2.7. Free radic al scaveng ing acti vity
Radical scavenging activity of the seven extracts
was measured using the stable radical DPPH . The
procedure followed was according to Brand-Williams,
Cuvelier, and Berset (1995) with some variations.
For each extract, different concentrations were tested.
At least 5 dilutions of each extract were prepared in
methanol using a 10 ml volumetric flask (methanol
for the control). Concentrations ranged from 48 to
1.51 mg/ml for less active extracts and from 10 to
0.33 mg/ml for more active ones. An aliquot of methanol (0.1 ml) solution containing different concentrations of orange peel extracts was added to 3.9 ml of
DPPH (104 M). Absorbance was measured at 515
nm until the reaction reached a plateau (each measurement repeated twice). After preliminary experiments,
the plateau was fixed at 4 h for all the extracts due
to the slow kinetics of certain extracts. The absorbance
of the DPPH solution, was measured daily. The
DPPH concentration in the reaction medium was calculated from the following calibration curve, determined by linear regression:
Að515 nmÞ ¼ 26 :501½DPPH T 0 :0244 ;
where [DPPH ] T as mg/ml and r2 = 0.9992.
The percentage of remaining DPPH was calculated as
follows:
% DPPHREM ¼ ½DPPH T =½DPPH T ¼ 0 :
The percentage of the % remaining DPPH against
mg dry extract/mg DPPH was plotted to obtain the
amount of antioxidant necessary to decrease the initial
DPPH concentration by 50% ( EC50), using the exponential model: ln[% DPPH rem] = b[mg antioxidant/
mg DPPH ] + a, where b is the slope and a is the intercept. For each of the extracts, the Antiradical Efficiency,
1/EC50was calculated. Values were also obtained for the
three standards.