LWT - Food Science and Technology 42 (2009) 11871192

Contents lists available at ScienceDirect

LWT - Food Science and Technology

journal homepage: www.elsevier.com/locate/lwt

Antioxidant activity, phenolic compounds and anthocyanins content of eighteen strains of Mexican maize
Leticia X. Lopez-Martinez a, Rosa M. Oliart-Ros a, Gerardo Valerio-Alfaro a, Chen-Hsien Lee b, Kirk L. Parkin b, Hugo S. Garcia a, *
a b

gico de Veracruz, M.A. de Quevedo 2779, Col. Formando Hogar, Veracruz, Ver. 91897, Mexico UNIDA, Instituto Tecnolo Department of Food Science, University of Wisconsin-Madison, 1605 Babcock Drive, Madison, WI 53706, United States

a r t i c l e i n f o
Article history: Received 1 February 2008 Received in revised form 15 October 2008 Accepted 20 October 2008 Keywords: Antioxidants Pigmented Corn Phenolics

a b s t r a c t
Free-radical scavenging, reducing activity and some phytochemical content (total phenolic, anthocyanin and ferulic acid) of eighteen Mexican maize phenotypes were determined. Total phenolic contents ranged from 215.8 to 3400.1 mg gallic acid/100 g of whole grain our and total anthocyanins ranged from 1.54 to 850.9 mg cyanidin-glucoside equivalents/100 g of whole grain our. Most of the phenolics in grain were in the bound form (ca. 85%), while anthocyanins were the major free phenolic compounds. Among the different samples, bound phenolic extracts of corn appeared to have greater anti-radical and reducing activities than free phenolic extracts from the same grain samples when tested at a normalized phenolic concentration. The phenotypes Veracruz 42 and AREQ516540TL exhibited the greatest activities and these purple-colored strains were most enriched in anthocyanins. Extracts from a red-colored phenotype Pinto were also among the most effective at exhibiting anti-radical activities. Differences in free-radical scavenging and reducing activities appeared to be dependent on the unique prole of anthocyanins and other phenolics in each phenotype. 2008 Elsevier Ltd. All rights reserved.

1. Introduction Corn (Zea mays L.) is a widely consumed cereal in Mexico and Central America mainly in the form of products such as tortilla and tortilla chips (De la Parra, Serna, & Liu, 2007), as well as typical beverages as chicha morada which has been linked to increased health benets in Peru (Brack-Egg, 1999). Pigmented corn contains anthocyanins or carotenoids, and phenolic compounds which are phytochemicals synthesized in the plant by secondary metabolism; although these compounds are considered nonnutritive, interest in antioxidant and bioactive properties has increased due to their health benets (Heinonen, Meyer, & Frankel, 1998; Rice-Evans & Miller, 1996; Setchell & Aedin, 1999). Epidemiological and in vitro research suggests an inverse relationship between consumption of fruits and vegetables and the incidence of various chronic and degenerative diseases that come with aging such as cancer, cardiovascular diseases, cataracts, and brain and immune dysfunction (Ames, Shigenaga, & Hagen, 1993). The health beneting properties of these plant metabolites have been related not only to high antioxidant and anti-radical activities, but also to several other biological properties, such as

* Corresponding author. Tel.: 52 229 934 5701; fax: 52 229 934 1478. E-mail address: hsgarcia@itver.edu.mx (H.S. Garcia). 0023-6438/$ see front matter 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2008.10.010

antimutagenic or estrogenic activities, inhibition of enzymes, and induction of detoxication enzymes such as glutathione transferase and quinone reductase (Rondini, Peyra-Maillard, Marsser-Baglieri, & Berset, 2002; Tsuda, Horio, Uchida, Aoki, & Osawa, 2003). Pigmented Mexican maize is a crop that to the best of our knowledge has not been often examined for properties related to health promotion in humans. Because maize is subjected to extensive processing into industrial (by)products, studies have been conducted on various fractions or derivatives of maize, including bran and oil fractions, as well as extracts of stigmata of owers (maize silk) (Maksimovic, Malencic, & Kovacevic, 2005). Maize of different phenotypes has been shown to exhibit different antioxidant activities and proles. Several studies have reported a range of bioactivities of maize components, the antioxidant and anticarcinogenic effect of white corn polyphenolics such as ferulic and r-coumaric acid along with their respective derivatives (Andreasen, Kroon, Williamson, & Garcia-Conesa, 2001; Kroon & Williamson, 1999; Rondini et al., 2002). It has been found that purple, blue and red pigmented maize inhibits colorectal carcinogenesis in male rats (Hagiwara et al., 2001), and possesses antimutagenic (Yoshimoto, Okuno, Kumagi, Yoshinaga, & Yamakawa, 1999) and radical scavenging activities (Oki et al., 2002). These bioactivities were associated with anthocyanins present in these crops.

1188

L.X. Lopez-Martinez et al. / LWT - Food Science and Technology 42 (2009) 11871192 Table 1 Description and origin of different maize strains and varieties. Maize variety name Black Negro normal OLI 04 PV compuesto 1 Negro normal OLI 04 PV compuesto 2 Negro IG04 PV Manuel 3 compuesto 2 Purple AREQ516540TL Veracruz 42 Oaxaca 337 IG04 PV Compuesto Red Pinto Rojo ancho Olinala oli 04 PV Manuel 5 IG04 PV Compuesto II Mazorca roja-morada ACATL-04 PV Rojo Olinala ACATL-04 PV Compuesto 3 Blue Orange Yellow White Code Black Mm04c1 N004C2 M3M2c2 Purple AREQ Ver 42 O337 Red Pinto RaO04PV M5IG04 Mr-m RO Blue Orange Yellow White Source Local market, Mexico City Colegio de Postgraduados Colegio de Postgraduados CIMMyT Local market, Mexico City Campo Cotaxtla, Veracruz Campo Cotaxtla, Veracruz CIMMyT Local market, Puebla Local market, Puebla Colegio de Postgraduados Colegio de Postgraduados Colegio de Postgraduados CIMMyT Local Local Local Local market, market, market, market, Mexico City Oaxaca Veracruz Veracruz Pigmentation Black Black Black Black Purple Purple Purple Purple Red Red Red Red Red Red Blue Orange Yellow Mome/trace

The antioxidant activity, phenolic acid and anthocyanin contents of some Mexican maize phenotypes have been reported (Del Pozo-Insfran, Brenes, Serna, & Talcott, 2006, 2007); however, a broad survey of antioxidant capacity of phenotypes that represents the diversity of Mexican maize germplasm has not been conducted extensively. Free-radical scavenging is a generally accepted mechanism for antioxidant activity related to the inhibition of lipid peroxidation (Arnao, 2000; Brand-Williams, Cuvelier, & Berset, 1995; Huang, Ou, & Prior, 2005). Some widely used antioxidant and anti-radical assays include those based on 1,1 diphenyl-2-picrylhydrazyl (DPPH ) and 2-20 -azino-bis(3-ethyl benzthiazoline-6-sulfonic acid) radical cation (ABTS ). With an absorbance maximum of 734 nm, the latter is often preferred for assaying antioxidant potential of pigmented compounds and extracts since interference in the assay can be avoided (Feet & Troncoso, 2004). The objective of this study was to determine the levels of phenolic compounds (anthocyanins and ferulic acid) and antioxidant activity of 18 different (mostly pigmented) Mexican phenotypes of maize (some accessed from CIMMyT (Centro Internacional del Mejoramiento de Maiz y Trigo)), grown using conventional agricultural practices in Mexico. 2. Materials and methods 2.1. Chemicals and reagents FolinCiocalteu reagent, ferulic acid and gallic were purchased from SigmaAldrich Chemical Co. (St. Louis, MO), while ethanol was obtained from Fisher Scientic, Chicago, IL. Sodium hydroxide, potassium hydroxide, hexane, ethyl acetate, magnesium carbonate and acetonitrile were obtained from Fisher Scientic (Pittsburgh, PA). Hydrochloric acid, methanol, ethanol, triuoroacetic acid and sodium carbonate were purchased from BAKER (Phillipsburg, NJ).  2-20 -Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS ),  DPPH (1,1-diphenyl-2-picrylhydrazyl), and 6-hydroxy-2,5,7,8-tetramethylcroman-2-carboxylic acid (Trolox) were purchased from Sigma Chemical Co. (St. Louis, MO). All water used was deionized. 2.2. Samples The source and coloration of the phenotypes used in this study are summarized in Table 1. Some maize samples representing different pigmented phenotypes were purchased from local markets in the cities of Puebla, Oaxaca, Veracruz, and Mexico City. Other maize samples were propagated from seeds obtained from CIMMyT (Texcoco), Campo Cotaxtla (Veracruz) and Colegio de Postgraduados (campus Iguala), using conventional cultivation methods in Mexico. Kernels obtained from harvested maize were allowed to dry in the sun to a water content of w20%. All samples were milled into whole grain our, passed through 250 mm screen mesh, thoroughly mixed and were stored at 20  C for no more than two days prior to analyses. 2.3. Extraction of free phenolic compounds Free phenolic compounds were extracted according to Dewanto, Wu, and Liu (2002): 5 g of ground whole grain was blended with 20 mL of chilled ethanolwater (80:20, v/v) for 10 min using a chilled Waring blender. Samples were then further homogenized using a Polytron homogenizer for an additional 3 min. The homogenate then was centrifuged at 2500 g (Sorvall RC5C, Sorvall Instruments, Dupont, Wilmington, DE) for 15 min. After centrifugation the supernatant was removed and extraction of the residue was repeated. The supernatants were pooled, then vacuumevaporated to 5 mL at 45  C and nally reconstituted with water to

a volume of 10 mL. The extracts were frozen and stored at 40  C until used. 2.4. Extraction of bound phenolic compounds Bound phenolic isolates were extracted according to the method of De la Parra et al. (2007). One gram of ground whole grain was extracted twice with ethanolwater (80:20, v/v) and subjected to centrifugation at 2500 g for 10 min. The resulting supernatant was discarded after each extraction. The residue was combined with 20 mL of 2 mol/L NaOH at room temperature, dissolved O2 was removed by ushing with nitrogen, and the samples were placed in an orbital shaker (Lab-Line Orbiton Environ, Model 3527, Melrose Plaza, IL) at 250 rpm for 1 h at room temperature. The mixture was neutralized with 4 mL of concentrated hydrochloric acid and then extracted six times with 10 mL of ethyl acetate. The ethyl acetate fractions were evaporated to dryness at 35  C with a nitrogen stream. The phenolic compounds were reconstituted in 10 mL of water, frozen and stored at 40  C until used. 2.5. Determination of soluble ferulic acid Ferulic acid was extracted according to Adom and Liu (2002) and Adom, Sorrells, and Liu (2005): 0.5 mL aliquots of free phenolic extracts were mixed with 2 mol/L sodium hydroxide at room temperature for 1 h in an orbital shaker at 250 rpm, under nitrogen gas. The mixture was neutralized with hydrochloric acid and extracted with hexane to remove lipids. The nal solution was extracted ve times with ethyl acetate. The ethyl acetate fraction was evaporated to dryness at 35  C with a stream of nitrogen. Ferulic acid was recovered in acetonitrilewater (20:80, v/v). Ferulic acid in sample extracts was quantied using an RP-HPLC procedure employing a Supelcosil LC-18-DB, 150 mm 4.6 mm 3 mm column. Isocratic elution was conducted in acetonitrile water (20:80, v/v) adjusted to pH 2 with triuoroacetic acid, at a ow rate of 1.0 mL/min, delivered by a Waters 515 HPLC pump (Waters Corp., Milford, MA), and a Waters 2487 dual wavelength absorbance detector (Waters) was used for UV detection at 280 nm. Ferulic acid concentration was calculated from a pure ferulic acid

L.X. Lopez-Martinez et al. / LWT - Food Science and Technology 42 (2009) 11871192

1189

standard curve. Ten microliter injections were made, and peak heights were used for all calculations. 2.6. Determination of total phenolic levels Total soluble phenolic levels were analyzed by a procedure adapted from Swain and Hillis (1959) and modied by Gao, Wang, Oomah, and Mazza (2002), using the FolinCiocalteu reagent. Briey, extracts of 100 mL were diluted with 500 mL of water. A volume of 700 mL of 0.2 mol/L equivalent FolinCiocalteu reagent was added and the mixture allowed to stand for 3 min at room temperature. Then 900 mL of 1 mol equivalent/L Na2CO3 was added and the mixture allowed to stand for 90 min, after which absorbance readings at 765 nm were taken in a photodiode array spectrophotometer (model 8452A; HewlettPackard Co., Waldbronn, Germany). Acidied methanol was used as the blank. Total phenolic contents were expressed as mg gallic acid/100 g, based on a standard curve of 0600 mg of gallic acid/mL. 2.7. Determination of anthocyanins content Analysis of total anthocyanins was carried out according to the method of Abdel-Aal and Hucl (1999), by measuring the absorbance of ethanolic extracts at pH 1.0. One gram of ground whole grain sample was homogenized in a 50 mL centrifuge tube with 25 mL of an acidethanol solution (0.225 mol equivalent/L HCl in ethanol water (95:5, v/v)). The tube was ushed with nitrogen gas, agitated for 30 min and then centrifuged at 3000 g (Sorvall RC5C, Sorvall Instruments, Dupont, Wilmington, DE) for 15 min and the supernatants collected. Absorbance readings at 535 nm were taken and corrected for background absorbance at 700 nm (due to turbidity) in a photodiode array spectrophotometer. Anthocyanins were expressed as mg of cyanidin-3 glucoside equivalents/100 g, using a molar extinction coefcient of 25,965 M1 cm1 and a molecular weight of 449.2 g/mol (Abdel-Aal & Hucl, 1999). Data were reported as mean standard deviation (SD) for three replicates. 2.8. 2-20 -Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) radical  cation (ABTS ) scavenging activity Stock ABTS was freshly produced by mixing 5 mL aliquots of  ABTS solution (7 mmol/L) and 88 mL of 140 mmol/L potassium persulfate, followed by standing in the dark for 612 h to produce a dark green solution (Pellegrini, Miller, & Rice-Evans, 1999). The solution was diluted with methanol until the absorbance reached 0.7 at 734 nm (Huang et al., 2005). Extracts of maize kernels were tested at a phenolic concentration of 0.1 mmol/L (gallic acid equivalent). Methanol (190 mL) was transferred into a quartz cuvette and the assay was started by addition of 1 mL of the  preformed ABTS solution. Trolox (at 0.02 mmol/L) was used as a positive antioxidant control. Absorbance at 734 nm was measured  after 10 min and the percent reduction of ABTS was calculated as


polypropylene tubes (BectonDickinson) at room temperature (25  C). Extracts of maize kernels tested at a phenolic concentration of 0.1 mmol/L (gallic acid equivalent) were added to 2.8 mL of  2,2-diphenyl-1-picrylhydrazyl radical (DPPH , 98.9 mmol/L in methanol) and vortexed for 15 s. The decrease in absorbance of  DPPH was measured at 515 nm in a photodiode array spectrophotometer, starting at the time the solution was added and then every hour until no further change in absorbance was measured (ca. 6 h). Methanol served as blank solution (equivalent methanol was added to test samples), Trolox (0.020 mmol/L) was a positive  antioxidant control; and 2.8 mL of DPPH plus 100 mL methanol served as a control. The antioxidant activity was expressed as %  inhibition (quenching) of DDPH absorbance. Data were reported as mean standard deviation (SD) for three replicates. 2.10. Statistical analyses All experiments used completely randomized block designs and the signicance (p < 0.05) of differences between treatment means was established using one-way ANOVA followed by Fishers pairwise comparison test run on the software MINITAB v. 10 (State College, PA). 3. Results and discussion 3.1. Component analysis of kernels of maize phenotypes The ranges of total phenolic levels observed among the 18 Mexican maize phenotypes were 1703400 mg/100 g of whole grain our for total phenolic compounds (Table 2). Among the groups of pigmented phenotypes where multiple members were examined, the ranges of total phenolics overlapped, and were (expressed in mg/100 g of our) purple (n 4), 4653400; black (n 4), 457565; red (n 6), 283617. The purple phenotype AREQ516540TL exhibited the greatest phenolic levels, while the Pinto and Mm04c1 phenotypes appeared to have the greatest phenolic levels among the respective red and black groups of phenotypes examined. The bound phenolic levels ranged 1362720 mg/100 g of our (Table 2). Among the groups of pigmented phenotypes the ranges of bound phenolics were (mg/100 g of our) purple (n 4) 381 2720, black (n 4) 354463, red (n 6) 232494. The purple phenotype AREQ516540TL contained the greatest levels of bound phenolics, while the Pinto and NO04C2 phenotypes appeared to have the greatest bound phenolic levels among the respective red and black groups of phenotypes examined. The free phenolic levels ranged 33680 mg/100 g of whole grain (Table 2). Among the groups of pigmented phenotypes the ranges of free phenolic levels (mg/100 g of our) were purple (n 4) 680 83, black (n 4) 101108, red (n 6) 50123. The purple phenotype AREQ516540TL exhibited the greatest levels of free phenolics, while the Pinto and NO04C2 phenotypes appeared had the greatest free bound phenolic levels among the red and black phenotypes examined. Several studies have investigated phenolic composition of cereals like wheat, barley, rice, oats and corn (Adom & Liu, 2002), and these studies demonstrated that most of the phenolics were bound or attached to cell wall structures. The relative contributions of free and bound phenolics to the total phenolic levels are shown in Table 2. These data clearly show that most phenolics in grain were in the bound form, with the free phenolic contributing 1823% of the total in the different phenotypes, which is in agreement with previous reports and conrmed that ca. 80% of total phenolics in corn is present in bound form (Adom & Liu, 2002; De la Parra et al., 2007; Del Pozo-Insfran et al., 2006).

% Reduction

 h Absorbanceinitial Absorbancefinal = i Absorbanceinitial 100

Data are reported as mean standard deviation (SD) for three replicates. 2.9. Anti-radical activity Measurement of anti-radical activity was adapted from BrandWilliams et al. (1995). Extracts were tested at a standardized phenolic concentration of 0.1 mmol/L (gallic acid equivalents). The reaction for scavenging DPPH radical was conducted in 15 mL

1190

L.X. Lopez-Martinez et al. / LWT - Food Science and Technology 42 (2009) 11871192 Table 3 Ferulic acid contents of maize strains. Anthocyanin contentq 76.2 2.2j 120 1.7e 113 3.8f 113 1.6f 93.2 1.1h 850 6.2a 389 3.9b 131 1.4d 85.2 2.2i 154 3.3c 93.0 3.9h 100 2.8g 76.2 2.2j 62.3 1.1k 99.5 1.8g 30.6 0.9l 70.2 0.9k 1.54 0.9m Code Black Mm04c1 NO04C2 M3M2c2 Purple AREQ Ver 42 O337 Red Pinto RaO04PV M5IG04 Mr-m RO Blue Orange Yellow White Total 151 1.3b 151 2.4b 151 1.9b 151 1.9b 154 1.9b 153 2.6b 154 1.3b 153 2.1b 153 2.3b 151 2.5b 151 1.7b 153 2.3b 151 1.9b 150 2.2b 152 1.3c 164 4.2a 140 1.1d 148 2.1c Free 1.87 0.3b 1.89 0.1b 1.91 0.2b 1.91 0.2b 1.97 0.09b 1.96 0.1b 1.89 0.2b 2.10 0.4b 2.02 0.5b 1.98 0.2b 2.02 0.1b 1.92 0.3b 1.97 0.5b 1.96 0.4b 2.02 0.4b 2.42 0.2ab 2.01 0.1b 1.57 0.6b Bound 150 1.4c 149 1.3c 149 3.3c 149 3.3c 152 1.5b 151 2.4b 152 1.9b 151 1.4b 151 2.9b 149 1.6d 149 2.1c 151 3.3b 149 2.4c 148 2.2c 149 2.2c 161 3.3a 138 1.1e 146 2.3d

Table 2 Anthocyanin and phenolic contents of maize strains. Code Black Mm04c1 NO04C2 M3M2c2 Purple AREQ Ver 42 O337 Red Pinto RaO04PV M5IG04 Mr-m RO Blue Orange Yellow White
an

Total 457 7.4gh 544 7.3f 565 9.1d 472.1 7.2g 465 9.8g 3400 10.9a 1760 10b 539 5.2f 465 4.4g 617 7.1c 456 4.1gh 406.76 9.1i 397 2.6i 283 5.1k 343 8.6j 215 5.1j 551 3.8e 170 1.1l

Free 103 2.6e 123 1.7c 101 3.6e 108 2.9d 83.7 2.5g 680 3.5a 417 7.2b 124 2.5c 82.3 0.8g 123 1.9c 82.1 3.1g 93.5 2.9f 71.4 2.3h 50.9 1.1i 73.1 1.4h 40.3 1.4j 104 2.2e 33.4 1.5k

Bound 354 3.1j 421 2.9f 463 4.5d 363 4.6i 381 4.4h 2720 9.2a 1343 7.9b 414 3.7g 384 7.1h 493 2.2c 374 3.3c 313 3.9l 325 4.3k 232 3.4n 271 4.5m 175 2.3o 447 4.3e 136 3.2p

Means within a column with the same superscript letter are not signicantly different (p > 0.05). p Concentration expressed in mg of gallic acid/100 g of sample. q Concentration expressed in mg cyanidin-3-glucoside/100 g of sample.

ad Means within a column with the same superscript letter are not signicantly different (p > 0.05). p Concentration expressed in mg of ferulic acid/100 g of sample.

3.2. Ferulic acid About 9498% of total ferulic acids were in the bound form in raw kernels (Table 3), in agreement with other authors (Adom & Liu, 2002; Lozovaya et al., 2000; Rouau et al., 2003). Adom and Liu (2002) compared levels of ferulic acid in corn, wheat, oats and rice, and found corn to contain the highest amount of ferulic acid (175 mg/100 g of grain). Additionally, they found that bound ferulic acid was the prevalent form present in the corn grain (>93%). The phenotype orange corn contained the highest amount of total ferulic acid with 162 mg/100 g of whole our grain (Table 3), while the white phenotype had the least bound ferulic acid with 139 mg/100 g from the phenotypes examined. There were no signicant differences in the content of free ferulic acid between all phenotypes. Ferulic acid is the most common phenolic acid found in the Gramineae family. It has been shown that ferulic acid occurs mostly as an ester, linked to cell wall and mainly located in the aleurone layer pericarp tissues (Cortes, Salinas, San Martin-Martinez, & Martinez-Bustos, 2006; Lozovaya et al., 2000). 3.3. Anthocyanin content Anthocyanin contents ranged from 1.54 to 860 mg cyanidinglucoside equivalents/100 g of whole grain for the 18 maize phenotypes examined (Table 2). The white, yellow and orange phenotypes had the least anthocyanin levels and this was expected as these colorations would indicate that carotenoids, particularly in the form of lutein and zeaxanthin, are the most abundant pigment type in these samples (Kurilich & Juvik, 1999). Among the groups of pigmented phenotypes, the ranges of anthocyanin levels, expressed as mg cyanidin-glucoside equivalents/per 100 g of whole grain, were purple, 93851 mg, black, 76120 mg; red, 85154 mg. The AREQ516540TL phenotype had the greatest anthocyanin levels among the purple phenotypes, and Pinto and Mm04c1 strains had the greatest anthocyanin levels among the red and black phenotypes examined, respectively. Thus, for the phenotypes examined, those that were most abundant in phenolics were also most abundant in anthocyanins (which contribute to the total phenolic levels). It has been found that polyphenolic compounds are one of the most effective antioxidant constituents in plant foods, including fruits, vegetables and grains (Velioglu, Mazza, Gao, & Oomah, 1998),

hence it is important to quantify polyphenolic contents and to assess their contribution to antioxidant activity. 3.4. Anti-radical capacity The DPPH quenching activity of isolates of the 18 phenotypes is  shown in Fig. 1, where DPPH radical scavenging varied from 40 to 100%. The highest radical scavenging activity was found in the varieties AREQ516540TL and Veracruz 42 (100%) in purple phenotypes, while the black NO04c2 and red M3M2c2 phenotypes also  had relatively high DPPH quenching capacity 92 and 69 (100%) respectively. Extracts from the non-pigmented phenotypes showed quenching capacity ranging 6040%. Thus, those maize isolates  exhibiting greatest DPPH quenching capacity appeared to be those with the greatest proportion of anthocyanins. These results are consistent with those of Miller, Rigelhof, Marquart, Prakash, and Kanter (2000), who reported relatively higher levels of antioxidant activity in highly pigmented berries, raisins, dates and prunes. Choi, Ku, Chang, and Lee (2005) also found that pigmented red sorghum and black rice showed relatively higher radical scavenging activity (9287%) than non-pigmented samples (767%).  DPPH quenching capacity in maize kernel extracts was different among different fractions (free and bound phenolics) of the same phenotypes. Free phenolic extracts exhibited signicantly lower  DPPH quenching activity (p < 0.05) (Fig. 1) compared to those of the bound phenolic extracts, as was also observed by Maillard and Berset (1995) and Del Pozo-Insfran et al. (2007). This is attributable to higher phenolic content and ferulic acid in bound extract compared to free extracts (Table 2). The highest radical scavenging activity for bound phenolic was found in the varieties AREQ516540TL and Veracruz 42 (80 and 79%) strains of the purple phenotypes, the black NO04c2 (78%) and red M3M2c2 (77%)  phenotypes also had high DPPH quenching capacity, while the non-pigmented phenotypes showed quenching capacity from 60 to 40(%) (Fig. 1). The highest radical scavenging activity among free phenolic isolates was found in the AREQ516540TL and Veracruz 42 (22 and 21%) strains of purple phenotypes, the black NO04c2 (22%)  and red M3M2c2 (13%) phenotypes also had high DPPH quenching capacities, while the non-pigmented phenotypes showed quenching capacities from 12 to 15% (see Fig. 1). The differences in the antioxidant capacity among colored phenotypes are possibly related to the specic composition of anthocyanin derivatives such as simple or acylated glycosides


L.X. Lopez-Martinez et al. / LWT - Food Science and Technology 42 (2009) 11871192

1191

% ABTS free radical cation reduction

100 80 60 40 20 0


Table 4 Correlation analysis of phenolics and free-radical scavenging. Phenolics Free extracts  ABTS DPPH Phenolics Bound extracts DPPH Phenolics Total  ABTS DPPH Phenolics
ad

Ferulic acid 0.009 0.005 0.17e 0.23 0.25c 0.21b 0.24 0.27c 0.23d

0.45 0.51b 0.57 0.65a 0.65 0.65a

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Fig. 1. ABTS reducing power by total, bound and free phenolic extracts of different strains of corn. Total (-), bound ( ), free ( ). 1. Black, 2. Mm04c1, 3. NO04c2, 4. M3M2c2, 5. Purple, 6. AREQ, 7. Ver 42, 8. O337, 9. Red, 10. Pinto, 11. RaO04PV, 12. M5IG04, 13. Mr-m, 14. RO, 15. Blue, 16. Orange, 17. Yellow, 18. White.

Means within a column with the same superscript letter are not signicantly different (p > 0.05).

of cyanidin, pelargonidin or peonidin (Meyer, Heinonen, & Frankel, 1998; Stintzing, Stintzing, Carle, Frei, & Wrolstad, 2002). The differences in the antioxidant capacity in free phenolic isolates among non-pigmented phenotypes and pigmented genotypes may be attributed to the presence of specic anthocyanins, since the free-radical scavenging properties of the phenolic hydroxy groups attached to ring structures are responsible for their antioxidant properties (Rice-Evans, Miller, & Paganga, 1996) in relation to the cinnamic acid derivatives. Although there was no  signicant correlation of ABTS reducing activity to the relative proportion of anthocyanins and phenolics in the extracts, the two strains most enriched in anthocyanins, purple phenotypes AREQ516540TL and Veracruz 42, were the most effective antiradical isolates (Table 3) of the samples, this behavior was found by Anagnostopoulou, Kefalas, Papageorgiou, Assimopoulou, and Boskou (2006) too. They investigated extracts and fractions of sweet orange peel and noted that the fractions with high radical scavenging activity showed a high phenolic content as well, but good correlations could not be found between them (r2 0.42). 3.5. Reducing power Results of ABTS reduction assays on extracts of kernels of the 18 phenotypes revealed that generally isolates were effective anti radical agents (Fig. 2). This is a similar pattern as observed for DPPH scavenging activity (Fig. 1) of these same isolates, and likely for the same reason as advanced earlier (the prole of extracted phenolics


100

% Antiradical capacity

80 60 40 20 0

is more effective reducing agents). Generally, the same phenotypes  showing greatest DPPH scavenging activity were also those with  greatest ABTS reducing activity, namely, the purple phenotypes AREQ516540TL and Veracruz 42 (100%), and the black NO04c2 and red Pinto (87 and 100%). In addition, the yellow (89.4%) was an  effective ABTS reducing preparation. Thus, anthocyanin-rich maize kernels were again associated with greatest anti-radical properties, with the only exception being the yellow phenotype.  A previous study that analyzed ABTS reducing properties of yellow maize aqueous extracts found about twice the reducing power of Trolox (0.016 mmol/L) at a 0.10 mmol/L level of phenolics tested (Wettasinghe, Boilling, Plhak, & Parkin, 2002), equivalent to w0.3 Trolox equivalents. In the present study the most effective  ABTS reducing isolates, AREQ516540TL and Veracruz 42, were similar in effectiveness as 0.020 mmol/L Trolox, or equivalent to w0.35 Trolox equivalents.  ABTS reducing power in the free phenolic extracts had the lower activity (4.535%) compared to that of bound phenolic extracts (30100%). The highest activity for bound phenolic extracts was found for the strains AREQ516540TL and Veracruz 42 (100%) for purple phenotypes, the black NO04C2 (92%) phenotype and the red phenotype Pinto (100%). Phenolic isolates of the nonpigmented white and orange showed the least capacity (2634%), respectively.  Although there was no signicant correlation of ABTS reducing activity to the relative proportion of anthocyanins and phenolics in the extracts, the two strains most enriched in anthocyanins, purple phenotypes AREQ516540TL and Veracruz 42, yielded the most effective anti-radical isolates (100%) of the samples examined (Table 3) and this result conrms that the phenolic compounds may be responsible for the major portion of the antioxidant activity of the corn. Better comparison between the anti-radical activities of the extracts of these strains would be obtained by using levels of isolates where less than 100% inhibition was produced (see Table 4). Moreover, factors such as polarity of the testing system, nature of the radicals and the type of substrate are known to inuence the overall effectiveness of an antioxidant (Del Pozo-Insfran, Brenes, & Talcott, 2004; Rice-Evans et al., 1996). Further work is in progress in our laboratory to elucidate the identity of compounds responsible for the antioxidant activity. 4. Conclusions

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Fig. 2. Anti-radical by total, bound and free phenolic extracts of different varieties of corn. (-) Total, ( ) bound, ( ) Free. 1. Black, 2. Mm04c1, 3. NO04c2, 4. M3M2c2, 5. Purple, 6. AREQ, 7. Ver 42, 8. O337, 9. Red, 10. Pinto, 11. RaO04PV, 12. M5IG04, 13. Mr-m, 14. RO, 15. Blue, 16. Orange, 17. Yellow, 18. White.

A broad range of pigmented and traditional maize phenotypes were assessed for various anti-radical activities. Although the specic proles of phenolic and anthocyanins in these samples are undoubtedly related to the antioxidant activities observed, there

1192

L.X. Lopez-Martinez et al. / LWT - Food Science and Technology 42 (2009) 11871192 Gao, L., Wang, Oomah, B. D., & Mazza, G. (2002). Wheat quality: antioxidant activity of wheat millstreams. In P. Ng, & C. W. Wrigley (Eds.), Wheat quality elucidation (pp. 233). St. Paul, MN: AACC International, ACCC Press. Hagiwara, A., Miyashita, K., Nakanishi, K., Sano, M., Tamano, S., Kadota, T., et al. (2001). Pronounced inhibition by a natural anthocyanin, purple corn color of 2amino-1-methyl-6-phenylimidazol [4,5-b]pyridine (PhIP)-associated colorectal carcinogenesis in male F344 rats pretreated with 1,2-dimethylhydrazine. Cancer Letters, 171, 1725. Heinonen, I., Meyer, A. S., & Frankel, E. N. (1998). Antioxidant activity of berry phenolics on human low-density lipoprotein and liposome oxidation. Journal of Agricultural and Food Chemistry, 46(10), 41074112. Huang, D., Ou, B., & Prior, R. L. (2005). The chemistry behind antioxidant capacity assays. Journal of Agricultural and Food Chemistry, 53(6), 18411856. Kroon, P. A., & Williamson, G. (1999). Hydroxycinnamates in plants and food: current and future perspectives. Journal of Agricultural and Food Chemistry, 79(3), 355361. Kurilich, A. C., & Juvik, J. A. (1999). Quantication of carotenoids and tocopherol antioxidants in Zea mays. Journal of Agricultural and Food Chemistry, 47, 19481955. Lozovaya, V. V., Gorshkova, T. A., Rumyantseva, N. I., Ulanov, A. V., Valieva, A. I., Yablokova, E. V., et al. (2000). Cell wall-bound phenolics in cells of maize (Zea mays, Gramineae) and buckwheat (Fagopyrum tataricum, Polygonaceae) with different plant regeneration abilities. Plant Science, 152, 7985. Maillard, M. N., & Berset, C. (1995). Evolution of antioxidant activity during kilning. Role of insoluble bound phenolic acids of barley and malt. Journal of Agricultural and Food Chemistry, 43, 17891793. Maksimovic, Z., Malencic, D., & Kovacevic, N. (2005). Polyphenol contents and antioxidant activity of Maydis stigma extracts. Bioresource Technology, 96, 873877. Meyer, A. S., Heinonen, M., & Frankel, E. N. (1998). Antioxidant interactions of catechin, cyanidin, caffeic acid, quercetin and ellagic acid in human LDL oxidation. Food Chemistry, 861(12), 7175. Miller, H. E., Rigelhof, F., Marquart, L., Prakash, A., & Kanter, M. (2000). Antioxidant content of whole grain breakfast cereals, fruits and vegetables. American Journal of Clinical Nutrition, 19(3), 312S319S. Oki, T., Masuda, M., Furuta, S., Nishiba, Y., Terahara, N., & Suda, I. (2002). Involvement of anthocyanins and other phenolic compounds in radical-scavenging activity of purple-eshed sweet potato cultivars. Journal of Agricultural and Food Chemistry, 67, 17521756. Pellegrini, G., Miller, N., & Rice-Evans, C. A. (1999). Screening of dietary carotenoids and carotenoid-rich fruit extracts for antioxidant activities applying 2, 2-Azinobis(3-ethylbenzthiazoline-6-sulfonic acid). In L Packer (Ed.), Methods in enzymology Vol. 299. Oxidants and antioxidants, Part A (pp. 379389). New York: Academic Press. Rice-Evans, C. A., & Miller, N. J. (1996). Antioxidant activities of avonoids as bioactive components of food. Biochemical Society Transactions, 24, 790795. Rice-Evans, C. A., Miller, N. J., & Paganga, G. (1996). Structureantioxidant activity relationship of avonoids and phenolic acids. Free Radical Biology & Medicine, 20(7), 933956. Rondini, L., Peyra-Maillard, M., Marsser-Baglieri, A., & Berset, C. (2002). Sulfated ferulic acid in the main in vivo metabolite found after short-term ingestion of free ferulic acid in rats. Journal of Agricultural and Food Chemistry, 50, 30373041. Rouau, X., Cheynier, V., Surget, A., Gloux, D., Barron, C., Meudec, E., et al. (2003). A dehydrotrimer of ferulic acid from maize bran. Phytochemistry, 63, 899903. Setchell, K. D. R., & Aedin, C. (1999). Dietary isoavones: biological effects and relevance to human health. Journal of Nutrition, 129, 758767. Stintzing, F. C., Stintzing, A. S., Carle, R., Frei, B., & Wrolstad, R. E. (2002). Color and antioxidant properties of cyanidin-based anthocyanin pigments. Journal of Agricultural and Food Chemistry, 50(21), 61726181. Swain, T., & Hillis, W. E. (1959). The phenolic constituents of Prunus domestica. I. The quantitative analysis of phenolics constituents. Journal of Agricultural and Food Chemistry, 10, 6368. Tsuda, T., Horio, F., Uchida, K., Aoki, H., & Osawa, T. (2003). Dietary cyanidin 3-O-b-Dglucoside-rich purple corn color prevents obesity and ameliorates hyperglycemia in mice. Nutri-Gene Interactions, 133(7), 21252130. Velioglu, Y. S., Mazza, G., Gao, L., & Oomah, B. (1998). Antioxidant activity and total polyphenolics in selected fruits, vegetables, and grain products. Journal of Agricultural and Food Chemistry, 46(10), 41134117. Wettasinghe, M., Boilling, B., Plhak, L., & Parkin, K. L. (2002). Screening for phase II enzyme-inducing and antioxidant activities of common vegetables. Journal of Food Science, 67(7), 25832588. Yoshimoto, M., Okuno, S., Kumagi, T., Yoshinaga, M., & Yamakawa, O. (1999). Distribution of antimutagenic components in colored sweet potatoes. Japan Agricultural Research Quarterly, 33, 143148.

appeared to be a general relationship between the enrichment of anthocyanins and the relative level of antioxidant activity. The most anthocyanin-rich strains were the purple AREQ516540TL and Veracruz 42 phenotypes, and these samples consistently exhibited the greatest antioxidant effects. In addition, extracts of the red Pinto strain were also among the most effective anti-radical isolates among the 18 strains examined. These three strains are the subject of further studies on various potential health-promoting biological effects with a goal of identifying the endogenous components primarily responsible for the antioxidant and related activities. Anthocyanins are among the groups of phenolic compounds that are prevalent in the human diet, and their strong antioxidant activities suggest a role in maintaining human health. Maize is not a traditional source of dietary anthocyanins in the US, but anthocyanin-rich strains warrant greater attention in this regard. References
Abdel-Aal, E. S. M., & Hucl, P. A. (1999). A rapid method for quantifying total anthocyanins in blue aleurone and purple pericarp wheats. Cereal Chemistry, 76, 350354. Adom, K. K., & Liu, R. H. (2002). Antioxidant activity of grains. Journal of Agricultural and Food Chemistry, 50(21), 61826187. Adom, K. K., Sorrells, M. E., & Liu, R. H. (2005). Phytochemical and antioxidant activity of milled fractions of different wheat varieties. Journal of Agricultural and Food Chemistry, 53, 22972306. Ames, B. N., Shigenaga, M. K., & Hagen, T. M. (1993). Oxidants, antioxidants, and the degenerative diseases of aging. Proceedings of the National Academy of Sciences of the United States of America, 90, 79157922. Anagnostopoulou, M. A., Kefalas, P., Papageorgiou, A., Assimopoulou, A., & Boskou, D. (2006). Radical scavenging activity of various extracts and fractions of sweet orange peel (Citrus sinensis). Food Chemistry, 94, 1925. Andreasen, M. F., Kroon, P. A., Williamson, G., & Garcia-Conesa, M. T. (2001). Esterase activity able to hydrolyze dietary antioxidant hydroxycinnamates is distributed along the intestine of mammals. Journal of Agricultural and Food Chemistry, 49, 56795684. Arnao, M. B. (2000). Some methodological problems in the determination of antioxidant activity using chromogen radicals: a practical case. Trends in Food Science & Technology, 11(11), 419421. Brack-Egg, A. (1999). Zea mays L.. In Diccionario enciclopedico de plantas utiles en de las Casas. Peru (pp. 537538) Cuzco, Peru: Imprenta del Centro Bartolome Brand-Williams, W., Cuvelier, M. E., & Berset, C. (1995). Use of free radical method to evaluate antioxidant activity. Lebensmittel Wissenschaft und Technologie, 28, 2530. Choi, Y., Ku, J. B., Chang, H. B., & Lee, J. (2005). Antioxidant activity of methanolic extracts from some grains consumed in Korea. Food Chemistry, 103, 130138. Cortes, G. A., Salinas, M. Y., San Martin-Martinez, E., & Martinez-Bustos, F. (2006). Stability of anthocyanins of blue maize (Zea mays L.) after nixtamalization of separated pericarp-germen tip cap and endosperm fractions. Journal of Cereal Science, 43, 5762. De la Parra, C., Serna, S. O., & Liu, R. H. (2007). Effect of processing on the phytochemical proles and antioxidant activity of corn for production of masa, tortilla and tortilla chips. Journal of Agricultural and Food Chemistry, 55(10), 41774183. Del Pozo-Insfran, D., Brenes, C. H., Serna, S., & Talcott, S. T. (2006). Polyphenolic and antioxidant content of white and blue corn (Zea mays L.) products. Food Research International, 39(6), 696703. Del Pozo-Insfran, D., Brenes, C. H., Serna, S., & Talcott, S. T. (2007). Polyphenolics and antioxidant capacity of white and blue corns processed into tortillas and chips. Cereal Chemistry, 84(2), 162168. Del Pozo-Insfran, D., Brenes, C. H., & Talcott, S. T. (2004). Phytochemical composition and pigment stability of Acai (Euterpe oleracea). Journal of Agricultural and Food Chemistry, 54, 15391545. Dewanto, V., Wu, X., & Liu, R. H. (2002). Processed sweet corn has higher antioxidant activity. Journal of Agricultural and Food Chemistry, 50, 49594964. Feet, R., & Troncoso, A. M. (2004). Actividad antioxidante de pigmentos antociania Alimentaria, 24(4), 691693. cos. Ciencia y Tecnolog