2.1. The Effect of MAE and PLE Parameters on the Total Phenolic Yield from Blackcurrant and Bilberry Leaves
To evaluate the effect of two green extraction techniques, MAE and PLE, on the total phenolic content (TPC) of blackcurrant and bilberry leaves, extractions were carried out using a 30% ethanol solution at different settings of temperature, extraction time and solvent/sample (SS) ratio., We used extraction temperatures of 60, 70 and 80 °C for MAE and of 100, 125 and 150 °C for PLE. Irradiation time for MAE and static extraction time for PLE were varied between 5 and 10 min, while the SS ratio was set to 20, 30 and 40 mL/g. The results of TPC determination in all experimental trials are shown in
Table 1.
The TPC in blackcurrant leaves ranged from 49.45 to 73.26 mg/g dry weight (dw) in MAE extracts and from 42.92 to 78.90 mg/g dw in PLE extracts. The ranges obtained were similar for both techniques, but it should be noted that PLE produced more extracts with phenolic yield at the upper end of the mentioned range. These values were slightly higher than the TPC of 39.96 mg/g dw in blackcurrant leaves extracted by ultrasound-assisted extraction, as reported by Nour et al. [
20], and that of 23.08 mg/g dry sample obtained by MAE, as reported by Cao-Ngoc et al. [
21], while they are in line with the TPC of 70.57 mg/g reported by Ziemlewska et al. [
22] for extracts produced by ultrasound-assisted extraction. The TPC range in bilberry leaves in this study, specifically, 33.74–70.55 mg/g dw, was slightly higher than the TPC of 45.18 mg/g dw reported by Stanoeva et al. [
23], while it was in line with or lower than the range of 54.7–106.9 mg/g dw, depending on the harvest year and vegetation period, reported by Bujor et al. [
24].
The results showing the effect of the MAE and PLE extraction parameters on the TPC of blackcurrant and bilberry leaves are shown in
Table 2.
In the case of MAE application for the isolation of phenols from blackcurrant leaves, only the SS ratio showed to be a significant parameter, while the effects of temperature and irradiation time were not statistically significant. For bilberry leaves, in addition to the SS ratio, temperature was also a significant factor during the extraction. In this study, temperature was selected as a controllable parameter, meaning that the constant microwave power of 400 W was applied only in respective periods of time required to maintain the set temperature. As can be seen from the results, the highest applied temperature of 80 °C was optimal for both blackcurrant and bilberry leaves’ phenol isolation. A similar conclusion was made by Dobroslavić et al. [
9] for the MAE of
Laurus nobilis L. leaves. Although the irradiation time was not a significant factor for MAE, some differences were observed between blackcurrant and bilberry. During the extraction of blackcurrant phenols, no difference was observed between 5 and 10 min of exposure to microwaves, while a 10 min exposure, on the other hand, produced a slightly higher phenolic yield from bilberry leaves. Generally, a prolonged microwave exposure favors the extraction of phenols; however, if excessive, it can lead to the degradation of the phenolic compounds [
25]. The SS ratio significantly affected the phenolic yield from both blackcurrant and bilberry leaves, leading to an increase with a higher amount of extraction solvent used. This trend is in an alignment with the general observation that an increase in solvent volume positively affects the extraction efficiency through the acceleration of the mass transfer between the solvent and the material and the promotion of the solubility of the target compounds [
26]. The increase in the concentration gradient between the samples and the solvent in this study benefited the phenolic yield, thereby resulting in the highest TPC when the highest SS ratio of 40 mL/g was applied. A positive effect of higher solvent volume for the isolation of phenols during MAE was also observed for grapevine and strawberry leaves [
27,
28], where SS ratios of 40 and 60 mL/g were set as optimal for maximal recovery. Regarding the variation in TPC between samples, it can be observed that the mean values obtained in different MAE conditions were higher for blackcurrant leaves than for bilberry leaves, indicating that blackcurrant leaves are a richer source of phenolics.
The mean TPC values in the blackcurrant and bilberry extracts obtained with PLE were slightly higher than those obtained with MAE. The higher effectiveness of PLE can be attributed to the application of high pressure during the extraction process. Specifically, high pressure allows for a better penetration of the solvent into the plant matrix and delays the formation of air bubbles in the matrix, which prevent the solvent to thoroughly reach the target compounds [
29]. For blackcurrant leaves, temperature significantly affected the TPC, while static extraction time and SS ratio had no effect. In the case of bilberry leaves, none of the examined parameters in the proposed ranges had a statistically significant effect. A temperature increase during PLE favored the extraction yield from blackcurrant leaves, which reached the highest value at 150 °C, while for bilberry leaves, the highest phenolic yield was obtained at 125 °C, as a further increase had a negative effect. Generally, temperature is considered the main factor in extraction processes, as it regulates the solubility of the target compounds in the extraction solvent through varying the molecular diffusivity and viscosity of the solvent [
30,
31]. Similar temperatures were found to be optimal for the PLE of polyphenols from strawberry leaves [
10],
Stevia rebaudiana leaves’ phenolic acids [
32] and
L. nobilis leaves’ polyphenols [
8].
In this study, only a variation in static extraction time between 5 and 10 min was evaluated, while all PLE extractions were performed in three consecutive extraction cycles. As the results indicate, no significant difference was observed in terms of PLE duration, although there was a slightly higher phenolic yield from bilberry leaves after 10 min of extraction. This observation may be related to the delivery of fresh solvent within each extraction cycle, which prevented the saturation of the solvent with the target compounds [
33]. The static extraction time range between 5 and 10 min was also reported to be optimal for chaga fungus phenolics, showing a constant increase in phenolic yield in single-factor experiments of PLE from 1 to 7 min [
34]. Similarly, the optimal static extraction time for the extraction of
L. nobilis leaves was 5 min within one extraction cycle [
8], while for nettle leaves, it was 10 min during three extraction cycles [
35]. Unlike what observed for MAE, the SS ratio was not a significant factor for PLE. The reason can again be found within the application of multiple extraction cycles and the supply of fresh solvent at each cycle, which minimized the effect of solvent saturation and extraction equilibrium. Although not significant, some differences can be outlined. During blackcurrant leave extraction, a slight decrease in TPC occurred when the SS ratio was increased from 30 to 40 mL/g, indicating the possible reaching of the extraction equilibrium. On the other hand, the TPC in bilberry leaves was the highest at the highest applied SS ratio of 40 mL/g. When the same PLE study design was applied for the isolation of strawberry leaves’ phenols, a similar effect was observed [
10].
Based on the made observations, optimal conditions were established for the extraction of blackcurrant and bilberry leaves’ polyphenols, as follows: for MAE, 80 °C/5 min/40 mL/g for blackcurrant leaves and 80 °C/10 min/40 mL/g for bilberry leaves, corresponding to the TPC values of 62.10 and 56.06 mg/g dw, respectively; for PLE, 150 °C/5 min/30 mL/g for blackcurrant leaves and 125 °C/10 min/40 mL/g for bilberry leaves, corresponding to the TPC values of 78.90 and 70.55 mg/g dw.
2.2. Profiling of the Optimized MAE and PLE Extracts of Blackcurrant and Bilberry Leaves
The optimized MAE and PLE extracts of blackcurrant leaves were analyzed by UPLC ESI MS
2, and the results of their polyphenolic composition are shown in
Table 3.
A total of 47 polyphenols were identified in the blackcurrant extracts, including 14 phenolic acids, 21 flavanols, 4 flavan-3-ols, 5 flavones and 3 procyanidins. Their fragmentation patterns were reported previously [
9,
36,
37,
38]. Among the phenolic acids, chlorogenic, caffeic, gallic and
p-hydroxybenzoic acids were present in the highest concentrations. The predominance of chlorogenic and caffeic acids in blackcurrant leaves was confirmed by Vagiri et al. [
2] and Raudsepp et al. [
3], while some studies, opposite to our observations, reported quinic [
39],
p-coumaric [
20] and salicylic acid [
40] as the main representatives. The concentrations of chlorogenic and caffeic acid in this study were significantly higher than 5.9–8.31 µg/g dw and 88.86 µg/g dw, respectively, reported by Nour et al. [
20] and than 20.9 µg/g dw for chlorogenic acid and 40.6 µg/g dw for caffeic acid reported by Chrzanowski et al. [
40]. On the contrary, Raudsepp et al. [
3] determined chlorogenic acid in significantly higher concentration, reaching even 14.93 mg/g dry leaves. Flavonols were found to be the predominant group of polyphenols in blackcurrant leaves in this study, with high proportions of quercetin, kaempferol, their glucosides and quercetin rutinoside. The presence of quercetin, quercetin-3-rutinoside and myricetin as representatives of flavonols in blackcurrant leaves was confirmed by Nour et al. [
20] and Chrzanowski et al. [
40], although in significantly lower concentrations. In line with our characterization, Vagiri et al. identified quercetin-3-
O-glucoside, kaempferol-3-
O-rutinoside and kaempferol-3-
O-glucoside, and Oszmianski et al. [
41] and Vagiri et al. [
42] isorhamnetin-3-
O-rutinoside, isorhamnetin-3-
O-glucoside and kaempferol acetylglucoside. Catechin and epicatechin were determined to be the major flavan-3-ols in blackcurrant leaves, similar to the reports of Raudsepp et al. [
3] and Cyboran et al. [
43]. This study also confirmed the presence of luteolin as the major flavone in blackcurrant leaves, as well as the presence of three procyanidins, with procyanidin B1 as the most abundant. To the extent of our knowledge, the individual characterization of flavones and procyanidins in blackcurrant leaves has not been reported previously; thus, this is the first report.
When observing the effect of the extraction technique, it can be concluded that PLE was more effective than MAE for the isolation of blackcurrant phenolic acids, in particular for quinic, 3-
p-coumaroylquinic, chlorogenic, syringic, galloylquinic, gallic and
p-coumaric acid, while 3,5-dicaffeoylquinic and ferruloylquinic acid were only identified in the PLE extract. The only exception was
p-hydroxybenzoic acid. Its higher recovery from MAE could be related to the general principle regulating the stability of phenolic acids during MAE, specifically the claim that stability under microwave irradiation is higher for compounds with fewer substituents in the structure of the aromatic ring [
44]. A similar trend could be observed for flavonoids and procyanidins. PLE was more effective for almost all flavonol representatives, except myricetin, quercetin and myricetin rhamnoside and arabinoside, noting that the differences in extraction efficiency in favor of MAE were not as pronounced. Additionally, the PLE extract had significantly higher amounts of luteolin and catechin, the main representatives of their respective phenolic classes, as well as a higher proportion of procyanidin B1. However, apigenin, luteolin glucoside and procyanidin B2, although in low concentrations, were only detected in the MAE extracts. On the other hand, the PLE extract contained quercetin and kaempferol pentoside, kaempferol acetylhexoside, quercetin pentosylhexoside, apigenin deoxyhexosyl hexoside, luteolin rutinoside and procyanidin trimer, which were not detected in blackcurrant leaves subjected to MAE. Although flavonoids and procyanidins would be expected to be more susceptible to thermal degradation due to their more complex structure than phenolic acids, PLE showed to be more effective than MAE for the isolation of all observed phenolic classes, despite the application of a significantly higher temperature than in MAE, namely, 150 versus 80 °C, resulting in an almost 3-fold higher concentration of total individual polyphenols. Flavonoids, being less polar than phenolic acids, may be more effectively isolated by PLE, regardless of the application of higher temperatures, which negatively affect them, as the pressure applied during PLE induces a reduction in solvent polarity and consequently promotes the solubility of less polar compounds [
45]. Moreover, the pressure application decreases the tension between the sample and the solvent, and compounds with more hydroxyl substituents increase the amount of hydrogen bonds with the solvent, which enhances their solubility [
45,
46]. These results are in accordance with those reported by Terpinc et al. [
10] for the comparison of MAE and PLE effects on individual polyphenols in strawberry leaves, where PLE was shown to be more effective than MAE for the isolation of flavonols, flavan-3-ols, flavones and procyanidins. Similarly, PLE provided a 2-fold higher polyphenolic yield from
Moringa oleifera leaves than MAE and was proven to be a more effective technique for the isolation of phenolic compounds with a higher number of hydroxyl-type substituents and those sensitive to high temperatures [
47]. In the same research, MAE allowed for a better recovery of quercetin and kaempferol, which is partially in line with our findings, as for blackcurrant leaves, MAE only benefited the extraction yield of quercetin.
The polyphenolic composition of the optimized MAE and PLE extracts from bilberry leaves is shown in
Table 4.
Bilberry extracts comprised a total of 46 compounds, including 11 phenolic acids, 26 flavonols, 4 flavan-3-ols, 3 flavones and 2 procyanidins. The predominant phenolic acids were caffeic, chlorogenic, gallic and syringic acid. The prevalence of chlorogenic acid and its derivates in bilberry extracts was confirmed previously [
4,
5,
41], as well as the presence of a significant amount of caffeic acid [
4]. However, there are variations in reports regarding the dominant phenolic acid. Our results showed that syringic acid was present in the highest concentration in the MAE extract, while caffeic acid was the predominant compound in the PLE extract. Değirmencioğlu et al. [
48] identified syringic acid as the most abundant compound in the leaves of bilberry from Turkey, Stefanescu et al. [
49] reported the predominance of feruloylquinic acid in extracts of wild bilberry from Romania, while Brezoiu et al. [
50] pointed out chlorogenic acid as the dominant compound in ethanolic bilberry extracts. The same authors identified caffeic acid only in hydroethanolic extracts, explaining its presence as a result of chlorogenic acid hydrolysis.
The predominant phenolic class in bilberry leaves determined in this study was that of flavonols, mainly represented by quercetin, kaempferol and their glycosides. In the MAE extract, the kaempferol aglycone was found in the highest concentration, while in the PLE extract the highest concentration was found for the quercetin glucoside. Similar to our results, although a lower amount was detected, Stanoeva et al. [
23] reported the quercetin glucoside concentration of 236 mg/100 g dw as the highest among those of flavonols in Macedonian bilberry. The differences in the obtained concentrations could be attributed to the extraction technique, the solvent used or the origin of the plant material. Besides quercetin and kaempferol glycosides, our results indicated the presence of myricetin glycosides as well, which were previously only reported for Turkish and Rovaniemi bilberry [
48,
51]. Regarding flavan-3-ols, the MAE extract was the most rich in catechin, while the PLE extract was rich in epicatechingallate, with both compounds confirmed previously as the most prevalent within their class by Stanoeva et al. [
23]. Flavones were detected in bilberry leaves as luteolin, apigenin and luteolin rutinoside. Reports on their presence in bilberry leaves are scarce, as they are mostly found in very low concentration [
52]. Regarding procyanidins, our results displayed the presence of procyanidin B1 as the most abundant and procyanidin trimer in low amount. These findings comply with reports on the prevalence of procyanidin B-type dimer and trimer compounds in bilberry leaves [
49].
When observing the full polyphenolic profiles of the bilberry leave MAE and PLE extracts, differences appeared not only in the quantitative, but also in the qualitative composition, indicating the PLE technique as the one providing a greater diversity of compounds. PLE provided significantly higher amounts of both hydroxycinnamic and hydroxybenzoic acids, while MAE was more effective only in the recovery of syringic acid. This observation is not in line with the results of Terpinc et al. [
10] for strawberry leaves, reporting that the syringic acid content was 63% higher in the PLE extract than in the MAE extract; however, it aligns with the phenolic acid stability principle during MAE, claiming a better stability of methoxyilates during exposure to microwaves [
44]. Regarding flavonoids, PLE was more effective for the isolation of all identified compounds apart from myricetin arabinoside, catechin and procyanidin B1, which were better recovered by MAE. These results are in accordance with observations made on blackcurrant leaves and may be related to the pressure effect on the enhancement of the solubility of flavonoid compounds during PLE [
45].
When comparing the two plant samples analyzed, blackcurrant and bilberry leaves, the yield of total polyphenols identified by UPLC ESI MS2 was similar for both techniques. The main phenolic acids in both plants were caffeic acid and chlorogenic acid, the main flavonols were quercetin and kaempferol aglycones, the main flavan-3-ols and procyanidins were catechin and procyanidin B1 and the main flavones were luteolin and its rutinoside. However, some special features can be deduced from their detailed composition. In comparison, blackcurrant leaves contained a higher total amount of phenolic acids, which accounted for about 15 and 8% of the total polyphenol content in the MAE and PLE extracts, while bilberry leaves had a phenolic acid content of 7 and 5% in the MAE and PLE extracts, respectively. Interestingly, syringic acid dominated in the MAE bilberry extract, whereas chlorogenic acid predominated in both the blackcurrant extracts and the PLE blueberry extracts. In terms of flavonoid content and composition, blackcurrant and bilberry leaves differed with regard to their flavonol profile and flavan-3-ol content. Blackcurrant leaves showed a higher proportion of flavan-3-ols and a higher amount of acylated flavonol glycosides, while the bilberry extracts had a higher concentration of myricetin glycosides. All these variations in the composition of the extracts could be related to differences in the preferred extraction conditions. For example, the significance of the temperature effect during MAE was only observed for the bilberry leaves, and that particular extract was characterized by syringic acid, which, as mentioned above, is a methoxylated phenolic acid with high stability under microwave exposure. On the other hand, blackcurrant leaves were preferentially processed at higher temperatures during PLE, which improved the solubility of flavonoid compounds under the applied pressure.
In addition, for both blackcurrant and bilberry leaves, regardless of the extraction technique, the yields obtained during extraction optimization, measured using the Folin–Ciocalteu method, were significantly higher than the yields resulting from the sum of the yields of all compounds identified using UPLC ESI MS
2. This discrepancy may be related to the low selectivity of the Folin–Ciocalteu reagent towards polyphenols, as it can also react with other compounds such as polysaccharides, organic acids, sugars [
53,
54] and, most importantly, chlorophylls [
55], which is of particular relevance for chlorophyll-rich material such as the leaves used in this study.
2.3. Antioxidant Capacity of the Optimized MAE and PLE Extracts of Blackcurrant and Bilberry Leaves
The obtained optimized blackcurrant and bilberry leave extracts were analyzed for their antioxidant capacity through four different assays in order to provide a reliable display of their properties (
Table 5).
The antioxidant capacity of blackcurrant leaves was significantly affected by the extraction technique employed, regardless of the assay applied. According to the FRAP, ABTS and ORAC assays, the PLE extract had a significantly higher antioxidant capacity than the MAE extract, which is in line with its higher TPC and individual polyphenolic content. Oppositely, the DPPH assay showed a higher antioxidant capacity of the MAE extract, although it had a lower TPC content and a lower concentration of all individual polyphenolic subclasses. As discussed for the polyphenolic characterization of the extracts, MAE favored the extraction of a limited number of compounds, including myricetin and its glycosides and the quercetin aglycone. As these flavonoids are known as strong antioxidants, the stronger DPPH radical scavenging capacity observed could be assigned to the aforementioned compounds, but the results of a Pearson’s correlation analysis between antioxidant capacity and the content of individual polyphenolic classes (
Table 6) showed no significance for the DPPH assay. Thus, this discrepancy could be attributed to the assay limitations, such as the non-linear reactivity of antioxidants with radicals [
56]. As the DPPH assay is a mixed assay, based on both HAT and ET mechanisms, its kinetics is strongly affected by the solvent, because the release of H atoms is limited by the strength of the solvent’s hydrogen bonding to the polyphenolic hydroxyl groups and DPPH radical, leading to a fast reaction in methanol and significantly slower one in acetone and ethanol [
56,
57]. As our extracts were prepared in an aqueous ethanol solution, this exception of the DPPH assay values could be explained considering the premise above. Regarding the range of antioxidant capacity obtained with the different assays, our values are slightly higher than those of Teleszko and Wojdylo [
58], who reported an antioxidant capacity for blackcurrant leaves of 329.1 and 191.6 µmol TE/g dw by the ABTS and FRAP assays, respectively, and than the DPPH value of 200.3 µmol TE/g dw reported by Katsube et al. [
59]. On the other hand, Ziobroń et al. [
60] reported significantly higher values obtained with the ABTS, DPPH and FRAP assays than most of the previously reported ones, reaching even 7877.82 µmol TE/g dw for the DPPH radical scavenging capacity of blackcurrant leaves, and attributed them to differences in the extraction process, as the authors carried out a 2 h extraction in a water bath shaker using acidified methanol.
In the case of the bilberry leaves extracts, the extraction technique influenced the antioxidant capacity determined by the FRAP, DPPH and ABTS assays, providing higher values when PLE was used, which is in line with the corresponding polyphenolic content, while there was no significant difference between the techniques as regards the ORAC values. Generally, the antioxidant capacity of bilberry leaves was slightly lower than the one of blackcurrant leaves, which is also consistent with their respective polyphenolic content. The literature reports an antioxidant capacity of bilberry leaves of 793.0 and 595.8 µmol TE/g dw obtained using the ABTS and FRAP assays [
61], which is close to our values. The ORAC values of bilberry leaves extracts were approximately 1.5-fold higher than the value of 251.6 µmol TE/g fresh weight reported for the bilberry fruit [
62]. When observing the contribution of total or individual polyphenolic classes to the antioxidant capacity of both blackcurrant and bilberry leaves extracts produced by MAE and PLE, it was shown that only flavan-3-ols and procyanidins had a significant and very strong positive correlation with the antioxidant capacity as determined by both ABTS and ORAC assays for flavan-3-ols and by only the ORAC assay for procyanidins. The share of flavan-3-ols in the total UPLC ESI MS
2 polyphenolic content was 4.73 and 6.08% in the blackcurrant leave MAE and PLE extracts, and 4.14 and 1.18% in the bilberry leave MAE and PLE extracts. Procyanidins were 7.76 and 9.50% of the total blackcurrant polyphenols extracted by MAE and PLE, and 15.41 and 1.96% of the total polyphenols in bilberry MAE and PLE extracts. Although those proportions are relatively low, compared to those of the flavonols, amounting to more than 50% of the total polyphenolic content, their significant contribution to the antioxidant capacity is a result of their structural characteristics. Soobrattee et al. [
63] reported the antioxidant capacity of reference polyphenolic compounds to decrease in the order procyanidin dimer > flavan-3-ols > flavonols > hydroxycinnamic acids > hydroxybenzoic acids. The highest capacity of procyanidins is attributed to their hydroxyl groups and conjugated double bonds, while for flavan-3-ols, the capacity is strongly dependent on the presence of the galloyl moiety in gallates, increasing the number of free hydroxyl groups. Furthermore, the dominance of flavan-3-ols over flavonols in terms of antioxidant capacity may be a result of different oxidation mechanisms, as flavan-3-ol oxidation results in the formation of semiquinons, which couple to form oligomeric compounds, thereby retaining the catechol and pyrogallol structures and preserving their scavenging ability. Flavonols, contrarily, form quinones, which are prone to the redox cycle and may act as pro-oxidants [
64,
65]. The observed significance of flavan-3-ol and procyanidin contribution to the antioxidant capacity of the extracts may explain the differences between the total polyphenolic content of the MAE and PLE extracts and their respective antioxidant capacity, especially in the case of bilberry leaves. The bilberry extract obtained by MAE was relatively poor in total polyphenols, with only 7.01 mg/g dw of total compounds identified by UPLC ESI MS
2. However, its ORAC value did not differ significantly from that of the PLE extract, with 27.99 mg/g dw of UPLC ESI MS
2 total polyphenols. Still, the MAE extract had a 2-fold higher content of procyanidins than the PLE extract, namely, 1.08 versus 0.55 mg/g dw, which made the procyanidin content share in the MAE extract higher than 15%. Therefore, despite the lower UPLC ESI MS
2 total polyphenol content, the bilberry extract obtained by MAE presented the same antioxidant capacity as the one produced by PLE.