JOURNAL OF MEDICINAL FOOD J Med Food 11 (1) 2008, 144–151 © Mary Ann Liebert, Inc. and Korean Society of Food Science and Nutrition DOI: 10.1089/jmf.2007.598
Antioxidant, Mutagenic, and Antimutagenic Activity of Frozen Fruits Patrícia D.S. Spada,1 Gabrielle Gianna Nunes de Souza,1 Giovana Vera Bortolini,1 João A.P. Henriques,1,2 and Mirian Salvador1 1Instituto
de Biotecnologia, Universidade de Caxias do Sul, Caxias do Sul; and 2Universidade Luterana do Brasil, Canoas, Rio Grande do Sul, Brazil
ABSTRACT Many studies have focused on the effect of fresh fruits on the risk of developing cancer and other diseases involved with reactive species and free radicals. The intake of frozen fruits has spread widely in the last years, but, until now, their biological activity is not completely known. In this study, 23 samples of frozen fruits were analyzed for their nutritional composition, total polyphenols, total carotenoids, and vitamin C content. Antioxidant, mutagenic, and antimutagenic effects were also evaluated. Antioxidant assays included 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) scavenging activity and determination of superoxide dismutase (SOD)- and catalase (CAT)-like activities. Mutagenic and antimutagenic evaluations were performed in eukaryotic cells of Saccharomyces cerevisiae yeast. Most samples (74%) showed antioxidant activity similar to vitamin C in the DPPH assay, and this activity was positively correlated (r 0.366; P .01) with carotenoid contents. All samples showed CAT-like activity. SOD-like activity was detected in 56% of samples assayed. Only four fruits (acai, cashew apple, kiwi fruit, and strawberry) showed mutagenic activity when tested in high (5%, 10%, and 15% [wt/vol]) concentrations. Twelve samples presented antimutagenic effects against hydrogen peroxide, and this effect was positively correlated with CAT-like activity (r 0.400; P .01). Evaluation of polyphenols, carotenoids, and ascorbic acid showed considerable levels of these compounds in frozen fruits, even after freezing. These data suggest that frozen fruits contribute to the prevention of biological damages. KEY WORDS:
•
antimutagenic
•
antioxidant
•
frozen fruits
INTRODUCTION
•
mutagenesis
On the other hand, some compounds present in fruits have been identified as being themselves mutagenic,12,13 pro-oxidant,12 and carcinogenic.13 Carcinogenic or genotoxic effects may be mediated either by the interaction of fruit components with transition metals or by by-products of the autooxidation of fruits. Vitamin C may act as a pro-oxidant through Fenton and Fenton-like reactions.9,12,14 Some phenolic compounds may increase DNA damage by acting as a topoisomerase poison15 or by binding to DNA.16 The intake of frozen fruits has spread widely because it is hard to market fresh fruits in places far from their harvesting site. Besides, frozen fruits are very important as a source of raw material. They are used in yogurts, candies, cookies, cakes, ice creams, fresh drinks, and children’s food. However, to our knowledge, until now, there are no significant data about the biological effects of frozen fruits as consumed by the general population. There is no doubt that epidemiological studies are more valuable than in vitro or animal studies. However, human studies need a large number of individuals, take more time to get ready, need to incorporate multiple end points, are rationalized to cover more than one type of disease, and include both plasma and DNA measurements. In addition, difficulties in standardizing the diet and the influence of lifestyle, which includes country of living, kind of job, and
M
ANY STUDIES HAVE SHOWN that the consumption of fruits and vegetables is associated with a reduced risk of many diseases, including cancer, atherosclerosis, and neurovegetative diseases, which are related to elevated levels of oxidative stress.1–4 Antioxidant compounds can decrease oxidative stress, minimizing the incidence of these diseases.5–8 Fruits supply several antioxidant compounds, such as vitamin C, -carotene, and/or polyphenols.9,10 The mechanisms of the antioxidant action of these compounds can include suppressing reactive species formation either by inhibition of enzymes or by chelation of trace elements involved in free radical production, scavenging reactive species, and up-regulating or protecting antioxidant defense.11 Some compounds can also act in a similar way to the enzymatic defenses, since they are able to neutralize reactive species such as superoxide anion and hydrogen peroxide.4
Manuscript received 24 September 2007. Revision accepted 7 December 2007. Address reprint requests to: Mirian Salvador, Instituto de Biotecnologia, Universidade de Caxias do Sul, Rua Francisco Getúlio Vargas 1130, Caxias do Sul, RS, Brazil, 95070560, E-mail:
[email protected]
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exercise practice, should be considered. In this way, some in vivo assays using Saccharomyces cerevisiae yeast cells have been successfully used, showing this system to be an attractive alternative to study many kinds of compounds.17,18 The purpose of this study was to investigate the antioxidant, mutagenic, and antimutagenic effects of frozen fruits. Besides, nutritional analysis, ascorbic acid, carotenoids, and total polyphenol compounds were determined.
MATERIALS AND METHODS Frozen fruits Frozen pulps of acerola (Malpighia glabra L.), apple (Malus domestica B.), acai (Euterpe oleracea L.), black mulberry (Morus nigra M.), cashew apple (Anacardium occidentale L.), coconut (Cocos nucifera L.), cupuacu (Theobroma grandiflorum W.), kiwi fruit (Actinidia chinensis P.), mango (Mangifera indica L.), melon (Cucumis melo L.), papaya (Carica papaya L.), passion fruit (Passiflora alata C.), peach (Prunus persica L.), pineapple (Ananas sativus L.), raspberry (Rubus idaeus L.), red guava (Psidium guajava L.), soursop (Annona muricata L.), strawberry (Fragaria vesca L.), and Surinam cherry (Eugenia uniflora L.) and frozen juices of red grape (Vitis vinifera L.), lemon (Citrus limon B.), orange (Citrus aurantium L.), and tangerine (Citrus reticulata L.) were obtained from the industry Mais Fruta (Antonio Prado, RS, Brazil). Pulps and juices were produced with fresh and clean fruits, free of filthy substances, parasites, and plant or animal debris. Only edible portions of the fruits were pressed in order to prepare pulps and juices. In the red grape, lemon, orange, and tangerine juices, flesh was separated from fluid obtained from pressing. After this procedure, pulps and juices were divided in aliquots of 100 g and kept frozen at 20°C. Immediately prior to the assays, frozen fruits were mixed with distilled water (in specific concentrations for each assay) in a blender and then sterilized by filtration through a filter (pore size, 0.45 m; catalog number SFGS 047LS, Millipore Corp., São Paulo, Brazil).
Main characteristics of the frozen fruits Carbohydrates, lipids, proteins, total acidity, and pH values were determined according to Association of Official Analytical Chemists official methods19 (methods 971.18, 983.23, 983.152, 942.15, and 943.02, respectively). All analyses were performed in duplicate. Caloric values expressed in kJ were obtained by multiplying levels of proteins and carbohydrates by 17 and contents of lipids by 37, with the later addition of these values.19 Total phenol content in frozen fruits was determined by using the modification of Singleton and Rossi20 of the FolinCiocalteu colorimetric method. Total phenol content was derived by comparison with a catechin standard curve (0.1–1 mg/mL catechin; Sigma Chemical Co., São Paulo) and expressed as mg of catechin equivalents/100 g. Total carotenoids were assayed by using a simplified method for
145
carotenoid distribution in natural compounds.21 Ascorbic acid determination was performed according to Association of Official Analytical Chemists official methods (967.21).19
Pesticide determination Organophosphorus and carbamate pesticides were determined in frozen fruit samples as methyl parathion-equivalent activity, which causes inhibition of acetylcholinesterase (AChE), as previously described by Bastos et al.22 and Lima et al.23 Methyl parathion (Folidol 600®, Bayer, São Paulo) calibration curve was used to express AChE activity in ppm of methyl parathion.
Antioxidant activity The antioxidant activity of frozen fruits was measured by in vitro assays: 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) scavenging activity and superoxide dismutase (SOD)- and catalase (CAT)-like activities. DPPH radical scavenging activity was measured using a method modified from that of Yamaguchi et al.24 in which frozen fruit solutions were added to Tris-HCl buffer (100 mM, pH 7.0) containing 250 M DPPH dissolved in ethanol to obtain final concentrations of 10, 20, and 40% (wt/vol). Tubes were stored in the dark for 20 minutes, after which absorbance was measured at 517 nm (model UV-1700 spectrophotometer, Shimadzu, Kyoto, Japan). Results were expressed in IC50 (amount of juice necessary to scavenge 50% of DPPH radical). Instead of antioxidant solutions, distilled water was used as the control. To evaluate enzyme-like activities, all frozen fruits were prepared in a concentration of 40% (wt/vol). SOD-like activity was spectrophotometrically determined in samples of frozen fruits by measuring the inhibition of self-catalytic adrenochrome formation rate at 480 nm, in a reaction medium containing 1 mmol/L adrenaline (pH 2.0) and 50 mmol/L glycine (pH 10.2). This reaction was performed at 30°C for 3 minutes.25 Results were expressed in IC50 (the amount of sample needed to reduce 50% of adrenochrome). The CAT-like activity assay was performed according to the method described by Aebi,26 by determining hydrogen peroxide decomposition rate at 240 nm. Results were expressed as mol of H2O2 decomposed/minute.
Mutagenic and antimutagenic activity An XV 185-14c strain (MAT, ade2-2, arg4-17, his1-7, lys1-1, trp5-48, hom3-10), kindly provided by Dr. R.C. Von Borstel (Genetics Department, University of Alberta, Edmonton, AB, Canada), was used for the mutagenicity assay. Rich liquid medium (YPD) was used for routine growth. The minimal medium contained 0.67% yeast nitrogen base with no amino acids, 2% glucose, 2% Bacto-agar, and 0.25% (NH4)2SO4. Synthetic complete (SC) medium was based on minimal medium and supplemented with 2% adenine, 5% lysine, 1% histidine, 2% leucine, 2% methionine, 2% uracil, 2% tryptophan, and 24% threonine (wt/vol). Deficient me-
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SPADA ET AL.
Statistical analysis
dia lacking lysine (SC-lys), histine (SC-his), or homoserine (SC-hom) were used for mutagenesis determination. A 0.9% NaCl solution was employed to dilute cell suspensions. A cell (stationary-phase) suspension of 2 108 cells/mL of the XV 185-14c yeast strain was incubated for 4 hours at 28°C with different frozen fruit concentrations (5%, 10%, and 15% wt/vol) and saline solution. Survival was determined in SC medium (3–5 days, 28°C), and mutation induction (LYS, HIS, or HOM revertants) was performed on corresponding deficient media (7–10 days, 28°C). Whereas his1-7 is a nonsuppressible missense allele and reversions result from mutations at the locus itself,27 lys1-1 is a suppressible ochre nonsense mutant allele,28 which can be reverted by either locus-specific or forward mutations in a suppressor gene.29 Both mutation types at the lys1-1 locus were differentiated according to Schuller and Von Borstel.30 It is believed that hom3-10 contains a frameshift mutation due to its response to several diagnostic mutagens.31 Plating was done in triplicate for each dose. Hydrogen peroxide (75 mM) was used as the positive control. For the antimutagenic assay, cells of the XV 185-14c yeast strain were pretreated (for 10 minutes) with the frozen fruit, before adding 75 mM hydrogen peroxide. The treatments were done for 4 hours at 28°C with shaking. After that, samples were plated onto the media described above (SC, SC-his, SC-lys, or SC-hom). Hydrogen peroxide (75 mM) was used as the control. Plating was done in triplicate for each dose.
TABLE 1. Sample Acai Acerola Apple Black mulberry Cashew apple Coconut Cupuacu Kiwi fruit Lemon Mango Melon Orange Papaya Passion fruit Peach Pineapple Raspberry Red grape Red guava Soursop Strawberry Surinam cherry Tangerine
pH 4.34 3.56 3.68 3.30 4.08 4.64 3.54 3.40 2.78 4.34 5.02 3.64 4.51 3.12 4.00 3.61 3.16 3.40 4.00 3.75 3.50 3.40 3.66
0.02b 0.02a 0.02j 0.07c 0.01d 0.02e 0.03a 0.04i 0.03k 0.10b 0.03m 0.03j 0.08l 0.08f 0.04g 0.02a 0.04f 0.04i 0.04g 0.05h 0.04n 0.08i 0.04j
Total acidity (%) 3.14 10.85 4.06 9.77 4.54 1.87 15.85 20.84 38.25 5.72 1.48 21.30 4.41 46.60 4.22 10.95 15.99 10.00 6.51 6.19 13.88 13.57 22.41
0.17b 0.30a 0.09d 0.44c 0.20d 0.13e 0.52f 0.28i 0.09j 0.08h 0.07e 0.03i 0.02d 0.61k 0.02d 0.33a 0.06f 0.38c 0.06g 0.15gh 0.11l 0.05l 0.20m
Values were determined as parametric or nonparametric by using the Kolmogorov-Smirnoff test. All assays were performed in triplicate. Data were subjected to analysis of variance, and means were compared using Tukey’s test. Relationships between variables were assessed with Pearson’s product-moment correlation coefficient. SPSS version 12.0 (SPSS, Chicago, IL) was used in all statistical analysis.
RESULTS Main characteristics of frozen fruits Values for total acidity, pH, proteins, lipids, and carbohydrates were determined in frozen fruits (Table 1), and their caloric contents were calculated. As expected, frozen fruits showed low levels of protein and total lipids. Carbohydrate contents ranged from 0.10 0.00 to 17.80 0.61 mg %. Tangerine juice samples showed the highest levels of caloric value and carbohydrate content. Contents of total phenolic compounds, carotenoids, and ascorbic acid are shown in Table 2. Cashew apple, soursop, red guava, orange, passion fruit, and Surinam cherry samples showed higher total phenolic values. Frozen pulps of acai, acerola, red guava, papaya, mango, and Surinam cherry showed more carotenoids than the others that were assessed. Acerola pulp showed a high ascorbic acid concentration (224.57 8.69 mg %).
MAIN CHARACTERISTICS Proteins (%) 0.55 0.65 0.15 0.47 0.42 0.48 0.37 0.53 0.26 0.29 0.19 0.57 0.34 0.50 0.40 0.35 0.27 0.31 0.49 0.40 0.64 0.50 0.64
0.03b 0.01b 0.02e 0.02c 0.03c 0.02c 0.03a 0.01b 0.04d 0.05d 0.02e 0.02b 0.01a 0.04c 0.02a 0.02a 0.02d 0.03a 0.03c 0.03a 0.03b 0.03b 0.04b
OF
FROZEN FRUITS
Lipids (%) 0.67 0.00 0.00 0.08 0.01 0.53 0.00 0.05 0.00 0.08 0.00 0.00 0.05 0.02 0.00 0.00 0.14 0.00 0.06 0.07 0.00 0.00 0.00
0.04b 0.00a 0.00a 0.01c 0.00a 0.03b 0.00a 0.02c 0.00a 0.02c 0.00c 0.00a 0.01c 0.00a 0.00c 0.00a 0.05c 0.00c 0.02c 0.01c 0.00c 0.00c 0.00c
Carbohydrates (%) 7.63 7.61 9.97 4.98 9.80 0.10 4.61 5.33 0.33 8.85 3.26 7.02 6.81 5.88 4.99 6.02 3.93 9.72 4.51 5.20 3.14 3.69 17.80
0.02b 0.03b 0.28b 0.01a 0.89a 0.00c 0.14b 0.56a 0.02a 0.13b 0.27a 1.04b 0.31b 0.61b 0.04a 0.28a 0.15b 1.33a 0.24b 0.71a 0.08a 0.02b 0.61d
Caloric values (kJ %) 163.25 138.14 171.63 96.28 171.63 29.30 83.72 100.46 8.37 154.88 58.60 125.58 121.39 108.84 92.09 104.65 75.35 167.44 87.91 96.28 62.79 71.16 309.76
0.03c 0.03c 0.24d 0.04a 0.02d 0.01e 0.07a 0.09a 0.19d 0.06d 0.03b 0.14c 0.43a 0.45c 0.23a 0.05a 0.10a 0.08d 0.20a 0.18a 0.34b 0.07a 0.04f
Data are mean SD values of three independent experiments. Different letters indicate a significant difference according to analysis of variance and Tukey’s post hoc test (P .05) for each parameter evaluated.
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ACTIVITY OF FROZEN FRUITS TABLE 2. Sample Acai Acerola Apple Black mulberry Cashew apple Coconut Cupuacu Kiwi fruit Lemon Mango Melon Orange Papaya Passion fruit Peach Pineapple Raspberry Red grape Red guava Soursop Strawberry Surinam cherry Tangerine
POLYPHENOLS, CAROTENOIDS,
Polyphenols (mg of catechin equivalent %) 1.19 1.36 0.15 1.04 1.70 0.18 1.29 0.42 0.28 0.95 1.29 1.44 0.46 2.28 1.18 1.08 0.64 0.41 1.93 1.53 1.33 1.77 1.41
0.20a 0.01a 0.05c 0.33a 0.13b 0.03c 0.09a 0.02c 0.05c 0.30a 0.40a 0.40ab 0.15c 0.05b 0.20a 0.14a 0.04c 0.14c 0.20b 0.20b 0.40a 0.20b 0.02b
AND
VITAMIN C
IN
FROZEN FRUITS
Carotenoids (mg %) 1.02 1.87 0.09 0.33 0.28 0.46 0.03 0.05 0.02 1.87 0.40 0.76 1.61 0.62 0.67 0.28 0.71 0.12 1.45 0.01 0.33 1.82 0.77
0.05b 0.02c 0.01i 0.02a 0.03a 0.01d 0.00e 0.00h 0.00e 0.03c 0.02d 0.06f 0.06g 0.01f 0.05f 0.02a 0.02f 0.01j 0.05g 0.00e 0.03a 0.02c 0.05f
Vitamin C (mg %) 15.70 224.57 1.19 19.11 77.00 2.56 27.65 57.85 46.76 34.47 25.94 42.87 49.15 27.65 6.08 49.83 18.09 1.33 21.16 30.38 45.05 10.58 42.32
0.97b 8.69c 0.01e 1.93b 0.19d 0.34e 5.31f 0.05g 0.48a 2.41f 4.83f 0.19a 2.90a 0.48f 0.10e 4.83a 0.48b 0.05e 0.97f 0.48b 1.93a 0.48e 5.79a
Data are mean SD values of three independent experiments. Different letters indicate a significant difference according to analysis of variance and Tukey’s post hoc test (P .05) for each parameter evaluated.
Pesticides determination No organophosphorus and carmabate pesticides were detected in the samples of frozen fruits (data not shown).
Antioxidant activity Antioxidant activity of frozen fruits was assayed by DPPH scavenging ability and SOD- and CAT-like activities. Antioxidant activity values and the ranking order (according to statistical differences for P .05) for each assay are shown in Table 3. Most samples (74%) showed antioxidant activity similar to vitamin C in the DPPH assay, and this activity was positively correlated (r 0.366; P .01) with carotenoid contents. Lemon, melon, and pineapple were the fruits with the poorest antioxidant activity observed in this assay. All samples showed CAT-like activity. SOD-like activity was detected in 56% of the samples.
Mutagenic and antimutagenic activity The mutagenicity test was performed with three higher, noncytotoxic, concentrations of frozen fruits (5%, 10%, and 15% [wt/vol]). Acai, cashew apple, kiwi fruit, and strawberry pulps showed mutagenic activity in all concentrations and loci assayed, in a dose-dependent manner (Table 4). Results for Lys-revertant yeast colonies (a locus-specific mutation) were positively correlated with content of carotenoids (r 0.793; P .01). In His-revertant yeast
colonies (a locus-specific mutation) a positive correlation with levels of polyphenols (r 0.688; P .01), carotenoids (r 0.654; P ( .01), and ascorbic acid (r 0.640; P .05) was found. In Hom-revertant yeast colonies (frameshift events) there was a positive correlation between levels of total carotenoids (r 0.701; P .05) and ascorbic acid (r 0.752; P .05). Antimutagenic effects of different samples against hydrogen peroxide were also analyzed in yeast cells at 5% (Table 5), 10%, and 15% (wt/vol) (data not shown). In all concentrations, coconut, cupuacu, raspberry, red guava, orange, apple, papaya, mango, peach, soursop, tangerine, and red grape frozen fruits showed an important antimutagenic effect. A positive correlation was observed with CAT-like activity (r 0.400; P .01).
DISCUSSION Epidemiological studies that analyze health implications of dietary components rely on intake estimates by sample populations. These data can be found in databases that list the component’s content in commonly consumed foods.6 However, complete databases on fruit pulp content are not yet available for all frozen pulps. Although geographical differences should be considered, this paper reports data about 23 frozen fruits of regular intake in most parts of the world. Polyphenols, carotenoids, and vitamin C are important components of fruits that provide protection against several
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SPADA ET AL. TABLE 3.
DPPH REDUCING
AND
SOD-
AND
DPPH a
CAT-LIKE ACTIVITIES
IN
DIFFERENT FROZEN FRUITS
SOD-like activityb
CAT-like activityc
Sample
Value
Rank
Value
Rank
Value
Rank
Acai Acerola Apple Black mulberry Cashew apple Coconut Cupuacu Kiwi fruit Lemon Mango Melon Orange Papaya Passion fruit Peach Pineapple Raspberry Red grape Red guava Soursop Strawberry Surinam cherry Tangerine
22.95 52.60 21.95 24.87 28.99 34.95 29.50 25.00 95.00 23.00 95.55 29.45 22.96 82.00 24.05 93.78 23.90 28.50 30.01 28.05 26.50 25.15 63.53
1 2 1 1 1 1 1 1 3 1 3 1 1 2 1 3 1 1 1 1 1 1 2
0.09 1.44 0.60 1.80 0.05 0.16 0.07 0.10 0.14 ND 3.38 0.11 0.08 ND ND ND 0.73 ND 0.12 0.40 ND 0.13 ND
1 3 2 3 1 1 1 1 1
0.90 108 3.00 108 0.60 108 1.50 108 1.89 108 1.30 108 0.59 108 1.70 108 0.90 108 1.70 108 1.90 108 0.60 108 0.90 108 0.90 108 2.30 108 1.29 108 1.92 108 1.91 108 2.60 108 4.30 108 1.28 108 1.27 108 0.60 109
4 2 4 3 3 3 4 3 4 3 3 4 4 4 3 3 3 3 3 1 3 3 4
4 1 1
2 1 2 1
Data are mean SD values of three independent experiments. ND, not detectable. value (% of amount of samples needed to scavenge 50% of DPPH, i.e., 125 M). The positive control used was vitamin C (IC50 value 26.09). bIC 50 value (mL of amount of samples needed to reduce by 50% the adrenochrome formation). cIn mol of decomposed H O /minute. 2 2 aIC 50
degenerative diseases in humans, including cancer, cardiovascular diseases, cataracts, and brain dysfunction.32,33 The best-described property of almost every group of polyphenols is their capacity to act as antioxidants able to scavenge free radicals and reactive oxygen species.34,35 Carotenoids are essential components of the photosynthetic apparatus in plants; they protect against photooxidative damage and contribute to light harvesting for photosynthesis. In humans, some carotenoids serve as precursors for vitamin A36 and possess antioxidant activity, acting as a singlet quencher.37 Vitamin C is a relevant micronutrient, mainly required as a co-factor for enzymes involved in oxidation-reduction reactions.38,39 It has been studied for its protective action against different diseases,40,41 and it is able to scavenge reactive oxygen and nitrogen species, protecting other biomolecules from oxidative damage.42 All pulps evaluated in this study showed significant values of polyphenols, carotenoids, and vitamin C (Table 2), turning out to be important nutritional choices for diets, mainly in places where the intake of fresh fruits is not feasible. The freezing process for fruits/juices may reduce vitamin C43 and carotenoid44 contents, but no significant differences were reported in total polyphenol levels after freezing of orange juice8 and raspberry pulp.43
DPPH scavenging is a widely used method to evaluate antioxidant activities in a relative short time when compared with other methods,45 and it is very useful for screening a large number of samples. Seventy-three percent of the samples presented activity similar to the positive control— vitamin C. A positive correlation between DPPH scavenging ability and carotenoid content was observed, suggesting that these compounds could have participation in the antioxidant activity reported. Pulps with reduced carotenoid and polyphenol levels (apple, coconut, kiwi, and red grape) also showed an important antioxidant activity in this work. Methanolic extract of red guava, cashew, acerola, soursop, and acai showed in vitro antioxidant activity, but without correlation with total phenolic content and vitamin C.46 For protection against free radicals and reactive species, aerobic organisms have developed intricate and interrelated processes, which include SOD and CAT enzymes. SOD catalyzes the dismutation of superoxide anion (O2) to oxygen and hydrogen peroxide, while CAT converts hydrogen peroxide to water and molecular oxygen.11 SOD and CAT enzymes have an important role in maintaining physiological redox equilibrium, avoiding or decreasing the oxidative stress. In this study, most samples (56%) showed SOD-like activity, and all presented CAT-like activity (Table 3). Vitamin C and some
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TABLE 4. REVERSION OF POINT MUTATIONS (HIS1-7 AND OCHRE ALLELE [LYS1-1]) AND FRAMESHIFT MUTATION (HOM3-10) IN A HAPLOID XV 185-14C STRAIN OF S. CEREVISIAE AFTER TREATMENT WITH FROZEN FRUITS IN THE STATIONARY PHASE OF GROWTH FOR 4 HOURS 1/107 survivors Treatment Controls Negative Positive Concentration (wt/vol) 5% Acai Cashew apple Kiwi fruit Strawberry 10% Acai Cashew apple Kiwi fruit Strawberry 15% Acai Cashew apple Kiwi fruit Strawberry
Survival (%)
Hisa
Lysb
Homa
100 (79) 62 (49)
5.04 0.76 (12) 95.80 1.98 (241)*
5.91 1.12 (14) 190.05 17.47 (452)*
3.02 0.88 (7) 140.05 14.50 (330)*
100 100 100 99
(81) (79) (80) (78)
42.45 53.27 33.85 47.00
2.76 6.60 7.42 8.63
(101)* (127)* (79)* (115)*
47.77 36.05 33.85 35.45
2.45 8.84 9.69 7.42
(113)* (84)* (70)* (97)*
65.25 37.35 41.75 50.35
7.00 9.11 2.52 7.71
(154)* (87)* (94)* (118)*
100 100 100 99
(82) (83) (85) (78)
64.80 53.60 47.30 53.35
2.76 6.60 7.42 8.63
(155)* (126)* (111)* (130)*
81.5 55.85 52.69 68.95
2.40 3.36 5.84 8.60
(192)* (131)* (100)* (166)*
99.45 60.05 67.10 67.45
6.43 0.92 8.20 5.16
(236)* (126)* (156)* (158)*
99 100 100 100
(95) (90) (80) (85)
139.85 54.80 52.15 47.55
7.00 7.96 2.55 1.77
(331)* (123)* (99)* (118)*
129.55 64.78 56.69 75.76
5.91 2.87 7.07 0.14
(305)* (152)* (133)* (174)*
154.91 102.05 66.10 81.54
8.81 3.96 6.36 7.11
(366)* (242)* (155)* (192)*
The negative control was saline; the positive control was 75 mM H2O2. Survival data are percentages of the total number of colonies for the plate (number of colonies). Revertant survivor data are mean SD values of three independent experiments. aLocus-specific revertants. bNon–locus-specific revertants (forward mutation). *Data significantly different in relation to the negative control group (saline) by analysis of variance and Tukey’s post hoc test (P .05).
TABLE 5.
REVERSION OF POINT MUTATIONS (HIS1-7 AND OCHRE ALLELE [LYS1-1]) AND FRAMESHIFT MUTATION (HOM3-10) HAPLOID S. CEREVISIAE STRAIN XV 185-14C AFTER CO-TREATMENT WITH THE FROZEN FRUITS (5%) (WT/VOL) AND HYDROGEN PEROXIDE (75 MM) IN THE STATIONARY PHASE OF GROWTH FOR 4 HOURS
IN A
1/107 survivors Treatment Controls Negative H2O2 Apple H2O2 Coconut H2O2 Cupuacu H2O2 Mango H2O2 Orange H2O2 Papaya H2O2 Peach H2O2 Raspberry H2O2 Red grape H2O2 Red guava H2O2 Soursop H2O2 Tangerine H2O2
Hisa
Survival (%) 100 45 82 100 92 91 95 100 88 92 100 100 96 100
(392) (173) (310) (406) (356) (345) (362) (413) (333) (350) (403) (398) (364) (430)
6.80 103.74 16.16 18.71 14.46 11.05 17.01 5.95 8.50 26.36 9.35 25.51 23.81 18.71
1.50 (8) 16.99 (122) 1.30 (19)* 1.54 (22)* 2.95 (17)* 1.56 (13)* 2.95 (20)* 1.25 (7)* 2.95 (10)* 3.90 (31)* 1.34 (11)* 5.10 (30)* 1.47 (28)* 1.53 (22)*
Lysb 5.10 106.29 17.01 2.55 2.55 7.65 1.70 5.95 9.35 1.70 8.50 5.10 4.25 7.65
0.35 (6) 24.51 (125) 1.74 (20)* 0.05 (3)* 0.05 (3)* 0.05 (9)* 0.59 (2)* 1.25 (7)* 2.31 (11)* 0.47 (2)* 1.47 (10)* 1.42 (6)* 1.38 (5)* 0.10 (9)*
Homa 3.40 113.95 14.46 7.65 3.40 5.10 5.10 17.01 8.50 6.80 12.76 1.70 8.50 11.05
1.01 5.89 1.56 0.98 0.95 0.04 0.96 1.50 1.54 1.43 0.06 0.76 1.31 1.47
(4) (134) (17)* (9)* (4)* (6)* (6)* (20)* (10)* (8)* (15)* (2)* (10)* (13)*
The negative control was saline; the H2O2 control was 75 mM H2O2. Survival data are percentages of the total number of colonies for the plate (number of colonies). Revertant survivor data are mean SD values of three independent experiments. aLocus-specific revertants. bNon–locus-specific revertants (forward mutation). *Data significant in relation to the control group (H2O2) by analysis of variance and Tukey’s post hoc test (P .05).
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flavonoids, such as catechin,4 have been reported as superoxide radical and hydrogen peroxide scavengers, suggesting that the antioxidant activity observed may be associated, at least in part, with the ability of scavenging reactive species. Only four (acai, cashew apple, kiwi fruit, and strawberry) of the 23 samples showed mutagenic effects (Table 4) when assayed in high concentrations, i.e., 5%, 10%, and 15% (wt/vol). Lys-revertants were positively correlated with content of carotenoids (r 0.793; P .01). His-revertants were positively correlated with contents of polyphenols (r 0.688; P .01), carotenoids (r 0.654; P .01), and ascorbic acid (r 0.640; P .05), and Hom-revertants were positively correlated with total carotenoids (r 0.701; P .05) and ascorbic acid (r 0.752; P .05). It is known that high concentrations of ascorbic acid15 and some polyphenols may induce mutagenic effects.47,48 In the presence of Cu(II) and Fe(III), these compounds may cause DNA degradation through the generation of hydroxyl radicals.9,12,47 Until now, there are no reports on mutagenic effects of carotenoids, and further studies are necessary to investigate the positive correlation we have observed between mutagenesis and carotenoid concentration. On the other hand, in this study, some fruits (acerola, orange, passion fruit, pineapple, soursop, and tangerine) also rich in vitamin C, polyphenol, and/or carotenoid contents did not show mutagenic effects under the assayed conditions. In fact, frozen orange and tangerine juices were antimutagenic. These contradictory effects should be further analyzed. Fifty-two percent of the frozen fruits showed antimutagenic activity against damages caused by hydrogen peroxide in S. cerevisiae yeast (Table 5). Antimutagenic effects and CAT-like activity were positively correlated (r 0.400, P .01), suggesting a role of this activity in antimutagenic effects. CAT is responsible for neutralizing hydrogen peroxide,11 avoiding hydroxyl radical formation and, consequently, damage to the DNA. In conclusion, in spite of the fact that some samples analyzed in high concentrations (acai, cashew fruit, kiwi fruit, and strawberry) showed mutagenic activity, the risk of mutagenic effects for humans is low, since the assayed concentrations were high. On the other hand, antimutagenic and antioxidant activities of frozen fruits shown in this work may prevent DNA damage,42,49,50 thus contributing to maintaining human health.
ACKNOWLEDGMENTS We thank the University of Caxias do Sul (Caxias do Sul, RS, Brazil) and the Research Council of the State of Rio Grande do Sul for their help and financial support. The authors are grateful to Lívia O. Soldatelli for her review and suggestions for improving the manuscript.
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