Acetonitrile

Analysis of the properties of the components contained in the extract was performed with an Agilent 1260 Infinity (Agilent Technologies, Santa Clara, CA, USA) ODS column (Cadenza CD-C18 column, 250 × 4.6 mm, particle size 3 µm, Imtakt Corporation, Kyoto, Japan) at a constant temperature of 40 °C. In the mobile phase, a 5-fold-diluted extract in ethanol of 30 µL was applied to the column, and elution was performed at a flow rate of 1 mL/min with a mixture of solvents A (0.4% formic acid (Fujifilm Wako Pure Chemicals, Osaka, Japan)) and B (100% acetonitrile (Kanto Chemical, Tokyo, Japan)). Elution was performed under the following conditions: gradient from initial conditions to 40 min (0 min (A:B = 93:7), 33 min (A:B = 60:40), 40 min (A:B = 0:100)), from 40 to 70 min (A:B = 0:100). Spectra were detected with a diode array detector (DAD) at 280 and 326 nm. In parallel with the analysis, fraction collection was also performed. The preparative schedule was as follows: Fr.1: 8–15 min, Fr.2: 19–25 min, Fr.3: 42–51 min, Fr.4: 51–60 min, Fr.5: 60–69 min.

Isoflurane, benzophenone, mannitol (D-mannitol), a Cholesterol E-Test Kit, and DDC were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). DIS powder and 2-hydroxypropyl-β-cyclodextrin (HPCD) were kindly donated by Ouchi Shinko Chemical Industrial Co., Ltd. (Tokyo, Japan) and Nihon Shokuhin Kako Co., Ltd. (Tokyo, Japan), respectively. A Mitochondrial Isolation kit, a Cytochrome c Oxidase Assay kit, and an ATP Bioluminescent Assay kit were purchased from Sigma Aldrich Japan (Tokyo, Japan). A Bio-Rad Protein Assay kit was provided by BIO-RAD (California, USA). We obtained 0.5% phenylephrine and 0.5% tropicamide from Santen Pharmaceutical Co., Ltd. (Osaka, Japan). An ELISA Insulin Kit and an RNA PCR Kit (AMV Ver 3.0) were purchased from Morinaga Institute of Biological Science Inc. (Kanagawa, Japan) and Takara Bio Inc. (Shiga, Japan), respectively. Methylcellulose type SM-4 (MC) was obtained from Shin-Etsu Chemical Co., Ltd. (Tokyo, Japan). Pentobarbital was provided by Sumitomo Dainippon Pharma Co., Ltd. (Toyo, Japan). Benzalkonium chloride (BAC), methanol, and acetonitrile was purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). LightCycler FastStart DNA Master SYBR Green I was provided by Roche Diagnostics Applied Science (Mannheim, Germany). All other used chemicals were of the highest purity commercially available.

Acetone, hexane, tetrahydrofuran (THF), methanol, chloroform, ethyl acetate (EtOAc), ethanol, acetonitrile, acryloyl chloride, sodium hydroxide, potassium hydroxide, N, N-dimethylformamide (DMF), anhydrous magnesium sulfate, and diethyl ether were purchased from Kanto Chemical. Copper (I) chloride (CuCl), lithium bromide (LiBr), ethylamine, and DIBOD were purchased from the Tokyo Chemical Industry. Sodium azide and dibenzocyclooctyne-amine (DBCO-amine) were purchased from Sigma-Aldrich. N, N-dimethylformamide (DMF) for preparing the GPC/SEC’s eluent and hydrochloric acid (12 N) were purchased from FUJIFILM Wako Pure Chemical Industry. Pentaerythritol tetra (2-chloropropionate) (PETCP) was commercially obtained as a custom-made product from the Tokyo Chemical Industry and used as an initiator. Ultrapure water (specific resistance value: 18.2 MΩ cm) obtained by purifying tap water with a Direct-Q 3UV manufactured by Millipore was used. In addition, N-isopropyl acrylamide (NIPA) and tris (2-dimethylamino ethyl) amine (Me₆TREN) were kindly provided by Kohjin and Mitsubishi Chemical, respectively. NIPA was purified by recrystallization using a mixture of hexane and Acetone. Me₆TREN (Mitsubishi Chemical) and DMF (Kanto Chemical) for synthesis were purified by vacuum distillation. Furthermore, other chemicals were used without purification.