Comparative Analysis of the Antioxidant Capacity of Selected Fruit Juices and Nectars: Chokeberry Juice as a Rich Source of Polyphenols

Abstract

The bioactive compounds, and hence the antioxidant capacity, of fruit juice are affected by a given type of juice and fruit processing conditions. In this study, some polyphenol-rich juices and nectars were compared, considering the manufacturing conditions (Stage I). Organic chokeberry juice was determined to possess the highest antioxidant capacity, which was 4- to 10-fold higher than in other popular juices. Subsequently, this juice was analyzed for selected flavonoids (flavanols, flavonols, flavones) and phenolic acids (Stage II). The results showed that chokeberry juice was a rich source of chlorogenic and neochlorogenic acids. The high total content of polyphenols and high antioxidant capacity encourage further clinical research on chokeberry juice, in the context of cardiovascular diseases prophylaxis.

Keywords:

• Antioxidants
• Aronia melanocarpa
• Flavonoids
• Phenolic acids
• DPPH

Introduction

Due to their high level of intake, fruit and vegetables are the main source of antioxidants in the human diet. Some amounts of antioxidants are also supplied by cocoa, chocolate, green tea, red wine, or herbs and spices,^[1,2] but these are not consumed as much as fruit, vegetables, and juices. The high antioxidant capacity of fruit and vegetables may arise from their content of polyphenols, including flavonoids (flavonols, flavones, flavanones, flavanols, isoflavones, anthocyanins) and carotenoids, vitamins C and E, zinc, and selenium.^[3–5] Many studies show that the presence in a dietary intake of polyphenol-rich vegetables and fruit significantly decreases the risk of cardiovascular and neoplastic diseases.^[6–8]

Berries are the richest source of polyphenols. For example, chokeberry, also known as aronia, contains 40–100 g polyphenols per kg. Slightly less of these compounds can be found in blueberries (30–38 g/kg), raspberries (12–27 g/kg), blackcurrants (22–28 g/kg), and cranberries and strawberries (22 and 12 to 24 g/kg, respectively).^[9–11] Differences in the content and composition of polyphenols arise from the variety of a plant, cultivation conditions and the harvesting time. Stone fruit, pome, or citruses contain less of polyphenols, which is why frequent consumption of berries is recommended, for example in the Nordic countries.^[12] Unfortunately, seasonal fluctuations in the consumption of fruit and vegetables appear in some countries with temperate climates. In Slovakia, for instance, the consumption of these products in the winter falls by half compared to summer, which reflects itself in the levels of oxidative stress markers.^[13] Seasonality also affects the Polish market, although there is no shortage of imported fruit in the winter. Our diet can be enriched with antioxidants by drinking fruit juices or taking supplements containing plant extracts from berries. The antioxidant capacity of juices varies, depending on the type and variety of fruit and the production technology, which may cause some loss of bioactive compounds.

Various methods have been proposed to determine the antioxidant capacity. Some use radicals (1,1-diphenyl-2-picrylhydrazyl [DPPH], 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) [ABTS], trolox equivalent antioxidant capacity [TEAC], total radical-trapping antioxidant parameter [TRAP], and oxygen radical absorbance capacity [ORAC]) and others use metal ions (ferric-reducing antioxidant power [FRAP], cupric reducing antioxidant capacity [CUPRAC], low-density lipoprotein [LDL] oxidation).^[14,15] The DPPH method is most popular because of the stability of a DPPH radical in alcohol solutions,^[16] simplicity of application and low cost.^[14] As well as being sensitive, this method is quick and easy. No expensive or sophisticated equipment is required.^[17] In addition, a high linear correlation has been demonstrated between the antioxidant capacity and the total flavonoid content^[11] or between results obtained by the DPPH assay and by other methods.^[15] Currently, the DPPH method is widely used to determine the antioxidant capacity of fruit and vegetables.^[18,19] Thus, the aim of the first stage of research was to compare the antioxidant capacity of polyphenol-rich juices and nectars determined by the DPPH method. The second stage was designed to examine the content of selected flavonoids and phenolic acids in the best juice identified during the first stage. This juice will be tested on patients in further clinical research.

Material and Methods

Study Design

Stage I

The analysis comprised seven fruit juices, five nectars, and two beverages, including six from organic farms. The organic juices were produced traditionally, using wicker hydraulic presses and mild flow pasteurization (temperature no more than 90ºC). Chokeberries, apples, blackberries, and blackcurrants originated from Polish plantations. Pomegranate and sea buckthorn juices were imported from Georgia. Other juices, nectars, and beverages came from industrial production. Blackcurrant nectars contain at least 20% of fruit, while commercial beverages have at least 20% of fruit or fruit concentrate. All the analyzed juices, nectars, and beverages were produced between September and October 2012, and had a 1-year best-before-date. The samples rich in polyphenols were additionally compared with some popular orange and apple juices. Table 1 presents the basic specification of the analyzed samples and abbreviations used in the article.

TABLE 1 Material

Sample	Abbreviation	Method of production	Country of origin	pH
Apple juice	A_c1	Commercial	Poland	3.58
Apple nectar	A_c2	Commercial	Poland	3.74
Cherry drink	Ch_c	Commercial	Poland	3.46
Forest fruits drink	FF_c	Commercial	Poland	3.10
Orange juice	O_c	Commercial	Spain	3.39
Blackcurrant nectar 1	BC_c1	Commercial	Poland	3.13
Blackcurrant nectar 2	BC_c2	Commercial	Poland	3.10
Blackcurrant nectar 3	BC_c3	Commercial	Poland	3.19
Blackcurrant nectar 4	BC_e	Ecological	Poland	3.28
Aronia juice 1	Ar_e1	Ecological	Poland	3.51
Aronia juice 2	Ar_e2	Ecological	Poland	3.71
Pomegranate juice	P_e	Ecological	Georgia	3.37
Sea buckthorn juice	SB_e	Ecological	Georgia	3.03
Apple and blackberry juice	A-B_e	Ecological	Poland	3.53

Stage II

On the basis of Stage I, the juice with the highest antioxidant capacity was chosen for further analysis. The total polyphenol content was determined in this product by the Folin–Ciocalteu assay (as mg of gallic acid equivalents).^[20] Moreover, the selected flavonoids and phenolic acids were examined by high-performance liquid chromatography (HPLC). This part of the study included determinations of flavanols (catechin and epicatechin), flavonols (quercetin and kaempferol), flavones (apigenin and luteolin), and phenolic acids (especially chlorogenic and neochlorogenic acid).

Analytical Methods

The analyses were carried out between March and May 2013. The pH of the tested samples of juices, nectars, and fruit beverages was measured with a glass electrode (temperature 18ºC).

Antioxidant Capacity by DPPH Method

The antioxidant capacity of the samples was determined by a modified Yen and Chen method,^[21] using the DPPH stable radical-cation (Sigma-Aldrich). The method is based on the reduction of a DPPH radical. Methods that employ the DPPH radical-cation are widely used to test the antioxidant capacity of fruits, vegetables, and juices. Recently, they have also been applied to determine the antioxidant capacity of human and animal plasma or serum.^[22–24] The absorbance was measured on a Hitachi U-3900 spectrophotometer at the wavelength λ = 517 nm after 30 min of incubation. For each juice, nectar, and beverage, five parallel samples in three replicates were analyzed, from which a mean value was calculated (A_m). The percentage of DPPH inhibition (I) was calculated from the formula:^[24]

$\mathrm{I}’=’100’\left(A_0-A_m\right)/A_0$

where A_m is the mean value of the sample’s absorbance and A₀ is the absorbance of the DPPH radical-cation.

The value thus achieved was then substituted into an equation of a previously prepared 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox–Sigma-Aldrich) calibration curve. The antioxidant capacity of the samples of juices, nectars, and beverages was expressed as milligrams of Trolox per 100 mL of sample.

Total Polyphenols Content

The total polyphenol content was determined by the Folin–Ciocalteu assay^[20] in the best sample selected at Stage I. The absorbance was measured on a Hitachi U-3900 spectrophotometer at the wavelength λ = 765 nm after 60 min. The results were expressed as mg of gallic acid equivalents per 100 mL of sample (mg GAE/100 mL).

HPLC Analysis of Flavonoids and Phenolic Acids

In Stage II, the selected flavonoids and phenolic acids were determined by HPLC methods for the best sample selected at Stage I. The analyses were performed using a Dionex LC system equipped with photodiode array detector (PAD, Dionex) and the absorption spectra were recorded in the range of 200–600 nm. The flow rate was 1 mL/min, the column temperature was 30°C, and the injection volume was 20 µL. Qualitative identification was done by comparing the retention times and spectra with the standards. Simultaneous monitoring was performed at 280 nm.

Phenolic acids were determined according the method described by Krygier et al.^[25] The separation was performed on a Hypresil Gold (Thermo Electron Corporation) C18 column (250 mm × 4,6 mm; 5 μm). The binary mobile phase consisted of 0.1% (v/v) formic acid in methanol (eluent A) and methanol-acetonitrile (80:20, v/v; eluent B). The gradient program was as follows: 0–5 min (0% B), 7–15 min (10% B), 25 min (25% B), 34 min (65% B), 35–39 min (100% B), 40–45 min (0% B).

Flavonoids were determined by a modified Hertog et al. method,^[26] after its acidic hydrolysis. The separation was performed on a Ascentis (Supelco) C18 column (250 mm × 4,6 mm; 5 μm). The binary mobile phase consisted of 0.1% (v/v) formic acid in water-methanol (75:25, v/v, pH 2.7; eluent A) and 0.1% (v/v) formic acid in methanol (eluent B). The gradient program was as follows: 0–2 min (0% B), 10–20 min (15% B), 30 min (40% B), 35–44 min (100% B), 47–51 min (0% B).

Statistical Analysis

All the results were statistically analyzed by calculating the mean, standard deviation, and relative standard deviation. The interpretation of the results was performed with MS Excel 2010 Analysis ToolPak software, one-way analysis of variance (ANOVA) and the Tukey’s test at p ≤ 0.05.

Results and Discussion

Stage I

Before determination of the antioxidant capacity, pH was measured in all the samples at 18°C. The pH-value of all the juices, nectars and beverages (commercial and organic ones) was in the range of 3.03 to 3.74 (Table 1). The lowest values were obtained for sea buckthorn juice (SB_e), and the highest values were achieved for apple nectar (A_c2). The pH of commercial drinks was probably kept low by adding ascorbic acid.

Next, the antioxidant capacity was compared. The percentage of inhibition was determined in the analyzed samples, converted to the antioxidant capacity and expressed as mg of Trolox equivalents per 100 mL of sample (mg Tx/100 mL). Among all the analyzed juices, nectars, and fruit beverages, the significantly highest antioxidant capacity was determined in chokeberry (aronia) juice (Ar_e1) from an organic farm (Table 2). Other organic juices, such as chokeberry juice (Ar_e2), sea buckthorn juice (SB_e), and pomegranate juice (P_e), were characterized by high antioxidant capacity, with no significant differences between them (at p ≤ 0.05). These juices provide more antioxidants than blackcurrant nectars and apple-blackberry juice. Orange juice (O_c) had a lower capacity than cherry drink and blackcurrant nectars. The lowest antioxidant capacity was assessed in the most popular types of fruit beverages: apple juice and nectar (A_c1 and A_c2) and forest fruit drink (FF_c). Similar results were reported by Seeram et al.^[27]

TABLE 2 Antioxidant capacity

Sample*	Xm [mg Tx/100 mL]	SD	RSD [%]
Ar_e1	403.1^a	21.3	5.3
Ar_e2	245.3^b	3.9	1.6
SB_e	228.7^b	1.8	0.8
P_e	209.4^b	5.9	2.8
BC_e	126.8^c	4.9	3.9
BC_c3	103.1^c	1.9	1.8
A-B_e	90.8^c	1.3	1.4
BC_c1	89.4^c	1.7	1.8
BC_c2	72.5^d	0.6	0.9
Ch_c	63.1^d	1.3	2.1
O_c	35.5^e	0.2	0.6
A_c1	29.2^e	0.3	0.9
FF_c	19.2^f	0.2	1.1
A_c2	14.6^f	0.1	0.1

*abbreviations as in Table 1;

Xm: mean value of antioxidant capacity per 100 mL of sample (n = 15);

SD: standard deviation;

RSD: relative standard deviation;

Means marked with successive letters a, b, c … are significantly different at p ≤ 0.05.

The current results showed that the juices from organic farms had a significantly higher antioxidant capacity than the commercial juices, nectars, and fruit beverages. Blackcurrant nectar was an exception in that it did not differ significantly (at p ≤ 0.05) from its commercial counterpart (Table 2). The lower antioxidant capacity of commercial products is due to their lower content of polyphenols because under industrial conditions juices are subjected to high temperatures, addition of water or contact with metal parts of technological equipment.

The above results are part of a larger study whose aim is to select juice with the highest antioxidant capacity. This one will be given to patients in clinical research concerning the prevention of cardiovascular diseases. Chokeberry juice has the highest antioxidant activity among all the analyzed juices, nectars, and fruit beverages, which predisposes it to further studies. The antioxidant capacity of chokeberry juice (depending on the origin) was 2- to 4-fold higher than that of blackcurrant nectar and up to 4- to 6-fold higher than in cherry drink. Similar results were reported by Jakobek et al.,^[11,28] who found out that chokeberry juice was twice as powerful in antioxidants as blackcurrant juice and six-fold more powerful than cherry juice.

Stage II

The total polyphenol content, phenolic acids, and selected flavonoids were determined in chokeberry juice chosen at Stage I (with the highest antioxidant capacity). The results (Table 3) showed that chokeberry juice was a rich source of polyphenols, whose total level determined by the Folin–Ciocalteu assay was about 560 mg GAE/100 mL of juice. Other researchers reported similar^[15] or slightly higher results.^[29]

TABLE 3 Total polyphenols, phenolic acids, and flavonoids in chokeberry juice

Compounds	Content (mg/100 mL)
Total polyphenols*	559.5 ± 20.5
Free phenolic acids	97.04
–neochlorogenic acid	54.26 ± 0.98
–chlorogenic acid	42.51 ± 0.62
–caffeic acid	0.27 ± 0.01
Bound phenolic acids	4.48
–gallic acid	1.13 ± 0.09
–vanillic acid	0.05 ± 0.01
–syringic acid	0.02 ± 0.01
–coumaric acid	2.68 ± 0.05
–ferulic acid	0.60 ± 0.03
Flavanols
–catechin	0.36 ± 0.03
–epicatechin	0.42 ± 0.08
Flavonols
–quercetin	3.00 ± 0.08
–kaempferol	nd
Flavones
–apigenin	nd
–luteolin	nd

*mg GAE/100 mL of juice (Folin–Ciocalteu method);

nd: not detected (detection limit 0.01).

Chokeberry juice is characterized by a high content of free phenolic acids especially chlorogenic and neochlorogenic acid (42.51 ± 0.62 and 54.26 ± 0.98 mg/100 mL of juice, respectively). Similar results were presented by Kujawska et al. (45.50 mg and 49.21 mg/100 mL, respectively).^[30] Whereas in the research done by Wilkes et al., a similar amount of chlorogenic acid (45.9 mg) but a lower level of neochlorogenic acid (27.9 mg) were determined, although the results were expressed in mg/100 g of juice.^[31] Other researchers also claimed that chokeberry was a rich source of free phenolic acids.^[11,32] Additionally, in our study, bound phenolic acids were analyzed after acid hydrolysis (Table 3). Coumaric acid was determined in the largest amount (2.68 ± 0.05 mg/100 mL of juice).

Among the analyzed group of flavonoids, quercetin (which is the principal flavonol) was detected in the highest amount (Table 3). Jakobek et al. also stated the highest amount of quercetin (over 93% of flavonols),^[11,28] although their research concerned the analysis of polyphenols in chokeberry fruit. Moreover, they identified a small content of kaempferol (about 7%), which is another compound that belongs to flavonols. Kaempferol was undetected in our study, which may be due to its concentration being below the detection limit (<0.01 mg/100 mL). Another group of the evaluated flavonoids was composed of flavanols, i.e., catechin and epicatechin (0.36 ± 0.03 and 0.42 ± 0.08 mg/100 mL of juice, respectively), but their levels were slightly lower than reported by Kujawska et al.^[30]

These studies showed that chokeberry juice was a rich source of polyphenols, especially free phenolic acids. The actual content of this compounds could be even higher, depending on the variety of fruit, the season of the year and exposure to the sun.^[10] However, the level of polyphenols in chokeberry juice is approximately half of that in fresh fruit.^[15,33] Processing and storage conditions, particularly freezing, blanching, and pressing, affected the total polyphenols content and their profile in chokeberry juice.^[31] Therefore, minimally invasive and traditional processing methods, such as wicker presses or mild pasteurization, will ensure better preservation of valuable biological antioxidant compounds. This suggestion was confirmed in our study by the antioxidant capacity of organic juices (Stage I), of which organic chokeberry juice (Ar_e1), submitted to a more detailed analysis during Stage II, the most valuable sample.

Conclusions

The antioxidant capacity of fruit juices and nectars depends on the type of fruit and the applied processing technology. Juices from fruits grown on organic farms and produced with traditional methods, had higher antioxidant capacity than commercial juices, nectars and fruit beverages (Stage I). Among all the analyzed samples, the highest antioxidant capacity was determined in organic chokeberry juice, in which it was 4-fold higher than in blackcurrant nectars and over 10-fold higher than in popular commercial orange and apple juice. The results (Stage II) showed that chokeberry juice was a rich source of polyphenols, especially free phenolic acids (chlorogenic and neochlorogenic) and flavonoids (in particular quercetin). Organic chokeberry juice owing to its high antioxidant capacity and high content of polyphenols could be useful in further clinical research concerning the prevention of cardiovascular diseases.