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Journal of Food Composition and Analysis 24 (2011) 307314

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Journal of Food Composition and Analysis


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Original Article

Bioactive compounds and antioxidant activities of different watermelon


(Citrullus lanatus (Thunb.) Mansfeld) cultivars as affected by
fruit sampling area
Imen Tlili a,b, Chak Hdider b, Marcello Salvatore Lenucci c,*, Ilahy Riadh a,b,
Hager Jebari b, Giuseppe Dalessandro c
a

Department of Biology, Faculty of Sciences of Bizerte, Zarzouna, 7021 Bizerte, Tunisia


Laboratory of Biotechnology and Plant Physiology, National Agricultural Research Institute of Tunisia, Rue Hedi Karray, 2049 Ariana, Tunisia
c
Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali (Di.S.Te.B.A.), Universita` del Salento, Via Prov.le Lecce Monteroni, 73100 Lecce, Italy
b

A R T I C L E I N F O

A B S T R A C T

Article history:
Received 19 November 2009
Received in revised form 12 April 2010
Accepted 12 June 2010

This investigation reports the variability of antioxidant components and antioxidant activities of six
watermelon cultivars (cvs) (four commercial cvs Aramis, Crimson Sweet, Dumara, Giza, and two new
selections P503 and P403 produced by the National Agricultural Research Institute of Tunisia) as
inuenced by sampling area. All cvs were simultaneously grown in an open-eld and subjected to
identical horticultural practices in order to minimize the effects of environmental conditions and
maximize those related to genotype. Signicant differences were found between watermelon cvs for
lycopene, phenolics, avonoids, ascorbic acid (AsA), dehydroascorbic acid (DHA) and total vitamin C
(AsA + DHA) contents, as well as in the antioxidant activity of their hydrophilic and lipophilic fractions.
P503 cv showed the highest lycopene and avonoid contents. Crimson Sweet and Giza cvs showed the
highest HAA and LAA when TEAC was used as assay method, while the highest HAA and LAA were
detected in Giza and Dumara cvs and in P503 cv, respectively, when FRAP assay was used. This study
demonstrates that the amount of each specic antioxidant, as well as the HAA and LAA, were both
inuenced by genotype and sampling area, emphasizing the need to evaluate watermelon biodiversity in
order to improve its nutritional value.
2010 Elsevier Inc. All rights reserved.

Keywords:
Citrullus lanatus (Thunb.) Mansfeld
Antioxidant activity
Flavonoids
Lycopene
Phenolics
Sampling area
Vitamin C
Watermelon
Biodiveristy and nutrition
Horticulture
Cultivar difference
Bioavailability
Food analysis
Food composition

1. Introduction
Today, there is an increasing grower and consumer demand for
high-quality, healthful foods and food products. Watermelon
(Citrullus lanatus (Thunb.) Mansfeld) is a popular fruit endowed
with high natural antioxidant capacity, an attribute which is
becoming an important qualitative factor for foods.
Watermelon is one of the main vegetable crops grown and
consumed all over the Mediterranean basin. It is much appreciated as
an excellent refreshing summer fruit. Besides vitamins (A, B, C and E),
mineral salts (K, Mg, Ca and Fe), and specic amino acids (citrulline

Abbreviations: AsA, ascorbic acid; b-CaE, b-carotene equivalents; cv(s), cultivar(s);


DHA, dehydroascorbic acid; GAE, gallic acid equivalents; HAA, hydrophilic
antioxidant activity; LAA, lipophilic antioxidant activity; RE, rutin equivalents;
TAA, total antioxidant activity.
* Corresponding author. Tel.: +39 0832 298612; fax: +39 0832 298858.
E-mail address: marcello.lenucci@unisalento.it (M.S. Lenucci).
0889-1575/$ see front matter 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.jfca.2010.06.005

and arginine), watermelon provides a wide variety of dietary


antioxidants such as carotenoids and phenolics (Perkins-Veazie
et al., 2002, 2007). Natural compounds, particularly lycopene,
ascorbic acid (AsA) and dehydroascorbic acid (DHA), avonoids and
other phenolics, have recently stimulated great attention because of
their antioxidant activity against free radicals, suggesting protective
roles in reducing the risk of certain types of cancers, cardiovascular
diseases and age-related degenerative pathologies (Rice-Evans et al.,
1996; Giovannucci, 1999; Rao, 2006).
The chromoplasts of watermelon mesocarp cells synthesize and
store lycopene as the major carotenoid (7090%). Lycopene is
responsible for the typical red-colour of the esh of the ripe fruits
(Tomes et al., 1963; Tadmor et al., 2005). This red pigment has the
highest antioxidant activity among all dietary antioxidants (Di
Mascio et al., 1989). Fresh watermelon constitutes an important
source of highly bioavailable lycopene for humans. Its bioavailability
from fresh watermelon juice is, in fact, similar to that of heat
processed tomatoes (Edwards et al., 2003). In addition, watermelon

308

I. Tlili et al. / Journal of Food Composition and Analysis 24 (2011) 307314

contains moderate but signicant quantities of phenolics (PerkinsVeazie et al., 2002; Brat et al., 2006). These key secondary metabolites
exhibit highly efcient peroxyl-radical scavenging activity and hence
potential pharmacological effects (Larson, 1988; Halliwell, 1994;
Manach et al., 1998). Among phenolics, avonoids reduce low
density lipoprotein (LDL) oxidation and quench reactive oxygen
radicals, decreasing thereby the risk of cardiovascular diseases and
cancers (Pietta, 2000; Lila, 2004). Although the biological effect of
avonoids is, in general, attributed to their antioxidant activity,
recent investigations indicate that they might affect signalling
pathways in animal cells (Williams et al., 2004).
Watermelon fruits have been identied as a good source of
vitamin C, mainly in the reduced form of AsA (Vanderslice et al.,
1990). In addition to its antioxidant activity against free radicals,
AsA has numerous biological functions, which include the
synthesis of collagen, steroid and peptide hormones and neurotransmitters (Cameron et al., 1979).
It is known that the amount of each antioxidant in fruits and
vegetables is strongly inuenced by genotype differences and
external factors such as agro-technical processes, environmental
conditions, ripening degree at harvest and post harvest manipulation (Waterman and Mole, 1994; Abushita et al., 2000; Dumas et al.,
2003). Few studies have been focused on the physicchemical
properties and antioxidant components of watermelon cvs.
Recently, Perkins-Veazie et al. (2006) and Perkins-Veazie and Davis
(2007) emphasized the importance of genotype and sampling areas
when assessing the lycopene and soluble solid content in watermelons. They also suggested the need to adopt standardized and
documented sampling methods when assessing quality attributes in
watermelon fruits. In fact, in large fruits, as watermelon, where only
a portion of the esh is feasibly tested for quality, an accurate and
reproducible sampling method must be developed.
The amount of a determined antioxidant compound in a food
does not necessarily correlate with its antioxidant activity. Due to
the complexity of the composition of foods, their antioxidant
power depends on the synergistic effects and redox interactions
among the different nutrient and non nutrient molecules which
together contribute to the supposed health benets. Therefore, the
use of methods to measure the total antioxidant activity (TAA) of
foods and biological samples is very appealing to researchers
(Huang et al., 2005).
The aim of this study was to investigate the inuence of
genotypic differences and fruit sampling area on the amounts of
lycopene, total phenolics, avonoids, AsA, DHA and total vitamin C
in the esh of different cvs of watermelon grown in Tunisia and to
characterize their in vitro hydrophilic and lipophilic antioxidant
activities.

4 L h 1 drippers placed at 0.4 m intervals along the irrigation line.


Drip irrigation ran for 13 h, at 12-day intervals, depending on
potential evapotranspiration of the research station, climate data
and crop coefcient. The production methods were in accordance
with the procedures utilized by the research and experimental
station of Teboulba, Monastir, Tunisia and recommended by INRAT.
They included fertilization with synthetic chemical fertilizers
(145 kg N ha 1, 140 kg P2O5 ha 1, 210 kg K2O ha 1). Chemical fertilizer solution was added to water irrigation by pump injection
twice a week. The production methods also included a handweeding control and plant-pathogen control with synthetic chemical pesticides. Imidacloprid (200 g L 1) was used to reduce aphids,
acetamipride (200 g L 1) was applied to reduce thrips and abamectine (18 g L 1) was used to reduce mites. All these pesticides were
applied once a cycle.
Ripe watermelons were harvested in June. Field ripeness was
judged by various methods, including tendril browning, yellowing
of the ground spot, and loss of surface gloss and by a thumping
sound which changes from a metallic ringing when unripe to a soft
hallow sound when ripe. Watermelons were selected randomly
from the different blocks. Four ripe fruits were harvested per block
per cvs. All the fruits were transported carefully to the laboratory
for analysis to avoid internal bruising.
2.1.2. Fruit tissue sampling
Fruit were cut longitudinally from the stem-end to the blossomend through the ground spot, and tissue samples were taken from
four different areas: blossom-end, heart, stem-end and peripheral
area (between locular and rind areas) (Fig. 1).
The soluble solid content (8Brix) was measured immediately as
described below. For further analysis, about 250 g of esh without
seeds per sampling area per fruit was collected, wrapped with
aluminium, placed into plastic bags and quickly frozen and stored
at 80 8C, until analysed.
2.2. Soluble solid content determination

()TD$FIG][of

Soluble solid content (8Brix) was measured by cutting a wedge


esh from all sampling areas and squeezing the juice into a

2. Materials and methods


2.1. Sampling
2.1.1. Plant culture
The eld-experiments were conducted in 2008 at the Research
and Experimental Station of Teboulba, Monastir, Tunisia. A total of
six watermelon cvs including four of the most important
commercial ones in the world [Crimson Sweet (Clause), Dumara
(Nunhems), Aramis (Nunhems) and Giza (Egyptian cv selected and
improved by the National Agricultural Research Institute of
TunisiaINRAT)] and two new cvs (P503 and P403) selected by
the INRAT, were used in this experiment.
Sowing was carried out on 19 February 2008 in plug-seedling
trays. Watermelons were transplanted on 29 March 2008 into a
sandy soil on black plastic mulch, with an in row spacing of 125 cm
and a between-row spacing of 150 cm. Four blocks were used with 10
plants per cv. After transplanting, drip irrigation was applied with

Fig. 1. Watermelon fruit esh sampling scheme.

I. Tlili et al. / Journal of Food Composition and Analysis 24 (2011) 307314

309

digital refractometer (Atago PR-100, NSG Precision Cells, Inc,


Farming dale, NY, USA) calibrated with a 10% sucrose solution.
Only fully ripe watermelons, with an average soluble solid content
8%, were further analyzed for lycopene to be sure the fruit was
fully ripened.

rather than a single method (Schlesier et al., 2002), in the present


study, the measurement of the HAA and LAA was performed using
two different methods, the Trolox equivalent antioxidant capacity
(TEAC) assay and the ferric reducing antioxidant power (FRAP)
assay.

2.3. Lycopene content determination

2.7.1. Trolox equivalent antioxidant capacity (TEAC) assay


The antioxidant activity was measured using the ABTS
decolouration method (Miller and Rice-Evans, 1997). Hydrophilic
and lipophilic antioxidants were extracted from 0.3 g homogenous
juice (three replicates) with 50% methanol or 50% acetone,
respectively, at 4 8C under constant shaking (300 rpm) for 12 h.
Samples were centrifuged at 10,000  g for 7 min and the different
supernatants were recovered and used for antioxidant activity
measurements. The antioxidant activities were measured at
734 nm in a Cecil BioQuest CE 2501 spectrophotometer. Two
different calibration curves were constructed, using freshly
prepared Trolox solutions for HAA and LAA determinations. Values
were obtained from three replicates as mmol Trolox per 100 g of
tomato fw (mmol Trolox/100 g fw).

Frozen samples of different sampling areas from every fruit


were rapidly homogenized with a laboratory blender (Waring
Laboratory and Science, Torrington, CT, US). Lycopene extraction
and determination were conducted as described by Fish et al.
(2002). The method uses a mixture of hexane/ethanol/acetone
(2:1:1 by vol.) containing 0.05% butylated hydroxytoluene (BHT).
The absorbance of the hexane extract was measured at 503 nm
with a Cecil BioQuest CE 2501 spectrophotometer (Cecil Instruments Ltd., Cambridge, UK). Hexane was used as blank. During the
extraction process and the analysis, some precautions were taken,
like working in a reduced luminosity room and wrapping glass
materials in aluminium foil to minimize lycopene loss by photooxidation. Lycopene molar extinction e = 17.2  104 M 1 cm 1 in
hexane was used for lycopene content determination and results
were expressed as mg/kg fw.
2.4. Phenolic content determination
Total phenolics were determined according to the colorimetric
method of FolinCiocalteu, as modied by Eberhardt et al. (2000)
and Singleton et al. (1999). Each sample (2 g) was extracted with
10 mL methanol for 24 h. 125 ml of the methanolic extract was
mixed with 500 ml distilled water in a test tube, followed by the
addition of 125 ml of FolinCiocalteu reagent, and allowed to stand
for 3 min. Then 1250 ml of 7% sodium carbonate solution was
added and the nal volume was made up to 3 mL with distilled
water. Each sample was allowed to stand for 90 min at room
temperature and measured at 760 nm against the blank on a Cecil
BioQuest CE 2501 spectrophotometer. The linear reading of
standard curve was from 0 to 300 mg of gallic acid per mL. Results
were expressed as mg gallic acid equivalent per kg of tomato fw
(mg GAE/kg fw).
2.5. Flavonoid content determination
The avonoid content was determined as described by Zhishen
et al. (1999) on triplicate aliquots of the homogenous juice (0.3 g).
Fifty microlitre aliquots of the methanolic extract were used for
avonoid determination. Samples were diluted with distilled
water to a nal volume of 0.5 mL, and 30 mL of 5% NaNO2 was
added. After 5 min, 60 mL of 10% AlCl3 was added and nally
200 mL of 1 M NaOH was added after 6 min. The absorbance was
read at 510 nm, using a Cecil BioQuest CE 2501 spectrophotometer,
and avonoid content was expressed as mg of rutin equivalents per
kg of fw (mg RE/kg fw).
2.6. Ascorbic acid and dehydroascorbic acid determination
Ascorbic acid (AsA) and dehydroascorbic acid (DHA) contents
were determined as reported by Kampfenkel et al. (1995) on
triplicate samples of the homogenate juice (0.1 g). AsA and DHA
were extracted using 6% metaphosphoric acid and detected at
525 nm in a Cecil BioQuest CE 2501 spectrophotometer.
2.7. Antioxidant activity determination
Since many authors recommend evaluating the antioxidant
activity of fruit and vegetable by a number of different methods

2.7.2. Ferric reducing antioxidant power (FRAP) assay


Hydrophilic and lipophilic antioxidants were extracted from
0.3 g of homogenate (three replicates) with absolute methanol or
hexane at 4 8C under constant shaking (300 rpm) overnight.
Samples were centrifuged at 10,000  g. The supernatants were
used for antioxidant activity measurement. Antioxidant activity
was measured in both hydrophilic and lipophilic fractions using
the FRAP assay method (Benzie and Strain, 1996). 50 mL of
hydrophilic or lipophilic tomato extract was added to 1.5 mL of
FRAP reagent [1 mM 2,4,6-tripiridyl-2-triazine (TPTZ) and 20 mM
ferric chloride in 0.25 M sodium acetate buffer, pH 3.6] and mixed
thoroughly. After 4 min at 4 8C, absorbance at 593 nm was read
against a blank of water using a Cecil BioQuest CE 2501
spectrophotometer. A calibration curve was prepared using freshly
prepared ammonium ferrous sulphate. Values were obtained from
three replicates as mM FRAP per g of tomato fw (mM FRAP/g fw).
2.8. Statistical analysis
The experimental design was a randomized complete block
with six factors (cvs) and three blocks (replicates). The analysis of
variance was performed according to the General Linear Models
(GLM) procedure developed by the Statistical Analysis Systems
Institute (SAS Inst., V.6.1, Cary, NC, US). Means and standard errors
were calculated. Correlations were estimated using Persons
correlation coefcient, P < 0.05. LSD test was applied to establish
signicant differences between means with a condence level of
95%.
3. Results and discussion
3.1. Lycopene, total phenolic and avonoid contents
The amounts of lycopene, total phenolics and avonoids of the
investigated watermelon cvs within different sampling areas are
shown in Table 1. The data, expressed on a fresh weight (fw) basis,
show statistically signicant differences (P < 0.01) among the
watermelon cvs in the amount of lycopene, total phenolics and
avonoids and in their distribution within the fruit esh. When
averaged across sampling areas, lycopene content reached the
highest value in P503 (102.4 mg/kg fw) and Giza (96.9 mg/kg fw)
cvs, whose means did not differ signicantly, and the lowest in
P403 (45.4 mg/kg fw) and Dumara (42.7 mg/kg fw) cvs. Intermediate values were found for Aramis (71.0 mg/kg fw) and Crimson
Sweet (53.5 mg/kg fw) cvs. These results are in agreement with

I. Tlili et al. / Journal of Food Composition and Analysis 24 (2011) 307314

310

those reported by Perkins-Veazie et al. (2006). These authors,


studying the carotenoid composition of 50 red-eshed ripe seeded
(2n) and seedless (3n) watermelon cvs grown in Oklahoma, found a
high variability of lycopene content among cultivars in a range
from 35.2 mg/kg fw to 112.4 mg/kg fw. These data conrm the
value of watermelon as a source of dietary lycopene for the
population of the Mediterranean area because of its availability
and high consumption, as previously reported by Vinson et al.
(1998) for Americans.
In most of the cvs under analysis, lycopene content was
signicantly different within the various sampling areas studied
(P < 0.01), with the exception of Crimson Sweet and P403 cvs in
which lycopene resulted uniformly distributed all over the fruit
esh.
The highest average total phenolic content was detected in Giza
cv (147.3 mg GAE/kg fw). Aramis (92.3 mg GAE/kg fw) and P503
(89.0 mg GAE/kg fw) cvs, whose values are not statistically
different, showed the lowest average content of total phenolics.
These results are consistent with those reported by Brat et al.
(2006) who found a moderate amount (116 mg GAE/kg fw) of
phenolics in watermelon fruits sampled from French national
markets. Much higher values ranging between 870 and
910 mg GAE/kg fw were obtained in red-eshed watermelon cvs
by Perkins-Veazie et al. (2002). In this case no statistically
signicant differences were found among cvs.
Although, the phenolic content in watermelons is moderate
compared to other fruits and vegetables, such as cranberry and
Table 1
Lycopene, total phenolic and avonoid contents in the fruit of the watermelon cvs
studied within different sampling areas.
Cultivars
Crimson Sweet
Blossom-end area
Stem-end area
Heart area
Peripheral area
Mean
Giza
Blossom-end area
Stem-end area
Heart area
Peripheral area
Mean
Dumara
Blossom-end area
Stem-end area
Heart area
Peripheral area
Mean
P403
Blossom-end area
Stem-end area
Heart area
Peripheral area
Mean
P503
Blossom-end area
Stem-end area
Heart area
Peripheral area
Mean
Aramis
Blossom-end area
Stem-end area
Heart area
Peripheral area
Mean

Lycopene
(mg/kg fw)

Total phenolics
(mg GAE/kg fw)

Flavonoids
(mg RE/kg fw)

52.7  0.2a
44.6  3.1a
60.3  6.9a
56.3  8.5a
53.5C

143.3  3.8ab
145.4  6.1a
92.8  1.8c
127.2  6.2b
127.2B

163.5  7.7b
194.6  2.4a
197.4  6.5a
148.9  6.4b
176.1A

90.1  5.6b
103.0  5.8ab
105.3  4.3a
89.1  1.7b
96.9A

180.1  2.0a
155.9  2.8b
127.2  6.0c
126.1  2.3c
147.3A

179.2  3.3ab
169.2  2.4bc
188.6  6.7a
162.6  8.3c
174.9A

41.2  0.7b
43.8  2.0ab
47.4  1.7a
38.3  0.8b
42.7D

128.6  2.3a
98.7  2.8a
94.2  5.2b
124.1  6.2b
111.4C

121.6  2.4a
130.3  3.7a
120.7  2.1ab
101.1  5.8b
118.4B

43.7  2.3a
46.5  3.6a
48.5  1.9a
42.7  0.4a
45.4CD

119.9  1.8b
135.5  3.7a
113.3  4.4b
92.1  4.3c
115.2C

120.6  3.2ab
94.2  6.5c
104.7  2.5bc
132.7  4.0a
113.0B

100.3  1.9b
112.7  0.7a
112.0  2.2a
84.5  3.7c
102.4A

69.3  4.0b
96.8  2.6a
89.7  4.7a
100.3  3.5a
89.0D

193.8  1.8a
177.3  6.1a
180.7  6.9a
149.1  2.7b
175.2A

92.4  4.7a
86.7  2.5a
95.9  4.5a
94.4  3.6a
92.3D

95.0  2.9b
105.0  2.1b
149.6  2.5a
95.7  1.9b
111.3B

67.0  3.9ab
77.7  9.1ab
81.2  2.2a
58.2  1.4b
71.0B

Lower case letters indicate mean separation within column and sampling area by
LSD test, P < 0.05. Capital letters indicate mean separation among means within
column by LSD test, P < 0.05.

onion, the large consumption of this fruit in the Mediterranean


diet, especially during summer, may considerably contribute to the
daily intake of phenols. Vinson et al. (2001) reported that
watermelon is classied fourth among the eight fruits providing
80% and 50% of the daily phenol intake in the American and
Spanish diet, respectively.
Total phenolic content varied signicantly between studied
sampling areas within all cvs, except for Aramis cv (P < 0.01).
When avonoid data results were averaged across the sampling
areas, Crimson Sweet was the cv with the highest amount
(176.1 mg RE/kg fw). Similar values were obtained for the newly
selected P503 cv (175.2 mg RE/kg fw) and for Giza cv (174.9 mg RE/
kg fw), which did not differ statistically from the latter. The lowest
values were recorded for Aramis (111.3 mg RE/kg fw), P403
(113.0 mg RE/kg fw) and Dumara (118.4 mg RE/kg fw) cvs. To
our knowledge, this is the rst report on avonoids in watermelon
cvs and provides evidence that watermelon constitutes a signicant source of avonoids, similar or even superior to red-ripe
tomatoes. In fact, the amounts of avonoids measured in the
watermelon cvs under analysis are higher than the average content
reported for tomatoes (50 mg RE/kg fw) by Stewart et al. (2000)
and in the range of the values reported for cherry and highpigment tomato cvs (134622 mg RE/kg fw) by Lenucci et al.
(2006). Flavonoids were signicantly different in the sampling
areas of all the cvs studied (P < 0.01).
When data were averaged across cvs, lycopene, total phenolic
and avonoid contents varied signicantly between sampling areas
(P < 0.05) (Table 2). For lycopene, the highest content was found in
the heart and stem-end areas with 75.8 and 71.4 mg/kg fw,
respectively. The lowest value was obtained for the peripheral area
with 61.5 mg/kg fw. Our results are in agreement with those of
Perkins-Veazie and Davis (2007), who reported that lycopene
content differs signicantly among sampling areas in watermelons.
As for phenolics, the highest value was obtained for stem and
blossom-end areas with 122.3 and 119.8 mg GAE/kg fw, respectively, followed by peripheral with 110.7 mg GAE/kg fw. The
lowest values were obtained in the heart area with 102.2 mg GAE/
kg fw. On the contrary heart was the esh area with the highest
average avonoid content (157.0 mg RE/kg fw), followed by the
blossom-end and stem-end areas (145.6 mg RE/kg fw), and,
lastly, peripheral area (131.7 mg RE/kg fw) (Table 2).
Table 2
Average distribution of lycopene, total phenolics, avonoids, AsA, DHA and total
vitamin C contents and of hydrophilic and lipophilic antioxidant activities within
the different sampling areas of the watermelon fruits.
Sampling areas

Antioxidant compounds
Lycopene (mg/kg fw)
Total phenolics
(mg GAE/kg fw)
Flavonoids
(mg RE/kg fw)
AsA (mg/kg fw)
DHA (mg/kg fw)
Total vitamin C
(mg/kg fw)

Blossomend

Stemend

Heart

Peripheral

65.8B
122.3A

71.37A
119.8A

75.8A
102.2C

61.5B
110.7B

145.6B

145.1B

157.0A

131.7C

76.2C
89.7B
165.9B

95.6A
90.4B
186.0A

63.8D
105.0A
168.7B

84.4B
103.3A
187.7A

281.5A
257.8A

193.2B
236.3B

206.0B
219.6C

30.2A
33.1B

22.6C
29.4C

23.9B
37.4A

Antioxidant activity
TEAC assay (mmol Trolox/100 g fw)
HAA
261.9A
LAA
236.5B
FRAP assay (mM FRAP/g fw)
HAA
22.9C
LAA
29.3C

In each row, values with the same letters are not signicantly different (LSD test,
P < 0.05).

I. Tlili et al. / Journal of Food Composition and Analysis 24 (2011) 307314


Table 3
Ascorbic acid (AsA), dehydroascorbic acid (DHA) and total vitamin C (AsA + DHA)
contents in the fruit of the watermelon cvs studied within different sampling areas.
Cultivars
Crimson Sweet
Blossom-end area
Stem-end area
Heart area
Peripheral area
Mean
Giza
Blossom-end area
Stem-end area
Heart area
Peripheral area
Mean
Dumara
Blossom-end area
Stem-end area
Heart area
Peripheral area
Mean
P403
Blossom-end area
Stem-end area
Heart area
Peripheral area
Mean
P503
Blossom-end area
Stem-end area
Heart area
Peripheral area
Mean
Aramis
Blossom-end area
Stem-end area
Heart area
Peripheral area
Mean

AsA
(mg/kg fw)

obtained for heart (168.7 mg/100 g


(165.9 mg/100 g fw) areas.

311

fw) and

blossom-end

DHA
(mg/kg fw)

Total vitamin C
(mg/kg fw)

3.3. Hydrophilic and lipophilic antioxidant activities


(TEAC and FRAP assays)

75.1  9.3ab
88.1  5.4a
67.7  4.1b
65.0  1.9b
73.9C

105.0  4.1a
69.3  12.9b
118.2  3.8a
65.1  6.8b
89.4C

180.1  16.6ab
157.4  8.6b
185.9  4.7a
130.1  0.4c
163.4C

160.9  5.8a
144. 7  9.4ab
84.0  1.2c
121.8  4.9b
127.8A

61.1  1.9c
107.9  7.0b
107.0  4.3b
140.0  1.9a
104.0B

222.0  16.3b
252.6  8.64a
191.0  6.84c
261.8  13.69a
231.8A

135.1  3.8b
146.2  4.4a
120.7  3.9c
127.7  1.8bc
132.4A

131.2  7.2a
85.0  4.3b
95.3  3.5b
117.8  7.1a
107.3B

266.3  10.61a
231.2  13.12bc
216.0  0.57c
245.6  8.81b
239.8A

22.5  0.4b
24.2  0.9b
10.8  1.2c
33.1  1.7a
22.7E

108.1  7.7c
150.0  8.4b
203.8  10.6a
214.6  6.8a
169.1A

130.6  13.06d
174.3  3.29c
214.6  10.34b
247.8  8.30a
191.8B

38.2  5.3c
113.1  6.8a
75.3  3.4b
110.2  1.2a
84.2B

72.8  10.4a
63.7  10.6a
22.7  5.2b
26.2  3.6b
46.4E

111.1  3.9c
176.8  5.6a
98.0  6.0c
136.4  3.4b
130.6D

60.0  9.2a
66.5  1.9a
82.6  2.0a
56.3  2.0a
66.4D

85.5  3.0a
123.7  9.2a
106.7  4.9a
104.8  1.5a
105.2E

The HAA and LAA, determined by both TEAC and FRAP


methods in the watermelon cvs within different fruit sampling
areas are shown in Table 4. Both TEAC and FRAP data showed
that HAA and LAA values varied signicantly among watermelon
cvs (P < 0.01). When TEAC data were averaged across sampling
areas, the highest HAA was registered in the Crimson Sweet and
Giza cvs (290.9 and 271.3 mmol Trolox/100 g fw, respectively),
whereas the lowest value was found in P503 cv (182.4 mmol Trolox/100 g fw). The LAA values were of the same order of
magnitude as HAA dwelling in a range of 210.7258.9 mmol Trolox/100 g fw, but less variable. Again, Crimson Sweet ranked
rst among the assayed watermelon cvs, whereas the last
position was held by P503 and Aramis cvs (210.7 and
206.0 mM Trolox/100 g fw, respectively). A different ranking
was obtained when HAA and LAA were measured by the FRAP
assay. In this case Giza and Dumara were the cvs with the
highest HAA (approx. 29.8 mM FRAP/100 g fw). Crimson Sweet
ranked 5th and P503 was conrmed the cv with the lowest HAA
(13.3 mM FRAP/100 g fw). Although the use of n-hexane as a
solvent for lipophilic antioxidants should limit the assessment
of LAA, because this solvent is considered incompatible with the
FRAP assay probably due to its immiscibility with the FRAP
reagent (Pellegrini et al., 2003), we obtained reliable results by
vigorously vortexing the samples. The highest LAA value was
obtained for P503 cv (40.3 mM FRAP/100 g fw), followed by
Dumara (34.1 mM FRAP/100 g fw), Aramis (33.1 mM FRAP/100 g
fw), Giza (29.2 mM FRAP/100 g fw), P403 (25.1 mM FRAP/100 g
fw) and Crimson Sweet (22.0 mM FRAP/100 g fw).
When cvs data were averaged, HAA and LAA varied signicantly
between sampling areas (P < 0.05) with both assays. With the
TEAC assay the stem-end area showed the highest HAA and LAA
values (281.5 and 257.8 mM Trolox/100 g fw, respectively),
whereas, the lowest HAA values were obtained for both the
peripheral and heart areas (206.0 and 193.2 mM Trolox Eq/100 g
fw) and the lowest LAA value was obtained for the peripheral area
(219.6 mM Trolox Eq/100 g fw).
The FRAP assay conrmed the stem-end as the esh area with
the highest HAA value (30.2 mM FRAP/100 g fw), while the
blossom-end (22.9 mM FRAP/100 g fw) and the heart (22.6 mM
FRAP/100 g fw) areas showed the lowest HAA values. With the
FRAP assay the highest LAA value was obtained for the peripheral
area (37.4 mM FRAP/100 g fw) and the lowest value was obtained
for the blossom and heart areas with 29.3 and 29.4 mM FRAP/100 g
fw, respectively.
To our knowledge the presented data are the rst report of HAA
and LAA in watermelon cvs and of their distribution within the
fruits esh. These results conrm that watermelon fruits, as well
as those from the Cucurbitaceae family, have a low antioxidant
activity. In fact, Pellegrini et al. (2003) reported that watermelon
ranked 30th and 28th for its TAA, measured with the FRAP and
TEAC assays, respectively, when compared to other 30 fruits
commonly consumed in the Mediterranean area. In all the
analysed cvs, the sum of HAA and LAA for each assay, was higher
than the TAA reported by the same authors for watermelon fruits
which were 69.0 mmol Trolox/100 g fw and 1.13 mmol Fe2+/kg fw
(corresponding to 31.5 mM FRAP/100 g fw), with the TEAC and
the FRAP assays, respectively.
These data highlight the importance of the sampling area in
determining the amount of antioxidant components as well as
their antioxidant activity in watermelon fruits. In large fruited

25.5  1.0c
75.2  2.3a
24.1  2.9c
48.6  1.5b
38.8D

Lower case letters indicate mean separation within column and sampling area by
LSD test, P < 0.05. Capital letters indicate mean separation among means within
column by LSD test, P < 0.05.

3.2. Vitamin C content


The contents of AsA, DHA and total vitamin C (AsA + DHA) of the
investigated watermelon cvs within the four different sampling
areas are shown in Table 3. Even in this case signicant variations
are evident among cvs and sampling areas within each cvs
(P < 0.01).
The average amount of total vitamin C in the watermelon fruit,
calculated as the mean of the values of all the sampling areas of
each cv, ranged from 105.2 mg/kg fw (Aramis cv) to 239.8 mg/kg
fw (Dumara cv). Considerably higher (576.2 mg/kg) and lower
values (38.269.8 mg/kg fw) were reported for other watermelon
cvs by Melo et al. (2006) and Leskovar et al. (2004), respectively.
Such variations are probably due to the differences in genotype,
environmental conditions and cultural practices.
The contribution of AsA and DHA to total vitamin C content was
also cv dependent. In fact, for Dumara cv, AsA and DHA accounted,
respectively, for 55% and 45% of the total vitamin C. However, they
accounted for 12% and 88% in P403 cv. In addition, the data showed
that total vitamin C levels were signicantly different in the
sampling areas of all cvs studied, except for Aramis (P < 0.01).
Hence, the effect of sampling areas on the total vitamin C content
was cv dependent. When data were averaged across cvs, vitamin C
content varied signicantly among sampling areas (P < 0.05). The
highest values were obtained for peripheral (187.7 mg/100 g fw)
and stem-end (186.0 mg/100 g fw) areas; the lowest were

312

I. Tlili et al. / Journal of Food Composition and Analysis 24 (2011) 307314

Table 4
Hydrophilic and lipophilic antioxidant activities of the watermelon cvs studied within different sampling areas.
Cultivars

Crimson Sweet
Blossom-end area
Stem-end area
Heart area
Peripheral area
Mean
Giza
Blossom-end area
Stem-end area
Heart area
Peripheral area
Mean
Dumara
Blossom-end area
Stem-end area
Heart area
Peripheral area
Mean
P403
Blossom-end area
Stem-end area
Heart area
Peripheral area
Mean
P503
Blossom-end area
Stem-end area
Heart area
Peripheral area
Mean
Aramis
Blossom-end area
Stem-end area
Heart area
Peripheral area
Mean

TEAC assay (mmol Trolox/100 g fw)

FRAP assay (mM FRAP/100 g fw)

Hydrophilic

Lipophilic

Hydrophilic

Lipophilic

311.4  7.8a
342.4  5.2a
215.1  5.2b
294.8  5.4a
290.9A

246.7  4.4b
271.9  4.4a
284.0  4.5a
233.2  4.7b
258.9A

13.4  0.3c
32.1  0.1a
24.1  0.4b
24.0  0.1b
23.4D

25.2  0.4a
21.9  0.6b
14.4  0.5c
26.6  0.3a
22.0E

325.0  4.0a
350.2  5.2a
201.1  4.3b
209.0  7.7b
271.3A

274.1  4.6a
259.7  5.7a
260.2  8.9a
230.1  9.1b
256.1AB

28.7  0.3c
32.6  0.3a
27.7  0.2d
30.6  0.1b
29.9A

23.9  2.6b
25.1  0.8b
33.8  0.8a
33.8  0.7a
29.2D

254.2  4.8a
229.0  4.9a
156.4  7.8b
170.5  5.1b
202.5D

247.8  3.2a
244.5  3.8a
256.1  5.4a
223.1  6.5b
242.9C

34.8  0.25a
32.1  0.26b
23. 6  0.25d
27.8  0.27c
29.6A

39.2  0.4b
41.7  0.6a
28.4  0.3c
27.1  0.2c
34.1BC

297.3  11.6a
267.2  5.7ab
185.2  8.7c
221.8  6.6bc
242.9C

269.2  4.2a
298.8  4.8b
195.2  7.6c
239.4  10.0d
250.7B

30.0  0.2b
32.2  0.1a
24.7  0.2c
23.0  0.2d
27.5B

31.4  2.6b
36.8  0.8a
33.9  0.6ab
38.4  0.5a
25.1B

182.2  3.2a
211.4  5.7a
161.5  6.1a
174.3  4.3a
182.4E

196.6  3.8a
208.3  5.5a
217.7  3.2a
201. 5  7.0a
206.0D

13.7  0.3b
13.2  0.2b
10.3  0.2c
15.9  0.2a
13.3E

33.5  0.5b
29.9  0.2c
34.7  1.1b
63.1  0.6a
40.3A

201.3  4.8bc
292.2  8.7a
239.9  5.6b
169.1  4.3c
225.7C

184.3  7.6c
263.5  5.3a
204.4  4.9b
190.5  3.3bc
210.7D

16.7  0.3d
39.2  0.1a
25.5  0.3b
21.9  0.3c
25.8C

22.3  0.8c
43.2  2.3a
31.5  1.1b
35.5  0.4b
33.1C

Lower case letters indicate mean separation within column and sampling area by LSD test, P < 0.05. Capital letters indicate mean separation among means within column by
LSD test, P < 0.05.

watermelon where only a portion of the fruit is feasibly tested for


quality, an accurate and reproducible sampling method must be
developed.
Correlations between bioactive compounds and antioxidant
activities in fruits and vegetables have been studied by many
authors, however little is known in watermelon fruit.
Although both TEAC and FRAP assays are, mechanistically,
based on a single electron transfer reaction, they often give results
which do not correlate well with each other, probably depending
upon the pH conditions in which the two assays are carried out
(TEAC in neutral and FRAP in acidic environment) and the free
radical or oxidant utilized (Halliwell and Gutteridge, 1995). The pH

values have an important effect on the reducing capacity of the


various classes of antioxidants depending on their dissociation
constant (Ka). Considering the data from all investigated watermelon cvs (Table 5), AsA did not signicantly correlate with the
HAA values measured by both TEAC and FRAP assays, whereas
good linear correlation between TEAC values and total phenolics
(R = 0.637; P < 0.01) was obtained in the present study. Signicant
but weak correlation was also obtained between FRAP values and
total phenolics (R = 0.400; P < 0.01). Differences between the two
assays were evidenced concerning the correlations with other
hydrophilic antioxidants. DHA and total vitamin C contents did not
correlate with the HAA measured by the TEAC assay but they did

Table 5
Correlation coefcients and related signicances between antioxidant compounds and antioxidant activities; n (sample size) = 72.
Antioxidants

HAA TEAC

HAA FRAP

Corr coeff
Ascorbic acid
Dehydroascorbic acid
Total vitamin C
Total phenolics
Flavonoids
Antioxidant

Lycopene
ns, no signicant correlation.

0.133
0.012
0.116
0.637
0.352

ns
ns
ns
<0.01
<0.01

LAA TEAC

Corr coeff
0.212
0.320
0.434
0.400
0.264

P
ns
<0.01
<0.01
<0.01
ns

LAA FRAP

Corr coeff

Corr coeff

0.230

ns

0.063

ns

I. Tlili et al. / Journal of Food Composition and Analysis 24 (2011) 307314

with the values found by FRAP. In addition, a negative but


signicant correlation was observed between HAA values, determined by TEAC but not by FRAP, and avonoids (R = 0.352;
P < 0.01). This negative correlation was not surprising, since a prooxidant activity of some avonoids had been previously demonstrated under certain conditions, e.g., in the presence of metal ions
(Cao et al., 1997). From these data, phenolics seem to be the major
hydrophilic compounds contributing to the HAA in watermelon,
nevertheless, it should always be kept in mind that the antioxidant
activity depends upon synergistic effects among all hydrophilic
antioxidants and their interaction with other components of the
fraction (Diplock et al., 1998). Other hydrophilic organic and
inorganic constituents of watermelon may play an important role
in dening the overall HAA of watermelon.
The LAA of fruits containing lycopene, such as tomatoes, has
been mainly attributed to the presence of this carotenoid
(Martinez-Valverde et al., 2002; Raffo et al., 2002; Cano et al.,
2003; George et al., 2004), however, considering our data, no
signicant correlation between TEAC or FRAP values and lycopene
content (R = 0.230 and R = 0.063, respectively; P > 0.05) was
found. Djuric and Powell (2001) reported that foods with the
highest levels of lycopene do not necessarily have the highest
antioxidant activity.
The antioxidant activities measured with both TEAC and FRAP
methods were similar in the hydrophilic and lipophilic fractions of
most analysed cvs, in agreement with what was reported by Djuric
and Powell (2001) in the watermelon.
4. Conclusions
This study has conrmed the important role played by the
genotype in determining the amount of bioactive compounds and
the in vitro antioxidant activity of fresh watermelon. Although the
capacity of such molecules in providing health benets by trapping
free radicals in vivo needs still to be proven, the daily dietary intake
of fruit and vegetable determines changes into their concentration
in human tissues where they may exert a positive effect acting as
antioxidants and/or with many other mechanisms.
In addition, the results highlight the importance of standardized and documented sampling methods when determining
lycopene, phenolics, avonoids, total vitamin C, as well as HAA
and LAA in watermelon cvs.
The variability detected in the four commercial watermelon cvs
and the two new selections emphasizes an existing unexploited
variability in watermelon germplasm and underlines the need to
evaluate more watermelon genotypes and to support conventional
breeding programs to improve the nutritional value of this fruit.
Watermelon could contribute substantially to the hydrophilic and
lipophilic antioxidant levels of human diets, and this excellent
refreshing summer fruit might be more widely adopted in
preventive medicine strategies by healthy consumers in preference to the antioxidant supplements publicized on the market.
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