ORAC and Total Phenolic Content

A separate extract was prepared for the determination, firstly, of the antioxidant capacity using the hydrophilic Oxygen Radical Adsorption Capacity (ORAC-FL) assay and secondly, the total phenolics in the sample. Twenty five milligrams (25 mg) of the freeze dried sample was placed in an ASE 350 cell and extracted with acetone:water:acetic acid (AWA) at the ratio of 70%:29.5%:0.5%. Parameters for the ASE 350 were: 80°C with 5 min static time; 60% flush; 60 s purge; 1 cycle; and 1500 psi. Aliquots of 0.2 ml were subsequently reacted with Folin-Ciocalteu phenol reagent (Sigma Aldrich, St. Louis, MO, United States) for the determination of total phenolics by the modified Folin-Ciocalteu assay (Prior et al., 2005). A series of known concentrations of gallic acid (Sigma Aldrich, St. Louis, MO, United States) was prepared and reacted with the same reagent to create a calibration curve that was in turn used to determine the concentration in the sample extracts. Consequently, the total phenolics were expressed as gallic acid (GA) equivalents. A diluted (1/50) AWA extract was subjected to the ORAC-FL assay as described by Wu et al. (2004) and Prior et al. (2005). Specifically, dilutions of the AWA extracted samples were prepared in a 75 mM phosphate buffer solution (pH 7.0). Aliquots of 20 μL of the diluted sample were placed (in triplicate) in a 96 well transparent flat bottom microplate (Thermo Fisher Scientific, Waltham, MA, United States), along with appropriately diluted Trolox [(±)-6-Hydroxy-2,5,7,8-tetramethylchromane- 2-carboxylic acid; Sigma-Aldrich, St Louis, MO, United States] standards (in triplicate), under subdued light. A “forward-then-reverse” pattern was used to place samples in the microplates, and edge wells were not used for standards or samples due to possible plate effects. Samples were analyzed on a BioTek Synergy Hybrid plate reader (BioTek, Winooski, VT, United States) which automatically added 200 μL of diluted fluorescein (Sigma-Aldrich, St. Louis, MO, United States) into each well, followed by 20 μL of the peroxyl radical generator (AAPH [2′2′azobis (2-amidinopropane)] Sigma-Aldrich, St. Louis, MO, United States). The assay was monitored every few minutes for 2 h, at 37°C. The ORAC-FL value was calculated from the area under the decay curve, and was reported as μmol Trolox equivalents (TE)/g dry weight.