Ferric reducing antioxidant power (FRAP) assay. The chemical composition of essential oil reveals four common compounds: eugenol, chavicol, linalool and a-terpineol. For the evaluation of the men. scavenging method 2,20 -diphenyl-1-picrylhydrazyl radical (DPPH) and ferric reducing/antioxidant power assay (FRAP). are performed: theDPPH method shows that free volatile aglycones possess good antioxidant properties comparable with that of the essential oil and well-known antioxidant butylated hydroxytoluene (BHT), but less than pure eugenol.This method was initially developed to assay plasma antioxidant capacity, but can be used for plantextracts too. The total antioxidant potential of the sample
was determined using a ferric reducing ability, (FRAP)assay (Benzie & Strain, 1996) as a measure of ‘‘antioxidant power’’. This assay measures the change in absorbance at 593 nm owing to the formation of a blue colored Fe 2+ pyridyltriazine compound from colorless oxidized, Fe3+form by the action of electron donating antioxidants. The
working FRAP reagent is prepared by mixing 10 partsof 300 mmol/l acetate buffer, pH 3.6, with 1 part of10 mmol/l TPTZ (2,4,6-tripyridyl-s-triazine) in 40 mmol/l
hydrochloride acid and with 1 volume of 20 mmol/l ferric chloride. Prepare fresh FRAP reagent (1.5 ml) and warm to 37 C and a reagent blank reading will be taken
at 593 nm (M1 reading). Subsequently, 50 ll of the sample (concentrations of stock solutions are 20, 10, 5 and 1 g/l) and 150 ll of deionized water is added to the FRAP reagent.Subsequently, 50 ll of the sample (concentrations of stock solutions were 20, 10, 5 and 1 g/l) and 150 ll of Final dilution of the sample in the reaction mixutre is 1:34.The sample is incubated at out the monitoring period. The change in absorbance between the final reading (4-min reading) and the M1 should de selected for the calculation of FRAP values. Standard curve can be prepared using different concentrations (0.1–5 mmol/l) of FeCl2 · 4H2O. A All solutions were used
The antioxidant capacity was determined following the procedure described by Benzie and Strain [4] with modifications.
The FRAP reagent was freshly prepared by adding 10 mM of 2,4,6-Tris (2-pyridyl)-1,3,5-triazine (TPTZ) (dissolved in 40 mM of HCl), 20 mM of FeCl3 in water and 300 mM of acetate buffer (pH 3.6) in the ratio of 1:1:10. A blank containing sample and solvents only was used for colour correction. The 96-well plates were then incubated at 37˚C for 90 minutes before absorbance was recorded at 600 nm. Vitamin C (L-ascorbic acid), gallic acid and quercetin were used as antioxidant standards and positive controls. The absorbance of the samples were compared to a FeSO4 standard curve and the FRAP values were expressed as Ferrous Equivalent (FE), the concentration of extract or chemical which gives the same absorbance as 1 mmol ferrous ion.
Please go through it following paper which will be useful for your work.
Ferric reducing antioxidant power of essential oils extracted from
Eucalyptus and Curcuma species
Durre Shahwar1*, Muhammad Asam Raza1,2, Sana Bukhariand Gulshan Bukhari3 , Asian Pacific Journal of Tropical Biomedicine (2012) , 1633-1636