*2.6. Antioxidant Properties*

### 2.6.1. Ferric Reducing Power (FRP)

The ferric reducing power of leaves and callus extracts was determined according to the method previously described by Peši´c et al. [37], with slight modifications. Briefly, an aliquot of the appropriately diluted sample (2.5 mL) was mixed with 2.5 mL of 0.2 M phosphate buffer (pH = 6.6) and 2.5 mL of 1% potassium ferricyanide. Then, the homogenized mixture was incubated at 50 ◦C, and after 20 min it was mixed with 2.5 mL of 10% TCA. Supernatant collected after centrifugation (2.5 mL) was mixed with 2.5 mL of milliQ water and 0.5 mL of 0.1% ferric chloride. After 10 min, the absorbance of the mixture was measured at 700 nm. Results for FRP were expressed as mg gallic acid equivalents per g dry weight of the sample.

### 2.6.2. ABTS•<sup>+</sup> and DPPH• Radical Scavenging Activity

ABTS•*+* scavenging activity was evaluated as previously reported by Peši´c et al. [37]. Prepared ABTS•± working solution (1 mL) was mixed with the sample (10 μL) and left in the dark for 7 min. Afterwards, the absorbance of the mixture was measured at 734 nm.

DPPH• scavenging activity was performed according to a method previously reported by Gawron-Gzella et al. [38], with small modifications. Briefly, 120 μL of prepared 150 μM DPPH• working solution was mixed with 15 μL of the sample and incubated for 30 min in the dark. Next, the absorbance was measured at 515 nm using a Plate Reader (Tecan Sunrise Spectrophotometer, Tecan Trading AG, Switzerland).

The percentage of ABTS•± and DPPH• quenched radicals was calculated using the following Equation (1):

$$\text{ABTS}^{\bullet\bullet} / \text{DPPH}^{\bullet} \text{ radical scavenging activity (\%)} = \text{[(Ac} - \text{As)} / \text{Ac]} \times 100 \tag{1}$$

where Ac is control absorbance (ABTS•± or DPPH• working solution mixed with 10 μL of methanol) and As is absorbance of the sample mixed with ABTS•*+* or DPPH• working solution.

The ABTS•*+* scavenging activity was expressed as μg of ascorbic acid equivalents per mL of sample (μg AAE/mL).
