Biomedicine & Pharmacotherapy 90 (2017) 179–186

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Original article

Assessment of Olea europaea L. fruit extracts: Phytochemical

characterization and anticancer pathway investigation
Amina Maaleja , Zouhaier Bouallaguia , Fatma Hadricha , Hiroko Isodab,c, Sami Sayadia,*
a
Environmental Bioprocesses Laboratory, Sfax Biotechnology Center, P.O. Box 1177, Sfax 3038, Tunisia
b
Alliance for Research on North Africa (ARENA), University of Tsukuba, Tennodai 1-1-1, Tsukuba City, Ibaraki 305-8587, Japan
c
Faculty of Life and Environmental Sciences, University of Tsukuba, Tennodai 1-1-1, Tsukuba City 305-8587, Japan

A R T I C L E I N F O A B S T R A C T

Article history:
Received 19 December 2016 Olea europaea L. has been widely used as an advantageous rich source of bioactive compounds of high
Received in revised form 5 March 2017 economic value leading to its use in pharmaceutical, cosmetic, and agriculture industries. Ethanolic
Accepted 14 March 2017 extracts of olive fruits from three different cultivars (OFE) were studied for their phytochemical contents
and were investigated for antioxidant activities and anticancer potential. Major polyphenols detected in
Keywords: these extracts were tyrosol, hydroxytyrosol, oleuropein, rutin, quercetin and glucoside forms of luteolin
Olive fruit extracts and apigenin. All these compounds have shown to significantly contribute to the antioxidant activity of
Polyphenols OFE, which was evaluated by DPPH and ABTS assays. Proliferation of hepatic and colon cancer cells,
Anticancer effect
HepG2 and Caco-2, were shown to be sensitive to OFE with IC50 less than 1.6 mg/ml for all tested extracts.
Antioxidant effect
Moreover, flow cytometry analysis showed that OFE induced cell cycle arrest in the S-phase within both
HepG2 cells
Caco-2 cells HepG2 and Caco-2 cells. This has triggered a cell death mechanism as shown by DNA fragmentation,
expression of p53 and phosphorylation level of Akt and Erk proteins. Interestingly, these extracts could be
further used as a potential source of natural compounds with both antioxidant and anticancer effects.
© 2017 Elsevier Masson SAS. All rights reserved.

1. Introduction the most common primary liver cancer, is the fifth most frequent
cancer globally and the third-leading cause of cancer death. In this
The olive tree is known as one of the oldest plants cultivated in context, HepG2 cells have been often used as a good model to study
the Mediterranean countries where more than 95% of the the in vitro toxicity to the liver, since they characterized the normal
worldwide olive production is concentrated [1,2]. Indeed, The human hepatocytes [6]. On the other hand, colon adenocarcinoma
beneficial impacts of olive products on human health have been is the most common histopamthological type of colorectal cancer
the subject of increasing scientific interest [3]. These products, in [7]. Such type of cancer, causing about 700,000 deaths per year is
particular, olive oil, are rich sources of promising nutritional and the fourth leading cause of cancer death [8].
bioactive molecules, such as phenolic compounds. Thanks to their Previous studies reported the effectiveness of some dietary
potent antioxidant activity, polyphenols exhibit several biological components such as isoflavones and polyphenols as inhibitors of
activities, including cardioprotective and anti-inflammatory prop- cancer cell growth by modulating cell signaling pathways.
erties [4]. Moreover, these compounds are believed to offer a Interestingly, these components activate cell death signals and
protection against the development of some diseases, including induce apoptosis in cancer cells, resulting in the prevention of
different forms of cancer. Indeed, a positive relationship between cancer development [9,10]. On this line, MAPKs (mitogen-
the consumption of olive fruits and the reduction of cancer activated protein kinases) have received increasing attention in
proliferation has been previously reported [5]. Nonetheless, cancer prevention and therapy. In addition, Akt (Protein kinase B),
studies dealing with the effect of olive fruits in both colon and known as a serine/threonine-specific protein kinase, has been
hepatic carcinoma cells have been scarcely investigated. also considered as an attractive target for cancer treatment [11]
Unfortunately, mortality rates related to cancer incidence are since several components of the PI3K–Akt pathway were
interestingly increasing in the world. Hepatocellular carcinoma, dysregulated in a wide spectrum of human cancers [12].
Moreover, the tumor suppressor and transcription factor p53
was considered as a critical regulator of many cellular processes
* Corresponding author. including cellular response to DNA-damage, cell cycle control,
E-mail address: sami.sayadi@cbs.rnrt.tn (S. Sayadi). and apoptosis [10]. It has been demonstrated that functional p53

http://dx.doi.org/10.1016/j.biopha.2017.03.034
0753-3322/© 2017 Elsevier Masson SAS. All rights reserved.
180 A. Maalej et al. / Biomedicine & Pharmacotherapy 90 (2017) 179–186

activated the transcription of some genes such as p21 and Bax to 2.5. High-performance liquid chromatography analysis
induce the apoptotic process, thus inhibiting the growth of cancer
cells [13,14]. Chromatographic analyses were performed according to
In this study, we have focused on the investigation of the Souilem et al. [17]. The instrument consists of an Agilent series
phytochemical composition and the antioxidant potential of olive 1260 HPLC-DAD (Agilent. Waldbronn. Germany). Compounds
fruit extracts from three different cultivars, namely Jerboui (JFE), separation was carried out on a ZORBAX Eclipse XDB-C18 column
Marsaline (MFE) and Ouesleti (OuFE). Based on the phytochemi- (4.6 mm I.D.  250 mm  3.5 mm particle size). The mobile phase
cal characterization, only one extract was selected for further was made of phase A (0.1% acetic acid in water) and phase B (100%
study dealing with the evaluation of its antiproliferative and acetonitrile). The elution conditions were: flow rate set at 0.5 ml/
apoptotic effects against liver and colon cancer cells (HepG2 and min, injection volume of 10 ml and operating temperature of 40  C.
Caco-2). The running gradient was as follows: 0–22 min, 10–50% B; 22–
32 min, 50–100% B; 32–40 min, 100% B; 40–44 min, 100-10% B. Re-
2. Material and methods equilibration duration lasted 6 min. The DAD detector scanned
from 190 to 400 nm and detection was achieved at 254, 280 and
2.1. Material 330 nm. Compounds were identified according to their UV,
retention times and mass spectra recorded using an ion trap mass
Dulbecco’s modified Eagle medium (DMEM) and Fetal bovine detector MSD trap XCT.
serum (FBS) were purchased from Gibco (Life Technologies, UK).
Penicillin (10.000 IU/ml)-Streptomycin (10.000 IU/ml) solution 2.6. LC–MS/MS analysis
was purchased from eurobio (France). 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetra-zolium bromide (MTT), trypan blue, Dime- Experiments were performed with an Agilent 1100 LC system
thylsulfoxide (DMSO), 1,1-phenyl-2-picrylhydrazyl (DPPH), 2,20 - according to Bouallagui et al. [18]. Briefly, the chromatographic
azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium separation was carried out using a Zorbax 300 Å Extend-C-18
salt (ABTS), catechin, gallic acid, Follin Cicalteu, acetonitrile and Column (2.1 150 mm). The column outlet was coupled to an
hexane were obtained from Sigma-Aldrich (France). Agilent MSD Ion Trap XCT mass spectrometer supplied with an ESI
ion source. Data acquisition and mass spectrometric evaluation
2.2. Olive fruit extracts preparation were accomplished on a personal computer with data Analysis
software (Chemstation).
Olive fruits from three different olive cultivars, namely Jerboui,
Ouesleti and Marsaline, were studied. Olive trees were grown in 2.7. Antioxidant activity assay
Tunisia and fruit samples with a maturation index of 2 were
collected. Fruit samples from five different trees were harvested for The antioxidant activity of OFE was determined using DPPH and
each cultivar. First, fresh fruits (200 g) were manually milled to ABTS methods. The DPPH radical scavenging activity was deter-
obtain a homogeneous paste which was subsequently extracted mined based on the method previously described by Chang et al. [19]
twice with 350 ml of ethanol (70%) under agitation (200 rpm in an with slight modifications. Briefly, In a 96 well plate, 25 ml of OFE
orbital shaker) for overnight at room temperature. The extract was solution were mixed with 200 ml of DPPH ethanolic solution
then concentrated and washed with hexane to remove the lipid (150 mM). The reaction was incubated for 1 h at room temperature
fraction. Finally, the resulting olive fruit ethanolic extract (OFE) in the dark and the absorbance was recorded at 517 nm. For the ABTS
was freeze-dried and stored for further analyses. The extraction assay, we applied the method previously reported by Bouaziz et al.
yield was in the order of 10%. [20]. In both methods, a reference curve was prepared with Trolox
(25–800 mM) under same conditions. The antioxidant activity was
2.3. Total phenols content expressed as Trolox equivalent (TE) per gram of extract.

Total phenols were measured using the method described by 2.8. Cell lines and culture conditions
Slinkard and Singleton [15], with some modifications. Appropri-
ately diluted extract (25 ml) or standard (gallic acid in the range of Two continuous human carcinoma cell lines, HepG2 and Caco-
0–400 mg/ml) were mixed with 25 ml Folin–Ciocalteu reagent and 2, were used for the anti-proliferative effect of OFE. HepG2 cells
incubated for 6 min. Later, 100 ml of Na2CO3 (75 g/l) were added to were grown in Dulbecco’s modified Eagle’s medium supplemented
the mixture which was allowed to stand for 90 min at room with 10% (v/v) FBS and 1% PS. This culture medium was
temperature before recording the absorbance at 765 nm. Total supplemented with 1% non-essential amino acid (NEAA) when
phenols content was expressed as mg gallic acid equivalent GAE used to grow Caco-2 cells. Cultures were maintained at a 5% CO2
per gram of OFE. atmosphere.

2.4. Total flavonoïds content 2.9. Viability assessment

Total flavonoïds content was measured as previously described Cell proliferation was assessed using the MTT assay following
by Meda et al. [16] with slight modifications. In a 96-well treatment of cells (3  104 live cells/ml) with different concen-
microplate, 25 ml of sample or standard solution and 10 ml of trations of OFE (0–2000 mg/ml) for different incubation times (24,
NaNO2 (50 g/l) were mixed and incubated for 5 min. Thereafter, 48 or 72 h). Cell viability was evaluated spectrophotometrically as
15 ml of AlCl3 (10%) were added to the reaction mixture and further previously reported by Bouallagui et al. [18].
incubated for 6 min. Finally, 50 ml of NaOH (1 M) and 50 ml distilled
water were added and the absorbance was measured at 510 nm. A 2.10. Flow cytometry analysis
blank sample and a reference curve using catechin (5–160 mg/ml)
were prepared under same conditions. Total flavonoïds in OFE Cell cycle distribution and multicaspases expression were
were expressed as mg of catechin equivalent (CE) per gram of determined using a Guava PCA instrument. Staining of cells was
extract. performed using a cell cycle and a multicaspases kits from Mersk-
 A. Maalej et al. / Biomedicine & Pharmacotherapy 90 (2017) 179–186 181

Table 1
Phytochemical characterization and antioxidant activity.a

Colorimetric assay Olive fruit extracts

JFE MFE OuFE

Total polyphenols (mg GAE/g extract) 110.6 (6.3) 55.8 (3.2) 80 (1.6)
Total flavonoïds (mg CE/g extract) 75.1 (1.3) 44.0 (2.6) 49.0 (1.1)
DPPH assay (mmol TE/g extract) 512.2 (2.3) 281.1 (1.6) 356.7 (1.4)
ABTS+ assay (mmol TE/g extract) 544.7 (2.5) 368.5 (1.6) 564.8 (2.2)
a
Values are means  SD (n = 3).

Millipore following the manufacturer instructions. Experiments Prism 6.0. The significance of multiple treatments was evaluated
were done at least in triplicate and data were analyzed with a based on the One-way ANOVA using Tukey’s multiple comparisons
Cytosoft Software. test at p < 0.05.

2.11. DNA fragmentation 3. Results

After being subjected to different treatments, cells were 3.1. Phytochemical characterization of OFE
collected by centrifugation (1200 rpm for 6 min) and lysed in
Tris-HCl buffer: Tris-HCl (10 mM, pH 8); EDTA (25 mM); SDS (0.5%); The quantification of total phenols and flavonoids contents of
proteinase K (50 mg/ml); RNase (20 mg/ml), at 37  C. DNA was the OFE is illustrated in Table 1. The amounts were in the range of
extracted following the method using phenol/chloroform/isoamyl 55.8  3.2 to 110.6  6.3 mg GAE/g and 44  2.06–75.1 1.3 mg CE/g
alcohol (25:24:1) [21]. Samples were then loaded onto agarose gel of extract, respectively. Among the three studied extracts, JFE
(1.8% agarose gel). DNA bands were visualized in presence of showed the highest total phenols and flavonoids contents with
ethidium bromide using a Gel Doc Imager from Bio-rad. respective concentrations of 110.6  6.3 mg GAE/g and
75.1 1.3 mg CE/g of extract.
2.12. Western blotting experiments The identification of phenolic compounds in OFE is given in
Table 2. All OFE showed globally a similar composition,
Cells were grown in 100 cm2 Petri dishes, washed twice with including phenolic compounds such as rutin, luteolin glucoside,
PBS and proteins were extracted using RIPA lysis buffer. Protein apigenin-7-glucoside, luteolin and oleuropein. Oleuropein was
quantification was performed according to the Bradford method. found to be one of the major phenolic compounds with a
Levels of target proteins including P-Akt, Akt, P-Erk, Erk, p53 and concentration range of 2.82  0.14 to 9.29  0.18 mg/g of extract.
b-actin were determined by western blot analysis. 30 mg of Once again, JFE sample showed the highest concentration of
extracted proteins were separated on 10% polyacrylamide gel and oleuropein. Using LC–MS analysis, it was possible to reveal the
transferred to a nitrocellulose blotting membrane. The membranes presence of other phenolic compounds such as comselogoside
were blocked for 30 min with 5% milk in TBS- Tween buffer and (data not shown).
incubated overnight at 4  C with primary antibodies at least for
90 min. Membranes were tagged with an HRP-conjugated second- 3.2. Scavenging DPPH free radical and ABTS activities
ary antibody for 1 h at room temperature. Immunoreactive bands
were detected using enhanced chemiluminescence reagents and The antioxidant activities of OFE using the DPPH free radical
densitometric analysis was performed with the use of Image J quenching and ABTS bleaching methods are shown in Table 1. JFE
System. b-actin was used to normalize the samples loading. has the highest DPPH radical scavenging activity of 512.2  2.3 TE/g
followed by OuFE and MFE extracts with 356.7  1.4 and 281 1.6
2.13. Statistical analysis mmol TE/g, respectively. Regarding the ABTS bleaching assay, JFE
and OuFE samples have similar ABTS activities with 544.7  2.5 and
The experimental design was randomized with three repli- 564.8  2.2 mmol TE/g, respectively, while MFE showed the lowest
cations. The statistical analysis was performed using Graph Pad activity with 368.5  1.6 mmol TE/g of extract.

Table 2
Quantification of major phenolic compounds identified in the olive fruit extracts.a

Compounds (mg/g of extract) RT (min) Olive fruit extracts

JFE MFE OuFE

Gallic acid 6.79 0.27 (0.02) 0.19 (0.02) 0.18 (0.008)
Hydroxytyrosol 10.04 2.08 (0.49) 1.09 (0.13) 1.09 (0.01)
Tyrosol 12.86 1.14 (0.22) 1.06 (0.03) 1.24 (0.17)
Caffeic acid 13.71 0.12 (0.06) – 0.08 (0.001)
Rutin 14.79 1.9 (0.12) 2.5 (0.28) 3.34 (0.21)
Verbascoside 15.29 5.7 (0.12) 0.11 (0.02) 0.33 (0.06)
p-Coumaric 16.31 0.42 (0.11) 0.03 (0.001) 0.1 (0.01)
Luteolin-7-Glucoside 15.76 5.5 (0.42) 1.31 (0.12) 0.35 (0.05)
Apigenin-7-Glucoside 17.46 2.76 (0.27) 1.06 (0.02) 0.42 (0.01)
Ferrulic acid 17.47 0.4 (0.03) 0.17 (0.02) 0.2 (0.01)
Oleuropein 18.93 9.29 (0.18) 2.82 (0.14) 7.19 (0.19)
Luteolin 22.21 0.45 (0.12) 0.4 (0.01) 0.41 (0.03)
Quercetin 22.58 1.2 (0.14) – 1.34 (0.08)
Apigenin 24.96 0.5 (0.23) – –
a
RT: Retention time; Values are means  SD (n = 3 independent analyses).
182 A. Maalej et al. / Biomedicine & Pharmacotherapy 90 (2017) 179–186

Fig. 1. Anti-proliferative effects of olive fruit extracts. Anti-proliferative effects of the olive fruit extracts from three different cultivars, namely Jerboui (JFE), Marsaline (MFE)
and Ouesleti (OuFE), on HepG2 and Caco-2 cells were tested for 24, 48 and 72 h. The data are expressed as percentages of control from three independent experiments.

3.3. Anti-proliferative activity applied for multicaspases expression (Fig. 2A and C). Results
showed a significant dose-dependent increase in apoptotic HepG2
The evaluation of cell proliferation after treatment with and Caco-2 cells following treatment with JFE. The differences
different concentrations of JFE, MFE and OuFE, indicated that all between the control and treated cells were statistically significant
extracts could inhibit the growth of both HepG2 and Caco-2 cells within all tested extract concentrations except for 600 mg/ml and
(Fig. 1). In fact, all studied OFE exerted their anti-proliferative effect 200 mg/ml treatments in HepG2 and Caco-2 cell lines, respectively.
in a time and dose-dependent manner. Furthermore, we deter- Indeed, total apoptotic HepG2 cells increased from 15.96  0.81 in
mined under the same experimental conditions (48 h of treatment) control cells to 72.46  4.5% after treatment with 1400 mg/ml of
the following IC50 values: 1000, 1800 and 1600 mg/ml for JFE, MFE JFE. Similarly, in Caco-2 cell line, total apoptotic cells increased
and OuFE, respectively with HepG2 cells, and 600, 800 and from 7.5  0.5 to 38.35  3.41% in response to 1000 mg/ml of JFE.
1000 mg/ml for JFE, MFE and OuFE, respectively with Caco-2 cells. Moreover, in all tested concentrations, late apoptotic cells showed
Based on these data, JFE was chosen to further deepen our the highest percentage compared to mid-apoptotic and dead cells
understanding of the in vitro effect of olive fruit extract. Therefore, with a more pronouncing effect on Caco-2 cells compared to
three concentrations were tested 600, 1000 and 1400 mg/ml for HepG2 cells. HepG2 and Caco-2 late apoptotic cells were
HepG2 cells, and 200, 600 and 1000 mg/ml for Caco-2 cells. approximately 1.8 times and 2.7 times greater than control cells,
when treated with 1000 and 600 mg/ml of JFE, respectively.
3.4. JFE induced cell cycle arrest in HepG2 and Caco-2 cells DNA fragmentation was also assessed as a marker of apoptosis
(Fig. 2). As compared to control cells where no DNA fragmentation
The mechanism of JFE-mediated cell proliferation inhibition
was examined by investigating the effect on cell-cycle distribution Table 3
using flow cytometry (Table 3). A significant dose-dependent Cell cycle distribution of HepG2 and Caco-2 cells treated with JFE at different
accumulation of cells was observed in the S phase, which increased concentrations for 48 h.a

from 28.2  1.95 to 29.6  2.6, 35.8  2.05 and 42  2.25% for JFE (mg/ml) G0/G1 S G2/M
HepG2 cells treated with 600, 1000 and 1400 mg/ml of JFE,
HepG2 cell cycle distribution (%)
respectively. A similar statistically significant trend was observed 0 (control) 62.2 28.2 (1.95) 7
for Caco-2 cells where the cell population in S phase increased 600 60.2 29.6 (2.6) 6
from 30.9  2.6 to 50.9  1.6% for cells treated with 600 mg/ml of 1000 52.9 35.8 (2.05)* 7.3
JFE. Furthermore, no obvious change was detected in the G2/M 1400 44.7 42 (2.25)* 7.3
Caco-2 cell cycle distribution (%)
phase for both cell lines when treated with JFE. 0 (control) 55.6 30.9 (2.6) 13.5
200 47.7 38.5 (2.2)* 13.8
3.5. JFE induced apoptosis in HepG2 and Caco-2 cells 600 32.4 50.9 (1.6)* 14.7
1000 30.3 53.8 (2.3)* 12.8
To assess whether or not cell cycle arrest induced by JFE was *
p < 0.05 compared to control.
associated with an apoptotic c cell death, flow cytometry was a
Results are the means of three repetitions.
 A. Maalej et al. / Biomedicine & Pharmacotherapy 90 (2017) 179–186 183

Fig. 2. Apoptosis detection by flow cytometry and DNA fragmentation. A and C: cells were analyzed by flow cytometry after 48 h exposure to different concentrations of olive
fruit extract from Jerboui cultivar (JFE). Data are presented as cell population (%) in the different apoptotic stages. B and D: effect of JFE on DNA fragmentation in HepG2 and
Caco-2 cells, respectively. Lanes 1 and 4: untreated cells; lanes 2 and 3: HepG2 cells treated with 1000 and 1400 mg/ml of JFE, respectively; lanes 5 and 6: Caco-2 cells treated
with 600 and 1000 mg/ml of JFE, respectively and lanes M: DNA molecular size marker. Results are representatives of three independent experiments. * P < 0.01 compared to
the untreated cells.

was detected, HepG2 and Caco-2 cells treated with JFE demon- considered to be regulated by p53 proteinwhich is inactive in normal
strated a relevant DNA fragmentation. This was noticeable in Caco- cells unless cells are exposed to stress signals [24].
2 cells after treatment with 600 mg/ml JFE and it became more Hence, HepG2 and Caco-2 cells were treated with JFE (0–
evident at 1000 mg/ml (Fig. 2D), confirming the concentration 1400 mg/ml and 0–1000 mg/ml, respectively) for 48 h, and the
dependent effect of the studied extract. However, for HepG2 cells, expression of P-Akt, Akt, P-Erk, Erk, and p53 were evaluated
except in lane 3 (Fig. 2B), no strand breaks were clearly detected. through western blot analysis (Fig. 3). The obtained results
The apoptotic mechanism triggered by JFE treatment was also suggested that JFE was able to significantly decrease the
studied through the phosphorylation level of Akt (protein kinase B) phosphorylation of Akt in both HepG2 and Caco-2 cells. However,
and Erk (extracellular signal-regulated kinases) as well as the the phosphorylation of Erk and the p53 expression were markedly
expression of p53. Indeed, Akt, one of the major signaling enzymes up-regulated in both cell lines. Changes in Akt, Erk and p53
involved in cell survival against oxidative stress, was shown to proteins expression was more pronounced in Caco-2 cells.
suppress apoptosis and promote cell survival [22]. Hence, the
influence of JFE on Akt phosphorylation was determined using an 4. Discussion
anti-phospho-Akt monoclonal antibody. Moreover, Erk belonging to
the MAPK subfamilies have been shown to be activated in response to Natural extracts with known antioxidant, antimicrobial and
oxidant injury, thus contributing to apoptosis [23]. Apoptosis is also anticancer properties are considered as products with potential to
184 A. Maalej et al. / Biomedicine & Pharmacotherapy 90 (2017) 179–186

Fig. 3. Effect of JFE on apoptotic markers expression in HepG2 and Caco-2 cells. Cells were treated for 48 h with different concentrations of JFE: 600, 1000 and 1400 mg/ml for
HepG2 cells, and 200, 600 and 1000 mg/ml for Caco-2 cells. An Equal amount of total protein from untreated and treated cells was used in western blot analysis. P-Akt, Akt, P-
Erk, Erk, and p53 were detected and quantified. Data are expressed as the mean  SD of three independent experiments and were analyzed using one-way ANOVA. ***
P  0.001, ** P  0.01, * P  0.05 compared to the untreated cells.

be used in pharmaceutical, food and cosmetic fields aiming to concentrations, papaya seed extracts lowered cell viability in
protect against diseases. In the case of cancer, much less is known HepG2 cell line. Our data are concomitant with previous studies
about the possible preventive effect of olive fruit extracts. On this demonstrating the anti-proliferative effect of olive phenolic
line, fruit extracts from Tunisian olive cultivars have attracted compounds on colon and breast cancer cell lines [31]. Furthermore,
attention thanks to their interesting biological activities including the Caco-2 cell line showed higher sensitivity to the antiprolifer-
antioxidant, antiproliferative and apoptotic activities. ative action of the three olive fruit extracts, especially with JFE. This
In this study, we demonstrated through DPPH and ABTS assays was previously shown by Chavez-Santoscoy et al. [32] who
that OFE exhibited an interesting antioxidant activity which may reported that the viability of colon cancer cells was the most
be related to their phenolic compound composition as proved by affected by the Gavia juice, among four tested cancer cell lines.
Sousa et al. [25]. The ROS scavenging effect is mainly related to the Similar data were reported by Loizzo et al. [33] who demonstrated
major phenolic compounds, in particular, oleuropein. In fact, it was that gallic acid showed the highest inhibitory effect on Caco-2 cells
demonstrated that oleuropein has antioxidant potential which has proliferation among nine tested human cancer cell lines.
been mostly related to its capacity to improve radical stability, ROS Further characterization of growth arrest induced by JFE was
scavenging effect and could, therefore, inhibit LDL oxidation carried out using flow cytometry to evaluate any potential
[26,27]. apoptotic effect. Conforti et al. [34] have mentioned that the
Several authors have used human cancer cell lines as a model effect of phenolic compounds on the cell cycle could probably
system to investigate antiproliferative activity in vitro [28]. Hereby, contribute to the tumor cell killing. Several studies demonstrated
we are demonstrating a dose and time-dependent cytotoxic effect that phenolic compounds from olive leaves could induce the cell
of the studied OFE in both HepG2 and Caco-2 cells. This might be cycle arrest at G1 phase after 48 h of treatment in MCF-7 cells [18].
attributed to the bioactive molecules content. A few studies have Others have reported that oleuropein inhibited smooth muscle
evaluated the antiproliferative effect of phenolic compounds on cells proliferation through a cell cycle arrest between the G1 and
HepG2 cancer cells [29]. Salla et al. [30] proved that at higher the S phases [35]. These findings may suggest that phenolic
 A. Maalej et al. / Biomedicine & Pharmacotherapy 90 (2017) 179–186 185

compounds inhibit the cell proliferation in a cell type-dependent Conflict of interest

manner. However, our results showed that cells treated with JFE
were remarkably accumulated in the S phase in a dose-dependent Authors declare that there are no conflicts of interest.
way. As previously reported by Chen et al. [36], the probable
explanation for cell cycle arrest in S phase could be the impact on
Funding
cyclins that regulate S-phase and/or DNA synthesis. Additionally,
the decrease in the proportion of cells in the G0/G1 phase was
This research was supported by the Ministry of Higher
detected in both HepG2 and Caco-2 cells, assuming following
Education and Scientific Research-Tunisia (LR01CBS2015) and
apoptotic phenomenon.
the JICA-JST Science and Technology Research Partnership for
Apoptosis is a programmed cell death process characterized by a
Sustainable Development (SATREPS) Project (A6A24087): “Valori-
series of biochemical and morphological changes, including ROS
zation of Bio-Resources in Semi-Arid and Arid land for Regional
generation, activation of caspases and chromosomal DNA fragmen-
Development”.
tation [37]. Caspases, cysteine aspartate-specific proteases, are the
central regulators of apoptosis. In fact, they could trigger a
programmed cell death by transducing the apoptotic signal cascade, Acknowledgements
connecting cellular targets and thus causing DNA fragmentation
[38]. In this study, analysis of the apoptosis induction potential of JFE Authors would like to thank Ms. Sirine Choura and Ms. Lobna
in tumor cells was evaluated by quantifying the proportion of Jlaiel for their assistance with LC-DAD and LC–MS/MS analyses.
apoptotic cells with multicaspases assay and indicating the DNA
fragmentation. In a previous study, an olive fruit extract was shown References
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