Anti-inflammatory Effects And Associated Molecular Mechanisms Of Polymethoxyflavones

It is well recognized that inflammation plays important roles in the initiation and progress ofmany diseases including cancers in multiple organ sites. Inflammation can be oncogenic byvarious mechanisms including induction of genomic instability, promoting angiogenesis, alteringthe epigenetic status and enhancing cell proliferation. Polymethoxyflavones (PMFs) refer toflavonoid compounds that have two or more methoxy groups on their basic benzo-γ-pyroneskeleton with a carbonyl group at the C4position. In nature, PMFs are found exclusively in theCitrus genus, particularly in the peel of sweet oranges (Citrus sinensis) and mandarin oranges(Citrus reticulate). PMFs have been of particular interest due to their documented broadspectrum of biological activities, including anti-inflammatory, anti-carcinogenic, and anti-atherogenic properties. Hydroxylated PMFs are derivatives of PMFs with one or more methoxygroup(s) replaced by hydroxyl group(s). Some hydroxylated PMFs have been isolated fromcitrus peels.In this study, four groups of PMFs were investigated for their inhibitory mechanisms on LPS-induced inflammation in RAW264.7macrophage cells, including nobiletin (NBN) andsulforaphane (SFN) in combination,5-demethylnobiletin (5HPMF) and its three majormetabolites, tangeretin (TMF) and its primary metabolite4’-demethyltangeretin (4’-OH-TMF),5-hydroxy-6,7,8,4’-tetramethoxyflavone (5HTMF) and its primary metabolite5,4’-dihydroxy-6,7,8-trimethoxyflavone (xanthomicrol). The research results are as follows:Combination of NBN and SFN synergistically inhibited LPS-induced inflammation in RAW264.7macrophage cells, which was evidenced by lowered NO production and decreased iNOS,COX-2, and IL-1levels. These effects were associated with synergistic induction of phase IIenzyme HO-1by the NBN+SFN co-treatment. Our results provided new knowledge on the interactions between different dietary bioactive components, which is an important butunderstudied area.A hydroxylated PMF,5HPMF has been isolated from aged orange peel and is a derivative ofNBN. Previous studies showed that5,3’-didemethylnobiletin (metabolite I),5,4’-didemethylnobiletin (metabolite II), and5,3’,4’-tridemethylnobiletin (metabolite III) were themajor metabolites of5HPMF in mouse urine. Compound5HPMF and its metabolites I, II, IIIsignificantly inhibited LPS-induced inflammation in RAW264.7macrophage cells, which wereevidenced by lowered NO production. The results showed that5HPMF and its metabolites II, IIIexerted their anti-inflammatory properties mainly by suppressing iNOS, COX-2, and IL-1geneexpression. The inhibitory effect of metabolite I was associated with increased phase II enzymeHO-1gene expression. Metabolites I, II and III showed much more potent activities than theirparent compound,5HPMF. To our knowledge, this is the first report to compare the inhibitorymechanisms of5HPMF with its metabolites I, II, III on LPS-induced inflammation in RAW264.7macrophage cells.It has been reported that TMF is a potent anticancer and antioxidant compound. From the HPLCprofile of mouse urine after the oral administration of TMF to the mice,4’-OH-TMF was foundto be the primary metabolite of TMF. Both TMF and its metabolite4’-OH-TMF inhibited LPS-induced inflammation in RAW264.7macrophage cells, which were evidenced by lowered NOand PGE2production and decreased iNOS and COX-2gene expression. Metabolite4’-OH-TMFshowed more potent activity than its parent compound, TMF. The results showed that TMF and4’-OH-TMF inhibited LPS-induced expression of iNOS and COX-2by regulating the signalingpathway, particularly affecting the activation of p38, JNK, PI3K/Akt, the degradation of IκB-,and the translocation of NF-κB. To our knowledge, this is the first report to show the inhibitorymechanism of4’-OH-TMF, the primary metabolite of TMF, on LPS-induced inflammation inRAW264.7macrophage cells.One of hydroxylated PMFs,5HTMF has been synthesized from TMF. From the HPLC profile ofmouse urine after the administration of5HTMF, xanthomicrol was found to be the primarymetabolite of5HTMF. Xanthomicrol significantly inhibited LPS-induced inflammation in RAW264.7macrophage cells, which was evidenced by lowered NO and PGE2production anddecreased iNOS gene expression and COX-2protein expression. These inhibitory effects were associated with inhibition on IL-1gene expression and increased phase II enzyme HO-1geneexpression. Xanthomicrol showed more potent activity than its parent compound,5HTMF.One metabolite of5HPMF,5,3’,4’-tridemethylnobiletin (metabolite III) showed the strongestanti-inflammatory effects in LPS-treated RAW264.7cells, compared with the other PMFs ortheir metabolites at concentrations tested. Metabolite III caused complete inhibition on theproduction of NO and PGE2by100%. The treatments with4’-OH-TMF and xanthomicrol led to95%and73%inhibition on NO production, respectively, and100%and96%inhibition onPGE2production, respectively. Compared with their parent compounds, the methoxyl group(s)on the B rings of these three metabolites was (were) demethylated and turned into hydroxylgroup(s). Their enhanced anti-inflammatory effects may be associated with the hydroxyl groupson the B rings. The differences in the number and positon of hydroxyl groups may provide thesecompounds different abilities in binding different signaling proteins or binding these proteinswith different affinity. These differences in proteins bindings can lead to differences in anti-inflammatory activities.