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), 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.

2 * 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. 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.

3 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. 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.)

4 , 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., 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.

5 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., 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.)

6 , 2007). MAPKs comprise three major subgroups, namely, p38 MAPKs, c-Jun N-terminal ki-nases (JNKs), and extracellular signal-regu-lated kinases (ERKs). 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. They are also used as antidotes and diuretics (Hong et al.

7 , 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., 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.

8 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. 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.

9 (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). All EXCLI Journal 2017;16:1103-1113 ISSN 1611-2156 Received: June 26, 2017, accepted: August 17, 2017, published: August 29, 2017 1105 other reagents were obtained from Sigma-Al-drich. Extraction of essential oil from the leaves of L. erythrocarpa The leaves of L. erythrocarpa were col-lected from Namwon (a region in Jeju Island, Korea) in May 2014 and LEO was extracted by hydrodistillation. Briefly, approximately 300 g of fresh L. erythrocarpa leaves was im-mersed in 3 l of distilled water in a 5-l flask.

10 Subsequently, the obtained essential oil was dried over anhydrous sodium sulfate, filtered, and stored in a sealed vial at 4 C until tested. The LEO yield was approximately % (v/w). Gas chromatography (GC)-mass spectrome-try (MS) analysis Analysis of the main components of the most active essential oil extracted from L. erythrocarpa leaves was carried out using a GC (Agilent 6890, Agilent Technologies Inc., Santa Clara, CA, USA) connected to an MS (Agilent 5975). The GC was equipped with a DB1-HT column (30 m mm; m film thickness). The oven temperature was programmed to increase from 40 to 100 C at a rate of 2 C/min, and then from 100 to 230 C at a rate of 5 C/min, after which it was held at 230 C for 5 min. The detector and injector temperatures were 280 C and 240 C, respectively.