Inhibitory action of eugenol compounds on the production of nitric oxide in RAW264.7 macrophages

Abstract

Effects of eugenol compounds on the production of nitric oxide (NO) in RAW264.7 macrophages were analyzed in relation to the anti-inflammatory action of these compounds. Eugenol and isoeugenol inhibited lipopolysaccharide (LPS)-dependent production of NO, which was due to the inhibition of protein synthesis of inducible nitric oxide synthase (iNOS). Isoeugenol showed the most effective inhibitory effect and eugenol was less effective. LPS-dependent expression of cyclooxygenase-2 (COX-2) protein was also inhibited markedly by isoeugenol, and less effectively by eugenol. Anti-inflammatory action of eugenol compounds may be explained by the inhibition of NO production and COX-2 expression, the pro-inflammatory mediators.Eugenol with a methoxyphenol structure is a principal constituent of bay leaves, allspice, and the oil of cloves that originate from the Syzygium species (7). Eugenol is widely used as a flavoring agent in cosmetic and food products (9), and is also utilized as an antiseptic drug in dentistry (2, 21). Isoeugenol, an isomer of eugenol is found in monkey orange (19), and shows anti-oxidant properties (15, 16). Recently we analyzed inhibitory effects of eugenol compounds on metal-mediated oxidation of membrane lipids and LDL (low density lipoprotein) that are an important risk factor for the development of atherosclerotic vascular disease (1, 22), and the metal-reducing activity of eugenol and isoeugenol may participate in the antioxidant action of these compounds (5). Development of oxidative damage is closely related to the reactive nitrogen species in addition to active oxygen: that is, peroxynitrite produced by the reaction of superoxide radical with nitric oxide shows potent cytotoxic effects. For further analyses of anti-oxidant, anti-inflammatory and anti-septic properties of eugenol compounds, we analyzed the effect of eugenol compounds on the production of NO and the expression of the iNOS protein. Eugenol compounds, in particular isoeugenol showed a potent inhibitory effect of LPS-mediated expression of iNOS protein, and COX2 protein in RAW264.7 macrophages. Anti-inflammatory action of eugenol compounds can be explained at least, in part by the inhibition of iNOS induction. MATERIALS AND METHODSReagents. The sources of materials used in this work were as follows: eugenol, isoeugenol, lipopolysaccharide (LPS), 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolidum bromide (MTT) from Sigma-Aldrich-Japan (Tokyo, Japan), rabbit anti-Cu/ Zn SOD and anti-nitric oxide synthase II (iNOS) antibodies from Stressgen Biotechnologies Corp (Victoria, BC, Canada), mouse anti-phospho-Iκ-Bα (Ser32/36) (5A5) antibody and mouse anti-COX2 antibody from Cell Signaling Technology (Heidel-

Mechanisms of gastroprotective effect of eugenol in indomethacin‐induced ulcer in rats

“…Peroxynitrite, produced by the reaction of NO with a superoxide radical, is a highly reactive nitrogen species showing toxic effects by oxidizing and nitrating cellular macromolecules (Moncada and Bolanos, 2006). Eugenol was found to inhibit the expression of inducible nitric oxide synthase (iNOS) and to decrease the production of NO, the powerful pro-inflammatory mediator (Li et al, 2006). The anti-inflammatory action of eugenol may be also attributed to its inhibitory effect on cyclooxygenase-2 and 5-lipoxygenase, the key enzymes in biosynthetic pathway of inflammatory prostaglandins and leukotrienes Raghavenra et al, 2006).…”

“…However, the mechanisms responsible for the anti-ulcer effect of eugenol are not yet clear. It was reported that eugenol is an antioxidant (Tiku et al, 2004;Ito et al, 2005), vasorelaxant (Nangle et al, 2006), transient receptor potential vanilloid 1 (TRPV1) agonist (Yang et al, 2003), and inducible nitric oxide synthase (iNOS) inhibitor (Li et al, 2006) which may contribute to its gastroprotective effect. This encouraged us to conduct the present study in order to clarify the possible mechanisms underlying the protective effect of eugenol against indomethacin-induced gastric ulceration in rats.…”

Protective effect of glutathione against lipopolysaccharide-induced inflammation and mortality in rats

“…NO upregulates the COX-2 expression through the cAMP-response element. Some antiinflammatory agents decreased NO production by inhibiting the expression of iNOS protein and further decreased COX-2 expression [8,9,33]. In addition, iNOS can bind and activate COX-2 [9].…”

The analgesic effects and mechanisms of orally administered eugenol

“…The agent has sedative and anodyne effects, but concomitantly shows an irritant action (Sneddon and Glew, 1973). It also has antioxidant, anti-inflammatory and antiseptic properties (Li et al, 2006). The analgesic action of eugenol had long been attributed to its action as a non-specific counter-irritant.…”