3.1. Extraction and Chemical Characterization
In chokeberry fruits, anthocyanins constitute the second largest group of phenolic compounds [
1]. The anthocyanins in
A. melanocarpa are mainly a mixture of four cyanidin glycosides: 3-galactoside, 3-glucoside, 3-arabinoside and 3-xyloside, of which cyanidin 3-galactoside is the main one (
Figure 1) [
17]. In order to isolate the major anthocyanins from Aronia berries, extraction with MeOH containing TFA was performed, since direct alcoholic extractions provide very poor yield, not keeping anthocyanins in the stable flavylium cationic form. The anthocyanins (
1–
4) were isolated as pure compounds, and their structures are shown in
Figure 2. Their chromatographic and spectral characteristics were in agreement with previous observations [
17]. Aronia berries were also extracted as shown in
Figure 3.
Figure 1.
High-performance liquid chromatography (HPLC) chromatogram of the isolated anthocyanins: cyanidin 3-galactoside (1), cyanidin 3-glucoside (2), cyanidin 3-arabinoside (3) and cyanidin 3-xyloside (4).
Figure 1.
High-performance liquid chromatography (HPLC) chromatogram of the isolated anthocyanins: cyanidin 3-galactoside (1), cyanidin 3-glucoside (2), cyanidin 3-arabinoside (3) and cyanidin 3-xyloside (4).
Figure 2.
Chemical structures of compounds isolated from berries and bark of Aronia.
Figure 2.
Chemical structures of compounds isolated from berries and bark of Aronia.
Figure 3.
Procedure for extraction and fractionation from berries of Aronia. Anthocyanins (
section 2.4.) and procyanidins (
section 2.5.) were extracted by different procedures.
Figure 3.
Procedure for extraction and fractionation from berries of Aronia. Anthocyanins (
section 2.4.) and procyanidins (
section 2.5.) were extracted by different procedures.
1H NMR and
13C NMR analysis revealed that proanthocyanidins were present in the 50% EtOH extract (70 °C). Subfractions of the extract were further shown to consist mainly of proanthocyanidins with epicatechin stereochemistry (
1H NMR: B-ring protons:
ca. 6.8 ppm, A-ring protons:
ca. 6.0 ppm [
21];
13C NMR: C-2 at
ca. 77 ppm, no signals at 80–82 ppm [
22]). However, fraction Seph II (eluted with 40% MeOH) did not contain proanthocyanidins.
In parallel to this procedure, dimeric (
5 and
6) and trimeric (
7) procyanidins (
Figure 2) were isolated from Aronia bark. The bark was found to be a simpler source for isolation of procyanidins compared to berries, as the complexity with respect to total phenolic structures was lower (e.g., no anthocyanins). The concentration of procyanidins was higher in Aronia bark, as well. It has previously been shown that procyanidins B2, B5 and C1 are present both in bark and berries of the Aronia plant [
2]. The isolated compounds were identified by co-chromatography against authentic substances (B2 and B5) and by mass spectrometry (
Figure 4,
Table 1). Thiolysis of crude extracts revealed that epicatechin was the major monomeric unit of the procyanidins, both as the starter and extender unit. Only minor amounts of catechin were detected. This might be due to epimerization of epicatechin.
Figure 4.
HPLC chromatogram of a bark extract of Aronia melanocarpa showing procyanidins B2 (5), B5 (6) and C1 (7).
Figure 4.
HPLC chromatogram of a bark extract of Aronia melanocarpa showing procyanidins B2 (5), B5 (6) and C1 (7).
Table 1.
Chromatographic and spectral characterization of anthocyanins and procyanidins from Aronia melanocarpa.
Table 1.
Chromatographic and spectral characterization of anthocyanins and procyanidins from Aronia melanocarpa.
| Compounds | TLC | HPLC | LCMS | [M + H]+ | Fragments (am→u) |
---|
| | rRF | tR (min) | tR (min) |
---|
1 | cyanidin 3-galactoside | | 12.9 | 22.0 | 449.104 | 287.044 |
2 | cyanidin 3-glucoside | | 13.9 | 22.2 | 449.102 | 287.044 |
3 | cyanidin 3-arabinoside | | 15.0 | 22.7 | 419.084 | 287.039 |
4 | cyanidin 3-xyloside | | 17.8 | 24.3 | 419.085 | 287.040 |
| epicatechin | 79 | 25.1 | 28.5 | 291.078 | |
5 | epi-(4β→8)-epi (B2) | 58 | 23.4 | 29.9 | 579.157 | 291.078 |
6 | epi-(4β→6)-epi (B5) | 67 | 32.5 | 35.6 | 579.168 | 291.084 |
7 | epi-(4β→8)-epi-(4β→8)-epi (C1) | 51 | 26.7 | 32.7 | 867.216 | 579.143, 291.080 |
It has previously been reported that polymeric procyanidins, composed predominantly of (−)-epicatechin units, are the major class of polyphenolic compounds in chokeberry [
1,
3]. The degree of polymerization of procyanidins varies from 2 to 23 units in the fruits, with clear domination of >decamers fraction and with flavan-3-ol subunits connected mainly with C4–C6 and C4–C8 bonds (B-type bonds) [
1]. Free epicatechin is also present in black chokeberries, although its concentration is significantly lower in comparison with polymeric procyanidins. Previous investigations have reported anthocyanin concentrations of 0.6%–2% (DW) and procyanidin concentrations of 4%–5% (DW) in Aronia berries [
2].
3.2. Biochemical Activities
The activity of crude extracts, subfractions and isolated compounds as DPPH scavengers, 15-LO and α-glucosidase inhibitors is shown in
Table 2. The 50% EtOH crude extract showed high radical scavenging activity, and the effect was strengthened in the subfractions enriched in procyanidins (Amb-MeOH, Seph I and Seph III fraction). Trimeric procyanidin (compound
7) showed higher radical scavenging activity than the dimeric procyanidins (compound
5 and
6). The radical scavenging ability of compound
5 and
7 is in good accordance with the literature [
23,
24]. Compound
6, however, seems to be previously unreported as a DPPH scavenger. Anthocyanins also possessed high radical scavenging capacity. IC
50 values could not be established for compounds
1–
4, since an increase in sample concentration resulted in a strongly colored mixture that influenced the UV-absorbance. For this reason, percent scavenging at a sample concentration of 10.4 µg/mL was measured. Compound
1–
3 were found to have the strongest DPPH scavenging capacity among the anthocyanins. The activity of compound
4 differed from the activity of the other anthocyanins, having the weakest DPPH radical scavenging capacity. Hence, sugar units linked to the anthocyanidin might have an influence on the biological effect. The radical scavenging activity of the anthocyanins is in fair accordance with previous investigations, although compound
3 has been reported to be slightly less active than
2 [
25].
Table 2.
Scavenging of the diphenylpicrylhydrazyl (DPPH) radical, 15-LO and α-glucosidase inhibitory activity of Aronia extracts, fractions and compounds.
Table 2.
Scavenging of the diphenylpicrylhydrazyl (DPPH) radical, 15-LO and α-glucosidase inhibitory activity of Aronia extracts, fractions and compounds.
Material | DPPH | α-Glucosidase | 15-Lipoxygenase |
---|
IC50a (µg/mL) #/ |
---|
IC50 a (µg/mL) | IC50a (µg/mL) |
---|
% scavenging at 10.4 µg/mL
^ |
---|
DCM | >167
# | Inactive | >83 |
EtOH | >167
# | Inactive | >83 |
50% EtOH | 25.0 ± 5.0
# | 3.5 ± 0.1 | >83 |
Amb-MeOH | 3.8 ± 0.2
# | 0.55 ± 0.01 | 56.7 ± 0.7 |
Seph I | 3.1 ± 0.5
# | nt
b | 30.3 ± 0.7 |
Seph II | 12.0 ± 2.8
# | nt
b | 91.0 ± 4.8 |
Seph III | 4.0 ± 0.5
# | nt
b | 33.0 ± 2.0 |
Compound
1 | 39.0 ± 2.9
^ | 1.54 ± 0.1 | 71.5 ± 1.8 |
Compound
2 | 37.0 ± 0.9
^ | 0.87 ± 0.2 | 73.3 ± 2.1 |
Compound
3 | 40.0 ± 0.4
^ | 0.37 ± 0.08 | 58.7 ± 2.5 |
Compound
4 | 25.0 ± 5.0
^ | 5.5 ± 1.6 | >83 |
Compound
5 | 4.7 ± 0.3
# | 4.7 ± 0.2 | 65.1 ± 2.6 |
Compound
6 | 5.2 ± 0.1
# | 5.5 ± 0.1 | 72.3 ± 5.7 |
Compound
7 | 3.2 ± 0.1
# | 3.8 ± 0.2 | 57.6 ± 2.0 |
Quercetin (control) | 3.0 ± 0.2
# | nt
b | 26.0 ± 2.0 |
Acarbose (control) | nt
b | 130.0 ± 20.0 | nt
b |
The 50% EtOH crude extract, the Amb-MeOH fraction and compound
1–
7 showed high activity in the α-glucosidase assay compared to the positive control acarbose, an anti-diabetic drug. In addition, the purified anthocyanins were more active than the isolated dimeric and trimeric procyanidins (compound
5–
7). It is known that some anthocyanin extracts from plants exert a potent
in vitro α-glucosidase inhibitory effect [
26]. Also, McDougall
et al. [
27] found that the extent of inhibition of α-glucosidase is related to the anthocyanin content in different soft fruits. Among the anthocyanins, compounds
2 and
3 showed the highest activity and compound
4 the lowest. The activity of substance
1 is consistent with the literature [
28]. To our knowledge, α-glucosidase inhibitory activity of substances
2–
7 has not been reported previously. Ma
et al. [
29] showed that the α-glucosidase inhibitory activity of flavan-3-ol monomer and oligomers increased as the molecular weight increased, with a significant difference in potency between the strongest ones (pentamers) and the weakest one (monomer). The Amb-MeOH fraction appeared to contain polymeric procyanidins, and this could explain its strong effect towards α-glucosidase. Trimeric procyanidin (compound
7) possessed stronger α-glucosidase inhibitory activity compared to the dimeric procyanidins (compound
5 and
6). It appeared that the activity increased with increasing molecular weight, which is in good accordance with previously reported results [
29]. For the anthocyanins, we found a highly significant correlation between α-glucosidase inhibition and DPPH radical scavenging activity (
p < 0.005,
R2 = 0.997). This is in good accordance with the literature [
6]. For the crude extracts, the Amb-MeOH fraction and the procyanidins, the correlation was not significant. The Sephadex LH-20 fractions (Seph I–III) could not be tested for α-glucosidase inhibitory activity due to lack of material.
The Amb-MeOH fraction showed high inhibitory activity toward 15-LO, and the effect was strengthened in the subfractions enriched in procyanidins (Seph I and Seph III fraction). Differences in activity between isolated anthocyanins and procyanidins were relatively small. Both groups of compounds possessed high 15-LO inhibitory ability, with compound
3 and
7 being the most active ones. The 15-LO inhibition of
1,
2,
3 and
7 is in accordance with previous investigations [
23,
30]. Substances
4,
5 and
6, however, seem to be previously unreported as 15-LO inhibitors.
The activity of crude extracts, subfractions and isolated compounds as XO inhibitors is presented in
Table 3.
Table 3.
Xanthine oxidase inhibitory activity of extracts, fractions and compounds from Aronia berries.
Table 3.
Xanthine oxidase inhibitory activity of extracts, fractions and compounds from Aronia berries.
Material | % inhibition at a concentration of 42 µg/mL |
---|
DCM | Inactive |
EtOH | Inactive |
50% EtOH | Inactive |
Amb-MeOH | 26.3 ± 3.4 |
Seph I | 32.2 ± 7.8 |
Seph II | 19.9 ± 7.6 |
Seph III | 46.5 ± 5.9 |
Compound
5 | 12.7 ± 2.8 |
Compound
6 | 6.6 ± 0.7 |
Compound
7 | 15.5 ± 1.5 |
| % inhibition at a concentration of 17 µg/mL |
Compound
1 | 11.9 ± 4.4 |
Compound
2 | 20.9 ± 3.4 |
Compound
3 | 39.1 ± 2.1 |
Compound
4 | 11.4 ± 2.8 |
The Amb-MeOH fraction possessed modest activity in the XO assay, and the effect was again strengthened in the subfractions enriched in procyanidins (Seph I and Seph III fraction). Due to absorbance above the upper detection limit of the spectrometer (sample concentrations >42 µg/mL for crude extracts, subfractions and procyanidins and sample concentrations >17 µg/mL for anthocyanins), higher concentrations of extracts and compounds could not be tested. Compound 7 was the strongest inhibitor among the isolated procyanidins, and compound 3 was the strongest among the anthocyanins. However, all were less efficient than the positive control quercetin (IC50 0.6 ± 0.1 µg/mL). To our knowledge, inhibition of XO of Aronia berry extracts and substances 1, 3, 4 and 6 have not been reported previously. The 50% EtOH crude extract showed no inhibitory activity toward XO.
Both the DCM and the EtOH crude extract were inactive as DPPH radical scavengers, 15-LO, XO and α-glucosidase inhibitors. Among the isolated anthocyanins, compound
3 possessed the strongest and compound
4 the weakest radical scavenging and enzyme inhibitory activity. These effects seem to be influenced by the sugar units linked to the anthocyanidin. Trimeric procyanidin (compound
7) showed higher activity in the biological assays compared to the dimeric procyanidins (compounds
5 and
6), and it appeared that the activity increased with increasing molecular weight. In addition, there was a difference in activity between the two dimeric procyanidins, with compound
5 being the most active one. Reactive oxygen species can be generated by the prooxidative enzyme, XO, and the peroxidative enzyme, 15-LO, in vascular cells [
31]. Components isolated from Aronia berries demonstrated inhibitory activity towards 15-LO and XO and may have a potential to alleviate oxidative stress. Until recently, anthocyanins were believed to have a very low bioavailability, but it has been demonstrated that the bioavailability of anthocyanins was underestimated [
32,
33]. In addition, anthocyanins are some of the few polyphenols that can be detected unmetabolized (e.g., as glycosides) in plasma [
32]. It has to be taken into consideration that the bioavailability of flavanols varies depending on the degree of polymerization. Low molecular weight oligomeric procyanidins (DP ≤ 3) are absorbed intact in the gastrointestinal tract, but polymerization greatly impairs intestinal absorption [
32,
33,
34]. In order to act as 15-LO and XO inhibitors
in vivo, constituents have to be absorbed from the gastrointestinal tract. In view of the known bioavailability of tested compounds, Aronia products and extracts containing anthocyanins and oligomeric procyanidins (DP ≤ 3) may have biologically relevant 15-LO and XO effects. Inhibition of α-glucosidase delays carbohydrate digestion and the absorption of monosaccharides from the intestine [
16]. Fractions enriched in procyanidins, the purified procyanidins and anthocyanins from
A. melanocarpa berries were potent α-glucosidase inhibitors, suggesting that they may have beneficial effects in reducing blood glucose level. In order to act as α-glucosidase inhibitors
in vivo, compounds do not have to be absorbed from the gastrointestinal tract, since it is a membrane-bound enzyme located at the epithelium of the small intestine [
20]. Therefore, anthocyanins and procyanidins with even a high degree of polymerization may exert local effects in the gastrointestinal tract as α-glucosidase inhibitors.