Journal of Applied Pharmaceutical Science Vol.

8(10), pp 032-038, October, 2018

Available online at http://www.japsonline.com
DOI: 10.7324/JAPS.2018.81005
ISSN 2231-3354

Evaluation of selected echinoderms from peninsular Malaysia for

cytotoxicity against HepG2 cells, antioxidant and antibacterial
activities, and their metabolites profiling

Yosie Andriani1*, Nurul Hazirah Mat Lazim2,3, Asnuzilawati Asari2, Faridah Mohamad4, Tengku Sifzizul Tengku Muhammad1,
Noraznawati Ismail1,2, Mariam Taib2, Hermansyah Amir5, Aziz Ahmad2, Habsah Mohamad1
1
Institute of Marine Biotechnology, Universiti Malaysia Terengganu, Terengganu, Malaysia.
2
School of Fundamental Science, Universiti Malaysia Terengganu, Terengganu, Malaysia.
3
Department of Chemistry Malaysia (JKM), Jalan Sultan, Selangor, Malaysia.
4
School of Marine and Environmental Sciences, Universiti Malaysia Terengganu, Terengganu, Malaysia.
5
Educational Chemistry Program, Faculty of Teacher Training and Education, Bengkulu University, Bengkulu, Indonesia.

ARTICLE INFO ABSTRACT

Received on: 12/06/2018 Study on the evaluation of Acanthaster planci, Echinaster luzonicus, and Echinothrix calamaris from Peninsular
Accepted on: 19/09/2018 Malaysia for cytotoxicity against human hepatocellular liver carcinoma cells (HepG2), antioxidant and antibacterial
Available online: 31/10/2018 activities were evaluated on their methanol extracts. The cytotoxicity, antioxidant and antibacterial activities were
conducted by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4sulfophenyl)-2H-tetrazolium assay,
1,1-diphenyl-2-picryl hydrazyl free radical scavenging assay, and disc diffusion method, respectively. In addition,
Key words:
thin layer chromatography (TLC) was done to profile the metabolites within the extracts using Dragendorff’s reagent
Acanthaster planci,
to identify the presence of alkaloids metabolite. The cytotoxicity result showed that the treatment of the extracts (100
Echinaster luzonicus,
mg/ml) inhibited the proliferation of HepG2 cells and the IC50 for all extracts exceeded 30 mg/ml indicating that the
Echinothrix calamaris,
extracts were not cytotoxic to the cells. For antioxidant activity, all extracts showed good antioxidant activity with
cytotoxicity, antibacterial,
the IC50 value obtained more than 50%. While, screening of bacterial properties using Gram-positive bacteria strains
antioxidant.
(Staphylococcus aureus, Bacillus cereus, and Micrococcus sp.) and Gram-negative bacteria strains (Escherichia
coli, Salmonella typhimurium, and Pseudomonas aeruginosa) showed that all samples have antibacterial activity
against Micrococcus sp. only. The TLC profiling of A. planci and E. luzonicus showed the presence of alkaloids.
Since the result found that A. planci, E. luzonicus, and E. calamaris have no cytotoxic activity against HepG2 cells
(no IC50 value), further study such as anti-atherosclerosis potential agent can be evaluated. Subsequently, a very good
antioxidant activity of all samples is also good to screen their potency as an anticancer agent against some cancer cells.

INTRODUCTION model chromosome numbers of 55. The cell line was adherent
Hepatocellular carcinoma is one of the common malignant and formed monolayer during cultivation. As an alternative to the
primary neoplasma where malignant tumors of the livers can be current treatment of this disease, many researchers are targeting
primary or secondary (Zakaria et al., 2009). In this study, HepG2 marine resources due to the fact that marine diversity holds the
cells which used were derived from the liver tissue with unique treasure for natural products that have a significant impact on
human healthcare. This study has focused on the organisms from
the phylum Echinodermata which are the A. planci, E. luzonicus,
and E. calamaris. The A. planci and E. luzonicus belong to the
*
Corresponding Author same class, Asteroidea whereas E. calamaris belong to the class
Yosie Andriani, Institute of Marine Biotechnology, Universiti Malaysia Echinoidea (Cleveland et al., 2007). This latter organism has a
Terengganu, Terengganu, Malaysia. E-mail: yosieandriani @ gmail.com
compact body enclosed in an endoskeleton test or shell and it is

© 2018 Yosie Andriani et al. This is an open access article distributed under the terms of the Creative Commons Attribution License -NonCommercial-
ShareAlikeUnported License (http://creativecommons.org/licenses/by-nc-sa/3.0/).
 Andriani et al. / Journal of Applied Pharmaceutical Science 8 (10); 2018: 032-038 033

said to have bioactive compounds in regulating the immune system 2-(4sulfophenyl)-2H-tetrazolium, where only 20 µl of the solution
(Smith et al., 1995). Four classes of echinoderms; Asteroidea, was put in each well and left incubated for one and a half hour in
Crinoidea, Holothuroidea, and Ophiuroidea have been reported to 37°C (adopted from Andriani et al., 2015). The viability of the
exhibit antibacterial and anticancer properties (Bryan et al., 1995; cell was measured using Glomax Multi detection (Promega) at
Cheng et al., 2009; Shamsuddin et al., 2010). These organisms absorbance 490 nm. The graph percentages of inhibition against the
are rich with steroid glycosides, gangliosides, thornasterol, Log10 concentration of crude extracts were plotted using GraphPad
sapogenols, and other branched fatty acid including lectins Prism 4.0 software. The value of IC50, the effective concentration
(Dong et al., 2011; Fleming et al., 1974; Ivanchina et al., 2011; of drug that is required for 50% inhibition, was determined with
Kelly, 2015; Kitagawa et al., 1975). Therefore, based on the potential non-linear regression. The bar chart of the viability of cells against
bioactivity of marine organisms from classes of echinoderms, crude concentration was presented in results.
the aims of this study were to describe the cytotoxicity of the
methanol extracts of selected samples (A. planci, E.luzonicus, and Antioxidant activity
E.calamaris) from East coast of Peninsular Malaysia towards the Antioxidant activity was analyzed using DPPH free
HepG2 cells, to evaluate their antioxidant capacity and antibacterial radical scavenging assay (Andriani et al., 2017) using the
property towards some selected bacteria (S. aureus, B. cereus, Quercetin as a positive control and DMSO as a negative control.
Micrococcus sp, E. coli, S. typhimurium, and P. aeruginosa). The Samples stock were diluted in DMSO and were prepared in
potential metabolites profiling within the methanol extracts were varying concentration by two-fold serial dilution in DMSO with
also identified in this study. concentrations of 10, 5, 2.5, 1.25, 0.625, 0.313, and 0.156 mg/
ml in 96 well plates. DPPH reagent was prepared with 0.04%
MATERIALS AND METHODS w/v) concentration by dissolving 2.37 mg of the DPPH powder
Sample preparation and extraction in 100 ml methanol solution. DPPH solution was shaken and
covered with aluminum foil to minimize the penetration of
The A. planci, E. luzonicus, and E. calamaris were
light. Two hundred microliter of methanolic DPPH solution
collected from the islands in Terengganu in the East Coast of
(6 × 10−5 M) was added to all wells and the mixture was
Peninsular Malaysia via scuba diving in the depth range from 5 to
covered with aluminum foil and incubated for 30 minutes at
10 m. The samples were kept in ice prior to reach the laboratory
room temperature. Then, the absorbance was measured at 517
and furthered with processing samples. The A. planci and E.
nm using Elisa reader (Multiskan Ascent, Thermo Electron
calamaris were separated into two parts which were the outer
Corporation). Free radical scavenging activity was determined
layer (skin) and visceral organs. The whole E. luzonicus was
according to the equation:
used and not separated into parts due to the small size. All the
Free radical scavenging activity was determined
samples were then individually freeze-dried and later ground
according to the equation:
into powder. Furthermore, the powdered samples were extracted
Ac - As
three times with methanol followed by solvent evaporation with a Free radical scavenging activity (%) = × 100%
Ac
rotary evaporator to obtain the crude extract. The weights of the
methanolic extracts were recorded. where AS is the absorbance of the sample and AC is the
absorbance of a negative control.
Reagents
Antibacterial assay
Minimal essential medium (MEM) was purchased from
Santa Cruz (Santa Cruz, CA) and Nacalai (Nacalai Tesque, Kyoto Antibacterial activity was determined against cultures
Japan). While Fetal Bovine Serum, 1% non-essential amino acid, of S. aureus, B. cereus, Micrococcus sp., E. coli, S. typhimurium,
1% sodium pyruvate, and 1% penicillin, streptomycin, and nutrient and P. aeruginosa using disc diffusion test (DDT), modified from
agar (NA) were purchased from Gibco Diagnostics (Madison, Andriani et al. (2017). DDT was used to test the production of
WI). Quercetin, 1,1-diphenyl-2-picryl hydrazyl (DPPH), and antibacterial compounds from the extracts. Before the plates were
others chemicals, solvents, and reagents were of analytical grade prepared, the target bacteria were inoculated in NA overnight.
and purchased from Sigma Aldrich (Steinheim, Germany). The petri dish was filled with NA and left for overnight. Then,
the broths were spread on the agar using sterile cotton bud. The
Cytotoxicity screening assay methanol crude extracts were diluted with appropriate solvents
The HepG2 cell was treated with a serial dilution of the and were put onto the sterile paper discs (Whatman; 6 mm
methanolic extracts. The extracts were diluted from the highest diameter) in about 50 µl for each disc. The discs were air-dried
concentration of 100 µg/ml to the lowest concentration at 0.39 µg/ before placed on the petri dish. Some antibiotics were used as
ml. Only 5 µl of extracts were treated on the cells. The treatments positive control followed by incubation at 37°C for 24 hours.
were carried out in eight replicates to ensure the accuracy of the The inhibitions zone were examined and measured. The extracts
results. The negative control consists of 20% dimethyl sulfoxide would be considered active when the diameter of the inhibition
(DMSO) and 80% of MEM. The positive control was made using zones was more than 6 mm.
vincristine sulfate as it was the standard drug used to treat liver
Thin layer chromatography
cancer. The cells were incubated for 72 hours in 5% carbon dioxide
incubator at 37°C. The cytotoxicity of the extracts was determined The extracts were diluted in the appropriate amount
using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)- of solvent and were spotted on thin layer chromatography
034 Andriani et al. / Journal of Applied Pharmaceutical Science 8 (10); 2018: 032-038

(TLC) Silica gel 60 F254 plastic plate (Merck 1.05735.0001) RESULTS AND DISCUSSIONS
which were pre-coated with silica gel 60 F254. Then, the plate
was placed in a developing chamber with a mixture of solvent Cytotoxicity screening assay
and the tank was closed. In this study, the best solvent system The cell viability of all extracts treated is more than 80%
that could be used for profiling was a mixture of hexane and which indicated the extracts were not cytotoxic to the cells. In
ethyl acetate (7:3) and dichloromethane with methanol (9:1). this study, the inhibition concentrations (IC50); the effective dose
Then, the plate was taken out after a while and left to dry. The required to inhibit the proliferative response by 50% (1), of all
plate was observed under UV light and the observed spot was extracts were reached more than 30 µg/ml. The IC50 values also
marked. The plates were also visualized using Dragendorff’s indicate the proliferative activities of the methanolic extracts of
reagent. A. planci, E. luzonicus, and E. calamaris towards the HepG2
cells. Cheng et al. (2009) described the isolated compound from
Data analysis an Asteroid; Culcita noveaguinea has the anticancer properties
All the experiments in this current study were conducted towards human glioblastoma (U87MG) cells. Since the species was
in triplicate and the data are presented as a mean values ± standard in the same class as A. planci and E. luzonicus, they were expected
deviation. to produce similar results if the same type of cells in the previous
study were used. In this study, all results (Figs. 1–3) signified that

Figure 1. The percentage of HepG2 cell viability against the concentration of methanolic extracts (µg/ml) of A. planci, IC50 > 30 µg/ml.

Figure 2. The percentage of HepG2 cells viability against the concentration of methanolic extracts (µg/ml) of E. calamaris, IC50 > 30 µg/ml.

Figure 3. The percentage of HepG2 cell viability against the concentration of methanolic extracts (µg/ml) of E. luzonicus, IC50 > 30 µg/ml.
 Andriani et al. / Journal of Applied Pharmaceutical Science 8 (10); 2018: 032-038 035

Figure 4. DPPH free radical scavenging activity of A. planci, E. luzonicus, and E. calamaris. The black arrow indicated for IC50 value.

Figure 5. TLC using solvent system of Hexane: Ethyl acetate (7:3) viewed under (a) naked eyes, (b) Iodine vapor, (c) UV longwave (365 nm), and (d)
Dragendorff’s reagent.

the extracts did not exhibit anticancer properties towards HepG2 and also no IC50 value until 100 µg/ml. Some studies reported that
cells. There was also a possibility if the extracts undergone the starfish E. luzonicus rich by steroid glycosides compounds such
isolation for a pure compound, they would give different results. as Luzonicosides A and B. They were found to have an inhibitory
However, higher inhibition of cell growth was observed at 100 activity against human malignant melanoma cells (RPMI-7951
µg/ml of the extract of the outer layer of A. planci in Figure 1, and SK-Mel-28 cells) (Malyarenko et al., 2017). Furthermore,
where the inhibition was almost 50%. This might be due to that cytotoxic activity, antibacterial, antitumor, antifungal, antifouling,
outer layer consists of venomous spines of the organisms that hold antiviral, and anticancer preventive effects of steroid glycosides
cytotoxic compounds for body protection. A previous study by from starfish E. luzonicus were also investigated by some
Watanabe et al. (2009) has figured that the plancitoxin I, the major researchers (Dong et al., 2011; Ivanchina et al., 2011; 2017;
lethal factor from the spines of A. planci has potent hepatoxicity. Minale et al., 1993).
They also reported that the toxin exhibits DNase activity
responsible for the hepatoxicity. This study has indicated that A. Antioxidant activity
planci has a potential in drug discovery studies. As shown in Fig. 2, Figure 4 shows samples of A. planci, E. luzonicus, and
the outer layer of E. calamaris inhibits the cell growth more than E. calamari exhibited DPPH free radical scavenging activity.
visceral organs. Therefore, the extracts showed anti-proliferative The highest activity was revealed by A. planci from the outer
activity with the highest concentration of extracts. However, layer with the IC50 value of 3.10 ± 0.13 µg/ml, followed by
the inhibitions were still not cytotoxic compared to the positive E. calamaris and E. luzonicus with the IC50 value range from
control, vincristine sulfate which is merely cytotoxic towards the 4.00 ± 0.65 µg/ml to 9.20 ± 0.20 mg/ml. The lowest antioxidant
cell with IC50 of 0.09 µg/ml. Although at the highest concentration activity was obtained by E. luzonicus with the IC50 value of
of extracts inhibit the cell, they were still not as cytotoxic activity 9.20 ± 0.20 mg/ml.
036 Andriani et al. / Journal of Applied Pharmaceutical Science 8 (10); 2018: 032-038

Table 1. Antibacterial activity of A. planci, E.luzonicus, and E. calamaris.

Bacteria strain
Name of sample
S. aureus B. cereus Micrococcus sp. E. coli S. typhimurium P. aeruginosa
A. planci
Outer layer - - 17 ± 0.23 - - -
Visceral organ - - 16 ± 0.55 - - -
E. calamaris
Outer layer - - 18 ± 0.26 - - -
Visceral organ - - 19 ± 0.33 - - -
E. luzonicus - - 11 ± 0.63 - - -
Antibiotic
Tetracycline - 19 ± 0.25 22 ± 0.91 19 ± 0.28 20 ± 0.21 14 ± 0.22
Penicilin 9.0 ± 0.28 14 ± 0.33 21 ± 0.33 20 ± 0.26 18 ± 0.22 -
Streptomycin - 20 ± 0.35 19 ± 0.46 14 ± 0.08 14 ± 0.66 -
Gentamycin 21 ± 0.23 20 ± 0.26 22 ± 0.66 19 ± 0.25 18 ± 0.97 23 ± 0.61
DMSO - - - - - -
*(-) No activity, weak activity (<10 mm halo), good activity (10–15 mm halo), and strong activity (≥15 mm halo).

According to Hseu et al. (2008), the DPPH can inhibit the growth of S. aureus, B. cereus, E. coli, S. typhimurium,
accommodate many samples and it is sensitive enough to detect and P. aeruginosa, they showed strong antibacterial activity only
active samples at low concentrations in a short period. Thus, against Micrococcus sp. (Table 1). The highest antibacterial activity
DPPH is well known and commonly used by researchers to detect was revealed by E. calamaris (19 ± 0.33 for visceral organ and 18
the antioxidant potency of the sample. Based on the TLC profiling ± 0.26 for outer layer) compared to two another samples, A. planci
in Figures 5 and 6 which indicate the presence of alkaloids and E. luzonicus. A study reported by (Shamsuddin et al., 2010)
could be correlated to the antioxidant capacity in this study. showed that the methanolic extracts of E. calamaris showed good
According to Liu et al. (2014) and Utkina (2009), alkaloids inhibition towards Gram-positive bacterium, S. aureus. In addition,
possessed very good antioxidant activity. Beside alkaloids, a previous study (Bryan et al., 1995) reported the antibacterial
phenols (Andriani et al., 2017; Farvin and Jacobsen, 2013), activity of ethanolic extracts from two asteroids, Goniaster
terpenes (Ranjith et al., 2007), and steroid glycosides were might tesselatus and Astrophyton muricatum towards different bacterial
also contribute in possess antioxidant activity (Dong et al., 2011; tested, namely Deleya marina (a marine bacterium). This indicates
Ivanchina et al., 2011; 2017; Minale et al., 1993). Thus, different the possibility that the extracts from echinoderms are strains-
visualization using other kinds of reagent spray will be needed specific and selective in terms of antibacterial properties. Besides
to analyze the presence of more chemicals constituents besides that, different solvent used for extraction produce different results in
alkaloids in the same species which could be correlated to their antibacterial properties. Although the methanolic crude extracts in
activities. our study did not show cytotoxic activity towards the HepG2 cells,
previous studies have indicated that these echinoderms have other
Antibacterial assay advantages such as anticoagulant factor (Karasudani et al., 1996) and
All the extracts gave negative results towards the tested isolated some bioactive compounds, including steroid glycosides,
bacteria strains, except Micrococcus sp. Although they did not thornasterols, and the carotenoids (Bhakuni and Rawat, 2005;

Figure 6. TLC using solvent system of Dichloromethane: Methanol (9:1) viewed under (a) naked eyes, (b) Iodine vapor, (c) UV longwave (365 nm), and (d)
Dragendorff’s reagent. *Methanolic extracts of; A: E. luzonicus, B: A. planci (outer layers), C: A. planci (visceral organs), D: E. calamaris (outer layers), and E: E.
calamaris (visceral organs).
 Andriani et al. / Journal of Applied Pharmaceutical Science 8 (10); 2018: 032-038 037

Fleming et al., 1974; Kelly, 2005; Maoka et al., 2010). According Post-Graduate and Horseshoe Crab Laboratories, Biotechnology
to Andriani et al. (2017), although the sample used come from the and Microbiology Laboratories’ staff for their hospitality and aid
same species, different activity achieved could probably be due to during this project.
the geographical area of sample collection. In addition, different
environmental and habitat of the samples could also effect on their CONFLICT OF INTERESTS
secondary metabolites produced and activities. The authors declare no conflict of interest.

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