Recent Advances in Biology
Insect Repellent Properties
of Melaleuca alternifolia
Recent Advances in Biology and Medicine, Vol. 2, Pages 57-61, 2016 57
ID: 293742 dx.doi.org/10.18639/RABM.2016.02.293742
Insect Repellent Properties of Melaleuca alternifolia
Mohamad Adib Bin Edris, Awang Soh Yusuff Mamat, Muhammad Shahzad Aslam*,
Muhammad Syarhabil Ahmad
School of Bioprocess Engineering, Universiti Malaysia Perlis,
Kompleks Pusat Pengajian Jejawi 3, 02600 Arau, Perlis, Malaysia.
Received: Jun 24, 2016; Accepted: Jul 25, 2016; Published: Aug 6, 2016
The aim of this study is to compare the use of plant-based insect repellents that are environment friendly with the use of insect repellents based
on chemical substances which can be harmful to the environment and human health. The plant studied here is “tea tree”; its scientific name
is Melaleuca alternifolia. Essential oil from this plant is extracted by steam distillation method. Based on the previous research, tea tree oil has
antimicrobial, antifungal, anti-inflammatory, and insect repellent properties. Some experiments were done on tea tree oil to determine its insect
repellent properties and the suitable concentration that can be used to make sure its repelling effect is optimum. The purpose of this determina-
tion is to avoid its harmful effect on humans because it can be toxic if it is used at high concentration. The results showed that tea tree oil repelled
Tribolium castaneum. Furthermore, the toxicity assays also gave positive result where the tea tree oil has toxic properties against Solenopsis invicta.
The lethal dose (LD) of tea tree oil to kill 50% of a group of S. invicta is 23.52 μL/mL. This LD50 is determined by using the arithmetic method of
Karber. Broadly, the results showed that M. alternifolia has insect repellent properties and shows toxicity against certain insects.
Keywords: Melaleuca alternifolia; Steam distillation; Repellency assay; Toxicity assay.
A long time ago, plant-based insect repellents were used in “old style” practice, especially as protective mediums. In recent
times, the wide marketing of a repellent product partially produced by a plant-based ingredient has shown increasing popularity
among users. Most plants have a compound that can be used as an insect repellent.
As known, M. alternifolia, simply called “tea tree,” is a small tree, is quite a beauty, can grow up to 5 m in height, and
can have a narrow and thin bark and fine leaves that can grow up to 20 mm in length . Generally, M. alternifolia live along
narrow rivers, marshy flats, beaches and “next to” the ranges. M. alternifolia is Australia’s native plant, and because of its
vigorous growth in Australia and its properties, this plant has been widely used in Australia. We can conclude that each part of
this plant can be used. In olden days, this plant was used to make medicinal ingredients, and sometimes it was used directly
on the human body. Despite its usefulness, wide use of M. alternifolia oil did not officially begin until early this century when its
antiseptic and eradicate properties were reported .
In recent years, an essential oil that has been extracted from the M. alternifolia has become increasingly used in medi-
cine. This essential oil, which can be called tea tree oil, is a whitish yellow viscous liquid with a distinctive spicy odour and is a
combination of a complex mixture of monoterpenes, 1-terpinen-4-01, cineole, and another hydrocarbon . This oil that has
been extracted is yielded by steam distillation process. The oil produced is comparatively low in concentration, and it is obtained
at a concentration estimated at 1%-2% of the weight of wet plant material.
Several factors affect the efficiency of the distillation process, and optimizing these factors is necessary to produce a
high yield of oil. Each year, the level of oil is different, and the harvesting time is an important thing that needs to be considered
for the best oil-level yield. The period from November to May is the time when the level of oil yield is high.
Moreover, one of the factors that effect the final quality of oil yield is the duration of the distillation process. The oil
has a number of properties which suggest its potential for use in wound treatments or as a protectant against flies. Besides,
it has documented insecticidal effects by which it can be used in the treatment of strikes by larvae, a repellent effect that can
help in protecting against new strikes or restrikes, and antimicrobial and anti-inflammatory effects that can help in healing
2. MATERIALS AND METHODS
2.1. Preparation of Essential Oil
The raw materials that have been used in this project are parts of the tea tree; its scientific name is M. alternifolia. The parts that
were used in this experiment were the leaves from which the essential oil was extracted. The leaves were placed in the upper
container of distillation apparatus with fair and tight packing. Some spaces were left to allow the steam to pass through the raw
material. A low flow of water was allowed into the condenser. This allowed the oil to flow into the tube. The heating mantle
58 Original Research Article
was turned on, and steam distillation began. The heating mantle was allowed to heat the water, and the water vapour passed
through the leaves. The process was carried out for a period of 30 minutes to 3 hours. The temperature was maintained at
165°C, as the boiling point of tea tree oil is 165°C. The heating mantle was turned off after the distillation process ended. The
distillate was extracted further by using “liquid-liquid extraction” technique. The essential oil obtained was poured into a small
2.2. Repellency Assays
Several test samples were prepared by dissolving different volumes of the essential oil in 1 mL of acetone (5, 10, 20, and 30 μL).
A Whatman filter paper was divided into two sections. Then, oil solution was applied on one half of the filter paper as uniformly
as possible using a micropipette. Acetone was placed on the other half of the filter paper as a controlling agent. The essential
oil–treated and acetone-treated halves were dried to completely evaporate the solvent.
After that, both treated and untreated halves were joined again by using tape and placed at the bottom of a Petri dish.
Twenty insects (stored-product beetle, T. castaneum) were placed at the center of the filter-paper disc, and then the petri dish
was covered and kept in a dark environment. For each concentration of the solution, this procedure was replicated about four
times. A number of the insects on both the treated and untreated halves were recorded after four hours in dimmer light .
2.3. Toxicity Assays
Disposable Petri dishes (100 25 mm2) were prepared by covering the Petri dishes with a white cloth to prevent the escape of
the ant under study and force the ant to remain on the treated paper. Several test samples were prepared by dissolving different
volumes of essential oil in 1 mL of hexane (25, 30, 40, and 50 μL).
Each concentration of the essential oil was applied to the Whatman filter-paper circle (90 mm in diameter). The paper
was then dried under a fume hood for 1 hour and placed in the bottom of a Petri dish. Control filter papers were treated with
1 mL of hexane. In each of five replicates, five S. Invicta ants were placed on top of the treated filter paper. The dish was left
covered with white cloth. This method allowed assessment of mortality due to contact toxicity, as opposed to fumigation. To
prevent desiccation while forcing ants to remain on the treated filter paper, water was provided in a plastic cap, cut from the top
of a 0.5 mL microcentrifuge tube and placed on top of the filter paper. Mortality was assessed after every 15 min for 2 h, then
finally after 24 h . Plates 3.5 and 3.6 show the toxicity assay test and S. Invicta ant preparation, respectively.
3. RESULTS AND DISCUSSION
3.1. Extraction of Essential Oil Using Steam Distillation
The product from the steam distillation will undergo a further process, that is, liquid-liquid separation technique. When solvent
(20 mL of diethyl ether) is added to the distillate (150 mL), three layers are formed—diethyl ether, essential oil, and water. From
Table 1: The productivity of tea tree oil by steam distillation apparatus.
Figure 1: Tea tree oil volume yield over time.
190 195 200 205 210 215
Extraction time (min)
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top to bottom are diethyl ether, essential oil, and water, respectively. This is due to the densities of each of them, which are
0.713 g/mL, 0.885-0.906 g/mL, and 1.000 g/mL, respectively. Water is discharged from the bottom of the separating funnel
(250 mL), and the process of liquid-liquid separation is repeated once again to make sure all the water is removed. Although,
there will be a small amount of water in the solution, most of the solution is solvent and essential oil. Anhydrous sodium hydrox-
ide is added to remove the small amount of water, and the solvent in the solution is removed by using a hot water bath because
of the low boiling point of the solvent. In steam distillation, typically the essential oil’s yield is in the range of 1% to 2% of weight
of wet plant material . Table 1 and Figure 1 show the productivity of tea tree oil by steam distillation apparatus and the graph
of tea tree oil volume yield per time, respectively.
As seen in Figure 1, the volume of essential oil yield increases as the time extraction increases, although the yield of
essential oil also depends on the moisture content of the raw material (leaves). Although the volume of essential oil yield increases
as the extraction time increases, it also depends on the temperature at which the mixture of raw material and steam is heated,
and the temperature of the surrounding also affects the yield of essential oil. Other than that, as you can see, the extraction speed
is fast at the beginning, slows down with time, and increase in the end. The average time needed to yield essential oil in this
experiment is 205 min, and the average volume of essential oil yield is 1.09 mL. The total amount of tea tree oil yield is 4.35 mL.
As we know, tea tree oil has more than 100 components; the main components are monoterpenes, sesquiterpenes and
their alcohols . These components have been identified using gas chromatography-mass spectrometry . But in the present
study, all of these components have not been identified because it is not compulsory to identify them in this project. One of the
objectives of this project is to extract the essential oil from M. alternifolia using steam distillation. Based on the result, we have
achieved the objective. The present study also proves that the yield of tea tree oil is in the range of 1% to 2% of the weight of
wet plant material which has been reported in the earlier journal .
3.2. Repellency Assays
This test was performed to determine the repellent activity of the tea tree oil. Chi-square test was performed to determine the
repellent activity of the essential oil tested. Table 2 shows the filter paper–repellency assays using M. alternifolia essential oil
against T. castaneum adults.
In this filter paper repellency assay, adults of T. castaneum were used. Four replications were carried out for each
concentration of essential oil, and 20 T. castaneum adults were used per replicate. The results were expressed as mean on the
untreated and treated halves in the assay. The essential oil of M. alternifolia tested as a repellent to T. castaneum adults can be
indicated by Chi-square distribution analysis. Figure 2 shows the graph for repellency assay.
Line Series 1 indicates the untreated section of filter paper, and line Series 2 indicates the treated section of filter paper
with tea tree oil.
From the result, it is obviously shown that M. alternifolia essential oil is a repellent to T. castaneum adults. This repels the
beetles sufficiently, even at very low concentration, and the hypothesis of the ratio 1:1 was rejected. Major constituents that have
Table 2: Filter paper repellency assays using M. alternifolia essential oil against T. castaneum adults.
Mean of insects SE
in untreated (%)
Mean of insects SE in
treated (%) χ2 value
Figure 2: Repellency assay.
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35
Percentage mean of insect
Concentration of tea tree oil (%) vol:vol
60 Original Research Article
repellent activity against insects are Terpinen-4-ol, y-terpinene, and α-terpinene. For repellent and insecticidal action against
T. castaneum, 1,8-cineole is the major constituent that is responsible for insecticidal action against T. castaneum. Based on Table
2, the χ2 value is the Chi-square test value, and it indicates the repellent activity of tea tree oil against T. castaneum. As you
can see that as the concentration of tea tree oil increases, the χ2 value also increases. The result means that as the concentra-
tion of tea tree oil used increases, the repellent activity of the tea tree oil against T. castaneum also increases. Tea tree oil has
been reported for its repellent behaviour against T. castaneum . These earlier reports have clearly supported the result of the
3.3. Toxicity Assays
In this test, the toxicity of the essential oil from tea tree was determined by its toxic effect against red imported fire ant,
S. invicta. Four different concentrations were used, and each concentration was replicated five times. Table 3 shows the
mean standard error (SE) cumulative mortality of red imported fire ants continuously exposed to filter paper treated with five
different concentrations of essential oil.
Toxicity was evaluated in continuous exposure tests with treated filter paper. Based on Table 3, the filter paper treated
with 50 μL concentration of essential oil is the most effective against S. invicta compared to the other concentrations. The
mortality of S. invicta has been determined by calculating the percentage mean death of the insects. As you can see in Table 3,
there is no insect death at the beginning. But the number of dead insects increases as the time increases. At 1440 min, for
the concentrations of 25 μL and 30 μL, 24% of the insects died, and for the concentration of 40 μL, 28% of the insects died.
Furthermore, at the concentration of 50 μL, the mean percentage of dead insects is about 44%. There is no dead insect in
control. From the earlier study, it is seen that the mortality of tea tree oil on S. invicta is not very efficient . The percentage
mean of dead insects is about 3.3% at the concentration of 25 μl . Even though the result from the earlier study is different
from the present study, it is also indicated that the toxicity of tea tree oil against S. invicta is not very efficient. Tables 4 and 5
show the mean of dead insects at different concentrations of tea tree oil and the mean mortality of tea tree oil, respectively.
Based on Tables 4 and 5, we can find the lethal concentration (LC50) using the arithmetic method of Karber. In this
toxicity assay, LC50 of tea tree oil against S. invicta is 23.52 μL/mL. From this result, we can assume that the lethal concentration
of tea tree oil needed to kill 50% of a certain number of insects is 23.52 μL/mL. Lee et al.  stated that the LC50 of tea tree oil
Table 3: Mean SE cumulative mortality of red imported re ants continuously exposed to treated lter paper.
(μL) 15 30 45 60 75 90 105 120 1440
Table 4: Mean of dead insects at different concentration of tea tree oil.
Group Concentration (μl/mL) Mean of dead insects
1 Blank 0
2 25 1.2
3 30 1.2
4 40 1.4
5 50 2.2
Table 5: Mean mortality of tea tree oil.
Mean mortality (b)
1 0 0 0
2 0 0.6 0
3 5 1.2 6
4 10 1.3 13
5 10 1.8 18
Recent Advances in Biology and Medicine, Vol. 2, Pages 57-61, 2016 61
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against S. invicta is 22.8 μL/mL. Although the result is quite different, we can make a conclusion that the previous report has
strongly supported the present study. The overall result, we can say, is that tea tree oil has a toxic effect on S. invicta.
Steam distillation is the best method to extract essential oil from M. alternifolia. The yield of essential oil depends on three fac-
tors: temperature, extraction time, and moisture content. As the extraction time increases, the yield of essential oil also increases.
The lower the moisture content, the greater is the yield of essential oil. In the repellency assays, the result shows that essential
oil from M. alternifolia is repellent and insecticidal against T. castaneum even at a low concentration of essential oil. The major
constituent that is responsible for the repellent and insecticidal actions is 1,8-cineole. It takes times for the essential oil to affect
the red flour beetle, T. castaneum. For the toxicity assay, the result indicates that essential oil from M. alternifolia is toxic against
red imported fire ants, S. invicta. In the beginning, there is no effect on the insect, but the effect becomes clearly visible in the
second hour. From these assays, it can be concluded that tea tree oil is toxic to S. invicta.
Each author has contributed equally in this study.
Source of Funding
Conflict of Interest
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Citation: Aslam MS, Edris MAB, Mamat ASY, Ahmad MS. Insect repellent properties of Melaleuca alternifolia. Recent Adv Biol Med. 2016;