Citrus unshiu water extract inhibits trypsin-induced TNF-α and tryptase productions by blocking the ERK phosphorylation and trypsin activity

Citrus unshiu (Rutaceae) has long been known as an anti-inflammatory and anti-allergic agent. In the present study, the inhibitory effect of CUWE (Citrus unshiu water extract) on the production of TNF-α and tryptase was examined. In addition, a possible mechanism for the inhibition of trypsin-stimulated human leukemic mast cell-1 (HMC-1) activation was determined. To do so, TNF-α production from the HMC-1 cells that were stimulated by trypsin (100 nM) in the presence or absence of CUWE (10, 100, and 100 μg/ml) was measured by enzyme-linked immunosorbent assay (ELISA) and reverse transcription-PCR. The tryptase production was evaluated by reverse transcription-PCR. Extracellular signal-regulated kinase (ERK) activation was analyzed by Western blot. Trypsin activity was measured by using Bz-DL-Arg-p-nitroanilide (BAPNA) as substrate. Results showed that the CUWE inhibited production of both TNF-α and tryptase from the trypsin-stimulated HMC-1 in a dose-dependent manner. The CUWE also inhibited the ERK phosphorylation and trypsin activity. These results indicate that the CUWE had an inhibitory effect on TNF-α and the tryptase productions by blocking the ERK phsphorylation and trypsin activity.