Structures and Inhibitory Activities for Interleukin-2 Production of Seasonally Variable Constituents in Flower Parts of Magnolia kobus at Different Growth Stages.

Magnolia Flower is a crude drug used for the treatment of headaches, toothaches, and nasal congestion. Here, we focused on Magnolia kobus, one of the botanical origins of Magnolia Flower, and collected the flower parts at different growth stages to compare chemical compositions and investigate potential inhibitory activities against interleukin-2 (IL-2) production in murine splenic T cells. After determining the structures, we examined the inhibitory effects of the constituents of the bud, the medicinal part of the crude drug, against IL-2 production. We first extracted the flower parts of M. kobus from the bud to fallen bloom stages and analysed the chemical compositions to identify the constituents characteristic to the buds. We found that the inhibitory activity of the buds against IL-2 production was more potent than that of the blooms. We isolated two known compounds, tiliroside (1) and syringin (2), characteristic to the buds from the methanol (MeOH) extract of Magnolia Flower. Moreover, we examined the inhibitory activities of both compounds against IL-2 production and found that tiliroside (1) but not syringin (2), showed strong inhibitory activity against IL-2 production and inhibited its mRNA expression. Thus, our strategy to examine the relationship between chemical compositions and biological activities during plant maturation could not only contribute to the scientific evaluation of medicinal parts of crude drugs but also assist in identifying biologically active constituents that have not yet been reported.

TRPM8 channel inhibitor AMTB suppresses murine T-cell activation induced by T-cell receptor stimulation, concanavalin A, or external antigen re-stimulation.

Transient receptor potential (TRP) channels are a family of non-selective cation channels that are functionally expressed in various organs and cells. Among them, transient receptor potential vanilloid (TRPV) 1 and TRPV4 channels are expressed in T cells, where they serve as Ca2+ channels for T-cell receptor signaling [Bertin et al., 2014, Majhi et al., 2015]. Here, we show that not only TRPV1 and TRPV4 channel inhibitors, but also a transient receptor potential melastatin (TRPM) 8 channel inhibitor can suppress murine T-cell activation. Mouse splenic lymphocytes pretreated with N-(3-aminopropyl)-2-[(3-methylphenyl)methoxy]-N-(2-thienylmethyl)benzamide hydrochloride (AMTB), a TRPM8 channel-selective inhibitor, showed significantly reduced IL-2 and IL-6 release from T cells after stimulation with anti-CD3ε/anti-CD28 antibodies or concanavalin A. AMTB also suppressed IL-2 mRNA expression and activation of extracellular signal-regulated kinase 1/2, which is involved in IL-2 production. Further, the increase of CD25 (IL-2 receptor alpha chain) expression after T-cell activation was suppressed by AMTB. TRPM8 channel was expressed in CD4+ T cells isolated from splenocytes, and we confirmed that the release of IL-2 from isolated CD4+ T cells was significantly suppressed by AMTB. In vitro re-stimulation of splenocytes from external antigen-immunized mice with the same antigen induced IL-2 and IL-6 production, which was significantly suppressed by AMTB. Thus, the TRPM8 channel inhibitor AMTB suppresses T-cell activation induced by various stimulants.