Mast cells play a central role in the immediate allergic reaction and in chronic inflammation associated with fibrosis. Mast cells also express several genetically encoded pattern recognition receptors including Toll-like receptors, which function in innate immunity. Thus, the roles of mast cells are complex, as they function in both immunity and inflammation. This discrepancy may be due to the fact that numerous bioactive chemical mediators including histamine, prostaglandins, and pro-inflammatory cytokines are released following the discrete reactions of mast cells against various stimuli. To understand the physiological roles of mast cells, studies have focused on the mechanisms of mast cell activation, as well as on the molecules that inhibit mast cell activation. Flavonoids, such as quercetin, are found in many plants and inhibit the activity of chemical mediators released from mast cells. Extracts of Citrus plants, which contain many flavonoids, exhibit anti-inflammatory, antiallergy, and anti-cancer effects. We isolated and identified two polymethoxyflavonoids--natsudaidain and nobiletin--from Citrus plants. Although nobiletin has an anti-inflammatory effect, the biological activity of natsudaidain, which closely resembles nobiletin, is not fully understood. Here we examined the inhibitory effect of natsudaidain on histamine release, tumour necrosis factor-α (TNF-α) production, and cyclooxygenase- 2 (COX-2) expression in Ca ionophore-stimulated rat basophilic leukemia cells (A23187-stimulated RBL-2H3 cells) using spectrofluorometric, ELISA, and immunoblotting methods. The percent of histamine release from A23187-stimulated RBL-2H3 cells pretreated with natsudaidain at 5, 25, or 50 µM was not altered compared with non-treated A23187-stimulated cells. Pretreatment with 100 or 200 μM natsudaidain resulted in slightly reduced levels of histamine release (89.8 ± 3.5% and 71.5 ± 5.6% histamine release at 100 and 200 μM, respectively). Thus, natsudaidain has little effect on histamine release from RBL-2H3 cells, except at high concentrations. On the other hand, natsudaidain inhibited TNF-α protein and mRNA levels in A23187-stimulated RBL-2H3 cells dose-dependently; a concentration of 6.8 µM was required for a 50% reduction. In addition, all levels of this compound tested also in A23187-stimulated RBL-2H3 cells treated with natsudaidain were also markedly reduced. The phosphorylated-p38 MAPK protein levels in A23187-stimulated RBL-2H3 cells were lower in natsudaidain-treated cells than in non-treated cells. These findings suggest that natsudaidain inhibits TNF-α and COX-2 production by suppressing p38 MAPK phosphorylation; thus, natsudaidain may alleviate inflammatory diseases. [ABSTRACT FROM AUTHOR]