Cytotoxic effect of Berberis libanotica roots extracts on human cancer cells and antioxidant activities

Abstract

In the present study, we evaluated the cytotoxic effect and the antioxidant activity of methanolic and ethanolic roots extracts obtained from Berberis libanotica a Lebanese medicinal tree. Cytotoxic activity was assessed on the colon cancer HT 29, HCT 116, Caco-2 and breast cancer MCF-7 and MDA-MB-231cell lines, using the MTT viability assay. Both extracts inhibited cancer cells proliferation in a doseand time depending manner without being cytotoxic against the normal MCF-10 cell line. Our results suggest that methanolic extract could induce a caspase-independent cell death in the colon and breast tumor cells HT 29 and MCF-7, respectively. DPPH and FRAP assays showed a moderate to strong antioxidant activity of the methanolic and ethanolic extracts with EC50 values of 0.13 ± 0.001 and 0.1± 0.002 mg/ml, respectively. Collectively, these findings suggest that Berberis libanotica roots could serve as a promising source of antioxidant and anticancer bioactive compounds.

Full text

311
The Journal of Phytopharmacology 2017; 6(6): 311-317
Online at: www.phytopharmajournal.com
Research Article
ISSN 2320-480X
JPHYTO 2017; 6(6): 311-317
November- December
Received: 07-10-2017
Accepted: 11-11-2017
© 2017, All rights reserved
Kamar Hamade
Laboratoire de Valorisation des
Ressources Naturelles et Produits de
Santé (LVRNPS), Faculty of Pharmacy,
Doctoral School of Sciences and
Technology, Lebanese University, Rafic
Hariri University Campus, P.O.BOX
14/6573, Hadath, Lebanon
Louna Karam
Laboratory of Genomic and Health,
Faculty of sciences, Doctoral School of
Sciences and Technology Lebanese
University, Rafic Hariri University
Campus, P.O.BOX 5, Hadath, Lebanon
Jean Habib
Laboratoire de Valorisation des
Ressources Naturelles et Produits de
Santé (LVRNPS), Faculty of Pharmacy,
Doctoral School of Sciences and
Technology, Lebanese University, Rafic
Hariri University Campus, P.O.BOX
14/6573, Hadath, Lebanon
Raghida Abou Merhi
Laboratory of Genomic and Health,
Faculty of sciences, Doctoral School of
Sciences and Technology Lebanese
University, Rafic Hariri University
Campus, P.O.BOX 5, Hadath, Lebanon
Assem Elkak
Laboratoire de Valorisation des
Ressources Naturelles et Produits de
Santé (LVRNPS), Faculty of Pharmacy,
Doctoral School of Sciences and
Technology, Lebanese University, Rafic
Hariri University Campus, P.O.BOX
14/6573, Hadath, Lebanon
Correspondence:
Assem Elkak
Laboratoire de Valorisation des
Ressources Naturelles et Produits de
Santé (LVRNPS), Faculty of Pharmacy,
Doctoral School of Sciences and
Technology, Lebanese University, Rafic
Hariri University Campus, P.O.BOX
14/6573, Hadath, Lebanon
Email: aelkak[at]ul.edu.lb
Cytotoxic effect of Berberis libanotica roots extracts on
human cancer cells and antioxidant activities
Kamar Hamade, Louna Karam, Jean Habib, Raghida Abou Merhi, Assem Elkak*
ABSTRACT
In the present study, we evaluated the cytotoxic effect and the antioxidant activity of methanolic and ethanolic
roots extracts obtained from Berberis libanotica a Lebanese medicinal tree. Cytotoxic activity was assessed on
the colon cancer HT 29, HCT 116, Caco-2 and breast cancer MCF-7 and MDA-MB-231cell lines, using the
MTT viability assay. Both extracts inhibited cancer cells proliferation in a doseand time depending manner
without being cytotoxic against the normal MCF-10 cell line. Our results suggest that methanolic extract could
induce a caspase-independent cell death in the colon and breast tumor cells HT 29 and MCF-7, respectively.
DPPH and FRAP assays showed a moderate to strong antioxidant activity of the methanolic and ethanolic
extracts with EC50 values of 0.13 ± 0.001 and 0.1± 0.002 mg/ml, respectively. Collectively, these findings
suggest that Berberis libanotica roots could serve as a promising source of antioxidant and anticancer bioactive
compounds.
Keywords: Berberis libanotica, Methanolic and ethanolic extracts, Cytotoxic and antioxidant activities.
INTRODUCTION
Free radicals, including highly reactive oxygen species (ROS) such as superoxide (O2-) and hydrogen
peroxide (H2O2), can be both helpful and harmful to human health. When found in moderate levels, ROS
mediates a beneficial effect on a broad range of physiological processes including cell growth and
immune responses. However, excessive ROS amounts can lead to oxidative stress, a toxic process that
can damage cellular lipids, proteins and DNA, thus causing cell death [1, 2]. Oxidative stress is reported to
be strongly associated with the pathogenic processes underlying several diseases including: cancer,
Alzheimer’s, autoimmune disorders and coronary vascular diseases [3-7]. To avoid oxidative stress,
human body relies on both endogenous and exogenous antioxidant systems. Endogenous antioxidants,
synthesized by the body, include different enzymes such as Superoxide dismutase, catalase and
glutathione peroxidase [8]. Exogenous ones are usually obtained from dietary natural sources such as
plants and fruits [ 9]. Plants have long been known for their pharmacological and therapeutic potential [10].
This is, mainly, attributed to their biologically active chemical components including flavonoids and
phenolic acids that exhibit a significant antioxidant activity [11]. Interestingly, a diet rich in plants
polyphenols has been associated with efficient antioxidant protection against different diseases including
cancer [12, 13].
Lebanon is endowed with a remarkable and very rich flora. Berberis libanotica or Lebanese berberry,
belonging to the Berberidaceae family, has been widely used in the Lebanese traditional medicine [14].
Phytochemical analysis of Berberis libanotica roots extracts identified a major presence of alkaloids [15].
Berberis libanotica roots extracts have been reported to exert important biological activities. For
instance, [16] showed that the ammonia-dichloromethane Berberis libanotica roots extract can induce a
significant reduction in cell viability and inhibit the proliferation of different human prostate cancer cell
lines. In addition, a recent study [17] showed that ethanol Berberis libanotica roots extract exert a potent
anti-proliferative and pro-apoptotic effects in human erythroleukemia cells (HEL and K562 cells). Yet,
little is known about the effect of alcoholic Berberis libanotica roots extracts on the proliferation and
viability of colon and breast cancer cell lines. In the present study, we investigated the cytotoxic effect
and the antioxidant activity of two Berberis libanotica root extracts. We also checked their efficacy in
reducing the viability of different human colon and breast cancer cell lines and the molecular
mechanism(s) mediating the cytotoxic effect of the methanolic root extract.

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MATERIALS AND METHODS
Plant material
The roots of Berberis libanotica were collected from Ehden, Bsharri
(north of Lebanon at an altitude of 1400m) in September 2015.
Botanical identification and authentication were performed and
voucher sample (AMB14) were deposited in the herbarium of Faculty
of Pharmacy, Lebanese University, Beirut, Lebanon. The plant roots
were open air dried under the shade at room temperature, then
pulverized using an electric blender and stored in amber airtight
bottles till further use.
Berberis libanotica roots extracts preparation
All chemicals used were of analytical grade and were purchased from
Sigma-Aldrich (St. Louis, MO, USA). Briefly, ethanol 80% and
methanol 80% extraction methods were inspired from the protocol
described in Berberis vulgaris monograph of the French
Pharmacopoeia. Briefly, 20 g of the plant root powder was extracted
using either ethanol/water 160:40 (v/v) or methanol/water 160:40(v/v)
as extracting solvents mixtures. The mixture was macerated for 24 h
under magnetic stirring, filtered using Whatman paper, then the
supernatant was collected and evaporated under reduced pressure at a
temperature of 40°C using a rotary evaporator (Heidolph, Schwabach,
Germany), and then subjected to freeze drying. The obtained dried
extracts were kept in amber airtight bottles at room temperature until
required and reconstituted for further analysis.
Evaluation of antioxidant activity
Diphenyl -1-picrylhydrazyl (DPPH) radical scavenging activity
The antioxidant activity of the methanolic and ethanolic extracts
(80/20) of the roots of Berberis libanotica were determined via 2,2-
diphenyl-1-picrylhydrazyl (DPPH) Assay. Firstly, 5 different
concentrations of each sample were prepared (0.5, 0.4, 0.2, 0.1, 0.05
mg/mL), the stock solution of each sample was diluted in the suitable
solvent and then the other concentrations were prepared by dilution
with water. The DPPH solution was prepared at a concentration of 5.2
mg / 100 mL. In separate tubes, we mixed vigorously 1mL of each
solution and 1 mL of the DPPH solution. The tubes were then left in
the dark for 30 min at room temperature. The absorbance was
measured at 517 nm. For each sample measurement, a blank solution
containing the same solvent was used for each preparation, and was
run in parallel and measured. The DPPH solution was used as a
negative control. Ascorbic acid solutions, having the same
concentrations as the sample preparations, were used as antioxidant
molecule reference. Percent activity was determined using the
following equation:
% Activity = [1-(Asample / ABlank)] × 100
Two replicate absorbance data were recorded. The EC50 value is the
extract concentration required to obtain a 50% antioxidant effect.
Ferric Reducing Antioxidant Power (FRAP) assay
The ferric reducing power of the extracts was performed. This assay is
normally based on the blue coloration that develops due to the
reduction of ferric iron to the ferrous. A serial dilution of solutions of
aqueous methanolic extract (80%), aqueous ethanolic extract (80%)
and ascorbic acid (0.5, 0.4, 0.2, 0.1, 0.05 mg / mL), were prepared and
diluted in water. An aliquot (200 µL) of each extract solution was
mixed with 200 µL of 0.2M phosphate buffer (pH 6.6) and 200 µL of
potassium ferrocyanate [KᴈFe (CN)6](1%). The mixture was
incubated at 50ºC for 20 min. After cooling, 200 µL of 10%
trichloroacetic acid (w/v) were added and the mixture was centrifuged
at 1000 rpm for 8 min. The upper layer (800 µL) was mixed with 800
µL of distilled water and 160µL of 0.1% ferric chloride. After a 10
min reaction time, the spectrometric absorbance was recorded at 700
nm and compared with ascorbic acid as positive control (higher
absorbance readings indicate higher reducing power). The data are
reported as the average of two measurements given as ± SEM.
In vitro cytotoxicity assay
Cell culture
A panel of five human cancer cell lines: estrogen receptor–positive
(MCF-7),estrogen receptor–negative (MDA-MB-231) human breast
adenocarcinoma cells, human colorectal adenocarcinoma HT 29,
human colon carcinoma HCT 116, and
human epithelial colorectal adenocarcinoma cancer cells (Caco-2)
were used. The MCF-10 cells were used as a non-tumorigenesis breast
epithelial cell line model for a comparison to the cancer cell
models. For MCF-7, MDA-MB-231 and Caco-2, cells were
maintained in dulbecco’s modified eagle’s medium (DMEM,
LONZA) where HT 29 and HCT 116 cells were grown in RPMI-1640
medium (LONZA). Both media were supplemented with 10% heat-
inactivated fetal bovine serum (FBS) (Sigma-Aldrich) and 1%
penicillin/streptomycin (LONZA). MCF-10 cells were grown in a 1:1
mixture of DMEM and Ham’s F12 medium containing 5% horse
serum with 20 ng/mL human epidermal growth factor,100 ng/ mL
cholera toxin,0.01 mg/mL bovine insulin and 500 ng/mL
hydrocortisone, 95% (Sigma-Aldrich). Cells were harvested by
trypsin-EDTA at 37 ºC, pelleted, re-suspended and grown in 75
cm2 culture flasks, under standard cell culture conditions at 37°C and
5% CO2 in a humidified incubator. The cell count was determined by
trypan blue exclusion.
Cell viability assay
Cell viability of methanolic and ethanolic Berberis libanotica roots
extracts was assessed by MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-
diphenyltetrazolium bromide)] assay from Sigma-Aldrich (St. Louis,
MO, USA).Cells were seeded in triplicates, in 100 µL complete
medium, into 96-well plates at a density of 7000 cells/well for HT 29,
HCT 116 Caco-2 and MDA-MB-231, 6500 cells/well for MCF-7 and
10 000 cells/well for the MCF-10 cell lines. After 24 h, cells were
treated with various extracts (methanol 80% and ethanol 80%) at
different concentrations (0, 5, 10, 20, 30, 50, 70 and 100 μg/mL) for
24, 48 and 72 h. MTT reagent was added to each well and incubated
for 3h at 37 ºC. Finally, the reaction was stopped by adding 100 µL of
SDS 10% with 0.01 N HCl into each well. The reduced MTT optical
density (OD) was measured by ELISA reader at 595 nm. The
percentage of cell viability was presented as an OD ratio between the
treated and untreated cells, at the indicated concentrations. Data are
expressed as IC50 ± SD obtained from triplicate determinations of
three independent experiments (n=3) for each cell line

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Statistical analysis
Values presented correspond to Mean ± SD. The IC50 and EC50
values were determined appropriately with graphPad prism software
(San Diego, USA).
Immunoblotting
HT 29 and MCF-7 cells were seeded in 6-well plates and treated for
48 h with various concentrations of methanol 80% extract at around
IC50. Treated cells were washed with PBS 1X and lysed by Laemmli
buffer (62.5 mMTris-HCl pH 6.8, 2% SDS, 10% glycerol, 100 mM
dithiothreitol, 0.01% bromophenol blue). Protein lysates were
centrifuged at 14000 rpm for 5 min at 4°C, to remove debris. Proteins
quantification was performed using lowry assay. Protein extracts (60
μg for each sample) were prepared, denatured and separated (80V,
120 min) by SDS-polyacrylamide gels electrophoresis and electro
transferred to nitrocellulose membranes (100 V, 120 min).
Membranes were blocked for 1h with 5% skimmed milk in Tris-
buffered saline/0.05% Tween-20 and incubated overnight under
shaking at 4˚C with primary antibody: PARP (ab6079), p53(ab26),
Bax(ab32503), Bcl-2(ab7973) and GAPDH (ab8245). After washing
twice, the membranes were probed with appropriate secondary
antibodies anti-rabbit (ab6721) and anti-mouse (cell signaling, ref
02/2015) conjugated to horseradish peroxidase. GAPDH was used as
loading control to ensure equal loading of proteins for each sample.
The signal was detected using an enhanced chemiluminescence
system ECL (Luminata Crescendo Western HRP substrate
(Millipore). Images were captured using an X-RAY film (MMXBE
Film, Care Stream, USA).
RESULTS AND DISCUSSION
Extraction yields of Berberis libanotica root extracts
The choice of the extraction procedure depends primarily on the class
of targeted compounds to be isolated as well as on
the type of solvents used with varying polarities [18]. The extraction
yields of aqueous methanolic and ethanolic roots extracts were
determined to be 5 and 5.6%, respectively (Table1).
Table 1: Extraction mass and yield of extracts obtained from Berberis
libanotica roots extracts.
Extract-Solvent
Initial mass used
(g)
Mass after extraction
(g)
Yield
(%)
Methanolic extract
20.0
0.92
5.0
Ethanolic extract
20.0
1.12
5.0
Antioxidant activity of Berberis libanotica root extracts
An antioxidant is defined as a substance that inhibits the oxidation
process, a reaction that can produce harmful free radicals [19]. Both
DPPH radical scavenging activity and the ferric reducing power
(FRAP) assays were carried out to evaluate the antioxidant activities
of methanolic and ethanolic Berberis libanotica roots extracts.
Ascorbic acid, a pure antioxidant compound, was used as reference
compound and showed the highest antioxidant activity. Interestingly,
both extracts exhibited substantial DPPH scavenging capacity after 30
min of the incubation period. This activity increased further upon
augmenting the extracts concentrations where they exhibited 90% of
activity at a concentration of 0.5mg/mL (Fig. 1). Remarkably, no
significant difference in the antioxidant activity displayed by each of
the two extracts in comparison to that of ascorbic acid was noted at
concentration of 0.5 mg/mL. The EC50 values are provided in Table 2.
Table 2: DPPH antioxidant capacities of the Berberis libanotica roots
extracts.
Samples
DPPH scavenging capacity EC 50 value (mg/ml)
Methanolic extract (80%)
0.1066 ± 0.001
Ethanolic extract (80%)
0.1309 ± 0.002
The Ec50 value is defined as the inhibitory concentration of the roots extracts necessary to
decrease the initial
DPPH radicals concentration by 50%. The EC50 values were obtained by interpolation
from linear regression. Analysis. Each EC50 value is determine as mean ± standard
deviation (n=3).
Figure 1: DPPH free radical scavenging activity of ethanol and methanol
Berberis libanotica roots extracts. Ascorbic acid (Vitamin C) was used as a
positive control. Each value is the average ±SD of two separate experiments
each done in duplicate (n=2). AA = ascorbic acid.
The FRAP assay was applied on the basis of evaluating the capacity
of both extracts to reduce ferric tripyridyltriazine (Fe +++) to ferrous
tripyridyltriazine (Fe++). The reducing power of the methanolic and
ethanolic extracts is shown in figure 2. Apparently, both extracts
showed a moderate reducing activity in comparison to that displayed
by the ascorbic acid. This activity increased in a manner dependent on
the extract concentration where it reached about half that of ascorbic
acid when the used concentration was augmented to 0.5mg/mL (Fig.
2).
Figure 2: Ferric reducing power of ethanol and methanol Berberis libanotica
roots extracts at various concentrations. Ascorbic acid (Vitamin C) was used as
a standard control. Each value is the average ± SD of two separate experiments
each done in duplicate (n=2). AA = ascorbic acid.
0
20
40
60
80
100
0.05 0.1 0.2 0.4 0.5
Scavenging effect on DPPH
radical (%)
Concentration (mg/ml)
MethOH
EthOH
AA
0
0.2
0.4
0.6
0.8
1
1.2
0.05 0.1 0.2 0.4 0.5
Absorbance at 700 nm
Concentration (mg/ml)
MethOH
EthOH
AA

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314
Generally, Berberis libanotica extracts have been reported to protect
against oxidative stress [20]. This substantial antioxidant potential may
be attributed to its chemical composition. Here, two different solvents
with different polarities were used, and probably extracted different
classes of chemical compounds. Polyphenolic compounds divided into
several classes, may react with free radicals in different ways,
depending on their chemical structure [21]. This could underline the
different behaviors observed for the same extraction a manner
depending on the antioxidant evaluation methodology.
Cytotoxic activity of Berberis libanotica root extracts
To assess the cytotoxic capacity of Berberis libanotica roots extracts,
MTT assay was carried out. Both extracts negatively affected the
proliferation of three colon cancer cell lines (HCT 116, HT 29, and
Caco 2) and two breast cancer cell lines MCF-7 and MDA-MB-231 in
a dose- and time-dependent manner (Fig. 3 and Fig. 4).
Methanolic extract exhibited the highest cytotoxic activity against HT
29 with an IC50=26.09μg/mL at 48 hours. It also displayed a
remarkable cytotoxic effect, after 48 hours, towards HCT 116, Caco 2
and MCF-7 with IC50 values of 35.08, 32.33 and 32.79 μg/mL,
respectively. On the other hand, methanolic extract was less efficient
in inhibiting MDA-MB-231cell proliferation as it exhibited higher
IC50 values (IC50= 61.48 ± 0.69 μg/mL) (Fig. 3 and Table 3).
Table 3: The growth inhibitory effects of the methanol and ethanol Berberis libanotica extracts against cancer selected cell lines expressed as
IC50 values (μg/mL).
Cancer cell line
MCF-7
MDA-MB-231
HCT 116
HT 29
Caco-2
IC 50a(µg/ml)
Methanolic extract
32.79 ± 0.96
1.48 ± 0.6
35.08 ± 0.9
26.09 ± 0.7
32.33 ± 0.8
Ethanolicextract
79.97 ± 0.93
2.61 ± 0.93
34.16 ± 0.7
48.94 ± 0.94
76.66 ± 0.6
a Each IC 50 value is determine as mean ± standard deviation(n=3)
Figure 3: Methanol roots extracts of Berberis libanotica treatment induced a time and dose dependent inhibition of proliferation in HCT 116, HT 29, Caco-2,
MCF-7 and MDA-MB-231 cancer cell lines, with a low cytotoxic activity on the normal breast cancer cell MCF-10. Cells were treated at 60% confluency with
different concentrations (5, 10, 20, 30, 50, 70 and 100 μg /mL) for 24, 48 and 72 h. Cell proliferation was assessed by MTT assay as described in materials and
methods. Results are expressed as percentage of control non treated cells. Each value is the average ± SD of three separate experiments each done in triplicates
(n=3).
0
20
40
60
80
100
Day 1 Day 2 Day 3
proliferation (% control)
MCF-7
0
20
40
60
80
100
Day 1 Day 2 Day 3
proliferation (% control)
HCT 116
CT
5ug/ml
10ug/ml
20ug/ml
30ug/ml
50ug/ml
70ug/ml
100ug/ml
0
20
40
60
80
100
Day 1 Day 2 Day 3
proliferation(% control)
MDA-MB-231
0
20
40
60
80
100
DAY 1 DAY 2 DAY 3
proliferation (% control)
HT 29
CT
5ug/ml
10ug/ml
20ug/ml
30ug/ml
50ug/ml
70ug/ml
100ug/ml
0
20
40
60
80
100
Day 1 Day 2 Day 3
proliferation(% control)
MCF-10
0
20
40
60
80
100
Day 1 Day 2 Day 3
proliferation (% control)
Caco-2
CT
5ug/ml
10ug/ml
20ug/ml
30ug/ml
50ug/ml
70ug/ml
100ug/ml

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The ethanolic extract also exerted a time and dose-dependent
inhibitory effect. The different tested cell lines exhibited differential
sensitivities to the ethanolic extract with MCF-7 and Caco-2 being the
least sensitive with IC50 values of 76.66 and 79.97 μg/mL,
respectively, followed byHT 29 (IC50=48.9μg/mL), HCT 116
(IC50=34.16μg/mL) and finallyMDA-MB-231 (IC50=32.61μg/mL).
It is noteworthy that the highest sensitivities were detected following
48 hours of ethanolic extract addition (Fig. 4).
Figure 4: Ethanol roots extracts of Berberis libanotica treatment induced a time and dose dependent inhibition of proliferation in HCT 116, HT 29, Caco-2, MCF-
7 and MDA-MB-231 cancer cell lines, with a low cytotoxic activity on the normal breast cancer cell MCF-10. Cells were treated at 60% confluency with different
concentrations (5, 10, 20, 30, 50, 70 and 100 μg /mL) for 24, 48 and 72 h. Cell proliferation was assessed by MTT assay as described in materials and methods.
Results are expressed as percentage of control non treated cells. Each value is the average ±SD of three separate experiments each done in triplicates (n=3)
Clearly, active compounds in both methanol and ethanol extracts from
Berberis libanotica root exhibit cytotoxic activity with variable
efficacy against colon and breast cancer cell lines. It is noteworthy
that only HCT 116 cell line showed similar sensitivity to both extract.
For a plant extract to act as successful anti-cancer drug, it should kill
cancer cells without causing excessive damage to normal cells.
Accordingly, we assessed the cytotoxic effect of Berberis libanotica
roots extracts on the non-tumorigenic breast epithelial cell line, MCF-
10. Interestingly, MCF-10 showed higher resistance to Berberis
libanotica root extracts compared to the cancer cell lines. In fact,
treating MCF-10 cells, for 72 hours, with a concentration up to 100
µg/mL, of either extract, reduced the growth rate by less than 30%
(Fig. 3 and Fig. 4). These observations indicate that both extracts were
more selective for cancer than normal cells.
To investigate the molecular mechanism(s) mediating the antitumor
activity of Berberis libanotica root extracts, we assessed, using
Western blot analysis, the protein levels of several apoptosis-related
components in HT 29 and MCF-7 being treated, for 48 hours, with
methanolic extract (Fig. 5). Interestingly, we observed an up-regulated
expression of tumor-suppressor protein p53 in both cell lines at the
indicated concentrations (Fig. 5). We also detected up-regulated levels
of the pro-apoptotic protein Bax but down-regulated expression of the
anti-apoptotic protein Bcl-2 after 48 hours (Fig. 5).It is noteworthy
that this effect was more pronounced in the HT 29 than MCF-7 cells
when treated with at low extract concentrations.
0
20
40
60
80
100
Day1 Day 2 Day 3
proliferation (% control)
MCF-7
0
20
40
60
80
100
Day 1 Day 2 Day 3
proliferation (% control)
HCT 116
CT
5ug/ml
10ug/ml
20ug/ml
30ug/ml
50ug/ml
70ug/ml
100ug/ml
0
20
40
60
80
100
Day 1 Day 2 Day 3
proliferation (% control)
MDA-MB-231
0
20
40
60
80
100
Day 1 Day 2 Day 3
proliferation (% control)
HT 29
CT
5ug/ml
10ug/ml
20ug/ml
30ug/ml
50ug/ml
70ug/ml
100ug/ml
0
20
40
60
80
100
Day 1 Day 2 Day 3
prolifration (% control)
MCF-10
0
20
40
60
80
100
Day 1 Day 2 Day 3
proliferation (% control)
Caco-2
CT
5ug/ml
10ug/ml
20ug/ml
30ug/ml
50ug/ml
70ug/ml
100ug/ml

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Figure 5: Expression of apoptosis related proteins in HT 29 and MCF-7
treated with increasing concentrations of methanolic 80% Berberis libanotica
roots extract. Cells were cultured and treated with 20, 30 and 50 μg/mLfor HT
29 and 20, 50 and 70μg/mL for MCF-7, compared to an untreated control for
48 h. Proteins expression were analyzed by western blotting. GAPDH was
used as a loading control.
We also analyzed poly-ADP-ribose-polymerase (PARP) cleavage.
Our results showed that methanolic Berberis libanotica root extract
did not stimulate cleavage of PARP in neither HT29 or MCF-7 cells
(Fig. 5).Activation of p53 induces the transcription of pro-apoptotic
genes such as Bax and represses the transcription of a number of anti-
apoptotic genes such as Bcl-2. In most cases, the activation of p53
proceeds through mitochondrial release of respiratory chain
component, cytochrome c, from the intermembrane space of the
mitochondria into the cytoplasm, where cytochrome c activates a
cascade of caspases [22, 23]. Moreover, poly-ADP-ribose polymerase
(PARP) is a substrate for caspases and it is typically cleaved and
inactivated during apoptosis, causing DNA fragmentation [24].
Apoptosis can also occur via alternative processes not
involving caspases [25]. For instance, AIF (Apoptosis inducing factor)
can be cleaved by activated poly (ADP-ribose) polymerase (PARP),
and yields truncated AIF (tAIF) which translocates from the
mitochondria to the nucleus where it leads to chromatin condensation
and DNA fragmentation [26, 27] Our findings suggest that the Berberis
libanotica methanolic extract induces non classical features of
apoptosis in HT 29 and MCF-7 cell lines due to the intact PARP.
These observations suggest that methanolic Berberis libanotica root
extracts might induce caspase-independent cell death in the colon and
breast tumor cells HT 29 and MCF-7, respectively.
CONCLUSSION
In conclusion, our results revealed that the methanolic and ethanolic
extracts of Berberis libanotica roots exhibit considerable antioxidant
activity. Moreover, both extracts showed a significant cytotoxic
capacity against cancer (colon and breast) cell lines without harming
normal cells. In addition, this study suggests that methanolic Berberis
libanotica root extract triggers a caspase-independent cell death in
human colon and breast tumor cells. In future work, we will assess, in
vivo, the effect of Berberis libanotica root extract on colon and breast
tumor cancer development. This, in turn would highlight new
therapeutic potential for Berberis libanotica plant especially in the
field of cancer treatment.
Acknowledgments
This research project has been done via the support of the scientific
research program of the Lebanese University. Research group:
"Valorisation des Ressources Naturelles et Produits de Santé "
(VRNPS)-EDST-UL.
The authors report no conflict of interest.
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HOW TO CITE THIS ARTICLE
Kamar H, Louna K, Jean H, Raghida AM, Assem E. Cytotoxic effect of
Berberis libanotica roots extracts on human cancer cells and antioxidant
activities J Phytopharmacol 2017; 6(6):311-317.