Time-Dependent Degradation of Polyphenols from Thermally-Processed Berries and Their In Vitro Antiproliferative Effects against Melanoma

Polyphenols are natural occurring micronutrients that can protect plants from natural weathering and are also helpful to humans. These compounds are abundantly found in fruits or berries. Because of berry seasonal availability and also due to their rapid degradation, people have found multiple ways to preserve them. The most common options are freezing or making jams. Polyphenol stability, during processing is a continuous challenge for the food industry. There are also multiple published data providing that they are sensitive to light, pH or high temperature, vectors which are all present during jam preparation. In this context the aim of this study was to assess phytochemical composition and bioactive compounds degradation after jam preparation. We also monitored their degradation during storage time and their in vitro antiproliferative potential when tested on melanoma cells. The obtained results revealed that when processed and stored in time, the bioactive compounds from berries jams are degrading, but they still exert antioxidant and antiproliferative potential. Prior to LC-MS analysis, polyphenolic compounds were identified as: flavonoids (anthocyanins (ANT), flavonols (FLA)) and non-flavonoid (hydroxycinnamic acids (HCA) and hydroxybenzoic acids (HBA)). The most significant decrease was observed for HCA compared to other classes of compounds. This variation is expected due to differences in constituents and phenolic types among different analyzed berries.


Introduction
Fruits and vegetables are important sources of bioactive compounds which were shown to have positive health benefits [1,2]. In recent years, food and nutrition sciences aimed to improve health through intelligent foods containing bioactive plant-based molecules that were proven to have positive health benefits, such as the prevention of cardiovascular diseases or cancer [3,4]. The demand for innovative and functional food products had increased lately due to the fact that consumers are more aware of their bodies and mental health. Diets rich in vegetables or fruits were proven to provide essential bioactive molecules which can play important roles in human health. These plant-based bioactive molecules include polyphenols (phenolic acids, flavonoids, anthocyanins, catechins), enzymes, amino acids, vitamins (vitamin C, folate, and provitamin A), minerals (potassium, calcium, and magnesium), and fibers (inulin, pectin, lignan). The related body of literature indicates a strong correlation between diet and degenerative diseases, and due to this fact, the use of natural compounds as ingredients in food has become a major concern for food technologists [5,6]. Berries and fruits are the main sources of bioactive compounds with many applications in the food, pharmaceutical, nutraceutical and cosmetic industries [7][8][9][10]. These bioactive compounds are mainly polyphenols, and among them, anthocyanins which have proved to have high nutritional and potential health    A decrease in the content of anthocyanins was observed immediately after processing and also, during storage time as it is shown in Figure 3.
A decrease in the content of anthocyanins was observed immediately after processing and also, during storage time as it is shown in Figure 3. This degradation can be attributed to the hydrolytic reactions that led to the conversion of anthocyanin glycosides into chalcones, whose form can be rapidly transformed into phenolic acids and aldehydes.
Moreover, heat stable forms of polyphenol oxidase or peroxidase may play roles in the reduction of anthocyanins [30,31]. Another mechanism that may be associated with anthocyanin degradation is related to the hydrolysis of the glycoside linkages. This hydrolysis is known as the first step towards anthocyanin degradation, because high temperatures can shift the anthocyanin equilibrium towards the colorless chalcones. Chalcone degradation can occur due to the presence of oxidation reactions which can generate brown compounds or pigments that have high molecular weights. Additionally, pH values can affect the flavylium salt degradation, which is stable under highly acidic conditions. At higher pH values, salts can lose a proton and are easily transformed into an unstable pigment (quinoidal base) that is bonded to water and forms a colorless compound, commonly known as chromanol [30,32]. During processing and at the end of the storage period, anthocyanin degradation ranged from 58.90% to 74.30%. The results of the loss of anthocyanin content were lower compared with data reported previously for processed and stored black carrot jam and marmalade (87.60-95.60%). Degradation was also observed in flavonoids immediately after processing and during storage. This finding is in agreement with the previously reported data for Rubus coreanus Miquel berry jams [33]. The results revealed that jams prepared at pH 2.0-3.0 lost 33-35% of their anthocyanins, while the total amount of anthocyanins in jams obtained at pH 3.5-4.0 were degraded by 40-48%. In another study on blueberry jam, in contrast to other polyphenolics, the level of total flavonols was stable in response to processing, with >94% retention, compared to levels found in fresh berries [31] (Figure 3). Our study showed a degradation of HBA compound ranging from 70 to 90%. This had the highest degradation rate comparing with other existing compounds.
Chokeberries are a rich source of anthocyanins compared with other fruits. Due to this, they are usually used in the food industry as colorants or as a supplementary source of antioxidants. A recent study showed that the supplementation of strawberry jams with chokeberries and flowering quince during processing increase in the content of phenolic components in final products, especially for proanthocyanidins [34].

Blackthorn
These berries are becoming very popular in the food industry due to their complex and valuable phytochemical composition and also due to their easily availability in nature. The chromatographic analysis of fresh berries or jams revealed the presence of five phenolic compounds ( Figure 4). A decrease in the content of anthocyanins was observed immediately after processing and also, during storage time as it is shown in Figure 3. This degradation can be attributed to the hydrolytic reactions that led to the conversion of anthocyanin glycosides into chalcones, whose form can be rapidly transformed into phenolic acids and aldehydes.
Moreover, heat stable forms of polyphenol oxidase or peroxidase may play roles in the reduction of anthocyanins [30,31]. Another mechanism that may be associated with anthocyanin degradation is related to the hydrolysis of the glycoside linkages. This hydrolysis is known as the first step towards anthocyanin degradation, because high temperatures can shift the anthocyanin equilibrium towards the colorless chalcones. Chalcone degradation can occur due to the presence of oxidation reactions which can generate brown compounds or pigments that have high molecular weights. Additionally, pH values can affect the flavylium salt degradation, which is stable under highly acidic conditions. At higher pH values, salts can lose a proton and are easily transformed into an unstable pigment (quinoidal base) that is bonded to water and forms a colorless compound, commonly known as chromanol [30,32]. During processing and at the end of the storage period, anthocyanin degradation ranged from 58.90% to 74.30%. The results of the loss of anthocyanin content were lower compared with data reported previously for processed and stored black carrot jam and marmalade (87.60-95.60%). Degradation was also observed in flavonoids immediately after processing and during storage. This finding is in agreement with the previously reported data for Rubus coreanus Miquel berry jams [33]. The results revealed that jams prepared at pH 2.0-3.0 lost 33-35% of their anthocyanins, while the total amount of anthocyanins in jams obtained at pH 3.5-4.0 were degraded by 40-48%. In another study on blueberry jam, in contrast to other polyphenolics, the level of total flavonols was stable in response to processing, with >94% retention, compared to levels found in fresh berries [31] (Figure 3). Our study showed a degradation of HBA compound ranging from 70 to 90%. This had the highest degradation rate comparing with other existing compounds.
Chokeberries are a rich source of anthocyanins compared with other fruits. Due to this, they are usually used in the food industry as colorants or as a supplementary source of antioxidants. A recent study showed that the supplementation of strawberry jams with chokeberries and flowering quince during processing increase in the content of phenolic components in final products, especially for proanthocyanidins [34].

Blackthorn
These berries are becoming very popular in the food industry due to their complex and valuable phytochemical composition and also due to their easily availability in nature. The chromatographic analysis of fresh berries or jams revealed the presence of five phenolic compounds ( Figure 4). A decrease in the content of anthocyanins was observed immediately after processing and also, during storage time as it is shown in Figure 3. This degradation can be attributed to the hydrolytic reactions that led to the conversion of anthocyanin glycosides into chalcones, whose form can be rapidly transformed into phenolic acids and aldehydes.
Moreover, heat stable forms of polyphenol oxidase or peroxidase may play roles in the reduction of anthocyanins [30,31]. Another mechanism that may be associated with anthocyanin degradation is related to the hydrolysis of the glycoside linkages. This hydrolysis is known as the first step towards anthocyanin degradation, because high temperatures can shift the anthocyanin equilibrium towards the colorless chalcones. Chalcone degradation can occur due to the presence of oxidation reactions which can generate brown compounds or pigments that have high molecular weights. Additionally, pH values can affect the flavylium salt degradation, which is stable under highly acidic conditions. At higher pH values, salts can lose a proton and are easily transformed into an unstable pigment (quinoidal base) that is bonded to water and forms a colorless compound, commonly known as chromanol [30,32]. During processing and at the end of the storage period, anthocyanin degradation ranged from 58.90% to 74.30%. The results of the loss of anthocyanin content were lower compared with data reported previously for processed and stored black carrot jam and marmalade (87.60-95.60%). Degradation was also observed in flavonoids immediately after processing and during storage. This finding is in agreement with the previously reported data for Rubus coreanus Miquel berry jams [33]. The results revealed that jams prepared at pH 2.0-3.0 lost 33-35% of their anthocyanins, while the total amount of anthocyanins in jams obtained at pH 3.5-4.0 were degraded by 40-48%. In another study on blueberry jam, in contrast to other polyphenolics, the level of total flavonols was stable in response to processing, with >94% retention, compared to levels found in fresh berries [31] (Figure 3). Our study showed a degradation of HBA compound ranging from 70 to 90%. This had the highest degradation rate comparing with other existing compounds.
Chokeberries are a rich source of anthocyanins compared with other fruits. Due to this, they are usually used in the food industry as colorants or as a supplementary source of antioxidants. A recent study showed that the supplementation of strawberry jams with chokeberries and flowering quince during processing increase in the content of phenolic components in final products, especially for proanthocyanidins [34].

Blackthorn
These berries are becoming very popular in the food industry due to their complex and valuable phytochemical composition and also due to their easily availability in nature. The chromatographic analysis of fresh berries or jams revealed the presence of five phenolic compounds ( Figure 4).
This degradation can be attributed to the hydrolytic reactions that led to the conversion of anthocyanin glycosides into chalcones, whose form can be rapidly transformed into phenolic acids and aldehydes.
Moreover, heat stable forms of polyphenol oxidase or peroxidase may play roles in the reduction of anthocyanins [30,31]. Another mechanism that may be associated with anthocyanin degradation is related to the hydrolysis of the glycoside linkages. This hydrolysis is known as the first step towards anthocyanin degradation, because high temperatures can shift the anthocyanin equilibrium towards the colorless chalcones. Chalcone degradation can occur due to the presence of oxidation reactions which can generate brown compounds or pigments that have high molecular weights. Additionally, pH values can affect the flavylium salt degradation, which is stable under highly acidic conditions. At higher pH values, salts can lose a proton and are easily transformed into an unstable pigment (quinoidal base) that is bonded to water and forms a colorless compound, commonly known as chromanol [30,32]. During processing and at the end of the storage period, anthocyanin degradation ranged from 58.90% to 74.30%. The results of the loss of anthocyanin content were lower compared with data reported previously for processed and stored black carrot jam and marmalade (87.60-95.60%). Degradation was also observed in flavonoids immediately after processing and during storage. This finding is in agreement with the previously reported data for Rubus coreanus Miquel berry jams [33]. The results revealed that jams prepared at pH 2.0-3.0 lost 33-35% of their anthocyanins, while the total amount of anthocyanins in jams obtained at pH 3.5-4.0 were degraded by 40-48%. In another study on blueberry jam, in contrast to other polyphenolics, the level of total flavonols was stable in response to processing, with >94% retention, compared to levels found in fresh berries [31] ( Figure 3). Our study showed a degradation of HBA compound ranging from 70 to 90%. This had the highest degradation rate comparing with other existing compounds.
Chokeberries are a rich source of anthocyanins compared with other fruits. Due to this, they are usually used in the food industry as colorants or as a supplementary source of antioxidants. A recent study showed that the supplementation of strawberry jams with chokeberries and flowering quince during processing increase in the content of phenolic components in final products, especially for proanthocyanidins [34].

Blackthorn
These berries are becoming very popular in the food industry due to their complex and valuable phytochemical composition and also due to their easily availability in nature. The chromatographic analysis of fresh berries or jams revealed the presence of five phenolic compounds ( Figure 4).  The identified compounds are summarized in Table 1. The mass spectra of peak 1 displayed a parent ion at m/z 181 and one fragment ion at m/z 163, which was identified as caffeic acid, while peak 2 had an ion at m/z 181 which was identified as neochlorogenic acid. Two more molecules were identified as quercetin (m/z 303) and quercetin-3-O-rutinoside (m/z 611). Anthocyanins were shown to be the most abundant class (fresh berries, jam), followed by flavonols and HCA. In contrast to the other berries in this study, blackthorn did not contain HBA. The identified anthocyanins were two glycosylated cyanidins and one acylated peonidin. For peak 5, in the case of cyanidins, the ESI-MS analysis indicated the presence of a molecular ion at m/z 449 corresponding to cyanidin-3-Ogalactoside.
The second isolated anthocyanin showed a molecular ion at m/z 595, suggesting the presence of cyanidin-3-O-rutinoside. In this case, the ion at m/z 449 showed a loss of one molecule of rhamnoside and an ion at m/z 287, confirming the presence of aglycone cyanidin. Further, the peaks, registered with a molecular ion at m/z 603 and a fragment ion at m/z 301, were identified as peonidin 3-(6"coumaroyl) glucoside. Peak 4 had a molecular ion at m/z 609 and two fragment ions at m/z 463 and m/z 301, which indicated the presence of peonidin 3-(6"-coumaroyl) glucoside ( Figure 5). This anthocyanin identification is in agreement with Stefǎnuț et al., who also reported the presence of peonidin-3-O-rutinoside [26]. These differences may be attributed to the climatic conditions or harvesting time. The identified compounds are summarized in Table 1. The mass spectra of peak 1 displayed a parent ion at m/z 181 and one fragment ion at m/z 163, which was identified as caffeic acid, while peak 2 had an ion at m/z 181 which was identified as neochlorogenic acid. Two more molecules were identified as quercetin (m/z 303) and quercetin-3-O-rutinoside (m/z 611). Anthocyanins were shown to be the most abundant class (fresh berries, jam), followed by flavonols and HCA. In contrast to the other berries in this study, blackthorn did not contain HBA. The identified anthocyanins were two glycosylated cyanidins and one acylated peonidin. For peak 5, in the case of cyanidins, the ESI-MS analysis indicated the presence of a molecular ion at m/z 449 corresponding to cyanidin-3-O-galactoside.
The second isolated anthocyanin showed a molecular ion at m/z 595, suggesting the presence of cyanidin-3-O-rutinoside. In this case, the ion at m/z 449 showed a loss of one molecule of rhamnoside and an ion at m/z 287, confirming the presence of aglycone cyanidin. Further, the peaks, registered with a molecular ion at m/z 603 and a fragment ion at m/z 301, were identified as peonidin 3-(6"-coumaroyl) glucoside. Peak 4 had a molecular ion at m/z 609 and two fragment ions at m/z 463 and m/z 301, which indicated the presence of peonidin 3-(6"-coumaroyl) glucoside ( Figure 5). This anthocyanin identification is in agreement with Stefǎnut , et al., who also reported the presence of peonidin-3-O-rutinoside [26]. These differences may be attributed to the climatic conditions or harvesting time. After processing, the bioactive compounds of blackthorn jam decreased ( Figure 6). The amount of anthocyanins in berries decreased by 50% immediately after thermal processing compared to fresh ones and reached 82.56% after 6 months of storage. The same results were reported for black carrot (Daucus carota) jams and marmalades [30]. After 20 weeks of storage, the preserved anthocyanins and antioxidant capacity in samples stored at 4 °C were 53.4-81.0% and 45.2-92.0%, respectively. Timedependent degradation of flavonols, ranging from 23 to 67%, was also observed. As in the case of HCA, the amounts of caffeic and neochlorogenic acid dramatically decreased from 41 to 81% during storage time. After processing, the bioactive compounds of blackthorn jam decreased ( Figure 6). The amount of anthocyanins in berries decreased by 50% immediately after thermal processing compared to fresh ones and reached 82.56% after 6 months of storage. The same results were reported for black carrot (Daucus carota) jams and marmalades [30]. After 20 weeks of storage, the preserved anthocyanins and antioxidant capacity in samples stored at 4 • C were 53.4-81.0% and 45.2-92.0%, respectively. Time-dependent degradation of flavonols, ranging from 23 to 67%, was also observed. As in the case of HCA, the amounts of caffeic and neochlorogenic acid dramatically decreased from 41 to 81% during storage time.

Elderberry
Thus far, elderberries were not very popular for consumers due to their alkaloid content. However, this can easily be neutralized by thermal processing. Recently, an interest in elderberries has developed due to their rich polyphenolic compound content and thus, high antioxidant potential. Consequently, these berries are now becoming a very popular crop in Europe, and thus, we included them in our study. The HPLC analysis revealed that elderberry has a simple phenolic fingerprint, characterized by the presence of six compounds: two anthocyanins and four hydroxycinnamic acids (HCA) (Figure 7). A decrease in the content of anthocyanins was observed immediately after processing and also, during storage time as it is shown in Figure 3. This degradation can be attributed to the hydrolytic reactions that led to the conversion of anthocyanin glycosides into chalcones, whose form can be rapidly transformed into phenolic acids and aldehydes.
Moreover, heat stable forms of polyphenol oxidase or peroxidase may play roles in the reduction of anthocyanins [30,31]. Another mechanism that may be associated with anthocyanin degradation is related to the hydrolysis of the glycoside linkages. This hydrolysis is known as the first step towards anthocyanin degradation, because high temperatures can shift the anthocyanin equilibrium towards the colorless chalcones. Chalcone degradation can occur due to the presence of oxidation reactions which can generate brown compounds or pigments that have high molecular weights. Additionally, pH values can affect the flavylium salt degradation, which is stable under highly acidic conditions. At higher pH values, salts can lose a proton and are easily transformed into an unstable pigment (quinoidal base) that is bonded to water and forms a colorless compound, commonly known as chromanol [30,32]. During processing and at the end of the storage period, anthocyanin degradation ranged from 58.90% to 74.30%. The results of the loss of anthocyanin content were lower compared with data reported previously for processed and stored black carrot jam and marmalade (87.60-95.60%). Degradation was also observed in flavonoids immediately after processing and during storage. This finding is in agreement with the previously reported data for Rubus coreanus Miquel berry jams [33]. The results revealed that jams prepared at pH 2.0-3.0 lost 33-35% of their anthocyanins, while the total amount of anthocyanins in jams obtained at pH 3.5-4.0 were degraded by 40-48%. In another study on blueberry jam, in contrast to other polyphenolics, the level of total flavonols was stable in response to processing, with >94% retention, compared to levels found in fresh berries [31] (Figure 3). Our study showed a degradation of HBA compound ranging from 70 to 90%. This had the highest degradation rate comparing with other existing compounds.
Chokeberries are a rich source of anthocyanins compared with other fruits. Due to this, they are usually used in the food industry as colorants or as a supplementary source of antioxidants. A recent study showed that the supplementation of strawberry jams with chokeberries and flowering quince during processing increase in the content of phenolic components in final products, especially for proanthocyanidins [34].

Blackthorn
These berries are becoming very popular in the food industry due to their complex and valuable phytochemical composition and also due to their easily availability in nature. The chromatographic analysis of fresh berries or jams revealed the presence of five phenolic compounds ( Figure 4). A decrease in the content of anthocyanins was observed immediately after processing and also, during storage time as it is shown in Figure 3. This degradation can be attributed to the hydrolytic reactions that led to the conversion of anthocyanin glycosides into chalcones, whose form can be rapidly transformed into phenolic acids and aldehydes.
Moreover, heat stable forms of polyphenol oxidase or peroxidase may play roles in the reduction of anthocyanins [30,31]. Another mechanism that may be associated with anthocyanin degradation is related to the hydrolysis of the glycoside linkages. This hydrolysis is known as the first step towards anthocyanin degradation, because high temperatures can shift the anthocyanin equilibrium towards the colorless chalcones. Chalcone degradation can occur due to the presence of oxidation reactions which can generate brown compounds or pigments that have high molecular weights. Additionally, pH values can affect the flavylium salt degradation, which is stable under highly acidic conditions. At higher pH values, salts can lose a proton and are easily transformed into an unstable pigment (quinoidal base) that is bonded to water and forms a colorless compound, commonly known as chromanol [30,32]. During processing and at the end of the storage period, anthocyanin degradation ranged from 58.90% to 74.30%. The results of the loss of anthocyanin content were lower compared with data reported previously for processed and stored black carrot jam and marmalade (87.60-95.60%). Degradation was also observed in flavonoids immediately after processing and during storage. This finding is in agreement with the previously reported data for Rubus coreanus Miquel berry jams [33]. The results revealed that jams prepared at pH 2.0-3.0 lost 33-35% of their anthocyanins, while the total amount of anthocyanins in jams obtained at pH 3.5-4.0 were degraded by 40-48%. In another study on blueberry jam, in contrast to other polyphenolics, the level of total flavonols was stable in response to processing, with >94% retention, compared to levels found in fresh berries [31] (Figure 3). Our study showed a degradation of HBA compound ranging from 70 to 90%. This had the highest degradation rate comparing with other existing compounds.
Chokeberries are a rich source of anthocyanins compared with other fruits. Due to this, they are usually used in the food industry as colorants or as a supplementary source of antioxidants. A recent study showed that the supplementation of strawberry jams with chokeberries and flowering quince during processing increase in the content of phenolic components in final products, especially for proanthocyanidins [34].

Blackthorn
These berries are becoming very popular in the food industry due to their complex and valuable phytochemical composition and also due to their easily availability in nature. The chromatographic analysis of fresh berries or jams revealed the presence of five phenolic compounds ( Figure 4). p < 0.001).

Elderberry
Thus far, elderberries were not very popular for consumers due to their alkaloid content. However, this can easily be neutralized by thermal processing. Recently, an interest in elderberries has developed due to their rich polyphenolic compound content and thus, high antioxidant potential. Consequently, these berries are now becoming a very popular crop in Europe, and thus, we included them in our study. The HPLC analysis revealed that elderberry has a simple phenolic fingerprint, characterized by the presence of six compounds: two anthocyanins and four hydroxycinnamic acids (HCA) (Figure 7).

Elderberry
Thus far, elderberries were not very popular for consumers due to their alkaloid content. However, this can easily be neutralized by thermal processing. Recently, an interest in elderberries has developed due to their rich polyphenolic compound content and thus, high antioxidant potential. Consequently, these berries are now becoming a very popular crop in Europe, and thus, we included them in our study. The HPLC analysis revealed that elderberry has a simple phenolic fingerprint, characterized by the presence of six compounds: two anthocyanins and four hydroxycinnamic acids (HCA) (Figure 7).  The identified anthocyanins in fresh fruits and prepared jam were exclusively cyanidin-based anthocyanins, quercetin derivatives, and chlorogenic acid. Peak 1 was assigned to chlorogenic acid because it has a parent ion at m/z 355 and two fragments ions at m/z 181 and 163. The next three peaks (2,3,4) were found to be quercetin-3-O-rutinoside (m/z 611, 303), quercetin-3-O-glucoside (465, 303), and quercetin (303) (Figure 8). Further, the ESI-MS analysis showed the presence of molecular ions at m/z 381 corresponding to cyanidin-3-O-sambubioside, while peak 6 was identified as cyanidin-3-O-glucoside (m/z 449, 287) (Table 1, Figure 8).
Three flavonols were identified in this sample, with quercetin-3-O-rutinoside (Figure 7) being the major one, followed by quercetin-3-O-glucoside. This rank was maintained after thermal processing and storage (Figure 9).

Quercetin-3-O-glucoside
Cyanidin-3-O-sambubioside The obtained results regarding phenolic changes during processing are in agreement with the available literature [35]. The current body of literature indicates that thermal processes have a large influence on flavonoid stability, especially for rutin which has higher stability compared to its aglycon form (quercetin) [32,33]. These findings were attributed to the presence of carbanion formation because of the glycosylation of the 3-hydroxyl group in the C-ring.
Other authors also reported that glycosylated form are more stable: luteolin was found more resistant to heat than rutin or luteolin-7-glucoside when heated at 180 °C for 180 min [34]. Moreover, the highest level of polyphenolic compounds was observed in the extraction of Orthosiphon stanmineus leaf, with 80% methanol at 40 °C and a significant degradation of the analytes recorded at temperatures above 60 °C [16]. The results of the previously cited study also showed a significant The obtained results regarding phenolic changes during processing are in agreement with the available literature [35]. The current body of literature indicates that thermal processes have a large influence on flavonoid stability, especially for rutin which has higher stability compared to its aglycon form (quercetin) [32,33]. These findings were attributed to the presence of carbanion formation because of the glycosylation of the 3-hydroxyl group in the C-ring.
Other authors also reported that glycosylated form are more stable: luteolin was found more resistant to heat than rutin or luteolin-7-glucoside when heated at 180 • C for 180 min [34]. Moreover, the highest level of polyphenolic compounds was observed in the extraction of Orthosiphon stanmineus leaf, with 80% methanol at 40 • C and a significant degradation of the analytes recorded at temperatures above 60 • C [16]. The results of the previously cited study also showed a significant reduction in the free radical-scavenging activity of the samples which were treated at temperatures above 60 • C. To conclude, the antioxidant capacity of the flavonoids, and therefore, their pathway to oxidative degradation is linked with their special structural features.
Molecules 2018, 23, x FOR PEER REVIEW 10 of 18 temperatures above 60 °C [16]. The results of the previously cited study also showed a significant reduction in the free radical-scavenging activity of the samples which were treated at temperatures above 60 °C. To conclude, the antioxidant capacity of the flavonoids, and therefore, their pathway to oxidative degradation is linked with their special structural features.

The Blackcurrant
This sample had the most complex matrix of polyphenol constituents. Prior to the ESI-MS analysis, we were able to identify 20 individual compounds ( Figure 10). The available literature data indicates that the polyphenolic compounds present in blackcurrant are caffeic acid, hydroxybenzoic acid, quercetin, myricetin, cyanidin, and delphinidin [28,29,36]. Table 1 presents the retention times, molecular ions, and fragmentation information for all polyphenolic compounds found in the present study. The mass spectra of peak 1 displayed a parent ion at m/z 341 and one fragment ion at m/z 139 which was identified as 4-hydroxybenzoic acid-4-O-glucoside. This sample was also characterized by the presence of 13 flavonols-mainly kaempferol-as well as quercetin or myricetin derivates. For anthocyanins, glucosides and rutinosides were the main sugar moieties of delphinidin and cyanidin identified (Table 1). Overall, the anthocyanins identified in the present study are in agreement with those detected in previous studies [12].
The first identified anthocyanin (peak 1) was delphinidin-3-O-glucoside (m/z 303), while the second isolated anthocyanin showed a parent ion at 611 and a molecular ion at m/z, suggesting the presence of delphinidin-3-O-rutinoside ( Figure 11). Moreover, the ion at m/z 449 indicated the loss of one molecule of rhamnoside, and the ion at m/z 287 confirmed the presence of aglycone cyanidin (Table 1). A decrease in the content of anthocyanins was observed immediately after processing and also, during storage time as it is shown in Figure 3. This degradation can be attributed to the hydrolytic reactions that led to the conversion of anthocyanin glycosides into chalcones, whose form can be rapidly transformed into phenolic acids and aldehydes.
Moreover, heat stable forms of polyphenol oxidase or peroxidase may play roles in the reduction of anthocyanins [30,31]. Another mechanism that may be associated with anthocyanin degradation is related to the hydrolysis of the glycoside linkages. This hydrolysis is known as the first step towards anthocyanin degradation, because high temperatures can shift the anthocyanin equilibrium towards the colorless chalcones. Chalcone degradation can occur due to the presence of oxidation reactions which can generate brown compounds or pigments that have high molecular weights. Additionally, pH values can affect the flavylium salt degradation, which is stable under highly acidic conditions. At higher pH values, salts can lose a proton and are easily transformed into an unstable pigment (quinoidal base) that is bonded to water and forms a colorless compound, commonly known as chromanol [30,32]. During processing and at the end of the storage period, anthocyanin degradation ranged from 58.90% to 74.30%. The results of the loss of anthocyanin content were lower compared with data reported previously for processed and stored black carrot jam and marmalade (87.60-95.60%). Degradation was also observed in flavonoids immediately after processing and during storage. This finding is in agreement with the previously reported data for Rubus coreanus Miquel berry jams [33]. The results revealed that jams prepared at pH 2.0-3.0 lost 33-35% of their anthocyanins, while the total amount of anthocyanins in jams obtained at pH 3.5-4.0 were degraded by 40-48%. In another study on blueberry jam, in contrast to other polyphenolics, the level of total flavonols was stable in response to processing, with >94% retention, compared to levels found in fresh berries [31] (Figure 3). Our study showed a degradation of HBA compound ranging from 70 to 90%. This had the highest degradation rate comparing with other existing compounds.
Chokeberries are a rich source of anthocyanins compared with other fruits. Due to this, they are usually used in the food industry as colorants or as a supplementary source of antioxidants. A recent study showed that the supplementation of strawberry jams with chokeberries and flowering quince during processing increase in the content of phenolic components in final products, especially for proanthocyanidins [34].

Blackthorn
These berries are becoming very popular in the food industry due to their complex and valuable A decrease in the content of anthocyanins was observed immediately after processing and also, during storage time as it is shown in Figure 3. This degradation can be attributed to the hydrolytic reactions that led to the conversion of anthocyanin glycosides into chalcones, whose form can be rapidly transformed into phenolic acids and aldehydes.
Moreover, heat stable forms of polyphenol oxidase or peroxidase may play roles in the reduction of anthocyanins [30,31]. Another mechanism that may be associated with anthocyanin degradation is related to the hydrolysis of the glycoside linkages. This hydrolysis is known as the first step towards anthocyanin degradation, because high temperatures can shift the anthocyanin equilibrium towards the colorless chalcones. Chalcone degradation can occur due to the presence of oxidation reactions which can generate brown compounds or pigments that have high molecular weights. Additionally, pH values can affect the flavylium salt degradation, which is stable under highly acidic conditions. At higher pH values, salts can lose a proton and are easily transformed into an unstable pigment (quinoidal base) that is bonded to water and forms a colorless compound, commonly known as chromanol [30,32]. During processing and at the end of the storage period, anthocyanin degradation ranged from 58.90% to 74.30%. The results of the loss of anthocyanin content were lower compared with data reported previously for processed and stored black carrot jam and marmalade (87.60-95.60%). Degradation was also observed in flavonoids immediately after processing and during storage. This finding is in agreement with the previously reported data for Rubus coreanus Miquel berry jams [33]. The results revealed that jams prepared at pH 2.0-3.0 lost 33-35% of their anthocyanins, while the total amount of anthocyanins in jams obtained at pH 3.5-4.0 were degraded by 40-48%. In another study on blueberry jam, in contrast to other polyphenolics, the level of total flavonols was stable in response to processing, with >94% retention, compared to levels found in fresh berries [31] (Figure 3). Our study showed a degradation of HBA compound ranging from 70 to 90%. This had the highest degradation rate comparing with other existing compounds.
Chokeberries are a rich source of anthocyanins compared with other fruits. Due to this, they are usually used in the food industry as colorants or as a supplementary source of antioxidants. A recent study showed that the supplementation of strawberry jams with chokeberries and flowering quince during processing increase in the content of phenolic components in final products, especially for proanthocyanidins [34].

Blackthorn
These berries are becoming very popular in the food industry due to their complex and valuable p < 0.001).

The Blackcurrant
This sample had the most complex matrix of polyphenol constituents. Prior to the ESI-MS analysis, we were able to identify 20 individual compounds ( Figure 10). The available literature data indicates that the polyphenolic compounds present in blackcurrant are caffeic acid, hydroxybenzoic acid, quercetin, myricetin, cyanidin, and delphinidin [28,29,36]. Table 1 presents the retention times, molecular ions, and fragmentation information for all polyphenolic compounds found in the present study. The mass spectra of peak 1 displayed a parent ion at m/z 341 and one fragment ion at m/z 139 which was identified as 4-hydroxybenzoic acid-4-O-glucoside. This sample was also characterized by the presence of 13 flavonols-mainly kaempferol-as well as quercetin or myricetin derivates. For anthocyanins, glucosides and rutinosides were the main sugar moieties of delphinidin and cyanidin identified (Table 1). Overall, the anthocyanins identified in the present study are in agreement with those detected in previous studies [12].
The first identified anthocyanin (peak 1) was delphinidin-3-O-glucoside (m/z 303), while the second isolated anthocyanin showed a parent ion at 611 and a molecular ion at m/z, suggesting the presence of delphinidin-3-O-rutinoside ( Figure 11). Moreover, the ion at m/z 449 indicated the loss of one molecule of rhamnoside, and the ion at m/z 287 confirmed the presence of aglycone cyanidin (Table 1).   Our results indicated that heating and prolonged storage time are influencing the contents of the individual phenolic compounds, and moreover, their content decreased overall ( Figure 12). These findings are in agreement with a previous study [37] which reported that flavonoid loss may depend on the preparation method used, such as boiling, frying with oil and butter, or microwaving.
Our results indicated that heating and prolonged storage time are influencing the contents of the individual phenolic compounds, and moreover, their content decreased overall ( Figure 12). These findings are in agreement with a previous study [37] which reported that flavonoid loss may depend on the preparation method used, such as boiling, frying with oil and butter, or microwaving.

Cell Proliferation
Cell proliferation was performed by evaluating the mitochondrial succinate dehydrogenase activity of both cell lines (normal and melanoma) after applying a 24 h treatment with RPE. In the case of a normal fibroblast cell line (HFL-1), all the extracts showed a stimulation of proliferation, especially elderberry extract, while blackthorn extract exerted a less significant influence on cell proliferation. These differences could be explained by their varied phytochemical compositions. In this context we can state that elderberry extract was characterised by the presence of anthocyanins and HCA, while blackthorn, in addition to its anthocyanin and HCA contents, was shown to contain flavonols as well. Further, for the human melanoma cell line (A375), the applied treatments reduced cell proliferation after 24 h at the highest applied concentration (100 μg/mL) ( Figure 13). The obtained results were well correlated with the dose concentration. Moreover, data showed that all of the applied treatments on the melanoma cell line have a cell proliferation effect ranging from 20-25%. The extract with the highest antiproliferative potential was the one obtained from blackthorn. This fact can be attributed to their phytochemical composition which is abundant in glycosylated and acylated anthocyanins (356 mg/100 g FW). In other words, there was no dose that inhibited 50% of the cultivated cells. The obtained results demonstrate that extracts rich in polyphenols have antiproliferative potential on tumour cells, while on normal cells, they have been proven to stimulate cell proliferation in a dose dependent manner. A decrease in the content of anthocyanins was observed immediately after processing and also, during storage time as it is shown in Figure 3. This degradation can be attributed to the hydrolytic reactions that led to the conversion of anthocyanin glycosides into chalcones, whose form can be rapidly transformed into phenolic acids and aldehydes.
Moreover, heat stable forms of polyphenol oxidase or peroxidase may play roles in the reduction of anthocyanins [30,31]. Another mechanism that may be associated with anthocyanin degradation is related to the hydrolysis of the glycoside linkages. This hydrolysis is known as the first step towards anthocyanin degradation, because high temperatures can shift the anthocyanin equilibrium towards the colorless chalcones. Chalcone degradation can occur due to the presence of oxidation reactions which can generate brown compounds or pigments that have high molecular weights. Additionally, pH values can affect the flavylium salt degradation, which is stable under highly acidic conditions. At higher pH values, salts can lose a proton and are easily transformed into an unstable pigment (quinoidal base) that is bonded to water and forms a colorless compound, commonly known as chromanol [30,32]. During processing and at the end of the storage period, anthocyanin degradation ranged from 58.90% to 74.30%. The results of the loss of anthocyanin content were lower compared with data reported previously for processed and stored black carrot jam and marmalade (87.60-95.60%). Degradation was also observed in flavonoids immediately after processing and during storage. This finding is in agreement with the previously reported data for Rubus coreanus Miquel berry jams [33]. The results revealed that jams prepared at pH 2.0-3.0 lost 33-35% of their anthocyanins, while the total amount of anthocyanins in jams obtained at pH 3.5-4.0 were degraded by 40-48%. In another study on blueberry jam, in contrast to other polyphenolics, the level of total flavonols was stable in response to processing, with >94% retention, compared to levels found in fresh berries [31] (Figure 3). Our study showed a degradation of HBA compound ranging from 70 to 90%. This had the highest degradation rate comparing with other existing compounds.
Chokeberries are a rich source of anthocyanins compared with other fruits. Due to this, they are usually used in the food industry as colorants or as a supplementary source of antioxidants. A recent study showed that the supplementation of strawberry jams with chokeberries and flowering quince during processing increase in the content of phenolic components in final products, especially for proanthocyanidins [34].

Blackthorn
These berries are becoming very popular in the food industry due to their complex and valuable phytochemical composition and also due to their easily availability in nature. The chromatographic analysis of fresh berries or jams revealed the presence of five phenolic compounds (Figure 4). served immediately after processing and also, ry jams during storage, (anthocyanins (ANT), xybenzoic acids (HBA)) Data represents the s (significant differences, ** , ••, ••, ▪▪ p < 0.01, *** , lytic reactions that led to the conversion of n be rapidly transformed into phenolic acids or peroxidase may play roles in the reduction be associated with anthocyanin degradation . This hydrolysis is known as the first step ratures can shift the anthocyanin equilibrium n can occur due to the presence of oxidation pigments that have high molecular weights. gradation, which is stable under highly acidic n and are easily transformed into an unstable rms a colorless compound, commonly known e end of the storage period, anthocyanin of the loss of anthocyanin content were lower and stored black carrot jam and marmalade lavonoids immediately after processing and previously reported data for Rubus coreanus s prepared at pH 2.0-3.0 lost 33-35% of their in jams obtained at pH 3.5-4.0 were degraded trast to other polyphenolics, the level of total % retention, compared to levels found in fresh n of HBA compound ranging from 70 to 90%. other existing compounds. mpared with other fruits. Due to this, they are pplementary source of antioxidants. A recent jams with chokeberries and flowering quince components in final products, especially for d industry due to their complex and valuable y availability in nature. The chromatographic of five phenolic compounds (Figure 4).
Molecules 2018, 23, x FOR PEER REVIEW 5 A decrease in the content of anthocyanins was observed immediately after processing and a during storage time as it is shown in Figure 3. This degradation can be attributed to the hydrolytic reactions that led to the conversion anthocyanin glycosides into chalcones, whose form can be rapidly transformed into phenolic a and aldehydes.
Moreover, heat stable forms of polyphenol oxidase or peroxidase may play roles in the reduc of anthocyanins [30,31]. Another mechanism that may be associated with anthocyanin degrada is related to the hydrolysis of the glycoside linkages. This hydrolysis is known as the first s towards anthocyanin degradation, because high temperatures can shift the anthocyanin equilibr towards the colorless chalcones. Chalcone degradation can occur due to the presence of oxida reactions which can generate brown compounds or pigments that have high molecular weig Additionally, pH values can affect the flavylium salt degradation, which is stable under highly ac conditions. At higher pH values, salts can lose a proton and are easily transformed into an unst pigment (quinoidal base) that is bonded to water and forms a colorless compound, commonly kno as chromanol [30,32]. During processing and at the end of the storage period, anthocya degradation ranged from 58.90% to 74.30%. The results of the loss of anthocyanin content were lo compared with data reported previously for processed and stored black carrot jam and marmal (87.60-95.60%). Degradation was also observed in flavonoids immediately after processing during storage. This finding is in agreement with the previously reported data for Rubus corea Miquel berry jams [33]. The results revealed that jams prepared at pH 2.0-3.0 lost 33-35% of t anthocyanins, while the total amount of anthocyanins in jams obtained at pH 3.5-4.0 were degra by 40-48%. In another study on blueberry jam, in contrast to other polyphenolics, the level of t flavonols was stable in response to processing, with >94% retention, compared to levels found in fr berries [31] (Figure 3). Our study showed a degradation of HBA compound ranging from 70 to 9 This had the highest degradation rate comparing with other existing compounds.
Chokeberries are a rich source of anthocyanins compared with other fruits. Due to this, they usually used in the food industry as colorants or as a supplementary source of antioxidants. A re study showed that the supplementation of strawberry jams with chokeberries and flowering qu during processing increase in the content of phenolic components in final products, especially proanthocyanidins [34].

Blackthorn
These berries are becoming very popular in the food industry due to their complex and valu phytochemical composition and also due to their easily availability in nature. The chromatograp analysis of fresh berries or jams revealed the presence of five phenolic compounds (Figure 4). p < 0.001).

Cell Proliferation
Cell proliferation was performed by evaluating the mitochondrial succinate dehydrogenase activity of both cell lines (normal and melanoma) after applying a 24 h treatment with RPE. In the case of a normal fibroblast cell line (HFL-1), all the extracts showed a stimulation of proliferation, especially elderberry extract, while blackthorn extract exerted a less significant influence on cell proliferation. These differences could be explained by their varied phytochemical compositions. In this context we can state that elderberry extract was characterised by the presence of anthocyanins and HCA, while blackthorn, in addition to its anthocyanin and HCA contents, was shown to contain flavonols as well. Further, for the human melanoma cell line (A375), the applied treatments reduced cell proliferation after 24 h at the highest applied concentration (100 µg/mL) ( Figure 13). The obtained results were well correlated with the dose concentration. Moreover, data showed that all of the applied treatments on the melanoma cell line have a cell proliferation effect ranging from 20-25%. The extract with the highest antiproliferative potential was the one obtained from blackthorn. This fact can be attributed to their phytochemical composition which is abundant in glycosylated and acylated anthocyanins (356 mg/100 g FW). In other words, there was no dose that inhibited 50% of the cultivated cells. The obtained results demonstrate that extracts rich in polyphenols have antiproliferative potential on tumour cells, while on normal cells, they have been proven to stimulate cell proliferation in a dose dependent manner. This fact is in agreement with other published data [38]. However, like all natural plant metabolites, anthocyanins are unstable and highly susceptible to degradation. Their stability is highly influenced by pH, temperature, light and also the presence of complexing compounds such as other phenolic acid and flavonoids or metals ions [39]. Moreover, it is not clear how exactly anthocyanins act at cellular level, but their proprieties seem to be closely related to their antioxidant activity [40]. Being unstable molecules, when exposed to high pH values, during the in vitro testing, anthocyanins can rapidly degrade or be breakdown in different metabolites. Very little is known about the details and the mechanisms of anthocyanin absorption and transportation when comparing with other flavonoid groups, such as flavonols. Anthocyanins exhibit complex biochemistry and much remained to be discovered about the biochemical activity of these compounds.
Currently, most investigations on anthocyanins are focusing on solving these problems, as well as anthocyanin bioavailability which seems to be very low with <1% absorption from the ingested dietary dose [41]. Moreover anthocyanins are subject to degradation in vivo, resulting in a breakdown to phenolic acids and aldehydes [42], such as protocatechuic acid (PCA) and phloroglucinol aldehyde (PGA) in the case of cyanidin. For other anthocyanins classes such as glycosylated or acylated form, the consumption and bioavailability of dietary phenolics have become a major concern in phenolic chemopreventive and cancer therapy research and these were not researched as far as we know.

Discussion
The obtained results revealed that during processing and storage, the bioactive compounds from all berry jams degraded over time, but they still exert antioxidant activity. Prior to the chromatographic analysis, polyphenolic compounds were identified as flavonoids (anthocyanins (ANT), flavonols (FLA)) or non-flavonoids (phenolic acids derivates of hydroxycinnamic acids (HCA) and hydroxybenzoic acids (HBA)). The polyphenolic compounds were identified by comparing the peak m/z of each molecule as well as their fragmentation and elution orders (retention time) with previously published values and available standards. This fact is in agreement with other published data [38]. However, like all natural plant metabolites, anthocyanins are unstable and highly susceptible to degradation. Their stability is highly influenced by pH, temperature, light and also the presence of complexing compounds such as other phenolic acid and flavonoids or metals ions [39]. Moreover, it is not clear how exactly anthocyanins act at cellular level, but their proprieties seem to be closely related to their antioxidant activity [40]. Bein unstable molecules, when exposed to high pH values, during the in vitro testing, anthocyanins can rapidly degrade or be breakdown in different metabolites. Very little is known about the details and the mechanisms of anthocyanin absorption and transportation when comparing with other flavonoid groups, such as flavonols. Anthocyanins exhibit complex biochemistry and much remained to be discovered about the biochemical activity of these compounds.
Currently, most investigations on anthocyanins are focusing on solving these problems, as well as anthocyanin bioavailability which seems to be very low with <1% absorption from the ingested dietary dose [41]. Moreover anthocyanins are subject to degradation in vivo, resulting in a breakdown to phenolic acids and aldehydes [42], such as protocatechuic acid (PCA) and phloroglucinol aldehyde (PGA) in the case of cyanidin. For other anthocyanins classes such as glycosylated or acylated form, the consumption and bioavailability of dietary phenolics have become a major concern in phenolic chemopreventive and cancer therapy research and these were not researched as far as we know.

Discussion
The obtained results revealed that during processing and storage, the bioactive compounds from all berry jams degraded over time, but they still exert antioxidant activity. Prior to the chromatographic analysis, polyphenolic compounds were identified as flavonoids (anthocyanins (ANT), flavonols (FLA)) or non-flavonoids (phenolic acids derivates of hydroxycinnamic acids (HCA) and hydroxybenzoic acids (HBA)). The polyphenolic compounds were identified by comparing the peak m/z of each molecule as well as their fragmentation and elution orders (retention time) with previously published values and available standards.
Compared to the other classes of quantified compounds, the most significant decrease was observed in HCA. This variation was expected due to the variation in the constituents and phenolic types among the different analyzed berries. These data correspond with similar conclusions of previous experiments reported regarding polyphenols antioxidant activity and their thermal degradation [43].
Moreover any processing of berries (especially thermal) and also storage were proved to be responsible for the significant losses of polyphenols. In a recent study, processing methods had insignificant effects on blueberry ellagitannins, but juice processing of berries resulted in total ellagitannin losses of about 70-82% [44]. Same author reported that storage at 25 degrees C of all processed products resulted in dramatic losses in monomeric anthocyanins with as much as 75% losses of anthocyanins throughout storage [45]. Another study, reported that thermal processes blackberries, showed a decreased content of anthocyanins (cyanidin-3-glucoside (by 52%) and cyanidin-3-malonyl glucoside (64%) respectively). They also reported that anthocyanins continue to decline during storage, especially when temperatures were high [46]. The obtained values for each type of berry of processed product are not easy to compare with literature available data due to the source of the berries or fruit. Their initial amount of polyphenols may vary with cultivation type, variety or climatic condition.
For the in vitro test we have obtained promising results which can be compared with available published data. Many studies have proved that anthocyanins present beneficial effects for human health [47]. Because of their physiological activities, the consumption of anthocyanins may play a significant role in preventing lifestyle-related diseases such as cancer, diabetes, cardiovascular and neurological disease. However, the exact roles of the anthocyanins in human health maintenance versus other phytochemicals in a complex mixture from a fruit extract or whole food have not been completely sorted out. In vitro studies have shown various beneficial effects of anthocyanins regarding human health, but without doubt, in vivo, epidemiological and clinical trials would be more accurate. However, to the best of our knowledge, no report exists on the effect of processing on the phenolic compounds content of homemade jams from chokeberry, elderberry, blackcurrant or blackthorn.

Sampling Procedure
Berry fruits (chokeberry, elderberry, blackcurrant, and blackthorn) were purchased from local farmers near Cluj-Napoca, Romania. Immediately after harvesting, the berries were frozen at −18 • C for future analyses. Basically, a common and simple jam-making procedure was followed in this study. Jams were prepared from berries using 250 g of sugar and 500 g of chopped berries, this being the most common ratio found in homemade jam products. The obtained mixture was heated in a gas stove (85 • C) for 15 min each day over 3 consecutive days. After boiling on the 3rd day, the jams were allowed to cool down to room temperature before being placed in glass jars (45 g). The obtained jams were analyzed immediately after preparation, and the remaining jams were divided into four batches and stored in the dark at 4 • C. Samples were analyzed after 1, 3, 6, and 9 months of storage.

Extraction of Anthocyanin and Non-Anthocyanin Phenolics from Fresh Berries and Berry Jam
For the extraction of polyphenols, 5 g of fresh berries of each type were ground using an ultraturrax (Miccra D-9 KT Digitronic, Heitersheim, Germany). Additionally, the same amount of berry jams was homogenized and weighed, followed by the addition of 10 mL of methanol containing hydrochloric acid (0.3% v/v). The obtained mixtures were sonicated for 20 min in the dark and then centrifugated at 5000 rpm for 5 min. The supernatant was collected, and the extraction process was repeated until the samples were colorless. The extracts obtained for each sample were concentrated at 35 • C under reduced pressure (Rotavapor R-124, Buchi, Flawil, Switzerland) and then filtered through a 0.45 µm Millipore filter. All the sample preparation steps were carried out in subdued light and under controlled conditions.

RP-HPLC-PDA Identification and Quantification of Phenolic Compounds
HPLC analysis was performed on a Shimadzu (Kyoto, Japan) system equipped with a binary pump delivery system (model LC-20 AT Prominence), a degasser (model DGU-20 A3 Prominence), a UV-VIS diode array detector (model SPD-M20), and a Luna C-18 column (film thickness, 5 µm; 25 cm, 4.6 mm) (Phenomenex, Torrance, CA, USA). The mobile phases were formic acid (4.5%) in double-distilled water (solvent A) and acetonitrile (solvent B). The gradient elution system was as follows: 10% B, 0-9 min; 12% B, 9-17 min; 25% B, 17-30 min; 90% B, 30-50 min; and 10% B, 50-55 min. The flow rate was 0.8 mL/min, and all analyses were performed at 35 • C. Identification and peak assignments were conducted based on their retention times UV-VIS spectra, standards and available literature. The chromatograms were monitored at 340 and 520 nm. Anthocyanin quantification was conducted using a cyanidin-3-O-galactoside standard curve while for flavanol and phenolic acid was assessed using a rutin or chlorogenic acid standard curve.

HPLC-PDA/-ESI-MS Identification and Quantification of Phenolic Compounds
To confirm the identified compounds, an ESI-MS analysis was also conducted. The ESI-MS analysis was performed using an Agilent 1200 system equipped with a binary pump delivery system (LC-20 AT, Prominence), a degasser (DGU-20 A3, Prominence), a diode array SPD-M20 A UV-VIS detector (DAD), and an Eclipse XDB C18 column (4 µm, 4.6 × 150 mm) was used. The mobile phases used the following solvents: (A) bidistilled water and 0.1% acetic acid/acetonitrile (99/1 v/v), and (B) acetonitrile and acetic acid 0.1%. The gradient elution system conditions were as follows: 0-2 min, isocratic with 5% (v/v) eluent B; 2-18 min, linear gradient from 5% to 40% (v/v) eluent B; 18-20 min, linear gradient from 40% to 90% (v/v) eluent B; 20-24 min, isocratic on 90% (v/v) eluent B; 24-25 min, linear gradient from 90% to 5% (v/v) eluent B; 25-30 min, isocratic on 5% (v/v) eluent B. The flow rate was 0.5 mL/min, and the column temperature was maintained at 25 • C. The chromatograms were monitored at 280 and 340 nm, respectively. The identification of compounds was conducted based on their retention times, UV-VIS spectra, standards (chlorogenic acid, caffeic acid, quercetin-rutinoside, quercetin-glucoside, ellagic acid, and myricetin, all purchased from Sigma-Aldrich, and published data. The mass spectrometric data were obtained using a single quadrupole 6110 mass spectrometer (Agilent Technologies, Chelmsford, MA, USA) equipped with an ESI probe with scanning range between 280 to 1000 m/z. The measurements were performed in the positive mode, with an ion spray voltage of 3000 V and a capillary temperature of 350 • C.

Cell Culture
The metastatic B16-F10 murine melanoma cell line was purchased from ATCC (Rockville, MD, USA) and grown under standard conditions. More specifically the cells were cultivated in DMEM (Dulbecco's Modified Eagle Medium) medium containing 4.5 g/L glucose, 10% FBS supplemented with 2 mM glutamine, 1% penicillin, and streptomycin. The non-tumor model (HFL-1 human fetal lung fibroblast cell line, ATCC) was cultivated in a F-12K (Kaighn's Modification of Ham's F-12 Medium) medium containing 10% FBS and 1% penicillin/streptomycin. Both cell lines were maintained under standard conditions at 37 • C, 5% CO 2 and 95% relative humidity.

Analysis of Cell Proliferation
For proliferation analysis, both cell lines were plated at a density of 8 × 10 3 cells/well in a 96-well microplate and cultured in complete medium for 24 h. The medium was then replaced with a complete medium containing, or not containing rich polyphenolic extracts (RPE) at various concentrations (0-100 µg/mL) for 24 h at 37 • C with 5% CO 2 . The RPE stock solution was prepared with a complete medium containing 0.3% DMSO. The treatment was applied for 24 h at 37 • C with 5% CO 2 . To assess the cell viability after treatment with RPE we used a standard procedure. Briefly, the cell culture medium was removed and freshly prepared MTT reagent (0.5 mg/mL) was added to each well. After 2 h of incubation at 37 • C, the MTT solution was carefully removed, and DMSO was added in order to dissolve the formazan crystals that had formed in the mitochondria. The solubilized formazan formed in the viable cells was measured at 550 and 630 nm (for the sample and background, respectively) using the microplate reader, HT BioTek Synergy (BioTek Instruments, Winooski, VT, USA). The results are expressed as the survival percentage with respect to an untreated control. The control cells were assessed to be 100% viable.

Statistical Analysis
Data are expressed as the mean ± standard error of mean (SEM) of three analyses of each sample. Analysis of variance (ANOVA) and Dunnett's multiple comparisons test were used to determine significant differences between values (p < 0.05).