Optimization for Ultrasound Extraction of Polyphenol from Aronia Melanocarpa (Chokeberry) with Antioxidant Activity

Gao Ningxuan1, Li Bin1*, Chou Shurui1, Yang Peiqing1, Li Enhui1, Zhang Ye1

1. College of Food, Shenyang Agricultural University, Shenyang City 110866, China E-mail: gaoningxuan1990@163.com

Abstract: Single-factor experiment and Box–Behnken design were applied to optimize the ultrasound-assisted extraction of chokeberry polyphenol. Under optimum conditions: ratio of liquid to raw material 39 mL/g, ethanol solvent concentration for extraction 56%, extraction temperature 46°C, extraction time 90 min, the chokeberry polyphenol yield was 1.95 ± 0.08%. The determination of DPPH, ABTS radical scavenging activity of chokeberry polyphenol exhibited the strongest antioxidant activity in vitro, and a concentration-dependent approach. Keywords: Ultrasound extraction; Chokeberry; Polyphenol; antioxidant activity. 1. Introduction

As the potential source antioxidant phenolic, berries are considered rich in polyphenols in plant material. Berries contain many different kinds of flavonoids and phenolic acids in order to show their antioxidant activity (J. Correa-Betanzo et al., 2014; Maria Handeland et al., 2014). In the major flavonoid constituents in the berries are anthocyanins, procyanidins, flavonol, and catechins. Phenolic acids constituents present in berries including hydroxyllated derivates of benzoic acid and cinnamic acid (Katarina Šavikin et al., 2014).

Compared with blueberries, cranberries and huckleberry, chokeberry is higher in polyphenols content and antioxidant activity. And anthocyanins content in the chokeberry also significantly higher than the other berries by comparison with blackberry, raspberry, blackcurrant (S.Benvenuti et al., 2004). Chokeberry anthocyanins content as high as 1% of dry weight, total phenol content is more than 20 mg/g (gallic acid equivalent)( Sabine E. Kulling et al., 2008 ). Owing to containing a large amount of polyphenols of phenol, flavonoids and procyanidins, chokeberry extracts showed stronger antioxidant activity than the blueberry extract, so the body to reduce damage from reactive oxygen free radicals (S. Medina et al., 2013; Seok Joon Hwang et al., 2014 ).

Cholesterol-lowering function of chokeberry has been widely praised in the clinical research, the study found that chokeberry could effectively restrain the pancreatic lipase, alpha amylase and alpha glycosidase enzymes, due to the fruit is rich in flavonoids. To regulate gastrointestinal digestion and absorption of lipids and carbohydrates. Flavonoid-rich chokeberry, as a functional food and a nutraceutical, modulating gastrointestinal carbohydrate and lipid digestion and absorption, maybe advocated as an exquisite and potential candidate for combinatorial obesity-diabetes prevention and phytotherapy (Entisar K. Al-Hallaq et al., 2013). In addition, through the seasonal influenza virus to antiviral experiment, the results showed that the extraction of chokeberry for a variety of influenza viruses have wide antiviral effect. Which contains ellagic acid and myricetin can limit the invasion of influenza virus and reduce 15-30% virus replication (Sehee Park et al., 2013).

The purpose of the present study aimed (1) to investigate the individual and mutual effect of ultrasonic-assisted extraction process parameters variables including ratio of liquid to raw material, ethanol solvent concentration for extraction, extraction temperature, and extraction time on the extraction yield of chokeberry polyphenols, (2) to establish the optimal ultrasonic-assisted extraction conditions for the extraction of polyphenol from chokeberry, (3) to evaluate antioxidant activity and toxicity of chokeberry polyphenols. 2. Material and methods 2.1 Samples and materials

Chokeberry were harvested by hand in the Zhuanghe, Liaoning Province, China, in June 2015, The fruits were washed thoroughly with deionized water, followed by placing in polyethylene bags and stored at -20°C prior to extraction. The plant was classified at the Food Science Laboratory of the College of Food, Shenyang Agriculture University, China, as chokeberry.

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2.2 Chemicals Dehydrated ethanol, ascorbic acid, gallic acid, Folin–Ciocalteu reagent, sodium carbonate (Na2CO3) were

purchased from Sinopharm Chemical Reagent Co., Ltd. Macroporous Adsorption Resin XAD-7 was obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). All other chemicals and solvents were reagent grade.

2.3 Ultrasound-assisted extraction of crude chokeberry polyphenol

Chokeberry was thaw at room temperature, and then the pulp for further use. The ultrasound-assisted extraction of chokeberry polyphenol was performed using an ultrasonic device (SB25-12DTN, Ningbo Scientz Biotechnology Co., Ltd, China). Each sample (5 g) was put into a 250 mL flat bottom flask. Then the extraction was carried out with a designed ratio of liquid to raw material, ethanol solvent concentration for extraction, extraction temperature and extraction time (Giovana Bonat Celli et al., 2015). After extraction, solutions were filtered through Whatman No. 2 filter paper under vacuum. The supernatants were concentrated with a rotary evaporator (RE-52AA, Shanghai Yarong Biochemistry Instrumenteactory, China) under reduced pressure at 45°C. Then determination of total phenolics in solutions. And the polyphenol yield (%) was calculated by the following equation:

Yield (%) = Polyphenol content of crude polyphenol (g) / Weight of pretreated chokeberry (g) × 100% (1)

2.4 Determination of total phenolics

The determination of total phenolic contents by colorimetric Folin-Ciocalteu method with some revamp (Singleton et al., 1999). Briefly, 1 mL of diluted chokeberry polyphenol solutions was mixed with 5 mL of deionized water and 1 mL 50% Folin-Ciocalteu reagent. After incubating for 6 min, 3mL 7% Na2CO3 solutions were added and the mixture was allowed to incubate for 2 h at room temperature in a dark place. The absorbance was measured at 765 nm using a visible spectrophotometer (V5800, METASH, China). Using gallic acid as standard, and drawing standard curve. The concentration of total phenolic solutions were determined as milligram gallic acid equivalents per 1 mL (mg/mL). And the polyphenol contents (mg) was calculated by the following equation:

Content (mg) = Concentration of total phenolic solutions × Volume of total phenolic solutions (2)

2.5 Single-factor design for chokeberry polyphenol extraction

The single-factor design was used to determine the preliminary range of the extraction factors including X1(ratio of liquid to raw material: 10, 15,20,25, 30,35, 40, 45 mL/g), X2(ethanol solvent concentration for extraction: 40,45,50,55,60,65,70 %), X3(extraction temperature: 30, 35,40,45,50,55,60°C), and X4(extraction time: 30, 60, 90, 120, 150 min). The extraction yield of chokeberry polyphenol was the dependent variables. Each experiment was conducted in triplicates. 2.6 Optimization experimental design

On the basis of the single-factor experiment results, experimental design, data analysis and model building were implemented by Design Expert software (Version 8.0.6.1). Choose three influence factors (ratio of liquid to raw material, ethanol concentration and extraction temperature) of the most significant influence on extraction chokeberry polyphenol yield. A three-level, three-factor Box–Behnken design (BBD) was applied to optimization. The whole experiment design contains 17 groups. Experimental run was carried out in a certain order as shown Table 1. All trials were performed in triplicate.

2.7 Purification of chokeberry polyphenols

The macroporous adsorption resin XAD-7 was employed to dynamic adsorption for enrichment of the crude chokeberry polyphenols as follows: pH 7.0 for adsorption sample solution, sample concentration of 3.6 mg/mL, at a flow rate of 2 mL/min. And using deionized water of 2 BV to remove polysaccharides, monosaccharides, protein and small molecule material. Then 95% ethanol with pH 7.0 as desorption solvent, at a flow rate of 2 mL/min. The ample polyphenols eluent were collected by a conical flask. The polyphenols eluent were concentrated with a rotary evaporator (RE-52AA, Shanghai Yarong Biochemistry Instrumenteactory, China) under reduced pressure at 45°C. And then freeze dried at −50°C to obtain chokeberry polyphenols.

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Table 1 Box–Behnken design with response values for the yield of chokeberry polyphenol.

2.8 In vitro antioxidant activity 2.8.1 DPPH radical scavenging activity

The DPPH radical scavenging activity of the chokeberry polyphenols was measured according to the method of Chun Chen (Chun Chen et al., 2015). With some modifications. Chokeberry polyphenols and ascorbic acid were dissolved in deionized water to obtain five different concentrations (20, 40, 60, 80, 100 μg/mL). The 1.9 mL of 0.1 mM DPPH in ethanol solution were mixed with 0.1 mL of chokeberry polyphenols or ascorbic acid standards solution at various concentrations. After the mixture was shaken evenly and incubated in the dark for 5 min at room temperature, and follow the determination of absorbance by a visible spectrophotometer (V5800, METASH, China) at 517 nm. The control used deionized water in place of sample solution, and the ethanol was used as the blank. Ascorbic acid was used as a positive standard. The experiment was performed in triplicate for each sample. The DPPH radical scavenging rate was calculated by the following equation:

Radical scavenging rate (%) = [A-(Ai-A0)] / A × 100% where, Ai was the absorbance of polyphenols/ascorbic acid reaction solution, A0 was the absorbance of the

solution including 0.1 mL of ethanol and 1.9 mL of DPPH, A was the absorbance of the solution including 0.1 mL of polyphenols/ascorbic and 1.9 mL of ethanol.

2.8.2 ABTS radical scavenging activity

The determination of ABTS radical scavenging activity of the chokeberry polyphenols according to the method of Kriengsak Thaipong (Kriengsak Thaipong et al., 2006) with some modifications. Mother liquor of ABTS: the 88 μL of 140 mM potassium persulfate solution were mixed with 5 mL of 7 mM ABTS solution, after the mixture was shaken evenly and incubated in the dark for 24 h at room temperature. The mother liquor

Factors Unit Symbols Level of factors

-1 0 1

ratio of liquid to raw material mL:g X1 35 40 45

ethanol solvent concentration % X2 50 55 60

extraction temperature °C X3 40 45 50

Std. order X1 X2 X3 PY (%)

1 -1 -1 0 1.49

2 1 -1 0 1.51

3 -1 1 0 1.78

4 1 1 0 1.60

5 -1 0 -1 1.55

6 1 0 -1 1.60

7 -1 0 1 1.86

8 1 0 1 1.75

9 0 -1 -1 1.28

10 0 1 -1 1.71

11 0 -1 1 1.59

12 0 1 1 1.57

13 0 0 0 2.08

14 0 0 0 2.08

15 0 0 0 2.05

16 0 0 0 2.12

17 0 0 0 2.11

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of ABTS were diluted deionized water to obtain working solution of ABTS, and follow the determination of absorbance by a visible spectrophotometer (V5800, METASH, China) at 734 nm. Chokeberry polyphenols and ascorbic acid were dissolved in deionized water to obtain five different concentrations (20, 40, 60, 80, 100 μg/mL). The 1.9 mL of 0.1 mM working solution ABTS were mixed with 0.1 mL of chokeberry polyphenols or ascorbic acid standards solution at various concentrations. After the mixture was shaken evenly and incubated in the dark for 6 min at room temperature, and follow the determination of absorbance at 734 nm. Ascorbic acid was used as positive standard. The ABTS radical scavenging rate was calculated by the following equation:

Radical scavenging rate (%) = [(A0-Ai) / A0] × 100% Where, A0 was the absorbance of working solution of ABTS, Ai was the absorbance of the solution

including 1.9 mL working solution ABTS and 0.1 mL of chokeberry polyphenols/ascorbic acid.

3. Results 3.1 Single-factor experiments of chokeberry polyphenols extraction 3.1.1 Effect of ratio of liquid to raw material on the extraction yield of chokeberry polyphenols

The effect of different ratio of liquid to raw material on the extraction yield of chokeberry polyphenols is shown in Figure 1. A, the extraction yield of chokeberry polyphenols was increased with the increase of the ratio, and reached the maximum value (2.10%) when the ratio of liquid to raw material was 40 mL/g. Then began to drop due to the higher ratio of liquid to raw material leads to the reduction of substrate. Thus, 40 mL/g was selected as the center point for the further experiment. 3.1.2 Effect of ethanol solvent concentration on the extraction yield of chokeberry polyphenols

As shown in Figure 1.B, the extraction yield of chokeberry polyphenols was increased with the increase of the ethanol solvent concentration, and reached the maximum value (2.04%). But extraction yield began to drop when ethanol solvent concentration more than 55%, because extraction yield of chokeberry polyphenols can reach the maximum value when polarity of extracting solvent is in close proximity to the polarity of the polyphenols. Thus, 55% was selected as the center point for the further experiment. 3.1.3 Effect of extraction temperature on the extraction yield of chokeberry polyphenols

As evident from Figure 1.C, the extraction yield increased rapidly with the increase in temperature from 30 to 45°C, and the maximum value of the chokeberry polyphenols was 2.01% at 45°C. But extraction yield began to drop when above 45°C. The reasons for this phenomenon due to the high temperature can increase the permeability of cell wall, and at the same time increase the solubility and diffusion coefficient of extract, reduce the viscosity of the solvent, and thus improve the extraction yield. However the higher temperature leads to the degradation, internal oxidation reduction and polymerization of phenolic compounds. Thus, 45°C was selected as the center point for the further experiment.

3.1.4 Effect of extraction time on the extraction yield of chokeberry polyphenols

As shown in Figure 1.D, which is proportional to the extraction yield of chokeberry polyphenols and extraction time from 30 to 90 min. When the extraction time was 90 min to reach the maximum value (2.04%). Thereafter extraction yield began to drop. Within a certain range, the longer ultrasound extraction time can increase extraction yield. At the same time, because of the heating effect of the ultrasonic, ultrasonic processing lead to degradation of heat sensitive polyphenols components. On the other hand, solvent evaporation results in the decrease of ethanol solvent concentration, indeed reduce the extraction yield. Due to the effects of different extraction time on the extraction yield of chokeberry polyphenols were not significant. Hence factor of extraction time will be ignored in the future experiment.

A B

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C D

Figure 1 Effects of different extraction parameters on the extraction yield of chokeberry polyphenols (A: ethanol solvent concentration; B: ratio of liquid to raw material; C: extraction temperature; D:

extraction time). 3.2 Optimization of chokeberry polyphenols extraction by Box–Behnken design 3.2.1 Model fitting and statistical analysis

The design matrix and the extraction yields of chokeberry polyphenols under different conditions were presented in Table 1. The analysis of variance was carried out on the test results in Table 1 by the Design - Expert 8.0 software. As shown in Table 2, the F-value was 55.29 and P-value was less than 0.001 which implied that the model was significant. In the analysis of variance (ANOVA) of the model, the linear coefficients (X2, X3), cross product coefficients (X2X3) and quadratic coefficients (X1

2, X22 and X3

2) were found significant effect (p < 0.05), whereas the other term coefficients (X1, X1X2 and X1X3) were insignificant (p > 0.05). Additionally, the lack of fit of the model was insignificant (p > 0.05) which was determined by lack-of-fit F-value of 5.14 and the P-value of 0.0738. And, the determination coefficient (R2 of 0.9861, R2

adj of 0.9683 and C.V. % of 2.64) also showed that the quadratic regression model fitting and predictability are good which can use the equation instead of the actual value for further analysis.

Table 2 Results of ANOVA of regression model for the extraction yield of chokeberry polyphenols

Source Sum of squares DF Mean square F-value P-value Model 1.06 9 0.12 55.29 <0.0001 X1 6.50E-003 1 6.50E-003 2.83 0.1363 X2 0.078 1 0.078 35.52 0.0005 X3 0.050 1 0.050 23.22 0.0019 X1 X2 0.010 1 0.010 4.68 0.0673 X1 X3 6.400E-003 1 6.400E-003 3.00 0.1271 X2 X3 0.051 1 0.051 23.70 0.0018 X1

2 0.12 1 0.12 57.12 0.0001 X2

2 0.44 1 0.44 205.30 <0.0001 X3

2 0.22 1 0.22 102.23 <0.0001 Residual 0.015 7 2.136E-003 Lack of fit 0.012 3 3.958E-003 5.14 0.0738 Pure error 3.080E-003 4 7.700E-004 Cor total 1.08 16 R2 0.9861 R2

adj 0.9683 R2

Pred 0.8193 Adeq precision 23.709 C.V.% 2.64

3.2.2 Analysis of response surface plot and contour plot

Three-dimensional response surface plot and two-dimensional contour plots provided a visual interpretation of the interactions between two variables, the relationship between variables and extraction yield, and also was used to locate the optimum conditions for maximum extraction yield of chokeberry polyphenols, as shown in Figure 2 and 3.

As shown in Figure 2.A the mutual interactions between the ratio of liquid to raw material (X1) and the ethanol solvent concentration (X2) on the extraction yield of chokeberry polyphenols for fixed extraction

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temperature of 45°C. A great increase in extraction yield (Y) resulted when the ratio of liquid to raw material from 35 to 39.41 mL/g and ethanol solvent concentration from 50 to 55.68% respectively; and then decreased. The counter plot in Figure 3.A confirmed that the two factors of the ratio of liquid to raw material and the ethanol solvent concentration were insignificant (p-0.0673 > 0.05).

As shown in Figure 2.B the mutual interactions between the ratio of liquid to raw material (X1) and the extraction temperature (X3) on the extraction yield of chokeberry polyphenols for fixed ethanol solvent concentration of 55%. The extraction yield (Y) increased with the increase in the ratio of liquid to raw material from 35 to 39.41 mL/g and extraction temperature from 40 to 45.75°C respectively; and then decreased. The counter plot in Figure 3.B confirmed that the two factors of the ratio of liquid to raw material and the extraction temperature were insignificant (p-0.1271 > 0.05).

In Figure 2.C and 3.C, the maximum extraction yield of chokeberry polyphenols 2.10% was obtained in the range of ethanol solvent concentration (X2) and extraction temperature(X3). In accordance the two-dimensional contour plots Figure 3.C and results shown in Table 2 indicates that the mutual interactions between ethanol solvent concentration and extraction temperature were significant (p-0.0018 <0.05).

A B

C

Figure 2 Response surface plots showing the effect of ratio of liquid to raw material (X1), ethanol solvent concentration (X2) and extraction temperature (X3) on the extraction yield of chokeberry polyphenols.

A B

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C

Figure 3 Response contour plots showing the effect of ratio of liquid to raw material (X1), ethanol solvent concentration (X2) and extraction temperature (X3) on the extraction yield of chokeberry polyphenols

3.2.3 Verification of predictive model

The optimum conditions obtained by the response surface methodology analysis: ratio of liquid to raw material 39.41 mL/g, ethanol solvent concentration 55.68%, extraction temperature 45.74°C. Under the conditions, the predicted value of extraction yield of chokeberry polyphenols was 2.10%. Considering the feasibility of the practical operation, conditions were carried out with slight modifications and ratio of liquid to raw material 39 mL/g, ethanol solvent concentration 56%, extraction temperature 46°C were used. Under these conditions, extraction yield of chokeberry polyphenols was 1.95 ± 0.08%. The actual value closely agreed with the predicted value, which shows that the optimum process condition is reliable by the response surface optimization method. 3.3 In vitro antioxidant activity 3.3.1 DPPH radical scavenging activity

As shown in Figure 4, DPPH radical scavenging activity of chokeberry polyphenols exhibited a positive correlation with concentration of chokeberry polyphenols. Ascorbic acid was used as positive standard. The chokeberry polyphenols showed stronger DPPH radical scavenging activity than ascorbic acid at every concentration point when concentration of chokeberry polyphenols and ascorbic acid within the range of 20-60 µg/mL. However, within the range of 60-100 µg/mL, the DPPH radical scavenging activities of chokeberry polyphenols were lower than that of the ascorbic acid at the same concentration, and with the increase of the concentration, the gap between DPPH radical scavenging activity of chokeberry polyphenols and ascorbic acid is gradually increasing. These findings exhibited that chokeberry polyphenols can donate electron or hydrogen to scavenge DPPH radical (A. Kumaran et al., 2006).

Figure 4 Scavenging rates of DPPH free radical

3.3.2 ABTS radical scavenging activity

The results of ABTS radical scavenging activity of chokeberry polyphenols are presented in Figure 5. Ascorbic acid was used as positive standard. ABTS radical scavenging activity of chokeberry polyphenols exhibited a positive correlation with concentration of chokeberry polyphenols. The chokeberry polyphenols showed stronger ABTS radical scavenging activity than ascorbic acid at each concentration point, when concentration of chokeberry polyphenols and ascorbic acid within the range of 20-60 µg/mL. These data exhibited that chokeberry polyphenols can effectively scavenge ABTS radical.

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Figure 5 Scavenging rates of ABTS free radical

4. Conclusions

This study provides evidence that ultrasonic-assisted extraction is an effective technique for the extraction of polyphenol from chokeberry berries using lower temperatures and in a reasonable time. Under the most favorable extraction conditions optimized by Box–Behnken designs as follows: ratio of liquid to raw material 39 mL/g, ethanol solvent concentration for extraction 56%, extraction temperature 46°C, extraction time 90 min, extraction yield of chokeberry polyphenols was 1.95 ± 0.08%. The predicted extraction yield of chokeberry polyphenols was 2.10%. The actual value closely agreed with the predicted value, which shows that the optimum process condition is reliable by the response surface optimization method.

By measuring the scavenging activity of polyphenols on DPPH and ABTS free radicals, research found that chokeberry polyphenols has strong antioxidant capacity, and can effectively scavenge DPPH and ABTS free radicals in vitro experiment of antioxidant activity. Through using the ascorbic acid as positive standard found that the chokeberry polyphenols showed stronger DPPH radical scavenging activity than ascorbic acid at each concentration point, when concentration of chokeberry polyphenols and ascorbic acid within the range of 20-60 µg/mL. On the scavenging ABTS radical hand, the chokeberry polyphenols also showed stronger ABTS radical scavenging activity than ascorbic acid at each concentration point. These are further evidence that chokeberry can be widely used in medicine and health products (Magdalena Kedzierska et al., 2013). 5. References [1]A. Kumaran, R. Joel karunakaran. Antioxidant and free radical scavenging activity of an aqueous extract of Coleus aromaticus. Food Chemistry, 2006:97,109-114. [2]Chun Chen, Li-Jun You, Arshad Mehmood Abbasi, Xiong Fu et al. Optimization for ultrasound extraction of polysaccharides frommulberry fruits with antioxidant and hyperglycemic activity in vitro. Carbohydrate Polymers, 2015: 130,122–132. [3]Entisar K. Al-Hallaq, Violet Kasabri, Shtaywy S. Abdalla, Yasser K. Bustanji et al. Anti-Obesity and Antihyperglycemic Effects of Crataegus aronia Extracts: In Vitro and in Vivo Evaluations. Food and Nutrition Sciences, 2013: 4, 972-983. [4]Giovana Bonat Celli, Amyl Ghanem, Marianne Su-Ling Brooks. Optimization of ultrasound-assisted extraction of anthocyanins from haskap berries (Lonicera caerulea L.) using Response Surface Methodology. Ultrasonics Sonochemistry, 2015:27,449-455. [5]J. Correa-Betanzo, E. Allen-Vercoe, et al. Stability and biological activity of wild blueberry (Vaccinium angustifolium) polyphenols during simulated in vitro gastrointestinal digestion, Food Chemistry, 2014:165, 522-531. [6]Katarina Šavikin, Gordana Zdunić et al. Berry fruit teas: Phenolic composition and cytotoxic activity. Food Research International, 2014: 62, 677–683. [7]Kriengsak Thaipong, Unaroj Boonprakob, Kevin Crosby, Luis Cisneros-Zevallos et al. Comparison of ABTS, DPPH, FRAP, and ORAC assays for estimating antioxidant activity from guava fruit extracts. Journal of Food Composition and Analysis, 2006: 19, 669-675. [8]Maria Handeland, Nils Grude, Torfinn Torp, Rune Slimestad. Black chokeberry juice (Aronia melanocarpa) reduces incidences of urinary tract infection among nursing home residents in the long term —a pilot study. Nutrition Research, 2014: 34, 518-525. [9]Magdalena Kedzierska, Joanna Malinowska et al. Chemotherapy modulates the biological activity of breast cancer patients’ plasma: The protective properties of black chokeberry extract. Food and Chemical Toxicology, 2013: 53, 126-132.

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[10]S. Benvenuti, F. Pellatl, M. Melegari, D. Bertelli, et al. Polyphenols, anthocyanins, ascorbic acid, and radical scavenging activity of Rubus, Ribes, and Aronia. Journa of Food Science, 2004: 69, 164-169. [11]Sabine E. Kulling, Harshadai M et al. Chokeberry (Aronia melanocarpa)-A Review on the Characteristic Components and Potential Health Effects. Planta Med, 2008: 74, 1625-1634. [12]Seok Joon Hwang, Won Byong Yoon, Ok-Hwan Lee, Seung Ju Cha et al. Radical-scavenging-linked antioxidant activities of extracts from black chokeberry and blueberry cultivated in Korea. Food Chemistry, 2014: 146, 71-77. [13]Sehee Park, Jin Il Kim, Ilseob Lee, Sangmoo Lee et al. Aronia melanocarpa and its components demonstrate antiviral activity against influenza viruses. Biochemical and Biophysical Research Communications, 2013: 440, 14-19. [14]S. Medina, R. Domnguez-Perles et al. Physical activity increases the bioavailability of flavanones after dietary aronia-citrus juice intake in triathletes. Food Chemistry, 2012: 135, 2133-2137. [15]Singleton, V. L., Orthofer, R. M., & Lamuela-Raventos, R. M. Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent. Methods in Enzymology, 1999: 299, 152-178.

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