After treatment with green tea infusions or solutions of pure EC and
EGCG, changes in the antioxidant capacity of insoluble wheat bran fraction
was measured by direct QUENCHER procedure using ABTS radical
probe. The ABTS•
+ radical solution was prepared according to Serpen,
Gökmen, and Fogliano (2009). A portion of insoluble wheat bran
(50 mg) was weighed into a test tube and the reaction was initiated
by adding 10 mL of green tea infusion, or aqueous solutions of EC or
EGCG prepared at different concentrations as mentioned above. The
tube was vigorously shaken in an orbital shaker at a speed of 350 rpm
at room temperature in dark. Aqueous solutions or green tea infusions
without insoluble wheat bran were also kept under the same condition
as control. After 30 min of reaction, the tube was centrifuged at 6080 g
for 2 min. The supernatant was filtered trough a 0.45 μm filter and analyzed
by LC–MS to monitor the changes in the concentrations of catechins.
The precipitate was washed out three times with 10 ml of
water to remove the remaining traces of catechins. For this purpose,
the precipitate was mixed with 10 mL of water. After washing cycles,final precipitate that was free of soluble catechins was lyophilized.
10 mg of this powder was mixed with 10 mL of ABTS•
+ working solution
in a test tube. The mixture was rigorously shaken for 27 min at
350 rpm in the dark using the orbital shaker. Then it was centrifuged
at 6080 g for 2 min. The optically clear supernatant was transferred
into a cuvette and absorbance was measured at 734 nm using a
Shimadzu model 2100 variable-wavelength UV–visible spectrophotometer
(Shimadzu Corp., Kyoto, Japan). The total antioxidant capacity of insoluble
wheat bran was calculated by means of a calibration curve built
for trolox in the concentration range between 0 and 600 μg mL−1
. The
results were expressed as mmol trolox equivalent antioxidant capacity
per kg insoluble dietary fiber.