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Nutrient Requirements

Total Antioxidant Capacity of Plant Foods, Beverages and Oils Consumed in Italy Assessed by Three Different In Vitro Assays¹

Department of Public Health, University of Parma, Parma, Italy and ^* Antioxidant Research Laboratory at the Unit of Human Nutrition, National Institute for Food and Nutrition Research, Rome, Italy

²To whom correspondence should be addressed. E-mail: mailnico{at}nemo.unipr.it.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION LITERATURE CITED

 
Epidemiologic studies have demonstrated an inverse association between consumption of fruits and vegetables and morbidity and mortality from degenerative diseases. The antioxidant content of fruits and vegetables may contribute to the protection they offer from disease. Because plant foods contain many different classes and types of antioxidants, knowledge of their total antioxidant capacity (TAC), which is the cumulative capacity of food components to scavenge free radicals, would be useful for epidemiologic purposes. To accomplish this, a variety of foods commonly consumed in Italy, including 34 vegetables, 30 fruits, 34 beverages and 6 vegetable oils, were analyzed using three different assays, i.e., Trolox equivalent antioxidant capacity (TEAC), total radical-trapping antioxidant parameter (TRAP) and ferric reducing-antioxidant power (FRAP). 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 actual foods. Among vegetables, spinach had the highest antioxidant capacity in the TEAC and FRAP assays followed by peppers, whereas asparagus had the greatest antioxidant capacity in the TRAP assay. Among fruits, the highest antioxidant activities were found in berries (i.e., blackberry, redcurrant and raspberry) regardless of the assay used. Among beverages, coffee had the greatest TAC, regardless of the method of preparation or analysis, followed by citrus juices, which exhibited the highest value among soft beverages. Finally, of the oils, soybean oil had the highest antioxidant capacity, followed by extra virgin olive oil, whereas peanut oil was less effective. Such data, coupled with an appropriate questionnaire to estimate antioxidant intake, will allow the investigation of the relation between dietary antioxidants and oxidative stress-induced diseases.

KEY WORDS: • antioxidant capacity • fruits • vegetables • beverages • oils

The consumption of fruits and vegetables has been inversely associated with morbidity and mortality from degenerative diseases (1–5). It is not known which dietary constituents are responsible for this association, but antioxidants appear to play a major role in the protective effect of plant foods (6–8). Epidemiologic studies that analyze the health implications of dietary components rely on the intake estimates in sample populations found in databases that list the component’s content in commonly consumed foods. Therefore, the availability of appropriate and complete food composition data is crucial. Due to the chemical diversity of antioxidant compounds present in foods, complete databases on food antioxidant content are not yet available. In addition, levels of single antioxidants in food do not necessarily reflect their total antioxidant capacity (TAC)²; this also depends on the synergic and redox interactions among the different molecules present in the food. Finally, geographical differences in food composition data should be considered when applying compositional databases to regional surveys.

Several methods were developed recently for measuring the total antioxidant capacity of food and beverages (9–11); these assays differ in their chemistry (generation of different radicals and/or target molecules) and in the way end points are measured (12). Because different antioxidant compounds may act in vivo through different mechanisms, no single method can fully evaluate the TAC of foods. The objective of this study was to assess the TAC of plant food, beverages and oils commonly consumed in Italy using different methods to obtain robust data useful for determining the potential intake of antioxidants in Italian population studies. To this aim, three methods, i.e., Trolox equivalent antioxidant capacity (TEAC) (12), total radical-trapping antioxidant parameter (TRAP) (13) and ferric reducing-antioxidant power (FRAP) (11), were selected. The TEAC assay measures the ability of antioxidants to quench a radical cation (ABTS·⁺) in both lipophilic and hydrophilic environments (12). The TRAP and FRAP assays evaluate the chain-breaking antioxidant potential (13) and the reducing power of the sample (11), respectively.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION LITERATURE CITED

 
Chemicals

The 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), and 2,4,6-tripyridyl-s-triazine (TPTZ) were purchased from Sigma-Aldrich (St. Louis, MO). R-Phycoerythrin (R-PE) was purchased from Prozyme (San Leandro, CA); 2,2'-azobis(2-amidinopropane) dihydrochloride (ABAP) was purchased from Waco Chemicals (Richmond, VA). All chemicals and solvents used were HPLC-grade and purchased from Carlo Erba (Milan, Italy). High-purity water was produced in the laboratory by using an Alpha-Q system (Millipore, Marlborough, MA).

Samples

The selection of the samples was based on food consumption data of the EPIC cohort of Varese province (Italy) kindly provided by the Epidemiology Unit of the National Cancer Institute based in Milan and developed on the basis of 24-h recalls recorded in the North Italy area (Dr V. Krogh, Department of Epidemiology, National Cancer Institute, Milan, Italy, personal communication). Once the main putative contributors of antioxidants in the Italian diet were identified, three food samples for each food item were purchased, selecting the three cultivars and/or brands with the highest sales in the Italian market. Samples were then prepared, mixed in equal proportions and analyzed for TAC, as reported below. Like other nutrients, the estimation of the overall dietary intake of TAC does not require an estimate of the variance for any single food item if the value of the given food as consumed by the responder is sufficiently close to the average value. The same approach was used previously to generate food TAC data (14).

    Beverages. The following beverages were purchased in local supermarkets: soft drinks (cola and lemon iced tea), fruit juices (orange, lemon, apple, pineapple, pear, grapefruit, peach, apricot, mixed fruits and tropical fruits), alcoholic drinks (beer, rum, whiskey, grappa, cognac, and red, rosé and white wines of different geographical origins) and vinegar from red wine. Teas and chamomile were prepared by brewing an infusion (2 g) in 250 mL boiling water for 5 min. Soluble coffee was prepared by solubilizing a coffee serving (2 g) in 40 mL of boiling water. Extracted coffee was prepared using an Italian Moka coffee machine. Espresso coffees were purchased in local coffee shops.

Before analysis, beverages were adequately diluted in high purity water, depending on their presumed activity. Carbon dioxide from cola and beer was removed completely by magnetic stirring under nitrogen. Diluted beverages were centrifuged for 5 min at 1000 x g, and the supernatant was collected and analyzed without further preparation.

    Oils. Oils (olive, extra virgin olive, sunflower, corn, soybean and peanut oils) were purchased in local supermarkets. Before analysis, oils were diluted in n-hexane, depending on their presumed activity.

    Fruits and vegetables. Fruits (n = 23; red Delicious and yellow Golden apples, apricot, banana, clementine, yellow grapefruit, orange, yellow peach, pear, honeydew and cantaloupe melons, pineapple, red plum, tangerine, water melon, kiwi fruit, black and white grapes, loquat, fig, prickly pear, black and green olives), berries (n = 7; cultivated and wild strawberries, blackberry, blueberry, raspberry, cherry, and redcurrant) and vegetables (n = 34; artichoke, arugula, asparagus, avocado, beet, red cooked beetroot, broccoli, green and Savoy cabbages, carrot, cauliflower, celery, chicory, cucumber, eggplant, endive, fennel, green bean, leek, green lettuce, mushroom, yellow onion, chili and red bell peppers, potato, pumpkin, radicchio, Swiss chard stalk, red radish, spinach, salad and sauce tomatoes, turnip tops and zucchini) were collected at three different local supermarkets. After washing and cutting, equal amounts of each food were pooled, mixed and homogenized under nitrogen in a high speed blender. A precisely weighed amount of the homogenized sample (1 g) was extracted with 4 mL of water under agitation for 15 min at room temperature, centrifuged at 1000 x g for 10 min and the supernatant collected. The extraction was repeated with 2 mL of water and the two supernatants were combined. The pulp residue was reextracted by the addition of 4 mL of acetone under agitation for 15 min at room temperature, centrifuged at 1000 x g for 10 min and the supernatant collected. The extraction was repeated with 2 mL of acetone and the two supernatants were combined. In the case of lipid-rich foods (e.g., avocados and olives), the pulp residue was reextracted twice by the addition of 2 mL of chloroform under agitation for 15 min at room temperature, centrifuged at 1000 x g for 10 min and the supernatant collected. All food extracts were adequately diluted in the appropriate solvent (depending on their activity) and immediately analyzed in duplicate for their antioxidant capacity. The variation in the TEAC, TRAP and FRAP values for replicates was always between 3 and 10% relative standard deviation (RSD). When the RSD was higher than 10%, the analyses were repeated to confirm the value.

    TEAC (Trolox equivalent antioxidant capacity) assay. The method is based on the ability of antioxidant molecules to quench the long-lived ABTS·⁺, a blue-green chromophore with characteristic absorption at 734 nm, compared with that of Trolox, a water-soluble vitamin E analog. The addition of antioxidants to the preformed radical cation reduces it to ABTS, determining a decolorization. A stable stock solution of ABTS·⁺ was produced by reacting a 7 mmol/L aqueous solution of ABTS with 2.45 mmol/L potassium persulfate (final concentration) and allowing the mixture to stand in the dark at room temperature for 12–16 h before use (12). At the beginning of the analysis day, an ABTS·⁺ working solution was obtained by the dilution in ethanol of the stock solution to an absorbance of 0.70 ± 0.02 AU at 734 nm, verified by a Hewlett-Packard 8453 Diode Array spectrophotometer (HP, Waldbronn, Germany), and used as mobile phase in a flow-injection system, according to Pellegrini et al. (15). Results were expressed as TEAC in mmol of Trolox per kg (solid foods and oils) or per L (beverages) of sample. All solid food extracts, obtained by water, acetone and chloroform, were analyzed by this assay.

    TRAP (total radical-trapping antioxidant parameter) assay. The TRAP was determined according to the method of Ghiselli et al. (13) based on the protection provided by antioxidants on the fluorescence decay of R-phycoerythrin (lag-phase) during a controlled peroxidation reaction. Briefly, 120 µL of diluted sample were added to 2.4 mL of phosphate buffer (pH 7.4), 375 µL of bidistilled water, 30 µL of diluted R-PE and 75 µL of ABAP; the reaction kinetics at 38°C were recorded for 45 min (or more, if necessary) by a LS-55 luminescence spectrometer (Perkin Elmer, Wellesley, MA). TRAP values were calculated from the length of the lag-phase due to the sample compared with that of Trolox and expressed as mmol of Trolox per kg (solid foods) or per L (beverages) of sample. All solid food extracts, with the exception of those in chloroform, were analyzed by this procedure.

    FRAP (ferric reducing-antioxidant power) assay. The FRAP was assessed according to Benzie and Strain (11) using a Hewlett-Packard 8453 diode array spectrophotometer. The method is based on the reduction of the Fe³⁺-TPTZ complex to the ferrous form at low pH. This reduction is monitored by measuring the absorption change at 593 nm. Briefly, 3 mL of working FRAP reagent prepared daily was mixed with 100 µL of diluted sample; the absorbance at 593 nm was recorded after a 30-min incubation at 37°C. FRAP values were obtained by comparing the absorption change in the test mixture with those obtained from increasing concentrations of Fe³⁺ and expressed as mmol of Fe²⁺ equivalents per kg (solid food) or per L (beverages) of sample. All solid food extracts, with the exception of those in chloroform, were analyzed by this procedure.

    Statistical analysis. To verify the association among methods, Pearson correlation analysis was performed using a statistical package running on a PC (Statistical Statsoft, Tulsa, OK); P-values < 0.05 were considered significant.

	RESULTS AND DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION LITERATURE CITED

 
The total antioxidant capacity of a variety of foods used in the Italian diet (i.e., vegetables, fruits, beverages and oils) was evaluated using three different assays. The food extracts had different antioxidant capacities in relation to the method applied; thus, the same item often ranked differently depending on the assay. Moreover, even for values obtained from the same assay, a comparison with the data in the literature was problematic due to the large variability within the food item and to the lack of standardization of the assays. For this discussion, the ranking order of the TAC values will be used. In the case of solid foods (i.e., vegetables and fruits), the water- and lipid-soluble extracts were analyzed separately and the overall TAC values were obtained from the sum of the two extract values. The approach of using only one solvent for fruits and vegetables extraction, generally used in TAC literature, may underestimate TAC values because antioxidant compounds at the extremities of the lipophilic or hydrophilic scale are incompletely extracted. In our case, food extracts obtained with two different solvents were analyzed separately and their sum reported in the tables. The approach of summing values of lipophilic and hydrophilic extracts permits the inclusion of the different contributors to the TAC of the food. However, it cannot be excluded that there may be a synergistic interaction between water and lipid-soluble antioxidants that is not evaluated by simply summing the components.

Total antioxidant capacity of vegetables.

The values of TAC of vegetables and the ranking order for each assay are shown in Table 1. In examining results of the FRAP and the TEAC assays, spinach was the vegetable with the greatest antioxidant capacity, followed by peppers (red bell for TEAC and chili pepper for FRAP assay), whereas cucumber and endive exhibited the lowest TAC values for the FRAP and TEAC assays, respectively. In the case of the TRAP assay, the highest TAC value was found for asparagus, whereas the TAC values of zucchini and cucumber were not detectable. The high TAC value assigned to spinach, when analyzed by the FRAP and TEAC assays, is in agreement with Proteggente et al. (14), whose ranking of TAC in 18 vegetables were similar to our results. The high antioxidant capacity of spinach is due to both the water- and lipid-soluble fractions; the former contains glucuronic acid derivates of flavonoids and derivates and isomers of p-coumaric acid (16), and the latter is rich in lutein and chlorophylls (17).

Finally, among salads, endive had the lowest antioxidant capacity, whereas radicchio exhibited a relatively high antioxidant capacity regardless of the applied method. This high antioxidant capacity of radicchio is consistent with the publication of Papetti et al. (24) and is possibly due to the presence of anthocyanins in the leaf.

In contrast to Ou et al. (22), who analyzing different varieties of vegetables using the FRAP and the oxygen radical absorption capacity (ORAC) assay, and did not find agreement among methods for the vegetables analyzed, with the exception of a few, our data were well correlated among assays. The data obtained using the FRAP and TEAC assays had the best correlation coefficients (r = 0.917, P < 0.0001), whereas the TEAC and TRAP values (r = 0.656, P < 0.0001) and TRAP and FRAP values (r = 0.744, P < 0.0001) were less well correlated. The differences observed in the ranking order between the TRAP and TEAC values of some vegetables could be the results of different variables, such as the sensitivity of the assays and the ability of TEAC assay, with respect to TRAP, to measure the antioxidant capacity of lipid-soluble antioxidants. Moreover, the different information furnished by the two assays (TRAP measures the chain breaking potential and TEAC the ability to scavenge the ABTS radical cation) could be the reason for the different results.

Total antioxidant capacity of fruits.

The TAC values of fruits and the ranking order for each assay are shown in Table 2. In general, berries had the greatest antioxidant capacity, with blackberry being the most effective. Its high antioxidant capacity, in agreement with the literature (18,25,26), is likely due to the high content of phenolic acids and flavonoids such as anthocyanins (27,28), which have demonstrated strong antioxidant activities in different model systems (9,29,30). In ranking TAC values of fruits, olives were second in antioxidant capacity, likely due to their high levels of hydroxytyrosol and tyrosol (31). Citrus fruits exhibited intermediate antioxidant capacity, with oranges as the most effective followed by grapefruit. This result is in agreement with the higher concentrations of phenolic compounds and vitamin C present in orange with respect to grapefruit (32). Among the fruits belonging to the Rosaceae family (i.e., plum, apricot, apple, pear and peach), plums had the highest antioxidant capacity for all methods. This observation is consistent with the higher phenolic content of plums with respect to other stone fruits (e.g., nectarine and peach) described by Gil et al. (33). Moreover, even if different rankings of antioxidant capacity were reported among these fruits, there is agreement in the literature that plums are the most effective (14,18,21,34). Finally, as already reported by other authors (17,18,35), fruits from the Cucurbitaceae family (i.e., honeydew and cantaloupe melons, and watermelon) had low TAC values.

Total antioxidant capacity of soft beverages.

The TAC values of soft beverages and the ranking order for each assay are shown in Table 3. Among these, citrus juices had the highest amount of antioxidants, even though different ranking orders were obtained from the three assays. The higher antioxidant capacity of citrus juices with respect to other soft drinks was observed by van der Berg et al. (36). Other fruit juices had intermediate TAC values; the values varied from 5.01 to 8.76 mmol Fe²⁺/L for the FRAP assay, and from 1.58 to 2.19 mmol Trolox/L and 1.50 to 2.56 mmol Trolox/L for the TRAP and TEAC assays, respectively. Cola drinks had the lowest TAC values, which were not detectable with the TRAP. This result is consistent with the publication of Karakaya et al. (37), who could not detect TAC activity for this beverage using the spectrophotometric TEAC assay. Finally, in contrast to the results for vegetables and fruits, the TAC values of soft beverages from the different assays were not correlated, with the exception of those obtained by the FRAP and TEAC assays (r = 0.941, P < 0.0001). The absence or presence of correlations among assays when applied to different food items highlights once again the importance of assessing TAC using a battery of different assays so as to obtain a detailed picture of the phenomenon.

The TAC of alcoholic and caffeine-rich beverages and the ranking order for each assay are shown in Table 4. Among the beverages analyzed, coffee drinks were the most effective, regardless of the assay applied, with espresso having the greatest antioxidant capacity. The removal of caffeine from the espresso coffee led to a decrease in TAC values of 25–30%, likely due to the antioxidant capacity of caffeine (38). During coffee making, the roasting process leads to profound changes in the chemical composition and biological activities of the coffee bean, resulting in the generation of compounds derived from the Maillard reaction, carbohydrate caramelization and pyrolysis of organic compounds (39). In roasted coffee most polyphenolic compounds are destroyed, but Maillard reaction products with antioxidant properties are generated (40), resulting in an increased antioxidant activity in the ß-carotene-linoleic acid model system (39).

Among alcoholic beverages, red wines had the most antioxidant capacity followed by rosé and white wines, in agreement with the literature (43–46). This is not surprising because phenolic compounds in wine derive mainly from the skin, seeds and stems of grapes, making them important sources of the polyphenols that are transferred to the juice at the first stage of wine fermentation. Thus, the content of polyphenols is high in red wine in which the contact between juice and pomace is prolonged; it is intermediate in rosé wine in which the contact is reduced compared with red wine and relatively low in white wine, which is usually made from the free-running juice without contact with the grape skins. The TEAC values of red and white wines were in the same range of those described by Simonetti et al. (43), whereas, in the case of red wines, other authors reported TEAC values slightly lower than ours (45). These differences could be due to the winemaking procedure as well as to the grape variety and growing conditions.

The TAC values of distilled spirits (i.e., cognac, grappa, rum and whiskey) were lower than those of wines, with the exception of whiskey; for the TRAP and TEAC assays, whiskey outranked some white and rosé wines. This higher antioxidant capacity of whiskey with respect to other distilled spirits could be due to its higher content of phenols and furans, as described by Goldberg et al. (47). Among distilled spirits, cognac followed whiskey in the ranking order obtained for all of the assays applied. This is likely due to polyphenols extracted from wood during the aging process (47). Finally, grappa and rum, distilled from grape pomace and molasses, respectively, but not aged in wood, had very low TEAC values with undetectable FRAP and TRAP values.

The TAC values of alcoholic beverages, teas and coffees obtained by different assays were well correlated (TRAP vs FRAP: r = 0.993, P < 0.0001; TEAC vs FRAP: r = 0.997, P < 0.0001; TEAC vs TRAP: r = 0.993, P < 0.0001). This is in keeping with Schlesier et al. (48) who analyzed some beverages, including different teas, and observed strong correlations between assays based on different chemistry (TEAC and DPPH).

Total antioxidant capacity of oils.

To assess the total antioxidant capacity of oils, samples were directly diluted in n-hexane; this procedure allows the contribution of all antioxidant compounds present, such as phenolic compounds, tocopherols, and carotenoids, to be considered (49). However, the use of this solvent limits the assessment of TAC of oils to the TEAC assay, because it is not compatible with the TRAP and FRAP assays. In Table 5, the TEAC values of the analyzed oils are shown. Soybean oil had the greatest antioxidant capacity, likely due to its high tocopherol content (50), whereas peanut oil was less effective. As already observed (51,52), the TEAC value of extra virgin olive oil (EVOO) was higher than that of olive oil (OO). The difference between OO and EVOO arises from the different manufacturing processes (49), leading to differences in the antioxidant composition. In particular, EVOO (obtained by cold pressure of the olive paste) is much richer in phenolic compounds than refined oils (obtained by solvent extraction), which are virtually devoid of phenols. OO is a vaguely defined mixture of refined olive oil and EVOO in which the amount of EVOO may vary from 33 to 95% (53), thus affecting the amount of antioxidants present.

The relevance of TAC as a new tool for investigating the relationship between dietary antioxidants and oxidative stress-induced pathologies seems confirmed by the data from a recent population-based case-control study, which showed an inverse correlation between the diet TAC and the risk of both cardia and distal gastric cancer (54). These relationships emerged in spite of the use of a very incomplete database of total antioxidant potential (12 items only among vegetables and fruits), highlighting the potentiality of the TAC as a descriptor of the diet.

In summary, the total antioxidant activities of 64 foods, 34 beverages and 6 oils were measured by three different methods. Such data, integrated with further food items of different classes (i.e., cereals, pulses and nuts), will result in a complete and versatile database of total antioxidant capacity. Coupled with an appropriate questionnaire, this will allow the evaluation of the overall intake of antioxidant-equivalents in selected groups of the Italian population in relation to the incidence of oxidative stress-induced diseases.

	ACKNOWLEDGMENTS

 
We thank Valeria Pala from the Epidemiology Unit of the National Cancer Institute (Milan, Italy) for the elaboration of food consumption data.

	FOOTNOTES

 
¹ Supported by the EC project Healthy Market (IST-2001-33204) and the National Research Council of Italy (CNR 01.00923. CT26/115.25178).

³ Abbreviations used: ABAP, 2,2'-azobis(2-amidinopropane) dihydrochloride; ABTS, 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid); ABTS·⁺, 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation; EVOO, extra virgin olive oil; FRAP, ferric reducing-antioxidant power; OO, olive oil; R-PE, R-phycoerythrin; RSD, relative standard deviation; TAC, total antioxidant capacity; TEAC, Trolox equivalent antioxidant capacity; TPTZ, 2,4,6-tripyridyl-s-triazine; TRAP, total radical-trapping antioxidant parameter; Trolox, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid.

Manuscript received 3 April 2003. Initial review completed 7 May 2003. Revision accepted 20 June 2003.

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