ORIGINAL ARTICLE

Bali Medical Journal (Bali MedJ) 2023, Volume 12, Number 2: 1163-1170
P-ISSN.2089-1180, E-ISSN: 2302-2914

Activity of enzyme Esterase,

Glutathione S Transferase and Inorganic Substance
of Dengue vector Aedes aegypti larvae against
Lansium domesticum leave extract
and fractionation

Devita Febriani Putri1*, Ismalia Husna2, Mala Kurniati3, Annisa Primadiamanti4

ABSTRACT

Backgound: Dengue vector control using chemical insecticides for a long time on the same target encourages the rapid
development of a resistant dengue vector population of Aedes aegypti. Therefore, an alternative method is needed in the
form of the use of plant-based insecticides, Lansium domesticum or duku has been proven to contain secondary metabolites
1
Department of Parasitology, Faculty that have the potential as insecticides. This study was conducted to measure the activity of enzyme esterase and glutathione
of Medicine, Universitas Malahayati, S-transferase, as well as to observe the concentration of inorganic substances such as Fe, Mg, Na, and K in Ae. aegypti larvae
Indonesia; exposed to L. domesticum extract using methanol as the solvent and fractionation using hexane, butanol, and ethyl acetate
2
Medical Education Study Program, as the solvents.
Faculty of Medicine Military, Universitas
Methods: This study used Ae. aegypti larvae and L. domesticum leaves extract and fraction. Esterase activity was measured
Pertahanan, Bogor, Indonesia;
3
Department of Biology, Faculty of
using a spectrophotometer at λ = 490 nm and obtained as light absorption per minute per mg protein. GST activity was
Medicine, Universitas Malahayati, measured using a spectrophotometer at λ = 340 nm and expressed as light absorption per minute per mg protein. Inorganic
Lampung, Indonesia; substance level was evaluated by the absorbance of the atoms using an atomic absorption spectrophotometer which was
4
Undergraduate Pharmacy Study analyzed at certain wavelengths of each atom according to Beer’s law equation.
Program, Faculty of Health Sciences, Results: The results showed LD50 and LD90 of the crude extract of L. domesticum were 2200ppm and 3200ppm after 24
Universitas Malahayati, Indonesia; hours of observation. Crude extract and the fraction of L. domesticum leaf influenced the development of Ae. Aegypti. It
reduced the activity of the esterase enzyme. Meanwhile, it increased the GST enzyme activity of Ae. aegypti larvae as well as
*Corresponding author: affects the levels of inorganic substances in Ae. aegypti larvae.
Devita Febriani Putri; Conclusion: Thus, L. domesticum as a plant-based insecticide is an alternative to control the vector whose target is more
Department of Parasitology, Faculty
selective and safe.
of Medicine, Universitas Malahayati,
Indonesia;
devita@malahayati.ac.id Keywords: Lancium domesticum, Aedes aegypti, enzyme activity, inorganic substance level.
Cite This Article: Putri, D.F., Husna, I., Kurniati, M., Primadiamanti, A. 2023. Activity of enzyme Esterase, Glutathione S
Received: 2023-11-29 Transferase and Inorganic Substance of Dengue vector Aedes aegypti larvae against Lansium domesticum leave extract and
Accepted: 2023-03-28 fractionation. Bali Medical Journal 12(2): 1163-1170. DOI: 10.15562/bmj.v12i2.3946
Published: 2023-04-12

INTRODUCTION
Dengue Hemorrhagic Fever (DHF) is
a common vector-borne disease in
Indonesia with a high endemicity level.
The Ministry of Health of the Republic
of Indonesia collaborated with the World
Health Organization (WHO) Country
Office Indonesia to establish the National
Strategic Program (NSP) for the Dengue
Fever Control Program (2021-2025). The
main indicator in the NSP related to dengue
infection is the percentage of regencies/
cities with an IR (incidence rate) below 49
per 100,000 population and a CFR of 0.5%,
reaching 90% by 2025. Therefore, in order
to reach this target, six strategies have been
prepared. Those strategies are increasing
effective, safe, and sustainable vector
management, such as increasing access
and quality of dengue cases treatment,
strengthening the comprehensive dengue
surveillance and responsive outbreak
management, promoting sustainable
community involvement, strengthening
government commitment, policy, program
management, and partnerships; and
improving the assessments, inventions,
innovations, and research as the basis for
managing evidence-based policies and
programs. In this case, vector control still
becomes an essential and effective strategy
in controlling dengue cases.1,2
The Ministry of Health of the
Republic of Indonesia has regulated
the management and control of dengue
vectors in Indonesia by implementing
Integrated Vector Management (IVM).
IVM is an integrated program that
uses all available techniques for dengue
vector control, including environmental
management, biological methods, physical
methods, and chemical methods. In this
case, chemical insecticides are still used
as a chemical method to control dengue
vectors quickly in the outbreak area. Using
insecticides for a long time for the same
target provides selection pressure that

Bali Medical
Open access:Journal 2023; 12(2): 1163-1170 | doi: 10.15562/bmj.v12i2.3946
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 ORIGINAL ARTICLE
encourages the development of Aedes
aegypti populations to become resistant
quickly. In addition, chemical insecticides
contain several harmful active ingredients,
such as dichlorvos, porpoxure, and
synthetic pyrethroids, which cause bad
side effects on human health.3,4
Plant-based insecticides are derived
from plants, an alternative for vector
control with a more selective and safe
target. Lansium domesticum or duku is
a tropical plant. In this case, a review
analysis has been conducted in the last 10
years and revealed that the extraction L.
domesticum plant has good potential as an
ovicide, larvicide, and even insecticide for
the adult stage of Ae. aegypti. L. domesticum
has also been proven to contain secondary
metabolites that have the potential as
insecticides, contain alkaloids, flavonoids,
saponins, and polyphenols.5–11 Alkaloids
with an indole structure such as strikhnin
and quinine have bitter taste and can be
used as insect repellents.12 Meanwhile,
saponins are also known to have anti-
insect effects, because this compound
can reduce digestive enzyme activity and
absorbs food. In addition, saponin can
also bind free sterols during food digestion
because sterols have a precursor role to
the hormone ecdysone. Therefore, in this
case, a decrease in the number of free
sterols will interfere with insect molting.13
Furthermore, another benefit of this
compound is that it also interacts with the
cuticle membrane of the larvae so that it
can damage the membrane of the larvae.14
However, research on duku extract
faced obstacles because this fruit only
grow in a certain season (once a year),
so research was carried out using duku
leaves that grow in any season. Research
conducted previously by Mayanti et
al. discovered that duku leaves contain
terpenoids, alkaloids, flavonoids, and
saponins.10 Furthermore, Monzon et al.
have also proven that the aqueous extract
of duku leaves was effective in killing Ae.
aegypti after 48 hours of exposure.11 In
this case, the duku leaves were used on
the 4th to 13th strands because they were
categorized as old duku leaves. As reported
by Dwinatari and Murti old leaves, the
formation of secondary metabolites
was complete and the phytochemicals
1164
contained in the leaves were stable.12
Ae. aegypti mosquito is a dengue
vector that manages toxicity through
their antioxidant defense mechanism,
consisting of enzymatic and non-
enzymatic antioxidant components.13,14
Increasing or modifying the activity
of esterase enzymes and glutathione
S-transferase (GST) is a detoxification
mechanism that causes immunity/
resistance to chemical insecticide toxicity.
In this case, research has been carried
out by Pethuan S et al. revealed that non-
specific esterases could play a role in Ae.
Aegypti, which was pyrethroid-resistant
and correlated with tolerant permethrin,
while the increased activity of GST plays
a role in dichlorodiphenyltrichloroethane
(DDT) resistance.15 A similar statement
was revealed by Hemingway that GST
played a role in DDT resistance, while
non-specific esterases played a role
in organophosphate, carbamate, and
pyrethroid resistance.15,16 Another study
also stated that the esterase enzyme had a
role in binding, and its insecticidal effect
slowly disappears, while the GST enzyme
has a role in xenobiotic detoxification.17
Another study stated that acetone
extract derived from the Lantara
camara plant significantly reduced
Acetylcholinesterase (AchE) and GST
enzyme activity in Culex pipiens larvae by
reducing the production of antioxidants,
thus leading to the death of the larval.
In their research, Yu et al. also employed
plant extracts and found that seaweed
extract (Bryopsis pennata) on anal papillae
could disrupt the regulation of ions or
inorganic substances in the larvae.18
This study was further carried out to
prove that the crude extract and fraction
of duku (L. domesticum) leaves affect
the activity of enzymes (esterase and
GST) and the concentration of inorganic
substances such as Fe, Mg, Na, and K.
The research was carried out using duku
leaves that grow in any season, so it was
not hampered in sampling because the
fruit only grow in a certain season (once a
year). The crude extract of L. domesticum
leaves was obtained using a solvent in
methanol, while the solvent used to make
the fraction is in the forms of hexane,
butanol, and ethyl acetates.
Bali Medical Journal 2023; 12(2): 1163-1170 | doi: 10.15562/bmj.v12i2.3946
MATERIALS AND METHODS
The research has passed the ethical test
with a statement of ethical clearance no.
2862/EC/KEP-UNMAL/VIII/2022 issued
by the Research Ethics Commission of
Universitas Malahayati.
Preparation of Ae. aegypti larvae at
the last third instar and early fourth
instar
The subject of this study was the eggs
of Aedes aegypti mosquito, which
resulted from rearing in the Entomology
Laboratorium of Universitas Indonesia.
In this case, the subject was prepared at
the Medical Chemistry of Universitas
Malahayati. The incubation was conducted
on the eggs in trays containing 1.5 – 2 L of
water reaching half the tray to obtain the
subjects. After the egg hatched, the larva
was maintained to survive by giving 0.5 g
of chicken liver feed on day 0, and 1 g of
chicken liver feed on the first to fifth day
or before becoming a complete pupa. In
this case, when there was a husk on the
water’s surface on the tray, it was cleaned
immediately before the feed was given. In
addition, the water was also changed 2-3
times a week. Furthermore, larvae were
maintained until they were at the late third
instar and early fourth instar of larvae.
Preparation, extraction, and
fractionation of L. domesticum leaves
L. domesticum leaves samples were
obtained from Mount Sugih, Central
Lampung. The leaves used were old
duku leaves of the 4th to 13th strands.18
FurthermoreL. domesticum leaves were
prepared, extracted, and fractionated at
the Medical Chemistry of Universitas
Malahayati. In this case, the preparation
was initially carried out by taking the 4th-
13th strands of the L. domesticum leaves
and drying them under the sun for one
week. After that, the leaves were mashed
using a blender to obtain 500 g of sample
powder. The next step is the extraction of
L. domesticum leaves. In order to obtain
the leaf extract, the sample powder was
soaked for 3x24 hours using absolute
methanol. In this case, the solvent was
changed every 1x24 hours, filtered, and
separated from the residue and filtrate.

Table 1. Analysis results of Probit LC50 and LC90 crude leaf extract of L. domesticum.
Time (hour)
Conc
24 2200
72 700
Table 2. Effect of crude extract and leaves fraction of L. domesticum on the
mortality of Ae. aegypti.
Types of Extract Total Mortality of
the Larvae (24 hours)
Control
Crude Extract
Hexane Fraction
Ethyl Acetate Fraction
Butanol Fraction
Sample Absorbance = Sample Mean Absorbance – Blank Mean Absorbance
The filtrate was evaporated using a rotary
evaporator to obtain a thick extract (crude
extract).19
Meanwhile, fractionation was carried
out after duku leaves extract was obtained.
This process was done by dissolving the
methanol duku leaves extract in water
and stirring until dissolved. After that, the
result was put into a separating funnel with
a capacity of 250 ml and fractionated using
120 ml of n-hexane as the solvent four
times. After separating and forming two
layers, the residue and n-hexane fraction
were taken. Furthermore, the methanol-
water residue was fractionated again using
120 ml of ethyl acetate solvent four times.
In this case, the residue and ethyl acetate
fraction was taken after separating and
forming two layers. Next, methanol-water
residue was added three times using 80
ml of butanol solvent. After separating
the two layers, the residue and butanol
fraction were taken. Then, each fraction
obtained was evaporated using a vacuum
evaporator.20
Examination of Esterase Activities
A test tube containing 0.5 ml of 0.1 M
potassium phosphate buffer at pH 7.5
was added by 15 µl of 0.02 M α-naphthyl
acetate substrate in methanol and 50 µl
of Ae. aegypti larvae homogenate. For
the blanks, the homogenate was replaced
with phosphate buffer. The mixture was
then incubated at 37oC for 30 minutes;
then the reaction was stopped by adding
Bali Medical Journal 2023; 12(2): 1163-1170 | doi: 10.15562/bmj.v12i2.3946
LC50 (ppm)
Lower limit Upper Limit Conc
1780 2520 3200
0 1600 1200
Larvae Mortality
(24 hours)
0 0%
15 60%
25 100%
12 48%
1 4%
2 ml of fast blue (0.2 mg/ml H2O). In this
case, the light absorption was measured
using a spectrophotometer at λ = 490 nm.
The esterase activity was obtained as light
absorption per minute per mg protein.21
Examination of GST Activities
In this case, a test tube containing 0.9
ml of 0.1 M phosphate buffer at pH 7.5
was added to 20 µl of Ae. aegypti larvae
homogenate, 100 µl glutathione 0.001 M,
and 10 µl CDNB (1- chloro -2, 4- dinitro
benzene). Light absorption was measured
using a spectrophotometer at λ=340 nm
at 30oC. Glutathione S-transferase activity
was expressed as light absorption per
minute per mg protein.21
Examination of Inorganic Substance
Level
Ae. aegypti larvae were fixed using
formalin. After that, it was aspirated into a
flame from the elements in the sample and
converted into atomic vapor so that the
flame contained the elements analyzed. In
this case, the amount of content of each
atom analyzed was obtained by looking
at the absorbance of the atoms using an
atomic absorption spectrophotometer
which was analyzed at certain wavelengths
of each atom according to Beer’s law
equation.22
Statistical Analysis
The research data was analyzed through
Probit Test using SPSS ver.24
ORIGINAL ARTICLE
LC90 (ppm)
Lower limit Upper Limit
2700 4180
0 2300
RESULTS
Before measuring the enzyme activity
and inorganic ion levels, a bioassay test
was conducted on L. domesticum leaves
extract at 2000ppm, 4000ppm, 6000ppm,
8000ppm, 10000ppm, and control using
distilled water. Bioassays were carried out
according to WHO regulations using late
III and early IV instar of 25 Ae. aegypti
larvaes that were added to 200 ml of
extract in each replication. Observations
were carried out for 24 hours and 48 hours,
then analyzed to obtain LC50 and LC90
from each concentration of L. domesticum
leaves extract.23
The fraction of duku leaves was made
based on the bioassay results on the crude
extract, which was 2200 ppm according
to the LC50 result of the extract (Table
1). Duku leaves fractionation has been
carried out using hexane, ethyl acetate,
and butanol as the solvents. The hexane
fraction was 0.246 g, the ethyl acetate
fraction was 0.261 g, and the butanol
fraction was 0.271 g, which were further
used to observe the activity of esterase and
GST enzymes as inorganic substances.
Table 2 presents the effect of crude
extract and the fraction of L. domesticum
leaves on the mortality of Ae. aegypti for
24 hours. It was obtained that no dead
larvae was found in the control group, but
the highest larval mortality was found in
the hexane fraction. This indicates that
the leaf fraction of L. domesticum has a
good effect in causing larval death, but
not all fractions have a better effect than
the crude extract. This result indicates that
the solvent used during fractionation also
influences the mortality of Ae. aegypti.
Measurement of Esterase and GST
Enzyme Activities
In this study, the enzyme activity of Ae.
aegypti larvae were measured in the control
and treatment groups (Table 3). In this
case, the enzymes measured were esterase
(Table 4) and Glutathione S Transferase
(GST) (Table 5), detoxification enzymes
1165
 ORIGINAL ARTICLE

Table 3. Total protein level on sample.

Absorbance at λ= 280 nm Total Protein Level Total Protein Level
Sample
Abs 1 Abs 2 Mean Abs (µg/ml) (mg/ml)
Control 0.460 0.463 0.4615 767.167 0.767167
Extract 0.394 0.341 0.3675 610.5 0.6105
Hexane Fraction 0.116 0.119 0.1175 193.83 0.19383
Ethyl Acetate Fraction 0.134 0.145 0.1395 230.5 0.2305
Butanol Fraction 0.319 0.278 0.2985 495.5 0.4955
Description: Abs= Absorbance

Table 4. The Absorbance of Sample on Esterase.

Absorbance at λ=490nm
Sample Abs of sample Incubation Period
Abs 1 Abs 2 Mean Abs
BL 0.011 0.008 0.0095 30 minutes
Control 0.175 0.176 0.1755 0.1660
Extract 0.126 0.127 0.1265 0.1170
Hexane Fraction 0.054 0.047 0.0505 0.0410
Ethyl Acetate Fraction 0.055 0.057 0.0560 0.0465
Butanol Fraction 0.113 0.106 0.1095 0.1000
Description: Abs= Absorbance, BL= blank

Table 5. Sample Absorbance on GST.

Absorbance at λ=340nm
Sample Abs of sample Incubation Time
Abs 1 Abs 2 Mean abs
BL 0.008 0.012 0.0100 20 minutes
Control 0.434 0.393 0.4135 0.4035
Extract 0.39 0.359 0.3745 0.3645
Hexane Fraction 0.142 0.123 0.1325 0.1225
Ethyl Acetate Fraction 0.146 0.15 0.1480 0.1380
Butanol Fraction 0.282 0.265 0.2735 0.2635
Description: Abs= Absorbance, BL= blank

affect the levels of inorganic substances

Enzyme Activities = Absorbance of Sample (OD)/minute of incubation
mg protein
in larvae when exposed to an insecticidal
substance. A total of 80 larvae were used to
determine the enzyme’s activity, including
a control group consisting of 16 larvae and
treatment groups including crude extract,
hexane fraction, ethyl acetate fraction, and
butanol fraction consisting of 16 larvae
each.
Figure 1 and Figure 2 show the activity
of esterase enzymes and GST in units
of OD (absorbance value) per minute
of incubation and per g of protein. The
activity of esterase enzymes in Ae. aegypti
larvae treatment was lower than the Ae.
aegypti larvae control, during the GST
enzyme activity of Ae. aegypti treatment
was higher than the Ae. aegypti larvae
control group. This result indicated that
the administration of crude extract and
leaf fraction of L. domesticum affected the
level of enzyme activity in Ae. aegypti.
Measurement of Inorganic Substance
Level
The levels of inorganic substances
possessed by the Ae. aegypti larvae were
examined in this study, including Fe, Mg,
Na, and K. The inorganic levels obtained
were as shown in Figure 3.
The inorganic levels were measured
so that damage to the anal papillae of Ae.
aegypti can be known and further can
in larvae. After the measurement, it
could be known whether the levels of
inorganic substances in the Ae. aegypti
larvae increased or decreased. This result
was presumably due to damage to the anal
papilla (an ion regulation tool in larvae)
due to the administration of crude extract
and leaves a fraction of L. domesticum.
DISSCUSION
The stems, leaves, and fruit of Lansium
domesticum (duku) contain alkaloids,
flavonoids, saponins, and polyphenols
that have a role as plant-based insecticides
against Ae. Aegypti.11–15 However, research
conducted on this plant is still rare,
thus further research was carried out to
determine the effect of duku, particularly

1166 Bali Medical Journal 2023; 12(2): 1163-1170 | doi: 10.15562/bmj.v12i2.3946


Figure 1. Effect of crude extract and the fraction of duku leaves on enzyme activities
Esterase of Ae. aegypti larvae.
Figure 2. Effect of crude extract and fraction of duku leaves on Enzyme Activities GST
of Ae. aegypti larvae.
the leaves, on the mortality of Ae. aegypti.
Aedes aegypti is a species that is
categorized as an Insect class and the
Culicidae family. Ae. aegypti has 4 stages
of change in its life: egg, larva, pupa, and
adult. Furthermore, the larvae formation
process consists of 4 stages, namely the first
instar larvae to the fourth instar larvae.24
According to WHO, 50 third and fourth-
instar larvae are not too small (4-6mm),
so they are easy to observe. In addition,
the third and fourth instar larvae are
actively looking for food in water media,
and the organ formation has been perfect.
Therefore, this study used these instars.
Bali Medical Journal 2023; 12(2): 1163-1170 | doi: 10.15562/bmj.v12i2.3946
Furthermore, the extraction of L.
domesticum leaves used methanol as the
solvent with a polarity index of 5.1 which
is believed to dissolve almost all secondary
metabolites from plants.23,25 In this case,
a study reported that using methanol
can isolate higher amounts of secondary
metabolites in leaves Artocarpus altilis F;
further compared the use of this solvent
with ethanol, obtaining that methanol as
a solvent can affect the larvicidal activity
of a plant extract.26 In addition, several
studies have also discovered that duku
extract contains alkaloids, flavonoids,
saponins, terpenoids, and polyphenols,
ORIGINAL ARTICLE
that were determined using TLC method
(thin layer chromatography).6,7,27 The
phytochemicals of duku leaves extract in
this study were tested using the GCMS
(gas chromatography) method so that
1-pentadecen-8-YNE (sesquiterpenes)
compounds were obtained by 13.32%
and linolenic acid (steroids) by 10.56%.
Furthermore, this study also employed
hexane, ethyl acetate, and butanol as
solvents to separate the components in the
methanol duku extract fraction.
As a result, the phytochemicals in
all fractions contained alkaloids and
saponins. Both of these compounds
include polar and semi-polar compounds.
In this case, previous studies have
revealed that alkaloids and saponins have
plant-based insecticide potential.7,18,21,27
Alkaloids with an indole structure such as
strikhnin and quinin taste bitter and can
be used as insect repellents.28 Meanwhile,
saponins are also known to have anti-
insect effects because this compound
can reduce digestive enzyme activity and
absorbs food. In addition, saponin can
also bind free sterols during food digestion
because sterols have a precursor role to the
hormone ecdysone. Therefore, in this case,
a decrease in free sterols will interfere with
insect molting.29 Furthermore, another
benefit of this compound is that it also
interacts with the cuticle membrane of the
larvae so that it can damage the membrane
of the larvae.30
The insect’s metabolic system will work
when an insecticide enters the insect’s
body. Biochemically, the insecticide that
enters the system will be detoxified by the
esterase and GTS enzymes in insects. The
esterase enzyme is present in the larvae of
Ae. aegypti is a detoxification enzyme that
plays a role in the mechanism of larval
resistance. Besides that, esterase is found
throughout the larval body, which plays
a role in larval metabolism and plays a
role in insect development and behavior,
such as its function in reproduction and
digestion.
The increase in the esterase enzyme
indicates that there has been insect
resistance to an insecticide that has
entered the body because the esterase
enzyme works very quickly in binding to
the toxin of an insecticide compared to
the insecticide metabolism itself.31 In this
1167
 ORIGINAL ARTICLE
Figure 3. Effect of crude extract and the fraction of duku leaves on Inorganic
Substance Ae. aegypti larvae.
case, the increase of esterase enzyme on
larvae indicates that the larvae is resistant
to the insecticide of organophosphates,
carbamates, and pyrethroids groups.32
However, the activity of the esterase
enzyme in the larvae treated was lower
than the enzyme activity in the control
larvae. This might be due to the crude
extract and the fraction of L. domesticum
leaves that did not affect the detoxification
activity through the esterase enzyme. It was
further suspected that crude extract and
the fraction of L. domesticum leaves did
not have ester groups, so the metabolism of
methanol extract and the fraction of duku
leaves can continue because the esterase
enzyme cannot carry out the catalytic
process. As a result, the treatment larvae
had esterase enzyme activity lower than
the control larvae, so the treated larvae
were susceptible to crude extract and the
fraction of L. Domesticum leaves.
Furthermore, Glutathione S been damaged and blackened. However,
Transferase (GST) is an enzyme that
catalyzes the conjugation reaction
between electrophilic compounds and
GSH. This GST enzyme is involved in
the inflammatory mechanism of forming
several inflammatory mediators. In
addition, the GST enzyme takes a role in
1168
detoxifying xenobiotics in insecticides of
the organophosphate group.32
This study showed that the GST activity
of Ae. aegypti exposed to crude extract and
the fraction of L. domesticum leaves was
higher than the control larvae. It indicates
that the provision of crude extract and the
fraction of L. Domesticum leaves affected
the detoxification activity of larvae and
indicated that the larvae were starting
to tolerate it. This is the adaptation of
larvae to the environment exposed to the
plant-based insecticide. Therefore, the
detoxification process is overexpressed,
leading to the treated larvae’ higher GST
enzyme than the controlled larvae.
Examination of inorganic substances of
Ae. aegypti was carried out to determine
the levels of ions in controlled and treated
larvae. Morphologically, the larval anal
papillae of the treated larvae, which
functioned as ion regulation tools, have
the larval anal papillae of the controlled
larvae were normal and transparent. This
indicates that the levels of substances,
including iron (Fe), magnesium (Mg),
sodium (Na), and potassium (K), were
affected by the exposure of crude extract
and the fraction of L. domesticum leaves.
Bali Medical Journal 2023; 12(2): 1163-1170 | doi: 10.15562/bmj.v12i2.3946
In the case of treated larvae, its
inorganic substances had different levels,
whereas most of the inorganic substances
examined had decreased levels but also
increased levels. This can happen because
the reactions possessed by the larvae may
vary. Fe content in controlled larvae was
0.03%, while in all treatment larvae, the
levels were above 0.03%. The increase was
probably due to the extracts and fractions
given containing high levels of Fe so that Fe
levels in the test larvae increased, although
there was damage to the anal papillae. A
study conducted by Waterhouse found
that the iron (Fe) content in the larvae of
Lucilia cuprina (green fly) accumulated
due to lack of nutrition in the midgut
larval epithelial cells.33 This was consistent
with this study because the larvae of
Ae. aegypti given methanol extract and
duku leaf fraction did not feed, so the
Fe examination in the larvae seemed to
increase even though the anal papillae of
the larvae were damaged.
Furthermore, sodium (Na) and
potassium (K) take a role in the nervous
system as impulse conductors, while the
alkaloids in the methanol extract and the
duku leaf fraction could be an insecticide by
inhibiting the enzyme acetylcholinesterase
(AChE). AChE, a sodium bridge, will
degrade its substrate into acetylcholine
(ACh) into acetic acid and choline.
Then, AChE will freely be binding to
other ACh. However, alkaloids can also
bind to AChE, so AChE cannot bind to
ACh anymore, and Na as an impulse will
continue to be carried on because ACh is
accumulating more and more. This further
causes nervous exhaustion and seizures
in larvae, leading to death.6,31,34 Natrium
levels in larvae exposed to L. domesticum
leaves crude extract and hexane fraction
were lower, which are 0.92 ppm and 0.83
ppm compared to control larvae with Na
levels of 1.79 ppm. This was because anal
papillae were indicated not to regulate
ions or inorganic substances, indicated by
the damaged and blackened anal papillae.
The Na levels in larvae given butanol and
ethyl acetate fractions of duku leaves were
1.81 ppm and 1.99, and K levels of butanol
fraction of duku leaves were 6 ppm. These
levels were higher than those of Na and K
in controlled larvae, which were 1.79 ppm
and 3.41 ppm. This might be caused by the

withdrawal of compounds in the butanol
and ethyl acetate fractions which was not
as much as in the methanol extract and
hexane fraction of duku leaves. Therefore,
it indicates that the butanol and ethyl
acetate fractions of duku leaves did not
affect the anal papillae in regulating ions
or inorganic substances such as Na and K.
Magnesium (Mg) levels in Ae. aegypti
was also obtained lower in all treated
larvae than controlled larvae, proving
that the extract and fraction of the duku
leaf used affected the anal papillae in
regulating ions or inorganic substances
of K in Ae. aegypti larvae. In this case, Ae.
aegypti larvae were indicated not drinking
during the research. This follows the
previous research by Kiceniuk & Phillips
who conducted research on Mg levels in
Ae. Aegypti campestris who stated that
Mg levels could decrease because the
larvae did not drink.35 In line with this
study, which showed that there were larval
obstacles in the feeding process, it was also
indicated that the provision of methanol
extract and duku leaves fraction affected
the drinking activities of the larvae, which
was indicated by the decreased Mg levels
in the larvae compared to the controlled
larvae.
Chemical insecticides are still
commonly used as a chemical method
to quickly control dengue vectors in
outbreaks. Whereas, the long-term
application of insecticides for the same
target creates selection pressure that
encourages the rapid development of
resistant Aedes aegypti populations. In
this case, plant-based insecticide Lancium
domesticum could be an alternative vector
control that has more selective and safe
target. Therefore, further research is
necessary by employing more fractions so
that a bioassay test can be carried out on
the leaves fraction of L. domesticum and
obtained LC50 and LC90 values from the
fraction.
CONCLUSION
Based on the current research, LD50 and
LD90 of the crude extract of L. domesticum
were 2200ppm and 3200ppm after 24
hours of observation, and it is summed
up that the methanol extract and duku
leaves fraction affect the development
of Ae. aegypti due to the exposure of
Bali Medical Journal 2023; 12(2): 1163-1170 | doi: 10.15562/bmj.v12i2.3946
crude extract and leaves fraction of L.
domesticum. In this case, the exposure
decreased the esterase enzyme activity
and increased the GST enzyme activity of
Ae. aegypti as well as affecting the levels
of inorganic substances in the Ae. aegypti
larvae.
ACKNOWLEDGMENTS
We thanked the Ministry of Education,
Culture, Research, and Technology for
supporting this research. This research
was funded by Hibah Penelitian Dasar
(0357/E5/AK.04/2022).
DISCLOSURE
The authors report no conflict of interest.
AUTHOR CONTRIBUTION
All authors contributed to this research.
DFP: Making initial drafts (originality) and
research concepts, formulating research
objectives as a whole, coordinating
each research activity according to its
stages, validating and visualizing, as
well as making initial drafts (originality)
of research articles. IH: Developing
or designing research methodologies,
coordinating the data collection,
writing, reviewing and editing research
articles. MK: Coordinating technical
and supervising the implementation of
the fundamental research, distributing
assignments to technical field
implementers, and carrying out data
processing and analysis. AP: Coordinating
technical research, especially experimental
research in the laboratory, collecting and
analyzing data, distributing assignments
to technical research implementers, and
preparing research publications.
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