Polyphenolic profile and antioxidant activity of five apple cultivars ...

International Journal of Food Science and Technology 2009, 44, 1167–1175

Original article Polyphenolic profile and antioxidant activity of five apple cultivars grown under organic and conventional agricultural practices Athanasios Valavanidis,* Thomie Vlachogianni, Antonios Psomas, Alexandra Zovoili & Vasilios Siatis Department of Chemistry, Laboratory of Organic Chemistry, University of Athens, University Campus Zografou, 15784 Athens, Greece (Received 11 September 2008; Accepted in revised form 10 February 2009)

Summary

The polyphenols and total antioxidant activities of five apple cultivars, grown by organic and conventional agricultural methods in neighbouring farms, were determined and compared. Total polyphenols in the whole fruit and in the peel were determined by the Folin-Ciocalteu method, and the total antioxidant activity was determined by three established methods, diphenyl picrylhydrazyl (DPPH), azinobis (3-ethylbenzthiazoline6-sulfonic acid) (ABTS) and ferric reducing ⁄ antioxidant power (FRAP). Polyphenolic content for the whole fruit was in the range of 80–196 and for the peel 165–400 (mg Gallic Acid Equivalent (GAE) ⁄ 100 g fresh weight) for both types of agricultural practices. Antioxidant activities of fruit extracts were also relatively similar and well correlated to their polyphenolic content. High-performance liquid chromatography (HPLC) analysis of the most important polyphenolics (chlorogenic acid, catechin, epicatechin, procyanidin B1 and B2, cyaniding 3-galactoside, phloridzin, quercetin 3-galactoside and quercetin 3-arabinoside) also showed that concentrations do not differentiate significantly between the organic and conventional apples. Statistical significance of differences in antioxidant activities among the same cultivars was relatively small (flesh + peel or peel only) for both types of apples. These results indicate that organic apples do not present higher antioxidant or nutritional value compared with conventionally grown ones, as far as polyphenolic content and total antioxidant activities are concerned.

Keywords

ABTS, antioxidant activity, apples, conventional agriculture, DPPH, FRAP, organic agriculture, polyphenols.

Introduction

Fruit, vegetables and grains contain high concentrations of phytochemicals with antioxidant, anti-inflammatory and anticancer activity, broadly termed polyphenolic compounds, which have been proved highly beneficial to human health (Manach et al., 2004). Concentrations of polyphenolic compounds in plants are strongly influenced mainly by cultivar, but also by soil type, growth stage and environmental conditions, especially light. In this respect, it is argued that organic agricultural practices enhance the nutritional value of plant foods compared with conventional agriculture (Brandt & Mølgaard, 2001). The last decade has seen significantly increased interest in organic food production, as conventional methods caused various environmental problems (Trewavas, 2001; Macllwain, 2004). Organic agricultural farming methods are more friendly to the environment and the wildlife, because they avoid the use of manmade fertilizers and pesticides (Heaton, 2001). *Correspondent: Fax: +30 210 7274761; e-mail: [email protected]

The popular perception that organically grown foods are ‘healthier’ is widespread among consumers in developing countries (Institute of Food Science and Technology, 1999; Magkos et al., 2003; Magkos et al., 2006). However, evidence that can clearly support or negate the higher levels of micronutrients remains equivocal, with the number of systematically-controlled studies that have compared organic vs. conventionally grown crops being very small (Williams, 2002). Scientific papers in this area have failed to draw definitive conclusions (Woese et al., 1997; Worthington, 1998; Dimberg et al., 2005). Published comparative measurements showed that organic fruit and vegetables contained more vitamin C, iron, magnesium and phosphorus and less nitrates than conventional crops (Worthington, 2001). A critical review on the issue emphasises that, ‘…with possible exception of nitrate content, there is no strong evidence that organic and conventional foods differ in concentrations of various nutrients. While there are reports indicating sensory qualities, the findings are inconsistent’ (Bourn & Prescott, 2002). Apples, and to a lesser extent other fruit, are a major source of flavonoids (polyphenols) in the Western diet and their health benefits have been reviewed (Boyer &

doi:10.1111/j.1365-2621.2009.01937.x 2009 The Authors. Journal compilation 2009 Institute of Food Science and Technology

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Polyphenolic profile and antioxidant activity of apples A. Valavanidis et al.

Liu, 2004). Polyphenols exert cardio- and cerebroprotective effects by decreasing oxidative damage (Hung et al., 2004). Quantitation of polyphenolic content of apples were performed mostly by the Folin-Ciocalteu method (Tsao et al., 2003, 2005; Vrhovsek et al., 2004; McGhie et al., 2005). Recent studies compared their total polyphenolics with their total antioxidant capacity (TAC) or radical scavenging capacities by various methods, but their results showed a large variety of values, probably reflecting different extraction methodologies, apple cultivars, growing regions, soil type and different time of light exposure (Vinson et al., 2001; Imeh & Khochar, 2002; Kondon et al., 2002; Lee et al., 2003). There are limited number of studies comparing organically and conventionally (or under integrated agricultural methods with controlled fertilizer and pesticide use) grown apples (Carbonaro et al., 2002; Van der Sluis et al., 2002; Asami et al., 2003; Chinnici et al., 2004; Veberic et al., 2005). A recent study with Golden Delicious apples, grown under organic and conventional methods, found relatively similar total polyphenols and antioxidant activity (Briviba et al., 2007). Comparative studies of antioxidant activity and polyphenolic content in fruits are believed to be difficult to evaluate, because of several methodological problems which are difficult to control (Wolfe et al., 2003). It is noteworthy to add that there are some environmental benefits from organic or the integrated apple production, especially in terms of sustainability, soil fertility and lower use of fertilizers and pesticides by organic or the so called integrated apple production systems (Reganold et al., 2001). The present study tried to overcome the extraneous variables by comparing organic and conventional apples from neighbouring farms, (purchased the same day and within a week of harvesting) in the period SeptemberOctober (for 2 years, 2006–07) when fruit are freshly produced. This study compared polyphenolic (FolinCiocalteu) content and antioxidant activity by three methods diphenyl picrylhydrazyl, azinobis (3-ethylbenzthiazoline-6-sulfonic acid), ferric reducing ⁄ antioxidant power (DPPH, FRAP and ABTS), in the whole fruit and peel, of five popular varieties of Greek apples, grown under organic and conventional agricultural conditions. Also, nine of the most important antioxidant polyphenolic compounds were analysed by HPLC in both types of apples in the whole fruit and in the peel. Materials and method

Materials

Folin-Ciocalteu phenol reagent, 2,2¢-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS), potassium persulfate, DPPH (2,2-phenyl-1-

International Journal of Food Science and Technology 2009

picrylhydrazyl), 2,4,6-tripyridyl-s-triazine (TPTZ) and other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA), Merck (Darmstadt, Germany) and Fluka Chemicals (Buchs, Switzerland). Standards of polyphenolics (HPLC analysis) were purchased: chlorogenic, catechin and epicatechin from Sigma Chemicals Co (St Louis, MO, USA); procyanidins B1 and B2, and cyaniding 3-galactoside from Indofine Chemicals Co (Hillsborough, NJ, USA), phloridzin, quercetin 3-arabinoside and quercetin 3-galactoside from Extrasynthese (Lyon, France). Biological material (fruits)

Apples (Malus pumila) cultivars: Red Delicious-Starking, Golden Delicious, Granny Smith, Jona Gold and Royal Gala. Apples of both types of agricultural practices were carefully selected to match area, region (neighbouring farms) and day of purchase, which was within a week of harvesting. Organic apples were purchased from established organic fruit producers in the period September–October (2006–2007) (certified products by Greek licensing authorities, regulated by the Ministry of Agriculture). The main growing region of Greece for apples is in the central-east of Greece, prefecture Magnesia, region Agia and Pilion. Also, we contacted interviews with accreditation agencies of organic products. All apples were kept in paper bags for 1–2 days under refrigeration at 4 C until used. Sample preparation and chemical analysis

The weight and water content of the whole fruit and peel was measured by freeze-drying from each cultivar of apple. Peels were separated from the flesh manually using a sharp metallic peeler (thickness of 0.2 cm). Samples were obtained from at least five (5) randomly selected fruits in each trial to minimise variation. Determination of total polyphenolic content in fruit extracts

Polyhenols were extracted by 90% aqueous methanol. 100 g of whole fresh fruit, and 100 g of fresh peel, were frozen in liquid nitrogen, grounded into powder and extracted twice with 50 mL of chilled 90% aqueous MeOH. The mixture was sonicated for 20 min under continuous stream of nitrogen gas (to prevent oxidation) and the slurry was filtered quickly (Whatman No. 1) under vacuum. The combined filtrates were evaporated (rotary evaporator) at 40 C to dryness. The dried extract was kept in a dark vacuum dessicator for 2 days and then weighted. The total amount of dried extract was made up to 50 mL with distilled water and frozen at – 25 C until analysis. All extracts were made at the same day in triplicate.

2009 The Authors. Journal compilation 2009 Institute of Food Science and Technology


The total phenolic contents (TP) were measured using the colorimetric Folin-Ciocalteu assay (F-C) as described by Dewanto et al. (2002). TP measurements (absorbance at 760 nm) were compared with a standard curve of Gallic acid solution and expressed as milligrams (mg) of gallic acid equivalents (GAE) per 100 g of fresh weight of fruit (f.w). The linearity was determined for concentrations of 1.0 to 10.0 mg L)1 GAE (R2 = 0.9979), and absorbance range of 0.10–0.80 AU. Measurements of antioxidant capacity

Extraction of polyphenols was performed as described above. The dried extracts were weighted and then dissolved in 50 mL of MeOH. The amount thus collected represents 100 g of fresh wet weight (f.w). The DPPH method

Antioxidant capacity was measured as IC50 (Inhibition Capacity 50%) by UV-Vis at 517 nm. This assay was performed as described in a previous paper by our research group (Valavanidis et al., 2004). IC50 was defined as the amount (mg) of dried extract (representing 100 g of f.w) required to lowering the initial absorbance of DPPH stable radical concentration by 50% per one mL of reaction mixture. IC50 was extrapolated from dose–response curves. Experiments were carried out in triplicate and results were expressed as mean values ± SD. All experimental data showed a linear correlation as to the amount of the antioxidants (straight line resulting from the fit by linear regression). The ABTS method

Experiments were performed combining the experimental methodology of two papers (Re et al., 1999; Herraiz & Galisteo, 2004) and is based on the oxidation of the 2,2¢-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS) by potassium persulfate to form a radical cation ABTS•+. An aqueous solution (25 mL) of ABTS (96.02 mg, 0.175 mmol) and potassium persulfate (16.55 mg, 0.0612 mmol) were mixed and left standing overnight (16 h) to develop the deep blue–green colour of ABTS•+. The IC50 is calculated as the mg of apple extracts per 1 mL of mixture (representing the initial fresh weight of 100 g corresponding to a 50% bleach of the radical cation at 734 nm). Experiments were always performed with freshly made up solutions in triplicate. The FRAP assay (ferric reducing ⁄ antioxidant power) was performed on a modified version of the method by Benzie & Strain (1999). It is based on the reducing power of antioxidants, which will reduce the Fe3+ to the Fe2+ in the form of a blue complex (Fe2+ ⁄ TPTZ). The reagent FRAP was prepared freshly every day. The FRAP value were calculated in lmol ascorbic acid equivalent per 100 g of fresh weight (f.w.) extracts,


instead of lmol kg)1 of extract which was used by Benzie & Strain (1999). The antioxidant capacity was calculated in comparison with ascorbic acid aqueous solutions, tested at five different concentrations (100– 1000 lmol). The 500 lmol concentration of ascorbic acid is equivalent to 1000 FRAP values. HPLC analysis of polyphenols

An HPLC system (Agilent Technology 1100 series, Hewlett-Packard, Waldbroun, Germany) with UV-Vis detector was used for the quantification of certain polyphenolic compounds. A Column Lichro 250 · 4 mm, Lichrospher 100 RP-18, 5 lm (25 cm · 4.6 mm) was used for the separation. The binary mobile phase consisted of 0.5% MEOH in aqueous 0.01 m phosphoric acid (solvent A) and 100% acetonitrile (solvent B). The linear gradient elution was as follows: from 96.5% A to 90.0% A in 11 min, then 80% A at 22 min, followed by re-equilibration at the initial conditions for 5 min. The flow rate was 1 mL min)1 and the injection volume was 10 lL. All standards were dissolved in MeOH. The detector was set at 280 nm for flavonols, at 320 and 350 for flavonol glycosides. Chlorogenic acid, catechin, epicatechin, procyanidins B1 and B2, cyaniding 3-galactoside, phloridzin, quercetin 3-galactoside and quercetin 3-arabinoside were quantified. Quantification was performed by the external standard method. Statistical analysis

The variability of polyphenolic content was measured in the whole fruit and in the peel of apples (organic and conventional). Statistical significance of the differences between organic and conventional grown fruits was determined by the one way analysis of variance (one way anova) and is shown as *P < 0.05; **P < 0.01; ***P < 0.001. The normality (Kolmogorov-Smirnov normality test) and homogeneity of variance (Levene ‘s test) of data was verified and data was applied as required to fulfil anova assumptions (Dytham, 1999). Descriptive statistics were estimated using the spss (SPSS Inc., Chicago, IL, USA) for Windows statistical package, version 10.0.1. Results and discussion

Organic and conventional apples

Results showed that conventionally grown apples are in general heavier (by 20–30%) than organically grown ones for most cultivars. Some organic apples contained blemishes on their skin (black spots, or damage). Water content for all apple cultivars was in the range of 82– 88%. Conventional apples showed a 2–3% lower water content than organic ones. Peels in most apples is 5–8%


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(a) Total phenols (mg GAE/100 mg f.w.)

of the weight of the whole apple. Prices of conventional apples were in the range of 0.75–1.60 Euro kg)1 and for organic apples were 2.0–3.0 Euro kg)1 (2006–2007). Organic apples were three times more expensive when imported (winter months) from Italy. Greek production per year is around 350.000 tones of apples, of which 15–20% are exported every year. Apples provide substantial amounts of antioxidants, approximately 20–25%, of the total polyphenols consumed per capita in the Western countries (Vinson et al., 2001; Imeh & Khochar, 2002). Consumption of cheap and indigenous apples and other fruit are very important for consumers, especially for families of low and medium income (Boyer & Liu, 2004; Lotito & Frei, 2004).

500


Organic

400 350 300 250 200 150 100 50 Red Delicious Starking

Comparison of polyphenolic content

The total polyphenolics in the whole fruit and in the peels of apples grown under organic and conventional agricultural practices results are presented in Fig. 1. Statistical analysis did not reveal any substantial difference in the polyphenolic content of conventional and organic apples. Most of our samples-fruit were purchased within a week of being harvested in the September–October period and must represent the highest concentration of fresh fruit. Results for total polyphenolic concentrations, for the whole fruit, showed concentrations in the range of 80–196 (mg GAE ⁄ 100 g f.w) and for the peels in the range of 165–400 (mgGAE ⁄ 100 g f.w). These results are relatively similar with results from other papers for conventional apples, although it is inevitable to have a broad variety of values in the scientific literature for apples from northern European countries or Canada and from the Mediterranean countries (Vinson et al., 2001; Lee et al., 2003; Vrhovsek et al., 2004; McGhie et al., 2005). Higher values of phenolics in the whole fruit were found by Imeh & Khochar (2002), in the range of 300– 535 mg (GAE ⁄ 100 g f.w.). Much lower total phenolics in apples and pears (48 and 38 mg GAE ⁄ 100 g f.w. respectively) were found in another study. The authors are aware of their low concentrations, but explained the huge difference as part of varietal differences and differences in phenol standards (Proteggente et al., 2002). The F.C. method for polyphenolic determination was compared with HPLC measurements in peels of eight apple cultivars grown in Ontario (Tsao et al., 2003). The concentrations were not exactly the same but they correlated well. In general, the concentrations were lower for the F.C. method than for the HPLC analysis. A similar comparative study of F.C. method and HPLC analysis (eight varieties of apples) showed that results by the two methods differed only by 2–3% (Vrhovsek et al., 2004). Polyphenol concentrations in apples change

Conventional

450

0

(b) Total phenols (mg GAE/100 mg f.w.)

1170

Golden Delicious

Granny Smith

Royal Gala Jona Gold

500 Conventional

450

Organic

400 350 300 250 200 150 100 50 0

Red Delicious Starking

Golden Delicious

Granny Smith

Royal Gala Jona Gold

Figure 1 Total phenolic content of the whole fruit (a) and peel (b), measured by the Folin-Ciocalteu method of apples grown under conventional and organic agricultural practice. Values, mean ± SD, in mg of Gallic acid equivalents (GAE) per 100 g of fresh weight (f.w). (n = 6, determinations in triplicate). Statistical differences are noted, *P < 0.05; **P < 0.01; ***P < 0.001.

during the early stages of development, but remain relatively constant in the maturation stage and during storage (Burda et al., 1990). Statistical analysis of the results in our study showed that conventional grown apples have similar content of total polyphenolics compared with organically grown apples. Polyhenols for the whole fruit, among the five apple cultivalrs, decreased in the order: Starking>Royal Gala‡Granny Smith‡Jona Gold> Golden Delicious. Starking apples (conventional) is at the top of the list in most studies with the highest polyphenolic content in the flesh and in the peel (Imeh & Khochar, 2002; Wolfe et al., 2003; Vrhovsek et al., 2004).



Polyphenols in the peels from conventional apples are relatively higher that organic apples. Peels in both types of apples have two- to threefold polyphenols than in the edible part of the apple. Apple peels contain additional flavonoids, such as quercetin glycosides (Wolfe et al., 2003). It is estimated that although the peel is only 5– 7% of the weight of the whole apple, contains 40–45% of polyphenols, making the consumption of the whole fruit very beneficial in terms of antioxidants (McGhie et al., 2005). Comparison of antioxidant capacities

The total antioxidant activity of organic and conventionally grown apples was measured by three standard methods, which are encountered frequently in the scientific literature. The results showed that the ABTS method was very consistent for repeated measurements and the range of error was less than 5%. DPPH and FRAP methods gave less consistent results, with errors for repeated measurements around 6–9%. The combination of three different methods is considered valid and useful to overcome errors and evaluate different samples

containing polyphenolic antioxidants (Sanchez Moreno, 2002). Results are presented in Table 1 (for the whole apple) and in Table 2 for the peel of the apples. The antioxidant activity of extracts of apples (whole fruit and peel), were well-correlated with the total polyphenolic contents measured by the F.C. method. Discrepancies between polyphenolic content and antioxidant activity were noted for the FRAP method, whereas ABTS proved to be the most reliable. Peels in all cultivars showed higher antioxidant capacity, especially for cultivars Starking and Granny Smith. Combining the results of the three methods we summarise our comparative results of antioxidant activity of conventional and organic cultivars. Results are presented in Table 3. Quantitative comparative results are expressed in a comprehensive manner by using ratios: IC50(org.) ⁄ IC50 (conv.) for DPPH and ABTS methods, whereas for the FRAP method we used the ratio: FRAP values (org.) ⁄ FRAP values (conv.).The majority of ratios are around unity, which is a strong indication for relatively small differences in the antioxidant activity for organic and conventional apples. Despite the inherent limitations of the quantitative

Table 1 Antioxidant activities (IC50) of the whole apple (flesh + peel) of apple extracts grown under organic and conventional agricultural practices

Apples

DPPH (IC50) Conventional

DPPH (IC50) Organic

ABTS (IC50) Conventional

ABTS (IC50) Organic

FRAP Conventional

FRAP Organic

Red Delicious Starking Golden Delicious Granny Smith Royal Gala Jona Gold

1.23 3.22 1.28 1.60 1.80

1.16 2.98 1.22 1.85 1.92

0.94 1.63 0.87 0.87 0.92

0.79 1.80 0.95 1.13 0.87

701 210 640 494 479

890 242 634 477 582

± ± ± ± ±

0.05 0.23 0.10 0.20 0.23

± ± ± ± ±

0.10 0.23 0.15 0.10 0.06

± ± ± ± ±

0.12 0.22 0.06 0.05** 0.04

± ± ± ± ±

0.1 0.01 0.13 0.07 0.06

± ± ± ± ±

56*** 17** 22 31 9**

± ± ± ± ±

28 8 14 12 14

DPPH, diphenyl picrylhydrazyl; ABTS, azinobis (3-ethylbenzthiazoline-6-sulfonic acid); FRAP, ferric reducing ⁄ antioxidant power. The DPPH and ABTS values are mg mL)1 per 100 g (f.w.) and for FRAP values are in lmol ⁄ 100 g (f.w.) of ascorbic acid equivalent. Values, mean ± SD (n = 6, determinations in triplicate). Statistical differences are noted with, *P < 0.05; **P < 0.01; ***P < 0.001.

Table 2 Antioxidant activities (IC50) of the peel of apples per 100 g extracts, grown under organic and conventional agricultural practices

Apples

DPPH (IC50) Conventional mg mL-1 per 100 g (f.w.)

DPPH (IC50) Organic mg mL-1 per 100 g (f.w.)

ABTS(IC50) Conventional mg mL-1 per 100 g (f.w.)

ABTS (IC50) Organic mg mL-1 per 100 g (f.w.)

FRAP Conventional lmol ⁄ 100 g (f.w.)

FRAP Organic lmol ⁄ 100 g (f.w.)


0.58 1.23 0.92 0.92 0.92

0.60 1.63 1.05 1.09 1.09

0.42 0.57 0.50 0.80 0.70

0.41 0.74 0.67 0.89 0.85

2756 381 1200 1032 1095

2850 350 728 1047 990

± ± ± ± ±

0.03 0.04*** 0.03** 0.01 0.10

± ± ± ± ±

0.02 0.04 0.02 0.11 0.08

± ± ± ± ±

0.04 0.03** 0.01*** 0.02 0.05

± ± ± ± ±

0.02 0.03 0.01 0.07 0.08

± ± ± ± ±

105 7** 24*** 29 27**

± ± ± ± ±

67 8 9 34 25

DPPH, diphenyl picrylhydrazyl; ABTS, azinobis (3-ethylbenzthiazoline-6-sulfonic acid); FRAP, ferric reducing ⁄ antioxidant power. Values for DPPH and ABTS in mg mL)1 per 100 g (f.w.), and for FRAP in lmol ⁄ 100 g (f.w.) (ascorbic acid equivalent). (Mean values ± SD (n = 6, determinations in triplicate). Statistical differences are noted with, *P < 0.05; **P < 0.01; ***P < 0.001.



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Table 3 Comparison of antioxidant activities of the whole fruit and peel

Apples

DPPH ratio whole fruit

DPPH ratio peel

ABTS ratio whole fruit

ABTS ratio peel

FRAP ratio whole fruit

FRAP ratio peel


0.94 0.92 0.95 1.18 1.06

1.03 1.32*** 1.14** 0.91 1.18

0.84 1.10 1.09 1.29** 0.94

0.97 1.30** 1.34*** 1.11 1.21

1.27*** 1.15** 0.99 0.96 1.20**

1.03 0.92** 0.60*** 1.01 0.90**

Ratios of mean values (only) of IC5o(org.) ⁄ IC50 (conv.) for DPPH and ABTS methods. Ratios of mean values (only) of FRAP values (org.) ⁄ FRAP values (conv.) for the FRAP method. a Ratios lower than one (1) indicate that organic is more antioxidant than conventional apple. Statistical differences are noted with, *P < 0.05; **P < 0.01; ***P < 0.001.

measurements the combination of three methods represents the best way for the comparison of the antioxidant capacity of five apple cultivars. Statistical analysis of our results showed that there are some statistically significant differences. Some organic apple cultivars have relatively higher antioxidant activities when compared with conventional apples. But in some cases conventional cultivars have also higher antioxidant capacity than organic ones. The antioxidant activities, performed by three different methods reflect to a great extent their polyphenolic content. Also, synergistic effects must be at work among polyphenolics and other chemical constituents which may contribute to the antioxidant potential, such as ascorbic acid and beta-carotene (Dragster et al., 1993). Comparisons of conventionally and organically grown fruit are very limited in the scientific literature. A relevant paper is by Chinnici et al. (2004) on the relationship between phenolic composition (analysed by HPLC) and radical scavenging activity (by DPPH) of organic and integrated agricultural practices (the authors give details of regulations, pesticide management and fertilization).of apple peel and pulp of Golden Delicious. It was found that integrated apples were richer in polyphenols and had relatively higher antioxidant activity than organic apples. They concluded from their results that radical scavenging activity depends on the polyphenolic composition in a qualitative and quantitative way. Another recent study with Golden Delicious apples showed that there is no statistically significant differences in the total phenolic compounds (308 lg g)1 for organic and 321 lg g)1 for conventional fresh weight) or in polyphenolic classes between the two agricultural methods. Consumption of both types of apples indicated similar antigenotoxic potential (Briviba et al., 2007). The complexity of chemical profiles in apple makes it difficult to evaluate the antioxidant activity based on


any one single assay method. There are indications that some polyphenols show different antioxidant activities depending on that antioxidant assay method (Tsao et al., 2005). This inconsistency reconfirms that it is necessary to have at least two methods with different modes of actions in one study to reflect accurately the antioxidant activity (Ozgen et al., 2006). HPLC polyphenolic content in the whole fruit and the peels

The mean polyphenolic composition (for nine polyphenols) of the whole fruit and the peel in both organically and conventionally grown apples is presented in Table 4. In the whole fruit, concentrations of the nine polyphenols differ according to cultivar with Starking apples having the highest concentrations. Statistical differences (noted with *) in polyphenol concentrations are relatively not significant between organic and conventional apples for all cultivars. Results showed that the highest concentrations in the whole fruit were (decreasing order): ())-epicatechin>> chlorogenic acid> phloridzin>procyanidin B2 > procyanidin B1 > quercetin 3-galactoide> (+)-catechin>cyaniding 3-galactoside> quercetin 3-arabinoside. Our results are similar to a great extent with the average concentrations determined in various cultivars by other authors (Lee et al., 2003; Tsao et al., 2003; Chinnici et al., 2004; Vrhovsek et al., 2004). It is suggested that polyphenolic content and polyphenol profile in apples depends mainly on cultivar, and on the type of soil, intensity of light and adequate nitrogen fertilization. (Asami et al., 2003). Polyphenol concentrations in the peel of the various apple cultivars were as expected much higher, in the range of 1.5- to 2.5-fold, compared with the whole fruit. The concentrations varied according to cultivar and their range in decreasing order were: ())-epicatechin> procyanidin B2 > procyanidin B1 > phloridzin>



Table 4 Average (±SD) concentrations of polyphenols in the whole fruit and peel of five apple cultivars: organic (a) and conventional (b), in milligrams per 100 g fresh weight (mg ⁄ 100 g f.w).

Polyphenolic compounds

Red Delicious, Starking

Chlorogenic acid Whole fruit A 8.4 ± B 11.2 ± Peels A 9.5 ± B 12.3 ± (+)-Catechin Whole fruit A 2.8 ± B 3.1 ± Peels A 4.3 ± B 4.9 ± ())-Epicatechin Whole fruit A 12.0 ± B 14.8 ± Peels A 28.6 ± B 32.4 ± Procyanidin B1 Whole fruit A 4.3 ± B 5.6 ± Peels A 18.3 ± B 22.1 ± Procyanidin B2S Whole fruit A 8.5 ± B 9.7 ± Peels A 19.4 ± B 20.3 ± Cyanidin 3-galactoside Whole fruit A 2.2 ± B 2.8 ± Peels A 9.8 ± B 10.7 ± Phloridzin Whole fruit A 8.4 ± B 9.6 ± Peels A 14.2 ± B 16.7 ± Quercetin 3-galactoside Whole fruit A 2.8 ± B 3.2 ± Peels A 10.5 ± B 12.4 ±


Golden Delicious

Royal Gala

Granny Smith

Jona Gold

1.2 1.8

9.5 ± 0.7 8.3 ± 1.1

6.5 ± 0.5* 4.7 ± 0.6

3.2 ± 0.4 4.5 ± 0.8

4.6 ± 0.7 3.8 ± 0.5

2.3 3.6

18.3 ± 3.8 16.8 ± 3.2

9.4 ± 1.4 7.5 ± 0.8

4.0 ± 0.7 5.3 ± 0.9

6.7 ± 0.8 5.8 ± 0.7

0.3 0.6

1.2 ± 0.3 0.8 ± 0.2

1.6 ± 0.6 1.9 ± 0.8

3.2 ± 1.1 3.6 ± 0.8

0.8 ± 0.3* 2.1 ± 0.6

0.9 1.1

2.4 ± 0.6 2.0 ± 0.5

2.8 ± 1.2 3.4 ± 1.4

4.6 ± 1.5 5.2 ± 1.8

2.3 ± 0.8* 5.4 ± 1.3

1.6 2.1

7.6 ± 0.8 9.2 ± 1.2

8.4 ± 1.5 7.7 ± 0.9

10.5 ± 1.8 12.4 ± 1.4

6.7 ± 0.9 7.8 ± 1.3

3.4 4.5

21.3 ± 3.8 24.1 ± 4.3

14.8 ± 2.1 13.6 ± 1.8

17.4 ± 1.9 19.3 ± 1.7

12.3 ± 2.2 14.7 ± 2.4

0.8 1.2

2.8 ± 0.4** 1.5 ± 0.3

1.7 ± 0.2* 1.1 ± 0.3

4.4 ± 1.3 3.2 ± 0.4

0.7 ± 0.1 0.4 ± 0.2

3.1 3.6

4.6 ± 0.8 3.8 ± 0.5

3.1 ± 0.4 2.5 ± 0.4

14.5 ± 3.2 12.4 ± 2.7

3.2 ± 0.4* 2.4 ± 0.3

1.3 1.8

7.8 ± 1.4 6.4 ± 0.8

6.3 ± 0.9 7.8 ± 1.2

12.6 ± 2.1 12.9 ± 1.4

6.4 ± 0.5 6.0 ± 0.4

3.1 2.8

15.6 ± 2.4 13.4 ± 2.1

14.2 ± 2.5 17.3 ± 2.8

23.4 ± 3.4 24.5 ± 3.7

12.3 ± 0.9 11.7 ± 0.7

0.4 0.5

Nd Nd

1.4 ± 0.3 1.8 ± 0.4

Nd Nd

0.4 ± 0.1 0.2 ± 0.1

0.9 0.9

Nd Nd

3.3 ± 0.6 4.8 ± 0.8

Nd Nd

1.2 ± 0.3 0.8 ± 0.2

1.3 1.6

4.5 ± 0.8 4.3 ± 0.6

1.2 ± 0.3 0.9 ± 0.2

1.6 ± 0.5 1.9 ± 0.3

0.8 ± 0.2 0.6 ± 0.1

2.3 2.8

9.8 ± 0.9 12.4 ± 1.2

2.3 ± 0.6 2.2 ± 0.6

3.1 ± 0.7 4.7 ± 0.9

2.1 ± 0.5 1.8 ± 0.4

0.4 0.5

3.3 ± 0.2 2.8 ± 0.3

3.2 ± 0.5 3.6 ± 0.3

1.8 ± 0.2 1.6 ± 0.3

1.4 ± 0.3 1.2 ± 0.2

1.2 1.6

13.1 ± 0.7* 11.2 ± 0.5

12.8 ± 0.8 13.5 ± 0.8

8.7 ± 0.7 8.5 ± 0.9

6.5 ± 0.6 5.9 ± 0.5


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Table 4 (Continued) Polyphenolic compounds

Red Delicious, Starking

Quercetin 3-arabinoside Whole fruit A 1.4 B 1.7 Peels A 7.5 B 10.4

Golden Delicious

Royal Gala

Granny Smith

Jona Gold

± 0.3 ± 0.4

1.0 ± 0.2 0.8 ± 0.1

1.3 ± 0.4 1.2 ± 0.3

0.8 ± 0.2 1.0 ± 0.2

0.6 ± 0.2 0.8 ± 0.3

± 0.7* ± 1.2

3.5 ± 0.5 3.2 ± 0.5

3.8 ± 0.7 3.3 ± 0.6

2.7 ± 0.7 3.3 ± 0.8

1.8 ± 0.4 2.3 ± 0.6

Nd, not detected. Statistical differences are noted, *P < 0.05; **P < 0.01; ***P < 0.001.

chlorogenic acid>cyaniding 3-galactoside>quercetin 3galactoside>quercetin 3-arabinoside (+)-catechin. Statistical analysis showed that there no differences between organic and conventional apples, in the whole fruit and peel. Conclusions

Quantitative measurements of extracts of five different apple varieties, grown in neighbouring farms under conventional and organic agricultural practices, did not show any significant differences in the total concentrations of antioxidant polyphenols. As expected, peels for all cultivars of apples showed around a 1.5- to 2.5-fold higher polyphenolic content than the whole fresh fruit. The TAC or radical scavenging activity, measured by three method (ABTS, DPPH and FRAP) showed that there are relatively small differences, reflecting to a great extent their polyphenolic concentrations. Statistical analysis of our results showed that there are no very clear differences, between organic and conventional cultivars as far as polyphenolic content and antioxidant activity is concerned, when all three methods are taken into account. Red Delicious-Starking apples showed that highest antioxidant activity among the five cultivars for both types of agricultural practices. The HPLC analysis and profile of nine of the most important and highly antioxidant polyphenolos differ for the whole fruit and the peels of the fruits. Results showed that there are no significant differences in the concentrations of polyphenols for organic and conventional grown apples. Our results for the average concentrations of nine polyphenolics showed that there are in the same range with other recent papers on extracts of apple cultivars (Lee et al., 2003; Tsao et al., 2003; Chinnici et al., 2004; Vrhovsek et al., 2004). Overall, our results showed that organically grown apples are not nutritionally superior when compared with apples grown under conventional agricultural practices, as far as antioxidant polyphenolic content and TAC are concerned. High prices of organically grown fruits, especially apples, do not reflect a richer nutritional value, but a higher cost of production and


distribution. Considering that apples are a major source of antioxidants in the Western diet and their contribution against chronic diseases is supported scientifically, lower prices can contribute substantially in their wider scale of consumption (Eberhardt et al., 2000; Sun et al., 2002). Despite consumer concern over the quality and safety of conventional fruit in recent years, organic fruits are not proved to be nutritionally superior. Acknowledgments

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