Comparison of Antioxidant Activity of Grape Seed Extract and Fruits Containing High β-Carotene, Vitamin C, and E

Abstract

Antioxidant activity of a potent antioxidant, grape seed extract, and fruits containing high β-carotene, vitamin C, and vitamin E was measured and compared by 2,2-diphengl-1-picrylhydrazyl radical and ferric reducing antioxidant power assay. The results showed that antioxidant activity of a 20-mg capsule of grape seed extract was approximately 10 to 20 times greater than 1 g of tomato, papaya, banana, and mango. However, if antioxidant activity was calculated per fruit or piece of fruit, the values were comparable. Therefore, consumption of fruit could be a cost-effective way of healthy eating when the price of a dietary supplement is relatively high.

Keywords:

• Grape seed extract
• Fruits
• DPPH
• FRAP
• β-Carotene
• Vitamin C
• Vitamin E

INTRODUCTION

Anti-aging and healthy eating are one of the emerging health issues in this century. Antioxidant vitamins and trace elements have been known to play an important role in the prevention of cancer, cardiovascular diseases, and age-related ocular diseases.[1] Therefore, there are numerous dietary supplements available in the market. Grape seed extract is one of the most popular products. The significant correlations between the antioxidant activities of grape seed extracts and polyphenols have been shown.[2] Proanthocyanidins, an active ingredient in grape seed extract, have been reported to exhibit a broad spectrum of pharmacological and protective effects, especially antioxidative properties.[3,4] Grape seed proanthocyanidins extract is a significantly more potent scavenger of oxygen free radicals as compared to vitamin C and vitamin E succinate.[5] Similar to other supplements, there is no standard recommended daily dose of grape seed extract. Kalin et al.[6] showed that grape seed extract (100 mg/day for a month) could reduce the inflammatory response and the oxidative stress developed in systemic sclerosis patients.

The epidemiological studies showed a positive correlation between the consumption of fruits and reduced risk of chronic diseases, such as coronary heart disease.[7] There are varieties of fruits in tropical countries, and various fruits are available all year round. The fruits are commonly consumed because of their good taste and affordable price. When total antioxidant activity of fruit is measured, the effect may be greater than the sum of the activity of the individual antioxidant compound in the fruit. The identification of one compound cannot exactly reflect the overall action.[8] Thus, high β-carotene, vitamin C, and vitamin E fruits may exhibit a synergistic effect. As a result, the antioxidant activity of these fruits may be comparable to a potent antioxidant, grape seed extract. In this study, scavenging of 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) and ferric reducing antioxidant power (FRAP) assay were used to determine and compare the antioxidant activities of grape seed extract, and fruits containing high β-carotene, vitamin C, and vitamin E.

MATERIALS AND METHODS

Sample Preparation

The grape seed extract product (Vitis vinifera) was purchased from the local pharmacy store. Each capsule contains 20 mg of grape seed extract. The content was dissolved in distilled water and the volume adjusted to 25 mL (800 μg/mL). Fruits included in this study were purchased from local markets. There were tomato (Solanum lycopersicum var. cerasiforme), ripe banana (Musa AA group), ripe papaya (Carica papaya), and ripe mango (Mangifera indica). These fruits were reported to contain a high level of β-carotene, vitamin C, and vitamin E according to the study of the Bureau of Nutrition, Department of Health, Ministry of Public Health, Thailand.[9] Table 1 shows β-carotene, vitamin C, and vitamin E content of the fruits. All fruits were washed under tap water before peeling. A portion of 2 g was homogenized with 10 mL of distilled water using a homogenizer (Ultra-Turrax T8, Ika-Werke, Germany). The homogenate was centrifuged for 5 min at 3000 rpm (Kokusan H-103N, Tokyo, Japan). The supernatant was recovered and used directly for DPPH and FRAP assay without storage.

Table 1 β-carotene, vitamin C, and vitamin E content in tomato (Solanum lycopersicum var. cerasiform e), ripe banana (Musa AA group), ripe papaya (Carica papay a), and ripe mango (Mangifera indica).*

Fruits	β-carotene content (mg/100 g)	Vitamin E content (mg/100 g)	Vitamin C content (mg/100 g)
Tomato	0.64	1.34	41
Ripe banana	0.88	1.1	15
Ripe papaya	0.48	0.19	56
Ripe mango	0.27	0.47	10
*Derived from Antioxidants in Fruits, 2006, reported by Bureau of Nutrition, Department of Health, Ministry of Public Health, Thailand.[9]

DPPH Assay

The DPPH assay was performed according to the method of Brand-Williams et al.[10] with some modifications. An amount of 0.24 g/mL of DPPH in methanol was used as a stock solution. The working solution was prepared by diluting 2.5 mL of stock solution with 25 mL of methanol. An aliquot of 20 μL of grape seed extract solution or fruit sample supernatant were mixed with 180 μL of DPPH solution in a 96-well plate. After 1 h of incubation, the absorbance was measured at 490 nm by a microplate reader (model 680 Bio-Rad, Hercules, CA, USA). Results were expressed in μmol Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) equivalents (TE)/capsule of grape seed extract and μmol TE/g fresh mass of fruit sample. Additional dilution was required if the measured value was over the linearity range of the standard curve (50–600 μM Trolox). The comparison was based on the amount of fruit people eat in daily life. For example, fresh fruit is consumed more than ten gram weight rather than in milligram weight. For the grape seed extract, results were expressed per capsule.

FRAP Assay

The FRAP assay was performed according to the method of Benzie and Strain[11] with some modifications. The stock solutions used in this study were 300 mM acetate buffer pH 3.6, 10 mM 2,4,6-tripyridyl-s-triazine (TPTZ) solution in 40 mM HCl and 20 mM FeCl₃.6H₂O solution. FRAP reagent (25 mL acetate buffer, 2.5 mL TPTZ, and 2.5 mL FeCl₃.6H₂O) was freshly prepared before use. An aliquot of 20 μL of grape seed extract solution or fruit sample supernatant was mixed with 180 μL of FRAP reagent in a 96-well plate. After 6 min of incubation, the absorbance was measured at 595 nm by a microplate reader (model 680 Bio-Rad). Results were expressed in μmol TE/capsule of grape seed extract and μmol TE/g fresh mass of fruit sample. Additional dilution was required if the measured value was over the linearity range of the standard curve (25–800 μM Trolox).

Statistical Analysis

Each antioxidant assay was done six times. The values were shown as mean ± standard deviation (SD). The difference in antioxidant activities among samples were analyzed by analysis of variance. Correlations among data were calculated as coefficient of determination (R ²).

RESULTS AND DISCUSSION

Comparison of TE of Fruits in DPPH and FRAP Assay

A large diversity of in vitro methods for determination of antioxidant capacity is available as reviewed by Magalhaes et al.[12] DPPH and FRAP assay have similar basic principles to determine antioxidant activity (the ability of antioxidant to reduce DPPH^• radical in the case of DPPH assay and reduce [Fe(III)-(TPTZ)₂]³⁺ in the case of FRAP assay). Therefore, a high correlation of determined values can be expected. In this study, DPPH and FRAP assay showed a strong linear correlation (R ² = 0.9971) as can be seen in Fig. 1. The two models are comparable to determine free radical-scavenging activity. Either DPPH or FRAP can be employed as a model for antioxidant activity determination. Both can be applied to a 96-well plate for high throughput and minimizing the use of reagents.

Figure 1 Comparison of TE of fruits in DPPH and FRAP assay. (Color figure available online.)

Antioxidant Activity of Grape Seed Extract and Fruits

Antioxidant activity of grape seed extract and fruits containing high β-carotene, vitamin C, and vitamin E was carried out by DPPH and FRAP assay. To standardize and compare the antioxidant activities, the values were expressed in μmol TE/capsule (20 mg) of grape seed extract or μmol TE/g fresh mass of fruit sample. The results are summarized in Table 2. On the basis of wet weight of fruits (peeled), tomato shows the highest antioxidant activity, followed by papaya, banana, and mango. There was no significant difference between the antioxidant activities of banana and mango (P > 0.05). Antioxidant activity of one capsule of grape seed extract was shown to be 10 to 20 times greater than 1 g of the fruit. However, if antioxidant activity was calculated per fruit or piece of fruit (Table 3), the values were comparable between grape seed extract and the fruits (tomato, papaya, and banana). The antioxidant activity of mango per fruit was considerably higher than 1 capsule of grape seed extract.

Table 2 Antioxidant activity of grape seed extract, tomato, ripe banana, ripe papaya, and ripe mango as determined by DPPH and FRAP assay

	Antioxidant activity (μmol TE/1 capsule of grape seed extract, μmol TE/g fresh mass of fruit)
Sample	DPPH	FRAP
Grape seed extract	79.73 ± 19.90	92.62 ± 14.39
Tomato	9.02 ± 0.99	9.68 ± 0.56
Ripe papaya	6.39 ± 0.30	6.63 ± 0.34
Ripe banana	4.22 ± 0.68	4.18 ± 0.47
Ripe mango	4.26 ± 0.36	3.89 ± 0.37
Data were expressed as mean ± SD.

Table 3 Antioxidant activity of grape seed extract (per capsule), tomato (per fruit), ripe banana (per fruit), ripe papaya (per piece), and ripe mango (per fruit)

Grape seed extract/fruit	Weight (g/fruit, g/piece)	DPPH (μmol TE)	FRAP (μmol TE)
Grape seed extract	—	79.73	92.62
Tomato	5	45.10	48.40
Ripe papaya	12 (per piece; 3 × 2.5 × 2 cm)	76.68	79.56
Ripe banana	40	168.80	167.2
Ripe mango	320	1363.20	1244.80

Since an organic solvent was not used in this study, vitamin E in fruits could not be extracted. As a result, vitamin E hardly contributed to antioxidant activity. The fruits in this study contained a high level of vitamin E as compared to other fruits,[9] though a good source of vitamin E is not fruits. Vegetable oils, nuts and nut oil seeds, egg yolk, margarine, cheese, soya beans, wheat germ, oatmeal, avocados, olives, and green leafy vegetables are a rich source of vitamin E.[13] Therefore, a water soluble antioxidant as vitamin C was supposed to contribute to antioxidant activity of fruits rather than vitamin E.

The quantity of one antioxidant compound in fruit could not indicate its total antioxidative property because there are various antioxidants contained in it. For instance, the contribution of vitamin C to antioxidant activity of mango, papaya, and banana varied widely from 4.4 to 62.3%.[8] Percent contribution of each antioxidant compound to the antioxidant activity was not measured in this study. The data of β-carotene, vitamin C, and vitamin E content in fruits was retrieved from the Bureau of Nutrition, Department of Health, Ministry of Public Health, Thailand.[9]

Due to the report of the World Health Organization and Food and Agriculture Organization (WHO/FAO),[14] daily intake of fresh fruits and vegetables in an adequate quantity (400–500 g per day) is recommended to reduce the risk of chronic disease, such as coronary heart disease, stroke, and hypertention. Recommended daily amounts of fruit from the United State Department of Agriculture (USDA)[15] is 2.5 cups. The recommended amount of fruits definitely has antioxidant activities higher than 1 capsule of grape seed extract. The consumption of a dietary supplement could be costly for low- and moderate-income people because these products are relatively expensive. Therefore, fruit consumption could be a cost-effective way of healthy eating. Fruits contain not only vitamins but also fibers and other micronutrients. However, one should be concerned about sugar content in some fruits.

The antioxidant activity of fruits could be affected by factors, such as geographical origin, cultivar, and harvest and storage time.[16] Our results also support this explanation. For example, mango and tomato from Equado showed 3.1 ± 0.6 and 0.8 ± 0.3 μmol Trolox/g fresh mass by DPPH assay, respectively,[17] whereas our study showed 9.02 ± 0.99 and 4.26 ± 0.36 μmol Trolox/g fresh mass of mango and tomato by the same assay model, respectively. Antioxidant activity of common fruits in Singapore has been reported recently.[18] The data showed that antioxidant activity of banana was 9.77 μmol Trolox/g fresh mass, mango was 8.48 μmol Trolox/g fresh mass, and papaya was 2.70 μmol Trolox/g fresh mass by hydrophilic-oxygen radical absorbance capacity assay. Whereas the authors’ study showed antioxidant activity of banana at 4.18 μmol Trolox/g fresh mass, mango at 3.86 μmol Trolox/g fresh mass, and papaya at 6.63 μmol Trolox/g fresh mass by FRAP assay. Antioxidant activity of fruits in India was also studied.[19] The data showed that antioxidant activity of banana, mango, and papaya are 41 ± 3.2, 211 ± 11.8, and 46 ± 5.1 mg Trolox/100 g fresh mass by DPPH assay, respectively. The unit μmol Trolox/g fresh mass by DPPH assay in our study had to be converted to mg Trolox/100 g fresh mass for comparison with the study of Vijaya Kumar Reddy et al.[19] The results showed that antioxidant activity of banana, mango, and papaya are 106, 107, and 160 mg Trolox/100 g fresh mass, respectively. The difference could be due to the reasons mentioned above and the assay models.

CONCLUSIONS

Antioxidant activity of grape seed extract and fruits containing high β-carotene, vitamin C and E was measured and compared by DPPH and FRAP assay. The results demonstrated that an adequate amount of fruits showed comparable antioxidant activity with a potent antioxidant, grape seed extract. As a variety of compounds contributes to antioxidant activity in fruits, it is difficult to pinpoint which substance is the most important.