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.
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.
Raghav
Sunil Kumar
SK
Proteomics and Structural Biology Division, Institute of Genomics and Integrative Biology, Delhi University Campus, Mall Road, Delhi-110 007, India.
Gupta
Bhawna
B
Shrivastava
Anju
A
Das
Hasi Rani
HR
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
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