Cycloalliin hydrochloride monohydrate

To analyze the cycloalliin, sample (or cycloalliin hydrochloride monohydrate as a standard, Fujifilm Wako Pure Chemical Co., Osaka, Japan) was dissolved in methanol at 0.1 g/mL. Agilent 6410 Triple Quad LC/MS (Agilent Technologies, Santa Clara, CA, USA) coupled to a MS QQQ mass spectrometer equipped with an electrospray ionization sources (Agilent Technologies) was used. Chromatographic separations were performed on a reversed phase C18 with polar end-capping (15 mm × 2 mm, Synergi^TM 4 μm Hydro-RP 80Å; phenomenex, Torrance, CA, USA). The operating temperature was set at 30 °C and the flow rate was 0.2 mL/min. Mobile phase A and B consisted of 0.1% formic acid in water and 0.1% formic acid in acetonitrile, respectively. The gradient system was as follows: 0 min, 5% B; 1 min, 5% B; 11 min, 100% B; 12 min, 100% B; 15 min, 5% B; and 20 min, 5% B. The mass spectrometer was operated using MS QQQ mass spectrometer for electrospray ionization (ESI). The general settings used were as follows: gas temperature, 300 °C; gas flow, 11 L/min; nebulizer, 15 psi; capillary, 4000 V. Regression equation (y) is 233.26x + 259.29 and correlation coefficient (R2) is 1.

As described in a previous study [20 ], the onion extract was prepared by cutting, extraction, column purification, and concentration with heat treatment prior to cutting so that they contained at least 6% cysteine sulfoxides. Nisshin Pharma Inc. (Tokyo, Japan) provided the onion extract. The onion extract was formulated to contain the following cysteine sulfoxides: 2.36% PCSO, 0.75% MCSO, and 4.13% CA. The resulting onion extract was dissolved in phosphate buffered saline (PBS) (Sigma-Aldrich, St. Louis, MO, USA). PCSO (Nagara Science Co., Gifu, Japan), MCSO (LKT Laboratories, St. Paul, MN, USA), and cycloalliin hydrochloride monohydrate (Wako Pure Chemicals, Osaka, Japan) were dissolved in PBS to prepare a stock solution (10 mM). H89 and forskolin (Sigma-Aldrich) were dissolved in dimethyl sulfoxide (Sigma-Aldrich) to prepare a stock solution (10 mM).