Total Antioxidant Capacity and Total Phenol Contents of Selected Commercial Fruit Juices in Turkey

Abstract

The in vitro total antioxidant capacity (TAC), and the total phenol (TP) contents of commercial fruit juice samples widely consumed in Turkey were determined. TAC methods were more recently developed methods (TAC₁ and TAC₂) using Fe⁺²-o-dianisidine complex and ABTS radical, FRAP (Ferric Reducing Ability of Plasma) method (TAC₃). Folin Ciocalteu assay were used for determining TP contents and the mean TP results were well correlated significantly and positively with the TAC₂ and TAC₃ results. Among the fruit juice, orange had the highest TAC₁, strawberry had the highest phenol, TAC₂, and TAC₃ values. Generally, strawberry and sour cherry samples had the highest TP, TAC₂, and TAC₃ values.

Keywords:

• Fruit juices
• Total antioxidant capacity
• Total phenols

INTRODUCTION

Recent studies have highlighted the importance of the antioxidant constituents of fruits. High consumption of fruits has proven to be associated with lower incidence and mortality rate of various degenerative diseases such as cancer, cardiovascular disease, and immune dysfunction by several human cohort and case-control studies.[1–3] The human body has a lot of defense systems to the harmful effects of free radicals and other reactive oxygen types. Normally, there is a balance between oxidant and antioxidant compounds in an organism. There are internal and external defense systems of antioxidants against the reactive oxygen radicals produced depending on internal and external factors. Any insufficiency in the antioxidant defense system changes the balance in favor of oxidants. High levels of antioxidants have an effective role of preventing atherosclerosis, cancer, early aging, and lipid peroxidation.[4]

The antioxidant vitamin content of fruits has attributed them the protective role before. However, recent interest in food phenolics has increased greatly, because of their antioxidant and free radical scavenging abilities. All of the most commonly sold fruit juices contain phenolic compounds showing a wide range of antioxidant activities in vitro.[5–7] Individual antioxidant compounds do not act alone.[8] They act in combination with other antioxidants, as interactions among them can affect total antioxidant capacity, producing synergistic or antagonistic effects.[9] Because plant foods contain many different classes and types of antioxidants, knowledge of their total antioxidant capacity, which is the cumulative capacity of food components to scavenge free radicals, would be useful for epidemiologic purposes.[10]

There are various methods developed recently for measuring TAC of food.[11–16] Previous studies revealed that antioxidant activities may differ by different measurement methods.[10,17,18] These assays differ in their chemistry (generation of different radicals and/or target molecules) and in the way end points are measured. Because different antioxidant compounds may act in vivo through different mechanisms, no single method can fully evaluate the TAC of foods. To accomplish this, total phenol (TP) and total antioxidant capacity (TAC) contents of fruit juice samples were measured and compared using different assays.

In this study, TP contents were measured with Folin Ciocalteu method and TAC of the fruit juice samples were analyzed using three different assays. These assays, based on different chemical mechanisms, were selected to take into account the wide variety and range of action of antioxidant compounds present in the beverages. In the literature available at present, there is a lack of information about the activity of antioxidants in commercial processed fruit juice. The purpose of this study was to investigate the TAC of apricot juice, peach nectar, orange juice, sour cherry juice, apple juice, strawberry juice and mixed fruit juice using the actual type of each beverage that is most commonly consumed and available on the Turkey market related with their total phenol contents, also allowing finding out which TAC method measures the phenolic antioxidants mostly and which measures antioxidants that are not phenolic.

MATERIALS AND METHODS

Materials

Fruit juice samples

Commercial samples of apricot juice, peach nectar, orange juice, sour cherry juice, apple juice, strawberry juice, and mixed fruit juice (n = 10 for each one) commonly consumed and produced by different firms in Turkey were purchased from local supermarkets in Sanliurfa, Turkey.

Chemicals

Gallic acid, Folin-ciocalteu reactive, O-diasidine, ABTS radical, Trolox, TPTZ were purchased from Sigma and Na₂CO₂, KCl, H₂O₂, sodium acetate, acetic acid were purchased from Merc Co. with maximum pureness. An Aeroset model automatic analyser (Abbott), UV/VIS spectrophotometer (Jasko V-530 model) and Universal 30 RF centrifuge (Hettich) were used.

Methods

Total phenol content determination

Total phenolic content was determined according to a modified method of Skerget et al.[19] that is based on a colorimetric oxidation/reduction reaction. For this purpose 200 μl sample was taken and mixed with 1000 μl of 10 fold diluted Folin–Ciocalteu reagent, 800 μl of sodium bicarbonate solution (7.5% w/w) was added, incubated at room temperature for 2 h; then absorbance was read at 750 nm in spectrophotometer. Water was used as blanked sample. 1 μM gallic acid was used in standard preparation and results were expressed as mM gallic acid equivalent/l.

Determination of total antioxidant capacity

Total antioxidant capacity of samples was determined using three methods and the results were given as TAC₁, TAC₂, and TAC₃. TAC₁ method: In this new method,[13] a standardized solution of Fe+2-o-dianisidine complex reacts with a standardized solution of hydrogen peroxide by a Fenton-type reaction, reducing OH*. These potent ROS oxidize the reduced colorless o-dianisidine molecules to yellow-brown colored dianisidyl radicals and further oxidation reactions develop. Since the antioxidants in the sample suppress the oxidation reactions and color formation, this reaction can be monitored by a spectrophotometer. The results are expressed as mM Trolox equivalent/l.

TAC₂ method: Total antioxidant capacity of the samples was measured by a new method developed by.[14] This method is based on the decolourization of ABTS [2-2 azinobis (3-methybenzothiazoline-6-sulfonate)] radical cation, which stays more stable for a long time in the acetate buffer solution. While it is diluted with a more concentrated acetate buffer solution at high pH values, the color is spontaneously and slowly bleached. Antioxidants present in the sample accelerate the bleaching rate to a degree proportional to their concentrations, which can be monitored spectrophotometrically, and the bleaching rate is inversely related with the TAC of the sample. The reaction rate is calibrated with Trolox, which is widely used as a traditional standard for TAC measurement assays, and the assay results are expressed in mM Trolox equivalent/l.

TAC₃ method: Total antioxidant capacity of the samples was measured by FRAP method developed by Benzie and Strain.[15] 20 μl sample is mixed with freshly prepared study reactive (10 volume 300mM acetate buffer+ 1 volume 10mM TPTZ solution), incubated at 37°C for 5 min and absorbance is read. The results are expressed as mM Trolox equivalent/l.

Statistical Analysis

Significant differences between the results were calculated by analysis of variance (ANOVA). Differences at p < 0.05 were considered to be significant. Where there were differences, a Duncan test was applied to indicate the samples between which there were differences. A multiple regression analysis was performed to study the influence of different factors on a given parameter and correlation analyses were done between variables. All the statistical analyses were performed using SPSS for Windows Release 10 (SPSS Inc.).

RESULTS AND DISCUSSION

For this study, TAC (Fig. 1A–C) and TP (Fig. 1D) values of fruit juice commonly consumed in Turkish diet and available from Turkish markets (apricot juice, peach nectar, orange juice, sour cherry juice, apple juice, strawberry juice and mixed fruit juice) were evaluated. TP and TAC results being important criteria from the nutritional point of view displayed in Table 1.

Figure 1 Total antioxidant capacity 1 (TAC₁), 2 (TAC₂), 3 (TAC₃) values (mM Trolox equivalent/l), Total phenol contents (mM gallic acid equivalent/l) of fruit juice samples (1: apricot; 2: peach; 3: orange; 4: sour cherry; 5: apple; 6: strawberry; and 7: mixed) (A,B,C,D, respectively).

Table 1 Total phenol (TP) and Total antioxidant capacity (TAC) values of the fruit juice samples (Mean ± SD)

Sample	Total phenol	TAC1	TAC2	TAC3
Apricot	3.0 ± 1.0(b)	26.5 ± 2.6(b)	3.0 ± 1.2(c)	2.2 ± 0.7(b)
Peach	3.4 ± 1.4(b)	21.2 ± 2.8(c)	3.4 ± 1.5(c)	2.4 ± 0.9(b)
Orange	3.1 ± 0.4(b)	33.3 ± 1.3(a)	3.8 ± 0.6(bc)	2.9 ± 0.5(ab)
Sour cherry	3.8 ± 0.5(ab)	20.9 ± 4.7(c)	5.4 ± 1.3(ab)	4.6 ± 1.0(a)
Apple	2.3 ± 0.5(b)	18.9 ± 6.6(c)	2.8 ± 0.5 (c)	2.0 ± 0.8(b)
Strawberry	5.0 ± 1.8(a)	28.3 ± 3.2(b)	5.9 ± 2.0(a)	4.1 ± 0.9(a)
Mixed	3.0 ± 0.5(b)	28.2 ± 2.3(b)	3.7 ± 1.0(bc)	2.8 ± 0.8(b)
Total	3.3 ± 1.0	25.4 ± 5.7	4.0 ± 1.5	2.9 ± 1.1
Unit of TP is mM gallic equivalent/l and unit of TAC is mM Trolox equivalent/l.

Orange juice samples exhibited the highest TAC₁ of 33.272 mM gallic acid equivalent/l. Orange has a high concentration of carotenoids, especially b-cryptoxanthin, which has a significant influence on TAC₁, and it is also rich in phenolic compounds (naringin, hesperetin), and consequently has a high antioxidant capacity. Zulueta et al.[20] showed that the contribution of vitamin C to total antioxidant capacity values was five times greater than were phenolic compounds which is similar to the result obtained by Gardner et al. [21] and Sa´nchez-Moreno et al.,[22] greatest antioxidant capacity in different orange juices was vitamin C. Wang et al.,[23] however, reported that the contribution of vitamin C to the TAC of a fruit was usually less than 15%, except for kiwi fruit and honeydew melon. This suggests that the major source of antioxidant capacity of most fruit and commercial fruit juices may not be from vitamin C.

Strawberry juice samples had the highest TP contents (5.007 mM gallic acid equivalent/l), and the highest TAC₂ (5.900 mM Trolox equivalent/l) values. Strawberry and sour cherry juice samples had higher TAC₃ values that were 4.093 mM Trolox equivalent/l and 4.093 mM Trolox equivalent/l respectively. Except for TAC₁ values, TP and TAC results were higher for strawberry and sour cherry samples. In a study,[24] it was reported that the antioxidant capacity of strawberries was the highest of the fruits he investigated. Previous studies showed that the antioxidant capacities of red fruits were relatively much higher than those of the other fruits[24,25] that can be explained that anthocyanins in red fruits are strong contributors to the total antioxidant capacity of fruits.[26] So, total phenolics and anthocyanins are suggested to be the major antioxidant compounds in sour cherry nectars while ascorbic acid is the major antioxidant in orange nectars.[21]

The samples had different antioxidant capacities in relation to the method applied; thus, the same item often ranked differently depending on the assay. The measurement of antioxidant activity of biological samples thus largely depends upon the free radical or the oxidants used in the assays and degree and type of antioxidants. Hence, it is pertinent to use different chemical and biochemical model assays in an efficient extraction medium, instead of relying on a single assay system to assess and compare the antioxidant activity in fruits and vegetable extracts. Synergistic effects and concentration may also change the results that are not observed when individual constituents are tested.[27] The novel TAC methods evaluated in the present study are reliable, fast, and easy-to-handle methods. They offer great advantages in monitoring TAC compared to analysis of all the individual antioxidant components in food samples.

The large variability of the antioxidant molecules within the fruit juice items and the lack of standardization of the TAC assays, therefore, some results of our investigation could be different from those of the other authors. For this discussion, the TP contents of the samples were compared with their TAC values. There existed a significant correlation between the TP of the samples and their mean TAC₂ and TAC₃ values having r = 0.832 and r = 0.731 values respectively. Similarly, Velioglu et al.[25] found a relationship between TP content and the antioxidant activity of plant materials using the β-carotene bleaching method. This was also found in other studies.[23,28,29] In spite of the variability of TAC results according to the method applied, fruit juice samples had very high values of TAC and phenol contents and our results of TAC₂, TAC₃, and TP values were comparable with the other research findings where fruits samples were investigated.[23,26,30]

CONCLUSIONS

The results of this study showed that, being more readily digestible than the other plant tissues, fruit juices are good sources of antioxidants. Significant differences in the TP and TAC values of the fruit juices and significant correlation of TP and some of the TAC methods were observed. Because the TAC of fruit juices is a function of the combined action of a number of compounds, evidently, the choice of the TAC method to be used must be done carefully taking into account the composition of the sample matrix and related factors. The concept of total antioxidant capacity of processed foods is gaining momentum and emerging as an important parameter to assess the quality of the product. With the expansion of the market and hence competition amongst the companies, the total antioxidant capacity parameter may soon take its place in nutritional labeling. So determining the total antioxidant capacity of food by a practical method and the development of consumer-relevant products with particular nutritive functionalities will become imperative and because the ability to act as a chemical antioxidant may not reflect its ability to act as an antioxidant in a biological situation, further studies about the TAC of commercial fruit juice samples in-vivo were needed.