4 aminoantipyrine

To assess glucose concentration in the urine, mice were restrained and urine was collected with a pipette and frozen at −20 °C. Samples were treated with glucose oxidase (G7141, Sigma-Aldrich) and peroxidase (P8250, Sigma-Aldrich), and the resulting H₂O₂ concentration was quantified by a colorimetric assay using glucose as standard and employing 4-aminoantipyrine (A4382, Sigma-Aldrich) and sodium 3,5-dichloro-2-hydroxybenzenesulfonate (D4645, Sigma-Aldrich) as coloring reagents. The plate was read at 520 nm on a Synergy H1 Hybrid Multi-Mode Reader (BioTek, Winooski, VT, USA).

Congo red (CR), acetone, hydrogen peroxide (H₂O₂), and 4-aminoantipyrine were all purchased from Sigma-Aldrich (St. Louis, MO, USA). Detailed properties of the dye along with the structure are presented in Table 1.

The hydrogen peroxide in the culture suspension was quantified as described previously (11 (link)). Briefly, 650 μl of culture supernatant was added to 600 μl of a solution containing 2.5 mM 4-amino-antipyrine (4-amino-2,3-dimethyl-1-phenyl-3-pyrazolin-5-one) (Sigma-Aldrich) and 0.17 M phenol. The reaction proceeded for 4 min at room temperature; horseradish peroxidase (Sigma-Aldrich) was then added to a final concentration of 50 mU/ml in 0.2 M potassium phosphate buffer (pH 7.2). After 4 min of incubation at room temperature, the optical density at 510 nm was measured with a Unico 2100 visible spectrophotometer (Unico, Shanghai, China). A standard curve was generated with known concentrations of chemical H₂O₂.

Neuro-2a (N2a) cell line was acquired from the Collection (ATCC, Manassas, VA, United States) whereas 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT), bovine serum albumin (BSA), penicillin, streptomycin, 2,2-diphenyl-1-picrylhydrazyl (DPPH), Trolox, Monoamine oxidase A (MAO-A), Trizma base, 4-aminoantipyrine, 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), tyramine, levodopa (L-DOPA), horseradish peroxidase, 2,2′-azobis (2-methyl-propionamidine)-di-hydrochloride (AAPH), hydrogen peroxide (30%), vanillic acid, tyrosinase, 2,4,6-Tris (2-pyridyl)-1,3,5-triazine (TPTZ), galantamine, 2′,7′-Dichlorodihydrofluorescein Diacetate (DCFH-DA), xanthine, ferrous sulfate (FeSO₄), acetylcholinesterase (AChE), acetylthiocholine iodide (ATCI) were obtained from Sigma-Aldrich (Madrid, Spain). Clorgyline and α-kojic were from Cymit química (Barcelona, Spain). Sodium carbonate (Na₂CO₃), HCl, NaCl, dimethyl sulfoxide (DMSO), methanol, and potassium phosphate were supplied from Panreac (Barcelona, Spain). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), Nitroblue Tetrazolium (NBT) and xanthine oxidase were purchased from Vidrafoc (Barcelona, Spain) and iron chloride (FeCl₃) from Laboaragon (Zaragoza, Spain). All reagents used were of analytical quality.

4-aminophenylacetic acid, 4-aminophenethyl alcohol, 4-fluoroaniline, 4-(heptadecafluorooctyl)aniline, 4-aminoantipyrine, 4-(4-aminophenyl)butyric acid, and 3,4,5-trimethoxyaniline, sodium nitrite, hydrogen peroxide 30%, sodium chloride, sodium citrate, and citric acid were obtained from Sigma-Aldrich (Taufkirchen, Germany). CuSO₄ anhydrous 98% was purchased from Alfa Aesar (Karlsruhe, Germany). Hydrochloric acid (37%) was obtained from Carl Roth (Karlsruhe, Germany). Sulphuric acid (98%) was purchased from Merck Millipore (Darmstadt, Germany). Chemically pure acetonitrile, ethanol, and purified water (18 MΩ cm⁻¹, Millipore Darmstadt, Germany) were used to prepare solutions.

Sucrose, H₂O₂, ethanol, sodium phosphate monobasic, sodium phosphate dibasic, and sodium carbonate (Cicarelli, Santa Fe, Argentina), lactic acid (Cicarelli, Origin in France, packed in Santa Fe, Argentina), fructose and acetone (Cicarelli, Origin in USA, packed in Santa Fe, Argentina), glucose (Anedra, Nantong, China), choline chloride (Sigma-Aldrich, Beijing, China), urea, ABTS, 4-aminoantipyrine, bromothymol Blue, quercetin, Folin and Ciocalteu′s phenol reagent, and gallic acid (Sigma Aldrich, St. Louis, USA), citric acid (Anedra, Bs As, Argentina), propylene glycol (Biopack, CABA, Argentina), aluminum chloride (Sigma-Aldrich, Taufkirchen, Germany), chloroform (Cicarelli, Origin in the Republic of Korea, packed in Santa Fe, Argentina), apomorphine hydrochloride (Merck, Darmstadt, Germany), phenol (Cicarelli, Origin in USA, packed in Santa Fe, Argentina), sulfuric acid (Cicarelli, Origin in Spain, packed in Santa Fe, Argentina).

Amylase inhibitory activity was measured using a microplate method: 10 μL of a sample solution in DMSO, 30 μL of phosphate buffer (pH 5.0), and 10 μL of amylase from Aspergillus niger (3 U mL⁻¹, Sigma) which were incubated for 20 min at 45°C. Then 10 μL of 2% starch solution, 40 μL of phosphate buffer (pH 5.0), and 100 μL of the reagent were added and incubated for 30 min at 50°C. Absorbance was measured at 510 nm. The reagent was a solution of K₂HPO₄ (0.8 mM), KH₂PO₄ (0.4 mM), phenol (220 mM), 4-aminoantipyrine (1.5 μM), glucose oxidase from Aspergillus niger (3 U mL⁻¹; Sigma), and peroxidase from horseradish (0.3 U mL⁻¹) in deionized water. A 2% solution of acarbose was used as a positive control (PC), and water was used as a negative control (NC). The experiment was carried out in triplicate and averaged. The ability to inhibit amylase was calculated using the following equation: inhibitory ability (%) = [(A₅₁₀^NC − A₅₁₀^PC) − (A₅₁₀^Sample − A₅₁₀^PC)/(A₅₁₀^NC − A₅₁₀^PC)] × 100, where A₅₁₀^NC is the absorbance of the negative control, A₅₁₀^PC is the absorbance of the positive control, and A₅₁₀^Sample is the absorbance of the sample solution. The IC₅₀ value is the effective concentration at which amylase activity was inhibited by 50%. Values are expressed as mean obtained from 5 independent experiments.