FERRIC REDUCING ANTIOXIDANT POWER (FRAP) ASSAY PRINCIPLE The FRAP assay uses antioxidants as reductants in a redox-linked colorimetric method employing an easily reduced oxidant, Fe(ll. Reduction of a ferric tripyridyttriazine complex to ferrous-(2,4,6-tripyridyl-s-triazine)2 ie: Ferric (Ill) [colourless] to Ferrous (1!) [blue] can be monitored by measuring absorbance at 593nm. The absorption readings are related to the reducing power of the electron- donating antioxidants present in the test compound. Hence the FRAP assay can rank the reducing power and the antioxidant potential of a wide range of test compounds. REAGENTS 300mM Acetate buffer >} Weigh out 3.10g sodium acetate trihydrate. © Add 16mL glacial acetic acid. > Adjust pH to 3.6. > Make up to a final volume of 1L using distilled deionised H20. 40mM HCl (i, 36.46; SG = 1.18; purity 35-38%) © Measure 3.4mL concentrated HCl. © Make up to a final volume of 1L using distilled deionised H20. The following reagents must be prepared fresh on the day of the assay: 10mM TPTZ 3.123g TPTZ/1L 40mM HCl. \ Dab O-D2i /W * 20mM FeCls.6H20 (make up fresh on the do ag ae 5.A06g/L using distilled deionised Hao. ¢ “4 = 0'0 (sa FRAP reagent Standard curve: 10:1:1 (acetate buffer: TPTZ: H:0) Samples: 10:1:1 (acetate buffer: TPTZ: FeCls.620) Standard Cuvette method: 0.001M FeSOs,7H20 (Dm b = Weigh 2.78gFes0,7H,0. “ U'0 22% 5 fx = Make up to a final volume of 1L using distilled deionised H:0. © Make a 1/10 dilution to obtain a 0.001M concentrationPROCEDURE STANDARD CURVE 1, Add following reagents into glass tubes (add standard last): Fe 1 2 3 4 5 6 7 3 #20 (ut) yooo_|9as_| 970 | 940 | a0 | 820 | 760 | 700 | Imm std (ut) [0 5 30 | 60 | 120 | 180 | 240 | 300 | FRAP (ml) 2 2 pcan arena 2 2 2 2 Std cone (uit) | _0 5 Torei|et320 40 60 30 | 100 2. For blank: 2mL FRAP reagent + ImLH20. 3. Intoa cuvette add 100pL sample + 900uL distilled HzO + 2mL FRAP reagent. 4, Invert tubes to mix. 5. Leave for 30min in the dark. 6. Transfer to cuvettes. 7. Zero spectrophotometer at 593nm using blank. 8. Read at 593nm. CALCULATIONS The FRAP equation is: FRAP value of sample (uM)= Abs(sample)xFRAP value of Std (4M) “Abs (td) 1. Drawa standard curve Abs V's Fe** Conc. (X) and obtain equation for best fit line. 2. Use sample absorbance values that have been zeroed (i.e. Abs sample ~ Abs blank) to express FRAP in molarity. Ascorbic Acid / aa Stock (10mM): Weigh 0.0176g Ascorbic acid and make up to a final volume of 10mL using methanol. (1). Dilute to 5mM by mixing 500uI of water and 500u of stock ascorbic acid. | (2) Dilute to 250M by mixing SOUL ascorbic acid (SmM) and 950,1L water.