Introduction
Dengue fever
is an infectious disease caused by the dengue
virus. �Dengue fever is a virus spread by the Aedes
aegypti mosquito, the world's fastest growing mosquito, which infects about
390 million people each year (Kementerian Kesehatan RI, 2018).
Data
obtained from the Ministry of Health of the Republic of Indonesia (2018), the
incidence of dengue fever in Indonesia has continued to increase since 1968 -
2015. This is due to several factors such as climate change, temperature,
humidity, and the direction of the air that is suitable for vectors of this
disease. In addition, the increasing population density and the lack of public
participation in vector prevention and eradication have caused the spread of
this disease to spread more quickly (Kementerian Kesehatan RI, 2018). The dengue fever
morbidity rate in East Java in 2019 was 47 per 100,000 population, an increase
compared to 2018 which was 24 per 100,000 population. The case fatality rate (CFR) for dengue fever
in 2019 was 1%, which indicates that the mortality rate due to dengue fever in
East Java is still above the target of <1%. Meanwhile, the larvae-free rate
was 78.2%, which was lower than the target that had been set, which was 95%.
Cases of dengue fever in Jember in 2019 were 988
cases, with a CFR of 0.7% (Dinkes Jawa Timur, 2020).
One method of
reducing or suppressing vector populations is with control. The use of
synthetic pesticides is not accompanied by consideration of the potential negative
effects. The use of sublethal doses stimulates the adaptation of insects to
insecticides. This feature will be passed on to future generations, resulting
in a new population of insecticide-resistant insects (Sembel, 2009).
Insecticides containing hazardous chemicals such as organochlorines have been
banned from use in Indonesia because of their persistent and bioaccumulative nature, which is stated in the Regulation
of the Minister of Agriculture No. 24/Permentan/SR.140/4/2011.
Insecticides generated from plants or other natural materials are one option
for dealing with these issues (Pertanian, 2011)
Breadfruit (Artocarpus altilis)
is one of Indonesia's most common plants and has the potential to be a mosquito
repellent. In people living in rural areas, breadfruit flowers are often used
as mosquito repellents (Narulita, W. Anggoro, B.S. And Novitasari, 2019). Based on the
research that has been done, the flowers and leaves of breadfruit contain several
compounds such as saponins, flavonoids, polyphenols, which have a sequential
mechanism effect, namely inhibiting the stimulation of eating insects, and
respiratory inhibitors. Meanwhile, saponin have a damaging effect on insect
cell membranes, causing them to activate enzymes and degrade cell proteins (Nikmah, 2016).
Maharani
(2014) found that the methanol extract of dried breadfruit leaves
includes alkaloids, flavonoids, tannins, phenols, and saponins, according to
the results of phytochemical screening (Maharani et al., 2014)
Research
conducted previously by Edi (2011), showed that breadfruit flowers
were able to immobilize 10 mosquitoes with an average time of 15.6 minutes for
5 repetitions (Hamsir & Fahmi, 2019). Lumowa (2011) found that breadfruit flower powder, which acts as an anti-inflammatory, was able to kill the most
mosquitoes with a content of 2 grams of breadfruit flowers, killing an average
of 15.6 insects (78%) (Lumowa, 2011). Kurniawan (2020) concluded that extracts from
breadfruit stems and leaves can be used as natural insecticides because they
show a fairly high mortality rate of Anopheles
sp mosquitoes. Although they are not as effective
as synthetic insecticides, breadfruit stem and leaf extracts are safer for
human health and environment (Kurniawan et al., 2020). The combination of several active
compounds which is a several extracts is able
to provide effects such as synergistic, antagonist, and addictive. The use of
plant extracts as vegetable insecticides can be used alone or in combination (Shaalan EA, Canyon D, Younes MW, Abdel-Wahab H, 2005). Previous research by Vastrad (2002)
explained that combining of two plant extracts that have synergistic bioactive
compounds is more effective as an agent for controlling insect pests and
disease vectors than a single extract (Taufika et al., 2020).
So if the active compounds from the leaves and flowers
of breadfruit which are toxic are combined, it is expected to increase the
mortality of Aedes aegypti larvae. Several previous studies only used Anopheles sp larvae as subjects, and only used a single extract. Therefore, this is attracted researchers
to compare the capacity each of extracts from breadfruit leaves and flowers, and combination extracts of breadfruit leaves and flowers, as a bioinsecticide to kill Aedes aegypti
larvae which are environmentally friendly and safe for humans.
Method
This is a true
experimental study with a post-test only control group design. This study used
a posttest-only control group design, in which two groups were randomly
assigned to each other. The subjects were Aedes aegypti instar III larvae taken
from 860 larvae reared in the Entomology Laboratory, with a total of 20 larvae
in each test for four treatments and three replications. Leaves and flower of
breadfruit were extracted at Phytochemical Laboratory UPT. Materia Medika, Batu City, East Java. Larval rearing and
effectiveness tests starting from 15 November 2021 to 02 December 2021, was
carried out at the Entomology Laboratory, Institute for Tropical Diseases, Airlangga University, Surabaya.
Breadfruit leaves
and flowers were collected from gardens in Banyuwangi,
East Java, and dried before being extracted with 96% ethanol using the
maceration method. To achieve a thick extract, the extraction process takes around
14 days. A total of 860 Aedes aegypti larvae in the third instar were collected
and fasted for 24 hours. The third instar larvae were chosen because they have
a stronger ability to neutralize harmful substances than the first and second
instar larvae. Whereas in instar IV it is closer to being a pupa, so there can
be bias during the study.
The third
instar of larvae that had been collected were then put into each plastic cup
containing 20 ml of distilled water along with breadfruit leaf extract, breadfruit
flower, and a combination of breadfruit leaf and flower extract with
concentrations of 1000, 2750, 4500, and 6250. ppm. Every 6, 9, and 24 hours
after treatment, the mortality of the larvae was observed and recorded. When a
stick is pressed against the larvae's body, and they stop moving and do not respond
to stimuli, indicating that they have died. The percentage of larval mortality
was determined using the Abbott formula:
One Way ANOVA test was used
to analyze the observed data. Duncan's follow-up test was used to determine the
significant difference in the effectiveness of each concentration of the test
extract if the results of the study demonstrated significance (Gomez, 2015). To find out the
values of LC50 and LT50 using Probit
Analysis in the SPSS program. To find out the value of the Combination Index
(CI) use the formula :
Description :
CI���������� ��
: Combination Index
LCx1 (cm) :
Lethal Consentration x Combination Extract on the First Single Extract
LCx2 (cm): Lethal Consentration x Combination
Extract on the Second Single Extract
LCx1������� :
Lethal Consentration x First
Single Extract
LCx2��� : Lethal Consentration x Second Single Extract.
After processing the data through the Kolmogorov-Smirnov normality test, it
was discovered that the larvae mortality data were normally distributed with a
significance value of >0.05. Combination of breadfruit leaf and flower
extracts was then analyzed using One Way ANOVA with a significance value of
0.05, indicating that it has an influence on the mortality of Aedes aegypti larvae. Duncan's test was
used to determine whether there was a significant difference in each extract
concentration, with the finding that at a concentration of 6250 ppm, there was
a significant difference with the highest value of 55.33. As a result, when
compared to other concentrations, the concentration of 6250 ppm had the highest
effect.
LC50 and LT50 Values
���� A Probit analysis was performed
using SPSS for Windows 16.0 software to determine the values of LC50
and LT50 in each extract. According to the results of the probit
analysis of breadfruit leaf extract, the estimated LC50, which can
kill 50% of larvae in 24 hours, is 1871 ppm, with an interval between 1253 and
2437 ppm, and the LT50, which can kill the larvae up to 50%, is
estimated to be 4.2 hours with a 0 to 8-hours interval after treatment. From
probit analysis of breadfruit flower extract, the estimated LC50,
which can kill 50% of larvae in 24 hours, is 2531 ppm, with an interval between
2120 and 2959 ppm, and the LT50, which can kill the larvae up to
50%, is estimated to be 10.8 hours with a 6 to 19.8 hours interval after
treatment. From probit analysis of combination breadfruit leaf and flower
extract, the estimated LC50, which can kill 50% of larvae in 24 hours,
is 903 ppm, with an interval between 558 and 1204 ppm, and LT50,
which can kill the larvae up to 50%, is estimated to be 3.7 hours with a 2 to 5
hours interval after treatment.
���� At the LC50,
the result for the combined index value is 0.82. According to Chou
& Martin (2004), this result shows that the combination of leaf
and flower extracts of Artocarpus altilis
has low synergistic properties in the category of interaction characteristics
of the combination extracts. Meanwhile, the LC90 result was 0.82,
indicating that the combination of breadfruit leaf and flower extracts has a
low synergistic character, according to Artocarpus
altilis.
Effect of
Breadfruit�s Leaf Extract on Mortality of Aedes aegypti Larvae
After giving the breadfruit leaf extract to the test
larvae, the maximum concentration of 6250 ppm caused the most mortality, with
86.6% of the test larvae was died. As a result,
breadfruit leaf extract can be utilized as a alternative bioinsecticide. The
high amount of active compounds such as alkaloids, tannins, and flavonoids was
assumed to be the cause of the test larvae's mortality. The alkaloids
contained in breadfruit leaves are able to slow down the growth hormone of the
larvae, which causes the larvae to be unable to metamorphose, causing death due
to a decrease in the formation of nitrate which is used to synthesize protein,
and is able to restrain the distribution of sucrose into the small intestine (War et al., 2012). There are two methods for tannin compounds to enter the body of a larva:
through penetrating the larva's body wall and through the digestive tract (Tiwari, 2012).
Flavonoids
work as inhibitors of the respiratory system in larvae. Flavonoids enter
through the respiratory tract of the larvae, it causes
nerve weakness and damages the respiratory tract which causes the larvae to be
unable to breathe. Flavonoids also play a role by slowing the action of the
acetylcholinesterase enzyme, the enzyme is useful in the breakdown of
acetylcholine into acetyl Co-A and choline on nerve cell impulses. Due to the
decrease in the work of the acetylcholinesterase enzyme, it causes a buildup of
acetylcholine which can result in disturbances in the impulse conducting system
from nerve cells to muscle cells, and causes muscles to spasm, paralysis and
then ends in the death of the larvae. (Rattan, 2010). According to Kurniawan (2014) research, the breadfruit leaf
can be utilized as a natural larvicide because it kills mosquito larvae. The
high rate of larval mortality indicates this, although it cannot be compared
with chemical larvicides. Bioinsecticides, such as those made from breadfruit
leaves, are better for the environment, animals, and humans (Kurniawan et al., 2020).
As a result, breadfruit leaf extract has the potential to be a natural
larvicide by reducing the number of mosquito larval Aedes aegypti mortality. According to a study conducted by Rosmawaty
(2013), the crude extract of the leaves of breadfruit contains
various secondary metabolite chemicals, including alkaloids, steroids,
terpenoids, and flavonoids. These chemicals are thought to be effective at
killing Aedes aegypti larvae (Rosmawaty, 2013). From this study which using breadfruit leaf
extract, it was found that breadfruit flower extract can be used as an
alternative bioinsecticides.
Effect of
Breadfruit�s Flower Extract on
Mortality of Aedes aegypti Larvae
The data from the experiment indicated from the treatment
of breadfruit flower extract showed that the highest consentration of 6250 ppm
was capable of killing 90% of the test larvae. As a result, breadfruit flower
extract can be used as a
alternative bioinsecticides. High amounts of saponins and flavonoids
were assumed to be the causes of the test larvae's mortality. Saponins
can have a bitter effect on the larvae, causing them to lose their interest in
food and eventually die. Saponin can harm the waxy coating that protects the
outer body of the larvae, causing the larvae to lose a lot of fluid and
eventually die (Prakoso, Gandung, 2016). In
larvae, flavonoids act as respiratory system inhibitors. This substance enters
the larvae's respiratory tract, causing neurological weak and damage the
respiratory tract, stopping the larvae from breathing (Rattan, 2010).
Jones
(2012) identified fatty acids that are more effective than
N,N-diethyl-m-toluamide (DEET) in an identification test of chemical compounds
found in male breadfruit flowers, suggesting that male breadfruit flower could
be one of the natural ingredients to repel mosquitoes (Jones AM, Klun JA, Cantrell CL, Ragone D, Chauhan KR, Brown PN, 2012).
DEET is the active ingredient commonly used in many insect repellent products (Medicine, 2021).
Breadfruit flower extract was shown to be a potential option for developing
bioinsecticides to control mosquito larvae Culex in a previous study by (Gladys & Bukola, 2019).
Effect of Combination Breadfruit�s Leaves and Flower
Extract on Mortality of Aedes
aegypti Larvae
The data from the experiment indicated from the
treatment of breadfruit flower extract showed that the highest dose of 6250 ppm
was capable of killing up to 98.3% of the test larvae. The
combination index calculation shows that the combination of leaf extract and
breadfruit flower in LC50 and LC90 is weakly synergistic.
However, at the maximum dose, the combined toxicity of the two extracts was
higher than the toxicity of each single extract. At the highest concentration,
a single extract of breadfruit leaves was able to kill an average of 86.6%, a
single extract of breadfruit flowers was able to kill an average of 90%, and
the combination of breadfruit leaves and flowers was able to kill up to
98.3%. The synergistic aspect of the two extracts may be due to the fact that
each extract has the same amount of active substances, such as flavonoids and
saponins. The toxic effect of combination extracts from breadfruit leaves
and flowers can be detected in the clinical symptoms that show in larvae, which
include slowed movement, shriveled, weak bodies, and eventually death.
Treatment with combination extracts of breadfruit leaves and flowers was
expected to get a more toxic effect than treatment with single extracts in this
research. Breadfruit leaves contain a variety of secondary metabolites, including
alkaloids, steroids, terpenoids, flavonoids, and saponins, according to
Rosmawaty (2013). Breadfruit flowers contain saponins, polyphenols, and
flavonoids, according to Nikmah (2016). Several types of toxic compounds
contained in single extracts of breadfruit leaves and flowers have similarities
because they are still the same type of plant, so that if the two single
extracts are mixed it is expected that the content of toxic compounds will be
stronger. Although the combination index value showed minimal synergistic
outcomes, the results of this study showed that the mortality of larvae was
higher when given a combination of two extracts when compared to the mortality
of Aedes aegypti larvae in a single
extract.
From the probit analysis, LT50 of combination extract leaves and
flowers breadfruit obtained at 2 hours after giving the treatment, the value is
much faster when compared to the LT50 for each single extract. The
results of this study are have similarity with the statement of Vastrad (2002)
in Taufika (2020), that the combination of two plant extracts containing
bioactive compounds that have synergistic properties is more effective as a
larvicide and controlling disease vectors when compared to a single extract.
Because plant sources of bioinsecticides are not always available in large
quantities in an area, the use of a synergistic combination of extracts
from bioinsecticides can reduce the amount of raw material used compared
to single extracts of bioinsecticides, allowing them to overcome if there is a shortage
of raw materials at the farm level. The use of a combination of bioinsecticides
at lower doses can also reduce unwanted effects on non-target organisms and the
environment. Resistance can also be prevented by using a combination of natural
pesticides with various modes of action (Abizar & Prijono, 2010).
Bioinsecticides have various advantages over chemical insecticides, including a
relatively simple manufacturing procedure and the ability to make them
individually. Bioinsecticides also leave less residue in the environment,
making them safer than chemical pesticides. Bioinsecticides have toxic
chemicals that decompose quickly, preventing resistance at the target.The
disadvantage of utilizing bioinsecticides is that they include various active
ingredients or complex active chemicals that are sometimes difficult to detect
and cannot be generalized due to the influence of diverse plant growth places,
temperatures, plant ages, soil types, and harvest periods. It makes the
active compounds become very varied (Hamsir & Fahmi, 2019).
Conclusions
Based on the study of Bioinsecticide from Combination Extract of Leaf and
flower of Breadfruit (Artocarpus altilis) on
Mortality of Aedes aegypti larvae, it concluded that using leaf extract, flower
extract, and combination of breadfruit leaf and flower extract has an effect on
Aedes aegypti larvae mortality. When compared to single extract, combination of
breadfruit leaf and flower extracts showed the highest mortality in killing
Aedes aegypti larvae after 24 hours of treatment with the same serial
concentration of each extract. At the highest concentration of 6250 ppm,
combination of breadfruit leaf and flower extracts was able to kill larvae up
to 98.3%. The LC50 value was reached at concentration 903 ppm in a combination
of breadfruit leaf extract and flower extract, and the LT50 value of breadfruit
leaf extract was 3.7 hours after treatment.
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