Tyrosinase from mushroom

Brassinin, 2,4,6-Tris(2-pyridyl)-s-triazine, Iron(II) sulfate heptahydrate, Iron(III) chloride hexahydrate, tyrosinase from mushroom, L-tyrosine, L-DOPA, phenylmethylsulfonyl fluoride, α-melanocyte stimulating hormone (α-MSH), and Triton X-100 were obtained from Sigma-Aldrich (St. Louis, MO, USA). All materials related to cell culture were purchased from Welgene (Daegu, Korea).

Acetone was purchased from Acros Organic, Fisher Scientific (Illkirch, France) and used for the extraction. Acetonitrile, methanol grade MS, and Milli-Q water, purchased from Carlo-Erba-SDS (Val de Reuil, France), were used for HPLC-DAD and HPLC–MS analyses. Standards (Sigma-Aldrich, St. Louis, MO, USA) were used to evaluate the biological activities. Reagents used to evaluate the extract activities were as follows: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide (MTT), 1,1-phenyl-2-picrylhydrazyl (DPPH), 2,4,6-tripyridyl-s-triazine (TPTZ), tyrosinase from mushroom, L-DOPA substrate, porcine pancreatic elastase, and N-succinyl-ala-ala-ala-p-nitroanilide, which were purchased from Sigma-Aldrich. Anhydrous methanolic HCl reagents and boron trifluoride (BF₃) were of reagent grade.

Wampee (Clausena lansium (Lour.) Skeels) (identified by Dr. Zhanping Gou, Department of pharmacognosy, Guangdong Medical University, China) was purchased from the local wholesale market (Maoming, Guangdong, China). D-Galacturonic acid monohydrate standard (purity ≥97%) was purchased from Sigma company (St. Louis, USA) and dialysis bags were obtained from United States union carbonization (Connecticut, USA). Citric acid, hydrochloric acid, sulfuric acid, and anhydrous ethanol were obtained from Sinopharm group chemical reagent co. LTD (Shanghai, China). EDTA-2Na was purchased from a chemical reagent factory (Guangzhou, China). All reagents are analytical grade. Carbazole (purity ≥96%) was purchased from Aladdin biochemical technology co., LTD (Shanghai, China). α-MSH, tyrosinase from mushroom, and L-DOPA were purchased from Sigma company (St. Louis, USA). TRY, tyrosinase-related protein 1 (TYRP-1), tyrosinase-related protein 2 (TYRP-2) and MiTF primary antibodies were purchased from Abcam company (Cambridge, UK). Creb (Phosopho-S133) antibodies (pCREB) were purchased from Signalway Antibody company (Maryland, USA).

RK, pHPA, 2pHP-IA, pHPE, and H₂O₂ were purchased from Wako Pure Chemical Industries (Osaka, Japan). RD and EDTA were purchased from Nacalai Tesque (Kyoto, Japan). MEHQ was purchased from Tokyo Chemical Industry (Tokyo, Japan), respectively. Tyrosinase from mushroom, neutral red, and Triton X-100 reduced were purchased from Sigma-Aldrich (St. Louis, MO, USA), and 2′,7′-dichlorofluorescin diacetate (DCFDA) was purchased from EMD Millipore (Billerica, MA). Hoechst33342 was purchased from Life Technologies (Carlsbad, CA). 5,5-Dimethyl-1-pyrroline-N-oxide (DMPO) was purchased from Labotec (Tokyo). Dimethyl sulfoxide (DMSO) was purchased from Pierce Biotechnology (Rockford, IL). Ferrous sulfate was purchased from United States Pharmacopeial Convention (Rockville, MD). Dulbecco's modified Eagle medium (DMEM) was purchased from Nissui Pharmaceutical (Tokyo). Fetal bovine serum (FBS) was purchased from Biological Industries Israel (Kibbutz Beit-Haemek, Israel).

Tyrosinase from mushroom (≥1000 U/mg solid) was purchased from Sigma-Aldrich (St. Louis, MO, USA). NR latex with low ammonia content (product name: Ulacol^®) was purchased from HR-TEC (Osaka, Japan). The composition of NR latex used in this study was as follows: NR, 61.5 wt%; ammonia, 0.1 wt%; and H₂O, 38.4 wt%. The other chemicals were used as received, without purification, unless otherwise noted.

Acetone (99.5%), acetic acid (99.5–99.9%), diphenylboryloxyehtylamine (NP) (≥97.0%), ethanol (96%), ethyl acetate (99.8%), 2-isopropyl-5-methyl-phenol (thymol) (≥98.5%), methanol (99.8%), o-phosphoric acid (85%), polyethylene glycol—4000 (PEG-4000), phosphate buffer, sodium hydroxide (≥98%), sulfuric acid (96–98%), and sodium acetate buffer were from POCH (Gliwice, Poland). P-Anisaldehyde (AS) (≥98%), bovine serum albumin (BSA) (≤100%), 2,2-diphenyl-1-picrylhydrazyl (DPPH) (≤100%), 3,4-dihydroxy-L-phenylalanine (L-DOPA) (>98.0%), α-glucosidase from Saccharomyces cerevisiae (≥10 units/mg protein), lipase from porcine pancreas (30–90 units/mg protein), 1-naphthyl acetate (≥98%), 2-naphthyl acetate (≤100%), 2-naphthyl α- D glucopyranoside ((≤100%), polyethylene glycol tert-octylphenyl ether (Triton X) (≤100%), rosavin (≥98.0%), salidroside (≥98.0%), p-tyrosol (98%), Fast Blue B salt (95%), tris(hydroxymethyl)aminomenthane hydrochloride (TRIS) buffer, and tyrosinase from mushroom (≥1000 unit/mg solid) were purchased from Sigma Aldrich (Poznań, Poland). Pure water was from the Millipore Q system (Millipore, Bedford, MA, USA). All reagents were of the analytical grade. Acetonitrile used in HPLC-UV/Vis-DAD quantification procedure was purchased from Romil (Cambridge, UK). TLC silica gel 60 F₂₅₄ (20 × 10 cm) plates were purchased from Merck, (Darmstadt, Germany).