The roots of A. cochinchinensis used in this study were obtained from the National Agricultural Cooperation Federation (Gochang, South Korea) and dried in a drying machine (FD5510S-FD5520S; Ilshin Biobase Co., Ltd., Seoul, South Korea) at 60°C. Voucher specimens of A. cochinchinensis roots (WPC-14-003) were deposited in the functional materials bank of the Wellbeing RIS Center at Pusan National University (Pusan, South Korea). Dried roots were reduced to a powder using a pulverizer (MF-3100S; Hanil Electric Co., Ltd., Seoul, Korea), after which the SEAC was obtained by extraction at 50°C for 24 h using a fixed liquor ratio (ratio of solid powdered A. cochinchinensis to ethyl acetate solvent, 1:10) using circulating extraction equipment (SHWB-30/45; Woori Science Instrument Co., Pocheon, South Korea). These extraction mixtures were subsequently passed through a 0.4-μm filter, after which they were concentrated by vacuum evaporation and lyophilization using circulating extraction equipment (IKA Labortechnik, Staufen, Germany). Finally, the collected SEAC powder was dissolved in 0.5% Tween-20 solution in distilled water (dH₂O) to 400 mg/kg, and then further diluted to the required concentration.

In addition, an aqueous extract of Platycodon grandifloras (AePG; Jangsaeng Doraji Co., Jinju-si, Korea) and dexamethasone (Dex) were used as positive controls. The AePG was obtained by extraction of P. grandifloras at 120°C for 45 min using a fixed liquor ratio (solid powdered P. grandifloras:dH₂O ratio, 75 g:500 ml) using circulating extraction equipment (SHWB-30/45). Dex was purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany).

The total phenolic contents of the SEAC were determined using the Folin-Ciocalteu method as previously described (12). Briefly, a mixture of SEAC solution (1 ml) and Folin-Ciocalteu reagent (5 ml; Sigma-Aldrich; Merck KGaA) was incubated at room temperature for 5 min. This mixture was subsequently added to 15 ml 20% Na₂CO₃ and vortexed for 30 sec, after which the absorbance was repeatedly measured at 765 nm using a VersaMax plate reader (molecular Devices, LLC, Sunnyvale, CA, USA). A standard calibration curve was generated using different concentrations of gallic acid (Sigma-Aldrich; Merck KGaA), and the concentration of the total phenolic compounds in the SEAC was presented as the gallic acid equivalent (mg) of the extract.

The flavonoid contents in the SEAC were measured as previously described (13). In brief, 20-μl samples of several different concentrations of SEAC were mixed with 60 μl 5% NaNO₂ and 60 μl 10% AlCl₃ (both from Sigma-Aldrich; Merck KGaA). Following incubation at 25°C for 5 min, the absorbance was measured using a VersaMax plate reader. A standard calibration curve was then created using different concentrations of catechin (Sigma-Aldrich; Merck KGaA). The flavonoid contents of the SEAC are presented as catechin equivalents (mg) of the extract.

Finally, the total saponin content was determined using the method previously described by Helaly et al (14). The SEAC (5 g) was dissolved in 0.5 ml 80% methanol, and then mixed with 0.5 ml 8% vanillin in ethanol and 5 ml 72% H₂SO₄ in water. These mixtures were placed in a 60°C water bath for 20 min, and then cooled at 0°C for 5 min, after which the absorbance was measured at 544 nm using a VersaMax plate reader. The saponin content was calculated from a calibration curve constructed using a purified saponin standard (Sigma-Aldrich; Merck KGaA).

The scavenging activity was evaluated using 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals as previously described (15). In brief, a 100-μl sample comprising one of eleven different concentrations of SEAC (0, 7.8, 15.6, 31.3, 61.5, 125, 250, 500, 1,000, 1,500 and 2,000 μg/ml) was mixed with 100 μl DPPH (0.1 mm; Sigma-Aldrich; Merck KGaA) in 95% ethanol solution or 100 μl 95% ethanol solution as a control, and then incubated for 30 min at room temperature. The absorbance of the reaction mixture was subsequently measured at 517 nm using a VersaMax plate reader. The DPPH radical scavenging activity of the SEAC was expressed as the percentage reduction in absorbance relative to the control. The half maximal inhibitory concentration (IC₅₀) was calculated as the concentration of substrate that caused a 50% loss in DPPH activity.

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

The accumulation of NO was measured in the culture medium of cells pretreated with the vehicle or SEAC (100 or 200 μg/ml) for 2 h at room temperature followed by LPS (1 μg/ml) for 24 h using Griess reagent [1% sulfanilamide, 5% phosphoric acid and 0.1% N-(1-naphthyl) ethylenediamine dihydrochloride; Sigma-Aldrich; Merck KGaA] as described previously (16).

The reactive oxygen species (ROS) levels of cells pretreated with the vehicle or SEAC (100 or 200 μg/ml) for 2 h followed by LPS (1 μg/ml) for 24 h were measured by staining with 2′,7′-dichlorodihydrofluorescein (DCFH) diacetate (Sigma-Aldrich; Merck KGaA), a cell permeable and nonfluorescent agent that is deacetylated by intracellular esterases to form nonfluorescent DCFH, and is converted to highly fluorescent 2′,7′-dichlorofluorescein in the presence of intracellular ROS.

The relative expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 mRNA in RAW264.7 cells that had been pretreated with the vehicle or SEAC (100 or 200 μg/ml) for 2 h followed by LPS (1 μg/ml) for 24 h were measured by reverse transcription-polymerase chain reaction (RT-PCR) using specific primers as previously described (17).

The animal protocols used in the present study were reviewed and approved for ethical and scientific care procedures by the Pusan National University-Institutional Animal Care and Use Committee (approval no. PNU-2015-0779). Six-week-old female BALB/c mice were purchased from Samtako Bio Korea Co., Ltd. (Osan, South Korea). Prior to the initiation of the animal experiment, the mice were allowed ≥1 week to adapt to the experimental environment. All mice were provided with ad libitum access to a standard irradiated chow diet (Samtako Bio Korea Co., Ltd.) and water throughout the 25-day study. During the experiment, mice were maintained in a specific pathogen-free state under a strict light cycle (lights on at 08:00 a.m. and off at 20:00 p.m.) at 23±2°C and 50±10% relative humidity. The BALB/c mice were housed in the Pusan National University-Laboratory Animal Resources Center accredited by the Korean Ministry of Food and Drug Safety in accordance with the Laboratory Animal Act (accredited unit no. 000231) and AAALAC International according to the National Institutes of Health guidelines (accredited unit no. 001525).

The airway challenge of the BALB/c mice was induced as previously described (18,19). In brief, 6-week-old Balb/c mice (female; n=48) were assigned to either an untreated group (n=8) or an OVA-treated group (n=40). The untreated group did not receive any treatment during the experimental period. In the other group, OVA-induced asthma was generated by sensitization for 20 days and challenge for 3 days. On days 0 and 14, the mice were sensitized by the intraperitoneal injection of OVA (albumin from chicken; 20 μg) emulsified with aluminum hydroxide (alum; both from Sigma-Aldrich; Merck KGaA) in 200 μl 1× PBS solution. On days 21–23, the mice were subjected to a 30-min airway challenge with 2% OVA in 1× PBS by inhalation through a nebulizer (NE-C28; Omron, Tokyo, Japan; Fig. 1). Additionally, the OVA-induced asthma group was further divided into a vehicle-treated group (OVA + vehicle; n=8), Dex-treated group (OVA + Dex; n=8), AePG-treated group (OVA + AePG; n=8), low dose SEAC-treated group (OVA + SEACL; n=8), and high dose SEAC-treated group (OVA + SEACH, n=8). The two OVA+SEAC-treated groups received 250 and 500 mg/kg body weight of SEAC, respectively, while the OVA + vehicle group received the same volume (200 μl) of 0.5% Tween-20 solution for 6 days. The therapeutic concentration of SEAC was determined based on the results from previous studies of skin inflammation (10). Furthermore, the OVA + Dex and OVA + AePG groups received 3 mg/kg body weight of Dex solution and 100 mg/kg body weight of AePG, respectively. At 48 h after the final treatment, all animals were euthanized using CO₂ gas, and tissue samples were acquired and stored in Eppendorf tubes at −70°C until assay.

Following anesthesia of the mice with Alfaxan^® (alfaxalone; Jurox Pty Ltd., Rutherford, Australia), BALF was obtained (yield, 80%; total volume, 0.8 ml) from the mice in each group via tracheal cannulation with cold 1× PBS. Total cells were then harvested from the BALF by centrifugation (2,000 × g) at 4°C for 5 min, after which they were resuspended in 100 μl hematoxylin. The eosinophils, macrophages and total cells in 1 mm² were counted on glass slides using the Leica Application Suite (Leica Microsystems, Heerbrugg, Switzerland).

The body weights of all mice in each group throughout the experimental period were measured using an electronic balance (Mettler Toledo Gmbh, Greifensee, Switzerland). The weights of the spleen and thymus were also determined following sacrifice using the electronic balance.

The concentration of IL-4 in the BALF was determined using an IL-4 ELISA kit (cat. no. 431107; BioLegend, Inc., San Diego, CA, USA) according to the manufacturer's protocol. In brief, following collection of the supernatant from the BALF, the assay buffer (50 μl) and the supernatant (50 μl) were added to a 96-well plate coated with anti-IL-4 antibody, and incubated for 2 h at room temperature. Following removal of unbound proteins with wash solution (0.05% Tween-20 in PBS, pH 7.4), 100 μl detection antibody solution was added and the samples were incubated for 1 h. After washing, 100 μl avidin-horseradish peroxidase (HRP) D solution was added to each well, and the plates were allowed to bind for 30 min. Next, the plate was washed and reacted with 100 μl substrate solution for 15 min. The reaction was then stopped by the addition of 100 μl blocking solution, after which the absorbance at 450 nm was determined with a VersaMax plate reader.

The OVA-specific IgE concentration in the serum of the mice was measured using an ELISA kit (cat. no. 439807; BioLegend, Inc.) according to the manufacturer's protocol. Briefly, wells were washed with washing solution (50 mM Tris, 0.14 M NaCl, 0.05% Tween-20, pH 8.0) four times, after which assay buffer (50 μl) and serum samples (50 μl) were added to wells coated with antibody and the plate was incubated with shaking for 2 h at room temperature. Next, the wells were washed with washing solution, after which 100 μl detection antibody solution was added and the samples were incubated for 1 h with shaking. After washing the wells, Avidin-HRP D solution (100 μl) was added to each well. The plates were then incubated at room temperature for 30 min, after which the wells were washed and an enzyme reaction was initiated by adding substrate solution and incubating the plates at room temperature in the dark for 15 min. Finally, the reaction was terminated by adding 2 M H₂SO₄ solution and the absorbance was measured at 450 nm using a VersaMax plate reader.

Lung tissues were collected from all mice of the various groups, fixed in 10% neutral buffered formalin, embedded in paraffin wax, routinely processed, and then sectioned into 4-μm slices. The tissue sections on a slide glass were stained with hematoxylin and eosin (H&E; IHC World, Woodstock, MD, USA) after which they were examined using light microscopy to observe the infiltration of inflammatory cells into the peribronchial region of the lung at ×400 magnification. The epithelial thickness and smooth muscle thickness of the bronchial tube were also measured using the Leica Application Suite (Leica Microsystems).

Hyperplasia of goblet cells for mucus production was detected by staining with periodic acid-Schiff. Following deparaffinization and dehydration of lung sections, samples were oxidized in periodic acid solution for 5 min at room temperature. The lung sections were then washed at warm water and placed in Schiff reagent for 5 min at room temperature. The sections were then washed with warm tap water, stained with hematoxylin solution (IHC world) for 30 sec, and examined by light microscopy to detect goblet cell hyperplasia and subepithelial fibrosis at ×400 magnification. The mucus score was then determined by four independent investigators in a single-blind analysis based on four different random locations using a microscope. The mucus scores were: 0, no mucus; 1, <5% of the epithelium; 2, 5–10% of the epithelium; 3, 10–20% of the epithelium; 4, 20–30% of the epithelium; 5, 30–40% of the epithelium

The relative quantities of IL-4 and IL-13 mRNA were measured using RT-PCR. In brief, frozen lung tissue was chopped with scissors and homogenized in RNA-Bee solution (Tel-Test, Inc., Friendswood, TX, USA). Total RNA was then isolated by centrifugation at 15,000 × g for 15 min, after which the RNA concentration was measured using UV spectroscopy. The expression of the target genes was assessed using RT-PCR with 3 μg total RNA from the tissue of each group. The RNA was annealed with 500 ng oligo(dT) 12–18 primer (Invitrogen; Thermo Fisher Scientific, Inc.) at 70°C for 10 min. Complementary DNA, which was used as the template for further amplification, was synthesized from the RNA by the addition of 2 mM dNTPs (Deoxynucleoside Triphosphate set; Roche Diagnostics, Basel, Switzerland) with 200 units of SuperScript™ II reverse transcriptase (200 U/μl; Invitrogen; Thermo Fisher Scientific, Inc.). PCR was then conducted with the mixture of 2.5 μl cDNA, 10 pmol sense and antisense primers (2.5 μl), Taq DNA polymerase (0.2 μl; Roche Diagnostics), 2 mM dNTP (2.5 μl; Deoxynucleoside Triphosphate set; Roche Diagnostics), and 10× reaction buffer containing 15 mM MgCl2 (2.5 μl), and the reaction mixture was subjected to 28–32 cycles of amplification in a thermal cycler (PerkinElmer, Inc., Waltham, MA, USA). The following temperature cycle was used for PCR: 30 sec at 94°C, 30 sec at 62°C and 42 sec at 72°C. The primer sequences used were as follows: IL-4 sense, 5′-CCA GCT AGT TGT CAT CCT GCT CTT C-3′ and antisense, 5′-GTG ATG TGG ACT TGG ACT CAT TCA TGG-3′; IL-5 sense, 5′-GAG AAG GAT GCT TCT GCA CTT GAG-3′ and antisense, 5′-CCA CTC TGT ACT CAT CAC ACC AAG G-3′; IL-13 sense, 5′-CCT TAA GGA GCT TAT TGA GGA GCT GAG-3′ and antisense, 5′-CAG TTG CTT TGT GTA GCT GAG CAG-3′; β-actin sense, 5′-TGG AAT CCT GTG GCA TCC ATG AAA C-3′ and antisense, 5′-TAA AAC GCA GCT CAG TAA CAG TCC G-3′. Three samples were analyzed in duplicate. The final PCR products were separated on 1% agarose gel, and then visualized by ethidium bromide staining. The band patterns were detected using a UV-transilluminator (ATTO, Tokyo, Japan) and analyzed Image saver 6 (ATTO).

Lung tissue (50 mg) collected from each group was homogenized using PRO-PREP™ Solution (Intron Biotechnology, Inc., Sungnam, Korea), after which total protein extracts were collected by centrifugation at 13,000 × g for 5 min at 4°C. The prepared proteins were subsequently subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis for 2 h at 100 V, after which they were transferred to a nitrocellulose membrane (GE Healthcare Life Sciences, Little Chalfont, UK) for 2 h at 40 V in transfer buffer (25 mM Trizma-base, 192 mM glycine and 20% methanol). Appropriate dilutions of the primary antibodies anti-COX-2 (1:1,000; #12282; Cell Signaling Technology, Inc., Danvers, MA, USA), anti-vascular endothelial growth factor (anti-VEGF; 1:1,000; #500-P131; PeproTech, Inc., Rocky Hill, NJ, USA) and anti-β-actin (1:3,000; #A5316; Sigma-Aldrich) were added to the membranes and allowed to hybridize overnight at 4°C. Following removal of the antibodies, the membrane was washed three times in a solution comprising 10 mM Trizma-base (150 mM NaCl and 0.05% Tween-20) for 10 min. The membrane was subsequently incubated with 1:3,000 diluted HRP-conjugated goat anti-rabbit IgG (#G21234; Invitrogen) for 1 h at room temperature, after which it was washed again as described above and developed using an enhanced chemiluminescence reagent plus kit (Amersham Biosciences; GE Healthcare Life Sciences). Finally, the results were quantified using an image analysis aystem (Fluorchem FC2; ProteinSimple, San Jose, CA, USA) and expressed as the fold-increase over control values.

One-way analysis of variance was used to identify significant differences between the untreated and LPS-treated cell groups or between the untreated and OVA-treated mouse groups. Additionally, differences between the vehicle + LPS-treated group and the SEAC + LPS-treated groups, as well between the OVA + vehicle-treated group and the OVA + Dex, OVA + AePG or OVA + SEAC-treated groups were evaluated using a post hoc Tukey's test of the variance and significance levels. All values were expressed as the mean ± standard deviation. The statistical analysis was conducted using SPSS for Windows, release 10.10, standard version (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

As shown Fig. 2A, SEAC contained high concentrations of three bioactive components associated with anti-inflammatory activity. Crude saponins were detected at a high level (57.2 mg/g) in the SEAC. The concentrations of total phenols and total flavonoids were 88.5 and 102.1 μg/g, respectively. Furthermore, the scavenging activity of SEAC for DPPH radicals increased in a dose-dependent manner, with an IC₅₀ of 708.56 μg/ml (Fig. 2B).

To examine the possibility that SEAC had anti-inflammatory properties, alterations in NO concentration, iNOS and COX-2 transcription and intracellular ROS level were measured in LPS-stimulated RAW264.7 cells following SEAC pretreatment. The NO concentration, iNOS and COX-2 expression levels (Table I) were significantly increased and intracellular ROS content was markedly increased in the vehicle + LPS-treated group compared with the untreated group (Fig. 2C). However, these values decreased in a dose-dependent manner in the cells pretreated with 100 and 200 μg/ml SEAC, although the iNOS level was maintained at a constant level in the low dose SEAC-treated group (Table I). These results provide strong evidence that SEAC pretreatment attenuates various types of inflammation in specific tissue by inhibiting the increase in NO concentration, COX-2 and iNOS mRNA expression and intracellular ROS production.

To investigate the toxic effects of SEAC on the whole body and immune-associated organs, the whole body, spleen and thymus weight were measured following SEAC treatment. The body weights of the mice in all groups were maintained at a constant level throughout the experimental period, and no significant differences between groups were observed. Furthermore, similar results were detected for the spleen and thymus weights in response to the inflammatory immune reaction, although insignificant increases in the spleen weights of the OVA + SEAC-treated groups were observed (Fig. 3). These results indicate the absence of toxic effects of SEAC on the whole body, including the immune organs.

To examine the suppressive effects of SEAC on the inflammatory response in the BALF, the total numbers of leukocytes, eosinophils and macrophages in the BALF were determined. The number of leukocytes was significantly higher in the OVA + vehicle group compared with the untreated group, reflecting the challenge of airway inflammation with OVA. These results imply that OVA antigen challenge triggers a marked influx of leukocytes into the BALF (Fig. 4A). Treatment with OVA + Dex, OVA + AePG or OVA + SEAC induced a significant reduction in the total number of leukocytes in the BALF when compared with the OVA + vehicle group (Fig. 4A). A similar pattern of changes was observed for eosinophils and macrophages (Fig. 4A). These findings indicate that SEAC inhibits the inflammatory response of the BALF to OVA inhalation.

The upsurge of inflammatory mediators, including serum IgE, serves as evidence of the onset of asthma (20,21). Therefore, OVA-specific IgE levels were measured in the serum of OVA-sensitized mice treated with vehicle, Dex, AePG or SEAC to evaluate the suppressive effects on IgE production. The OVA-specific IgE level was increased in the OVA + vehicle-treated group compared with the untreated group, suggesting that OVA induction was successful for creation of the asthma model (Fig. 4B). Furthermore, the OVA-specific IgE levels were decreased significantly in the 250 mg/kg SEAC (OVA + SEACL), 500 mg/kg SEAC (OVA + SEACH), Dex and AePG treatment groups compared with the OVA + vehicle-treated group (Fig. 4B). These results suggest that SEAC treatment successfully reduced OVA-specific IgE levels in the serum.

To discern whether SEAC affects the recovery of pathological structures in the airways, alterations in the histopathological structure of the lungs were evaluated in the OVA + SEAC-treated group. Lung tissue sections from mice that were sensitized with OVA (OVA + vehicle-treated group) displayed a significant expansion in the thickness of the respiratory epithelium and smooth muscle. However, these thicknesses in the OVA + SEACH-treated groups, were significantly decreased compared with those in the OVA + vehicle-treated group. A similar reduction was observed in the infiltration of inflammatory cells in the peribronchiolar region (Fig. 5). These findings demonstrate that SEAC has the potential to alleviate the histopathological structural changes, including the increased thickness of the respiratory epithelium and smooth muscle, in the airways of the OVA asthma model.

The levels of the Th2 cytokines IL-4, IL-5 and IL-13 in the lung were measured using RT-PCR analysis to evaluate differences in the expression of inflammatory mediators among the groups. The transcript levels of these cytokines were higher in the OVA + vehicle-treated group compared with the untreated group. However, the mRNA levels of only IL-4 and IL-13 were significantly decreased in the OVA + SEACH group compared with the OVA + vehicle-treated group (Fig. 6A), while the level of IL-5 mRNA was constantly maintained in the same groups (data not shown). Similar effects were observed in the OVA + Dex and OVA + AePG-treated groups (Fig. 6A). Furthermore, to verify whether these changes at the transcriptional level were maintained at the translational level, IL-4 was selected as one of the key mediators and the protein level of this protein was measured in the BALF. The reduction of IL-4 mRNA detected in the OVA + SEACH group was also observed for IL-4 protein, although the extent of the reduction differed (Fig. 6B).

The expression of another inflammatory mediator, COX-2, was measured in the lung tissue of the OVA + SEAC-treated group. The level of COX-2 protein in the OVA + vehicle-treated group was significantly higher than that of the untreated group. However, COX-2 levels were significantly reduced in the OVA + SEACL and OVA + SEACH groups compared with the OVA + vehicle-treated group, and these reductions were slightly dependent on the concentration of SEAC (Fig. 7). These results indicate that SEAC is able to inhibit the production of inflammatory mediators, including several cytokines and COX-2 in the lung tissue and BALF.

To determine the effect of SEAC on airway remodeling, measurements of goblet hyperplasia, the thickness of the peribronchiolar collagen layer and VEGF expression levels were made in the OVA-sensitized mice treated with SEAC. A high mucus score indicative of goblet cell hyperplasia was observed in the bronchial airways of mice that were sensitized with OVA (OVA + vehicle-treated group). However, the mucus scores in the OVA +SEACL and OVA + SEACH-treated groups were significantly lower than that in the OVA + vehicle-treated group (Fig. 8).

Similar results were observed for the thickness of the peribronchiolar collagen layer and subepithelial fibrosis, as may be expected, since collagen deposition in the airways is associated with severe asthma and a reduction in pulmonary function (22) (Fig. 9A and B). In addition, the expression level of VEGF, which is a stimulator of angiogenesis and structural changes in asthma (23), was significantly decreased in the OVA + SEAC-treated groups compared with the OVA + vehicle-treated group (Fig. 9C). These findings demonstrate that SEAC has the potential to stimulate airway remodeling through the regulation of goblet hyperplasia, peribronchiolar collagen layer thickness, subepithelial fibrosis and VEGF expression in the airways of the mouse model of OVA-induced asthma.