PHARMACEUTICAL BIOLOGY, 2016

VOL. 54, NO. 5, 747–751

http://dx.doi.org/10.3109/13880209.2015.1079222

ORIGINAL ARTICLE

Evaluation of Pistacia lentiscus seed oil and phenolic compounds for in vitro
antiproliferative effects against BHK21 cells
Faten Mezni1, Sarra Shili2, Nejia Ben Ali2, Mohamed Larbi Khouja1, Abdelhamid Khaldi1,
and Abderrazak Maaroufi2
1
National Institute for Research in Rural Engineering, Water and Forests, INRGREF, Ariana, Tunisia and 2Laboratory of Epidemiology and
Microbiology, Bacteriology and Biotechnology Development Group, Pasteur Institute of Tunisia (IPT), Tunis, Tunisia

ABSTRACT ARTICLE HISTORY

Context: Within the global context of increasing cancer diseases, natural products are important in Received 2 December 2014
devising new drugs and providing unique ideas in cancer therapy. In Tunisian folk medicine, Revised 30 May 2015
Pistacia lentiscus L. (Anacardiaceae) fixed oil is used for cancer treatment. Accepted 29 July 2015
Objective: This investigation studied, for the first time, the antiproliferative effect of Pistacia lentiscus Published online 5 October
fixed oil and its phenolic extract on BHK21 cancer cells. 2015
Materials and methods: Oil was extracted from fruits harvested in northwest Tunisia and the KEYWORDS
phenolic fraction was obtained by mixing with methanol. The anti-proliferative activity of the two Fixed oil, immortal cells,
tested substances on BHK 21 cells were investigated in vitro using trypan blue assays. Cells were mastic tree, phenols, viability
treated with different concentrations of P. lentiscus oil (0.009, 0.018, 0.036, and 0.09 g/mL) and the
phenolic extract (0.007, 0.014, 0.03, and 0.07 g/mL) for 24, 48, and 72 h.
Results: The inhibitory effect of Pistacia lentiscus fixed oil increases with the increase in dose. The
IC50 value was estimated at 0.029 g/mL. The percentage of cell viability was 42.46 ± 3.4% at a dose
of 0.09 g/mL and was significantly lower than that of the untreated control (96.24 ± 2.5%, p50.01).
The phenolic extract demonstrated a dose- and time-dependent inhibitory effect on BHK21 cell
growth. After 48 h of incubation, the IC50 value was estimated at 0.15 g/mL.
Discussion and conclusion: The results demonstrated the potential of Pistacia lentiscus fixed oil in
treating cancer, as it is used in traditional medicine.

Introduction properties. Extract of different parts of the plant show

Cancer is an often deadly disease that affects a consid-
erable number of people worldwide. Despite numerous
recent advances in the cancer research, this disease
remains a leading cause of death in developed countries.
Throughout the world, ongoing research seeks effect-
ive treatments for cancer, including using plants to
relieve and treat patients. The treatment used here makes
use of compounds naturally present in plants known to
inhibit or kill carcinogenic cells.
Natural products hold great importance in devising
new drugs and providing unique ideas in cancer therapy.
Currently, some herbs and spices have already been
approved for their anticancer activity. For example,
extracts from Origanum vulgare L. (Lamiaceae) and
Rosmarinus officinalis L. (Lamiaceae) have been reported
to be a factor linked to a lower incidence of some cancers
(Cheung & Tai, 2007; Grbović et al., 2013).
Many other herbs have long been used for various
health benefits. Pistacia lentiscus L. (Anacardiaceae) is
one of the plants used most often for its medicinal
various activities such as anti-inflammatory, antioxidant,
antimicrobial, and anticancer. Several studies have
reported that P. lentiscus mastic gum inhibited prolifer-
ation of different cancer cells in vitro. Balan et al. (2007)
reported that ethanolic extracts of mastic gum of this
plant inhibited proliferation and induced death in
human colon cancer cells. Dimas et al. (2009) reported
that hexane extracts of mastic gum are used in the
treatment of colorectal tumors. Mastic oil was also used
to treat prostate cancer (He et al., 2006).
In Tunisia, the fixed oil extracted from P. lentiscus
fruits is used in traditional medicine for healing wounds,
alleviating stomach problems, and treating cancer
(Mezghani, 1992).
To the best of our knowledge, there has been no
research that has examined the anticancer effect of
this oil.
This investigation studied, for the first time, the
antiproliferative activity of Pistacia lentiscus seed oil and
its methanolic extract on BHK 21 cancer cells.

Correspondence: Faten Mezni, National Institute for Research in Rural Engineering, Water and Forests, INRGREF, BP 10, Ariana 2080, Tunisia.
Tel: +21671230039. faten-mez@hotmail.com
ß 2015 Taylor & Francis
748 F. MEZNI ET AL.
Materials and methods
Plant material
Pistacia lentiscus fruits were harvested in November 2012
from plants growing wild in Feija region in northwest of
Tunisia (36 300 18.2800 N, 8 150 13.8700 E). The plant was
identified by Dr A. Khaldi from I.N.R.G.R.E.F-Tunisia
and certified specimens (VS1-PL2009) were deposited at
the Herbarium run by I.N.R.G.R.E.F.
Oil extraction
The fruits were ground to a paste using an ordinary
grinder. A small quantity of cold water (about 250 mL/kg
of paste) was added to the paste and mixed for 30 min in
a hot water bath (60  C). The mixed paste was then
placed in a hydraulic press to separate the liquid phase
from the meal and finally put in a centrifuge to recover
floating oil.
Phenolic extract preparation
The phenolic compounds were extracted from the
P. lentiscus oil according to the method described by
Cert et al. (2007). The oil (2 g) was weighed and mixed
with 5 mL of methanol/water (80/20; v/v) for 1 min in a
vortex apparatus. The mixture was then isolated in an
ultrasonic bath for 15 min at room temperature and
centrifuged at 5000 rpm for 25 min. The methanolic
phase was removed and stored in a dark cool
environment.
Cell culture conditions
Baby hamster kidney cells (BHK-21) were purchased
from the Pasteur Institute, Ariana, Tunisia. Cells were
regrown from stock which had been frozen about 20
generations from the original cloning. The cells were
grown in Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 10% tryptose phosphate broth, 10%
fetal bovine serum (FBS), and penicillin and strepto-
mycin (100 units/mg) in an incubator at 37 C with 5%
CO2. They grew rapidly in monolayer, with a mean
doubling time in the exponential phase of about 12 h,
and were subcultured at 2-d intervals.
The phenolic extract was evaporated and diluted with
saline solution so that the final methanol concentration
in the dishes was less than 0.1%. Cells (106cells/well)
were seeded in the supplemented medium (5% fetal
bovine serum) containing different concentrations of
P. lentiscus oil (0.009, 0.018, 0.036, and 0.09 g/mL) and
the phenolic extract (0.007, 0.014, 0.03, and 0.07 g/mL)
and were incubated in a humidified atmosphere with 5%
CO2 at 37  C for 24, 48, and 72 h. Control cultures were
handled in the same manner as the treated cultures.
Cell viability assay
The anti-proliferative potential of the fixed oil and its
phenolic extract was evaluated using the trypan blue
exclusion method. The cell suspension was mixed with a
0.4% trypan blue solution. The mixture (10 mL) was then
applied to the edge of the hemocytometer counting
chamber. This method depended on the ability of
Trypan blue dye to stain the dead cells blue thus
making them easier to count on a hemocytometer slide
(under a microscope).
Cell counts in the samples treated with the test
compounds were normalized to a percentage of the
control, and the means and standard errors (SEM) were
calculated from three independent experiments. The IC50
value of the anti-proliferative effects was determined by
estimating linear regression.
Statistical analysis
Data were analyzed using the GLM procedure (General
Linear Models) of the SAS (9.0) program (SAS Inc.,
Cary, NC). An analysis of variance was performed on the
studied parameters and significant correlations were
retained.
Results
The results of this investigation showed that both
P. lentiscus fixed oil and its phenolic extract inhibited
proliferation of BHK 21 cells. Incubation of BHK 21 cells
with P. lentiscus fixed oil and its phenolic extract for 24 h
was demonstrated to reduce viability of the cells at all
concentrations levels. Dead cells increased with an
increase in concentration of both tested substances.
Cell proliferation was significantly lower (p 5 0.01) than
that of untreated control cells. After 24 h of incubation,
the highest concentration killed more than 50% of cells
for the fixed oil (0.09 g/mL) and more than 20% for the
phenolic extract (0.069 g/mL).
In the presence of the fixed oil, no incubation time
effect was observed; the effect was pronounced 24 h after
supplementation (Figure 1A). For the phenolic extract,
both the dose and the time of incubation affected the
inhibitory effect and the highest inhibitory effect was
observed after 48 h of incubation (Figure 1B). Cell
viability rates at doses of 0.007, 0.014, 0.03, and
0.07 g/mL were 71.27 ± 0.6%, 76.18 ± 1.2%, 85.5 ± 1.5%,
and 86.64 ± 1.0%, respectively.
 ANTIPROLIFERATIVE EFFECT OF PISTACIA LENTISCUS EXTRACTS 749

Figure 1. Effects of Pistacia lentiscus fixed oil (A) and its phenolic compounds (B) on the proliferation of BHK21 cells; (C) untreated
control cells. The data are mean ± SD of three independent experiments.

Figure 2. The antiproliferative effect of Pistacia lentiscus fixed oil (A) and its methanolic extract (B) on BHK21 cells, 24, 48, and 72 h
after exposure. The effect was measured by Trypan blue cell viability assay. The data are mean ± SD of three independent
experiments.

From the graphs of the P. lentiscus seed oil
(Figure 2A), the lowest IC50 values were determined to
be for 24 h of incubation, and from those of its phenolic
extract (Figure 2B), for 48 h of incubation.
Thus, P. lentiscus seed oil showed potent anti-prolif-
erative effects with the IC50 value of 0.029 g/mL whereas
the phenolic extract produced an IC50 value of 0.15 g/mL
in BHK21 cells. These IC50 values indicated that the anti-
proliferative activity of P. lentiscus seed oil on BHK21
cells was higher than that of the phenolic extract.
Compared with the fixed oil, the methanolic extract
showed a lower antiproliferative effect.
Discussion
The antiproliferative effect of P. lentiscus fixed oil against
BHK21 cells is probably related to its fatty acid
composition. A few studies on the biochemical compos-
ition of this oil have shown that it contains high amounts
of oleic, palmitic, and linoleic acids (Mezni et al., 2012;
Tej Yaakoubi & Dhaou, 2007; Trabelsi et al., 2011).
Several studies have also shown that fatty acids inhibit
cancer cell proliferation. Oleic acid, which is the major
component of this oil, is known for its significant anti-
proliferative activity. Lior et al. (2003) demonstrated that
it causes cell apoptosis in some cancer cell lines.
Furthermore, Pierre et al. (2013) showed the significant
anticancer effect of linoleic acid. These findings may
explain the reduced cell viability when supplemented
with P. lentiscus fixed oil. Other studies suggest that this
fatty acid has a positive effect on the proliferation and
differentiation of cancer cells in relation to the model of
used cell lines (Reddy et al., 1991; Singh et al., 1997).
This anti-proliferative effect against BHK21 cells
could also be explained by the presence of antioxidants,
such as tocopherols. Dhifi et al. (2013) studied the
biochemical composition of P. lentiscus seed oil and
showed that it contains a large amount of a-tocopherol.
This compound has been shown to induce cell death by
apoptosis (McIntyre et al., 2000). Chatelain et al. (1993)
750 F. MEZNI ET AL.
linked the anti-proliferative effect of a-tocopherol to the
inhibition of protein kinase C activity. In addition, it has
been demonstrated that combinations of some forms of
vitamin E, such as g-tocopherol and d-tocopherol,
exhibit additive or synergistic inhibitory effects (Jiang
et al., 2004).
In comparison with the fixed oil, the methanolic
extract showed a lower anti-proliferative effect. This
could be due to the anti-proliferative activity of phenols
contained in the extract.
Several studies have shown that these compounds
inhibit the proliferation of different cancer cell lines such
as prostate, colon, and breast (Bennani et al., 2007;
Janicke et al., 2011; Kampa et al., 2003). This
antiproliferative effect of phenols, which usually gener-
ates programmed death of cancer cells, depends on the
nature of the phenolic compound (Cho et al., 2013).
Yáñez et al. (2004) suggest that there is a correlation
between the structural state of oxidation and the
position, the number, and the nature of the substituent
of the phenolic compound and its anti-proliferative
effects.
Pistacia lentiscus seed oil may be more effective than
its methanolic extract because of a correlation between
the oil’s fatty acids, phenols and tocopherols, whereas the
phenolic extract only has the effect of phenols.
Conclusions
The results obtained in this study indicated that
P. lentiscus seed oil, the ingredient of Tunisian folk
medicine to treat cancer patients, was active against
BHK21 cancer cells. This study highlights the anti-
proliferative effect of this natural product; further studies
are required to examine the anti-proliferative effect of
this oil on other cancer cell lines.
Declaration of interest
The authors wish to kindly thank IRDC – Canada (105568-
006) for its financial support.
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