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ORIENTAL JOURNAL OF CHEMISTRY
www.orientjchem.org
An International Open Access, Peer Reviewed Research Journal
ISSN: 0970-020 X
CODEN: OJCHEG
2019, Vol. 35, No.(4):
Pg. 1420-1425
This is an Open Access article licensed under a Creative Commons license: Attribution 4.0 International (CC- BY).
Published by Oriental Scientific Publishing Company © 2018
The Activity of d-Limonene from Sweet Orange Peel
(Citrus sinensis L.) Exctract as a Natural Insecticide
Controller of Bedbugs (Cimex cimicidae)
SRI MURSITI1*, NANDA AYU LESTARI2, ZERLINDA FEBRIANA3,
YUAN MAYLIA ROSANTI4 and TRI WAHYU NINGSIH5
1,2,3,4Chemistry Department, Faculty of Mathematics and Natural Sciences,
Universitas Negeri Semarang, Indonesia.
5Biology Department, Faculty of Mathematics and Natural Sciences,
Universitas Negeri Semarang, Indonesia.
*Corresponding auhtor E-mail: kumalasari_berliana@yahoo.com
Doi http://dx.doi.org/10.13005/ojc/350424
(Received: November 13, 2018; Accepted: July 05, 2019)
ABSTRACT
Bedbugs are harmful human blood-sucking ectoparasites. The disadvantages of a bedbug bite
cause itching, redness bumps, secondary infections, and anemia in children. Bedbugs controller so
far uses harmful chemical pesticides, but reappearance of bedbugs indicates less effective chemical
pesticides, and becomes a serious problem. So it needs safer controller for human health. This study
aims to analyze the d-Limonene activity of orange peel (Citrus sinensis L.) in controlling the bedbug
infestation (Cimex cimicidae). The study was conducted through 5 stages: d-Limonene isolation, GC-MS
characterization, bioassay Y test d-Limonene nanoparticles, SEM characterization, and d-Limonene
activity test on bedbug. The results showed that the essential oil of orange peel contained the main
compounds as d-Limonene (90.018%), 1,6-Octadien-3-ol, 3,7-dimethyl- (3,017%), β-Myrcene (2.071%),
Octanal (0.349%), 1-Octanol (0.589%), 1R-α-Pinene (0.688%), 6-Octen-1-ol, 3,7-dimethyl- (0.667%),
3-Cyclohexene-1- methanol, α, α, 4-trimethyl-, (S)-(1.453%), and Bicyclo [3.1.1] heptane, 6.6-dimethyl-
2-methylene-, (1S) - (1.147%). The Bioassay Y test shows that d-Limonene was both repellent and
fumigant, d-Limonene nanoparticles had uneven and irregular surface morphology, and in the flea
activity test showed more than 50% bedbugs death. These research concluded that d-Limonene
compounds from sweet orange peel had activity to control the bedbugs.
Keywords: Citrus sinensis L, Cimex cimicidae, d-Limonene, Natural Insecticide.
INTRODUCTION
Bedbug (Cimex cimicidae) is a human blood-
sucking extoparasite found in subtropical and tropical
countries. The problem of bedbugs found in many
homes, theaters, and hotels in the late 1970s. The
bedbugs were controlled using insecticides, so there
was almost no information about the bedbug attacks in
1980-2000. Reemergence of bedbug infestation in the
last five years in several star hotels, inns, dormitories,
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and residential houses.1 This appearance was due to
the lack of effective chemical insecticides.
Bedbugs live in groups and easily to
be founded in dormitories, hospitals, and hotels.
Preferred residences are bed bugs in crevices,
cracks, furniture (chairs and beds), rear wallpaper,
bedding, wood panels, or under carpets. Bedbugs
are active at night (nocturnal), but when hungry
during the day bedbugs can also appear and
approach to the host.2
Infestation of bedbugs had a negative
impact on health and the economy.3 The bitting
bedbugs can cause itching, reddish bumps,
secondary infections caused by repeated cardiac
itching, and result in anemia in children at high
infestation. This is very harmful to humans. The flea
controller used by people today was chemically
controlled because it was considered practical and
powerful to kill bedbugs.
The d-Limonene, an active ingredients
present in the essential oil destroy the wax layer of
the insect respiratory system so that once applied
directly, the insects will suffocate.4 d-Limonene
bioassay results of tests undertaken after treatment
of the fabrics, Bioassay results show the toxic
activity of MCT-b-CD finished cotton fabrics treated
with different doses of limonene, and also show the
effect of exposure time against mosquitoes. The
results show that the repellent, knockdown and killing
action against mosquitoes increased with increasing
concentration of limonene in MCT-b-CD finished
cotton fabrics within the range studied (250–1500
mg/m2). Also, results show that the corrected activity
(repellency, knockdown and mortality) increased by
increasing the exposure time.5
Existing research results showed that total
of 24 compounds were identified and the major
components were d-Limonene (86.75%), linalool
(1.96%), α-pinene (1.63%), trance-limonene oxide
(1.39%), and γ-terpinene (1.03%).6 Extensive studies
on the chemical composition of various C. sinensis
species have also been conducted. d-Limonene is the
most abundant component in citrus peels volatile extract
and constitutes 51.97% -95.35% of the total amount.7
Extensive studies on the chemical
composition of various species of Citrus Sinensis
L. had been performed. The antimicrobial activity
of sweet oranges (Citrus Sinensis L.) had not been
fully investigated, and little was known about its
activity against fungi that produced toxins, such as
Aspergillus flavus and Aspergillus parasiticus.6
d-Limonene is used as insecticide to control
ectoparasites of pet animals, but it has activity
against many insects, mites, and microorganisms.
Possible attractive effects of limonene to natural
enemies of pests may offer novel applications to use
natural compounds for manipulation of beneficial
animals in organic agriculture.8
The potential of volatile oil against insects
was resistant (repellent), fumigant (fumigant), reduce
appetite (antifeedant), and attract (attractant).9 The
advantage of d-Limonene over synthetic pesticides
in this regard is its biodegradability, which makes it
more environmentally friendly for both fumigation
and contact applications. The broad range of
pharmacological properties of d-limonene coupled
with low toxicity offers the possibility of incorporating
this compound into various medical and cosmetic
formulations.10 Natural plant products and materials
have been used by local people as insecticides to
control insects such as mosquito. Among the most
notable plants having such insecticidal properties
are Azadirachta indica (neem) and Citrus sinensis
(sweet orange).11
Research had been carried out on citrus
plants isolated to produce essential oils from orange
peel (Citrus nobilis L.), was carried out using the
steam distillation method.12 has conducted a study
to test the activity of biothermal activity in the leaves
of citrus Nobilis lour against termites (Coptotermes
curvignathus sp) showing a concentration of 20%
volatile oil with 100% mortality toxic.
This research about activity of d-Limonene
from sweet orange peel as a natural insecticide
controller of bedbugs and there was no conflict of
interest in this study.
MATERIALS AND METHODS
Instruments, Tools and Materials
These research used GC-MS instruments,
glasses tool set such erlenmeyer, beaker, flakon
bottle, analytical balance, Y tube, microliter syringe,
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stopwatch, magnetic strirrer, scanning electron
microscope (SEM), glass spray diameter 1.8 cm
and height 3 cm, tally counter, desiccator, separating
funnel, and oven. The materials were include the
cemetery colony (Cimex cimicidae), hexane, glacial
acetic acid, Whatmann N°1 (15 cm diameter) disc,
Whatman paper No. 41, sterile sand, plaster paris,
regent, black cloth, strimin woof, and Na2SO4.
Preparation of Bedbugs and d-Limonene
Compounds
Bedbugs (Cimex cimicidae) obtained from
boarding houses around the State University of
Semarang. Sampling of the extract of d-Limonene was
carried out by using a simple distillation apparatus for 8
h to obtain sweet orange peel oil. The sweet orange peel
oil was separated from the water using a separating
funnel then separated back using Na2SO4. The extract
be used directly or stored at room temperature until
the next analysis process (Bioassay Y).
Bioassay Y Compound d-Limonene
Bioassay was done under standard
conditions with 90% relative humidity and room
temperature of 28°C. The two extracts produced
(d-Limonene) were tested using bioassay Y on
Whatmann N°41 disc paper, with an intermediate
angle of 120°. In Y (3 cm) and one Y (7 cm) branch,
artificial trail was drawn using a microliter syringe in
which there was 1 mL of extract per 1 cm marker.
Other extracts or pure solvents (hexane) as controls,
were placed in the same conditions on the basis of
Y and other Y branches. Then a bedbug was placed
in a 55 mm diameter small bottle at the bottom of
Y, and the distance of bedbugs was measured. The
time spent between introducing bedbugs to bottles
and bottle relics by bedbugs was also measured to
determine the role of the marker in determining the
behavior of bedbugs.
The d-Limonene compound was tested 3
replicates (n = 3) and each repeated using a new bed
bug and filter paper to prevent the effect of precondition
conditioning behavior. The activity threshold for both
synthetic extract and d-Limonene was determined
as the minimum concentration inducing bedbugs to
cover an average distance of more than 3 cm. While
the maximum response was 10 cm.
Preparation of Nanoparticles d-Limonene with
Chitosan
Chitosan of 1 g dissolved in 100 mL glacial
1% acetic acid using magnetic stirer to obtain 1%
chitosan parent solution. Take 80 mL of 1% chitosan
solution and added bitwise to 100 mL d-Limonene
over magnetic stirer. Stirring was continued for
30 min to obtain a stable solution of d-Limonene
nanoparticles. The nanoparticles were looked at their
size and morphology under an electron scanning
microscope (SEM).
Anti-Bleed Activity Test
Fig. 1. Anti-Bleed Activity Test
Anti-flea activity of sweet orange peel
extract in the form of d-Limonene compound was
tested using force feeding test which has been done
by some modifications. Test glass made of plastic
material (diameter 5 cm, height 5 cm). The bottom
of the test glass was a porous plaster of paris and
contained sterile sand 10 g weight.
The feed paper (diameter 2 cm; Whatman
paper No. 41) was stirred at 60°C for 1 h stored
in desiccators for 24 h then weighed for initial
weight. The feed paper was immersed in a solution
of d-Limonene nanoparticles for 1 h with 0%
concentration (negative control), 4%, and 8% (w/v).
Negative controls no treatment of addition of extract
(solvent only).
After immersion, the feed paper was dried
to evaporate the solvent. The 4 cm (4 cm) diameter
strimine cloth (modified) was placed in a test glass
and on a strimin cloth is placed in the feed paper.
Test glass was placed in a container with the bottom
of which was given wet cotton. A total of 35 active
bedbugs were inserted into the test glass. The test
glass was covered with a black cloth and kept in a
place protected from light for 3 days. The feed paper
was stirred at 60°C for 1 h and stored in the desiccator
for 24 h then the paper was weighed for final weights.
The test was done with three repetitions (triplo).
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Observation Parameters
Bug Mortality
The mortality observation was performed by
counting the number of bedbugs that died every day
for 4 days, the accumulated amount calculated on
the 4th day. Percentage mortality of bedbug per glass
of test was calculated using the following formula:
% Mortality = (Number of Bedbugs Dead) / (Number
of Bedbugs Early) x 100%.
Percentage Weight Bait Paper Feed
The percentage weight loss (PB) of feed
paper after 4 days was calculated using the following
formula:
% Pb = (B1-B2) / B1 x 100%
Description:
B1 = dry paper weight prior to feed (g)
B2 = dry weight of filter paper after feed (g)
RESULTS AND DISCUSSION
Sweet Orange Peel Extract
The sampling of orange peel extract
was done by steam distillation method made from
sweet orange peel. The isolation was carried out for
8 h and the result was 24 ml of sweet orange peel
extract of 2.6 kg orange peel. The essential oils
produced was clear and contain a wide variety of
terpen groups, sesquiterpenes, aldehydes, esters
and sterols. Then the essential oil was stored in a
bottle and in -15oC. The result of extract was taken
1 ml for its d-Limonene concentration using GC-MS
in Engineering Faculty Universitas Negeri Semarang.
Here is the table of compounds from sweet orange
peel extract.
Test Result of Bioassay Y d-Limonene Compound
The bioassay Y test of the d-Limonene
compound aims to recognize the interest of the
insect on the d-Limonene compound. On the Y rods
a n-hexane solution as a control and a d-Limonene
compound. It was placed in the same condition
on the basis of Y and other Y branches. Then a
bedbug was placed inserted. Once inserted and
measured the time and distance traveled by foul
decay, the result was in less than five minutes the
bedbug dies and the distance traveled by ± 3-5 cm.
This test was done three times and had the same
result. This means that bedbugs were not interested
in d-Limonene compounds, and the d-Limonene
compound was very powerful to turn off the bedbugs.
The test was supported by 9 which revealed that the
potential of volatile oils on insects was also resistant
(repellent), fumigant, and antifeedant.
SEM Test (Scanning Electron Microscopy)
Nanoparticles d-Limonene with Chitosan
Nanoparticles chitosan with d-Limonene
were tested by SEM (Scanning Electron Microscopy)
analysis conducted at the Physics Laboratory of
the Faculty of Mathematics and Natural Sciences
Universitas Negeri Semarang. SEM test aims to
know the three-dimensional structure of a compound
produced. Fig. 1, 2, 3, 4 and 5 show SEM test results
with magnification 500, 1000, 5000 and 10000.
Table 1: Compunds from sweet orange peel extract
Compound Content
(%)
D-limonena 90,018
1,6-Octadien-3-ol, 3,7-dimethyl- 3,017
β-Myrcene 2,071
3-Cyclohexene-1-methanol, α,α,4-trimethyl-, (S)- 1,453
Bicyclo[3.1.1]heptane, 6,6-dimethyl-2-methylene-, (1S)- 1,147
6-Octen-1-ol, 3,7-dimethyl- 0,667
1R-α-Pinene 0,688
1-Octanol 0,589
Octanal 0,349
The orange peel extract contains (dominant) 90.01% d-Limonene
(C10H16), so the d-Limonene was very dominant in the sweet
orange peel. The results of other studies of citrus peel oil in
Iran had been identified d-Limonene (92.42%) and β-myrcene
(3.89%).5
Fig. 2. Results of SEM Test with Magnification 500 times
Fig. 3. Results of SEM Test with Magnification 1000 times
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From the test results obtained, surface
morphology of d-Limonene nanoparticles showed
irregular and rectangular surface conditions. This
condition also illustrates that d-Limonene nanoparticles
were not uniform and have uneven surfaces.13
Anti Bugs Activity Test Results
Anti-lice activity test using force feeding
method. The principle of this forced feed was the
diesel feed paper at 60°C for 1 h and stored in
the desiccator for 24 hours. The feed paper was
immersed in a solution of d-Limonene nanoparticles
for 1 h then dried to evaporate the solvent. The
4 cm (4 cm) diameter strimine cloth (modified) was
placed in a test glass and on a strimin cloth was
placed in the feed paper. Test glass was placed in
a container with the bottom of which was given wet
cotton. A total of 35 active bedbugs were inserted
into the test glass. Test glass covered with black cloth
and observed the activity of bedbugs. The results
showed that d-Limonene nanoparticles were able
to control bedbugs. The number of bedbugs that
died for 4 days was 23 out of a total of 35 bedbugs
with a concentration of 8% and 20 out of a total of
35 bedbugs with a concentration of 4%.
Using of synthetic chemicals in pest control
continuously caused directly detrimental to humans,
wildlife, aquatic life, and the environment as a whole.
Besides the negative effects of synthetic insecticides,
most remote villages are unreliable and often forged
or otherwise dissolved into ineffective, outdated
concentrations due to their toxicity to humans and
Fig. 4. SEM Test Results with Magnification 5000 times
Fig. 5. SEM Test Results with Magnification 10000 times
the environment, and ineffective due to the rapid
evolution of pesticide resistance.14, 15, 16
However, this could be due to the necrosis
or cytolysis of the hepatocytes by various toxic
phytochemicals present in both Azadirachta indica
and Citrus sinensis such as Azadirachtin and
d-Limonene which probably cause leakage of the
liver enzymes into the blood. This implies that
these local insecticides cause hepatic injury which
means that they have toxic effect on the liver of the
experimental animals. This finding is consistent with
the findings of Ashafa et al., (2012) who reported
the toxic effects of Azadirachta indica and Tarkang
et al., (2012) who reported the toxic effects of
Citrus sinensis on the liver and other vital organs of
experimental rats.17,18
CONCLUSION
Isolation of 2.6 kg of sweet orange peel
produces 24 ml of essential oil. Characterization
of GC-MS essential oils of sweet orange peel
containing the main component compounds
were d-Limonene (90.018%), 1,6-Octadien-3-ol,
3,7-dimethyl- (3,017%), β-Myrcene (2.071%),
Octanal (0.349%), 1-Octanol (0.589%), 1R-α-Pinene
(0.688%), 6-Octen-1-ol, 3,7-dimethyl- (0.667%),
3-Cyclohexene-1- methanol, α,α, 4-trimethyl-,
(S)-(1.453%), and Bicyclo [3.1.1] heptane,
6.6-dimethyl-2-methylene-, (1S)- (1.147%). Bioassay
Y test between d-Limonene and N-hexane compounds
showed that d-Limonene is resistant (repellent) and
fumigant (fumigant). Characterization of d-Limonene
nanoparticles had an uneven and irregular surface
morphology. Anti-flea activity test by using force-feed
method indicates more than 50% dead blight and
showed that orange peel extract (Citrus Sinensis
L.) can be used as a natural insecticide for bed bug
controller (Cimex Cimicidae).
ACKNOWLEDGEMENT
Acknowledgments author to convey to:
1) Directorate General of Learning and Student
Affairs, Ministry of Research, Technology and
Higher Education, Indonesia with contract
number 1020/B3.1/KM/2018 dated March 09th,
2017 for research funding that had been given.
2) Supervisor of PKM-PE, Dr. Sri Mursiti, M.Si.
and Supervisor PKM Universitas Neger i
Semarang who had provided advice, input,
motivation, and prayer.
3) Universitas Negeri Semarang as our
institution to gain knowledge.
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4) Chemistry Laboratory FMIPA Universitas
Negeri Semarang who had served the
purposes of research.
5) Chemical Engineering Laboratory FT
Universitas Negeri Semarang who had
served the purposes of research.
6) Physics Laboratory FMIPA Universitas Negeri
Semarang who had served the purposes of
research.
Conflicts of Interest
The authors declare no conflict of interest.
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