Phenolic Content and Antioxidant Activity during the Development of

Article pubs.acs.org/JAFC

Phenolic Content and Antioxidant Activity during the Development of ‘Brookfield’ and ‘Mishima’ Apples Mayara C. Stanger,*,† Cristiano A. Steffens,† Cristina Soethe,† Marcelo A. Moreira,† and Cassandro V. T. do Amarante† †

Centre for Agrooveterinary Sciences, University of Santa Catarina State, Luiz de Camões Avenue 2090, Conta dinheiro, Lages 88520-000, Santa Catarina, Brazil ABSTRACT: The aim of this study was to characterize the changes in the contents of total (TPC) and individual (IPC) phenolic compounds, the total antioxidant activity (TAA) in the peel and pulp, and total anthocyanins (TAN) in the peel during the development of the fruits of ‘Brookfield’ and ‘Mishima’ apple trees. ‘Brookfield’ apples were harvested from the 49th to the 138th days after full bloom (DAFB) and ‘Mishima’ apples from the 45th to the 172th DAFB. In the pulp, the IPC, TPC, and TAA rapidly reduced at 75 and 79 DAFB for the ‘Brookfield’ and ‘Mishima’ apples, respectively, and then remained constant until commercial maturity. In the peel of ‘Brookfield’ apples there was a reduction in the TPC and TAA at 79 DAFB. The quercetin 3-galactoside, epicatechin, and procyanidin B2 contents reduced up to 107 DAFB with a subsequent increase in the values at commercial maturity. In the peel of ‘Mishima’ apples there was a reduction in the TPC, TAA, epicatechin, and procyanidin B1 and B2 contents at 130 DAFB, with a subsequent increase until commercial maturity. The TAN content in the peel increased during the 2 and 4 weeks prior to commercial maturity for ‘Brookfield’ and ‘Mishima’ apples, respectively. In the pulp and peel of both cultivars there was a reduction in the IPC, TPC, and TAA as the development proceeded. On nearing commercial maturity, there was an increase in the contents of quercetin 3-galactoside, epicatechin, procyanidin B2, and TAN in the peel for both cultivars. KEYWORDS: Malus domestica, days after full bloom, liquid chromatography, polyphenols, anthocyanins

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fruit is close to maturity.11,12 The TPC content can also vary depending on the apple cultivar.8,9,15 In relation to the edaphoclimatic conditions of production in southern Brazil, there are no studies reporting the TPC dynamics and the total antioxidant activity (TAA) during the development of fruits of clones of ‘Gala’ and ‘Fuji’ cultivars, which represent 90% of the total apple production in this region.16 The ‘Brookfield’ and ‘Mishima’ cultivars are clones of ‘Gala’ and ‘Fuji’, respectively, and their main characteristic is an intense red color, which is of interest to farmers, traders, and consumers. The aim of this study was to characterize changes in the individual and total phenolic compounds and total anthocyanins contents, as well as the total antioxidant activity, occurring in the peel and pulp of ‘Brookfield’ and ‘Mishima’ apples during their development, and it provides information on the content of these compounds to farmers, traders, and consumers.

INTRODUCTION Apples are the most popular fruit grown in regions of temperate climate in terms of cultivated area and volume consumed. Recent studies report the positive effects of apple consumption, with benefits to human health including the prevention of chronic diseases.1,2 These beneficial effects have been attributed to phenolic compounds and their antioxidant effect.3,4 Phenolic compounds have at least one aromatic ring and one or more hydroxyl groups.5,6 They originate from secondary metabolism and perform essential functions related to the cellular biochemistry, reproduction, growth, defense mechanisms (against pathogens, parasites, and predators), color, and taste of fruits.1,7 The main groups of phenolic compounds in apples are the phenolic acids, dihydrochalcones, flavonols, flavanols (flavan-3ols), and anthocyanins.8,9 According to studies by Ceymann et al.,8 Jakobek et al.,9 and Tsao et al.,10 the phenolic acids, dihydrochalcones, and flavonols contribute to the content of total phenolic compounds (TPC) present in apples, with values of 3−30, 1−5, and 2−10%, respectively. Flavan-3-ols in monomeric [(+)-catechin and (−)-epicatechin] and oligomeric (proanthocyanidins) forms are the main flavanols present, and they represent 55−85% of the TPC in apples. The anthocyanins are present in cultivars of red or partially red apples, and they represent between 1 and 7% of the TPC. Studies have demonstrated that the TPC contents of peel11,12 and pulp13,14 decrease as the fruit develops and then remain relatively constant until commercial maturity. In the peel, some specific compounds, such as the anthocyanins in colored cultivars and glycosylated quercetins, increase when the © XXXX American Chemical Society

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MATERIALS AND METHODS

‘Brookfield’ and ‘Mishima’ apples were collected from a commercial orchard, located in the municipality of São Joaquim, in Santa Catarina state, in southern Brazil (28°17′ S and 49°55′ W, altitude 1360 m), during the 2014/2015 harvest. The orchard was composed of 7-yearold trees, grafted on Marubakaido rootstock, with spacings of 2.0 m × 6.0 and 2.5 m × 6.0 m for ‘Brookfield’ and ‘Mishima’ cultivars, respectively. The fruits were harvested at 49, 79, 107, 134, and 138 days after full bloom (DAFB) in the case of the ‘Brookfield’ cultivar Received: Revised: Accepted: Published: A

December 16, 2016 April 13, 2017 April 17, 2017 April 17, 2017 DOI: 10.1021/acs.jafc.6b04695 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry and at 45, 75, 103, 130, 145, 165, and 172 DAFB for the ‘Mishima’ cultivar. Reagents, Standards, and Solvents. The reagents 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), Folin−Ciocalteu reagent, sodium acetate, and potassium persulfate were acquired from Sigma Chemical Co. (St. Louis, MO, USA) at analytical grade (PA). Standard solutions of chlorogenic acid, catechin, epicatechin, procyanidin B1, procyanidin B2, quercetin 3-galactoside, and phloridzin and the solvents acetonitrile, acetic acid, and methanol were obtained from Sigma Chemical Co., all of HPLC grade. Gallic acid, sodium carbonate, acetone, and ethyl alcohol were obtained from Vetec (Rio de Janeiro, Brazil) at analytical grade (PA). Preparation of Samples. For each harvest date and each cultivar four homogeneous sample units composed of 20 fruits were prepared in the laboratory in a randomized form; fruits with signs of rotting, bruising, or defects were discarded. The peel was removed from the entire surface with the aid of a sharp blade (1 mm thick). The pulp sample was obtained by removing a longitudinal slice, of around 1 cm, from the middle part of the fruit, discarding the endocarp region and conserving each side of the slice. The pulp samples were processed with the aid of a vertical grinder (model RI1364), manufactured by Philips Walita (Varginha, Brazil), and the peel samples were macerated in a mortar with liquid nitrogen. Obtainment of Extracts for the Quantification of TPC and TAA. The extract for the quantification of TPC and TAA was obtained as described by Rufino et al.,17 adapted from Larrauri et al.18 In this procedure, 10.0 g of pulp and 2.5 g of peel were used. The sample was placed in a Falcon tube (Zollstr, Switzerland) with the addition of 10 mL of methanol/distilled water (50:50, v/v), followed by homogenization in an Ultra-Turrax disperser (model D-91126) manufactured by Heidolph (Schwabach, Germany), and left to rest for 60 min at room temperature. The samples were then centrifuged in an Eppendorf centrifuge, model 5810R (Hamburg, Germany) at 12880g for 20 min at 4 °C. The supernatant was transferred to a 25 mL volumetric flask, and 10 mL of acetone/distilled water (70:30, v/v) was added to the residue of the first extraction, followed by homogenization, and the sample was then left to rest for 60 min at 20 °C. The samples were again submitted to centrifugation, under the same conditions. The second supernatant was transferred to a volumetric flask containing the first supernatant, and the volume was made up to 25 mL with distilled water. Determination of Total Phenolic Compounds (TPC) in the Peel and Pulp. The TPC content was determined with the use of a modified version of the Folin−Ciocalteu spectrophotometric method, as described by Roesler et al.19 In this procedure, 2.5 mL of a mixture of Folin−Ciocalteu reagent/distilled water (25:75, v/v) was added to 0.5 mL of a known dilution of the hydroalcoholic extract, in triplicate, in a test tube, followed by homogenization in a test tube shaker manufactured by Arsec, model ATS-100 (São Paulo, Brazil; the sample was maintained for 3 min at 20 °C for the reaction. In the next step, 2.0 mL of sodium carbonate solution (10 g 100 mL−1) was added, and the samples were shaken again and left to rest for 1 h protected from light. The absorbance was determined at the wavelength (λ) of 765 nm (nm) on a UV−visible spectrophotometer, manufactured by Bel Photonics, model BEL2000-UV (Piracicaba, Brazil). The TPC content was calculated from the calibration curve obtained with gallic acid, and the results were expressed in milligrams of gallic acid equivalent per 100 g of fresh mass (mg GAE 100 g−1 FM). Determination of Total Antioxidant Activity (TAA) in the Peel and Pulp. The TAA was determined using the methodologies based on the capacity of the extract to sequester 2,2′-azinobis(3ethylbenzothiazoline-6-sulfonic acid) radicals (ABTS method)17 and 1,1-diphenyl-2-picrylhydrazyl radicals (DPPH method).20 For the ABTS method, the radicals were generated from the reaction of the stock solution of ABTS (7 mM) with potassium persulfate (140 mM) kept in the dark for 16 h, at 20 °C. Before the analysis, the radicals were diluted with ethylic alcohol until an absorbance of 0.70 ± 0.05, at λ = 734 nm, was obtained. Three

different dilutions of the hydroalcoholic extract were prepared, in triplicate, in test tubes. Aliquots (30 μL) of each dilution of the extract were transferred to test tubes with 3.0 mL of the ABTS radicals and homogenized in a tube shaker. The readings were taken on a UV− visible spectrophotometer (model BEL2000-UV), manufactured by Bel Photonics, at λ = 734 nm, after 6 min of reaction. Ethylic alcohol was used as the blank to calibrate the spectrophotometer. From the absorbance values obtained for the different extract dilutions, a linear equation was obtained. Curves for standard solutions of Trolox were constructed, and the TAA results were expressed in Trolox equivalent antioxidant capacity (TEAC) per gram of fresh mass (μmol TEAC g−1 FM). In the DPPH method, the radical (0.06 mM) was prepared through dilution in methanol on the day of the test. An aliquot (0.1 mL) of the hydroalcoholic extract was transferred to the test tubes with 3.9 mL of the DPPH radical, in triplicate, and the sample was homogenized in a tube shaker. The absorbance measurement was carried out at λ = 515 nm, after 30 min of reaction with the addition of the sample. Methyl alcohol was used as the blank to calibrate the spectrophotometer. Curves for standard solutions of Trolox were constructed, and the TAA results were expressed in micromoles of TEAC 100 g−1 FM. Determination of Total Anthocyanin (TAN) Content in the Peel. The TAN content was determined according to the methodology adapted by Fuleki and Francis.21 For the TAN determination, a 5.0 g sample of the peel was added to 15 mL of ethanol/distilled water (95:5, v/v) and acidified with ethanol/hydrochloric acid (1.5 N HCl) (85:15, v/v). The samples were homogenized in an homogenizer (model SilentCrusher M), manufactured by Heidolph, kept for 24 h at 4 °C, and then centrifuged for 20 min at 12880g, at 4 °C, in an Eppendorf centrifuge, model 5810R. In the next step, 2 mL of the supernatant was transferred to a volumetric flask, and the volume was made up to 50 mL with extractor solvent. The readings were carried out on a UV−visible spectrophotometer (model BEL2000-UV), manufactured by Bel Photonics, at λ = 535 nm. The TAN content was expressed in milligrams of cyanidin 3-glucoside per 100 g of fresh mass (mg cyanidin 3-glucoside 100 g−1 FM). Determination of Individual Phenolic Compounds (IPC) in the Peel and Pulp. The analysis to determine the contents of IPC was carried out using high-performance liquid chromatography (HPLC) as described by Tsao et al.22 The sample preparation was the same as that used for TPC and TAA. Each sample was transferred to a beaker with methanol/ultrapure water (70:30, v/v), in the proportion of 1:1 (w/v). The samples were homogenized using an homogenizer, model SilentCrusher M, manufactured by Heidolph. The filtering was carried out using a quantitative filter under vacuum, following by a 0.45 μm syringe filter, manufactured by Kasvi (Curitiba, Brazil). The final sample was stored at −20 °C until the analysis was carried out. The quantification of the IPC content was carried out through HPLC using a chromatograph manufactured by Shimadzu (Tokyo, Japan), equipped with an SCL-10AVP controller, an FCV-10AL VP quaternary gradient mixer, an LC-10ADVP pump, an SIL 10-ADVP autoinjector, an SPD-10A VP ultraviolet detector, and CLASS VP software (version 6.14). An analytical C18 column (250 × 4.6 mm; particle size = 5 μm), manufactured by Restek (Bellefonte, PA, USA) was used. The mobile phase was acetic acid/ultrapure water (6:94, v/ v) in 2 mM sodium acetate buffer (solvent A, pH 2.55, v/v) and acetonitrile (solvent B). The gradient program was as follows: from 0 to 15% of B in 45 min, from 15 to 30% of B in 15 min, from 30 to 50% of B in 5 min, and from 50 to 100% of B in 5 min. The time for the return to the initial condition was 10 min. The flow rate was 1.0 mL min−1 for a total running time of 80 min. The detector was set at λ = 280 nm, and the injection volume was 20 μL for all samples. All standards were dissolved in methanol. The identification of the phenolic compounds was based on standard retention times: procyanidins B1 = 10.9 min; catechin = 17.4 min; procyanidin B2 = 22.0 min; chlorogenic acid = 22.7 min; epicatechin = 35.2 min; quercetin 3-galactoside = 52.6 min; and phloridzin = 67.0 min. For identification purposes, a spectral library was constructed comprising the retention times and light-absorbing B

DOI: 10.1021/acs.jafc.6b04695 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Figure 1. Total phenolic compounds and total antioxidant activity determined by DPPH and ABTS methods in the peel and pulp tissues during the development of ‘Brookfield’ apples. Different letters in the graph show significant differences according to the DAFB at which the fruits were analyzed, as determined by the Tukey test (p < 0.05). Vertical bars represent the standard error of the mean (SEM). spectra at 280 nm of authenticated standards under the chromatographic conditions specified above. External calibration curves, in concentrations of 0−100 mg g−1, with appropriate standards prepared in methanol were used for the quantification. A mixture of standards was injected at the beginning and at the end of the batch to check the quality of the analyses and to detect possible errors. The recoveries of these standards were between 97 and 110%. All samples were prepared and analyzed in duplicate. Analysis of the Maturation Attributes of the Fruits. For the collection carried out at 134 and 138 DAFB for ‘Brookfield’ apples and at 165 and 172 DAFB for ‘Mishima’ apples, four replicates of 10 fruits were additionally analyzed to determine the background color of the peel, pulp firmness, starch−iodine index, soluble solids (SS), and titratable acidity (TA). The background color of the peel was determined using an electronic colorimeter (model CR400), manufactured by Konica Minolta (Tokyo, Japan). The readings were taken in the equatorial region of the fruits and expressed in terms of hue angle (h°) (0° = red; 90° = yellow; 180° = green; and 270° = blue). The pulp firmness (newtons, N) was determined in the equatorial region of the fruits, at two opposite sides, where a small portion of the peel had been removed with the aid of an electronic penetrometer (GÜ SS Manufacturing Ltd., Cape Town, South Africa), with an 11 mm diameter tip. The starch−iodine index was evaluated using a scale of 1 (pulp cross section stained by the starch−iodine complex, indicating high starch content and unripe fruit) to 5 (pulp cross section not stained by the starch−iodine complex, indicating starch content close to zero and ripe fruit). The SS and TA values were determined using a 5 mL sample of fruit juice previously extracted from transversal slices removed from the equatorial region of the apples with the aid of an electric centrifuge (model 1260-01), manufactured by Mondial (Conceiçaõ do Jacuı ́pe, Brazil). The TA (% of malic acid) was determined by the titration of 5 mL of juice, diluted in 45 mL of

distilled water with a 0.1 N sodium hydroxide solution, to pH 8.1, using an automatic titrator (model TitroLine easy), manufactured by Schott Instruments (Mainz, Germany). The SS content (°Brix) was measured using a digital refractometer (model PE201α), manufactured by Atago, with automatic temperature compensation (Tokyo, Japan). Statistical Analysis. A completely randomized experimental design was used, with four replicates of 20 fruits for each harvest date, except for the analysis to determine the background color of the peel, pulp firmness, starch−iodine index, SS, and TA, for which four replicates of 10 fruits were used. The IPC, TPC, and TAA data were submitted to analysis of variance (ANOVA), using a statistical analysis system (SAS Institute, 2002).23 The effects of the harvest date were analyzed by applying the Tukey test (p < 0.05). Pearson’s correlation analysis of the relationship between the TPC and TAA values (obtained through DPPH and ABTS methods) for the fruit peel and pulp was also carried out with the SAS program.

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RESULTS AND DISCUSSION In the analysis carried out to characterize the commercial maturity, the ‘Brookfield’ apples, at 134 and 138 DAFB, respectively, showed the following values: h° for the peel, 78.8 and 80.6; pulp firmness, 75.8 and 70.6 N; TA 0.5 and 0.4%; SS, 10.8 and 10.7 °Brix; and starch−iodine index 4.0 and 4.5. On the other hand, the ‘Mishima’ apples, at 165 and 172 DAFB, respectively, showed the following values: h° for the peel, 83.8 and 85.3; TA, 0.5 and 0.4%; SS, 12.6 and 12.6 °Brix; and starch−iodine index, 3.5 and 3.8. As the development of the ‘Brookfield’ apples progressed, a reduction in the values for the TPC and TAA (ABTS and DPPH methods) was observed for the peel and pulp (Figure 1). In the peel, the TPC reduced by half, when the fruits C

DOI: 10.1021/acs.jafc.6b04695 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Table 1. Phenolic Compounds Content (mg kg−1 FM) in the Peel and Pulp during the Development of ‘Brookfield’ Applesa DAFBb 49 79 107 127 134 138 CVc (%) 49 79 107 127 134 138 CV (%)

chlorogenic acid Peel 104.12 38.77 26.00 32.16 24.67 42.13 21.08 Pulp 106.50 6.51 1.93 1.27 1.25 0.66 68.77

phloridzin

quercetin 3-galactoside

a b b b b b

201.12 58.79 14.25 12.38 13.23 33.18 23.74

a b c c c bc

231.65 173.71 114.22 155.06 158.34 152.68 19.02

a b b b b b

72.33 0.02 0.02 0.02 0.04 0.03 64.19

a b b b b b

ndd nd nd nd nd nd

a ab b ab ab ab

epicatechin

catechin

procyanidin B1

198.59 112.77 73.68 143.80 129.84 183.03 25.26

a ab b ab ab a

204.04 14.61 8.31 10.08 6.74 9.14 51.23

a b b b b b

145.27 37.31 22.95 30.84 24.81 36.22 33.90

a b b b b b

11.94 0.19 0.11 0.06 0.08 nd 141.72

a b b b b

15.40 0.57 0.45 0.21 0.29 0.38 110.82

a b b b b b

25.37 0.96 0.37 0.48 0.44 0.35 121.58

a b b b b b

procyanidin B2 181.9 ab 121.00 b 81.51 b 192.71 ab 167.71 ab 302.47 a 32.09 nd nd nd nd nd nd

a c

Means followed by different lower case letters in a column differ by Tukey’s test at 5% of error probability (p < 0.05). bDays after full bloom. Coefficient of variation. dNot detected.

Figure 2. Total phenolic compounds and total antioxidant activity (determined through DPPH and ABTS methods) in the peel and pulp tissues during the development of ‘Mishima’ apples. Different letters in the graph show significant differences according to the DAFB at which the fruits were analyzed, as determined by the Tukey test (p < 0.05). Vertical bars represent the standard error of the mean (SEM).

harvested at 49 DAFB were compared to those harvested at 134 and 138 DAFB. During this period, the TAA decreased by factors of 3.0 and 2.5 (according to the DPPH and ABTS methods, respectively). The contents of chlorogenic acid, catechin, and procyanidin B1 were highest at 49 DAFB, decreasing until 79 DAFB, and subsequently they remained relatively constant until commercial maturity (Table 1). There was a reduction in the contents of phloridzin, quercetin 3-

galactoside, epicatechin, and procyanidin B2 from 49 DAFB until 107 DAFB, followed by an increase, reaching values at 138 DAFB similar to those quantified at 49 DAFB (Table 1). For the pulp, there was a major reduction in the TPC and TAA values between 49 and 79 DAFB, and from 107 DAFB these values remained relatively constant. The observed reductions were by factors of 6.5 for TPC and 7 and 10 for TAA, quantified using the DPPH and ABTS methods, respectively. D

DOI: 10.1021/acs.jafc.6b04695 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry Table 2. Phenolic Compounds Content (mg kg−1 FM) during the Development of ‘Mishima’ Applesa DAFBb 45 75 103 130 145 165 172 CVc (%) 45 75 103 130 145 165 172 CV (%)

chlorogenic acid 29.66 a 15.92 bc 4.117 c 4.67 bc 10.63 bc 16.26 b 15.87 bc 30.80 160.73 11.98 5.13 9.31 2.50 3.83 2.20 21.88

a b b b b b b

phloridzin

quercetin 3-galactoside

61.32 38.04 13.72 8.59 20.06 18.76 20.03 21.30

a b c c c c c

178.51 252.59 161.59 128.30 264.93 380.39 398.44 25.25

21.00 0.77 0.18 0.70 nd nd nd 57.64

a b b b

ndd nd nd nd nd nd nd

epicatechin Peel 95.81 a 30.89 b 5.67 b 3.71 b 32.6 b 25.90 b 75.19 a 34.61 Pulp 30.73 a 0.57 b 0.26 b 0.62 b 0.04 b 0.39 b 0.19 b 79.02

b ab b b ab ab a

catechin 39.88 23.38 4.59 3.05 9.26 9.29 8.47 26.22

a b c c c c c

39.42 0.10 0.18 0.35 0.29 0.26 0.28 52.72

a b b b b b b

procyanidin B1

procyanidin B2

26.79 a 11.01bc 3.49 c 1.75 c 8.17 bc 9.34 bc 13.27 b 30.31 362.80 0.53 0.26 0.11 0.10 0.08 0.10 118.33

444.34 216.55 57.89 41.47 184.69 225.53 293.50 24.89

a b b b b b b

a bc c c bc bc ab

nd nd nd nd nd nd nd

a c

Means followed by different lower case letters in a column differ by Tukey’s test at 5% of error probability (p < 0.05). bDays after full bloom. Coefficient of variation. dNot detected.

The IPC value was highest at 49 DAFB, decreasing until 79 DAFB and remaining relatively constant from then onward (Table 1). For the ‘Mishima’ cultivar, a reduction in the TPC and TAA (ABTS and DPPH methods) was also observed as the development of the fruits progressed (Figure 2). In the peel, when fruits harvested at 45 DAFB were compared with those harvested at 165 and 172 DAFB, reductions in the TPC (by a factor of 1.3) and TAA (by factors of 1.5 and 2.0 according to the DPPH and ABTS methods, respectively) were observed. There was a rapid reduction in the TPC and TAA up to 130 DAFB, followed by an increase in the TPC and in the TAA quantified through the DPPH method (Figure 2). There were reductions in the contents of all phenolic compounds evaluated, with the exception of the epicatechin and procyanidin B2 contents, which showed reductions up to 130 DAFB, followed by an increase in the subsequent evaluations, showing contents similar to those quantified at 45 DAFB (Table 2). After 145 DAFB, there was an increase in the quercetin 3-galactoside content (Table 2). For the pulp, the behavior was similar to that observed for the ‘Brookfield’ cultivar. There was a major reduction between 45 and 75 DAFB, whereas values remained stable after 103 DAPF. The reductions observed were by factors of 4.5 for TPC and 5.0 and 9.0 for the TAA quantified through DPPH and ABTS methods, respectively. The contents of all individual phenolic compounds were higher for the first harvest date (45 DAFB) in comparison to the subsequent dates, remaining relatively constant from 75 to 172 DAFB (Table 3). At commercial maturity, for both cultivars, the IPC content in the pulp represented approximately 1% of the amount quantified in the early stages (45 or 49 DAFB). The reduction in the TAA can be attributed to the decrease in the contents of the IPC, mainly catechin, epicatechin, procyanidin B1, and chlorogenic acid, because these are the compounds mainly responsible for the TAA of apple pulp.8,9,24 Behavior similar to that reported in this paper for TPC, IPC, and TAA was observed by Zheng et al.14 during the development of ‘Fuji’ apples (25−185 DAFB). The authors attributed the fast

Table 3. Pearson’s Correlation Coefficient between Phenolic Compounds and Total Antioxidant Activity (TAA), Quantified through ABTS and DPPH Methods, in the Peel of ‘Brookfield’ and ‘Mishima’ Apples during Developmenta ‘Brookfield’

‘Mishima’

correlation

DPPH

ABTS

DPPH

ABTS

chlorogenic acid phloridzin quercetin 3-galactoside epicatechin catechin procyanidin B1 procyanidin B2

0.71** 0.78*** 0.55* 0.11ns 0.61* 0.67* 0.28ns

0.65** 0.72*** 0.50* 0.13ns 0.71** 0.48* 0.12ns

0.70** 0.82*** 0.10ns 0.578 0.85*** 0.12ns 0.69*

0.56* 0.76*** 0.14ns 0.45ns 0.83*** 0.54ns 0.53*

***, significant at 0.0001; **, significant at 0.001; *, significant at 0.01; ns, not significant. a

reduction in the TPC to the high degree of cell expansion, which occurs up to approximately 85 DAFB. Zhang et al.13 suggested that the phenolic compounds are synthesized and/or accumulate at a slower rate in relation to the growth of the fruits. In the peel of ‘Brookfield’ apples, the increase in the procyanidin B2, epicatechin, and quercetin 3-galactoside contents was not reflected in an increase in the TPC and TAA at commercial maturity (134 and 138 DAFB). According to the correlation analysis, the main phenolic compounds responsible for TAA are phloridizin, chlorogenic acid, catechin, procyanidin B1, and quercetin 3-galactoside (Table 3). However, in the case of ‘Mishima’ apples, the increase in procyanidins B1 and B2, epicatechin, and quercetin 3galactoside did result in an increase in the TPC and TAA at commercial maturity (165 and 172 DAFB). In the correlation analysis between IPC and TAA, higher correlation coefficients were found for catechin, phloridzin, chlorogenic acid, and procyanidin B2 (Table 4). Tsao et al.10 reported that procyanidin B2 is one of the compounds that most contribute to antioxidant activity of apples. There is a characteristic TPC content for each cultivar,8,9,15,24 and the contribution of the E

DOI: 10.1021/acs.jafc.6b04695 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

A positive significant correlation (p < 0.001) was observed between the TPC and TAA contents (obtained through ABTS and DPPH methods) and between the TAA determination methods, for the peel and pulp of both cultivars (Table 4). The results obtained in this study confirm those reported by Zheng et al.,14 Panzella et al.,15 and Kevers et al.,30 suggesting that TPC is the main TAA contributor in apples. Many studies show that the TPC and TAA contents are higher in the peel than in the pulp of apples.9,24 Besides a higher TPC content, specific compounds are found in higher quantities in the peel, such as glycosylated quercetin, demonstrating that the peel has greater bioactivity in relation to the pulp.15,31 However, on a weight basis, the percentage in the peel is much lower than that in the pulp. Thus, the pulp may represent the part that mostly contributes antioxidants, mainly because the peel tends to be frequently discarded by consumers. Values for the individual and total phenolic compounds and total antioxidant activity of ‘Brookfield’ and ‘Mishima’ apples decrease as the fruits develop. This reduction occurs with greater intensity in the pulp compared with the peel. For both cultivars, as the fruits develop there is an increase in the quercetin 3-galactoside, epicatechin, and procyanidin B2 contents of the peel, coinciding with an increase in the total anthocyanin content. In the case of ‘Mishima’ apples, this increase is reflected in an increase in the TPC and TAA contents. Our results show that in apple fruit, polyphenols and antioxidant activity are influenced by cultivar, fruit developmental stage, and tissue (peel or pulp). The different concentrations of phenols in apple fruit tissues affect processed products’ phenolic quality, and it has been well demonstrated that, during processing of primary products, the antioxidant capacity of apples may be lost.32 For processed apple products, the most popular is apple juice. During its production, only a fraction of phenolic compounds are extracted, whereas the others in the pomace.33 Due to the fact that the peel (and also seeds) is discharged during juice production, phenolic compounds are found in small amounts in apple juice.33 Apple pomace is the “waste” product generated during apple juice processing. Therefore, apple pomace represents a potential source of natural polyphenols for use as dietary or food antioxidants.34

Table 4. Pearson’s Correlation Coefficient between Total Phenolic Compounds (TPC) and Total Antioxidant Activity (TAA), Quantified through ABTS and DPPH Methods, in the Peel and Pulp of ‘Brookfield’ and ‘Mishima’ Apples during Developmenta ‘Brookfield’

a

‘Mishima’

correlation

peel

pulp

peel

pulp

TPC × DPPH TPC × ABTS DPPH × ABTS

0.92*** 0.81*** 0.83***

0.98*** 0.99*** 0.98***

0.94*** 0.81*** 0.90***

0.96*** 0.98*** 0.97***

***, significant at 0.0001.

different groups of phenolic compounds leads to variations in the TAA.10,14 However, in complex samples such as fruits, TAA is a result of the presence of several molecules and their synergistic effects, so TAA cannot be related to a single class of compounds due to the synergistic and additive effects they present,25−27 as well as a possible effect of other antioxidants such as ascorbic acid.10 Thus, the smaller reduction in flavonoids in the peel in relation to the pulp is an important mechanism of protection against solar radiation in fruits and provides an excellent source of these compounds in the human diet. For both cultivars, the TAN content increased as the development of the fruits progressed (Figure 3). This increase occurred in the second and fourth weeks prior to commercial maturity for ‘Brookfield’ and ‘Mishima’ cultivars, respectively. Bizjak et al.11,12 obtained similar results for Braeburn apples. The anthocyanins are a subclass of flavonoids responsible for the production of the red color in apple peel,28 which is an important attribute in terms of stimulating consumer interest in buying the fruit. Bi et al.29 stated that cyanidin 3-galactoside, the main anthocyanin in apple peel, is more effective in the removal of hydrogen peroxide (H2O2) than other polyphenols. The authors also suggest that in humans the anthocyanins in foods come into direct contact with H2O2 during digestion, acting effectively in the removal of this compound. The IPC and TAN data for the peel suggest that in the second and fourth weeks prior to commercial maturity, for ‘Brookfield’ and ‘Mishima’ cultivars, respectively, the biosynthetic production of flavonoids is intensified, increasing the concentration of quercetins, in glycosylated forms, and leucoanthocyanidins (flavan-3,4-diol), which are the common precursors of flavan-3-ols and anthocyanidins. Bizjak et al.11 observed similar behavior in the peel of ‘Braeburn’ apples.

Figure 3. Changes in the total anthocyanins composition (mg cyanidin 3-glucoside 100 g−1 FM of peel) during the development of ‘Brookfield’ and ‘Mishima’ apples. Different letters in the graph show significant differences according to the DAFB at which the fruits were analyzed as determined by the Tukey test (p < 0.05). Vertical bars represent the standard error of the mean (SEM). F

DOI: 10.1021/acs.jafc.6b04695 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

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AUTHOR INFORMATION

Corresponding Author

*(M.C.S.) E-mail: [email protected]. Phone: +55 49 99487349. ORCID

Mayara C. Stanger: 0000-0002-2456-5626 Funding

We thank the National Council for the Improvement of Higher Education (CAPES) and the National Council for Scientific and Technological Development (CNPq) for financial support of this work. Notes

The authors declare no competing financial interest.

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DOI: 10.1021/acs.jafc.6b04695 J. Agric. Food Chem. XXXX, XXX, XXX−XXX