P nitrophenyl α d glucopyranoside pnpg

Acarbose, acetic acid, 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH), bovine serum albumin (BSA), dichlorofluorescin diacetate (DCFH-DA), dimethylsulfoxide, fluorescein, α-glucosidase (EC 3.2.1.20, Saccharomyces cerevisiae type I, ≥10 Units/mg protein), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox), luteolin, methanol, p-nitrophenyl-α-D-glucopyranoside (p-NPG), thiazolyl tetrazolium bromide, and vernodalol were purchased from Merck Life Science S.r.l. Milan, Italy. The purity of the standards was ≥97%, while for the other compounds it was at least of analytical grade. Specifically, the purity of luteolin was 98% and that of vernodalol was 95%.

Ethanol 96%, citric acid, acetic acid, trichloroacetic acid (TCA), formic acid, acetonitrile (ACN), sodium phosphate salts (Na₂HPO₄, NaH₂PO₄), p-nitrophenyl-α-D-glucopyranoside (p-NPG), α-glucosidase from rice, sodium carbonate, acarbose and cyaniding were purchased from Merck (Darmstadt, Germany). All reagents used in the experimental part were of analytical purity.

Gallic acid, ascorbic acid, and ellagic acid (>95% purity) were sourced from Sigma Aldrich (Sydney, NSW, Australia). DPPH [2,2-diphenyl-1-picryl hydrazyl], acarbose, sodium carbonate, metaphosphoric acid, acetic acid, ethylenediaminetetraacetic acid (EDTA), formic acid, hydrochloric acid, p-nitrophenyl-α-d-glucopyranoside (pNPG), α-glucosidase, acarbose, organic solvents (HPLC-grade), and other reagents used throughout the study were supplied by Merck or Sigma Aldrich (Sydney, NSW, Australia). In the cytotoxicity assessment, the Caco-2 cell line was purchased from the American Type Culture Collection (Manassas, VA, USA), and HepG2 cell lines were purchased from Sigma Aldrich (Sydney, NSW, Australia). Nunc cell culture flasks and 96-well plates were purchased from Sigma-Aldrich (Sydney, NSW, Australia). Hank’s Balanced Salt Solution (HBSS), penicillin and streptomycin, fetal bovine serum (FBS), glutamate, trypsin-EDTA, non-essential amino acids (NEAA), Dulbecco’s modified eagle medium (DMEM), Dulbecco’s phosphate-buffered saline without calcium and magnesium (PBS), trypan blue exclusion dye, and CyQUANT^® NF Cell Proliferation Assay reagent (Molecular Probes™) were purchased from Invitrogen (Thermo Fisher Scientific Corporation, Waltham, MA, USA).

Acarbose, α-glucosidase (EC 3.2.1.20, Saccharomyces cerevisiae type I, 10 U/mg protein), dimethylsulfoxide (DMSO), luteolin (3’,4’,5,7-tetrahydroxyflavone), methanol, p-nitrophenyl-α-D-glucopyranoside (pNPG), buffered phosphate saline (PBS) and magnolol (5,5′-diallyl-biphenyl-2,2′-diol) were purchased from Merck KGaA, Darmstadt, Germany. The purity of the reference standards was ≥97%, while other chemicals were of at least analytical grade.

To measure β-galactosidase or β-glucuronidase activity cultures were grown in YPD overnight, shifted to SC media with 3% glycerol, 2% ethanol, 3% acetate or without any added carbon source at OD₆₀₀ 0.1 to induce transcription and grown further to OD₆₀₀ = 0.8 to 1.6. Cells were harvested and glass bead-disrupted in β-galactosidase buffer (5.0 mM Tris-HCl pH7.8; 5% glycerol; 10 mM KCl) or β-glucuronidase buffer (10 mM β-mercaptoethanol; 10 mM Na₂EDTA; 0.1% sodium lauroyl sarcosinate; 0.1% Triton X-100; 10 mM sodium phosphate pH 7.0), respectively. Substrates were 4 mg/ml O-nitrophenyl-β-D-galactopyranoside (ONPG (Sigma), molar extinction coefficient ε = 4.5 x 10⁶ M^-1cm^-1 at 420 nm) for the measurement of β-galactosidase activity and 4 mg/ml p-nitrophenyl-α-D-glucopyranoside (PNPG (Sigma), ε = 8800 M^-1cm^-1 at 415 nm [58 (link)]) for the measurement of β-glucuronidase activity. The kinetics of product formation over time were followed and the specific enzyme activities [mU/mg protein] were calculated by (ΔE/min x 10⁶) / (ε [M^-1cm^-1] x protein concentration [mg]).

The fruiting bodies of Inonotus obliquus were purchased from Greater Khingan Range, ground in a tight disintegrator (Micron Co. Ltd., Beijing, China), passed through a rough 40-mesh screen and stored at 4 °C during experiments. DEAE-52 column chromatography was purchased from the Pharmacia Chemical Co. (Trenton, NJ, USA) and Sephadex G-100 was purchased from Waters Chemical Co. (Milford, MA, USA). Monosaccharide standards, insulin, α-Glucosidase, p-nitrophenyl-α-d-glucopyranoside (pNPG) and STZ were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Foetal bovine serum (FBS), Dulbecco’s Modified Eagle’s medium (DMEM), penicillin, streptomycin and phosphate-buffered saline (PBS) were purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). A glucose test kit was purchased from Shanghai Rongsheng Biotech Co., Ltd. (Shanghai, China). All other chemicals and solvents were of analytical grade.