Determination of total flavonoid content
Total flavonoid content (TFC) was determined by the alu-
minum chloride colorimetric method described by Chang
et al. [
10
]. Each extract solution (0.5 mL) was separately
mixed with 95% methanol (1.5 mL), 10% aluminum chloride
(0.1 ml), 1 M potassium acetate (0.1 mL) and deionized
water (3.0 mL). After 30 min, the absorbance of the resultant
mixture was measured by UV–vis double-beam spectropho-
tometer (Analytik Jena, Jena, Germany) at 415 nm against
a blank sample without reactants. Quercetin was used as a
reference standard and the results were expressed as milli-
gram quercetin equivalents (mg QE)/g of extract. The assay
was performed in triplicate.
Determination of antioxidant activity
DPPH scavenging assay
DPPH radical scavenging assay of different extracts was per-
formed by the method reported by Sun et al. [
18
], with slight
modifications. Briefly, an equal volume of methanolic solu-
tion of DPPH (0.1 mmoL/L) and extracts at different con-
centrations (6.25, 12.5, 25, 50 and 100 µg/mL) were mixed
with vortex shaker. The mixture was left in the dark at room
temperature for 15 min. BHA was used as a positive standard
control. A control was considered for each extract concen-
tration. The absorbance of the resulting solution was read at
517 nm against a blank sample. The percentage of the con-
sumed DPPH was calculated from the following equation:
where, A
0
is the initial absorbance (no antioxidant) and A
1
is the absorbance in the presence of the extracts. The IC
50
value was reported from the plots as the effective concentra-
tion of extract needed scavenging 50% of DPPH free radi-
cals. All tests were carried out in triplicates.
Reducing power assay
Antioxidant activities of the extracts were also determined
by reducing power assay following the protocol described
by Yen and Duh [
19
]. First, different concentrations (3.125,
6.25, 12.5, 25, 50, 100, 200 and 400 μg/mL) of the extracts
were prepared. Then, 1 ml of each extract was dissolved
in 1% potassium ferricyanide and 0.2 M sodium phosphate
buffer (pH 6.6) and incubated in a bain-marie for 20 min at
50 ºC. Afterward, 10% trichloroacetic acid was added fol-
lowed by centrifugation at 5 ºC. The upper layer was diluted
with an equal volume of deionized water and 0.1% ferric
(2)
Radical scavenging activity (%) =
[ A
0
− A
1
A
0
]
× 100,
chloride. Ascorbic acid was used as a positive standard
control. The sample OD was measured at 700 nm against
a blank sample (including all of the components without
the extract).
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