Effects of Shiranuhi Flower Extracts and Fractions on Lipopolysaccharide-Induced Inflammatory Responses in Murine RAW 264.7

Home , Hesperidin, Narirutin, Neohesperidin

Turk J Biochem 2018; 43(4): 375–384

Research Article

Min-Jin Kim, Sang Suk Kim, Ji Hye Ko, Young Il Moon, Kyung-Jin Park, Hyun Joo An, Young Hun Choi, Nam Ho Lee and Chang-Gu Hyun* Effects of Shiranuhi flower extracts and fractions on lipopolysaccharide-induced inflammatory responses in murine RAW 264.7 cells https://doi.org/10.1515/tjb-2016-0209 Keywords: Cyclooxygenase-2; Inducible Nitric Oxide Syn- Received January 12, 2017; accepted February 9, 2017 thase; Inflammation; MAPK; Shiranuhi Flower. Abstract Özet Objective: In this study, we evaluated the anti-inflammatory effect of Shiranuhi flower in RAW 264.7 cells. Amaç: Bu çalışmada, RAW 264.7 hücrelerinde Shiranuhi Methods: The effects of the extracts and solvent fractions çiçeğinin anti-inflamatuar etkisini değerlendirdik. on cell viability and LPS-induced inflammatory responses Yöntem: Ekstraktların ve çözücü fraksiyonlarının hücre were investigated in RAW 264.7 cells. sağlığı ve LPS ile uyarılan inflamatuvar tepkileri üzerin- Results: The results showed that the ethyl acetate fraction deki etkileri, RAW 264.7 hücrelerinde araştırılmıştır. (HEF) significantly decreased NO production in RAW 264.7 Bulgular: Sonuçlar etil asetat fraksiyonunun (HEF) RAW cells; however, cell viability was not affected. In addition, 264.7 hücrelerinde NO üretimini önemli ölçüde azalttığını ELISA assay revealed that HEF significantly inhibited gösterdi; ancak hücre canlılığı etkilenmemiştir. Buna ek the productions of PGE , TNF-α, and IL-6. As well, using 2 olarak, ELISA tahlili, HEF’in PGE2, TNF-α ve IL-6 üretim- Western blot analysis, it was observed that HEF signifi- lerini önemli ölçüde inhibe ettiğini ortaya koymuştur. cantly reduced the expression levels of iNOS and COX-2 Sonuçlar etil asetat fraksiyonunun (HEF) RAW 264.7 hüc- in a dose dependent manner. Furthermore, we detected relerinde NO üretimini önemli ölçüde azalttığını gösterdi; a reduced phosphorylation of mitogen-activated protein Ancak hücre canlılığı etkilenmemiştir. Buna ek olarak, kinases such as p38, JNK, and ERK1/2. This indicates that ELISA tahlili, HEF’in PGE2, TNF-α ve IL-6 üretimlerini HEF regulates LPS-induced inflammatory responses, at önemli ölçüde inhibe ettiğini ortaya koymuştur. Bunun least in part, via suppressing the MAPK signaling pathway. yanı sıra, Western blot analizi kullanılarak, Hef’in doza Correlation analysis also showed that anti-inflammatory bağımlı olarak iNOS ve COX-2 ekspresyonu anlamlı bir activities were highly correlated to antioxidant activities şekilde azalttığı gözlenmiştir. Dahası, p38, JNK ve ERK1/2 in this study. Characterization of the Shiranuhi flowers for gibi mitojenle aktive olan protein kinazların fosforilasyo- flavonoid contents using HPLC showed varied quantity of nunun azaldığını tespit edilmiştir. Bu bulgular, HEF’in, en narirutin and hesperidin. azından kısmen MAPK sinyal yolunu baskılayarak, LPS Conclusion: Overall, the results demonstrate that HEF ile uyarılan inflamatuvar tepkileri regüle ettiğini gösterir. may be a potential anti-inflammatory agent. In addition, Ayrıca bu çalışmada yapılan korelasyon analizi, anti-inf- our findings contribute to understanding the molecular lamatuvar aktivitelerin antioksidan aktivitelerle yüksek mechanism underlying the anti-inflammatory effect of oranda korele olduğunu göstermiştir. Shiranuhi çiçeğinin Shiranuhi flower. flavonoid içeriği HPLC kullanarak karakterize edildiğinde çeşitli miktarlarda narirütin ve hesperidin bulunmuştur. Sonuç: Genel olarak, sonuçlar HEF’in potansiyel bir *Corresponding author: Chang-Gu Hyun, Department of Chemistry anti-inflamatuar ajan olabileceğini göstermektedir. Buna and Cosmetics, Jeju, Korea (the Republic of), e-mail: [email protected] ek olarak, bulgularımız Shiranuhi çiçeğinin anti-infla- Min-Jin Kim, Ji Hye Ko and Nam Ho Lee: Department of Chemistry matuar etkisinin altında yatan moleküler mekanizmanın and Cosmetics, Jeju, Korea (the Republic of) anlaşılmasına katkıda bulunmaktadır. Sang Suk Kim, Young Il Moon, Kyung-Jin Park, Hyun Joo An and Young Hun Choi: Citrus Research Institute, Seogwipo, Anahtar Sözcükler: Siklooksijenaz-2; Indüklenebilir nitrik Korea (the Republic of) oksit sentaz; Inflamasyonu; MAPK; Shiranuhi çiçeği. 376 Min-Jin Kim et al.: Effects of Shiranuhi flower extracts and fractions on lipopolysaccharide

Introduction anti-inflammatory [21], antioxidant [22], anti-obesity [23], and anti-angiogenic effects [24]. In addition, the Inflammation is a highly regulated defensive process that peel has been noted to inhibit breast cancer cell migra- results from tissue injury or external stimuli. It leads to tion. However, potential biological activities of Shiranuhi the release of numerous inflammatory mediators and flower have not been explored. Therefore, in this study, ultimately triggers the restoration of tissue structure the Shiranuhi flower was investigated for its potential and function [1–3]. However, uncontrolled and excessive anti-inflammatory activity. To the best of our knowledge, inflammatory responses can cause severe tissue damage this is the first study to demonstrate that Shiranuhi flower and secondary inflammatory injuries, such as sepsis, shows an anti-inflammatory activity. asthma, rheumatoid arthritis, vascular diseases, and cancer [4, 5]. Macrophages play a crucial role in provid- ing an immediate defense by directly counteracting the aforementioned stimuli by releasing cellular signaling Materials and methods molecules such as inflammatory mediators and cytokines [6, 7]. In a normal state, these inflammatory mediators Preparation of Shiranuhi flower extracts and cytokines released from macrophages are essential for host survival and tissue repair [8]. Lipopolysaccha- Shiranuhi flowers were collected from Citrus Research ride (LPS) is a molecule obtained from Gram-negative Institute (RDA, Jeju Island) and were identified by Dr. bacteria. It induces the release of many pro-inflamma- Young Hun Choi. A voucher specimen was prepared and tory mediators and cytokines such as nitric oxide (NO), deposited at the Cosmetic Science Center at Jeju National prostaglandin E2 (PGE2), tumor necrosis factor (TNF)-α, University (Korea). interleukin (IL)-6, and IL-1β from macrophages [9, 10]. The dried flowers (1.39 kg) were soaked in 5 L of Therefore, a decrease in LPS-induced expression of pro- 80% ethanol at room temperature for 24 h, after which inflammatory mediators and cytokines in macrophages the solvent was evaporated under vacuum. The residue may be considered a significant therapeutic property obtained was freeze-dried and stored in a desiccator at 4°C during the development of anti-inflammatory agents before use. The weight of the dried sample was 52.48 g; [11–13]. Accumulating evidence indicates that mitogen- therefore, the yield of the extraction was 3.78%. The dried activated protein kinases (MAPKs) also induce the pro- extract (20 g) was then sequentially suspended in water duction of inflammatory molecules and immune-related (1 L) and extracted with ethyl acetate (1 L). The recoveries cytotoxic factors via a sequence of phosphorylation of the extract from the ethyl acetate and water fractions events. The MAPK family is composed of subfamilies, were 12.5% (2.49 g) and 70.5% (14.1 g), respectively. which include members such as extracellular signal- For the water extract, the dried flowers (700 g) were regulated kinase (ERK), c-Jun N-terminal kinase (JNK), ground into powder and extracted with 3 L of water in and p38. Furthermore, many studies have demonstrated a water bath at 80°C for 5 h. Next, the mixture was fil- that MAPKs are important in modulating the expressions tered and the filtrate was concentrated to about 250 mL. of cyclooxygenase (COX)-2 and inducible NO synthase The water extract of the Shiranuhi flower was thereafter

(iNOS) and the productions of NO, PGE2, and pro-inflam- obtained by lyophilizing the concentrate. The yield was matory cytokines, such as IL-1β, TNF-α, and IL-6 [14–16]. 25.4 g (3.6%). In this study, each fraction and extracts were Thus, regulating the activation of MAPK pathways may be dissolved in DMSO and filtered through syringe filters a useful strategy in modulating inflammatory responses (0.45 μm pore size). [17–19]. Shiranuhi [(Citrus unshiu Marcov × C. sinensis Osbeck) × C. reticulata Blanco] is a seedless and sweet Reagents and cell culture variety of mandarin orange that has been cultivated on Jeju Island (Korea) since 1988, after its development in Murine RAW 264.7 macrophages were purchased from Japan in 1972. Its trade name is ‘Hallabong’, named after American Type Culture Collection (Manassas, VA, USA). the Hallasan Mountain located on Jeju Island. Shiranuhi Dulbecco’s Modified Eagle’s Medium (DMEM) and fetal is also the name of a genus of flowering plants in the bovine serum (FBS) were purchased from Gibco (Grand Rutaceae family. The Shiranuhi fruit is primarily used Island, NY, USA). LPS, antibiotics (penicillin and strepto- for its juice; however, its peel has diverse pharmacologi- mycin), and bovine serum albumin (BSA) were obtained cal properties including anti-tumor, anti-metastatic [20], from Sigma-Aldrich (St. Louis, MO, USA). Lactate Min-Jin Kim et al.: Effects of Shiranuhi flower extracts and fractions on lipopolysaccharide 377 dehydrogenase (LDH) cytotoxicity detection kit and Determination of cytokine levels enzyme-linked immunosorbent assay (ELISA) antibody sets were purchased from Promega (Madison, WI, USA) The concentrations of PGE2, TNF-α, and IL-6 in the and R&D Systems Inc. (Minneapolis, MN, USA), respec- supernatant were assayed using a commercial mouse tively. Primary antibodies for iNOS, COX-2, phospho-ERK, ELISA kit according to the manufacturer’s instructions. ERK, phosphor-p38, p38, phospho-JNK, JNK, and horse- The RAW 264.7 cells were plated at 1.8 × 105 cells/mL in radish peroxidase-conjugated secondary antibodies were 24-well plates and incubated for 18 h, and pretreated in purchased from Cell Signaling Technology Inc. (Danvers, absence or presence the ethyl acetate fraction (HEF; 25, MA, USA). β-actin antibody was purchased from Santa 50, or 100 μg/mL) for 2 h, after which they were stimulated Cruz Biotechnology (Santa Cruz, CA, USA). Nitrocellu- with LPS (1 μg/mL) for 24 h. Supernatant samples were lose membrane was obtained from Invitrogen Inc. (Seoul, obtained 24 h later and frozen until analysis by ELISA. Korea). The RAW 264.7 cells were maintained at subcon- Cytokine concentrations were measured from standard fluence at 37°C in a humidified atmosphere of 95% air curves that were constructed using known concentrations and 5% CO2. The medium used for routine subculture was of recombinant TNF-α, IL-6, and IL-1β. DMEM containing 10% FBS, penicillin (100 units/mL), and streptomycin (100 μg/mL). Western blot analysis

Cell viability assay Western blot analysis was performed as previously described with some modifications [25]. First, for iNOS Cell viability was analyzed by using the LDH cytotoxic- and COX-2 protein expression analysis, RAW 264.7 macro ity detection kit according to the manufacturer’s instruc- phages were pre incubated for 18 h in a 6-well culture tions. The RAW 264.7 macrophages (1.8 × 105 cells/mL) plate at a density of 1.0 × 106 cells/well. And then, the were plated in 24-well plates and incubated for 18 h, cells were pretreated stimulated with LPS (1 μg/mL) in after which they were treated with the extract samples the absence or presence of HEF (25, 50, or 100 μg/mL) for for 2 h. The macrophages were then challenged with LPS 24 h, after which they were stimulated with LPS (1 μg/mL) (1 μg/mL) for an additional 24 h. The release of LDH from for 24 h. Next, for MAPKs protein expression analysis, the macrophages was used to assess the cytotoxicity of RAW 264.7 macrophages were pre incubated for 18 h in a each sample. This method determines LDH activity from 6-well culture plate at a density of 1.0 × 106 cells/well. And the production of NADH during the conversion of lactate then, the cells were pretreated in the absence or presence to pyruvate. Absorbance was measured at 450 nm using of HEF (25, 50, or 100 μg/mL) for 2 h, after which they were an ELISA multiplate reader (Sunrise, Tecan, Switzerland). stimulated with LPS (1 μg/mL) for 10 min. After incuba- Percentage cytotoxicity was determined relative to the via- tion, the cells were collected and washed twice with cold bility of the control cells. All experiments were performed phosphate-buffered saline. The macrophages were lysed in triplicate. in a lysis buffer (radioimmunoprecipitation assay buffer, 1% Nonidet P-40, and 1% protease inhibitor cocktail) for 1 h, collected into microtubes, and then centrifuged at Measurement of NO levels 15,000 rpm for 15 min at 4°C. The protein contents of the cell lysates were then determined by the Bradford Assay The RAW 264.7 cells were plated at 1.8 × 105 cells/mL in (Bio-Rad, Richmond, CA, USA). BSA was used as the 24-well plates and incubated for 18 h, and pretreated standard for the analysis. Equal amounts of the lysates with the various samples for 2 h, after which they were (30–50 μg of protein) were separated on a 4–12% Bis-Tris stimulated with LPS (1 μg/mL) for 24 h. The nitrite levels mini gel. After electrophoresis, the proteins were trans- in the culture media were determined using Griess ferred onto a nitrocellulose membrane (Invitrogen Inc.). reagent (1% sulfanilamide in 5% phosphoric acid and The membrane was washed in Tris-buffered saline (TBS) 0.1% N-(1-naphthyl)ethylenediamine dihydrochloride in containing 0.1% Tween 20 (TTBS) and blocked in TTBS distilled water) and incubated at room temperature for containing 5% BSA solution for 2 h. The membrane was 5 min. Absorbance was then measured at 540 nm using then incubated overnight at 4°C with primary antibodies the ELISA multiplate reader. The quantity of nitrite in (iNOS, COX-2, p-JNK, JNK, p-ERK, ERK, p-p38, and p38) the samples was calculated using sodium nitrite as the diluted in TTBS (1:1000) and washed 4 times in TTBS. standard. Each membrane was incubated for 1 h with secondary 378 Min-Jin Kim et al.: Effects of Shiranuhi flower extracts and fractions on lipopolysaccharide peroxidase-conjugated goat immunoglobulin G (IgG, Statistical analysis 1:5000) and washed again with TTBS. The target proteins were detected using enhanced chemiluminescence solu- The results have been expressed as mean ± SD. All the tion. The immunoreactive bands were detected and then quantitative data are representative of at least three exposed to X-ray film. Protein levels were quantified by independent experiments. Student’s t-test was used to scanning the immunoblots. determine statistically significant differences between each treated group and the negative control (LPS group). Statistical significance was considered at p < 0.05 (*) or 2,2-diphenyl-1-picrylhydrazyl (DPPH assay) p < 0.01 (**).

The modified method of Kim et al. [26] was used. Twenty microliter of sample solution was mixed with 180 μL of 0.2 mM DPPH solution in a microtiter plate. After incuba- Results and discussion tion for 15 min in the dark, the absorbance was measured at 517 nm. The % DPPH scavenging activity of samples are Effect of Shiranuhi flower on NO synthesis calculated as: in activated macrophages Inhibition (%)[=−1(AbsA− bs )/ sample sample blank In murine RAW 264.7 macrophages, bacterial LPS alone (Abs −×Abs)]100 controlcontrol blank can induce iNOS transcription, protein synthesis, and subsequent NO production. Therefore, the RAW 264.7 cell line is a common model for evaluating anti-inflam- 2,2′-azino-bis(3-ethylbenzothiazoline-6- matory agents by inhibiting pathways that trigger iNOS sulphonic acid) (ABTS assay) and NO productions [1, 2, 10]. To investigate the effect of Shiranuhi flower extract on NO production, we measured The modified method of Yang et al. [27] was used. To make the levels of nitrite, a stable oxidized product of NO, in ABTS + solution, 7 mM ABTS and 2.45 mM potassium culture media containing RAW 264.7 cells that had been persulfate were mixed and left in the dark for 16 h. one stimulated with LPS in the presence or absence of the Shi- hundred and eighty microliter of ABTS+ solution was ranuhi flower extract. added to 20 μL of sample solution in a microtiter plate. It was observed that the nitrite level in the medium After incubating for 15 min in the dark, the absorbance containing the LPS-stimulated cells increased signifi- was measured at 700 nm. The % ABTS scavenging activity cantly and was higher than that in the medium containing of samples are calculated as: the control cells. To evaluate whether Shiranuhi flower

Inhibition (%)[=−1(AbsAsample − bssample blank ) can modulate NO production by activated macrophages,

/(AbsAcontrolc−×bs ontrol blank )] 100 we examined the effects of the hot water and ethanolic extracts, as well as those of the ethyl acetate and water fractions on NO production in the murine RAW 264.7 mac- HPLC fingerprint rophages. As well, the effects of 2-amino-4-methylpyridine (10 μM) and dexamethasone (20 μM) on the cells Chromatographic analysis of the Shiranuhi flower per- were investigated. As shown in Figure 1A, among the four formed using a HPLC system (Waters, Milford, MA, USA) Shiranuhi flower preparations, HEF (100 μg/mL) mark- with a YMC Pro-Triart C18 RS column (250 × 4.6 mm, edly inhibited LPS-induced NO production in the RAW S-5 μm, 8 nm; YMC Co., Kyoto, Japan) and a Waters 2489 264.7 cells by 85%. Furthermore, 2-amino-4-methylpyri- UV visible detector. The mobile phases for the HPLC dine, which was used as an NO inhibitor and the posi- system were acetonitrile (A) and water containing 20 mM tive control, also inhibited LPS-induced NO production phosphoric acid (2:8) (B) at a flow rate of 2 mL/min. The by 91%. As shown in Figure 1B, the numbers of viable samples were filtered through 0.22 μm polyvinylidene activated macrophages were not altered by HEF, 2-amino- difluoride membrane filter. The following flavonoid 4-methylpyridine, or dexamethasone, as were observed compounds were analyzed in the citrus sample: rutin, from the LDH assays. This was an indication that the inhi- naringin, hesperidin, neohesperidin, and narirutin. UV bition of NO production by HEF was not simply due to detection was performed at 280 nm. cytotoxic effects. Min-Jin Kim et al.: Effects of Shiranuhi flower extracts and fractions on lipopolysaccharide 379

A 120 * * * * * * *** 100 *

80 * * 60 ** 40 ** 20 No production (%) ** 0 LPS – + + + + + + + + + + + + + + + + Sample – – Vehicle A B 25 50 100 25 50 100 25 50 100 25 50 100 (µg/mL) 80% EtOH Ex. EtOAcFr. Water Fr. Water Ex.

B 20

10 ** ** ** ** ** ** 0 ** ** ** ** ** ** –10 ** ** ** LDH release (%) –20 LPS – + + + + + + + + + + + + + + + + Sample – – Vehicle A B 25 50 100 25 50 100 25 50 100 25 50 100 (µg/mL) 80% EtOH Ex. EtOAcFr. Water Fr. Water Ex.

Figure 1: Inhibitory effects of Shiranuhi flower extracts and fractions on cytotoxicity and nitric oxide (NO) production in murine RAW 264.7 cells. RAW 264.7 cells were pretreated with the Shiranuhi flower extracts and fractions (each at concentrations of 25, 50, and 100 μg/mL), 2-amino-4-methylpyridine (A, 10 μM), dexamethasone (B, 20 μM), and vehicle (DMSO, the solvent in which the dried extracts and fractions are dissolved) for 2 h, and then treated with lipopolysaccharide (1 μg/mL) for 24 h. (A) The nitrite levels in the culture media were determined using Griess reagent. (B) Cell viability was analyzed by using the LDH cytotoxicity detection kit according to the manufacturer’s instructions. The data are expressed as mean ± SD (n = 3). *indicates p < 0.05 and **indicates p < 0.01.

COX-2 expression, which is induced by cytokines and Effect of HEF on PGE2 production in LPS-induced RAW 264.7 cells endotoxins such as LPS, results in the release of large amounts of PGE2 at sites of inflammation [1, 2, 10]. There-

Prostaglandins are small pro-inflammatory molecules fore, we examined the effects of HEF on PGE2 production in derived from arachidonic acid that play roles in a mul- the LPS-stimulated RAW 264.7 macrophages. As shown in titude of biological processes including inflammation. Figure 2, LPS increased PGE2 production by approximately

120

100 * 80 * *

60 production (%)

2 40

PGE 20

0 LPS –+ + ++(1 µg/mL) HEF –– 25 50 100 (µg/mL)

Figure 2: Inhibitory effect of the ethyl acetate fraction of Shiranuhi flower extract (HEF) on prostaglandin (PG)E2 production in murine RAW 264.7 cells. HEF was prepared by suspending the residue obtained after drying the ethanolic extract of Shiranuhi flower in water, followed by extraction with ethyl acetate. The cells were stimulated with of LPS alone or with a combination of LPS and various concentrations (25, 50, or 100 μg/mL) of HEF for 24 h. PGE2 produced and released into the culture media were determined using ELISA Kit according to the manufacturer’s instructions. The data are expressed as mean ± SD (n = 3). *indicates p < 0.05 and ** indicates p < 0.01. 380 Min-Jin Kim et al.: Effects of Shiranuhi flower extracts and fractions on lipopolysaccharide

12-fold, whereas pretreatment with HEF (25, 50, or the inhibitory effects of HEF on LPS-induced NO and PGE2

100 μg/mL) markedly reduced LPS-induced PGE2 produc- productions are the results of the suppression of iNOS and tion in the cells in a dose-dependent manner. Furthermore, COX-2 expression levels, respectively. dexamethasone, which is a COX-2 selective inhibitor, had a significant inhibitory effect on PGE production. 2 Effect of HEF on the production of pro-inflammatory cytokines Effects of HEF on LPS-induced expression Because TNF-α and IL-6 are markers of early-phase inflam- of iNOS and COX-2 proteins mation, elevated levels of these molecules can be detected during several acute and chronic inflammatory diseases. Western blot analyses were performed to determine Therefore, we evaluated the effects of HEF on the levels of whether the inhibitory effects of HEF on NO and PGE 2 these cytokines in the LPS-treated cells by enzyme immu- productions were related to modulations of iNOS and noassay. As expected, the LPS-treated RAW 264.7 cells dis- COX-2 expressions, respectively. As shown in Figure 3, we played notable increases in the levels of TNF-α and IL-6. did not detect iNOS and COX-2 protein expressions in the Moreover, the induction of pro-inflammatory cytokines control cells. However, iNOS and COX-2 levels were mark- was significantly decreased by HEF in a dose-dependent edly upregulated by the LPS treatment. On the other hand, manner (p < 0.05). As shown in Figure 4, the productions HEF did not affect the expression of β-actin, which is a of TNF-α and IL-6 were decreased at the IC values of HEF housekeeping gene. In general, these results suggest that 50 (41 and 35 μg/mL, respectively). These indicate that HEF efficiently suppressed LPS-induced TNF-α and IL-6 pro- LPS – + + + + + + (1 µg/mL) ductions, which suggests that HEF inhibited the initial HEF – – A B 25 50 100 (µg/mL) phase of the LPS-induced inflammatory response. COX-2 72 kDa

iNOS 130 kDa Effect of HEF on the phosphorylation of MAPKs

β-actin 42 kDa in LPS-stimulated RAW 264.7 cells

MAPKs play critical roles in the regulation of cell growth Figure 3: Inhibitory effects of the ethyl acetate fraction of Shiranuhi flower extract (HEF) on the expression levels of cyclooxygenase (COX)-2 and differentiation, as well as in the control of cellular and inducible nitric oxide synthase (iNOS) in murine RAW 264.7 cells. responses to cytokines and stressors. One of the most The RAW 264.7 cells were plated at 1.0 × 106 cells/mL in 24-well extensively investigated transduction pathways involved in plates and incubated for 18 h, and pretreated with HEF (25, 50, or inflammation is the MAPK pathway. Previous studies have μ μ 100 g/mL), 2-amino-4-methylpyridine (A, 10 M), or dexametha- shown that MAPKs play significant roles in regulating the sone (B, 20 μM) for 2 h, after which they were stimulated with LPS (1 μg/mL) for 24 h. COX-2 and iNOS protein level was determined by levels of COX-2 and iNOS and the production of pro-inflam- immunoblotting. matory cytokines by modulating nuclear factor κB (NF-κB)

120 120 * 100 100 80 80 * 60 60 * * 40 40 *

20 IL-6 production (%) 20 **

TNF- α production (%) 0 0 LPS– ++++(1 µg/mL) LPS –+ +++(1 µg/mL) HEF ––25 50 100 (µg/mL) HEF ––25 50 100 (µg/mL)

Figure 4: Inhibitory effects of the ethyl acetate fraction of Shiranuhi flower extract (HEF) on the productions of tumor necrosis factor (TNF)-α and interleukin (IL)-6 in murine RAW 264.7 cells. The cells were stimulated with of LPS alone or with a combination of LPS and various concentrations (25, 50, or 100 μg/mL) of HEF for 24 h. TNF-α and IL-6 produced and released into the culture media were determined using ELISA Kit according to the manufacturer’s instructions. The data are expressed as mean ± SD (n = 3). *indicates p < 0.05 and **indicates p < 0.01. Min-Jin Kim et al.: Effects of Shiranuhi flower extracts and fractions on lipopolysaccharide 381 and activator protein 1 (AP-1) signals in LPS-stimulated JNK MAPKs were unaffected in cells that were treated with macrophages [3, 8, 13]. Therefore, Western blot analy- either LPS alone or both LPS and HEF. These results suggest ses were performed to determine whether the inhibitory that the phosphorylation of MAPKs may be involved in the effects of HEF on the phosphorylations of ERK1/2, p38, and mechanisms underlying the inhibitory effects of HEF on JNK MAPKs were related to the modulations of NF-κB and LPS-induced inflammation in murine RAW 264.7 cells. AP-1 signals. As shown in Figure 5, the LPS treatment significantly promoted the phosphorylations of ERK1/2, JNK, and p38 MAPKs in the RAW 264.7 cells. At all the concen- Investigation of the relationship between the trations studied (25, 50, and 100 μg/mL), HEF dramatically anti-inflammatory and antioxidant activities reduced the phosphorylations of ERK1/2, p38, and JNK MAPKs, each in a dose-dependent manner. However, the It has been shown that reactive oxygen species (ROS) expression levels of unphosphorylated ERK1/2, p38, and trigger the phosphorylation of MAPKs and the production

ABLPS –++ ++(1 µg/mL) LPS –++ ++(1 µg/mL) HEF ––25 50 100 (µg/mL) HEF ––25 50 100 (µg/mL)

54 kDa p-JNK p-ERK 44 kDa 46 kDa 42 kDa 54 kDa 44 kDa JNK 46 kDa ERK 42 kDa

β-actin 42 kDa β-actin 42 kDa

120 120 100 100 80 80 60 60 40 40 (p-JNK, %) (p-ERK, %) Relative fold 20 Relative fold 20 0 0 LPS ++ ++(1 µg/mL) LPS ++ ++(1 µg/mL) HEF –2550 100 (µg/mL) HEF –2550 100 (µg/mL)

C LPS –++ ++(1 µg/mL) HEF ––25 50 100 (µg/mL)

p-p-38 43 kDa

p-38 43 kDa

β-actin 42 kDa

120 100 80 60 40 (p-p38, %) Relative fold 20 0 LPS ++++(1 µg/mL) HEF –2550 100 (µg/mL)

Figure 5: Effect of the ethyl acetate fraction of Shiranuhi flower extract (HEF) on lipopolysaccharide (LPS)-induced protein expression of p-JNK (phosphorylated-JNK)/JNK, p-ERK (phosphorylated-ERK)/ERK, and p-p-38 (phosphorylated-p-38)/p-38 in murine RAW 264.7 macrophages. RAW 264.7 cells (1.0 × 106 cells/mL) were pre-incubated for 18 h, incubated for 2 h with HEF, and then stimulated for 10 min with LPS (1 μg/mL). Immunoblotting was used to determine the levels of p-JNK (phosphorylated-JNK)/JNK, p-ERK (phosphorylated-ERK)/ERK, and p-p-38 (phosphorylated-p-38)/p-38. 382 Min-Jin Kim et al.: Effects of Shiranuhi flower extracts and fractions on lipopolysaccharide

AB120 120

100 IC50 = 171.19 µg/mL 100 IC50 = 149.33 µg/mL

80 80

60 60

40 40

20 20 DPPH radical scavenging activity (%) DPPH radical scavenging activity (%) 0 0 15.625 31.25 62.5 125250 500 1000 15.625 31.25 62.5 125 250 500 1000 (µg/mL) (µg/mL)

CD120 120 IC = 63.91 µg/mL IC = 54.57 µg/mL 100 50 100 50

80 80

60 60

40 40

20 20 ABTS radical scavenging activity (% ) 0 ABTS radical scavenging activity (% ) 0 15.625 31.25 62.5 125250 500 1000 15.625 31.25 62.5 125 250 500 1000 (µg/mL) (µg/mL)

Figure 6: Antioxidant activities of 70% ethanol extracts and HEF. DPPH and ABTS scavenging activities of 70% ethanol extracts (A and C) and HEF (B and D) at different concentrations. Each value represents mean ± SEM of three replicates. of pro-inflammatory cytokines [28, 29]. Therefore, 70% predominantly composed of narirutin (3.13 ± 0.43 g) and ethanol extracts and HEF of Shiranuhi flower was used hesperidin (6.03 ± 0.59 g) in 100 g of HEF. On the other to investigate the relationship between the data sets of hands, the 70% ethanol extract is composed of narirutin anti-inflammatory and the anti-oxidant activities. In this (0.69 ± 0.07 g) and hesperidin (1.75 ± 0.98 g). study, 70% ethanol extracts and HEF were screened free radical-scavenging effects such as DPPH and ABTS. Two extracts, HEF was showed good anti-oxidative effects with Conclusions

IC50 of 149.44 μg/mL for DPPH and 54.57 μg/mL for ABTS (Figure 6). The anti-oxidative evaluation demonstrates The present study was undertaken to identify the anti- that the same fraction was the best to inhibit the pro- inflammatory effects of the hot water extract, ethanolic inflammatory cytokines and MAPK mechanism. extract, and ethyl acetate and water fractions of the ethanolic extract of Shiranuhi flowers in murine RAW 264.7 macrophages. The results indicate that, in LPS-stimu- HPLC fingerprint of 70% ethanol extracts lated murine RAW 264.7 cells, HEF inhibits the productions and HEF of pro-inflammatory molecules such as NO, iNOS, PGE2, and COX-2, and cytokines such as TNF-α and IL-6. Further- Finally, in other to apply 70% ethanol extracts and HEF more, the phosphorylation of MAPKs such as p38, ERK, and for anti-inflammatory agents in cosmetic industry, stand- JNK was suppressed by HEF in a concentration-dependent ard and/or functional materials have to be investigated. manner. Furthermore, HEF containing rutin, narirutin, Therefore, the flavonoid constituents of the 70% ethanol hesperidin, naringenin, and hesperetin could be used as extracts and HEF were also investigated using HPLC anti-inflammatory agents given their ability to suppress analysis in the present study. As shown in Figure 7, a inflammatory cytokines and mediators. Based on these total of 2 citrus flavonoids were identified on the basis of results, we suggest HEF as a potential anti-inflammatory the compared to those in our HPLC library. The HEF is a candidate for pharmaceutical and cosmetic applications. Min-Jin Kim et al.: Effects of Shiranuhi flower extracts and fractions on lipopolysaccharide 383

A 0.35 HEF 0.30

0.25 Narirutin Hesperidin

0.20

AU 0.15

0.10

0.05

0.00 B 0.25 Ethanol Extract 0.20

0.15 Hesperidin AU

0.10 Narirutin

0.05

0.00

C Narirutin 0.10 Standards Naringi n

0.08 Neohesperidin Hesperidin

0.06 Rutin

AU 0.04 0.02 0.00 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 min

Figure 7: HPLC fingerprinting analysis of the HEF(A) and 70% ethanol (B) extracts. The lower side (C) represents standard flavonoids. The wavelength of flavonoids is 280 nm.

Acknowledgments: This work was supported by the 3. Jeong DH, Kim KB, Kim MJ, Kang BK, Ahn DH. Skipjack tuna (Kat- Cooperative Research Program for Agriculture Science & suwonus pelamis) eyeball oil exerts an anti-inflammatory effect by inhibiting NF-κB and MAPK activation in LPS-induced RAW Technology Development (Project no. PJ010934082015) 264.7 cells and croton oil-treated mice. Int Immunopharmacol through the Rural Development Administration (Republic 2016;40:50–6. of Korea). 4. Lee WS, Shin JS, Jang DS, Lee KT. Cnidilide, an alkylphthalide isolated from the roots of Cnidium officinale, suppresses LPS- induced NO, PGE , IL-1β, IL-6 and TNF-α production by AP-1 and Conflict of interest statement: The authors have no con- 2 NF-κB inactivation in RAW 264.7 macrophages. Int Immunophar- flict of interest. macol 2016;40:146–55. 5. Chun J, Tosun A, Kim YS. Anti-inflammatory effect of corymbo- coumarin from Seseli gummiferum subsp. corymbosum through References suppression of NF-κB signaling pathway and induction of HO-1 expression in LPS-stimulated RAW 264.7 cells. Int Immunophar- 1. Jeong YH, Oh YC, Cho WK, Yim NH, Ma JY. Anti-inflammatory macol 2016;31:207–15. effect of Rhapontici radix ethanol extract via inhibition of NF-κB 6. Chen L, Teng H, Fang T, Xiao J. Agrimonolide from Agrimo- and MAPK andi of HO-1 in macrophages. Mediators Inflamm nia pilosa suppresses inflammatory responses through 2016;2016:7216912. down-regulation of COX-2/iNOS and inactivation of NF-κB in 2. Jang KJ, Choi SH, Yu GJ, Hong SH, Chung YH, Kim CH, et al. Anti- lipopolysaccharide-stimulated macrophages. Phytomedicine inflammatory potential of total saponins derived from the roots of 2016;23:846–55. Panax ginseng in lipopolysaccharide-activated RAW 264.7 mac- 7. Intayoung P, Limtrakul P, Yodkeeree S. Anti-inflammatory activi- rophages. Exp Ther Med 2016;11:1109–15. ties of crebanine by inhibition of NF-κB and AP-1 activation 384 Min-Jin Kim et al.: Effects of Shiranuhi flower extracts and fractions on lipopolysaccharide

through suppressing MAPKs and Akt signaling in LPS-induced 19. Yoon WJ, Moon JY, Song G, Lee YK, Han MS, Lee JS, et al. Artemi- RAW 264.7 macrophages. Biol Pharm Bull 2016;39:54–61. sia fukudo essential oil attenuates LPS-induced inflammation by 8. Cha SM, Cha JD, Jang EJ, Kim GU, Lee KY. Sophoraflavanone G suppressing NF-kappaB and MAPK activation in RAW 264.7 mac- prevents Streptococcus mutans surface antigen I/II-induced pro- rophages. Food Chem Toxicol 2010;48:1222–9.

duction of NO and PGE2 by inhibiting MAPK-mediated pathways 20. Lee EH, Park HR, Shin MS, Cho SY, Choi HJ, Shin KS. Anti- in RAW 264.7 macrophages. Arch Oral Biol 2016;68:97–104. tumor metastasis activity of pectic polysaccharide purified 9. Wu J, Zhang H, Hu B, Yang L, Wang P, Wang F, et al. Coptisine from the peels of Korean Citrus Hallabong. Carbohydr Polym from Coptis chinensis inhibits production of inflammatory 2014;111:72–9. mediators in lipopolysaccharide-stimulated RAW 264.7 murine 21. Herath KH, Bing SJ, Cho J, Kim A, Kim GO, Lee JC, et al. Citrus macrophage cells. Eur J Pharmacol 2016;780:106–14. hallabong [(Citrus unshiu × C. sinensis) × C. reticulata)] exerts 10. Park JY, Moon JY, Park SD, Park WH, Kim H, Kim JE. Fruits extracts of potent anti-inflammatory properties in murine splenocytes Hovenia dulcis Thunb. Suppresses lipopolysaccharide-stimulated and TPA-induced murine ear oedema model. Pharm Biol inflammatory responses through nuclear factor-kappaB pathway 2016;22:1–12. in Raw 264.7 cells. Asian Pac J Trop Med 2016;9:357–65. 22. Chang YH, Seo J, Song E, Choi HJ, Shim E, Lee O, et al. Biocon- 11. Ham YM, Yoon WJ, Lee WJ, Kim SC, Baik JS, Kim JH, et al. Anti- verted Jeju Hallabong tangor (Citrus kiyomi × ponkan) peel inflammatory effects of isoketocharbroic acid from brown alga, extracts by cytolase enhance antioxidant and anti-inflammatory Sargassum micracanthum. EXCLI J 2015;14:1116–21. capacity in RAW 264.7 cells. Nutr Res Pract 2016;10:131–8. 12. Kim MJ, Yang KW, Kim SS, Park SM, Park KJ, Kim KS, et al. Chemi- 23. Lim H, Seo J, Chang YH, Han BK, Jeong JK, Park SB, et al. Anti- cal composition and anti-inflammation activity of essential oils obesity effects of Jeju Hallabong tangor (Citrus kiyomi × ponkan) from Citrus unshiu flower. Nat Prod Commun 2014;9:727–30. peel extracts in 3T3-L1 adipocytes. J Korean Soc Food Sci Nutr 13. Kim MJ, Yang KW, Yang EJ, Kim SS, Park KJ, An HJ, et al. Citrus 2014;43:1688–94. unshiu flower inhibits LPS-induced iNOS and COX-2 via MAPKs 24. Park JY, Shin MS, Kim SN, Kim HY, Kim KH, Shin KS, et al. Poly- in RAW 264.7 macrophages Orient J Chem 2015;31:1915–22. saccharides from Korean Citrus hallabong peels inhibit angio- 14. Cho YH, Kim NH, Khan I, Yu JM, Jung HG, Kim HH, et al. Anti- genesis and breast cancer cell migration. Int J Biol Macromol inflammatory potential of quercetin-3-O-β-D-(“2”-galloyl)- 2016;85:522–9. glucopyranoside and quercetin isolated from Diospyros kaki 25. Kim MJ, Kim SS, Park KJ, An HJ, Choi YH, Lee NH, et al. Methyl calyx via suppression of MAP signaling molecules in LPS- jasmonate inhibits lipopolysaccharide-induced inflammatory induced RAW 264.7 macrophages. J Food Sci 2016;81:C2447–56. cytokine production via mitogen-activated protein kinase and 15. Wu L, Li X, Wu H, Long W, Jiang X, Shen T, et al. 5-Methoxyl nuclear factor-κB pathways in RAW 264.7 cells. Die Pharmazie aesculetin abrogates lipopolysaccharide-induced inflammation 2016;2016:540–3. by suppressing MAPK and AP-1 pathways in RAW 264.7 cells. Int 26. Kim MJ, Hyun JM, Kim SS, Seong KC, Lim CK, Kang JS, J Mol Sci 2016;17:315. et al. In vitro screening of subtropical plants cultivated in 16. Jin SE, Kim OS, Yoo SR, Seo CS, Kim Y, Shin HK, et al. Anti- Jeju island for cosmetic ingredients. Orient J Chem inflammatory effect and action mechanisms of traditional 2016;32:807–15. herbal formula Gamisoyo-san in RAW 264.7 macrophages. BMC 27. Yang EJ, Hyun JM, Lee NH, Hyun CG. In vitro screening of korean Complement Altern Med 2016;16:219. halophytes for cosmeceutical ingredients. Int J ChemTech Res 17. Kim MJ, Kim SJ, Kim SS, Lee NH, Hyun CG. Hypochoeris radicata 2016;9:541–7. attenuates LPS-induced inflammation by suppressing p38, ERK, 28. Chanput W, Krueyos N, Ritthiruangdej P. Anti-oxidative assays as and JNK phosphorylation in RAW 264.7 macrophages. EXCLI J markers for anti-inflammatory activity of flavonoids. Int Immu- 2014;13:123–36. nopharmacol 2016;40:170–5. 18. Yoon WJ, Moon JY, Kang JY, Kim GO, Lee NH, Hyun CG. Neolitsea 29. Diaz P, Jeong SC, Lee S, Khoo C, Koyyalamudi SR. Antioxidant sericea essential oil attenuates LPS-induced inflammation in and anti-inflammatory activities of selected medicinal plants RAW 264.7 macrophages by suppressing NF-kappaB and MAPK and fungi containing phenolic and flavonoid compounds. Chin activation. Nat Prod Commun 2010;5:1311–6. Med 2012;7:26.