The roots of A. cochinchinensis used in this study were harvested from plantations in the Go-Chang area in Korea and dried in a drying machine (FD5510S-FD5520S, Ilshinbiobase Co., Dongducheon, Korea) at 60℃. 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. Dried roots of A. cochinchinensis were reduced to powder using a pulverizer (MF-3100S, Hanil Electric Co., Seoul, Korea), after which EaEAC was obtained at 50℃ 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). These solutions 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) (Figure 1A). Finally, the collected EaEAC powder was dissolved in 0.5% Tween 20 Solution (in dH₂O) to 400 mg/kg, then further diluted to the required concentration.

Polyphenolic contents in EaEAC were measured by the Folin-Ciocalteu method as previously described [42]. Briefly, a mixture of EaEAC solution (1 mL) and Folin-Ciocalteu reagent (5 mL, Sigma-Aldrich Co., St. Louis, MO, USA) was incubated at room temperature for 5 min. The mixture was subsequently added to 15 mL of 20% Na₂CO₃ and vortexed for 30 sec, after which the absorbance was repeatedly measured at 765 nm using a Versa-max plate reader (Molecular Devices, Sunnyvale, CA, USA). A standard calibration curve was made using different concentrations of gallic acid (Sigma-Aldrich Co.), and the concentration of polyphenolic contents in EaEAC was presented as mg gallic acid equivalent of extract.

The flavonoid contents were measured as previously described [43]. Briefly, 200 µL of several different concentrations of EaEAC were mixed with 60 µL of 5% NaNO₂ (Sigma-Aldrich Co.) and 60 µL of 10% AlCl₃ (Sigma-Aldrich Co.). Following incubation at 25℃ measured as previously described [43]. A standard calibration curve was then made using different concentrations of catechin (Sigma-Aldrich Co.). The flavonoid contents in EaEAC are presented as mg catechin equivalent of extract.

Total saponins content was measured by the Helaly method as previously described [44]. The extract of n-butanol was redissolved in 0.5 mL of 80% MeOH, then mixed with 0.5 mL of 8% vanillin in ethanol and 5 mL of 72% H₂SO₄ in water. These mixtures were placed in a 60℃ water bath for 20 min, then cooled at 0℃ for 5 min, after which the absorbance was measured at 544 nm using a Versa-max plate reader (Molecular Devices). The saponin content was calculated from a calibration curve constructed using purified saponin standard (Sigma Sigma-Aldrich Co.).

IL-4/Luc/CNS-1 Tg mice were kindly provided by the Department of Laboratory Animals Resources at the National Institute of Food and Drug Safety Evaluation (Osong, Korea), and HR1 mice of the same age for mating were purchased from Central Lab Animal Inc. (Seoul, Korea). All mice were provided with ad libitum access to standard irradiated chow (Samtako, Osan, Korea) and autoclaved water throughout the experimental period. Moreover, the mice used in this study were maintained in a specific pathogen-free state under a strict light cycle (lights on at 08:00 hours and off at 20:00 hours) at 23±2℃ and 50±10% relative humidity. The mice were housed in the Pusan National University-Laboratory Animal Resources Center accredited by the Korea Food and Drug Administration (FDA) in accordance with the Laboratory Animal Act (Accredited Unit Number-000231) and AAALAC International according to the National Institutes of Health guidelines (Accredited Unit Number; 001525).

The protocols for the animal experiment used in this study were carefully reviewed for ethical and scientific care procedures and approved by the Pusan National University-Institutional Animal Care and Use Committee (PNU-IACUC; Approval Number PNU-2015-0976). IL-4/Luc/CNS-1 Tg mice (eight-week-old, n=20) were randomly divided into one of two groups. In the first group (AOO, n=5), 100 µL of AOO (4:1 acetone: olive oil, v/v: AOO) was spread on the dorsum of the ears every day for 2 weeks as a control. In the second group (PA, n=15), 100 µL of 15% PA solution in AOO was repeatedly spread on the dorsum of the ears every day for 2 weeks. The second group was further divided into three treatment groups, PA+Vehicle, PA+EaEAC2, and PA+EaEAC4, which received PA in combination with a comparable volume of 0.5% tween 20 solution (in dH₂O) and 200 or 400 mg/kg EaEAC, respectively, via oral administration for the same period.

Production and identification of IL-4/Luc/CNS-1 Tg mice were performed as previously described [4546]. Briefly, founder mice were produced by breeding IL-4/Luc/CNS-1 Tg mice and HR1 mice. The IL-4/Luc/CNS-1 transgenes in the founder mice were then identified by DNA-PCR of tail-derived genomic DNA using specific primers. For DNA-PCR, 10 pmol each of sense (5'-CTC GCA TGC CAG AGA TCC TA-3') and antisense (5'-CCA CAA CCT TCG CTT CAA AA-3') primers were added into the genomic DNA template mixture. Amplification was then conducted in a thermal cycler (Perkin-Elmer, Waltham, MA, USA) by subjecting the samples to 25 cycles of 30 sec at 94℃, 30 sec at 62℃, and 45 sec at 72℃. Next, the amplified PCR products were separated by 1% agarose gel electrophoresis, after which the band patterns were detected using a Kodak Electrophoresis Documentation and Analysis System 120 (Eastman Kodak, Rochester, NY, USA).

The ear morphology and ear thickness were measured as previously described [1415]. Changes in ear color, ear vein thickness and other morphological characteristics of IL-4/Luc/CNS-1 Tg mice were analyzed using photographs. Additionally, ear thickness was measured to investigate the degree of skin inflammation induced by PA+Vehicle and PA+EaEAC treatment using a thickness gauge (Digimatic Indicator, Matusutoyo Co., Tokyo, Japan).

The body weights of all mice were measured throughout the experimental period using an electronic balance (Mettler Toledo, Greifensee, Switzerland). Additionally, lymph nodes were collected from sacrificed mice and weighed using an electronic balance (Mettler Toledo).

IgE concentration in the serum of IL-4/Luc/CNS-1 Tg mice was measured using an enzyme-linked immunosorbent assay (ELISA) kit (Shibayagi Inc., Gunma, Japan) according to the manufacturer's instructions. Briefly, serum samples and standards diluted 20 fold with dilution solution (50 µL) were added to wells coated with antibody, after which the plate was incubated for 2 h at room temperature. Next, the wells were washed with washing solution (50 mM Tris, 0.14M NaCl, 0.05% Tween 20, pH 8.0), after which 50 µL of biotin-conjugated avidin was added and the samples were incubated for 2 h. Horseradish peroxidase-conjugated detection antibodies were then diluted 5,000 fold with conjugate diluent (50 mM Tris, 0.14 M NaCl, 1% BSA, 0.05% Tween 20, pH 8.0) and transferred to each well. Following incubation of the plates at room temperature for 1 h, an enzyme reaction was initiated by adding substrate solution and incubating the plates at room temperature in the dark for 20 min. Finally, the reaction was terminated by adding 2 M H₂SO₄ solution and the absorbance was measured at 450 nm.

Ear tissues were removed from mice of subset groups, fixed in 10% formalin, embedded in paraffin wax, routinely processed, and then sectioned into 4 µm thick slices. Next, the ear tissue sections were stained with hematoxylin and eosin (H&E), after which they were examined by light microscopy for the presence of immune cell accumulation at 400× magnification. The thickness of the epidermis and dermis were also measured using the Leica Application Suite (Leica Microsystems, Wetzlar, Germany). In addition, the infiltration of mast cells into the ear tissue was detected by staining with toluidine blue as described in previous studies [47]. After deparaffinization and dehydration, ear skin sections were stained with 0.25% toluidine blue (Sigma-Aldrich Co.) and examined by light microscopy for the presence of mast cells at 400× magnification. The number of cells per specific area was determined using the Leica Application Suite (Leica Microsystems).

The relative quantities of mRNA for TNF-α and IL-1β were measured by RT-PCR. First, lymph nodes were frozen in liquid nitrogen, then chopped with scissors and homogenized in RNAzol B solution (Tet-Test Inc., TX, USA). Total RNA molecules were isolated by centrifugation at 15,000×g for 10min, then measured by UV spectroscopy. The expression of target genes was assessed using RT-PCR with 3 µg of total RNA from tissue of each group. Next, 500 ng of oligo-dT primer (Invitrogen, Carlsbad, CA, USA) were annealed at 70℃ for 10 min. The complementary DNA (cDNA), which was used as the template for further amplification, was synthesized by the addition of deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), and deoxythymidine triphosphate (dTTP) with 200 units of reverse transcriptase (Superscript II, Invitrogen, 200 U/µL). Next, 10 pmol of the sense and antisense primers were added, and the reaction mixture was subjected to 28–32 cycles of amplification in a Perkin-Elmer Thermal Cycler. The following temperature cycle was used for PCR: 30 sec at 94℃, 30 sec at 62℃, and 45 sec at 72℃. The primer sequences for target gene expression identification were as follows: TNF-α, sense primer: 5'-CCT GTA GCC CAC GTC GTA GC-3', anti-sense primer: 5'-TTG ACC TCA GCG CTG ACT TG-3'; IL-1β, sense primer: 5'-GCA CAT CAA CAA GAG CTT CAG GCA G-3', anti-sense primer: 5'-GCT GCT TGT GAG GTG CTG ATG TAC-3'; β-actin, sense primer: 5'-TGG AAT CCT GTG GCA TCC ATG AAA C-3', anti-sense primer: 5'-TAA AAC GCA GCT CAG TAA CAG TCC G-3'. The experiment was repeated three times, and all samples were analyzed in triplicate. The final PCR products were separated on 1% agarose gel, then visualized by ethidium bromide staining.

In vivo imaging analysis to detect the luciferase-derived signal was conducted using an IVIS imaging system (Xenogen, Oakland, CA, USA) as previously described [1415]. Briefly, IL-4/Luc/CNS-1 Tg mice were anesthetized with Alfaxan and injected i.p. with 150 mg/kg of D-luciferin (Sigma-Aldrich Co.). Ten minutes after D-luciferin injection, images of the whole body and several organs (lung, kidney, spleen, heart, mesenteric lymph node (ML), submandibular lymph node (SL), thymus and pancreases) of mice from subset groups were taken for 3 min using an IVIS imaging system, after which the photons emitted from specific regions and organs were quantified using the Living Image software (Xenogen). The in vivo luciferase activity was then expressed in photons per second.

One-way ANOVA was used to identify significant differences between the PA and AOO treated groups (SPSS for Windows, Release 10.10, Standard Version, Chicago, IL, USA). Additionally, differences between the PA+Vehicle treated group and the PA+EaEAC treated group were evaluated by a post hoc test (SPSS for Windows, Release 10.10, Standard Version) of the variance and significance levels. All values were expressed as the means±SD. A P value of <0.05 was considered significant.

First, the concentration of two important antioxidants was determined in EaEAC to evaluate the potential for use as an antioxidant. As shown in Figure 1B, EaEAC contained 88.5 µg/mL of flavonoids and 102.1 µg/g of polyphenol, as well as a high concentration of total saponin (57.2±1.47 mg/g).

We first determined if EaEAC treatment suppresses changes in ear phenotypes induced by PA treatment. To accomplish this, the ear thickness and morphology were observed in IL-4/Luc/CNS-1 Tg mice over 2 weeks. The ear thickness was higher in the PA treated group than the AOO treated groups; however, this level decreased rapidly in the PA+EaEAC2 and PA+EaEAC4 treated groups, although no significant difference was observed in either group (Figure 2A). Furthermore, the outline of the ear vein, ear color and scab were dramatically recovered in the PA+EaEAC treated group (Figure 2A). Therefore, these findings demonstrate that EaEAC treatment may successfully relieve the increase of ear thickness and morphology including vein diameter and color induced by PA treatment.

We investigated the effects of EaEAC treatment on body weight, lymph node weight and IgE concentration in the PA-induced skin inflammation of IL-4/Luc/CNS-1 Tg mice. Lymph node weight increased dramatically in response to topical application of PA, although the body weight remained at a constant level (Figure 2B, 2C). However, the lymph node weight decreased significantly by 17% and 70%, respectively, in the PA+ EaEAC2 and PA+EaEAC4 group (Figure 2C).

Additionally, repeated topical application of PA solution induced a rapid increase of serum IgE concentrations in IL-4/Luc/CNS-1 Tg mice. However, these levels decreased significantly by 19% and 25% in the PA+EaEAC2 and PA+EaEAC4 treated groups, respectively (Figure 3). Taken together, these results suggest that EaEAC treatment may contribute to the decrease in the weight of lymph nodes, as well as recovery of the IgE concentration in IL-4/Luc/CNS-1 Tg mice treated with PA.

To investigate the suppressive effects of EaEAC treatment on histological structure of ear tissue, the histopathological features of ear tissue of IL-4/Luc/CNS-1 Tg mice were evaluated after EaEAC treatment for two weeks. The thickness of the epidermis and dermis of the ear tissue was increased in the PA+Vehicle treated group relative to the AOO treated group. However, these levels decreased significantly by 62–72% (dermis) and 29% (epidermis) after EaEAC treatment, and this decrease was does dependent (Figure 4A, 4B). In addition, the PA+EaEAC treated group showed the disappearance of infiltrated lymphocytes, while the PA+Vehicle treated group showed a large number of lymphocytes (Figure 4A). Furthermore, large numbers of mast cells were stained blue in the dermis in the PA+Vehicle group relative to the AOO treated group. However, the number of stained cells was significantly decreased in the PA+EaEAC2 and PA+EaEAC4 (15%) treated groups (Figure 4). Therefore, the present results indicate that EaEAC treatment may improve the histological changes in PA-induced skin inflammation, as well as contribute to the suppression of mast cells infiltration in the dermis of ear skin.

To determine if EaEAC treatment could induce alterations in the inflammatory cytokines in PA-induced skin inflammation, the level of IL-1β and TNF-α transcripts were measured in the lymph nodes of AOO, PA+Vehicle and PA+EaEAC treated group. The pattern of IL-1β and TNF-α expression in AOO and PA treat group was similar to the intensity of the luciferase signal, although the rate of decrease varied among groups. The expression of IL-1β and TNF-α was generally higher in the PA+Vehicle treated group than the AOO treated group. However, these levels in the PA+EaEAC2 and PA+EaEAC4 groups were dramatically decreased to the level of the AOO treated group (Figure 5). Overall, the present results suggest that suppression of IL-1β and TNF-α expression by EaEAC treatment contributes to the relief of skin inflammation.

Finally, we investigated the role of IL-4 cytokines during the anti-inflammatory action of EaEAC in IL-4/Luc/CNS-1 Tg mice. To achieve this, the luciferase signal, which is a marker indicating the skin inflammation in PA treated IL-4/Luc/CNS-1 Tg mice, were measured throughout the body and in eight organs of IL-4/Luc/CNS-1 Tg mice after EaEAC treatment using the Living Image software. In case of the whole body image, the luciferase signals were higher in the abdominal region of the PA+Vehicle group than that of the AOO treated group. However, the increase in the signal with PA treatment decreased significantly as the dose of EaEAC increased (Figure 6A, 6C). As shown in the organ image, high luciferase signals were observed in SL and ML among the eight investigated organs of the PA+Vehicle group, while extremely low levels of signal were observed in the same organs of the AOO treated group. However, the luciferase signal was reduced in the ML and SL of the PA+EaEAC2 and PA+EaEAC4 group, and complete recovery was observed in the PA+EaEAC4 group (Figure 6B, 6C). Therefore, these results indicate that overactivation of the IL-4 promoter can successfully reflect alterations in the phenotypes of skin inflammation, and that IL-4 may play an important role in the suppression of EaEAC in PA-induced skin inflammation.