*2.7. Extraction of Phytochemicals*

The extraction procedure of phytochemicals was carried out according to Blackhall et al. [29] after some modifications. Frozen cherries (three replicates of 10 cherries each) were de-stoned and homogenized in a blender (Model 38BL40, Waring commercial, New Hartford, CT, USA) for 15 s. Approximately 2 g of cherry pulp and 20 mL of methanol containing 0.1% 10 N HCl were homogenized using an Ultra-Turrax (Model T25, Ika Labortechnik, Germany) for 1 min at 9500 rpm min<sup>−</sup>1. The homogenate was incubated in a supersonic bath for 60 min at 37 ◦C, centrifuged at 4000 rpm for 6 min and the supernatant was recovered and used for the analyses.

#### *2.8. Determinations of Total Phenolics, Flavonoids, Anthocyanins and Antioxidant Capacity*

Total phenolics (TP) was measured by the Folin–Ciocalteu method according to Tsantili et al. [30], recording the absorbance at 750 nm versus a blank using a spectrophotometer (Model Cary 50, Varian Inc., Walnut Creek, CA, USA). Total flavonoids (TF) were measured by a colorimetric method using a 0.3 mL cherry extract for reactions and absorbance recording at 510 nm [31], as described by Tsantili et al. [30]. Total anthocyanins (TAN) were measured according to Meyers et al. [31], as described by Tsantili et al. [30], recording absorbance at 510 and 700 nm in buffers at pH 1.0 and 4.5, and converted to cyanidin 3-rutinoside (keracyanin) equivalents (c-3-rut) using a molar extinction coefficient of 28,840 L mol–1 cm–1. Total antioxidant capacity (TAC) was evaluated using both ferric reducing antioxidant power (FRAP) [30] and radical scavenging capacity (2,2-diphenyl-1-picrylhydrazyl, DPPH) [32] assays according to Christopoulos and Tsantili [33]. For all

determinations, triplicate reactions per replicate were performed, and the results of TP, TF, TAN and TAC were expressed as equivalents of gallic acid (GAE), catechin (CAE), c-3-rut and Trolox acid (TAE), respectively, all on a DW basis.
