Anti-Inflammatory Effect of the Oligomeric Stilbene α-Viniferin and its Mode of the Action through Inhibition of Cyclooxygenase-2 and Inducible Nitric Oxide Synthase

• Abstract
• Introduction
• Materials and Methods
• Materials and animals
• Measurement of carrageenin-induced paw edema in mice
• Assay of COX activity
• Measurement of NO production
• RT-PCR of COX-2 and iNOS transcripts
• Statistical analysis
• Results
• Discussion
• References

Abstract

The anti-inflammatory activity of α-viniferin, a trimer of resveratrol, has been demonstrated in an animal model of carrageenin-induced paw edema, and inhibitory effects of the compound on cyclooxygenase (COX) and inducible nitric oxide synthase (iNOS) have been investigated in order to understand the mode of the observed action. α-Viniferin at doses > 30 mg/kg (p. o.) or > 3 mg/kg (i. v.) showed significant anti-inflammatory activity on carrageenin-induced paw edema in mice. α-Viniferin showed an inhibitory effect with an IC₅₀ value of 4.9 μM on COX-2 activity but a very weak inhibitory effect with 55.2 ± 2.1 % of the control (100 %) at 100 μM on COX-1 activity. α-Viniferin at doses of 3 μM to 10 μM inhibited the synthesis of COX-2 transcript in lipopolysaccharide (LPS)-activated murine macrophages Raw264.7. α-Viniferin showed an IC₅₀ value of 2.7 μM on nitric oxide (NO) production in LPS-activated Raw264.7 cells when α-viniferin and LPS were treated simultaneously, but did not inhibit the NO production when α-viniferin was treated at 12 h after LPS stimulation. α-Viniferin inhibited synthesis of iNOS transcript with an IC₅₀ value of 4.7 μM. Consequently, the inhibitory effect of α-viniferin on the release of prostanoids and NO could play an important role to show anti-inflammatory action.

Introduction

α-Viniferin (Fig. [1]) from Caragana chamlagu Lam., an oriental folk medicine with anti-arthritic and anti-neuralgic effects, was reported to show anti-inflammatory effect on croton oil-induced paw edema [1]. The compound was also identified from Caragana sinica (Buchoz) Rehd., Carex humilis Leysser, Iris clarkei Linne, and Sophora hemsleyana Sieb. et Zucc. [2], [3], [4]. α-Viniferin has been reported to inhibit protein kinase C, acetylcholinesterase and keratinocyte proliferation, and to antagonize 20-hydroxyecdysone action [2], [3], [4].

In this study, we have demonstrated the anti-inflammatory activity of α-viniferin on the carrageenin-induced edema model in mice. Furthermore, the effects of α-viniferin on cyclooxygenase (COX) isozymes and inducible nitric oxide synthase (iNOS) have been investigated in order to gain a better insight on mechanism of the observed anti-inflammatory action.

Materials and Methods

Materials and animals

Dulbecco's modified Eagle's medium, arachidonic acid, lucigenin, ibuprofen and NS-398 were purchased from Sigma-Aldrich, fetal bovine serum (FBS) from HyClone, and lipopolysaccharide (LPS, E. coli 0127:B8) from Difco. A Fast RNA kit and an RNA PCR kit were obtained from Bioneer. α-Viniferin ([α]_D ²⁵: 51.4; EtOH, c 1.0) was isolated from Carex humilis (Cyperaceae) as described in our previous paper [5], and its purity was > 95 %. Mice of the ICR strain were purchased from Jeil Animal Care Center, Kyeongki, Korea.

Measurement of carrageenin-induced paw edema in mice

Male ICR mice were accustomed at an animal room in our College of Pharmacy with free access to a standard diet and water ad libitum. Seven mice with 25 ± 3 g body weight were used per group for the experiments. Paw edema in the mice was induced by carrageenin as described previously [6]. Briefly, samples were given to the mice by oral administration or intravenous injection. After 1 h, 10 μL of 2 % carrageenin or saline were injected to a subplantar site of right or left hind paw, respectively. Volumes of right and left hind paws of mice were measured at 1 h, 2 h, 4 h and 6 h after carrageenin challenge. Paw edema was represented as 100 % × [(Rt - Ro)-(Lt - Lo)]/Ro, where Ro or Rt is the volume of right hind paw at zero time or at indicated time after carrageenin injection and Lo or Lt is the volume of left hind paw at zero time or at indicated time after saline injection.

Assay of COX activity

COX activity was measured by chemiluminescence in a total of 200 μL reaction mixture consisting of 0.1 M Tris-HCl (pH 8.0) containing 100 μM arachidonic acid and 25 μM luminol. The COX-1 source was prepared from resting murine macrophages Raw264.7, and the COX-2 source from LPS-stimulated Raw264.7 cells. Murine macrophages Raw264.7 were cultured in DMEM (10 mg/mL Dulbecco's modified Eagle's medium, 24 mM NaHCO₃, 10 mM HEPES, 143 units/mL benzylpenicillin potassium, 100 μg/mL streptomycin sulfate, pH 7.1) containing 10 % FBS at 37 °C with 5 % CO₂ for 48 h and then collected for preparation of COX-1 source. The Raw264.7 cells were cultured in DMEM containing 10 % FBS and 300 μM aspirin at 37 °C with 5 % CO₂ for 24 h. After washing twice with PBS, the cells were added with DMEM containing LPS (10 μg/mL). After incubation at 37 °C with 5 % CO₂ for 24 h, the Raw264.7 cells were collected and sonicated in 0.1 M Tris-HCl (pH 8.0) to obtain the COX-2 source.

Measurement of NO production

Two hundred μL of murine macrophages Raw264.7 (1 × 10⁶ cells/mL) in DMEM containing 10 % FBS were dispensed into a 96-well culture plate and incubated at 37 °C with 5 % CO₂ for 24 h. After washing twice with PBS, the Raw264.7 cells were added with 100 μL each of LPS (20 μg/mL) and the sample dissolved in DMEM, and then incubated at 37 °C with 5 % CO₂ for 24 h. After centrifugation at 700 × g for 30 min at 4 °C, 100 μL of the supernatant were reacted with 100 μL of the Griess reagent (1 % sulfanilamide and 0.1 % naphthylethylenediamine dihydrochloride in 2.5 % phosphoric acid) and then the nitrite content was measured by the absorbance at a wavelength of 540 nm with sodium nitrite as a standard.

RT-PCR of COX-2 and iNOS transcripts

Two mL of murine macrophages Raw264.7 (5 × 10⁵ cells/mL) were dispensed into a 6-well plate, and then incubated at 37 °C with 5 % CO₂ for 48 h. After washing twice with PBS, the Raw264.7 cells were treated with sample at 2 h before stimulation with LPS (10 μg/mL). After incubation at 37 °C with 5 % CO₂ for 6 h, the Raw264.7 cells were collected to purify total RNA by using a FastRNA kit according to the supplier's instruction (Bioneer). The total RNA was subjected to RT-PCR using an RNA PCR kit according to the supplier's instruction (Bioneer). Briefly, total cellular RNA (2 μg) was incubated with avian myeloblastosis virus reverse transcriptase at 50 °C for 25 min. The resulting cDNA samples were subjected to 25 cycles of PCR, one cycle with 30-sec denaturation at 94 °C, 30-sec annealing at 65 °C, and 2-min extension at 72 °C. The PCR primers were designed as follows; COX-2 (583 base pairs) with forward 5′-ACTCACTCAGTTTGTTGAGTCATTC-3′ and reverse 5′-TTTGATTAGTA CTGT-AGGGTTAATG-3′, iNOS (457 base pairs) with forward 5′-GTCAACTGCAAGAGAACGGAGAAC-3′ and reverse 5′-GAGCTCCT-CCAGAGGGTAGGCT-3′, and β-actin (745 base pairs) as an internal control with forward 5′-CACCACACCTTCTACAATGACCTGC-3′ and reverse 5′-GCTCAGGAGGAGCAATGATCTTGAT-3′. The RT-PCR products were resolved on 1.2 % agarose gel by electrophoresis, and quantified by scanning densitometry (Kodak).

Statistical analysis

Paw edema % is expressed as mean ± SEM of two independent experiments with seven mice per group. Effects on COX isozymes and NO production are expressed as control %, mean ± SEM of three independent tests. Data were analyzed by ANOVA followed by Student's t-test. Probability of less than 0.05 (P < 0.05) was considered significant.

Results

α-Viniferin showed dose-dependent anti-inflammatory activity on carrageenin-induced paw edema in mice (Fig. [2]). Paw edema in the control group increased as a function of time, 25.0 ± 1.1 % to 25.2 ± 2.2 % at 1 h, 33.3 ± 1.7 % to 34.4 ± 1.1 % at 2 h, 48.3 ± 1.5 % to 48.8 ± 1.6 % at 4 h and 45.9 ± 1.3 % to 46.3 ± 1.6 % at 6 h after carrageenin challenge. When administered orally, α-viniferin (30 mg/kg dose) showed a significant anti-inflammatory effect with paw edema, 29.8 ± 1.1 % at 2 h, 42.4 ± 1.2 % at 4 h and 39.4 % ± 1.2 % at 6 h; and α-viniferin (100 mg/kg dose) with 19.5 ± 1.3 % at 1 h, 26.4 ± 1.3 % at 2 h, 36.5 ± 1.1 % at 4 h and 34.5 ± 1.8 % at 6 h after carrageenin challenge (Fig. [2] A). When injected intravenously, α-viniferin (3 mg/kg dose) showed a significant anti-inflammatory effect with paw edema, 42.7 ± 1.1 % at 4 h and 39.9 ± 1.2 % at 6 h; α-viniferin (10 mg/kg dose) with 38.4 ± 0.9 % at 4 h and 36.9 ± 0.6 % at 6 h; and α-viniferin (30 mg/kg dose) with 27.1 ± 1.5 % at 2 h, 32.5 ± 1.6 % at 4 h and 31.9 ± 1.1 % at 6 h after carrageenin challenge (Fig. [2] B). However, α-viniferin showed a weaker anti-inflammatory potency than ibuprofen, a non-steroidal anti-inflammatory drug (NSAID) as a positive control.

α-Viniferin showed a dose-dependent inhibitory effect on COX-2 activity with 55.1 ± 6.4 % of the control (100 %) at 3 μM, 36.9 ± 2.2 % at 10 μM, and 7.6 ± 2.6 % at 30 μM but showed a very weak inhibitory effect on COX-1 activity with 67.1 ± 3.4 % of the control (100 %) at 50 μM and 55.2 ± 2.1 % at 100 μM (Fig. [3] A). As a positive control, NS-398 showed an IC₅₀ value of 1.5 μM on COX-2 activity but did not inhibit COX-1 activity. Ibuprofen, an NSAID known as non-selective inhibitor of COX-1 and 2, showed IC₅₀ values of 13.4 μM and 8.6 μM on COX-1 and 2 activities, respectively. α-Viniferin at concentrations of 3 μM to 10 μM showed inhibitory effects on the synthesis of COX-2 transcript in LPS-activated murine macrophages Raw264.7, which was analyzed by RT-PCR (Fig. [3] B). A density ratio of COX-2 versus β-actin signal as an internal standard was 1.5 % in the resting Raw264.7 cells, and was increased to 49.0 % in LPS-activated Raw264.7 cells. The density ratio of COX-2 versus β-actin signal in LPS-activated Raw264.7 cells was decreased to 44.7 %, 34.9 % and 25.8 % by pretreatment with α-viniferin at doses of 1 μM, 3 μM and 10 μM, respectively.

The effect of α-viniferin on NO production in LPS-activated murine macrophages Raw264.7 was analyzed by measurement of the nitrite content in the culture medium (Fig. [4] A). Nitrite content was 8.0 ± 0.4 μM in the resting Raw264.7 cells, and increased to 45.3 ± 1.5 μM after LPS stimulation. α-Viniferin showed dose-dependent inhibitory effects with 39.1 ± 4.0 % of the control (100 %) at 3 μM, 8.7 ± 0.6 % at 10 μM and 1.3 ± 0.5 % at 30 μM with an IC₅₀ value of 2.7 μM on NO production when α-viniferin was treated with LPS simultaneously. However, α-viniferin did not show significant inhibitory effects on the NO production when α-viniferin (1 μM to 30 μM) was treated at 12 h after LPS stimulation. α-Viniferin inhibited the synthesis of iNOS transcript in LPS-activated murine macrophages Raw264.7, which was analyzed by RT-PCR (Fig. [4] B). A density ratio of iNOS versus β-actin signal as an internal standard was 1.3 % in the resting Raw264.7 cells, and was increased to 60.7 % in LPS-activated Raw264.7 cells. The density ratio of iNOS versus β-actin signal in LPS-activated Raw264.7 cells was decreased to 56.2 %, 39.2 % and 2.6 % by pretreatment with α-viniferin at 1 μM, 3 μM and 10 μM, respectively.

Discussion

α-Viniferin given by oral administration (30 mg/kg and 100 mg/kg doses) or intravenous injection (3 mg/kg, 10 mg/kg and 30 mg/kg doses) significantly reduced the carrageenin-induced paw edema in a dose-dependent manner (Fig. [2]). In order to obtain a better insight into the mechanism of the observed anti-inflammatory action, we have investigated the inhibitory effects of α-viniferin on COX isozymes and iNOS.

COX catalyzes a rate-limiting step of prostanoid biosynthesis, which is a pharmacological target of NSAIDs. There are two isozymes of COX; COX-1 is constitutively expressed in various tissues including the stomach, whereas COX-2 does not appear to be expressed in most tissues and is rapidly up-regulated in response to cytokines and growth factors [7], [8]. Gastric intestinal damage due to NSAIDs is caused by inhibition of COX-1 but not COX-2 [9]. α-Viniferin showed a potent inhibitory effect on COX-2 activity with an IC₅₀ value of 4.9 μM but a very weak inhibitory effect on COX-1 activity (Fig. [3] A). Therefore, α-viniferin was identified as a selective inhibitor of COX-2 activity, which could show a similar mode of anti-inflammatory action as NSAIDs without causing unwanted side effects. In addition, α-viniferin inhibited the synthesis of COX-2 transcript in LPS-activated murine macrophages Raw264.7 (Fig. [3] B).

Physiological and normal production of NO from phagocytes is beneficial for the host's defense against microorganisms, parasites and tumor cells [10]. However, overproduction of NO can be harmful and result in several diseases. Macrophages can increase production of NO and superoxide anion simultaneously in pathological conditions including inflammation [11]. NO is produced by iNOS in activated macrophages with L-arginine as a substrate, and reacts with superoxide anion to form peroxynitrite, a cytotoxic oxidant species [12]. Nitrite concentration in the supernatant, an index of NO production, of LPS-activated murine macrophages Raw264.7 was decreased when α-viniferin and LPS were treated simultaneously (Fig. [4] A). However, no inhibitory effect on NO production was shown by treatment of α-viniferin, when iNOS synthesis could be completed already, at 12 h after LPS stimulation. This result would indicate that α-viniferin did not inhibit iNOS activity. RT-PCR analysis was performed to examine whether the inhibitory effect of α-viniferin on NO production was influenced by down-regulation of iNOS synthesis (Fig. [4] B). The iNOS transcript in murine macrophages Raw264.7 was induced by LPS stimulation, which was decreased by pretreatment of α-viniferin with an IC₅₀ value of 4.7 μM. Therefore, α-viniferin seems to down-regulate iNOS synthesis in LPS-activated murine macrophages Raw264.7.

α-Viniferin inhibited the synthesis of both COX-2 and iNOS transcripts, where the compound showed a stronger down-regulation of iNOS transcript than COX-2 transcript (Figs. [3] B and [4] B). The promoter region of murine iNOS gene contains several consensus sequences for binding of transcription factors such as NF-κB, STAT, C/EBP, CREB and OCT [13]. α-Viniferin did not inhibit NF-κB transactivation in LPS-stimulated murine macrophages Raw264.7, which was identified by a reporter gene assay with triplicated NF-κB consensus sequence connected to alkaline phosphatase gene as a reporter plasmid (data not shown). The signaling pathways affected by α-viniferin in the LPS-induced iNOS synthesis will be elucidated in a future study.

α-Viniferin is an oligomeric stilbene with resveratrol as the building block. Resveratrol was first detected in Polygonum cuspidatum Sieb. et Zucc., and is presumed to be beneficial for human health [14]. Anti-inflammatory activity of resveratrol was demonstrated by reduction of carrageenin-induced paw edema [15]. Resveratrol was reported to inhibit COX-1 activity preferentially and is known to suppress COX-2 synthesis [16]. Resveratrol was reported to inhibit NO production through interference of NF-κB signaling [17]. Thereby, α-viniferin seems to display modes of anti-inflammatory action different from resveratrol.

In this study, we have demonstrated the anti-inflammatory action of α-viniferin, an oligomeric stilbene compound from Carex humilis Leysser as a source. α-Viniferin reduced carrageenin-induced paw edema in mice by intravenous injection with 3 mg/kg, a tested minimal dose, which could correspond to a whole-body concentration of about 10 μM. The compound showed IC₅₀ values of 4.9 μM on COX-2 activity and of 2.7 μM on NO production in LPS-stimulated murine macrophages Raw264.7. Therefore, the inhibitory effects of α-viniferin on the release of prostanoids and NO may play an important role to show anti-inflammatory action in vivo even though the compound will not be distributed evenly throughout the body. These findings expand the importance of α-viniferin as a beneficial agent, and will help to clarify protective mechanisms of the compound against inflammatory conditions.

References

• 1 Kitanaka S, Ikezawa T, Yasukawa K, Yamanouchi S, Takido M, Sung H K. et al . Alpha-viniferin, an anti-inflammatory compound from Caragana chamlagu root.  Chem Pharm Bull. 1990;  38 432-5
  CrossrefPubMedSearch in Google Scholar
• 2 Kulanthaivel P, Janzen W P, Ballas L M, Jiang J B, Hue C Q, Darges J W. et al . Naturally occurring protein kinase C inhibitors; isolation of oligomeric stilbenes from Caragana sinica .  Planta Med. 1995;  61 41-4
  PubMedSearch in Google Scholar
• 3 Keckeis K, Sarker S D, Dinan L. Resveratrol-type oligostilbenes from Iris clarkei antagonize 20-hydroxyecdysone action in the Drosophila melanogaster B (II) cell line.  Cell Mol Life Sci. 2000;  57 333-6
  PubMedSearch in Google Scholar
• 4 Sung S H, Kang S Y, Lee K Y, Park M J, Kim J H, Park J H. et al . Alpha-viniferin, a stilbene trimer from Caragana chamlague, inhibits acetylcholinesterase.  Biol Pharm Bull. 2002;  25 125-7
  CrossrefPubMedSearch in Google Scholar
• 5 Lee S -H, Shin N -H, Kang S -H, Park J S, Chung S R, Min K R. et al . α-Viniferin: a prostaglandin H₂ synthase inhibitor from root of C. humilis .  Planta Med. 1998;  64 204-7
  PubMedSearch in Google Scholar
• 6 Min K R, Hwang B Y, Lim H -S, Kang B -S, Oh G -J, Lee J. et al . (-)-Epiafzelechin: cyclooxygenase-1 inhibitor and anti-inflammatory agent from aerial parts of Celastrus orbiculatus .  Planta Med. 1999;  65 460-2
  PubMedSearch in Google Scholar
• 7 Eberhart C E, Dubois R N. Eicosanoids and the gastrointestinal tract.  Gastroenterology. 1995;  109 285-301
  PubMedSearch in Google Scholar
• 8 Feng L, Xia Y, Garcia G E, Hwang D, Wilson C B. Involvement of reactive oxygen intermediates in cyclooxygenase-2 expression induced by interleukin-1, tumor necrosis factor-alpha, and lipopolysaccharide.  J Clin Invest. 1995;  95 1669-75
  PubMedSearch in Google Scholar
• 9 Brzozowski T, Konturek P C, Konturek S J, Sliwowski Z, Pajdo R, Drozdowicz D. et al . Classic NSAID and selective cyclooxygenase (COX)-1 and COX-2 inhibitors in healing of chronic gastric ulcers.  Microsc Res Tech. 2001;  53 343-53
  CrossrefPubMedSearch in Google Scholar
• 10 Maeda H, Akaike T. Nitric oxide and oxygen radicals in infection, inflammation, and cancer.  Biochemistry. 1998;  63 854-65
  PubMedSearch in Google Scholar
• 11 Salvemini D, Wang Z Q, Wyatt P S, Bourdon D M, Marino M H, Manning P T. et al . Nitric oxide: a key mediator in the early and late phase of carrageenan-induced rat paw inflammation.  Br J Pharmacol. 1996;  118 829-38
  CrossrefPubMedSearch in Google Scholar
• 12 Huie R E, Padmaja S. The reaction of NO with superoxide.  Free Radic Res Commun. 1993;  18 195-9
  CrossrefPubMedSearch in Google Scholar
• 13 Xie Q W, Whisnant R, Nathan C. Promoter of the mouse gene encoding nitric oxide synthase confers inducibility by interferon gamma and bacterial lipopolysaccharide.  J Exp Med. 1993;  177 1779-84
  CrossrefPubMedSearch in Google Scholar
• 14 Bhat K PL, Kosmeder J W, Pezzutto J M. Biological effects of resveratrol.  Antioxid Redox Signal.. 2001;  3 1041-64
  CrossrefPubMedSearch in Google Scholar
• 15 Gentilli M, Mazoit J X, Bouaziz H, Fletcher D, Casper R F, Benhamou D. et al . Resveratrol decreases hyperalgesia induced by carrageenan in the rat hind paw.  Life Sci. 2001;  68 1317-21
  CrossrefPubMedSearch in Google Scholar
• 16 Subbaramaiah K, Chung W J, Michaluart P, Telang N, Tanabe T, Inoue H. et al . Resveratrol inhibits cyclooxygenase-2 transcription and activity in phorbol ester-treated human mammary epithelial cells.  J Biol Chem. 1998;  273 21 875-82
  PubMedSearch in Google Scholar
• 17 Tsai S H, Lin-Shiau S Y, Lin J K. Suppression of nitric oxide synthase and down-regulation of the activation of NFkappaB in macrophages by resveratrol.  Br J Pharmacol. 1999;  126 673-80
  CrossrefPubMedSearch in Google Scholar

Prof. Youngsoo Kim, Ph. D.

College of Pharmacy

Chungbuk National University

Cheongju 361-763

Korea

Fax: +82-43-268-2732

Email: youngsoo@cbucc.chungbuk.ac.kr

References

• 1 Kitanaka S, Ikezawa T, Yasukawa K, Yamanouchi S, Takido M, Sung H K. et al . Alpha-viniferin, an anti-inflammatory compound from Caragana chamlagu root.  Chem Pharm Bull. 1990;  38 432-5
  CrossrefPubMedSearch in Google Scholar
• 2 Kulanthaivel P, Janzen W P, Ballas L M, Jiang J B, Hue C Q, Darges J W. et al . Naturally occurring protein kinase C inhibitors; isolation of oligomeric stilbenes from Caragana sinica .  Planta Med. 1995;  61 41-4
  PubMedSearch in Google Scholar
• 3 Keckeis K, Sarker S D, Dinan L. Resveratrol-type oligostilbenes from Iris clarkei antagonize 20-hydroxyecdysone action in the Drosophila melanogaster B (II) cell line.  Cell Mol Life Sci. 2000;  57 333-6
  PubMedSearch in Google Scholar
• 4 Sung S H, Kang S Y, Lee K Y, Park M J, Kim J H, Park J H. et al . Alpha-viniferin, a stilbene trimer from Caragana chamlague, inhibits acetylcholinesterase.  Biol Pharm Bull. 2002;  25 125-7
  CrossrefPubMedSearch in Google Scholar
• 5 Lee S -H, Shin N -H, Kang S -H, Park J S, Chung S R, Min K R. et al . α-Viniferin: a prostaglandin H₂ synthase inhibitor from root of C. humilis .  Planta Med. 1998;  64 204-7
  PubMedSearch in Google Scholar
• 6 Min K R, Hwang B Y, Lim H -S, Kang B -S, Oh G -J, Lee J. et al . (-)-Epiafzelechin: cyclooxygenase-1 inhibitor and anti-inflammatory agent from aerial parts of Celastrus orbiculatus .  Planta Med. 1999;  65 460-2
  PubMedSearch in Google Scholar
• 7 Eberhart C E, Dubois R N. Eicosanoids and the gastrointestinal tract.  Gastroenterology. 1995;  109 285-301
  PubMedSearch in Google Scholar
• 8 Feng L, Xia Y, Garcia G E, Hwang D, Wilson C B. Involvement of reactive oxygen intermediates in cyclooxygenase-2 expression induced by interleukin-1, tumor necrosis factor-alpha, and lipopolysaccharide.  J Clin Invest. 1995;  95 1669-75
  PubMedSearch in Google Scholar
• 9 Brzozowski T, Konturek P C, Konturek S J, Sliwowski Z, Pajdo R, Drozdowicz D. et al . Classic NSAID and selective cyclooxygenase (COX)-1 and COX-2 inhibitors in healing of chronic gastric ulcers.  Microsc Res Tech. 2001;  53 343-53
  CrossrefPubMedSearch in Google Scholar
• 10 Maeda H, Akaike T. Nitric oxide and oxygen radicals in infection, inflammation, and cancer.  Biochemistry. 1998;  63 854-65
  PubMedSearch in Google Scholar
• 11 Salvemini D, Wang Z Q, Wyatt P S, Bourdon D M, Marino M H, Manning P T. et al . Nitric oxide: a key mediator in the early and late phase of carrageenan-induced rat paw inflammation.  Br J Pharmacol. 1996;  118 829-38
  CrossrefPubMedSearch in Google Scholar
• 12 Huie R E, Padmaja S. The reaction of NO with superoxide.  Free Radic Res Commun. 1993;  18 195-9
  CrossrefPubMedSearch in Google Scholar
• 13 Xie Q W, Whisnant R, Nathan C. Promoter of the mouse gene encoding nitric oxide synthase confers inducibility by interferon gamma and bacterial lipopolysaccharide.  J Exp Med. 1993;  177 1779-84
  CrossrefPubMedSearch in Google Scholar
• 14 Bhat K PL, Kosmeder J W, Pezzutto J M. Biological effects of resveratrol.  Antioxid Redox Signal.. 2001;  3 1041-64
  CrossrefPubMedSearch in Google Scholar
• 15 Gentilli M, Mazoit J X, Bouaziz H, Fletcher D, Casper R F, Benhamou D. et al . Resveratrol decreases hyperalgesia induced by carrageenan in the rat hind paw.  Life Sci. 2001;  68 1317-21
  CrossrefPubMedSearch in Google Scholar
• 16 Subbaramaiah K, Chung W J, Michaluart P, Telang N, Tanabe T, Inoue H. et al . Resveratrol inhibits cyclooxygenase-2 transcription and activity in phorbol ester-treated human mammary epithelial cells.  J Biol Chem. 1998;  273 21 875-82
  PubMedSearch in Google Scholar
• 17 Tsai S H, Lin-Shiau S Y, Lin J K. Suppression of nitric oxide synthase and down-regulation of the activation of NFkappaB in macrophages by resveratrol.  Br J Pharmacol. 1999;  126 673-80
  CrossrefPubMedSearch in Google Scholar

Prof. Youngsoo Kim, Ph. D.

College of Pharmacy

Chungbuk National University

Cheongju 361-763

Korea

Fax: +82-43-268-2732

Email: youngsoo@cbucc.chungbuk.ac.kr