Antioxidant Capacity of Binary and Ternary Mixtures ...

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Antioxidant Capacity of Binary and Ternary Mixtures of Orange, Grape, and Starfruit Juices Raúl Siche1,*, Carmen Ávalos1, Hubert Arteaga1, Erick Saldaña2 and Thais M. F. S. Vieira2 1

Instituto Regional de Investigación Agraria, Universidad Nacional de Trujillo, Av. Juan Pablo II s/n. Ciudad Universitaria, Trujillo, Peru; 2Department of Agro-industry, Food and Nutrition, “Luiz de Queiroz” Agricultural College, University of São Paulo, Brazil Abstract: Background: The growing interest in new functional foods with special characteristics and health properties has led to the development of new beverages based on fruit juice mixtures. The proliferation of ready-to-drink beverages has driven the market to focus its interest on these products. The aim of this study was to evaluate the antioxidant capacity of mixtures formed by orange (Citrus sinensis L. var. Washington navel), grape (Vitis vinifera L. var. Lavallet Alfonso) and starfruit (Averrhoa carambola L. var. Golden Star) juices.

Raúl Siche

Methods: Samples were prepared with different proportions of each juice according to simplex centroid mixture design (SCMD). The antioxidant capacity of each sample was determined by 2,2 - diphenyl -1 - picrylhydrazyl (DPPH) radical scavenging ability in vitro and expressed as the amount of sample needed to scavenge 50% of the DPPH (IC50). Results: The antioxidant capacity was higher in treatments of mixtures of these fruits than those who used pure juices, indicating binary mixtures of orange and starfruit juices and also ternary mixtures including grape juice in different proportion presents higher antioxidant capacity than pure juices. Conclusion: These results provide important information for the juice industry, an industry that could design mixtures of fruit juices instead of pure juice, with the intention to improve their functional properties.

Keywords: Vitamin C, flavonoids, statistical mixture design, DPPH, antioxidant capacity. 1. INTRODUCTION In recent years the relationship between diet and health has emerged, and products that promote health and wellbeing, mainly related to cardiovascular and neurodegenerative diseases, cancer, and obesity reduction are available worldwide [1, 2]. Fruits and vegetables contain many bioactive compounds with antioxidant activities, such as vitamins A, C and E [3]. Phenolic compounds in food stuff are related to antioxidant capacity [4]. Within the variety of fruits and vegetables, many have been studied extensively due to their high antioxidant activity, such as avocado, broccoli, cabbage, sprouts, carrots, berries (blackberries, strawberries, raspberries, blueberries, etc.), onions (especially the purple one), spinach, tomatoes, grapes and citrus [5]. Orange juice is a rich source of vitamin C, which is an important antioxidant [6]. The concentration of vitamin C is a significant indicator of orange juice quality, and it may serve as an indicator of thermal processing, to ensure desirable nutritional level. Orange juice also contains phenolic compounds and carotenoids, which have been shown to be good contributors to the total antioxidant capacity of foods [7-9]. Another fruit

*Address correspondence to this author at the Facultad de Ciencias Agropecuarias, Universidad Nacional de Trujillo, Av. Juan Pablo II s/n. Ciudad Universitaria, Trujillo, Peru; Tel/Fax: +51-44294778; E-mail: [email protected] 1573-4013/16 $58.00+.00

with significant antioxidant content is grape. Antioxidant activities of grapes are due to the presence of flavonoids, phenolic acids, anthocyanins, and carotenoids. Carotenoids play an important role in human nutrition through their provitamin A activity, but also by acting as antioxidant, to prevent age-related macular degeneration or to protect the skin from UV radiation [10]. Starfruit, also called carambola (Averrhoa carambola L.), is a popular juicy fruit grown widely in the tropical and subtropical regions of the world. Fresh fruits (mature with greenish-yellow tinge) are widely used as a basic raw material for juice, jelly and jam preparation [11]. Star fruit is reported to be low in sugar, sodium and acid, and rich in phenolic compounds like () epicatechin, vitamin C, proanthocyanidins and carotenoids [12, 13]. The growing interest in the study of natural antioxidant compounds has been accompanied by an increase in the market presence of what are known as functional foods or nutraceuticals or healthy foods [14]. International trade in tropical fruit juices is a business with 1,359 million dollars a year and a volume of 1,142 t and presents a high revenue growth of 16% and volume growth of 7% annually. This growth is a sustained trend in global markets, especially in the European Union and the United States of America. Because of the health considerations and the higher life expectancy for American and European consumers, the consumption of fruit juices with tropical blends is constantly increasing [15]. © 2016 Bentham Science Publishers

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Current Nutrition & Food Science, 2016, Vol. 12, No. ?

The measurement of isolated components from natural sources does not allow to know the total antioxidant capacity of a biological fluid, due to the combined and synergistic effect of a large number of compounds [16]. The investigation of the effect of the composition of binary and ternary mixtures should be done by techniques of experimental design for mixtures. It helps to minimize the number of experiments to be performed and maximize the amount of acquired information. These designs lead to the identification of synergistic and antagonistic interaction effects between different components or pseudocomponents. Though in the latest years some studies appeared on the investigation of the composition through this technique [17, 18], scarce information regarding the synergistic effect of fruits mixtures is available in the literature.

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Fig. 1 shows the composition of the 10 mixtures investigated. Since the components are linearly related to the volume proportions, model interpretation is easily converted into laboratory values. The left-hand part of the plot shows a situation where the three components X1, X2 and X3 are allowed to vary between 0 and 1. Each vertex of the mixture region corresponds to a pure component.

The objective of this investigation was to evaluate the antioxidant capacity, as well as the content of vitamin C and flavonoids of binary and ternary mixtures of orange (Citrus sinensis L. var. Washington navel), grape (Vitis vinifera L. var. Lavallet Alfonso), and starfruit (Averrhoa carambola L. var. Golden Star) juices using response surface methodology (RSM) experimental design to increase data useful in future works. 2. MATERIALS AND METHODS 2.1. Raw Material Orange (var. Washington navel), red grape (var. Alfonso Lavallet), and starfruit (var. Golden Star) samples were obtained from the cities of Huaral (Lima, Peru), Gran Chimu (La Libertad, Peru) and Viru (La Libertad, Peru), respectively, and immediately were subjected to juice preparation. 2.2. Process for Obtaining Juices The raw materials were selected based on their state of maturity and macroscopic high quality. Selected fruits were washed to remove foreign material. Juices were extracted by cold compression or crushing (for oranges) (Pulper DFV 1940 I/C, Vulcano, Peru). Subsequently, in order to remove traces of bark, seeds and excess of pulp in the juice, raw juices were filtered under vacuum (through a 110 mm diameter Buchner funnel using Whatman No. 40). Finally, glass bottles were filled with the juices, covered with aluminum foil to prevent oxidation and stored in domestic refrigerator (7±1 ºC) until further analysis. 2.3. Sample Preparation Mixtures were prepared following the experimental design presented in Table 2, where the composition was orange (X1), grape (X2) and starfruit (X3) juices in the designated proportions. 2.4. Statistical Experimental and Analysis A statistical mixture design, which corresponds to a triangle that represents the entire universe of possibilities of mixtures of orange juice, grape juice and starfruit juice, was applied to indicate the values of antioxidant capacity (performed in triplicate), expressed as IC50. The results of IC50 were analyzed using the Statistica 12.0 (StatSoft, USA) software, with a confidence interval of 95%.

Fig. (1). An overview of the simplex-shaped mixture region for a three-component mixture.

A three component, simplex-centroid mixture design was chosen for the experiments because all the components had the same range, between 0 and 1, and there were no constrains on the design space [19]. The mixture components consisted of orange (X1), grape (X2) and starfruit (X3) juices. Component proportions were expressed as fractions of the mixture with a sum (X1+ X2+ X3) of one. These three components (i.e. orange, grape and starfruit juice) levels and experimental design are presented in Table 2, with three singleingredient treatments, three two-ingredient mixtures and four three ingredient mixtures, as explained in Fig. 1. Each run was performed in triplicate. 2.5. In vitro Antioxidant Capacity of Mixtures Pure samples and mixtures in study were subjected to analysis in order to measure the in vitro antioxidant capacity by a method developed by Brand-Williams et al. [20], adapted by the Department of Pharmacognosy and Pharmacobotany of National University of Trujillo, that is based on the reduction of absorbance at 517 nm of 0.1 mM DPPH radical. The reactions were performed using 5 mL of DPPH ethanol solution at 0.1 mM as volume, and 50 μL of sample. The mixture was homogenized carefully; it was left to stand at room temperature protected from light over 30 minutes before absorbance at 517 nm reading. A spectrophotometer (SpectroQuest 4802 UV-Vis, UNICO, USA) was used and methanol and 0.1 mM DPPH solutions were used as negative and positive control. The percentage of DPPH radical scavenging (% DPPHRadical_scavenging) of the tested sample was calculated by the Equation 1: % DPPHRadical_scavenging = (Abs control – Abs sample)*100 / (Abs control) (Eq. 1) The results were evaluated in triplicate and were expressed as IC50, which represents the amount of sample (mL)

Application of Bifidobacterium lactis as the Single Strain Probiotic Starter


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Table 1. Physicochemical characteristics of juices extracted from orange, grape and star fruit.

a b

Fruit

pH

TSS (a) (°Brix)

Acidity (%) (b)

Orange

4.11±0.01

10.49±0.03

0.51±0.01

Grape

3.19±0.02

12.48±0.06

0.74±0.01

Starfruit

2.15±0.02

4.50±0.02

0.37±0.01

TSS (Total soluble solids) Expressed in percentage of tartaric acid.

that reduces the initial concentration of DPPH radical solution by 50% [21]. The IC50 obtained by this process is called the experimental IC50 (IC50_exp). 2.6. Obtaining the Estimated IC50 Values for the Mixtures The calculation of IC50 estimated values was based on IC50 values obtained experimentally for each of the treatments consisting 10 treatments including pure juice samples, binary and ternary mixtures of orange, grape and starfruit juices in different proportions, according to simplex centroid mixture design (SCMD) presented in Table 2, for which we used the equation 2: IC50_Estimate = A*X1 + B*X2 + C*X3

(Eq. 2)

Where: A: IC50 experimental value from pure juice of orange; B: IC50 experimental value from pure juice of grape; C: IC50 experimental value from pure juice of starfruit; X1: proportion of orange juice used in the mixture; X2: proportion of grape juice used in the mixture; X3 : proportion of starfruit juice used in the mixture. 2.7. Physicochemical Analysis The pH of pure juices was determined by AOAC Official Method 981.12, and titratable acidity, using tartaric acid as the predominant acid, by the AOAC Official Method 942.15, and total soluble solids content by AOAC Official Method 932.12. 2.7.1. Determination of Vitamin C Content The quantification of vitamin C was performed on the sample mixtures with the higher antioxidant capacity. Tillman’s method modified by Bessey and King [22] was used based on an assessment redox using the DCPI (2,6– dichlorophenol indophenol), as described by AOAC Official Method 967.21 (Eq. 3) Ascorbic acid + DCPI oxidized acid + SCPI reduced

(blue)

Dehydroascorbic (Eq. 3)

The DCPI in alkaline conditions is blue, pink at acidic pH, and colorless when it is reduced by the ascorbic acid. The assessment is carried out in acidic medium, so that the color change at the endpoint is colorless to pink. 2.7.2. Determination of Total Flavonoids The determination of total flavonoids was determined in the higher antioxidant capacity samples. The flavonoids extraction method was as suggested by Chinapongtitiwat et al. [23] with slight modifications. 80 mL sample was refluxed

with 80 mL ethanol and 80 mL 10% sulfuric acid w/v. The ethanolic extract was vacuum filtered and the residue was washed with ethanol. The filtrate was taken to waterbath at 50 °C until 50% volume reduction. The extract was filtered and the residue was washed with bi-distilled water. The filter paper containing flavonoids was carried to the oven (40°C), solubilized in methanol and transferred into a 100 mL vial for absorbance reading at 258 nm. Quercetin solution was used as standard. The concentration of total flavonoids expressed as quercetin equivalents was calculated using the equation 4: x = (Am*PR*5)*100/AR

(Eq. 4)

Where x: total flavonoid content expressed in quercetin (%); Am: absorbance of the sample solution (nm); AR: absorbance of the reference solution (nm); PR: weight of reference substance (g). 3. RESULTS AND DISCUSSION 3.1. Pure Juices, Mixtures, and Antioxidant Capacity The physicochemical characteristics (pH, total soluble solids and acidity) of the pure juices are presented in Table 1. The pH and acidity are important criteria for fruit juice processing as they can prolong the shelf life of the product and can be used as a reliable indicator to evaluate juice overall quality, whereas the °Brix can be used to evaluate the amount of sugars present in fruits and their juices [11]. In the present study, physicochemical characteristics of orange juice presented pH values of 4.11±0.01, TSS values of 10.49±0.03 and acidity values of 0.51±0.01, expressed as percent tartaric acid. All values are very close to those presented by Legua et al. (2013), where juice pH varied from 3.69 to 3.90 (Cleopatra mandarin), and total soluble solids (TSS) ranged from 8.18 to 11.77 °Brix (slight variations could be explained because phenolic compounds are secondary metabolites produced and accumulated in plant tissues). Changes in phytopathogenesis, among others factors, may result in different concentrations of these compounds in plant organs [24], consequently resulting in changes in the physicochemical properties. Moreover, according to Table 1, grape juice pH, TSS and acidity values agreed with those reported by Danii et al. [25], where juice showed total acidity between 0.40 and 0.96 g/100 mL expressed as tartaric acid, and pH values varied from 3.21 to 3.60. Starfruit juice values reported in Table 1 are in agreement with the results found by Bhat et al. [11], with a small differ-

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Table 2. Experimental IC50, Modeled IC50, Relative dewtion and Estimated IC50 for each treatment. Independent Variables Treatment

Answer

IC50_Mod (b) (L)

Relative Deviation (%)

IC50_Est (c) (L)

X1

X2

X3

IC50_Exp (L)

T1

0

0

1

34.33 ± 0.04

35.84

4.40 ± 0.13

34.33

T2

1

0

0

52.33 ± 0.00

51.85

0.92 ± 0.00

52.33

T3

0

1

0

42.27 ± 0.04

40.96

3.10 ± 0.08

42.27

T4

0

1/2

1/2

35.96 ± 0.00

35.49

1.31 ± 0.00

38.30

T5

1/2

1/2

0

42.14 ± 0.04

39.68

5.85 ± 0.08

47.30

T6

1/2

0

1/2

31.34 ± 0.00

31.70

1.14 ± 0.00

43.33

T7

1/3

1/3

1/3

33.61 ± 0.28

33.20

1.31 ± 0.65

42.98

T8

1/6

1/6

4/6

35.51 ± 0.00

31.92

10.09 ± 0.00

38.66

T9

4/6

1/6

1/6

36.26 ± 0.67

38.65

6.61 ± 1.97

47.65

T10

1/6

4/6

1/6

31.41 ± 0.00

36.29

15.52 ± 0.00

42.62

X1: Proportion of orange juice; X2: Proportion of grape juice; X3: Proportion of starfruit juice. IC50_Exp: IC50 obtained by the experimental procedure detailed in Materials and methods. (b)

IC50_Modeled: 51.85*X1 + 40.96*X2 + 35.84*X3 - 48.62*X1*X3 - 26.92*X1*X2 - 11.64*X2*X3 (R2: 0.86; p < 0.001)

(c)

IC50_Estimated: A*X1 + B*X2 + C*X3; where IC50: Necessary quantity of the sign to reduce in a 50% the radical's initial concentration DPPH; A: IC50_Experimental of the pure orange juice (T2); B: IC50_Experimental of the pure grape juice (T3); C: IC50_Experimental of the pure starfruit juice (T1).

ence in pH and TSS values (the pH ranged from 4.39 to 4.37 and the °Brix values ranged between 9.13 and 8.87), maybe due to the fruit maturity stage, soil composition and other factors. Table 2 shows the experimental design and antioxidant capacity results for pure juices and their mixtures. As shown in Table 2, the experimental antioxidant capacity values ranged from 31.34 L to 52.33 L, being the binary mixture of orange and starfruit juices and ternary mixture with a higher proportion of grape juice better able to capture the DPPH radical, while pure orange juice showed a lower ability to capture DPPH radical (IC50_exp = 52.33 L). Differences were verified among estimated and experimental results, indicating the original components interaction in the mixtures can lead to an unexpected IC50 value and so, the importance of trying real mixtures. Higher antioxidant capacity of orange and starfruit binary mixtures could be due to the high concentration of phenolic compounds in starfruit (131 mg of total phenols / 100 g and 5.2 mg of ascorbic acid / 100 g) and high concentration of ascorbic acid in orange (75 mg of total phenols / 100 g and 67 mg of acid ascorbic / 100 g) [26]. While the antioxidant properties of ternary mixture (1/6 orange juice, 4/6 grape juice and 1/6 starfruit juice) could be due to the high presence of total phenolics compounds, mainly anthocyanins, quercetin, rutin, catechin, and resveratrol, in grape (5422.8 mg / 100 g average of four varieties studied) [27]. Studies showed grape as fruit with excellent antioxidant properties [28], although this study found that grape juice (IC50_exp = 42.27 L) has lower antioxidant capacity than starfruit juice (IC50_exp = 34.33 L), but higher than that of orange juice (IC50_exp = 52.33 L). Maybe this statement: although different process conditions are used for grape juice production, the results are in agreement to other authors. When grape

juices are produced industrially, the pulp is heated along with the skin, resulting in the incorporation of skin phenolic compounds into the juice [29]; Leong and Shui [30] suggested that the strawberry and grape are fruits with high antioxidant capacity. Starfruit can be classified as moderated antioxidant capacity product exceeding orange, lemon, passion fruit, papaya and others. However, antioxidant capacity is lower than camu camu, noni, and yacon [31]. Table 2 also shows that the treatments consisting of a mixture of juices presented higher antioxidant capacity than pure juices. It was observed that the IC50_exp values (from 31.34 L to 42.13 L) are lower than the IC50_est values (from 38.29 L to 47.65 L), suggesting the existence of synergism. This increase in antioxidant capacity is due to the combined and synergistic effect of the compounds in the mixture of juices (vitamins, carotenoids, polyphenols, minerals, terpenes, etc.). Yang et al. [32] studied the synergistic antioxidant capacity of medicinal plants, demonstrating that there are significantly better pharmacological effects when using various herbs in combination than using them separately. For his part Alonso et al. [33] evaluated the antioxidant capacity and potential synergism between the main constituents of some antioxidants in foods, confirming the wellknown cooperation between alpha-tocopherol and ascorbic acid, suggesting a similar cooperation between tocopherol and quercetin, rutin and ascorbic acid, and caffeic acid and ascorbic acid, but also found an antagonism between quercetin and ascorbic acid. Parker et al. [34] found similar results when they investigated the ability to pro-antioxidant and antioxidant, and the synergistic potential of various compounds such as rutin, p-coumaric acid, abscisic acid and ascorbic acid.



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material; while flavonoids depend on the variety, fruit growing conditions, geographical origin, harvest time, among others [36]. 3.3. Optimization for the Antioxidant Capacity Expressed as IC50 We found that the quadratic model best fits the experimental conditions (R2 = 86% and p-value < 0.05), being possible to build the response surface for values IC50 (Fig. 2).

Fig. (2). Contour graph for the values of IC50.

3.2. Vitamin C and Flavonoids Orange and starfruit binary mixture (1/2 orange juice and 1/2 starfruit juice) presented the lowest experimental IC50 values (31.34 L) and it was subjected to further analysis. Quantitative analysis of vitamin C showed 2.42 mg of ascorbic acid / 100 mL of juice mix, while for total flavonoids, a value of 102.4 mg of flavonoids / 100 mL of juice mix expressed as quercetin. Hours et al. [35] found an average mean value of ascorbic acid in W. Navel orange of 72.97 mg / 100 mL of juice, the lowest when compared to four other varieties, indicating that the ascorbic acid content tends to increase with time of harvest. Hours et al. [35] found that the hesperidin content is between 50 and 100 mg / 100 mL juice for Florida oranges. Although ascorbic acid levels in the mixture is low, the total flavonoid content is high. It is known that the polyphenolic compounds, including flavonoids, phenolic acids and stilbenes, are the most abundant antioxidants in foods, whose capacity for sequestration of free radicals is significantly higher than that of vitamins and carotenoids [4]. According to Hours et al. [35], antioxidant capacity does not depend only on the concentration of polyphenols, but also on the position of the hydroxyl group in these substances; which would have implications on the results obtained in the different mixtures. Variations in ascorbic acid content is due to factors such as variety, cultural practices, maturity and the evolution of temperature at fruit harvest [35], as well as temperature and storage time the raw

From an analysis of these graphs, the region of interest could be defined, which becomes the region where the IC50 values of the variable are minimal. This region can be more easily visualized in the graph of contour (shaded area) and is defined as follows for each variable: 0.15 to 0.55 of orange juice, 0 to 0.35 of grape juice and 0.35 to 0.85 of starfruit juice. It should be noted that the sum of the proportions of the juices that make up the mix, whether it is composed of two or three juices, must always be equal to one (i.e. 100%). In this definition we see that with higher concentration of starfruit juice and much lower concentration of grape juice and orange juice, IC50 values decrease. Also states that at different concentrations of starfruit juice, minimum values for IC50 can be obtained, confirming the important contribution of this juice in the antioxidant capacity of mixtures. In addition, binary mixture of orange and starfruit juices (1/2 orange juice and 1/2 starfruit juice) is considered in the region of interest. The optimal IC50 value can be achieved with ratios of 0.34 for orange juice, 0.66 for starfruit juice and without grape juice, which would give us a minimum value of 30.38 L (Fig. 3). The adjusted model indicates that grape juice has no significant influence on the antioxidant capacity for both binary and ternary mixtures. This would be explained in this study because the antioxidant compounds found in grape are mostly in the skin and it is necessary to make a hot soaking or fermentation to improve its extraction and incorporation in the juice [37]. Experimentally pure grape juice presented a high antioxidant capacity (as shown in Table 2), but when combined with orange and/or starfruit juice a reduced antioxidant ca-

Fig. (3). Optimization of IC50 using the desirability function.

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pacity was observed, which could indicate an antagonism between quercetin and ascorbic acid, or as evidence of prooxidant role of flavonoids [33]. According to PérezTrueba [38] prooxidation is influenced by factors such as structural conformation, test conditions, the stability of the flavonoid radical, the pH of the medium; the effective concentration is reached at the site where reactive oxygen species is formed, and generally high concentrations of flavonoids are needed to develop mutagenicity and cytotoxicity.

[9]

4. CONCLUSION

[13]

It was found that the binary mixtures of orange and starfruit juices result in high antioxidant capacity when compared to pure juices. Fitted mathematical model indicates the importance of applying mixture design in order to identify synergistic effects of the components. The experimental results were different from estimated results. From the adjusted model it was found that a mixture with ratios of 0.34 orange juice and 0.66 starfruit juice, without the presence of grape juice, would achieve the optimal IC50 (30.38 L). These results provide important information for the juice industry, an industry that could design mixtures of fruit juices instead of pure juice, with the intention to improve their functional properties.

[10] [11]

[12]

[14] [15]

[16] [17]

[18] [19]

CONFLICT OF INTEREST The authors confirm that this article content has no conflict of interest.

[20] [21]

ACKNOWLEDGEMENTS R. Siche thank Universidad Nacional de Trujillo - UNT (PIC2-2013 / UNT) for funding. Erick Saldaña thank the “Ministerio de Educación del Perú” for the scholarship granted by the program “Programa Nacional de Becas y Crédito Educativo” (PRONABEC).

[22] [23]

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Received: July 30, 2015

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Revised: August 26, 2015

Accepted: September 01, 2015