# Changes in Elderberry (_Sambucus nigra_ L.) Juice Concentrate Polyphenols during Storage
## 3 Results and Discussion
### Effect of Storage on Individual Phenolic Compounds  
The content of individual phenolic compounds in the juice concentrates was followed by HPLC-DAD over the seven months of storage. The chromatograms of concentrate I after seven months of storage are presented in Figure 2c-f. A decrease in the areas of anthocyanin peaks at refrigerated temperature and an almost disappearance of anthocyanin peaks at room temperature were observed. Figure 4 shows the evolution of the content of individual phenolic compounds in concentrate I and concentrate II over storage. After seven months, at 5 \({}^{\circ}\)C the concentrates presented a composition of anthocyanins that represented 61-76% of the initial concentration, while for room temperature, the values represented 3-21%--Figure 4a-c. Chlorogenic acid showed a slight decrease around 10% over the time--Figure 4d, and the amount of rutin and quercetin-3-\(O\)-glucose remained quite stable, with a tendency to increase after five months--Figure 4e,f. For both concentrate I and concentrate II there is a marked loss of anthocyanin content at room temperature over the storage time--Figure 4a-c. Degradation was more pronounced for cyanidin-3-O-sambubioside and cyanidin-3-\(O\)-glucoside--Figure 4a,b, respectively--than for cyanidin-3,5-\(O\)-diglucoside and cyanidin-3-\(O\)-sambubioside-5-\(O\)-glucoside--Figure 4c. In the case of concentrate I, after seven months of storage at room temperature, 21.5% of the initial concentration of cyanidin-3,5-\(O\)-diglucoside + cyanidin-3-\(O\)-sambubioside-5-\(O\)-glucose was found, whereas only 9.0% of cyanidin-3-\(O\)-sambubioside and 9.5% of cyanidin-3-\(O\)-glucose were maintained. A similar result was obtained for concentrate II with retained concentrations of 19.4%, 2.5%, and 3.4%, respectively for cyanidin-3,5-\(O\)-diglucoside + cyanidin-3-\(O\)-sambubioside-5-\(O\)-glucose, cyanidin-3-\(O\)-sambubioside, and cyanidin-3-\(O\)-glucose, respectively. It has been described that anthocyanins' stability is influenced by the glycosylation site, and by the type and number of glycosyl moieties attached [34]. Our findings are in line with other studies that showed lower stability of 3-\(O\)-glycos compared to the 3,5-\(O\)-diglycosides counterparts [35; 36]. The lower stability of 3-\(O\)-glycosides can be explained by the mesomeric effect of the hydroxyl group at C-5 position that favors the electrophilic attack at C-6 and C-8 positions. The glycosyl substitution at C-5 reduces the nucleophilicity of C-6 and C-8 positions which makes 3,5-\(O\)-diglycosides more stable to electrophilic attack [37]. The contents of chlorogenic acid also decreased substantially during storage at room temperature in both concentrates. After seven months, 65.0% and 68.4% of its initial concentration was obtained for concentrate I and II, respectively. This indicates a possible involvement of chlorogenic acid in anthocyanins polymerization, which agrees with the observations of other authors [24]. In the present study, concentrate II, with about 80.6 \({}^{\circ}\)Brix, showed a faster degradation of some compounds at room temperature when compared with concentrate I, which had 55.8 \({}^{\circ}\)Brix. For instance, after three months of storage, concentrate I presented 39% of the initial concentration of cyanidin-3-O-sambubioside, while in concentrate II only 19% was  
Figure 4: Changes in (**a**) cyanidin-3-O-sambubioside, (**b**) cyanidin-3-O-glucose, (**c**) cyanidin-3,5-O-diglucoside + cyanidin-3-O-sambubioside-5-O-glucose, (**d**) chlorogenic acid, (**e**) rutin, and (**f**) quercetin-3-O-glucose of elderberry concentrates I and II during storage at 5 \({}^{\circ}\)C and at room temperature (RT). retained. A similar result was obtained for cyanidin-3-O-glucoside. Conversely, [PERSON] et al.