oxicity of Barringtonia racemosa (L.

) Kernel Extract on Pomacea

canaliculata(Ampullariidae)
Musri Musman*

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Abstract
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INTRODUCTION

Barringtonia racemosa (L.) Spreng (Lecythidaceae) is a tropical higher plant that is widely
spread in East Africa, Southeast Asia and the Pacific islands (Strey 1976; Chantaranothai
1995). It grows in the moist lower countries where it is exposed to high sunlight intensities
and for extended periods. It is locally known aspenteut ie and its fruits are used as a
traditional medicine to treat asthma, cough and diarrhoea. The seed of this plant contains
saponins and flavonoids (Ojewole et al. 2004; Gowri et al. 2009). These compounds are
widely distributed amongst plants and show a wide range of biological properties (Sparg et
al. 2004). Saponins are glycosides with a distinctive foaming characteristic when dissolved in
water (Hostettmann & Marston 1995); this is assumed to be due to their amphiphilic nature
(Xu et al. 1996; Francis et al. 2002). Saponins consist of polycyclic aglycone and sugar
moieties. The ability of a saponin to foam is caused by the combination of the nonpolar
aglycone and the water soluble sugar portion. Saponins are highly toxic to cold-blooded
animals due to their ability to lower surface tension. Saponins serve as plant immune systems,
acting as a natural antibiotic to protect the plant against microbes and fungi (Estrada et al.
1997). The saponin class of natural products induce red blood cell lysis in animal systems
(Francis et al. 2002). It has been reported that B. racemosa seed kernel has a wide range of
therapeutic applications (Thomas et al. 2002; Gowri et al. 2007; Hussin et al. 2009).
Flavonoids are hydroxylated phenol substances that occur as C6-C3-C6 moieties. These
compounds are widely distributed in plants and possess a number of pharmacological
properties (Whiting 2001).

Pomacea canaliculata (Ampullariidae) (Ghesquiere 2007) is a well known golden apple

snail, which is originally from South America. It was introduced into Asia through the pet
trade and as a potential food source for humans. The rapid invasion of P. canaliculata accross
Asia during the last decades has been terrifying. The snails have been causing damage to rice
production in Indonesia and other countries (Hirai 1988; Halwart 1994; Wada et al.
2002; Darby et al. 2008; Joshi et al. 2008). Some approaches, either mechanical (Teo 2003)
or biological (Sumangil 1989) methods to control the snails have been conducted, but with
unsatisfactory results due to the widespread distribution of this pest. The chemical approach,
using synthetic molluscicides, such as metaldehyde and niclosamide, has been applied (Dela
Cruz et al. 2000) to control this pest. Unfortunately, the synthetic substances can pollute
water bodies, causing the deaths of fishes and livestock (Calumpang et al. 1995; Wada 2004).
The advances in the battle against the snails using natural molluscicides must be encouraged
in order to minimise the negative side effects to the environment. A number of tropical plants
have been investigated for their molluscicidal activity (Kardinan & Iskandar
1997; Arunlertaree et al. 2003; Musman 2004, 2006, 2009;Joshi et al. 2005; San Martin
2007; Plan et al. 2008). The compound groups from plants identified as having active
molluscicide activity are saponin, tannin, alkaloid, and flavonoid (Perry 1980; Kloos &
McCullough 1987; Huang et al. 2003). Literature searches revealed that there have been
scientific studies carried out studying the cytotoxic activity of the seed kernel of B.
racemosa on P. canaliculata. Hence, the present study is focused on evaluating the toxicity
potentials of different seed kernel extracts of B. racemosa on P. canaliculata.

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MATERIALS AND METHODS

The experiment was conducted in the Marine Laboratory of the Department of Marine
Science, Coordinatorate of Marine Science and Fishery at Syiah Kuala University, Banda
Aceh, Indonesia.

Extraction of B. racemosa Seed Kernels

B. racemosa fruits were collected from Lam Neuhen village, in the Aceh Besar District on
October 2008. The harvested fruits were kept in the Marine Laboratory of the Department of
Marine Sciences, Syiah Kuala University. Subsequently, fruits were decorticated to remove
the kernels and air dried in the shade for seven days. The seed kernels (1.8 kg, dry) were
pulverised with an electric blender and sieved with a 40 mm mesh screen to obtain fine
powder. 500 g of the powder was extracted twice sequentially, on each occasion with 2 l of
heptane (hp), dichloromethane (DCM), ethyl acetate (EtOAc), and methanol (MeOH), at
room temperature for 48 hours with shaking. In each case, the filtrates were filtered through
Whatman filter paper and were evaporated to dryness by placing the container in a rattling
water bath (40C). The extracts were further dried by placement in a vacuum desiccator. The
extracts were stored in labeled specimen bottles for bioassays.

Snail Collection

The identification and characterisation of P. canaliculata was performed based on previously

published data (Ghesquiere 2007). P. canaliculata with 79 in3 in shell volume (meanSD
was 7.890.88) were collected from the rice field at Gla Deyah village, Aceh Besar District,
and subsequently acclimated in a glass aquarium for 7 days in the Marine Laboratory of the
Department of Marine Sciences, Syiah Kuala University.

Preliminary Phytochemical Analysis

Qualitative phytochemical screening of B. racemosa seed kernel extracts was carried out
using standard laboratory techniques. The Molisch, Fehling, and Benedict tests were applied
for carbohydrates analysis (Evans 1989; Raman 2006), a frothing test was carried out for
saponin analysis (Harborne 1984; Evans 1989), Schinodas test was applied for flavonoid
analysis (Evans 1989; Raman 2006), Lieberman-Burchards test was carried out for
triterpenoid analysis (Harborne 1984; Evans 1989), the Mayers, Wagners and Dragendorffs
tests were applied for alkaloids (Evans 1989), and ferric chloride and alkaline tests were
carried out for tannin analysis (Evans 1989). The percentage of yield for each extraction was
calculated according to Kennedy (1990). For each solvent, the yield % was derived following
the equation shown below:

%yield=Extract(g)Sample(500g)100
Bioassay Technique

The extracts of B. racemosa seed kernel were evaluated at 50, 100, 200, 400 and 800 ppm
concentrations in an aqueous solution. A sample size of 480 individual P. canaliculata snails
were collected based on the specified size (Ghesquiere 2007) and screened for molluscicidal
bioassay with a protocol adapted from the Food and Agriculture Organisation (FAO) method
(Reish & Oshida 1987). Twenty four glass aquariums measuring 45 x 28 x 35 cm (l x w x h)
were arranged in 4 groups (in order to apply the hp, DCM, EtOAc, and MeOH extracts)
consisting of 6 aquariums (to apply solutions at 0, 50, 100, 200, 400, and 800 ppm). The
aquariums were filled up with paddy-field water (pH 6.4 and temperature 31C) taken from
the location where the P. canaliculata were collected. The water was filled into the aquarium
to a depth of 10 cm, as measured from the bottom. Each aquarium was then filled with as
many as 20 individual P. canaliculata. The tested organisms were allowed a free moving
period of 30 minutes. The aquaria groups were arranged in order first for hp, second for
DCM, third for EtOAc, and fourth for MeOH extract applications, in which each group
consisted of 6 rows of aquariums. The 6 rows of aquariums consisted of a first row for the
control, second row for 50 ppm, third row for 100 ppm, fourth row for 200 ppm, fifth row for
400 ppm, and sixth row for 800 ppm concentrations. The aqueous extracts of B.
racemosa were poured into the aquariums in the first, second, third, and fourth groups,
according to the row position, in volumes of up to 100 ml. The mixture had a pH of 6.1 at the
end of the bioassay. P. canaliculata mortality was assessed after 48 hours of exposure by
probing the snail secreted mucus through an operculum gap. The toxicity of the compound
towards the snails is reflected in the release of mucus from the tested snails. The signs of
mortality were taken to be rigidity and lack of movement. Lethal concentration at 50% (LC50)
calculations were used for the determination of lethal concentration endpoints in the acute
toxicity bioassay tests. Mortality data were used to calculate LC50 values using Trimmed
Spearman-Karber (TSK) program (Hamilton et al. 1997) version 1.5 software downloaded
from the US Environmental Protection Agency.

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RESULTS AND DISCUSSION

The phytochemical analysis results are show in Table 1. Application of the extract to the
tested organisms showed a significant effect on the mortality rate of P. canaliculata (Table
2). The molluscicidal character, in this case is toxicity of the extract to apple snails. The
extract was predicted to demonstrate toxicity towards snails due to the presence of saponins
(Hostettmann 1984; Sparg et al. 2004; Ojewole et al. 2004) and flavonoids (Macheix et al.
1990) in the extracts (Table 1). The molluscicidal activity of saponin was due to its ability to
effect the cell membrane and its ability to lower the surface tension of water. The extract
caused various responses in the tested subjects as a result of direct contact between the tested
organisms and the saponin (Hostettmann et al. 1982; Mahato & Nandy 1991; Hutchings et al.
1996). The tested P. canaliculatademonstrated a response to the molluscicidal action of
saponin. This response was initiated by the production and secretion of mucus in order to
reduce further contact of their body surfaces with the molluscicide (Brainet al. 1990). During
a period of 48 hours, the data revealed that the mortality rate of the tested P. canaliculatawas
higher in extracts containing both saponins and flavonoids compared to the extracts
containing only flavonoids (Fig. 1). It was expected that both substances could block the
process of breathing. This probably is due to diffusion of oxygen through the gills of the
snails, which is subsequently obstructed by the mucus.

Figure 1:
Number of mortalities of P. canaliculata in solutions of B. racemosa.

Table 1:
Phytochemical constituents of the B. racemosa seed kernel extract.

Table 2:
The effect of the B. racemosas extract on the mortality of P. canaliculata.

The study showed that a 50% mortality rate of P. canaliculata was reached within 48 hours
of exposing theP. canaliculata to 50 ppm of the aqueous solution of the MeOH extract. A
100% P. canaliculata mortality rate was observed within 48 hours of incubation of the snails
in 200, 400, and 800 ppm of the aqueous solution of DCM and MeOH extracts (Fig. 1). It is
predicted that a variety of active compounds dissolved in the same solvents are responsible
for the different effects observed in the mortality of the tested species. This experiment also
demonstrated that the mortality of the tested P. canaliculata was different depending on the
time (48 hours after exposure) at which the extract was applied (Table 2). This is such as
saponin will break down into sugar and aglycone whereas flavanoid will undergo dissociation
when both compounds are dissolved in water leading to inactiveness. The inactiveness in the
biological properties depends on time, as shown in Table 2, where the higher is the
concentration used, the higher is the number of mortality, at 48 hours after exposure. This
suggested that the active compounds of the extract were unstable in water.

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CONCLUSION

In this study, we determined that the LC50 values (lower-upper limits) of the B.
racemosa seed kernel extract in the hp, DCM, EtOAc, and MeOH solvents were 672.72
(366.571234.53), 70.71 (41.33120.97), 186.84 (129.21270.17), and 94.39 (62.48142.59)
(ppm/48 hours), respectively, at 95% C.I. Furthermore, we found that the dichloromethanic
extract showed the strongest cytotoxic activity (LC50 = 70.71 ppm) and that the heptanic
extract had the least cytotoxic activity (LC50 = 672.72 ppm). It is assumed that the observed
biological effects of the extracts are largely due to the saponins and flavonoids present in the
seed. Thus, these results support that the B. racemosa seed kernel extract is an attractive
compound for further studies leading to molluscicidal development.