Plant Foods Hum Nutr (2007) 62:79–84
DOI 10.1007/s11130-007-0045-9
Antioxidative and Anticancer Activity of Extracts of Cherry
(Prunus serrulata var. spontanea) Blossoms
Bo-Bae Lee & Mi-Ran Cha & Soo-Yeon Kim &
Eunju Park & Hae-Ryong Park & Seung-Cheol Lee
Published online: 19 June 2007
# Springer Science + Business Media, Inc. 2007
Abstract Organic solvent (methanol, ethanol, and acetone)
extracts and water extracts of cherry (Prunus serrulata var.
spontanea) blossoms were prepared, and antioxidant
activities of the extracts were evaluated. Methanolic CBE
(100 μg/ml) showed the highest total phenol content
(104.30 μM), radical scavenging activity (34.2%), and
reducing power (0.391). The effect of CBE on DNA
damage induced by H2O2 in human leukocytes was
evaluated by Comet assay. All CBE was a potent dose
dependent inhibitor of DNA damage induced by 200 μM of
H2O2, methanolic CBE showed the most strong inhibition
activity. The methanolic CBE of 500 μg/ml showed 38.8%
inhibition against growth of human colon cancer cell line
HT-29. These results indicated that cherry blossoms could
provide valuable bioactive materials.
Keywords Prunus serrulata var. spontanea . Extract .
Antioxidant . Anticancer
Abbreviations
CB cherry blossoms
CBE cherry blossom extract
DMSO dimethyl sulfoxide
DPPH 1,1-diphenyl-2-picrylhydrazyl
LMA low melting agarose
B.-B. Lee : M.-R. Cha : H.-R. Park : S.-C. Lee (*)
Department of Food Science and Biotechnology,
Kyungnam University,
Masan 631-701, Republic of Korea
e-mail: sclee@kyungnam.ac.kr
S.-Y. Kim : E. Park
Department of Food and Nutrition, Kyungnam University,
Masan 631-701, Republic of Korea
MTT 3-(4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyl-
tetrazolium bromide
PBS phosphorous buffered saline
ROS reactive oxygen species
RP reducing power
RSA radical scavenging activity
TPC total phenolic content
Introduction
Free radicals can be generated in biological systems in the
form of reactive oxygen species (ROS), and these are
removed by the antioxidant system in the body. There are
several antioxidants including glutathione S-transferase,
glutathione peroxidase, superoxide dismutase, and catalase
[1]. However, when the level of free radicals exceeds the
ability of the antioxidant system, lipid peroxidation and DNA
and protein damage occur, which result in aging and various
diseases, including inflammation, cancer, Parkinson’s disease,
cardiovascular diseases, multiple sclerosis, and lupus [2].
Among these various diseases, the incidence of cancer is
increasing worldwide and it is the single most common
cause of death in both developed and developing countries
[3]. Although surgery, chemotherapy, and irradiation are the
mainstream therapeutic approaches for cancer, undesirable
chemotherapy reactions and poor prognosis in advanced
cancer patients are still major problems in cancer therapy
[4]. Natural products and their derivatives already play
critical roles in cancer chemotherapy [5].
There are two types of commercial antioxidant supple-
ments which are in use for the reduction of oxidative
damage in the human body. There are natural antioxidants
such as ascorbic acid, tocopherol, epigallocatechin gallate,
and synthetic antioxidants such as butylated hydroxy anisol,
80
butylated hydroxy toluene, and tert-butylhydroquinone [6].
Synthetic antioxidants however, are known to induce
carcinogenesis by mutagenicity and toxicity against human
enzymes and lipids [7]. For this reason, studies of natural
antioxidants are attractive to investigators for use in foods or
medicinal materials to replace synthetic antioxidants. Partic-
ularly, much research is focused on natural products
including vegetables and wild plant sources [8].
The cherry tree (Prunus serrulata var. spontanea), which
belongs to the Rosaceae family, is found throughout Korea.
The cherry tree has been used as a medicinal plant for a
long time. In particular, red cherry fruits are used in a
traditional herbal remedy for various diseases such as heart
failure, beriberi, dropsy, and mastitis. This herbal remedy is
an emmenagogue and is also used for toothache. Bark and
stems of the cherry tree are used for detoxification and
relaxation [9]. Several researchers have investigated the
source and level of antioxidant activity and anticancer
activity in the cherry tree [9, 10] however, there are few
reports regarding cherry blossoms. The objective of this
study was to evaluate the antioxidant and anticancer
activity of various solvent extracts of cherry blossoms.
Materials and Methods
Materials
Cherry (Prunus serrulata var. spontanea) blossoms (CB)
were collected in early April 2006 at the campus of
Kyungnam University, Masan City, Korea. 1,1-Diphenyl-2-
picrylhydrazyl (DPPH), dimethyl sulfoxide (DMSO), 3-(4,5-
Dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide
(MTT), Histopaque 1077, fetal calf serum, low-melting-
point agarose, methylene blue and 1,2-methylhydrazine
were purchased from Sigma Chemical Co. (St. Louis, MO,
USA). Folin-Ciocalteu reagent was from Wako Pure Chem-
ical Industries, Ltd (Osaka, Japan). RPMI 1640 medium,
fetal bovine serum and penicillin/streptomycin were pur-
chased from Gibco BRL (Grand Island, NY, USA). All
organic solvents and other chemicals were of analytical
grade or complied with the standards needed for cell culture
experiments.
Preparation of Extracts from Cherry Blossoms
Each 10 g of fresh CB were extracted with 1 L of solvents
(methanol, ethanol, acetone, or distilled water) in a shaking
incubator (100 rpm) for overnight at room temperature and
filtered through a Whatman No. 1 filter paper (Advantec,
Tokyo, Japan). Solvents were then removed by evaporation
in vacuo, and the dried extracts were obtained. The solvent
Plant Foods Hum Nutr (2007) 62:79–84
extracts from CB were named as CBE. The CBE were then
dissolved in DMSO in a concentration of 50 mg/ml for
experiments, and diluted with DMSO when needed.
Total Phenolic Content (TPC)
TPC of CBE was determined according to the method of
Gutfinger [11]. Each CBE (1 ml) was mixed with 1.0 ml of
2% Na2CO3 and added 0.2 ml of 50% Folin-Ciocalteu
reagent after standing for 5 min, and centrifuged at
13,400×g for 5 min after 30 min incubation at room
temperature. The absorbance was measured with a spectro-
photometer (Shimadzu UV-1601, Tokyo, Japan) at a
wavelength of 750 nm. TPC was expressed as gallic acid
equivalents.
DPPH Radical Scavenging Activity (RSA)
The DPPH RSA of CBE was determined according to the
method of Lee et al. [12]. After 0.1 ml of each
concentration of CBE had been mixed with 0.9 ml of
0.041 mM DPPH in ethanol for 10 min, the absorbance of
the sample was measured at a wavelength of 517 nm by a
spectrophotometer.
Reducing Power
Reducing power (RP) of CBE was determined according to
the method of Oyaizu [13]. CBE (1.0 ml), sodium phosphate
buffer (1 ml, 0.2 M, pH 6.6) and potassium ferricyanide
(1.0 ml, 10 mg/ml) were mixed and incubated at 50°C for
20 min. Trichloroacetic acid (1.0 ml, 100 mg/ml) was added
to the mixture and centrifuged at 13,400×g for 5 min. The
supernatant (1.0 ml) was mixed with distilled water (1.0 ml)
and ferric chloride (0.1 ml, 1.0 mg/ml), and then its
absorbance was measured at a wavelength of 700 nm.
Determination of DNA Damage (Comet Assay)
CBE was dissolved in DMSO and added to culture medium
so that the final concentration of DMSO was less than 1%.
Blood was obtained from three healthy male volunteers and
leukocytes were isolated using Histopaque 1077. Leuko-
cytes (2×104 cell/ml) were incubated with CBE diluted into
concentrations 0, 5, 10, 25 and 50 μg/ml for 30 min at 37°C
in a dark incubator. For oxidative stimulus they were then
resuspended in phosphorous buffered saline (PBS) with
200 μM H2O2 for 5 min on ice. The experiments were
replicated at least three times independently. 1% DMSO
without oxidative stimulus was treated for negative control.
The alkaline comet assay was conducted according to
Singh et al. [14] with little modification. The cell
suspension was mixed with 75 μl of 0.5% low melting
Plant Foods Hum Nutr (2007) 62:79–84 81

Table 1 Antioxidant activity of cherry blossom extracts

Extraction solvents Positive control

MeOH EtOH Acetone DW Ascorbic acid

TPC (μM GAE) 104.30a 79.18c 95.77b 51.20d –

DPPH (RSA %) 34.2a 25.7c 39.9b 19.1d 85.1
RP (OD) 0.397a 0.216b 0.345a 0.169b 0.911

Each 10 g of cherry blossom were extracted with 1 L of solvents (methanol, ethanol, acetone, or distilled water). Solvents were then removed, and
the dried extracts were dissolved in DMSO with a concentration of 100 μg/ml for determination of antioxidant activity. All measurements were
done in triplicate, and analysis of variance was conducted by of the General Linear Model using SAS software [15].
GAE, gallic acid equivalents
a–d
Different letters within a row are significantly different (P<0.005), n=3.

agarose (LMA), and added to the slides precoated with
1.0% normal melting agarose. After solidification of the
agarose, slides were covered with another 75 μl of 0.5%
LMA, and then immersed in lysis solution (2.5 M NaCl,
100 mM EDTA, 10 mM Tris, and 1% sodium lauryla-
sarcosine; 1% Triton X-100 and 10% DMSO) for 1 h at 4°C.
The slides were next placed into an electrophoresis tank
containing 300 mM NaOH and 10 mM Na2EDTA (pH
13.0) for 40 min for DNA unwinding. For electrophoresis
of the DNA, an electric current of 25 V/300 mA was
applied for 20 min at 4°C. The slides were washed three
times with a neutralizing buffer (0.4 M Tris, pH 7.5) for
5 min at 4°C, and then treated with ethanol for another
5 min before staining with 50 μl of ethidium bromide
(20 μg/ml). Measurements were made by image analysis
(Kinetic Imaging, Komet 5.0, U.K) and fluorescence
microscope (LEICA DMLB, Germany), determining the
percentage of fluorescence in the tail (tail intensity, TI; 50
cells from each of two replicate slides). Cell viability
measured by trypan blue exclusion test was above 95% for
all treatments.
Cell Culture and Treatments
HT-29 human colon carcinoma cells were provided from
Korean Cell Line Bank. The cells were maintained in RPMI

Fig. 1 Preventive effects of

supplementation in vitro with
different concentration of cherry
blossom extracts (CBE) on
200 μM H2O2-induced DNA
damage in isolated human
leukocytes. Human leukocytes
were preincubated for 30 min
with a methanol, b ethanol, c
acetone and d water extract from
cherry blossoms. DMSO,
DMSO-treated negative control;
Values are expressed as mean±
standard error of duplicate
experiments with leukocytes
from each of two different
donors. Values not sharing the
same letter are significantly
different from one another
(P<0.05)
82
1640 medium supplemented with 10% heat-inactivated
fetal bovine serum, penicillin (100 U/ml), streptomycin
(100 mg/ml) and 2 mg/ml NaHCO3 at 37°C incubator with
5% CO2. CBE was dissolved in DMSO and added to
culture medium so that the final concentration of DMSO
was less than 1%.
MTT Reduction Assay for Cell Viability
Cell viability was measured with blue formazan that was
metabolized from MTT by mitochondrial dehydrogenase,
which are active only in live cells. HT-29 cells preincubated
in 96-well plates at a density of 1.0×105 cells per well for
24 h. Cells were pretreated with various concentrations of
CBE. After incubation for 24 h, MTT reagent (5 mg/ml)
was added to each of the wells, and the plate was incubated
for an additional 1 h at 37°C. The media was then
removed, and the intracellular formazan product was
dissolved in 100 μl of DMSO. The absorbency of each
well was then measured at a wavelength of 540 nm using
the ELISA reader (BioRad, Model 680, USA), and the
percentage viability was calculated.
Statistical Analysis
All measurements were done in triplicate, and analysis of
variance was conducted according to the procedure of the
General Linear Model using SAS software [15]. Student–
Newman–Keul’s multiple-range tests were used to compare
the significant differences of the mean value among
treatments (P<0.05). The data for Comet assay were the
means of three determinations and were analyzed using the
SPSS package for Windows (Version 11.5). The mean
values of the DNA damage (tail intensity) from each
treatment were compared using one-way analysis of
variance (ANOVA) followed by Duncan’s multiple range
test. P value of less than 0.05 was considered significant.
Results and Discussion
Antioxidant Activity of CBE
Phenolic compounds are known to act as antioxidants not
only because of their ability to donate hydrogen atoms or
electrons but also because of their stable radical inter-
mediates, which prevent the oxidation of various food
ingredients, particularly fatty acids and oils [16]. TPC of
each extract (100 μg/ml) of CB is listed in Table 1.
Methanol extract (104.30 μM) and acetone extract
(95.77 μM) had higher TPC values, while the water
extract showed the lowest value (51.20 μM) among the
studied solvents.
Plant Foods Hum Nutr (2007) 62:79–84
Radical scavengers were evaluated by their reactivity
towards a stable free radical, DPPH. DPPH RSA of each
CBE is also shown in Table 1. Acetone extract (39.9%) and
methanol extract (34.2%) also showed higher DPPH RSA,
and water extract showed the lowest activity (19.1%). RSA
of each CBE was lower than that of ascorbic acid (85.1%)
at the same concentration.
The power of certain antioxidants is associated with their
RP. Duh [17] reported that the reducing properties of
antioxidants are generally associated with the presence of
reductants. RP of methanol and acetone extracts of CB were
0.391 and 0.345, respectively, which were higher than those
of ethanol and water extracts (Table 1). RP of ascorbic acid at
same concentration (50 mg/ml in DMSO) was 2.29–2.64-fold
higher than that of methanol and acetone extracts of CB.
RSA and RP were highly related to TPC of CBE.
Several kinds of flavonoids such as prunetin, genistein, and
quercetin [9], and phenolic glucosides including pursargen-
toside and chlorogenic acid [18], which show strong
antioxidant activity peroxynitrite scavenging activity, have
been identified from the cherry species used in this study.
Fig. 2 Effects of cherry blossom extracts (CBE) on cell viability of
HT-29 cells. The cells were exposed to the indicated concentrations of
CBE for 24 h (a methanol extract, b ethanol extract, c acetone extract,
d water extract). Cell viability was measured with the MTT reduction
assay. After a MTT assay, the MTT reduction rate was calculated by
setting each of control survivals. Data (means±SD of triplicate
determinations) are representative of at least three independent
experiments
Plant Foods Hum Nutr (2007) 62:79–84
Jung et al. [9] reported a considerable antioxidant activity
of methanol extract of CB. Although it is difficult to
compare the antioxidant activity of CBE with a pure
antioxidant, such as ascorbic acid, the significant antioxi-
dant effects of CBE could be due to the synergistic effects
of various phenolic compounds in CBE.
Protective Effect of CBE on Oxidative DNA Damage
in Human Leukocytes
The comet assay, which measures the breaking of the DNA
strand at the level of single cells, is very easily applied to
lymphocytes and therefore lends itself to human bio-
monitoring studies. It has become a standard technology
for the measurement of oxidative DNA damage both in
vitro and in vivo [19]. H2O2 is believed to cause DNA
strand breakage by generating the hydroxyl radical (OH·)
close to the DNA molecule, via the Fenton reaction [20].
The genotoxic effects of H2O2 and the protective ability
of various solvent extracts of CB were assessed in normal
human leukocytes by the comet assay. The percentage of
the fluorescence tail DNA intensity of leukocytes treated
for 30 min with DMSO (negative control) was significantly
different from that of leukocytes treated for 30 min with
200 μM H2O2 in PBS (positive control; Fig. 1). This
increase of the DNA damage induced by H2O2 was
significantly inhibited in a dose dependent manner by
pretreatment of the cells for 30 min with methanol (A),
ethanol (B), acetone (C) or water (D) extracts of CB at
concentrations of 5, 10, 25 and 50 μg/ml in DMSO. The
highest concentration (50 μg/ml) of ethanol and acetone
extracts resulted in percentage fluorescence densities that
were not statistically different to the DMSO-treated
negative control.
The possible mechanism by which CBE inhibited
oxidative DNA damage in human leukocytes might be
ascribed to the chemical structure of the phenolic com-
pound contained in CBE. Although the exact phenolic
compounds in CBE need to be isolated in a further study,
generally the flowers of plant contain polyphenolics
including flavonoids and anthocyanins, which may have
anti-oxidative activity [21]. The phenolic compounds in CBE
may work by providing hydrogen atoms from their phenolic
hydroxyl groups to scavenge hydroxyl radical generated from
H2O2 [22]. The protective effect of polyphenolic compounds
(tannic, ellagic, and gallic acid) on oxidative DNA damage
measured by comet assay has been demonstrated by
Labieniec and Gabryelak [23]. Yeh et al. [24] reported the
protective effect of naringenin and rutin on ultraviolet A
induced DNA damage. Schaefer et al. [25] also found that
rutin from apple juice reduced oxidative DNA damage in
Caco-2 cells. Heo et al. [26] reported that the ethanol extract
of the flowers of Prunus persica (50–200 μg/ml), which is
83
also a member of the Rosaceae family (Prunus yedoensis),
was found to inhibit ultraviolet (UV) B as well as UVC-
induced DNA damage measured by the comet assay in skin
fibroblast cells (NIH/3T3).
Cytotoxic Effects of CBE on HT-29 Human Colon
Carcinoma Cells
The effects of CEB on the inhibition of the growth and
proliferation of HT-29 human colon carcinoma cells were
determined via MTT reduction assay. HT-29 cells were
originally derived from a human colon carcinoma, and
were chosen because they represent a hypovascular tumor
[27]. HT-29 cells treated with various concentrations of
CBE for 24 h demonstrated significant decrease in cell
viability compared to control (Fig. 2). The highest
inhibition in this study was determined as 38.8% at a
concentration of 500 μg/ml of methanolic CBE. The results
indicated that CBE had cytotoxic activity on HT-29 cells.
The mechanism of action of this cytotoxicity of CB
remains unclear and the anti-proliferation effect of CB has
not been reported up to now. Although additional research
is needed to delineate the relative contribution of this
pathway to cytotoxicity, our results suggest that the CB
may be a potential candidate for the development of novel
therapeutic agents to induce cell death in human colon
carcinoma cells.
Acknowledgements This study was supported by Kyungnam
University Research Fund, 2007.
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