Starch azure

Pheaotheca triangularis EXF-206, T. salinum EXF-295, and Wallemia ichthyophaga EXF-5676 were obtained from the University of Ljubljana, Biotechnical Faculty, Department of Biology (Ljubljana, Slovenia). Carbon dioxide 2.5 (purity 99.5%) was supplied by Messer MG (Ruše, Slovenia). Peptone from meat, potassium phosphate, potassium dihydrogen phosphate, sodium carbonate, sodium bicarbonate, and acetic acid were purchased from Merck (Darmstadt, Germany). Sodium pyrophosphate decahydrate (≥99.0%), sodium phosphate monobasic (≥99.0%), sodium phosphate dibasic (≥99.0%), albumin from bovine serum (BSA) (≥98.0%), malt extract, agar, d-(+)-glucose, sodium acetate, Sigmacell, glucose assay reagent, Casein, Hammarsten bovine, trichloroacetic acid (TCA), d-(−)-Salicin (≥99.0%), starch azure, and sodium chloride were supplied from Sigma (Schnelldorf, Germany).

The ability of the extracts to inhibit α-amylase was studied according to the spectrophotometric method described by Kladar et al. [5 (link)]. For the purposes of the experiment, porcine 𝛼-Amylase (final reaction mixture activity 0.6 U/mL), Starch azure^® (Sigma Aldrich, Wien, Austria) and sodium phosphate buffer (pH = 7.2) with NaCl were used. Two different concentrations of extracts were added to the reaction mixture; after incubation for 10 min, the reaction was stopped by the addition of acetic acid (50%, m/m). The inhibition percentage was calculated based on the control measurement that contained water instead of the extract. Acarbose was used as a positive control.

Carvacrol (≥97%, GC); p-cymene (≥97%, GC); acetylcholinesterase (AChE) type VI-S from Electrophorus electricus, EC 3.1.1.7, 245 U/mg solid; butyrylcholinesterase (BChE) from equine serum 8.8 U/mg; DTNB [5,5′-dithiobis-(2-nitrobenzoic acid)]; acetylthiocholine iodide (AChI); butyrylthiocholine iodide (BChI); galantamine (USP, reference standard); starch azure; porcine pancreatic amylase; calcium chloride; acetic acid; gallic acid, hydrogen peroxide (1 M); hydrocloridric acid; acetone; N,N-dimethylformamide, 1,10-phenanthroline; ferrous ammonium sulfate; RPMI; l-glutamine; FBS; penicillin/streptomycin; MTT; SDS; primary anti-caspase-3 antibodies were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-GAPDH antibody and enhanced chemiluminescence reagents were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA); horseradish peroxidase-conjugated secondary antibody was purchased from Amersham Biosciences (Pittsburgh, PA, USA). Nitrocellulose was purchased from Biorad (Hercules, CA, USA).