Sains Malaysiana 43(12)(2014): 1895–1900
Antiproliferative Potential of Extracts from Kappaphycus Seaweeds
on HeLa Cancer Cell Lines
(Potensi Antiproliferatif Ekstrak Rumpai Laut Kappaphycus ke atas Titisan Sel Kanser HeLa)
TIEK YING LAU, DELBORA FENNY VITTAL, CASSANDRA SZE YII CHEW
& WILSON THAU LYM YONG*
A review of the current literature indicates that natural seaweeds are an excellent source of bioactive compounds with
antioxidant, antimicrobial and antitumor properties. In the present study, 90% methanolic, 70% acetonic and aqueous
extracts from Kappaphycus alvarezii (strains Crocodile, Giant and Brown) and Kappaphycus striatum were used to inhibit
the growth of HeLa cell lines. MTS assay was carried out to determine the proliferation of HeLa cells in the presence of
different seaweed extracts. Both 500 µg/mL of aqueous and methanolic extracts from K. striatum demonstrated highest
anti-proliferative activity against HeLa cells with cell growth inhibition of 53.5 and 43.7%, respectively. Treatment with
the aqueous extracts from three strains of K. alvarezii did not show any growth inhibition against HeLa cell lines. The
acetonic extract of Kappaphycus seaweeds exhibited a very poor cell growth inhibition with inhibitory activity observed
under the treatment of 300 to 500 µg/mL of K. alvarezii strain Brown only. Further studies are suggested to identify and
purify the specic anti-tumoral compounds for potential use in cancer therapy.
Keywords: Antiproliferation; growth inhibition; Kappaphycus alvarezii; Kappaphycus striatum
Suatu kajian kepustakaan semasa menunjukkan bahawa rumpai laut semula jadi adalah sumber komponen bioaktif
yang sangat baik dengan aktiviti antioksidan, antimikrobial dan antitumor. Dalam kajian ini, ekstrak metanol 90%,
aseton 70% dan akueus daripada Kappaphycus alvarezii (strain Buaya, Giant dan Brown) dan Kappaphycus striatum
telah digunakan untuk merencat pertumbuhan titisan sel HeLa. Asai MTS telah dijalankan untuk mengkaji pertumbuhan
sel HeLa dalam kehadiran pelbagai ekstrak rumpai laut. Kedua-dua 500 µg/mL ekstrak akueus dan metanol daripada
K. striatum menunjukkan aktiviti antiproliferasi yang tertinggi terhadap sel HeLa masing-masing dengan perencatan
pertumbuhan sel sebanyak 53.5 dan 43.7%. Rawatan dengan ekstrak akueus daripada tiga strain K. alvarezii tidak
menunjukkan perencatan pertumbuhan terhadap titisan sel HeLa. Ekstrak aseton daripada rumpai laut Kappaphycus
mempamerkan perencatan pertumbuhan sel yang lemah dengan aktiviti perencatan hanya boleh diperhatikan dengan
rawatan 300 hingga 500 µg/mL daripada K. alvarezii strain Brown sahaja. Kajian lanjutan adalah dicadangkan untuk
mengenal pasti dan menulenkan komponen khusus antitumor untuk pembangunan terapi kanser.
Kata kunci: Antiproliferasi; Kappaphycus alvarezii; Kappaphycus striatum; perencatan tumbuhan
Seaweeds are considered to be a source of bioactive
compounds as they are able to produce a variety of
secondary metabolites characterized by a broad spectrum of
biological activities. Compounds with cytostatic, antiviral,
anthelmintic, antifungal and antibacterial activities have
been detected in green, brown and red algae (Lindequist
& Schweder 2001; Newman et al. 2003). More recently,
seaweeds are reported to be a rich source of antioxidant
compounds (Duan et al. 2006; Kuda et al. 2005; Lim et al.
2002). For example, chlorophylls, carotenoids, tocopherol
derivatives such as vitamin E and related isoprenoids,
which are structurally related to plant-derived antioxidants,
were found in some marine organisms including seaweeds
(Takamatsu et al. 2003). Antioxidants in biological systems
have multiple functions, including defense against oxidative
damage and participating in the major signaling pathways
of cells. Besides, some compounds from the seaweeds
have antibacterial activities with potential use as mosquito
control agents. Extracts from Eucheuma denticulatum have
exhibited antibacterial activity on Gram positive organisms
including Staphylococcus aureus and Streptococus pyogenes
(Al-Haj et al. 2009).
Seaweeds also contain bioactive substances with great
potential as antitumoral drugs, which lead to emerging
interests in the biomedical research in seaweeds (Michio et
al. 1984; de Sousa et al. 2007). Several species of seaweeds
are rich sources of polysaccharides and glycoproteins with
immune-stimulant, anticancer or antiviral activity (Abdel-
Fattah et al. 1974; de Sousa et al. 2007; Michio et al. 1984;
Nishino et al. 1989; Smit 2004). Certain algae have long
been used in traditional Chinese herbal medicine in cancer
treatment (Yamamoto et al. 1984). Red and green algae
have been shown to demonstrate protective effects against
mammary, intestinal and skin carcinogenesis (Yuan & Walsh
2006). Zandi et al. (2010) reported that cold water extract
of red alga, Gracilaria corticata, possessed biological
activity against tumor cells replication. In recent years,
much attention has been focused on fucoidan, a sulphated
polysaccharide derived from brown seaweeds. Recent
studies evidenced that fucoidan has strong antitumor activity
and exhibited important roles against human cancer cell lines
(Ly et al. 2005; Matsuda et al. 2010). Fucoidan was found
to be able to suppress the growth of tumor cells in vivo and
activate the immune system against tumors (Itoh et al. 1993;
Maruyama et al. 2003; Noda et al. 1990; Usui et al. 1980;
Yamamoto et al. 1984; Zhuang et al. 1995).
The two red seaweed species, K. alvarezii and K.
striatum, which are extensively distributed in Sabah, have
been uncovered as a novel source for a variety of compounds
such as dietary bers, vitamin C, α-tocopherol, minerals,
fatty acid and protein (Matanjun et al. 2008). However,
there is limited information about their biological activity
on cancer cell growth inhibition. The objectives of this study
were to screen and evaluate the anti-proliferative activities of
crude methanolic, acetonic and aqueous extracts of selected
strains of K. alvarezii and K. striatum. The information
compiled during the course of this study can be of use for
further development of cancer therapy.
MATERIALS AND METHODS
Kappaphycus alvarezii (strains Crocodile, Giant and
Brown) and Kappaphycus striatum were collected from
Semporna, Sabah. The samples were washed with fresh
water and dried at room temperature for 1 week. The dried
seaweed samples were separately milled and subjected
to compound extractions. For aqueous extraction, dry
powder of seaweed was macerated with de-ionized water
and ltered through cotton wool and Whatman (No. 1)
lter paper to remove debris. The ltrate was lyophilized
using freeze dryer for 3 days. For each of extraction using
90% methanol and 70% acetone, approximately 100 g of
powdered seaweed samples were extracted using a soxhlet
apparatus. The methanol and acetone were purchased from
Sigma-Aldrich (St. Louis, MO, USA). About 500 mL of
each solvent was used to carry out the extraction in soxhlet
apparatus for a period of 24-72 h until the solvent becomes
colorless at 65±2°C. The solvent was evaporated using a
rotary vacuum evaporator to make the nal volume one-
fourth of the original volume. The methanolic, acetonic and
aqueous extracts were stored in -10°C for further analysis
of anti-proliferative assay in triplicate.
CELL LINE AND CULTURE CONDITION
HeLa Cancer Cell Line CCL-23TM was purchased from
American Type Culture Collection (ATCC®, USA). Cells
were seeded and grown in RPMI (Roswell Park Memorial
Institute) media. They were maintained in 12.5 cm3 BD
FalconTM cell culture ask (California, USA) at 37°C in a
humidied atmosphere with 5% CO2. The RPMI medium
was replaced once every two days and passaging was
performed to maintain the adherent cell lines.
GROWTH INHIBITION ASSAY
In order to observe the seaweed extracts responsiveness,
a cell proliferation assay was carried out. The inhibition
effects of methonolic, acetonic and aqueous extracts
on the growth of HeLa cells were evaluated in vitro
by the MTS assay. This method relies on the ability
of dehydrogenase enzymes in the metabolically
active cells to convert 3-(4,5-dimethylthiazol-2-yl)-
tetrazolium or MTS to a formazan precipitate. The Cell
Titer 96® Aqueous Non-Radioactive Cell Proliferation
Assay purchased from Promega (Madison, USA) was
used to determine the cell proliferation of HeLa cells in
the presence of different types of extracts (methanolic,
acetonic and aqueous) at different concentrations (50,
100, 200, 300, 400 and 500 μg/mL). The different
concentrations of each extracts were prepared from the
stock solutions by serial dilution.
A known number of HeLa cells (104) were incubated
in 96-well plates in a volume of 200 μL of culture medium
and permitted to adhere for 24 h before addition of test
compounds. About 100 μL of different concentrations
(50, 100, 200, 300, 400 and 500 μg/mL) of each extracts
(methanolic, acetonic and aqueous) were added to the
cells. After 48 h of exposure, the cells were washed with
100 μL of phosphate-buffered saline (PBS) and replaced
with fresh medium. Approximately 20 μL of CellTiter
96® AQueous One Solution Reagent was added into each
well of the 96-well assay containing the samples in
100 μL of culture medium. The plates were incubated
at 37°C in a humidified atmosphere with 5% CO2.
Following incubation for 4 h, the plates were read with
SPECTRAMax M2 ROM (Molecular Devices) microplate
reader at absorbance of 490 nm. The experiments were
performed twice in triplicate. The results were evaluated
by comparing the absorbance of the treated cells with the
absorbance of wells containing cell treated by the solvent
control. Conventionally, cell viability was estimated to
be 100% in the solvent control.
Percentage of cell growth inhibition versus extracts
concentration was calculated according to Patel et al.
(2009) as follow:
Percentage of cell growth inhibition = 100-[(A-B)/
where A is the absorbance of sample; B is the absorbance
of blank and C is the absorbance of control.
MTS assay was carried out to investigate the inhibition
effects of methonolic, acetonic and aqueous extracts of
Kappaphycus seaweeds on the growth of HeLa cells and
the results are represented in Figures 1 to 4. Six different
concentrations (50, 100, 200, 300, 400 and 500 μg/
mL) of each extract (methanolic, acetonic and aqueous)
were applied. Figure 1 shows the percentage of growth
inhibition against 90% methanolic, 70% acetonic and
aqueous extracts of K. alvarezii strain Crocodile. Among
the three types of extracts, only methanolic extract from
200 to 500 μg/mL showed obvious anti-proliferative
activity against the HeLa cells. The highest percentage
(31.7%) of growth inhibition was observed with the
treatment using 300 μg/mL of methanolic extract. This was
followed by the treatment using methanolic extracts at 400,
500 and 200 μg/mL with 24.6, 16.5 and 4.0% of growth
inhibition, respectively. Treatments with 50 and 100 μg/mL
methanolic extracts and all acetonic and aqueous extracts
did not show any growth inhibition but they promoted
growth of the treated cells instead.
For the treatment with different extracts from K.
alvarezii strain Giant, only methanolic extract at 400 μg/
mL demonstrated growth inhibition (8.5%) as summarized
in Figure 2. Methanolic extract with other concentrations
and all the acetonic and aqueous extracts from K. alvarezii
strain Giant did not show any cell growth inhibition. While
for the treatment with K. alvarezii strain Brown extracts,
the highest growth inhibition (30.4%) was observed with
50 μg/mL of methanolic extract, as shown in Figure 3.
Increment of methanolic extract concentrations resulted
in decrement of growth inhibition as seen with 100 and
200 μg/mL of extracts demonstrated 7.9 and 5.9% of
growth inhibition, respectively. On the other hand, only
acetonic extract from this strain demonstrated positive anti-
proliferative activity against the HeLa cells as compared
with the other two strains (Crocodile and Giant). Increment
of acetonic extract from 300 to 500 μg/mL had parallel
increment in the inhibition percentage from 17.5 to 29.7%.
Figure 4 shows the percentage of cell growth inhibition
against concentrations of methanolic, acetonic and aqueous
extracts of K. striatum. The results indicated that all the
methanolic and aqueous extracts have positive inhibition
on the HeLa cell lines. Treatment with the aqueous extract
ranging from 50 to 500 μg/mL exhibited concentration
dependent anti-proliferative activity against HeLa cells
with 17.8 to 53.5% of cell growth inhibition. Whereas,
the inhibition effect of methanolic extract on cells growth
ranged from 6.2 to 43.7%. All acetonic extracts from K.
striatum did not inhibit, but promote, cell growth.
Marine algae contain many unidentied useful components
and physiologically active substances. Studies on
bioactivity of marine algae against cancer cell lines
have been reported in previous researches, where the
ndings have brought great promise to the development
of cancer treatment activities (Albano et al. 1990;
Berlinck et al. 1996). Some studies involved general
extractions of seaweeds while others applied extraction of
specic metabolites such as carotene, bromophenols and
carrageenan (Ly et al. 2005; Xu et al. 2004). In the present
study, 90% methanolic extracts, 70% acetonic extracts
and aqueous extracts of K. alvarezii and K. striatum were
studied for their potential to inhibit the growth of HeLa
cell lines. The most effective concentration to inhibit cell
growth was found to be 500 µg/mL of aqueous extract of
K. striatum followed by 500 µg/mL of methanolic extract
of same species, with 53.5 and 43.7% of growth inhibition,
respectively. These differences in antitumor activities may
be attributed to their different molecular weights, charge
characteristics and monosaccharide distributions (Dias et
FIGURE 1. Percentage of growth inhibition of HeLa cell lines in the presence of 90%
methanolic, 70% acenotic and aqueous extracts of K. alvarezii strain Crocodile.
Data points show the mean ± SE for a minimum of three experiments
% Growth Inhibition
FIGURE 2. Percentage of growth inhibition of HeLa cell lines in the presence of
90% methanolic, 70% acenotic and aqueous extracts of K. alvarezii strain Giant.
Data points show the mean ± SE for a minimum of three experiments
% Growth Inhibition
FIGURE 3. Percentage of growth inhibition of HeLa cell lines in the presence of
90% methanolic, 70% acenotic, and aqueous extracts of K. alvarezii strain Brown.
Data points show the mean ± SE for a minimum of three experiments
% Growth Inhibition
FIGURE 4. Percentage of growth inhibition of HeLa cell lines in the presence of
90% methanolic, 70% acenotic and aqueous extracts of K. striatum. Data points
show the mean ± SE for a minimum of three experiments
% Growth Inhibition
Previous studies reported that alcoholic extracts
from plant samples exhibited several bioactivities such as
adaptogenic, anti-ammatory, anticonvulsant, sedative,
androgenic and immunopromoting activities (Xu et al.
1992). This might be the reason why methanolic extracts
from Kappaphycus seaweeds generally showed positive
growth inhibition to the HeLa cell lines as compared
with acetonic and aqueous extracts. Studies by Shao et al.
(1996) also reported that alcoholic extract from asparagus
shoots exhibited antitumor activities and Singh et al. (1992)
reported their fruit to be the source of bile-stimulating agent.
Reports from World Intellectual Property Organization
(2010) also indicated that methanolic extracts from various
seaweed species have demonstrated cytotoxic effect on
human cancer cell lines including HeLa, MCF-7 and MDA-
MB-231. Alcohol is found to be effective to extract active
compounds such as biophenols, lipids, saccharides, minerals
and small peptides due to their polarity. The potential
bioactive compounds in seaweed may interact with special
cancer associated receptors or cancer specic molecules to
trigger the mechanisms leading to cancer cell death.
Previous researches show that acetone-water mixtures
are good solvent systems for the extraction of polar
antioxidants (Lu & Yeap Foo 1999; Luximon-Ramma et
al. 2005; Sun 2002). Literature also describes that acetone
and water extracts of plant owers presented the best total
phenolic content (Liu et al. 2009). Nyenje and Ndip (2011)
suggested that an organic solvent, in particular, acetone
is a good solvent as it extracts more active compounds
from plant material. Flavonoids and steroids have also
been reported to be extracted using acetone according to
Abdulmalik et al. (2011) and Eloff (1998). Besides, van
Slambrouck et al. (2007) demonstrated that crude aqueous
extracts of L. tridentata (Creosote Bush) and J. communis
L. (Juniper Berry) have signicantly decreased the growth
of MCF-7/AZ breast cancer cells. Traditional medicines
are often prepared by water extraction, but water-soluble
impurities present challenges for conventional isolation
methods, such as chromatography or crystallization (Bart
2011). Water preferentially extracts polar compounds but
they need some special post treatment such as ion exchange
or caustic wash for further purication (Jones & Kingkorn
Further studies are suggested to identify the specic
anti-tumoral compounds in the targeted extracts.
Purication can be carried out to obtain the bioactive
compounds for the development of cancer therapy.
Besides, identication of specic metabolites such as
carotene, bromophenols and carrageenan from seaweeds
is also recommended for the discovery of potential anti-
proliferative or anticancer compounds.
The authors wish to thank the Ministry of Education (MOE),
Malaysia for funding the research under the Fundamental
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& Wilson Thau Lym Yong*
Biotechnology Research Institute
Universiti Malaysia Sabah, Jalan UMS
88400 Kota Kinabalu, Sabah
Delbora Fenny Vittal
School of Science and Technology
Universiti Malaysia Sabah, Jalan UMS
88400 Kota Kinabalu, Sabah
*Corresponding author; email: firstname.lastname@example.org
Received: 10 March 2013
Accepted: 24 April 2014
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