*2.6. Statistical Analysis*

All reported data are presented as the mean values ± the standard deviation obtained from the three replicates. Statistical analysis was conducted using XLSTAT Release 10 (Addinsoft, Paris, France) software, and the level of significance was evaluated by one-way analysis of variance (ANOVA), considering *p*-values < 0.05 to be significant.

#### **3. Results and Discussion**

Cornelian cherry fruits have been reported as a valuable source of anthocyanins, compounds with high antioxidant activities that contribute to the beneficial biological properties of these fruits. A diet rich in anthocyanins results in downregulating disease markers, improves the antioxidant function, and protects cells against the deleterious effects of oxidative stress [27]. The antioxidant activity of the Cornelian cherries' extract was evaluated using the ABTS assay and was found to be 553 μM Trolox. The anthocyanin content was determined by the pH differential method and was 360 mg Cy-3-glu equivalents/L. The HPLC analysis of the Cornelian cherry fruit extract revealed the presence of three anthocyanins (Figure 2). The results were in agreemen<sup>t</sup> with those previously reported by Moldovan et al. [11]. The three main anthocyanin peaks detected at 506 nm were identified by comparing the retention times and the UV absorption spectra with authentic anthocyanin standards and were assigned to cyanidin-3-*O*-galactoside (Peak 1), pelargonidin-3-*O*-glucoside (Peak 2), and pelargonidin-3-*O*-rutinoside (Peak 3).

**Figure 2.** HPLC chromatograms (520 nm) of Cornelian cherries' anthocyanins before and after in vitro digestion: (**a**) crude extract; (**b**) gastric digestion; (**c**) intestinal digestion.

The content of the three anthocyanins is presented in Table 1. The main anthocyanin of the Cornelian cherry fruits was pelargonidin-3-*O*-glucoside (62.99% from total anthocyanin content), followed by cyanidin-3-*O*-galactoside (35.68% from total anthocyanin content). Pelargonidin-3-*O*-rutinoside has been identified in very small amounts (<2%) in the crude fruit extract.


**Table 1.** Anthocyanin identification and quantification (mg/L) of Cornelian cherry fruit extract samples before and after in vitro digestion.

a,b Numbers in rows followed by different letters are significantly different (*p* < 0.05).

Anthocyanins are generally not very stable compounds. Many factors can a ffect the stability of these antioxidant compounds, such as pH, oxygen, chemical structure, and the presence of enzymes [28]. Thus, investigating their stability during their passage through the gastrointestinal tract is very important, in order to understand the bioavailability of these compounds. In the present study, the stability of Cornelian cherry anthocyanins was investigated using an in vitro simulated gastrointestinal digestion model. The Cornelian cherry fruit extract was incubated with pepsin at pH = 2 for the gastric digestion and, after that, with pancreatin and bile salts at pH = 7.5, to simulate small intestine digestion. After each step of the in vitro digestion, samples were analyzed in terms of their total anthocyanin content and antioxidant capacity. HPLC analysis was conducted to monitor changes in anthocyanin composition and content after each digestion step. Stomach digestion did not significantly change the qualitative or quantitative composition of the anthocyanin compounds. Figure 2b presents the HPLC chromatogram after simulated gastric digestion, and no di fferences between crude extract and that subjected to digestion in gastric conditions were observed. After subjecting the anthocyanin-rich extract to simulated gastric digestion, a slight increase of the total anthocyanin content to 386 mg/<sup>L</sup> was noticed. The same anthocyanins as in the original sample were recovered after stomach digestion, the recovery being 107.23%. Our findings are in good agreemen<sup>t</sup> with those obtained in other studies. Ryu et al. reported a slight increase after gastric digestion of Bokbunja anthocyanins [15], while Sun et al. observed the same result in the case of purple rice anthocyanins subjected to in vitro simulated digestion [29]. These findings strongly sugges<sup>t</sup> that Cornelian cherry anthocyanins are highly stable when exposed to stomach conditions. The low pH value in stomach mainly contributes to the high stability of anthocyanins, which at this pH (pH = 1.5–2), occurs in the chemical structure of a stable flavylium cation [30]. The increase of the anthocyanin content could also sugges<sup>t</sup> that an acidic environment and digestive enzymes improve the release of the monomeric anthocyanins from the polymeric ones, by disrupting macromolecules. Additionally, an increase in the antioxidant activity during simulated gastric digestion occurred (Figure 3). Changes in total anthocyanin content and radical scavenging capacity were not significant during the in vitro gastric digestion as compared to undigested extract (*p* > 0.05).

**Figure 3.** Changes in total anthocyanin content and antioxidant activity of Cornelian cherry fruit extract before and after in vitro gastrointestinal digestion. Means (bar value) with di fferent letters are significantly di fferent (*p* < 0.05).

The samples obtained after gastric digestion were further transferred to a mild alkaline intestinal environment, and pancreatic enzymes and bile salts were added in order to simulate the intestinal digestion process. After small intestine digestion, a clear decrease of the total anthocyanin content up to 26.46 mg cyanidin-3- *O*-glucoside equivalents/<sup>L</sup> was observed, reduced by more than 70% compared to the undigested sample. The low recovery of anthocyanins after intestinal digestion (26.46%) can be explained by the low stability of these compounds under alkaline conditions (pH = 7.5) attributed to the structural changes of the flavylium cation to a colorless, less stable chalcone [30]. Other studies also found that simulated intestinal digestion had a dramatic impact on the anthocyanins from di fferent fruits. Pomegranate juice anthocyanins decreased by ~97% during this stage of digestion [31], while blueberry anthocyanins decreased more than 80% after intestinal digestion [32]. All the anthocyanin compounds present in the Cornelian fruit extract presented a significant decrease during incubation under small intestine conditions. Cyanidin-3- *O*-galactoside was the most unstable of the three investigated anthocyanins, its content being the most a ffected by the intestinal digestion compared to the crude extract.

The pancreatic-bile digestion significantly decreased the antioxidant capacity of the anthocyanin-rich extract of Cornelian cherries. The ABTS•<sup>+</sup> scavenging activity decreased by ~43%, as compared to the undigested or gastric digested sample. The changes in the antioxidant capacity of digested Cornelian cherry fruit anthocyanins were consistent with those previously observed for red cabbage anthocyanins [33]. While the bioaccessibility of Cornelian cherry anthocyanins largely decreased during intestinal digestion, the antioxidant capacity of the extract was less a ffected by this phase of digestion. This fact could be explained by the formation of new antioxidant metabolites from anthocyanins degraded from the simulated intestinal fluid, which can further exert their beneficial effects on human health.
