Fatty Acid Methyl Ester Mix (DPPH)

1359 Words6 Pages
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…show more content…
(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…show more content…
(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

More about Fatty Acid Methyl Ester Mix (DPPH)

Open Document