHT extract, cultured for 24 hours, andthen MTT sample (Sigma, USA) was added and the cells were incubated for 4 hours. After removing the medium, 200 μg DMSO (Sigma, USA) was mixed and the absorbance was measured at 570 nm using a microplate reader (Labsystems, Finland).

The concentration of NO was measured using Griess reagent composed of 1% sulfanilamide, 0.1% naphthylethylene diamine dihydrochloride and 2% phosphoric acid. The stabilized RAW 264.7 macrophages were treated with 1 μg/mL LPS and DGHT extracts at 10, 50 100 and 200 μg/mL, respectively, and cultured for 18 hours. Cultured supernatant 100 μg and Griess reagent 100 μg were mixed and measured at 540 nm using a microplate reader.

The concentration of PGE₂ was measured using a commercial competitive enzyme immunoassay kit (R&D, USA). RAW 264.7 macrophages were treated with 1 μg/mL LPS and DGHT extracts at 10, 50, 100 and 200 μg/mL, respectively, and cultured for 18 hours. Cultured supernatant 100 μg was loaded on a 96-well plate coated with goat anti-mouse, primary antibody solution and PGE₂ conjugate 50 μg were added and incubated overnight at 4°C. A 200 μg substrate solution was added and left to react for 15 minutes, treated with 50μg stop solution, and the absorbance was measured at 450nm using a microplate reader.

ELISA kit (R&D, USA) was used to measure the amounts of IL-6, IL-1ß and TNF-α. RAW 264.7 macrophages were treated for 1 hour with 10, 50, 100 and 200 μg/mL DGHT, respectively, and then treated with 1 μg/mL of LPS for 18 hours. Treated solutions were loaded on a 96-well plate coated with capture antibody for IL-6, IL-1β, and TNF-α, and incubated overnight at 4°C. After washing 3 times with washing buffer, antibody reagent 100 μg was added to each well and reacted at room temperature for 1 hour. Then, it was washed 3 times again, tetramethylbenzidine (TMB) substrate 100 μg was added for 30 minutes, and stop solution 100 μg was added to stop the reaction. The absorbance was measured at 450 nm using a microplate reader.

DGHT extracts were diluted with methanol (Honeywell, USA) using 10, 50, 100 and 200 μg, mixed with 80 μg of 0.15mM DPPH solution (Sigma, USA) and 96-well plate. After blocking the light at room temperature and reacting for 30 minutes, the absorbance was measured at 520 nm using a microplate reader. DPPH radical scavenging activity was calculated by subtracting the control group from the treated group, dividing by the control group, and then multiplying by 100.

Statistical analyses were performed with IBM SPSS program Ver. 22.0 (IBM Corp., USA). It was performed using Kolmogorov-Smirnov test, followed by Student’s t-test. All measurements were expressed as mean ± SD, and statistical significance level was p < 0.05.

Cell viability of the normal untreated group was 100 ± 1.52%, while cell viability of the LPS-activated group was 99.16 ± 0.52%, 98.76 ± 0.44%, 95.01 ± 0.51%, 86.60 ± 1.72%, and 65.63 ± 2.9% in the 10, 50, 100, 200, and 400 μg/mL DGHT groups, respectively. There was no significant cytotoxicity in the 10, 50, 100, and 200 μg/mL DGHT treated groups (Fig. 2).

NO production was found to be 100 ± 0.68% in LPS-activated alone group (control group), compared with 98.07 ± 1.83%, 95.75 ± 1.41%, 84.56 ± 1.60%, and 73.86 ± 1.63% in the 10, 50, 100, and 200 μg/mL DGHT treated groups was observed. There was a significant decrease of NO production in the 200 μg/mL DGHT treated group (Fig. 3).

PGE₂ production was found to be 100 ± 1.63% in the control group, compared with 98.43 ± 0.53%, 94.14 ± 1.23%, 80.31 ± 0.71%, and 72.71 ± 0.71% in the 10, 50, 100, and 200 μg/mL DGHT treated groups. There was significant decrease of NO production in the 100 and 200 μg/mL DGHT treated groups (Fig. 3).

IL-6 production was found to be 100 ± 1.33% in the LPS-activated control group, compared with 98.78 ± 1.26%, 98.59 ± 1.37%, 92.41 ± 0.20%, and 86.74 ± 1.25% in the 10, 50, 100, and 200 μg/mL DGHT treated groups. There was a significant decrease in NO production in the 100 and 200 μg/mL DGHT treated groups (Fig. 4).

IL-1ß production was found to be 100 ± 0.28% in the LPS-activated control group, compared with 99.25 ± 1.05%, 98.80 ± 0.81%, 96.48 ± 0.68%, and 93.74 ± 0.70% in the 10, 50, 100, and 200 μg/mL DGHT treated groups. There was a significant decrease of NO production in the 100 and 200 μg/mL DGHT treated groups (Fig. 4).

TNF-α production was found to be 100 ± 2.53% in the control group, compared with 99.60 ± 1.26%, 98.58 ± 0.76%, 95.69 ± 1.14%, and 90.42 ± 1.28% in the 10, 50, 100, and 200 μg/mL DGHT treated groups. There was a significant decrease in NO production in the 100 and 200 μg/mL DGHT treated groups (Fig. 4).

The DPPH radical scavenging rate was found to be 3.96 ± 1.79%, 13.93 ± 0.74%, 31.46 ± 1.24%, and 49.26 ± 0.86% in the 10, 50, 100, and 200 μg/mL DGHT treated groups. There was DPPH free radical scavenging capability at all of concentrations, but this did not exceed 50% (Fig. 5).