*2.1. Chemicals*

Cyanidin-3-*O*-galactoside, pelargonidin-3-*O*-glucoside, and pelargonidin-3-*O*-rutinoside standards were purchased from Extrasynthese (Lyon, France), and pancreatin from porcine pancreas and pepsin from porcine stomach were purchased from Alfa Aesar (Karsluhe, Germany). All other chemicals and solvents were obtained from Merck (Darmstadt, Germany) and were of analytical or HPLC purity. A TYPDP1500 water distiller (Techosklo LTD., Drzkov, Czech Republic) was used to obtain the distilled water.

#### *2.2. Preparation of Fruit Extract*

Cornelian cherry (*Cornus mas* L.) fruits were manually collected at ripening stage in August 2018 in Chinteni, Cluj County, Romania. The picked fruits were selected according color, mass, and shape uniformity and stored at −18 ◦C until further investigation. The Cornelian cherries' anthocyanins were isolated by extraction applying an already published procedure [11]. Briefly, 90 g of homogenized fruit pulp were treated with 300 mL of food-grade acetone. The extraction was carried out at room temperature, under vigorous stirring for 60 min. The mixture was vacuum filtered through Whatman No. 1 filter paper, and the obtained solution was concentrated to a 65-mL final volume at 40 ◦C by a rotary evaporator to remove the acetone. The obtained concentrate was further subjected to determination of total anthocyanin content by the pH differential method, to identify anthocyanins present in the extract by HPLC analysis and in in vitro simulated gastrointestinal digestion.

#### *2.3. Quantification and Identification of Anthocyanins*

The total anthocyanin content of the Cornelian cherries' anthocyanin-rich extract was determined using the well-known spectrophotometric pH differential method [22] as previously described [23]. Briefly, samples of fruit extract were mixed with 0.025 M potassium chloride buffer solution (pH = 1) and 0.04 M sodium acetate buffer solution (pH = 4.5), respectively. The absorbance of each resulting solution was measured at 506 nm and 700 nm, respectively, against distilled water as a blank, using a UV-Vis Perkin Elmer Lambda 25 double-beam spectrophotometer (Perkin Elmer, Shelton, CT, USA). The total anthocyanin content was expressed as cyanidin-3-glucoside equivalents/liter. All measurements were performed at room temperature, in triplicate.

The identification of anthocyanins in the Cornelian cherry fruit extract was accomplished by HPLC analysis. For this purpose, an Agilent 1200 (Agilent Technologies Inc.; Santa Clara, CA, USA) equipped with a diode array detector HPLC system was used. The separation of anthocyanins from the fruit extract was performed on an Eclipse XTB-C18 (Agilent) column (150 × 4.6 mm inner diameter, particle size 5 μm), maintained at 20 ◦C. Samples of the extract were 2-fold diluted with 0.1% formic acid, filtered through a 0.2-μm PTFE membrane disk filter, and 20 μL were injected into the column. A mixture of two solvents, 0.1% formic acid aqueous solution (Solvent A) and 0.1% formic acid acetonitrile solution (Solvent B), was used as a mobile phase at a flow rate of 0.4 mL/min. The elution was performed by applying the following gradient profile: 0–16 min 95% A, 16–17 min 60% A, 17–20 min 5% A, and 20–21 min 95% A. The detection was performed at 506 nm. The main anthocyanins from the Cornelian cherry fruit extract were identified according to the consistency of the retention times compared to authentic anthocyanin standards. The quantification of each anthocyanin present in the extract was performed using a calibration curve of cyanidin-3-*O*-galactoside by an external standard method. All samples were injected in triplicate.

#### *2.4. Evaluation of the Antioxidant Activity Using the ABTS Assay*

The antioxidant capacity of the Cornelian cherry fruit extract was determined by the ABTS radical scavenging activity method [24] with some modifications [25]. The ABTS•<sup>+</sup> stock solution was freshly diluted with distilled water to prepare a working solution with an absorbance between 0.6 and 0.8 recorded at 734 nm against distilled water using a spectrophotometer. The free radical scavenging capacity of the fruit extract was evaluated by adding 0.1 mL of extract sample to 6 mL diluted ABTS solution followed by incubation at room temperature, in the dark, for 15 min. After 15 min, the absorbance of the sample was monitored at 734 nm, and the antioxidant activity was calculated using a calibration curve of Trolox standard and expressed in μmol Trolox equivalents/L.

#### *2.5. In Vitro Simulated Digestion Process*

The gastrointestinal digestion was simulated according to a slightly modified protocol of Gil-Izquierdo [26]. Samples of 50 mL were subjected to an in vitro gastric digestion process by adjusting the pH of the extract to 2, by adding 1 M HCl solution. Thereafter, pepsin from porcine gastric mucosa (15.750 units EC 3.4.23.1) was added, and the samples were incubated in the absence of light, in a shaker (200 rpm), at 37 ◦C, for 2 h. After the simulated gastric digestion, aliquots of 5 mL were collected, rapidly cooled at −20 ◦C, and used further for the evaluation of anthocyanin composition and antioxidant activity of the gastric digested samples. The remaining solution from the gastric digestion step was further submitted to the in vitro simulated intestinal digestion process. The pH of the remaining solution was adjusted to 7.5 with 1 M NaHCO3 solution. Then, 22.5 mL of pancreatin (2 mg/mL) from porcine pancreas (8 × USP specifications) and bile salts (25 mg/mL) solution were added, and the obtained mixture was further incubated at 37 ◦C for another 2 h. The obtained digested samples were properly diluted with 0.1% formic acid (3-fold for the gastric digestion samples and 1.6-fold for the intestinal digestion samples) and analyzed directly by HPLC for the determination of the anthocyanin profile, and also, the antioxidant capacity and the total anthocyanin content were evaluated. The simulation of the in vitro digestion process was performed in triplicate.
