SHORT COMMUNICATION

Assessment of in vivo and in vitro cytotoxic activityof 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 LC50 for

the acute toxicity (LC50 = 4.67 mg/ml) than chronic tox-

icity (LC50 = 2.10 mg/ml), which means that it was

essentially non-toxic. The hydrolysable tannin showed

cytotoxicity effects on HepG2 cancer cells (IC50 =

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 findings demonstrate

that hydrolysable tannin has high LC50 and low IC50 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 scientific

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 identification 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

123

World J Microbiol Biotechnol (2011) 27:2737–2740

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 (LC50) 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 finely ground to 1 mm and stored at 4�C

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 ± 2�C).

The acetone was removed using a rotary evaporator under

pressure and the resultant extract was then concentrated to

dryness and then freeze–dried 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 filtration by

using the filter paper (Whatman No. 1). The filtrate

obtained was concentrated using rotary evaporation and

then freeze-dried. The powder (formed of hydrolysable

tannin) obtained was kept at 4�C 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 artificial

sea water prepared by dissolving 38 gram of sea salt in

1.0 l of distilled water, and then diluted to obtain final

concentrations of 0.5–8.0 mg/ml.

Brine shrimp (Artemia salina) eggs were hatched in

artificial sea water after 48 h incubation at room tempera-

ture (25–30�C), 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 artificial 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 (LC50) 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-confluent stock cultures of cells were

harvested with 0.05% (w/v) Trypsin–EDTA and resus-

pended in complete medium with 10% FCS to

1 9 105 cells/ml. Then, the cells were plated into 96-well

plates (Costar, Albany, NY, USA) and were incubated at

37�C in a CO2 incubator (5% (v/v) CO2) for a further

24–48 h. When the cells reached confluency 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

(1.0–500.0 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 37�C for further 72 h.

Cell survival was determined by a procedure using

methylene blue staining (Yamazaki et al. 1986; Li and

Hwang 1991). Briefly, glutaraldehyde was added to each

well to a final concentration of 2.5% (v/v) and the sur-

viving cells were fixed for 15 min. After washing with

0.15 M sodium chloride and removing the dead cells, the

fixed 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.

IC50 values for growth inhibition was derived from a

nonlinear regression model (curve fit) 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) 27:2737–2740

123

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 modified 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 LC50 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 LC50 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 significant in vitro

cytotoxic activity against HepG2 cancer cell lines with an

IC50 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 IC50 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 find 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 finding 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 first 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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Log concentration (µg/ml)

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