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9 protocols using distilled water

1

Extraction and Characterization of Eleocharis dulcis

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The fresh Eleocharis dulcis were purchased from the central agricultural product market of Hezhou (Hezhou, Guangxi Province, China) and cleaned with water, and then the peels without decay and deterioration were collected and dried with a DHG series heating and drying oven (DHG-9140A, Keelrein, China) at 60 °C for 36 h. After this, the dried peels were ground by a universal high-speed grinder (DFY-600, Linda Machinery Co., Ltd, China) and stored for further extraction.
α-Glucosidase (EC 3.2.1.20) from yeast was purchased from Oriental Yeast Co., Ltd (Tokyo, Japan), and dissolved in 0.1 M sodium phosphate buffer of pH 7.4. Methanol, acetonitrile and distilled water as HPLC grade chemicals were purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). Acarbose and p-nitrophenyl-α-d-glucopyranoside (pNPG) were purchased from Tokyo Chemical Industry Co., Ltd (Tokyo, Japan). A Glucose C-II Test Wako Kit was obtained from Fujifilm Wako Pure Chemical Industries, Ltd (Osaka, Japan). Pyridine-d5 for NMR spectroscopy and maltose were purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). All other reagents were of analytical purity.
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2

Evaluation of Kampo Herbal Medicines

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A total of 147 oral Kampo prescriptions, which are covered by health insurance in Japan, were purchased from Ohsugi Pharmaceutical Co., Ltd. (Osaka, Japan), Kracie Holdings, Ltd. (Tokyo, Japan), Kotaro Pharmaceutical Co., Ltd. (Osaka, Japan), Sanwa Shoyaku Co., Ltd. (Tochigi, Japan), Taikoseido Pharmaceutical Co., Ltd. (Hyogo, Japan), Tsumura & Co. (Tokyo, Japan), and Toyo-Kampo Pharmaceutical Co., Ltd. (Osaka, Japan), respectively. The Albumin Glycation Assay Kit, Glyceraldehyde (Cat. No. AAS-AGE-K01) was purchased from Cosmo Bio Co., Ltd. (Tokyo, Japan). Sodium azide was purchased from Sigma-Aldrich Japan Co. (Tokyo, Japan). Ephedrae herba was purchased from Tanuma Shokai Co., Ltd. (Chiba, Japan) (Lot. 131017). Methylglyoxal and Glyceraldehyde were purchased from Nacalai Tesque, Inc. (Kyoto, Japan). An amount of 0.1 moL/L Phosphate buffer, acetic acid, and dimethyl sulfoxide were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). LC-MS-grade acetonitrile, methanol, formic acid, and distilled water were purchased from Kanto Chemical Co., Inc. (Tokyo, Japan).
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3

Preparation and Purification of FAD-GDH Enzyme

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Distilled water, potassium dihydrogen
phosphate, disodium hydrogen phosphate, d-glucose, l-ascorbic acid, uric acid, acetonitrile, and anionic surfactant sodium
cholate (SC) were purchased from Kanto Chemical Co., Inc. (Tokyo,
Japan). Single-walled carbon nanotubes (SWNT; 1.2–1.7 nm in
diameter, 0.5 mm average length, Super Pure Tubes) were purchased from Nanointegris Inc. (Boisbrin,
Canada). FAD-GDH (EC 1.1.5.9) from thermophilic T.
emersonii
was identified by our group.8 (link) Recombinant T. emersonii FAD-GDHs were expressed by the bacterium E. coli and yeasts P. pastoris(8 (link)) and S. cerevisiae. The S. cerevisiae INVSc1 strain
and pYES2 expression vector were purchased from Thermo Fisher Scientific
(Waltham, USA). In both yeast expression systems, the α-factor
secretion signal was fused to FAD-GDH, and FAD-GDH was secreted into
the culture media. FAD-GDH was purified by methods from our group.8 (link)
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4

Quantification of Steroid Metabolites

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PREGS sodium salt and E2S sodium salt were purchased from Sigma-Aldrich Japan (Tokyo, Japan), DHEAS hydrate was from Tokyo Kasei Kogyo (Tokyo, Japan), and CRTS potassium salt was from Toronto Research Chemicals Inc. (TRC, North York, ON, Canada). Their stable isotope-labeled compounds, PREGS-13C2-d2 (isotopic purity >98%, Sigma-Aldrich, St. Louis, MA, USA), DHEAS-d5 (isotopic purity >90%, Sigma-Aldrich), CRTS-d4 (isotopic purity >95%, TRC), and E2S-d4 (isotopic purity 95%, TRC), were used as internal standards (IS). These standard and IS compounds (Figure S2) were dissolved in LC–MS-grade methanol to a concentration of 0.1 mg mL−1 and diluted with LC–MS-grade distilled water to the required concentration prior to use. The mixed standard solution consisted of 20 ng mL−1 each of PREGS, DHEAS, and CRTS and 100 ng mL−1 E2S, whereas the mixed IS solution consisted of 2 ng mL−1 each of PREGS-13C2-d2, DHEAS-d5, and CRTS-d4 and 10 ng mL−1 of E2S-d4. All of these solutions were stored at 4 °C. LC−MS-grade acetonitrile and distilled water used as mobile phases were purchased from Kanto Chemical (Tokyo, Japan); all other chemicals were of analytical reagent grade.
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5

Quantitative Analysis of GM1 Ganglioside in CNS

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High-performance liquid chromatography-MS/MS (HPLC-MS/MS) was used for the quantification of GM1-ganglioside in the CNS. Total lipids were extracted using the Folch method.48 (link) Briefly, tissues were homogenized with distilled water, followed by extraction with chloroform:methanol (2:1, v/v). The extracted samples were analyzed using LC-MS/MS (LCMS-8040, Shimadzu). A Synergi MAX-RP (150 mm × 4.6 mm, Phenomenex, Torrance, CA) was used for separation. The flow rate was 0.4 mL/min. Mobile phase A was distilled water (Kanto Chemical, Tokyo, Japan) containing 2 mM ammonium acetate (Fujifilm Wako Pure Chemical) and 0.1% formic acid (Nacalai Tesque, Kyoto, Japan). Mobile phase B consisted of methanol (Fujifilm Wako Pure Chemical) containing 2 mM ammonium acetate and 0.1% formic acid. The mobile phase gradients were as follows: 0–2 min, 100% B; 2–30 min, 100% B; and 30.1–31 min, 0% B. GM1 ganglioside isoforms were quantified by multiple reaction monitoring (MRM) in negative ion mode. The transitions are as follows: C16 (d18:1/16:0); m/z 1,517.35 > 290.1, C18 (d18:1/18:0); m/z 1,545.85 > 290.1, C20 (d18:1/24:0); m/z 1,573.45 > 290.1. The concentrations of these isoforms were calculated using a calibration curve of GM1 ganglioside (860065, Avanti Polar Lipids, Birmingham, AL).
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6

Pharmaceutical Synthesis and Characterization

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PS300 was generously provided by Tomita Pharmaceutical Co., Ltd. (Tokushima, Japan). CaSiO3 was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). IBU was generously provided by Kaken Pharmaceutical Co., Ltd. (Tokyo, Japan). Zirconia beads of 0.3 mm in diameter were purchased from Nikkato Co., Ltd. (Osaka, Japan). Ethanol, 1 mol/L sodium hydroxide solution, calcium chloride, phosphoric acid, acetonitrile, and distilled water were purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). All received chemicals and solvents were of analytical reagent grade.
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7

Neonicotinoid Pesticide Quantification

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Acetamiprid, dinotefuran, imidacloprid, nitenpyram and thiacloprid were purchased from Kanto Chemical Corp. (Tokyo, Japan). Clothianidin, Clothianidin-d3, dinotefuran-d3, imidacloprid-d4, thiacloprid-d4, Thiamethoxam-d4, and N-desmethylacetamiprid (DMAP) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Hydroxyimidacloprid was purchased from Hypha Discovery (Slough, UK). Acetamiprid-d6 and nitenpyram-d3 were purchased from Hayashi Pure Chemical Ind. (Osaka, Japan). Thiamethoxam was purchased from Dr. Ehrenstorfer. Acetonitrile, dichloromethane formic acid, ammonium acetate and distilled water were all HPLC grade and were purchased from Kanto Chemical.
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8

Lipid Analysis Protocol Using SFC

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Ammonium acetate was obtained from Sigma–Aldrich (#73594, St. Louis, MO, U.S.A.). LC/MS-grade methanol and distilled water were purchased from Kanto Chemical Co. (#11307-2B, Tokyo, Japan). HPLC-grade chloroform was obtained from Kishida Chemical (#140-16013, Osaka, Japan). All synthetic lipid standards were purchased from Avanti Polar Lipids Inc. (Alabaster, AL, U.S.A.). Carbon dioxide (99.9% grade; Yoshida Sanso Co., Fukuoka, Japan) was used as the SFC mobile phase.
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9

Y5 Receptor Antagonist Synthesis and Characterization

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N-(((1r,4r)À4-( (6-fluorobenzo[d]oxazol-2-yl) amino) cyclohexyl) methyl)À2-methylpropane-2-sulfonamide was designed as a Y5 receptor antagonist of neuropeptide Y by Shionogi & Co. Ltd. (Osaka, Japan), which was used as the active pharmaceutical ingredient (API) in this study. The physicochemical properties of the compounds were calculated using ACD/Percepta 2019. 2. 2 (Advanced Chemistry Development, Toronto, Canada). Ethylene glycol, ethanol, and ethyl acetate were purchased from the Fujifilm Wako Pure Chemical Corporation (Osaka, Japan). Acetonitrile and distilled water were obtained from Kanto Chemical Co., Inc. (Tokyo, Japan) and Otsuka Pharmaceutical Co. Ltd. (Tokyo, Japan), respectively.
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