International Food Research Journal 20(2): 551-556 (2013)
Journal homepage: http://www.ifrj.upm.edu.my
Rabeta, M. S.,
Neda, G. D.,
Lam, K. L. and
Ong, M. T.
Plants, particularly fruits and vegetables, have many phytochemicals that possess various
bioactivities, including antioxidant and anticancer properties. In this study, the aim was to
investigate the antiproliferative properties of Syzygium fruits, namely water apple (Syzygium
against two types of cancer-origin cells, namely MCF-7 (hormone dependent breast cancer
cell line) and MDA-MB-231 (nonhormone-dependent breast cancer cell line). Two solvent
methods were prepared using aqueous and methanol extraction. Antiproliferation activities
of these extracts were evaluated by employing colorimetric MTT (3-(4,5-dimethylthiazol-2-
yl)2,5 diphenyltetrazolium bromide) assay through time periods of 24, 48, and 72 hours. The
result showed that extracts from the three fruits had no significant effects for 24 and 48 hours
time periods (p >0.05) but extracts of Water apple and Malay apple displayed antiproliferation
effects on MCF-7 cell lines (p <0.05) in 72 hours, also there were no effects on the non-cancer
origin cell line. The methanolic extracts of the malay apple was more significant with 79% cell
viability in the case of MCF7 (IC
=632.3 µg/ml). However, extracts of the milk apple did
in 72 hours. This finding revealed that fruits extract exhibit antiproliferative activity against
MCF-7, which is strongly estrogen-dependant, probably due to extract compound responsible
for its anticancer properties. Many studies had shown that plant polyphenols may prevent the
metastasis of breast cancer cells through common pathway.
Recent studies focusing on the exploitation
of natural compounds from fruits for medicinal
purposes has drawn much attention to the effective
extraction of the desired bioactive ingredients
from natural products. Plants, particularly fruits
and vegetables, have many phytochemicals that
possess various bioactivities, including antioxidant
and anticancer properties. Fruits can add important
vitamins, minerals, and other bioactive compounds to
the human diet (Vasco et al., 2008). Some promising,
but very under-utilized fruits belonging to Syzygium
genus of the Myrtaceae family found in Malaysia
have been recognized as having the potential to be
more useful for nutritional and medical purposes.
Syzygium is a genus in the Myrtaceae family that
includes a number of popular species cultivated for
their colorful, edible fleshy fruit. The genus name
Syzygium is derived via Latin from the Greek word
‘syzygos’, meaning yoked together, possibly referring
to the paired leaves (Janick and Paull, 2008). Their
fleshy fruit are eaten as such or added to fruit salads,
or are cooked or preserved in various ways for home
use (Wong and Lai, 1996). The three fruits focused
on water apple, Syzygium aqueum, which is also
called jambu air by the local community, milk apple,
Khoo et al. (2008), milk apple contains a low level
of total carotene content of (about 3.35 mg/100 g),
however beta-carotene was not found in milk apple.
In addition, Syzygium species possess antibacterial
activity (Chattopadhyay and Sinha, 2000). Also, the
bark of the malay apple tree has a variety of interesting
biological activities. It inhibited four species of
viruses, three species of fungus and provides
experimental verification for its use in traditional
medicine (Locher et al., 1995). Syzygium species
has the potential to be more useful for nutritional and
Breast cancer is one of the major causes for the
increasing mortality among women. In Malaysia,
there has been an increased admission rate of patients
diagnosed with breast cancer in government hospitals
(Abdullah and Yip, 2003). Breast cancer is the most
common cancer among females in all ethnic groups
and all age groups in females from the age of 15. It
is also the most important cancer regardless of sex in
Peninsular Malaysia (Zainal et al., 2006).
According to the study done by Norsa’adah et al.
(2005), the main factors associated with high risk of
breast cancer in women are nulliparity (the condition
of not bearing offspring), overweight/obesity, family
history of breast cancer, and oral contraceptives
(birth control pills) usage. Abdominal obesity has
been shown to be correlated with breast cancer risk
in the Klang Valley, Malaysia (Rabeta et al., 2007)
and estrogen hormone modifying breast cancer risk
(Oldenburg et al., 2007).
Many evidences from researches have
demonstrated that many natural products isolated
from plant sources possess antitumor properties (Wu
the antioxidant properties of these fruits. The main
objective of this study was to investigate the anti
proliferative properties of selected underutilized
fruits namely water apple, milk apple, and malay
apple against cancer-origin MCF-7 (hormone
dependent breast cancer cell line), MDA-MB-231
(nonhormone-dependent breast cancer cell line) and
noncancer origin HS27 (human foreskin fibroblast
Water apple, milk apple, and malay apple were
harvested in October 2010. HS27 (ATCC® CRL-
1634™, human foreskin fibroblast cell line), MCF-7
(ATCC® HTB-22™, hormone-dependent breast
cancer cell line) and MDA-MB-231 (ATCC® HTB-
26™, non hormone-dependent breast cancer cell line)
were purchased from the American Type Culture
Collection (ATCC), USA. Phosphate Buffer Solution
(PBS) tablets were obtained from AMRESCO
INC, Cleveland, Ohio, USA. The media used was
Dulbecco’s Modified Eagle Medium (DMEM with
low glucose, and high glucose) and Foetal Bovine
Serum (FBS), penicillin–streptomycin and trypsin
were from Gibco®, InvitrogenTM, USA. MTT
labelling reagent was obtained from Molecular
Probes®, InvitrogenTM, Oregon, USA.
The fruits were harvested from Kuala Kurau,
Perak, Malaysia. The fruits were identified by the
Herbarium Unit of the Forest Research Institute
Malaysia (FRIM) in Kepong, Selangor. Fruits of
water apple, milk apple and malay apple were
cut into small pieces and dried by using a freeze
drier (ALPHA Freeze Drier Model 1-2 LD plus,
Vacuubrand, Germany) for four days. Ground fruits
were kept in -20°C prior to extraction. This was done
at the School of Industrial Technology, USM Penang,
Based on the method proposed by Huang et al.
(2003), ground fruits were extracted with boiling
distilled water in the proportion of 1:20 (w/v) for 4
hours. The resulting crude extracts were filtered with
Whatman filter. The filtrate was lyophilized down to
dry powder by using freeze drier. The dried extracts
were kept in -20°C.
Methanol extraction of the plant was performed
according to the method described by Wicaksono et
al. (2009). Firstly, we had use 100 g of ground fruits.
The sample were weighed and then soaked in 300
mL absolute methanol for 24 hours. Subsequently
the crude extract was filtered with Whatman
filter. Residual solvent of methanolic extract was
removed under reduced pressure at 40
Model N-1000, Tokyo Rikakika Co., Ltd, Japan).
Evaporation was continued by storing the methanolic
extract at room temperature for 2 days. The extract
was diluted in PBS before assays. Final dilution was
made in DMEM containing 20% FBS.
This was carried out at the Institute for Research
in Molecular Medicine (INFORMM), USM based
on method from Freshney (1994). The cells were
observed under inverted phase-contrast microscope
and split using trypsin-EDTA after incubation at 37
in 5% CO
incubator for 5 mins. Cell suspension of
cells /ml was added into the T-25 flask containing
inverted microscope and the subculture was fed by
removing the existing media and replenished with
fresh complete media.
Cell growth was observed under inverted
phase-contrast microscope. Firstly, the cells were
trypsinized and then centrifuged at 1000 rpm for 4
mins. Subsequently, the supernatant was discarded
and the cells were resuspended with 1 mL PBS.
Again, the cells were centrifuged at 1000 rpm for 4
mins. Once the supernatant was discarded, the cells
were resuspended with 3 mL of incomplete media
(incomplete media = basal medium + 1% penicillin-
streptomycin). Then, the cell suspension was well
mixed and 15 µL of cells was added to 15 µL of
trypan blue in a small vial for cell counting using the
hemocytometer. The cells were diluted to obtain 3000
cells in each 60 µL using incomplete media. Cells
of 60 µL were dispensed into each well of a 96-well
microtitre plate and incubated at 37
C and 5% CO
Each cancer cell line was grown in a 96-well
microtiter plate (Nunc, Denmark) in a final volume of
120 μL culture medium per well. Each well contained
3 x 10
cells/well and was incubated for 24 hours in a
incubator at 37
C. The cells were then treated
with extracts of the fruits at doses of 0.78, 1.56, 3.125,
6.25, 12.5, 25, 50 and 100 μg/mL and maintained
C with 5% CO
for 24,48 and 72 hours. After
the incubation period, 0.5 mg/ml of MTT labelling
reagent was added to each well. The microtiter plate
was then incubated again for 4 hours at 37
C with 5%
. Then, the formazan crystals were solubilised
microlitre distilled water was added into each well
for further colour development. Absorbance of viable
cells was measured using a spectrophotometric plate
reader (Multiskan spectrum, Thermo Electron Co.,
Waltham, Massachusetts, USA) at 570 nm (Freshney,
Results for percentage cell viability were
reported as means ± standard error from triplicate
determinations. Significant differences for multiple
comparisons were determined by one-way analysis
of variance (ANOVA) followed by Duncan test by
SPSS statistical package (ver.17.0). The p value less
than 0.05 were considered as statistically significant.
Figures 1 and Figure 2 showed the effects of
water and methanol extracts in water apple using
MCF-7 and MDA-MB-231 cell lines and non cancer-
origin cells at 72 hours, respectively. In examining
the antiproliferative effect of aqueous extract from
water apple, it was found to have the strongest
inhibitory effect when non cancer-origin cell lines
were compared with cancer-origin cell lines. It was
observed that the viability of MCF-7 and MDA-
MB-231 cells were reduced in extracts at high
concentration, the viability of MCF-7 cells were
significantly (p < 0.05) reduced in methanol extract.
Based on results obtained, it is apparent that
water apple possesses inhibiting activity on two
breast cancer origin cell lines, especially MCF-7 that
is hormone/estrogen dependent breast cancer cell
line, and its activity is more prominent after 72 hours
incubation with the extract. Some studies reported
a strong correlation between phenolic content and
antioxidant activity in fruits, vegetables and grains
(Ismail et al., 2004; Dasgupta and De, 2007; Osman
water apple contains natural antioxidants.
Studies also showed that the volatile oils isolated
from Syzygium species by vacuum distillation contain
a high percentage of terpenoids and γ-terpinene.
Terpenoids may act by affecting the farnesylation
of ras gene product in premalignant and malignant
cells (Smith and Yang, 1994). Limonene has been
found to be effective in inhibiting the promotion or
progression stage of carcinogenesis and significant
(p <0.05) in inhibiting rat mammary tumors.
Limonene has also been found to cause inhibition
to primary differentiated mammary tumors by 7,12-
Dimethylbenz(a)anthracene (DMBA) which is a
Results from this study indicate that the water
apple has antiproliferative effects on MCF-7. This
may be due to the involvement of polyphenols
on estrogen metabolism, hence inhibiting the
proliferation of MCF-7 which is strongly estrogen-
dependant. Furthermore, it has also been suggested
that polyphenols may act as estrogen agonists or
antagonists in different contexts. Thus, several
factors may play a role in determining the effect of
polyphenols on breast cancer cell growth (Hakimuddin
et al., 2008).
Growth inhibition with milk apple extract
The percentage of cell viability of MCF-7, MDA-
MB-231 and HS-27 cell line for treatment with
different concentrations of aqueous and methanol
extract of milk apple in 72 hours are presented in
Figure 3 and Figure 4, respectively.
According to the figures, there is a significant
increase (p <0.05) in cell viability for HS27 cell and
decrease in cell viability for MCF-7 and MDA-MB-
231 cells at low concentration in 72 hours incubation
with aqueous extract of milk apple. However, after
incubation with methanol extract of milk apple for
72 hours, the cell viability of noncancer-origin cell
lines (HS-27) showed significant decrease (p <0.05)
starting from a concentration of 1.56 µg/mL, while
cell viability for cancer-origin cell lines does not
show significant changes (p>0.05).
Based on the results obtained, it can be concluded
that milk apple possesses antiproliferation effects
against HS27 cell and growth-promoting effects
against MCF-7 and MDA-MB-231 cells. This study
can supports the finding by Khoo et al. (2008) that milk
apple does not contain beta-carotene, which acts as
an anticancer compound as discussed in the previous
section. This can be explained by the whitish colour
of milk apple, as beta-carotene is mostly present in
fruits with yellow-orange colour.
The effect of aqueous and methanol extracts of
malay apple was tested against MCF-7 and MDA-
MB-231. Figures 5 and 6 exhibit the effect of these
Figure 1. Cell viability for treatment with different concentrations of
aqueous extract of water apple for 72 hours. Values are expressed as
mean ± standard error (SE) of triplicate measurements.
a-c represents means for each concentration labelled with different letters
were significantly different at p<0.05.
Figure 2. Cell viability for treatment with different concentrations of
methanol extract of water apple for 72 hours. Values are expressed as
mean ± standard error (SE) of triplicate measurements.
a-b represents means for each concentration labelled with different letters
were significantly different at p<0.05.
Figure 3. Cell viability for treatment with different concentrations of
aqueous extract of milk apple for 72 hours. Values are expressed as
a-b represents means for each concentration labelled with different
letters were significantly different at p<0.05.
Figure 4. Cell viability for treatment with different concentrations of
methanol extract of milk apple for 72 hours. Values are expressed as
Figure 5. Cell viability for treatment with different concentrations of
aqueous extract of malay apple for 72 hours. Values are expressed as
a represents means for each concentration were not significantly different
Figure 6. Cell viability for treatment with different concentrations of
methanol extract of malay apple for 72 hours. Values are expressed as
Both figures indicated that for both examined
cancerous cell lines, the viability of cells reduced
constantly by increasing malay apple concentration.
Cell viability for HS-27 showed significant increase
(p <0.05) starting from concentration 25 µg/mL; while
MDA-MB-231 did not show significant changes
(p>0.05) after treatment with methanol extract.
Based on the results obtained, the methanol
extract of malay apple displays antiproliferative
effects against MCF-7 cell line. The effect is more
obvious after 72 hours of incubation (Figure 6). This is
corroborated by the findings of Wongwattanasathien
malay apple on MCF-7 cell line with The methanolic
extracts of the malay apple was more significant
value of 632.3 µg/ml. The polyphenilic
effect of malay apple on estrogen metabolism is
presumed to be the antiproliferative cause of MCF-7,
which is known to be strongly estrogen dependent
(Hakimuddin et al., 2008).
Based on results obtained, the water apple, malay
apple, and milk apple possess antiproliferative activity
against cancer origin cell lines of MDA-MB-231
and MCF-7. The results of the study demonstrated
that both water and methanolic extracts of the fruits
decrease the viability of the mentioned cancerous
cell lines. However, the influence of the methanol
extracts of the Malay apple was more significant with
79% cell viability in the case of MCF7 at 72 hours
estrogen dependent breast cancer cell line. Extracts of
the three fruits did not exhibit significant effects for
24 and 48 hour periods (p>0.05). Furthermore, it can
be concluded that the fruits have anticancer activity
as demonstrated in previous studies on different
cancerous cell lines. However, knowing the exact
compound responsible for its anticancer properties
will help in making appropriate formulations that can
be used as anticancer agents in future.
The authors are thankful to Universiti Sains
and School of Industrial Technology, USM.
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