4 nitrophenyl β d glucuronide

Gastrodin (>98%) was purchased from Tokyo Chemical Industry. 4-HBA (≥98%), erythromycin, oxytetracycline hydrochloride, cefadroxil, 4-nitrophenyl β-d-glucuronide, 4-nitrophenyl sulfate, and 4-nitrophenyl β-d-glucopyranoside were purchased from Sigma-Aldrich. The anaerobe gas-generating system was purchased from Becton, Dickinson and Company. HPLC-grade methanol was purchased from J. T. Bakers (Central Valley, PA, USA). All other chemicals used were of reagent grades and were used as received.

Leucine aminopeptidase (EC 3.4.1.2) microsomal from porcine kidney (LAP), β-glucuronidase (EC 3.2.1.31) from helix pomatia type H-2 (GLU), N-(tert-Butoxycarbonyl)glycine, glycine, triglycine, σ-pinene, triphosgene, triethylamine, N-carboxyanhydride (NCA), 2-aminoethyl methacrylate hydrochloride (2-AMEA), allylamine, 4-nitrophenyl- β-D-glucuronide (PNPG), hydroxybenzotriazole (HOBt), N,N’-diisopropylcarboiimide (DIC), acrylic acid N-hydroxysuccinimide (Ac–NHS ester), deuterium oxide (D₂O), dimethylsulfoxide-d₆ (DMSO-d), chloroform-d (CDCl₃), 2,2-dihydroxyindane-1,3-dione (Ninhydrin) and tris(hydroxymethyl)aminomethane hydrochloride (Tris–HCl) was purchased from Sigma Aldrich (St. Louis, MO, USA). GGRSK was custom-synthesized from Lifetein (Somerset, NJ, USA) and Cosmogenetech (Seoul, South Korea). Urokinase (EC 3.4.4.21)-type plasminogen activator (uPA) was purchased from Fortunachem (Wuhan, China). Acrylic acid (AAc), sodium hydroxide (NaOH), ethyl acetate anhydrous (EA), dimethylformamide (DMF), tetrahydrofuran (THF), chloroform (CCl₄), methanol (MeOH), n-butanol (BuOH), acetic acid, acetonitrile, and trifluoroacetic acid were purchased from Daejung (South Korea). 2-hydroxy-2-methlypropiophenone (Darocur 1173) and dimethylsulfoxide anhydrous (DMSO) were purchased from TCI (Tokyo, Japan).

Benzo[a]pyrene (BaP, Seoul, South Korea, 98% pure), gallic acid, methanol (HPLC-grade), N-methyl-N-(trimethylsilyl)-trifluoroacetamide (MSTFA), and 4-Nitrophenyl β-D-glucuronide (PNPG) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Folin–Ciocâlteu phenol reagent was obtained from Merck (Darmstadt, Germany), and peanut oil was obtained from Sekem company (Belbeis, Egypt). BaP was handled in accordance with NIH safety guidelines [133 ] and dissolved in peanut oil. Fresh solutions were prepared weekly and were stored at room temperature in the dark.

The fixed colon tissue was stained with methylene blue (0.2%) in saline for 2–3 min. Samples were morphologically examined under an optical microscope (Olympus, Optical Co. Ltd., Tokyo, Japan) at 40 × magnification, and ACF were counted.
Faecal pre-treatment: fresh faecal samples were fully suspended in 0.1 M potassium phosphate buffer (pH 7.0), pre-cooled by a homogenizer, and then homogenized. The supernatant was obtained by ultrasound at 4°C for 10 s (six times in total) and centrifugation at 2,000 g for 15 min (18 (link)).
β-glucuronidase assay: (18 (link), 19 (link)) β-glucuronidase activity in rat faeces was determined according to the consumption of 4-nitrophenyl-β-D-glucuronide (Sigma-Aldrich, St Louis, MO, USA) in the reaction system. The 1 ml reaction system contained 0.02 M potassium phosphate buffer (pH 7.0), 0.1 mm EDTA, 2 mm 4-nitrophenyl-β-d-glucosidic acid solution and 100 μl faecal supernatant; incubation was performed for 30 min at 37°C. Then, 0.2 M NaOH solution was added to stop the reaction, and the release of 4-nitrophenol was measured at 405 nm.
β-glucosidase assay: (18 (link), 19 (link)) the procedure and reaction system for the β-glucosidase assay were the same as described above for the β-glucuronidase assay. The reaction substrate was 4-nitrophenyl-β-D-glucopyranoside (Bailingwei Technology Co., Ltd., Beijing, China).