Determination of total antioxidant activity of fruit juices widely consumed in Syria

 

Nour Al Asaad¹, Dima Al Diab²

¹MSc in Food Control, Department of Analytical and Food Chemistry, Faculty of Pharmacy,

Al-Andalus University for Medical Sciences, Tartus, Syria

²Ph.D. in Food Control, Assistant Professor at the Department of Analytical and Food Chemistry,

Faculty of Pharmacy, Tishreen University, Latakia, Syria.

 

ABSTRACT:

Fruits are considered essential constituents of a healthy diet. They possess antioxidant properties which can be attributed mainly to their phenolic compounds. Two samples of the following fruit juices (orange, grapefruit, blackberry, black mulberry, black sweet cherry, strawberry, pomegranate, organic tomato, greenhouse tomato, black plum, black grape and red grape) were investigated for their phenolic content and total antioxidant activity. Total phenolic content was determined according to Folin-Ciocalteu method. Black mulberry juice showed the highest results with amounts from 8.6g_GAE/L_juice to 11.2g_GAE/L_juice, while the lowest was red grape juice with amounts from 0.3 g_GAE/L_juice to 0.7g_GAE/L_juice. Total Antioxidant activity was subsequently determined using Ferric Reducing Antioxidant Power (FRAP) assay and reducing power assay. The results were in accordance with Folin-Ciocalteu method since black mulberry juice had the highest antioxidant power, while red grape juice had the lowest. The study indicates that black mulberry juice is the richest source of phenolic compounds and provides the highest antioxidant potential compared with other fruit juices in this study. Reducing power assay gave a similar order compared to FRAP assay. Both assays depend on reducing the ferric complex to its ferrous form which could explain the similarity in juice order considering their antioxidant activity.

 

 

 

INTRODUCTION:

The apparent health effects of the Mediterranean diet are probably due to the natural antioxidants which are available in high content in vegetables, fruits and olive oil. One of these natural Antioxidants are phenolic compounds. Phenolic compounds comprise a large group of plant secondary metabolites widely studied due to their beneficial effects on human health. These effects include protection against cancer, cardiovascular diseases and neurodegenerative diseases like Alzheimer’s disease and dementia ^1-5.

 

The beneficial effects of phenolic compounds are related to their rich structure in hydroxyl groups which enable phenolic compounds to act as antioxidants and scavenge free radicals. Thus, a diet rich in fruits and vegetables is highly encouraged because they are a major source of phenolic compounds, especially when taking into account that nutrition therapy has become an interest for consumers after the growing popularity of functional food  ^6,7,8.

 

Phenolic compounds are also potential substitutes of synthetic antioxidants ⁹ such as butylatedhydroxyanisole (BHA) and butylatedhydroxytoluene (BHT) in food products with emerging evidence of the potential toxic and carcinogenic effects of BHA and BHT ^1,4,10.

The Syrian Mediterranean diet is rich in fruits consumed directly or as fresh, non-processed juice. Juice has been
used for decades as an easy way to obtain water, energy and nutrients as well as natural antioxidants namely, phenolic compounds¹¹.For example, chokeberry juice showed the ability to reduce oxidative damage, while pomegranate juice can protect against cardiovascular diseases and cancer ¹². Another example is grape juice which due to its content of phenolic compounds can inhibit low density lipoprotein (LDL) oxidation and reduce native plasma protein oxidation ¹³.

 

The aim of this study was to determine the total antioxidant activity  and total phenolic content of frequently consumed fruit juices in Syria due to the lack of research which compare the total antioxidant activity in different types of fruit juices considering that the antioxidant activity is related mainly to the phenolic compounds.

 

MATERIALS AND METHODS:

Chemicals and apparatus:

Di Sodium Phosphate was purchased from Merck, Germany, Ferric Chloride anhydrous was purchased from Qualikems, India, Ferrous sulfate was purchased from BDH, England, Folin-Denis’ reagent and 2,4,6-Tris(2-pyridyl)-s-triazine were purchased from Fluka (Sigma-Aldrich), Gallic Acid was purchased from Biotech LTD, Potassium ferricyanide was purchased from May and Baker LTD, England, Sodium Acetate was purchased from BDH, England, Anhydrous Sodium Carbonate was purchased from BDH, England, Sodium Phosphate was purchased from Riedel-De Haen AG, Germany, Trichloroacetic acid was purchased from Riedel-De Haen AG, Germany, Analytical balance (RADWAG, AS 220/C/2), Micropipette was purchased from Labkit (Chemelex, S.A., Spain), Spectrophotometer (Jasco V-530 UV), Water bath (K and H Industries), Oven (Carbolite).

 

Sample preparation:

Two samples of each of the following fruits were purchased from local markets: orange, grapefruit, blackberry, black mulberry, black cherry, strawberry, pomegranate, red grape, organic tomato, greenhouse tomato, black plum and black grape. Juice samples were prepared by the following steps: each type of fruit was washed and then squeezed using home blender. The obtained  juice was filtered and kept in the freezer           (-20°C) until analysis⁷. All samples were diluted properly in distilled water prior to analysis.

 

Total phenolic content:

Total phenolic content was determined according to the Folin-Ciocalteu method¹⁴. Two ml of freshly prepared sodium carbonate solution 2% (w/v) were added to 0.1 ml of the properly diluted sample, mixed then allowed to stand for 5 min. Then 0.1 ml of 1:1 diluted Folin-Denis’ reagent was added to the mixture and left to stand for 30 min. Absorbance was then read using a spectrophotometer at 750 nm. A blank solution was prepared in the same way like samples using 0.1 ml of distilled water instead of the sample. A standard curve was obtained using gallic acid solutions in concentrations between (0.08-0.6 g/L). The total phenolic content was expressed as grams of gallic acid equivalents (GAE) per liter of juice. All experiments were carried out in duplicate.

 

Ferric reducing antioxidant power:

The Ferric Reducing Antioxidant Power (FRAP) was measured spectrophotometrically¹⁵. FRAP reagent was prepared by mixing 300 mM acetate buffer (pH 3.6), 10 mM 2,4,6-tripyridyl-s-triazine (TPTZ) solution in 40 mM HCl and 20 mM ferric chloride solution at a ratio of 10:1:1 (v/v/v) respectively. One ml of FRAP reagent was added to 100 µl of sample. The mixture was then incubated for 4 min at room temperature. Absorbance was read at 593 nm and the results were calculated according to a calibration curve obtained using FeSO₄ solutions in concentrations between (100-600 µM). The results were expressed as mM Fe².All experiments were carried out in duplicate.

 

Reducing power:

The reducing power of the diluted sample was determined by adding 0.5 ml of the diluted sample to 0.5 ml of phosphate buffer 0.2 M, pH 6.6 and 0.5 ml of 1% potassium ferricyanide solution. The mixture was incubated at 50 ºC for 20 min in a water bath then 0.5 ml of 10% (w/v) trichloroacetic acid was added to the mixture. Finally, 1 ml of the mixture was mixed with 1 ml of distilled water and 0.2 ml of 0.1% ferric chloride solution. Absorbance was read at 700 nm. Increased absorbance indicated increased reducing power¹⁶.  All experiments were carried out in duplicate.

 

Statistical analysis:

All results were presented as means ± standard deviations (SD). The differences between some results were determined by applying the Student’s t-test.

 

RESULTS AND DISCUSSION:

Total phenolic content:

The total phenolic content was expressed as g_GAE/L_juice as shown in table 1. Black mulberry juice had the highest phenolic content among all studied juices. Black mulberry juice is particularly rich in anthocyanidins¹⁷. The obtained results were higher than some results which were (2.05 g_GAE/Kg_juice)¹⁸.

 

 

Blackberry juice is rich in ellagic acid, gallic acid, anthocyanidins, cyanidins and quercetin¹⁹, which led to a high total phenolic content. These results were higher than multiple results indicating that total phenolic content of blackberry juice is (0.52 g_GAE/L_juice)²⁰, (1.83 g_GAE/L_juice)²¹ and (0.64 g_GAE/L_juice)²². The difference in results among studies could be due to climate change and the stage of ripening when fruits were harvested²³.

Cherry contains multiple phenolic compounds like cyanidin, catechin and quercetin. These compounds play a role in promoting apoptosis and cell differentiation. They also inhibit cyclooxegenase II, reducing the inflammatory response²⁴. Phenolic content of black cherry juice was higher than some results (1.57 g_GAE/L_juice)²¹. Environmental and genetic factors affect the concentration of phenolic compounds which may explain the difference in results²⁴.

 

Strawberry is a rich source of ellagic acid²², p-coumaric acid, flavonoids and anthocyanidins²⁵. Other studies revealed that content of phenolic compounds in strawberry juice is (0.54 g_GAE/L_juice)²², and (1.27 g_GAE/L_juice)²¹. Panico et al. (2009) indicated the role of temperature and light exposure on the accumulation of anthocyanidins in strawberry fruits which explain the difference in results.

 

Pomegranate juice is rich in anthocyanidins and ellagitannins²⁶, as well as gallic acid, chlorogenic acid²⁷, cyaniding and pelargonidin²⁸. Several studies obtained similar results such as (2.05 – 2.91 g_GAE/L_juice)²⁹, (2.46 – 2.55 g_GAE/L_juice) ²⁷and (2.12 g_GAE/L_juice)²⁸. Some other studies obtained notably lower results with a total phenolic content of (0.12 g_GAE/L_juice)³⁰ and (0.73 g_GAE/L_juice)³¹.

 

A study by Moreno-Montoro et al. (2015) showed that total phenolic content was (0.54 – 0.65 g_GAE/L_juice) in grapefruit juice and (0.54 – 0.76 g_GAE/L_juice) in orange juice which were relatively lower than the results found in this study.

 

Grape juice is a rich source of flavonoids¹³, alongside with quercertin and resveratrol which have anti-tumor properties³². Grape juice may reduce the risk of cancer, ulcer, inflammation and atherosclerosis³³. Black grape juice had a higher phenolic content than red grape juice. Black grape juice results were in accordance with several studies. One study showed that homemade black grape juice phenolic content was between (1.28 – 2.26 g_GAE/L_juice)³¹. Another study reported phenolic content of black grape juice was (0.18g_GAE/L_juice)³⁰ and (0.22 g_GAE/L_juice)³². Red grape juice showed the lowest phenolic content among all studied juices. Moreno-Montoro et al. (2015) reported that total phenolic content in red grape juice ranges between (0.28 – 1.73 g_GAE/L_juice), while the results obtained by Davalos et al. (2005) showed a range between (0.7 – 1.2 g_GAE/L_juice), another study showed that total phenolic content in red grape juice could reach concentrations as high as (1.6 g_GAE/L_juice)³⁴. It is important to note that phenolic compounds in grape are mainly found in seeds followed by peel then leaves. Grape juice has the lowest content compared to other parts of the plant^31,35.

 

Plum is a rich source of phenolic compounds which show different distribution in whole fruit compared with pulp and skin as reported by Gil et al.(2002). Their study showed that the skin contained the highest phenolic content (3.18g/kg). Another study arrived at higher results than the ones obtained in this study with phenolic content ranging between (2.8 – 9.2 g/kg)³⁶. These high content could be attributed to the fact that their study used the whole fruit while this study used only juice.

 

Phytochemicals in tomato are carotenoids and phenolic compounds e.g. flavanones and flavonol³⁷ and hydroxycinnamic acids³⁸. Light exposure increases phenolic compounds synthesis by stimulating the enzymes. That would make phenolic content higher in organic tomato compared to greenhouse tomato³⁹. However, student’s t-test showed no significant difference between the two. This could be explained by the warm sunny winter in which this study was carried out. The climate in the year of this study provided growth conditions for greenhouse tomato similar to organic tomato which led to similar phenolic content.

 

Table 1: Total phenolic content of different types of fruit juices

Type of Juice	Sample No.	Total Phenolic Content gGAE/Ljuice ± SD
Black mulberry	1	8.6 ± 0.9
	2	11.2 ± 0.7
Blackberry	1	3.1 ± 0.09
	2	3.9 ± 0.2
Black Cherry	1	3.7 ± 0.4
	2	3.8 ± 0.3
Strawberry	1	4.3 ± 0.5
	2	3.5 ± 0.1
pomegranate	1	4.0 ± 0.02
	2	2.6 ± 0.07
Grapefruit	1	1.1 ± 0.08
	2	1.6 ± 0.06
Orange	1	1.4 ± 0.2
	2	0.9 ± 0.06
Red Grape	1	0.7 ± 0.1
	2	0.3 ± 0.02
Organic Tomato	1	0.8 ± 0.08
	2	0.6 ± 0.03
Greenhouse Tomato	1	0.7 ± 0.04
	2	0.6 ± 0.02
Black Plum	1	2.2 ± 0.1
	2	1.9 ± 0.1
Black Grape	1	1.3 ± 0.2
	2	0.8 ± 0.04

 

Difference in results among the two samples of the same juice could be attributed to the different conditions these fruits underwent. The fruits were obtained from different shops, which means they could have been grown in different geographic areas with different climate and soil, the time in which fruits were harvested and the storing conditions are also unknown⁴⁰,chemical composition of the fruit also depends on factors such as genetics and fruit maturity⁴¹.

 

Ferric reducing antioxidant power:

Table 2 shows total antioxidant activity of different types of fruit juices according to FRAP method. Results were expressed as (mM Fe⁺²).

 

Black mulberry juice possessed the highest antioxidant activity among all juices studied. This high antioxidant activity could be attributed to its high content in  cyanidin and pelargonidin compounds^18,42.

 

The antioxidant activity of blackberry was higher than the results obtained by Ivanovic et al. (2014), which were (0.19 mM Fe⁺²/L) of blackberry extract. The low results could be due to the poor yield of extraction method used in their study.

 

Black cherry juice contains cyanidins and hydroxycinnamate compounds which have higher antioxidant activity than those isolated from blackberry or strawberry²⁴. Nevertheless, strawberry juice possessed a higher antioxidant activity than black cherry. Anthocyanidins present in strawberry are considered high in antioxidant activity due to the oxonium ion on the C ring²⁵.

 

As can be noted in table 2, the antioxidant activity of juice was in the following order (strawberry > black plum>> orange > grapefruit). This order was in accordance with the study conducted by Gorinstein et al. (2006) on the above mentioned fruits.

 

It’s important to note that carotenoids in black plum are mainly found in the skin, which diminish their role in the antioxidant activity of black plum juice²⁸. Besides,  phenolic compounds were correlated with the antioxidant activity in black plum fruit. However, ascorbic acid and carotenoids were not⁴³.

 

Pomegranate juice can protect LDL from oxidation, it also reduces the risk of Alzheimer’ disease and colon cancer²⁷. The antioxidant activity of pomegranate juice and its content of punicalagin –a phenolic compound which is characteristic of pomegranate fruit – are responsible for its previously mentioned health benefits¹². Moreno-Montoro et al. (2015) mentioned that antioxidant activity of pomegranate juice ranges between (8.6 – 10 mM Fe⁺²/L_juice).

 

Grapefruit juice showed antioxidant activity relatively lower than orange juice. Although the phenolic content of both juices were close, and relatively higher in grapefruit juice, the antioxidant activity was in favor of orange juice. In fact, grapefruit is rich in naringin, while orange is rich in hesperidin. Even though these two compounds have similar structures, the antioxidant activity of hesperidin is much higher than the antioxidant activity of naringin⁴⁰. A study arrived at similar results, stating that the antioxidant activity of orange reached values as high as (20.5 mM Fe⁺²/Kg_juice), while it only reached (8.3 mM Fe⁺²/Kg_juice) in grapefruit⁴⁰.

 

Table 2: Total antioxidant activity of different types of fruit juices according to FRAP method

Type of Juice	Sample No.	Total antioxidant activity M Fe+2/Ljuice ± SD
Black mulberry	1	65.7 ± 0.3
	2	108.2 ± 0.1
Blackberry	1	34.1 ± 0.2
	2	36.4 ± 0.3
Black Cherry	1	22.4 ± 0.2
	2	20.1 ± 0.3
Strawberry	1	26.1 ± 0.1
	2	21.7 ± 0.1
pomegranate	1	35.8 ± 0.3
	2	24.6 ± 0.4
Grapefruit	1	9.5 ± 0.07
	2	13.1 ± 0.1
Orange	1	20.3 ± 0.1
	2	13.9 ± 0.08
Red Grape	1	1.8 ± 0.06
	2	1.4 ± 0.07
Organic Tomato	1	5.4 ± 0.2
	2	4.1 ± 0.1
Greenhouse Tomato	1	4.8 ± 0.1
	2	3.97 ± 0.07
Black Plum	1	13.3 ± 0.1
	2	11.02 ± 0.1
Black Grape	1	3.8 ± 0.04
	2	3.5 ± 0.05

 

 

 

Grape juice can reduce oxidative stress, it can also inhibits DNA synthesis in breast cancer cells^35,44. Black grape juice showed higher antioxidant activity than red grape juice. The main antioxidant compounds responsible for this higher activity of black grape juice are proanthocyanidins, anthocyanins, flavonoids and catechins²⁶. In contrast, red grape juice showed the lowest antioxidant activity among all studied juices. A study reported that the antioxidant activity of grape juice was (32 mM Fe⁺²/L_juice) which is significantly higher than our results³⁵. The type of the grape was not specified in that study which could explain the difference.

 

The antioxidant activity of tomato juice can be attributed to its content of ascorbic acid, lycopene and phenolic compounds³⁸. A recent study found a strong correlation between the phenolic content and the antioxidant activity. They also arrived at results stating that the phenolic content in tomato can reach levels 10 times greater than its lycopene content⁴⁵. Besides, Jacob et al. (2010) found no correlation between lycopene content and the antioxidant activity.

 

Finally, it is important to mention that many studies considered that the antioxidants activity of fruits is related mainly to the phenolic compounds^16,46,47 while ascorbic acid plays only a minor role in this activity^17,48. Therefore, our study focused on determining total antioxidant activity and phenolic content in the studied fruit juices without measuring ascorbic acid content.

 

Reducing power:

The total antioxidant activity of the studied juices according to the absorbance measured at 700 nm was in the following order: mulberry > blackberry > pomegranate > cherry > strawberry > orange > plum > grapefruit > black grape > organic tomato > greenhouse tomato > red grape. This order was similar to FRAP method with slight difference. Methods of determining antioxidant activity usually differ in results and their order. This could be attributed to the difference in the method’s principle. Some methods are based on measuring oxygen intake or binding free radicals and so forth⁴⁹. Since FRAP assay and reducing power assay are both based on reducing the ferric complex to its ferrous form, the results were close to each other in order.

 

CONCLUSIONS:

In the present study mulberry juice possessed the highest phenolic content and the highest antioxidant activity among other juices under investigation, which indicates that this high antioxidant activity could be attributed to its high phenolic content. FRAP assay and reducing power assay showed similar order in antioxidant activity of the studied juices.

 

Abbreviations:

2,4,6-tripyridyl-s-triazine (TPTZ)

butylatedhydroxyanisole (BHA)

butylatedhydroxytoluene (BHT)

ferric reducing antioxidant power (FRAP)

gallic acid equivalents (GAE)

low density lipoprotein (LDL)

standard deviations (SD)

 

ACKNOWLEDGMENT:

Authors would like to thank Al-Andalus University for Medical Sciences and Tishreen University for the financial support.

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Accepted on 20.09.2016           © RJPT All right reserved