The FRAP assay was performed as previously described (Benzie and Strain,
1996) to test the reducing activity of MPEE and green tea extract. Briefly, the extract
was centrifuged for 10 min at 2830gprior to use and the supernatant was saved.
FRAP reagent consisted of 25 ml of 300 mM acetate buffer at pH 3.6, 2.5 ml of
10 mM 2,4,6-tripyridyl-s-triazine and 2.5 ml of 20 mM FeCl3. FRAP reagent
(900ll) was heated at 37C for 10 min, then 90ll of Milli-Q water and 30llof
sample were added to it and mixed well. The absorbance of the reaction mixture
at 593 nm was measured spectrophotometrically after incubation at 37C for
4 min. FeSO47H2O of 1 mM was used as the standard solution (standard solution
ranged from 0 to 1 mM). The antioxidant activity of the tested MPEE or green tea
extract was expressed as FRAP value/g dry pericarp weight (DW) as shown in the
equation below:
FRAP valueðlmol=LFeSO4equivalentsðFEÞ=gDWÞ
¼½ðAbsorbance at 593 nm=slopeÞdilution factor
volume of supernatantðmlÞ=equivalent DWðgÞ=1000