Gingerol Metabolite and a Synthetic Analogue Capsarol™ Inhibit Macrophage NF-κB-Mediated iNOS Gene Expression and Enzyme Activity

 

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Abstract

Ginger (Zingiber officinale) is widely used in traditional Chinese medicine, with beneficial effects reported in numerous diseases, including inflammation. Inducible nitric oxide synthase (iNOS), a proinflammatory enzyme responsible for the generation of nitric oxide (NO), has been implicated in the pathogenesis of inflammatory diseases. Gingerols, the main pungent principles of ginger, have anti-inflammatory properties in vitro. In this study we examine the inhibitory effect of a stable [6]-gingerol metabolite, rac-[6]-dihydroparadol ([6]-DHP) and a closely related gingerol analogue, rac-2-hydroxy-1-(4-hydroxy-3-methoxyphenyl)dodecan-3-one [a capsaicin/gingerol (Capsarol™) analogue referred to as ZTX42] on NO production, inducible nitric oxide synthase (iNOS) activity and protein expression levels in a murine macrophage cell line, RAW 264.7. Both ZTX42 and [6]-DHP significantly inhibited lipopolysaccharide-induced NO production in a concentration-dependent manner, with an IC₅₀ of 1.45 ± 0.03 μM and 7.24 ± 0.22 μM, respectively (P < 0.05). Although both compounds partially inhibited the catalytic activity of iNOS, their inhibitory effect was predominantly due to attenuation of iNOS protein production. This occurred at the transcriptional level, since the gingerol compounds decreased LPS-induced IκB-α degradation, prevented nuclear translocation of NF-κB p65 and reduced NF-κB activity in a concentration-dependent manner. Taken together, these results show that ZTX42 and [6]-DHP suppress NO production in murine macrophages by partially inhibiting iNOS enzymatic activity and reducing iNOS protein production, via attenuation of NF-κB-mediated iNOS gene expression, providing a rationale for the anti-inflammatory activity reported for this class of compounds.

Abbreviations

ZTX42:rac-2-hydroxy-1-(4-hydroxy-3-methoxyphenyl)dodecan-3-one

COX:cyclo-oxygenase

[6]-DHP:rac-[6]-dihydroparadol

LPS:lipopolysaccharide

MTT:3-(3,4-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

NF-κB:nuclear factor-κB

NO:nitric oxide

NOS:nitric oxide synthase

PDTC:pyrrolidine dithiocarbamate

TRPV1:vanilloid receptor 1

References

• 1 Bredt D S. Endogenous nitric oxide synthesis: biological functions and pathophysiology.  Free Radic Res. 1999;  31 577-96
  PubMedGoogle Scholar
• 2 Hobbs A J, Higgs A, Moncada S. Inhibition of nitric oxide synthase as a potential therapeutic target.  Annu Rev Pharmacol Toxicol. 1999;  39 191-220
  CrossrefPubMedGoogle Scholar
• 3 Alderton W K, Cooper C E, Knowles R G. Nitric oxide synthases: structure, function and inhibition.  Biochem J. 2001;  357 593-615
  CrossrefPubMedGoogle Scholar
• 4 Abramson S B, Amin A R, Clancy R M, Attur M. The role of nitric oxide in tissue destruction.  Best Pract Res Clin Rheumatol. 2001;  15 831-45
  CrossrefPubMedGoogle Scholar
• 5 Koo K LK, Ammit A J, Tran V H, Duke C C, Roufogalis B D. Gingerols and related analogues inhibit arachidonic acid-induced human platelet serotonin release and aggregation.  Thromb Res. 2001;  103 387-97
  CrossrefPubMedGoogle Scholar
• 6 Nurtjahja-Tjendraputra E, Ammit A J, Roufogalis B D, Tran V H, Duke C C. Effective anti-platelet and COX-1 enzyme inhibitors from pungent constituents of ginger.  Thromb Res. 2003;  111 259-65
  CrossrefPubMedGoogle Scholar
• 7 Tjendraputra E, Tran V H, Liu-Brennan D, Roufogalis B D, Duke C C. Effect of ginger constituents and synthetic analogues on cyclooxygenase-2 enzyme in intact cells.  Bioorg Chem. 2001;  29 156-63
  CrossrefPubMedGoogle Scholar
• 8 Kiuchi F, Shibuya M, Sankawa U. Inhibitors of prostaglandin biosynthesis from ginger.  Chem Pharm Bull (Tokyo). 1982;  30 754-7
  PubMedGoogle Scholar
• 9 Caterina M J, Schumacher M A, Tominaga M, Rosen T A, Levine J D, Julius D. The capsaicin receptor: a heat-activated ion channel in the pain pathway.  Nature. 1997;  389 816-24
  CrossrefPubMedGoogle Scholar
• 10 Roufogalis B D, Tran V H, Duke C C. unpublished results. 
  Google Scholar
• 11 Bhattarai S, Tran V T, Duke C C. The stability of gingerol and shogaol in aqueous solutions.  J Pharm Sci. 2001;  90 1658-64
  CrossrefPubMedGoogle Scholar
• 12 Ippoushi K, Azuma K, Ito H, Horie H, Higashio H. [6]-Gingerol inhibits nitric oxide synthesis in activated J774.1 mouse macrophages and prevents peroxynitrite-induced oxidation and nitration reactions.  Life Sci. 2003;  73 3427-37
  CrossrefPubMedGoogle Scholar
• 13 Roufogalis B D, Duke C C, Tran V H. Medicinal uses of phenylalkanols and derivatives. US Patent 6,518,315 2003
  Google Scholar
• 14 Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays.  J Immunol Methods. 1983;  65 55-63
  CrossrefPubMedGoogle Scholar
• 15 Aktan F, Henness S, Roufogalis B D, Ammit A J. Gypenosides derived from Gynostemma pentaphyllum suppress NO synthesis in murine macrophages by inhibiting iNOS enzymatic activity and attenuating NF-kappaB-mediated iNOS protein expression.  Nitric Oxide. 2003;  8 235-42
  CrossrefPubMedGoogle Scholar
• 16 Hong S H, Seo S H, Lee J H, Choi B T. The aqueous extract from Artemisia capillaris Thunb. inhibits lipopolysccharide-induced inflammatory response through preventing NF-κB activation in human hepatoma cell line and rat liver.  Int J Mol Med. 2004;  13 717-20
  PubMedGoogle Scholar
• 17 Schreck R, Meier B, Mannel D N, Droge W, Baeuerle P A. Dithiocarbamates as potent inhibitors of nuclear factor kappa B activation in intact cells.  J Exp Med. 1992;  175 1181-94
  CrossrefPubMedGoogle Scholar
• 18 Xie Q W, Kashiwabara Y, Nathan C. Role of transcription factor NF-kappa B/Rel in induction of nitric oxide synthase.  J Biol Chem. 1994;  269 4705-8
  PubMedGoogle Scholar
• 19 Sreejayan R MN. Nitric oxide scavenging by curcuminoids.  J Pharm Pharmacol. 1997;  49 105-7
  PubMedGoogle Scholar
• 20 Matsuda H, Kagerura T, Toguchida I, Ueda H, Morikawa T, Yoshikawa M. Inhibitory effects of sesquiterpenes from bay leaf on nitric oxide production in lipopolysaccharide-activated macrophages: Structure requirement and role of heat shock protein induction.  Life Sci. 2000;  66 2151-7
  CrossrefPubMedGoogle Scholar
• 21 Sheu F, Lai H H, Yen G C. Suppression effect of soy isoflavones on nitric oxide production in RAW 264.7 macrophages.  J Agric Food Chem. 2001;  49 1767-72
  PubMedGoogle Scholar
• 22 Dedov V N, Tran V H, Duke C C, Connor M, Christie M J, Mandadi S. et al . Gingerols: a novel class of vanilloid receptor (VR1) agonists.  Br J Pharmacol. 2002;  137 793-8
  CrossrefPubMedGoogle Scholar
• 23 Chen C W, Lee S T, Wu W T, Fu W M, Ho F M, Lin W W. Signal transduction for inhibition of inducible nitric oxide synthase and cyclooxygenase-2 induction by capsaicin and related analogs in macrophages.  Br J Pharmacol. 2003;  140 1077-87
  CrossrefPubMedGoogle Scholar
• 24 Kim C S, Kawada T, Kim B S, Han I S, Choe S Y, Kurata T. et al . Capsaicin exhibits anti-inflammatory property by inhibiting IκB-α degradation in LPS-stimulated peritoneal macrophages.  Cell Signal. 2003;  15 299-306
  CrossrefPubMedGoogle Scholar

Alaina J. Ammit

Faculty of Pharmacy

Building A15, Room S222

University of Sydney

Sydney

NSW 2006

Australia

Phone: +61-2-9351-6099

Fax: +61-2-9351-4391

Email: ajammit@pharm.usyd.edu.au