Antioxidant activity determination

The DPPH radical scavenging activity was determined using a method described by Xu et al. [15] with some modifications. Each extract (1 mL) was mixed with 2 mL of 0.2 mmol/L DPPH solution, which was vigorously shaken and allowed to stand at room temperature for 30 min in the dark. DPPH radical scavenging activity was calculated as the percentage decrease of the absorbance at 517 nm relative to a blank (as 100%). The positive control was 0.05% gallic acid, 0.05% ascorbic acid, and 0.05% α-tocopherol. The concentrations of positive control were selected based on the content of gallic acid present in the spent coffee extract (0.01–0.05%). The percentage of the scavenging activity of the DPPH radical was calculated using the following equation:

In this equation, A_blank and A_sample are the absorbances of the control and extract, respectively. All samples were analyzed in triplicate.

The ferric reducing antioxidant power (FRAP) assay was determined using the redox-linked colorimetric method of Ranic et al. [16] with slight modifications. A FRAP reagent was freshly prepared using 300 mmol/L acetate buffer (pH 3.6), 20 mmol/L ferric chloride, and 10 mmol/L tripyridyltriazine in 40 mmol/L HCl. These three solutions were mixed together at a ratio of 10:1:1 (v/v/v). A ferrous sulfate solution was then prepared at 1000 μmol/L in distilled water. An aliquot of 0.1 mL of working solutions (0, 100, 200, 300, 400, 500, 700, 900, and 1000 μmol/L) were mixed with 3 mL of the FRAP reagent and incubated at 37 °C for 30 min before absorbance was measured at 593 nm. The extract (0.1 mL) was analyzed as described above. The antioxidant capacity, based on the ability to reduce ferric ions in the sample, was calculated from the linear calibration curve and expressed as mmol FeSO₄ equivalents per mL of sample. All samples were analyzed in triplicate.

The superoxide dismutase (SOD)-like activity was determined using the method described by Koh and Surh [17] with some modifications. An aliquot of 0.1 mL of each extract was mixed with 2.8 mL reducing solution (250 μmol/L xanthine in 0.1 mmol/L EDTA phosphate buffer pH 7.4 and NBT). Xanthine oxidase (0.2 mL, 0.1 unit/mL) was added and then incubated at 37 °C for 40 min. Absorbance was measured at 550 nm. The radical scavenging activity was calculated as the percentage inhibition of the reduced NBT relative to the blank (as 100%). The positive controls of 0.05% gallic acid, 0.05% ascorbic acid, and 0.05% α-tocopherol were used for comparison. The scavenging activity was calculated using the equation described for DPPH.