Anti-inflammatory effect from extracts of Red Chinese cabbage and Aronia in LPS-stimulated RAW 264.7 cells.

A chronic inflammatory environment facilitates tumor growth and proliferation. Fruits and vegetables are important sources of anthocyanins, polyphenols, and other biologically active substances that can favorably affect the pathogenesis of cancer. The objective of the study was to investigate the anti-inflammatory effects of Red Chinese cabbage (RC) and mixture of commercial Red Chinese cabbage leaves and Aronia fruits (ARC) in LPS-stimulated RAW 264.7 cells. The RAW 264.7 cells were cultured and measured the cytotoxicity by using an MTT assay. The inflammatory markers, such as nitrite, IL-6, and TNF-alpha expression, were evaluated using ELISA, and protein expression of inflammatory markers like iNOS and COX-2 was analyzed using Western blot. MTT assays showed that pretreatment of RAW 264.7 cells with RC and ARC did not change cell growth or cytotoxicity. We also found that ARC extracts reduced inflammation-related biomarker (TNF-a, IL-6, and NO) production and gene expression (iNOS, COX-2). Our results suggested that ARC has good anti-inflammatory properties compared with RC that maybe used as potential nutrients for treating inflammatory diseases.

. Recently, Red Chinese cabbages (RC) (Brassica rapa L) are produced by crossing red cabbage (Brassica oleracea L. var. captita f. rubra) with Chinese cabbage (Brassica rapa L. ssp. pekinensis) (Jiang et al., 2013). It is rich in anthocyanins and characterized by red color on the outside and inside.
Aronia fruits, with the common name chokeberry, belong to the Aronia genus of the Rosaceae family, Maloideae subfamily. It is a rich source of polyphenolic compounds, which are one of the most important and vital natural antioxidants (Dei Cas & Ghidoni, 2018;Jurikova et al., 2017).
A review study reported that the synergistic effects of phytochemical extracts from fruits and vegetables have strong antioxidant and antiproliferative activities, and the major part of total antioxidant activity is from the combination of phytochemicals (Liu 2004).
Studies for each single food such as Red Chinese cabbage, or Aronia related to anti-inflammation and antioxidation are reported (Joo et al., 2018;Kokotkiewicz, Jaremicz, & Luczkiewicz, 2010), but the anti-inflammatory effects with the mixture of Red Chinese cabbage and Aronia fruits have not been studied. Therefore, our objective was to investigate the anti-inflammatory effect of mixtures of Red Chinese cabbage and Aronia (ARC) in LPS-stimulated RAW 264.7 cells.

| Sample preparation
We obtained Aronia (Aronia melanocarpa (Michx.) Elliot) frozen fruits from a plantation farm located in Yongin (Gyeonggi-do, Korea) and ground it in a grinder (FM-909W, Hanil Co., Sejong, Korea). And RC was purchased from a plantation farm located in Bugil-myeon (Haenam-gun, Jeollanam-do, Korea), cultivated using the seeds of commercial cultivar (Kwonnong 3 Ho), distributed by Kwonnong Seed Company (Chungju, Korea), and removed the foreign matter, and cut the whole RC sample into one centimeter pieces.

| Preparation of extracts
RC extraction was diluted with 95% fermented ethanol at a sample:alcohol ratio (1:10) and extracted the filtrate for 24 hr. We prepared the ARC mixture (Red Chinese cabbage and Aronia fruit extracts at a ratio of 2:8) further, diluted with 95% fermented ethanol at a sample:alcohol ratio (1:10), and extracted the filtrate for 24 hr. To remove impurities, the solvent extract was filtered through a cotton fabric (No. 1, Whatman International. Ltd., Leicestershire, England). The extract was allowed to evaporate at room temperature using a rotor evaporator (EYELA/N1000, Tokyo Rikakikai Co.) under reduced pressure to obtain dry residue, and further, it is stored in a cool and dark condition. Finally, the dry residue is dissolved in dimethyl sulfoxide (DMSO) and stored at −20°C.

| Cell culture
The RAW 264.7 murine cell line of macrophage (Korean cell-line bank) was cultured in Dulbecco`s modified Eagle`s medium (DMEM, Gibco) supplemented with 10% inactivated fetal bovine serum and 1% penicillin-streptomycin. And the cell culture is maintained at humidified atmosphere with 5% CO 2 at 37°C.
After incubation for 2 hr in CO 2 incubator, the supernatant was removed after centrifugation at 4℃ at 1747 g for 10 min. Each well then had dimethyl sulfoxide added (Sigma-Aldrich Co.), and the plates were identified by a microplate reader (Model 550, Bio-Rad) at 540 nm.

| Nitrite measurement
The generation of nitric oxide (NO) was measured by Griess reaction.
The RAW 264.7 cells (1 × 10 6 cells/ml) were aliquot in six-well plate and incubated them for 20 hr. Followed by pretreatment of the cells with different concentrations of RC and ARC, 1 μg/ml of lipopolysaccharide (LPS) was added to 250 μg/ml of each sample. After 24 hr incubation, 100 μl culture was mixed with an equal volume of Griess reagent at room temperature for 10 min. The absorbance of the mixture was measured by a microplate reader at 540 nm. We performed all of the experiments in triplicate.

| Cytokine expression by ELISA
Enzyme-linked immunosorbent assay (ELISA) was used to evaluate tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) expression. The RAW 264.7 cells (1 × 10 6 cells/ml) were aliquot into six-well plate and incubated for overnight; 1 μg/ml lipopolysaccharide (LPS) was added to 250 μg/ml of each sample. After incubation for 24 hr, the concentration of TNF-α and IL-6 in the cell-free supernatant (i.e., CM) was assessed by using the ELISA kit (Cloud-Clone Crop) according to the manufacturer's instructions.

| Western blot analysis
Initially, the cell pellets were washed in ice-cold PBS buffer, followed by lysis using Radioimmunoprecipitation assay (RIPA) buffer (Pierce Biotechnology) and then measured protein concentration by DC TM assay (Bio-Rad). A protein sample with 30 μg was subjected to electrophoresis using 10% SDS-PAGE in running buffer at 120 V for 2 hr and electroblotting onto a PVDF (polyvinylidene difluoride) membrane. The membrane was then blocked in the solution that contains 5% nonfat milk, 0.1% (v/v) Tris-buffered saline, and Tween-20 (TBS-T) for 1 hr at room temperature. After blocking, we then washed the membranes thrice with TBS-T for 10 min. Then, each membrane was added with primary antibodies (1:1,000; diluted with 1% nonfat milk in TBS-T) specific with inducible nitric oxide synthase (iNOS) (AVIVA) and cyclooxygenase-2 (COX-2) (Santacruz) and incubated overnight at 4°C on shaker. β-actin was used as the internal control.
After incubation, the membrane was washed with TBS-T thrice (10 min/time) and again incubated by adding secondary antibodies (anti-rabbit and anti-mouse IgG horseradish peroxidase) at a ratio of 1:5,000 (3% nonfat milk: TBS-T) for 2 hr. Followed by washing the membrane in TBS-T thrice, the last washing was done using TBS for 10 min. Finally, the protein expression was detected by an enhanced chemiluminescence (ECL) immunoassay and Western blotting detection reagents (Amersham, GE Healthcare).

| Statistical analysis
Data analyses were done using SPSS software version 17.0 and represented data as means ± standard deviation (SD). An independent t test was used to compare means, and p < .05 was considered to indicate statistically significant difference. Figure 1 shows the effect of RC and ARC at various concentrations (0,15,31,62,125,250,500, and 1,000 μg/ml) on cell viability of RAW 264.7 cells. The nonspecific cell toxicity of RC and ARC was measured using the MTT assay. From the results of cell viability assay, it was evident that even at high concentration, RC and ARC did not show cytotoxicity on RAW 264.7 cells. When LPS was treated with RC and ARC, the results were similar to those of RC and ARC alone. In addition, LPS treatment alone showed 90% cell viability at high concentrations. Figure 2 presents the effect of RC and ARC on NO production in RAW 264.7 cells. Cells treated with LPS increased NO production gradually, and the production reduced with treatment of RC and ARC extracts. The degree of NO production was remarkably decreased in ARC-treated cells more than by RC-treated cells.

| D ISCUSS I ON
The anti-inflammatory efficacy of RC and ARC in LPS-stimulated RAW 264.7 cells was evaluated. The inflammatory markers, such as NO, IL-6, and TNF-α expression, were evaluated using ELISA, and protein expression of inflammatory markers like iNOS and COX-2 was analyzed using Western blot. In conclusion, our findings indicate that ARC could attenuate the inflammatory response by suppressing the iNOS and COX-2 genes and mediators in RAW 264.7 cells.
According to a review study, Lee et al. reported on the effects of anthocyanins-rich food on attenuating inflammation in cells, animals, and humans . Particularly, anthocyanin mixtures such as red cabbage, microgreens, blueberry, blackcurrant, cherry, and chokeberry had higher clinical efficacy than single anthocyanins.
In our study, the results were similar to those reported in previous reviews.
The synergistic effects of phytochemical extracts from fruits and vegetables have strong antioxidant and antiproliferative activities, and the major part of total antioxidant activity is from the combination of phytochemicals (Liu 2004 (Chiou, Li, Ho, & Pan, 2018). Therefore, synergistic research of naturally derived fruit and vegetable mixtures is very important and necessary.
Although studies on single foods of Red cabbages or Aronia have been reported, the effects of the complex have not been studied.
We verified the effect at the cellular level.
Chronic inflammation may promote the development of tumor growth through a variety of mechanisms, such as stimulating angiogenesis, preventing apoptosis, and promoting proliferation and migration (Allen & Jones, 2015). Inflammatory response regulated by inflammatory mediators (e.g., NO) and proinflammatory cytokines (e.g., TNF-α, IL-6). Under inflammatory conditions, iNOS and COX-2 protein expression are elevated, which facilitates the generation of NO and Prostaglandin F2 alpha (PGF 2α ). iNOS is a target of inflammation-associated tissue damage (Southan & Szabo, 1996), and COX-2 is a mediator of inflammation, angiogenesis, and cancer progression (Wang et al., 2017). Also, cytokines that include TNF-α and IL-6 are related to the inflammation process and known to typical proinflammatory cytokine with tumor growth effect (Landskron, De la Fuente, Thuwajit, Thuwajit, & Hermoso, 2014).
The findings in our study evidence that ARC extracts significantly inhibit the production of NO, TNF-α, and IL-6. Additionally, these were associated with decreased expression of iNOS and COX-2 protein. To the best of our knowledge, this study is the first to examine will be necessary to validate the results. In addition, accurate content analysis of polyphenols and anthocyanins in single and mixed extracts should be preceded in further study.
In conclusion, our findings indicate that ARC could attenuate the inflammatory response by suppressing the iNOS and COX-2 genes and mediators in RAW 264.7 cells. However, further studies should be performed to confirm the effect at different concentrations and to verify the effect on the single substance as well as the mixture.

ACK N OWLED G M ENTS
This work was supported by the National Research Foundation of Korea (NRF) and Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries through the Golden Seed Project.

CO N FLI C T O F I NTE R E S T
The authors declare no potential conflicts of interest.

AUTH O R CO NTR I B UTI O N S
The authors' responsibilities were as follows: JHK and YK designed and created the study concept; HJL acquired the data and performed the statistical analysis; JHK wrote the paper; SIR, ML, HJL, and YPL contributed critical advice and revisions of the manuscript; JKP had responsibility for the entire contents of the manuscript and obtained funding; JKP supervised the study; and all authors had full access to the study data and read and approved the final manuscript.

E TH I C A L A PPROVA L
This study does not involve any human or animal testing.

F I G U R E 4
Effect of RC and ARC on LPS-induced COX-2 and iNOS expression in RAW 264.7 cells, which were treated with LPS (1 μg/ml), RC (250 μg/ml), and ARC (250 μg/ml) concentrations for 24 hr. The data are expressed as mean ± SD (n = 3). ***p < .001, statistically different from LPS control using t test