Summary
Chokeberries (Aronia melanocarpa) are rarely used in diet in Croatia but they have high content of polyphenolic compounds and one of the highest in vitro antioxidant activities among fruits. The aim of this study is to compare the quality, phenolic content and antioxidant capacity of different chokeberry products (juices, powders, fruit tea, capsules and dried berries). It can be expected that processing influences antioxidant activity and phenolic content of final products reaching consumers. Characterisation of phenolic compounds was carried out by using spectroscopic methods (Folin–Ciocalteu and pH differential methods). Antioxidant activity of chokeberry products was determined using 2,2-diphenyl-2-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP) methods. The results show that the investigated products contain high amount of phenols (3002 to 6639 mg per L and 1494 to 5292 mg per 100 g of dry matter) and lower amount of total anthocyanins (150 to 1228 mg per L and 141 to 2468 mg per 100 g of dry matter). The examined juices and other chokeberry products possess high antioxidant capacity (12.09 to 40.19 mmol per L or 58.49 to 191.31 mmol per 100 g of dry matter, respectively) and reducing power (38.71 to 79.86 mmol per L or 13.50 to 68.60 mmol per 100 g of dry matter, respectively). On the basis of phenolic content and antioxidant activity, capsules and powders stand out among other products. The study indicates that there are significant differences (p<0.05) in the quality, phenolic content and antioxidant capacity among examined products.
Key words: chokeberry products, quality, phenols, anthocyanins, DPPH, FRAP
Introduction
Increased consumption of fruits and vegetables is recommended in dietary guidelines worldwide (1). Among different fruit species, berries have attracted great attention for their bioactivity. In addition to nutritive dietary components (vitamins, minerals, sugars, organic acids, dietary fibres and unsaturated fats), berries are also a good source of different classes of phytochemicals such as flavonoids (anthocyanins, flavonols and flavanols), tannins (proanthocyanidins, ellagitannins and gallotannins), stilbenoids (e.g. resveratrol), phenolic acids (hydroxybenzoic and hydroxycinnamic acid derivates) and lignans (2). Berry fruits are popularly consumed not only in fresh and frozen forms but also as processed and derived products including canned fruits, yogurts, beverages, jams and jellies. In addition, there has been a growing trend in the intake of berry extracts as ingredients in functional foods and dietary supplements, which may or may not be combined with other colourful fruits, vegetables and herbal extracts (1, 2). In Croatia, berries like red raspberries, blackberries, blueberries and strawberries are commonly used in diet, but black chokeberry is almost unknown fruit (3).
Black chokeberry (Aronia melanocarpa (Michx.) Elliott) belongs to the Rosaceae family, subfamily Maloideae, and is a deciduous shrub originating from the eastern part of North America (4, 5) where it has been used for the treatment of cold by native Americans (Abnakians and Potawatomians). Today, chokeberry is also cultivated in Eastern European countries and Russia (6), where it is used for production of homemade or commercial juices, jams, fruit tea, wine and natural food colourants (5, 7). It shows high resistance to frost, mechanized harvesting, damage during transportation and cold storage. Due to these advantages, popularity of chokeberry has increased recently (8). Chokeberries have very high contents of polyphenols, namely phenolic acids, proanthocyanidins, anthocyanins, flavonols and flavanones (9–12). In a study where 143 different plant samples were analysed for polyphenols, the highest contents of these compounds were found in chokeberry (13). The high content and composition of the phenolic constituents of Aronia melanocarpa seem to be responsible for the wide range of the fruit’s potential medicinal and therapeutic effects. Chokeberries have one of the highest in vitro antioxidant activities among fruits. The mechanisms of the in vivo antioxidant activity of their phenolics after absorption spread out far beyond radical scavenging and include suppressing the formation of reactive oxygen species (ROS) and reactive nitrogen species (RNS), inhibition of prooxidant, and restoration of antioxidant enzymes, and probably also cellular signalling to regulate the level of antioxidant compounds and enzymes (14). Although recent studies have pointed out different positive effects of chokeberry juices and extracts (15–21), current evidence of effectiveness does not yet meet the accepted standards that would secure chokeberry products an indisputable place in therapy. Promising indications from laboratory and clinical data need to be confirmed in more rigorous studies before putative therapeutic uses can be confidently recommended for chokeberry products (22).
There are no studies on compositional and physical properties of Aronia melanocarpa products present on the Croatian market. Therefore, in this study 22 chokeberry products are evaluated. The objective of this study is to evaluate the physicochemical properties, the content of phenolics (total phenolics, flavonoids, nonflavonoids and anthocyanins) as well as antioxidant properties of different chokeberry products present on the market.
Materials and Methods
Chokeberry products (Table 1) were purchased on Croatian markets during February 2014. There was only one requirement: they had to contain only chokeberry, without added sugar or other fruits. Products were stored at 4 °C until analysis.
Table 1. Producer, country of origin, fruit content and composition of chokeberry products.
| Producer | Country of origin |
w(fruit) % |
Composition |
|---|---|---|---|
| Juices | |||
| Aronia Original Naturprodukte GmbH | Germany | 100 | Chokeberry fruit |
| Aronija Live d.o.o. | Croatia | 100 | Chokeberry fruit |
| Vitanea LTD | Bulgaria | 100 | Chokeberry fruit |
| Alnavit GmbH | Germany | 100 | Chokeberry fruit |
| Biotta AG | Germany | 100 | Chokeberry fruit |
| NA | Poland | 100 | Chokeberry fruit |
| Bobica d.o.o. | Croatia | 100 | Chokeberry fruit |
| Armedina d.o.o. | Serbia | 100 | Chokeberry fruit |
| Aronija Vita d.o.o. | Serbia | 100 | Chokeberry fruit |
| Voelkel GmbH | Germany | 100 | Chokeberry fruit |
| Medicura Naturprodukte AG |
Germany | 100 | Chokeberry fruit |
| Powders | |||
| Aronia Original Naturprodukte GmbH | Germany | 100 | Chokeberry pulp |
| Bobica d.o.o. | Croatia | 100 | Chokeberry fruit |
| Aronija Vita d.o.o. | Serbia | 100 | Chokeberry pomace |
| Capsules | |||
| Darvitalis d.o.o. | Serbia | 100 | Chokeberry extract |
| Bobica d.o.o. | Croatia | 100 | Chokeberry extract |
| Fruit tea* | |||
| Aronija Live d.o.o. | Croatia | 100 | Chokeberry pomace |
| Darvitalis d.o.o. | Serbia | 100 | Chokeberry pomace |
| Bobica d.o.o. | Croatia | 93 | Chokeberry pomace; chokeberry leaves |
| Vitanea LTD | Bulgaria | 100 | Chokeberry pomace |
| Dried berries | |||
| Aronia Original Naturprodukte GmbH | Germany | 100 | Chokeberry fruit |
| Bobica d.o.o. | Croatia | 100 | Chokeberry fruit |
*Dried and ground chokeberry pomace. The pomace consists of chokeberry skin and pips that are left when chokeberries are processed to become chokeberry juice NA=data about producer not available, imported by Biovega d.o.o.
Determination of physicochemical parameters
Total solid content of the chokeberry products was determined using a gravimetric method. A mass of (2± 0.0001) g of chokeberry sample was mixed with about 5 g of sea sand and dried at 105 °C until constant mass. Soluble solid content was determined with a digital refractometer (Atago PAL-3, Tokyo, Japan) and expressed as °Brix. Sample pH was determined at room temperature using an MA 5740 pH meter (ISKRA, Kranj, Slovenia). Two-point calibration was obtained using buffers at pH=7.0 and 4.0. Titratable acidity was determined by titration of the water solution of chokeberry product with 0.1 M NaOH to end point of neutral pH (8.1). The volume of 0.1 M NaOH required to reach pH=8.1±0.2 was determined. The total titratable acidity was expressed as percentage of citric acid using a conversion factor of 0.070 (23).
Determination of juice colour
The colour of the chokeberry juices was measured in a transmitted mode through Konica Minolta CM-3500d spectrophotometer (Konica Minolta, Inc., Tokyo, Japan). Measurements were conducted in CIE L*a*b* system. L* is a measure of lightness, where values range from completely opaque (0) to completely transparent (100), a* is a measure of redness (or −a* of greenness) and b* of yellowness (or −b* of blueness) on the hue circle. The hue angle, h°, (Eq. 1) describes the relative amounts of redness and yellowness where 0°/360° is defined for red/magenta, 90° for yellow, 180° for green and 270° for blue colour:
 |
Chroma (C*) gives further information on the saturation or intensity of colour (24, 25):
 |
Extraction of phenolics
Phenolics were extracted according to the modified method by Benvenuti et al. (26). Exactly 6 g of samples were weighed out and mixed with 20 mL of methanol/2% HCl (95:5, by volume). After 60 min the solution was filtered under vacuum in a 50-mL volumetric flask. Extraction of the residue was repeated using the same conditions. The filtrates were combined and adjusted to 50 mL in a volumetric flask with methanol/2% HCl (95:5, by volume). The obtained extract was used for determination of total phenolic content (TPC), total nonflavonoids (TN), total anthocyanins (TA) as well as for antioxidant capacity assay by DPPH method and reducing power assay using FRAP method.
Determination of total phenolics
For determination of TPC, a method with Folin-Ciocalteu reagent was used (27). An aliquot (20 µL) of diluted chokeberry extract or standard solutions of gallic acid (25–500 mg/L) was mixed with 1580 µL of distilled water and 100 µL of Folin-Ciocalteu reagent. A volume of 300 µL of sodium carbonate solution (200 g/L) was added to the mixture which was then shaken. After incubation at room temperature for 2 h, the resulting absorbance was measured by the spectrophotometer Pye Unicam SP6-500 (Pye Ltd., Philips, Cambridge, UK) at the wavelength of 765 nm against the blank sample, which was used as reference. The results were calculated according to the calibration curve for gallic acid as follows:
 |
where y is the absorbance at 765 nm and x is the concentration of gallic acid in mg/L; R2=0.9973. Total phenolics were expressed as mg of gallic acid equivalents (GAE) per L of chokeberry juices and as mg of GAE per 100 g of dry matter (dm) of other chokeberry products.
Determination of total flavonoid and nonflavonoid contents
Determination of total flavonoid (TF) content was performed by the indirect method using formaldehyde to precipitate these compounds, as described by Ough and Amerine (28). A mixture of 3 mL of chokeberry extract solution, 1.5 mL of aqueous solution of hydrochloric acid (1:4, by volume) and 3 mL of formaldehyde was prepared in a 25-mL flask. In order to remove air, nitrogen gas was injected and the stoppered flask was left in the dark for 24 h at 22 °C. The next day it was filtered and the clear liquid was used in the same procedure (27) as the one used to prepare samples for TPC determination. The amount of TF was calculated as the difference between total phenolics and total nonflavonoids (TN). The results were expressed as mg of GAE per L and as mg of GAE per 100 g of dm for chokeberry juices and other products, respectively.
Determination of total anthocyanins
Total anthocyanin (TA) content, calculated as cyanidin-3-glucoside, was determined by the pH differential method of Giusti and Wrolstad (29). Two dilutions of each chokeberry extract were prepared, one with potassium chloride buffer (pH=1.0), and the other with sodium acetate buffer (pH=4.5). After 15 min of incubation at room temperature, the absorbance was measured simultaneously at the wavelengths of 510 and 700 nm. The content of TA was calculated using Eqs. 4 and 5 with molar absorption coefficient of cyanidin-3-glucoside of 26 900 L/(mol·cm) and molar mass of 449.2 g/mol:
 |
 |
where A is absorbance, ε is molar absorption coefficient of cyanidin-3-glucoside equivalents (CGE) (L/(mol·cm)), L is cell pathlength (1 cm), Mr is molecular mass of CGE, DF is dilution factor, V is final volume (mL), and m is mass of the sample (mg). Results were expressed as mg of CGE per L of chokeberry juices and as mg of CGE per 100 g of dm of other products.
Determination of total antioxidant capacity by DPPH method
The effect of chokeberry products on 2,2-diphenyl-2-picrylhydrazyl (DPPH) radical was determined according to the method of Brand-Williams et al. (30). The method was based on the reduction of stable DPPH radical in the presence of antioxidants. A volume of 2 mL of diluted chokeberry extract or methanol solution of Trolox (25–200 µmol/L) was mixed with 2 mL of methanol and 1 mL of 0.5 mM DPPH methanolic solution. The mixture was vortexed and kept in the dark for 20 min. After incubation, the absorbance was measured at the wavelength of 517 nm against a blank of methanol without DPPH. The results were calculated according to the calibration curve for Trolox:
 |
where y is the absorbance at 517 nm and x is the concentration of Trolox in µmol/L; R2=0.9817. DPPH values were expressed as mmol of Trolox equivalents (TE) per L and mmol of TE per 100 g of dm for chokeberry juices and other products, respectively.
Determination of FRAP
The ferric reducing antioxidant power (FRAP) assay was conducted according to Benzie and Strain (31). The method is based on the reduction of the Fe3+-2,4,6-tripyridyl-s-triazine (TPTZ) complex to the ferrous form at low pH. This reduction is monitored by measuring the absorbance change at 595 nm. The FRAP reagent was prepared from 5 mL of TPTZ solution (10 mmol/L) in hydrochloric acid (40 mmol/L) and 5 mL of FeCl3 solution (20 mmol/L) mixed with 50 mL of acetate buffer (0.3 mol/L, pH=3.6). For the determination of the antioxidant capacity, the FRAP reagent (2.08 mL) was mixed with 240 µL of water and 80 µL of the appropriately diluted sample or standard solution of FeSO4·7H2O (0.125–2.000 mmol/L). The mixture was allowed to stand for 5 min at 37 °C before the absorbance was measured at 595 nm. FRAP values were calculated according to the calibration curve for FeSO4·7H2O:
 |
where y is the absorbance at 595 nm and x is the concentration of FeSO4·7H2O in mmol/L; R2=0.9987, and they were expressed as mmol of Fe2+ equivalents (FE) per L and as mmol of FE per 100 g of dm for chokeberry juices and other products, respectively.
Statistical analysis
The data were analysed using STATISTICA v. 12.0 (Statsoft Inc, Tulsa, OK, USA). Analysis of variance (ANOVA) was used to establish significant differences between and within groups of chokeberry products. Differences were considered significant at p≤0.05. Values were expressed as means (N=3). For comparison of the contents of TPC, TF, TN, TA and DPPH or FRAP assays and also for comparison of colour parameters and TPC, TF, TN or TA contents, the coefficients of correlation were determined for each combination.
Results and Discussion
Chokeberry fruits are not popular as table fruits but they are generally consumed as processed chokeberry products including juice, jam, syrup and nutritional supplements. Data on the phenolic contents of chokeberry have been reported in several studies (3, 6, 26, 32–35), and the present study contributes to the existing knowledge by providing new data on different chokeberry products such as powders, capsules, fruit tea and dried berries.
Physicochemical parameters and juice colour
The analysis of chokeberry samples indicated different physicochemical properties among groups of products as well within groups (Table 2). In case of chokeberry juices, the total solid content ranged from 13.42% in juice sample J10 to 21.54% in juice sample J2, while in the other samples it was much higher. Chokeberry capsules had the highest total solid content among groups (mean value 93.78%) followed by chokeberry powders (mean value 92.35%), fruit tea (mean value 91.49%) and dried berries (mean value 83.31%). In research of Mayer-Miebach et al. (36) the dry matter content of berries ranged from 17.9 to 26.0%, in juices from 11.1 to 17.4% and in pomace from 44.6 to 50%. The mean value of total solid content of chokeberry capsules (93.78%), fruit tea (2.35%) and powders (91.49%) present on the market is very similar to the results of Sójka et al. (37), who investigated chokeberry pomace obtained in an industrial-scale processing of fruit into juice. The lowest value of soluble solid content (13.70 °Brix) was in juice sample J10, while the highest value characterised capsules, i.e. sample C1 (83.71 °Brix). The soluble solid content in chokeberries depends on numerous factors: weather, environmental conditions, crop period and variety, and it amounts to 12.4 or 18.3% (5). Chokeberry products had a mean pH value of 3.90 ranging from 3.54 (sample J10) to 4.28 (sample DB1). The mean total titratable acidity (TTA) of all products was 1.42 (as percentage of citric acid) ranging from 0.29 (sample J2) to 4.66% (sample C1). Comparing the groups of products, it is evident that capsules have the highest total titratable acidity and juices the lowest. Ochmian et al. (5) reported similar values for titratable acidity in the range from 0.75 to 1.05 g of citric acid per 100 g of berries. The °Brix/TTA ratio is a quality attribute used by the fruit industry to indicate the tartness of fruits and fruit juices (38). This ratio increases with maturity of the fruit and is used to identify the optimum maturity for harvesting to produce maximum product quality (39). The mean °Brix/TTA ratio was 20.42 and ranged from 11.24 in the fruit tea sample FT2 to 78.54 in the juice sample J2 (Table 2). ANOVA showed significant differences of physicochemical properties among juices, powders, fruit tea, capsules and dried berries and also among individual samples within groups, with the exception of total solid content and total titratable acidity of samples of dried berries.
Table 2. Physicochemical properties of chokeberry products.
| Sample | pH | TTA/% | Total solid content/% | °Brix | °Brix/TTA |
|---|---|---|---|---|---|
| Juices | |||||
| J1 | 3.90±0.02 | 0.67±0.07 | 14.70±0.04 | 14.56 | 24.72 |
| J2 | 3.90±0.02 | 0.29±0.07 | 21.54±0.03 | 20.99 | 78.54 |
| J3 | 3.86±0.02 | 0.85±0.07 | 18.98±0.14 | 18.52 | 23.98 |
| J4 | 3.71±0.03 | 1.13±0.12 | 15.75±0.11 | 15.47 | 15.20 |
| J5 | 3.80±0.03 | 1.06±0.07 | 15.79±0.15 | 15.27 | 16.08 |
| J6 | 3.68±0.02 | 1.26±0.07 | 14.50±0.16 | 14.09 | 12.52 |
| J7 | 3.74±0.02 | 0.87±0.07 | 14.61±0.22 | 13.99 | 18.06 |
| J8 | 3.75±0.02 | 1.32±0.07 | 17.01±0.21 | 17.34 | 14.61 |
| J9 | 3.92±0.01 | 0.84±0.07 | 14.32±0.11 | 13.69 | 18.28 |
| J10 | 3.54±0.01 | 1.30±0.07 | 13.42±0.05 | 13.30 | 11.48 |
| J11 | 3.89±0.02 | 0.97±0.07 | 14.27±0.01 | 13.79 | 16.01 |
| Powders | |||||
| P1 | 4.10±0.02 | 1.67±0.07 | 94.797±0.001 | 26.75 | 16.02 |
| P2 | 4.02±0.01 | 2.30±0.07 | 91.82±0.32 | 37.53 | 16.36 |
| P3 | 4.13±0.01 | 2.17±0.07 | 90.44±0.15 | 37.34 | 17.24 |
| Capsules | |||||
| C1 | 3.31±0.02 | 4.66±0.07 | 93.96±0.27 | 83.71 | 17.97 |
| C2 | 4.10±0.01 | 2.10±0.07 | 93.60±0.26 | 31.91 | 15.23 |
| Fruit tea | |||||
| FT1 | 4.13±0.02 | 1.34±0.06 | 91.90±0.26 | 38.22 | 28.52 |
| FT2 | 4.01±0.01 | 1.08±0.06 | 88.32±0.27 | 12.17 | 11.24 |
| FT3 | 4.01±0.02 | 1.60±0.07 | 89.74±0.19 | 33.42 | 20.86 |
| FT4 | 4.04±0.02 | 1.37±0.06 | 96.01±0.14 | 25.48 | 18.63 |
| Dried berries | |||||
| DB1 | 4.28±0.02 | (1.13±0.06)a | (84.61±0.92)b | 25.49 | 22.62 |
| DB2 | 4.01±0.02 | (1.37±0.11)a | (82.00±0.55)b | 20.93 | 15.30 |
The values are presented as mean±standard deviation (S.D.). The same letter in the superscript in the same column indicates no significant differences (p>0.05). TTA=total titratable acidity as citric acid, J1–J11=chokeberry juices, P1–P3=chokeberry powder, C=chokeberry capsules, FT=chokeberry fruit tea, DB=chokeberry dried berries
Since the colour of the product, especially juices, is extremely important feature that contributes to the overall quality, one of the aims of this paper was to determine colour parameters of chokeberry juices (Table 3). Values of variable L* were low in all samples, from 0.52 (juice sample J10) to 15.00 (juice sample J7), which indicates that samples were very dark since the variable L* varies from 0 representing black to 100 representing white. Similar values of the parameter L* of chokeberry juices were observed by Ochmian et al. (5). The a* value, providing information of the position in the colour gamut between green and red, measured on the juice surface ranged from 3.74 (juice sample J10) to 46.42 (juice sample J7). The juice surface colour defined by the b* parameter, indicating the location on the axis between yellow and blue colours, ranged from 0.88 (juice sample J10) to 25.84 (juice sample J7), which means that yellow colour is present. Positive a* values were also observed in chokeberry juices, pulp and fruit by Ochmian et al. (5) and in chokeberry powders by Horszwald et al. (40). In a research of Horszwald et al. (40) yellow colour was present in chokeberry powders, while in the work of Ochmian et al. (5) b* values were negative, which indicates the presence of blue colour. Parameters L*, C* and h° are related to the physiological attributes of visual response (41). Hue describes the visible colour and chroma describes the brightness or intensity of the hue. Indices of L*, C* and h° are usually useful for tracking colour changes (42). The decrease in chroma means an increase in the tonality of the fruit colour (43).
Table 3. Colour parameters of chokeberry juice samples (J1–J11).
| Colour parameters | Sample | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| J1 | J2 | J3 | J4 | J5 | J6 | J7 | J8 | J9 | J10 | J11 | |
| L* | 1.20 | 5.93 | 8.87 | 4.27 | 13.85 | 2.38 | 15.00 | 5.91 | 9.75 | 0.52 | 5.31 |
| a* | 8.14 | 33.95 | 39.29 | 27.76 | 44.92 | 16.64 | 46.42 | 33.51 | 39.44 | 3.74 | 31.58 |
| b* | 2.04 | 10.19 | 15.21 | 7.35 | 23.79 | 4.08 | 25.84 | 10.15 | 16.79 | 0.88 | 9.12 |
| h° | 14.1 | 16.7 | 21.2 | 14.8 | 27.9 | 13.8 | 29.1 | 16.9 | 23.1 | 13.2 | 16.1 |
| C* | 8.4 | 35.4 | 42.1 | 28.7 | 50.8 | 17.1 | 53.1 | 35.0 | 42.9 | 3.8 | 32.9 |
Table 4 shows the correlation coefficients between the colour parameters and TPC, TN, TF and TA, from which a negative correlation of colour parameters with the content of total nonflavonoids and of colour parameters with the content of total anthocyanins is evident.
Table 4. Correlation coefficients (R) between phenolics and colour parameters of chokeberry juices.
| Colour parameters | TPC | TN | TF | TA |
|---|---|---|---|---|
| L* | –0.21 | –0.61a | –0.10 | –0.76b |
| a* | –0.02 | –0.52a | –0.08 | –0.81b |
| b* | –0.21 | –0.61a | –0.10 | –0.76b |
| h° | –0.28a | –0.59a | –0.17 | –0.70b |
| C* | –0.06 | –0.54a | –0.04 | –0.81b |
a,bsignificant at p≤0.05 and p≤0.001, respectively Contents of total phenolics (TPC), total nonflavonoids (TN), total flavonoids (TF) and total anthocyanins (TA) are expressed as mg per L. TPC, TN and TF are expressed as mg of gallic acid equivalents (GAE), while TA is expressed as mg of cyanidin-3-glucoside equivalents (CGE)
Total phenolics, flavonoids, nonflavonoids and anthocyanins
The content of total phenolics (TPC), total flavonoids (TF) and total nonflavonoids (TN) in twenty-two chokeberry products is given in Table 5. TPC ranged from 1494 mg of GAE per 100 g of dm in fruit tea sample FT3 to 5292 mg of GAE per 100 g of dm in capsule sample C2. Comparing the results of our research with the results of other authors, the mass fraction of TPC in chokeberry juices was lower than in the findings of others (3, 14, 35, 43). Some authors noticed higher values of phenolics in black chokeberry fruit in comparison with our results (14, 26, 32–34), while Jurgoński et al. (44) reported much higher values of total phenolics in commercial chokeberry extract. Different cultivars of chokeberries were analysed and total phenolic values ranged from 8563.8 to 12055.7 mg of GAE per kg of fresh mass (fm) (34). Lower or higher values reported in the literature might have resulted from different extraction methods used for analysis, differences in analytical procedures applied, different processing technologies and storage conditions, or differences in chokeberry cultivars (14). It was demonstrated that the total phenolics in hot-air-dried tomatoes increased up to 29% compared to the corresponding levels in fresh tomatoes (45). In comparison with other products, chokeberry juices had lower phenolic content, which might be related to the differences in their moisture content (46). In total phenolic content, flavonoids were predominant, and their amounts varied from 867 mg of GAE per 100 g of dm in DB1 sample to 3317 mg of GAE per 100 g of dm in P2 sample. Average total flavonoid content in chokeberry juices was 3180 mg of GAE per L. It was calculated that percentages of TF in TPC varied between 36.06 and 80.46%. The obtained results suggest that flavonoids were the most abundant phenolics in chokeberry products. Chokeberries are a rich source of anthocyanins, proanthocyanidins and hydroxycinnamic acids (14). Oszmianski and Wojdylo (35) showed that polymeric proanthocyanins are the major class of polyphenolic compounds in chokeberry and represent 66% of polyphenols in fruits. Their content ranged between 1578.79 mg per 100 g of dm of chokeberry juice up to 8191.58 mg per 100 g of pomace. In a research of Kapci et al. (47) the content of total flavonoids was higher in chokeberry juices and in dried chokeberries. According to the literature, the main contributor of total flavonoid content is quercetin. Quercetin and several quercetin glycosides (quercetin-3-galactoside, quercetin-3-glucoside and quercetin-3-rutinoside) were also detected in chokeberries but in relatively low mass fractions of about 71 mg per 100 g of fm (14).
Table 5. Total phenolics (TPC), total nonflavonoids (TN), total flavonoids (TF) and total anthocyanins (TA) in chokeberry products.
| Sample | TPC | TN | TF | TA |
|---|---|---|---|---|
| Juices | ||||
| J1 | 5202±252 | 1383±124 | 3819±160 | 526±20 |
| J2 | 5448±479 | 1064±92 | 4384±571 | 592±24 |
| J3 | 3908±682 | 1088±240 | 2819±451 | 216±10 |
| J4 | 3358±702 | 1090±161 | 2267±550 | 434±13 |
| J5 | 4672±644 | 1156±259 | 3515±384 | 154±6 |
| J6 | 4083±490 | 1415±174 | 2667±330 | 504±16 |
| J7 | 3002±388 | 808±52 | 2193±386 | 150±4 |
| J8 | 6639±455 | 1368±83 | 5271±527 | 541±29 |
| J9 | 3759±692 | 1370±238 | 2389±618 | 235±11 |
| J10 | 3500±338 | 1320±213 | 2180±519 | 1228±5 |
| J11 | 5002±572 | 1527±417 | 3474±587 | 303±19 |
| Powders | ||||
| P1 | 4434±153 | 1602±124 | 2831±189 | 1641±24 |
| P2 | 4951±230 | 1634±67 | 3317±240 | 1576±74 |
| P3 | 4233±234 | 1906±139 | 2327±373 | 1165±10 |
| Capsules | ||||
| C1 | 4511±184 | (2051±184)a | 2459±31 | 2468±102 |
| C2 | 5292±243 | (2300±231)a | 2992±265 | 1997±138 |
| Fruit tea | ||||
| FT1 | 3436±242 | 1113±86 | 2322±168 | 675±17 |
| FT2 | 2435±75 | 1557±52 | 878±124 | 459±34 |
| FT3 | 1494±179 | 574±55 | 919±125 | 282±11 |
| FT4 | 1504±90 | 479±22 | 1024±110 | 353±16 |
| Dried berries | ||||
| DB1 | 1954±54 | (1086±74)b | 867±109 | (141±9)c |
| DB2 | 2466±91 | (1072±84)b | 1394±20 | (147±17)c |
The values are presented as mean±standard deviation (S.D.). The same letter in the superscript in the same column indicates no significant differences (p>0.05). J1–J11=chokeberry juices, P1–P3=chokeberry powders, C=chokeberry capsules, FT=chokeberry fruit tea, DB=chokeberry dried berries. Contents of TPC, TN, TF and TA are expressed as mg per 100 g of dry matter (dm) in powder, capsule, fruit tea and dried berry samples. Contents of TPC, TN, TF and TA in juice samples are expressed as mg per L. TPC, TN and TF are expressed as mg of gallic acid equivalent (GAE), while TA are expressed as mg of cyanidin-3-glucoside equivalents (CGE)
All samples had lower content of TN (808 to 1527 mg of GAE per L and 479 to 2300 mg of GAE per 100 g of dm) and TA (150 to 1228 mg of CGE per L and 141 to 2468 mg of CGE per 100 g of dm). Chlorogenic and neochlorogenic acids are the major non-flavonoid polyphenolic compounds in chokeberries, and according to Oszmianski and Wojdylo (35) they represent about 7.5% of chokeberry fruit polyphenols. The hydroxycinnamic acids are represented by significant amounts of chlorogenic (61 to 193 mg per 100 g of fm) and neochlorogenic acids (85 to 123 mg per 100 g of fm) (14). Higher contents of TA in chokeberry juice were reported by Jakobek et al. (34) and others (26, 47), while Horszwald et al. (40) reported higher content of TA in chokeberry powders. Results of all chokeberry samples were found to be lower, which can be explained by using pH differential method instead of HPLC method. Anthocyanins represented significant fraction of total phenolics in powder and capsule samples (from 27.53 in P3 to 54.72% in C1 sample). Chokeberries contain relatively higher amounts of anthocyanins compared to other fruits including blueberry, blackberry, raspberry, grape and cherry, which are known as rich sources of anthocyanins (14). In research of Jakobek et al. (3) the fraction of anthocyanins in chokeberry was 41%, which was much higher compared to the fraction in red raspberry (19%) and strawberry (23%). Similar to total phenolic content, Jurgoński et al. (44) reported considerably higher concentration of anthocyanins. Compared to other berries, the aronia anthocyanin profile is very simple, consisting almost exclusively of cyanidin glycosides, namely cyanidin-3-arabinoside, cyanidin-3-galactoside, cyanidin--3-glucoside and cyanidin-3-xyloside. Cyanidin-3-galactoside and cyanidin-3-arabinoside are predominant in the berries with a cumulative content >90% (14). Lower levels of total anthocyanins in chokeberry products can be the result of factors such as pH, chemical composition, temperature, light and oxygen. These factors may change easily during processing of fruits into juice and other products. It was reported that anthocyanins are affected at several steps of juice processing, namely pressing, clarification and pasteurisation (47, 48).
Total antioxidant capacity and reducing power
The total antioxidant capacity (TAC) and reducing power (RP) of different chokeberry samples are shown in Table 6. Examined products possess high antioxidant capacity (12.09 to 40.19 mmol of TE per L and 58.49 to 191.31 mmol of TE per 100 g of dm) and reducing power (38.71 to 79.86 mmol of Fe2+ per L and 13.50 to 68.60 mmol of Fe2+ per 100 g of dm). Highest TAC was reported in dried berries (mean value 187.41 mmol of TE per 100 g of dm), followed by fruit tea (mean value 144.54 mmol of TE per 100 g of dm) and powder (mean value 110.58 mmol of TE per 100 g of dm) samples. The reducing power (FRAP assay) in this study was determined as reduction of Fe3+ to Fe2+. The highest RP was observed in P2 sample (68.60 mmol of Fe2+ per 100 g of dm), followed by C1 sample (65.82 mmol of Fe2+ per 100 g of dm), P1 sample (60.66 mmol of Fe2+ per 100 g of dm) and C2 sample (60.35 mmol of Fe2+ per 100 g of dm). High antioxidant activity of chokeberry fruit and products has been reported in numerous studies (3, 6, 14, 34, 35). Walkowaik-Tomczak (48) showed that antioxidant activity of chokeberry juices is under the influence of pasteurisation and storage. Oxygen availability rate during pasteurisation and storage and storage temperature were found to have the biggest effect on the antioxidant activity of chokeberry juices. Reducing power is generally linked to the presence of reducing substances, which have been shown to exert antioxidant activity by breaking the free radical chain by donating a hydrogen atom (49). Antioxidant activity of chokeberry juice concentrate against DPPH radical was stronger than that of black currant, elderberry, red currant, strawberry, red raspberry and cherry concentrate (3, 26).
Table 6. Total antioxidant capacity (TAC) and reducing power (RP) of chokeberry products.
| Sample | TAC | RP |
|---|---|---|
| Juices | ||
| J1 | 33.37±0.54 | 76.14±0.36 |
| J2 | 12.09±0.93 | 51.50±0.24 |
| J2 | 23.03±0.53 | 48.76±0.48 |
| J4 | 19.47±0.33 | 72.43±0.52 |
| J5 | 18.29±0.68 | 48.64±0.43 |
| J6 | 20.66±2.45 | 79.86±0.14 |
| J7 | 16.51±0.14 | 38.98±0.25 |
| J8 | 34.22±1.61 | 71.50±0.28 |
| J9 | 26.25±0.28 | 38.71±0.41 |
| J10 | 40.19±2.13 | 62.92±0.35 |
| J11 | 28.12±0.88 | 60.13±0.29 |
| Powders | ||
| P1 | 95.00±2.94 | 60.66±2.17 |
| P2 | 105.68±5.58 | 68.60±0.99 |
| P3 | 131.06±0.47 | 47.38±2.68 |
| Capsules | ||
| C1 | 58.49±7.30 | (65.82±4.20)a |
| C2 | 80.93±4.56 | (60.35±1.70)a |
| Fruit tea | ||
| FT1 | 149.44±0.89 | 32.74±1.66 |
| FT2 | 111.43±2.01 | 43.12±0.91 |
| FT3 | 163.33±4.23 | 13.50±0.22 |
| FT4 | 153.96±2.99 | 15.94±1.32 |
| Dried berries | ||
| DB1 | 183.52±4.20 | 21.51±2.330 |
| DB2 | 191.31±0.38 | 17.4±1.0 |
The values are presented as mean±standard deviation (S.D.). The same letter in the superscript in the same column indicates no significant differences (p>0.05). TAC is expressed as mmol of Trolox equivalent (TE), while RP is expressed as mmol of Fe2+ equivalents (FE). For chokeberry juices TAC and RP are expressed as mmol of TE per L and mmol of FE per L, respectively. For powder, capsule, fruit tea and dried berry samples TAC and RP are expressed as mmol of TE per 100 g of dry matter (dm) and mmol of FE per 100 g of dm, respectively J1–J11=chokeberry juices, P1–P3=chokeberry powders, C=chokeberry capsules, FT=chokeberry fruit tea, DB=chokeberry dried berries
The correlation between the antioxidant activity measured by DPPH and FRAP method and total phenolics is presented in Table 7. Different groups of polyphenolic compounds may contribute differently to total antioxidant activity and, therefore, it is necessary to observe the existence of a correlation between the antioxidant activity and individual groups of polyphenolic compounds. The antiradical activity was mostly affected by the content of phenolic compounds. To see the relationship between the phenolic compounds in chokeberry products and their antiradical activity, TAC and RP values were correlated with the amount of phenolic compounds. This showed that the highest correlation of phenolic compounds and total antioxidant acitivity was between TA and TAC, and between TN and TAC in powder samples, followed by TN and TAC in fruit tea samples. High correlation was also found between TF and RP in powders and between TN and RP in fruit tea. These results imply that flavonoids and nonflavonoids were the major contributors to the antioxidant capacity of the investigated chokeberry products, especially in the case of powders, fruit tea and capsules. Acording to the data presented by others, TPC of various small fruits correlates better with the antioxidant activity than TA does (3, 9). ANOVA showed significant differences between TAC and RP values between groups of chokeberry products and also among individual samples within groups, with the exception of the RP of samples of capsules.
Table 7. Correlation coefficients (R) between phenolics and total antioxidant capacity (TAC) or reducing power (RP) in chokeberry products.
| Phenolics | TAC | RP |
|---|---|---|
| Juices | ||
| TPC | 0.19* | 0.29** |
| TN | 0.47 | 0.37* |
| TF | 0.09** | 0.21** |
| TA | 0.59* | 0.47** |
| Powders | ||
| TPC | –0.45* | 0.80 |
| TN | 0.74 | –0.72* |
| TF | –0.60** | 0.86 |
| TA | –0.94** | 0.84 |
| Capsules | ||
| TPC | 0.85 | –0.67* |
| TN | 0.52 | –0.25* |
| TF | 0.84 | –0.76* |
| TA | –0.86* | 0.70* |
| Fruit tea | ||
| TPC | –0.33** | 0.70 |
| TN | –0.89** | 0.98 |
| TF | 0.20** | 0.23* |
| TA | –0.10** | 0.48 |
| Dried berries | ||
| TPC | 0.76** | –0.78* |
| TN | 0.10* | –0.32 |
| TF | 0.71** | –0.68* |
| TA | 0.02* | 0.09 |
*, **significant at p≤0.05 and p≤0.001, respectively Contents of total phenolics (TPC), total nonflavonoids (TN), total flavonoids (TF) and total anthocyanins (TA) are expressed as mg per 100 g of dry matter (dm) for powder, capsule, fruit tea and dried berry samples. Contents of TPC, TN, TF and TA for juice samples are expressed as mg per L. TPC, TN and TF are expressed as mg of gallic acid equivalent (GAE), while TA is expressed as mg of cyanidin-3-glucoside equivalents (CGE). TAC and RP are expressed as mmol per 100 g of dm for powder, capsule, fruit tea and dried berry samples. TAC and RP for juice samples are expressed as mmol per L. TAC is expressed as mmol of Trolox equivalent (TE), while RP is expressed as mmol of Fe2+ equivalents (FE)
Conclusion
In this investigation, very high contents of phenolic substances and high values of antioxidant properties were observed in different chokeberry products. The presented data show differences in the quality and phenolic composition of chokeberry juices, powders, capsules, fruit tea and dried berries found on the market. Chokeberry capsules and powders have considerably higher amount of total phenolics and total anthocyanins in comparison with other products. Different levels of antioxidants might be related to the differences in the variety and growing conditions of the fruits. To fully understand the effect of processing, research focused on different processing techniques starting from the same material should be done. Chokeberry products can become a valuable source of nutritionally important substances in human nutrition. Due to the high content of natural antioxidants, their consumption could bring health benefits. Besides studies focusing on different processing techniques, future studies should include additional analyses to obtain a complete evaluation of the quality of chokeberry products and also in vivo and in vitro bioavailability studies. Data from these studies will be helpful to understand the bioaccessibility and bioavailability of nutritive compounds of chokeberry and its products.
Acknowledgements
This work was supported by the Croatian Ministry of Science, Education and Sports (Project number 058-0580696-2808).
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