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Antioxidant Activity of Brazilian Vegetables and Its Relation Ewith Phenolic Composition

Antioxidant Activity of Brazilian Vegetables and Its Relation Ewith Phenolic Composition

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Int. J. Mol. Sci. 2012, 13, 8943-8957; doi:10.


International Journal of

Molecular Sciences
ISSN 1422-0067 www.mdpi.com/journal/ijms Article

Antioxidant Activity of Brazilian Vegetables and Its Relation with Phenolic Composition
Ana P. Tiveron, Priscilla S. Melo, Keityane B. Bergamaschi, Thais M. F. S. Vieira, Marisa A. B. Regitano-d’Arce and Severino M. Alencar * Department of Agri-Food Industry, Food and Nutrition, “Luiz de Queiroz” College of Agriculture, University of São Paulo, Piracicaba, SP, Av. Pádua Dias, 11, CEP 13418-900, Brazil; E-Mails: anapaulativeron@gmail.com (A.P.T.); priscilla_esalq@yahoo.com.br (P.S.M.); keityberga@yahoo.com.br (K.B.B.); tvieira@usp.br (T.M.F.S.V.); marisadarce@usp.br (M.A.B.R.A.) * Author to whom correspondence should be addressed; E-Mail: smalencar@usp.br; Tel.: +55-19-34294150; Fax: +55-19-34294288. Received: 21 February 2012; in revised form: 9 June 2012 / Accepted: 10 July 2012 / Published: 18 July 2012

Abstract: Vegetables are widely consumed in Brazil and exported to several countries. This study was performed to evaluate the phenolic content and antioxidant activity of vegetables commonly consumed in Brazil using five different methods, namely DPPH and ABTS free radical, β-carotene bleaching, reduction of Fe3+ (FRAP), oxidative stability in Rancimat, and the chemical composition using gas chromatography-mass spectrometry (GC-MS). The content of phenolic compounds ranged from 1.2 mg GA/g (carrot) to 16.9 mg GA/g (lettuce). Vegetables presenting the highest antioxidant activity were lettuce (77.2 µmol Trolox/g DPPH•; 447.1 µmol F2+/g FRAP), turmeric (118.6 µmol Trolox/g ABTS•+; 92.8% β-carotene), watercress and broccoli (protective factor 1.29—Rancimat method). Artichoke, spinach, broccoli, and asparagus also showed considerable antioxidant activity. The most frequent phenolic compounds identified by GC-MS were ferulic, caffeic, p-coumaric, 2-dihydroxybenzoic, 2,5-dihydroxybenzoic acids, and quercetin. We observed antioxidant activity in several vegetables and our results point out their importance in the diet. Keywords: antioxidant activity; phenolic compounds; vegetables; GC-MS

Fe3+-TPTZ: ferric tripyridyltriazine. effectively delays or inhibits oxidation of the substrate [6]. On the one hand. ABTS•+: 2. are easier to apply. Phenolic compounds are secondary metabolites commonly found in plants. a: absorbance at initial time (0 min). Although synthetic antioxidants are very effective and stable. Vitamins and phytochemicals. Many methods are available for analyzing antioxidant activity.10]. carotenoids. DW: dry weight. but present some limitations. indirect methods. J. In the last few years. Several techniques have been used to determine the in vitro antioxidant activity in order to allow rapid screening of promising substances and/or mixtures. the methods evaluate the free radical scavenging ability of antioxidant compounds. GLM: general linear models. and applications [12–14]. PIa: IP of the oil with the vegetable extracts or standards. Recent studies have shown the importance of vegetables in a healthy diet and to prevent degenerative diseases caused by oxidative stress [1]. their advantages and limitations are still under discussion and no consensus has been reached to define a unique standard method capable of encompassing all the peculiarities exhibited by the different classes of antioxidants [11]. evaluation of the antioxidant potential of food. and FRAP. PF: protection factor. in some circumstances. although.2'-azinobis-3-ethylbenzotiazoline-6-sulfonic acid.Int. and biological fluids have also been improved [5. as well as natural. it is also highly interesting to find new and safe antioxidants from natural sources. natural products. GA: gallic acid. AA: antioxidant activity. useful in the defensive function against pathogens and radiation. The interest on this field began to expand in the 1990s based on the observation that many natural products have beneficial effects on human health. 13 Abbreviations 8944 GC-MS: gas chromatography-mass spectrometry. such as ABTS•+. with different concepts. and this does not necessarily correspond to the real oxidative degradation. Furthermore. Sci. such as ascorbic acid. For the food industry. TPC: total phenolic content. they have limited use in many countries due to the possibility of causing adverse effects on human health [7. and are directly involved in the antioxidant activity [3–5]. IC50: inhibitory concentration. the . BHT: butylated hydroxytoluene. UV: ultraviolet. present in low concentration compared to an oxidized substrate. 1. and fiber have been regarded as the bioactive substances responsible for these effects [2]. Brazil is a country that presents great agro-ecological diversity and a large production of vegetables. pharmaceutical. TMS: trimethylsilyl. 2012. b: absorbance at final time (120 min). DPPH•: 2. and cosmetic products has been increasing. ways of expressing results. polyphenols.8]. IP: induction period. Antioxidants can be defined as any substance that. DPPH•. SPE: solid phase extraction. Mol. Introduction Vegetables are widely produced in Brazil both for local consumption and export to several countries. MSTFA: N-methyl-N-(trimethylsilyl)trifluoroacetamide. DRc: control degradation rat. the research and validation of methodologies for evaluating the antioxidant activity in complex matrices such as food. Plc: IP of the control (oil without the vegetable extracts or standards).2-diphenyl-1-picryl-hydrazine. Although many methods have been developed and tested in the literature. In this case.9. mechanisms of action. FRAP: ferric reducing antioxidant power. DRs: degradation rate in the presence of standard or vegetable extract. involving electron transfer reactions.

according to Becker et al. pollution. According to Gobbo-Neto and Lopes [24]. soil nutrients.05) by Tukey test. Several analytical methods have been used to evaluate antioxidant activity by free radical scavenging.16]. [23]. Results and Discussion Phenolic compounds are responsible for the antioxidant activity of vegetables. means followed by different letters differ statistically (p < 0. such as phenolic compounds. which allows the analyses of both lipophilic and hydrophilic compounds [17].9 mg GA/g sample (dry weight—DW). using the same method. very few studies have been carried out to assess vegetables grown in Brazil and consumed locally or exported to several countries. Content of phenolic compounds (mg GA/g of DW) in several vegetables grown in Brazil.2'-azinobis-3-ethylbenzotiazoline-6-sulfonic acid radical). J. since these chemically distinct methods are based on different reaction mechanisms. on oxygen depletion. UV radiation. Although in recent years the antioxidant analysis of vegetables has been extensively researched worldwide [18–22]. in carrot and lettuce. . temperature.2 to 16. but besides being one of the fastest. This study evaluates the antioxidant activity properties of the main vegetables produced and consumed in Brazil. Llorach et al. [15]. ABTS•+ method also provides good solubility. The content of phenolic compounds found in our samples ranged from 1. β-carotene bleaching and oxidative stability by the Rancimat method. water availability. Sci. based on measuring changes in the concentration of compounds being oxidized. Therefore. direct methods. 2012. 2. since in their study lettuce was one of the vegetables that presented the lowest total phenolic content.2-diphenyl-1picrylhydrazyl radical) and ABTS (2. [22] found higher levels of phenolic compounds in red-leafed varieties of lettuce compared to green ones. several factors such as seasonality. and pathogen attack can affect the content of secondary metabolites in vegetables. On the other hand.Int. such as β-carotene and Rancimat. including antioxidant activity by scavenging abilities on DPPH (2. Content and profile of potential antioxidant in these vegetables are also examined. respectively (Figure 1). it is important to use different methods in order to obtain a more thorough assessment of the antioxidant potential of a sample [10. Antioxidant properties are determined in various test systems with different mechanisms of action. reducing power test. are characterized by their ability to inhibit or halt lipid oxidation in model systems. Figure 1. These results were different from the findings reported by Chu et al. or on formation of oxidation products. Mol. 13 8945 donation of hydrogen atoms (or electrons) correlates with the antioxidant activity.

1. One factor that may influence this analysis is the medium where the reaction occurs. a result similar to our findings.1 μmol Trolox/g) and lettuce (85. and watercress (77.3 µmol Fe2+/g) (Table 1).4%) presented the lowest result.29) and chives (1. The Rancimat method is widely used for the determination of the oxidative stability of natural fats and oils. phenolic compounds found in plants have antioxidant and anticancer activities. Studying wild artichoke from Slovakia. depending on the polarity of the sample.Int. had a longer induction period compared to the control (pure soybean oil.7 µmol Fe2+/g).2. Sci. respectively [20]. In this regard.1 µmol Fe2+/g). 48. without added antioxidants). Turmeric (92. 50. All vegetables tested. 2012.6. consequently. The results presented in Table 1 show that using the ABTS•+ method. Brazilian artichoke showed much lower ability to reduce Fe3+ (98.29).8%) and lettuce (90%) showed the highest antioxidant activity by the β-carotene bleaching method. Kukić et al.0 µmol Trolox/g. the termination phase.8 μmol Trolox/g). respectively. followed by watercress (97. J. The analysis of the antioxidant activity of commonly consumed vegetables grown in Colorado (USA) using the ABTS•+ method showed that spinach and broccoli presented antioxidant activity of approximately 50 µmol Trolox/g and 40 µmol Trolox/g.4 µmol Fe2+/g). thus delaying the propagation phase and. and 340 µmol Fe2+/g for aqueous extracts. 57. while escarole showed no protection against lipid oxidation. watercress (277. [28] reported 350 µmol Fe2+/g in dry basis for ethanol extracts. except for escarole. artichoke. 13 8946 According to the literature. escarole. 70. soybean oil added to vegetable extracts required a longer time to form free radicals. with a protection factor and consequent antioxidant activity (Table 1). spinach.2 µmol Trolox/g and 43.24).0 µmol Trolox/g. characterized as an emulsion. with polar and non-polar regions simultaneously.1. in the present study. turmeric. broccoli (1.9. some authors highlighted the antioxidant activity of curcumin. . The highest antioxidant activities by the DPPH• method were found in lettuce. of 41. In contrast. it can interact more or less intensely with the emulsion. The highest ability to reduce Fe3+ to Fe2+ was found in lettuce (447. and spinach (273. while turnip (3. except the escarole. The process is carried out under high temperatures and constant aeration. The induction period is characterized by change in conductivity of deionized water due to oxidation-generated products. It could be inferred that. a naturally occurring phenolic compound of turmeric that is responsible for its peculiar color and important antioxidant and anticancer activities [25–27]. respectively) (Table 1). turmeric had the highest antioxidant activity (118. Therefore. The vegetables that showed the highest protection factors were watercress (1. and 44. Mol.6 μmol Trolox/g). reactive molecules that trigger the initiation phase of the oxidation process.

5 ±3.7 jk 49.2-diphenyl-1-picryl-hydrazine.4 ± 1.07 mn 3.02 ± 0.98 ± 0.33 lmn 9.7 m 70.5 ± 2.5 ± 4.22 hij 8.23 n 48.01 f 12.04 ± 0.03 no 25. J.39 jkl 61.6 ± 0. Values represent the average of triplicates ± standard deviation.94 1.82 ab •+ 8947 β-carotene (%) 57.095 klmn 26.40 g 11.5 ±0.3 ±1.9 ± 0.04 cd 188.04 ± 0.2 ± 1.26 lmn 50. Antioxidant activity of commonly consumed vegetables in Brazil.8 ± 0.26 * b 273.77 * h 90. ABTS•+: 2.4 ± 0.6 lm 90.07 * gh 113.64 d 1.7 ± 6.5 ± 0.83 a 10.0 de 1.01 cde 169.1 ± 0.21 ef 0.10 ± 0.10 ± 0.97 f 8.7 ± 4.3 ± 0.7 ± 1.8 ± 0.1 ± 0.78 1.39 1.9 ± 4.3 ± 2. 1507.2 ± 2. 11762.1 ± 0.09 ± 0.05 a i 49.05 def jk 15.8 ± 1.3 ± 1.0 de 82.5 ± 0.2 ± 5.1 ± 5. * Induction period not calculated due to the large amount of samples required to achieve the concentration of 100 ppm of total phenolic compounds.1 ± 2.0 ± 0.38 lmno 54.36 1.2 i 82.12 mn 77.090 (Rancimat).05 ± 0.2 ± 2.2 ± 0.5 bc 65.5 ± 0.16 ± 0.0 ± 1.03 ab 16. .44 b 15.5 ± 0.5 ± 7.53 de 25.21 cd 2.0 ± 3.6 ± 1.06 k * 447.0 ± 2.6 ± 3.42 klmn 97.43 * b 277.0 ± 4.8 ± 0.0 ± 1.0 ± 4. 3462.5 ± 9.8 ± 0.8 ± 1.14 ijkl 2.34 bc 30.8 ± 4.3 ± 1.04 def jk 15. α-tocopherol (100 ppm)—2855.0 µmol Trolox/g (ABTS•+).47 ij 5.7 jk 54.0 ± 2.17 ± 0.1 ± 5.2'-azinobis-3-ethylbenzotiazoline-6-sulfonic acid.4 ± 0.58 ijk 0.9 ± 0.8 ± 5.12 ijk 57.01 bc ij 43.1 ± 4.9 c 28.2 ± 4.9 ± 0.8 ± 1.29 ± 0.0 j 15.7 gh 16.29 ± 0.7 ± 3.9 def 92.2 µmol Trolox/g (DPPH).3 µmol Trolox/g (DPPH).7 ± 0. means followed by different letters in the same column differ statistically (p < 0.48 jklm 19.3 ± 5.4 ± 0.5 h 36.24 ± 0.6 ± 2.80 efg 43.Int.4 ± 0.10 1.12 ghi 118.1 ± 1.7 ± 1.44 gh 1.4 ± 8. Mol.42 defg 25.53 e Reference values: BHT (100 ppm)—1666.4 µmol Trolox/g (FRAP).7 ± 2.005 cde h 95.67 k * c 217.7 ± 0.92 * 13.7 ± 2.0 ± 1.64 jk * 148.7 ± 2. Sci.11 ± 0.11 1.93 ijk 8.51 lmno 41.55 a 1.2 gh 67.77 a 6.9 ± 3.6 ± 3.21% (β-carotene).75 ghi 3.9 c 9.6% (β-carotene).0 ± 3.54 defg 37.8± 5.33 jklm 44.3 fgh 88.13 hij 11.06 fghi 10. 93.31 d 9. 88 ± 1.9 µmol Trolox/g (ABTS•+).38 h 33.05) by the Tukey test. 2 ± 6. DPPH: 2.6 d Antioxidant activity FRAP (µmol Fe2+/g DW) Rancimat (protection factor) 98.002 ef 104. 1. FRAP: ferric reducing antioxidant power. 200.21 jklmn 85.3 de 85.0 ab 60.32 * k 13.3 gh 22.10 h 1. Vegetable Artichoke Asparagus Broccoli Cabbage Carrot Celery Chicory Chives Cucumber Escarole Leek Lettuce Parsley Pumpkin Radish Red beet Rocket Snap beans Spinach Swiss chard Turmeric Turnip Watercress ABTS (µmol Trolox/g DW) 39.1 ± 0.6 ± 3. 2012.2 ± 0.01 a DPPH (µmol Trolox/g DW) 70.5 ± 1.6 ± 0.61 de 3.1 ± 6.5 µmol Trolox/g (FRAP).48 def 15.58 fg 1. 13 Table 1.85 * 130.9 ± 1.01 def jk 19.2 ± 0.5 ± 0.26 hi 3.8 abc 54.8 i 67.2 ± 1.3 ± 6.2 ± 0.13 o 2.3 ± 0.98fgh 11.2 ± 6.9 ± 4.9 ± 3.39 c 7.3 a 3.1 ± 4.35 ijk 3.8 ± 0.05 bc i 50.9 ± 1.4 ± 2.1 efg 54.8 ± 3.0 ± 1.

All vegetables except for escarole have considerable amounts of total phenolic compounds and consequent significant antioxidant activity.47%). Table 2. J. ABTS TPC vs. 2012.14 mg/mL). The vegetable that showed the lowest extract concentration to reduce the initial amount of DPPH radical by 50% was lettuce (17.2-diphenyl-1-picryl-hydrazine. Figure 2. followed by artichoke (18.2 mg/mL). The correlation between them becomes important and is presented in Table 2.89 0. and escarole were calculated and are shown in Figure 2. ABTS: 2. spinach. artichoke. spinach (22. and escarole (32.14 mg/mL).82 0. 13 8948 Based on the highest results using the DPPH• method. DPPH TPC vs. The amount of phenolic compounds found in the vegetables evaluated in this study showed no direct relationship with their antioxidant activity using some methods. β-carotene TPC vs. Rancimat r 0. Correlation TPC vs. Vegetables presenting the lowest IC50 values can be considered better in terms of antioxidant activity. turmeric. Mol.18 r2 (%) 80.64 27. Sci. whereas the DPPH• and FRAP methods (80. Pearson’s correlation coefficient between total phenolic compounds (TPC) and total antioxidant activity. turmeric (21.85 68.87 mg/mL).49 61.79 0. The lowest correlation between phenolic compound content and antioxidant activity was observed for the Rancimat method (r2 = 3.64% and 68. the IC50 of lettuce.47 TPC: total phenolic content. respectively) showed the highest correlations. since a lower concentration to reduce the DPPH free radical by 50% is required. . DPPH: 2. This fact could be explained by the different characteristics and mechanisms of action of the bioactive compounds present in the samples as well as the different principles used to detect antioxidant properties in each method. FRAP: ferric reducing antioxidant power.52 0.2'-azinobis-3-ethylbenzotiazoline6-sulfonic acid. The results reveal lack of consistency among these methodologies.60%. Concentration of vegetable extract required to reduce the initial DPPH radical by 50%. FRAP TPC vs.Int.07 mg/mL).60 3. Table 3 shows the correlation between the methodologies used in this study to evaluate antioxidant activity in vegetables.

60%). since this is a compound with high electron donation capacity. validated. gallic. Several compounds derived from benzoic acid (3-hydroxybenzoic. while the Rancimat vs. β-carotene 0. The most abundant phenolic compound derived from cinnamic acid present in the samples assessed in this study was caffeic acid. J. 2-hydroxybenzoic. Rancimat 0. and 2.56%) chives (0. syringic. The method that best correlated with Rancimat was β-caroten (19.52 27.93 β-carotene vs.5-dihydroxybenzoic.09 0.65 DPPH vs.74 56.75 56. and escarole (3. ABTS 0.09 DPPH: 2. Mol.22 4. snap beans (3. [22] detected luteolin and quercetin derivatives in lettuce.71 51. FRAP (56. ABTS 0.58 34.08%).4-dihydroxybenzoic acids) and from cinnamic acid (sinapic. FRAP (56. both considered direct methods.54%) showed the greatest amount of this compound. DuPont et al.32%).41%). [29] analyzed the flavonoids of Cichorium endivia in the United Kingdom. cabbage.08 ABTS vs. 13 8949 Table 3.65%) and ABTS•+ vs. . p-coumaric.02%). asparagus (5.36 DPPH vs. The best correlations were found for DPPH• vs.03 DPPH vs. quercetin was found in lettuce (2. Sci.2-diphenyl-1-picryl-hydrazine. and standardized methods.44 19. FRAP 0. and watercress. The ascorbic acid identified in some of our samples may have contributed to the antioxidant activity of vegetables. FRAP did not show a good correlation (0. Rancimat 0. 2012. with more information available should be chosen.Int.09%). Correlation r r2 (%) DPPH vs.9%). Rancimat 0. Llorach et al. and spinach (53. Regarding flavonoids.97 FRAP vs. Rancimat 0. despite the difficulty to choose the most appropriate method for the evaluation of antioxidant activity. ABTS: 2. parsley.67%) and kaempferol in chives (4. FRAP 0.19%). and caffeic acids) were found. The results of the phenolic composition obtained using GC-MS are presented in Table 4.03 0. FRAP: ferric reducing antioxidant power.07 β-carotene vs. the most commonly accepted. corroborating the results found in this work. rocket. and also identified the presence of kaempferol. already known for its high antioxidant activity (Table 4). 2. chives. chicory (1. FRAP 0. Therefore.9 ABTS vs. Pearson’s correlation coefficient between the different antioxidant activity methodologies. such as in the cases of broccoli.2'-azinobis-3-ethylbenzotiazoline-6-sulfonic acid.53 28. in amounts that varied according to the cultivar.93%) and snap beans (8.67 β-carotene vs.

.13 – – – – – 0.14 – – – – – 0.28 – 0. 2012.60 0.21 – – – – – – – 1. 7: Sinapic acid.08 – – 0.43 14. J.61 0. 9: Ferulic acid. 2: 3-hydroxybenzoic acid.12 – – – 0.19 – – – – – – – 3.60 – 3.66 11 – – – – – – – 4.41 – – – – 1.63 0.63 – – – – – 12.32 0. 3: Syringic acid.35 – – 8950 1: Ascorbic acid.4-dihydroxybenzoic acid.39 – 47.32 – – – – – 0. Mol.50 – – – – – – – – 1.90 – – – – – 0.31 – – 0.38 – – – – – – – – – – – – 0.57 – – 14 – – – – – 0.68 – – – – 1.15 – – – 2.89 – – – – – 1. Concentration (%) of compounds identified in ethanol extracts of the vegetables analyzed using GC-MS.16 0.81 1.51 – 6.Int.56 – – – – 1.71 0. 10: Caffeic acid.14 – – 2.72 – 1.60 – 26.46 – – – – 0. 5: 2-hydroxybenzoic acid.41 – – – – – 12 – 5.82 – 5.82 – – – – 0.40 1.72 15. 12: Quercetin.81 1.22 42.22 – – – – – – – – – 0.93 – – – 2.13 0. 14: 2. 11: Kaempferol.42 0.06 0.08 9. GC-MS: gas cromatography-mass spectrometry. Sci.67 – – 0.12 – – – – – – – – – 9.99 – – – – – 0.10 2.31 0.92 1.14 – – 0.61 0. Vegetable ethanol extract Artichoke Asparagus Broccoli Cabbage Carrot Celery Chicory Chives Cucumber Escarole Leek Lettuce Parsley Pumpkin Radish Red beet Rocket Snap beans Spinach Swiss chard Turmeric Turnip Watercress Area of the component (%) 1 2 3 4 5 6 7 8 9 10 – – – 0.88 – – – – – – – 0.54 – – – – 0. 8: p-coumaric acid.78 – 10.23 – – – 0.42 – 0.49 – – – 1.43 0. 13: Isovanillic acid.26 – – – – – 0.67 – – – – – 13 – – – – 0.41 12.60 53.54 0.85 – 1. 6: Gallic acid.61 1.48 0.24 – 10. 13 Table 4.60 0.01 – – – – – 32.53 – – – – – – – 0.95 0.19 0.96 – – – – – 0.04 0. 4: 2.73 – – 0.78 – – 0.5-dihydroxybenzoic acid.52 – – – 0.18 – – 9.02 – – – – – 8.56 – – – 1.96 – – – – – – – – 0.

USA). 2. ferrous sulfate. capitata). italic Plenck). (18) snap beans (Phaseolus vulgaris L.). the tubes were sonicated for 5 min. (13) parsley (Petroselinum crispum (Mill. and shaken thoroughly. The mixture was allowed to sit for 3–8 min. β-carotene. . Japan). (21) turmeric (Curcuma longa L. Mol. (19) spinach (Tetragonia expansa L. (22) turnip (Brassica rapa L.).).). the absorbance was measured at 740 nm using a UV-mini 1240 spectrophotometer (Shimadzu. [30.31]. wet tropical climate. (11) leek (Allium porrum L. tripyridyltriazine. (15) radish (Raphanus sativus L. A blank test was also performed under the same conditions and the results of total phenolic compounds were expressed as gallic acid equivalent (mg GA/g sample DW). (4) cabbage (Brassica oleracea L.).).).3.1. J.) Nym. (12) lettuce (Lactuca sativa L. (3) broccoli (Brassica oleracea L.).). Experimental Section 3. (16) red beet (Beta vulgaris L. Samples were first frozen at −18 °C and then lyophilised. Sample Collection and Extraction All the vegetables used were produced in the São Paulo State. MO. from January to October.5 mL of Folin Ciocalteau reagent diluted in water at 1:10. mixed with 20 mL of 80% ethanol (v/v). var. (7) chicory (Cichorium intybus L. 22°43'31'' S and 47°38'57'' W. and standards for GC-MS were purchased from Sigma-Aldrich (St. 2012.).5 mL of the solution obtained was transferred to a tube with 2.). then 2 mL of sodium carbonate 4% was added.). in Southeastern Brazil. The other reagents were purchased from local sources. (6) celery (Apium graveolens L.Int. Sci. All samples were extracted in triplicate. ferric chloride. The cartridges used for SPE-LC18 chromatographic analysis were obtained from Supelco (Bellefonte. (8) chives (Allium fistolosum L. The extraction of the compounds of interest from the vegetable samples was performed according to the method described by Kähkönen et al. linoleic acid.). USA). (17) rocket (Eruca sativa L. (2) asparagus (Asparagus officinalis L.). Subsequently. [3] with some modifications. 2.). (9) cucumber (Cucumis sativus L. var. Afterwards. var.). Chemicals and Reagents 8951 Gallic acid. (14) pumpkin (Cucurbita maxima Duch. 3. 13 3. Louis. based on a calibration curve of gallic acid in the concentration range of 5 to 80 μg/mL. cicla).). the material was ground and 1 g of each vegetable powder was placed in Falcon tubes. The vegetable extracts were diluted in ethanol 80% and 0. (5) carrot (Daucus carota L. After that. N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA). and the tubes were kept in the dark for 2 h. (10) escarole (Cichorium endivia L. The following vegetable species were assessed: (1) artichoke (Cynara scolymus L.2'-azinobis-3-ethylbenzotiazoline-6-sulfonic acid (ABTS•+).2.2-diphenyl-1-picryl-hydrazine (DPPH•). (20) Swiss chard (Beta vulgaris L. and the supernatant collected for analysis. Determination of Total Phenolic Content Total phenolic content analysis was performed using the Folin Ciocalteau spectrophotometric method described by Singleton et al. (23) watercress (Nasturtium officinale). PA.). 3. centrifuged for 15 min at 5000× g.).

0 mL of pure ethanol. 3. Mol.700 ± 0. The chloroform was removed using a stream of nitrogen and the residue obtained was redissolved in 100 mL of water aerated for 30 min..5. Japan) at 470 nm. i. using a UV-mini 1240 spectrophotometer (Shimadzu. The emulsion oxidation was monitored using a UV-mini 1240 spectrophotometer (Shimadzu. The ABTS radical was formed through the reaction of ABTS•+ solution 7 mM with potassium persulfate solution 140 mM. which was incubated at room temperature for 45 min. and readings were monitored at 517 nm. Once formed. The antioxidant activity measured by DPPH free radical method can be expressed as IC50. The absorbance was read at 734 nm after 6 min of the reaction using ethanol as a blank.020 at 734 nm. The antioxidant activity was expressed as percentage of relative inhibition compared to control samples after 120 min and calculated as: % AA = [(DRc − DRs)/DRc] × 100 where AA = antioxidant activity.0 mL of ABTS radical in the dark.e.6. Aliquots of 3 mL of the resultant β-carotene/linoleic acid emulsion were mixed with 50 μL of the vegetable extracts and incubated in a water bath at 50 °C. 3. b = absorbance at final time (120 min). at baseline and at 20-min intervals for 2 h. a synthetic water-soluble antioxidant analogue of vitamin E.Int. incubated at 25 °C in the dark for 12–16 h. J. and 0. The concentration of vegetable samples required to reduce the initial DPPH radical by 50% is expressed in mg/mL. Control samples contained only solvent in place of the vegetable extracts. [33] with some modifications. a = absorbance at initial time (0 min).3 mL of DPPH radical in ethanol solution 0.4. 2012. 30 μL of each vegetable extract dilution were transferred to test tubes with 3. . [17] with modifications. DRc = control degradation rate (ln (a/b)/120).5 mL of standards or vegetable extracts. Antioxidant Activity Using β-Carotene Bleaching Method The antioxidant activity assessment was performed according to the method described by Emmons et al. The calibration curve was constructed with the standard Trolox in the concentration range of 0 to 200 µM Trolox. the radical was diluted with ethanol P. Several vegetable extract concentrations were used. depending on the sample. BHT and α-tocopherol (200 ppm) were used as reference standards. DPPH Free Radical Scavenging Assay and IC50 8952 The reaction mixture consisted of 0. every 20 min for 140 or 160 min. An emulsion was prepared by dissolving 10 mg of β-carotene in 100 mL of chloroform PA and aliquots of 3 mL were added to 40 mg of linoleic acid and 400 mg of Tween 40. After that.2'-azinobis-3-ethylbenzotiazoline-6-sulfonic acid) was assessed according to the method described by Re et al. DRs = degradation rate in the presence of standard or vegetable extract (ln (a/b)/120). Japan).5 mM. until a constant reading was obtained. to an absorbance of 0. Three different dilutions of each vegetable extract were prepared in triplicate. 3. and the activity was expressed in µmol Trolox/g of sample DW [32]. Trolox. Sci. the antioxidant concentration required to reduce the initial DPPH radical by 50%. 13 3. Antioxidant Activity Using ABTS•+ Assay The antioxidant activity by the ABTS•+ method (2.A.

1. which could mask the compounds of interest.6). The protection factor was calculated as: % PF = (PIa/PIc) × 100 where PF = protection factor. PA. in acidic medium (pH 3.Int. 3. LC-18 SPE cartridges (2 g. Ferric Reducing Antioxidant Power Assay (FRAP) To determine the antioxidant activity by iron reduction. we followed the methodology described by Benzie and Strain [34] with some modifications. forming an intense blue color as the ferric tripyridyltriazine (Fe3+-TPTZ) complex is reduced to the ferrous (Fe2+) form. based on the content of phenolic compounds. The oxidative stability index of this mixture was measured by the Rancimat method according to AOCS Cd 12b-92 [35] at 110 ± 1 °C and a flow rate of 9 L/h of dry air. Mol. and 2.9.7. using the ferric reducing antioxidant power (FRAP) assay. Bellefonte. 2. After this time.8. Sci. J. FRAP reagent was prepared immediately before analysis by mixing 25 mL of acetate buffer (300 mM. A control was prepared with pure soybean oil without added antioxidants and samples containing synthetic antioxidant BHT (100 ppm) were also submitted to this analysis. The conductivity increase due to the accumulation of oxidized compounds plotted as a function of the reaction time allowed the construction of the curve and the calculation of the induction period (IP).0). without added antioxidants) were mixed with the vegetable extracts at a concentration of 100 ppm. 3. 4 mL of each vegetable ethanol extract once again evaporated and redissolved in 4 mL of water were added to their respective cartridges.9. 13 8953 was used as reference at concentrations ranging from 100 to 2000 µM and the results were expressed as µM Trolox/g sample. Removal of Outliers from Samples Using the Solid Phase Extraction (SPE) Technique The Solid Phase Extraction (SPE) technique was employed for the removal of sugars. 3. Switzerland).5 mL of TPTZ solution (10 mM TPTZ in 40 mM HCl). FRAP measures the ferric reducing ability of the samples. After the vegetable extract completely passed through. efficient. Chromatographic Analysis 3. and requires very small volumes of samples and solvents. CH-9100 Herisau. Plc = IP of the control (oil without the vegetable extracts or standards). using 743 Rancimat (Metrohm AG. Oxidative Stability—Rancimat Samples of 5 g of pure soybean oil (supplied by Cargill. the absorbance was measured using a UV-mini 1240 spectrophotometer (Shimadzu. The calibration curve was constructed using ferrous sulfate (100–2000 μM) and the results were expressed in µmol Fe2+/mg.5 mL of FeCl3 (20 mM) in aqueous solution. the column . Japan) that was reset with FRAP solution. This technique has been increasingly used because it is quick. Subsequently. pH 3. USA) were conditioned with methanol and acidic water (pH = 2. An aliquot of 100 µL of the vegetable extracts was added to 3 mL of FRAP reagent and incubated in a water bath at 37 °C for 30 min. Supelco. 2012.6). PIa = IP of the oil with the vegetable extracts or standards.

presenting high molecular weight compounds and/or strongly polar functional groups. Derivatization—Formation of Trimethylsilyl Derivatives (TMS) Prior to the GC-MS analysis. 13 8954 was washed with sufficient acidic water to remove the sugars. Samples not showing this profile. presenting characteristics suitable for analysis [36]. remaining at 310 °C for 5 min (5 min 40 s).3. 3. remaining at 300 °C for 5 min (13 min 20 s). Derivatized samples were separated using a capillary column (RTX-5MS 30 m × 0. The reaction mixture was homogenized and incubated at 70 °C for 10 min. ferulic acid. Kyoto. phenolic acids. totaling 40 min of analysis.9. and thermally stable substances.. Compounds of interest were eluted with methanol into coded glass vials and their bands were identified under UV light.. the supernatant was transferred to a vial and injected into the GC-MS system. remaining at 320 °C for 10 min (10 min 30 s). volatile.Int. Gas Chromatography-Mass Spectrometry (GC-MS) GC-MS analyses of the vegetable extracts were performed on a gas chromatograph GC 2010 (Shimadzu Corp. Data integration was performed using the LabSolutions-GCMS software. rutin. 3. Japan) coupled to a mass spectrometer QP 2010 Plus (Shimadzu Corp. . After homogenization. require a derivatization procedure.25 µm). resveratrol. the samples were submitted to a crucial stage called derivatization. Kyoto.25 mm × 0. The fractions obtained after purification were added to 100 µL of derivatizing reagent MSTFA. and derivatives were identified by comparing their retention time and ion fragmentation with coded and authentic standards (quercetin. epicatechin. The reagent was evaporated under a stream of nitrogen and trimethylsilyl (TMS) derivatives were rediluted in hexane (800 µL). increasing at 15 °C/min to 310 °C. cinnamic acid) eluted under the same conditions as well as with the Wiley Version 8 library. kaempferol. remaining at 250 °C for 1 min (9 min 30 s). catechin. Flavonoids. GC-MS is only useful for the analysis of gases. The interface was maintained at 280 °C and the detector was operated in the scanning mode (m/z 40–800). increasing at 6 °C/min to 300 °C. 3.9.5 µL in splitless mode. a reaction which transforms a substance of interest into a product of similar chemical structure.10. the injector temperature was 280 °C. apigenin. caffeic acid. 2012.2. The temperature program started at 80 °C (1 min). Chemical derivatization is widely used to reduce the polarity of functional groups and facilitate their separation during GC-MS analysis. Mol. increasing at 20 °C/min to the final temperature of 320 °C. and the injection volume was 0. kaempferide. called a derivative. Japan). The Tukey test at 5% probability was used for mean comparison. p-coumaric acid. Statistical Analysis The results obtained were statistically analyzed using the SAS software [37] and the analysis of variance using the general linear models (GLM) procedure. Helium was used as the carrier gas. J. increasing at 20 °C/min to 250 °C. Sci.

J. Benzie. Several phenolic compounds with high antioxidant activity could be identified using GC-MS in vegetables. some of them present in high concentrations.. Antioxidant activity of plant extracts containing phenolic compounds. Due to the great chemical diversity of the compounds found in this study. C. Sci. 235–254. tubers and vegetables commonly consumed in India. The highest correlation of the amount of phenolic compounds was found using the DPPH• method. 1999. Hopia. Food Chem. Scalbert.C. Pokorný.C. 3.. 2010. J. Kähkönen. 1017–1020. such as caffeic acid in spinach.. and that the presence of high levels of certain classes of compounds in vegetables does not always guarantee higher antioxidant power. The samples did not present the same results in all methodologies and no defined order of antioxidant activity could be found. 6. Antioxidants from Spices and Herbs. 92. Food Res. Ed.. Antioxidant activity and phenolic content of roots. Lissi. Review of methods to determine chain-breaking antioxidant activity in food. Polyphenols: Food sources and bioavailability. M.P. Heinonen. Technol. J.. pp. F. 3954–3962. mainly phenolic acids.J. Shahidi. A. Nutrition 2004. K. L. and escarole.. 64–65. Rémésy. Sreeramulu. A. Based on this finding. the use of more than one method is more suitable for the analysis of in vitro antioxidant activity of these vegetables.. M. 8. 79. 2012. Are natural antioxidants better—and safer—than synthetic antioxidants? Eur. J..A. 2th ed. C. 2007. Int. 1989. References 1. J. C. Agric. turmeric.. One possible explanation for this could be the difference in the chemical composition of vegetables and different mediums and principles of analyses. Clin. Brazaca for her assistance in freeze-drying samples and the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) (Process No. Roginsky. 47. 629–642. M. UK. Kwok.P. Szeto. E. 2005. Rauha. Vuorela. Raghunath. 20. 7. Morand. USA. Manach.M.F. H. and broccoli presented the highest antioxidant activity among the Brazilian vegetables analyzed. 13 4. Am. J.. Food Chem. Mol.T.. B.S. 2. IL.. C. Effects of a long-term vegetarian diet on biomarkers of antioxidant status and cardiovascular disease risk. Health Effects and Applications. Pihlaja. T. V. 4. D. T. 2004.... Gutteridge. I. Lipid Sci. 109. Nutr. Kujala. . it is possible to affirm that different phenolic compounds and some non-phenolic substances present different antioxidant activities. 863–866. chicory.. 2008/52834-4). watercress. Conclusions 8955 The amount of phenolic compounds present in the vegetables analyzed showed no direct relationship with the antioxidant activity in a large number of samples. Lettuce. 43. Jiménez. 1996... N.I. Clarendon Press: Oxford. J.Int. Halliwell. In Natural Antioxidants: Chemistry. 5. 727–747. AOCS Press: Champaign. Free Radicals in Biology and Medicine. Acknowledgments The authors thank Solange G. Y. Nakatani.

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