In Vitro Anti-Inflammatory Activity of Panduratin A Isolated from Kaempferia pandurata in RAW264.7 Cells

• Abstract
• Introduction
• Materials and Methods
• Plant material
• Extraction and isolation
• Cell culture
• Treatment of macrophages with LPS
• MTT assay for cell viability
• Determination of PGE₂ production
• Determination of nitrite production
• SDS-polyacrylamide gel electrophoresis and Western blot analysis
• Reverse transcriptase-polymerase chain reaction (RT-PCR)
• NF-κB-driven reporter gene assay
• Statistical analysis
• Results
• Cell viability
• Inhibition of LPS-induced PGE₂ production by panduratin A
• Inhibition of LPS-induced COX-2 protein and mRNA expression by panduratin A
• Inhibition of LPS-induced nitrite production by panduratin A
• Inhibition of LPS-induced iNOS protein and mRNA expression by panduratin A
• Inhibition of LPS-induced phosphorylation and degradation of IκBα
• Blocking of NF-κB-regulated gene expression by panduratin A
• Discussion
• References

Abstract

An active compound identified as panduratin A was isolated from a methanol extract of Kaempferia pandurata (Zingiberaceae). We examined the effect of panduratin A on nitric oxide (NO) and prostaglandin E₂ (PGE₂) production induced by lipopolysaccharide (LPS) in RAW264.7 cells. Modulations of iNOS and COX-2 enzyme expression were evaluated by Western blotting. Panduratin A strongly inhibited both NO (IC₅₀ : 0.175 μM) and PGE₂ (IC₅₀ : 0.0195 μM) production and suppressed both iNOS and COX-2 enzyme expression without any appreciable cytotoxic effect on RAW264.7 cells in a dose-dependent manner. Panduratin A also suppressed the phosphorylation of inhibitor κBα (IκBα) and degradation of IκBα associated with nuclear factor κB (NF-κB) activation. Furthermore, panduratin A inhibited LPS-induced NF-κB transcriptional activity in a dose-dependent manner. These results suggest that panduratin A could exert its inhibitory effects on the production of NO and PGE₂ through the suppression of NF-κB activation, indicating its potential for use as an anti-inflammatory agent.

Introduction

Prostaglandins (PGs) and nitric oxide (NO) are involved in various pathophysiological processes including inflammation and carcinogenesis. Prostaglandins (PGs) are lipid mediators, which are involved in many processes, including inflammation, and are produced by many cell types [1]. Especially, prostaglandin E₂ (PGE₂) affects cell proliferation, tumor growth and suppresses the immune response to malignant cells [1]. NO plays an important role in the regulation of many physiological functions, such as host defense, neurotoxicity, and vasodilation [2]. However, the excess production of NO has been implicated for immunological and inflammatory diseases including septic shock, rheumatoid arthritis, graft rejection, and diabetes [3]. The inducible isoform of cyclooxygenase (COX-2) and nitric oxide synthase (iNOS) are mainly responsible for the production of large amounts of these mediators (PGE₂ and NO) [4]. Inhibition of PGE₂ and NO production is apparently an important therapeutic consideration in the development of anti-inflammatory agents.

Macrophages play an important role in a host defense mechanism against bacterial and viral infection. When macrophages are activated by various stimuli, such as lipopolysaccharide (LPS) and interferon-γ (IFN-γ), macrophages inhibit the growth of a wide variety of tumor cells and microorganisms via release of factors such as NO, cytokines, and eicosanoid mediators of the immune response [5]. PGE₂ production by COX-2 and NO production by iNOS are mainly regulated at the transcriptional level [4]. NF-κB is a transcription factor that regulates the expression of multiple immune and inflammatory genes [6]. LPS activates transcription factor NF-κB in macrophages, which leads to induction of expression of iNOS and COX-2. The cis-acting NF-κB element is present in the 5′-flanking regions of both the COX-2 and iNOS genes [4].

Kaempferia pandurata Roxb. (Zingiberaceae) is a herb cultivated in some tropical countries, including Indonesia and Thailand. Its rhizome has been used as a condiment and occasionally as a folk medicine for treatment of various ailments, including colic disorder, fungal infections, dry cough, rheumatism, and muscular pains [7], [8]. Panduratin A (Fig. [1]) is a chalcone derivative isolated from K. pandurata. Recently, Tuchinda et al. [8] reported that panduratin A showed significant topical anti-inflammatory activity in an experimental model of TPA-induced ear edema in rats. However, the mechanism of the anti-inflammatory action of panduratin A in macrophage cells is unknown.

To investigate the cellular mechanism involved in the anti-inflammatory activity of panduratin A, we used the murine macrophage cell line RAW264.7, which can be stimulated with bacterial LPS to mimic a state of infection and inflammation. In this study, isolated panduratin A from methanol extracts of K. pandurata was examined for its effects on LPS-induced NO and PGE₂ production and LPS-induced expression of iNOS and COX-2. In addition, we conducted more detailed investigations to elucidate the in vitro mechanism of the observed inhibition of iNOS and COX-2.

Materials and Methods

Plant material

Kaempferia pandurata Roxb. (syn. Boesenbergia pandurata, Zingiberaceae) was collected in January 2000 from the Biofarmaka Research Center of Bogor Agricultural University (Indonesia), and identified by Dr. Latifah Kadarusman, Department of Pharmacy. The plant material was shade-dried and ground to powder. A voucher specimen (No. H082) is deposited at 4 °C in the Bioproducts Research Center, Yonsei University, Seoul, Korea.

Extraction and isolation

Dried rhizomes (1 kg) of K. pandurata were ground and extracted twice with 75 % methanol (4 L, v/v) for 24 h at room temperature. The extract was concentrated, frozen, and lyophilized (22.2 g). The methanol extract was further fractionated successively with ethyl acetate, n-butanol, and water. Each fraction was evaporated and dried under reduced pressure (ethyl acetate fraction 11.2 g, butanol fraction 2 g, water fraction 8.8 g). The ethyl acetate fraction was further separated using silica gel column chromatography (5 × 45 cm, 650 g of silica gel; 70 - 230 mesh, Merck, USA). A sample was mixed with silica gel and loaded on a silica gel packed column using slurry packing. The sample was eluted with a solution of n-hexane/chloroform/ethyl acetate (15 : 5:1.5, v/v/v): 1.9 mL/min, divided into four fractions (fraction I - fraction IV) on a silica TLC plate (Silica gel 60 F₂₅₄; Merck, USA), then each fraction was assayed for anti-inflammatory activity. The active fraction (5.2 g, 5 L - 5.8 L; fraction II) was then further separated. Fraction II yielded fraction II-B (2.5 g) after passing through a silica gel column (2.5 × 45 cm, 400 g of silica gel) when eluted with n-hexane/ethyl acetate/methanol (18 : 2:1, v/v/v; 1.5 L - 2 L). Fraction II-B yielded fraction II-B-2 (0.42 g) when eluted with n-hexane/chloroform (3 : 10, v/v ; 0.8 L - 1.2 L). Panduratin A (R_f 0.38, n-hexane/chloroform = 3 : 10, v/v ) was finally purified from fraction II-B-2 by recycling preparative HPLC using a JAIGEL W-252 column [(i. d. 20 × 500 mm, Japan Analytical Industry Co., Japan); detection by UV absorption at 365 nm; mobile phase used was 100 % MeOH; flow rate 3 mL/min] and was eluted at 35 min as a single peak. ¹H-NMR and ¹³C-NMR spectra were measured on an Avance-600 (Bruker Co., Germany) spectrometer at 600 MHz for proton and 150 MHz for carbon spectra. Fast atom bombardment (FAB) mass spectra were obtained using a JMS-700 Mstation (JEOL Ltd., Japan). Panduratin A (Fig. [1] ) was identified by comparison of the spectral (¹H-NMR, ¹³C-NMR, and FAB-MS) properties with published values [13], [14]. Copies of the original spectra are obtainable from the author of correspondence. Optical rotation was measured with a Perkin-Elmer 241 polarimeter as [α]_D ²¹: + 0.0066° (c 0.1, CHCl₃).

Cell culture

RAW264.7 murine marcrophages (KCLB No. 40 071) were purchased from the Korea Cell Line Bank (Korea). The macrophages were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco/BRL, USA) containing 10 % fetal bovine serum, 100 units/mL of penicillin, and 100 μg/mL of streptomycin (Gibco/BRL, USA). Macrophages were then incubated in 24-well tissue plates at a density of 1 × 10⁶ cells/mL for 24 h at 37 °C. Cells were maintained at 37 °C in a humidified atmosphere containing 5 % CO₂.

Treatment of macrophages with LPS

Macrophages were incubated with 10 μg/mL of LPS to stimulate COX-2 and iNOS gene expression. Each sample was dissolved in dimethyl sulfoxide (DMSO) and added to the incubation medium 1 h prior to addition of LPS. The final concentration of DMSO was adjusted to 0.1 % (v/v).

MTT assay for cell viability

Cell viability was determined using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) reagent [9]. After culturing on 96 well plates for 24 h, the cells were washed twice and incubated with 110 μL of 0.5 mg/mL MTT for 2 h at 37 °C. The medium was discarded and 100 μL DMSO were then added. After 30 min incubation, the absorbance at 570 nm was measured using a microplate reader (Molecular Devices Co., USA).

Determination of PGE₂ production

Macrophages were pretreated with aspirin (250 μM) for 2 h to inactivate the COX-1 enzyme prior to a COX-2 activity assay. The cells were washed three times with serum-free DMEM, then LPS (10 μg/mL) was added to induce COX-2 expression. The supernatant was used 20 hours later for measurement of PGE₂ concentration. The concentration of PGE₂ in culture supernatants was determined using ELISA kits (R&D System, USA) according to the manufacturer’s instructions.

Determination of nitrite production

The nitrite concentration in the medium was measured as an indicator of NO production according to the Griess method as described previously [10]. An amount of 100 μL of each culture supernatant was mixed with the same volume of Griess reagent (1 % sulfanilamide in 5 % phosphoric acid, and 0.1 % naphthylethylenediamine dihydrochloride in water) and the absorbance of the mixture at 550 nm was measured using a microplate reader (Molecular Devices Co., USA). Nitrite concentrations were calculated from a standard curve of sodium nitrite prepared in the culture medium.

SDS-polyacrylamide gel electrophoresis and Western blot analysis

RAW264.7 cells (1 × 10⁶ cells/mL), grown in 60 mm dishes to confluence, were incubated with or without LPS in the absence or presence of tested samples for 24 h, respectively. Cells were washed with ice-cold phosphate-buffered saline and stored at -70 °C until further analysis. Macrophages were collected and an equal amount of protein (30 μg/lane) was loaded and electrophoresed on 8 % SDS-polyacrylamide gel (for iNOS and COX-2) and 10 % SDS-polyacrylamide gel (for phospho-IκBα and IκBα). Gels were then transferred to polyvinylidene difluoride (PVDF; Millipore Co., USA) membranes. Membranes were blocked and incubated for 1 h at room temperature with 1 : 1000 dilution of rabbit IκBα polyclonal antibody, mouse phospho-IκBα monoclonal antibody, rabbit iNOS polyclonal antibody, and goat COX-2 polyclonal antibody (Santa Cruz Biotechnology, USA). α-Tubulin (Santa Cruz Biotechnology, USA) was used as an internal control. The immunoreactive protein was detected using a chemiluminescent system (ECL kit; Amersham Pharmarcia Co., USA). After exposure to X-ray film, the intensity of the band was calculated from the optical density using an image analyzer (Vilber Co., France).

Reverse transcriptase-polymerase chain reaction (RT-PCR)

Total RNA from RAW264.7 cells (treated as indicated) was isolated using RNAquous^TM (Ambion Inc, USA). Complementary DNA (cDNA) was made by reverse transcription (RT) of 1 μg each total RNA by using one step RNA PCR kit (Takara Co., Japan). PCR primers for mouse COX-2 and iNOS were synthesized according to the following oligonucleotide sequences (Corebiosysytem Co., Korea). COX-2: forward primer 5′-GGAGAGACTATCAAGATAGTGATC-3′ (1094 - 1117), reverse primer 5′-ATGGTCAGTAGACTTTTACAGCTC-3′ (1931 - 1954). iNOS: forward primer 5′-CCCTTCCGAAGTTTCTGGCAGCAGC-3′ (2944 - 2968), reverse primer 5′-GGCTGTCAGAGAGCCTCGTGGCTTTGG-3′ (3416 - 3440). cDNAs of iNOS and COX-2, respectively, were amplified with the one step RNA PCR kit (Takara Co., Japan) for 40 cycles of 2 min at 94 °C, 1 min at 62 °C, and 2 min at 72 °C followed by an extension at 72 °C for 2 min (1 cycle). Equal amounts of PCR product were electrophoresed on 2 % agarose gel and visualized by ethidium bromide staining. The intensity of the band was calculated from the optical density using an image analyzer (Vilber Co., France).

NF-κB-driven reporter gene assay

An NF-κB reporter construct, consisting of the firefly luciferase gene under the control of the consensus NF-κB site, was used to quantify NF-κB transcriptional activity. A Renilla luciferase reporter was used as an internal control to normalize the reporter gene activity. RAW264.7 cells were seed in 60 mm dishes at a density of 1 × 10⁵ cells/mL. The RAW264.7 cells were transiently transfected with the reporter construct (3 μg) using Fugene 6 reagent (Roche, USA) according to the manufacturer’s instructions. After 3 h post-transfection, the cultures were treated with various concentrations of compounds tested and stimulated with 10 μg/mL of LPS. After 24 h incubation, each 60 mm culture dish was washed twice with cold phosphated-buffed saline. And then cells were collected and subjected to dual luciferase assays. Dual-luciferase assays were performed with the Dual-Luciferase Reporter Assays System (Promega, USA), according to the manufacturer’s instructions. Luciferase activities were determined with a luminometer (FLUOstar; BMG Labtechnologies, Germany).

Statistical analysis

Each experiment was performed at least in triplicate. Results are expressed as the means value ± standard deviation (SD). Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Dunnett’s t-test for multiple comparisons. Statistical significance is expressed as *p < 0.05.

Results

Cell viability

The effects of panduratin A on RAW264.7 cell viability were determined by a MTT assay. Panduratin A did not exhibit cytotoxicity at concentrations less than 2.5 μM compared to an LPS-treated control (data not shown).

Inhibition of LPS-induced PGE₂ production by panduratin A

The amounts of PGE₂ in the supernatants of the macrophages were determined in order to study the inhibitory effect on COX-2 activity (Table [1]). LPS (10 μg/mL) increased the PGE₂ concentration in the culture medium by 13-fold compared to the control. Cells that were incubated with various concentrations of panduratin A (0.01, 0.02, 0.025, 0.12, 0.25, and 2.5 μM) together with LPS (10 μg/mL) for 24 h exhibited 33.5, 50.9, 56.3, 68.8, 75.0, and 81.7 % inhibition, respectively. Panduratin A inhibited LPS-induced PGE₂ production in RAW264.7 cells with an IC₅₀ value of 0.0195 μM. A concentration of 2.5 μM indomethacin (a non-steroidal anti-inflammatory drug; Sigma, USA) was used as a positive control, which resulted in a 96.4 % inhibition of PGE₂ production in the macrophages.

Inhibition of LPS-induced COX-2 protein and mRNA expression by panduratin A

The levels of COX-2 protein and COX-2 mRNA were analyzed by Western blotting (Fig. [2]) and RT-PCR (Fig. [3]), respectively, to determine the inhibitory mechanism of PGE₂ production by panduratin A in LPS-stimulated RAW264.7 cells. Expression of the COX-2 protein was barely detectable in unstimulated cells, but markedly increased after LPS (10 μg/ml) treatment (Fig. [2] A). Treatment with panduratin A (0.025, 0.12, 0.25, and 2.5 μM) for LPS-stimulated macrophages caused dose-dependent inhibition of COX-2 protein expression, which was consistent with PGE₂ production shown in Table [1]. Evalulation of signal intensities by densitometric analysis showed decreased COX-2 protein levels of 93.0, 89.1, 79.8, and 84.3 %, respectively (Fig. [2] B). Expression of COX-2 mRNA was hardly detectable in unstimulated cells, but LPS (10 μg/mL) induced COX-2 mRNA expression (Fig. [3] A). COX-2 mRNA induction by LPS was markedly attenuated by panduratin A. The evaluation of signal intensities by densitometric analysis showed decreased COX-2 mRNA levels of 97.0, 80.0, 43.9, and 47.3 %, respectively (Fig. [3] B).

Inhibition of LPS-induced nitrite production by panduratin A

The effect of panduratin A on LPS-induced NO production in macrophages was assessed. The cell culture medium was harvested and the concentration of accumulated nitrite, the oxidative product of NO, was determined by the Griess method (Table [2]). LPS (10 μg/mL) increased the nitrite concentration in the culture medium by four-fold compared to the control. Macrophages incubated with different concentrations of panduratin A (0.025, 0.012, 0.25 and 2.5 μM) together with LPS (10 μg/mL) for 24 h resulted in 29.2, 50.6, 56.0, and 62.5 % inhibition, respectively. Panduratin A inhibited LPS-induced nitrite production in macrophages in a concentration-dependent manner with an IC₅₀ value of 0.175 μM. A concentration of 2.5 μM L-NAME (NOS inhibitor, Nω-nitro-L-arginine methyl ester; Sigma, USA) was used as a positive control, resulting in 64.1 % inhibition of nitrite production in RAW264.7 cells.

Inhibition of LPS-induced iNOS protein and mRNA expression by panduratin A

The levels of iNOS protein and iNOS mRNA were analyzed by Western blotting (Fig. [4]) and RT-PCR (Fig. [5]), respectively. A concentration of 2.5 μM was used for iNOS gene expression analysis because preliminary data showed that panduratin A markedly inhibited nitrite production at a concentration of 2.5 μM. Cells expressed only a slightly detectable concentration of iNOS protein when incubated in the medium alone for 24 h, but LPS treatment elevated the iNOS protein expression. The elevated expression of iNOS protein was inhibited by 2.5 μM panduratin A (Fig. [4]). Also, iNOS mRNA induction by LPS was markedly attenuated by panduratin A (Fig. [5]).

Inhibition of LPS-induced phosphorylation and degradation of IκBα

We assessed whether COX-2 and iNOS gene expression due to panduratin A was processed by regulation of a transcriptional factor. The inhibitory effect of panduratin A on phosphorylation and degradation of IκBα in LPS-activated macrophages was, therefore, examined (Fig. [6], Fig. [7]). The cytoplasmic levels of phospho-IκBα and IκBα were determined by Western blotting. Phospho-IκBα protein concentration in RAW264.7 cells increased after LPS (10 μg/mL) treatment, but decreased in a dose-dependent manner after pandurtin A treatment. Treatment of macrophages with 2.5 μM panduratin A completely blocked LPS-induced IκBα phosphorylation (Fig. [6]). Concentrations IκBα proteins also decreased to almost undetectable levels after LPS (10 μg/mL) treatment and recovered after panduratin A treatment. Treatment of macrophages with 0.12 μM panduratin A completely blocked LPS-induced IκBα degradation (Fig. [7]).

Blocking of NF-κB-regulated gene expression by panduratin A

We performed an NF-κB-driven luciferase reporter gene assay to show that panduratin A is able to block NF-κB-regulated gene expression (iNOS and COX-2) as shown in Fig. [8]. Incubation of RAW264.7 cells with 10 μg/mL LPS for 24 h increased NF-κB transcription activity strongly. Induction of NF-κB transcription activity by LPS was markedly inhibited by panduratin A in a dose-dependent manner.

Discussion

Panduratin A (Fig. [1]) is a cyclohexenyl chalcone derivative isolated from K. pandurata. Chalcones are natural products which have been reported to possess a variety of biological properties, including anti-inflammatory, analgesic and antioxidant activities [11]. Recent studies demonstrated that several prenylated chalcones and hydroxychalcones were potent free radical scavengers, and also inhibited the expression of adhesion molecules and iNOS by blocking the activation of NF-κB [12], [13]. In a previous study [8], panduratin A from K. pandurata showed significant anti-inflammatory activity in a skin inflammatory animal model, however, the mechanism has not yet been examined in vitro. In order to delineate the mechanism involved in the anti-inflammatory activity of panduratin A, we studied the effects of panduratin A on NF-κB mediated iNOS and COX-2 expression and on NO and PGE₂ production by LPS. Our results indicate that panduratin A inhibits LPS-induced nitrite (IC₅₀ : 0.175 μM) and PGE₂ production (IC₅₀ : 0.0195 μM) in macrophages without appreciable cytotoxic effects. We demonstrated that panduratin A potently inhibits COX-2 and iNOS expression and that down-regulation of COX-2 and iNOS expression is related to the reduced production of PGE₂ and NO production in RAW264.7 cells. Panduratin A inhibits PGE₂ production more than NO production.

The eukaryotic transcription factor NF-κB plays a central role in inflammation since its activation is a prerequisite for expression of numerous target genes, including iNOS and COX-2 [4], [6]. Yin et al. [14] reported that the anti-inflammatory properties of aspirin have been linked to suppression of NF-κB activation through stabilization of IκB. The phosphorylation and degradation of IκB proteins are critical in NF-κB activation. We showed that phosphorylation and degradation of IκBα, required for NF-κB activation, are inhibited by panduratin A. The levels of phosphorylation (2.5 μM) and degradation (0.12 μM) of IκBα by LPS were strongly decreased in cells treated with panduratin A. Also, the luciferase assay revealed that panduratin A suppresses LPS-mediated gene expression mainly via inhibition of NF-κB activity. These results indicate that inhibition of COX-2 and iNOS gene expression by panduratin A may be, at least in part, related to the NF-κB inhibition pathway. Recently, the anti-inflammatory properties of aspirin have been linked to inhibition of IKK, thereby preventing activation of NF-κB [15]. A number of upstream kinases including NF-κB inducing kinase (NIK) and MAPK/ERK kinase (MEKK) are involved in regulation of IKK activation. Therefore, panduratin A probably inhibits IKK activity either directly or by inhibiting the activation of an upstream kinase that ultimately causes activation of IKK. However, the exact molecular mechanism of NF-κB down-regulation by panduratin A needs to be investigated.

These results support the pharmacological basis of K. pandurata used as a herbal medicine for treatment of inflammation. Our study also indicates the possibility for using panduratin A in cancer chemoprevention or anticancer therapy since elevated COX-2 levels have been found in certain cancers.

References

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Prof. Jae-Kwan Hwang

Department of Biotechnology

Yonsei University

Seoul 120-749

Republic of Korea

Phone: +82-2-2123-5881

Fax: +82-2-362-7265

Email: jkhwang@yonsei.ac.kr

References

• 1 Marnett L J. Aspirin and the potential role of prostaglandins in colon cancer.  Cancer Res. 1992;  52 5575-89
  PubMedSearch in Google Scholar
• 2 Moncada S, Palmer R M, Higgs E A. Nitric oxide: physiology, pathophysiology, and pharmacology.  Pharmacol Rev. 1991;  43 109-42
  PubMedSearch in Google Scholar
• 3 Anggard E. Nitric oxide: mediator, murderer, and medicine.  Lancet. 1994;  343 1199-206
  CrossrefPubMedSearch in Google Scholar
• 4 Surh Y J, Chun K S, Cha H H, Han S S, Keum Y S, Park K K. Molecular mechanisms underlying chemopreventive activities of anti-inflammatory phytochemicals: down-regulation of COX-2 and iNOS through suppression of NF-κB activation.  Mutat Res. 2001;  480 - 481 243-68
  PubMedSearch in Google Scholar
• 5 Hibbs J B, Taintor R R, Vavrin Z. Macrophage cytotoxicity: role of l-arginine deiminase and imino nitrogen oxidation to nitrite.  Science. 1987;  235 473-6
  CrossrefPubMedSearch in Google Scholar
• 6 Baeuerle P A, Baichwal V R. NF-κB as a frequent target for immunosuppressive and anti-inflammatory molecules.  Adv Immunol. 1997;  65 111-37
  PubMedSearch in Google Scholar
• 7 Trakoontivakorn G ., Nakahara K, Shinmoto H, Takenaka M, Onishi-kameyama M, Ono H, Yoshida M, Nagata T, Tsushida T. Structural analysis of a novel antimutagenic compound, 4-hydroxypanduratin A, and antimutagenic activity of flavonoids in a Thai spice, fingerroot (Boesenbergia pandurata Schult.) against mutagenic heterocyclic amines.  J Agric Food Chem. 2001;  49 3046-50
  PubMedSearch in Google Scholar
• 8 Tuchinda P, Reutrakul V, Claeson P, Ponprayoon U, Sematong T, Santosuk T, Taylor W C. Anti-inflammatory cyclohexenyl chalcone derivatives in Boesenberia pandurata .  Phytochemistry. 2002;  59 169-73
  CrossrefPubMedSearch in Google Scholar
• 9 Tubaro A, Florio C, Luxich E, Vertua R, Loggia R D, Yasumoto T. Suitability of the MTT-based cytotoxicity assay to detect okadaic acid contamination of mussels.  Toxicon.. 1996;  34 965-74
  CrossrefPubMedSearch in Google Scholar
• 10 Schmidt H HHW, Kelm M. Determination of nitrite and nitrate by the Griess reaction. In: Methods in Nitric Oxide Research. London; John Wiley & Sons Ltd 1996: 491-7
  Search in Google Scholar
• 11 Hsieh H K, Tsao L T, Wang J P, Lin C N. Synthesis and anti-inflammatory effect of chalcones.  J Pharm Pharmacol. 2000;  52 163-71
  CrossrefPubMedSearch in Google Scholar
• 12 Madan B, Batra S, Ghosh B. 2′-hydroxychalcone inhibits nuclear factor-κB and blocks tumor necrosis factor-α and lipopolysaccharide-induced adhesion of neutrophils to human umbilical vein endothelial cells.  Mol Pharmacol. 2000;  58 536-34
  PubMedSearch in Google Scholar
• 13 Cheng Z J, Lin C, Hwang T, Teng C. Broussochalcone A, a potent antioxidant and effective suppressor of inducible nitric oxide synthase in lipopolysaccharide-activated macrophages.  Biochem Pharmacol. 2001;  61 939-46
  CrossrefPubMedSearch in Google Scholar
• 14 Yin M J, Yamamoto Y, Graynor R B. The anti-inflammatory agents aspirin and salicylate inhibit the activity of IκB-β.  Nature. 1998;  396 77-80
  CrossrefPubMedSearch in Google Scholar
• 15 Chan M M, Fong D. Modulation of the nitric oxide pathway by natural products. In: Laskin J, Laskin D, editors Cellular and Molecular Biology of Nitric Oxide. New York; Marcel Dekker 1999: pp. 333-51
  Search in Google Scholar

Prof. Jae-Kwan Hwang

Department of Biotechnology

Yonsei University

Seoul 120-749

Republic of Korea

Phone: +82-2-2123-5881

Fax: +82-2-362-7265

Email: jkhwang@yonsei.ac.kr