Optimization of a DNA nicking assay to evaluate Oenocarpus bataua and Camellia sinensis antioxidant capacity

• Published in: International journal of molecular sciences 2014-10, Vol.15 (10), p.18023-18039
• Main Authors: Leba, Louis-Jérôme, Brunschwig, Christel, Saout, Mona, Martial, Karine, Vulcain, Emmanuelle, Bereau, Didier, Robinson, Jean-Charles
• Format: Article
• Language: English
• Subjects: Amazonian palm; antioxidant; Antioxidants; Antioxidants - chemistry; Antioxidants - metabolism; Arecaceae - chemistry; Arecaceae - metabolism; Camellia sinensis; Camellia sinensis - chemistry; Camellia sinensis - metabolism; Chromans - chemistry; Chromans - metabolism; Deoxyribonuclease I - metabolism; DNA Breaks, Single-Stranded; DNA nicking assay; Enzyme Assays; Fenton; Flowers & plants; Gallic Acid - chemistry; Gallic Acid - metabolism; Hydroxyl Radical - chemistry; Oenocarpus bataua; Phytochemicals; Plant Extracts - chemistry; Plant Extracts - metabolism; Plant Leaves - chemistry; Plant Leaves - metabolism; Plasmids - genetics; Plasmids - metabolism; prooxidant; pUC18; Quercetin - chemistry; Quercetin - metabolism
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms151018023

This study was aimed at assessing the DNA damage protective activity of different types of extracts (aqueous, methanolic and acetonic) using an in vitro DNA nicking assay. Several parameters were optimized using the pUC18 plasmid, especially FeSO4, EDTA, solvent concentrations and incubation time. Special attention has been paid to removing the protective and damaging effect of the solvent and FeSO4 respectively, as well as to identifying the relevant positive and negative controls. For each solvent, the optimal conditions were determined: (i) for aqueous extracts, 0.33 mM of FeSO4 and 0.62 mM of EDTA were incubated for 20 min at 37 °C; (ii) for acetone extracts, 1.16% solvent were incubated for 15 min at 37 °C with 1.3 mM of FeSO4 and 2.5 mM of EDTA and (iii) for methanol extracts, 0.16% solvent, were incubated for 1.5 h at 37 °C with 0.33 mM of FeSO4 and 0.62 mM of EDTA. Using the optimized conditions, the DNA damage protective activity of aqueous, methanolic and acetonic extracts of an Amazonian palm berry (Oenocarpus bataua) and green tea (Camellia sinensis) was assessed. Aqueous and acetonic Oenocarpus bataua extracts were protective against DNA damage, whereas aqueous, methanolic and acetonic extracts of Camellia sinensis extracts induced DNA damage.