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Phenolic Composition and Related Antioxidant Properties in Differently Colored Lettuces: A Study by Electron Paramagnetic Resonance (EPR) Kinetics Usue Pérez-López,*,† Calogero Pinzino,§ Mike Frank Quartacci,# Annamaria Ranieri,# and Cristina Sgherri# †

Departamento de Biologı ́a Vegetal y Ecologı ́a, Facultad de Ciencia y Tecnologı ́a, Universidad del Paı ́s Vasco, UPV/EHU, Apartado 644, E-48080 Bilbao, Spain § Istituto di Chimica dei Composti OrganoMetallici (ICCOM) − CNR, Area della Ricerca del CNR di Pisa, Via G. Moruzzi 1, I-56124 Pisa, Italy # Dipartimento di Scienze Agrarie, Alimentari e Agro-ambientali, Università di Pisa, Via del Borghetto 80, I-56124 Pisa, Italy ABSTRACT: Differently colored lettuce (Lactuca sativa L.) cultivars (green, green/red, and red) were studied to correlate their phenolic composition with their antioxidant kinetic behavior. Electron paramagnetic resonance (EPR) was employed to monitor decay kinetics of 1,1-diphenyl-2-picrylhydrazyl (DPPH•), which allowed the identification of three differently paced antioxidants. The results showed that as long as lettuce had higher red pigmentation, the hydrophilic antioxidant capacity increased together with the contents in free and conjugated phenolic acids, free and conjugated flavonoids, and anthocyanins. EPR allowed the identification of slow-rate antioxidants in green and green/red cultivars, intermediate-rate antioxidants in green, green/red, and red cultivars, and fast-rate antioxidants in green/red and red cultivars. At present, the different kinetic behaviors cannot be attributed to a specific antioxidant, but it is suggested that the flavonoid quercetin accounted for the majority of the intermediaterate antioxidants, whereas the anthocyanins accounted for the majority of the fast-rate antioxidants. KEYWORDS: ABTS•+, anthocyanins, antioxidant capacity, DPPH•, electron paramagnetic resonance, flavonoids, Lactuca sativa, phenolic acids

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INTRODUCTION Several epidemiological studies indicate that diets rich in fruits and vegetables are related to lower incidences of some types of cancer and cardiovascular diseases.1 These health-promoting effects have been associated with the antioxidant action of some compounds present in natural foods. Antioxidative effects are a result of the capacity of antioxidants to inhibit the initiation of free radical processes or to interrupt the chain reactions in the propagation of oxidation. In plant cells, two different types of antioxidants can be distinguished: lipophilic, such as chlorophylls, carotenoids, and vitamin E, and hydrophilic, such as vitamin C and phenolic compounds.2 Phenolic compounds such as flavonoids, phenolic acids, and anthocyanins are widely distributed in fruits, herbs, and vegetables, and their antioxidant capacity has been extensively reported.3,4 Indeed, polyphenols have greater antioxidant capacity than vitamins C and E.5 Moreover, they may appear in free or conjugated form, which could influence their bioavailability.6 Previous studies demonstrated the importance of the chemical nature of these conjugates and suggested that the degree of hydroxylation and the relative position of the hydroxyl groups5 determine their antioxidant capacity. The profile and concentration of antioxidant compounds are susceptible to variation among species7 and varieties of the same species.8,9 Together with tomatoes, lettuce (Lactuca sativa L.) is the most important salad vegetable, known as a source of phytonutrients.9 Indicators of lettuce quality include color, texture, and, from a nutraceutical point of view, other attributes © XXXX American Chemical Society

such as antioxidant capacity. Several authors have studied the antioxidant capacity and the bioactive compounds of different cultivars of lettuce demonstrating a higher antioxidant power in red versus green lettuce9,10 and indicating the importance of eating a particular variety of food sources such as colored foods. However, it has not yet been clarified to what extent the differences in the antioxidant compounds among the cultivars can contribute to explaining the higher antioxidant capacity of red lettuce.5,9,11 Indeed, studies made until now on lettuce correlate the contents of polyphenolic compounds with the total antioxidant capacity performed only by the steady-state spectrophotometric determination. Previous research carried out on vegetable oils12 and apple juices13 indicated that a kinetic approach to the study of the free radical scavenger capacity was able to add useful information such as the speed of antioxidant disappearance. Sgherri et al.2,14 applied this kinetic approach to the determination, by electron paramagnetic resonance (EPR), of the antioxidant capacity of lipophilic extracts from basil. Thus, slow and fast antioxidants could be identified. By using an EPR spectrometer equipped with a data acquisition system and a software package especially designed for the analysis and simulation of spectra, Sgherri et al.14 were able to monitor EPR kinetics of the stable radical 1,1-diphenylReceived: July 8, 2014 Revised: November 6, 2014 Accepted: November 11, 2014

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dx.doi.org/10.1021/jf503260v | J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry

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2-picrylhydrazyl (DPPH•) and to distinguish the kinetic behavior of different antioxidants, quantifying the type of antioxidants and determining the molar ratio of antioxidant to DPPH•. Oszmianski et al.13 also proved that EPR spectroscopy appeared to be a much better method than UV−visible spectrophotometry, the first methodology reflecting more accurately the content in procyanidins. Moreover, EPR detection is not affected either by the turbidity of samples or by the presence of colored samples, thus avoiding the necessity for corrections for background absorbance.13 With the present work we aimed at studying more in depth the behavior of hydrophilic extracts from lettuce related to a different phenolic composition. In fact, from preliminary analyses, the hydrophilic extract showed to be more interesting than the lipophilic one having an antioxidant capacity many fold higher (data not shown). To better discriminate the antioxidant capacity of phenolics, we focused our study on differently colored lettuces [green (G); green/red (G/R); and red (R)] with the aim of identifying the antioxidant kinetic behavior of the different antioxidant-rich cultivars.15 The three cultivars of lettuce have been characterized for the profile and concentrations of phenols, including conjugated forms, which until now have been less studied.8 Even if several authors have tried to correlate the antioxidant capacity of food with a specific antioxidant compound,11,16 this is the first approach in lettuce that aimed at correlating the kinetic behavior of hydrophilic extracts from different pigmented cultivars with their relative antioxidant composition. This study could contribute to fully characterize plant antioxidants, adding useful information about the different importance of each hydrophilic antioxidant in the human diet.

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12100g for 15 min, supernatants were filtered by Sartorius (Goettingen, Germany) filters (Minisart 0.45 μm) to remove any suspended material. Quantification of Antioxidant Capacity by EPR Analysis. The antioxidant capacity was measured using DPPH• quenching. DPPH• is a commercially available free radical compound that is soluble and stable in ethanol. All operations were performed in the dark to avoid photochemical effects on DPPH• as well as on leaf extracts. EPR spectra were recorded with a Varian E112 (X-band) spectrometer equipped as previously reported2 at the conditions specified in Sgherri et al.14 Computer-based simulations of EPR spectra were performed using the Winsim program.18 All data analyses were performed using the CurveExpert software (version 1.34). The incubation mixture contained DPPH• at a final concentration of 400 μM in ethanol. This concentration was chosen by preliminary experiments because it was much in excess to the initial concentration of antioxidants in the sample, thus allowing all of the antioxidants to react with the radical.12 In these conditions the reactions with DPPH• were completed after several hours. To determine the decay of the radical before measuring antioxidant capacity, decay kinetics of DPPH• without the extract were recorded. The amplitude of the central line of DPPH• spectra was taken for registration of kinetics, which started soon after the addition of the sample to the radical solution. In this way, the scan time was 1 min, and many experimental points could be monitored. Both rate constant and number of DPPH• molecules reduced by extracts in the tube were obtained by fitting of the kinetic curves. Antioxidant capacity was the result of the number of DPPH• molecules reduced by 1 g of plant material and of the EPR decay rate constants (M−1 s−1). The following equation, 1, which is a special form of the pseudofirst-order kinetic model previously applied to the determination of antioxidant capacity of lipid extracts of basil14 as well as of oil fractions,12 was employed in the analyses of the experimental kinetic data:

A = A S exp( − k S′t ) + A1 exp( − kI′t ) + AF exp( − kF′t ) + AR (1) • A is DPPH molar concentration at time t, AS, AI, and AF are the initial DPPH• molar concentrations that can be reduced by the slow, intermediate, and fast fractions, respectively, and AR is the remaining DPPH• concentration in the medium because of the antioxidant depletion. kS, kI, and kF represent the rate constants of the slow, intermediate, and fast fractions, respectively, and were calculated as follows:

MATERIALS AND METHODS

Chemicals and Reagents. All solvents were of highest purity and were purchased from Sigma-Aldrich (Milan, Italy). Water was of MilliQ grade. All solvents and water were accurately degassed before use in the analyses. The standards luteolin-7-O-glucoside, rutin, apigenin-7O-glucoside, quercetin, quercitrin, luteolin, kaempferol, quercetin-3-Oglucuronide, cyanidin, and cyanidin-3-O-glucoside were purchased from Extrasynthèse (Genay, France). The standards gallic, protocatechuic, p-hydroxybenzoic, chlorogenic, vanillic, caffeic, syringic, pcoumaric, ferulic, and chicoric acids as well as free stable DPPH• and 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) were purchased from Sigma (Milan, Italy). Plant Material and Growing Conditions. Seeds of three commercially available cultivars of lettuce, a green-leaf cultivar (Blonde of Paris Batavia, G), a green/red-leaf cultivar (Marvel of Four Seasons, G/R), and a red-leaf cultivar (Oak Leaf, R) (Clemente Viven, VitoriaGasteiz, Spain) were planted in plastic pots (13 cm side × 15 cm high) containing a mixture of perlite/vermiculite (3:1, v/v). Seven days after sowing (DAS), the most uniformly sized lettuce plants were selected, leaving one plant per pot. Seedlings were grown in a controlled growth chamber maintained at 24/20 °C (day/night) with a relative humidity of 60/80% (day/night); seedlings were watered with Hoagland’s solution17 every 2 days. Photosynthetic active radiation was 400 μmol photons m−2 s−1 with a photoperiod of 14 h. To minimize the effects of intrachamber environmental gradients, plants were randomly repositioned within the chamber each week. At the end of the experiment (46 DAS) the antioxidant capacity and the concentration of the different antioxidant compounds were measured in the fully expanded external leaves chosen at random. Sample Extracts. Fresh leaves (0.5 g) were pulverized with liquid nitrogen by a tissue homogenizator (MM301, Fisher Bioblock Scientific, France). Extraction was performed in the dark with 70% methanol containing 1% HCl using a mortar. Homogenization was completed with sonication for 30 min. After centrifugation at 4 °C at

k S = k S′/[DPPH]t 0

kI = kI′/[DPPH]t 0 and kF = kF′/[DPPH]t 0 Equation 1 is the solution of more than one differential equation of second order assuming that the initial molar concentration of DPPH• ([DPPH]t0) is greater than the molar concentration of antioxidants in the reactions.12 Quantification of Antioxidant Capacity by Spectrophotometric Analysis. The radical cation 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS•+) was generated as described by Pellegrini et al.19 The radical solution was diluted in ethanol to obtain an absorbance at 734 nm of 0.7 ± 0.05. After addition of the extract, the decrease in absorbance was monitored and compared to that of the trolox standard. Antioxidant capacity was expressed in terms of trolox equivalent antioxidant capacity (TEAC) per gram dry weight (DW) of plant material. Determination of Total Phenols. Determination of total phenolic compounds was performed on methanolic extracts following the method reported by Nguyen and Niemeyer20 based on the reaction between phenols with Folin−Ciocalteu reagent. Calculations were performed using a calibration curve prepared with gallic acid as standard. Determination of Total Anthocyanins. Approximately 0.01 g of lettuce fresh weight was homogenized in liquid nitrogen and extracted B



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with 3 M HCl/H2 O/MeOH (1:3:16 v/v/v) using a tissue homogenizer. The extracts were centrifuged at 16100g for 5 min, and anthocyanin levels were estimated (as cyanidin-3-glycoside equivalents) using a molar extinction coefficient of 33000 M−1 cm−1.21 Absorbance of anthocyanins at 524 nm was corrected by subtracting the interference due to pheophytin as A524 − 0.24A653.22 Analysis of Flavonoids. Qualitative and quantitative analyses were performed by RP-HPLC. Twenty microliters of extract was injected into a Waters model 515 HPLC system fitted with a 4.6 mm × 250 mm Prodigy ODS column (Phenomenex, Bologna, Italy). Detection was at 360 nm, using a Waters 2487 dual λ UV−visible detector. Mobile phase A contained water acidified with formic acid (pH 2.7), and mobile phase B contained methanol. A linear gradient of 10−90% mobile phase B was run for 26 min at 1 mL min−1. The identity of the free flavonoids was confirmed by cochromatography with authentic standards. Quantification was performed using standard curves in the range of 10−500 ng of a standard mixture containing luteolin-7-O-glucoside, rutin, apigenin-7-O-glucoside, quercitrin, quercetin-3-glucuronide, quercetin, luteolin, and kaempferol. Chromatogram analysis was performed by the software Millennium 32 (Waters). Hydrolyzable flavonoids were detected after hydrolysis of methanolic extracts at 85 °C for 30 min with 4 N HCl containing 0.1% (w/v) ascorbic acid. Detection of cyanidin and cyanidin-3-O-glucoside was performed in the dark similarly to that of flavonoids with the exception of absorbance, which was changed to 520 nm. The quantification of anthocyanins was obtained after cochromatography with cyanidin and cyanidin-3-O-glucoside in the range 0.1−0.5 μg. Analysis of Phenolic Acids. Qualitative and quantitative analyses were performed by RP-HPLC as previously reported in Sgherri et al.23 The identity of the free phenolic acids was confirmed by cochromatography with authentic standards, and quantification was performed using a standard curve in the range 0.1−0.5 μg of standard mixtures containing gallic, protocatechuic, p-hydroxybenzoic, chlorogenic, vanillic, caffeic, syringic, p-coumaric, ferulic, and chicoric acids. Chromatogram analysis was performed as previously reported for flavonoid determination. Detection of conjugated phenolic acid was performed after alkaline hydrolysis of methanolic extracts. Samples were added of 4 N NaOH (1:1, v/v) containing 1% ascorbic acid and 10 mM ethylenediaminetetraacetic acid (EDTA) to prevent the degradation of phenolic acids during alkaline hydrolysis. After incubation for 1 h in the dark under a continuous flux of nitrogen, samples were acidified using 12 N HCl to achieve a pH value of about 2. The resulting mixtures were extracted three times with 1 mL of ethyl acetate. The organic phases were collected after centrifugation at 3000g for 5 min and evaporated to dryness at 35 °C in a rotary evaporator. Immediately before analysis, the residue was redissolved in 50% (v/v) acetonitrile and then passed through a Sartorius (Goettingen, Germany) filter (Minisart 0.45 μm) to remove any suspended material. Statistical Analysis. The results are the means from three replicates of three independent experiments (n = 9). All data are reported as mean values ± SE. The significance of differences among mean values was determined by one-way ANOVA. Comparisons among means were performed using Duncan’s multiple-range test. Means in tables and figures accompanied by different letters are significantly different at p ≤ 0.05. When necessary, an arc sin or angular transformation was applied before statistical analysis was performed.

capacity, and the radical reactions involved during its scavenging by an antioxidant are well detailed in Espı ́n et al.12 Decay kinetics of DPPH• following the addition of the hydrophilic extracts from lettuce leaves (G, G/R, and R) are shown in Figure 1. The decay kinetics of DPPH• in the

Figure 1. EPR decay kinetics of 400 μM 1,1-diphenyl-2-picrylhydrazyl (DPPH•) after the addition of hydrophilic extracts from green (G), green/red (G/R), and red (R) lettuces.

presence of the hydrophilic extract were due to the contribution of two or three pseudo-first-order kinetics, depending on the cultivar: two for G and R and three for G/R. This is related to the presence of different antioxidants or groups of antioxidants that differ in their velocity to scavenge the radical with the rate constants (k) to be considered as a measure of the speed of DPPH• disappearance.12 G cultivar was characterized by an intermediate and a slow rate constant and R by a fast and an intermediate rate constant, whereas G/R was characterized by a fast, an intermediate, and a slow rate constant (Table 1). These k values, which characterize the decay kinetics of DPPH•, are indicative of the presence of antioxidants in the hydrophilic extract distinguishable for a fast, intermediate, and slow action. For this reason, we can calculate (by EPR kinetics) a fast, an intermediate, and a slow antioxidant capacity of the hydrophilic extract. Each antioxidant capacity, detected by EPR, is expressed as the ability to reduce DPPH• molecules by 1 g of lettuce leaves (Table 1). Substantial increases in the fast antioxidant capacity of the hydrophilic extract were observed in G/R compared to G (not detectable) and in R compared to G/ R. A significant increase in the slow antioxidant capacity of the hydrophilic extract was also observed in G/R compared to G, whereas R lettuce did not show any slow antioxidant capacity. Although G and G/R showed similar intermediate antioxidant capacities, in R lettuce intermediate antioxidant capacity was about 5 times higher than in G and G/R (Table 1). Data obtained by the discoloration of ABTS•+ (Figure 2A) confirmed what is reported in Table 1, namely, that antioxidant capacity of hydrophilic extracts increased with the increase in red pigmentation. In particular, the enhancement in antioxidant capacity was about 120% in G/R and 400% in R, compared to G lettuce. The enrichment in total phenols (Figure 2B) and total anthocyanins (Figure 2C) followed a similar trend. In fact, compared to G lettuce, the contents of total phenols and total anthocyanins increased by 3- and 5-fold in G/R and by 4- and 12-fold in R, respectively.

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RESULTS The EPR spectrum of DPPH•, previously reported by Sgherri et al.,2,14 is characterized by five lines that are narrow enough, compared to the spectrum acquisition time, to be used for registration of kinetics with many points close. DPPH• shows a well-resolved quintet EPR spectrum with aN1 = 0.927 mT and aN2 = 0.846 mT at g = 2.0036, and its unimolecular decay constant was 2.06 × 10−6 s−1.14,24 The model of scavenging stable DPPH• is a widely used method to evaluate antioxidant C



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Table 1. Decay Rate Constants and Number of Antioxidants Obtained from EPR Decay Kinetics of DPPH after the Addition of Hydrophilic Extract from Green (G), Green/Red (G/R), and Red (R) Lettucesa decay rate constant (M−1 s−1) G G/R R

no. of antioxidants (no. DPPH• reduced × 1019/g)

kF

kI

kS

FRA

IRA

SRA

nd 2.75 ± 0.09 2.55 ± 0.06

0.51 ± 0.04 0.32 ± 0.03 0.41 ± 0.02

0.017 ± 0.003 0.023 ± 0.005 nd

nd 1.43 ± 0.08 3.70 ± 0.07

2.34 ± 0.07 2.71 ± 0.05 14.58 ± 0.09

3.85 ± 0.09 4.62 ± 0.05 nd

a

kF, fast rate constant; kI, intermediate rate constant; kS, slow rate constant; nd, not detectable. FRA, fast-rate antioxidants; IRA, intermediate-rate antioxidants; SRA, slow-rate antioxidants.

Among phenols, Tables 2 and 3 showed that both flavonoids (in free and conjugated form) and phenolic acids (in free and conjugated form) were significantly affected by cultivar type, increasing their total amounts as a function of red pigmentation. In G the main free flavonoids were represented by quercetin and kaempferol, in G/R by kaempferol and quercetin-3-O-glucuronide, whereas in R by quercetin, quercetin-3-O-glucuronide, and luteolin-7-O-glucoside (Table 2). In each lettuce cultivar the most abundant conjugated flavonoid was quercetin, with an average contribution between 56 and 80%. Other hydrolyzable flavonoids identified in lettuce were luteolin and kaempferol (Table 2). In comparison with G, the G/R cultivar showed about double values of total free and hydrolyzable flavonoids. With regard to free flavonoids, this was due to a general increase in all flavonoids, with the exception of quercetin, which remained constant. The increase in the total amount of conjugated flavonoids was instead ascribable to a 2fold increment of quercetin and kaempferol. R lettuce showed values 15- and 5-fold higher than G lettuce for total free and hydrolyzable flavonoids, respectively. In the R cultivar, each flavonoid took part in the increase in free flavonoids, whereas, similarly to what happened for G/R, only quercetin and kaempferol were responsible for the increase in hydrolyzable flavonoids, luteolin remaining almost constant (Table 2). For all cultivars the composition in free phenolic acids was characterized by the prevalence of chlorogenic acid, contributing for 63−73% to the total (Table 3). Noteworthy was also the presence of ferulic and chicoric acids. After alkaline hydrolysis, the most representative phenolic acid became caffeic acid, contributing 70−80% to the total amounts of conjugated phenolic acids (Table 3). In comparison with G, higher contents in free phenolic acids showed by G/R and R were mainly related to the increases in chlorogenic, p-coumaric, and chicoric acids, whereas those of conjugated phenolic acids were prevalently ascribable to gallic, caffeic, p-coumaric, and chicoric acids (Table 3). With regard to anthocyanins, we detected both cyanidin and cyanidin-3-O-glucoside, with cyanidin being prevalent (Figure 3). In G none of these compounds was observed, whereas in R lettuce values for cyanidin and cyanidin-3-O-glucoside were 22and 12-fold higher than in G/R, respectively.

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DISCUSSION Although in our previous studies DPPH• was employed to analyze the antioxidant behavior of lipid extracts,2,14,24 the use of DPPH• in an ethanol mixture makes it suitable also for the determination of the antioxidant power of hydrophilic extracts (Figure 1; Table 1). Moreover, because without extract DPPH• shows a decay constant many times lower than that of Fremy’s salt,24 it is more suitable for monitoring long-lasting decay kinetics.

Figure 2. Antioxidant capacity determined by discoloration of 2,2′azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (A), total phenols (B), and total anthocyanins in leaves of green (G), green/red (G/R), and red cultivars (R) of lettuce. All data are reported as mean values ± SE. Means accompanied by different letters are significantly different at p < 0.05. TEAC, trolox equivalent antioxidant capacity; GAE, gallic acid equivalent.

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Table 2. Changes in the Composition (Percent of the Total) and in the Total Amount (Micrograms per Gram Dry Weight) of Free and Conjugated Flavonoids in Different Cultivars of Lettucea G cultivar free ± ± ± ± ± ± ± ±

luteolin-7-O-glucoside rutin apigenin-7-O-glucoside quercitrin quercetin luteolin kaempferol quercetin-3-O-glucuronide

5.77 2.30 10.97 5.77 32.40 9.85 30.66 2.30

total

21.33 ± 0.98c

G/R cultivar conjugated

0.42b 0.33c 0.98a 0.05 3.84b 0.33a 1.55a 0.23b

free 15.30 14.31 6.96 tr 11.52 5.77 24.26 21.87

55.85 ± 0.79c 17.39 ± 0.51a 26.76 ± 0.59a

147.66 ± 1.29c

R cultivar

conjugated

± 1.45a ± 1.26a ± 0.54b ± ± ± ±

1.58c 0.12b 1.33b 1.65a

67.10 ± 1.98b 10.39 ± 0.32b 22.51 ± 0.42b

58.75 ± 1.54b

271.96 ± 4.58b

free 17.56 6.87 2.22 tr 43.82 1.05 4.36 24.13

conjugated

± 0.37a ± 0.29b ± 0.17c ± ± ± ±

0.79a 0.03c 0.28c 0.72a

79.74 ± 1.14a 4.69 ± 0.14c 15.56 ± 0.17c

319.10 ± 5.15a

706.14 ± 7.34a

All data are reported as mean values ± SE. For each compound, in free or conjugated form, means accompanied by different letters are significantly different at p ≤ 0.05 among cultivars. G, green; G/R, green/red; R, red. a

Table 3. Changes in the Composition (Percent of the Total) and in the Total Amount (Micrograms per Gram Dry Weight) of Free and Conjugated Phenolic Acids in Different Cultivars of Lettucea G cultivar free gallic acid protocatechuic acid chlorogenic acid caffeic acid p-coumaric acid ferulic acid chicoric acid total

73.78 ± 3.50a tr 15.55 ± 0.12a 10.67 ± 0.98b 111.10 ± 4.95c

G/R cultivar conjugated

7.19 3.15 3.68 76.59 3.98 3.88 1.52

± ± ± ± ± ± ±

0.31b 0.22b 0.26b 2.05b 0.25b 0.27a 0.11a

759.51 ± 17.98c

free

R cultivar

conjugated

68.80 ± 3.68a 2.85 ± 0.67b 9.51 ± 0.11b 18.84 ± 1.65a 135.16 ± 7.95b

16.54 0.77 1.89 71.04 5.52 2.66 1.58

± ± ± ± ± ± ±

0.53a 0.03c 0.12c 1.89b 0.25a 0.18b 0.11a

1141.82 ± 19.58b

free

63.34 ± 0.93b 10.97 ± 0.42a 5.96 ± 0.34c 19.73 ± 0.12a 1166.18 ± 11.87a

conjugated 3.03 4.01 6.99 81.08 2.38 1.38 1.13

± ± ± ± ± ± ±

0.14c 0.35a 0.54a 2.57a 0.12c 0.11c 0.13a

2980.61 ± 20.86a

All data are reported as mean values ± SE. For each compound, in free or conjugated form, means accompanied by different letters are significantly different at p ≤ 0.05 among cultivars. G, green; G/R, green/red; R, red. a

intermediate-, and fast-rate antioxidants (Figure 1; Table 1). For the moment, we cannot attribute the kinetic behavior to a specific antioxidant, but only state that G/R contains antioxidants belonging to both G and R lettuce (SRA, IRA, FRA) and that G possesses the slowest antioxidants, whereas R the fastest ones (Table 1). The next step will be the identification of a specific kinetic behavior for each hydrophilic antioxidant (mostly representative for lettuce composition) by the study of the decay kinetics of authentic standards, which is currently underway in our laboratory and which was, in part, already performed for lipophilic antioxidants.24 Our results suggest that the different decay rates (Table 1) were related to the presence or absence of some antioxidants (Tables 2 and 3; Figure 3). Noteworthy is the relationship between the presence of cyanidin and cyanidin-3-O-glucoside and the fastest antioxidant behavior of R and R/G compared to G, which does not contain those antioxidants. Thus, as red pigmentation increased (see inset of Figure 1), the hydrophilic antioxidant capacity was higher (Figure 2A). This result from EPR analysis was further confirmed by spectrophotometric determinations (Figures 1 and 2; Table 1). In the red lettuces (R and G/R) the higher antioxidant capacity, compared to G, could be also related to the presence of higher amounts of phenols (Figure 2B; Tables 2 and 3). In fact, the lettuce cultivars analyzed showed a positive correlation between the presence of phenols and the antioxidant potential (Figures 1−3; Tables 1−3). However, it is known that not all phenols have an antioxidant capacity,25 making difficult the

Figure 3. Contents of cyanidin (mg g−1 DW, left axis; grey) and cyanidin 3-O-glucoside (μg g−1 DW, right axis; black) in leaves of green (G), green/red (G/R), and red cultivars (R) of lettuce. All data are reported as mean values ± SE. Means accompanied by different letters are significantly different at p < 0.05.

The idea that a different pigmentation of plants is related to a different antioxidant capacity of their extracts is not new,9,10,15 but the possibility to monitor and simulate EPR decay kinetics of DPPH•, as previously done for lipophilic extracts,14 may add information about the kinetic behavior of the different antioxidants present in lettuce. Applying this methodology14 to hydrophilic extracts, we could distinguish among slow-, E



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contribute mainly to the antioxidant capacity recorded in R lettuce (Figure 1; Table 1). About 70% (in R) and 90% (in G and G/R) of the phenolic acids detected in lettuce were in the bound form (Table 3). The same pattern was detected for flavonoids (Table 2). Reasons for conjugation of phenolic compounds are related to their potential toxicity and their sequestration in the plant vacuoles.30 Because it was previously demonstrated that the antioxidant capacity of a free phenolic acid mixture was higher than that of a mixture of bound phenolics,31 we can assume that the higher antioxidant capacity of R lettuce, compared to G and G/R, could be also related to the decreased presence of the conjugated forms on the total (Tables 2 and 3). However, these results are in contrast with those reported for some barley varieties by Maillard and Berset,32 who attributed a higher antioxidant capacity to bound phenolic acids. Moreover, it has been demonstrated that also flavonol glycosides can be absorbed as intact molecules.33 Thus, it is advisible to give attention to the identification and quantification of both free and conjugated forms. In conclusion, as long as lettuce had higher red pigmentation, the hydrophilic antioxidant capacity increased together with the contents in free and conjugated phenolic acids, free and conjugated flavonoids, and anthocyanins. The study of EPR decay kinetics allowed the identification of slow-, intermediate-, and fast-rate antioxidants in the three different lettuce cultivars. At present, we cannot attribute a kinetic behavior to a specific antioxidant, but we can suggest that the flavonoid quercetin accounted for the majority of the intermediate-rate antioxidants, whereas anthocyanins accounted for the majority of the fast-rate antioxidants.

understanding of the relative contribution of the different antioxidant compounds to the higher antioxidant capacity of R lettuce. Because phenols represent the most important hydrophilic antioxidants in lettuce,10,26 they are likely the most responsible for its antioxidant capacity. Chlorogenic and chicoric acids being the most representative free phenolic acids for all cultivars (Table 3), it was expected that after basic hydrolysis caffeic acid prevailed in all samples, reaching about 70−80% of the total (Table 3). In fact, chlorogenic acid is the ester of caffeic and quinic acid, whereas chicoric acid results from the esterification of caffeic with tartaric acid. The fact that other authors10,11 found different values of phenolic acids in lettuce might be attributed to the different growing conditions, as environment affects significantly the phenolic composition.10 Another source of variation might be represented by the extraction method and, in particular, by the solvent used in the extraction.27 Among the variety of phenolic compounds, phenolic acids have attracted considerable interest in the past few years due to their many potential health benefits.7 The antioxidant nature of phenolic acids and their esters is related to the number of hydroxylations and methoxylations on their aromatic rings.25 The increase, among others, in chlorogenic acid from G to R could account for the enhancement in antioxidant capacity because Zlotek et al.11 found that the DPPH• scavenging ability of lettuce was significantly and positively correlated with the presence of chlorogenic acid. In lettuce the most commonly found flavones and flavonols, having dihydroxylations in the 3′- and 4′-positions of the B ring, were represented by quercetin, quercetin-3-O-glucuronide, rutin, luteolin, and luteolin-7-O-glucoside (Table 2). Less commonly present were instead those with the lone B-ring hydroxyl group in the 4′-position, that is, kaempferol and apigenin-7-O-glucoside (Table 2). It is known that aglycones such as quercetin, luteolin, and kaempferol are more potent antioxidants than their corresponding glycosides.28 Furthermore, according to Rice-Evans et al.5 the antioxidant capacity of flavonoids is related to the number and positions of hydroxyl groups, and Soobrattee et al.4 found that quercetin (3,5,7,3′,4′OH) has a higher TEAC value than luteolin (5,7,3′,4′-OH) and kaempferol (3,5,7,4′-OH). Thus, also the increase in flavonoids as well as in their glycoside forms (Table 2) could account for the enhancement in the antioxidant capacity of R lettuce compared to G and G/R. From the results of the present study and the finding of Zlotek et al.11 that the DPPH• scavenging ability of lettuce is significantly and positively correlated with the presence of quercetin, we could hypothesize that this flavonoid might be one of the antioxidants responsible for the intermediate-decay activity. In fact, G/R and G contained similar concentrations of quercetin, showing the same levels of intermediate-rate antioxidants (Table 1), whereas in R, where quercetin approached a value 20-fold higher (Table 2), the concentration of intermediate-rate antioxidants was much higher (Table 1). In the R cultivar cyanidin mostly contributed to the amount of total phenols, different from its glucosidic form cyanidin-3O-glucoside (Figures 2 and 3). Anthocyanins are particularly important food bioactive compounds having a double interest: one technological (sensorial characteristics of food products) and the other one related to their health properties.29 Because glycosylation of anthocyanidins in the 3-position diminishes the antioxidant capacity of its aglycone, 25 cyanidin should

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AUTHOR INFORMATION

Corresponding Author

*(U.P.L.) Phone: +34 94 601 3374. Fax: +34 94 601 35 00. Email: [email protected]. Funding

This research was financially supported by the University of Pisa (Fondi di Ateneo) and by the following grants: UFI11/24, EHUA14/19 and GRUPO Gobierno Vasco-IT577-13. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED ABTS, 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid); DAS, days after sowing; DPPH•, 1,1-diphenyl-2-picrylhydrazyl; EPR, electron paramagnetic resonance; G, green; G/R, green/ red; GAE, gallic acid equivalent; R, red; TEAC, trolox equivalent antioxidant capacity

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REFERENCES

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