Anti-Inflammatory Activity of Sonchus oleraceus Extract in Lipopoly saccharide-Stimulated RAW264.7 Cells

The anti-inflammatory activity and non-toxicity of Sonchusoleraceusextract (J6) were tested by measuring its effect on the levels of nitric oxide (NO), prostaglandin E2 (PGE2), and the pro-inflammatory cytokines,interleukin-1b (IL-1b), interleukin-6 (IL-6), and tumor necrosis factor-a (TNF-a), in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. We treated the RAW264.7 cells with various concentrations (50, 100, or 200 μg/mL) of J6. Our results showed that J6 inhibited the production of NO, PGE2, and pro-inflammatory cytokines in a concentrationdependent manner, without compromising cell viability. In addition, we provided supporting evidencethat the inhibitory activity of J6 on the production of NO and PGE2occurredvia the downregulation of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), respectively. Our findings suggestthat J6 is a new source for anti-inflammatory drugs and ingredients for healthcare products that include functional cosmetics.

Lipopolysaccharide (LPS), a well-known endotoxin, is found in the outer membraneof Gramnegative bacteria.LPS induces the expression of inflammatory mediators in macrophages, such as RAW264.7 cells, and monocytes 1 and increases the production of nitric oxide (NO) and cytokines in the early stage of inflammation 2 .
Nitric oxide synthase (NOS), which mediates inflammation, exists as three isoforms: neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS). eNOS is mainly expressed in endothelial cells and n NOS expression is restricted mostly to the nervous system and pancreatic beta cells. These two isoenzymes are constitutively expressed and play a key role in homeostasis,including vasodilation, blood flow control, and nerve signal transmission 3 . iNOS, in contrast, is induced by LPS and enzymatically generatesthe pro-inflammatory mediator, NO 4,5 . Another inflammatory enzyme, cyclooxygenase (COX), exists astwo isoforms,namely COX-1 and COX-2. COX-1 is expressed in almost every tissue and is involved in homeostasis and protection of body organs. COX-2 is mostly involved in inflammatory responses 6,7 , and its expression is induced by LPS and inflammatory cytokines 8 . The expression of iNOS and COX-2, their production of NO and prostaglandin E2(PGE 2 ), respectively, and the generation of pro-inflammatory cytokines, such as tumor necrosis factor-a (TNF-a), interleukin (IL)-1b, and IL-6, are common markers of inflammation 9 .
Sonchus oleraceus is a biennial, conicalshaped plant thatbelongs to the Asteraceae family. It can grow up to 1 m high and has been utilized asa medicinal compound and food source for livestock and humans.Its seedling leaves can be eaten uncooked. This plant contains a high content of flavonoid compounds, such as kaempferol, luteolin. quercetin, quercimeritrin, and chrysanthemin [10][11][12][13] .
Previous studies mainly investigated the polyphenol content and antioxidant activity of S.oleraceuswhen grown at different locations, with seasonal changes, and after exposure to various environmental factors [10][11][12][13] . However,there has been little study on the pharmacological and functional properties of S.oleraceus and its use as an ingredient in functional cosmetics. Some widely used synthetic materials such as oxybenzone, mineral oil, and parabens can reportedly cause adverse effects such as liver damage, abnormal body growth, and gastrointestinal bleeding 20 . As a result, interest in biotic materials with minimal side effects is increasing. Thus, the aim of the present study was to investigate the inhibitory activity of J6 S. oleraceus extraction the expression of inflammatory mediators in LPS-stimulated macrophages and to explore its potential as a biotic material.

J6 procurement and cell culture
J6 was obtained from the Jeju Biodiversity Institute 19 . The murine macrophage cell line RAW264.7 was purchased from American Type Culture Collection (Manassas, VA, USA) and cultured in Dulbecco's Modified Eagle Medium (DMEM) (Gibco, Grand Island, NY, USA) supplemented with 100 units/mL penicillinstreptomycin and 10% (v/v) fetal bovine serum (FBS) at 37°C and 5% CO 2 .

Cytotoxicity assay
An MTT (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide) assay was implemented to assess the cytotoxicity of the J6 extract. RAW264.7 cells were plated at 1.8×10 5 cells/200uL/wellin a 96-well plate and incubated for 18 h prior to the 24 h-treatment of 1 µg/mL LPS and different concentrationsof J6 extract (50, 100, and 200 µg/mL) at 37°C and 5% CO 2 . Fifty microliters of MTT reagent was then added and incubated for 4 h. The spent culture medium was completely removed and 200 µL dimethylsulfoxide (DMSO, Sigma, MO, USA) was added to dissolve the formazan precipitate.The absorbance at 540 nm was then measured using a microplate reader (Bio-TekInstrument Inc., Vermont, WI, USA). The average absorbance for each sample group was used to evaluate cell viability. NO assay RAW264.7 cells were plated at 1.8×10 5 cells/200uL/wellin a 24-well plate and incubated for 18 h prior to the 24 h-treatment of 1 µg/mL LPS and different concentrationsof J6 extract (50, 100, and 200 µg/mL) at 37°C and 5% CO 2 .One hundred microliters of culture mediumfrom each well and an equal volume of Griess reagent were mixed and incubated at room temperaturefor 10 min 15 followed by the measurement of absorbance at 540 nm using anenzyme-linked immunosorbent assay (ELISA) reader. The Griess reagent (1% (w/v) sulfanilamide, 0.1% (w/v)N-(1-naphthyl) ethylenediaminein 2.5% (v/v)phosphoric acid) reacts with nitrite in the culture medium to estimate the amount of NO produced. Values were calculated by using a standard curve of sodium nitrite (NaNO 2 ). PGE 2 , IL-1b, IL-6, and TNF-a ELISAs RAW264.7 cells were cultured as described above and treated for 24 h with 450 µL of 1 µg/mL LPS and 50 µL of 10-times-concentrated 1 mg/mL J6 extract. The culture medium was then centrifuged at 12,000 rpm for 3 min and the resulting supernatant was used to measure the content of PGE 2 , IL-1b, IL-6, and TNF-a. All samples were kept at 20°C until quantification. PGE 2 and the other three pro-inflammatory cytokines were quantified using specific ELISA kits (R&D Systems Inc., Minneapolis, MN, USA); the R-squared values for the standard curves were 0.99.

Statistical analysis
All data are expressed as means ±standard deviations (SDs). Statistical differences between samples were resolved by Student's t-tests.

Effect of J6 on cell viability
For the cytotoxicity assay, murine RAW264.7 macrophage cells were treated for 24 h with 1 µg/mL LPS alone or in combination with various concentrations of J6 extract (50, 100, or 200 µg/mL). Anincrease in cell viability and no toxicity were observed in the J6-treated samples relative to that of thecontrol group (Fig. 1, line plot), which was consistent with the results ofaprevious study   h. TNF-a production was measuredby ELISA. The data represent the means ± SD of triplicate experiments. *p <0.005, **p<0.001 versus LPS alone on the anti-inflammatory effect of Scutellariaeradix extract 16 . Up to 200 µg/mL of J6 extract, near the cytotoxic threshold concentration, was used further to assay the inhibitory activity of J6 on the generation of NO, PGE 2 , and pro-inflammatory cytokines.

Effect of J6 on NO generation
The effect of J6 extract on the generation of NO, a critical mediator of the inflammatory response, was examined (Fig. 1, bar graph). RAW264.7 cells were treated with LPS and J6 extract as described above and the generated NO was quantified asnitriteusing a Griess assay.
Relative to that of the LPS-only-treated group, NO production was decreased by 64% at the peak concentration of 200 µg/mL J6 extract. This confirmed the potent inhibitory activity of J6 on NO production and itspotential to inhibit iNOS expression. Effect of J6 extracton PGE2 production in LPSstimulated RAW264.7 cells RAW264.7 cells were plated and treated with LPS and J6 extract as described in the NO assay and PGE 2 generation was thenassessedusing a PGE 2 ELISA kit. The cells were treated with J6 at various concentrations of 50, 100, and 200 µg/ mL.Relative to that of the cells treatedwith LPS only, PGE 2 production was substantially decreased over the J6 concentration range of 50-200 µg/ mL;this reduction reached 95.5% at the peak concentration of 200 µg/mL J6 (Fig. 2). Therefore, J6 extract inhibited LPS-induced PGE 2 production concentration-dependently.

Effect of J6 extract on pro-inflammatory cytokine (IL-1b, IL-6, and TNF-a) generation
Cytokines act as mediators in the activation, proliferation, and differentiation of immune cells and typical pro-inflammatory cytokines, such asIL-1b, IL-6, and TNF-a, are well recognized as modulators of inflammatory responses both in vitro and in vivo 17 . The effect of J6 extract on the production of these three pro-inflammatory cytokines in the presence and absence of LPS was examined. The secretionof IL-1b, IL-6, and TNF-a was increased in the medium of LPS-treated cells (Fig. 3, 4, 5). Relative to that of the LPS-only-treated group, IL-1b and IL-6 production were concentration-dependently decreased by J6 extract, reaching 43.5% (Fig.  3) and 80% (Fig. 4) reduction, respectively,at 200 µg/mL J6. Similarly, TNF-áproduction was decreased in a concentration-dependent manner (Fig. 5). This confirmed the anti-inflammatory effect of J6 extract on secretion of the three proinflammatory cytokines,IL-1b, IL-6, and TNF-a, in LPS-stimulated RAW264.7 cells.

Effect of J6 on protein expression of iNOS and COX-2
iNOS and COX-2 are known as key players in the synthesis of NO and PGE 2 during inflammation 18 . Based on our findings that J6 inhibits the production of NO and PGE 2 in a concentration-dependent manner, we used western blotting to examine whether the reduced production of NO and PGE 2 was associated with reduced protein expression of iNOS and COX-2. Our results demonstrate that J6 extract had a suppressive effect on the LPS-induced expression of iNOS and COX-2 in a concentration-dependent manner (Fig. 6, 7). This also showed that J6 inhibits NO and PGE 2 generation via down regulation of iNOS and COX-2 expression, respectively.

CONCLUSIONS
S. oleraceus has been valued for animal and human consumption but there have been fewstudies on its pharmacological activity and toxicity. Our study confirmed that J6 extract was not toxic and exerted an inhibitory effect on the production of NO, PGE 2 , and pro-inflammatory cytokines in LPS-stimulated macrophages.We found that during LPS-stimulation J6 extract inhibited the generation of NO and PGE 2 in a concentration-dependent manner. We also examined whether J6 was involved in modulating the production of pro-inflammatory cytokines and founditto concentration-dependently inhibit the secretionof three pro-inflammatory mediators(i.e., IL-1b, IL-6, and TNF-a),suggesting that it has potent anti-inflammatory effects. Moreover,the protein level of bothiNOS and COX-2 was drastically decreased with increasing concentrations of J6 extract. Further research is required to clarify that the down-regulation of iNOS and COX-2 expression is through the downregulation of NF-kB activation and MAPK phosphorylation 21,22 . These results provide strong support that J6 is a potential new source for anti-inflammatory drugs and ingredients for healthcare products, including functional cosmetics.