suppression of inducible nitric oxide synthase expression by nyasol and broussonin a, two phenolic...

Suppression of Inducible Nitric Oxide Synthase Expression by Nyasol andBroussonin A, Two Phenolic Compounds from Anemarrhena asphodeloides,

through NF-kB Transcriptional Regulation in vitro and in vivo

by Eun Jin Leea)1), Hwa-Jin Chungb)1), Yuna Pyeeb), Ji-Young Hongb), Ui Joung Youna),Eun-Kyoung Seoa), and Sang Kook Lee*b)

a) College of Pharmacy, Ewha Womans University, Seoul 120-750, Koreab) College of Pharmacy, Seoul National University, Seoul 156-742, Korea(phone: þ82-2-8802475; fax: þ82-2-7628322; e-mail: [email protected])

Anemarrhena asphodeloides is widely used in traditional Chinese medicine, and is known to possessantidiabetic and anti-inflammatory properties. Because inducible nitric oxide synthase (iNOS) plays animportant role in inflammation, we investigated the inhibitory effects of two known phenolic compounds,nyasol (1) and broussonin A (2), from A. asphodeloides, on iNOS and its plausible mechanism of action.Compounds 1 and 2 exhibited inhibitory effects on nitric oxide (NO) production in lipopolysaccharide(LPS)-stimulated RAW 264.7 macrophage cells. Compounds 1 and 2 also suppressed the expressions ofiNOS protein and mRNA. Moreover, compounds 1 and 2 suppressed the expression of inflammatorycytokines such as interleukin-1b (IL-1b) and interferon-b (IFN-b). They also inhibited the transcrip-tional activity of NF-kB and degradation of IkB-a, as well as the activation of Akt and ERK in LPS-stimulated RAW 264.7 cells. In in vivo animal model, compounds 1 and 2 significantly inhibited TPA-induced mouse ear edema. These results suggest that 1 and 2 suppress LPS-stimulated iNOS expressionat the transcriptional level through modulating NF-kB and down-regulation of the Akt and ERKsignaling pathways. Taken together, these findings indicate that the suppressive effects of 1 and 2 oniNOS expression might provide one possible mechanism for their anti-inflammatory activities.

Introduction. – Nitric oxide synthase (NOS) catalyzes the synthesis of a free radicalnitric oxide (NO) and l-citrulline from l-arginine [1]. Especially, NO has manyphysiological functions such as vasorelaxation, neurotransmission, tissue homeostasis,wound healing, inflammation, and cytotoxicity [2] [3]. At least, three isoforms of NOShave been identified, including constitutively expressed endothelial NOS (eNOS) andneuronal NOS (nNOS), and inducible NOS (iNOS) [1]. Although eNOS and nNOSare essential for maintaining tissue homeostasis and constitutively expressed, iNOS, inparticular, mediates several inflammatory responses by the overproduction of NO andis induced in response to various proinflammatory stimuli such as tumor necrosis factor-a (TNF-a), interferon-g (IFN-g), interleukin-6 (IL-6), and lipopolysaccharide (LPS)[4] [5]. Since the iNOS is highly involved in the inflammatory responses, iNOS isconsidered a new target for developing new substances for the treatment ofinflammatory diseases [6].

Anemarrhena asphodeloides Bunge (Liliaceae), a medicinal plant widely distrib-uted in mainland China, Japan, and Korea, has been traditionally used as antidepres-

CHEMISTRY & BIODIVERSITY – Vol. 11 (2014) 749

� 2014 Verlag Helvetica Chimica Acta AG, Z�rich

1) These authors contributed equally to this work.

sant, antidiabetics, anti-inflammatory, anti-platelet aggregation, and antipyretic agent[7] [8]. Previous phytochemical investigation of the roots of A. asphodeloides led to theisolation of steroidal saponins, xanthones, and lignans [9 – 11]. Recently, we reportedthe isolation of steroidal saponin timosapoinin-III that exhibits cytotoxic and antitumoractivities against human cancer cells [12]. However, the precise mechanism of action ofthe anti-inflammatory activity of the A. asphodeloides constituents has beeninsufficiently investigated. We herein report the anti-inflammatory effects of isolates1 and 2 (Fig. 1) from the rhisomes of A. asphodeloides in vitro cell culture and in vivoanimal model. Our findings demonstrated that the anti-inflammatory potentials of 1and 2 might be associated with the suppression of iNOS and NF-kB transcriptionalregulation.

Results and Discussion. – Inhibition of NO Production. To investigate whether 1and 2 (Fig. 1) alter NO production in LPS-stimulated murine macrophage RAW 264.7cells, the production of nitrite, the stable metabolite of NO, was determined in culturesupernatant. As shown in Fig. 2, a, treatment of LPS (1 mg/ml) with 1 drasticallyincreased the NO production from a basal level of 1.8 to 22.8 mm. Co-treatment of 1 or 2with LPS significantly inhibited NO production in a concentration-dependent mannerwith IC50 values of 4.0 and 3.7 mm, respectively. Under the same assay conditions, l-NMMA (l-monomethylarginine), a positive control for a nonselective inhibitor ofNOS, exhibited an IC50 value of 6.5 mm. Cell viability was also determined by MTT (¼3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) assay; no signifi-cant cytotoxic effect was observed. Therefore, an additional study was designed toinvestigate the anti-inflammatory activity at the various concentrations of 1 (2.5 –10 mm) and 2 (1.5– 15 mm). These results indicated that the inhibition of NO productionby 1 and 2 was not related with a cytotoxic effect (Fig. 2, b).

Suppression of iNOS Protein and Gene Expression. To further elucidate themechanism of action on the inhibition of NO production, the iNOS protein and mRNAexpression were investigated. RAW 264.7 Cells were stimulated with LPS (1 mg/ml) inthe absence or presence of various concentrations of 1 (2.5 – 10 mm) and 2 (1.5– 15 mm)for 16 h. As shown in Fig. 3, treatment of LPS enhanced the expression of iNOSprotein, but co-treatment with 1 (Fig. 3, a) or 2 (Fig. 3,b) suppressed iNOS proteinexpression in a concentration-dependent manner. In addition, to further investigatewhether these two phenolic compounds affect to the iNOS mRNA expression, iNOSmRNA levels were determined using RT-PCR (¼ reverse transcription polymerasechain reaction) and semi-quantitative real-time PCR analysis. As illustrated in Fig. 4,compound 1 suppressed the LPS-induced iNOS mRNA expression in a concentration-

CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)750

Fig. 1. Chemical structures of nyasol (1) and broussonin A (2)

dependent manner as revealed by RT-PCR (Fig. 4,a) and real-time PCR (Fig. 4, b).Treatment of 2 also suppressed the iNOS expression in a concentration-dependentmanner at the transcriptional levels of iNOS (Fig. 4, c, RT-PCR; Fig. 4,d, real-timePCR). These data suggest that the inhibition of NO production by 1 and 2 might beassociated with the suppression of iNOS expression at translational and transcriptionallevels.

Suppression of the Expression of Pro-Inflammatory Cytokines. To determinewhether 1 and 2 affect the expression of pro-inflammatory cytokines, the levels ofsteady-state transcripts of interleukin-1b (IL-1b) and interferon-b (IFN-b) wereanalyzed by RT-PCR. As shown in Fig. 5, treatment with LPS (1 mg/ml) for 4 hmarkedly increased the expression of IL-1b and IFN-b mRNA, but co-treatment with 1or 2 dose-dependently suppressed the expression of these pro-inflammatory cytokines.

Inhibition of LPS-Induced NF-kB Transcriptional Activity and IkB-a Degradation.It is well-known that NF-kB is a major transcription factor for the induction of theiNOS gene by LPS [13]. To further investigate whether NF-kB is an important target ofthe action of 1 and 2 in LPS-stimulated RAW 264.7 cells, a reporter gene assay for NF-kB transcriptional activity (SEAP) was used. Treatment with LPS for 18 h elicited a 4 –4.2-fold increase in NF-kB transcriptional activity, but treatment with 1 or 2 markedly


Fig. 2. Effects of 1 and 2 on NO production in LPS-stimulated macrophage cells. a) RAW 264.7 Cellswere stimulated with 1 mg/ml LPS in the presence or absence of the test compounds. After 20 h, culturedmedia were collected and analyzed for nitrite using the Griess reaction. Data are expressed as mean�S.D. of three independent experiments. * p<0.01 was considered statistically significant. b) Cell viability

was determined by MTT assay as described in the Exper. Part.

and dose-dependently inhibited LPS-induced increases in NF-kB transcriptionalactivity (Fig. 6, a). Since the activation of NF-kB signaling is preceded by thedegradation of the inhibitory subunit IkB-a, we next investigated whether 1 and 2affect the degradation of IkB-a induced by LPS. As shown in Fig. 6,b, LPS treatmentfor 30 min markedly degraded the IkB-a, but pre-treatment of 1 and 2 for 30 min priorto LPS stimulation inhibited the degradation of IkB-a in a concentration-dependentmanner.

Inhibition of LPS-Induced Activation of Akt and MAP Kinases. It is known that theactivation of NF-kB by LPS is associated with the stimulation of signal transductionpathways including MAP kinases and Akt signaling [14] [15]. To further elucidate how1 and 2 inhibit NF-kB activation, the signaling molecules such as Akt, p38, and ERK1/2influenced by NF-kB were examined in LPS-stimulated RAW 264.7 cells. Compound 1suppressed LPS-stimulated activation of Akt and MAP kinases including ERK1/2 andp38 (Fig. 7,a). Treatment with 2 also suppressed LPS-induced activation of Akt andERK1/2, but p38 was not inhibited by 2 (Fig. 7, b).

Inhibition of TPA-Induced Ear Edema in Mice. Mouse ear edema was induced bythe applications of TPA (¼12-O-tetradecanoylphorbol-13-acetate). Topically appliedTPA for 4 h increased the ear edema, and the mean weight of ear in the control groupwas ca. 9.2 mg/mouse. However, the administration of 1 and 2 (10 or 20 mg/kg)significantly (p<0.01) and dose-dependently inhibited the TPA-induced ear edema(Fig. 8,a). Under the same experimental conditions, indomethacin (20 mg/kg) wasshown to exert 52.8% inhibition at 4 h. The potency of anti-inflammatory effects of 1


Fig. 3. Effect of 1 (a) and 2 (b) on iNOS protein expression in LPS-stimulated RAW 264.7 cells. Cellswere treated with LPS (1 mg/ml) and test compounds for 16 h. After incubation, cell extracts wereobtained and subjected to Western blot analysis as described in the Exper. Part. Data are representative

of three independent experiments. b-Actin was used as an internal standard.

(20 mg/kg; 70.8% inhibition) and 2 (20 mg/kg; 68.1% inhibition) were stronger thanthat of indomethacin. In addition, compounds 1 and 2 inhibited the expressions of anti-inflammatory biomarkers iNOS protein in TPA-induced mouse ear (Fig. 8, b). Thesedata suggest the inhibition of TPA-induced mouse ear edema by 1 and 2 might beassociated with the suppression of TPA-induced expression of iNOS in this acuteinflammation mouse ear model.

Conclusions. – Compounds 1 and 2 from A. asphodeloides inhibit NO production inLPS-stimulated RAW 264.7 macrophage cells through the suppression of iNOS proteinand mRNA expression. The suppression of iNOS expression is, at least in part,associated with the regulation of NF-kB activation, and Akt and ERK1/2 signalingpathway. Moreover, compounds 1 and 2 significantly inhibited the TPA-induced earedema in vivo. Thus, compounds 1 and 2 might be potential candidates for clinicallyuseful anti-inflammatory agents.


Fig. 4. Effect of 1 (a and b) and 2 (c and d) on iNOS mRNA expression in LPS-stimulated RAW 264.7cells. RAW 264.7 Cells were treated with LPS (1 mg/ml) and test compounds for 4 h. Total RNA wasisolated, and 1 mg of RNA was used for reverse-transcription. The gene expression level of iNOS wasmeasured using RT-PCR (a and c) and real-time RT-PCR (b and d) methods, as described in the Exper.Part. Data are representative of three separate experiments. b-Actin was used as an internal standard.Values are expressed as mean�S.D. of three independent experiments. *: p<0.01 was considered

statistically significant.

This work was supported by a grant (12172MFDS989) from the Ministry of Food and Drug Safety in2014 and a grant of the National Research Foundation of Korea (NRF) funded by the KoreanGovernment (MEST; MRC No. 2009-0083533).

Experimental Part

General. Dulbecco�s Modified Eagle�s Medium (DMEM), fetal bovine serum (FBS), sodiumpyruvate, l-glutamine, and antibiotics-antimycotics soln., and SYBR� safe DNA gel stain werepurchased from Invitrogen (Grand Island, NY, USA). AMV reverse transcriptase, dNTP mixture,random primer, RNasin, and Taq polymerase were purchased from Promega (Madison, WI, USA). iQTM

Supermix for TaqMan real-time PCR was purchased from Bio-Rad (Hercules, CA, USA). Pre-designedand pre-labeled TaqMan PCR primer and probe sets were purchased from Metabion International AG(DE-Martinsried). Anti-mouse, -goat, and -rabbit IgG-HRP antibodies, rabbit polyclonal antibodiesagainst iNOS, IkB-a, ERK, p38, and mouse monoclonal anti-phospho-ERK antibody were from SantaCrutz Biotechnology Inc. (Santa Crutz, CA, USA). Rabbit polyclonal anti-phospho-38 antibody waspurchased from Cell Signaling Technology (Beverly, MA, USA). Rabbit polyclonal anti-hemeoxygenase-1 antibody was from Stressgen Bioreagents (Victoria, Canada). Lipopolysaccharide (LPS),


Fig. 5. Effect of 1 (a) and 2 (b) on LPS-induced pro-inflammatory cytokine expression in macrophagecells. RAW 264.7 Cells were treated with LPS (1 mg/ml) and test compounds for an additional 4 h. TotalRNA was isolated, and the gene expression levels of IL-1b were IFN-b was measured by RT-PCR. Data

were representative of three independent experiments. b-Actin was used as an internal standard.

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), CF3COOH (TFA), bovineserum albumin (BSA), bicinchoninic acid (BCA), b-sulfanylethanol, TRI reagent, mouse monoclonalanti-b-actin antibody, and other agents, unless otherwise indicated, were purchased from Sigma�Aldrich(St. Louis, MO, USA). Compounds 1 and 2 (Fig. 1) were isolated from the rhizomes of A. asphodeloidesas described in [16].

Cell Culture. RAW 264.7 Murine macrophage cells were cultured in DMEM supplemented with 10%heat-inactivated FBS and antibiotics-antimycotics (100 U/ml penicillin G sodium, 100 mg/ml streptomy-cin sulfate, and 0.25 mg/ml amphotericin B). RAW 264.7 Cells stably transfected with pNF-kB-SEAP-NPTreporter plasmid (SEAP-RAW cells) were kindly provided by Dr. Yeong Shik Kim (Seoul National


Fig. 6. Effects of 1 and 2 on LPS-induced NF-kB transcriptional activity and IkB-a degradation. a) RAW264.7 Cells stably transfected with an NF-kB-SEAP-NPT reporter plasmid were pretreated with variousconcentrations of test compounds for 2 h, and then stimulated with LPS (1 mg/ml) for an additional 18 h.The supernatants were analyzed for secreted alkaline phosphatase activity (SEAP) as described in theExper. Part. Values are expressed as mean�S.D. of three independent experiments. *: p<0.01 wasconsidered statistically significant. b) RAW 264.7 Cells were treated with test compounds for 30 minprior to LPS treatment (1 mg/ml). After incubation for an additional 30 min, Western blot analysis was

performed to determine the Ik-Ba degradation.

University, Korea). SEAP-RAW Cells were maintained in DMEM containing 500 mg/ml G418. All cellswere incubated at 378 in 5% CO2 in a humidified atmosphere.

Measurement of NO Production and Cytotoxicity Assay. RAW 264.7 Cells were placed in a 24-wellculture plate (5�105 cells/ml) and incubated for 24 h. The cells were washed twice with PBS and pre-incubated with phenol red-free medium containing various concentrations of compounds for 15 min,followed by LPS treatment (1 mg/ml) for 20 h. The amount of NO released into the cultured medium wasmeasured by the Griess reagent (1 : 1 mixture (v/v) of 1% sulfanilamide and 0.1% N-(naphthalen-1-yl)ethylenediamine in 2.5% H3PO4) [17]. The absorbance was measured at 540 nm. The standard curvewas created by using known concentrations of NaNO2. % Inhibition was expressed as [1� (NO level oftest samples/NO levels of vehicle treated control)]�100. The IC50 value, the sample concentrationresulting in 50% inhibition of production, was determined using nonlinear regression analysis (%inhibition vs. concentration). To evaluate the cytotoxic effect of test compound in RAW 264.7 cells, anMTT assay was performed. After the Griess reaction, MTT soln. (final concentration, 500 mg/ml) wasadded to each well and further incubated for 4 h at 378. Media were discarded, and DMSO was added toeach well to dissolve generated formazan. The absorbance was measured at 570 nm, and % survival wasdetermined by comparison with the control group.

Preparation of Total Cell Lysates. RAW 264.7 Cells (5�105 cells/ml in 60-mm dish) were incubatedwith or without various concentrations of test compound and LPS (1 mg/ml) for indicated times. Cellswere washed with PBS and lysed in boiling sample loading buffer (250 mm Tris · HCl pH 6.8, 4% SDS,10% glycerol, 0.006% bromophenol blue, 2% b-sulfanylethanol, 50 mm NaF, and 5 mm Na3VO4). Celllysates were boiled for an additional 5 min and stored at �208. Protein concentration of each lysate wasdetermined by BCA (¼ bicinchominic acid) assay.

Western Blot Analysis. Equal amount of cell lysates (30–50 mg) were subjected to 8 and 10% SDS-PAGE. Separated proteins were electrically transferred onto polyvinylidene difluoride (PVDF)


Fig. 7. Effect of 1 (a) and 2 (b) on LPS-induced Akt and MAP kinase pathway. RAW 264.7 Cells weretreated with test compounds for 30 min prior to LPS treatment (1 mg/ml). After incubation for anadditional 30 min, Western blot analysis was performed to determine the activation of Akt and MAPkinase pathway. Data were representative of three independent experiments. b-Actin was used as an

internal standard.

membranes (Milipore, Bedford, MA, USA). Membranes were blocked with blocking buffer (5% non-fatdry milk in PBS containing 0.1% Tween-20 (PBST)) for 1 h at r.t. After washing three times with PBST,membranes were incubated with primary antibodies overnight at 48. Membranes were washed threetimes with PBST, and incubated with corresponding secondary antibodies for 90 min at r.t. Membraneswere washed three times with PBST, and then exposed to enhanced chemiluminescence (ECL) detectionkit (LabFrontier, Suwon, Korea). Blots were detected by LAS 3000 (Fuji Film Corp., Tokyo, Japan).

Reverse Transcription Polymerase Chain Reaction (RT-PCR) and Real-Time Polymerase ChainReaction. RAW 264.7 Cells were stimulated with 1 mg/ml LPS in the presence or absence of testcompound for 4 h. Total cellular RNA was extracted using TRI reagent (Sigma) according tomanufacturer�s recommendation. One mg of total RNA was reverse-transcribed using oligo-(dT)15

primers and avian myeloblastosis virus (AMV) reverse transcriptase (Promega, Madison, WI, USA).PCR was performed in a mixture containing the obtained cDNA, 0.2 mm dNTP mixture (Promega), 10pmol of target gene-specific primers, and 0.25 unit of Taq DNA polymerase (Promega) using a GeneAmpPCR system 2400 (Applied Biosystems, Foster, CA, USA). Each of PCR steps was performed as follows:


Fig. 8. Effect of 1 and 2 on the TPA-induced ear edema. a) Compounds 1 and 2 was administered 30 minprior to the TPA (1.0 mg/ear) application in the right ear of ICR mice. The mice were sacrificed 4 h aftertopical TPA treatment, and ear biopsies were conducted for determination of edema formation bymeasurement of ear weight and analysis of Western blot. Data represent the mean�S.D. (n¼6). *: p<0.01 indicates statistically significant differences from the TPA treated group. b) The expression of iNOSwas determined in the prepared homogenate of ear biopsies by Western blot analyses. Data were

representative of three independent experiments. b-Actin was used as an internal standard.

initial denaturation step for 4 min at 948 ; 25–30 cycles of amplification step consisting denaturation for30 s at 948, annealing for 30 s at 558, and elongation for 30 s at 728 ; and final extension step for 5 min at728. PCR Products were separated by 2% agarose gel electrophoresis, stained with SYBR� safe DNA gelstain (Invitrogen, Grand Island, NY, USA), and visualized by UV transillumination. Forward and reverseprimers used for this study were detailed in Table 1.

Real-time PCR was conducted on a MiniOpticon system (Bio-Rad, Hercules, CA, USA), using 5 mlof reverse transcription product, 10 ml of iQTM Supermix (Bio-Rad, Hercules, CA, USA), 0.5 ml ofTaqman PCR primer, and probes in a total volume of 20 ml. The standard thermal cycler conditionsemployed were: 958 for 5 min before the first cycle, 958 for 10 s, and 568 for 30 s, repeated 60 times,followed by 308 for 1 min. The threshold cycle (Ct), indicating the fractional cycle number at which theamount of amplified target gene reaches a fixed threshold from each well, was determined using by MJOpticon Monitor software. The mean threshold cycle (Ct) value was normalized by the mean Ct value forthe house-keeping gene b-actin (DCt). The normalized transcript levels were expressed relative tosample obtained from LPS (�) control (DDCt). The magnitude change of test gene mRNA wasexpressed as 2�DDCt [18]. Sequences of Taqman real-time PCR primer and probe sets are compiled inTable 2.

Reporter Gene Assay for NF-kB Transcriptional Activity. To determine the effect of the testcompound on the activation of NF-kB, reporter gene assay was performed as described in [19] with somemodifications. The cells were treated with test compound for 2 h and then further stimulated with LPS(1 mg/ml) for an additional 18 h. Cell culture supernatants were heated at 658 for 5 min, and reacted withSEAP assay buffer (2m diethanolamine, 1 mm MgCl2, 500 mm 4-methylumbelliferyl phosphate (MUP)) indarkness at 378 for 1 h. Fluorescence from the product of the SEAP/MUP rection was measured inrelative fluorescence units (RFU) by using a 96-well plate fluorometer with excitation at 360 nm andemission at 449 nm, and normalized by protein concentration. Data are expressed as the proportion tovehicle-treated control cells without LPS.

Animals. Male ICR mice (18 –20 g, 5-weeks-old) were purchased from Central Laboratory AnimalInc. (Seoul, Korea). Animals were housed under standard laboratory conditions with free access to food


Table 1. Sequences of Gene-Specific Primers Used in RT-PCR

Target gene Sequences

Mouse iNOS Sense 5’-ATGTCCGAAGCAAACATCAC-3’Antisense 5’-TAATGTCCAGGAAGTAGGTG-3’

Mouse IL-1b Sense 5’-TGCAGAGTTCCCCAACTGGTACATC-3’Antisense 5’-GTGCTGCCTAATGTCCCCTTGAATC-3’

Mouse IFN-b Sense 5’-GAGTTACACTGCCTTTGCC-3’Antisense 5’-GATTCACTACCAGTCCCAGA-3’

Mouse b-actin Sense 5’-TGTGATGGTGGGAATGGGTCAG-3’Antisense 5’-TTTGATGTCACGCACGATTTCC-3’

Table 2. Taqman Real-Time PCR Primers and Probes

Genes Sequences

Mouse iNOS Forward 5’-CTCACCTACTTCCTGGACATTACG-3’Reverse 5’-CATTGTACTCTGAGGGCTGACAC-3’Probe 5’-FAM-ATCCGTCTCGTCCGTGGCAAAGCG-BHQ-1-3’

Mouse b-actin Forward 5’-ACAGCTTCTTTGCAGCTCCTTC-3’Reverse 5’-CGACCAGCGCAGCGATATC-3’Probe 5’-HEX-CACACCCGCCACCAGTTCGCCAT-BHQ-1-3’

and water. The temp. was thermostatically regulated to 22�28, and a 12-h light/dark schedule wasmaintained. Prior to their use, animals were allowed one week for acclimatization within the work areaenvironment. All animal experiments were carried out in accordance with Institutional Animal Care andUse Committee Guidelines of Seoul National University (SNU-201110-4).

TPA-Induced Ear Edema. 12-O-Tetradecanoylphorbol 13-acetate (TPA)-induced mouse ear edemamodel was performed as described in [20]. TPA (1.0 mg) dissolved in acetone (20 ml) was applied to theinner and outer surfaces of the right ear of ICR mice. Nyasol (1) and broussonin A (2), or vehicle wasadministered intraperitoneally injection 30 min prior to the TPA application. The animals were sacrificedby cervical dislocation after 4 h, and ear biopsies were obtained with a punch (a diameter of 5 mm) andweighed. The increase in the weight of the right ear punch over the left indicated the edema [21]. The earsections were homogenized in lysis buffer and centrifuged at 1500�g for 10 min at 48. iNOS Proteinlevels of the supernatants were determined by Western blot.

Statistics. All experiments were repeated at least three times. Data are expressed as mean� standarddeviation (S. D.) for the indicated number of independently performed experiments. Statisticalsignificance (p<0.05) was assessed by one-way analysis of variance (ANOVA) coupled with theDunnett�s t-test.

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Received January 2, 2014


suppression of inducible nitric oxide synthase expression by nyasol and broussonin a, two phenolic...

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