130429 (402)

Biosci. Biotechnol. Biochem., 77 (12), 130429-1–6, 2013

Anti-Inflammatory Activities of Methanol Extract of arborea C.B. Clarke as to Mouse Macrophage and Paw Edema

Hui-Seong KIM,1;4;* Ok-Kyoung KWON,2;4;* Ji-Won PARK,3;4 Hye Gwang JEONG,2 Sei-Ryang OH,1;4 y Hyeong-Kyu LEE,1;5 Tran The BACH,6 Do Van HAI,6 and Kyung-Seop AHN4;

1Biomolecular Science, University of Science and Technology, Daejon 305-806, Korea 2Department of Toxicology, College of Pharmacy, Chungnam National University, Daejeon 305-764, Republic of Korea 3Department of Biotechnology, Korea University, Anam Campus, Anam-dong 5-ga, Seongbuk-gu, Seoul 136-713, Republic of Korea 4Natural Medicine Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejon 363-883, Korea 5Targeted Medicine Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejon 363-883, Korea 6Department of Botany, Institute of Ecology and Biological Resources, Vietnam Academy of Science and Technology, 18 Hoang Quac Viet, Cau Giay, Hanoi, Vietnam

Received May 28, 2013; Accepted September 29, 2013; Online Publication, December 7, 2013 [doi:10.1271/bbb.130429]Advance View The biological activity of Mastixia arborea (MA) central target in anti-inflammation therapy.5,6) Inhibition relates to inflammation, but the underlying mechanisms of NO, PGE2, IL-1, and IL-6 by blocking the are largely unknown. We confirmed the anti-inflamma- expression of iNOS and COX-2 in macrophages is a tory effects of a methanol extract of MA extract on strategy in the development of anti-inflammatory agents. lipopolysaccharide (LPS)-stimulated RAW264.7 mouse Intracellular signaling events activated by LPS regu- macrophage cells and carrageenan-induced mice paw late the phosphorylation of mitogen-activated protein edema. The MA extract significantly inhibited nitric kinases (MAPKs), including extracellular signal-related oxide (NO), prostaglandin E2 (PGE2), interleukin-1 kinase (ERK), p38, and c-Jun N-terminal kinase (JNK), (IL-1 ), and IL-6 production in LPS-stimulated and the activation of transcription factors such as RAW264.7 cells. In vitro expression of inducible nitric nuclear factor NF-ProofsB.7) These signaling events lead oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) macrophages to be transcriptionally activated and to was suppressed by the extract. The extract attenuated express proinflammatory genes, including ones for acute inflammatory responses in carrageenan-induced cytokines, iNOS, and COX-2.8) mice paw edema. A mechanism study indicated that Mastixia arborea C.B. Clarke (MA) is a tree that translocation of the NF-B (p65) subunit into the grows up to 20 m tall belonging to the Cornaceae family nucleus and phosphorylation of ERK and JNK were whose range extends from India through Southeast Asia inhibited by the extract. These results indicate that the and New Guinea to the Solomon Islands.9) An early extract is an effective suppressor of the inflammatory report indicated that MA contains a compound, loganin, response, blocking the phosphorylation of ERK and in the bark,10) but the mechanisms underlying the anti- JNK and the translocation of NF-B in macrophages, inflammatory properties of a methanol extract of MA thereby producing an anti-inflammatory effect in vivo. have not been identified. In this study, we aimed to confirm the anti-inflammatory effects of a methanol Key words: inflammation; Mastixia arborea; MAPK; extract of MA in in vitro and in vivo inflammatory nitric oxide; NF-B models. There have been no studies focusing on the anti- inflammatory activity of methanol extracts of MA in Lipopolysaccharide (LPS) is a potent activator of cells or animal models. macrophages that produces a variety of proinflammatory mediators, including nitric oxide (NO), prostaglandins, Materials and Methods and cytokines.1,2) Among these proinflammatory mole- cules, NO, produced by inducible nitric oxide synthase Preparation of MA extract (Mastixia arborea). were (iNOS), is associated with damage to normal cells and collected at Quang Binh, Quang Ninh, Truong Son, Vietnam in 3) 2008. The samples were identified by Tran The Bach of the Institute of tissues. Prostaglandin E2 (PGE2), synthesized from arachidonic acid by cyclooxygenase-2 (COX-2), also Ecology and Biological Resources. A voucher specimen (KRIB 0019270) has been deposited in the herbarium of the Korea Research plays a major role as a mediator of the inflammatory Institute of Bioscience and Biotechnology. Leaves and branches of 4) response. Similarly, interleukin-1 (IL-1) and IL-6 M. arborea (67 g) were treated with MeOH and sonicated several times act significantly as proinflammatory cytokines, and are a at room temperature for 3 d to produce an extract (4.13 g). A stock

y To whom correspondence should be addressed. Tel: +82-43-240-6113; Fax: +82-43-240-6129; E-mail: [email protected] * Hui-Seong Kim and Ok-Kyoung Kwon contributed equally to this study 130429-2 H.-S. KIM et al. solution (20 mg/mL) of the extract was prepared in DMSO, and this 30) and COX-2 (forward 50-GAA GTC TTT GGT CTG GTG CCT G- was stored at 20 C before use. 30, reverse 50-GTC TGC TGG TTT GGA ATA GTT GC-30) was done by GeneAmp PCR System 9700 (Applied Biosystems) at 94 C for Cell Culture. The RAW264.7 mouse macropharge cell line was 30 s, and at 56 C 30 s, and 72 C for 30 s. As internal control, -actin 0 0 0 maintained under a 95% air, 5% CO2 (v/v) atmosphere in Dulbecco’s (forward 5 -AGG CTG TGC TGT CCC TGT ATG C-3 , reverse 5 - Modified Eagle’s Medium (Gibco, Grand Island, NY) supplemented ACC CAA GAA GGA AGG CTG GAA A-30) was amplified routinely. with 10% (v/v) heat-inactivated fetal bovine serum (Hyclone, Logan, The amplified products were resolved in agarose gel at a 1.2% (w/v), UT) and a 1% (w/v) antibiotic–antimycotic solution (Invitrogen, stained with Red Safe (Intron Biotechnology, Daejon, Korea), and Grand Island, NY). photographed under ultraviolet light.

Animal experiments. Specific pathogen-free female BALB/c mice Western blot analysis. Cells were lysed in 100 mL of lysis buffer (6 weeks old) were purchased from Koatech (Seoul, Korea), and were containing protease inhibitors (50 mM Tris–HCl pH 7.4, 150 mM used after 2 weeks of quarantine and acclimatization. They were NaCl, 1 mM EDTA, 0.5% v/v NP-40, 0.1% w/v SDS, 1 mM EGTA, allowed sterilized tap water and standard rodent chow. All exper- 100 mg/mL of PMSF, 10 mg/mL of pepstatin A, and 100 mM Na3VO3). imental procedures were carried out in accordance with the NIH The cell lysate were centrifuged at 14,000 rpm at 4 C for 20 min, and Guidelines for the Care and Use of Laboratory Animals, and were the protein concentrations in supernatants were determined with approved by the Institutional Animal Care and Use Committee of the Bradford reagent (Bio-Rad, Hercules, CA). Twenty mg of protein was Korea Research Institute of Bioscience and Biotechnology, and the loaded into each well of an SDS-polyacrylamide gel prior to animals were cared for in accordance with the guidelines of the electrophoresis at 100 V for 90 min, and the separated proteins were 11) National Animal Welfare Law of Korea. transferred to PVDF membranes (Amersham Biosciences, Piscataway, NJ). The membranes were blocked with 5% (w/v) nonfat dry milk Cell viability. Cell viability under various concentrations of MA dissolved in TBST buffer (10 mM Tris–HCl pH 7.5, 150 mM NaCl, was determined with 3-(4,5-dimethylthiaxol-2yl)-2,5-diphenyltetrazo- 0.1% v/v Tween-20) overnight at 4 C and then incubated with lium bromide (MTT) (CAS#298-93-1; Amresco, Solon, OH). Briefly, primary antibodies that recognized iNOS (1:1,000, Enzo, Farmingdale, an MTT solution (5 mg/mL) was added to a cell supernatant at final NY), COX-2 (1:1,000, Santa Cruz Biochemicals, Santa Cruz, CA), concentration of 0.5 mg/mL. After 4 h of incubation at 37 C, the -actin (1:2,000, Abcam, Cambridge, UK), poly (ADP-ribose) poly- medium was removed and DMSO was added. The optical density of merase (PARP), Nuclear-factor-B subunit 65 (NF-B p65), extrac- formazanAdvance was measured with a microplate reader (Benchmark; View Bio- ellular signal-regulated protein kinase (ERK)2, p38 mitogen-activated Rad Laboratories, Hercules, CA) at 570 nm. The level of formazan protein kinase (MAPK), c-Jun N-terminal kinase (JNK)1/3 (1:1,000, generated by the untreated cells was chosen as the 100% value. Santa Cruz Biochemicals, Santa Cruz, CA), phospho-forms of p38 MAPK and JNK1/2 (1:1000, Enzo, Farmingdale, NY), and phospho- Nitric oxide assay. RAW264.7 cells were plated at a density of forms of IB-, ERK, or total IB- (1:1,000, Cell Signaling : 5 2 5 10 cells/mL in 96-well plates and incubated with and without Technology, Boston, MA). Immunoreactive bands were visualized LPS (1 mg/mL) in the absence and the presence of various concen- with ECL reagent (Amersham Pharmacia Biotech, Uppsala, Sweden) trations of MA extract for 24 h. Nitrite accumulation in the super- in RAS-4000mini (Fujifilm, Tokyo). Densitometry band values were natants was assessed by the Griess reaction. Aliquots (100 mL) of the determined with image J image analysis software (version 1.43, culture supernatants were mixed with equal volumes of Griess reagent National Institutes of Health), and analyzed statistically. (0.1% w/v N-(1-naphthyl)-ethylenediamine, with 1% w/v sulfanila- mide in 5% v/v phosphoric acid) and incubated at room temperature Immunofluorescence. RAW264.7 cells were cultured on Permanox for 10 min. the absorbance at 540 nm was measured with a microplate plastic chamber slidesProofs (Nunc, Rochester, NY) and fixed in ethanol at reader, and a series of known sodium nitrite concentrations served as 4 C over 30 min. The slides were washed 3 times with PBS and blocked standards. with 3% (w/v) BSA in PBS for a further 30 min. Then they were incubated for 24 h at 4 C with anti-iNOS, anti-COX-2 (rabbit PGE2 assay. PGE2 levels in the supernatants were determined with polyclonal IgG, 1:200 dilution, Santa Cruz Biotechnology), and anti- a PGE2 EIA kit (Cayman Chemical, Ann Arbor, MI) employing the NF-B p65 subunit (rabbit polyclonal IgG, 1:500 dilution, Assay manufacturer’s protocol. Briefly, 50 mL doses of diluted standards and samples were pipetted into the wells of a 96-well plate precoated with Designs, Ann Arbor, MI) antibodies. After washing to remove excess primary antibody, the slides were further incubated with anti-rabbit goat polyclonal anti-mouse IgG. Aliquots of a PGE2 monoclonal Alexa fluor 488-conjugated or anti-rabbit Texas Red conjugated antibody solution and a PGE2 acetylcholine esterase conjugate solution secondary antibody (Santa Cruz Biotechnology) for 2 h at room tempera- were added to each well, and incubation at 4 C for 18 h followed. The wells were washed 6 times with buffer containing 0.05% (v/v) Tween- ture, washed with PBS, and mounted with ProLong Gold Antifade reagent containing 40,6-diamidino-2-phenylindole (DAPI) (Invitrogen) 20, followed by the addition of 200 mL doses of Ellman’s reagent for 5 min, prior to localization and quantification of nuclei. Subse- containing acetylthiocholine and 5,50-dithio-bis-(2-nitrobenzoic acid). quently, the slides were coverslipped and visualized by confocal laser PGE2 concentrations were measured by the absorbance at 405 nm. scanning microscopy (LSM510; Carl Zeiss, Oberkochen, Germany). Cytokine assays. The inhibitory effects of the MA extract on IL-6 All samples were photographed under the same exposure conditions. and IL-1 production were measured by enzyme-linked immunosorb- ent assay (ELISA) of the culture supernatants. Samples were analyzed Carrageenan-induced mouse-paw edema. Carrageenan-induced 12) with a mouse cytokine–Quantikine kit, following the manufacturer’s paw inflammation was achieved by a method described previously. recommendations (Biosourse, Minneapolis, MN). The concentrations Female Balb/c mice (body weight 20 g) were selected randomly and of IL-6 and IL-1 were calculated with reference to standard curves divided into groups of five to six animals. MA was dissolved in PBS generated with the recombinant cytokines provided in the ELISA kit. and administered at doses of 40 and 80 mg/kg. The other groups were injected with the reference drug (indomethacin, 5 mg/kg, per oral) or Reverse transcription (RT)-PCR analysis. Total RNA was isolated the control (PBS). Thirty min later, edema was induced by injection of from RAW264.7 cells with TRIzol (Invitrogen, Carlsbad, CA), 20 mL of 1% v/v carrageenan solution in PBS into an animal’s left hind following the manufacturer’s instructions. Briefly, the purified RNA paw. Measurements of the paw volume were done by caliper was resuspended in diethylpyrocarbonate-treated water and its integrity immediately prior to the carrageenan injection and after 4 h. Paw was confirmed by agarose-formaldehyde electrophoresis. cDNA was thickness was determined as the difference between the final and the synthesized from total RNA previously treated with DNA-free DNase initial thickness. (Ambion, Austin, TX). One mg of RNA were incubated with oligo dT 18mer (Bioneer), RNase OUT (Invitrogen), and the omniscript RT kit Statistics. For statistical analysis, values were expressed as (Qiagen, Hilden, DE) for 60 min at 37 C, following the manufacturer’s means SEM. Statistical significance was determined by two-tailed instructions. Amplification of iNOS (forward 50-GGA GCG ACT TGT Student’s t test for independent means. p values of less than 0.05 were GGA TTG TC-30, reverse 50-GTG AGG GCT TGG CTG AGT GAG- considered statistically significant. Anti-Inflammatory Activities from MA as to Mouse Macrophage and Paw Edema 130429-3

Results the amounts of PGE2 released and COX-2 level were assessed with a PGE2 ELISA kit and Western blotting. Effects of MA extract on NO production and iNOS As shown in Fig. 2, LPS increased the PGE2 (Fig. 2A) activity in LPS-stimulated RAW264.7 cells and COX-2 levels (Fig. 2B and C), but these changes iNOS is a key enzyme in the pathophysiological were attenuated significantly by the MA extract, in a process of inflammation and the induction of NO. MA dose-dependent manner. These results suggest that inhibited NO production (Fig. 1A) by inhibiting expres- MA extract can inhibit LPS-induced PGE2 release and sion of the iNOS protein (Fig. 1B and 1D) and mRNA COX-2 production in RAW264.7 cells. (Fig. 1C). Western blot and immunofluorescence analy- ses indicated that LPS treatment significantly increased Effects of the MA extract on IL-1 and IL-6 release in expression of the iNOS protein. Pretreatment of the cells LPS-stimulated RAW264.7 cells with MA attenuated the LPS-induced increases in iNOS Cytokines are produced during the inflammatory protein expression in a dose-dependent manner. RT- process and cytokine levels are indicative of inflamma- PCR analysis indicated that the expression of iNOS tory progression. To determine whether MA extract mRNA was augmented markedly in the presence of LPS would affect the release of cytokines in LPS-stimulated alone. This suggests that the MA extract inhibited NO RAW264.7 cells, we used an IL-1 and IL-6 sensitive production and suppressed iNOS gene expression in Quantikine kit (BioSource International, Camarillo, LPS-stimulated RAW264.7 cells. CA). As shown in Fig. 3, incubation of RAW264.7 cells with LPS for 24 h increased the levels of IL-1 Effects of MA on PGE2 and COX-2 production in (Fig. 3A) and IL-6 (Fig. 3B) in the culture medium. LPS-stimulated RAW264.7 cells RAW264.7 cells were pretreated with various concen- Next we examined the effects of MA on PGE2 release trations of the MA extract (5, 10, 20, or 30 mg/mL) for andAdvance COX-2 levels. Cells were stimulated with View LPS and 1 h and continuously incubated with LPS for 18 h. The A B

µ − 12 LPS (1 g/mL) ++ + + + # MA (µg/mL) − 0 5 10 20 30 10 * iNOS * 8 ) β-Actin M µ µ 6 ** C Nitrite ( 4 Proofs − *** LPS (1 µg/mL) ++ + + +

2 MA (µg/mL) − 0 5 10 20 30

iNOS 0 µ − +++++ LPS (1 g/mL) β-Actin MA (µg/mL) − 0 5 10 20 30

D Control LPS LPS+MA

iNOS

DAPI

Merge

Fig. 1. LPS-Induced NO Production and iNOS Expression Were Inhibited by an Extract of MA in RAW264.7 Cells. (A) RAW264.7 cells were pretreated with MA extract (5, 10, 20, and 30 mg/mL) for 1 h, and then stimulated with LPS (1 mg/mL). Culture medium was collected at 24 h, and NO concentrations were measured by the Griess reaction. RAW264.7 cells were pretreated with MA extract for 1 h and stimulated with LPS for 6 h or 24 h. Total RNA and protein were isolated, and iNOS protein (B) and mRNA (C) were measured. - Actin expression was used as internal control in RT-PCR and Western blotting. Immunocytochemistry analysis of iNOS expression was done. After fixation, cells were stained green. Nuclei were visualized using DAPI (blue), and observed at a magnification of 400. Control, untreated cells; LPS, LPS only (1 mg/mL); MA, MA extract (30 mg/mL) and LPS. Three independent experiments were done, and data are presented as means SEM. # p < 0:05, p < 0:05, p < 0:01, and p < 0:001 compared to control (#) and to cells treated with LPS alone (). 130429-4 H.-S. KIM et al. AB

1200 LPS (1 µg/mL) − +++++

MA (µg/mL) − 0 5 10 20 30 1000 # COX-2 800 * β -Actin 600

(pg/mL) ** 2 ** C ** PGE 400 LPS (1 µg/mL) − ++ +++ 200 MA (µg/mL) − 0 5 10 20 30

COX-2 0 LPS (1 µg/mL) − +++++ β -Actin MA (µg/mL) − 0 5 10 20 30

Fig. 2. MA Extract Inhibited LPS-Induced PGE2 Production and COX-2 expression in RAW264.7 Cells. (A) RAW264.7 cells were pretreated with MA extract (5, 10, 20, and 30 mg/mL) for 1 h and stimulated with LPS (1 mg/mL). Culture media were collected at 24 h to measure PGE2 concentrations by enzyme immunoassay. Data are presented as means SEM of three independent experiments, # p < 0:05, p < 0:05, and p < 0:01 compared to control (#) and to cells treated with LPS alone (). RAW264.7 cells were pretreated with MA extract for 1 h and stimulated with LPS for 6 h or 24 h. Total RNA and protein were isolated, and COX-2 protein (B) and mRNA (C) were measured. -actin expression was used as internal control in both experiments. Representative results of four independent Advanceexperiments are shown. View A B 80 500 # ** # *** 400 60 ** ** 300 40

*** (pg/mL) β β ** 200 IL-6 (pg/mL)

IL-1 ** 20 ** Proofs 100

0 0 LPS (1 µg/mL) − +++++ LPS (1 µg/mL) − +++++ MA (µg/mL) − 0 5 10 20 30 MA (µg/mL) − 0 5 10 20 30 C 160 ***

140

120

100

80

60 Cell viability(%) 40

20

0 MA (µg/mL) 0 5 10 20 30

Fig. 3. MA Extract Inhibited IL-1 and IL-6 Production in LPS-Stimulated RAW264.7 Cells. (A) IL-1 and (B) IL-6 cytokines were measured by ELISA of culture supernatants collected from treated cells. Samples were analyzed following the manufacturer’s protocols. RAW264.7 cells were treated with 0–30 mg/mL the MA extract for 1 h, and continuously incubated with LPS (1 mg/mL) for a further 18 h. (C) Cells were incubated in the presence and the absence of 0–30 mM of MA extract, and cell viability was determined by MTT assay. Data are presented as means SEM of three samples. (#Significant difference from control, Significant difference from LPS treated only. # p < 0:05, p < 0:05, p < 0:01, and p < 0:001). Anti-Inflammatory Activities from MA as to Mouse Macrophage and Paw Edema 130429-5 MA extract blocked increases in the cytokine level, but treated with LPS (1 mg/mL) for 5–30 min, and Western did not influence the cell viability of the RAW264.7 blotting was used to detect all MAPK phosphorylation. cells (Fig. 3C). The MA extract suppressed LPS-induced phosphoryla- tion of ERK and JNK in a dose-dependent manner Effects of MA on NF-B translocation and phosphor- (Fig. 4B). ylation of ERK and JNK in LPS-stimulated RAW264.7 cells Effects of the MA extract on carrageenan-induced We examined the effects of the MA extract on LPS- paw edema induced NF-B and MAPK activation. NF-B induces To determine the inhibitory effects of MA on acute both iNOS and COX-2, and is translocated into the inflammation in vivo, we used the carrageenan-induced nucleus by phosphorylation of IB and subsequent paw edema model.12) Paw edema formation appeared at degradation of IB subunit. Treatment of RAW264.7 1 h after exposure to carrageenan. Administration of the cells with MA for 1 h markedly inhibited LPS-induced MA extract (40 or 80 mg/kg over 4 h) significantly increase, in the nuclear levels of NF-B (Fig. 4A). This reduced the increase in paw thickness induced by suggests that MA prevents inflammatory responses such carrageenan. Indomethacin (5 mg/kg), an anti-inflam- as iNOS and COX-2 expression by blocking the trans- matory agent inhibiting prostaglandin (PG) synthesis in location of NF-B into the nucleus. We also examined vivo and in vitro, was used as positive control. Figure the effects of MA extract on LPS-stimulated phospho- shows the effect of the MA extract at 4 h after exposure rylation of ERK, JNK, and p38 MAPK. Cells were to carrageenan, corresponding to its maximum activity. Discussion A Control LPS LPS+MA Inflammation is a process regulated by a variety of Advance Viewimmune cells and effector molecules. NO, PGE2, and p65 proinflammatory cytokines are important mediators of macrophage-mediated inflammation.13,14) In the present study, we evaluated the anti-inflamma- tory effects of an MA extract both in vivo and in vitro. Carrageenan-induced paw edema is an established

DAPI method of studying inflammatory states and screening for the anti-inflammatory activity of experimental com- pounds in animal models. A reduction of paw swelling is a good indicator of the protective action of an anti- inflammatory agent.12) As shown in Fig. 5, MA (40 or 80 mg/kg) inhibitedProofs the formation of paw edema at 4 h Merge after treatment. In an in vitro study, we examined the potential anti-inflammatory effects of MA by measuring LPS-induced changes in the levels of iNOS, COX-2, NO, and PGE2 and the production of cytokines such as B LPS LPS+MA IL-1 and IL-6 in RAW264.7 macrophage cells. During 05 15 300 5 15 30 (min) pJNK1/2 1.2 JNK1/2 # 1

pp38 0.8 ** ** ** p38 0.6 pERK1/2 0.4

ERK Edema thickness (mm) 0.2

0 Fig. 4. MA Extract Suppressed the Translocation of NF-Bto Car (20 µL) − ++++ Nucleus and the Phosphorylation of MAPK Molecules in the LPS- MA (mg/kg) − − − stimulated RAW264.7 Cells. 40 80 (A) Cells were treated with MA extract (30 mg/mL) for 1 h and IM (5 mg/kg) − − − − + stimulated with LPS for a further 24 h. Translocation of NF-B to the nucleus was confirmed by immunocytochemistry. After Fig. 5. Acute Inflammation in Carrageenan-Induced Paw Edema Was fixation, the cells were stained green. The nuclei were visualized Inhibited MA Extract. using DAPI (blue), and were observed at a magnification of 400. These data reflect post-injection paw thickness and pre-injection Control, untreated cells; LPS, LPS only (1 mg/mL); MA, MA extract paw thickness SE. Administration of MA (40 or 80 mg/kg) (30 mg/mL) and LPS. (B) Cells were pretreated with MA extract markedly inhibited carrageenan-induced paw edema (0.36 mm (30 mg/mL) for 1 h, and then stimulated with LPS (1 mg/mL) for 0, and 0.38 mm), and the percentages of inhibition of edema thickness 5, 15, and 30 min. Cellular proteins were evaluated for detection of were calculated (40.1% and 42% respectively). IM, indomethacin phosphorylated patterns of MAPK molecules: ERK, JNK, and p38 (5 mg/mL); Car, 20 mL of 1% v/v carrageenan solution in PBS. MAPK. Representative results of three independent experiments are (#Significant difference from control, Significant difference from shown. LPS treated only. # p < 0:05, p < 0:05, and p < 0:01). 130429-6 H.-S. KIM et al. the inflammatory process, macrophages participate in Acknowledgments inflammatory responses by producing inflammatory cytokines IL-1 and IL-6 and other inflammatory This study was supported by grants from FGC mediators, such as NO and PGE2, which recruit addi- (no. 1011231) and KGM (no. 1221312) awarded to the tional immune cells to the site of infection or tissue Korea Research Institute of Bioscience and Biotechnol- injury.13) As shown in Fig. 3A and B, the MA extract ogy (KRIBB) of the Republic of Korea. reduced the production of proinflammatory cytokines IL-1 and IL-6 dose dependently without cytotoxicity References (Fig. 3C). iNOS activates its effector molecule NO, a 1) Fujihara M, Muroi M, Tanamoto K, Suzuki T, Azuma H, and free radical synthesized from L-arginine that causes 15) Ikeda H, Pharmacol. Ther., 100, 171–194 (2003). cellular damage in inflammatory sites. Like iNOS, 2) Kuroda E and Yamashita U, J. Immunol., 170, 757–764 (2003). 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Dicotyledons: Celastrales, iNOS and COX-2 expression by downregulating NF-B Oxalidales, Rosales, , Ericales (The Familes and translocation to the nucleus (Fig. 4A) and the phospho- Genera of Vascular ) (v.6),’’ Springer, New York (2004). 10) Wieffering JH, Phytochemistry, 5, 1053–1064 (1966). rylation of MAPKs, including ERK and JNK (Fig. 4B). Advance View11) Yuk JE, Lee MY, Kwon OK, Cai XF, Jang HY, Oh SR, Lee HK, We used two approaches to show the effects of MA: a and Ahn KS, Int. Immunopharmacol., 11, 273 (2011). mouse model of carrageenan-induced inflammation, and 12) Handy RL and Moore PK, Br. J. Pharmacol., 123, 1119–1126 RAW264.7 macrophage cells induced by LPS. The MA (1998). extract attenuated the paw swelling induced by carra- 13) Bosca L, Zeini M, Traves PG, and Hortelano S, Toxicology, geenan and suppressed the activation of inflammatory 208, 249–258 (2005). pathways in macrophages by preventing induction of 14) Moncada S, J. R. Soc. Med., 92, 164–169 (1999). 15) Connelly L, Palacios-Callender M, Ameixa C, Moncada S, and iNOS and COX-2 expression, by attenuating NO and Hobbs AJ, J. Immunol., 166, 3873–3881 (2001). PGE2 production by inhibiting the translocation of NF- 16) Cuccurullo C, Fazia ML, Mezzetti A, and Cipollone F, Curr. B into the nucleus, and by reducing the phosphoryla- Med. Chem., 14, 1595–1605 (2007). tion of ERK and JNK. To date mastixia have been 17) Park JW, Kwon OK, Jang HY, Jeong H, Oh SR, Lee HK, Han reported only for loganin and loganic acid.10,18) The SB, and Ahn KS,ProofsInflammation, 35, 321–331 (2012). iridoid compound especially loganin inhibited COX-2 18) Hegnauer R, Pure Appl. Chem., 14, 173–187 (1967). enzyme activities, TNF-, and NO production in vivo19) 19) Ryu KH, Rhee HI, Kim JH, Yoo H, Lee BY, Um KA, Kim K, 20) Noh JY, Lim KM, and Chung JH, Biosci. Biotechnol. Biochem., and in vitro. Firstly we identified a biological effect of 74, 2022–2028 (2010). methanol extract of MA, and it is possible if extract 20) Park KS, Kim BH, and Chang IM, Evid. Based Complement. inhibits inflammatory disease due to constituents in- Alternat. Med., 7, 41–45 (2010). cluding loganin.