2.5. DPPH free radical scavenging assay

From the citrus leaf powder, the antioxidant activity and capacity were measured by using the DPPH (2,2-diphenyl-1-picrylhydrazyl) technique as discussed previously (Ozgen et al., 2010). A total of 100 mg of citrus leaf samples had been homogenized in 1 ml of extraction solution (ethanol 70%, water 29%, and acetic acid 1%). After that, the leaves sample was centrifuged at 10,000 rpm for 8 min at room temperature. The supernatant (0.03 ml) were collected in a fresh tube followed by the addition of DPPH (0.1 mM) 2.97 ml in each sample accompanied by incubation in the dark, for 30 min, at room temperature. The absorbance reading was taken at 517 nm on the UV-1800 (Shimadzu, Japan) spectrophotometer. The different concentrations of Trolox were used as a standard, and the standard curve was generated to calculate the antioxidant capacity described in millimolar of Trolox/100 mg. Moreover, the antioxidant activity (free radical scavenging %) was determined by using the following formula: