Objective: To investigate the contribution of beverages to the intake of lipophilic and hydrophilic antioxidants in the Spanish diet.
Design: This includes the following (i) estimation of the daily intakes of beverages in Spain, from national food consumption data obtained from annual surveys of 5400 households, 700 hotels and restaurants and 200 institutions; (ii) determination of total antioxidant capacity in the selected beverages using two complementary procedures: ferric reducing ability of plasma (FRAP), which measures the ferric reduction capacity, and ABTS, which measures the radical scavenging capacity; (iii) determination of the antioxidant capacity in both lipophilic and hydrophilic extracts of the beverages; (iv) determination of the antioxidant efficiency of the lipophilic and hydrophilic phase of the beverages; and (v) estimation of the intake of dietary antioxidants from beverages in comparison with the daily requirements of antioxidant vitamins C and E.
Results: The contribution of beverages to the antioxidant intake in the Spanish diet is estimated at 1623 mg of vitamin E and 598 mg of vitamin C by FRAP, and 1521 mg of vitamin E and 556 mg of vitamin C by ABTS. Coffee is the main contributor (66 and 61% by FRAP and ABTS, respectively), followed by red wine (16 and 22%), fruit juices (6 and 5%), beer (4 and 5%), tea (3 and 5%) and milk (4 and 1%).
Conclusions: Beverages account for a very high proportion of dietary antioxidant intake as compared to intake of antioxidant vitamins C and E. Although their metabolic effect must be affected by the bioavailability of the antioxidants, the significance of this intake for antioxidant status and health should be considered.
Reactive oxygen species are formed in vivo during metabolism. They attack target molecules (lipids, proteins and DNA) inducing oxidative modifications. Oxidative stress is involved in the pathology of many diseases, such as atherosclerosis, diabetes, neurodegenerative diseases, ageing and cancer (McBrian & Slater, 1982; Nakagami et al, 1995; Hollman et al, 1996; Aruoma et al, 1997; Meyer et al, 1998). Protection against this degenerative illness has been attributed to endogenous antioxidants (superoxide dismutase, catalase and various peroxidases) and also to dietary antioxidants of fruits and vegetables (Gey et al, 1991; Frei, 1994; Mackerras, 1995; Schwartz, 1996; Abushita et al, 1998; Aruoma, 1998).
The most important plant substances presenting antioxidant activity are carotenoids, flavonoids and other simple phenolic compounds and vitamins (A, C and E). Some antioxidants, like ascorbic acid, are hydrophilic, while others, like carotenoids and vitamin E, are clearly lipophilic. It is not easy to separate polyphenols (PPs) into hydrophilic and lipophilic because they are a complex group of substances with a wide range of molecular mass and are found either free or bound to protein or dietary fibre. Hydrophilic and lipophilic antioxidants each have their own function in the organism. They act at different points but work in collaboration. PPs and their metabolites exert antioxidant protection in vivo through a cascade involving reactive oxygen species and physiologic antioxidants. Based on their daily intake, which greatly exceeds that of other antioxidants (vitamin C, vitamin E and β-carotene), PPs may be a major factor in assuring the antioxidant potential of the diet, and may constitute an important exogenous defence against an imbalance of pro-oxidants and antioxidants (oxidative stress). In addition, vitamins have shown less antioxidant activity than PPs in different in vitro studies (Sánchez-Moreno et al, 1998, 2000a; Pulido et al, 2000).
Dietary antioxidants are found in cereals, fruits, legumes, spices, vegetables and beverages (Ganthavorn & Hughes, 1997; Lin et al, 1998; Kalt et al, 1999; Ewald et al, 1999; Che Man & Tan, 1999; Markus et al, 1999). The contribution of total PPs to antioxidant capacity has been extensively studied in fruits and vegetables (Furuta et al, 1997; Gazzani et al, 1998; Heinonen et al, 1998; Saleh et al, 1998; Wang et al, 1999).
Also, there are many references in the literature to the total antioxidant capacity of drinks such as fruit juices (Chambers et al, 1996; Wen et al, 1999), beverages (Abu-Amsha et al, 1996; Benzie & Szeto, 1999; Hodgson et al, 1999, 2000; Prior & Cao, 1999; Richelle et al, 2001) and alcoholic drinks (Criqui, 1998; Sánchez-Moreno et al, 1999; Denke, 2000; Lorimier, 2000). These studies were intended to determine the contribution of whole PPs to total antioxidant capacity, but they do not consider either the hydrophilic and lipophilic nature of food PPs or their contribution to the total antioxidant capacity of a specific diet. To our knowledge, only a few articles have addressed the hydrophilic and lipophilic contribution to total antioxidant capacity, and these analysed only specific samples, such as vegetable soups or juices (Bonilla et al, 1999; Arnao et al, 2001).
There has been no analysis of the contribution of hydrophilic and lipophilic components to total antioxidant activity of dietary antioxidants in beverages. The aim of this study was to determine the total antioxidant activity of hydrophilic and lipophilic components of the most representative beverages in the Spanish Mediterranean diet and their contribution to dietary antioxidant intake.
Materials and methods
Beverage intake in Spain
Estimates of beverage intakes in the Spanish diet are based on national consumption data (MAPA, 2000). These data are obtained annually from daily budget questionnaires. In total, 5400 households are surveyed, along with 700 hotels and restaurants and 200 institutions such as schools, hospitals and the armed forces (confidence level 95.45%; error range 3% in amount of food). The samples described below were selected from these data.
Two commercial alcoholic beverages, two nonalcoholic beverages, two infusions and milk were analysed. Beer: Aguila-Amstel (5% alcohol) from Heineken Spain S.A. (Sevilla, Spain); milk: UHT full-fat milk from Leche Pascual S.A. (Burgos, Spain); coffee: Colombian chicory coffee (roasted with sugar) from Cafés la Mexicana Rodriguez y Mateus S.A. (Madrid, Spain); tea: Lipton yellow label quality no. 1 from Unilever Belgium N.V. (London, England); red wine: Los Molinos (12% alcohol) from Bodegas Felix Solis (Valdepeñas, Spain); cola: Coca-Cola from Coca-Cola S.A. (Madrid, Spain); orange juice: 100% orange juice from Juver Alimentación S.A. (Murcia, Spain).
Commercially available infusions were prepared as follows: one tea bag (1.5 g) was infused for 5 min in 250 ml of hot water; soluble coffee was prepared with 26.2 g of ground coffee in 325 ml of hot water.
Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), a water-soluble analogue of vitamin E, was from Aldrich Co. (St Louis, MO, USA). DL-α-tocopherol was from Sigma Chemical Co. (St Louis, MO, USA). TPTZ (2,4,6-tri(2-pyridyl)-s-triazine) was from Fluka Chemicals (Madrid, Spain). ABTS (2,2′-azinobis(3-ethylbenzothiazoline-6-sulphonic acid, diammonium salt) was from Fluka Chemicals (Steinheim, Germany) and potassium persulphate was obtained from Sigma-Aldrich (Steinheim, Germany). Ascorbic acid, FeCl3·6H2O, acetone, ethanol and methanol were from Panreac Química S.A. (Madrid, Spain). All reagents used were of analytical grade.
Extraction and separation of components
Drinks and solvents (ethyl acetate, n-hexane and dichloromethane) were mixed in a 1:1 ratio (v/v), and after 1 h of gentle shaking the samples were centrifuged at 1800 × g for 10 min. Two phases were formed: aqueous and organic. The aqueous phase was collected to measure hydrophilic PPs and antioxidant activity, while the organic phase was collected to measure lipophilic PPs and antioxidant activity. All extraction procedures were carried out at room temperature and the determinations were performed as soon as possible.
of all drinks were estimated by the Folin–Ciocalteau method (Montreau, 1972) both in the total and the hydrophilic and lipophilic extracts, using gallic acid as standard, expressing the results as gallic acid equivalent (GAE).
Antioxidant activity assays
Samples obtained as described above were used to determine the antioxidant capacity of all drinks, both in the total and the hydrophilic and lipophilic extracts, by two different methods.
The antioxidant capacity of each sample was estimated according to the procedure described by Benzie and Strain (1996), with some modifications introduced by our laboratory: the readings at 30 min were selected to calculate ferric reducing ability of plasma (FRAP) values (Pulido et al, 2000). Methanolic solutions of known Trolox and vitamin C and E concentrations were used for calibration.
The antioxidant capacity was estimated following the procedure described by Re et al (1999) with some modifications. ABTS radical cation (ABTS+•) was produced by reacting 7 mM ABTS stock solution with 2.45 mM potassium persulphate and allowing the mixture to stand in the dark at room temperature for 12–16 h before use. The ABTS+• solution (2 days stable) was diluted with methanol to an absorbance of 0.70±0.02 at 658 nm. After addition of 100 μl of sample or Trolox standard to 3.9 ml of diluted ABTS+• solution, absorbance readings were taken every 20 s using a Beckman DU-640 spectrophotometer (Beckman Instruments Inc., Fullerton, CA, USA). The reaction was monitored during 6 min. The percentage inhibition of absorbance vs time was plotted and the area below the curve (0–6 min) was calculated. Methanolic solutions of known Trolox and vitamin C and E concentrations were used for calibration.
Results are expressed as mean values±s.d. Comparison of the means of three measurements using a significance level of P<0.05 was performed by one-way analysis of variance (ANOVA) using the Statgraphics Computer System, version 5.1.
The intake of beverages in Spain is shown in Table 1. The beverage most consumed is milk. The alcoholic beverages most consumed are wine and beer. Red wine accounts for 57% of all wine consumed. Coffee accounts for 97% of all infusions consumed in Spain. Orange juice and cola account for 23 and 54%, respectively, of all fruit juices and all soft drinks consumed.
To extract and separate both the hydrophilic and lipophilic components of these beverages, we had tested three solvents with different polarity: ethyl acetate, n-hexane and dichloromethane Ethyl acetate was selected because it does not interfere with analytical procedures to determine PPs and measure antioxidant capacity in the aqueous and organic phases.
Of the samples studied for total PP content (Table 2), Colombian chicory coffee had both the highest PP content and antioxidant activity. Red wine also had high PP content and antioxidant activity followed by tea, beer, orange juice and cola in that order.
In the hydrophilic phases (Table 3), Colombian chicory coffee and red wine again had more PPs and higher antioxidant capacity than the others. Orange juice contained more PPs than tea or beer, although the antioxidant capacity determined by the FRAP method was similar to that of tea.
As Table 4 shows, the lipophilic contribution of coffee was the highest, as noted before in both the total and hydrophilic extract, although a few differences were observed between the lipophilic contribution to the total PPs in studied drinks and their hydrophilic components. In fact, there were no differences between the lipophilic components of tea and red wine, but red wine had higher hydrophilic PP content and antioxidant capacity than tea. The lipophilic component of beer and orange juice was similar, but less than that of tea, red wine or coffee. The behaviour of antioxidant capacity was similar for all the drinks studied. Coffee had the highest antioxidant capacity followed by tea and red wine, beer and orange juice. The lipophilic extract was negligible in milk and cola.
The antioxidant efficiency, by the FRAP method, expressed as μmol equivalent of Trolox per mg of PPs, of each fraction (total, hydro and lipo) is shown in Figure 1. Similar results were obtained by using the ABTS procedure. Tea presents the highest efficiency in both hydrophilic and lipophilic extracts, followed by orange juice and red wine. Although the total PP content of commercial orange juice was similar to that of beer, it had higher antioxidant activity. Indeed, the commercial orange juice presented five times more efficiency in the total FRAP value than beer (10.3 μmol/mg PP orange juice vs 2.9 μmol/mg PP beer). This juice had four times more power in terms of the hydrophilic FRAP value than beer (8.2 μmol/mg PP orange juice vs 2.2 μmol/mg PP beer). The hydrophilic component of tea also had five times more activity than beer (13.3 μmol/mg PP tea vs 2.2 μmol/mg PP beer). Cola, the most widely consumed soft drink, had the lowest PP content and the lowest antioxidant activity.
Correlation coefficients between PP content and antioxidant capacity determined by FRAP and ABTS in all the fractions studied (total, hydro and lipo) are shown in Figure 2. There is a high correlation (range 0.849–0.981) in all the cases.
Figure 3 shows the contribution of beverages and drinks to the total intake of antioxidant capacity in the Spanish diet. The largest single contributor is coffee (66 and 61% by FRAP and ABTS, respectively), considering the high intake and high antioxidant capacity, followed by red wine (16 and 22% by FRAP and ABTS, respectively). The antioxidant capacity equivalent to vitamins C and E of the daily per capita intake of drinks, as measured by FRAP and ABTS, is shown in Table 5. The vitamin-E-equivalent antioxidant capacity of the analysed drinks was in the range 7–1135 mg by FRAP and 6–958 mg by ABTS. The vitamin C-equivalent antioxidant capacity of these drinks was in the range 2–420 and 2–356 mg by FRAP and ABTS, respectively.
In the last decade, a large number of assays have been conducted to measure total antioxidant capacity in food and biological matrixes (Robards et al, 1999; Frankel & Meyer, 2000; Ghiselli et al, 2000). Each of them has its own characteristics; there are differences in the free radical-generating system, molecular target, end point, kinetic, biological matrix, residence in lipo- and hydrophilic compartment and physiological relevance. The influence of all relevant parameters cannot be evaluated using only one assay protocol. A comparison of antioxidant capacity assays was previously performed in a European interlaboratory study (Serafini et al, 2002). On this basis, two systems were chosen to evaluate the antioxidant activity of the extracts. ABTS and FRAP, respectively, measure the radical scavenging activity and the total reduction power. Trolox (an analogue of vitamin E) was used as standard because, owing to its amphoteric properties, it can be dissolved in aqueous (as a salt) or organic media (as an acid) (Arnao et al, 2001).
The high correlation coefficients (0.849–0.981) indicate a relatively strong relation between PP content and antioxidant capacity. Nevertheless, we have found some exceptions; orange juice, for instance, presents greater antioxidant capacity than beer although it has less PP content. This could be explained by qualitative differences between PPs, the presence of specific components in orange juice such as carotenoids, terpenes, vitamins and minerals, and possibly synergic effects between these compounds. Information given by the correlation coefficient between the PP content and the antioxidant capacity will be reliable only if one kind of drink is being studied, as is the case for wines (Frankel et al, 1995; Simonetti et al, 1997; Prior et al, 1998; Larrauri et al, 1999; Sánchez-Moreno et al, 1999, 2000b). Any comparison of different drinks must take into account both the kind of PPs and the synergic and antagonic effects with other components. Red wine has high PP content and antioxidant activity as reported elsewhere (Larrauri et al, 1999; Sánchez-Moreno et al, 1999, 2000b).
Full-fat milk had more antioxidant activity than the hydrophilic plus the lipophilic components, possibly as a result of precipitation and elimination of the protein in extraction with ethyl acetate. Total milk was therefore taken as the hydrophilic plus lipophilic phases to analyse the contribution of other molecules to antioxidant activity since protein is hydrolysed in the digestive tract. Milk contains peptides derived from caseins with bioactive properties, such as antihypertensive, antimicrobial and antithrombotic activities (Mazza, 1998; Clare & Swaisgood, 2000; Shah, 2000), and maybe they would show antioxidant capacity. Milk also contains small proportions of vitamin E and carotenoids (Pérez Gavilán & Pérez Gavilán, 1984), which could be responsible for the antioxidant activity of the lipophilic phase of milk. It also contains vitamin C (ascorbic acid) (Pérez Gavilán & Pérez Gavilán, 1984), which may contribute to antioxidant activity in the hydrophilic phase.
Given the heavy intake of coffee and the fact that high concentrations of antioxidants may exert pro-oxidant effects (Long et al, 1999), it is important to elucidate its contribution to the diet as antioxidant.
In conclusion, drinks of Spanish diet showed a potential antioxidant capacity equivalent that largely exceeded (see Table 5) the recommended dietary amounts of antioxidant vitamins C and E (60 and 8–10 mg per day/person, res-pectively). These values are tested in vitro so they must be considered potential values, since the real effect is modulated by the bioavailability of antioxidant compounds. Although the bioavailability of phenolic compounds is low (Bravo, 1998), it seems that the intake of nonvitamin antioxidants from tested drinks may contribute significantly to the antioxidant status in humans along with vitamins C and E. By extracting and determining the hydrophilic and lipophilic antioxidants in beverages, it is possible to ascertain the contribution of each of them to the total antioxidant capacity.
We have calculated the antioxidant capacity derived from beverages in the Spanish diet; however, this is only part of the overall dietary intake of antioxidants, an important part of which comes from fruit and vegetables.
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RP thanks the Comunidad de Madrid for granting her a scholarship.
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Pulido, R., Hernández-García, M. & Saura-Calixto, F. Contribution of beverages to the intake of lipophilic and hydrophilic antioxidants in the Spanish diet. Eur J Clin Nutr 57, 1275–1282 (2003). https://doi.org/10.1038/sj.ejcn.1601685
- dietary antioxidants
- lipophilic antioxidants
- hydrophilic antioxidants
- antioxidant capacity
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