European Journal of Scientific Research

ISSN 1450-216X Vol.37 No.3 (2009), pp.376-387

EuroJournals Publishing, Inc. 2009
http://www.eurojournals.com/ejsr.htm

In Vitro Antioxidant and Antiproliferative Activities of Three

Malaysian Sea Cucumber Species
Osama Y. Althunibat
Corresponding Author,Kulliyyah of Science, International Islamic University Malaysia
Jalan Istana, Bandar Indera Mahkota, 25200, Kuantan, Pahang, Malaysia
E-mail: thnibatjust2001@yahoo.com
Ridzwan Bin Hashim
Kulliyyah of Science, International Islamic University Malaysia, Jalan Istana
Bandar Indera Mahkota, 25200, Kuantan, Pahang, Malaysia
Muhammad Taher
Kulliyyah of pharmacy, International Islamic University Malaysia, Jalan Istana
Bandar Indera Mahkota, 25200, Kuantan, Pahang, Malaysia
Jamaludin Mohd. Daud
Kulliyyah of Science, International Islamic University Malaysia, Jalan Istana
Bandar Indera Mahkota, 25200, Kuantan, Pahang, Malaysia
Masa-Aki Ikeda
Graduate School, Tokyo Medical and Dental University, 1-5-45, Yushima
Bunkyo-ku, Tokyo 113-8549, Japan
Zali B.I
Lembaga Ikan Malaysia, Kuantan,Pahang, Malaysia
Abstract
The antioxidant and antiproliferative activities of aqueous and organic extracts from
three sea cucumber species, Holothuria scabra, Holothuria leucospilota and Stichopus
chloronotus were studied. The result showed that aqueous extract of H. leucospilota
contains the highest level of total phenols (9.7 mg GAE/g), while the lowest level was
obtained from organic extract of H. scabra (1.53 mg GAE/g). An aqueous extract of S.
chloronotus was the most efficient extract in scavenging of DPPH free radical, giving IC50
= 2.13 mg/ml. The aqueous extracts (50mg/ml) of H. scabra, H. leucospilota and S.
chloronotus exhibited high antioxidant activities (77.46%, 64.03% and 80.58%,
respectively) against linoleic acid free radical. Only an aqueous extract of S. chloronotus
inhibited growth of C33A (human cervical cancer) and A549 (human non-small lung
carcinoma) cancer cells, giving IC50 equal to 10.0 g/ml and 28.0 g/ml, respectively. On
the other hand, the dose dependent antiproliferative effects of all organic extracts studied
were observed on C33A and A549 cancer cells. This study, therefore, revealed that all the
three species of Malaysian sea cucumber are potential sources of natural antioxidant and
anticancer agents.

In Vitro Antioxidant and Antiproliferative Activities of Three Malaysian Sea Cucumber Species

377

Keywords: Sea cucumber, Antioxidant, Antiproliferation.

1. Introduction
Sea cucumber is a marine invertebrate of the phylum Echinoderm and the class Holothuroidea, which
is found on the sea floor worldwide. There are about 45 identified sea cucumber species in Malaysia
water. Some of these species are being used in traditional medicine to treat wound, eczema, arthritis or
hypertension (Ridzwan, 2007). Several studies have shown multiple biological activities of sea
cucumber species. These include wound healing promoters and exhibiting antinociceptive,
antibacterial, antifungal and antioxidant properties (Ridzwan et al., 1995; Fredalina et al., 1999; Hawa
et al., 1999, Ridzwan et al., 2001, Hing et al., 2007). They are also rich in saponin glycosides that
exhibit anticancer activity (Kaswandi et al., 2004).
A number of published reports have revealed that some of sea cucumber species contain several
potentially antiproliferative compounds. Ogushi et al., (2005) demonstrated that a high molecular
fraction of hot water extracts from sea cucumber, Stichopus japonicus, inhibited the growth of human
colon adenocarcinoma cells in a dose dependent manner and concluded that the observed decreased in
the growth of Caco-2 cells further exposure to S. japonicus extract resulted from induction of apoptosis
Ogushi et al., (2006). In addition, the antitumor and antiangiogenic effects of sulfated saponins and
triterpene glycosides which were partially purified from sea cucumber species were also reported (Tian
et al., 2005; Tong et al., 2005).
Despite of several worldwide studies that have revealed the efficacy of some sea cucumber
species as potential sources of anticancer and antioxidant compounds, there is lack in information
about levels of these activities in most Malaysian sea cucumber species. Therefore, this study was
propose to investigate the antiproliferative and antioxidant activities of extracts from three Malaysian
sea cucumber species; i.e., Holothuria scabra, Holothuria leucospilota and Stichopus chloronotus as
part of a research hopes to find new potential anticancer compounds in Malaysian sea cucumbers.

2. Methods
2.1. Chemicals
Sodium carbonate, Folin-ciucalteu phenol reagent, -tocopherol gallic acid, free radical 2,2 diphenyl 1picrylhydrazyl (DPPH), Linoleic acid, Tween 20, -carotene and Trypsin-EDTA were purchased from
Sigma (St. Louis, MO, USA). Fetal bovine serum (FBS), Dulbecco`s modified Eagle medium and
Penicillin streptomycin were from Gibco Invitrogen Co. (Scotland, UK). Methanol, Dichloromethan,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and trypan blue were from
Merck KGaA, Germany.
2.2. Sample Collection and Extraction
Samples of the three sea cucumber species ie, Holothuria scabra, Holothuria leucospilota and
Stichopus chloronotus were collected from Terengganu coastal areas in Malaysia. The samples were
dissected to remove internal organs, and packed immediately with ice prior sending to the lab and kept
at 80C until extracted. The taxonomic identity of the samples was confirmed by Prof. Dr. Ridzwan
Bin Hashim from the Department of Biomedical Science, International Islamic University Malaysia.
Voucher specimens have been deposited at the museum in the Faculty of Science, International Islamic
University, Malaysia.
Bioactive compounds were extracted as a function of their polarity using water and organic
solvents according to method of Rashid et al. (2001) with slight modification. About 200 g of frozen
samples were homogenized with deionized water. The mixture was continuously stirred in the dark at 4
C for 24 h. Then it was centrifuged at 5000 rpm for 15 min, the supernatant was collected and
filtrated. The collected aqueous extracts were freeze-dried and kept at -80 C, while the insoluble solid
materials were re-extracted with MeOHCH2Cl2 (1:1 v/v), followed by methanol (100%). The organic

378

Osama Y. Althunibat, Ridzwan Bin Hashim, Muhammad Taher,

Jamaludin Mohd. Daud, Masa-Aki Ikeda and Zali B.I

extracts were combined and the solvents removed by rotary evaporation at 40 C under low pressure to
avoid degradation of compounds. The yield of each extract was calculated as weight of dried extract to
weight of frozen sample (Table 1).
2.3. Evaluation of Extracts Antioxidant Activities
2.3.1. Determination of Total Phenols
Total phenolic contents of aqueous and organic extracts were assessed approximately by using the
FolinCiocalteau phenol reagent according to methods of Mamelona et al. (2007) with some
modifications. 0.1 ml of extracts (50 mg/ml) was transferred into the test tubes and their volumes made
up to 0.5 ml with distilled water. After addition of 0.25 ml FolinCiocalteu reagent diluted with
distilled water (1:10 v/v) and 1.25 ml of 20% aqueous sodium carbonate solution, tubes were vortexed.
The absorbance of blue colored mixtures recorded after 40 min at 725nm against a blank containing
same mixture except FolinCiocalteu reagent replaced with distilled water. The total phenolic contents
were expressed as gallic acid equivalents (GAE) in milligrams per gram of extract, using a standard
curve generated with 10 100 g of gallic acid. All determinations were performed in triplicate.
2.3.2. DPPH Scavenging Assay
Antioxidant activity of the extracts was measured in terms of radical scavenging ability by using the
stable radical 1,1-diphenyl-2-picrylhydrazyl (DPPH), as described by Blois (1958). 0.1 ml of different
concentrations of extracts and standards (-tocopherol) were taken in different test tubes. 2.5 ml of 0.1
mM methanolic solution of DPPH was added to these tubes and shaken vigorously. The tubes were
allowed to stand at room temperature for 20 minutes. The control was prepared as above using the
vehicles (distilled water or DMSO) instead of the extract. 100 l of extracts in 2.5 ml methanol was
used as blank. The changes in the absorbance of the samples were measured by spectrophotometer at
517 nm. Radical scavenging activity was expressed as the inhibition percentage and was calculated
using the following formula:
% radical scavenging = (Control OD - Sample OD/Control OD)* 100
Antioxidant capacity was expressed as IC50; extract concentration (mg/ml) that required
scavenging 50% of DPPH. All measurements were carried out in triplicate.
2.3.3. -Carotene Bleaching Assay
Total antioxidant activity of sea cucumber extracts and -tocopherol was measured according to the
method of Jayaprakasha et al. (2001) with some modifications. One mililitre of -carotene solution
(0.2 mg/ml chloroform) was pipetted into a round-bottom flask (100 ml) containing 0.02 g of linoleic
acid and 0.2 g of 100% Tween 20. The mixture was then evaporated at 40C for 10 min by using a
rotary evaporator to remove chloroform. After evaporation, an emulsion was immediately formed by
dilution and vigorously agitation of the mixture with 100 ml of distilled water. 2.5 ml aliquots of the
emulsion were transferred into different test tubes containing 0.15 ml of samples (50 mg/ml), tocopherol (50g/ml) or vehicles as a control. The tubes were then gently mixed and placed at 45C in
a water bath for 3 h. Absorbance of the samples was measured at 470 nm at initial time (t= 0) against a
blank, consisting of a mixture of samples with emulsion without -carotene. The measurement was
carried out at 30 min intervals. All determinations were performed in triplicate.
Antioxidant activity (AA) was measured in terms of inhibition of successful bleaching of carotene by using the following formula:
AA = (1 [(A0 - At) / (A0 - At )]) * 100
where A0 and A0 are the absorbance values measured at initial time of the incubation for samples and
control, respectively, while At and At are the absorbance values measured in the samples and control
at t = 180 min.

In Vitro Antioxidant and Antiproliferative Activities of Three Malaysian Sea Cucumber Species

379

2.4. Evaluation of the Extracts Antiproliferative Effects

2.4.1. Cell Cultures
Two human cancer cell lines; C33A (human cervical cancer) and A549 (human non-small lung
carcinoma); were kindly provided by Dr. Masa-aki Ikeda; Department of Molecular and Carniofacial
Embryology, Tokyo Medical and Dental University; were maintained in DMEM medium (with high
glucose and glutamine) supplemented with 10% heat inactivated FBS and 1% penicillin streptomycin,
at 37C in a humidified atmosphere containing 5% CO2.
2.4.2. MTT Assay
The 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to evaluate
the antiproliferative activities of the tested extracts against the cancer cell lines. The assay depends on
the cleavage of the tetrazolium salt (MTT) into formazan blue by the mitochondrial enzyme succinate
dehydrogenase. The conversion takes place only in living cells and the amount of formazan produced
is proportional to the number of viable cells present. Thus, the MTT assay is potentially useful for
assaying antiproliferative activities of materials.
For this purpose the cancer cells were seeded in complete medium in a 96-well plate at a
density of 1x105 cells/ml. After reaching confluent, the cells were incubated with different
concentrations of the sample (900- 3g/ml) for 48 hr. DMSO and PBS (the vehicles) were used as
controls. The medium was then discarded and the adherent cells were washed twice with phosphate
buffer solution (PBS), then 20 l of MTT stock solution (5 mg/ml in PBS) were added to each well and
the plates were further incubated overnight at 37 C. 100 l of DMSO were added to each well to
solubilize the formazan crystals produced by viable cells. After complete dissolving of formazan blue,
the absorbance was measured at 570 and 690 nm, as reference wavelength, using Versa MaxTM
Tunable microplate reader. The percentage of cell viability was calculated according to the equation
described in Moongkarndi et al. (2004).
% of cell viability = (OD of treated cells / OD of control cells) 100
The concentrations required for inhibition of 50% of cell viability (IC50) were calculated.

3. Results
3.1 Total Phenols
The mean values of total phenols in extracts varied from 1.53 to 9.7 mg GAE/g of extract, depending
on species and extracts involved (Table 1). Aqueous extract of H. leucospilota showed the highest
level of total phenols (9.7 mg GAE/g), while the lowest level was obtained from organic extract of H.
scabra (1.53 mg GAE/g). There were more concentrated total phenols in aqueous extract of all species
compared to their organic extract. However, the total phenols of aqueous extracts varied from 4.85 mg
GAE/g of H. scabra to 9.7 mg GAE/ g of H. leucospilota extract with mean value of 7.61 2.49. On
the other hand, the mean values of total phenols of organic extracts varied from 1.53 GAE/g of H.
scabra to 2.91 mg GAE/ g of H. leucospilota with average of 2.03 0.76.
3.2. DPPH Scavenging Activity
The antioxidant activity of extracts was evaluated by their ability to scavenge free radicals by using
DPPH assay. The extract concentration that caused scavenging of 50% of DPPH (IC50) was detected.
As shown in (Table 2), IC50 of extracts varied from 2.01 mg/ ml to out of range of the examined
concentrations (>10 mg/ml) giving significantly lower potencies than -tocopherol (IC50 = 9.02
g/ml). Generally, aqueous extracts exhibited higher DPPH scavenging capacity than organic extracts.
Aqueous extract of S. chloronotus showed the highest antioxidant activity among all extracts by using
DPPH assay, with IC50 equal to 2.13 mg/ml. Both aqueous and organic extracts of H. scabra in

380

Osama Y. Althunibat, Ridzwan Bin Hashim, Muhammad Taher,

Jamaludin Mohd. Daud, Masa-Aki Ikeda and Zali B.I

addition to organic extract of S. chloronotus couldnt scavenge 50% of DPPH even at high
concentration (>10mg/ml).
3.3. Inhibition of -carotene Bleaching
The antioxidant activity of sea cucumber extracts was also detected by -carotene-linoleate model
system and compared with -tocopherol activity (Table 2). Antioxidant activities of extracts in this
system varied from 35.92 to 80.58%. Aqueous extracts of the three tested species have shown higher
activities than the organic extracts. The mean values of antioxidant activity of aqueous extracts were
from 64.3 to 80.58 %, with average of 74.02 8.79 %, while the organic extracts were from 35.92 to
73.87 %, with average of 55.21 18.98 %. As shown in Fig.1, aqueous extracts of H. scabra, S.
chloronotus and H. leucospilota exhibited high antioxidant activities (77.46, 80.58 and 64.03 %,
respectively) and gave reaction kinetics curves against -carotene similar to each other and close to tocopherol kinetics curves (A.A = 86.87 %). On the other hand, organic extracts of H. scabra and H.
leucospilota showed lower antioxidant activities in comparison with -tocopherol and S. chloronotus
(A.A = 73.87 %) which gave reaction kinetics curve close to -tocopherol kinetics curve (Fig. 2).
Table 1:

Sea Cucumber Species Used in the Study

Species
Voucher Specimens % Yield of Aqueous Extracts*
4.03
Holothuria scabra
Tg4
4.29
Holothuria leucospilota
Tg8
5.25
Stichopus chloronotus
Tg7
*%Yield = (weight of dried extract / weight of frozen sample) *100

Table 2:

% Yield of Organic Extracts*

0.25
0.25
0.17

Total phenolic contents, DPPH scavenging capacities and antioxidant activities of three Malaysian
sea cucumber species

Species
Holothuria scabra
Holothuria leucospilota
Stichopus chloronotus

Extracts
Aqueous
Organic
Aqueous
Organic
Aqueous
Organic

Inhibition of -Carotene
Total Phenols
Bleaching (%Antioxidant
(GAE)***
Activity)**
> 10
77.46 5.16
4.85 0.04
> 10
35.92 2.87
1.53 0.05
3.91 0.12
64.03 6.24
9.7 0.09
5.44 0.15
55.85 3.38
2.91 0.10
2.13 0.05
80.58 4.92
8.27 0.04
> 10
73.87 3.04
1.66 0.11
0.0090 0.0004
86.87 2.88
ND
of extract that required for scavenging of 50% of DPPH) in mg extract/ ml
DPPH Scavenging
Capacity (IC50)*

-tocopherol
*Data expressed as IC50 (the concentration
DPPH, mean SD (n = 3).
**Data expressed as antioxidant activity (A.A) according to formula in methods, mean SD (n = 3).
***Data expressed as gallic acid equivalents (GAE) mg/g extract, mean SD (n = 3).

3.4. Antiproliferative Effects

The effects of aqueous and organic extracts of the three sea cucumber species on growth of two human
cancer cell lines, i.e. human non-small lung carcinoma (A549) and cervical cancer cells (C33A), were
evaluated by MTT assay. Curves of percentage viability of treated cells were plotted against extracts
concentrations. The inhibitory concentrations required to reduce 50% of cell viability (IC50) as
compared to untreated control were detected.
The dose dependent decreasing in the percentage of viability of treated cancer cells comparing
to controls was represented in the Figures 3-6. As shown in Figures 3 and 4, among the three aqueous
extracts, only S. chloronotus exhibited antiproliferative effects against the cancer cells. However, S.
chloronotus aqueous extract was more toxic against C33A cells (IC50 = 10.0 g/ml) than A549 (IC50 =

In Vitro Antioxidant and Antiproliferative Activities of Three Malaysian Sea Cucumber Species

381

28.0 g/ml) while the aqueous extracts of H. scabra and H. leucospilota had no obvious effect on the
growth of the cancer cells in concentration range presented. On the other hand, all organic extracts
inhibited growth of A549 (Fig. 5) and C33A (Fig. 6) cell lines in different degree. The organic extract
of H. scabra exhibited the highest antiproliferative effects against both C33A and A549 cell lines,
giving IC50 equal to 3.0g/ml and 15.5g/ml, respectively. In addition S. chloronotus organic extract
was highly cytotoxic against C33A cells giving IC50 = 6.0 g/ml but less activity against A549 cells
(IC50 = 21.0 g/ml). From the curves presented in the Figures 5 and 6, the IC50 values of H.
leucospilota organic extract against A549 (93.5 g/ml) and C33A (38.5 g/ml) were higher than the
other extracts, indicating that it has less activity than organic extracts of both H. scabra and S.
chloronotus.
Figure 1: Reaction kinetics of Malaysian sea cucumber aqueous extracts (50 mg/ml) with -carotene in
compression with -tocopherol (5 mg/ml) and negative control. Each value represents mean of three
independent experiments SD.

Figure 2: Reaction kinetics of Malaysian sea cucumber organic extracts (50 mg/ml) with -carotene in
comparison with -tocopherol (5 mg/ml) and negative control. Each value represents mean of three
independent experiments SD.

382

Osama Y. Althunibat, Ridzwan Bin Hashim, Muhammad Taher,

Jamaludin Mohd. Daud, Masa-Aki Ikeda and Zali B.I

Figure 3: Dose-dependent antiproliferative effects of aqueous extracts from three Malaysian sea cucumber
species; Holothuria scabra, Holothuria leucospilota, Stichopus chloronotus; against C33A cell.
Each value represents mean of three independent experiments SD.

Figure 4:

Dose-dependent antiproliferative effects of aqueous extracts from three Malaysian sea cucumber
species; Holothuria scabra, Holothuria leucospilota, Stichopus chloronotus; against A549 cell.
Each value represents mean of three independent experiments SD.

In Vitro Antioxidant and Antiproliferative Activities of Three Malaysian Sea Cucumber Species

383

Figure 5: Dose-dependent antiproliferative effects of organic extracts from three Malaysian sea cucumber
species; Holothuria scabra, Holothuria leucospilota, Stichopus chloronotus; against C33A cell.
Each value represents mean of three independent experiments SD.

Figure 6: Dose-dependent antiproliferative effects of organic extracts from three Malaysian sea cucumber
species; Holothuria scabra, Holothuria leucospilota, Stichopus chloronotus; against A549 cell.
Each value represents mean of three independent experiments SD.

4. Discussion
Free radicals; mainly the reactive oxygen species (ROS); are involved in initiation, promotion and
progression of carcinogenesis (Ray and Husain, 2002). ROS induce oxidative damage of DNA and
other cellular components leading to cancer related mutations (Valko et al., 2004). Consequently,
antioxidants play an important role in the protection of human body against damage by reactive oxygen
species (Govindarajan, 2005). Furthermore previous epidemiological studies have shown that the
intake of natural antioxidants has been associated with reduced risks of cancer and other diseases
associated with oxidative damages (Rietveld and Wiseman, 2003). Accordingly, the present study was

384

Osama Y. Althunibat, Ridzwan Bin Hashim, Muhammad Taher,

Jamaludin Mohd. Daud, Masa-Aki Ikeda and Zali B.I

conducted to evaluate antioxidant and anticancer properties of extracts from three Malaysian sea
cucumber species, Holothuria scabra, Holothuria leucospilota and Stichopus chloronotus.
Two different free radical systems were used to evaluate antioxidant activities of the tested
extracts, stable radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) and linoleic acid free radical mediated
-carotene bleaching. DPPH produces a violet solution in methanol and it is reduced in the presence of
antioxidant molecules, giving rise to no color, which has been used to evaluate the antioxidant activity
of the extracts (Blois, 1958; Abdille et al., 2005). In free radical mediated -carotene bleaching system,
linoleate-free radical is formed by abstraction of hydrogen atom from one of its diallylic methylene
groups. The radical formed then oxidizes the double bonds of -carotene molecules and resulting in loss
of their characteristic orange color. The presence of antioxidant molecules can neutralize linoleate-free
radical and then delay the extent of -carotene bleaching (Jayaprakasha et al., 2001).
Findings of this study revealed that Malaysian sea cucumbers as promising sources of natural
antioxidants. However, extracts from different species showed varying degrees of free radical
scavenging ability by using both free radical systems, where almost aqueous extracts found to be more
efficient than organic extracts. Antioxidant properties of natural extracts are generally ascribed to
redox reactions with some bioproducts present in the extracts, including phenolic compounds, salt,
sugars, carotenoids, ascorbic acid, glutathione, peptides in addition to some pigments such as gadusol.
(Prior et al., 1998, Bandaranayake and Des Rocher, 1999, Ehlenfeldt and Prior, 2001). These
compounds may neutralize free radicals by acting as rapid donators of a hydrogen atom to radicals.
However, our results come in support to the antioxidant potential previously observed in ceolomic
fluid from other sea cucumber Malaysian species (Hawa et al., 1999).
The main sources of sea cucumbers food are phenolic rich materials such as phytoplankton and
particles derived from degrading marine macro-algae. Therefore, it is expected to find phenolic compounds
in tissue of sea cucumbers. However, levels of total phenols and flavonoids from Atlantic sea cucumber,
Cucumaria frondosa, have been reported by Mamelona et al., (2007). Our method for extraction of the
phenols and other antioxidant compounds involved extraction of polar (hydrophilic) compounds with water
first. Thereafter, the non soluble compounds (hydrophobic) were successively extracted with organic
solvents with different polarities. Tested extracts were found to have various amounts of total phenolic
compounds according to species and solvent used. Concentrations observed in aqueous extracts were
significantly higher compared to those observed in organic extracts which means most of phenolic
compounds in sea cucumber are hydrophilic compounds. These findings agreed with previous study by
Mamelona et al., (2007), where aqueous extract of muscle and gonads of Atlantic sea cucumber were found
to have slightly higher phenolic compounds than ethyl acetate extracts.
The results of this study showed variation in level of scavenging capacity of individual extract
against different testing radicals. This difference may be explained by the different mechanisms
involved in the radical-antioxidant reactions. Other factors, such as selectivity of the radicals or the
solubility of extracts in different testing systems, may also affect the capacity of extract to react and
quench different radicals. These results were in accordance with others that marine natural products
were conflicting in activities against different radicals (Takamatsu et al., 2003;; Moncheva, et al.,
2004).
A weak correlations were found between total phenols and free radical scavenging capacities in
tested extracts by using DPPH and -carotene bleaching methods (R2 = 0.31 and 0.1 respectively).
This demonstrated a low contribution of phenolic compounds in antioxidant properties of sea
cucumber extracts comparing with other possible molecules such as carotenoids, ascorbic acid,
glutathione and gadusol. However, contribution of phenols to radical scavenging capacity was found to
be fluctuated among chemical species and depends upon their concentration (Zheng and Wang, 2003).
Inhibition effect of the extracts from the three sea cucumber species on the growth of two
cancer cell lines, i.e. A549 and C33A, was demonstrated by using MTT assay. The variation in the
activity among the species was indicated by determining the IC50 of each extract against the particular
cell line. Among Aqueous extracts only S. chloronotus exhibited antiprolifrative effects against A549

In Vitro Antioxidant and Antiproliferative Activities of Three Malaysian Sea Cucumber Species

385

and C33A cells, giving IC50 equal to 28.0 and 10.0 g/ml, respectively, whereas in organic extracts, H.
scabra was found to be the most active cytotoxic extract against both A549 and C33A cells, giving
IC50 equal to 15.5 and 3.5g/ml.
The strong growth inhibitory activity found in aqueous extract of S. chloronotus resulted from
hydrophilic antiprolifrative compounds which might be not available or in lower levels in H.
leucospilota and H. scabra extracts. However, Rodriguez et al. (1991) isolated five triterpene
glycosides from sea cucumber species, Holothuria forskalii, and reported their anticancer and antiviral
activities. As triterpene glycosides are water soluble compounds, therefore, they might be the main
active compounds in the aqueous extract of S. chloronotus. Furthermore, Adrian and Collins (2005)
manage to extract the anticancer compounds, frondoside A and B, from aqueous fraction of Cucumaria
frondosa extract.
Organic extracts of the three species have exhibited antiprolifration effects against the cancer
cell lines. Since organic extracts in this study were mainly hydrophobic compounds, they could be rich
with sphignoid bases which have been found to have anticancer properties (Sugwara et al. 2006). The
present study found that C33A cell line was more sensitive to the active extracts than A549 cells. This
is in agreement with Itharat et al. (2004) who reported that different cell lines exhibited different
responses to the cytotoxic compounds.

5. Conclusion
The results from this study show that the levels of natural antioxidant and antiproliferative compounds
vary among species of Malaysian sea cucumbers. Aqueous extracts from tested samples showed higher
antioxidant activities than organic extracts, meaning that most of antioxidant products were hydrophilic
chemical nature. In contrast, antiproliferative activity was found to be higher in organic extracts than
aqueous extracts. These findings have encouraged us to continue our work to determine the active
ingredients. Therefore further isolation and identification of active structures from the active extracts is
in progress. In addition, further investigations are needed to clarify intracellular pathways involved in
the mechanism of the growth inhibition activity.

6. Acknowledgement
Authors are thankful to the Research Management Center, IIUM for the funding this project (EDW B
0902-210). Dr. Shahbudin Saad (INOCEM), Mr. Mohamad Salleh and Miss Nor Azira Johari for their
technical support.

References
[1]
[2]
[3]
[4]
[5]

Abdille, M. d., Singh, R, Jayaprakasha, G., Jena, B. (2005) Antioxidant activity of the extracts
from Dillenia indica fruits. Food Chemistry 90: 891896.
Adrian TE, Collins P. 2005. Anticancer Glycoside Compounds. United State patent.
WO/2005/072528.
Bandaranayake, W. M., Des Rocher, A. (1999).Role of secondary metabolites and pigments in
the epidermal tissues, ripe ovaries, viscera, gut contents and diet of the sea cucumber
Holothuria atra. Marine Biology 133: 163-169.
Blois, M. S. (1958). Antioxidants determination by the use of a stable free radical. Nature,
4617: 11991200.
Ehlenfeldt, M. K., Prior, R. L. (2001). Oxygen radical absorbance capacity (ORAC) and
phenolic and anthocyanin concentrations in fruit and leaf tissues of highbush blueberry. Journal
of Agricultural and Food Chemistry, 49, 22222227.

386
[6]
[7]
[8]
[9]

[10]
[11]
[12]

[13]
[14]

[15]

[16]
[17]
[18]
[19]
[20]
[21]

Osama Y. Althunibat, Ridzwan Bin Hashim, Muhammad Taher,

Jamaludin Mohd. Daud, Masa-Aki Ikeda and Zali B.I
Fredalina, B. D., Ridzwan, B. H., Zainal Abdin, A.A., Kaswandi, M.A., Zaiton, H., Zali, I.,
Kittakoop, P. and Jais, A.M. (1999). Fatty acid composition in local sea cucumber, Stichopus
chloronotus for wound healing. Gen. pharmacol.,33: 337-340.
Govindarajan, R., Vijayakumar, M., Pushpangadan, P. (2005). Antioxidant approach to disease
management and the role of Rasayana herbs of Ayurveda. Journal of Ethnopharmacology 99:
165178.
Hawa, I., Zulaikah, M., Jamaludin, M., Zainal Abidin, A. A., Kaswandi, M. A., & Ridzwan, B.
H. (1999). The potential of the coelomic fluid of sea cucumber as an antioxidant. Malaysian
Journal of Nutrition, 5, 5559.
Hing, H.L., Kaswandi, M. A., Azraul-Mumtazah, R., Hamidah,S.A.,Sahalan, A. Z.,
Normalawati S., Samsudin, M. W. and Ridzwan, B. H. (2007). Effect of Methanol Extracts
from Sea Cucumbers Holothuria edulis and Stichopus chloronotus on Candida albicans.
Microsc Microanal 13(Suppl 2).
Itharat, A., Houghton,P.J., Eno-Amooquayec, E., Burke, P.J., Sampson, J. H., Raman,
A.,(2004). In vitro cytotoxic activity of Thai medicinal plants used traditionally to treat cancer.
Journal of Ethnopharmacology .90: 3338
Jayaprakasha GK, Singh RP & Sakariah KK (2001). Antioxidant activity of grape seed (Vitis
vinifera) extracts on peroxidation models in vitro. Food Chem 73: 285-290.
Kaswandi M.A. Hing H.L.,Sahalan A.Z., Farah F., Ridzwan B.H., Samsudin M.W., Yasin
M.S.M and Ali A. (2004). Saponin from sea cucumber Stichopus badionotus sluiter as potential
cytotoxic agent on CEM-SS T-lymphoblastic cell. Journal of Microscopy Society of Thailand
18: 79-84.
Mamelona, J., Pelletier, E.M., Lalancette, K.G., Legault,J., Karboune, S. and Kermasha,
S.(2007). Quantification of phenolic contents and antioxidant capacity of Atlantic sea
cucumber, Cucumaria frondosa. Food Chemistry 104 10401047.
Moncheva, S. Trakhtenberg, S. Katrichc, E. Zemserd, M. Gosheve, I., Toledof, F., ArancibiaAvilaf, P., Donchevaa, V., Gorinsteind, S.,(2004). Total antioxidant capacity in the black
mussel (Mytilus galloprovincialis) from Black Sea coasts. Estuarine, Coastal and Shelf Science
59: 475-484.
Moongkarndi P, Kosem N, Kaslungka S,Luanratana O, Pongpan N, Neungton N. 2004.
Antiproliferation, antioxidation and induction of apoptosis by Garcinia mangostana
(mangosteen) on SKBR3 human breast cancer cell line. Journal of Ethnopharmacology 90:161
166.
Ogushi, M., Yoshie-stark, M. and Suzuki, T.,(2005). Cytostatic Activity of Hot Water Extracts
from the Sea Cucumber in Caco-2. Food Sci. Technol. Res., 11, 2, 202-206.
Ogushi, M., Yoshie-stark, M., and Suzuki, T., (2006).Apoptosis-inducing Activity of Hot
Water Extracts from the Sea Cucumber in Human Colon Tumor Cells. Food Sci. Technol. Res.,
12 (4): 290-294.
Prior, R. L., Cao, G., Martin, A., Sofic, E., McEwen, J., OBrien, C., et al. (1998). Antioxidant
capacity as influenced by total phenolic and anthocyanin content, maturity, and variety of
Vaccinum species. Journal of Agricultural and Food Chemistry, 46, 2686 2693.
Rashid, M. A., Gustafson, K. R., Cartner, L. K., Pannell, L. K., and Boyd, M. R. (2001) New
nitrogenous constituents from the South African marine ascidian Pseudodistoma sp.
Tetrahedron 57, 57515755.
Ray G, Husain SA. 2002. Oxidants, antioxidants and carcinogenesis. Indian J Exp Biol, 40(11):
1213-32.
Ridzwan BH .2007. Sea Cucumber: The Malaysian Heritage. Research Centre, IIUM. Kuala
Lumpur, Malaysia.

In Vitro Antioxidant and Antiproliferative Activities of Three Malaysian Sea Cucumber Species
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]

387

Ridzwan, B. H. Kaswandi, M. A., Azman, Y. and Fuad, M. (1995). Screen for antibacterial
agents in three species of sea cucumber from coastal areas of sabah. Gen. pharmacol., 26:15391543.
Ridzwan, B.H., Zarina M.Z, Nadirah, M., Kaswandi, M.A. and Fuad, M. (2001). The
antinociceptive effects of extracts from Stichopus chloronotus Brandt. Pakistan J. Biol. Sc.
(4(3):244-246).
Rietveld, A., Wiseman, S. (2003). Antioxidant effects of tea: evidence from human clinical
trials. J. Nutr. 133, 3285S 3592S.
Rodriguez J, Castro R, Riguera R. 1991. Holothurinosides: new anti-tumour non sulphated
triterpenoid glycosides from the sea cucumber Holothruia forskalii. Tetrahedron 47:4753-4762.
Sugwara. T., Zamia, N., Yamamoto, A., Sakai, S., Noguchi, R., Hiraia, T., 2006. Isolation of
shignoid bases of sea cucumber cerebrosides and their cytotoxic activity against colon cancer
cells. Bioscience, Biotechnology and Biochemistry. 2906-2912.
Takamatsu, S., Hodges, T.W., Rajbhandari, I., Gerwick, W.H., Hamann, M.T., Nagle, D.G.,
2003. Marine natural products as novel antioxidant prototypes. Journal of Natural Products 66:
605-608.
Tian, F., Zhang, X, Tong, Y., Yi, Y., Zhang, S., Li, L., Sun, P., Lin, L. and Ding, J. (2005). PE,
a new sulfated saponin from sea cucumber, exhibits anti-angiogenic and anti-tumor activities in
vitro and in vivo. Cancer Biol. Ther. 4: 874-882.
Tong, Y., Zhang, X., Tian F., Yi, Y., Xu, Q., Li, L., Tong. L., Lin, L. and Ding, J. (2005).
Philinopside A, a novel marine-derived compound possessing dual anti-angiogenic and antitumor effects. Int J Cancer, 114: 843-853.
Valko M., Izakovic M., Mazur M., Rhodes CJ., and Telser J. (2004) Role of oxygen radicals in
DNA damage and cancer incidence. Molecular and Cellular Biochemistry; 266: 3756.
Zheng W, Wang SY. (2003). Oxygen radical absorbing capacity of phenols in blueberries,
cranberries, chokeberries, and lingonberries. Journal of Agricultural and Food Chemistry, 51,
502509.