2.8. Hydroxyl radical scavenging activity
Co (II)/EDTA-induced luminol chemiluminescence
measurements were carried out on a model 6200 Fluorimeter, JENWAY (Jenway Gransmore Green Felsted
Dunmow Essex CM6 3 LB), keeping the lamp off and
using only the photo multiplier of the apparatus. The
procedure used is the one described by Parejo, Codina,
Petrakis, and Kefalas (2000) with some variations.
1 ml boric acid buffer solution (0.05 M, adjusted to
pH 9 with NaOH 1 M) containing 1 mg/ml EDTA
and 0.2 mg/ml CoCl2 Æ 6H2O was vortexed for 15 s with
100 ll of luminol solution (5.6 · 104 M) in boric acid
(0.05 M, adjusted to pH 9 with 1 M NaOH). Then, 25
ll of H2O2 aqueous solution (5.4 · 10 3 M) were added
and the mixture was vortexed again for 30 s and fast taken into a glass cuvette. The CL intensity (I0) was recorded when the plateau was reached (the lifetime of
the plateau is 30 s). Immediately afterwards, 25 ll of
the sample solution were added with a Pasteur pipette
for thorough mixing and the instantaneous decrease of
the plateau was recorded (I) (each measurement repeated three times).
The instantaneous reduction in luminol intensity elicited by the addition of the sample extract was symbolized as I. The light intensity in the absence of the
sample was symbolized as I0.
The ratio I0/I was plotted against lg dry extract/ml
and a linear regression was established I0/I = a[ lg dry
extract/ml] + b, where a is the slope and b is the intercept, in order to calculate the IC50, which is the amount
of sample necessary to decrease by 50% the initial CL
intensity. Again, the Antiradical Efficiency was calculated which is the 1/IC50.
Comparison was made with the three standards,
which have already been mentioned in the DPPH test.