cochinchinensis roots fermented with Lactobacillus plantarum (BAL). The roots of A. cochinchinensis used in this study were collected from plantations in the Go-Chang area of Korea and dried in a drying machine (Ilshinbiobase, Dongducheon, Korea) at 60°C. Voucher specimens of A. cochinchinensis roots (WPC-14-003) were deposited in the functional materials bank of the PNU-Wellbeing RIS Center at Pusan National University. These samples were also identified by Dr. Shin Woo Cha at the Herbal Crop Research Division, National Institute of Horticultural & Herbal Science.

Firstly, to prepare aqueous fractions of unfermented A. cochinchinensis (UnF), 20 g of freeze-dried A. cochinchinensis roots were ground to a powder and a hot water extract prepared with 1.2 l of deionized distilled water (dH₂0) for 2.5 h in a hot water extractor (DW-290, Daewoong, Kyunggi, Korea). After finishing aqueous extraction, samples were filtered through filter paper (Whatman No. 2, Whatman, Brentford, UK) and then evaporated by using a rotary vacuum evaporator (EYELA, N-1100 series, Tokyo, Japan). Finally, UnF was used for fermentation after freeze-drying. Yield from the hot water extraction process was 60.7%.

The two bacterial strains (W. cibaria and Lactobacillus plantarum) used in the fermentation process were provided by Professor Hong Joo Son, Department of Life Science and Environmental Biochemistry, Pusan National University. To prepare fermented products, UnF powder was dissolved with 1% (w/v) in dH₂O (pH 5.3), and the mixture sterilized at 121°C for 15 min. After cooling at room temperature, W. cibaria or L. plantarum precultivated in lactobacilli MRS broth (Difco Laboratories, Detroit, MI) until the final cell density approached 10⁷ CFU/ml (OD600=0.1) were inoculated [5% (v/v)] to the UnF mixture. The mixture was then incubated in a shaking incubator (VS-8480; Vision Scientific, Bucheon, Korea) at 37°C at 200 rpm/min for 4.3 d. Fermented A. cochinchinensis products of W. cibaria (FPW) or L. plantarum (FPL) were obtained from the mixtures solution fermented with W. cibaria or L. plantarum by using centrifugation at 12,000 × g for 10 min.

To obtain the n-butanol fractions of UnF (BUnF), FPW (BAW) and FPL (BAL), an equal volume of butanol was firstly added to UnF, FPW or FPL. After vigorous mixing and incubating, the butanol phase was collected from each mixture by centrifugation at 12,000 × g for 10 min. Butanol extraction was repeated three times, after which all butanol phases were combined, evaporated under a rotary vacuum evaporator, freeze-dried, and stored at −20°C until use.

The inhibition activity of hyaluronidase against BUnF, BAW, and BAL was investigated by using a colorimetric assay based on the modified Morgan-Elson method (17–19) which measures the amount of N-acetyl-D-glucosamine produced from sodium hyaluronate. Briefly, 12 µl of BUnF, BAW, or BAL were mixed with 12 µl of hyaluronidase (10 mg/ml) in 0.1 M sodium acetate buffer (pH 3.5), then incubated in a water bath at 37°C. After pre-incubation for 20 min, the sample and hyaluronidase solution mixture was added to 12 µl of 12.5 mM calcium chloride to activate the hyaluronidase and then incubated for 20 min. To the activated hyaluronidase solution, 24 µl of sodium hyaluronate (6 mg/ml) dissolved in 0.1 M sodium acetate buffer (pH 3.5) was added and incubated for an additional 40 min. Next, the solution was amended with 12 µl of 0.4 N NaOH and 0.4 M potassium tetraborate, then vortexed, boiled for 3 min, and placed on ice to terminate the hyaluronidase activity. Subsequently, 360 µl of DMAB solution (p-dimethylaminobenzaldehyde 4 g in a solution of acetic acid 350 ml mixed with 50 ml of 10 N HCl) was added, and the sample placed in a 37°C water bath for 20 min. The absorbance of each test tube at 540 nm was measured by using a microplate reader (Tecan Sunrise, Tecan, Hombrechtikon, Switzerland). Hyaluronidase inhibition activity was estimated by determining differences in the absorbance values of the sample solutions.

The concentration of three bioactive compounds including total phenols and crude saponins in BUnF and BAW were measured as previously described. The amount of total phenols in BAW was determined according to the Folin-Ciocalteu method (20). Briefly, BUnF and BAW (20 µl) was mixed with 100 µl of 0.2 N Folin-Ciocalteu reagent for 5 min, after which 300 µl of 20% sodium carbonate was added. Following incubation at room temperature for 2 h, the absorbance of the reaction mixture was measured at 765 nm. A calibration curve was generated with Gallic acid as the standard. Total phenolic content was expressed in milligrams of gallic acid equivalents per gram of BAW.

The total crude saponins amount was detected as described previously (21). Briefly, BUnF and BAW dissolved in 30 ml dH₂O was repeatedly extracted with ethyl ether (50 ml) to remove lipid soluble substances. After collection of the aqueous layer, samples were further extracted with n-butanol (30 ml) three times. This layer was then concentrated by vacuum evaporation and lyophilization by using circulation extraction equipment (IKA Labortechnik, Seoul, Korea). Finally, total crude saponins was calculated by using the equation: crude saponin (mg/g)=A-B/S, where, A is the dry weight of the n-butanol layer (mg), B is the weight of the flask (mg), and S is the weight of the solid volume of the sample (g).

Protodioscin, a saponin, was identified in BUnF and BAW by using an ILC 3000 HPLC system (Interface Engineering Co. Ltd., Seoul, Korea) equipped with a Corona^® CAD^® detector (ESA Biosciences, Inc., Chelmsford, MA, USA). Chromatographic separation was performed on a CapCell PAK MG C18 (4.6×250 mm, particle size 5 µm; Shiseido Co., Ltd., Tokyo, Japan). The mobile phase consisted of solvent A (dH₂O) and solvent B (acetonitrile) prepared by using a gradient elution program: 0–25 min with 30–90% solvent B and 25–40 min with 90% solvent B. A flow rate of 1.0 ml/min was used for sample analysis. The nebulizer gas was compressed nitrogen. Gas flow rate and gas pressure were maintained at 1.53 l/min and 35±2 psi, respectively. The output signal of the detector was recorded by using Clarity™ chromatography software (DataApex, Prague, Czech Republic).

The scavenging activity of the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical against BAW was measured as previously described (22). Briefly, each sample (100 µl) in eight different concentrations of BAW (7.8 to 1,000 µg/ml) was mixed with 100 µl of 0.1 mM DPPH (Sigma-Aldrich Co., St. Louis, MO, USA) in 95% ethanol solution or 100 µl of 95% ethanol solution, then incubated for 30 min at room temperature. Next, the absorbance of the reaction mixture was measured at 517 nm by using a Versa-max plate reader (Molecular Devices, Sunnyvale, CA, USA). The DPPH radical scavenging activity of the BAW was expressed as the percent decrease in absorbance relative to that in the control. The IC₅₀ value is defined as the concentration of substrate that produces a 50% loss in DPPH activity.

The RAW264.7 cell line used in this study consists of macrophage cells originating from the Abelson murine leukemia virus-induced tumor provided by the Korean Cell Line Bank (Seoul, Korea). The RAW264.7 cells were grown with Dulbecco's Modified Eagle's Medium (DMEM, Thermo Scientific, Waltham, MA, USA) containing 10% fetal bovine serum (FBS, S001-01, Welgene, Gyeongsan-si, Korea), L-glutamine, penicillin, and streptomycin (Thermo Scientific) in a humidified incubator at 37°C under 5% CO₂ and 95% air.

Cell viability was determined by using tetrazolium compound 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich Co.). To determine cell viability, RAW264.7 cells were seeded at a density of 5×10⁴ cells/0.2 ml and grown for 24 h in a 37°C incubator. When the cells attained 70–80% confluence, they were either untreated (No) or treated with DMSO (Vehicle), or pretreated with 100 µg/ml (BAWLo) or 200 µg/ml (BAWHi) of BAW dissolved in DMSO. Following incubation for 24 h, the supernatants were discarded, after which 0.2 ml of fresh MEM media and 50 µl of MTT solution [2 mg/ml in phosphate buffered saline (PBS)] were added to each well. The cells were then incubated at 37°C for 4 h. Formazan precipitate was dissolved in DMSO, after which the absorbance at 570 nm was read directly in the wells by using a Molecular Devices VERSA max Plate reader (Sunnyvale, CA, USA). The morphological features of RAW264.7 cells in each treated group were observed by using a microscope (Leica Microsystems, Switzerland).

NO concentration in the culture supernatant of RAW264.7 cells was measured by using Griess reagent [1% sulfanilamide, 5% phosphoric acid, 0.1% N-(1-naphthyl) ethylenediamine dihydrochloride; Sigma-Aldrich Co.] as described previously (23). Briefly, RAW264.7 cells were treated with Vehicle or BAW (100 or 200 µg/ml) for 2 h followed by LPS (1 µg/ml) for 24 h. Following 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 by using a VersaMax microplate reader (Molecular Devices).

Intracellular ROS levels in RAW264.7 cells were measured by staining with 2′,7′-dichlorofluorescein diacetate (DCFH-DA; Sigma-Aldrich Co.), which is a cell permeable, nonfluorescent agent that can be deacetylated by intracellular esterases to form nonfluorescent DCFH. In the presence of ROS, DCFH is converted to highly fluorescent DCF intracellularly. Briefly, RAW364.7 cells were seeded at 5×10⁵ cells/2 ml in 6-well plates, then grown with two different concentrations of BAW for 2 h in a 37°C incubator. After washing once with 1× PBS, the cells were incubated with 1 µg/ml of LPS for another 24 h. Next, cells were incubated with 25 µM DCFH-DA for 30 min at 37°C. Finally, the cells were washed twice with PBS, after which the green fluorescence was observed at 200× magnification via fluorescence microscopy (Eclipse TX100, Nikon, Tokyo, Japan).

Total homogenate of RAW264.7 cells obtained by using Pro-Prep Protein Extraction Solution (iNtRON Biotechnology, Seongnam, Korea) was centrifuged at 13,000 rpm/min for 5 min, then the concentration of total protein was quantified by using a SMARTTM BCA protein assay kit (Thermo Scientific) for western blotting. Briefly, the proteins were separated by 4–20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for 2 h, after which resolved proteins were transferred to nitrocellulose membranes for 2 h at 40 V. Each membrane was then incubated overnight separately at 4°C with the following primary antibodies: SAPK/JNK antibody (Cell Signaling Technology, Danvers, MA, USA), p-SAPK/JNK (Thr183/Tyr185) antibody (Cell Signaling Technology), ERK1 (K-23) antibody (Santa Cruz Biotechnology, Inc. Santa Cruz, CA, USA), p-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) antibody (Cell Signaling Technology), p38 MAPK antibody (Cell Signaling Technology), p-p38 MAP Kinase (Thr180/Tyr182) antibody (Cell Signaling Technology), and anti-actin antibody (Sigma-Aldrich Co.). Next, the membranes were washed with washing buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, and 0.05% Tween 20) and incubated with 1:1,000 diluted horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Invitrogen, Carlsbad, CA, USA) at room temperature for 1 h. Finally, membrane blots were developed by using Amersham ECL Select western blotting detection reagent (GE Healthcare, Little Chalfont, UK). The chemiluminescence signals that originated from specific bands were detected by using FluorChemi^®FC2 (Alpha Innotech, San Leandro, CA, USA).

The concentration of cytokine IL-6 in the culture supernatant of RAW264.7 cells treated with BAW was determined by using an IL-6 ELISA kit (Biolegend, San Diego, CA, USA) according to the manufacturer's instructions. Briefly, RAW264.7 cells were treated with two different concentrations of BAW (100 or 200 µg/ml) for 2 h, followed by 1 µg/ml of LPS for 24 h. After collection of the supernatant, 100 ml of serial dilutions of the culture supernatant were added to a 96-well plate coated with anti-IL-6 antibody and then incubated for 2 h at room temperature. After five washes with wash solution (PBS, 0.05% and Tween-20, pH 7.4), 100 µl of avidin-horseradish peroxidase solution was added to each well, and the plates were allowed to develop at 37°C for 2 h. After five washes with wash solution, the plate was maintained at 37°C for 30 min to react with 100 µl of substrate solution. The reaction was then stopped by the addition of 100 µl of blocking solution, after which the absorbance at 450 nm was read with a VersaMax microplate reader (Molecular Devices).

The mRNA levels of iNOS, COX-2, TNF-α, IL-1β, and IL-6 were measured by RT-PCR as previously described (24). First, total RNA molecules were purified by removing media from a subset group of cells and then homogenizing the cells in RNAzol CS104 (Tel-Test Inc., Friendswood, USA). The isolated RNA was then quantified by using UV spectroscopy. Expressions of the target genes were assessed by using RT-PCR with 5 µg of total RNA from cells of each group. Next, 500 ng of oligo-dT primer (Invitrogen, Carlsbad, CA, USA) were annealed at 70°C 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. Amplification was conducted in a Perkin-Elmer Thermal Cycler and used the following cycle: 30 sec at 94°C, 30 sec at 62°C, and 45 sec at 72°C. The primer sequences for target gene expression identification were as follows: iNOS, sense primer: 5′-CACTTGGAGTTCACCCAGT-3′, anti-sense primer: 5′-ACCACTCGTACTTGGGATGC-3′; COX-2, sense primer: 5′-CAGGTCATTGGTGGAGAGGTGTATC-3′, anti-sense primer: 5′-CCAGGAGGATGGAGTTGTTGTAGAG-3′; TNF-α, sense primer: 5′-CCTGTAGCCCACGTCGTAGC-3′, anti-sense primer: 5′-TTGACCTCAGCGCTGACTTG-3′; IL-1β, sense primer: 5′-GCACATCAACAAGAGCTTCAGGCAG-3′, anti-sense primer: 5′-GCTGCTTGTGAGGTGCTGATGTAC-3′; and IL-6, sense primer: 5′-TTGGGACTGATGTTGTTGACA-3′, anti-sense primer: 5′-TCATCGCTGTTGATACAATCAGA-3′. The experiment was repeated three times, and all samples were analyzed in triplicate. The final RT-PCR products were separated on 1–2% agarose gel and then visualized by ethidium bromide staining. The densities of specific bands were quantified by using the Kodak Electrophoresis Documentation and Analysis System 120 (Eastman Kodak, Rochester, NY).

One-way ANOVA was used to identify significant differences between No-treated and LPS-treated groups (SPSS for Windows, Release 10.10, Standard Version, Chicago, IL, USA). Differences in the responses of the Vehicle+LPS-treated group and the BAW+LPS-treated groups were evaluated by using a post hoc test (SPSS for Windows, Release 10.10, Standard Version). All values are reported as the mean ± standard deviation (SD) and a P-value of <0.05 was considered significant.

To enhance the anti-inflammatory activity of A. cochinchinensis roots, fermentation technique was firstly applied to these roots using two bacterial strains (W. cibaria and L. plantarum). Anti-inflammatory activity and toxicity were measured in the two final products (BAW and BAL) obtained from fermented A. cochinchinensis roots using butanol extraction. As shown Table I, a significance alternation was detected in hyaluronidase inhibition rate and suppression rate of NO concentration. Hyaluronidase inhibition rate was dramatically enhanced after fermentation although this level was slightly higher with 18–21% in BAW than BAL. A similar pattern to that observed for the hyaluronidase inhibition rate was also observed in the suppression rate of NO concentration. After fermentation, the suppression rate of NO concentration was increased regardless of their concentration. But, the increase of these level was greater with 57–64% in BAW than BAL. Meanwhile, any significance differences were not detected in cell viability of group treated with BUnF, BAW and BAL. Therefore, the present results suggest that the fermentation with W. cibaria can successfully change from the roots of A. cochinchinensis to the fermented products with high anti-inflammatory activity and non-toxicity. Also, above results showed that BAW can be considered as the better candidate for suppressing the inflammatory response.

To identify the key components altered by fermentation, the concentration of several key components were analyzed in BUnF and BAW. As shown in Fig. 1A, BUnF contained significantly different concentrations of total phenolic compounds (1.82 mg/g) and total crude saponins (150 mg/g). After fermentation, the total phenolic and crude saponin concentrations in BAW were significantly increased with 9.3, 126.6%, respectively. Furthermore, the protodioscin concentration, detected as a sharp specific peak in the HPLC curve, was also higher in BAW (Fig. 1B). These results demonstrate that concentrations of several key components that associated with high anti-oxidative capacity can be increased in the extracts of roots of A. cochinchinensis after fermentation with W. cibaria.

To investigate the antioxidative capacity of BAW, alterations in DPPH scavenging activity and the inhibitory activity of intracellular ROS production were measured in RAW264.7 cells treated with BAW. The scavenging activity against the DPPH radical was gradually enhanced with the increase in BAW concentration (7.8 and 1,000 µg/ml), and the IC₅₀ value for BAW was 31.62 µg/ml (Fig. 2A). In addition, the intracellular level of ROS was notably enhanced after LPS treatment. However, the ROS level markedly decreased with depending on BAW concentration increase in LPS+BAWLo and LPS+BAWHi treated group (Fig. 2B). These results indicate that BAW may have a high anti-oxidative capacity that is obtained via suppression of intracellular ROS production and DPPH radical scavenging activity.

The signaling pathway for iNOS-mediated COX-2 induction has a key role during the inflammation process (25). Thus, we investigated the suppressive effects of BAW on the potential iNOS-mediated COX-2 induction pathway in LPS-activated Raw264.7 cells after BAW pretreatment. Firstly, we observed that the enhanced NO concentration after LPS treatment was dramatically decreased in the BAW-treated group (Fig. 3A). Also, the levels of the iNOS and COX-2 gene transcripts were higher in the Vehicle+LPS-treated group than that in the No-treated group. However, these levels were slightly decreased in BAWHi+LPS-treated Raw264.7 cells (Fig. 3B). These results suggest that BAW pretreatment can recover the signaling pathway for iNOS-mediated COX-2 induction during the inflammatory process.

To investigate whether alteration of iNOS and NO level accompanies a change in the MAP kinase pathway, the phosphorylation levels of ERK, JNK and p38 were measured in LPS-stimulated RAW 264.7 cells treated with BAW. As shown in Fig. 4, the phosphorylation levels of the three members were higher after LPS activation with various ratios than they were after No treatment. However, the levels of only ERK and JNK were significantly decreased after BAW pretreatment, while the level of p38 was continuously increased with depending on BAW concentration (Fig. 4). Therefore, the present data suggest that the suppression of iNOS and NO induced by BAW treatment may exert it regulatory activity by controlling the ERK and JNK in MAP kinase pathway.

To investigate alteration of the expression of pro-inflammatory and anti-inflammatory cytokines during an anti-inflammatory response induced with BAW treatment, the transcript levels of TNF-α, IL-1β, and IL-6 were measured by performing RT-PCR of LPS-activated Raw264.7 cells. Among the three cytokines, IL-1β and IL-6 showed a suppression pattern in the BAW+LPS-treated group. In contrast, the TNF-α transcript maintained a constant level (Fig. 5A and B). Furthermore, the concentration of IL-6 in the culture supernatant of LPS-activated Raw264.7 cells was measured by ELISA in order to confirm the RT-PCR results. The IL-6 concentrations in the culture supernatant completely reflected the results for the IL-6 transcripts, although a few differences were observed in the BAWLo+LPS-treated group (Fig. 5B). Therefore, these results indicate that BAW pretreatment suppresses the enhancement of pro-inflammatory cytokine expressions that were induced by LPS treatment in RAW264.7 cells.