5 5 dithio bis nitrobenzoic acid dtnb

Alanine aminotransferase (ALT), alkaline phosphatase (ALP), aspartate aminotransferase (AST), total protein, albumin, urea, creatinine, total bilirubin, triglycerides, total cholesterol assay kits, and GOD-POD glucose analysis kit were purchased from Aggappe Diagnostics, Ernakulam, India. Reduced glutathione (GSH) and 5,5-dithio(bis)nitrobenzoic acid (DTNB) were purchased from Sigma-Aldrich, Bangalore, India. All the chemicals and reagents used in the study were of analytical grade.

Dichloromethane, ethyl acetate, hexane, sodium bicinchoninate, 7, 12-dimethylbenz(α)anthracene, 5-5-dithiobisnitrobenzoic acid (DTNB), sodium dithionate, L-γ-glutamyl-4-nitroanilide, H₂O₂, and malondialdehyde (MDA) were purchased from Sigma-aldrich, Bangalore, India. Xylenol orange, ammonium ferrous sulfate, cyclohexane, 2, 6-dichlorophenol-indophenol (DCPIP), reduced nicotinamide adenine dinucleotide (NADH), reduced nicotinamide adenine dinucleotide phosphate (NADPH), bovine serum albumin (BSA), potassium ferricyanide, 1-chloro-2,4-dinitrobenzene (CDNB), glycylglycine, reduced glutathione (GSH), oxidized glutathione (GSSG), ascorbic acid, guaiacol, Triton-X, nitroblue tetrazolium chloride (NBT), hydro-xylamine hydrochloride, and pyruvate were purchased from Himedia Laboratories, Mumbai, India. DMBA (7, 12- dimethylbenz(α)anthracene) was obtained from Sigma-aldrich, Bangalore, India. Autopack kit for analyzing bilirubin was purchased from Beacon Diagnostics, Gujarat, India, while alkaline phosphatase, serum glutamic oxaloacetic transaminase and serum glutamic pyruvic transaminase autopack kits were purchased from Delta Labs, Sindhudurg, India. Rest of the chemicals used in the present study were of analytical grade.

Electric eel acetylcholinesterase (type-VI-S, Sigma-Aldrich USA), Aquine butyrylcholinesterase (Sigma-Aldrich USA), Acetylthiocholine Iodide (Sigma-Aldrich UK), Butyrylthiocholin iodide (Sigma-Aldrich Switzerland), 5,5-dithio-bis-nitrobenzoic acid (DTNB) (Sigma-Aldrich Germany), Potassium phosphate buffer (pH 8.0), Galantamine from Lycoris Sp. (Sigma-Aldrich France).

The chemicals used for the extraction process were methanol (99.9% purity, Merck, Darmstadt, Germany), n hexane (99% purity, Sigma-Aldrich, Darmstadt, Germany), distilled water, ethanol (95% purity, Merck, Darmstadt, Germany), acetone (Sigma-Aldrich, Darmstadt, Germany), dichloromethane (Sigma-Aldrich, Darmstadt, Germany), and carbon dioxide (99.9% purity, Alpha Gas Solution, Selangor, Malaysia). For the bioassay tests, the chemicals and reagents used were gallic acid (99% purity, Merck, Darmstadt, Germany), 1,1-diphenyl-2-picrylhydrazyl (≥85% purity, Sigma-Aldrich, Burlington, MA, USA), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (≥98% purity, Sigma-Aldrich, Burlington, MA, USA), methanol and ethanol (99.9% purity, Merck, Darmstadt, Germany). Acetylthiocholine iodide (ATCI), AChE, bovine serum albumin (BSA), 5,5-Dithio(bis)nitrobenzoic acid (DTNB) and galanthamine were obtained from Sigma-Aldrich, Steinheim, Germany). The AChE used in the assay was from electric eel (type-VI-S, EC 3.1.1.7, Sigma-Aldrich, St. Louis, MO, USA), BChE was from horse (EC 3.1.1.8) and butyrylthiocholin chloride was purchased from Sigma (St. Louis, MO, USA). The HIV-1 protease inhibitor screening kit (fluorometric) was purchased from Biovision Incorporated (Milpitas, CA, USA). All chemicals and reagents used in the study were of analytical grade.

l-Ornithine, putrescine and pyridoxal 5-phosphate (PLP), polyacrylamide solution and 5,5-dithiobisnitrobenzoic acid (DTNB) were purchased from Sigma–Aldrich Co. (Saint Louis, MO, USA). 3-Methyl-2-benzothiazolinone hydrazone hydrochloride was obtained from Merck Co. (Darmstadt, Germany). All other chemicals were of analytical grade.

A 96-well microplate assay was developed from the modification of the methods described previously [5 (link)]. The frozen cell pellets were re-suspended in 200–300 µL of 50 mM Tris buffer (pH 7.4 at 37 °C). The cell pellets were sonicated using a Labsonic^®M (Sartorius Stedim Biotech, Göttingen, Germany). Between 15–30 µg of protein was assayed in duplicate spectrophotometrically, using 1 mM acetylthiocholine iodide (Sigma-Aldrich, St. Louis, MO, USA) as the substrate for AChE activity and 2 mM S-Butyrylthiocholine iodide (Sigma-Aldrich, St. Louis, MO, USA) as the substrate for BChE activity. The chromagen 5,5′-dithio-bis(nitrobenzoic acid) (DTNB, Sigma-Aldrich, St. Louis, MO, USA) was used at a final assay concentration of 0.3 mM, and 10 µM of eserine sulfate (Sigma-Aldrich, St. Louis, MO, USA) was used in duplicate parallel samples to correct for non-enzymatic substrate hydrolysis. The microplate containing the sample, chromagen, and an inhibitor (if required) was pre-incubated for 10 min at 37 °C with shaking. The substrate was added, and the microplate was read at 412 nm for 20 min at 41-sec intervals with mixing. The specific activity was calculated as nmoles product formed per minute per mg protein using the extinction coefficient 14,150 M⁻¹ cm⁻¹.

Potassium phosphate buffer (pH 8.0), Galantamine from Lycoris Sp. (Sigma-Aldrich France), Butyrylthiocholin iodide (Sigma-Aldrich Switzerland), Acetylthiocholine Iodide (Sigma-Aldrich UK), 5,5-dithio-bis-nitrobenzoic acid (DTNB) (Sigma-Aldrich Germany), Electric eel acetylcholinesterase (type-VI-S, Sigma-Aldrich USA) and Aquine butyrylcholinesterase (Sigma-Aldrich USA) were used in enzyme inhibition assays. For antioxidant assays, DPPH (Sigma Aldrich CHEMIE GmbH USA), H₂O₂ (Riedel-de Haen Germany) and ABTS (Sigma-Aldrich Germany) were used. All solvents used in the assays were of analytical grades purchased from Sigma-Aldrich.