. TU-100 contains multiple TRP activators. In the present study, therefore, we examined the involvement of TRP channels in the ADM-mediated vasodilatatory effect of TU-100. Rats were treated intraduodenally with the TRP vanilloid type 1 (TRPV1) agonist capsaicin (CAP), the TRP ankyrin 1 (TRPA1) agonist allyl-isothiocyanate (AITC), or TU-100, and jejunum IBF was evaluated using laser-Doppler blood flowmetry. All three compounds resulted in vasodilatation, and the vasodilatory effect of TU-100 was abolished by a TRPA1 antagonist but not by a TRPV1 antagonist. Vasodilatation induced by AITC and TU-100 was abrogated by anti-ADM antibody treatment. RT-PCR and flow cytometry revealed that an IEC-6 cell line originated from the small intestine and purified IE cells expressed ADM and TRPA1 but not TRPV1. AITC increased ADM release in IEC cells remarkably, while CAP had no effect. TU-100 and its ingredient 6-shogaol (6SG) increased ADM release dose-dependently, and the effects were abrogated by a TRPA1 antagonist. 6SG showed similar TRPA1-dependent vasodilatation in vivo. These results indicate that TRPA1 in IE cells may play an important role in controlling bowel microcirculation via ADM release. Epithelial TRPA1 appears to be a promising target for the development of novel strategies for the treatment of various gastrointestinal disorders.daikenchuto; TU-100; vasodilatation; 6-shogaol; inflammatory bowel diseases TRANSIENT RECEPTOR POTENTIAL (TRP) channels are nonselective calcium ion channels ubiquitously expressed in many tissues and are known to participate in a broad range of physical, chemical, and environmental stimuli such as taste, temperature, changes in osmolarity, pressure, stretch, and light.

Key Points • TRPA1 expresses in enterochromaffin cells (ECC) and may be involved in gut motility. • Here, we investigated the effect of TRPA1 agonists, including high-quality traditional Japanese medicine daikenchuto (TU-100), in three different systems: (i) a manipulation-induced postoperative ileus model, (ii) in vitro culture of small intestinal (SI) segments and (iii) a model ECC. • TRPA1 was found to be indispensable for coordinated peristaltic motility and TRPA1 agonists improve SI transit. Further research is required to determine whether ECC are involved. Abstract Background Stimulation of transient receptor potential ankyrin 1 (TRPA1), which abundantly expressed in enterochromaffin cells (ECC), has been reported to exert apparently contradictory results in in vitro contractility and in vivo gastrointestinal (GI) transit evaluations. The pharmaceutical-grade Japanese traditional medicine daikenchuto (TU-100) has been reported to be beneficial for postoperative ileus (POI) and accelerate GI transit in animals and humans. TU-100 was recently shown to increase intestinal blood flow via stimulation of TRPA1 in the epithelial cells of the small intestine (SI). Methods The effects of various TRPA1 agonists on motility were examined in a manipulation-induced murine POI model, in vitro culture of SI segments and an ECC model cell line, RIN-14B. Key Results Orally administered TRPA1 agonists, aryl isothiocyanate (AITC) and cinnamaldehyde (CA), TU-100 ingredients, [6]-shogaol (6S) and c-sanshool (GS), improved SI transit in a POI model. The effects of AITC, 6S and GS but not CA were abrogated in TRPA1-deficient mice. SI segments show periodic peristaltic motor activity whose periodicity disappeared in TRPA1-deficient mice. TU-100 augmented the motility. AITC, CA and 6S increased 5-HT release from isolated SI segments and the effects of all these compounds except for CA were lost in TRPA1-deficient mice. 6S and GS induced a release of 5-HT from RIN-14B cells in a dose-and TRPA1-dependent manner. Conclusions & Inferences Intraluminal TRPA1 stimulation is a potential therapeutic strategy for GI motility disorders. Further investigation is required to determine whether 5-HT and/or ECC are involved in the effect of TRPA1 on motility.

Cerebrosides, mainly glucocerebrosides and galactocerebrosides, are the simplest glycosphingolipids (GLSs) and known to be the biosynthetic precursors of most other GLSs. 1) While glucocerebrosides have been isolated from various starfishes, occurrence of galactocerebrosides seems to be limited to three species: Stellaster equestris, 2) Culcita novaeguineae, 3) and Oreaster reticulatus. 4) In our continuing research on GLSs from echinoderms, a galactocerebroside molecular species (PNC-1) was purified from the starfish Protoreaster nodosus (kobuhitode in Japanese) collected in Okinawa. Reversed-phase HPLC separation of PNC-1 afforded sixteen new galactocerebrosides (3-7, 9-17, 19, 21), along with five known galactocerebrosides (1, 2, 8, 18, 20). Further, we developed one-pot GC-MS analysis for cerebrosides which consist of phytosphingosinetype long chain base (LCB). Generally, the trimethylsilyl (TMS) derivatives of LCB are used for GC-MS analysis and give a useful structural information, 5) however, the preparation of phytosphingosine-type LCB from the parent cerebroside is in a quite low yield and not quantitative, because the glycosidic linkage between sugar and LCB is hard to cleavage against methanolysis. One-pot reaction following mathanolysis and periodate oxidation, and subsequent GC-MS analysis gave efficient structural information. In this paper, the isolation and structure elucidation of galactocerebroside molecular species (PNC-1) and constituent cerebrosides (1-21) of PNC-1 from the starfish Protoreaster nodosus are described.Isolation and Structure of PNC-1 The lipid fraction, obtained from CHCl 3 /MeOH extract of pyloric caeca, which was dissected from the fresh material of the starfish P. nodosus, was subjected to repeated silica gel, reversed-phase and Sephadex LH-20 column chromatography to give a less polar GLS, named PNC-1, showing a single spot on silica gel TLC plate.The positive ion FAB-MS spectrum of PNC-1 exhibited a series of homologous pseudomolecular ion peaks [MϩNa] )] in the total correlation spectroscopy (TOCSY) spectrum. Thus, a structural fragment from H 2 -1 to H 2 -6 was established, suggesting the LCB of ceramide was phytosphingosine-type. Analysis of COSY and TOCSY spectra revealed another structural fragment (H-2Ј to H 2 -4Ј) in the fatty acid part of ceramide. H-2Ј (d H 4.52) signal showed a heteronuclear multiple-bond correlation spectroscopy (HMBC) cross peak with the amide carbon signal [d C 175.7 (C-1Ј)], indicating the presence of a-hydroxy fatty acid. Further, H-2 (d H 5.18) signal in LCB showed a cross peak with amide carbon (C-1Ј), and the ceramide moiety was depicted as shown in Fig. 1.Identification of the compositions of fatty acid and LCB of ceremide moiety was conducted by GC-MS analysis following methanolysis and periodate oxidation of PNC-1 (see Experimental). The mixture of fatty acid methyl esters (FAMEs) obtained from methanolysis and a mixture of long chain aldehydes generated by periodate oxidation were simultaneously subjected to GC-MS, which revealed ...

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