Inhibitory Effect of Agrimonia pilosa Ledeb. on Inflammation by Suppression of iNOS ROS Production

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Immunological Investigations

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Inhibitory Effect of Agrimonia pilosa Ledeb. on Inflammation by Suppression of iNOS and ROS Production

Chang Hwa Jung, Jeong-Hyun Kim, SunJu Park, Dae-Hyuk Kweon, Sung-Hoon Kim & Seong-Gyu Ko

To cite this article: Chang Hwa Jung, Jeong-Hyun Kim, SunJu Park, Dae-Hyuk Kweon, Sung-Hoon Kim & Seong-Gyu Ko (2010) Inhibitory Effect of Agrimonia pilosa Ledeb. on Inflammation by Suppression of iNOS and ROS Production, Immunological Investigations, 39:2, 159-170To link to this article:

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Immunological Investigations, 39:159–170, 2010Copyright ? Informa Healthcare USA, Inc.ISSN: 0882-0139 print / 1532-4311 online

DOI: 10.3109/08820130903501790

Inhibitory Effect of Agrimonia pilosa Ledeb. on Inflammation by Suppression of iNOS and ROS Production

Chang Hwa Jung,1 Jeong-Hyun Kim,1 SunJu Park,1

Dae-Hyuk Kweon,2 Sung-Hoon Kim,1 and Seong-Gyu Ko 1

1

Cancer Preventive Material Development Research Center, College of Oriental medicine, Kyunghee University, Seoul, Republic of Korea 2

Department of Genetic E ngineering, School of Biotechnology and Bioengineering,Sungkyunkwan University, Suweon, Republic of Korea

Herbal medicines including Ag rimonia pilosa Ledeb. (APL) have been traditionally used to treat inflammations including allergic disease as valuable medicinal properties.To investigate the attenuating ability of APL on inflammation, the NO release and ROS production, which play a key role in inflammatory and immune responses, was first tested using in vitro assay. The 80% ethanol extract of APL showed a significant activity to inhibit NO release and ROS production. In additional extracts from 80%ethanol extract of APL, n -butanol (BuOH) extract displayed the most potent anti-inflammatory effects based on in vitro assay. The extract also significantly reduced nitric oxide in lipopolysaccharide-activated RAW 264.7 macrophage cells (p < 0.05),and suppressed the nitric oxide synthase (iNOS) expression, whereas the extract showed no inhibitory effect on cyclooxygenase-2 (COX-2) expression, suggesting that the BuOH extract of APL could reduce the NO production through suppression of iNOS, but not COX-2. The BuOH extract also showed a significant effect in a carrageenan-induced rat paw edema in vivo model, consistent with our in vitro results. Our findings suggest that the BuOH extract of APL shows a potential anti-inflammatory activity, substantiating its traditional use in medicine.Keywords

Ag rimonia pilosa Ledeb. (APL), iNOS, COX-2, Nitric oxide, Reactive oxygen species.

Chang Hwa Jung and Jeong-Hyun Kim contributed equally to this work.

Address correspondence to Seong-Gyu Ko, Cancer Preventive Material Development Research Center, College of Oriental Medicine, Kyunghee University, Seoul 130-701,Republic of Korea; E-mail: epiko@khu.ac.kr

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INTRODUCTION

Macrophages are responsible for homeostasis and defense against injury.Although stimulation of macrophages protects cells and tissues from invading pathogens and injury, chronic macrophage activation and consequent overpro-duction of pro-inflammatory mediators can damage cells and exacerbate the primary insult (Klebanoff, 1993). Activated macrophages enhances the pro-duction of several pro-inflammatory mediators such as nitric oxide (NO),tumor necrosis factor-alpha (TNF-a ), and interleukins (Hanisch, 2002; Kundu and Surh, 2005) These pro-inflammatory mediators are induced by increasing the activity of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2(COX-2) (Surh et al., 2001). Therefore, suppression of iNOS and COX-2 activity can be an important approach to preventing inflammation of organs.

Recently, a number of natural plants used as traditional anti-inflammatory medicines have been shown to inhibit pro-inflammatory genes in vitro (Setty and Sigal, 2005; Kaileh et al., 2007). Ag rimonia pilosa Ledeb. (Rosaceae ) is widely distributed in Asia and is used as an herbal medicine with little under-standing of the underlying mechanism for its therapeutic effects. Previous studies have shown that A. pilosa possesses anti-bacterial, anti-tumor, and hepatoprotective activities (Gao et al., 2000; Park et al., 2004). The pharmaco-logical activities of A. pilosa may be primarily due to phenolic compounds such as agrimonin, catechin, querectin, and rutin (Miyamoto et al., 1988; Xu et al.,2005).

However, in spite of its legacy of medication, the anti-inflammatory effects of A. pilosa is not fully understood. In this study, we investigated the inhibitory effects of APL on macrophage-mediated inflammatory phenomena including NO release, functional activation of adhesion molecules, and oxidative stresses in an attempt to understand the anti-inflammatory properties. In addition,effects of APL were evaluated using a carrageenan-induced rat paw edema test.

MATERIALS AND METHODS

Extraction and Fractionation of Plant Materials

Whole plants were purchased from Omniherb (Yeongcheon, Korea) and a voucher specimen (No. 012) was deposited in the herbarium of the College of Oriental Medicine of Kyunghee University, Seoul, Korea. Dried and finely powdered Ag rimonia pilosa leaves (100 g) were extracted twice by repeat sonication (30 min with 80% ethanol). The 80% ethanol extract (EtOH) was filtered and then dried using a vacuum evaporator. The dried ethanol extract was resuspended in distilled water and extracted with diethyl ether, chloro-form (CHCl 3), E thyl acetate (E tOAC) and n-butanol (BuOH) in order of increasing polarity.

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Total Phenolics and Flavonoid

Total phenolic contents in Ag rimonia pilosa extracts were determined using Folin-Ciocalteu’s reagent according to the method (Vinson et al., 2001)with modifications. One milliliter of diluted extract and 1.0 mL of diluted Folin-Ciocalteu reagent were mixed; after 3 min, 1.0 mL of 10% sodium carbonate was added. After 1 h of reaction, the concentration of total phenolic contents was measured by reading the absorbance at 760 nm and compensating the reading to standard gallic acid. Total flavonoid content was calculated using a calibration curve of catechin. After each 0.5 ml of ethanol extract and 2%AlCl 3 ethanol solution was added and incubated for 1 hr at room temperature,and then absorbed at 420 nm.

Liquid Chromatography–Mass Spectrometry (LC-MS) Analysis

An Agilent (Palo Alto, CA) 1100 series quadrupole LC-MS with an atmo-spheric pressure chemical ionization (APCI) interface was used in the negative and positive ionization mode. Data was collected using Chemstation software version A.09.03. A GE MINI 5-μm C18 110A column (150 mm × 4.60 mm)(Phenomenex, CA) was used with an injection volume of 10 μL for the HPLC separation. The mobile phases consisted of (A) water and (B) methanol at a flow rate of 0.7 ml/min. Gradient elution was applied as follows: 0–70 min, 5–90%(B). Gallic acid, (+)-catechin, epicatechin, (-)-epigalocatechin gallate (EGCG),quercetin, quercitrin, luteolin, and kaempferol were used as standards.

Radical Scavenging Activity

The antioxidant activities of extracts and the positive control were assessed on the basis of radical scavenging effects of the stable 1,1-Diphenyl-2-Picrylhydrazyl (DPPH) free radical. Extracts (20 μL) were added to 180 μL of DPPH (100 μM) solution in a 96-well microplate. After incubation at 37°C for 20 min, the absorbance of each solution was determined at 517 nm using an enzyme-linked immunosorbent assay (E LISA) plate reader (Molecular devices, CA). Hydroxyl radical scavenging activity was measured according to the method (Lim et al., 2001) with some modifications .

The reaction mixture was comprised of 0.30 mL 0.02 M sodium phosphate buffer (pH 7.0), 0.15 mL 10 mM 2-deoxyribose, 0.15 mL 10 mM FeCl 3, 0.15 mL 10 mM EDTA, 0.15 mL 10 mM H 2O 2, 0.15 mL 10 mM ascorbic acid, 0.40 mL H 2O, and 0.05 mL of the test sample solution. Reaction mixtures were incu-bated at 37°C for 2 h and the thiobarbituric acid reactive substances (TBARS)formed were measured. After incubation, 0.5 mL of 2.8% trichloroacetic acid and 0.5 mL of 1.0% thiobarbituric acid (TBA) were added to the test tubes and boiled for 20 min. After cooling, absorbance was measured at 532 nm. Radical scavenging activity (%) = [1?(A sample /A control )] ×100.

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Cell Culture

The murine macrophage RAW 264.7 cells were obtained from the Korea Cell Line Bank (Seoul, Korea). The macrophages were cultured in DMEM medium supplemented with 2 mM glutaminne, antibiotics (100 U/mL of penicillin and 100 μg/mL of streptomycin) and 10% heat-inactivated fetal bovine serum and maintained at 37 °C in a humidified incubator containing 5% CO 2.

Cell Viability by MTS Assay

Cell viability assessment was performed by using the MTS assay. The samples were dissolved in DMSO and diluted in cell culture medium. The final concentration of DMSO was 0.1%, a concentration that did not demon-strate any effects on the measured parameters in previous control experi-ments. The cells were cultured in a 96-well plate at a concentration of 5 × 104cells/well. After 24h of preconditioning, culture medium was aspirated and the cells were exposed to a variety of concentrations of sample for 24 h.Subsequently, 20 μL of MTS dye (1 mg/mL) was added to the cultures and further incubated for 2 h at 37°C. The index of cell viability was calculated by measuring the optical density of color produced by MTS dye reduction at 490 nm.

Nitrite Determination

Cells (5 × 104 cells/well) were treated with various concentrations (5–100μg/mL) of samples and stimulated with LPS (1 μg/mL) for 18 h. The nitrite concentration in the medium is an indicator of NO production according to the Griess reaction. One hundred microliters of each supernatant from each well were transferred to another 96-well plate and 100 μL of the Griess reagent (1% sulfanilamide in 5% phosphoric acid and 0.1% naphth-ylethylenediamine dihydrochloride in water) was added. The mixtures were incubated at room temperature for 10 min. The absorbance of the mixtures (at 550 nm) was determined with an ELISA plate reader (Molecular Devices, CA).

Intracellular Reactive Oxygen Species Assay

Cells (5 × 104 cells/well) were pretreated with a variety of concentrations of extract for 24 h, at which time LPS was added to cells and further incubated according to the specified time course at 37°C. At the end of the oxidation treatment, cells were incubated with 50 μM of fluorescent probe DCHF-DA for 30 min at 37°C. The degree of fluorescence, corresponding to intracellular ROS, was determined using Fluoroscan Ascent FL (Type 374, Labsytems,Finland) (490 nm excitation and 526 nm emission).

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Western Blot and Reverse Transcriptase Polymerase Chain

Reactions (RT-PCR)

Fifty μg of protein was electrophoresed in SDS-PAGE and transferred to a nitrocellulose membrane. The membranes were immunobloted with monoclonal anti-iNOS and anti-COX-2 antibodies and horseradish peroxidase-conjugated secondary antibody, then developed by enhanced chemiluminescence (E CL).RNA was extracted using TRIzol reagent (Invitrogen, Australia) according to the manufacturer’s instructions. The sense and antisense primers for iNOS were 5′-AATGGCAACATCAGGTCGGCCATCACT-3′ and 5′-GCTGTGTGTCA-CAGAAGTCTCGAACTC-3′, and for COX-2 were 5′-GGAGAGACTATCAA-GATAGT-3′ and 5′-ATGGTCAGTAGACTTTTACA-3′, for GAPDH as a control were 5′-TGAAGGTCGGTGTGAACGGATTTGGC-3′ and 5′-CATGTAGGC-CATGAGGTCCACCAC-3′, respectively. The 1 μg of cDNA was amplified by PCR for 20 cycles of denaturation (95°C for 1 min), annealing (iNOS, 40°C for 1min or COX-2, 60°C for 1 min), polymerization (72°C for 1 min), and a final elon-gation step of 5 min at 72°C.

Animals

Male Sprague–Dawley rats (150–200 g) were housed in plastic cages under controlled conditions (temperature: 20 ± 2°C, humidity: 40–60%, 12 h light/dark cycle) and acclimatized for 1 week. For 24 h before the experiment,only water was offered to the animals. All animal experiments were conducted in accordance with the “Guide for the Care and Use of Laboratory Animals”established by the Korea National Institute of Health.

Carrageenan-Induced Rat Paw Edema

The carrageenan-induced rat paw edema test was performed according to the method (Cutler et al., 1998). The rats were divided into four groups (n = 5).Before any treatment, the average volume of the right paw of each animal was determined (V 0, basal volume) using a plethysmometer. The different groups were treated orally with BuOH extract (50 and 100 mg/kg b.w.), indomethacin (10 mg/kg b.w), and vehicle control. All of samples were dissolved in DMSO.One hour after the administration, paw edema was induced by injection of 10μl of 2% carrageenan into the right hind paw. The paw volumes were measured at 5 h after the injection, and the volume (V t) of the edema was measured with a plethysmometer.

Statistical Analyses

All experimental data were examined by analysis of the variance (ANOVA) and significant differences among the means from triplicate deter-minations assumed at p < 0.05. Further statistical analyses were performed

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using Duncan’s multiple range tests and the Statistical Analysis System,version 8.2 (SAS Institute Inc., NC, USA).

RESULTS

Extractions and Screening for Anti-inflammatory Effects

To investigate anti-inflammatory effects, 91 herbs of oriental medicines including APL were first extracted with 80% ethanol and screened for NO inhibitory ability in LPS-induced macrophage cells and DPPH radical scav-enging activity. As a result, APL showed potential anti-inflammatory effects than other herbals. To further study the effects of APL, ethanol extract was additionally extracted with various solvents. The fractional yields of the extracts were as follows (w/w): diethyl ether (2.25 g), CHCl 3 (0.79 g), EtOAC (0.44 g), n-BuOH (5.0 g) and water (4.01 g) from 12.48 g of the dried EtOH extract. In analysis of the radical scavenging activity, DPPH and hydroxyl radical scavenging activities of the 80% ethanol extract and its solvent extracts were increased in the order of BuOH (IC 50 = 15.3 and 30.9 μg/mL) >EtOAC (15.6 and 48.1 μg/mL) > 80% ethanol (25.0 and 87.6 μg/mL) > water extracts (35.2 and 73.2 μg/mL) (Table 1). Furthermore, BuOH extract con-tained higher contents of total phenolics and flavonoids than other extracts (Table 1).

Inhibitory Effect of APL on NO and ROS Production

To further investigate the antioxidant activity in inflammation, the cell viability of EtOAC and BuOH extracts was first was evaluated. No significant cytotoxicity up to 100 μg/mL of both extracts was observed when the RAW 264.7 macrophages were stimulated with LPS, or not (Figure 1A). To investi-gate whether E tOAC and BuOH extracts inhibit NO and ROS production,

Table 1:Total phenolics, flavonoids and radical scavenging activities (IC 50) of ethanol

extract and its solvent extracts from Agrimonia pilosa Ledeb.

Total phenolics (g/100 g of extract,

dry weight)

Total flavonoids (g/100 g of extract,

dry weight)

DPPH ·

(m g/ml)

OH ·

(m g/ml)

EtOH

15.81 ± 0.77c 1.44 ± 0.13a 25.02 ± 1.71b

87.68 ± 8.21c

Diethyl ether 5.13 ± 0.13e 0.65 ± 0.47bc >100>200CHCl 3 4.48 ± 0.10f 0.41 ± 0.17c >100>200EtOAC 22.16 ± 1.22b 0.70 ± 0.38b 15.63 ± 1.10a 48.17 ± 2.21a n-BuOH 27.47 ± 0.60a 1.76 ± 0.38a 15.31 ± 0.05a 30.93 ± 3.21b Water

7.37 ± 1.02d 0.68 ± 0.39bc 35.21 ± 1.21c 73.27 ± 9.25c

Each value is the mean ± S.D. a-f Mean values with different superscript letters are significantly different (P < 0.05) by Duncan’s multiple-range test. IC 50 values were defined as the extract concentrations required to scavenge 50% of the DPPH and hydroxyl radicals.

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Figure 1:Effects of EtOAC and BuOH extracts from Agrimonia pilosa Ledeb. on cell viability

(A), nitrite (B) and ROS (C) production in LPS-induced macrophage RAW 264.7 cells. RAW 264.7 cells were treated with LPS (open) or without LPS (solid) for up to 24 min. *p < 0.05 com-pared to each LPS alone.

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LPS-induced RAW 264.7 macrophages were pretreated with various concen-trations of both extracts for 1 h before the stimulation with LPS for 18 h. LPS treatment transiently enhanced the NO and ROS levels, whereas both extracts significantly inhibited LPS-induced NO and ROS production in a dose-dependent manner (Figure 1B and 1C, p < 0.05). Moreover, the BuOH extract exhibited higher inhibitions of NO and ROS than EtOAC extract.

Effect of APL on the Expression of iNOS and COX-2

Since NO and ROS are mediators in inflammatory reactions, it was exam-ined whether the BuOH extract could directly suppress the expression of iNOS and COX-2 in LPS-induced RAW 264.7 macrophages. The proteins of iNOS and COX-2 were increased in treatment with LPS in the cells. Despite of no change in COX-2, the protein expression of iNOS was significantly decreased in treatment of EtOAC and BuOH extracts of APL (Figure 2A, p <0.05). However, in consistent with the result of NO and ROS inhibition, BuOH extract showed higher suppression than E tOAC extract. Then, the mRNA expression of iNOS and COX-2 was also determined. Consistently, BuOH extract significantly reduced the transcriptional level of iNOS, but not COX-2,in a dose-dependent manner (Figure 2B, p < 0.05)

Figure 2:Effects of EtOAC and BuOH extracts on iNOS and COX-2 protein and mRNA expression in LPS-induced macrophage RAW 264.7 cells. Cells were pretreated with different concentrations of EtOAC and BuOH extracts for 1 h before LPS treatment (1 μg/mL), and the cells further incubated for 18 h. The protein levels were determined by Western blotting (Figure 2A). The relative levels of mRNA were assessed by RT-PCR (Figure 2B). Each bar repre-sents the means of duplicate ± S.D.

iNOS COX-2iNOS COX-2GAPDH

0.2

0.40.60.81.01.21.4+++

–++

100

––50

BuOH (μg/ml)

LPS (1μg/ml)+

+

–++

iNOS COX-2

iNOS COX-2

(i N O S o r C O X -2)/G A P D H r a t i o

*

*

*

*

*

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Inhibition of Paw Edema in Rat by BuOH Extract of APL

To elucidate the anti-inflammatory effect of APL, in vivo study was employed using carrageenan-induced rat paw edema. Consistent with these in vitro results, the BuOH extract reduced carrageenan-induced rat paw edema in a dose-dependent manner (Table 2). Treatment with 50 mg/kg and 100 mg/kg of BuOH extract reduced paw edema levels by 42.2% and 65.3%,respectively, at 5 hr, compared to 78.12% reduction by indomethacin (10 mg/kg)as a positive control (p < 0.05).

DISCUSSION

In this study, it was found that BuOH extract from Agrimonia pilosa suppressed the LPS-induced NO production by inhibiting iNOS expression at mRNA and protein levels. BuOH extract of APL also inhibited the acute inflammatory process when administered orally, which was demonstrated by animal model using carrageenan-induced rat paw edema. Therefore, our findings suggest that the significant scavenging capacity against free radical by BuOH extract from APL plays an important mechanism for its anti-inflammatory activity.In addition, APL might be a promising candidate as an agent to prevent inflammation or to exert its combinational therapeutic effect with anti-inflammatory drugs.

Ag rimonia pilosa has been traditionally used to treat diseases, such as hemorrhage and liver troubles, in Asia. The antitumor and hepatoprotective activities of Agrimonia pilosa were also observed (Miyamoto et al., 1987; Park et al., 2004). More recently, it was found that Ag rimonia pilosa has a high antioxidant activity among 45 traditional herbal medicines (Liao et al., 2008).Therefore, based on previous and our results, it is strongly suggested that Agrimonia pilosa , as a herbal or alternative medicine, could play an important role to restore cellular damages arising from free radicals and the imbalanced scavenging systems in diseases including inflammation.

Table 2:Inhibitory effect of BuOH extract from Agrimonia pilosa on

l -carrageenan-induced paw edema of rats.

Group

Dose (mg/kg)

Increased paw volume (ml)

Inhibition (%)

Control –0.692 ± 0.028d –BuOH

500.400 ± 0.034c 42.191000.210 ± 0.021a 69.65Indomethacin

10

0.172 ± 0.172b

75.14

Values represent means ± S.D. (n = 5). a-d Mean values with different superscript letters are significantly different (P < 0.05) by Duncan’s multiple-range test.Percentages refer to the change in edema size relative to the negative control groups.

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Free radicals have been regarded to be responsible for various diseases including inflammation (Knight, 1995; Petrone et al., 1980). The stable DPPH radical has also been used to evaluate the radical quenching capacities of extracts (Suja et al., 2004). It is well known that antioxidant activity offers anti-inflammatory action. Since inflammation elevates the levels of free radicals,antioxidants is considered to possibly display anti-inflammatory effects (Rahman et al., 2006).

In this study, EtOAC and BuOH extracts showing the most powerful radical scavengers were selected in subsequent experiments designed to determine their anti-inflammatory activities. The higher scavenging activities of BuOH and E tOAC extracts compared to others might be attributed to the high amount of phenolic and flavonoid compounds. It was reported that the concen-tration of phenolic compounds contributes directly to the antioxidant action (Awika et al., 2003). More recently, the antioxidant and free radical scavenging activities of APL have been determined. Especially, five phenolic compounds of aromadendrin (AD), dihydrokaempferol 3-O -b -D -glucoside (DK3-O -glc),quercitrin (QC), aglimonolide-6-O-b -D -glucoside (AG6-O -glc) and loliolide (LL)showed the scavenging activity of NO (He et al., 2009; Taira et al., 2009).

NO production is a pro-inflammatory mediator in many different acute and chronic inflammatory diseases (Bosca et al., 2005). Also, ROS levels are known to contribute to inflammatory reactions (Tsao et al., 2005). The genera-tion of the initial ROS mediates the production of NO via mitogen activated protein kinases and nuclear factor-k B (NF-k B) activities in the macrophages (Schoonbroodt and Piette, 2000; Tsao et al., 2005; Kim et al., 2006). In addition,the antioxidant activity of phenolics inhibits NF-kB activation by reducing ROS (Bremner and Heinrich, 2002). In the present study, the pretreatment of the cells with BuOH and E tOAC extracts significantly attenuated the LPS-induced ROS production. Therefore, it is suggested that the antioxidant activity of both extracts is involved with the mechanism responsible for the inhibition of ROS production.

The protein expression of iNOS was significantly reduced to 19% and 32%by the EtOAC extract (p < 0.05) and to 62% and 81% by the BuOH extract in concentrations of 50 μg/mL and 100 μg/mL, respectively (Figure 2A, p < 0.05).The inhibitory effect on iNOS protein expression by the BuOH extract was higher than that of the EtOAC extract, indicating that the reduced expression of iNOS by exposure to BuOH extract was responsible for the inhibition of NO production. However, there was no significant reduction of COX-2 protein expression with a consistent result that BuOH extract inhibited LPS-induced iNOS mRNA expression in a concentration-dependent manner, but not COX-2mRNA expression. Therefore, the inhibition of NO production might be attributed to the inactivated expression of not COX-2 but iNOS.

The anti-edema activity of the BuOH extract might also be related to the antioxidant activity, which is possibly responsible attenuated inflammatory

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action. Indeed, flavonoids exhibit the anti-inflammatory effects on the acute inflammatory process in carrageenan-induced rat paw edema (Rotelli et al.,2003). In conclusion, our findings revealed that Ag rimonia pilosa Ledeb.appears to have the potential to prevent inflammatory diseases by suppression of iNOS.

ACKNOWLEDGMENT

This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MEST) (No. 2009-0063466).Declaration of Interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

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