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Veterinary Parasitology 152 (2008) 264270

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Molluscicidal activity of Carica papaya and Areca catechu

against the freshwater snail Lymnaea acuminata
Preetee Jaiswal, D.K. Singh *
Department of Zoology, DDU Gorakhpur University, Gorakhpur 273009, UP, India
Received 19 September 2007; received in revised form 19 December 2007; accepted 21 December 2007

Abstract
The molluscicidal activity of seed and lyophilized latex powder of Carica papaya and seed powder of Areca catechu against the
vector snail Lymnaea acuminata was studied. The toxicity of these plant products was time and dose dependent. The toxicity of C.
papaya lyophilized latex powder (LC50 at 96 h: 8.38 mg/l) was more pronounced than that of A. catechu seed powder (LC50 at 96 h:
12.32 mg/l) and C. papaya seed powder (LC50 at 96 h: 61.56 mg/l). Ethanolic extracts of C. papaya seed and A. catechu seed were
more toxic than their other extracts. The ethanolic extract of A. catechu seed (LC50 at 24 h: 17.21 mg/l) was more effective than the
ethanolic extract of C. papaya seed (LC50 at 24 h: 53.38 mg/l). The LC50 of column-purified fraction of A. catechu seed at 96 h was
3.99 mg/l, whereas that of C. papaya seed was 7.06 mg/l. C. papaya and A. catechu may be used as potent molluscicides since the
concentrations used to kill the snails were not toxic for the fish Colisa fasciatus which shares the same habitat with the snail L.
acuminata.
# 2008 Elsevier B.V. All rights reserved.

Keywords: Lymnaea acuminata; Carica papaya; Areca catechu; Molluscicidal activity

1. Introduction the control of the vector snail population by the use of

Fascioliasis is a common parasitic disease of
ruminants, especially cattle, buffalo, sheep and goats
(Mas-Coma et al., 2005). Human cases of fascioliasis
have also been reported from India (Narain et al., 1997).
This disease is mainly caused by two digenetic
trematodes Fasciola hepatica and Fasciola gigantica
(Singh and Agarwal, 1981; Marcos et al., 2006).
Infection of Fasciola causes reductions in meat and
milk production. The freshwater snails of the genus
Lymnaea (Saleha, 1991) act as the intermediate host of
these flukes (Cucher et al., 2006; Velusamy et al., 2004).
One of the major preventive steps against fascioliasis is
* Corresponding author. Tel.: +91 551 2202187 (O)/0509 (R).
E-mail address: dksingh_gpu@yahoo.co.in (D.K. Singh).
molluscicides (Agarwal and Singh, 1988; Sukumaran
et al., 2004). The development of biomolluscicides as a
possible substitute for chemical molluscicides is gaining
wide attention because they are effective, less expensive
and environmentally acceptable (Marston and Hostett-
mann, 1985). The search for less dangerous mollusci-
cides, at lower costs stimulated the search for potential
active substances from native plants (Mott, 1987). Many
plant products have been found to have a high
molluscicidal potential (Hostettmann and Lea, 1987;
Singh et al., 1996; Silva et al., 2006). In the present study
we evaluated the molluscicidal activity of Carica papaya
Linn. (Caricaceae) seeds and latex and Areca catechu
Linn. (Palmae, Arecaceae) seeds against the target snail
Lymnaea acuminata, and toxicity of these plant products
was also studied against the non-target freshwater fish
Colisa fasciatus which shares the same habitat.

0304-4017/$ see front matter # 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.vetpar.2007.12.033
 P. Jaiswal, D.K. Singh / Veterinary Parasitology 152 (2008) 264270 265

2. Materials and methods
C. papaya seeds and latex were collected locally
from cultivated crops in the field, and A. catechu seeds,
traditionally called Supari were purchased from a
local market in Gorakhpur district. Specimens were
identified by Prof. S.K. Singh, Taxonomist, Botany
Department, DDU Gorakhpur University, Gorakhpur
where samples (Voucher specimens number C-3125, A-
4115) have been deposited.
toxicity study. Ethanol was evaporated under vacuum
and the remaining solids were used for the determina-
tion of molluscicidal activity.
2.4. Pure compounds
Papain (Cysteine protease) and arecoline hydro-
bromide (Methyl 1,2,5,6-tetrahydro-1-methyl-3-pyridi-
necarboxylate hydrobromide) were purchased from
Sigma Chemical Co. USA.

2.1. Preparation of dried powder of latex and seeds 2.5. Thin layer chromatography

Latex: The white milky latex of C. papaya was
drained into a graduated tube by cutting the
epidermis of unripe fruits and lyophilized at
40 8C. The lyophilized powder was stored in
airtight desiccator for further use. The wet weight of
1 ml of latex of C. papaya was 905 mg and the
lyophilized powder was 385 mg.
Seeds: The seeds from ripe fruits of C. papaya were
dried in an incubator at 37 8C. The dried seeds of
C. papaya and A. catechu were pulverized separately
with a grinder, and the crude powders thus obtained
were used for the toxicity experiment.
Molluscicidal component present in seeds and latex
of C. papaya and seeds of A. catechu were identified by
thin layer chromatography. TLC was done on
20 cm  20 cm precoated silica gel (Precious Electro-
chemindus Private Limited, Mumbai). The solvent used
was benzene/ethyl acetate (9:1). Spots of column-
purified fractions of C. papaya seed, A. catechu seed
and aqueous extract of lyophilized latex and pure papain
and arecoline hydrobromide were made on TLC plates.
TLC plates were developed by iodine vapour.
2.6. Collection of snails

2.2. Organic solvent extracts Adult L. acuminata (2.25 cm  0.20 cm in length)

were collected from different ponds of Gorakhpur
Five grams of seed powders of C. papaya and A. district, UP, India. The animals were acclimatized in
catechu were extracted separately with 100 ml of the laboratory conditions for 72 h. They were kept in a
following solvents: ethanol, ether, chloroform, carbon glass aquarium containing dechlorinated tap water at
tetrachloride and acetone at room temperature for 24 h.
The solvents were removed under vacuum and the Table 1
remaining dried parts were used for the determination of Concentration of different preparations of seed powder, lyophilized
molluscicidal activity. For C. papaya seeds we got 1.2 g latex powder and active compound of Carica papaya and seed powder
and active compound of Areca catechu used in toxicity trial against
of chloroformic extract, 2.55 g of carbon tetrachloride Lymnaea acuminata
extract, 2.37 g of ether extract, 1.41 g of acetone extract,
1.35 g of ethanolic extract and for A. catechu seeds we Plant material used Test concentration (mg/l)
got 625 mg of chloroformic extract, 1.0 g of ether Carica papaya seed powder 50, 70, 90, 120
extract, 2.06 g of acetone extract and 2.09 g of ethanolic Chloroform extract 20, 30, 50, 70
Carbontetrachloride extract 20, 30, 50, 70
extract.
Ether extract 30, 40, 50, 70
Acetone extract 30, 40, 50, 70
2.3. Column chromatography Ethanol extract 10, 20, 30, 40
Column purified 5, 7, 10, 15
One hundred milliliters of ethanolic extract of C. Lyophilized latex powder 5, 10, 15, 20
Papain 7, 9, 12,15
papaya seed and A. catechu seed powder were subjected
Areca catechu seed powder 10, 15, 20, 30
to silica gel (60120 mesh, Qualigens glass, Precious Chloroform extract 5, 10, 15, 20
Electrochemindus Private Limited, Mumbai, India) Ether extract 9, 12, 15, 18
chromatography through a 5 cm  45 cm column. Five Acetone extract 10, 15, 20, 30
milliliter fractions of 50 elutents were eluted with 95% Ethanol extract 7, 9, 12, 15
Column purified 3, 5, 7, 9
ethanol. In C. papaya seed elutent nos. 2027 and in A.
Arecoline 0.1, 0.2, 0.3, 0.4
catechu seed elutent nos. 2330 were used for the
266 P. Jaiswal, D.K. Singh / Veterinary Parasitology 152 (2008) 264270

2224 8C. The pH of the water was 7.17.3 and dissolved
oxygen, free carbon dioxide and bicarbonate alkalinity
were 6.57.2, 5.2/6.3 and 102105 mg/l, respectively.
2.7. Treatment protocol for concentrationresponse
relationship
Toxicity experiments were performed by the method
of Singh and Agarwal (1984). Ten experimental animals
were kept in a glass aquarium containing 3 l of
dechlorinated tap water. The test animals were exposed
continuously for 96 h to different concentrations of
plant products (Table 1). Six aquaria were set up for
each concentration. Control animals were kept in an
equal volume of water under similar conditions without
treatment. The toxicity was also studied in mixed
population of fish C. fasciatus and snails. Ten
experimental fish (7.5  0.09 cm length) and ten snails

Table 2
Toxicity of Carica papaya seed powder, its different organic solvent extracts, column-purified fractions, lyophilized latex powder and active
compound papain against the snail Lymnaea acuminata at different exposure period
Exposure period (h) Tested materials LC50 (mg/l) Limits Slope value t-ratio g-value Heterogeneity
LCL UCL
24 Seed powder 127.26 108.62 174.86 3.36  0.69 4.85 0.16 0.33
Chloroform extract 111.93 90.50 177.22 2.86  0.62 4.64 0.18 0.26
Carbon tetrachloride extract 81.20 67.21 116.35 2.16  0.50 4.35 0.20 0.17
Ether extract 111.89 89.57 183.05 2.67  0.59 4.53 0.19 0.23
Acetone extract 97.75 73.47 241.72 2.53  0.71 3.55 0.31 0.18
Ethanol extract 53.38 40.91 95.34 2.29  0.50 4.60 0.18 0.27
Column purified 27.33 18.00 106.25 2.03  0.58 3.53 0.31 0.20
Lyophilized latex powder 19.92 16.90 25.99 2.94  0.50 5.87 0.11 0.46
Papain 16.63 14.55 21.55 4.46  0.86 5.17 0.14 0.28
48 Seed powder 102.87 89.23 132.50 2.82  0.62 4.53 0.19 0.19
Chloroform extract 103.89 79.58 214.13 1.85  0.50 3.67 0.28 0.13
Carbon tetrachloride extract 58.58 47.79 73.57 1.97  0.47 4.18 0.22 0.15
Ether extract 93.49 75.10 152.22 2.09  0.51 4.14 0.22 0.18
Acetone extract 66.30 55.18 104.39 2.38  0.64 3.72 0.28 0.22
Ethanol extract 42.97 32.53 80.41 1.65  0.40 4.10 0.23 0.19
Column purified 20.14 14.47 53.97 1.87  0.51 3.63 0.29 0.13
Lyophilized latex powder 16.52 13.68 22.14 2.14  0.41 5.26 0.14 0.37
Papain 15.07 13.14 19.75 3.50  0.74 4.77 0.17 0.13
72 Seed powder 80.93 68.62 97.03 2.46  0.60 4.10 0.23 0.20
Chloroform extract 65.70 53.45 89.70 1.83  0.47 3.86 0.26 0.17
Carbon tetrachloride extract 42.20 29.06 51.78 1.85  0.47 3.95 0.25 0.14
Ether extract 67.64 55.00 94.45 1.81  0.47 3.82 0.26 0.15
Acetone extract 41.83 34.56 48.55 2.59  0.63 4.13 0.23 0.16
Ethanol extract 23.01 17.32 31.11 1.47  0.37 3.95 0.25 0.21
Column purified 12.46 10.15 18.83 1.97  0.48 4.10 0.23 0.08
Lyophilized latex powder 11.86 9.78 14.61 2.05  0.39 5.32 0.14 0.40
Papain 12.74 11.30 15.51 3.27  0.69 4.73 0.17 0.14
96 Seed powder 61.56 51.79 69.03 3.53  0.64 5.51 0.13 0.27
Chloroform extract 41.19 33.09 47.72 2.74  0.49 5.58 0.12 0.22
Carbon tetrachloride extract 30.75 21.09 37.49 2.58  0.51 5.06 0.15 0.33
Ether extract 13.14 44.64 151.71 2.41  0.48 5.04 0.15 0.19
Acetone extract 31.65 25.23 35.96 3.62  0.70 5.17 0.14 0.17
Ethanol extract 15.93 11.93 19.36 2.00  0.38 5.27 0.14 0.25
Column purified 7.06 5.57 8.35 2.25  0.48 4.66 0.18 0.12
Lyophilized latex powder 8.38 6.61 9.98 2.23  0.39 5.77 0.12 0.51
Papain 9.74 8.65 10.81 3.71  0.69 5.39 0.13 0.18
Six batches of ten snails were exposed to different concentrations of the above treatments. Mortality was recorded every 24 h. Concentrations given
are the final concentration in the glass aquarium water. Significant ( p < 0.05) negative regression was observed between the exposure time and
different LC50 values. Testing significance of the regression coefficient of Carica papaya seed powder (27.59+), chloroform extract (6.18+), carbon
tetrachloride extract (9.48+), ether extract (5.45+), acetone extract (8.36++), ethanol extract (7.91+), column-purified fraction (19.89+), lyophilized
latex powder (22.70+) and papain (10.09+). (+) Linear regression between x and y. (++) Non-linear regression between log x and log y.
 P. Jaiswal, D.K. Singh / Veterinary Parasitology 152 (2008) 264270 267

were taken in 5 l of dechlorinated tap water. These
mixed populations were exposed to previously deter-
mined LC90 at 24 h of purified active molluscicidal
component against snail L. acuminata. Control groups
did not receive any treatment.
Mortality of snail was recorded at interval of 24 h each
up to 96 h. Dead animals were removed immediately to
avoid contamination in aquarium water. Snail mortality
was established by the contraction of the body within
shell; no response to a needle probe was taken as evidence
of death. LC values, lower and upper confidence limits
(LCL and UCL), slope values, t-ratio, g-values and
heterogeneity factors were calculated by using the POLO
computer software of Russell et al. (1977). The regression
coefficient between exposure time and different values of
LC50 was determined by the method of Sokal and Rohlf
(1973).
3. Results
The toxicity of seed and lyophilized latex powder of
C. papaya and their organic solvent extracts were time
and dose dependent. The LC50 of dried seed powder and
lyophilized latex powder of C. papaya at 24 h were
127.26, 19.92 mg/l and at 96 h 61.56 and 8.38 mg/l,
respectively (Table 2). Maximum toxicity among
different organic solvent extracts of C. papaya seed
powder was observed in ethanolic extract (Table 2). The
column-purified fraction of seed powder was highly
toxic. The LC50 of column-purified fraction of C.

Table 3
Toxicity of seed powder of Areca catechu, its different organic solvent extracts, column-purified fractions and active compound arecoline against the
snail Lymnaea acuminata at different exposure period
Exposure period Tested materials LC50 (mg/l) Limits Slope value t-ratio g-value Heterogeneity
LCL UCL
24 h Seed powder 27.23 23.74 33.87 3.46  0.58 5.99 0.11 0.30
Chloroform extract 37.19 25.15 113.44 2.07  0.54 3.83 0.26 0.32
Ether extract 17.70 16.19 20.51 5.43  0.94 5.77 0.12 0.23
Acetone extract 36.00 28.76 58.34 2.75  0.59 4.70 0.17 0.26
Ethanol extract 17.21 14.75 23.74 3.97  0.83 4.81 0.17 0.26
Column purified 11.25 9.04 18.13 2.76  0.60 4.61 0.18 0.23
Arecoline 0.49 0.38 0.80 2.33  0.49 4.79 0.17 0.36
48 h Seed powder 23.32 20.56 27.88 3.27  0.53 6.13 0.10 0.21
Chloroform extract 28.47 20.71 62.38 1.91  0.46 4.15 0.22 0.16
Ether extract 15.90 14.39 18.61 4.01  0.79 5.07 0.15 0.16
Acetone extract 27.95 22.86 42.25 2.23  0.51 4.39 0.20 0.14
Ethanol extract 14.08 12.58 17.04 3.99  0.74 5.37 0.13 0.21
Column purified 8.41 6.97 12.06 2.25  0.50 4.47 0.19 0.16
Arecoline 0.34 0.28 0.46 2.05  0.41 5.02 0.15 0.13
72 h Seed powder 17.45 15.38 19.82 3.21  0.51 6.27 0.10 0.25
Chloroform extract 25.52 17.76 75.33 1.38  0.40 3.41 0.32 0.12
Ether extract 13.59 12.42 14.99 4.36  0.78 5.61 0.12 0.17
Acetone extract 20.32 16.97 26.25 2.09  0.49 4.31 0.21 0.11
Ethanol extract 11.31 10.13 12.97 3.47  0.69 5.07 0.15 0.11
Column purified 6.73 5.50 9.23 1.86  0.47 3.93 0.25 0.14
Arecoline 0.21 0.17 0.27 1.74  0.38 4.62 0.18 0.17
96 h Seed powder 12.32 9.70 14.32 2.75  0.52 5.32 0.14 0.22
Chloroform extract 14.74 11.56 21.92 1.54  0.38 4.07 0.23 0.16
Ether extract 10.95 9.77 11.89 4.93  0.80 6.14 0.10 0.27
Acetone extract 12.95 10.71 14.77 3.09  0.53 5.89 0.11 0.14
Ethanol extract 8.05 6.79 8.96 3.88  0.72 5.42 0.13 0.32
Column purified 3.99 3.13 4.66 2.63  0.49 5.38 0.13 0.23
Arecoline 0.14 0.09 0.17 1.79  0.38 4.74 0.17 0.31
Six batches of ten snails were exposed to different concentrations of the above treatments. Mortality was recorded every 24 h. Concentrations given
are the final concentration in the glass aquarium water. Significant ( p < 0.05) negative regression was observed between the exposure time and
different LC50 values. Testing significance of the regression coefficient of Areca catechu seed powder (18.64+), chloroform extract (6.92+), ether
extract (16.90+), acetone extract (71.01+), ethanol extract (47.62+), column-purified fraction (14.86+) and arecoline (9.10+). (+) Linear regression
between x and y. (++), non-linear regression between log x and log y.
268 P. Jaiswal, D.K. Singh / Veterinary Parasitology 152 (2008) 264270
papaya seed was 27.33 and 7.06 mg/l at 24 and 96 h,
respectively. The aqueous extract of lyophilized latex
powder of C. papaya was more toxic than its other
organic solvent extracts. Toxicity of different prepara-
tions of A. catechu seed was also time and dose
dependent against L. acuminata. The LC50 of seed
powder was 27.23 and 12.32 mg/l at 24 and 96 h,
respectively. Among the organic solvent extracts, the
ethanolic extract of seed powder was more toxic
(Table 3). The LC50 at 24 h of ethanolic extract of A.
catechu seed powder against L. acuminata was
17.21 mg/l. The LC50 of column-purified fractions of
A. catechu seed powder at 24 h was 11.25 mg/l and at
96 h was 3.99 mg/l. The LC50 at 24 h of arecoline
hydrobromide was 0.49 mg/l. There was a significant
negative regression between LC50 of A. catechu seed
and exposure periods. There was no mortality in fish C.
fasciatus exposed to LC90 at 24 h of purified active
molluscicidal component against snail L. acuminata.
TLC analysis demonstrated that the Rf values of
spots of column-purified fraction of C. papaya seed and
aqueous extract of lyophilized latex (0.05 and 0.05,
respectively) show a correspondence to the Rf value of
papain (0.05). The Rf value of arecoline hydrobromide
(0.97) was equivalent to the Rf value of column-purified
fraction in A. catechu seed (0.97). LC50 at 24 h of
papain and arecoline hydrobromide were 16.63 and
0.49 mg/l, respectively (Tables 2 and 3). The slope
value given in the Tables 2 and 3 were steep and the
separate estimates of LC based on each of the six
replicates were found to be within the 95% confidence
limits of LC50. The t-ratio was higher than 1.96 and
heterogeneity factor was less than 1.0. The g-value was
less than 0.5 at all probability level (Tables 2 and 3).
There was significant negative regression ( p < 0.05)
between the exposure time and LC50 of the treatments
(Tables 2 and 3).
4. Discussion
The results of the present study indicate that the
seeds and latex of C. papaya and seeds of A. catechu are
potential source of botanical molluscicides. Their toxic
effects are time and dose dependent as evident from
negative regression between exposure period and LC50
of different treatments. The time dependent toxic effect
of these plant products may be either due to the uptake
of the active moiety which progressively increases the
amount of active component in snail body with increase
in exposure period or it may be possible that the active
compound (s) could change into more toxic forms in the
aquarium water or in the snail body by the action of
different enzymes. Among organic solvent extracts, the
higher toxicity of ethanolic extract of A. catechu seed
and C. papaya seed indicates that the molluscicidal
components present are more soluble in ethanol than
other organic solvents. It is evident from co-migration
on thin layer chromatographic plates that the mollusci-
cidal activity of C. papaya seeds and latex may be due to
the presence of papain. C. papaya seed extracts contain
antibacterial activity that could be useful in treating
chronic skin ulcers to promote healing (Dawkins et al.,
2003). Benzyl isothiocyanate is the main bioactive
compound responsible for the anthelmintic activity of
papaya seeds (Kermanshai et al., 2001). Papain protects
the tree from many herbivorous insects and pests
(Konno et al., 2004). Papain has potential anthelmintic
activity against the rodent gastrointestinal nematode
Heligmosomoides polygyrus (Stepek et al., 2005).
Although the antibacterial (Dawkins et al., 2003),
antiprotozoan (Ekanem et al., 2004), immunostimula-
tory, anti-inflammatory (Mojica-Henshaw et al., 2003),
antiamoebic (Tona et al., 1998) and antifertility (Lohiya
et al., 2006; Pathak et al., 2000) activities of C. papaya
seeds and larvicidal (Thomas et al., 2004) and
antifungal (Giordani et al., 1996) activities of C.
papaya latex were reported yet there was no report on
their molluscicidal activity.
Molluscicidal activity of A. catechu seed is due to the
presence of an alkaloid arecoline hydrobromide as
evident from the individual toxicity and identification
by TLC. Arecoline hydrobromide, a commercial salt, is
a stronger stimulant to the salivary glands than
pilocarpaine and a more energetic laxative than eserine
(Grieve, 2006).
Toxicity of C. papaya seed and latex and A. catechu
seed extracts against L. acuminata is in the range of high
molluscicidal activity, the LC50 value being less than
100 ppm (Singh and Singh, 1996a,b). Thus, the LC50 at
96 h of column-purified fraction of C. papaya seed
(7.06 mg/l), A. catechu seed (3.99 mg/l) and lyophilized
latex of C. papaya against L. acuminata are lower than
those of synthetic molluscicidescarbaryl (14.4 mg/l),
phorate (15.0 mg/l), formothion (8.5 mg/l) and niclosa-
mide (11.8 mg/l) (Singh and Agarwal, 1983). LC50 at
96 h of crude powder of C. papaya seed (61.56 mg/l) and
A. catechu seed (12.32 mg/l) against L. acuminata are
lower than the crude powder of Abrus precatorius seed
(93.83 mg/l), Punica granatum bark (62.62 mg/l), Canna
indica root (359.02 mg/l) and Lawsonia inermis seed
(125.84 mg/l) (S. Singh and D.K. Singh, 1999; Tripathi
and Singh, 2000; A. Singh and D.K. Singh, 2001).
It is evident from the steep slope values that a small
increase in the concentration of different treatments
 P. Jaiswal, D.K. Singh / Veterinary Parasitology 152 (2008) 264270
causes a marked mortality in snails. A t-ratio value
greater than 1.96 indicates that the regression is
significant. Values of heterogeneity factor less than
1.0 denote that in the replicate tests of random samples
the concentration response lines would fall within 95%
confidence limits and thus the model fits the data
adequately. The index of significance of potency
estimation g-values indicates that the values of the
mean are within the limits at all probability levels (90,
95, 99) as it is less than 0.5.
On the basis of above study it can be concluded that
the seeds and latex of C. papaya and seeds of A. catechu
may be used as potent molluscicides since the
concentration (LC90) used to kill 90% snail was not
toxic for C. fasciatus fish, which shares the same habitat
with snails. Nevertheless, further studies are required to
determine the mechanism of action of these plant
products in snail body.
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