Roots of A. cochinchinensis were collected from plantations in the Go-Chang county of North Jeolla (Korea) and were dried in a drying machine (model FD5510S-FD5520S; IlShinBioBase Co., Dongducheon, Korea) at 60°C. Voucher specimens of A. cochinchinensis (WPC-14-003) were deposited in the Functional Materials Bank of the PNU-Wellbeing RIS Center at Pusan National University (Pusan, Korea). Dry roots of A. cochinchinensis were reduced to powder using a pulverizer (model MF-3100S; Hanil Electric Group Co., Ltd., Seoul, Korea), followed by extraction of EaEAC at 50°C for 24 h in a fixed liquor ratio (solid powder of A. cochinchinensis/ethyl acetate solvent ratio, 1:10) using circulating extraction equipment (SHWB-30/45, Woori Science Instrument Co., Pocheon, Korea). Extract solutions were subsequently passed through a 0.4 µm filter, and concentrated by vacuum evaporation and lyophilized using circulating extraction equipment (IKA Labortechnik, Staufen im Breisgau, Germany). Finally, the EaEAC powder was dissolved in dimethyl sulfoxide (DMSO; Duchefa Biochemie B.V., Haarlem, Netherlands) to 1 mg/ml, then further diluted to the required concentration.

The scavenging activity of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical was measured as previously described (30). Briefly, each 100 µl sample in eight different concentrations of EaEAC (250–2,000 µg/ml) was mixed with 100 µl DPPH (0.1 mM; Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) in 95% ethanol solution or 100 µl 95% ethanol solution, then incubated for 30 min at room temperature. The absorbance of the reaction mixture was measured at 517 nm using a VersaMax plate reader (Molecular Devices, LLC, Sunnyvale, CA, USA). The DPPH radical scavenging activity of the EaEAC was expressed as the percent decrease in absorbance relative to the control. The half maximal inhibitory concentration (IC₅₀) is defined as the concentration of substrate that causes a 50% loss in DPPH activity.

The RAW264.7 cell line used in the present study is an Abelson murine leukemia virus-transformed macrophage cell line, provided by the Korean Cell Line Bank (Seoul, Korea). RAW264.7 cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal bovine serum (FBS; cat. no. S001-01; Welgene, Gyeongsan, Korea), L-glutamine, penicillin, and streptomycin (Thermo Fisher Scientific, Inc.) in a humidified incubator at 37°C with 5% CO₂ and 95% air.

Cell viability was determined using the tetrazolium compound 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich; Merck Millipore). To determine cell viability, RAW264.7 cells were seeded at a density of 5×10⁴ cells/0.2 ml and cultured for 12 h in a 37°C incubator. When the cells reached 70–80% confluence, they were either untreated (NC group), treated with vehicle (DMSO), or pretreated with 100 or 200 µg/ml EaEAC dissolved in DMSO for 2 h. Cells were subsequently incubated for 24 h with 1 µg/ml LPS (derived from Escherichia coli, serotype 055:B5; Sigma-Aldrich; Merck Millipore), then the supernatants were discarded and 0.2 ml fresh DMEM and 50 µl MTT solution [2 mg/ml in phosphate-buffered saline (PBS)] was added to each well. Cells were incubated at 37°C for a further 4 h. Formazan precipitate was dissolved in DMSO and the absorbance at 570 nm was read directly in the wells using a VersaMax plate reader (Molecular Devices, LLC). The morphological features of RAW264.7 cells in each treated group were also observed using an inverted light microscope [Leica Microsystems (Schweiz) AG, Heerbrugg, Switzerland].

NO accumulation was measured in the cell culture medium using Griess reagent [1% sulfanilamide, 5% phosphoric acid, 0.1% N-(1-naphthyl) ethylenediamine dihydrochloride; Sigma-Aldrich; Merck Millipore] as described previously (31). Briefly, RAW264.7 cells were treated with EaEAC (100 and 200 µg/ml) for 2 h followed by LPS (1 µg/ml) for 24 hr. Following the collection of the supernatant, each sample (100 µl) was mixed with the same volume of Griess reagent and incubated at room temperature for 10 min. The absorbance was read at 540 nm using a VersaMax microplate reader (Molecular Devices, Sunnyvale, CA, USA).

ROS levels in cells were measured by staining with 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA; Sigma-Aldrich, Merck Millipore), a cell permeable and nonfluorescent agent that can be deacetylated by intracellular esterases to nonfluorescent DCFH, and is converted to highly fluorescent 2′,7′-dichlorofluorescein (DCF) in the presence of intracellular ROS. When the RAW264.7 cells reached 70–80% confluence, they were either untreated, treated with vehicle, or pretreated with 100 or 200 µg/ml EaEAC dissolved in DMSO for 2 h. Cells were subsequently incubated for 24 h with 1 µg/ml LPS. Cells were then incubated with 25 µM DCFH-DA for 30 min at 37°C, before they were washed twice with PBS. Green fluorescence was visualized at ×200 and ×400 magnification using an Eclipse TX100 fluorescent microscope (Nikon Corporation, Tokyo, Japan).

When the RAW264.7 cells reached 70–80% confluence, they were either untreated, treated with vehicle, or pretreated with 100 or 200 µg/ml EaEAC dissolved in DMSO for 2 h. Cells were subsequently incubated for 24 h with 1 µg/ml LPS. Total protein for western blotting was extracted from RAW264.7 cells using Pro-Prep Protein Extraction Solution (iNtRON Biotechnology, Seongnam, Korea), centrifuged at 11,000 × g for 5 min, and quantified using a SMART bicinchoninic acid assay kit (Thermo Fisher Scientific, Inc.). Proteins were separated by 4–20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis for 2 h (30 µg per gel), then resolved proteins were transferred to nitrocellulose membranes for 2 h at 40 V. After the blocking with 3–5% skim milk in TNT solution (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄ and 0.05% Tween-20) and incubating at room temperature for 1 h, the membranes were incubated overnight at 4°C with the following primary antibodies: Anti-SAPK/JNK (cat. no. 9252; dilution, 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), anti-phosphorylated (p-) SAPK/JNK (Thr183/Tyr185; cat. no. 9251; dilution, 1:1,000; Cell Signaling Technology, Inc.), anti-ERK1 (K-23; cat. no. sc-94; dilution, 1:1,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti-p-ERK1/2 (Thr202/Tyr204; cat. no. 9101; dilution, 1:1,000; Cell Signaling Technology, Inc.), anti-p38 mitogen-activated protein kinase (MAPK; cat. no. 9212; dilution, 1:1,000; Cell Signaling Technology, Inc.), anti-p-p38 MAPK (Thr180/Tyr182; cat. no. 9211; dilution, 1:1,000; Cell Signaling Technology, Inc.) and anti-β-actin (cat. no. A5316; dilution, 1:3,000; Sigma-Aldrich; Merck Millipore). Membranes were then washed with TNT solution and incubated with 1:1,000 diluted horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (cat. no. 81–6120; Zymed; Thermo Fisher Scientific, Inc.) at room temperature for 1 h. Blots were developed using Amersham ECL Select Western Blotting detection reagent (GE Healthcare Life Sciences, Chalfont, UK). Protein bands were visualized using the FluorChem^® FC2 Imaging system (Alpha Innotech Corporation, San Leandro, CA, USA) and the band density was semi-quantified using the AlphaView Program (version, 3.2.2; Cell Biosciences, Inc., Palo Alto, CA, USA). The target protein expression levels of three samples from each group were analyzed in three separate western blot analyses.

The concentration of IL-6 secreted from RAW264.7 cells was determined using an IL-6 ELISA kit (cat. no. 431304; Biolegend, Inc., San Diego, CA, USA) according to the manufacturer's protocols. Briefly, RAW264.7 cells were treated with two different concentrations of EaEAC (100 and 200 µg/ml) for 2 h, followed by 1 µg/ml of LPS for 24 h. Cell supernatant was collected and 100 ml serial dilutions of the standard or the supernatant were added to a 96-well plate coated with anti-IL-6 antibody, then incubated for 2 h at room temperature. Following five washes with wash solution (PBS, 0.05% Tween-20, pH 7.4), 100 µl avidin-HRP solution was added to each well, and the plates incubated at 37°C for 2 h. Following five washes, 100 µl substrate solution was added and the plate was incubated at 37°C for a further 30 min. The reaction was terminated by the addition of 100 µl stop solution (2N H₂SO₄) and the absorbance at 450 nm was determined with a VersaMax plate reader (Molecular Devices, LLC).

The relative expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), TNF-α, IL-1β, IL-6 and IL-10 mRNAs were measured by RT-qPCR as previously described (32). Total RNA was purified by removing media from each cultured sample and homogenizing the cells in RNAbee (cat. no. CS104; Tel-Test Inc., Friendswood, TX, USA). The isolated RNA concentration was then determined using a NanoDrop spectrophotometer (BioSpec-Nano, Shimadzu Scientific Instruments, Columbia, MD, USA). Total RNA (5 µg) was used to synthesize cDNA in a reaction mixture consisting of oligo (dT) primers (500 ng; Invitrogen; Thermo Fisher Scientific, Inc.), 10 mM deoxyadenosine, deoxycytidine, deoxyguanosine and deoxythymidine triphosphates, 0.1 M dithiothreitol, 5X reaction buffer and 1 µl Superscript II reverse transcriptase (200 U/µl; cat. no. 18064–014; Invitrogen; Thermo Fisher Scientific, Inc.). Thermal cycling conditions for reverse transcription consisted of 70°C for 10 min followed by 42°C for 50 min. RNA was removed from cDNA samples using 3.2 U/µl RNase H (cat. no. 18021071; Invitrogen; Thermo Fisher Scientific Inc.) at 37°C for 20 min. In order to amplify target genes, 2.5 µl cDNA, 10 pmol specific sense and antisense primers (Macrogene Co., Seoul, South Korea), 5 U/µl Taq polymerase (0.2 µl; PCR Core kit; Roche Diagnostics, Basel, Switzerland), 2 mM deoxynucleotide triphosphate (2.5 µl), and 10X reaction buffer containing 15 mM MgCl₂ (2.5 µl) were combined. A thermal cycler (Perkin-Elmer, Inc., Waltham, MA, USA) was used to amplify target genes using the following parameters: A pre-denaturation step of 7 min at 94°C, followed by 25–32 cycles of 30 sec at 94°C, 30 sec at 62°C, and 45 sec at 72°C and a final post-elongation step of 7 min at 72°C. The primer sequences for target gene expression identification were as follows: iNOS, sense 5′-CACTTGGAGTTCACCCAGT-3′ and anti-sense, 5′-ACCACTCGTACTTGGGATGC-3′; COX-2, sense 5′-CAGGTCATTGGTGGAGAGGTGTATC-3′ and anti-sense, 5′-CCAGGAGGATGGAGTTGTTGTAGAG-3′; TNF-α, sense 5′-CCTGTAGCCCACGTCGTAGC-3′ and anti-sense, 5′-TTGACCTCAGCGCTGACTTG-3′; IL-1β, sense 5′-GCACATCAACAAGAGCTTCAGGCAG-3′ and anti-sense, 5′-GCTGCTTGTGAGGTGCTGATGTAC-3′; IL-10, sense 5′-CCAAGCCTTATCGGAAATGA-3′ and anti-sense, 5′-TTTTCACAGGGGAGAAATCG-3′; IL-6, sense 5′-TTGGGACTGATGTTGTTGACA-3′ and anti-sense, 5′-TCATCGCTGTTGATACAATCAGA-3′. The final PCR products were separated on 1% agarose gel and then visualized by ethidium bromide staining. The band signal for each sample was visualized using the AE-9000 E-Graph gel documentation system (ATTO Corporation, Tokyo, Japan) and the band density was quantified using Image Saver 5 software (version 5.0; ATTO Corporation). The experiment was repeated three times and all samples were PCR-analyzed in triplicate.

The cell cycle was measured using a Muse™ Cell Cycle kit (cat. no. MCH100106; EMD Millipore, Billerica, MA, USA) according to the manufacturer's instructions. Briefly, RAW264.7 cells were divided into 100 mm² dishes (2.5×10⁶ cells/dish), then pretreated with 100 or 200 µg/ml of EaEAC for 2 h. Following treatment with 1 µg/ml of LPS for 24 h, cells from subset groups were harvested by centrifugation at 3,000 × g for 5 min, then fixed with 70% ethanol at −20°C for 3 h. The fixed cells were washed with 1X PBS, then added to 200 µl Cell Cycle Reagent. Following incubation at 37°C in a CO₂ incubator for 30 min, cell cycles were analyzed using a Muse^® Cell Analyzer with the Muse 1.3.1 analysis program (EMD Millipore).

Statistical tests were performed using SPSS version 10.10 (SPSS, Inc., Chicago, IL, USA). One-way analysis of variance was used to identify significant differences between NC- and LPS-treated groups. Differences in the responses of the Vehicle + LPS treated group and EaEAC + LPS treated groups were evaluated using a post hoc Tukey's test. All values are reported as the mean ± standard deviation. P<0.05 was considered to indicate a statistically significant difference.

The anti-oxidant activity of EaEAC was measured by DPPH scavenging analysis. Scavenging activity of EaEAC against DPPH radical was increased in a dose-dependent manner, with an IC₅₀ of 781.1497 µg/ml (Fig. 1). This indicated that EaEAC exhibited DPPH radical scavenging activity.

To determine the toxicity of EaEAC, cell viability was measured by MTT assay. No marked differences in morphological features (Fig. 2A) or significant changes to cell viability (Fig. 2B) were observed in RAW264.7 cells treated with 100 and 200 µg/ml EaEAC for 24 h compared with NC or vehicle group cells, indicating EaEAC is not toxicity at concentrations up to 200 µg/ml.

To examine the anti-inflammatory properties of EaEAC, alterations in NO concentration, and iNOS and COX-2 transcription were measured in LPS-stimulated RAW264.7 cells following EaEAC pretreatment. The concentration of NO was significantly increased in the vehicle + LPS-treated group compared with the NC group (P=0.01; Fig. 3A). However, the concentration of NO decreased by 18 and 33% in cells pretreated with 100 and 200 µg/ml EaEAC, respectively, with the concentration in the cells that were pretreated with 200 µg/ml EaEAC significantly reduced compared with the vehicle + LPS group (P=0.002; Fig. 3A).

Similarly, increased levels of iNOS and COX-2 mRNA expression were detected in the vehicle + LPS-treated group compared with the NC group (P=0.011 and P=0.001, respectively; Fig. 3B). However, these levels were reduced by 21% (COX-2) and 33% (iNOS) in LPS-stimulated RAW264.7 cells that were pretreated with 200 µg/ml of EaEAC compared with the vehicle + LPS group (P=0.012 and P=0.003, respectively; Fig. 3B). EaEAC pretreatment was, therefore, demonstrated to inhibit the increase of NO concentration, and COX-2 and iNOS mRNA expression in LPS-activated RAW264.7 cells.

EaEAC-induced alterations to the mRNA expression levels of pro-inflammatory (TNF-α and IL-1β) and anti-inflammatory (IL-10 and IL-6) cytokines in LPS-activated RAW264.7 cells were then investigated by semi-quantitative RT-PCR. The mRNA levels of the cytokines were significantly increased in the vehicle + LPS-treated group compared with the NC group (P=0.002; Fig. 4A). Pretreatment with EaEAC had no significant effect on the mRNA expression levels of TNF-α at either 100 or 200 µg/ml (Fig. 4A). However, pretreatment with 100 and 200 µg/ml EaEAC significantly reduced mRNA expression of IL-1β (P=0.003 and P=0.003, respectively; Fig. 4A), while 200 µg/ml EaEAC significantly reduced mRNA levels of IL-10 (P=0.008) and IL-6 (P=0.002) compared with vehicle + LPS (Fig. 4A).

The concentration of IL-6 in the culture supernatant of LPS-activated RAW264.7 cells was also measured by ELISA. Similarly to the mRNA expression levels, the concentration of IL-6 was significantly increased in the Vehicle + LPS treated group compared with the NC group (P=0.005; Fig. 4B). However, no significant differences were observed between the Vehicle + LPS group and the EaEAC + LPS groups, despite a trend towards lower concentrations following EaEAC treatment (Fig. 4B). EaEAC pretreatment is, therefore, suggested to suppress enhanced anti- and pro-inflammatory cytokine expression in RAW264.7 cells stimulated by LPS treatment.

The MAPK pathway is critical in the regulation of cell growth and differentiation, and in the control of cellular responses to various cytokines and stresses (33). To investigate whether EaEAC pretreatment influences activation of the MAPK pathway, the phosphorylation levels of ERK, JNK and p38 were assessed in LPS-stimulated RAW264.7 cells by western blotting (Fig. 5A and B). Levels of phosphorylated ERK and p38 were significantly higher in cells stimulated with LPS (vehicle + LPS group) compared with untreated control cells (P=0.001 and P=0.002, respectively; Fig. 5B). EaEAC pretreatment significantly suppressed the LPS-induced phosphorylation of ERK at concentrations of 100 (P=0.002) and 200 µg/ml (P=0.003) compared with the vehicle + LPS group (Fig. 5B). JNK phosphorylation was also suppressed by 100 (P=0.015) and 200 µg/ml EaEAC (P=0.033) compared with the vehicle + LPS group (Fig. 5B), however, the JNK phosphorylation level was not induced by LPS stimulation, as demonstrated by the non-significant difference between the NC and vehicle + LPS group (Fig. 5B). EaEAC pretreatment resulted in no significant difference in LPS-induced phosphorylation of p38 at concentrations of 100 or 200 µg/ml compared with the vehicle + LPS group (Fig. 5B). Therefore, EaEAC attenuated the enhanced phosphorylation of some MAPK proteins in LPS-stimulated RAW264.7 macrophage cells.

To examine the effect of EaEAC treatment on the cell cycle, the number of cells in each stage of the cell cycle was counted (Fig. 6A and B). The number of cells in the G0/G1 stage was increased in the vehicle + LPS group compared with untreated control cells (P=0.003; Fig. 6B), whereas those in the S and G2/M stage were decreased (P=0.001; Fig. 6B). However, a significant increase in the number of cells in the G2/M stage was induced by treatment with 100 µg/ml EaEAC compared with the vehicle + LPS group (P=0.023; Fig. 6B), and a similar but non-significant trend towards increased G2/M stage cells was induced by treatment with 200 µg/ml EaEAC (Fig. 6B). A significant decrease in the number of S stage cells was also observed following treatment with 100 (P=0.05) and 200 µg/ml EaEAC (P=0.008) compared with the vehicle + LPS (Fig. 6B). EaEAC treatment was, therefore, demonstrated to induce arrest of the cell cycle in the G2/M stage and stimulate progression from the S stage to the G2/M stage.

Finally, the inhibitory effects of EaEAC against LPS-induced ROS production in RAW264.7 macrophage cells were determined using a fluorescent oxidation-sensitive dye, DCFH-DA, to measure ROS levels. ROS production increased rapidly in the vehicle + LPS-treated group compared with the NC group. However, the level of ROS production decreased in an apparently concentration-dependent manner, despite cell morphologies remaining consistent. Therefore, increased ROS levels induced by LPS stimulation were effectively suppressed by EaEAC pretreatment.