Int.J.Curr.Microbiol.App.Sci (2014) 3(6): 1-09
ISSN: 2319-7706 Volume 3 Number 6 (2014) pp. 1-09
http://www.ijcmas.com
Original Research Article
Insecticidal effects of Fortunella crassifolia essential oil used
against house fly (Musca domestica)
Gehad T. El-Sherbini1* and Nancy Osman hanykamel2
1
Department of Parasitology, Faculty of Pharmacy, 2Department of Parasitology, Faculty of
Medicine October 6 University Cairo, Egypt
*Corresponding author
ABSTRACT
Keywords
Fortunella
crassifolia,
essential oil,
chemical
composition,
insecticidal
activity,
Musca
domestica L.
Growing patterns of insecticidal drug resistance laid the foundation for
research in exploring novel anti-insect agents from medicinal plants
particularly with fumigant action for ease of application.The aim of this
study was to determine the some constituents of the essential oil isolated
from Fortunellacrassifolia Swingle peel by hydro-distillation, and to test the
efficacy of the essential oil on insecticidal activity against house fly,
Muscadomestica. Two Fortunellafractions obtained by steam distillation,
Soxhlet n-hexane extraction, extraction with aqueous ethanol, and with
ethanolic ammonia solution were evaluated in vitro for activity against
house fly, Musca domesticaL.These data warrant further study on
identifying the components of the extracts with the highest activities.The
essential oil showed potent insecticidal activity .It was suggested that the
essential oil from Fortunella crassifolia Swingle peel might be used as a
natural insecticide against house fly.
Introduction
The house fly, MuscadomesticaL. is
considered as one of the most important
pests which cause health problems in the
environment as it accompanies humans
during their daily activity everywhere,
both indoors and outdoors. It is recognized
as a public health pest causing a serious
threat to human and livestock by vectoring
several pathogenic organisms such as
protozoa cysts, helminthic parasites,
enteropathogenic bacteria, and enterovirus
(Emerson, et al., 1999; Douglass and
Jesse, 2002; Mian, etal., 2002; Barin, et
al., 2010). Many insecticides such as
organochlorines and organophosphates,
and more recently pyrethroids and
spinosad, have been used for housefly
control. However, houseflies can develop
resistance to these pesticides (Scott, et al.,
2000; Shono and Scott, 2003) and health
and environmental risks are associated
with these compounds; thus, investigators
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Int.J.Curr.Microbiol.App.Sci (2014) 3(6): 1-09
continue to search for alternative methods
of fly management. The use of plants in
medicine can be dated back to before
humans understood the cause of infectious
disease (Douglass and Jesse, 2002). In this
sense, essential oils (EOs) are potential
alternatives and environmental friendly
insecticides. Kumquats (Figure.1) have
been used in Chinese folk remedies for
centuries. Kumquats are preserved using
salt to draw out the juice. The juice is then
steeped in hot water to relieve sore throats
and suppress coughing. Most traditional
medicines are still folk remedies and have
not been confirmed. Technological
advances in the 20th century left traditional
medicines to be forgotten. Antioxidant,
antimicrobial, and cytotoxic properties
have been demonstrated in several Yemeni
traditional medicinal plants (RegnaultRoger, 1997). Kumquats (Fortunella spp.)
belong to the Citrus genus are fruit,
usually eaten raw as a whole fruit together
with the peel, excluding the seeds. The
peel is sweet and edible with a typical
aroma due to the presence of flavonoids
and terpenoids (Koyasako and Bernhard,
1983). Kumquats are also an excellent
source of nutrients and phytochemicals,
including ascorbic acid, carotenoids,
flavonoids and essential oils. F.
crassifolia, known as jingdan or meiwa
kumquat, has a typical citrus character. As
with other citrus fruits, F. crassifolia can
be candied, prepared as marmalade, added
to fruit salad and preserved as a whole in
sugar syrup in food industry.It is well
known that F. crassifolia is rich in
flavonoids. Previously, 3 , 5 -di-C- gluco- pyranosylphloretin, a characteristic
flavonoid, was well-studied in the
genus Fortunella (Ogawa, et al., 2001).
Recently, the antioxidant activity of F.
crassifolia has been reported (Kondo, et
al., 2005). However, studies on the
chemical composition and biological
activities of F. crassifolia peel oils are
limited. From a dietary viewpoint,
essential oils represent added value for
kumquat fruit because, in addition to their
contribution to the flavor, they play an
important role in human health, as with
other non-nutritive phytochemicals such as
polyphenols and flavonoids (Schirra,
2008).
The Citrus essential oils have various
functional properties, such as an attractive
aroma, a repellant agent against insects
and animals, and antioxidant activities. A
previous study has reported the
antimicrobial action of citrus oils (Subba,
et al., 1967). Meanwhile, citrus oils are not
only available to the food industry, but are
also generally recognized as safe and have
been found to be inhibitory, in both oil and
vapor form, against a range of both Grampositive and Gram-negative bacteria
(Fisher and Phillips, 2008). Moreover,
there are a large number of studies on
plant essential oils regarding their
antimicrobial properties in order to
develop a source of antimicrobial
ingredients for the food industry
(Mkaddem, et al., 2009). To our
knowledge, there are no reports in the
literature concerning the insecticidal
activity of the essential oil from F.
crassifolia until now. The aim of this
study was therefore to determine the main
constituents of the essential oil of F.
crassifolia peel,
together
with
an
evaluation of its insecticidal activity
against the house fly larvae for the
possibility of using these oils as larvicides
for controlling the insects by treating
insect breeding places which create serious
problems mostly in the developing
countries. It is odorous components and
secondary metabolites that can be
extracted from plant tissues through steam
distillation. Essential oils and their volatile
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Int.J.Curr.Microbiol.App.Sci (2014) 3(6): 1-09
constituents are widely used in the
prevention and treatment of human
illnesses. They are also documented for
exhibition of acute toxicity, anti-feeding
and ovi position deterrents against a wide
variety of insect-pests. The risk lower
level of the volatile essential oils to the
environment and their minimal residual
activity against predator, parasitoid and
pollinator insect populations, making
essential-oil-based pesticides compatible
with
integrated
pest
management
programs (Regnault-Roger, 1997; Isman,
2006; Koul, et al., 2008).
sugar, petri dishes containing cotton pads
soaked in milk powder dissolved in water
(10% w/v) and jars (500 mL) containing
larval media for egg laying. Larval media
consisted of yeast, dry milk powder, wheat
bran and water according to the method
described by Pavela (2006) (Pavela, 2006).
The jars were removed from cages after 23 days when eggs were visible and were
provided with wood dust for pupation and
kept in separate cages for fly emergence.
Fumigant toxicity bioassays
Fumigant toxicity of the essential oil was
determined by sealing ten adult flies in a
120ml jar with a 1 cm diameter cotton ball
treated with 100 µl of 100% essential oil.
Essential oil was injected into the center of
each ball to allow its volatilization while
preventing the insects from coming in
contact with the oil. Mortality was
recorded at 15 minutes, 30 minutes, 1 hour
and 2 hours, respectively for the house
flies. Control jar had insects and an
untreated cotton ball. The experiment is
repeated thrice.
Materials and Methods
Chemicals Isolation of the Essential Oil
Fresh F. crassifolia fruit was purchased
from a local market in Cairo in December
2012. The plant was initially identified by
morphological features and the database
present in the Department of Biology,
October 6University. The individual fruits
were washed with water and then
separated into peel and flesh by hand. The
fresh peelings (250 g) were homogenized
with a mixer, followed by hydrodistillation using a Clevenger-type
apparatus for 4 h. The essential oil was
collected over water, dried over anhydrous
sodium sulfate and stored at 4°C until
analyzed. The oil yield was about 2% (5
g/250 g of fresh peel).
Biological studies
The second instar larvae used in this
experiment were 3-days-old after hatching
from the same egg batch. Larval treatment
carried out in petri dishes according to the
method explained by Brady 1966, in
which interior of each petri dish treated
with 1 ml from each of the aforementioned
concentrations of the tested essential oils.
Each experiment was conducted in four
replicates (20 larvae/ replicate) along with
the control group. After treatment, the
larvae were transferred to the rearing jar
and the mortality assessed by touching
each larva with a paint brush (no. 0), and
those not responding were considered
dead. The LD50 and LD75 toxicity
determined based on mortality data at 24 h
assessments. Living larvae were further
Collection and maintenance of flies
Adults of M. domestica were collected
from the garbage site of the Abu Arish
area (Cairo), by using a sweeping net and
transported into a small cage to the
Department of Parasitology, Faculty of
Medicine, October 6
University, for
identification and reared in laboratory for
four generations before experiment. Adult
flies were maintained in cages (30x 30 x
30 cm) and provided with granulated
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Int.J.Curr.Microbiol.App.Sci (2014) 3(6): 1-09
examined daily to estimate the effect on
the larval duration after treatment,
percentage of pupation and the
successfully emerged adults according to
Sripongpun 2008 and Peydro et al. 1995.
In
addition,
any
morphological
abnormalities recorded were photographed
at all developmental stages (Fig. 3).
duration of the control larvae of M.
domesticawas 4.46 ± 0.51 days; a
significant prolongation was observed in
the duration of larvae treated with LC50
and LC75 of the examined oils, which
were 5.61 ± 0.69 and 5.56 ± 0.73 days.
Pupation percent and pupal duration:
Data in Table 4 showed a high significant
reduction in pupation percent (62.50 and
42.50%) at LC50 and LC75 of F.
crassifolia, comparable to 95.00% for
control group. Data in the same table
revealed that the pupal duration of the
control group was 4.43 ± 0.50 days which
significantly prolonged in groups treated
with LC50 and LC75 of tested oils and
reached 5.44 ± 0.53 and 5.00 ± 0.67 days
at LC75 .The current results are in
agreement with findings of Kumar et al.
2012 during their evaluation of insecticidal
activity of E. globulus (Myrtales:
Myrtaceae) against the house fly.
Statistical analysis of Data
The results were recorded as means ±
standard deviation. Statistical analysis was
performed using one-way analysis of
variance (1-way ANOVA) using SPSS
tatistical package (SPSS 16) and the
statistical significance was determined at
P< 0.05.
Results and Discussion
Essential oil was obtained from the fresh
peel of F. crassifolia by hydro- The
amount of the major compound, limonene,
in the essential oil obtained from F.
crassifolia peel is high (74.6%), followed
by myrcene (8.11%), -pinene (0.34%). A
previous report showed that the major
chemical component of citrus oils is
limonene, ranging from 32 to 98%, with
sweet orange containing 68 98%, lemon
45 76% and bergamot 32 45% (14-24).In
another study on the characteristic odor
components in the essential oil from F.
japonica peel, limonene was the most
dominant component, amounting to
93.73% (Choi, 2005). The limonene
content of F. crassifolia peel oil was
comparatively less than that reported
for F. japonica peel oil. The -pinene
content of the essential oil was similar to
that reported for F. japonica peel oil
(Choi, 2005). However, the contents of
myrcene, camphene and -selinene were
found to be higher than those reported
for F. japonica peel oil (Choi, 2005).
The adult emergence percentage
Adult emergence reduced from 95% for
control group to 57.5 and 30% for
groups treated with LC50 and LC75 of
F. crassifolia (Table 5). High reduction
in M. domestica emergence was also
reported by Abdel Halim and Morsy
2005 after using volatile oils of
C.macrocarpa and A. officinarum
against Synthesio myianudiseta. Also,
Kumar et al. 2011 found that crude oils
of M. piperita and E. globulus
suppressed the emergence of adult flies
by 100%. Fortunella essential oil has
shown good insecticidal as shown in the
(Table2,3,4,5). Itpossesses moderate
toxicity to variousinsects. The mortality
ranged from 25 to100%during the first
12hours in the fumigant toxicity
bioassay of the essential oil, mortality
ranged from 25 to 55% during the first
3 hours.
Larval duration
Results in Table 4 revealed that the
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Int.J.Curr.Microbiol.App.Sci (2014) 3(6): 1-09
Fig.1 Fortunella margarita, kumquat,is in the Rutaceae family
Table.1 The main chemical components of the essential oil in the
fresh Fortunella crassifolia Swingle peel.
NO
1
2
3
RIa
936
993
1035
Compound
-Pinene
Myrcene
Limonene
Identificationb
MS,RI
MS,RI
MS,RI
(%)c
0.34
8.11
74.6
a
RI, retention indices as determined on TR-5 capillary column using homologous series of n-alkanes.
Methods of identification: MS, by comparison of the mass spectrum with those of the computer mass libraries;
RI, by comparison of RI with those from the literature.(%)crelative percentage obtained from peak area.
b
Table.2 Contact toxicity of the essential oil of fortunella to adult M. domestica
Essential
oils
Limonene
Myrcene
-Pinene
Control
Conc.%
5
10
100
5
10
100
5
10
100
15min
0
0
33.3
0
0
15,1
0
0
8.5
0
30min
0
25
53.3
0
20
34
0
18
25
0
60min
25
50
63.3
20
25
45.3
20
20
33.3
0
5
Mean mortality (%)
2 hr
3 hr
6 hr
35
55
75
65
75
80
70
100
100
40
50
65
54
65
85
60
75
100
34
44
55
44
55
67
50
62
80
0
0
0
12 hr
100
100
100
100
100
100
100
100
100
0
24 hr
100
100
100
100
100
100
100
100
100
0
Int.J.Curr.Microbiol.App.Sci (2014) 3(6): 1-09
Table.3 Fumigant toxicity of the essential oil of fortunella to M. domestica insect stages
Time
15 min
30 min
60 min
Larva
0
0
10
Mortality %
Pupa
Adult Control
5
4
0
10
8
0
15
12
0
2 hrs
12
20
25
0
3 hrs
25
25
33.5
0
6 hrs
30
50
50
0
12hrs
100
100
100
0
24hrs
100
100
100
0
.
Table.4 Effect of the tested plant oils on the larval duration of M.domestica
treated as 3rd larval in star at 29°C
Oil
Dose
Larval duration
fortunella
LC50
LC75
5.61 ± 0.69b
5.56 ± 0.73 b
4.46 ± 0.51 a
Control
Data expressed as mean± SD, significance at p<0.05 is between different superscripts
Table.5 Effect of the tested plant oils on the pupulation percentage, and pupal duration
(days) of M. domestica treated as 3rd larval in star at 29°C
Es. oil
Dose
fortunella
LC50
LC75
Pupation
(%±SD
62.50±9.57b
42.50±9.57b
95.00±5.77a
Control
Pupal
duration
4.82± 0.53b
5.00 ± 0.67b
4.43 ± 0.50a
Data expressed as mean± SD, significance at p<0.05 is between different superscripts
Table.6 Effect of the tested plant oils on the adult emergence percent
M. domestica treated as 3rd larval instar at 29°C
Es. oil
Dose
fortunella LC50
LC75
Control
Adult emergence (%±SD)
57.50±9.57b
30.00±8.16b
95.00±5.77a
Data expressed as mean± SD, significance at p<0.05 is between different superscripts
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Int.J.Curr.Microbiol.App.Sci (2014) 3(6): 1-09
Plate.1 Adult Pupa, Larva and adult house fly
Plate.2 The control figure represented the normal pupa and figures (1-11)
represented the malformations
Plate.3 The control figure represented the normal larva and Figures 1-7 represented the
malformations at larval stage
.
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Int.J.Curr.Microbiol.App.Sci (2014) 3(6): 1-09
Although, the insecticidal activity of
essential oil is much lessthan that of
synthetic insecticides such as dichlorvos,
propuxorand permethrin, they are quite
harmless. They are not toxic to mammals.
Their volatility as well as their nontoxicity makes them an ideal candidate to
be used in restaurants, hospitals and
schools against various insects. There is a
need to assess various essential oils for
insecticidal activity against insects that
cause diseases and damage food products.
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