Anti-Inflammatory Effects of Fermented Bark of Acanthopanax sessiliflorus and Its Isolated Compounds on Lipopolysaccharide-Treated RAW 264.7 Macrophage Cells

The fermentation was carried out on the bark of Acanthopanax sessiliflorus (AS). Acanthopanax species have been used in traditional medicine as tonics, sedatives, and antispasmodics. An activity-guided isolation of the fermented bark of A. sessiliflorus (FAS) yielded several phytochemicals: acanthoside D (1), acanthoside B (2), daucosterol (3), protocatechuic acid (4), chlorogenic acid methyl ester (5), ciwujiatone (6), syringaresinol (7), farnesol (8), 3,4-dicaffeoylquinic acid (9), and falcarindiol (10). HPLC analysis showed that content of lignan glycoside (1) was decreased and 4 and 7 were increased after fermentation. Anti-inflammatory activities on FAS showed the decrease of nitric oxide (NO) production, and inhibitory activities of iNOS and COX-2, proinflammatory cytokines (IL-6 and tumor necrosis factor-α), and collagenase. The aglycone, syringaresinol (7), which was increased through fermentation showed enhanced activity than 1. Thus, FAS may have the potential to treat inflammatory disorders, such as arthritis.

e major active component in these plants is lignan, and it has been determined that lignan plays a key role in the therapeutic effects [9,10].
Various molecules are involved in the induction and maintenance of inflammatory responses. In addition to major cytokines, such as interleukin-(IL-) 1, IL-6, and tumor necrosis factor-(TNF-) α, prostaglandin and nitric oxide (NO), which is synthesized by inducible NO synthase (iNOS), are important chemical mediators of inflammation [11]. Prostaglandin is synthesized by two cyclooxygenase (COX) isoforms, COX-1 and inducible COX-2. erefore, the inhibition and/or downregulation of these proinflammatory molecules may exert anti-inflammatory effects in arthritis [12].
Recent studies have been conducted to enhance the physiological activity of materials by fermentation using microorganisms such as fungi, yeast, lactic acid bacteria, and mushrooms. e benefits include nonsecondary pollution, a mild reaction, and the low cost of biotransformation. Microorganisms have many bioconversion elements. As a part of our previous related work [13], inhibitory activities of nitric oxide (NO) production, iNOS and COX-2, proinflammatory cytokines (IL-6 and tumor necrosis factor-α), and collagenase in lipopolysaccharide-(LPS-) treated RAW 264.7 macrophage cells were evaluated on the biotransformation product of A. sessiliflorus.

Plant Material.
In April 2018, the bark of A. sessiliflorus was collected from Pyeongtaek, Republic of Korea, and certified by Dr. Kim (National Arboretum, Pocheon). In addition, a voucher specimen (201804-AS) was deposited at the herbarium of the College of Pharmacy, Chung-Ang University.

Fermentation.
Lactobacillus plantarum subsp. argentoratensis was selected for this study, on the basis of a previous study [14]. L. plantarum subsp. argentoratensis was purchased from the Korean Agricultural Culture Collection (Seoul, Korea), inoculated into MRS broth, and grown at 37°C and 200 rpm for 48 h. en, 1% (v/v) L. plantarum subsp. argentoratensis was inoculated into MRS medium with 1% (w/v) A. sessiliflorus extract, and fermentation was performed at 37°C and 200 rpm for 5 days. After fermentation, centrifugation was performed for 20 min at 3000 rpm, and the supernatant was evaporated to obtain the fermented A. sessiliflorus (FAS) extract.

HPLC Analysis.
e compounds in the FAS extract were compared with those in the AS extract by using highpressure liquid chromatography (HPLC). e mobile phase consisted of solvent A (0.2% acetic acid in H 2 O) and B (acetonitrile (ACN); Table 1). e extracts were dissolved in 50% MeOH.

Measurement of Cell Viability.
e cytotoxicity was measured by the mitochondrial-dependent reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT; Sigma, St. Louis, MO, USA) to formazan. RAW 264.7 cells were seeded in 96-well plates at a density of 1 × 10 5 cells/mL. After incubation for 16 h at 37°C, the cells were treated with 20 μL of each sample in serum-free DMEM and incubated at 37°C in a humidified atmosphere for 24 h. e medium was then removed, and MTT solution (100 μL; 0.5 mg/mL) was added to each well. After incubation for 4 h, the supernatant was aspirated. e formazan crystals in each well were dissolved in 100 μL dimethyl sulfoxide, and the absorbance at 540 nm was measured by using an enzymelinked immunosorbent assay (ELISA) plate reader (Tecan Co. Ltd., Salzburg, Australia). Relative cell viability was evaluated from the quantity of MTT converted to the insoluble formazan salt compared with distilled water as the control.
e cell viability was calculated as given in the following: 2.8. Analysis of Inhibition of NO Production. RAW 264.7 cells (2 × 10 5 cells/well) were seeded in 96-well plates and incubated for 16 h at 37°C in a humidified atmosphere (approximately 5% CO 2 ). e cells were incubated in a serum-free medium containing the sample and 1 μg/mL LPS (Sigma, St. Louis, MO, USA). After incubation for an additional 20 h, the NO content was evaluated by using the Griess assay. e supernatant was obtained, Griess reagent (0.1% naphthylethylenediamine and 1% sulphanilamide in 5% H 3 PO 4 solution; Sigma, St. Louis, MO, USA) was added, and the absorbance at 540 nm was recorded. N G -monomethyl-L-arginine monoacetate salt (L-NMMA) was used as the positive control. e inhibition of NO production was calculated from equation (2). e IC 50 value was defined as the concentration that inhibited 50% of NO production: 2 Evidence-Based Complementary and Alternative Medicine

Determination of Proinflammatory Cytokine Levels.
e concentrations of IL-6 and TNF-α were determined by using ELISA. RAW 264.7 cells (1 × 10 6 cells/well) were seeded in 6-well plates and preincubated for 16 h; subsequently, the samples were treated with LPS (1 μg/mL) for 24 h to induce the production of cytokines. e supernatant was evaluated using an ELISA kit (Youngin Frontier, Seoul, Korea) in accordance with the manufacturer's instructions. e cytokine concentrations were quantified by measuring the absorbance at 450 nm. e cell lysates were prepared in RIPA buffer (50 mM/L Tris-HCl [pH 7.4], 150 mM/L NaCl, 1% Triton X-100, 0.1% sodium dodecyl sulfate (SDS), and 1 mM/L EDTA; ermo Fisher Scientific, MA, USA) for 30 min on ice. e cell lysates were centrifuged at 13, 000 × g for 15 min at 4°C, and 30 μg of the cell lysate was separated by to 7.5% SDS-polyacrylamide gel electrophoresis. e separated proteins were transferred onto a PVDF membrane (Bio-Rad, CA, USA). Nonspecific binding to the membrane was blocked by incubation with blocking buffer ( ermo Fisher Scientific) for 60 min at room temperature. en, the membrane was incubated with anti-mouse iNOS (1 : 500; Santa Cruz, CA, USA) and anti-mouse COX-2 (1 : 1000; BD Biosciences Pharmingen, CA, USA) overnight at 4°C. After washing, the blots were incubated with horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody (1 : 1000; Santa Cruz) for 60 min at room temperature. e bands were visualized by using the LAS-4000 luminescent image analyzer (GE Healthcare Life Sciences, NJ, USA) and ECL detection reagent (GE Healthcare Life Sciences).

Collagenase Assay.
e collagenase assay was performed as described previously [15]. Collagenase (5 μg) and 4-phenylazobenzyloxycarbonyl-L-Pro-L-Leu-Gly-L-Pro-D-Arg (0.5 mg), as a substrate of collagenase, were added to 0.1 M Tris buffer (pH 7.4) in the presence or absence of samples to a total volume of 1.7 mL. e mixture was incubated at 37°C for 30 min, and 1 mL of 25 mM citric acid solution was added to terminate the enzyme reaction. Ethyl acetate (5 mL) was added, and the absorbance of the organic layer was measured by using UV spectrophotometry at 320 nm to calculate the inhibitory activity. e collagenase inhibitory activity was calculated from equation (3), where OD control � OD of the control with collagenase-OD of the control without collagenase, and OD sample � OD of the test sample with collagenase-OD of the test sample without collagenase: 2.12. Statistical Analysis. All data were expressed as mean ± SD values and were evaluated by using one-way analysis of variance (ANOVA), followed by the Student-Newman-Keuls (S-N-K) test; statistical analyses were computed using Statistical Package for the Social Sciences (SPSS, Chicago, IL, USA) software package. Values were considered significantly different if the p value was less than 0.05. Different superscripts in the same column indicate that values are significantly distinct from the other data.

HPLC Analysis Results.
e compounds in the FAS were compared with AS by using HPLC. In this study, the remarkable differences were found in the contents of several compounds that acanthoside D (1) was lower and syringaresinol (7) and protocatechuic acid (4) were higher in FAS than in AS (Figure 2, Table 2).

Cell Viability.
Prior to the biological assays, the MTT assays were performed using the extracts and each compound from the FAS extract. e extracts and compounds did not affect the cell viability (>80%) at the experimental doses ( Figure 3). ese results demonstrated that the concentration used in the inhibition experiments of the production of NO and cytokines and inflammatory molecules by the extracts and compounds was not cytotoxic.

Inhibition of iNOS and COX-2 Levels.
To further characterize the mechanisms underlying the inhibition of the LPS-induced production of NO by the extracts and   Evidence-Based Complementary and Alternative Medicine compounds, western blotting assays were performed. Four compounds (7-10) that showed strong inhibitory activity in the NO production assay were selected for the test of iNOS and COX-2; in addition, compound 1 was selected for comparison with its aglycone, 7. e levels of iNOS and COX-2 were significantly increased in the LPS-stimulated cells compared with the control cells ( Figure 4). Treatment with AS, FAS, and the selected compounds significantly inhibited iNOS expression in a concentration-dependent manner; similar results were obtained for COX-2. e FAS extract strongly inhibited iNOS and COX-2 levels compared with AS. Most of the compounds decreased iNOS and COX-2 production, especially 9 and 10, and the aglycone (7) showed better activity than the glycoside (1). ese results indicated that the extracts and compounds inhibited NO production through a decrease in iNOS and COX-2 expression in the LPS-stimulated RAW 264.7 cells.

Effects on Cytokine
Production. e inhibition of cytokine (IL-6 and TNF-α) production was analyzed by using ELISA. RAW 264.7 macrophage cells were exposed to LPS, and the cytokine levels in these cells were measured for their evaluation of the inhibitory effects of AS, FAS, and the isolated compounds. In the LPS-treated group (control), IL-6 and TNF-α levels were detected to be 548.12 pg/mL and 777.89 pg/mL, respectively. In the AS and FAS treated groups, IL-6 and TNF-α levels were decreased (Figures 5(a) and 5(b)). e FAS inhibited cytokines to a greater extent than AS did. RAW 264.7 cells were exposed to LPS, and the inhibitory effects of compounds (1 and 7-10) on cytokine production (IL-6 and TNF-α) were measured. In the LPStreated group (control), IL-6 and TNF-α levels were 262.07 pg/mL and 302.88 pg/mL, respectively. IL-6 and TNF-α levels were decreased by treatment with the selected compounds. And the 9, 10, and the aglycone 7 showed better activity than the glycoside (Figures 5(c) and 5(d)).

Discussion
ere are many reports that the linkage of glycosyl groups of the aglycone can alter their bioactivities [27,28]. e variation of natural products and their bioactivities by fermentation is also reported. For example, some researchers reported that rare ginsenosides could be produced from major ginsenosides by microorganisms [27,29]. e aglycones of ginsenosides are more easily absorbed from the small intestine than glycosides and have altered physiological actions in vitro and in vivo [30,31]. is has been reported for not only ginsenosides, but also other compounds, including tannins and fatty acids. L. plantarum subsp. argentoratensis has tannase and can decompose tannin; thus, it has been used for the initial degradation of complex tannins [32]. e fermentation of Vigna sinensis L., cowpea, increased phenolic compound content and enhanced antioxidant activity [33]. In another study, microorganisms converted linoleic and linolenic acids to unsaturated hydroxy fatty acids [34]. Syringaresinol, the aglycone form of lignan (7), has been reported to more strongly inhibit proinflammatory molecules (prostaglandin E 2 , TNF-α, iNOS, and COX-2) compared with acanthoside D, the glycoside form of lignan (1) [3]. In addition, caffeic acid, quinic acid, and protocatechuic acid, which are common in plants, have antioxidant, antitumor, and antiinflammatory activities [35]. In the present study, FAS showed stronger anti-inflammatory activities than AS. Moreover, the activity of syringaresinol (7) was better than that of acanthoside D (1). e HPLC analysis showed that the content of syringaresinol, the aglycone (7), increased during fermentation and the content of acanthoside D, the glycoside (1), decreased during fermentation. Protocatechuic acid (4) content also increased by bioconversion, and these compounds had good inhibitory effects on inflammatory-related factors. ese results indicated that the variations due to bioconversion led to significant differences in the anti-inflammatory effects of the FAS and AS.

Conclusion
e biotransformation of the A. sessiliflorus extract via fermentation was performed. e inhibitions of the production of NO and inflammatory molecules by AS, FAS, and the isolated compounds were measured in vitro. e results showed that the content of the compounds was different in FAS and AS. e increased compounds, syringaresinol (7) and protocatechuic acid (4), showed better activity than acanthoside D (1). Moreover, the anti-inflammatory activities of FAS were stronger than those of AS. ese results indicated that FAS and the isolated compounds may be a potential treatment for inflammatory disorders such as arthritis.

Data Availability
e data used to support the findings of this study are included within the article.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this paper.

Supplementary Materials
Supplementary materials file includes NMR spectra of the 10 compound isolated from FAS. Figure S1