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Pak. J. Pharm. Sci., Vol.27, No.3, May 2014, pp.587-592 587
REPORT
Investigation of anti-cancer effects of cherry in vitro
Recai Ogur1*, Hakan Istanbulluoglu2, Ahmet Korkmaz3, Asli Barla4,
Omer Faruk Tekbas1 and Emin Oztas5
1Department of Public Health, Division of Environmental Health, Gulhane Military Medicine Faculty, Etlik, Ankara, Turkey
2Department of Public Health, Gulhane Military Medicine Faculty, Etlik, Ankara, Turkey
3Department of Physiology, Gulhane Military Medicine Faculty, Etlik, Ankara, Turkey
4Extraction Department Responsible, AROMSA Co., Istanbul, Turkey
5Department of Histology and Embriology, Gulhane Medical Faculty, Ankara, Turkey
Abstract: Cherry (Prunus Cerasus) is still one of the most popular preserve in Turkish cuisine. Cherry has been
traditionally used for the treatment of inflammatory-related symptoms. Recent researches have proved that cherry is a
valuable natural source of some important bioactive compounds in human health preservation. Evidence suggests that,
cherry consumption may decrease the risk of chronic diseases and cancer. The aim of the present study was to determine
the effects of cherry on breast cancer cells lines, asymmetric dimethylarginine (ADMA) level and certain multidrug-
resistant bacteria. The cancer cell proliferation activity and analysis of apoptotic-necrotic cells was evaluated by using
the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and scoring of apoptotic cell nuclei.
Measurement of ADMA and the minimum inhibitory concentration was accomplished by HPLC and the micro dilution
broth method. The results showed that, extracts of cherry exhibit anti-proliferative activity in mammary adenocarcinoma
(MCF-7) & mouse mammary tumor cell (4T1) breast cancer cells lines as well as induction of apoptosis, lower ADMA
concentrations in cell cultures treated with cherry extract and antibacterial effects against certain multidrug-resistant
bacteria in vitro. These findings may open new horizons for traditional anti-inflammatory product as prophylactic-
therapeutic agent from cancer, cardiovascular diseases and multidrug-resistant infections.
Keywords: Cherry, Breast Cancer Cells, Anticancer Effect, Minimum Inhibitory Concentration, ADMA.
INTRODUCTION
Cherry (Prunus cerasus) is a sour fruit strain that is like
berry from the rosaceae family. The region between the
Caspian Sea and North Anatolia is regarded as the
possible homeland of cherry in most of the resources.
Cherry especially is quite rich in terms of mineral matter.
By the reason of fruit juice’s efficiency’s (70-75%) and
total acidity’s (3%) being high cherry is very convenient
to be processed as fruit juice (Turkey Fruit Juice Industry
2011; Krishkov 2009). In Turkey the production of cherry
that is 24.000 ton in 1965 reached 85.000 ton in 1985 and
120.000 ton in 1991. In this aspect Turkey ranks as the
fifth country in cherry production. Differently from other
countries, in our country the mostly consumed fruit juices
are peach and cherry. The consumption of fruit juice that
is 0,4 liter per person in the beginnings of 1970s
increasing 10 times reached 3,9 liter per person in 1996
and today the rate is around 4,5 liter (Turkey Fruit Juice
Industry 2011).
According to the researches, antioxidant capacity of the
cherry is quite high and can be defined as nutriceuticals. It
has been denoted that anthocyanins in cherry have the
effects of decreasing rheumatism, the risk of colon cancer
and inhibiting fever, inflammation and tumor progression.
Cherry being rich in terms of melatonin that is a powerful
antioxidant and well sleeping regulator produced in the
brain backs up growth and cell renewal (Jafari et al.,
2008; Kim et al., 2005; Burkhardt et al., 2001; Wang et
al., 1999).
Nitric oxide (NO) is the most important endothelium
borne vasodilator. In addition to NO’s vascular smooth
muscle proliferation’s positive effects on thrombocyte
aggregation and vascular superoxide productions, it has
anti-atherosclerotic features. ADMA is the major inhibitor
of NO biosynthesis in human body. It is thought that the
increase of ADMA leading endothelial dysfunction has a
significant role in atherogenesis and cardiovascular
diseases (Vallance et al., 1992; Boger 2004).
Some bacteria like methicillin-resistant Staphylococcus
aureus, carbapenem-resistant Pseudomonas aeruginosa
and Acinetobacter baumannii developed resistance ways
against drugs’ effects. Along with surveying resistance
mechanisms and new drugs, these resistant
microorganisms, pose serious problems for health care
workers especially nosocomial infection at the top (Jeong
et al., 2006; Chastre and Trouillet, 2000).
*Corresponding author: e-mail: rogur@gata.edu.tr
Investigation of anti-cancer effects of cherry in vitro
Pak. J. Pharm. Sci., Vol.27, No.3, May 2014, pp.587-592
588
The purpose of this study is to investigate the potential
anticancer effects of cherry on human breast mammary
adenocarcinoma (MCF-7) and mouse mammary tumor
cell (4T1). To evaluate ADMA concentrations in cell
cultures treated with cherry extract and to examine
whether cherry has an antibacterial effect against certain
multidrug-resistant bacteria in vitro.
MATERIALS AND METHODS
Reagents
Maltodextrin Non-GMO was purchased from Tate & Lyle
Corporate and Investor Relations, Company, Slovakia.
Chemical and reagents; Folin-Ciocalteu reagent 2N,
DPPH (1,1-diphenyl-2-picrylhydrazyl), gallic acid, alpha
tocopherol, BHA (3-tert-butyl-4-hydroxyanisole) were
purchased from Sigma-Aldrich (St. Louis, MO) and
sodium carbonate was from Alfa Aesar GmbH & Co, KG.
All other chemicals were of food grade.
Plant material and preparation of extracts
Extracts of cherry pulps were used in the study. Cherry
pulp concentrations were provided by an international
aroma producer (Aromsa, Turkey). Cherry concentrate
was macerated with water and ethanol (1:1 w/w) for 4
times at room temperature. Evaporation of the solvent
under vacuum yielded 40 BX cherry extract, which used
in spray dry. Another concentrates from cherry
concentrates were used as a concentrate at 65 BX in all
tests.
Preparation of microcapsules by spray drying
Microencapsulation of flavors in carrier matrices can
provide protection prevent loss of volatile flavors and
enhance stability of the flavor core materials. The most
common and economical way to apply microencap-
sulation is spray drying. There are many papers were
published about the encapsulation of flavors in liquid by
spray drying. In this study, microencap-sulation was
performed by Aromsa Co. Inc and maltodextrin Non-
GMO used as a wall material. The total concentration of
cherry extract was 40% (w/w) and that was blended of
maltodextrin 30% (w/w; MD:Water). The mixture was
emulsified in a Sylverson, (Sylverson, Chesham,
England) for 5 min at 4000 rpm until complete dispersion
of the extract. The resulting slurry was spray dried in
APV-Anhydro, Denmark. The microcapsules prepared
were collected from the collecting chamber and filled in
air tight self-sealable polyethylene pouches.
Studied activity
Total phenolic content was measured by using the Folin-
Ciocalteu’s reagent. Results are expressed as microgram
of gallic acid equivalents per mg of extract. Free radical
scavenging activity was determined by 1,1-diphenyl-2-
picryl-hydrazil (DPPH) assay. The data on all antioxidant
activity tests were triplicated.
Cancer cell lines
Dulbecco's Modified Eagle Medium (DMEM) and fetal
bovine serum (FBS) was purchased from Invitrogen
Corporation (Carlsbad, CA 92008 USA). Dimethyl
sulphoxide (DMSO), phosphate buffer saline (PBS
solution) and MTT were purchased from Sigma-Aldrich
Chemical Co. (St. Louis, MO). MCF-7 and 4T1 cell lines
were used, in order to determine antitumoral activity,.
Cancer Cell proliferation inhibitory assay
The cancer cell proliferation activity of the cherry
extraction was tested by MTT colorimetric assay. Cancer
cell cultures (MCF-7 & 4T1) were maintained at the
Public Health Laboratory at Gulhane Medicine Faculty
Ankara, Turkey. MCF-7 & 4T1 were cultured in 10%
FBS, 2 µM L-glutamine, 100 µg/ml streptomycin and 100
µg/ml penicillin were added in DMEM medium in a
humidified incubator at 37°C under 5% CO2. MCF-7 &
4T1 cell lines were harvested, counted and moved into
96-well plates and incubated for 24 hours before
treatment. By melting away the cherry extracts in DMSO
the samples were made ready. Samples at 50 and
100µg/mL, were supplemented to DMEM medium. The
ending concentration of DMSO in the assay was 0.1%.
Samples at 50 and 100µg/mL concentrations were
supplemented to the wells. Into each well 25/µL MTT
solution was added after 48 hours incubation of samples
with cancer cells. Plates were incubated for 3 h at 37°C.
For dissolving the formed formazan crystals, content of
each well removed and 200/µL DMSO was added to each
well, and then the plates were shaken. Plates were
incubated for 5 min at room temperature, and finally the
optical density (OD) of the wells was ascertained using a
micro-plate reader at 570 nm. Samples' cell viability at
each concentration was calculated related to solvent
control. At each concentration, cell viability was
evaluated by dividing the optical density of samples with
the optical density of solvent control.
Analysis of apoptotic and necrotic cells
After treating cell lines with different amounts cherry
extract for 72 hours, all of the cells (attached and
detached cells) in the flask were brought together. After
this step, cells were washed with PBS for three times and
stained with Hoechst dye (2mg/ml), propodium iodide
(PI) (1mg/ml) and DNAse free-RNAse (100mg/ml) at the
25°C for 15 minutes and analyzed under a confocal
fluorescence microscope (LSM-GB200: Olympus, Tokyo,
Japan). The nuclei of apoptotic cells were marked blue by
Hoechst dye where necrotic cells were marked red by PI.
Ten areas in microscope, chosen by chance, were counted
and the result presented as a ratio of apoptotic and
necrotic to normal cells.
Measurement of ADMA
Measurement of ADMA was performed by HPLC (high
pressure liquid chromatography), refer to the method
described by Chen et al., (Chen et al., 1997). In short, 1
Recai Ogur et al.
Pak. J. Pharm. Sci., Vol.27, No.3, May 2014, pp.587-592 589
ml cell culture medium and 20/mg 5-sulfosalicylic acid
was mixed and the mixture was kept waiting in an ice
bath for 10 min. After centrifugation for 10 min at 2000
rpm the precipitated protein was removed. 10/µl of the
supernatant that was filtered and mixed with 100/µl of
extraction reagent (10/mg o-phthaldialdehyde in 0.5/ml of
methanol and 2/ml of 0.4/m borate buffer) then injected
into HPLC.
The minimum inhibitory concentration
Antibacterial characteristic of cherry evaluated on two
standard (Staphylococcus aureus ATCC 29123,
Pseudomonas aeruginosa ATCC 27853) and three clinical
(MRSA, Carbapenem-resistant Acinetobacter baumannii,
Carbapenem-resistant Pseudomonas aeruginosa) strains
of bacteria. The micro dilution broth method was used for
carrying out the minimum inhibitory concentration
(MIC). By using Mueller Hinton Broth sequential two
times dilutions of cherry extract were done in sterile 96-
well micro-plates with concentrations between ½ and
1/1024. Bacterial suspensions were regulated to 0.5 Mc
Farland standards. From an agar plate culture, at least
three well-separated colonies were chosen and they were
incubated at 37°C for 48 hr. Bacterial growth was
analyzed by 625 nm. absorbance measurement after
incubation. DMSO was used as a control. Ascertain the
late antimicrobial activity of cherry all assays were also
incubated for 48 hr at 37°C aerobically. Bacterial growth
was analyzed by 625 nm. absorbance measurement after
incubation. Lowest concentration of cherry which limited
bacterial growth to a <0.05 level at 625 nm was accepted
as MIC.
STATISTICAL ANALYSIS
Statistical analysis of variance was determinate by
ANOVA, significant differences between means were
determined by Student’s-t test, P<0.05 were noted as
significant.
RESULTS
Activity result
The cherry extract encapsulated was found to be the most
active extract in tests. Previously, the cherry extract
encapsulated was found to be rich in phenolic
compounds. Total phenolic content of cherry
encapsulated extracts were varied between 93.7 mg and
115.6 mg (mg GEs/100 g extract). Total phenolic content
of the other cherry concentrates were varied between 54.3
mg and 79.0/mg (mg GEs/100 g extract). As far as DPPH
assay, the cherry extract encapsulated was showed higher
antioxidant activity than Ascorbic acid and all tested
extracts of cherry (table 1). Konya and Tokat Cherry
concentrate were showed similar activity in DPPH assay
and all other extracts showed higher activity of Ascorbic
acid (fig. 1).
Results of MTT
The cell proliferation inhibitory effects of encapsulated
cherry extracts that have 50 and 100ug/ml concentration
respectively has been examined in the MCF-7 and 4T1
cancerous cell series. It has been concluded that the
extracts that are added to cell culture not only decreased
the cell proliferation in both MCF-7 and 4T1 cells but
also decreased in the concentration. It has been stated that
above mentioned extracts the one that has 50 ug/ml
concentrations inhibited cell growth 37% in MCF-7 cell
series and 48% in 4T1 series. Similarly, it has been stated
that the extract that has 100 ug/ml concentration inhibited
cell proliferation 54% in MCF-7 series and 53% in 4T1
series. It has been specified that the decrease ascertained
in the proliferation in cell series were significance
statistically (fig. 2).
DPPH ASSAY RESULTS
0,00
10,00
20,00
30,00
40,00
50,00
60,00
70,00
80,00
90,00
100,00
BHA
Ascorbic acid
Sour cherry extract Encapsulated
Kütahya Sour cherry Concentrate
Konya Sour cherry Concentrate
Tokat Sour cherry Concentrate
Afyon Sour cherry Concentrate
Sour Cherry Extracts and Standarts
% INHIBITION (IC5 0
)
Series1
Fig. 1: DPPH assay results of cherry extracts.
0
20
40
60
80
100
120
050100
Conc. (µg/mL)
% Viability
MCF-7
4T1
Fig. 2: In vitro cell proliferation inhibitory results of
cherry against cancer cell lines
Results of apoptotic and necrotic cells analysis
The effects of encapsulated cherry extracts on cell
development and its survival have been analyzed by
staining the MCF-7 and 4T1 cells that were treated with
cherry extract with Hoechst and Propodium Iodid. In
consequence of microscopic examination, it has been
stated that the greater part of examined cells cannot be
stained by PI and they are at late apoptosis phase that is
characterized by cells whose nucleuses are separated into
smaller organisms in cytoplasmic membrane. In addition
to these analyzed fields, the existence of red painted early
Investigation of anti-cancer effects of cherry in vitro
Pak. J. Pharm. Sci., Vol.27, No.3, May 2014, pp.587-592
590
and late necrotic cells have been specified. In the light of
findings above, it has been resulted that cherry extracts
induced anticancer activity through apoptosis (fig. 3).
Fig. 3: A general view of apoptotic and necrotic cells by
fluorescence microscopy. Blue points represent the
apoptotic cells, red points represent necrotic cells.
Table 1: Minimal inhibitory concentration (µL/mL)
results of cherry against clinical and standards strains of
bacteria.
24 hr of
incubation
(µL/mL)
48 hr of
incubation
(µL/mL)
Clinical isolates 200 100
Methicillin-resistant
Staphylococcus aureus
100 12.25
Carbapenem-resistant
A. baumannii
100 12.25
Carbapenem-resistant
Ps.aeruginosa
100 12.25
Standard strains
S. aureus ATCC 29123 200 100
Ps. aeruginosa ATCC 27853 100 12.25
Results of ADMA
ADMA levels of cell cultures supplemented with cherry
extract were significantly lower than the Ø control and
control cell culture values. ADMA levels of cell cultures
supplemented with cherry extract, Ø control and control
cell culture are 1.21, 2.54 and 2.79µmol/l respectively.
Minimal inhibitory concentration (µL/mL)
The results obtained from the MIC examination show that
cherry extract is effective on three clinical (MRSA,
Carbapenem-resistant Acinetobacter baumannii,
Carbapenem-resistant Pseudomonas aeruginosa) and two
standard (Staphylococcus aureus ATCC 29123,
Pseudomonas Aeruginosa ATCC 27853) examined strains
of bacteria. Following 24 hours incubation, MIC
concentration has been found 200ug/ml for gram-positive
microorganisms and 100ug/ml for gram-negative
microorganisms. Similarly, following 48 hours incubation
it has been stated that MIC rate fell 100ug/mg for gram-
positive microorganism and 12.25ug/ml for gram-
negative microorganism. At the same period there has
been obtained increase in the number of microorganisms
at the control wells.
DMSO, used as negative control, its effect on bacteria
proliferation has been researched. At the ½ diluted well of
Carbapenem-resistant Pseudomonas aeruginosa except
detected inhibition at the end of 48 hours incubation there
aren’t detected any inhibition in other wells. Including
positive controls, all of the results obtained from MIC
examination are in the border of CLSI quality control
parameter.
DISCUSSION
This is the first study in which the effect of cherry on
MCF-7 and 4T1 human and mouse breast cancer cell lines
was surveyed. The results obtained from this study is in
accordance with the results of a study in which the effects
of cyanine, giving the bright color of cherry and classified
as subgroup of anthocyanins, on human colon cancer was
surveyed. Furthermore, it has been stated that cherry
inhibits the formation and development of intestinal
cancer on ApcMin mice that are developed as human
cancer model. In the stated study, the number and size of
adenoma have been stated respectively less and small in
the mice whose diet cherry is added compared to other
mice (Kamei et al., 1998; Kang et al., 2003).
NAG-1 has been ascertained to be a gene that shows anti-
tumorigenic activity by triggering the apoptosis in the cell
lines. Studies proved that NAG-1 is a significant
promising target gene in inhibiting the cancer
development. The “wild-cherry’’ extract sometimes called
as “black-cherry’’ has been stated to increase NAG-1
expression and inhibit cell reproduction in the cancer cell
lines by increasing apoptosis as well (Baek et al., 2001;
Liu et al., 2003).
The surveys conducted revealed that wild-cherry extract
respectively suppresses B-catenin/TCF signal. It has been
stated that in the colorectal adenokarsinoms cyclin D1
protein expression increases. Similarly, the results of wild
cherry treatment made in the colorectal adenokarsinom
cell lines verify the decrease of cyclin D1 protein
expression with treatment. In the light of this information
it has been assessed that wild cherry suppresses B-catenin
signal in cancer cells and in consequence of this
suppression cyclin D1 protein expression decreases and
cancer cell development regresses (Yamaguchi et al.,
2006; Fu et al., 2004).
Genetic surveys states that genetic features of different
cherry kinds resemble each other very much. NAG-1
Recai Ogur et al.
Pak. J. Pharm. Sci., Vol.27, No.3, May 2014, pp.587-592 591
gene’s and cyclin D1 protein’s being covalent in the
analyzed kinds is possible. It has been thought that anti-
tumoral effect stated in this survey occurred by gene and
proteins in question (Mariette et al., 2010; Stockinge et
al., 1996).
When we consider the results of the survey, cherry is
specified to have shown antimicrobial action against the
gram-negative and gram positive bacteria examined in the
cope of survey. Examined bacteria’s causing nosocomial
infection and their being antibiotic resistant increases
importance of obtained results of the survey. Cherry along
with showing antimicrobial action on both gram-negative
and gram-positive bacteria, its effect on gram-negative
bacteria is stronger. In the previous studies cherry has
been mentioned to shown antibacterial action by
inhibiting the adhesion of bacteria. Mechanism of action
of the cherry is a subject that has not been sufficiently
researched yet. It is possible that the difference of the
action that cherry showed towards gram-negative and
gram-positive bacteria kind results from the difference of
the protein and lipid layer in the membrane structure of
these bacteria kinds. There is needed further study on this
subject (Klevens et al., 2007; Hebeler et al., 1973;
Rothfield et al., 1964).
In a recent study it is stated that much of fruit juices and
extracts especially those that are rich of anthocyanins and
acidic show antibacterial effect. It is ascertained that the
action in question disappears with the disappearance of
the acidic structure. However, in the same study it is also
ascertained that cherries and raspberries extracted with
water and blackcurrant extracted with methanol show
highly antibacterial effect. Effect showed in cherry,
raspberry and blackcurrant is stated to have been
independent from low acidic level and resulted from
anthocyanins and ellagitannin that are intensive in these
fruits (Galgóczy et al., 2009).
In this study ethanol was used in the preparation of cherry
extract. This situation made us think that the strong
antibacterial effect ascertained in cherry extracts, as
showed in some other studies, depends on both used
extract’s being acidic and pigments included in cherry’s
ingredient like anthocyanins and etc (Lee et al., 2003;
Harborne and Williams 2000).
This is the first study in which ADMA level increase
observed in MCF-7 and 4T1 breast cancer lines treated
with cherry extract. After showing its inhibiting NO
synthesis, in a short time lots of study was carried out
about ADMA’s pathophysiology (Valkonen et al., 2001;
Böger at al 2003). Increased ADMA levels by inhibiting
NO synthesis activity, inhibits NO formation and vascular
structure is preserved. In recent studies it is stated that
anthocyanins in cherry have positive effects on vascular
endothelium cells. It is expressed that mentioned effects
were controlled by plenty factors and enzymes partaking
in ADMA catabolism like Dimetilarginin dimetil amimo
hidrolaz are among these factors (Ding et al., 2000; Mc
Carty 2004).
The effects of cherry on cancer development, antibiotic-
resistant gram-positive and gram-negative pathogen
microorganisms and ADMA levels is of great concern to
human health because it is an ingredient in foods
consumed daily. Whereas, little is known about the
metabolic outcome of ingested cherry and its compounds
in humans in order to correlate its health benefits in vivo
as compared to in vitro studies.
ACKNOWLEDGEMENTS
This project was supported by the Aromsa Besin Aroma
ve Katki Maddeleri San ve Tic. AS and MEYED (Turkey
Fruit Juice Industry Association).
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Oncol. Rep., 15(1): 275-281.
