Inducible nitric oxide synthase (iNOS) is involved in pathological processes related to overproduction of nitric oxide (NO), and is expressed in response to pro-inflammatory agents such as interleukin (IL)-1b, tumor necrosis factor (TNF)-a and lipopolysaccharide (LPS) in various cell types including macrophages, endothelial cells, and smooth muscle cells.1) Nuclear factor (NF)-kB is a major transcription factor involved in iNOS, TNF-a, IL-1b, and IL-8 genes expression. NF-kB activation involves dissociation of an inhibitory subunit, IkB, which keeps NF-kB in the cytoplasm, thereby preventing activation of the target gene in the nucleus. Cellular signals lead to phosphorylation of IkB following elimination of IkB from NF-kB by proteolytic degradation. Then, the activated-NF-kB is released and translocated into the nucleus to activate transcription of its target genes.2) Inhibition of iNOS enzyme activity or iNOS induction and inhibition of NF-kB activation may be of therapeutic benefit in various types of inflammation. 10) In the course of our studies on constituents with NO production inhibitory activity from natural medicines, 11) the methanolic extract from the dried bark of M. obovata was found to inhibit nitrite (NO 2 Ϫ , a product of NO) accumulation in LPS-activated mouse macrophages (IC 50 ϭ25 mg/ml).Previous reports demonstrated that two neolignans [magnolol (1), honokiol (2)] showed inhibitory effects on NO production from LPS-activated RAW 264.7 cells.12) However, effects of other constituents on NO production from LPS-activated mouse macrophages and their cytotoxicities for macrophages have not been examined. This report describes the effects of the constituents from the bark of M. obovata on NO production in LPS-stimulated macrophages. In addition, we describe the effects of principal active neolignan constituents [magnolol (1), honokiol (2), obovatol (3)] on iNOS enzyme activity, induction of iNOS, and activation of NF-kB to clarify their action mechanisms.
Results and DiscussionIsolation of Chemical Constituents from the Dried Bark of M. obovata The bark of Japanese M. obovata was extracted with methanol under reflux. The methanolic extract was subjected to ordinary-and reversed-phase silica gel column chromatography and finally HPLC to furnish five neolignans, magnolol (1, 13) 2.1% from the natural medicine), honokiol (2, 14) 0.43%), obovatol (3, 15) 0.26%), 4-O-methylhonokiol (4, 16) 0.0031%), and 6Ј-O-methylhonokiol (5, 10,16) 0.0031%), seven sesquiterpene-neolignans, eudesmagnolol (6, 17) 0.096%), clovanemagnolol (7, 18) 0.0061%), caryolanemagnolol (8, 10) 0.0044%), eudeshonokiols A (9, 10) 0.0056%) and B (10, 10) 0.0054%), eudesobovatols A (11, 19) 0.043%) and B (12,19) 0.024%), a trineolignan, magnolianin (13,20) 0.27%), a phenylpropanoid glycoside, syringin (14, 21) 0.39%), two lignan glycosides, liriodendrin (15,22) 0.15%) and (ϩ)-syringaresinol 4Ј-O-b-D-glucopyranoside (16,23) 0.029%), and four sesquiterpenes, caryophyllene oxide (17, 24) 0.0049%), a-eudesmol (18, 11g,24) 0.096%), b...