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Various concentrations of chestnut extracts (2.5 ml)
were mixed with 2.5 ml of 200 mM sodium phosphate buf-
fer (pH 6.6) and 2.5 ml of 1% potassium ferricyanide. The
mixture was incubated at 50 C for 20 min. After 2.5 ml of
10% trichloroacetic acid (w/v) was added, the mixture was
centrifuged at 1000 rpm for 8 min (Centorion K24OR-2003
refrigerated centrifuge). The upper layer (5 ml) was mixed
with 5 ml of deionised water and 1 ml of 0.1% of ferric
chloride, and the absorbance was measured spectrophoto-
metrically at 700 nm (Barros et al., 2007). The extract con-
centration providing 0.5 of absorbance (EC50) was
calculated from the graph of absorbance at 700 nm against
extract concentration. BHA and a-tocopherol were used as
standards.
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