SHORT COMMUNI CATI ON

Assessment of in vivo and in vitro cytotoxic activity

of hydrolysable tannin extracted from Rhizophora apiculata barks
Lim Sheh Hong

Darah Ibrahim

Jain Kassim
Received: 29 December 2010 / Accepted: 17 March 2011 / Published online: 27 March 2011
Springer Science+Business Media B.V. 2011
Abstract Rhizophora apiculata is a common mangrove
tree in Malaysia. The bark of this tree has been reported to
contain a chemical constituent such as tannin that exhibited
antimicrobial activity. Recently hydrolysable tannins have
been studied for their potential effects against pathogenic
microorganisms and cancer cells through different mech-
anisms. The essence of the present study was to focus on
the in vivo and in vitro cytotoxicity of hydrolysable tannin
which was extracted from barks of R. apiculata. Cytotox-
icity of the hydrolysable tannin obtained was tested in an in
vivo brine shrimp lethality assay, and in vitro anticancer
cells assay. The results of the in vivo assay demonstrated
that hydrolysable tannin showed a higher value of LC
50
for
the acute toxicity (LC
50
= 4.67 mg/ml) than chronic tox-
icity (LC
50
= 2.10 mg/ml), which means that it was
essentially non-toxic. The hydrolysable tannin showed
cytotoxicity effects on HepG2 cancer cells (IC
50
=
12.26 lg/ml). It was found that the number of surviving
HepG2 cancer cells became less as the concentration of the
hydrolysable tannin increased. These ndings demonstrate
that hydrolysable tannin has high LC
50
and low IC
50
val-
ues, and could be used as potential source for pharmaco-
logically useful products.
Keywords Brine shrimp lethality Artemia salina
Lethality concentration-50 HepG2 cancer cells
Introduction
In recent times, focus on plant research has increased all
over the world and a large body of evidence has been
collected to show the immense potentials of medicinal
plants used in various traditional systems. Various medic-
inal plants have been studied using modern scientic
approaches (Dahanukar et al. 2000; Auddy et al. 2003).
The results from these plants have revealed the potential of
medicinal plant in the area of pharmacology (Somova et al.
2003; Fayehi et al. 2003), especially the tannin which can
be extracted from the barks of mangrove trees. Although
the tannin obtained showed antimicrobial activity (Lim
et al. 2006) and antioxidant activity (Suraya et al. 2010),
there is none of recorded data for clinical studies or for
toxicity against cancer cell lines or brine shrimp. Toxicity
studies are an important step for identication and isolation
of new compounds from plant extracts (Ramachandran
et al. 2011).
In this study, the hydrolysable tannin obtained from the
barks of mangrove tree (Rhizosphora apiculata) was tested
against the two most common toxicity assays namely the
brine shrimp lethality test and the cancer cell line inhibition
test. The Artemia salina assay was developed by Michael
et al. (1956). It is a preliminary toxicity test, the brine
shrimp being highly sensitive to various chemical sub-
stances. This method has been used for the detection of
fungal toxins (Harwig and Scott 1971), cyanobacterial
toxins (Jaki et al. 1999), and also plant extract toxicity
(McLauglin et al. 1991). The cancer cell line test in this
work is to evaluate natural remedies for different pharma-
cological activities, taking into account the basic premise
that pharmacology is simply toxicology at a lower dose.
Toxicity to brine shrimp coincides with cytotoxicity to
mammalian cells in many cases. However, Meyer et al.
L. S. Hong (&) D. Ibrahim
Industrial Biotechnology Research Laboratory (IBRL),
School of Biological Sciences, Universiti Sains Malaysia,
11800 Penang, Malaysia
e-mail: limshehhong77@gmail.com
J. Kassim
School of Chemical Sciences, Universiti Sains Malaysia,
11800 Penang, Malaysia
1 3
World J Microbiol Biotechnol (2011) 27:27372740
DOI 10.1007/s11274-011-0727-1
(1982) and Solis et al. (1993) have reported there is no
correlation in the degree of toxicity between the two
systems.
Cytotocity study of the extract provides us important
preliminary useful data for selecting the natural remedies
with potential antimicrobial properties for future work.
Therefore, the objectives of present work were to evaluate
and to determine the lethality concentrations (LC
50
) of
hydrolysable tannin extracted from R. apiculata barks
against brine shrimp nauplii and human hepatocellular
carcinoma (HepG2) cells.
Materials and methods
Preparation of extract
The bark samples of R. apiculata were collected from
Kuala Sepetang, Daerah Larut Matang, Perak, Malaysia.
The samples were washed with running tap water, dried
under the sun and nely ground to 1 mm and stored at 4C
prior to use. Hundred grams of the pulverized barks of the
plant were macerated in 300 ml of 70% aqueous acetone
for 3 consecutive days at room temperature (30 2C).
The acetone was removed using a rotary evaporator under
pressure and the resultant extract was then concentrated to
dryness and then freezedried to form a tannin powder. The
tannin powder (1.5 g) was then defatted with hexane
(50 ml), followed by extraction with ethyl acetate (50 ml).
A fraction of the aqueous phase was concentrated to dry-
ness in a rotary evaporator and freeze-dried. Ten grams of
the powder was then dissolved in methanol: water (1:1) and
mixed with Sephadex LH 20. Removal of the Sephadex LH
20 from solvent was done by vacuum pump ltration by
using the lter paper (Whatman No. 1). The ltrate
obtained was concentrated using rotary evaporation and
then freeze-dried. The powder (formed of hydrolysable
tannin) obtained was kept at 4C until further used.
In vivo cytotoxicity test against the brine shrimp nauplii
The bioactivity of hydrolysable tannin was carried out by
the brine shrimp lethality test (Meyer et al. 1982; Rama-
chandran et al. 2011). Samples were dissolved in articial
sea water prepared by dissolving 38 gram of sea salt in
1.0 l of distilled water, and then diluted to obtain nal
concentrations of 0.58.0 mg/ml.
Brine shrimp (Artemia salina) eggs were hatched in
articial sea water after 48 h incubation at room tempera-
ture (2530C), the larvae (nauplii) were then found to be
attracted to one side of the vessel (with a light source) and
collected by pipette. The nauplii were transferred into a
universal bottle containing 5.0 ml of various concentrations
of hydrolysable tannin. The number of survivor nauplii was
counted after 6 h (acute toxicity) and 24 h (chronic toxicity)
of exposure to the hydrolysable tannin. A universal bottle
with articial sea salt water served as a drug-free control or
negative control. The surviving shrimps were counted and
the concentration that could kill 50% of larvae (LC
50
) was
assessed (Geran et al. 1972).
In vitro assay for cytotoxicity
HepG2 (Human hepatocellular carcinoma) was used
throughout this study. The cell was purchased from Amer-
ican Type Culture Collection (ATCC; Rockville, MD,
USA). HepG2 cells were cultured in MEM/EBSS with
0.1 mM non-essential amino acids and 1.0 mM sodium
pyruvate. The medium was supplemented with 10% of fetal
calf serum (FCS), 2 mM L-glutamine, 100 units strepto-
mycin/ml and 100 units penicillin/ml. The cellular viability
was estimated by the Trypan blue dye exclusion.
In this study, near-conuent stock cultures of cells were
harvested with 0.05% (w/v) TrypsinEDTA and resus-
pended in complete medium with 10% FCS to
1 9 10
5
cells/ml. Then, the cells were plated into 96-well
plates (Costar, Albany, NY, USA) and were incubated at
37C in a CO
2
incubator (5% (v/v) CO
2
) for a further
2448 h. When the cells reached conuency between 80
and 90%, the medium was removed and replaced with
medium which contained only 0.5% (v/v) FCS. The cells
were then incubated for approximately 4 h before testing.
The cells were then treated with different concentrations
(1.0500.0 lg/ml) of hydrolysable tannin extracted from R.
apiculata. Cells cultured in 0.5 (v/v) FCS-containing
medium alone served as negative control. After treatment,
the plates were incubated at 37C for further 72 h.
Cell survival was determined by a procedure using
methylene blue staining (Yamazaki et al. 1986; Li and
Hwang 1991). Briey, glutaraldehyde was added to each
well to a nal concentration of 2.5% (v/v) and the sur-
viving cells were xed for 15 min. After washing with
0.15 M sodium chloride and removing the dead cells, the
xed cells were stained with 0.1 ml of 0.05% (w/v)
methylene blue solution for 15 min. After washing off the
excess dye with 0.15 M sodium chloride solution, dye
elution was carried out with 0.2 ml of 0.33 M HCl. After
shaking the plates, the dye content was determined by
measuring absorbance at 650 nm by using Vmax Kinetic
Microplate Reader (Molecular Devices, USA).
Experiments were performed in triplicate. Results were
expressed as percentage growth inhibition of the control.
IC
50
values for growth inhibition was derived from a
nonlinear regression model (curve t) based on a sigmoidal
dose response curve (variable) and computed using
GraphPad Prism version 3.00 for Windows, Graph Pad
2738 World J Microbiol Biotechnol (2011) 27:27372740
1 3
Software, San Diego, CA, USA (www.graphpad.com).
Data were given as mean standard error mean (SEM).
Results and discussion
Isolation of hydrolysable tannin from R. apiculata requires
toxicity information on the constituent of interest. It should
be emphasized that the toxic effects of the antimicrobial
agent on the host cell must be considered, since any anti-
microbial activity may be a consequence of its toxic effect
on the cells. The general principle of cytotoxicity assay is
based on the assessment of a special characteristic of most
cytotoxic agents, which inhibit mammalian cell division in
culture at effective concentrations of the agent.
The brine shrimp is well characterized as a primary test
for biological evaluation. In this procedure we assumes that
any nauplii that survived to a given dose would also had
survived to any lower dose, and also any cell that died at a
certain dose would also died at any higher dose (Chavez
et al. 1997). The test period was taken as the exposure of
the nauplii to various concentration of hydrolysable tannin
for a period of 6 h for acute toxicity and 24 h for chronic
toxicity. The brine shrimp toxicity assay was developed by
Michael et al. (1956) and adapted and modied by others
(Meyer et al. 1982; Solis et al. 1993). It is a convenient
toxicity test, since the brine shrimp is sensitive to a variety
of chemical compounds. The assay is considered a useful
tool for assessment of toxicity (Solis et al. 1993) and is
widely used. The live brine shrimp shows internal and
external movement. The dead nauphii however show no
movement at all when seen under the light microscope.
The LC
50
values obtained from in vivo cytotoxicity
assay of the hydrolysable tannin against brine shrimp were
4.67 mg/ml (acute cytotoxicity) and 2.10 mg/ml (chronic
toxicity), respectively. According to Venugopal et al.
(2002), bioactive compounds which exhibit an LC
50
value
more than 1.0 mg/ml are considered not toxic to the nauplii
of Artemia salina. Therefore, the cytotoxicity result
obtained in this study indicated that the hydrolysable tannin
extracted from R. apiculata showed no toxicity against
brine shrimp. The brine shrimp assay is a useful tool for the
isolation of bioactive compounds from plant extracts (Sam
1993). Thus, the results suggested that the hydrolysable
tannin could be a potential candidate to be used as an
antibacterial and antifungal agent.
Hydrolysable tannin exhibited a signicant in vitro
cytotoxic activity against HepG2 cancer cell lines with an
IC
50
value of 12.06 lg/ml (Fig. 1). From this study, it was
found that the number of surviving HepG2 cancer cells
became less as the concentration of the hydrolysable tannin
increased (Fig. 1). The American National Cancer Institute
(NCI) has made guidelines where IC
50
values less than
30 lg/ml of the crude extract are accepted as criteria for
cytotoxicity. The reduction in growth of HepG2 cancer
cells was possibly due to interference by the active prin-
ciple of the extract. A toxic substance might indeed elicit,
at lower toxic dose, interesting pharmacological effects
McLauglin (1991). After detailed in vivo and in vitro
evaluation of toxicological studies, hydrolysable tannin
extracted from R. apiculata may nd use as an antimi-
crobial agent in known dosages for developing new drugs.
Moreover, the toxicity screening model also provides
important data to help us for selecting natural remedies
with potential antimicrobial properties for the future work.
Conclusion
The hydrolysable tannin extracted from R. apiculata barks
was not toxic against brine shrimp but toxic to the in vitro
cancer cell line. Hence, this nding indicates that the
presence of potent cytotoxic compounds which warrants
further investigation. Further studies are needed to verify
the medicinal importance of hydrolysable tannin which
could then be used to treat pathogenic microbial infectious
diseases.
Acknowledgments The rst author thanks the Ministry of Science,
Technology and Innovation of Malaysia for awarding her a PASCA
scholarship throughout her Masters degree. The authors also would
like to thank the Ministry of Science, Technology and Innovation of
Malaysia for providing the support under the IRPA research grant
(09-02-05-2086 EA001).
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