Biosci. Biotechnol. Biochem., 77 (3), 482–486, 2013

Kuromoji ( umbellata) Essential Oil Inhibits LPS-Induced Inflammation in RAW 264.7 Cells

y Hayato MAEDA, Mao YAMAZAKI, and Yohtaro KATAGATA

Faculty of Agriculture and Life Science, Hirosaki University, 3 Bunkyo-cho, Hirosaki, Aomori 036-8561, Japan

Received September 5, 2012; Accepted December 10, 2012; Online Publication, March 7, 2013 [doi:10.1271/bbb.120692]

Kuromoji (Lindera umbellata) essential oil (KEO) has tion of prostaglandins causes inflammatory disorders. long been used in Japan as a traditional medicine. It Therefore, inhibition of iNOS and COX-2 under contains linalool (C10H18O), a naturally occurring small inflammatory disorder condition by macrophages is a . For this study, we investigated the anti- useful strategy to screen anti-inflammatory drugs.6) inflammatory effect of KEO in lipopolysaccharide Essential oils diluted from several plants have been (LPS)-stimulated RAW 264.7 cells. Mouse macro- used for a long time in perfumery, , food, phage-like RAW 264.7 cells were stimulated with LPS. and flavors. Many essential oils are known to be potent Then they were treated with 25 or 50 g/mL of KEO antibacterial and antifungal agents.7) Some essential for 24 h. KEO suppressed LPS-induced pro-inflamma- oil components reportedly have anti-inflammatory ac- tory cytokine production such as that of nitric oxide tivity.8) The advantage of these components in anti- (NO), interleukin-6 (IL-6), and tumor necrosis factor- inflammatory therapy is their low or negligible toxicity. (TNF- ) in a dose-dependent manner. In addition, Therefore, such components are useful in the long-term inducible NO synthase (iNOS) and cyclooxygenase-2 chemoprevention and chemotherapy of inflammation. (COX-2) mRNA expression and protein levels were Kuromoji, a deciduous shrubby tree native to cool and suppressed by treatment with KEO cells. In addition, by warm temperate areas of Japan, is known as a spicebush treatment with 25 or 50 g/mL of linalool showed the because its twigs and leaves contain aromatic compo- same anti-inflammatory effect. The results suggest that nents. Consequently, kuromoji branches are used as KEO and linalool can be regarded as a natural resource indoor decorations and to produce shavings for aromatic for use in anti-inflammatory therapeutic products. baths. Furthermore, kuromoji essential oil (KEO) ob- tained through steam distillation is used traditionally as Key words: essential oil; Kuromoji (Lindera umbellata); a medicine for neuralgia, stiff neck, and back pain. The RAW 264.7 cells; linalool main KEO components are and esters, of which linalool is the most abundant component. Inflammation is a complex process mediated by the Geranyl acetate, an ester in KEO, has a relaxing and activation of various immune cells. Macrophages play a calming effect on humans. Other terpenes ( and central role in mediating various immunopathological -) and ( and cineol) in essential phenomena during inflammation, including the over- oils have beneficial activities in various diseases, production of pro-inflammatory cytokines and inflam- supporting anti-inflammatory and anti-obesity effects.8,9) matory mediators such as IL-6, TNF-, and NO.1–4) NO These terpenoid components become ligands in nuclear has been identified as an important molecule involved in receptors, regulate gene expression, and ameliorate regulating biological activities in vascular, neural, and conditions related to these diseases.10) immune systems. NO produced by activated macro- Linalool, a compound that is reportedly phages has been found to mediate host defense func- a major volatile component of the essential oils of tions, including antimicrobial and antitumor activities, several aromatic species, is used in traditional medicine but excess production of it causes tissue damage systems to relieve symptoms and to cure various associated with acute and chronic inflammation.5) iNOS ailments, both acute and chronic. It was evaluated is key enzyme producing large amounts of NO through recently for its psychopharmacological activity in mice, macrophages. Increased NO production has been im- revealing marked dose-dependent sedative effects on plicated as a cause of diverse inflammatory diseases, the central nervous system,11,12) including protection including rheumatoid arthritis and ulcerative colitis. against pentylenetetrazol, picrotoxin, and transcorneal Moreover, COX-2 promotes prostaglandin E2 (PGE2) electroshock-induced convulsion, and its hypnotic and synthesis from . Cyclooxygenase hypothermic properties.13) Furthermore, several recent (COX) exists in two major isoforms, COX-1 and reports have stated that linalool shows anti-proliferative COX-2. COX-1 is expressed constitutively in many activity against some solid tumor cells such as melanoma tissues and COX-2 is an inducible enzyme expressed in cells and renal cell adenocarcinoma cells14) and inflammation-related cells such as macrophages, pro- HepG2.15) It induces apoptosis in human leukemia ducing large amounts of prostaglandins. Excess produc- cells.16) Our previous study indicated that KEO mainly

y To whom correspondence should be addressed. Tel/Fax: +81-172-39-3790; E-mail: [email protected] Abbreviations: LPS, lipopolysaccharide; NO, nitric oxide; IL-6, interleukin-6; TNF-, tumor necrosis factor-; NO, inducible NO synthase; COX-2, cyclooxygenase-2; NF-B, nuclear factor-B; PPAR, peroxisome proliferator-activated receptor Kuromoji Inhibits LPS-Induced Inflammation 483 containing linalool showed antitumor activity against cleavage of the tetrazolium salt WST-1 by mitochondrial dehydrogen- several human tumor cell lines.17) In addition, Peana ase of viable cells. et al. have reported that linalool and the corresponding 5 ester (linalyl acetate) exhibited anti-inflammatory activ- Quantification of NO production. RAW 264.7 cells (5 10 cells/ 18) mL) were cultured in a 24-well culture plate with 500 mL medium and ity in rat inflammation model experiments. Therefore, incubated as described previously. The nitrite-containing culture KEO containing linalool and other is neces- medium was measured as an indication of NO production based on sary to elucidate anti-inflammatory principles. the Griess reaction. Briefly, 100 mL of cell culture medium was mixed In murine macrophage RAW 264.7 cells, LPS with 100 mL of Griess reagent (Cayman Chemical, Ann Arbor, MI) in a stimulation alone can induce the production of pro- 96-well culture plate. After incubation of 10 min at room temperature, inflammatory cytokines and inflammatory mediators. the optical density was determined at 550 nm using a microplate reader. Fresh culture medium was used as the blank in all experiments. Thus, this experimental model is useful for drug The nitrite concentration in the samples was measured by serial screening and for the evaluation of potential inhibitors dilution the standard curve of NaNO2. of the inflammatory response. The present study was undertaken to investigate the anti-inflammatory activity Extraction of total RNA and quantitative RT-PCR analysis. The of KEO by adjusting the production of pro-inflammatory total RNA of the cultured cells was extracted (Quick Gene mini 80; cytokines and inflammatory mediators by LPS-stimu- Fujifilm, Tokyo) following to the manufacturer’s instructions. Then lated RAW 264.7 cells. In addition, the effect was total RNA was reverse-transcribed by High Capacity cDNA Reverse Transcription (Applied Biosystems Japan, Tokyo) for cDNA synthesis. compared to that of linalool of which is a major IL-6, TNF-, COX-2, and iNOS mRNA expression was measured component of KEO. using a real-time PCR detection system (Opticon 2; Bio-Rad Laboratories, Hercules, CA). Thunderbirdtm SYBER qPCR Mix Materials and Methods (Toyobo, Osaka, Japan) was used in the PCR reaction. PCR cycling was done at 95 C for 1 min at 95 C for 15 s and 60 C for 1 min for 40 Chemical reagents and cells. For use in this study, Mouse cycles. The primer sequences used for RT-PCR were the following: 0 0 0 macrophage-like cell line RAW 264.7 cells were purchased from DS 5 -CCA CGG CCT TCC CTA CTT C-3 (forward) and 5 -TTG GGA 0 Pharma Biomedical (Osaka, Japan). Linalool was from Tokyo Chemi- GTG GTA TCC TCT GTG A-3 (reverse) for mouse IL-6 0 0 cal Industry (Tokyo). Fetal bovine serum (FBS) was from Biological (NM 031168.1); 5 -CAC AAG ATG CTG GGA CAG TGA-3 0 0 Industries (Kibbutz Beit, Israel). Dulbecco’s Modified Eagle’s Medium (forward) and 5 -TCC TTG ATG GTG GTG CAT GA-3 (reverse) 0 (DMEM) was from Nissui Pharmaceutical (Tokyo). ELISA kits for for mouse TNF- (NM 013693.2); 5 -TGC CTC CCA CTC CAG ACT 0 0 0 TNF- and IL-6 were from Immuno-Biological Laboratories (Gunma, AGA-3 (forward) and 5 -CAG CTC AGT TGA ACG CCT TTT-3 0 Japan) and Shibayagi (Gunma, Japan). Anti-iNOS, anti-COX-2, and (reverse) for mouse COX-2 (NM 011198.3); 5 -GGA TCT TCC CAG 0 0 anti--actin antibodies were from Santa Cruz Biotechnology (Santa GCA ACC A-3 (forward) and 5 -CAA TCC ACA ACT CGC TCC 0 0 Cruz, CA), Cayman Chemical (Ann Arbor, MI), and AbD SeroTec AA-3 (reverse) for mouse iNOS (NM 010927.3); 5 -CAT GGC CTT 0 0 (Raleigh, NC) respectively. All other chemicals, guaranteed to be of CCG TGT TCC TA-3 (forward) and 5 -GCG GCA CGT CAG ATC 0 reagent or tissue-culture grade, were from Sigma-Aldrich Japan CA-3 (reverse) for mouse GAPDH (NM 008084.2). PCR reactions (Tokyo) or Wako Pure Chemical Industries (Osaka, Japan). were normalized to GAPDH.

Isolation of Kuromoji essential oil (KEO). KEO was donated by Detection of IL-6 and TNF-a in the supernatants. The concen- Enjyakudo (Aomori, Japan). Fresh kuromoji leaves and branches were trations of IL-6 and TNF- in the culture supernatants were determined collected in the mountains near the city of Aomori, and were heated to by ELISA, conducted using a Mouse IL-6 ELISA Kit (Immuno- boiling. Then the hydro-distilled volatile fraction was collected and Biological Laboratories, Gunma, Japan) and a Mouse TNF- ELISA separated (upper layer) from the aqueous portion. Analysis of KEO Kit (Shibayagi, Gunma, Japan) in accordance with the manufacturers’ constituents was conducted by gas chromatography-MS at the Aomori instructions. Prefectural Industrial Technology Research Center, as described in an earlier report.17) The GC–MS system (GC-17A/QP-5000; Shimadzu, Western blot analysis. Cells were lysed with cold RIPA buffer Kyoto, Japan) was equipped with a DB-1701 column (30 mm (pH 7.4) containing 20 mM Tris–HCl, 150 mM NaCl, 1% NP-40, 0.5% 0:25 mm 0:25 mm; J&W Scientific). The carrier gas was helium. The sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 0.1 mg/mL program was operated with the following oven temperature program: of phenylmethylsulfonyl fluoride, 50 mg/mL of aprotinin and 1 mM 50 C held for 5 min, rising at 5 C/min to 100 C, rising at 2 C/min to Na3VO4 for 1 h on ice. Cell lysates were centrifuged at 12,000 rpm for 150 C, rising at 10 C/min to 250 C, and the injection temperature 20 min at 4 C. Protein concentrations were measured by D.C. Protein and volume were 270 C and 1.0 mL respectively. Constituents were assay (Bio-Rad Laboratories, Tokyo, Japan). The supernatant (20 mg identified by computer matching of mass spectral data with data from protein/lane) was separated 10% SDS-polyacrylamide gels electro- the NIST62 mass spectral database (Shimadzu). The major constituents phoresis and transferred onto polyvinylidene fluoride (PVDF) mem- of KEO were linalool (65.78%), geranyl acetate (17.59%), geraniol branes (Bio-Rad Laboratories). The membranes were incubated with (5.29%), cineol (2.34%), limonene (2.11%), 3-carene (1.78%), blocking solution for 2 h at room temperature with subsequent -pinene (1.43%), and carvone (1.18%). incubation for 1 h with specific primary antibodies. The membranes were further incubated 1 h at room temperature with secondary Cell culture. RAW 264.7 cells were cultured in DMEM medium antibodies. They were treated with reagents (Chemi-Lumi One L; supplemented with 10% heat-inactivated FBS, 100 mg/mL of strepto- Nacalai Tesque, Kyoto, Japan) following the manufacturer’s instruc- mycin, and 100 IU/mL of penicillin at 37 C in a 5% CO2 humidified tions. Band intensities were quantified using Scion image software incubator. After 24 h of pre-incubation, KEO or linalool was added to (Scion; Scion, Frederick, MD). -Actin was used as control. the medium as ethanol (EtOH) solution. The final concentration of ethanol was less than 0.1% (v/v). Then LPS was added to the culture Peroxisome proliferator-activated receptor (PPAR) ligand medium at 0.1 mg/mL in the presence of KEO or linalool, and activity. Agonistic activity for PPAR was examined using a nuclear RAW264.7 cell were stimulated for an additional 24 h. receptor cofactor assay system (EnBio RCAS for PPAR; Cosmobio, Tokyo). This is a cell-free assay system using a nuclear receptor and Measurement of cell viability by WST-1 assay. RAW 264.7 cells cofactor to screen chemicals. Changes in absorbance (450 nm) at KEO (1:0 106 cells/mL) were cultured in a 96-well culture plate with (25, 50 mg/mL) and troglitazone (10 mM) were measured. 100 mL medium and incubated as described previously. Cell viability was expressed as percentage of control by measuring the density of the Statistical analysis. Results were expressed as mean SE. Stat- color produced by WST-1 dye at 450 nm. The WST-1 assay is based on istical analysis between multiple groups were done by ANOVA. 484 H. MAEDA et al. Statistical comparisons were made by Dunnett’s multiple comparison with linalool (50 mg/mL) significantly (p < 0:05) sup- test. Differences were considered significant for p < 0:05 and at pressed TNF- mRNA expression (Fig. 2B). IL-6 and p < 0:01. The analyses were performed using Stat View-J ver. 5.0 TNF- production in the culture supernatant was software; Abacus Concepts, Berkeley, CA. measured by ELISA. IL-6 and TNF- concentrations in the media were suppressed significantly (p < 0:01) Results and dose-dependently in the cells treated with KEO and linalool (Fig. 2C and D). Inhibitory effects of Kuromoji essential oil (KEO) and linalool on LPS-induced NO production Inhibitory effects of KEO and linalool on LPS- The cytotoxity of KEO or linalool on RAW 264.7 induced iNOS and COX-2 expression cells stimulated by LPS was investigated by WST-1 iNOS and COX-2 expression in the cells treated with assay. Treatment with sample cells showed no cell KEO and with linalool was determined by real-time RT- toxicity on LPS stimulated RAW 264.7 cells (LPS PCR and Western blotting. iNOS and COX-2 mRNA Control cells: 100 1:8%, LPSþ Control cells: 100 expression was markedly downregulated in the cells 1:1%, LPSþ 25 mg/mL KEO cells: 106:6 1:8%, treated with KEO as compared to control cells treated LPSþ 50 mg/mL KEO cells: 99:5 2:2%, LPSþ 25 with LPS (Fig. 3A and B). In addition, protein expres- mg/mL linalool cells: 98:5 0:6%, LPSþ 50 mg/mL sion in the cells were determined Western blot. The cells linalool cells: 107:0 1:9%). treated with KEO (25, 50 mg/mL) suppressed COX-2 To evaluate anti-inflammatory effects on LPS-in- and iNOS protein expression as compared with the LPS- duced NO production, RAW 264.7 cells were stimulated stimulated control cells. (Fig. 4A, B and C). Linalool with LPS (0.1 mg/mL) in the presence of KEO or treated with the same concentration of KEO showed linalool for 24 h. Control cells treated with LPS showed similar effects on iNOS and COX-2 mRNA and protein increased NO production as compared with the non- expression. treated LPS Control cells. The cells treated with 10 mM troglitazone, chemosynthetic ligands of PPAR, showed suppressed NO production. Similarly, the cells treated 25 with KEO and linalool (25, 50 mg/mL) exhibited

) 20 significantly (p < 0:01) suppressed NO production, in M µ ** a dose-dependent manner (Fig. 1). ** ** 15 ** **

Inhibitory effects of KEO and linalool on LPS- ( production

- 10 induced IL-6 and TNF- expression 2 To investigate the anti-inflammatory effects of KEO NO 5 or linalool on IL-6 and TNF-, which are pro-inflam- matory cytokines mRNA, were quantified in RAW 0 264.7 cells stimulated with LPS. Control cells stimulated Control Control TR KEO KEO Linalool Linalool 25 50 25 50 with 0.1 mg/mL of LPS showed markedly increased IL-6 LPS − + and TNF- mRNA expression as compared with non- treated control cells. However, those cells treated with Fig. 1. Inhibitory Effects of KEO and Linalool on LPS-Induced KEO (25, 50 mg/mL) exhibited significantly (p < 0:01) Nitrite Production in RAW264.7 Cells. attenuated IL-6 mRNA expression of dose-dependently RAW264.7 cells were maintained in the culture medium (Con- trol), 10 mM troglitazone (TR), 25 mg/mL KEO (KEO25), 50 mg/mL (Fig. 2A). TNF- mRNA expression did not change KEO (KEO50), 25 mg/mL linalool (Linalool 25), 50 mg/mL linalool significantly, but it tended to be down regulated treated (Linalool 50) stimulated with 0.1 mg/mL of LPS for 24 h. with KEO (25, 50 mg/mL). On the other hand, treatment p < 0:01 vs. control treated with LPS.

A B C D

3,000 2,500

60 4.0 2,500 ** ** 2,000 50 3.5 2,000 3.0 ** ** 40 ** 1,500 2.5 * 1,500 ** **

** /GAPDH 30 ** α α 2.0 1,000 ** ** puroduction (ng/mL) puroduction

** 1,000

IL-6/GAPDH 1.5 α α

20 TNF-

1.0 (ng/mL) IL-6 production 500 500 10 TNF- 0.5 0 0 0 0 ControlControl KEO KEO Linalool Linalool Control Control KEO KEO Linalool Linalool Control Control KEOKEO Linalool Linalool Control Control KEO KEO Linalool Linalool 25 50 25 50 25 50 25 50 25 50 25 50 25 50 25 50

LPS − + LPS − + LPS − + LPS − +

Fig. 2. Inhibitory Effects of KEO and Linalool on LPS-Induced IL-6 and TNF- mRNA Expression, and Protein Secretion into the Culture Medium. RAW 264.7 cells were stimulated with 0.1 mg/mL of LPS for 24 h in the presence of 25 mg/mL KEO (KEO25), 50 mg/mL KEO (KEO50), 25 mg/mL linalool (Linalool 25), 50 mg/mL linalool (Linalool 50): (A) IL-6 mRNA expression, (B) TNF- mRNA expression, (C) IL-6 concentration in the supernatant, and (D) TNF- concentration in supernatant. p < 0:01 vs. control treated with LPS. p < 0:05 vs. control treated with LPS. Kuromoji Inhibits LPS-Induced Inflammation 485 PPAR ligand activity of KEO 50 mg/mL of KEO cells reduced NO production in LPS- The PPAR agonistic activity of KEO was examined stimulated RAW 264.7 cells (Fig. 1). In addition, pro- using a nuclear receptor cofactor assay system. The inflammatory cytokines such as IL-6 and TNF- were absorbance of the non-treated well (control) was suppressed by treatment with KEO cells (Fig. 2). NO, estimated to have a 100% change of absorbance derived from arginine after activation of iNOS, is an percentage. The absorbance the well treated with important effector molecule involved in immune regu- Troglitazone (10 mM) increased by 286:7 14:5% com- lation and defense. COX-2 catalyzes the production pared with the non-treated well (control; 100:0 2:9%). of prostaglandins, which play important roles in the In addition, the PPAR agonistic activities of the wells inflammatory process. Prostaglandin production in LPS- treated with KEO (25, 50 mg/mL) were significantly treated macrophages is primarily attributable to tran- higher (p < 0:05) than those of the non-treated wells scriptional activation of the COX-2 gene. Hence iNOS (KEO 25 mg/mL; 126:6 9:3%, KEO 50 mg/mL; and COX-2 inhibitors are regarded as potential anti- 132:5 5:9%), but activity was considerably lower than inflammatory agents.21–24) with troglitazone. In this study, KEO suppressed iNOS and COX-2 mRNA expression. Protein expression was also sup- Discussion pressed. The results suggest that KEO is a natural product that can prevent the production of pro-inflam- Macrophages play important roles in the initiation and matory cytokines in RAW264.7 macrophages. A major amplification of various inflammatory diseases. There- constitute of KEO is linalool a monoterpene alcohol. fore the development of methods to reduce the number Linalool similarly reduced NO, IL-6, and TNF- of activated macrophages, to inhibit activation signals, production. Peana et al. reported that linalool inhibited and to prevent the production of inflammatory mediators NO production,25) but this was not associated with iNOS is a promising therapeutic approach to the treatment of expression. In this study, KEO and linalool suppressed various inflammatory diseases.19,20) iNOS and COX-2 expression. It was assumed that This study demonstrated the anti-inflammatory effects difference cell types and LPS stimulation levels lead to of KEO in RAW 264.7 cells. Treatment with 25 or different results. NO produced by activated macrophages is associated with acute and chronic inflammation. Toll-like receptor A B 4 (TLR4) is regarded as a cell-surface receptor neces- 26) 8 sary for the recognition of LPS macrophages. TLR4 6 7 regulates several transcription factors encoding inflam- 5 6 matory mediators, such as NF-B, an important tran- ** 4 5 ** scription factor adjusting pro-inflammatory mediator ** 4 ** 3 ** ** ** ** production, including iNOS, IL-6, TNF- and NO 3 iNOS/GAPDH production in activated macrophages.27) Normally, NF- COX-2/GAPDH 2 2 B forms a heterodimer of p65/p50 binding to inhibitor 1 1 proteins IB. After stimulation, p65/p50 is released 0 0 Control Control KEOKEO Linalool Linalool Control Control KEO KEO Linalool Linalool 25 50 25 50 25 50 25 50 from IB, resulting in p65 translocation into the nucleus

LPS − + LPS − + to regulate gene transcription of these pro-inflammatory mediators. In this study, the level of the NF-B p65 Fig. 3. Inhibitory Effects of KEO and Linalool on LPS-Induced subunit and the IB protein under treatment with KEO COX-2 and iNOS mRNA Expression in RAW264.7 Cells. were analyzed by Western blot. KEO slightly suppressed RAW 264.7 cells were stimulated with 0.1 mg/mL LPS for 24 h in NF-B p65 levels, but no suppressing effect of IB the presence of 25 mg/mL KEO (KEO25), 50 mg/mL KEO (KEO50), 25 mg/mL linalool (Linalool 25), 50 mg/mL linalool degradation was detected (data not shown). (Linalool 50): (A) COX-2 mRNA expression and (B) iNOS mRNA Recently, PPAR ligands have been found to suppress expression. p < 0:01 vs. control treated with LPS. the intranuclear NF-B pathway, engendering suppres-

A B C 1.6 2.5 1.4 * * 2.0 1.2 * * *

COX-2(72 kDa) -Actin 1.0 -Actin *

1.5 β β β 0.8 iNOS(130 kDa) 1.0 0.6

iNOS/ 0.4 0.5 β-Actin(43 kDa) COX-2/ 0.2 0 0 Control Control KEOKEO Linalool Linalool Control Control KEOKEO Linalool Linalool Control Control KEOKEO Linalool Linalool 25 50 25 50 25 50 25 50 25 50 25 50

LPS − + LPS − + LPS − +

Fig. 4. Inhibitory Effects of KEO and Linalool on LPS Induced COX-2 and iNOS Protein Expression in RAW264.7 Cells. RAW 264.7 cells were stimulated with 0.1 mg/mL of LPS for 24 h in the presence of 25 mg/mL KEO (KEO25), 50 mg/mL KEO (KEO50), 25 mg/mL linalool (Linalool 25), 50 mg/mL linalool (Linalool 50): (A) COX-2, iNOS, and -actin protein expression determined by Western blotting, (B) COX-2 protein expression level quantified fold of the level the control. (C) iNOS protein expression level quantified fold of the level in the control. p < 0:05 vs. control treated with LPS. 486 H. MAEDA et al. sion of the release of pro-inflammatory mediators. References PPARs are members of the nuclear receptor super- 12;14 1) Gordon S, Nat. Rev. Immunol., 3, 23–35 (2003). family activated by 15-deoxy- -prostaglandin J2 converted from arachidonic acid.10,28) Troglitazone, a 2) Mosser DM and Edwards JP, Nat. Rev. Immunol., 8, 958–969 (2008). 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