Fatty Acid Methyl Ester Mix (DPPH)

Materials and methods
Standards and reagents
Fatty acid methyl ester mix (PUFA No 3 from menhaden oil) was purchased from Supelco (USA). Folin–Ciocalteu reagent, gallic-acid, phloroglucinol, 1, 1-Diphenyl-2-picrylhydrazyl (DPPH), ascorbic acid and BHT were purchased from Sigma-aldrich, (Germany); sodium carbonate, YPD medium and Mueller-Hinton agar were purchased from Merck (Germany); Chloramphenicol, Ciprofloxacin and McFarland scale were purchase from Biomerieux (France). Methanol, n-Hexane and dichloromethane were purchased from Fisher Scientific (U. K). Dimethyl sulfoxide, 3-[4, 5-dimethylthiazol-2-yl]-2 5-diphenyl tetrazolium bromide (MTT) and cisplatin were purchased from Sigma-Aldrich (U.S.A.); isopropanol was purchased from Panreac

(2006), after slight modifications. The fundamental principle of the DPPH method is the reduction of the DPPH radical in an ethanolic solution by an H-donator antioxidant (AH) to form the non-radical form DPPH-H. In a microtube, 10 µL of each fraction at different concentrations (10 - 1000 µg/mL) were mixed with 990 µL of a DPPH solution (0.1mM) prepared daily. The reaction was allowed to develop for 30 minutes in the dark at room temperature, and then the absorbance was read at 515 nm with a spectrophotometer (Spectronic Helios Alpha UV-Visible, Thermo Electron Corporation, U.S.A). The analysis was done in triplicate for each

(2004) as follows: The reaction was carried out in 75 mM phosphate buffer (pH 7.4), and the final reaction mixture was 200 µL. Sample (20µL) and fluorescein (120 µL; 70 nM, final concentration) were placed in the well of the microplate. The mixture was pre-incubated for 15 min at 37 °C. AAPH solution (60 µL; 12 mM, final concentration) was added rapidly using a multichannel pipet. The microplate was immediately placed in the reader and the fluorescence recorded every minute for 240 min. The microplate was automatically shaken prior each reading. A blank using phosphate buffer instead of the fluorescein and eight calibration solutions using Trolox (1-8 µM, final concentration) as antioxidant were also carried out in each assay. All the reaction mixtures were prepared in duplicate, and at least three independent assays were performed for each