Pharmaceutical Biology

ISSN: 1388-0209 (Print) 1744-5116 (Online) Journal homepage: https://www.tandfonline.com/loi/iphb20

Antioxidant activity and in vitro inhibition of tumor

cell growth by leaf extracts from the carob tree
(Ceratonia siliqua)

Luísa Custódio, Eliana Fernandes, Ana Luisa Escapa, Sandra López-Avilés,

Alba Fajardo, Rosa Aligué, Fernando Alberício & Anabela Romano

To cite this article: Luísa Custódio, Eliana Fernandes, Ana Luisa Escapa, Sandra López-Avilés,
Alba Fajardo, Rosa Aligué, Fernando Alberício & Anabela Romano (2009) Antioxidant activity
and in�vitro inhibition of tumor cell growth by leaf extracts from the carob tree (Ceratonia�siliqua),
Pharmaceutical Biology, 47:8, 721-728, DOI: 10.1080/13880200902936891

To link to this article: https://doi.org/10.1080/13880200902936891

Published online: 24 Jun 2009.

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Pharmaceutical Biology, 2009; 47(8): 721–728

RESEARCH ARTICLE

Antioxidant activity and in vitro inhibition of tumor

cell growth by leaf extracts from the carob tree
(Ceratonia siliqua)
Luísa Custódio1, Eliana Fernandes1, Ana Luisa Escapa1, Sandra López-Avilés2, Alba Fajardo2,
Rosa Aligué2, Fernando Alberício3, and Anabela Romano1
1
Institute for Biotechnology and Bioengineering, Center for Molecular and Structural Biomedicine, University
of Algarve, Campus of Gambelas, Faro, Portugal, 2School of Medicine, Department of Cell Biology, University of
Barcelona, Barcelona, Spain, and 3Institute for Research in Biomedicine, Barcelona Science Park, Barcelona, Spain

Abstract
The methanol leaf extracts of female cultivars of the carob tree [Ceratonia siliqua L. (Fabaceae)] and of
hermaphrodite and male trees were investigated for their contents of phenolic compounds, their in vitro
antioxidant activity, measured by 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging and linoleic
acid system assays, and their in vitro tumor growth inhibition on HeLa cells. The different cultivars and
trees showed high levels of phenols, and considerable variations in the amount of these compounds. The
extracts showed significant radical scavenging activity (RSA), which was not significantly affected by the
gender of the tree. From the female cultivars tested, Galhosa exhibited the highest RSA. Gender signifi-
cantly affected the antioxidant activity of the extracts measured by the linoleic acid system assay, and
males and hermaphrodites showed the highest activities. The extracts displayed a remarkable ability to
inhibit tumor cell proliferation, and their bioactivity varied with different cultivars or trees tested. Extracts
from male and hermaphrodite trees exhibited higher capacity to inhibit the proliferation of HeLa cells than
the female cultivars.
Keywords:  Cytotoxicity; flavonoids; HeLa cells; polyphenols; reactive oxygen species

Introduction and free radical scavengers (Sugihara et  al., 1999).

Free radicals (reactive oxygen species, ROS, and reactive
nitrogen species, RNS) are products of normal cellular
metabolism, and are extremely reactive and potentially
damaging transient chemical species. The delicate bal-
ance between beneficial and harmful effects of free
radicals is very important to living organisms and is
maintained by “redox regulation” defense mechanisms,
which include preventive and repair mechanisms,
physical defenses, and antioxidant defenses provided
by vitamins, carotenoids, sterols, and phenols (Valko
et al., 2007).
There have been numerous studies on the biologi-
cal activities of phenols, which are potent antioxidants
Furthermore, many isolated phenol compounds or
extracts rich in these compounds have been reported to
possess anticancer, anti-carcinogenic, anti-mutagenic,
antibacterial, antiviral, or anti-inflammatory activities
(Yamagishi et al., 2000; Greenspan et al., 2005; Lambert
et al., 2005; Moreno et al., 2006; Song et  al., 2007).
Phenolic compounds may also have antioxidant effects
used for managing oxidation stress-related chronic
diseases such as diabetes and hypertension (Kwon
et al., 2008).
The carob tree [Ceratonia siliqua L. (Fabaceae)] is
one of the most useful trees of the Mediterranean basin.
It is mainly used in the food industry as a source of gum
extracted from the seeds (LBG; E410). The pulp is used

Address for Correspondence:  A. Romano, Institute for Biotechnology and Bioengineering, Center for Molecular and Structural Biomedicine, University of
Algarve, Campus of Gambelas, Faro, Portugal. Tel: +351 289 800910. Fax: +351 289 818419. E-mail: aromano@ualg.pt
(Received 29 July 2008; revised 15 September 2008; accepted 15 September 2008)
ISSN 1388-0209 print/ISSN 1744-5116 online © 2009 Informa UK Ltd
DOI: 10.1080/13880200902936891 http://www.informapharmascience.com/phb
722   Luísa Custódio et al.
in infants for the treatment of diarrhea of bacterial and
viral origin (Loeb et al., 1989). The leaves and pulps have
antioxidant (Kumazawa et  al., 2002; Makris & Kefalas,
2004, Owen et al., 2003; Papagiannopoulos et al., 2004),
antiproliferative (Corsi et  al., 2002), and antimicro-
bial activities (Kivçak et  al., 2002). Leaf extracts have
potential anxiolytic and sedative effects (Avallone et al.,
2002).
The chemical composition of pods varies among
the different cultivars, and includes a high amount of
different types of polyphenolic compounds (Avallone
et  al., 1997; Corsi et  al., 2002; Kumazawa et  al., 2002;
Owen et  al., 2003; Papagiannopoulos et  al., 2004),
which are also present in leaves in higher amounts
(Corsi et al., 2002). Some previously isolated constitu-
ents include gallic acid, hydrolyzable and condensed
tannins, flavonolglycosides, flavonoids (Owen et  al.,
2003; Papagiannopoulos et  al., 2004), and pinitol
(Baumgartner et al., 1986).
As far as our literature survey could ascertain, there is
no information about the phenolic profile or biological
activities of leaf extracts of Portuguese female cultivars
of this species; nor are there any comparative studies
of the variation in chemical composition between the
three genders. In this context, we assessed the amounts
of phenol compounds from the methanol leaf extracts
of six representative Portuguese female cultivars of the
carob tree growing in the Algarve region, and the leaf
extracts of two male and two hermaphrodite individu-
als. The antioxidant activity of the extracts was assessed
using two established in vitro methods, and this activity
was compared to the activity of pure phenolic com-
pounds. Furthermore, we investigated the antitumor
activity of the extracts using HeLa cells, a cell line of
human uterine cervix adenocarcinoma, as a model
system.
Materials and methods
Collection of plant material and preparation of
extracts
Samples from six of the most representative female
Portuguese cultivars of the carob tree in terms of
fruit productivity, namely Mulata, Galhosa, Aida,
Costela/Canela, Gasparinha, and Preta de Lagos, and
from two hermaphrodite (H1 and H2) and two male
trees (M1 and M2) were sampled during August and
September of 2005. The different cultivars and trees are
from a cultivar collection from the Ministry of Agriculture,
Rural Development and Fisheries (Direcção Regional
de Agricultura do Algarve, Delegação de Tavira), and
were identified by J. Graça. Fully expanded leaves
were randomly collected from the middle third of the
branches in all canopy orientations, dried at 40°C for 2
days, crushed and milled in a laboratory-scale hammer
mill, and stored in the dark at −20°C until extraction.
The extracts were prepared as described by Owen et al.
(2003). Samples (10 g) were extracted in a Soxhlet appa-
ratus first with n-hexane (2 × 3 h), to remove lipids, and
then with methanol (5 h). The methanol extracts were
centrifuged at room temperature (RT) during 10 min at
3000 rpm, the supernatant was collected, and the sol-
vent was removed by rotary evaporation with vacuum.
The extracts were resuspended in methanol and kept at
−20°C in the dark until analysis. Every extraction was
performed at least twice.
Chemicals used for experimentation
Dulbecco’s modified Eagle medium (DMEM),
l-glutamine, penicillin, and streptomycin were pur-
chased from Biological Industries; fetal bovine serum
(FBS) was from PAA Laboratories and n-hexane was
from Labscan. Sigma Chemical Co. supplied DPPH
(1,1-diphenyl-2-picrylhydrazyl), chlorogenic acid,
(+)-catechin, linoleic acid, chloroform and -carotene,
whereas WST-1 [2-(4-iodophenyl)-3-(4-nitrophenyl)-5-
(2,4-disulfophenyl)-2H-tetrazolium], monosodium salt)
was purchased from Roche. Folin–Ciocalteu reagent,
(–)-epicatechin, gallic acid, methanol (MeOH), Tween
40, p-dimethylaminocinnamaldehyde (DMACA), alumi-
num chloride (AlCl3), and sodium carbonate (Na2CO3)
were from Fluka Biochemika. Catechol was purchased
from Acros organics. All the other chemicals where of
analytical grade.
Measurement of total phenol content
The total phenol content of the extracts was determined
in samples (2 mg/mL) by the Folin–Ciocalteu colorimet-
ric method (Julkunen-Tiitto, 1985). Briefly, aliquots of the
extracts (0.1 mL, 2 mg/mL) were added to 5 mL of distilled
water and 0.5 mL of Folin–Ciocalteu reagent and vigor-
ously shaken. After 3 min, 1 mL of a saturated solution of
sodium carbonate (Na2CO3) was added, and the volume
made up to 10 mL with distilled water. The mixtures were
allowed to stand for 60 min at RT for complete reaction,
and the total phenols were determined by colorimetry
at 720 nm (Shimadzu UV-160A). The amount of total
polyphenols was calculated as a gallic acid equivalent
from the calibration curve of gallic acid standard solu-
tions, and expressed as gallic acid equivalents (GAE) in
milligrams per gram of initial dry plant material.
Determination of total condensed tannins content
The amount of condensed tannins was determined
by the p-dimethylaminocinnamaldehyde (DMACA)

method, according to Arnous et  al. (2001). Samples
(0.4 mL, 2 mg/mL) were mixed with 2 mL of DMACA
solution (0.1% in 1 N HCl in MeOH). The mixture
was vortexed, allowed to react at RT for 10 min, and
the absorbances were read at 640 nm on a Shimadzu
UV-160A spectophotometer. The concentration of
condensed tannins was estimated from a calibration
curve, constructed by plotting known solutions of
(+)-catechin, and results were expressed as catechin
equivalents (CE) in milligrams per gram of initial dry
plant material.
Determination of total flavonoids
The flavonoid content was estimated in the extracts
by the aluminum chloride (AlCl3) method (Lamaison
& Carnat, 1990). In short, 1 mL of methanol extract
(2 mg/mL) was added to 1 mL of 2% methanol
AlCl3.6H2O. The absorbance was measured 10 min
later at 430 nm (Shimadzu UV-160A). The results were
expressed as rutin equivalents (RE) in milligrams per
gram of initial dry plant material by comparison with
the values obtained from standard rutin treated in the
same conditions.
Determination of antioxidant activity
DPPH radical scavenging activity
The effect of extracts on the DPPH radical was
­estimated using the method of Brand-Williams et  al.
(1995). Aliquots (0.1 mL) of test sample at concentra-
tions ranging from 400 to 50 µg/mL were reacted with
2.5 mL of methanol DPPH solution (100 µM) for 30 min
in the dark, and the decrease in absorbance at 517 nm
was measured spectrophotometrically (Shimadzu
UV-160A). The DPPH radical scavenging activ-
ity (RSA) was calculated by the following equation:
RSA (%) = [(control absorbance − sample absorbance)]/
(control absorbance) × 100. Results were expressed
as IC50 value (µg/mL), which is the amount of sample
necessary to decrease by 50% the absorbance of DPPH,
and as antiradical efficiency (AE), which is 1000-fold
the inverse IC50 value (Parejo et al., 2003).
Linoleic acid system
The activity of the extracts was determined according to
Tepe et al. (2006). A stock solution of -carotene–linoleic
acid mixture was prepared by dissolving -carotene
(0.5 mg) in chloroform (50 mL), and adding linoleic
acid (25 µL) and Tween 40 (200 mg). Chloroform was
completely evaporated using a vacuum evaporator,
and 100 mL of distilled water was added with vigor-
ous shaking. Aliquots (3 mL) of this reaction mixture
were dispensed to test tubes, the extracts (0.1 mL) were
added, and the emulsion system was incubated for
Antioxidant and antitumor activities of carob tree   723
60 min at 50°C. Oxidation of the emulsion was assessed
spectrophotometrically (Shimadzu UV-160A) by meas-
uring absorbance at 470 nm after the 60 min period. The
antioxidant activity is expressed as percent inhibition
relative to the control after a 60 min incubation using
the following equation: AA = 100(DRC – DRS)/DRC,
where AA is the antioxidant activity, DRC is the
­degradation rate of the control [= ln(a/b)/60], DRS
is the degradation rate in the presence of the sample
[= ln(a/b)/60], a is the initial absorbance at time 0, and
b is the absorbance at 60 min. Extracts and polyphenol
compounds were evaluated at the final concentrations
of 1 mg/mL in the assay mixture. Both the scaveng-
ing and the antioxidant activities of the extracts were
compared to those of pure phenolic compounds,
namely gallic acid, (+)-catechin, chlorogenic acid,
(–)-­epicatechin, and catechol.
Antitumor activity
In vitro bioassay test
Human cervical adenocarcinoma cells (HeLa line) from
the American type culture collection (Rockville, MD)
were grown and maintained in Dulbecco’s ­modified
Eagle medium (DMEM) containing 1 g/mL of glucose,
and supplemented with 10% (v/v) fetal bovine serum
(FBS), l-glutamine (2 mM), penicillin (50 U/mL),
and streptomycin (50 µg/mL). The cells were grown
in an incubator at 37°C, in a 5.1% CO2 humidified
atmosphere.
Tumor growth inhibition was determined by the
WST-1 assay (Choudhary et al., 2006; Wang et al., 2006).
Exponentially growing cells were seeded on 96-well
plates at a density of 7 × 103 cells/well. After incubation
for 24 h at 37°C, cells were treated with several dilutions
of the extracts (400–25 µg/mL), and incubated for 24, 48,
and 72 h. Two hours before the end of incubation, 20 µL
of WST-1 were added to each well and further incubated
for 2 h at 37°C, and the optical density (OD) was meas-
ured at 450 nm on a microplate reader (Power Wave XS
spectrophotometer). The cytotoxicity of the extracts was
expressed as the percentage viability of the cells, half
maximal inhibitory concentration (IC50), and maximal
degree of inhibition.
Statistical analysis
The experimental results are expressed as mean ±
­standard deviation (SD). All experiments were con-
ducted in triplicate, and all tests and measurements
were repeated at least three times. The data were ana-
lyzed using the analysis of variance (ANOVA) method
to assess differences with the SPSS statistical package
for Windows (release 15.0; SPSS Inc.), and significance
between means was tested by Duncan’s new multiple
724   Luísa Custódio et al.

range test (p = 0.05). The correlation between antioxi-
dant and free-radical scavenging activities versus the
polyphenol, tannin, and flavonoid contents was deter-
mined using the correlation and regression program of
Microsoft Excel. IC50 values were calculated by sigmoidal
fitting of the data in the GraphPad Prism V 4.0 micro-
computer program.
of extracts from different genders of tree, significant
differences were observed between female culti-
vars (p < 0.05) (Table 1). Among the female cultivars,
Galhosa and Mulata had the highest and lowest values,
respectively, while among hermaphrodites, flavonoids
were present in the highest amounts in leaves from
tree H2 (Table  1). Among males, M2 had the highest
content of these compounds (Table 1).

Results
Radical scavenging activity (RSA)
Polyphenol contents The extracts showed a significant RSA (Table 2), which
was concentration-dependent (data not shown). There
The total content of phenol compounds was not dif-
was no gender-based variation in RSA (p > 0.05), but
ferent between extracts from trees of different genders
there was a significant variation in RSA between extracts
(p > 0.05). Among female trees, the total amount of
taken from different cultivars and different individual
phenols varied among different cultivars; the levels of
trees (p < 0.001). Among the female cultivars, Galhosa
these compounds were markedly higher in the female
exhibited the highest activity, denoted by the lowest
cultivars Galhosa, Aida, and Preta de Lagos than in the
IC50 and highest antiradical efficiency values, while
cultivars Mulata and Gasparinha (Table 1).
among the male and hermaphrodite trees, M1 and
The amount of condensed tannins varied signifi-
H2 exhibited the highest RSA, respectively (Table  2).
cantly between the different genders and cultivars
Furthermore, the RSAs of extracts from the three gen-
(p < 0.05), ranging from 1.8 to 23.7 mg CE g−1, with
ders were similar to those displayed by chlorogenic acid
the lowest amount obtained from extracts from
and (+)-catechin.
the female cultivar Preta de Lagos and the highest
A correlation analysis was conducted between the
amount obtained from the female cultivar Galhosa
RSA and the amount of phenols, tannins, and flavonoids
and hermaphrodite tree H1 (Table 1). We found
in all of the trees studied, and in the female cultivars
that the leaves of the hermaphrodites were richer
(Table 3). For all trees studied, the RSA was closely cor-
in tannins, and the level of these compounds was
related to the total amount of phenols and flavonoids,
markedly higher in tree H1. In female cultivars,
but was not correlated to the amount of tannins in the
tannins were present in highest amounts in the
cultivar cultivar Galhosa (Table 1). While there was
no significant difference in total flavonoid contents Table 2.  DPPH radical scavenging activity of methanol leaf extracts
from female, male and hermaphrodite carob trees, and pure
polyphenolic compounds.
Table 1.  Contents of total phenol compounds, condensed tannins IC50 (µg/mL−1) AE
and total flavonoids in methanol leaf extracts of male, female and Female
hermaphrodite carob trees.
  Mulata > 400 nd
Total phenols Condensed
  Galhosa 94.6 ± 0.7 10.5 ± 0.1
(mg GE g−1 tannin Flavonoids
Cultivar/tree DW) (mg CE g−1 DW) (mg RE g−1 DW)   Aida 217.3 ± 4.6 4.6 ± 0.1
Female   Gasparinha 273.2 ± 11.9 3.6 ± 0.1
  Mulata 16.4 ± 1.5 c 16.7 ± 0.1e 2.1 ± 0f   Costela/Canela 199.1 ± 5.5 5.0 ± 0.1
  Galhosa 39.4 ± 2.3 a 23.7 ± 1a 13.4 ± 0.4a   Preta de Lagos 175.7 ± 6.5 5.6 ± 0.2
  Aida 34.2 ± 7.4 a 17.8 ± 0.1c 7.0 ± 1.0c Hermaphrodite
  Gasparinha 24.0 ± 0.5b 17.7 ± 0.7c 7.2 ± 0.3c   H1 167.2 ± 8.1 5.9 ± 0.3
  Costela/Canela 29.8 ± 3.2ab 19.9 ± 0.1b 6.9 ± 0.2c   H2 159.8 ± 18.7 6.3 ± 0.8
  Preta de Lagos 34.1 ± 3.1a 1.8 ± 0.3f 7.7 ± 0.5b Male
Hermaphrodite   M1 153.2 ± 17.0 6.5 ± 0.7
  H1 31.6 ± 2.7a 21.07 ± 0.2a 5.1 ± 0e   M2 159.6 ± 49.7 6.7 ± 2.3
  H2 29.7 ± 1.1ab 19.5 ± 0.8b 6.3 ± 0.2d Phenolics
Male   Gallic acid 84.3 ± 15.6 12.0 ± 2.2
  M1 28.7 ± 2.4ab 16.1 ± 0.3d 6.0 ± 0.5d   Chlorogenic acid 143.2 ± 2.5 6.9 ± 0.1
  M2 28.9 ± 2.8ab 16.3 ± 0.2d 7.9 ± 0.1b   (+)-catechin 158.9 ± 15.6 6.3 ± 0.6
Values represent means ± SD of 3 assessments. For each column   (-)-epicatechin 288.5 ± 28.4 3.4 ± 0.3
and gender, statistical analysis was made between cultivars or trees.   Catechol 49.1 ± 7.8 20.7 ± 3.5
Values followed by different letters are significantly different at Values represent means ± SD of 3 measurements of IC50 and
P < 0.05 (one-way ANOVA, Duncan’s New Multiple Range Test). antiradical efficiency (AE).
 Antioxidant and antitumor activities of carob tree   725

extracts. In extracts from female trees, the amount of not between the AA and the total amount of phenols or
total phenols and of flavonoids was very closely corre- tannins. When all samples were considered, there was
lated with the RSA. no correlation between the analyzed parameters.

Antioxidant activity in the linoleic acid system Antitumor activity

Significant gender-based differences in the antioxidant Except for the female cultivar Galhosa, whose extracts
activity of the extracts were observed (p < 0.05), with had no effect on HeLa cell proliferation, carob extracts
males and hermaphrodites having the highest activities significantly inhibited HeLa cell proliferation in a dose-
(Figure 1). Overall, extracts from males and hermaph- dependent manner (p < 0.05) (Figure 2). The arrest of
rodites are better than or as good at preventing the dis- HeLa cell growth was not time-dependent and was
coloration of -carotene as pure phenolic compounds.
As for the RSA, a correlation analysis was conducted
Aida
between the AA and the amount of phenols, tannins, 100 Galhosa
Mulata
and flavonoids in extracts of all trees studied, and in Preta de Lagos
extracts from the female cultivars (Table 3). For extracts Gasparinha
80 Costela/Canela
from females, high correlation coefficients were found
between the AA and the total amount of flavonoids, but
60

Table 3.  Correlation coefficients (R) between radical scavenging 40

activity (RSA), antioxidant activity (AA) and contents of total phenols
(GAE), tannins (CE) and flavonoids (RE).
R 20
Plant RSA/ RSA/ RSA/ AA/ AA/ AA/
Material N GAE RE CE GAE RE CE
Females 18 0.88a 0.95b 0.15 0.53 0.81a 0.19
All the trees 22 0.85b 0.71a 0.29 0.49 0.47 0.36 100 H2
H1
  mixed
Note: GAE- gallic acid equivalents; CE- catechin equivalents; RE-
Cell viability (% of control)

80
rutin equivalents.
a
p<0.05; b p<0.01
60

Mulata e 40
Galhosa d
Aida c
20
Preta de Lagos d
Costela/Canela c
Gasparinha d 0
M1 ab
M2 b
H1 a
H2 ab 80
Catechin d
(−)−Epicatechin d
60
Gallic acid b
Chlorogenic acid a
Catechol e 40
0 25 50 75 100
Antioxidant activity(%) 20

Figure 1.  Antioxidant activity of methanol leaf extracts from female,

0
male, and hermaphrodite carob trees, and of pure polyphenol com-
pounds. Values represent means ± SD of three assessments of the 0 100 200 300 400
percent inhibition of autoxidation of the linoleic acid/-carotene Concentration (µg/mL)
emulsion. Extracts and phenol compounds were evaluated at final
concentrations of 1 mg/mL. Values followed by different letters are Figure 2.  Effect of treatment with methanol leaf extracts from female
significantly different at p < 0.05 (one-way ANOVA, Duncan’s new (A), hermaphrodite (B), and male (C) carob trees on cell proliferation.
multiple range test). Each point represents the mean of three independent experiments.
726   Luísa Custódio et al.
Table 4.  Inhibitory concentrations (IC50) and maximal degree of
inhibition (Max.) of methanol leaf extracts of female, hermaphrodite
and male carob trees on HeLa cells.
Cultivar/tree IC50 (µg/mL−1) Max. (%)
Female
  Mulata 79 ± 3.7
  Galhosa > 400
  Aida 150.3 ± 21.6 96.9 ± 0.7
  Gasparinha 82.8 ± 8.9
  Costela/Canela 67.01 ± 15.2
  Preta de Lagos 143.82 ± 54.5 79.8 ± 16.9
Hermaphrodite
  H1 149.9 ± 70.3 95.3 ± 1.5
  H2 < 25
Male
  M1 81.6 ± 5
  M2 50.6 ± 0.5
already evident after a treatment lasting 24 h. For the
highest concentration tested (400 µg/mL), cell survival
was generally below 25% (Figure 2). There was signifi-
cant variation in bioactivity between the extracts from
different genders of tree. Regardless of the concentra-
tion, treatment with extracts from males and hermaph-
rodites resulted in a significantly higher decrease in cell
proliferation than treatment with extracts from females
(p < 0.01). Extracts from the hermaphrodite tree H1 pro-
duced the highest inhibition capacity (IC50 < 25 µg/mL),
while extracts from the female cultivar Galhosa were
the least effective at preventing the proliferation of
HeLa cells (IC50 > 400 µg/mL) (Table 4). The maximal
inhibition values were high (generally 100%) (Table 4).
A correlation analysis was performed between cyto-
toxic activity and RSA for all the trees studied and for
the female cultivars, but no relationship was observed
(data not shown).
Discussion and conclusions
The idea of using leaves of carob, a typical Mediterranean
tree, as a source of polyphenols, was raised after the evi-
dence that carob leaves contain a high amount of these
compounds (Corsi et al., 2002). Our results support these
findings since in this study, except for female cv. Mulata,
all the cultivars and trees displayed remarkably high lev-
els of total phenolic content, with GAE values >20 mg/g
dry weight (DW) (Kähkönen et al., 1999). On the other
hand, the amount of total phenols in leaf extracts from
the carob tree were higher than those observed in Salvia
spinosa L. (Lamiaceae), a plant species that is well known
as a rich source of polyphenols (Alali et al., 2007).
Cross-varietal screening tests have repeatedly shown
that certain genotypes within a plant species can have
widely divergent levels of inherent antioxidants (Lila,
2006). In this work, we observed a general variation in
the amount of phenolic compounds between extracts
from different carob tree genders and different cultivars
(Table 1). Working with the same species, Avallone et al.
(1997) observed different levels of total polyphenols in
100 different organs of female trees, and among trees from
– different locations. Emmons and Peterson (2001) also
found significant differences among cultivars in con-
100
centrations of the majority of the phenol compounds
100
measured in Avena sativa L. (Poaceae). Similar results
were found by other authors in different plant species
(Cetkovic et al., 2004; Maksimović et al., 2005; Gattuso
et al., 2007; Li et al., 2007).
100
In this study, the free radical scavenging activity
100
(RSA) of leaf extracts was assessed against DPPH radi-
100 cals, and their antioxidant activity (AA) was assessed
using the -carotene–linoleic acid system. The extracts
showed a significant RSA (Table 2), suggesting that they
are capable of scavenging free radicals, and thus, may
be able to prevent the initiation of free radical-mediated
chain reactions by preventing the abstraction of hydro-
gen from susceptible polyunsaturated fatty acids. On the
other hand the RSA varied between extracts from dif-
ferent genotypes. There was a significant gender-based
difference in the AA of the extracts, and overall, extracts
from males and hermaphrodites were equally or better
able to prevent the discoloration of -carotene than pure
phenolic compounds (Figure 1).
Extracts with a higher phenol or flavonoid content
generally show higher antioxidant activity, and good
correlations have been found among these parameters
(Alali et al., 2007; Li et al., 2007). Generally, the RSA was
closely correlated to the total amount of phenols and
flavonoids suggesting that these compounds are major
contributors to the RSA of the extracts (Table 3). These
results agree with previous reports which found that
phenols and flavonoids contribute significantly to the
RSA of different plant species (Parejo et al., 2003; Alali
et al., 2007).
Interestingly, despite the fact that the extracts have a
higher content of tannins than flavonoids, no significant
correlation was found between RSA and the amount of
tannins in an extract (Table 3), suggesting that flavonoids
may be the major contributor to the RSA of the extracts.
Flavonoids do indeed show strong antioxidant and radi-
cal scavenging activities (Pietta, 2000), and the use of
flavonoid-containing drugs is associated with a reduced
risk for certain chronic diseases, some cardiovascular
disorders, and certain types of cancerous processes
(Kris-Etherton et  al., 2002). Their antiradical proper-
ties stem from targeting ROS that are implicated in the
initiation of lipid peroxidation. Moreover, flavonoids are
soluble chain-breaking inhibitors of the peroxidation
process. They scavenge intermediate peroxyl and alkoxyl
radicals and chelate metal ions, the latter of which are

among the major components in the initiation of radical
reactions (Pietta, 2000).
For female cultivars, high correlation coefficients were
found between the AA and the total flavonoid content,
but not with total phenol or tannin content (Table  3).
When we considered all samples together, there was no
correlation between the analyzed parameters (Table 3).
These results are consistent with the findings of other
authors in the same species (Alali et al., 2007), and sug-
gest that apart from phenols and flavonoids, other com-
pounds in the extracts may also contribute significantly
to the antioxidant activity of carob tree leaf extracts.
Moreover, a plant with high antioxidant activity, for
which phenolic content versus antioxidant activity falls
above the regression line, should be a plant in which
novel antioxidants may be found, and the carob tree is
such a plant (Alali et al., 2007).
The search for new therapeutic drugs for the treat-
ment of cancer from natural products is based mainly
on the cytotoxic properties of the natural samples
(Goldin et  al., 1981). In this sense, the incubation
of HeLa cells with carob leaf extracts resulted in a
remarkable concentration-dependent decrease of
cell proliferation. Such an effect has been previously
reported in several cancer cell cultures, after the appli-
cation of different plant extracts (Wedge et al., 2001;
Chen et al., 2004; Ramos et al., 2005). Cytotoxicity and
the inhibition of cell proliferation are also displayed
by several anticancer drugs derived from natural
materials that are in wide clinical use, such as vinc-
ristine and vinblastine from Catharanthus roseus G.
Don (Apocynaceae), paclitaxel (Taxol®) and taxo-
tere from species of yew (Taxus), etoposide derived
from lignans of Podophyllum spp. (Berberidaceae),
and camptothecin analogs, such as topotecan, from
Camptotheca acuminate D. (Nyssaceae) (Houghton
et al., 2007).
Interestingly, the arrest of HeLa cell growth after
treatment was not time-dependent and was already
evident after a treatment lasting 24 h. This indicates
that the polyphenols in the extracts promptly initiated
a series of cellular events leading to the inhibition of
cell proliferation and/or the induction of cell death.
However, it should be noted that by using the WST-1
assay it is not possible to differentiate between cell
growth inhibition and an increase in cell death. In this
regard, data from studies focusing on elucidation of
the molecular basis of the putative anticancer activity
of polyphenols indicate that both the inhibition of cell
growth and the induction of cell death play a role in the
antitumor activity of polyphenols (Wenzel et al., 2000;
Lazzè et al., 2004).
The bioactivity of the extracts varied between differ-
ent cultivars and trees tested (Figure 2). Similarly, the
extracts from male and hermaphrodite trees showed a
Antioxidant and antitumor activities of carob tree   727
higher antitumor activity, which also corresponds to the
AA results. The RSA of the different extracts was directly
correlated to the total amount of phenols and flavonoids,
but there was no correlation relationship between RSA
and antitumor activity (data not shown). These results
suggest that the inhibition of tumor cell proliferation
in vitro by the methanol leaf extracts of carob cannot
be solely explained by the concentration of phenol/fla-
vonoid compounds. Other phytochemicals may play a
significant role in the antiproliferative activity.
This is the first time that the carob tree has been
investigated for antitumor activity against human can-
cer cells. Taken together with results for the phenolic
profile and antioxidant activities, our results suggest
that the methanol leaf extracts possess exploitable anti-
oxidants, and that they could be a source of phenolic
compounds with potential antitumor activity. Our find-
ings therefore serve as a prelude to in vitro and in vivo
studies that may lead to verification of the efficacy of the
antitumor activities of carob leaf extracts against vari-
ous human cancers, elucidation of the mechanisms of
their cytotoxic activity, and identification of the active
components.
Declaration of interest: This work was partially sup-
ported by CICYT (CTQ2006-03794/BQU), Instituto
de Salud Carlos III (CB06_01_0074 and PI060624),
the Generalitat de Catalunya (2005SGR 00662),
the Institute for Research in Biomedicine, and the
Barcelona Science Park. One of the authors (L.C.)
thanks the Portuguese Foundation for Science and
Technology (FCT) for a post-doctoral grant (grant
SFRH/BPD/20736/2004).
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