Original article: ANTI-INFLAMMATORY EFFECT AND …

1 EXCLI Journal 2017;16:1103-1113 ISSN 1611-2156 Received: June 26, 2017, accepted: August 17, 2017, published: August 29, 2017 1103 Original article : ANTI-INFLAMMATORY EFFECT AND MECHANISM OF ACTION OF LINDERA ERYTHROCARPA ESSENTIAL OIL IN LIPOPOLYSACCHARIDE-STIMULATED CELLS Yeong-Jong Koa,1, Ginnae Ahnb,1, Young-Min Hama, Sang-Mock Songa, Eun-Yi Koc, Su-Hyeon Choc, Weon-Jong Yoona,*, Kil-Nam Kimc,d,* a Jeju Biodiversity Research Institute (JBRI), Jeju Technopark (JTP), Jeju 699-943, Republic of Korea b Department of Marine Bio-Food Sciences, Chonnam National University, Yeosu 550-74, Republic Korea c Chuncheon Center, Korea Basic Science Institute (KBSI)

2 , Chuncheon 200-701, Republic of Korea d Department of Marine Biotechnology, University of Science and Technology, Daejeon 305-350, Republic of Korea 1 These authors contributed equally to this work. * Corresponding authors: Tel.: +82-33-815-4607, E-mail: ( Kim) Tel.: +82-64-720-2840, E-mail: ( Yoon) This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( ). ABSTRACT The aim of this study was to investigate the chemical constituents of Lindera erythrocarpa essential oil (LEO) by gas chromatography-mass spectrometry and evaluate their inhibitory EFFECT on the expression of pro-inflammatory mediators in lipopolysaccharide (LPS)-stimulated cells.

3 Fifteen compounds, accounting for % of the composition of LEO, were identified. The main compounds were nerolidol ( %), caryophyllene ( %), -humulene ( %), germacrene-D ( %), and -pinene ( %). LEO significantly inhibited the expression of inducible nitric oxide (NO) synthase and cyclooxygenase-2, and subsequent production of NO and prostaglandin E2. In addition, it reduced the release of pro-inflammatory cytokines in LPS-activated cells. The molecular mechanism underlying the EFFECT of LEO was associated with inhibition of the phosphoryla-tion of mitogen-activated protein kinase (MAPK). Furthermore, LEO inhibited LPS-induced phosphorylation and degradation of inhibitor of kappa B- , which is required for the activation of the p50 and p65 nuclear factor (NF)- B subunits in cells.

4 Taken together, these data suggest that LEO exerted its ANTI-INFLAMMATORY EFFECT by downregulating LPS-induced production of pro-inflammatory mediators through the inhibition of NF- B and MAPK signaling in cells. Keywords: Lindera erythrocarpa, ANTI-INFLAMMATORY , essential oil, NF- B, MAPK INTRODUCTION Prolonged inflammation is involved in the pathogenesis of a variety of diseases, includ-ing pulmonary fibrosis, atherosclerosis, chronic hepatitis, rheumatoid arthritis, and in-flammatory brain diseases (Chung et al., 2007). Inflammation is caused by pro-inflam-matory mediators, including both pro-inflam-EXCLI Journal 2017;16:1103-1113 ISSN 1611-2156 Received: June 26, 2017, accepted: August 17, 2017, published: August 29, 2017 1104 matory cytokines such as tumor necrosis fac-tor (TNF)- , interleukin (IL)-1 , and IL-6, and pro-inflammatory factors such as nitric oxide (NO), prostaglandin E2 (PGE2), induci-ble nitric oxide synthase (iNOS), and cy-clooxygenase (COX)-2 (Ljung et al.)

5 , 2006; Walsh et al., 2005). Therefore, inhibition of these inflammatory mediators is an important target pathway in the treatment of diseases with ANTI-INFLAMMATORY components. Nuclear transcription factor kappa-B (NF- B) regulates a variety of genes associated with immune and acute-phase inflammatory responses. The activation of NF- B due to pro-inflammatory stimulation is indicated by rapid phosphorylation and degradation of in-hibitors of kappa B (I Bs) (Rajapakse et al., 2008). Freed NF- B dimers from this process translocate to the nucleus and bind to the pro-moter regions of target genes (Lee et al.

6 , 2003). They then induce the transcription of pro-inflammatory mediators such as iNOS, COX-2, IL-6, IL-1 , and TNF- (Makarov, 2000; Yoshimura, 2006). Recent studies have reported that the ANTI-INFLAMMATORY EFFECT of phytochemicals occurs through blocking of the NF- B signaling pathway (Ham et al., 2015; Hsieh et al., 2011). Mitogen-activated protein kinases (MAPKs) are one of the major kinases involved in cellular processes such as apoptosis, stress responses, differentiation, and immune defense (Liu et al., 2007). MAPKs comprise three major subgroups, namely, p38 MAPKs, c-Jun N-terminal ki-nases (JNKs), and extracellular signal-regu-lated kinases (ERKs).

7 Their activation plays an important role in the expression of iNOS and COX-2 and in the production of cytokines (Rajapakse et al., 2008; Rao, 2001). There-fore, MAPK and NF- B may be effective as ANTI-INFLAMMATORY agents. Lindera erythrocarpa is widely distrib-uted in the Republic of Korea, Japan, and China (Sun and Chung, 1988). Lindera spe-cies, including L. strychnifolia, L. lucida, L. chunii, and L. aggregate are important medic-inal plants. The fruit and leaves of L. erythro-carpa are used in folk medicine for treating digestive disorders, thirst, pain, and neuralgia.

8 They are also used as antidotes and diuretics (Hong et al., 2009; Oh et al., 2005; Sun and Chung, 1988). Recently, L. erythrocarpa was reported to suppress adipogenesis and mela-nin synthesis, attenuate obesity, as well as ex-hibit antioxidant, ANTI-INFLAMMATORY , and an-tifungal activities (Hsieh and Wang, 2013; Hwang et al., 2007; Kumar et al., 2010; Wang et al., 2008). Essential oils extracted from me-dicinal and aromatic plants are known to have biological effects, most notably anti-inflam-matory, antioxidant, antifungal, and antibac-terial activities (Chaieb et al., 2007; Pinheiro et al.)

9 , 2011). These properties are a driving in-terest in the use of essential oils in the cos-metic, pharmaceutical, and food industries (Chaieb et al., 2007; Lang and Buchbauer, 2012; Tumen et al., 2010). However, the mechanism by which L. erythrocarpa essen-tial oil (LEO) exerts its ANTI-INFLAMMATORY ef-fect has not been elucidated. Therefore, in this study, we examined the ANTI-INFLAMMATORY ef-fects of LEO and its constituents on lipopoly-saccharide (LPS)-stimulated cells. MATERIALS AND METHODS Reagents LPS, phosphate buffered saline (PBS), di-methyl sulfoxide (DMSO), 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphen yltetrazolium bro-mide (MTT) and radio-immunoprecipitation assay RIPA lysis buffer were bought from Sigma Aldrich (St.

10 Louis, MO, USA). Fetal bovine serum (FBS) and Dulbecco s modified Eagle s medium (DMEM) were purchased from Invitrogen-Gibco (Grand Island, NY, USA). Enzyme-linked immunosorbent assay (ELISA) kits for the analyses of TNF- , IL-6, and PGE2 were obtained from BD Biosci-ences (San Diego, CA, USA) and R&D Sys-tems, Inc. (St. Louis, MO, USA). Antiphos-phorylated I B- (anti-p-I B- ), anti-NF- B, anti-JNK, anti-p38, anti-ERK1/2, anti-phos-phorylated JNK (anti-p-JNK), anti-phosphor-ylated p38 (anti-p-p38) and anti-phosphory-lated ERK1/2 (anti-p-ERK1/2) mouse or rab-bit antibodies were bought from Cell Signal-ing Technology (Beverly, MA, USA).