title: Inhibition of lipopolysaccharide-inducible nitric oxide synthase and IL-1beta through suppression of NF-kappaB activation by 3-(1'-1'-dimethyl-allyl)-6-hydroxy-7-methoxy-coumarin isolated from Ruta graveolens L.
abstract: The Ruta graveolens L. plant is used in traditional medicine to treat a large number of diseases. The methanol (50%) extract of the whole plant was observed to inhibit the expression of inducible nitric oxide synthase (iNOS) and the cycloxygenase-2 (COX-2) gene in lipopolysaccharide (LPS)-induced macrophage cells (J774A.1, [Raghav, S.K., Gupta, B., Agrawal, C., Goswami, K., Das, H.R., 2006b. Anti-inflammatory effect of Ruta graveolens L. in murine macrophage cells. J. Ethnopharmacol. 104, 234-239]). The effect of whole plant extract on the expression of other pro-inflammatory genes such as tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), IL-12, interferon-gamma (IFN-gamma) and the activation of nuclear factor-kB (NF-kappaB) were investigated in LPS stimulated macrophage cells. An active compound was isolated from this methanol extract by further solvent fractionation and reverse phase high performance liquid chromatography (RP-HPLC). The purified compound was identified as 3-(1'-1'-dimethyl-allyl)-6-hydroxy-7-methoxy-coumarin having IUPAC nomenclature of 6-hydroxy-7-methoxy-3-(2-methyl but-3-en-2yl)-2H-chromen-2-one by ESI-MS, MALDI, FT-IR and NMR. Effect of this purified compound was assessed on iNOS, COX-2 and various pro-inflammatory cytokine genes and was observed to inhibit both the protein and mRNA expression of iNOS and IL-1beta in LPS challenged macrophages. Electrophoretic mobility shift assay (EMSA) and Western blot analyses indicated that the plant extract and the isolated active compound blocked the LPS-induced activation of NF-kappaB through the prevention of inhibitor-kB (IkB) degradation. The purified compound also showed the anti-oxidant activity. The active compound at a dose of 40 mg/kg body weight was observed to inhibit the iNOS and IL-1beta gene expression significantly in endotoxin-induced inflammatory model of BALB/c mice. The low level of nitric oxide production was also observed in the sera of compound treated mice. The normal behavioral condition in LPS challenged BALB/c mice was noticed when these were treated with active compound.
authors.affiliation: Proteomics and Structural Biology Division, Institute of Genomics and Integrative Biology, Delhi University Campus, Mall Road, Delhi-110 007, India.
authors.forename: Sunil Kumar
authors.initials: SK
authors.lastname: Raghav
authors.forename: Bhawna
authors.initials: B
authors.lastname: Gupta
authors.forename: Anju
authors.initials: A
authors.lastname: Shrivastava
authors.forename: Hasi Rani
authors.initials: HR
authors.lastname: Das
mesh_terms: D000818:Animals; D000893:Anti-Inflammatory Agents / Q000494:pharmacology*; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D003374:Coumarins; D016207:Cytokines / Q000037:antagonists & inhibitors / Q000378:metabolism; D004195:Disease Models, Animal; D024202:Electrophoretic Mobility Shift Assay; D015870:Gene Expression / Q000187:drug effects; D020672:I-kappa B Proteins; D053583:Interleukin-1beta / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects / Q000201:enzymology; D051379:Mice; D008807:Mice, Inbred BALB C; D016328:NF-kappa B / Q000378:metabolism; D009569:Nitric Oxide / Q000096:biosynthesis; D052247:Nitric Oxide Synthase Type II / Q000037:antagonists & inhibitors*; D008517:Phytotherapy; D010936:Plant Extracts / Q000494:pharmacology*; D032105:Ruta
chemical_list: C519548:3-(1'-1'-dimethyl-allyl)-6-hydroxy-7-methoxy-coumarin; D000893:Anti-Inflammatory Agents; D003374:Coumarins; D016207:Cytokines; D020672:I-kappa B Proteins; D053583:Interleukin-1beta; D008070:Lipopolysaccharides; D016328:NF-kappa B; D010936:Plant Extracts; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II