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Antioxidant action of Rosmarinic acid (Ros A), a natural phenolic ingredient in many Lamiaceae herbs such as Perilla frutescens, sage, basil and mint, was analyzed in relation to the Ikappa-B activation in RAW264.7 macrophages. Ros A inhibited nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) protein synthesis induced by lipopolysaccharide (LPS), and also effectively suppressed phorbol 12-myristate 13-acetate (PMA)-induced superoxide production in RAW264.7 macrophages in a dose-dependent manner. Peroxynitrite-induced formation of 3-nitrotyrosine in bovine serum albumin and RAW264.7 macrophages were also inhibited by Ros A. Moreover, Western blot analysis demonstrated that LPS-induced phosphorylation of Ikappa-Balpha was abolished by Ros A. Ros A can act as an effective protector against peroxynitrite-mediated damage, and as a potent inhibitor of superoxide and NO synthesis; the inhibition of the formation of reactive oxygen and nitrogen species are partly based on its ability to inhibit the serine phosphorylation of Ikappa-Balpha.

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-

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In the plasma membrane fraction from Caco-2 human colon carcinoma cells, active Nox1 (NADPH oxidase 1) endogenously co-localizes with its regulatory components p22(phox), NOXO1, NOXA1 and Rac1. NADPH-specific superoxide generating activity was reduced by 80% in the presence of either a flavoenzyme inhibitor DPI (diphenyleneiodonium) or NADP(+). The plasma membranes from PMA-stimulated cells showed an increased amount of Rac1 (19.6 pmol/mg), as compared with the membranes from unstimulated Caco-2 cells (15.1 pmol/mg), but other components did not change before and after the stimulation by PMA. Spectrophotometric analysis found approx. 36 pmol of FAD and 43 pmol of haem per mg of membrane and the turnover of superoxide generation in a cell-free system consisting of the membrane and FAD was 10 mol/s per mol of haem. When the constitutively active form of Rac, Rac1(Q61L) or GTP-bound Rac1 was added exogenously to the membrane, O(2)(-)-producing activity was enhanced up to 1.5-fold above the basal level, but GDP-loaded Rac1 did not affect superoxide-generating kinetics. A fusion protein [NOXA1N-Rac1(Q61L)] between truncated NOXA1(1-211) and Rac1-(Q61L) exhibited a 6-fold increase of the basal Nox1 activity, but NOXO1N(1-292) [C-terminal truncated NOXO1(1-292)] alone showed little effect on the activity. The activated forms of Rac1 and NOXA1 are essentially involved in Nox1 activation and their interactions might be responsible for regulating the O(2)(-)-producing activity in Caco-2 cells.