Professional Documents
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Orange Peel
Orange Peel
Orange Peel
DOI 10.1007/s00436-012-3021-8
ORIGINAL PAPER
Abstract Mosquitoes are the carriers of severe and wellknown illnesses such as malaria, arboviral encephalitis,
dengue fever, chikunguniya fever, West Nile virus and yellow fever. These diseases produce significant morbidity and
mortality in humans and livestock around the world. The
present study explored the effects of orange peel ethanol
extract of Citrus sinensis on larvicidal, pupicidal, repellent
and adulticidal activity against Anopheles stephensi, Aedes
aegypti and Culex quinquefasciatus. The orange peel material was shade dried at room temperature and powdered
coarsely. From orange peel, 300 g powdered was macerated
with 1 L of ethanol sequentially for a period of 72 h each
and filtered. The yields of the orange peel ethanol crude
extract of C. sinensis 13.86 g, respectively. The extracts
were concentrated at reduced temperature on a rotary vacuum evaporator and stored at a temperature of 4 C. The
larvicidal, pupicidal and adult mortality was observed after
24 h of exposure; no mortality was observed in the control
group. For C. sinensis, the median lethal concentration
values (LC50) observed for the larvicidal and pupicidal
activities against mosquito vector species A. stephensi first
to fourth larval instars and pupae were 182.24, 227.93,
291.69, 398.00 and 490.84 ppm; A. aegypti values were
92.27, 106.60, 204.87, 264.26, 342.45, 436.93 and
K. Murugan : P. Mahesh Kumar : K. Kovendan (*) :
D. Amerasan : J. Subrmaniam
Division of Entomology, Department of Zoology, School of Life
Sciences, Bharathiar University,
Coimbatore 641 046 Tamil Nadu, India
e-mail: gokulloyo@yahoo.co.in
J.-S. Hwang
Institute of Marine Biology, National Taiwan Ocean University,
Keelung 202-24, Taiwan
Introduction
Insect-transmitted disease remains a major source of illness
and death worldwide. Diseases that are healthcareassociated transmission of viruses to human from mosquitoes are an expanding problem in tropical and subtropical
regions. Some of them such as dengue, malaria and West
Nile virus (WNV) are now the most frequent arboviral
diseases in the world (Wilder-Smith et al. 2009). Female
mosquitoes are one of the most world-wide important insect
pests that affect the health of human being and domestic
animals. Anopheles stephensi and Anopheles culicifacies are
the important vectors of malaria while Aedes aegypti and
Culex quinquefasciatus are the vectors of dengue and filariasis, respectively. All these mosquito species have been
identified as primary vectors of the above diseases in this
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Taxonomy
Kingdom: Plantae
Phylum: Tracheophyta
Subphylum: Euphyllophytina
Class: Magnoliopsida
Subclass: Rosidae
Superorder: Rutanae
Order: Sapindales
Family: Rutaceae
Subfamily: Aurantioideae
Tribe: Aurantieae
Subtribe: Citrinae
Genus: Citrus
Species: sinensis
Botanical name: Citrus sinensis (Zipcode zoo 2012)
Essential oils are vegetable products whose constituents
are basically complex mixture of terpenic hydrocarbons and
oxygenated derivatives such as aldehydes, alcohols and
esters. These are accumulated mainly in secretary cavities
scattered throughout the fruit peels and leaf. Among many
sources, citrus fruit peels are the most familiar and rich
source of essential oils. Peels of citrus fruit comprise of
two layers, red outer layer as flavedo and inner white layer
as albedo (Nagi et al. 1977). The flavedo layer contains
essential oils in the range of 0.5 to 3.0 kg/ton of fruit (Sattar
and Mahmud 1992). Some of the Citrus species have been
reported as a source of botanical insecticides: as a variety of
these plants contain secondary metabolites that show insecticidal activity against several coleopteran and dipteran (Su
et al. 1972; Sheppard 1984; Salvatore et al. 2004; Shrivastava et al. 2010).
The present investigation was to explore the mosquito
control agent under laboratory conditions. The orange peels
extract C. sinensis for the control of malarial vector, A.
stephensi, dengue vector, A. aegypti, and filarial vector, C.
quinquefasciatus, was evaluated mosquito species.
The pupae were collected from the culture trays and transferred to plastic containers (1212 cm) containing 500 mL
of water with the help of a dipper. The plastic jars were kept
in a 909090-cm mosquito cage for adult emergence.
Mosquito larvae were maintained at 27+2 C, 7585 %
relative humidity, under a photoperiod of 14:10 (light/dark).
A 10 % sugar solution was provided for a period of 3 days
before blood feeding.
Blood feeding of adult mosquito vectors
The adult female mosquitoes were allowed to feed on
the blood of a rabbit (a rabbit per day, exposed on the
dorsal side) for 2 days, to ensure adequate blood feeding for 5 days. After blood feeding, enamel trays with
water from the culture trays were placed in the cage as
oviposition substrates.
Collection of plant and preparation of extract
The fresh orange fruit brought from Coonoor, Nilgiris district, Tamil Nadu, were peeled and shopped with knife and
the soaked in distilled water. The fresh orange peels of C.
sinensis were shade dried at room temperature (282 C)
for 10 to 15 days. The ground materials were obtained by
grinding the dry peels into a fine powdered separately using
commercial electrical blender. From orange peel, 300 g
powdered was macerated with 1 L of ethanol sequentially
for a period of 72 h each and filtered. The yields of the
orange peel ethanol crude extract of C. sinensis 13.86 g,
respectively. The extracts were concentrated at reduced temperature on a rotary vacuum evaporator and stored at a
temperature of 4 C. One gram of the plant residue was
dissolved in 100 mL of acetone (stock solution) considered
as 1 % stock solution. From this stock solution, concentrations were prepared ranging from 100, 200, 300, 400 and
500 ppm, respectively.
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Corrected mortality
Percentage mortality
Protection
fNo: of bites received by control armg fNo: of bites received by treated armg
100
No: of bites received by control arm
Adulticidal bioassay
Sugar-fed adult female mosquitoes (5 to 6 days old) were
used. The orange peel extract were diluted with acetone to
make different concentrations. The diluted plant extracts
were impregnated on filter papers (140120 mm). A blank
paper consisting of only ethanol was used as control. The
papers were left to dry at room temperature to evaporate off
the ethanol overnight. Impregnated papers were prepared
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Results
Larvicidal and pupicidal activity of orange peel ethanol
extract of C. sinensis at various concentrations against the
malarial vector, A. stephensi is given in the Table 1. Considerable mortality was evident after the treatment of C.
sinensis for all larval instars and pupae. Mortality was
increased as the concentration increased, for example, in
first instars stage at 100 ppm concentration the larval mortality was 39 %; whereas at 500 ppm concentration, it was
increased to 97 %. In pupal mortality at 100 ppm concentration, it was 17 % increased to 52 % at 500 ppm (Fig. 1).
The LC50 and LC90 values were represented as follows:
LC50 value of first instar was 182.24 ppm, second instar
was 227.93 ppm, third instar was 291.69 ppm and fourth
Table 1 Larval and pupal toxicity effect of orange peel ethanol extract of C. sinensis against malarial vector, A. stephensi
Mosquito
life stages
LC50 (LC90)
First instar
Second
instar
Third instar
Fourth
instar
Pupa
95 % confidence limit
LC50
(LFL-UFL)
LC90 UFL-UFL
x2
(df04)
100
200
300
400
500
391.41
321.32
521.85
461.41
661.01
581.72
831.32
741.85
971.16
891.15
182.24 (452.44)
227.93 (544.72)
(147.11210.44)
(192.86257.57)
(412.40508.77)
(490.98624.33)
4.52*
1.35*
261.01
221.41
381.32
291.01
491.35
351.72
641.41
521.32
781.16
621.85
291.69 (659.31)
398.00 (858.92)
(257.35325.23)
(354.80458.14)
(583.27779.67)
(728.241,095.55)
0.36*
1.08*
171.01
221.16
291.85
411.41
521.60
490.84 (987.28)
(432.00591.37)
(817.281,318.52)
0.41*
Control nil mortality, LFL lower fiducidal limit, UFL upper fiducidal limit, x2 Chi-square value, df degrees of freedom
*P<0.05, significant level; each value (meanSD) five replicates
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Fig. 1 Larval and pupal toxicity effect of orange peel ethanol extract of C. sinensis against mosquito vectors
was 385.32 ppm and fourth instar was 452.78 ppm, respectively. The LC90 value of first instar was 566.96 ppm, second
instar was 729.84 ppm, third instar was 806.57 ppm and
fourth instar was 890.08 ppm, respectively. The LC50 value
of pupae was 530.97 ppm, and the LC90 value of pupae was
967.19 ppm, respectively.
In the present observation, the results from the skin
repellent activity of orange peel ethanol extracts of C.
sinensis against blood starved adult female of A. stephensi,
A. aegypti and C. quinquefasciatus are given in Tables 4.
The present result shows that the percentage protection in
relation to dose and time (minutes). The highest concentrations of 450 ppm provided over 180 and 150 min protection
in ethanol extracts of C. sinensis, and over 90 and 120 min
protection in ethanol extracts of C. sinensis against mosquito
Table 2 Larval and pupal toxicity effect of orange peel ethanol extract of C. sinensis against dengue vector, A. aegypti
Mosquito
life stages
LC50 (LC90)
First
instar
Second
instar
Third
instar
Fourth
instar
Pupa
95 % confidence limit
LC50
(LFL-UFL)
LC90
UFL-UFL
x2
(df04)
100
200
300
400
500
352.0
491.41
631.72
771.85
921.16
204.87 (509.72)
(168.22234.67)
(460.97580.90)
1.60*
291.01
401.85
521.67
691.32
831.41
264.26 (607.02)
(230.24295.22)
(542.25706.18)
0.09*
231.85
311.41
421.16
591.35
701.72
342.45 (734.98)
(307.38382.58)
(642.38887.13)
0.05*
191.72
261.54
311.41
431.87
611.16
436.93 (891.63)
(390.09507.51)
(754.53.1,140.62)
1.94*
141.01
201.41
250.63
361.32
541.85
497.41 (938.06)
(442.85586.25)
(791.901,205.04)
1.69*
Control nil mortality, LFL lower fiducidal limit, UFL upper fiducidal limit, x2 chi-square value, df degrees of freedom
*P<0.05, significant level; each value (meanSD) five replicates
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Table 3 Larval and pupal toxicity effect of orange peel ethanol extract of C. sinensis against filarial vector, C. quinquefasciatus
Mosquito
life stages
LC50 (LC90)
First
instar
Second
instar
Third
instar
Fourth
instar
Pupa
x2
(df04)
95 % confidence limit
LC50
(LFL-UFL)
LC90
UFL-UFL
100
200
300
400
500
321.16
411.41
561.35
691.93a
891.72
244.70 (566.96)
(210.98274.23)
(509.94652.10)
3.57*
261.41
351.16
431.01
581.85
741.32
324.04 (729.84)
(287.75363.51)
(635.46887.18)
1.37*
201.32
291.72
381.41
511.16
651.49
385.32 (806.57)
(346.34436.38)
(694.83998.69)
.274*
160.48
221.85
331.41
421.01
571.72
452.78 (890.08)
(405.48524.86)
(756.651,128.82)
.353*
111.41
161.01
261.16
311.85
491.72
530.97 (967.19)
(470.76632.78)
(813.451,251.91)
1.13*
Control nil mortality, LFL lower fiducidal limit, UFL upper fiducidal limit, x2 Chi-square value, df degrees of freedom
*P<0.05, significant level; each value (meanSD) five replicates
Discussion
Singhi et al. (2006) have reported the latex of C. procera has
shown larvicidal efficacy against all three important vector
Table 4 Repellent activity of orange peel ethanol extract of C. sinensis against mosquito vectors
Mosquito species
Concentration (ppm)
% of repellencySD
Time post application of repellent (min)
30
60
90
120
150
180
A. stephensi
50
150
250
350
450
99.40.8
1000.0
1000.0
1000.0
1000.0
891.0
981.6
99.60.4
1000.0
1000.0
83.81.6
89.81.4
961.6
1000.0
1000.0
74.61.7
79.2 1.1
87.61.8
911.4
1000.0
67.21.4
72.81.7
80.21.1
86.61.8
92.41.8
58.41.3
631.4
71.81.4
77.61.0
88.61.2
A. aegypti
50
150
250
350
450
50
150
250
350
450
94.41.3
990.8
1000.0
1000.0
1000.0
91.21.1
97.21.9
1000.0
1000.0
1000.0
87.81.7
92.41.8
1000.0
1000.0
1000.0
83.81.4
90.61.8
94.81.7
1000.0
1000.0
791.4
85.61.6
92.61.7
98.41.3
1000.0
76.41.0
821.4
881.2
910.8
97.61.0
72.81.6
74.21.7
83.41.4
87.61.8
98.21.7
64.61.8
72.21.5
82.21.1
85.41.8
910.8
64.21.5
671.4
76.21.1
78.20.7
88.61.4
56.21.6
621.4
721.3
76.61.3
82.41.3
541.4
60.60.4
68.61.0
71.60.8
77.21.1
490.6
56.62.0
63.61.3
67.81.7
73.21.5
C. quinquefasciatus
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Table 5 Adulticidal activity of
orange peel ethanol extract of C.
sinensis against mosquito
vectors
Mosquito
species
Concentration
(ppm)
A. stephensi
180
260
340
24.22.0
50.42.7
67.83.4
420
82.52.2
500
Control
95.11.7
0.00.0
180
260
340
21.71.4
47.82.3
62.62.2
420
76.22.6
500
Control
91.42.5
0.00.0
180
260
17.22.3
37.42.1
340
55.92.0
420
500
Control
72.41.4
86.82.9
0.00.0
A. aegypti
C. quinquefasciatus
Mortality (%)
(Mean SD)
LC50, ppm
(LFL-UFL)
LC90, ppm
(LFL-UL)
x2
272.19
(250.93290.73)
457.14
(429.68494.00)
1.27*
289.62
(267.53309.24)
494.88
(462.58539.43)
2.19*
320.38
(300.29339.55)
524.57
(490.31571.84)
0.49*
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mosquito larvae in India are Cleome viscosa, Ocimum basilicum, Vitex negundo, Delonixregia, Oligo chaetaramosa, Azadirachta indica, Quassia amara, Anacardium occidentale,
Thevetianerii folia etc. Natural products are preferred because
of their biodegradability and less toxic compared to the synthetic ones (Datta et al. 2010).
Benzene and methanol extracts of Artemisia vulgaris
have been reported to have repellent activity against A.
aegypti 17 (Yit et al. 1985). Quelling, the insect repellent
produced in China, derived from the extract of the lemon
grass and eucalyptus plants were evaluated against mosquitoes. Essential oil obtained from Vitex negundo was used as
repellent against A. aegypti (Hebbalkar et al. 1992). Neem
products are good mosquito repellents showing 90 % to
100 % protection against malaria vectors and about 70 %
against C. quinquefasciatus (Ansari and Razdan 1994). One
controlled study evaluated the efficacy of a cream formulation containing 5 % neem oil against C. quinquefasciatus
and A. culicifacies. The cream (45 g) was applied to the
exposed skin areas of human volunteers in Ghaziabad, India
in the summer months of May/June and the monsoon
months of August/September. Neem cream was found to
offer 82 % protection against Culex bites and 100 % protection against Anopheles bites, as compared to untreated
controls (Nagpal et al. 2001). The ethanolic extracts of the
orange peel (C. sinensis) were tested for the toxicity effect
on the larvae of the yellow fever mosquito A. aegypti
(Amusan et al. 2005); susceptibility tests were carried out
in C. quinquefasciatus larvae using peel oil extracts of
Citrus aurantium, C. sinensis and Citrus limon (Mwaiko
1992); volatile extracts of C. sinensis had insecticidal activity against mosquito, cockroach and housefly (Ezeonu et al.
2001). Garcia and Desrochers (1979) observed appreciable
mortality only with high concentrations (1107cells/ml) of
B. thuringiensis var. israelensis. The biocide at 1 to 10 kg/ha
(0.25 to 2.5 ppm) caused 18 % to 88 % mortality of midges
during a 4-week evaluation period. Mahesh Kumar et al.
(2012) have reported that the LC50 values of B. thuringiensis against the first to fourth instar larvae and pupae were
133.88, 157.14, 179.44, 206.80 and 240.74 ppm; and the
LC90 values were 321.04, 346.89, 388.86, 430.95 and
492.70 ppm, respectively
Ansari et al. (2000) reported that the peppermint oil gave
94.1 % protection for 6 h, while mylol oil gave 95 %
protection for 7.2 h. They also reported that mylol oil and
peppermint oil gave 100 % protection for 11 h against
Anopheles annularis. Mylol oil gave 95.4 % protection for
8.7 h for the Anopheline species, whereas the peppermint oil
gave 86.3 % protection for 8 h. Phukan and Kalita (2005)
showed that Litsea salicifolia recorded 70 % and 50 %
repellency for 3 and 4 h, respectively, against A. aegypti,
but they failed to show much activity against C. quinquefasciatus. Mullai et al. (2008) have also reported that the
Parasitol Res
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