Inhibitory Effect of Essential Oil from Agastache Rugosa Against Nitric Oxide

Full Length Research Paper

Inhibitory effect of essential oil from Agastache rugosa against nitric oxide (NO) production induced by inducible nitric oxide synthase (iNOS) over-expression through NF-κB and mitogen-activated protein kinase (MAPK) activation in lipopolysaccharide (LPS)- stimulated RAW264.7 cells

Se Chul Hong1, Jin Boo Jeong2* and Jin Suk Koo2

1International Ginseng and Herb Research Institute, Geumsan, 312804, Korea. 2Medicinal Plant Resources Major, Andong National University, Andong, 760749, Korea.

Accepted 22 June, 2012

In this study, the anti-inflammatory effect of the essential oil from Agastache rugosa (EOA) in RAW264.7 cells was evaluated. The effect of EOA on nitric oxide (NO) production in lipopolysaccharide (LPS)- stimulated RAW264.7 cells was examined. EOA inhibited NO over-production, which was accompanied by the down-regulation of inducible nitric oxide synthase (iNOS) expression, as evaluated by western blot. To elucidate the action mode of EOA for inhibiting iNOS expression, the effects of EOA on phosphorylation and degradation of IκB-α, p65 nuclear translocation, and NF-κB activation was evaluated. EOA suppressed NF-κB activation through inhibiting the hyper-phosphorylation and degradation of IκB-α. In addition, EOA also inhibited the LPS-induced hyper-phosphorylation of mitogen-activated protein kinases (MAPKs), p38, extracellular signal-regulated kinase-1/2 (ERK1/2), and N-terminal kinase (JNK).

Key words: Essential oil, Agastache rugosa, anti-inflammation, inducible nitric oxide synthase (iNOS), NF-κB, mitogen-activated protein kinase (MAPK).

INTRODUCTION

Plants are good sources for natural preparations Essential oils of herbs and their components have many containing effective bioactive compounds used for applications in ethno-medicine, food, beverages, different applications, particularly as food additives and presservation, cosmetics as well as in the fragrance and health promoting ingredients in the formulations of pharmaceutical industries (Tavares et al., 2008). Infla- functional foods and nutraceuticals (Meskin et al., 2002; mmation is a major feature of many patho-physiological Gibson and Williams, 2000; Shahidi, 1997). Especially, conditions in response to tissue injury and host defense there is an increasing interest in medicinal plants as an against invading pathogens (Li et al., 2001). Activated alternative to synthetic drugs (Tavares et al., 2008). macrophages by lipopolysaccharide (LPS) play an important role in inflammatory diseases via production of nitric oxide (NO), an important inflammatory mediator linking chronic inflammation (Fujiwara and Kobayashi, *Corresponding author. E-mail: [email protected]. Tel: +82 54 2005). Macrophages are the main proinflammatory cells 820 7757. involved in the inflammatory response and release many Hong et al. 4495

inflammatory mediators, including NO (Petros et al.,1991; by the Bonghwa Alpine Medicinal Plant Experiment Station, Korea. Vodovotz et al., 1996). NO is produced endogenously One kilogram of A. rugosa was extracted by the steam distillation during arginine metabolism by different isoforms NOS apparatus (SDA) at 80°C for 4 h, and then the extract was dehydrated with sodium sulfate anhydrous. The extracted essential (Moncada et al., 1991). During inflammation, induced oil was concentrated by a vacuum evaporator at 30°C and was expression of inducible nitric oxide synthase (iNOS) in stored at -20°C until use. macrophages leads to production of NO. The expression of iNOS and the level of NO have been shown to be elevated in various inflammatory diseases including Cell viability rheumatoid arthritis, atherosclerosis, asthma, and RAW264.7 cells were seeded in a 96-well plate at a density of 5 pulmonary fibrosis (Bharat and Reddanna, 2009). ×104 cells/well. EOA was added for 24 h. Control group was treated Agastache rugosa, a perennial herb ubiquitous in the with an equal amount of dimethyl sulfoxide (DMSO), which resulted fields of Korea, has been used as a wild vegetable and in a final concentration of 0.3% DMSO in the medium. At 24 h after an herbal drug in traditional therapies (Shin, 2004) and the treatment of EOA, a MTT solution was added, and then the cells has a variety of physiological and pharmacological were incubated for another 4 h at 37°C. After removing the medium, activities (Hong et al., 2001; Lee et al., 2002). It has been 100 µl of DMSO was added to the cells. The absorbance was measured by using a microplate reader at 590 nm. The control reported that A. rugosa extracts inhibited cytokine group consisted of untreated cells, considered as 100% of viable induced vascular cell adhesion molecule 1 (VCAM-1) in cells. human umbilical vein endothelial cells (HUVECs) (Hong et al., 2001), and inhibited apoptosis in leukemia cells (Lee et al., 2002). From these reports, A. rugosa may be Determination of NO generation valuable source for the treatment of anti-inflammatory 5 and oxidative stress-induced disorders (Oh et al., 2006). RAW 264.7 cells (2 × 10 cells/well) in 10% FBS-DMEM without phenol red were seeded in a 6-well plate for 24 h at 37°C. Cells However, anti-inflammatory effect of its essential oil has were washed with 1 × phosphate buffered saline (PBS), replaced not yet been studied. In this study, we evaluated the with fresh media, and then treated with EOA for 24 h. The cells inhibitory effect of the essential oil against NO were treated with LPS (1 μg/ml) for 24 h at 37°C. After 24 h, 200 μl overproduction as an inflammatory mediator and of the medium were placed in a 96-well plate and an equal amount elucidated its possible mechanism. of Griess reagent (1% sulfanilamide and 0.1% N-1-(naphthyl) ethylenediamine-diHCl in 2.5% H3PO4) was added. The plate was incubated for additional 5 min at the room temperature and then the MATERIALS AND METHODS absorbance was measured at 540 nm with a microplate reader. The amount of NO was calculated using sodium nitrite standard curve. Chemical regents

LPS (Escherichia coli 055:B5) and 3-(4,5-dimethylthiazol-2-yl)-2.5- SDS-PAGE and western blot diphenyltetrazolium bromide (MTT) were purchased from Sigma- Aldrich (St. Louis, MO, USA). Antibodies recognizing iNOS and p65 The cell pellets were resuspended in lysis buffer (50 mM Tris-HCl were purchased from Santa Cruze Biotechnology (Santa Cruz, CA, pH 7.4, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid USA). IĸB-α, phospho-IĸBα, p-p38, p-extracellular signal-regulated (EDTA), 1 mM egtazic acid (EGTA), 10 μg/ml aprotinin, 10 μg/ml kinase1/2 (ERK1/2), and p-N-terminal kinase (JNK) antibodies were leupeptin, 5 mM phenylmethylsulfonyl fluoride (PMSF), and 1 mM purchased from Cell Signaling Technology (Danvers, MA, USA). dithiothreitol (DTT)) containing 1% Triton X-100 for 30 min at 4°C The secondary antibody, goat anti-rabbit immunoglobin G (IgG)- and was centrifuged to yield whole-cell lysates. The proteins (50 horseradish peroxidase (HRP) conjugated, was purchased from μg) were separated by sodium dodecyl sulfate polyacrylamide gel Santa Cruz Biotechnology (Santa Cruz, CA). Nuclear Extraction Kit electrophoresis (SDS-PAGE) and transferred into polyvinylidene and NF-ĸB Combo Transcription Factor Assay Kit were purchased fluoride (PVDF) membranes. The PVDF membranes were then from Cayman Chemical (Ann Arbor, MI, USA). All electrophoresis blocked for non-specific binding in blocking buffer for 1 h and then it chemicals were purchased from Bio-Rad Labs (Hercules, CA, USA). was washed with 1 × TBS solution (1 × Tris buffered saline (TBS) containing 0.1% Tween-20). Subsequently, the membranes were

Cell culture incubated with each primary antibody with gentle shaking at 4°C for 18 h. After washing, the membranes were incubated with The murine macrophage cell line RAW264.7 was purchased from Phototope-HRP Western Blot Detection System, Anti-rabbit IgG, the Korean Cell Line Bank (Seoul, Korea). RAW264.7 cells were HRP-linked antibody as the secondary antibody for 1 h at room cultured in Dulbeco’s modified Eagle’s medium (DMEM) temperature and then they were washed again. After washing, the supplemented with 100 U/ml of penicillin, 100 µg/ml of streptomycin, membranes were treated with the detection agent (Amersham and 10% fetal bovine serum (FBS). The cells were incubated in an Biosciences) and immediately developed in Polaroid film. atmosphere of 5% CO2 at 37°C and subcultured every 2 days. In all experiments, cells were grown to 80 to 90% confluence and were subjected to no more than 20 cell passages. NF-ĸB p65 activation assay

NF-ĸB p65 activation assay was carried out using Cayman’s NF-ĸB Preparation of the essential oil from A. rugosa p65 Transcription Factor Assay Kit. RAW264.7 cells (2 ×105 cells/well) were seeded in a 6-well plate for 24 h at 37°C. Cells Essential oil from A. rugosa (EOA) was prepared according to the were washed with 1 × PBS, and replaced with fresh media, and literature (Jeong et al., 2009). Briefly, A. rugosa was kindly provided then treated with EOA for 24 h. LPS (1 μg/ml) was treated for 30 4496 J. Med. Plants Res.

Gas chromatography/ mass spectrometry (GC/MS) analysis

GC/MS analysis for the determination of the essential oil was performed using GC/MSD equipped with a Supelcowax 10 fused silica capillary (30 m length × 0.25 mm i.d. supelco, USA). For analysis, helium was used as the carrier gas and the constant flow rate of helium was 1.0 ml/min. The oven temperature was initially held at 50°C, and then it was raised to 240°C at a rate of 2°C min-1, and finally held at 240°C for 5 min. The temperatures of injector and detector were 200 and 240°C, respectively. The mass detector was operated in electron impact mode with ionization energy of 70 eV, a scanning range of 33 to 550 a.m.u., and a scan rate of 1.4 scans/s. Component of essential oil was identified with the aid of the Wiley 275 Imass spectral database (Hewlett-Packard, 1995).

(A) Statistical analysis

For statistical analysis, data were analyzed by One-way analysis of variance (ANOVA), followed by Dunnett’s test. In western blot analysis, percentage relative density was calculated by the density using the software Un-SCAN-IT gel Version 5.1 (Silk Scientific, Inc). Experiments were repeated at least three times.

RESULTS AND DISCUSSION

Inhibition of EOA against NO generation through suppressing iNOS expression in LPS-stimulated RAW264.7 cells (B) Excessive NO production has been implicated in the pathogenesis of inflammatory tissue injury and in several disease states (Petros et al., 1991; Vodovotz et al., 1996). Recent studies have shown that inflammation of many of these tissues is accompanied by upregulation of an

inducible isoform of NO and iNOS (Sawa et al., 2006). The level of iNOS expression is well correlated with the degree of inflammation (Kimura et al., 1998). Thus, inhibition of NO production by suppressing iNOS

viability(%) overexpression has been regarded as promising targets

ell ell for the inflammatory disorders (Petros et al., 1991; C Vodovotz et al., 1996). To determine whether EOA inhibits LPS-induced NO production, RAW264.7 cells were pretreated with EOA for 24 hs and then stimulated with LPS (1 μg/ml). After stimulation for 24 h, the cell medium was harvested, and the production of NO was measured EOA (µg/ml) (C) using the Griess assay. As shown in Figure 1A, RAW264.7 cells without LPS secreted basal level of NO, (C) while LPS treatment without EOA increased NO Figure 1. Effect of EOA on (A) NO production, (B) iNOS protein generation by 28.2 μM. EOA significantly inhibited the NO expression, and (C) cell viability, in RAW264.7 cells. Relative ratio production by LPS by 37.1% at 25 μg/ml and 86.4% at 50 in the plot was calculated by the density using the software Un- μg/ml. To investigate whether the inhibitory effect of EOA SCAN-IT gel Version 5.1 (Silk Scientific, Inc). Cell viability by EOA on NO overproduction was mediated by down-regulating was measured by MTT assay. the iNOS expression, western blot of iNOS protein was performed on the LPS-treated lysates of RAW264.7 cells with or without EOA. As shown in Figure 1B, LPS- min. The cells were washed and scraped into cold 1 × PBS and treatment without EOA induced over-expression of iNOS, were centrifuged at 500 g at 4°C. The nucleus fractions were extracted using Cayman’s Nuclear Extraction Kit. NF-ĸB p65 while pretreatment of EOA down-regulated the iNOS activation assay was evaluated according to the manufacturer’s expression. To exclude the cytotoxic effect of EOA from protocol. EOA induced NO production, the cytotoxicity of EO against Hong et al. 4497

5activation (%)

6 P

(A) (B)

Figure 2. Effect of EOA on (A) LPS-induced NF-ĸB activation shows the images of Western blot for phosphorylation and degradation of IĸB-α, and nucleus translocation of p65, and (B) shows NF-kB p65 activation. Cells were pre-treated with EOA for 24 h and then 1 µg/ml of LPS was treated for 30 min. Relative ratio in western blot was calculated by the density using the software Un-SCAN-IT gel Version 5.1 (Silk Scientific, Inc).

RAW264.7 cells was evaluated. As shown in Figure 2C, with NF-ĸB activation. To evaluate whether inhibitory EOA did not exhibit cytotoxicity against RAW264.7 cells. effects of EOA against NO overproduction and iNOS These results demonstrated that EOA significantly overexpression were mediated by the inhibition of NF-ĸB inhibited NO production by suppressing iNOS over- activation, we examined the effect of EOA against LPS- expression in RAW264.7 cells. induced hyper-phosphorylation and degradation of IĸB-α, which are essential steps in NF-ĸB activation (Sung et al., 2009). As shown in Figure 2A, LPS treatment induced Inhibition of EOA against NF-ĸB activation by hyper-phosphorylation and degradation of IĸB-α. blocking the phosphorylation and degradation of IĸB- However, EOA treatment inhibited the degradation of IĸB- α in LPS-stimulated RAW264.7 cells α through suppressing its hyper-phosphorylation induced by LPS. Also, translocation of p65, the major component NF-ĸB regulates gene expressions involved in innate and of NF-ĸB, into the nucleus is necessary for NF-ĸB adaptive immunity, apoptosis, proliferation, cancer activation by IĸB-α degradation. As shown in Figure 2A, progression, and inflammation (Gilmore, 2006). In the the amount of p65 in the nucleus was markedly increased presence of external stimuli like LPS, NF-ĸB can be upon exposure to LPS alone, but EOA suppressed LPS- activated via disassociation with IĸB. Once disassociated mediated nuclear translocation of p65. From these results, with IĸB, NF-ĸB is translocated into the nucleus where it it is though that EOA inhibits LPS-induced NF-ĸB acti- controls transcription of distinct but overlapping genes vation, which was confirmed by NF-ĸB p65 activation (Jobin and Sartor, 2000). NO overproduction and iNOS assay (Figure 2B). These results suggested that EOA overexpression by LPS in RAW264.7 cells are associated might block LPS-induced NF-κB activation by suppression 4498 J. Med. Plants Res.

Figure 3. Effect of EOA on LPS-induced MAPK activation. Western blot analysis shows the effect of EOA on the phosphorylation of MAPKs such as p38MAPK, ERK1/2 and JNK. Cells were pretreated with EOA for 24 h and then 1 µg/ml of LPS was treated for 5 min. Relative ratio in western blot was calculated by the density using the software Un-SCAN-IT gel Version 5.1 (Silk Scientific, Inc).

of IκB-α phosphorylation and degradation. Analysis of the bioactive compound from the essential oil

Inhibition of EOA against mitogen-activated protein Identification of the active components for anti- kinase (MAPK) activation by blocking the inflammation from EOA was analyzed by GC/MS, leading phosphorylation of ERK1/2, JNK, and p38 in LPS- to comparison of the mass spectra from data library. In stimulated RAW264.7 cells this study, patchouli alcohol was identified from EOA. Figure 4A and B shows the chromatogram and mass Activations of MAPKs such as JNK, p38, and ERK1/2 are spectrum of identified patchouli alcohol from EOA. Mass known to be involved in the regulation of iNOS spectrum of identified patchouli alcohol showed a similar expression. In addition, they play a critical role in the homology (99%) with that of patchouli alcohol in the Wiley modulation of NF-κB activity (Surh et al., 2001). To further 275 Imass spectral database (Hewlett-Packard, 1995). investigate whether the inhibition of EOA on the Patchouli alcohol, a tricyclic sesquiterpene, has been expression of iNOS via blocking NF-ĸB activation is widely studied as a cognition enhancer, learning related to the modulation of MAPK pathway, we impairment attenuating and neuroprotective agent evaluated the effects of EOA on phosphorylation of p38, (Huang et al., 2008, 2009). Recently, it is reported that ERK1/2, and JNK in LPS-stimulated RAW264.7 cells by patchouli alcohol has anti-inflammatory effect (Xian et al., western blot. As shown in Figure 3, hyper- 2011; Li et al., 2011; Sah et al., 2011). From these phosphorylation of p38, ERK1/2, and JNK was observed literatures, it is thought that the inhibitory effect of EOA in RAW264.7 cells treated with LPS alone compared to against NO production and iNOS expression via the untreated cells. However, EOA suppressed LPS- suppressing NF-ĸB and MAPK activation may be induced phosphorylation of p38, ERK1/2, and JNK. mediated by the identified patchouli alcohol contained in These results suggest that EOA blocks the EOA. phosphorylation of p38, ERK1/2, and JNK to suppress In conclusion, from our data, it is thought that EOA the inflammatory response in LPS-stimulated RAW264.7 inhibits LPS-induced NO production and iNOS expression cells. in RAW264.7 cells. These effects might be mediated by Hong et al. 4499

bundance A

Time(A)

bundance A

m/z (B)

Figure 4. Chromatogram (A) and mass spectrum (B) of by GC/MS.

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