Repellent and Fumigant Effects of Essential Oil from Clove Buds Syzygium aromaticum L. against Tribolium castaneum (Herbest) (Coleoptera: Tenebrionidae)

Abstract

Essential oil from clove buds Syzygium aromaticum L. Merr. & Perry (Myrtaceae) was extracted using petroleum ether in Soxhlet apparatus. The resultant oil contained eugenol (48.92%), caryophyllene (18.55%), α-caryophyllene (3.25%), eugenol acetate (23%), cis-13-docosenamide (3.21%), presenting more than 96% of the oil. This oil was examined as a fumigant and repellent agent against adults of the red flour beetle, Tribolium castaneum. Data showed that various concentrations of the oil have revealed dramatic repellent activity against the tested insect, where it gave 100% repellency by 1.0, 0.8, and 0.2% clove oil at 4, 8, 10 min, respectively. The RT 50 and RT 95 were 1.1 and 8.0 min at 0.2% and 0.4, 2.6 min at 0.8%, respectively. In addition, fumigation assay has also exhibited strong fumigant activity toward the adults of T. castaneum. At 100 µL oil/L air, mortality was 75, 80 and 100% after 6, 7, and 8 days exposure period with LC 50 and LC 95 17 and 70 µL/L air, respectively. The utilization of clove oil for its potential effects against stored product insect is discussed.

Full text

Journal of Agricultural Science and Technology A 1 (2011) 613-620
Earlier title: Journal of Agricultural Science and Technology, ISSN 1939-1250
Repellent and Fumigant Effects of Essential Oil from
Clove Buds Syzygium aromaticum L. against Tribolium
castaneum (Herbest) (Coleoptera: Tenebrionidae)
M. M. Abo-El-Saad1, A. M. Al Ajlan2, M. A. Al-Eid2 and I. A. Bou-Khowh1
1. Date Palm Research Center (Center of Excellence ), King Faisal University, P.O. Box 400, Al-Hassa 31982, Saudi Arabia
2. College of Agriculture and Food Science, King Faisal University, P.O. Box 400, Al-Hassa 31982, Saudi Arabia
Received: December 11, 2010 / Published: August 20, 2011.
Abstract: Essential oil from clove buds Syzygium aromaticum L. Merr. & Perry (Myrtaceae) was extracted using petroleum ether in
Soxhlet apparatus. The resultant oil contained eugenol (48.92%), caryophyllene (18.55%), α-caryophyllene (3.25%), eugenol acetate
(23%), cis-13-docosenamide (3.21%), presenting more than 96% of the oil. This oil was examined as a fumigant and repellent agent
against adults of the red flour beetle, Tribolium castaneum. Data showed that various concentrations of the oil have revealed dramatic
repellent activity against the tested insect, where it gave 100% repellency by 1.0, 0.8, and 0.2% clove oil at 4, 8, 10 min, respectively.
The RT50 and RT95 were 1.1 and 8.0 min at 0.2% and 0.4, 2.6 min at 0.8%, respectively. In addition, fumigation assay has also
exhibited strong fumigant activity toward the adults of T. castaneum. At 100 µL oil/L air, mortality was 75, 80 and 100% after 6, 7,
and 8 days exposure period with LC50 and LC95 17 and 70 µL/L air, respectively. The utilization of clove oil for its potential effects
against stored product insect is discussed.
Key words: Essential oils, clove, Syzygium aromaticum, Tribolium castaneum, fumigation, repellency.
1. Introduction
Stored products of agricultural and animal origin
are attacked by more than 600 species of beetle pests
causing quantitative and qualitative losses [1]. Control
of these insects relies heavily on the use of synthetic
insecticides and fumigants, which has led to problems
such as disturbances of the environment, increasing
costs of application, pest resurgence, pest resistance to
insecticides and lethal effects on non-target organisms
in addition to direct toxicity to users [2, 3]. Thus,
repellents, fumigants, feeding and insecticides of
natural origin are rational alternatives to synthetic
insecticides. In stored product insect control, essential
oils may have numerous types of effects [4, 5]: they
may have a fumigant activity [6, 7], they may
Corresponding author: M. M. Abo-El-Saad, professor,
research field: insect toxicology. E-mail:
maboelsaad@gmail.com.
penetrate inside the insect body as contact insecticides
[8] and they may act as repellents [9-11].
Accordingly, essential oils are potential alternatives
to current stored-grain fumigants and repellents
because of their low toxicity to warm-blooded
mammals and their high volatility [7, 12]. Pest control
in many storage systems depends on fumigation with
methyl bromide. The use of methyl bromide is being
restricted and will be phased out by 2015 because of
its potential to damage the ozone layer [13].
Many alternatives have been tested to replace methyl
bromide fumigation for stored product and quarantine
uses. During recent years, some plants have been
receiving global attention and their secondary
metabolites have been formulated as botanical pesticides
for plant protection since they do not leave residues
toxic to the environment, have lower toxicity to
mammals and medicinal properties for human uses [14].

Repellent and Fumigant Effects of Essential Oil from Clove Buds Syzygium aromaticum L. against
Tribolium castaneum (Herbest) (Coleoptera: Tenebrionidae)
614
In nature, essential oils play an important role in the
protection of plants as antibacterials, antivirals, anti-
fungals, insecticides and also as antifeedants against
herbivores. At present, approximately 3,000 essential
oils are known, 300 of which are commercially
important, especially for the pharmaceutical,
agronomic, food, sanitary, cosmetic and perfume
industries [15].
The essential oil isolated from the clove buds,
Syzygium aromaticum is widely used and well known
for its medicinal properties. Traditional uses of clove
oil include use in dental care, as an antiseptic and
analgesic, where the undiluted oil may be rubbed on
the gums to treat toothache. It is active against oral
bacteria associated with dental caries and periodontal
disease [16]. Hussein et al. [17] found that S.
aromaticum extract was highly active at inhibiting
replication of the hepatitis C virus. Kurokawa et al.
[18] isolated and identified an anti-HSV compound,
eugenin, from the extracts of S. aromaticum, which
showed specificity in inhibiting HSV-1 DNA
polymerase activity. Clove essential oil has been
reported to show anticarcinogenic property [19].
The biological activity of S. aromaticum oil has
been investigated against several pests. It was shown to
inhibit the emergence of Culex pipiens larvae [20] and
to display insecticidal activity against Pediculus capitis
[21], Anopheles dirus mosquitoes [22] and some stored
product insects and suppresses progeny development
of T. castaneum and Sitophilus zeamais with
isoeugenol being particularly active [23]. A previous
study has confirmed that clove oil could be used as a
novel fumigant against Japanese termites [24].
T. castaneum is a major pest of stored grains and
grain products in the tropical countries. One of the
most popular foods in Saudi Arabia and other tropics is
dates which are attacked in storehouses by the red flour
beetle T. castaneum as a major insect [25, 26]. As part
of future strategies for stored-product insect control,
essential oils with repellent and/or insecticidal
properties should be studied. Therefore, the aim of the
present work was conducted to evaluate the potential
repellent and fumigant effects of essential oil extracted
from clove buds, S. aromaticum against the red flour
beetle, T. castaneum (Coleoptera: Tenebrionidae).
2. Materials and Methods
2.1 Extractions of Essential Oil
Essential oil from clove buds S. aromaticum was
extracted according to the method of Wenqiang et al.
[27]. Clove buds, 40-50 gm were ground with an
electric blender to fine powder. The powder was
packed in extraction thimbles cellulose 33 × 100 mm.
Each packed material was then extracted in 200 mL of
petroleum ether using a Soxhlet apparatus under
cooling for 6 hours. After extraction, the extract was
filtered and then evaporated using a rotary evaporator
(at 70 °C and 100 rpm) under vacuum to avoid
chemical degradation of extracted materials. The
extracts were stored at 10 °C until further use.
2.2 Gas Chromatography-Mass Spectrometry (GC-MS)
GC-MS QP 2010 model Shimadzu was used. The
mass data were analyzed by a GCMS solution. The
GC conditions were as follows: column, RTX(r), 30 m
× 0.32 mm; 0.25 mm; He carrier gas flow rate, 1.4
mL/min; split less: temperature program, 3 min at
100 °C, 3 °C min gradient to 200 °C, 20 °C/min
gradient to 280 °C; 13 min; injection temperature,
260 °C; detector temperature, 270 °C. The EI was
70eV. The NIST/EPA/NIH standard mass-spectrum
data bank was used for reference.
2.3 Insects
Adults of T. castaneum were obtained from
laboratory cultures maintained in dark incubators at
28-30 °C and 70-80% r.h. at the Date Palm Research
Center, King Faisal University, Saudi Arabia.
2.4 Repellent Activity
The experimental method was performed as
described by Jilani and Saxena [28] with some

Repellent and Fumigant Effects of Essential Oil from Clove Buds Syzygium aromaticum L. against
Tribolium castaneum (Herbest) (Coleoptera: Tenebrionidae)
615
modifications. Whatman No.1 filter papers (diameter
9 cm) were cut in half. Test solutions were prepared
by dissolving 2, 4, 6, 8 and 10 μL of S. aromaticum oil
in 1 mL acetone. Each solution was applied to half a
filter-paper disc as uniformly as possible with a
micropipette. The other half of the filter paper was
treated with acetone alone as a control. The treated
and control half discs were dried in air for 5 min to
evaporate the solvent completely. Treated and
untreated halves were attached to their opposites using
adhesive tape and placed in Petri dishes. Twenty
unsexed adult (7-10 day old) flour beetles were
released separately at the centre of each filter paper
disc. Three replications were used for each
concentration. Observations on the number of insects
present on both the treated and untreated halves were
recorded at different time intervals 2, 4, 6, 8, and 10
min. Percent repellency is defined as the percentage of
insects that avoided the treated section and was
calculated as described in methods of Ngoh et al. [29]
as follows:
% Repellency = 100 − [(100 ×Nt)(Nt + Nc)]
Nt = Number of insects in the treated side;
Nc = Number of insects in the control side.
2.5 Fumigant Activity
The fumigant activity of S. aromaticum oil was
tested as described by Tripathi et al. [30] with some
modification. For adults, 0, 20, 40, 60, 80 and 100 μL
of S. aromaticum oil were dissolved in 1 mL acetone
and applied to Whatman No.1 filter paper (diameter 9
cm) were then were dried in air for 5 min. Treated
filter papers were attached with the lid of 1 L glass
jars using adhesive tape. Twenty adults (10 days old)
were then placed in 1 L glass jar containing 5 g of
wheat flour with 15% broken grains. The jars were
then sealed with air-tight lids. Three replicates of each
control and treatment were performed. Mortality was
observed every 24 h thereafter until end-point
mortality, which was reached after 8 days. Probit
analysis was used to estimate concentration (LC50, and
LC95) and lethal time (LT50, and LT95) values as
described by Finney [31].
2.6 Statistical Analysis
Data were analyzed using MSTAT-C software and
means were compared by Duncan’s multiple range
tests at 5% level.
3. Results and Discussions
3.1 Chemical Analysis of S. aromaticum Oil
Essential oil from clove buds contained five major
compounds: eugenol (48.92%), caryophyllene
(18.55%), α-caryophyllene (3.25%), eugenol acetate
(23%) and cis-13-docosenamide (3.21%), presenting
more than 96% of the oil. In addition, the oil contained
five other minor compounds: copaene (0.66%),
cycloheptasiloxane (0.5%), caryophyllene oxide
(0.61%), n-hexatriacontane (0.47%), n-dotriacontane
(0.83%) (Table 1). Our results are consistent with data
reported by Bensky et al. [32] showing that Eugenol
comprises 72-90% of the essential oil extracted from
cloves, and is the compound most responsible for the
cloves’ aroma. Furthermore, Wenqiang et al. [27]
showed that the maximum content of eugenol in
extracts oil from clove buds is 58.77%, whereas, the
highest percentage of eugenol acetate reached to
approximately 21%, which is also the main antioxidant
ingredients in clove oil [33]. They also reported that the
highest percentage of active antioxidant ingredients,
eugenol together with eugenol acetate, in the extracted
Table 1 Chemical composition of S. aromaticum essential
oil.
Compound Retention index Abundance (%)
Eugenol
Copaene
Caryophyllene
α- Caryophyllene
Eugenol acetate
Cycloheptasiloxane
Caryophyllene oxide
cis-13-Docosenamide
n-hexatriacontane
n-Dotriacontane
1392
1221
1494
1597
1552
1447
1507
2625
3600
3202
48.92
0.66
18.55
3.25
23.0
0.5
0.61
3.21
0.47
0.83

Repellent and Fumigant Effects of Essential Oil from Clove Buds Syzygium aromaticum L. against
Tribolium castaneum (Herbest) (Coleoptera: Tenebrionidae)
616
clove oil was 78% which are in agreement with our
results showing eugenol together with eugenol acetate;
reached to 72%.
3.2 Repellent Activity
The extracted essential oil from clove buds, S.
aromaticum was tested as a repellent agent against
adults of red flour beetle, T. castaneum which is a
major insect in Saudi Arabia and other tropical
countries [25, 26] attacking dates in storehouses
causing significant loss. The filter paper assay
exhibited the behavior of the insect once they were
exposed to oil extract. At the onset, insects were
agitated, moved frequently and showed exceptional
excitement. Within 10 min, almost all experimental
insects gathered together in one spot in the Petri dish.
The number of insects in both treated and untreated
area was recorded and the percent repellency was
calculated as shown in Table 2. Fig. 1 shows that the
extracted oil exhibited highly significant repellent
effect against adults of T. castaneum. At 0.2, 0.6 and
1.0% of extracted oil, the percent repellency was 63%,
75%, 87% after 2 min of exposure and it reached
100% repellency at various concentrations after 10 min.
The corresponding RT50 values were 1.1, 0.68 and 0.4
min. respectively at concentration 0.2, 0.6 and 0.8%,
whereas, RT95 values were 8.0, 4.0 and 2.0 min
respectively for the same exposure periods (Table 2).
Thus, S. aromaticum oil has potential for use against
some stored-product insects as a repellent agent.
Present data are consistent with those reported by
Ngoh et al. [29] who indicated that eugenol, methyl
eugenol and isoeugenol are better toxicants and
repellents to the insects than monoterpenes.
Furthermore, Huang et al. [34] was tested insecticidal
property of eugenol derivatives; eugenol, isoeugenol
and methyleugenol against T. castaneum, the order of
potency of these chemicals compared by the LD50
level was isoeugenol > eugenol > methyleugenol.
Recently, there have been many reports concerning
the repellent properties of many kinds of essential oils;
however, most of results came from artificial (in vitro)
testing methods using filter paper, insect, animal or
olfactometry, but some came from more realistic (in
vivo) using animal, insect or human subjects [35]. The
repellency effect of many essential oils included
egunol from S. aromaticum against medicinal insects
was evaluated by using an arm-in-cage test, where, it
was found that eugenol posses strong effect as a
repellent agent [22].
Based on our results, adults of T. castaneum were
found to be repelled by S. aromaticum oil at very low
concentration. We believe the strong repellent activity
of S. aromaticum oil that is presented here could be
due to eugenol the main constituent of the oil
(48.92%). In previous research, this compound has
shown to be powerful repellent towards many pests
[22, 29, 34-36].
Table 2 Estimated RT50 and RT95 values of various
concentrations of S. aromaticum essential oil against adults
of Tribolium castaneum.
Conc. % (V/V) RT50 (min) RT95 (min)
0.2
0.4
0.6
0.8
1.0
1.10
0.90
0.68
0.40
ND
8.0
6.8
4.0
2.6
ND
RT = the time required of clove oil that provides 50% and 95%
repellency; ND = not detected values because too far beyond
the tested exposure ranges to be reliable.
Fig. 1 Repellency percent of the red flour beetle,
Tribolium castaneum adults exposed to S. aromaticum
essential oil for various periods of times.

Repellent and Fumigant Effects of Essential Oil from Clove Buds Syzygium aromaticum L. against
Tribolium castaneum (Herbest) (Coleoptera: Tenebrionidae)
617
However, to clarify repellent activity of these pure
constituents against T. castaneum future studies
should be done.
3.3 Fumigant Activity
The analysis was extended to see whether S.
aromaticum oil has a fumigant effect against adults of
red flour beetle T. castaneum. Fig. 2 showed that the
oil has a weak mortality up to three days from the
exposure at various concentrations. The mortality
reached 20% and 40% after 4 and 5 day of exposure at
the concentration 100 µL/L air. However, by
increasing the exposure period up to 7 and 8 days,
significant mortality (P < 0.05) was achieved, it
reached 80% and 100% respectively. The lethal
concentrations values LC50 and LC95 were determined
based on the method of Finney [31]. LC50 values were
156.89, 54.59 and 10.7 µL/L air after respectively 5, 6,
and 8 days of exposure, whereas, LC95 values were
1,182.76, 357.7, and 254.87 µL/L air respectively for
the same exposure periods (Table 3). Moreover, the
median effective time causing mortality of 50% of
tested insects (LT50) at various concentrations (20, 40
and 100 µL/L air) were 7.2, 6.8 and 5.12 min
respectively, whereas the LT95 were 16.8, 15.9 and
10.34 min. respectively (Table 4). The weak insect
mortality of this oil for up to three days from the
exposure at various concentrations against T.
castaneum may be due to that the insect in the early
days of exposure to essential oil is closing the
spiracles, which reduces the opportunity for toxic
substance to enter. So, this study it is clear indicated
that the fumigation with natural substances is one of
the most important ways to control stored-product
pests without use of insecticides and the problems
arising from its application.
The widespread use of synthetic insecticides has led
to many negative consequences resulting in an
increasing attention being given to natural products [36,
37]. Among biopesticides, botanical pesticides are
experiencing a revival because some have favorable
eco-toxicological properties (low human toxicity, rapid
degradation and reduced environmental impact), which
make them suitable insecticides for organic agriculture
and store-products pests as well. In the case of
stored-products pests, the biological activity of S.
aromaticum oil has been investigated against several
pests. It was shown to suppress progeny development
0.00 20.00 40.00 60.00 80.00 100.00
Concentration (ul/l air)
0.00
20.00
40.00
60.00
80.00
100.00
Mortality (%)
Day 8
Day 7
Day 6
Day 5
Day 4
Day 3
000 0 0
10
Fig. 2 Mortality percent of the red flour beetle, Tribolium castaneum adults exposed to S. aromaticum essential oil for
various periods of times.

Repellent and Fumigant Effects of Essential Oil from Clove Buds Syzygium aromaticum L. against
Tribolium castaneum (Herbest) (Coleoptera: Tenebrionidae)
618
Table 3 LC50 and LC95 values of S. aromaticum essential oil against adults of Tribolium castaneum after exposed to various
periods of time.
Chi square (X2)Slope (± SE)
Degrees of
freedom
LC95 (μL/L air)
(95% fiducial limits)
LC50 (μL/L air)
(95% fiducial limits)
Exposure time
(days)
7.81 1.96 ± 0.46 3 1182.76
(2270.81-332.34)
156.89
(119.5-87.6)
5
7.81 1.60 ± 0.38 3 357.7
(282.1-165.3)
54.59
(95.54-36.96)
6
7.81 1.29 ± 0.36 3 329.48
(510.42-139.06)
35.27
(55.52-18.4)
7
7.81 0.93 ± 0.36 3 254.87
(167.60-92.5)
10.70
(21.93-0.254)
8
LC = the lethal concentration for a 50% and 95% of treated insects.
Table 4 LT50 and LT95 values of various concentrations of S. aromaticum essential oil against adults of Tribolium
castaneum.
Chi square (X2)Slope (± SE) Degrees of freedom
LT95 (min)
(95% fiducial limits)
LT50 (min)
(95% fiducial limits)
Conc. µL/L
5.99
7.4 ± 2.01
2 16.8
(14.5-8.5)
7.2
(8.5-6.6)
20
5.99
1.96 ± 2.1 2 15.9
(15.4-8.4)
6.8
(7.7-6.3)
40
7.81 5.8 ± 1.36
3 12.7
(17.1-8.6)
6.3
(7.2-5.6)
80
9.49 6.79 ± 1.09
4 10.34
(9.8-7.0)
5.12
(5.6-4.6)
100
LT = the time required for a 50% and 95% kill.
of T. castaneum with isoeugenol being particularly
active [23]. Our results are consistent with that data
reported by Wang et al. [38] showing that some
essential oils containing eugenol derivatives have
strong fumigant activity against adults of T.
castaneum.
It could be concluded that the oil extracted from
clove buds has potential effects against T. castaneum,
however, better understanding at the pure constituent
level along with structure-activity relationships are
required to develop S. aromaticum oil as a fumigant or
repellent agent against stored-product insect pests.
Further research on how to use S. aromaticum oil
effectively for the control of insects in stored products
is required.
Acknowledgments
Authors would like to thank the Deanship of
Scientific Research, King Faisal University, Saudi
Arabia for funding the project No. 90024.
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