*2.4. Reducing Power*

Reducing power was determined spectrophotometrically as described previously [13]. \_The RP0.5AU value showing the powder concentration that caused an absorbance of 0.5 at 700 nm was calculated from the graph plotting absorbance against powder concentration. Vitamin C was used as a positive control. At least two independent experiments in triplicate were performed for each tested sample.

#### *2.5. Evaluation of Relative Antioxidant Capacity Index (RACI)*

In order to find out which samples exhibited the highest antioxidant activity in all antioxidant assays, the RACI was evaluated for each sample as described in Sun and Tanumihardjo [13]. RACI is the mean value of standard scores evaluated by initial data generated with di fferent methods for a sample. A standard score was calculated according to the following equation:

$$\text{Standard score} = (\mathbf{x} - \mu) / \sigma \tag{3}$$

where x was the raw data, μ was the mean of all samples' values of each method, and σ was the standard deviation.

Since in all antioxidant assays the raw data were IC50 values, the lower the RACI value the higher the antioxidant capacity.

#### *2.6. Cell Culture Conditions*

As previously described [14], human endothelial EA.hy926 cells gifted from Prof. Koukoulis (University of Thessaly, Greece) were cultured in normal Dulbecco's modified Eagle's medium (DMEM) in plastic disposable tissue culture flasks at 37 ◦C in 5% carbon dioxide.

#### *2.7. XTT Cell Viability Assay*

The antioxidant activity of the powders in EA.hy926 cells was examined using non-cytotoxic concentrations. In order to select these concentrations, the cytotoxicity of the powders was checked using the XTT cell viability assay kit (Sigma) as previously described [14]. Briefly, EA.hy926 cells were seeded into a 96-well plate (1 × 10<sup>4</sup> cells per well) in DMEM containing 10% fetal bovine serum FBS. After 24 h incubation at 37 ◦C in 5% CO2, the cells were treated with di fferent concentrations of the powders in FBS-free DMEM and incubated for another 24 h. Then, 50 μL of XTT test solution was added to each well. After 4 h of incubation, absorbance was measured at 450 nm and also at 630 nm as a reference wavelength in a Bio-Tek ELx800 microplate reader (Winooski, VT, USA). Negative control was DMEM serum-free medium. The absorbance values of the control and powders were used for calculating the percentage inhibition of cell growth caused by the powder treatment. All experiments were carried out in triplicate and on two separate occasions.

#### *2.8. Treatment of EA.hy926 Cells with the Powders*

The cells were cultured until 75%–80% confluence of the flask. Afterwards the medium was replaced with serum-free medium containing the tested powders at non-cytotoxic concentrations. The cells were treated with the powders for 24 h, and then they were trypsinized, collected, and centrifuged twice at 300× *g* for 10 min at 5 ◦C. At the end of the first centrifugation, the supernatant fluid was discarded and the cellular pellet was resuspended in PBS. After the second centrifugation, the cell pellet was collected and used for measuring the glutathione (GSH) and reactive oxygen species (ROS) levels.

#### *2.9. Assessment of GSH and ROS Levels in EA.hy926 Cells by Flow Cytometry*

The GSH and ROS levels in EA.hy926 cells were assessed using mercury orange and 2,7-dichlorofluorescein diacetate (DCF-DA), respectively, as described previously [9]. In brief, the cells were re-suspended in PBS (10<sup>6</sup> cells/mL) and incubated in the presence of mercury orange (10 μM) or DCF-DA (40 μM) respectively, in the dark at 37◦ C for 30 min. Then, the cells were washed, re-suspended in PBS, and subjected to flow cytometric analysis using a FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) with excitation and emission wavelengths at 488 and 530 nm for ROS, and at 488 and 580 nm for GSH. Data were analyzed using 'BD Cell Quest' software (Becton Dickinson). Each experiment was repeated at least three times.
