DETERMINATION OF LETHAL AND FEEDING DETERRENT ACTIVITIES OF SAPONIN FROM PHALERIA MACROCARPA AGAINST POMACEA MACULATA

Abstract

Apple snail is one of the major pest of rice crop and saponin proved to be the most promising bioactive compound to control it. This study was carried out to quantify saponin from God's Crown, Phaleria macrocarpa and to evaluate its efficacy against the biological activities of black apple snail, Pomacea maculata. Fruits, leaves and stem-barks of P. macrocarpa were quantified for saponin using HPLC. The toxicity of leaf and fruit crude extracts was evaluated through mortality and feeding deterrent bioassays using complete randomized design and data were analyzed by ANOVA for LSD test. The highest saponin contents 24.67 ppm was detected in fruits followed by 22.67 ppm in leaves and 5.94 ppm in stem-bark. Bioassays showed the highest mortality percentage (44%) after 24 hours exposure at the concentration of 1000 ppm of a leaf extract followed by 36% and 28% @ 750 and 1000 ppm of leaf and fruit extracts, respectively. After the exposure of 48 hours, mortality percentage increased to 100% @ 1000 and 750 ppm of both crude extracts while the mortality percentage recorded at the concentration of 500 ppm of leaves and fruits were 56% and 52% respectively. Mortality percentage at the concentration of 500 ppm was increased to 80% and 68% in leaf and fruit extracts after exposure of 72 hours, respectively. In terms of feeding deterrent, 1000, 750 and 500 ppm concentration of both crude extracts were not significantly different from the positive control niclosamide (p>0.05). The results obtained from the study revealed that saponin extracted from fruits and leaves of P. macrocarpa has a potential to control black apple snails.

Full text

Roonjho et al., The J. Anim. Plant Sci., 31 (4) 2021
1070
DETERMINATION OF LETHAL AND FEEDING DETERRENT ACTIVITIES OF
SAPONIN FROM PHALERIA MACROCARPA AGAINST POMACEA MACULATA
A. R. Roonjho, R. Muhamad*and D. Omar
Department of Plant Protection, Faculty of Agriculture Universiti Putra Malaysia
Corresponding Author’s email: rita@upm.edu.my
ABSTRACT
Apple snail is one of the major pest of rice crop and saponin proved to be the most promising bioactive compound to
control it. This study was carried out to quantify saponin from God’s Crown, Phaleria macrocarpa and to evaluate its
efficacy against the biological activities of black apple snail, Pomacea maculata. Fruits, leaves and stem-barks of P.
macrocarpa were quantified for saponin using HPLC. The toxicity of lea f and fruit crude extracts was evaluated through
mortality and feeding deterrent bioassays using complete randomized design and data were analyzed by ANOVA for LSD
test. The highest saponin contents 24.67 ppm was detected in fruits followed by 22.67 ppm in leaves and 5.94 ppm in stem-
bark. Bioassays showed the highest mortality percentage (44%) after 24 hours exposure at the concentration of 1000 ppm
of a leaf extract followed by 36% and 28% @ 750 and 1000 ppm of leaf and fruit extracts, respectively. After the exposure
of 48 hours, mortality percentage increased to 100% @ 1000 and 750 ppm of both crude extracts while the mortality
percentage recorded at the concentration of 500 ppm of leaves and fruits were 56% and 52% respectively. Mortality
percentage at the concentration of 500 ppm was inc reased to 80% and 68% in leaf and fruit extracts after exposure of 72
hours, respectively. In terms of feeding deterrent, 1000, 750 and 500 ppm concentration of both crude extracts were not
significantly different from the positive control niclosamide (p>0.05). The results obtained from the study revealed that
saponin extracted from fruits and leaves of P. macrocarpa has a potential to control black apple snails.
Key words: Phaleria macrocarpa,Pomacea maculata, HPLC, botanical molluscicides, saponins
https://doi.org/10.36899/JAPS.2021.4.0304
Published online December 18, 2020
INTRODUCTION
Pomacea spp., generally known as apple snail,
belong to Family Ampullariidae from the phylum
Mollusca. Apple snail is a well-known pest of rice crops in
many Asian countries (Mokhtar, 2016). Historically, the
pest is a South American native and introduced in Asia
around 1980s as a food resource, but later it becomes a
grievous pest of rice crop in many rice growing East Asian
countries (Cowie, 2005; Naylor, 1996). Currently, apple
snail has extended from Asia to USA, Australia and latest
being in Spain, which makes it the first recorded
infestation in Europe (Cowie, 2005, 1998; Eldredge, 1994;
Rawlings et al., 2007). Apple snail has been acknowledged
as one of the 100 invasive alien species found in the world
(Lowe et al., 2000). Pomacea maculata (Black apple snail)
and Pomacea canaliculata (Golden apple snail) mostly
infest rice fields throughout the world (Hayes et al., 2012;
Yahaya et al., 2007). Both species can be differentiated
through the colour and suture structure of the shells,
whereby, P. canaliculata is yellowish to golden with short
and deeply channelled suture whereas, P. maculata is
black with longer suture (Hayes et al., 2012). Black apple
snails are more abundantly distributed in Malaysia than
golden apple snails (Arfan et al., 2014). Mostly synthetic
molluscicides are being used against the snails but
unfortunately; these molluscicides are also well known for
their adverse effects on human and environment (Mokhtar,
2016). Among the botanicals, saponin is the most
promising and widely studied bioactive compound against
apple snails (Mokhtar, 2016; Hostettmann et al., 1982; San
Martin, 2007). Numerous studies have revealed that the
haemolytic properties of saponin affect biological
activities, thus, making it highly toxic to most cold -
blooded pests. In snails, it causes apoptosis, which leads to
uncontrolled cell death (De Geyter et al., 2007; Sparg et
al., 2004). Phaleria macrocarpa also known as mahkota
dewa or God’s crown, it is a medicinal plant indigenous to
Indonesia and Malaysia. The leaves, stem barks and fruits
of P. macrocarpa are widely known for its medicinal
purpose and have been used since years as traditional
medicines in Indonesia and Malaysia to treat breast cancer,
bone cancer, heart and liver diseases, tumours and diabetes
(Kim et al., 2010; Hending, 2009). The fruits of P.
macrocarpa are good source of saponin (Altaf et al., 2013;
Gotama et al., 1999). The stem-barks and leaves of P.
macrocarpa have also been reported to have saponin
bioactive compound (Altaf et al., 2013; Andrean et al.,
2014; Gotama et al., 1999; Tjandrawinata et al., 2011).
Thus, this study was conducted to quantify saponin
bioactive compound in P. macrocarpa and its efficacy on
the biological activities of P. maculata.
The Journal of Animal & Plant Sciences, 31(4): 2021, Page: 1070-1077
ISSN (print): 1018-7081; ISSN (online): 2309-8694

Roonjho et al., The J. Anim. Plant Sci., 31 (4) 2021
1071
MATERIALS AND METHODS
Collection and preparation of plant materials: Leaves,
fruits and stem-barks of P. macrocarpa were collected in
the month of June 2016 from Taman Pertanian Universiti,
Universiti Putra Malaysia. The collected materials were
properly washed using tap water, and then followed by
distilled water. Plant materials were then dried in oven at
45 °C for one week. The dried plant materials were then
pulverized using a grinder, and the powdered material was
passed through a 0.7 mm sieve (attached with grinder) to
obtain a finer dust.
Extraction: The extraction of saponin was performed
through the maceration extraction method described by
Takeuchi et al. (2009) with slight modifications. Methanol
was used as a solvent to isolate the bioactive compound
from plant samples (Mustarichie et al., 2012). A hundred
grams of each sample was placed into a 1000 ml beaker
and mixed with 700 mL of the solvent. The mixture was
shaken for four days using an orbital shaker (Protech) and
left to stand for the next 24 hours. Subsequently, the
mixtures were filtered twice, once through a fine cloth and
again through Whatman No.1 filter paper and finally the
filtrates were ensured to be solvent free using the
Rotavapour R-215 that was connected to a heating bath B-
149 at 40 °C and vacuum pump V-700 (BUCHI, United
Kingdom) at 100 RPM. The crude extracts in the flask
were transferred into glass vials and stored in a refrigerator
at -4 oC until their next use.
Quantification of saponin bioactive compound: The
quantification of saponin bioactive compound was
performed on Agilent 1100 series HPLC system with DAD
Diode Array Detector (Agilent Technologies, USA). The
method used was modified from Guo et al. (2011) and
standard saponin of analytical grade from Sigma-Aldrich,
USA was used as external reference of saponin. The
Waters C18 column (250 mm × 4.6 mm, 5 µm) was used
for separation at 254 nm wavelength and 25 °C column
temperature. The flow rate was set at 0.7 ml/min with
injection volume of 10 µl. The mobile phase was consisted
of solvent A (Methanol) and solvent B (Water + 0.5%
phosphoric acid) at a ratio of 50:50 v/v for 7 minutes. The
signals were acquired and processed in a computer (HP)
using the software ChemStation.
Rice cultivation and snails rearing: The rice was
cultivated continuously in the glasshouse at Field 2,
Universiti Putra Malaysia to feed the snails and to be used
in bioassay experiments. Rice variety MR 219 was directly
seeded into plastic containers (28 cm × 39 cm × 11 cm)
filled with clay soil. The snails were collected from
Tanjung Karang paddy field, Selangor, Malaysia
(3°25′27″ N 101°11′05″ E). The eggs, juveniles
(hatchlings) and adults were handpicked. The egg masses
were placed in a separate tank as they had to be kept away
from water. The snails were reared in a plastic aquarium
(15 cm × 41 cm × 20 cm) in the glasshouse under natural
condition. Throughout the study, the aquariums were
washed and the water was changed every two days to avoid
contamination. Adult snails were provided with rice leaves
of up to 28 days old for their consumption, whereas, one
to 20 days old hatchlings were fed with algae. The black
apple snails were identified based on their shell
morphology (Cowie et al., 2006). Snails with shell height
of 4 cm were used in all experiments.
Mortality bioassay: Mortality bioassay was carried out
based on the guidelines for molluscicide evaluation
(WHO, 1983) with slight modifications. The crude
extracts of fruits and leaves were tested on P. maculata at
five different concentrations (1000, 750, 500, 250 and 100
ppm) along with positive control synthetic molluscicide
Niclosamide (1.12 ml in 200 ml of water) and negative
control distilled water. For each treatment, 200 ml of the
respective solution was added to a plastic aquarium
containing five apple snails. The snails were starved for 24
hours prior to the experiment, and 0.5 g of rice leaves were
provided to the snails for feeding during the experiment.
The experiment was done under Completely Randomized
Design with five replications. The mortality was assessed
at 24, 48 and 72 hours. If the apple snails failed to show
coordinated movements when softly pushed were
considered dead.
Feeding deterrent bioassay: Feeding deterrent bioassay
was conducted through leaf dip bioassay method described
by Dawidar et al. (2012) with slight modifications. Five
concentrations (1000, 750, 500, 250 and 100 ppm) of both
crude extracts were tested on black apple snails.
Niclosamide and water were used as positive and negative
controls, respectively. Five apple snails per aquarium were
used in all five replications. Apple snails were starved for
24 hours before the experiment. The leaf area and weight
of rice leaves were measured using LI -3100 Leaf Area
Meter (LI-COR, USA) and Sartorius BT224S analytical
balance (Sartorius, Germany) before and after exposure.
Statistical analysis: The experiments were carried out in
CRD, and data were analysed by ANOVA for LSD test at
0.05 probability level using SAS 9.4 computer software
(SAS Institute Inc. 2009). The data obtained from
mortality bioassay were normalized using arcsine
transformation.

Roonjho et al., The J. Anim. Plant Sci., 31 (4) 2021
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RESULTS
Extraction and quantification of saponin bioactive
compound: The crude extract of fruits, leaves and stem-
barks of P. macrocarpa were used for saponin
quantification. Before HPLC analysis of samples, the
external standard system was used to optimize the method
as it was necessary for the quantification of the compound
in samples (Mradu et al., 2012). The standard saponin
from Sigma-Aldrich at five different concentrations (30,
25, 20, 15, and 10 ppm) were used to establish and
calibrate the HPLC method at R2(0.999) for saponin
quantification. The retention time for saponin detection
was recoded from 2.12 to 2.17 minutes. Table 1 shows the
retention time, peak area, peak height, saponin content in
30 ppm concentration of sample and saponin yield
percentage obtained from the crude extracts of each plant
parts that were quantified using HPLC analysis.
The highest saponin contents were 24.7 ppm with
the yield percentage of 14.8 %. Saponin was detected in
the fruit extracts at the retention time of 2.134 minutes
with peak area of 150 µV*sec and peak height 7.46 µV.
The second highest saponin contents were 22.7 ppm with
the yield percentage of 14.4 % and that were detected at
the retention time of 2.112 minutes with peak area of 139.7
µV*sec and peak height 5.72 µV in the leaf extract.
Meanwhile, the crude extracts of stem barks recorded the
least amount of saponin which was 5.9 ppm with yield
percentage of 2.4 %. Saponin in stem barks of P.
macrocarpa was detected at the retention time of 2.108
minutes with peak area of 53.48 µV*sec and peak height
3.11 µV.
Mortality bioassay: Table 2 shows the mortality
percentage of P. maculata in different concentrations of
fruits and leaves extracts of P. macrocarpa at 24, 48 and
72 hours. At 24 hours, both plants parts showed positive
molluscicidal activities against P. maculata at 500, 750
and 1000 ppm concentrations. The highest mortality
percentage recorded at 24 hours was 44% in 1000 ppm of
leaves extract followed by 36% in 750 ppm of same crude
extract and 28% in 1000 ppm of fruits extract as compared
to 100% mortality in positive control niclosamide.
All treatments were significantly different from
positive control niclosamide at P<0.05. The lowest
mortality percentage recorded was 8% in 750 ppm of fruits
extracts. Meanwhile, no dead snails were found at 24 hours
in 100 and 250 ppm of both crude extracts and negative
control water. After 48 hours, the highest mortality was
100% recorded at 1000 ppm and 750 ppm of both crude
extracts followed by 56% and 52% in 500 ppm of leaves
and fruits extracts respectively. There were no de ad snails
found in 250 and 100 ppm of leaves and fruits extracts, as
well as in untreated control at 48 hours. Leaves and fruits
extract applied in higher concentrations at 1000 pm and
750 ppm were not significantly different from positive
control niclosamide at 48 hours. This meant that the
molluscicidal effects of both plants’ parts were similar to
niclosamide. After exposure of 72 hours, the mortality
percentage in 500 ppm of leaves and fruits crude extracts
reached 80% and 68% respectively. However, no mortality
was recorded in 250 ppm and 100 ppm of both crude
extracts and water until 72 hours.
Feeding deterrent bioassay: Table 3 illustrates the mean
weight of rice leaves consumed by P. maculata when
exposed to the crude extracts of leaves and fruits. After 24
hours, the mean leaf weight consumed by black apple
snails for positive control treated with niclosamide was
0.00 ± 0.00 g. Similarly, leaves showed no reduction in
weight when treated with 1000 ppm concentration of
leaves extract. The lower the consumed leaf weight, the
better the effects of crude extracts in disrupting the feeding
behaviour of P. maculata.
The consumed leaf weight for extracts from both
fruits and leaves at 1000, 750 and 500 ppm concentrations
were not significantly different from niclosamide at
P>0.05, therefore, it showed similar antifeedant effects as
niclosamide. The lowest consumed weight of leaves was
0.00 ± 0.00 g in 1000 ppm concentration of leaves extracts
followed by 0.002 ± 0.002 g at 1000 ppm concentration of
fruits extract. Meanwhile, the 250 and 100 ppm
concentrations of leaves and fruits extracts were
significantly different from control niclosamide at P<0.05.
The highest mean consumed weight recorded was 0.096 ±
0.006 and 0.096 ± 0.009 g at 100 ppm concentrations of
leaves and fruits extracts respectively. The consumed
weight of leaves at 250 and 100 ppm of both crude extracts
were also significantly different at P<0.05 from negative
control water which was 0.37 ± 0.008 g. Table 4 shows the
leaf area consumed by P. maculata when exposed to 1000,
750, 500, 250 and 100 ppm concentrations of fruits and
leaves extracts. After 24 hours, the lowest mean leaf area
was 0.00 ± 0.00 cm2when treated with positive control
niclosamide. Lower leaf area means, reflected better
effects of crude extracts in disrupting the feeding
behaviour of P. maculata.
In terms of leaf area consumption, the three
highest concentrations of both crude extracts were not
significantly different with the control niclosamide at
P>0.05. This proved that 1000, 750 and 500 ppm
concentrations of both crude extracts have the same
antifeedant effects as niclosamide. However, the lowest
mean area of leaves for crude extracts recorded was 0.02
± 0.02 cm2at 1000 ppm concentration of leaves extracts
followed by 0.122 ± 0.122 cm2at 1000 ppm concentration
of fruit extract respectively. T he 250 ppm and 100 ppm
concentrations of both crude extracts showed significant
difference with negative control water at P <0.05. The
mean highest consumed leaf area recorded was 6.474 ±
0.24 cm2followed by 5.538 ± 0.56 for 100 ppm
concentrations of leaves and fruits extracts, respectively.

Roonjho et al., The J. Anim. Plant Sci., 31 (4) 2021
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Table 1: Saponin contents (ppm) in 30 ppm concentration and saponin yield (%) from different parts of P.
macrocarpa.
Plant Parts
Ret. Time
(min)
Area (µV*sec)
Height
(µV)
Area %
Saponin contents
(ppm)
Saponin yield
%
Fruits
2.134
150.01
7.46
100
24.7
14.8
Leaves
2.112
139.73
5.72
100
22.7
14.4
Stem-barks
2.108
53.48
3.11
100
5.9
2.4
Table 2: Mortality Percentage (%) of P. maculata when treated with crude extracts of leaves and fruits at 24, 48,
and 72 hours.
Treatments
Concentration (ppm)
Mean±SE
24h
48h
72h
Fruit Extract
1000
28 ±4.89cd
100 ±0.0a
100 ±0.0a
750
8 ±8.0ef
100 ±0.0a
100 ±0.0a
500
12 ±4.89ef
52 ±10.19b
68 ±4.8c
250
0±0.0f
0±0.0c
0±0.0d
100
0±0.0f
0±0.0c
0±0.0d
Leaves Extract
1000
44 ±7.48b
100 ±0.0a
100 ±0.0a
750
36 ±7.4bc
100 ±0.0a
100 ±0.0a
500
20 ±6.32de
56 ±7.4b
80 ±8.9b
250
0±0.0f
0±0.0c
0±0.0d
100
0±0.0f
0±0.0c
0±0.0d
Control
Niclosamide
100 ±0.0a
100 ±0.0a
100 ±0.0a
Untreated
0±0.0f
0±0.0c
0±0.0d
Means with same letters within column are not significantly different at P > 0.05
Table 3: Mean leaf weight (g) consumed by P. maculata when treated with crude extracts of leaves and fruits.
Treatments
Concentration (ppm)
Mean±SE
Weight
Fruit Extract
1000
0.002 ±0.002e
750
0.004 ±0.002e
500
0.01 ±0.003e
250
0.072 ±0.015c
100
0.096 ±0.006b
Leaves Extract
1000
0.00 ±0.00e
750
0.006 ±0.004e
500
0.012 ±0.005e
250
0.05 ±0.004d
100
0.096 ±0.009b
Control
Niclosamide
0.00 ±0.00e
Untreated
0.37 ±0.008a
Means with same letters within column are not significantly different at P > 0.05

Roonjho et al., The J. Anim. Plant Sci., 31 (4) 2021
1074
Table 4: Mean leaf area (cm2) consumed by P. maculata when treated with crude extracts of leaves and fruits.
Treatments
Concentration (ppm)
Mean±SE
Area
Fruit Extract
1000
0.122 ±0.122e
750
0.404 ±0.24e
500
0.714 ±0.28e
250
3.902 ±0.33c
100
6.474 ±0.24b
Leaves Extract
1000
0.02 ±0.02e
750
0.482 ±0.288e
500
0.884 ±0.38e
250
2.306 ±0.58d
100
5.538 ±0.56b
Control
Niclosamide
0.00 ±0.00e
Untreated
15.982 ±0.48a
Means with same letters within column are not significantly different at P > 0.05
DISCUSSION
Extraction and quantification of saponin bioactive
compound: Previous study on the crude extract from the
fruits of P. macrocarpa showed the presence of bioactive
compounds such as saponin glycosides, tannins, phenols,
and flavonoids (Lay et al. 2014). Altaf et al. (2013) and
Andrean et al. (2014) had also highlighted that the fruits
of P. macrocarpa are rich in saponin bioactive compound.
Similarly, the HPLC analysis results of this study
supported this observation. The fruit extracts of P.
macrocarpa contained 82.4% of saponin while the yield of
crude extract from dried powder of fruits of P. macrocarpa
was 18 % so the total yield of saponin from fruits was
recorded 14.8 %. This strongly supports the P.
macrocarpa fruits as a main source of saponin bioactive
compound, thus, confirming that saponin was the main
bioactive compound in the fruits of P. macrocarpa. This
finding is crucial, as future extraction of saponin from this
plant can be targeted on the fruits as it can yield high
quantity.
As detected by the HPLC analysis, the leaves
extracts were found to be the second richest in saponin
content. Total saponin amount was 75.7 % while the yield
of crude extract from dried powder of Leaves of P,
macrocarpa was 19% therefore; the saponin yield % was
14.4 in leaves. Previous studies have reported the presence
of saponin bioactive compound in the leaves of P.
macrocarpa, together with tannins and flavonoids
(Shodikin, 2010; Faried et al., 2016).Previously, some
other studies also reported presence of saponin and
alkaloids in leaf extracts of P. macrocarpa and proposed
the use of P. macrocarpa leaves for antibacterial purposes
(Elianora et al., 2017; Altaf et al., 2013). Furthermore, the
yield of crude extract from leaves was higher comparing
with fruits. This further proved that the leaves are also a
suitable source of saponin.
The HPLC analysis of the stem-barks extracts
revealed the lowest saponin content in comparison to all
the other plant parts. Although previous studies have
reported the presence of saponin bioactive compound in
the stem-barks of P. macrocarpa (Gotama et al., 1999;
Altaf et al., 2013). But based on the HPLC analysis in this
study, only 5.94 ppm of saponin was detected in 30 ppm
of crude extract which were only 19.8 % and the yield of
crude extracts was also low as it was only 12% and total
saponin yield from stem-barks was only 2.4%. The low
saponin content in the barks reports as an inefficient source
of saponin as compared to the leaves and fruits of P.
macrocarpa.
Mortality bioassay: The results obtained from mortality
bioassay proved that saponin extracted from fruits and
leaves of P. macrocarpa have the potential to kill apple
snails as early as 24 hours. Previous studies had also
recorded the molluscicidal effects of saponin from
Furcraea spp.on apple snails at 24 hours (Osman et al.,
2011; Jose et al., 2013; Mokhtar, 2016). It was also
observed that saponin from fruits and leaves could show
100% mortality after 48 hours. Similarly, some previous
studies also revealed that saponin from Entada
phaseolides could achieve 80 to 100% mortality against
apple snails after 48 hours of exposure (Morallo-Rejesus
and Maini, 1991; Morallo-Rejesus et al., 1995; Rejesus
and Punzalan, 1997). As observed in this study, the
effectiveness of both crude extracts was directly relative to
the time of exposure to the saponin concentration. If the
exposure time to saponin increased the egg laying
capacity, growth rate and survival rate of freshwater apple
snails also decreased (Mahato et al., 1982).

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1075
Feeding deterrent bioassay: The results of this study from
feeding deterrent bioassay revealed that apple snails avoid
the saponin treated diet, some previous studies also
recorded 0% leaf damaged in lettuce caused by snails at 4
hours when treated with 3% ethanolic Myrrh (Ali, 2005).
Another study also reported 19% damage in rice seedlings
when treated with methanolic extract of dried neem leaves
(Latip et al., 2017). Higher saponin contents lowered the
feeding activities of snails, as saponin displayed
molluscicidal properties against apple snails (Huang et al.,
2003). Additionally, the noxious odour produced can also
prevent apple snails from consuming rice leaves. Higher
concentration of crude extracts of saponin containing
plants, reduced the feeding attraction of apple snails
towards rice leaves (Mokhtar, 2016). Besides of saponin’s
direct molluscicidal and insecticidal activities, it is also
very well-known for its antifeedant properties. Therefore,
plants that containing saponin could also serve as an
antifeedant to prevent molluscs from feeding on living
plants. (Mason et al., 1994; Chaieb et al., 2009), This
supported the finding in the anti-feedant bioassays
conducted in this research.
Conclusion: Methanolic extracts of fruits and leaves of P.
macrocarpa are rich in saponin and showed strong
bioactivities against black apple snails. Therefore, it is
recommended that it should be further investigated as an
eco-friendly cost effective botanical molluscicide.
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