2 2 azobis 2 methylpropionamidine dihydrochloride abap

Tris-HCl (Trizma hydrochloride), ethylenediaminetetraacetic acid (EDTA), 2,2′-azobis(2-methylpropionamidine) dihydrochloride (ABAP), glutathione (GSH), adenosine triphosphate (ATP), glutamate, borate, serine, cysteine, cumene hydroperoxide (CHP), and xylenol orange were purchased from Sigma-Aldrich (St. Louis, MO, USA). KCl, MgCl₂, and FeSO₄ were purchased from Labsynth (Diadema, SP, Brazil). 2′,7′-Dichlorofluorescein-diacetate (H₂DCF-DA) and naphthalene-2,3-dicarboxaldehyde (NDA) were purchased from Molecular Probes (Eugene, OR, USA). Biuret protein assay kit was purchased from Doles (Goiânia, GO, Brazil).

This analysis was proceeded based on Amado et al. [68 (link)]. One aliquot of the homogenate was centrifuged at 14,000 rpm at 4 °C for 10 min to collect the supernatant. From the supernatant, 200 μL was incubated with a peroxyl radical generator (2,2′-azobis-2-methylpropionamidine dihydrochloride (ABAP; 4 mM; Sigma-Aldrich, San Louis, MO, USA) in triplicate to verify the oxidation of ABAP and generate fluorescence. After the reaction with ABAP, the generated fluorescence was measured on a fluorimeter (Victor X3, Perkin Elmer, Waltham, MA, USA) at a controlled temperature of 35 °C. After the first reading to determine the fluorescence value, a total volume of 10 µL of 2′,7′ H2DCF-DA (40 nM) was added to the microplate, with cyclic readings every 5 min for 60 min. High relative areas under the curve established by a second-degree polynomial curve indicate a low antioxidant capacity to neutralize peroxyl radicals. The inverse relative difference between the area with and without ABAP was considered a measure of antioxidant capacity. The results were later expressed as the percentage of control.

Ampelopsis grossedentata leaves (moisture content 9.42%) were purchased from Zhangjiajie, Hunan province, China and were crushed through a 200-mesh sieve. Dihydromyricetin (≥98%), CC (≥98%), citric acid (≥99.5%), glycerol (≥99%), and glucose (≥99%) were obtained from Aladdin (Shanghai, China). In addition, 2,2′-azobis (2-methylpropionamidine) dihydrochloride (ABAP), fluorescein sodium salt, Trolox, and 2′,7′-dichlorofluorescein diacetate (DCFH-DA) were purchased from Sigma-Aldrich LLC (Shanghai, China). Annexin V-FITC/PI apoptosis, 2′,7′-dichlorofluorescein diacetate (DCFH-DA), PI cell cycle, and JC-1 detection kits were obtained from Becton, Dickinson Company (Franklin Lakes, NJ, USA). Human hepatoma (HepG2), hepatocyte (LO-2), breast cancer (MDA-MB-231), and colorectal cancer (Caco-2) cell lines were obtained from the Cancer Institute of Sun Yat-Sen University (Guangzhou, China). Cell cultural media, penicillin, and streptomycin were purchased from Gibco Life Technologies (Grand Island, NY, USA). Fetal bovine serum (FBS) was obtained from Tianhang Biotech Co. Inc (Zhejiang, China). HPLC grade methanol was purchased from Fisher Chemical (Ottawa, ON, Canada). Ultrapure water was obtained from a Thermo Gen Pure ultraviolet (UV)/ultrafiltration water system (Waltham, MA, USA). All other chemicals were of analytical grade.