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Phenolic composition and free radical scavenging activity of different apple

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Food Chemistry 127 (2011) 493–500

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Phenolic composition and free radical scavenging activity of different apple

varieties in relation to the cultivar, tissue type and storage
K. Carbone a,⇑, B. Giannini a, V. Picchi b, R. Lo Scalzo b, F. Cecchini a
a
Agricultural Research Council – Research Unit for Enology in Central Italy (CRA-ENC), Via della Cantina Sperimentale 1, 00049 Velletri, Rome, Italy
b
Agricultural Research Council – Food Technology Research Unit (CRA-IAA), Via Venezian 26, 20133 Milano, Italy

a r t i c l e i n f o a b s t r a c t

Article history: The aim of this research was to evaluate the influence of genotype, tissue type and cold storage on the
Received 12 August 2010 bioactive compounds content and on the antiradical activity (AA) of different apple cultivars (Golden
Received in revised form 15 November 2010 cl. B, Fuji cl. Kiku8, Braeburn cl. Hillwell). The content of analysed phyto-compounds depended on the
Accepted 6 January 2011
clone, on the part of fruit, and to a minor extent, on the storage. For EC50 data, the cultivar represented
Available online 9 January 2011
the main source of variation and the interaction with the type of tissue, was significant. The AA of apples,
measured by means of the DPPH test, was highly correlated to the flavan-3-ols content, which represents
Keywords:
a good predictor of the apple antiradical power. The new Braeburn’s clone, the Hillwell, had the worst AA
Apple
Cultivars
related to a minor phyto-chemical content. Also, its phenolic content was dramatically reduced after cold
Bioactive compounds storage (flesh: 50%; peels: 20%; p < 0.05). Obtained results underlined the key role of the genotype on
Phenols the content of the nutraceutical power of apples, which is important to improve their quality and con-
DPPH sumption benefits, suggesting to the breeders to pay more attention to the potential healthy compounds
in the development of new hybrids.
Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction
Phenolics are ubiquitous secondary metabolites in plants. They
include a large group of biologically active components, from sim-
ple phenol molecules to polymeric structures with molecular mass
above 30 kDa (Dreosti, 2000). The content of phenolic compounds
in plants varies with the species, the variety, the considered organ,
the physiological stage and the pedoclimatic conditions (Scalbert &
Williamson, 2000). Phenols in foods have gained much attention,
owing to their antioxidant activities and their possible beneficial
implications to human health, a consequence of their proven bio-
logical activity in the prevention of cancer and cardiovascular dis-
eases (Pearson, Tan, German, Davies, & Gershwin, 1999). A major
class of polyphenols found commonly in fruit and vegetables are
the flavonoids. Apples are one of the most important dietary
sources of this kind of phytochemicals in people’s diet, both in
the US and in Europe (Boyer & Liu, 2004). From an agronomic point
of view, the range of varieties of apples has greatly expanded com-
mercially: the old duopoly Golden Delicious–Red Delicious, and its
large corollary of clones are now challenged. This is due to the
European markets becoming increasingly exposed to the aggres-
sive penetration of apples from many competing countries that,
as a matter of fact, are heavily influencing even the European
⇑ Corresponding author. Tel.: +39 6 9630222; fax: +39 6 9634020.
E-mail address: katya.carbone@entecra.it (K. Carbone).
choice of varieties: from Gala that, in these later few years, with
its new mutants, has now become the most appreciated summer
apple by consumers, followed temporally by Braeburn, Fuji, Pink
Lady and their new hybrids. These new genotypes by plant breed-
ing are not always sufficiently evaluated and tested, particularly in
relation to their nutraceutical properties. Literature data almost al-
ways refer to a restricted number of polyclonal cultivars (i.e. Gold-
en Delicious), while there are few studies on the bioactive
compounds content, their composition and antiradical activity of
the new hybrids, resulting from the oldest cultivars (Wojdyło,
Oszmiański, & Laskowski, 2008), the importance of which is grow-
ing rapidly on the market. This is a key point, as the market is
increasingly orientated towards the production of hybrids, adding
to the better sensory attributes (i.e. colour), including an improved
content of healthy compounds (Bassi & Ferraro, 2005). In fact, one
of the main issues is to obtain fruit with more intense staining and
virus resistance, that would suggest an increase in phytochemical
concentrations and subsequently a higher antioxidant power. In
order to verify these assumptions, the aim of this study was to
evaluate the effect of the genotype, type of tissue and storage on
the phytochemical composition of apple peel and flesh and to
correlate it to the antiradical activity of these tissues. Moreover,
to our knowledge, there is no phytochemical and nutritional
characterisation of Hillwell apples, making this study quite
relevant for providing breeders with experimental data on this
emerging clone.

0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2011.01.030
 Author's personal copy
494 K. Carbone et al. / Food Chemistry 127 (2011) 493–500
2. Materials and methods
2.1. Chemicals
All used reagents were of analytical spectrophotometric grade
(Carlo Erba, Rome, Italy). All chromatographic solvents (methanol,
glacial acetic acid and water) and phenolic standards were HPLC
grade and were purchased from Sigma–Aldrich (Milan, Italy). Fo-
lin–Ciocalteu reagent, and vanillin were purchased from Carlo Erba
(Italy), while 2,2-diphenyl-1-picrylhydrazyl (DPPH) was purchased
from Sigma–Aldrich (Milan, Italy).
2.2. Apple sampling, harvest and storage conditions
During this research, two relatively new hybrids of different ap-
ple cultivars were analysed: Fuji cl. Kiku8 and Hillwell, a hybrid
belonging to the Braeburn cv., which were compared with one of
the most established genotypes: Golden cl. B. Apples were har-
vested from an orchard situated at the experimental farm ‘‘ F. Dot-
ti’’, at Arcagna, near Lodi (Northern Italy). Fruit trees were on
rootstocks M9. The rows of fruit trees were 50 feet, the age of
the plants were 8 years old and the cultivation method consisted
of conventional farming with an integrated control. For each culti-
var, apples were harvested from two different rows, sampling all
the fruit in the portion of the plant between 1 and 2.50 m, avoiding
the top and the bottom of the trees. Ten plants per row were sam-
pled. Apples were harvested at commercial maturity: 12–14°Brix,
starch index (Eurofru Scale) 6–7, flesh firmness 7–8 kg cm 2. From
harvested fruit, seven apples were chosen at random and in tripli-
cate (named H group). Also, in cold storage, two boxes of apples
harvested from each cultivar were kept for 3 months in a refriger-
ator at 0–1 °C (named CS group). At the end of cold storage apples
were sampled exactly as in the case of H group ones.
2.3. Polyphenolic apple extract
From each fruit, which both belonged to the H and CS group,
after cleaning with water, a slice (about 30 g) was taken, which
was immediately separated into flesh and peel that were frozen
separately in a forced air tunnel at 50 °C. Frozen samples were
then lyophilised and stored at 20 °C until extraction. The lyophi-
lised sample (0.3 g) was extracted at 2–4 °C with 15 ml of water
followed by 1 min of vortexing and 10 min of centrifugation at
5600g at 2–4 °C. The supernatant was divided in aliquots of 3 ml,
which were quickly frozen and lyophilised. All extracts were made
in triplicate.
2.4. Determination of total phenolic compounds
Total polyphenols of dried samples from H and CS groups were
determined using the Folin–Ciocalteu (F–C) method (Slinkard &
Singleton, 1977), with some modifications. Briefly, the dried sam-
ples were completely dissolved in 6 ml of a 70:30 ethanol/water
solution by vigorous vortexing until the dried material completely
dissolved, followed by centrifugation at 800g for 15 min. In order
to perform the assay, a volume of 5 ml of deionised water and
1 ml of a known dilution of the extract were added to a test tube.
F–C reagent (1 ml) was added to the solution and allowed to react
for 3–5 min. Then, 4 ml of 10% sodium carbonate solution was ali-
quoted into the test tubes, and the mixture was diluted up to 20 ml
with deionised water. The colour developed for 90 min, and the
absorbance was read at 760 nm, against a blank (substituting the
phenol solution in the reaction mix with water), using a UV-1700
PharmaSpec spectrophotometer (SHIMADZU, Milan, IT). TPC was
calculated from a calibration curve, using catechin as standard. Re-
sults were expressed as milligrammes of catechin equivalents
(CTE) per g of fresh apple tissue. Every analysis was replicated from
4 to 6 times.
2.5. Determination of flavan-3-ols
The vanillin assay was performed as described by Sun, Ricardo
da Silva, and Spranger (1998), with some modifications. Briefly, a
volume of 3 ml of 4% (w/v) vanillin in methanol and 0.5 ml of a
known dilution of the extract were added to a test tube. After 3–
5 min, 1.5 ml of concentrated HCl was added to the solution, mixed
and incubated for 15 min at 20 °C. The absorbance was read at
500 nm, using a UV-1700 PharmaSpec spectrophotometer (SHIMA-
DZU, Milan, IT). A blank was prepared by substituting the vanillin
solution in the reaction mix with methanol. FLC was calculated
from a calibration curve, using catechin as standard. Results were
expressed as milligrammes of catechin equivalents (CTE) per g of
fresh apple tissue. Every analysis was replicated from 4 to 6 times.
2.6. DPPH scavenging activity
The effect of the ethanolic extracts on the content of 2,2-diphe-
nyl-1,picrylhydrazyl radical (DPPH) was estimated according to
the method of Lo Scalzo, Iannoccari, Summa, Morelli, and Rapi-
sarda (2004), with some modifications. Briefly, an aliquot of the
DPPH solution (0.5 ml; 0.48 mM) was diluted in 1.0 ml of metha-
nol, and 1.0 ml of the ethanolic extracts at various concentrations
was added. The mixture was shaken vigorously and allowed to
stand for 90 min in the dark. The decrease in absorbance was mea-
sured at 517 nm against a blank (using methanol to replace the ex-
tract) in a UV-1700 PharmaSpec spectrophotometer (SHIMADZU,
Milan, IT), at room temperature. All the measurements were made
in triplicate. EC50 was calculated according to Sanchez-Moreno,
Larrauri, and Saura-Calixto (1998), from a calibration curve ob-
tained with different amounts of ethanolic extracts.
2.7. Single phenol analysis
The rationale for the single phenol analysis of apple extract was
taken from the previous works of Lister, Lancaster, and Sutton
(1994) and van der Sluis, Dekker, de Jager, and Wim Jongen,
2001. The used methods have been subjected to some variations,
as here described. The lyophilised aliquot of apple aqueous extract
(3 ml) previously obtained was treated with 0.3 ml of EtOH/HCl
0.02 N 1:1 v/v. The resulting mixture was vortexed for 1 min at
room temperature, and subsequently cleaned by centrifugation
(10,000g, 5 min), filtered on 0.45 lm filter and HPLC injected. The
HPLC analysis was performed with a JASCO system equipped with
a diode array detector (MD-910 JASCO). The pump (PU-980 JASCO)
was coupled with a ternary gradient unit (LG-1580-02 JASCO). The
analytical data were evaluated using a software-management sys-
tem of chromatographic data (ChromNAV, Jasco). The separation
was performed by a reversed phase, using an ODS-3 Lichrosorb
250  4 mm column. The flow rate was 0.8 ml/min, the injection
volume 20 ll, and the oven temperature was 40 °C. The mobile
phase consisted of water with 10% of glacial acetic acid (solvent
A), methanol (solvent B) and water with 50% of glacial acetic acid
(solvent C). The gradients were as follows (A/B/C): 95/0/5 for 0-
10 min, from 95/0/5 to 75/20/5 in 10 min, from 75/20/5 to 45/50/
5 in 10 min, 45/50/5 for 5 min, from 45/50/5 to 95/0/5 in 10 min,
95/0/5 for 12 min. Total analysis time was 57 min. Peak identifica-
tion was performed both by the direct comparison with commer-
cial standards and by their spectral and chromatographic
properties with respect to previous literature data from the ratio-
nale. The identified compounds in apple flesh and peel, ordered
by retention time (Rt), were plotted according to the maximum
 Author's personal copy

K. Carbone et al. / Food Chemistry 127 (2011) 493–500 495

of their absorbance: (+)catechin (Rt = 9.2 min, kmax = 280 nm), Table 1
chlorogenic acid (Rt = 13.4 min, kmax = 325 nm), ( )epicatechin Phenolic composition (mg CTE/g of fresh weight) and free radical scavenger activity of
the apple peel and pulp extracts (mean ± SE).
(Rt = 16.6 min, kmax = 280 nm), coumaryl quinic ester (Rt =
19.4 min, kmax = 310 nm), and phloretin glycoside (Rt = 32.0 min, Tissue Total phenols Flavan-3-ols EC50a
kmax = 280 nm). In the peel extracts, also quercetins glycosides, Peels 1.66 ± 0.058a 0.79 ± 0.036a 19.72 ± 0.744a
with maximum absorbance at 360 nm, have been identified and Flesh 0.79 ± 0.033b 0.18 ± 0.013b 41.60 ± 2.479b
quantified as their sum: 3-galactoside (Rt = 30.1 min), 3-glucoside a
EC50 = mg of tissue (on fresh weight basis) required to obtain 50% DPPH scav-
(Rt = 30.5 min), 3-xyloside (Rt = 31.1 min), 3-arabinoside (Rt = enging. Values with different letters in a column are significantly different (Mann–
32.1 min) and 3-rhamnoside (Rt = 32.7 min). The quantification Whitney test, p < 0.05).
was made by external standard calibration response, made by
authentic standards for chlorogenic acid, (+)catechin and ( )epi-
catechin. Solutions of p-coumaric acid was used for the quantifica- proved appearance, due to an increased pigmentation of the fruit,
tion of coumaryl quinic acid ester, phloretin for the phloretin as with Hillwell. This is a protected cultivar, with high productivity
glycosides, and quercetin-3-rhamnoside was used for quercetins and extended red striated pigmentation of fruit, of good quality
quantification. and resistant to handling (Donati, Palara, Berra, Guerra, &
Sansavini, 2004). The three genotypes selected for this study were
characterised by an over-colour of the peels that ranged from
2.8. Statistical analysis
green-yellow (Golden) to deep red (Fuji and Hillwell). In Table 1,
the average data on the distribution of polyphenols, flavan-3-ols
Statistical analysis was performed with SPSS 17.0 software
and antiradical activity between the peels and the flesh of analysed
(SPSS, Inc., Chicago, Illinois). Data were reported as means ± stan-
apples are reported, independently from the cultivar. Both TPC and
dard error of the mean (SE). An exploratory data analysis was made
FLC were significantly higher in the apple peel extract than in the
to check the data normal distribution (Shapiro–Wilkinson test) and
flesh one (TPC = +110%; FLC = +339%). A similar result for TPC was
the equal variances (Levene’s test). For TPC both ANOVA assump-
found by Lata (2007). The average ratio between FLC and TPC is
tions were violated, even after mathematical transformation of
more than twice as much in the peel as in the flesh (0.48 vs.
data (log-transformed meets only the equality of variances). For
0.23), in agreement with previous reports by other authors
FLC the equality of variances was met but not the normal distribu-
(Drogoudi, Michailidis, & Pantelidis, 2008). The results are consis-
tion of values, while EC50 data matched the ANOVA assumptions
tent with those reported in the literature. In fact, it is well known
entirely. In light of the obtained results, we analysed obtained data
that the content of phytochemicals is higher in apple peel (Chinnici,
through the Kruskal–Wallis non parametric test to test the overall
Bendini, Gaiani, & Riponi, 2004; Tsao, Yang, Young, & Zhu, 2003),
distribution of total phenols, flavan-3-ols and antiradical power of
such as catechins in particular (Burda, Oleszek, & Lee, 1990), and
apple tissues, while significant mean differences were established
some phytochemicals (i.e. quercitin glycosides) are only found here
using the Mann–Whitney test for independent and non parametric
(van der Sluis et al., 2001). Presented results emphasise the
procedures (p < 0.0167 for Bonferroni’s correction, where not spec-
properties of apple phytochemicals as such, regardless of specific
ified differently, Field (2005, chap. 13)). For single HPLC phenols
agronomic parameters (cultivars, storage, etc.), stressing the
analysis both the normal distribution and the equal variances were
importance of consumption and/or use of the whole fruit including
met. Data were subjected to one-way analysis of variance and
the peel, which is generally discarded by peeling the apple or in the
comparisons between means were determined according to Bon-
manufacturing of applesauce and canned apple.
ferroni’s test. Significant differences were accepted at p < 0.05
and represented by different letters. Spearman’s correlation coeffi-
3.2. Varietal differences and storage implications: phenolic compounds
cient was used to determine the correlation among variables in the
non parametric analysis, while Pearson’s one was used in the para-
Table 2 shows the flesh and peel phytochemicals content (to be
metric test. The regression between examined parameters was
considered as an index of the whole fruit properties) of different
made using the log-transformed values (both for EC50 and FLC
cultivars. TPC was the highest in Fuji cv. and significantly different
data) to match the normal and equal variance assumptions re-
from that of Hillwell (p < 0.05), while there were no significant dif-
quired by the model. In order to test the distribution of the antiox-
ferences in FLC between cultivars, although Golden apples showed
idant activity between the apple tissues as well as the influence of
a slightly higher value for this parameter. Moreover, we found that
the cultivar, storage and tissues on it, obtained results were elabo-
the average ratio between FLC and TPC in the Hillwell samples was
rated by the three way factorial analysis of variance (ANOVA), as
about twice as much as in the Fuji one (0.51 vs. 0.29; p < 0.05).
EC50 values were normally distributed and the variances were
Vrhovsek, Rigo, Tonon, and Mattivi (2004) reported an opposite
equal. The significance of the differences in the means of main ef-
trend of values for TPC of analysed cultivars, being higher for Gold-
fects (cultivar, storage, type of tissue, and interactions) was evalu-
en Delicious than for the Fuji one (+30%), but much less than that
ated using Bonferroni’s test, at the 5% probability level.
we obtained for clone B (about 35%), which are closer to those re-
ported by other authors (Chinnici et al., 2004; Tsao, Yang, Xie,
3. Results and discussion Sockovie, & Khanizadeh, 2005). Hillwell was found to have a higher
TPC with respect to the parental Braeburn (about +23%, Vrhovsek
3.1. Distribution of polyphenols within the apple

Table 2
It is well-known that phenolic and flavonoid contents vary Comparison of phenolic composition (mg CTE/g of fresh weight) of three apple
amongst different cultivars of fruit and vegetables, and within dif- cultivars (mean ± SE).
ferent tissues (van der Sluis et al., 2001). In respect to these consid-
Cultivar Total phenols Flavan-3-ols
erations, for this study, the cultivars were selected in order to well
represent the new orientation of the Italian market: an autumn Fuji, Kiku8 1.41 ± 0.109a 0.41 ± 0.065a
Braeburn, Hillwell 0.92 ± 0.780b 0.47 ± 0.084a
variety (Golden cl. B) and two winter ones (Fuji cl. Kiku8 and Brae-
Golden cl. B 1.33 ± 0.098a 0.53 ± 0.068a
burn cl. Hillwell). Among Braeburn’s hybrids, a general preference
in Northern Italy is given to those of more recent origin and im- Values with different letters in a column are significantly different (p < 0.05).
 Author's personal copy
496 K. Carbone et al. / Food Chemistry 127 (2011) 493–500
et al., 2004), which may be consistent with a more pronounced pig-
mentation of the hybrid. With respect to this, recent studies have
demonstrated the influence of apple cultivar on the fruit phyto-
chemical content (Imeh & Khokhar, 2002), as well as the possible
link between the colour of different cultivars and their nutritional
properties (Drogoudi et al., 2008). The influence of the cultivar on
the FLC was found to be in agreement with that of Vrhovsek et al.
(2004), showing slightly lower, but not statistically different val-
ues. In the present study, the flavanols represent 29%, 40% and
51% of the TPC in the apple, respectively, for Fuji, Golden and Hill-
well. Fig. 1(a) and (b) show the distribution of bioactive com-
pounds in apple tissues of the analysed cultivars. Fuji’s peel
showed the highest TPC (Fig. 1a), followed by the Golden one
(1.92 ± 0.051 and 1.85 ± 0.043 mg CTE/g fresh peel, respectively),
but the difference was not significant (Fig. 1a). Drogoudi et al.
(2008) and Lata (2007) reported a similar trend between Fuji and
Golden, although in their case the values were expressed on dry
basis. The Golden TPC reported in our study was closer to that re-
ported on fresh weight by Chinnici et al. (2004) and Tsao et al.
(2003). There are no data in the literature on the Hillwell cv. to al-
low a direct comparison with our results, although Yuri, Neira,
Quilodran, Motomura, and Palomo (2009) reported no statistical
differences between the parental (Braeburn) and the Fuji TPC of
the peels. Our data instead showed a marked difference between
the two cultivar (p < 0.05), being the TPC of Fuji higher. Drogoudi
et al. (2008) conducted an interesting study on the possible corre-
lation between harvest quality characteristics and nutritional
properties of several apple varieties. Their results show the exis-
tence of a significant link between the degree of red peel colour
and its polyphenolic content (i.e. anthocyanins), being the TPC
higher in Fuji (a bi-coloured cultivar) then in Golden (a green cul-
tivar). This result may be mainly due to the greater extent of red
over-colour of the peel in the first cultivar, caused by anthocyanins,
as reported by Kunradi Vieira et al. (2009). This could be particu-
larly interesting in our case in which we studied apple cultivars
with different peel colouration, having the Braeburn hybrid, the
Hillwell, a more red over-colour of the peel with respect to
the other cv. In our case, however, no correlation was found as the
TPC of Hillwell was even lower than that of the yellow Golden. In
this respect, Wojdyło et al. (2008) reported, for some red varieties,
an anomalous content of anthocyanins, lower than that expected
from the peel colour. The same results were reported by Lata
Fig. 1a. Concentration of phytochemicals (mg CTE/g of fresh weight) in the peels of different apple cultivars (mean ± SE). Different letters denote significant differences
(Mann–Whitney test, p < 0.0167 for Bonferroni correction, Field, 2005).
(2007). This is a key point as new farming techniques are based
on the production of clones from a parent of a more intense colour,
which in many cases, however, could produce a substantial loss in
high impact nutraceutical phytocompounds. Fig. 1a also shows
that there was no significant influence of the cultivar on the peel’s
FLC. It is interesting to note that Chinnici et al. (2004) reported an
average FLC for Golden Delicious of 0.234 mg/g fresh peel (HPLC
determination), which raised up to a value more similar to ours
(0.86 mg CTE/g fresh peel) if we added the procyanidins content
(0.731 mg/g fresh peel – HPLC determination). The same results
were found by Tsao et al. (2003) for the same cultivar. Fig. 1b also
shows a marked difference between TPC of Hillwell’s flesh and the
other analysed cultivar, while Golden’s flesh showed the highest
FLC (p < 0.05). Other important aspects in the apple production
chain are the harvest and storage conditions, which might cause
a loss of bioactive components. In the present study, the cold stor-
age for a short period of time (i.e. 3 months) did not seem to affect
the phytochemicals content of whole apples (flesh and peel, inde-
pendently from the cultivar) in a significant manner (TPC(H) =
1.31 ± 0.082 vs. TPC(CS) = 1.13 ± 0.087 and FLC(H) = 0.42 ± 0.050
vs. FLC(CS) = 0.52 ± 0.068). This is consistent with the general
observations of van der Sluis et al. (2001), who reported that cold
storage (4 °C) did not appear to substantially alter the content of
the main phenolic subgroups up to 25 weeks, except for a minor ef-
fect on total catechin concentrations, which tended to decrease
over time. In the light of these general results, analysis was carried
out into whether there was any influence of storage on phyto-
chemical distribution related to the dependence of bioactive com-
pounds by the part of fruit considered and/or the hybrid chosen. An
effect of cold storage on the peel’s FLC (Fig. 2) was found. In this
case the level of flavanols increased significantly by about +30%
after 3 months of cold storage, while the level of total phenolics re-
mained about the same. The same trend, although not significant,
was found in the flesh. Golding, McGlasson, Grant Wyllie, and
Leach (2001) analysed the fate of different apple peel phenolic sub-
groups during storage in air at 0 °C. The authors found an increase
in phenolics during the first months of storage and the influence of
the cultivar on them. Regarding the influence of cold storage on the
bioactive compounds of the different cultivars, some significant
differences were found only for Hillwell’s TPC, which was lower
than about 32% with respect to the harvest (TPC(H) = 1.10 ± 0.095
vs. TPC(CS) = 0.748 ± 0.104; p = 0.05). Looking at the influence of
 Author's personal copy

K. Carbone et al. / Food Chemistry 127 (2011) 493–500 497

Fig. 1b. Concentration of phytochemicals (mg CTE/g of fresh weight) in the flesh of different apple cultivars (mean ± SE). Different letters denote significant differences
(Mann–Whitney test, p < 0.0167 for Bonferroni correction, Field, 2005).

Fig. 2. Effect of cold storage on bioactive compounds distribution of apple peels and flesh (mean ± SE). Different letters denote significant differences (Mann–Whitney test,
p < 0.0167 for Bonferroni correction, Field, 2005).

storage on the different parts of the fruit of this cultivar, a dramat-
ical effect of the time on the flesh TPC was found, with a reduction
of above 50% after 3 months, while the reduction on peel TPC was
lower ( 20%). To our knowledge there are no literature references
about the effect of cold storage on Hillwell cv. In this case our re-
sults point out that its phenolics are not stable and undergo a
net metabolic turnover during storage, while flavanols do not seem
to be affected in this way as their content remained at the harvest
level.
3.3. Varietal differences and storage implications: free radical
scavenging activity
In order to evaluate the capacity of apple bioactive components
to scavenge free radicals we used one of the most easy-to use as-
says, consisting in the measurement of the ability of a molecule
to reduce a synthetic radical, DPPH (Brand-Willams, Cuvelier, &
Berset, 1995). This assay is based on a mixed mechanism of free
radical stabilisation: hydrogen atom transfer (HAT) and electron
transfer (ET). The method presents several analytical critical points
(Prior, Wu, & Schaich, 2005), but it has the great advantage of being
easy to implement and not requiring special equipment (just a
simple spectrophotometer). The results are generally expressed
in an indirect way, measuring the quantity of antioxidant neces-
sary to reduce the initial DPPH concentration by 50%, which is a
value generally defined as EC50: the higher the antioxidant activity,
the lower this value is. Fig. 3 shows an example of the graph we
have obtained for our apple samples to be employed for EC50 esti-
mation. Table 1 reports the average AA of the different part of fruit.
Obtained results showed that, in general, flesh and peel have an
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498 K. Carbone et al. / Food Chemistry 127 (2011) 493–500
Fig. 3. Example of % residual DPPH calculated at the steady state as a function of the
antioxidant quantity (mg apple fresh tissue).
intermediate activity like a scavenger, the peels being the best
scavengers (19.7 ± 0.744 mg fresh peel) and the flesh the worst
ones (41.6 ± 2.479 mg fresh flesh). In the latter case we also found
a great variability of the data, as we can see from SE values. These
results were consistent with those reported by Wolfe, Wu, and Liu
(2003). A highly significant relationship was found between the
antiradical power of apple and its phytochemical content, espe-
cially with flavanols (qs: 0.819; p < 0.001). The correlation is neg-
ative because when the phytochemical content of fruit increases
there is a corresponding decrease of the percentage of antioxidant
necessary to reduce the radical concentration by 50%. The correla-
tion with TPC was not as strong as the previous one (qs: 0.550;
p < 0.01). In literature, the correlation between TPC and AA is not
well-established because the coefficient is either high or low
depending on the study taken into consideration. Tsao et al.
(2003) reported similar results using both FRAP and b-CLAMS as-
says to test the antioxidant power of apples, underlining the strong
power as radical scavenger of the flavan-3-ols. The authors also
found a correlation between FRAP and TPC higher than that re-
ported in this study. This is probably due to the different mecha-
nisms underlying the tests used, as well as the presence of
interfering substances (Prior et al., 2005). As the correlation be-
tween antiradical power and flavonoids was very good, we used
the latter as a predictor in a linear regression model, using the
log-transformed data for both variables (EC50 and FLC) to meet dis-
tributional conditions (equality of variances for FLC data). Overall,
the regression model predicted the antiradical activity significantly
well (log EC50 = 1.237 0.433 log FLC; corrected R2 = 0.673;
p < 0.001). Table 3 shows the main effects of all principal factors
(cultivar, tissue type and storage), as well as their interactions,
on EC50 values. There was a strong effect of the type of tissue
and of the cultivar on the AA of the fruit as well as of their interac-
tion (p < 0.05). In fact, we found that the AA of the selected tissue
type strongly depended on the chosen cultivar. Golden cl. B peel + -
flesh possessed the best scavenging activity (22.6 ± 0.756 mg of
fresh weight apple), followed by Fuji (28.3 ± 0.862) and Hillwell
Table 3
Summary of analysis of variance (ANOVA) for EC50.
Component Source of variation
Cultivar (A) Tissue type (B)
dfc 2 1
EC50d 146.2a 570.8a
a
Significant at a = 0.05.
b
NS, not significant.
c
Degree of freedom.
d
F values.
(40.6 ± 0.756) and these differences resulted statistically signifi-
cant (p < 0.05), demonstrating a considerable variability in the va-
lue of the AA in relation to the genotype. These results were
consistent with the observed TPC for these cultivars. However, in
this regard it was noted that Golden possessed an antiradical
power that was approximately twice as much as the Hillwell type,
although it had a TPC only 30% higher. The observations we have
reported here suggest the importance of genotype for determine
AA and TPC. As a consequence, variety manipulation may be a
powerful tool for modifying antioxidant fruit patterns and con-
tents. Peel and flesh EC50 values of Fuji, Hillwell and Golden are gi-
ven in Table 4. All cultivars showed a total AA of the peels
significantly greater than that of the flesh. According to their phe-
nolic content, Fuji’s peel showed the highest antiradical activity,
while Hillwell had the lowest. Moreover, Golden EC50 peel/flesh
ratio was approximately twice as much as in the Hillwell one,
which may reflect the highest flavan-3-ols content of Golden flesh
with respect to the flesh of the other analysed cultivars
(Golden = 0.26 ± 0.018; Fuji = 0.12 ± 0.010; Hillwell = 0.15 ± 0.018
mg CTE/g fresh flesh; Mann–Whitney test, p < 0.0167 for Bonfer-
roni’s correction). ANOVA analysis (Table 3) pointed out that pro-
longed cold storage did not significantly influence the antiradical
power of apples. The same results were found by other authors
(van der Sluis et al., 2001). However, little effect was found when
the interactions between the main factors were considered. The
more pronounced interaction effect was found when the interac-
tions between all the main factors was considered (tissue type, cul-
tivar and cold storage), while the effect of cold storage on the
antiradical activity of both peels and flesh was minor (p = 0.025)
and similar to that due to the interaction between the storage
and the cultivars.
3.4. Quantification of bioactive compounds by HPLC
Results of the single phenols content generally confirmed what
was previously found in TPC and FLC analysis. In the flesh samples
(Table 5), the major phenol found in the extracts was chlorogenic
acid, ranging from 2.94 to 7.52 mg/100 g fw. Unlike what was
found by Lata, Trampczynska, and Paczesna (2009), the highest
concentration of this organic acid was found in the peels with
the exception of the Hillwell, for which the acid is concentrated
Table 4
Free radical scavenging activity in apple peels and flesh depending on cultivar
(mean ± SE).
Cultivar EC50
Peel Flesh
Fuji, Kiku8 17.34 ± 1.310a 39.263 ± 1.122b
Braeburn, Hillwell 22.67 ± 1.070b 58.485 ± 1.070b
Golden cl. B 18.667 ± 1.070a 26.596 ± 1.070b
EC50 = mg of tissue (on fresh weight basis) required to obtain 50% DPPH scavenging.
Values with different letters in a column are significantly different (p < 0.05).
Interactions
Storage (C) AB AC BC ABC
1 2 2 1 2
2.1b 84.9a 6.4a 5.3a 18.2a
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K. Carbone et al. / Food Chemistry 127 (2011) 493–500 499

Table 5
Single phenols composition (mg/100 g fw) of apple flesh in three cultivars after harvest and cold storage, (mean of three replicates ± SE).

Chlorogenic acid Coumaryl quinic ester (+)Catechin ( )Epicatechin Phloretin glucoside

Kiku8 H 5.76 ± 1.22ab 0.66 ± 0.10a 0.98 ± 0.31b 1.75 ± 0.64a 1.09 ± 0.06ab
CS 4.62 ± 1.81ab 0.57 ± 0.24a 0.75 ± 0.04b 0.98 ± 0.22a 0.82 ± 0.18bc
Hillwell H 5.32 ± 1.33ab 0.82 ± 0.11a 0.91 ± 0.18b 1.91 ± 0.33a 0.75 ± 0.09bc
CS 2.94 ± 0.49b 0.44 ± 0.23a 1.21 ± 0.37ab 1.34 ± 0.17a 0.58 ± 0.16c
cl. B H 7.52 ± 0.90a 0.67 ± 0.15a 2.20 ± 1.34ab 1.46 ± 0.17a 1.00 ± 0.11abc
CS 4.98 ± 0.10ab 0.79 ± 0.34a 3.65 ± 1.75a 1.78 ± 0.41a 1.41 ± 0.25a

Values with different letters in a column are significantly different (p < 0.05).

Table 6
Single phenols composition (mg/100 g fw) of apple peels in three cultivars after harvest and cold storage, (mean of three replicates ± SE).

Chlorogenic acid Coumaryl quinic ester (+)Catechin ( )Epicatechin Phloretin glucoside Quercetin* glycosides
Kiku8 H 8.96 ± 1.22a 0.99 ± 0.16a 12.91 ± 0.50a 9.94 ± 0.76a 1.04 ± 0.25a 14.92 ± 2.32ab
CS 8.05 ± 2.38a 0.75 ± 0.31abc 8.64 ± 0.90c 5.59 ± 0.92bc ND 13.687 ± 4.23ab
Hillwell H 2.82 ± 0.38bc 0.36 ± 0.04bc 9.81 ± 1.73abc 5.12 ± 0.65bc 0.77 ± 0.40a 11.54 ± 5.27b
CS 0.96 ± 0.22c 0.20 ± 0.07c 8.06 ± 1.37c 4.43 ± 0.86bc ND 14.889 ± 1.35ab
cl. B H 8.64 ± 2.12a 0.87 ± 0.35ab 12.182 ± 1.11ab 6.03 ± 1.11bc 1.05 ± 0.72a 23.51 ± 2.78a
CS 7.33 ± 0.13ab 1.03 ± 0.08a 9.06 ± 1.06bc 6.65 ± 2.40b ND 20.91 ± 5.11ab

ND, not determined. Values with different letters in a column are significantly different (p < 0.05).
*
Quercetin glycosides are represented by the sum of the peaks of: quercetin-3-glucoside, galactoside, arabinoside, xyloside and rhamnoside.

in the flesh (+89%), indicating again that the range of differences Table 7
between apple’s tissues were highly cultivar dependent. The other Correlation coefficients obtained by simple regres-
polyphenol compounds gave an amount in the order of about sion analysis (rxy) between single polyphenols of
apple and antiradical capacity, expressed as EC50
1 mg/100 g fw, with interesting values for (+)catechin (3.65 mg/
from DPPH quenching.
100 g fw in Golden after storage) and ( )epicatechin (1.91 mg/
100 g fw in Hillwell at harvest). In the peel samples (Table 6), the Compound

amount of single phenols was higher when compared to the flesh. Chlorogenic acid 0.76b
Moreover there was a good relationship with our previous spectro- Coumaryl quinic ester 0.51b
(+)Catechin 0.82a
photometric data, especially for the increasing amount of (+)cate-
( )Epicatechin 0.75b
chin and ( )epicatechin and for the exclusive presence of Phloretin glucoside 0.25b
quercetin glycosides. Regarding the cultivar, Golden cl. B was the Quercetins glycosides 0.73b
richest in phenolics both in the peels and in the flesh. These data Catechin + epicatechin 0.80a
are consistent with those reported by Lata et al. (2009). The highest a
Significant at a = 0.05.
values for (+)catechin, were found in Fuji and Golden at harvest b
NS, not significant.
(12.9 and 12.2 mg/100 g fw), and the greatest amount of ( )epicat-
echin resulted in Fuji at harvest (9.94 mg/100 g fw). The quercetin
glycosides content resulted very high respect to previous works phloretin glycoside in flesh with respect to peel, where it was com-
(van der Sluis et al., 2001), particularly in Golden samples (Felici- pletely depleted; (ii) some increase in catechins after storage both
ano et al., 2010). However, our data are consistent with those re- in flesh and in peel; and (iii) the very good stability of chlorogenic
ported by Mari et al. (2010) on apples of Italian origin, acid and quercetins, in peel samples.
suggesting that Italian apples possess a tendency towards a high
quercetin content. Also, flavonols are known to increase in re-
sponse to full sunlight or in other oxidative conditions (Guidi, 4. Conclusions
Degl’Innocenti, Remorini, Massai, & Tattini, 2008), while the quer-
cetin content of apple fruit is increased upon postharvest irradia- Apples are recognised as a good source of nutraceutical com-
tion (Hagen et al., 2007). It can be assumed that a high level of pounds. The essential role of flavan-3-ols as radical scavengers
these compounds is prevalent due to the particular environmental has been pointed out here, taking into account that cold storage
conditions of the Mediterranean cultivation area. This increased may be a possible cause in the variation between different geno-
amount in peel phytochemicals accounts for the increased index types, which considered here as an added variable. The study re-
of antiradical capacity. Catechins and quercetins are known as ported herein describes for the first time the phytochemical
the best free radical scavengers after a comparison with other phe- composition and antiradical activity of the new clone Hillwell. Re-
nol classes (Rice-Evans, Miller, & Paganga, 1996), a very good cor- ported data point out the role played by the genetic features of the
relation was found between EC50 values and (+)catechin and the fruit, underlying the importance of apple varietal selection, based
sum (+)catechin and ( )epicatechin (Table 7), with values very not only on high productivity and organoleptic properties (i.e. col-
close ( 0.82 and 0.81, respectively) to the relationship found our), but also on the nutraceutical characteristics of the fruit. As a
for the flavonoid index previously discussed ( 0.819). These data consequence, the introduction of new commercial hybrids should
are consistent with those reported by Serra et al. (2010). The stor- not only take into consideration the parameters concerned with
age effect generally confirmed what was previously found: the to- the appearance and resistance to pathogens, but also those related
tal amount of apple phenolics is not so greatly affected by storage. to the content of secondary metabolites responsible for the
However, some exception must be pointed out: (i) the stability of enhancement of the health value of apples, which are not always
 Author's personal copy
500 K. Carbone et al. / Food Chemistry 127 (2011) 493–500
positively correlated with the former. In light of this, the classic
question ‘‘does an apple a day keep the doctor away?’’ should be
reformulated as ‘‘which apple clone does it best?’’.
Acknowledgements
This research was supported by the funding released by Na-
tional Council of Agricultural Research (CRA resolution 9431/2,
14/11/2008), in the framework of ANTIOX-ITA project.
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