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Postharvest Biology and Technology 49 (2008) 348–354

Effect of cold storage on vitamin C, phenolics and antioxidant activity

of five orange genotypes [Citrus sinensis (L.) Osbeck]
Paolo Rapisarda ∗ , Marisol Lo Bianco, Paolo Pannuzzo, Nicolina Timpanaro
CRA-Centro di Ricerca per l’Agrumicoltura e le Colture Mediterranee, Corso Savoia 190, 95024 Acireale, Italy
Received 25 October 2007; accepted 2 February 2008

Abstract
The effect of cold storage on antioxidant profile and the antioxidant activity of five sweet orange genotypes [Citrus sinensis (L.) Osbeck], three
blood (pigmented) varieties with different anthocyanin contents (‘Tarocco Messina’, ‘Tarocco Meli’ and ‘Moro’) and two blond varieties (‘Ovale’
and ‘Valencia late’), stored at 6 ± 1 ◦ C for 65 d was investigated. During fruit storage, anthocyanins, hydroxycinnamic acids, vitamin C, flavanones
and total phenolics were determined, and juice antioxidant capacity was measured by two different in vitro tests (DPPH scavenging activity and
inhibition of induced linoleic acid peroxidation). The results showed an increase in anthocyanins, flavanones and hydroxycinnamic acids and a
slight decrease in vitamin C in the blood oranges. Cold storage negatively affected flavanone concentration, while positively influenced vitamin
C in blond orange varieties. Both antioxidant activity tests showed an increase in antioxidant capacity during storage caused mainly by phenolic
accumulation (blood oranges) and vitamin C increase (blond oranges). Finally, correlations between antioxidant activity and total or individual
phenolic components were examined.
© 2008 Elsevier B.V. All rights reserved.

Keywords: Blood oranges; Anthocyanins; Flavanones; Hydroxycinnamic acids

1. Introduction diuretic, analgesic and hypolipidemic activities (Galati et al.,

Clinical trials and epidemiological studies have established
an inverse correlation between fruit and vegetable dietary intake
and the occurrence of diseases such as inflammation, cardiovas-
cular diseases, cancer and aging-related disorders (Doll, 1990;
Ames et al., 1993; Willet, 1994). Dietary antioxidants, including
vitamin C, polyphenols and carotenoids, are believed to be effec-
tive nutrients in the prevention of these oxidative stress-related
diseases (Kris-Etherton et al., 2002).
Citrus fruit is rich in vitamin C and other bioactive com-
pounds including flavonoids and phenolic acids, all potentially
health-promoting (Widmer and Montanari, 1996). Several
studies have shown that certain cancers can be prevented
by selective food consumption; citrus flavonoids being one
of several possible cancer-preventing agents (Attaway, 1994).
Moreover, citrus flavonoids, particularly hesperidin and dios-
min, have shown a wide range of therapeutic properties in
medical and clinical applications such as antihypertensive,
∗ Corresponding author. Tel.: +39 095 7653134; fax: +39 095 7653113.
E-mail address: paolo.rapisarda@entecra.it (P. Rapisarda).
1994; Monforte et al., 1995). Citrus fruit also contains signifi-
cant quantities of hydroxycinnamic acids (HCA) such as ferulic,
p-coumaric, sinapic and caffeic (Risch and Hermann, 1988).
Recently, the role of hydroxycinnamic acids as antioxidants
(Silva et al., 2000) and orange juice markers (Rapisarda et al.,
1998) has been pointed out.
‘Tarocco’, ‘Moro’ and ‘Sanguinello’ are the most widely cul-
tivated sweet orange varieties [Citrus sinensis (L.) Osbeck] in
Italy. They have unique flesh and rind color, due to the red
pigments belonging to the anthocyanin class, and higher con-
centration of vitamin C, flavanones and hydroxycinnamic acids
than blond oranges (Rapisarda and Giuffrida, 1992; Rapisarda
and Intelisano, 1996; Rapisarda et al., 1998; Postorino and
Gionfriddo, 1999).
Anthocyanins have been associated with potentially bene-
ficial effects on various diseases such as capillary fragility, dia-
betic retinopathy and human platelet aggregation (Rapisarda
et al., 2001). The main anthocyanins in blood (pigmented)
orange juice are cyanidin-3-glucoside (Cy3G) and cyanidin-
3-(6 -malonyl)-glucoside (Cy3MG) (Maccarone et al., 1998).
Recently, it was reported that Cy3G has a higher antioxidant
activity than other more common anthocyanins (Wang et al.,

0925-5214/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.postharvbio.2008.02.002
 P. Rapisarda et al. / Postharvest Biology and Technology 49 (2008) 348–354
1997), and that Cy3MG protects plant cells against UV-induced
damage (Takahashi et al., 1991). In addition, Bonina et al. (1998)
found that a standardized extract of blood orange juice contain-
ing 3% anthocyanins has strong in vitro antioxidant activity and
in vivo photoprotective effects against UVB-induced skin ery-
thema. Lastly, other studies have demonstrated that blood orange
juices have higher antioxidant efficiency than blond orange
juices (Rapisarda et al., 1999).
These health benefits have prompted research into pre- and
postharvest citrus fruit treatments to maintain or enhance their
biological activity. Postharvest storage at low temperature for
more or less extended periods is necessary to extend the com-
mercial shelf-life of citrus fruit. Some blood orange genotypes
such as ‘Tarocco Messina’ and ‘Tarocco Meli’ are late varieties
that reach maturity in May. Therefore, postharvest management
of these varieties could extend the presence of blood oranges in
the market until the summer months.
Although oranges are considered to be non-climateric fruit,
storage treatment can increase fruit respiration because of pos-
sible chilling injury (CI) (Grierson and Ben-Yehoshua, 1986).
Blood oranges are highly susceptible to CI when stored below
7–8 ◦ C and they can undergo internal metabolic changes during
prolonged storage (Pratella et al., 1969).
The current study describes the responses of five orange
genotypes (three pigmented varieties with different anthocyanin
content and two blond varieties) to storage at 6 ± 1 ◦ C for
65 d, focusing on phenolic and vitamin C content and their
antioxidant activity. Anthocyanins, hydroxycinnamic acids, fla-
vanones, total phenolics and vitamin C, were determined and
juice antioxidant capacity was assessed by means of two
different in vitro tests (DPPH scavenging activity and inhi-
bition of induced linoleic acid peroxidation). Therefore, the
objective of this study was to investigate the effects of cold
storage on the levels of phenolics, vitamin C and the antioxi-
dant capacity in blood and blond oranges in order to evaluate
if this treatment may enhance the health functionality of
fruit.
2. Materials and methods
2.1. Chemicals
Ascorbic acid (AA), hesperidin, p-coumaric, ferulic, caffeic
and sinapic acids, Folin-Ciocalteu (FC) reagent, 1,1-diphenil-
2-picrylhydrazyl radical (DPPH), linoleic acid, lipoxygenase
from soybean (E.C.1.13.11.12) were purchased from Sigma
Chemical Co. (St. Louis, MO). Water, acetonitrile and methanol
(HPLC grade) were obtained from Merck (Darmstadt, Ger-
many). The solvents and reagents not expressly specified had a
high degree of purity and were supplied by Carlo Erba (Rodano,
Italy).
2.2. Fruit treatments
The study was carried out on fruit of five sweet orange geno-
types [C. sinensis (L.) Osbeck], three pigmented varieties with
different anthocyanin content (‘Tarocco Messina’, ‘Tarocco
349
Meli’ and ‘Moro nucellar 58-8D-1’) and two blond varieties
(‘Ovale nucellar 55-7C-5’ and ‘Valencia late’). The fruit were
harvested in 25 February (‘Moro’), in 13 May (‘Tarocco’
clones) and 10 June 2005 (‘Ovale’ and ‘Valencia’), respec-
tively, at the Palazzelli experimental farm of the CRA-Centro
di Ricerca per l’Agrumicoltura e le Colture Mediterranee
(Acireale, Italy). Once in the laboratory, fruit were treated
with 1000 mg/L of Imazalil to minimize decay during storage.
Fruit were then kept dry at room temperature for about 6 h
before being placed randomly in boxes (20 fruit per box).
Twelve boxes were used for each variety (four samples in
triplicate form) and stored in temperature-controlled chambers
at 6 ± 1 ◦ C, relative humidity (RH) 90–95% and complete air
change every hour. Fruit sampling (three boxes per each sample)
was carried out before storage (time 0) and at about 20-day
intervals, for a total storage period of 65 days. Fruit of each
box were squeezed by a domestic juicer and the juice obtained
used for physicochemical analyses and antioxidant activity
determination.
2.3. Physicochemical analysis
Total soluble solids (TSS) and titrable acidity (TA, as %
of anhydrous citric acid) were determined by standard meth-
ods (Kimball, 1999). Vitamin C (l-ascorbic acid) concentration
was determined by liquid chromatography using a Waters
Alliance 2695 HPLC equipped with a Waters 996 photodiode
array detector (PDA) and Waters Empower software (Rapisarda
and Intelisano, 1996). The column was a C18 Hypersil ODS
(150 mm × 4.6 mm i.d., 5 ␮m; Phenomenex, Torrence, CA)
maintained at 35 ◦ C. The elution was performed with a buffer
solution of 0.1 M KH2 PO4 /H3 PO4 (pH 2.3) at a flow rate
1 mL/min. Wavelength was set at 260 nm.
Anthocyanin content was evaluated spectrophotometrically
(Varian UV-vis spectrophotometer mod. Cary 100 Scan) by the
pH differential method (Rapisarda et al., 2000) and expressed
as cyanidin-3-glucoside equivalents (mg/L).
Flavanone glycosides, expressed as hesperidin equivalents
(mg/L), were determined by an HPLC procedure (Rouseff et
al., 1987) using the HPLC-PDA equipment described above.
A sample of centrifuged juice was diluted 1:1 with the mobile
phase, filtered through a 0.45 ␮m filter and injected directly into
the column. The solvent system was water/acetonitrile/acetic
acid (79.5:20:0.5), with flow rate 1 mL/min. The column effluent
was monitored at 280 nm. Total flavanone glycosides content
(the most abundant being hesperidin, narirutin and didymin) was
calculated by comparing the integrated peak areas of individual
compounds to that of standard curve prepared using hesperidin
standard.
Hydroxycinnamic acids (p-coumaric, ferulic, caffeic and
sinapic acids) were extracted from the juice using a solid-
phase extraction (SPE) procedure, after alkaline hydrolysis of
hydroxycinnamic esters (Fallico et al., 1996). Ten milliliters of
centrifuged juice was added to 10 mL of 2 N NaOH and stored
at room temperature in the dark. Complete hydrolysis of bound
hydroxycinnamic acids occurred in 4 h. The solution was then
acidified with 2N HCl to pH 2.5 and passed through a C18 SPE
350 P. Rapisarda et al. / Postharvest Biology and Technology 49 (2008) 348–354

cartridge (Waters, Milford, MA). Hydroxycinnamic acids were
eluted with 0.1% HCl in methanol. Chromatographic analysis
was performed by HPLC equipped as above. The eluents were
water/acetic acid (98:2), solvent A and methanol, solvent B with
a gradient transition from 95 to 70% of solvent A during 40 min.
The flow rate was 1 mL/min and detection was performed at
300 nm. Total hydroxycinnamic acids content was calculated
using peak areas from standard curves of p-coumaric, ferulic,
caffeic and sinapic acids.
The orange juices were analyzed for total phenolics by the
Folin-Ciocalteu (FC) colorimetric method (Singleton et al.,
1999). Appropriately diluted samples (1 mL) were mixed with
5 mL FC commercial reagent (previously diluted with water
1:10, v/v) and 4 mL of a 7.5% sodium carbonate solution. The
mixture was stirred for 2 h at room temperature away from strong
light. The absorbance of the resulting blue solution was mea-
sured spectrophotometrically at 740 nm and the concentration
of total phenolics was expressed as (±) catechin equivalents
(mg/L).
2.4. Antioxidant activity
The DPPH-scavenging activity (DPPH-SA) of orange
juice was tested as bleaching of the stable 1,1-diphenil-
2-picrylhydrazyl radical (Brand-Williams et al., 1995). The
reaction mixture contained 4 mL 10−4 M DPPH methanolic
solution and different amounts of orange juice. After 10 min at
room temperature, the absorbance of the resulting solution was
recorded at 517 nm and the results were expressed as percentage
decrease against controls. Percent inhibition vs. sample volume
(␮L) curves were used to determine the concentration at which
50% radical scavenging occurred (IC50 ). Inhibition percentage
for each sample was calculated as follows:
A 0 − Ax
%inhibition = 100
A0
where A0 is the absorbance of a DPPH blank and Ax is the
absorbance of juice solution. Stronger radical quenching results
in a lower IC50 value.
Antioxidant activity was also determined through inhibi-
tion of induced linoleic acid peroxidation (InLAP) assay. The
enzymatic oxidation of linoleic acid was obtained by adding
lipoxygenase (Lo Scalzo et al., 2004) and monitored in the
absence (blank test) and presence (sample test) of orange
juice by recording the linear increase in absorbance at 234 nm
(Amax of conjugated diene peroxides from linoleic acid oxida-
tion). In the sample test, InLAP was measured in a solution
containing 2.65 mL 0.1 M phosphate buffer (pH 7.0), 0.3 mL
2.28 mM linoleic acid–water emulsion, 0.05 mL orange juice
and 0.025 mL lipoxygenase solution (15 mg lipoxygenase stan-
dard in 25 mL 0.1 M phosphate buffer at pH 7.0). In the blank
test orange juice was substituted with 0.05 mL 0.1 M phosphate
buffer (pH 7.0).
Antioxidant activity was expressed as the percentage protec-
tion from linoleic acid enzymatic degradation, where the blank
represents 100% degradation.
2.5. Statistical analysis
One-way ANOVA was performed for each cultivar to obtain
a statistical assessment of the evolution of physicochemical
parameters and antioxidant capacity during storage. Means were
separated by Tukey’s test at p ≤ 0.05 and at p ≤ 0.01 significance.

Table 1
Change of standard quality parameters of different orange genotypes stored at 6 ± 1 ◦ C for 65 d
Orange variety Storage (days) TA (%) TSS (%) TSS/TA pH

0 1.38 A 8.07 D 5.85 C 3.70 B

20 1.36 A 9.82 C 7.22 B 3.52 D
‘T. Messina’
40 1.37 A 9.92 B 7.24 B 3.57 C
65 1.09 B 10.12 A 9.28 A 3.73 A
0 1.40 A 8.62 D 6.16 C 3.46 B
20 1.43 A 9.42 C 6.57 B 3.31 C
‘T. Meli’
40 1.46 A 9.97 B 6.85 B 3.46 B
65 1.25 B 10.62 A 8.49 A 3.51 A
0 1.57 A 10.57 D 6.73 D 3.44 C
20 1.53 AB 10.62 C 6.94 C 3.36 D
‘Moro’
40 1.52 B 11.07 B 7.28 B 3.53 B
65 1.34 C 11.47 A 8.56 A 3.63 A
0 0.90 A 10.58 B 11.80 B 3.74 b
20 0.88 A 10.54 B 11.98 B 3.76 ab
‘Ovale’
40 0.83 B 10.94 A 13.18 A 3.76 ab
65 0.80 B 10.95 A 13.70 A 3.78 a
0 1.11 B 10.47 C 9.43 A 3.59 A
20 1.11 B 10.81 B 9.74 A 3.60 A
‘Valencia’
40 1.13 AB 11.21 A 9.89 A 3.56 B
65 1.18 A 10.51 C 8.88 B 3.58 AB

Values in the same column for each orange variety having different letters are significantly different: p ≤ 0.01, capital letters or p ≤ 0.05, small letters.
 P. Rapisarda et al. / Postharvest Biology and Technology 49 (2008) 348–354 351

Correlation analysis of antioxidant activity values vs. vitamin
C or phenolic components (anthocyanins, flavanones, HCA and
total phenolics) was performed by MSTAT WIN 10.
3. Results and discussion
Changes of fruit quality parameters for ‘T. Messina’ and ‘T.
Meli’, ‘Moro’, ‘Ovale’ and ‘Valencia’ oranges stored at 6 ± 1 ◦ C
for 65 d are shown in Table 1. Fruit weight loss after 65 d of stor-
age was less than 5% with respect to the initial weight (data not
shown). Titratable acidity remained constant in the early stages
of storage, then decreased in all the varieties, except for ‘Valen-
cia’ orange which increased slightly. This trend is reflected on
pH levels which significantly increased at the end of storage,
whereas a slight decrease in ‘Valencia’ fruit was observed. Cit-
ric acid has been reported to decrease in stored citrus fruit and
this decline may be in part due to the use of organic acids for
energy production and alcoholic fermentation (Echeverria and
Valich, 1989). In addition, organic acids may provide carbon
skeletons for the synthesis of phenolics, including anthocyanin
and non-anthocyanin phenolics (Kalt et al., 1999). An increase
in TSS during storage occurred in all samples, although both
‘Tarocco’ clones showed a more noticeable rise than the oth-
ers. In ‘Valencia’ orange the increase was followed by a decline
at the last interval of storage. The same TSS change in cold-
stored ‘Valencia’ orange was observed by El-Zeftawi (1976).
Previous studies have demonstrated that de novo synthesis of
sugars from organic acids is a possible mechanism involved
in the continuous increase of sugar levels in harvested citrus
fruit but the presence and increase of glycolytic enzymes during
later stage of ‘Valencia’ orange storage can explain the differ-
ent behavior of this variety (Echeverria and Valich, 1989). As a
result of concomitant changes in TSS and TA, the TSS/TA ratio
increased during storage in all varieties with the exception of
‘Valencia’ orange where a drop was observed on the 65th day
of storage.
Table 2 shows the changes in the antioxidant components of
different orange genotypes during storage. In orange fruit the
predominant form of vitamin C is ascorbic acid whereas dehy-
droascorbic acid (DHA) is less than 10% of total vitamin C
content. Moreover, the change during fruit storage of DHA val-
ues is negligible (Wills et al., 1984), thus the amount of ascorbic
acid can be assumed as vitamin C content in juice.
Vitamin C slightly decreased in ‘T. Meli’ and ‘Moro’ oranges.
In ‘Valencia’, this component rose after 40 days of storage,
whereas in ‘T. Messina’ and ‘Ovale’ it increased initially and
then declined. However, at the end of storage, the vitamin C
levels in all the genotypes (48.86–63.66 mg/100 mL) were not
such as to lead to an excessive reduction in fruit antioxidant
protection (Scandalios, 1993).
A marked increase of juice anthocyanins in both ‘Tarocco’
clones was observed during storage. Anthocyanin levels rose
almost 5-fold (from 4.89 to 23.83 mg/L) in ‘T. Meli’ and 9-
fold (from 1.09 to 10.26 mg/L) in ‘T. Messina’, whereas ‘Moro’
pigment increased only 2-fold (from 78.85 to 151.32 mg/L).
Recently, it has been reported that anthocyanin production in
blood oranges continues after harvesting when fruit are stored
at low temperatures and this accumulation depends on activa-
tion of the enzymes involved in phenylpropanoid metabolism
such as phenylalanine ammonialyase (PAL), chalcone synthase
(CHS), dihydroflavonol 4-reductase (DFR), and UDP-glucose
flavonoid glucosyl transferase (UFGT) (Lo Piero et al., 2005).

Table 2
Change in antioxidant components in juice of different orange genotypes stored at 6 ± 1 ◦ C for 65 d
Orange variety Storage (days) Vitamin C (mg/100 mL) Anthocyanins (mg/L) Flavanones (mg/L) HCAa (mg/L) T. phenolics (mg/L)

0 55.37 B 1.09 B 107.55 C 54.35 C 523.74 D

20 58.73 A 2.07 B 139.49 B 79.81 A 673.57 A
‘T. Messina’
40 59.88 A 9.23 A 133.55 B 81.45 A 647.37 B
65 50.87 C 10.26 A 175.55 A 70.80 B 580.64 C
0 64.78 A 4.89 D 78.48 C 39.51 C 507.13 B
20 61.64 C 7.46 C 85.13 C 60.41 A 621.58 A
‘T. Meli’
40 63.79 B 11.30 B 95.59 B 57.29 AB 620.99 A
65 63.00 B 23.83 A 109.37 A 54.34 B 597.31 A
0 55.11 A 78.85 C 201.69 B 69.15 C 696.43 C
20 50.27 B 122.60 B 213.59 B 78.87 BC 693.80 C
‘Moro’
40 52.63 AB 130.44 B 292.59 A 90.42 A 835.08 B
65 48.87 B 151.32 A 306.82 A 80.10 AB 875.96 A
0 57.13 C 310.58 AB 51.63 B 636.90 B
20 61.44 B 312.50 A 53.49 B 666.61 AB
‘Ovale’
40 67.36 A 304.82 B 62.54 A 717.74 A
65 55.56 D 286.89 C 59.16 A 631.29 B
0 61.33 B 186.97 A 67.68 A 571.05 A
20 61.97 B 149.67 B 61.80 AB 578.57 A
‘Valencia’
40 62.74 AB 132.85 BC 62.77 AB 557.78 B
65 63.66 A 127.66 C 57.06 B 540.29 C

Values in the same column for each orange variety having different letters are significantly different, p ≤ 0.01.
a Hydroxycinnamic acids.
352 P. Rapisarda et al. / Postharvest Biology and Technology 49 (2008) 348–354

Total flavanone glycoside concentration (the most abundant Table 3

being hesperidin and narirutin) changed during storage in all the Change in antioxidant activity of different orange genotypes stored at 6 ± 1 ◦ C
for 65 d
genotypes. Fresh fruit flavanone levels ranged from 78.5 mg/L
for ‘T. Meli’ to 310.6 mg/L for ‘Ovale’. Low temperatures Orange variety Storage (days) DPPH-SAa (␮L) InLAPb (%)
stimulated flavanones biosynthesis in all the pigmented oranges, 0 38.14 A 57.00 B
whereas in ‘Ovale’ and ‘Valencia’ oranges flavanone content 20 30.57 B 57.30 B
‘T. Messina’
decreased significantly during 65 d storage which could be 40 29.77 B 57.46 B
attributed to storage-related degradation. 65 30.45 B 65.75 A
The orange varieties showed different trends for total 0 38.34 A 56.70 b
hydroxycinnamic acids with ‘T. Messina’, ‘T. Meli’ and ‘Moro’ 20 29.86 B 57.93 ab
‘T. Meli’
40 25.76 C 62.19 a
demonstrating an increase during early storage and a decline at
65 24.73 C 62.12 a
the end of storage. This change could be related to the possible
involvement of HCA in anthocyanin biosynthesis (Heller and 0 26.66 A 59.45 B
20 27.06 A 63.86 AB
Forkman, 1988) which reached a maximum in the last period ‘Moro’
40 27.52 A 62.66 AB
of storage. In ‘Ovale’, the blond orange variety with the highest 65 21.47 B 66.46 A
total phenolics, HCA slightly increased. Finally, phenolic acids
0 33.72 A 55.27 B
in ‘Valencia’ decreased, probably due to senescence phenomena 20 28.42 B 51.36 B
during storage. ‘Ovale’
40 29.53 B 61.30 A
Initial total phenolics ranged from 507.1 mg/L for ‘T. Meli’ 65 33.56 A 64.21 A
to 696.4 mg/L for ‘Moro’. The amount of total phenolics in 0 36.30 A 58.08 C
‘Moro’ orange depends on the high anthocyanin, flavanone and 20 28.48 B 62.31 BC
‘Valencia’
HCA concentrations, typical of this cultivar, while in ‘Ovale’ 40 28.30 B 62.84 B
the total phenolics was high because of the high flavanone con- 65 26.29 B 68.01 A
centration. During storage, there was an initial increase and Values in the same column for each orange variety having different letters are
subsequent decrease in total phenolics in both ‘T. Messina’ and significantly different: p ≤ 0.01, capital letters or p ≤ 0.05, small letters.
a Volumes (␮L) of juice sample yielding 50% inhibition of radical absorbance
‘Ovale’ reflecting the trend of vitamin C content. In these culti-
(IC50 ). A stronger radical quenching capacity results in a lower IC50 value.
vars, the correlation coefficients for total phenolics and vitamin b Inhibition (%) of linoleic acid hydroperoxide formation referred to the blank,
C were r = 0.640 (p ≤ 0.05) and r = 0.925 (p ≤ 0.001) respec- considered as 100% degradation.
tively. In addition, components were negatively correlated in
‘T. Meli’ (r = −0.740; p ≤ 0.01) and ‘Valencia’ (r = −0.868;
p ≤ 0.001). Folin-Ciocalteu reagent is nonspecific for phenolic synergistic effect with phenolic components. ‘Tarocco Meli’ and
compounds since it measures sample reducing capacity through ‘T. Messina’, having similar total phenolics, exhibited compa-
electronic transfer-based antioxidant capacity and, thus it can rable antioxidant activity during the initial storage period (0–20
be reduced by many non-phenolic compounds such as vitamin days), after which, ‘T. Meli’ showed more efficient DPPH-SA
C (Huang et al., 2005). Thus, due to the contribution of vita- than ‘T. Messina’, probably owing to higher ascorbic acid and
min C, the response of different genotypes to the FC assay anthocyanin content. The InLAP values of ‘T. Messina’ were
was high and comparable. ‘Moro’ total phenolics correlated the highest at the end of storage where flavanone concentra-
strongly with flavanones (r = 0.957; p ≤ 0.01) and anthocyanins tion was highest. ‘Moro’ samples reached maximum DPPH-SA
(r = 0.781; p ≤ 0.01). High interaction between total phenolics and InLAP only in the last storage period corresponding to the
and HCA was also found in ‘T. Messina’ (r = 0.938; p ≤ 0.001) highest concentration of anthocyanins, flavanones and total phe-
and ‘T. Meli’ (r = 0.946; p ≤ 0.001), whereas the correlation was nolics. A different behavior was noted for ‘Ovale’ which, after
relatively weak in ‘Valencia’ (r = 0.560; p ≤ 0.05) and ‘Ovale’ an initial increase in radical quenching capacity, a significant
(r = 0.561; p ≤ 0.05). decline followed, attributable mainly to decreasing vitamin C
The antioxidant activity of five orange genotypes determined in the last stage of storage. The antioxidant activity of ‘Valen-
as DPPH-SA and InLAP is given in Table 3. ‘Moro’ fresh cia’ oranges, determined by two methods, significantly increased
fruit showed major DPPH-SA as expected (IC50 = 26.66 ␮L), probably due to an increase in vitamin C during storage.
since it contained the highest concentration of anthocyanins, DPPH-SA was negatively correlated with anthocyanins, fla-
hydroxycinnamic acids and total phenolics. Thereafter, ‘Ovale’ vanones, HCA (only in ‘T. Messina’ and ‘T. Meli’ juices) and
orange juice showed good initial DPPH-SA due to high fla- total phenolics in pigmented varieties, showing a significant
vanones, total phenolics and vitamin C. There was no significant relationship between radical scavenging activity and phenolic
difference in the initial InLAP values of the different oranges compounds. Significant correlation was also observed between
(data not shown). Antioxidant activity for all the genotypes, DPPH-SA and total phenolics in both blond varieties and DPPH-
measured by two methods, increased significantly during cold SA vs. HCA and flavanones in ‘Valencia’ oranges. Besides,
storage. Recent studies have shown that the antioxidant effi- DPPH-SA was directly associated with vitamin C in ‘Moro’,
ciency of orange juice may be attributed, in a significant part, to ‘Ovale’ and ‘Valencia’ oranges (Table 4). Regardless of InLAP,
their total phenolic content (Rapisarda et al., 1999). However, a high correlation was found with flavanones in all cultivars. This
according to Kähkönen et al. (2001), ascorbic acid could exert a is probably because flavanone glycosides (hesperidin, narirutin
 P. Rapisarda et al. / Postharvest Biology and Technology 49 (2008) 348–354 353

Table 4
Correlation coefficients between radical scavenging activity (DPPH-SA) and vitamin C, or phenolic components (anthocyanins, HCAa , flavanones and total phenolics)
in fruit of different orange genotypes stored at 6 ± 1 ◦ C for 65 d
Orange variety DPPH-SA vs. vitamin C DPPH-SA vs. anthocyanins DPPH-SA vs. HCAa DPPH-SA vs. flavanones DPPH-SA vs. T. phenolics

‘T. Messina’ −0.184 −0.656* −0.881*** −0.701** −0.793**

‘T. Meli’ 0.483 −0.757** −0.797** −0.858*** −0.851***
‘Moro’ 0.579* −0.651* 0.053 −0.553* −0.613*
‘Ovale’ −0.759** – −0.177 −0.481 −0.737**
‘Valencia’ −0.755** – 0.782** 0.922*** 0.525*

Significant at *p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001.

a Hydroxycinnamic acids.

Table 5
Correlation coefficients between antioxidant activity (InLAP) and vitamin C, or phenolic components (anthocyanins, HCAa , flavanones and total phenolics) in fruit
of different orange genotypes stored at 6 ± 1 ◦ C for 65 d
Orange variety InLAP vs. vitamin C InLAP vs. anthocyanins InLAP vs. HCAa InLAP vs. flavanones InLAP vs. T. phenolics

‘T. Messina’ −0.817** 0.636* 0.020 0.842** −0.210

‘T. Meli’ −0.050 0.671* 0.467 0.746** 0.525*
‘Moro’ −0.813** 0.821** 0.309 0.707** 0.582*
‘Ovale’ −0.060 – 0.783** −0.828** 0.011
‘Valencia’ 0.875*** – −0.820** −0.815** −0.783**

Significant at *p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001.

a Hydroxycinnamic acids.

and dydimin), being lipophilic substances, are more active than
other phenolics in a linoleic emulsion system (Benavente-Garcia
et al., 1997). Positive correlations were observed between InLAP
and anthocyanins in pigmented cultivars, the highest correlation
being in ‘Moro’, the blood orange with the highest antho-
cyanin content. Vitamin C and InLAP correlated negatively in
‘T. Messina’ and ‘Moro’ and positively in ‘Valencia’, since dur-
ing storage it decreased in the former two cultivars and rose
in the last. Total phenolic and HCA correlated weakly or not
with InLAP values in almost all the cultivars with the excep-
tion of ‘Valencia’ for both components and ‘Ovale’ for HCA
(Table 5).
The relationship between the antioxidant activity and pheno-
lic compounds or vitamin C depends on several factors such as
chemical structure of individual component, synergistic inter-
action among them and specific conditions applied in different
assay (Huang et al., 2005). The scavenging of phenolic com-
pounds against DPPH radical depends mainly on the number
of hydroxyl groups present in molecules and on their electron-
donating ability. Therefore, the fruit juices richest in polyphenols
such as blood orange and ‘Ovale’ juices have shown a closer
correlation between DPPH-SA and total phenolics. In a lipid
system such as the InLAP assay, the polarity of compounds
plays an important role on their antioxidant efficiency. Besides,
hydrophilic antioxidants such as anthocyanins possess a strong
antioxidant activity also in lipid phase probably due to the ability
of hydrophilic compounds to accumulate into the water phase of
the emulsion and therefore to maintain the antioxidant efficiency
(Kähkönen and Heinonen, 2003).
In conclusion, the results of this study indicate that cold stor-
age at 6 ± 1 ◦ C increased anthocyanins, flavanones, HCA and
total phenolics in blood oranges. Significant increases in vita-
min C and HCA were observed also in ‘Ovale’ and ‘Valencia’
varieties, respectively. Furthermore, postharvest storage treat-
ment also enhanced antioxidant efficiency and, therefore, the
health functionality of orange fruit. The antioxidant activity
of orange juice seems not to be the property of a single phy-
tochemical compound but is widely distributed among vitamin
C and phenolics constituents. The two antioxidant tests sig-
nificantly correlated with anthocyanins, indicating that, the
biosynthesis of anthocyanins during storage may contribute to
increase antioxidant activity in blood orange varieties. Finally,
the high correlation coefficients for the InLAP test and fla-
vanone content in all the cultivars indicate that the affinity of
this phenolic class with lipid substrates may be important in their
activity.
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