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REPELLENT ACTIVITY OF CARROT SEED ESSENTIAL OIL AND ITS PURE
COMPOUND, CAROTOL, AGAINST MOSQUITOES
ABBAS ALI, MOHAMED M. RADWAN, AMIRA S. WANAS AND IKHLAS A. KHAN
National Center for Natural Products Research, The University of Mississippi, University, MS 38677
ABSTRACT. In our natural products screening program for mosquitoes, carrot seed essential oil showed high
repellency. The gas chromatography (GC)/flame ionization detector and GC/mass spectrometry analysis of the
essential oil revealed the presence of 47 compounds. Carotol was more than 75% w/w, followed by muurolene
(4.86%), (Z)-b-farnesene (2.9%), and diepicedrene (1.1%). Systematic bioassay-guided fractionation of the essential
oil was performed to identify active repellent compounds. In both Klun and Debboun (K&D) and Ali and Khan
(A&K) bioassays, carotol showed biting deterrent activity similar to N,N-Diethyl-3-methylbenzamide (deet) and
carrot seed essential oil against both Aedes aegypti and Anopheles quadrimaculatus, while in in vivo cloth patch
bioassay, the minimum effective dose (MED) of deet was lower (12.5 lg/cm
2
) than the essential oil and carotol (25
lg/cm
2
) against Ae. aegypti. In the A&K bioassay, the MED values were similar, whereas the values of the mixtures
of deet with essential oil and carotol was lower (6.25 þ6.25 ¼12.5 lg/cm
2
) than their individual treatments (25 lg/
cm
2
). In direct skin application bioassay, both the essential oil and carotol provided good repellency. The mixtures
of deet and essential oil or carotol significantly increased the residual activity, indicating synergism. Mosquito
repellency of the essential oil and carotol is reported for the 1st time. These data indicate the potential of these
natural products to be developed as commercial repellents.
KEY WORDS Aedes aegypti,Aedes albopictus,Anopheles quadrimaculatus, biting deterrent, repellent, carrot
seed essential oil, carotol
INTRODUCTION
Mosquitoes are important in global public health
because they transmit many disease pathogens. Aedes
aegypti (L.) and Ae. albopictus (Skuse) are consid-
ered the primary and secondary vectors of Zika virus,
respectively, as well as other arboviruses (Ali et al.
2017a). Anopheles spp. transmit malaria (Sanders et
al. 1996, Meslin 1997) and Culex quinquefasciatus
Say transmits West Nile virus (Godsey et al. 2005).
The use of synthetic insecticides in mosquito control
has proved to be one of the major components for
prevention and reduction of mosquito-borne disease
incidence (Bhatt et al. 2015).
Insect repellents play an important role in the
reduction of disease incidence by preventing infected
mosquitoes from biting humans (Leal 2006). More-
over, repellents have always been used against host-
seeking mosquitoes as they provide immediate,
localized, personal protection. The most widely used
repellent, N,N-Diethyl-3-methylbenzamide (deet),
has been in use for .60 years and is the standard
to which all repellents are measured in the market-
place (Frances 2007). The discovery of novel
repellents against mosquito vectors from nontoxic
plant sources that are biodegradable and safe for
humans and the environment is continued by several
researchers (Wedge et al. 2006, Tabanca et al. 2013,
Cantrell et al. 2016).
Carrot (Daucus carota sativus L.) is a biennial root
vegetable that belongs to the family Apiaceae. In
addition to its use as a vegetable, carrot seeds have
medicinal properties and are used in many ways to
cure certain medical conditions in humans, while
their essential oils have multiple uses including the
treatment of skin conditions (Sumit et al. 2012).
Carotol is the major compound present in carrot seed
essential oils (Ozcan and Chalchat 2007). In this
study we report the chemical composition of carrot
seed essential oil and document its repellent activity
as well as its major component, carotol, against adult
Ae. aegypti,Ae. albopictus, and Anopheles quad-
rimaculatus Say.
MATERIALS AND METHODS
Pure therapeutic essential oils of carrot were
purchased from Edens Garden (1322 Calle Avanza-
do, San Clemente, CA). Deet was purchased from
Sigma-Aldrich (St. Louis, MO). Thin-layer chroma-
tography (TLC) plates were purchased from Merck
(Darmstadt, Germany) and silica gel 60 for column
chromatography (CC) were obtained from Silicycle
(Quebec, Canada).
Spectroscopic analysis of carrot oil and carotol
Proton nuclear magnetic resonance (NMR) and
carbon-13 NMR spectra of carrot essential oil were
measured using a 400-MHz Bruker NMR spectrom-
eter (Brucker, Billerica, MA). In addition, gas
chromatography/flame ionization detector (GC/FID)
and gas chromatography/mass spectrometry (GC/
MS) analyses were performed on this material with a
Varian CP-3380 (Varian, Walnut Creek, CA) and
Thermo Finnegan Trace, respectively. Both instru-
272
Journal of the American Mosquito Control Association, 34(4):272–280, 2018
Copyright Ó2018 by The American Mosquito Control Association, Inc.
ments were equipped with autosamplers. The tem-
perature programming, column dimensions, detector
types, and other experimental parameters were
followed after Wanas et al. (2016). Column chro-
matographic separations were performed on silica gel
60 (0.04–0.063 mm; Silicycle) for the column. Thin-
layer chromatography was carried out on precoated
silica gel TLC plates 60 F254 (0.2 mm; Merck),
using n-hexane, ethylacetate (EtOAc) (95:5) as the
TLC developing system.
Bioassay-guided fractionation, using 700 mg of
carrot essential oil, was performed on silica gel 60
(0.04–0.063 mm; Silicycle) eluted with EtOAc with
increasing polarity to 5%. In addition, TLC was
carried out on precoated silica 60 F254 gel plates
(0.2 mm), using EtOAc (95:5) as the TLC
developing system. Carrot seed essential oil (6.0
g) was subjected to silica gel CC, and 7 fractions
were collected, concentrated under vacuum, and
screened for biting deterrent activity. The active
fraction was rechromatographed on silica gel CC
and isocratically eluted with 2% EtOAc/hexane to
yield 3.51 g of an active compound identified as
carotol.
Insects
Adults of Ae. aegypti,Ae. albopictus, and An.
quadrimaculatus used in these studies were from the
laboratory colonies maintained at the Mosquito and
Fly Research Unit at the Center for Medical,
Agricultural and Veterinary Entomology, USDA-
ARS, Gainesville, FL. For biting deterrence and
repellent bioassays, the larvae hatching from the eggs
were reared to adults in the laboratory maintained at
27 628C and 60 610% RH with a photoperiod
regimen of 12 h light and 12 h dark. Adult females 8–
18 days old were used in these bioassays.
General methodology
Both in vitro and in vivo bioassays were
conducted to determine the repellent activity of
carrot seed essential oil, its fractions, carotol, and
deet.Ininvitrobioassays,afeedingsolution
consisting of citrate phosphate dextrose adenine-1
(CPDA-1) and adenosine triphosphate (ATP) was
used instead of blood as described by Ali et al.
(2012). Details of the feeding solution (CPDA-1 þ
ATP and green fluorescent tracer dye), experimen-
tal procedures, and data collection were followed
after Ali et al. (2012, 2015). These bioassay
systems are based on the concept that mosquitoes
are attracted to warm temperatures. The tempera-
ture of feeding solution in the reservoirs was
maintained at 378C by continuously passing the
warm water through the reservoir using a circula-
tory bath. All the treatments were freshly prepared
in molecular biology–grade 100% ethanol (Fisher
Scientific Chemical Co., Fairlawn, NJ) at the time
of bioassay. In in vitro Ali and Khan (A&K)
bioassay, a series of dosages were tested to achieve
1% biting as the minimum effective dose (MED).
Two hundred (65%) female mosquitoes were
transferredinto30330 330-cm test cages using
an aspirator (John W. Hock Company, Gainesville,
FL). To ensure proper landing and biting, 3–4 cages
were used at a time and only 1 treatment replication
of individual samples was completed in a single
cage. A total of 5–10 replicates were completed in
each bioassay.
For in vivo bioassays, 500 (65%) female
mosquitoes were transferred into 45 345 345-cm
test cages using an aspirator. The test started when
the arm or hand with treated muslin cloth or direct
skin application was inserted into the mosquito
cage. After 1 min of exposure, the hand was gently
shaken and the number of biting females (feeding
females do not fly) was recorded. Any treatment
with 5 females biting during 1 min of exposure
was considered as passed, whereas a treatment with
.5 bites out of 500 mosquitoes was considered as
failed. A next lower or higher serial dose was tested
to reach the MED. Residual repellent activity was
determined by exposing the females to the treated
muslin cloth or treated skin at an interval of 30 min.
Solvent control treatment was tested first to observe
the response of the mosquitoes. The test was started
only if .20 females successfully landed in 20 sec in
solvent control. The caged mosquitoes were tested
against ethanol control after every 5 successive
exposures to determine the response of the mosqui-
toes. A protocol approved by the University of
Mississippi Human Use Institutional Review Board
(IRB protocol no. 15-070) was followed in both the
in vivo bioassays.
In vitro Klun and Debboun biting deterrent
bioassay
Bioassays were conducted using a 6-celled in
vitro Klun and Debboun (K&D) module bioassay
system developed by Klun et al. (2005) for
quantitative evaluation of biting deterrence. Briefly,
the assay system consists of a 6-well reservoir with
each of the 3 34-cm wells containing 6 ml of
feeding solution. The reservoirs were covered with
a layer of collagen membrane (Devro, Sandy Run,
SC). The test compounds were applied to 6 4 35-
cm marked areas of organdy cloth (G Street
Fabrics, Rockville, MD) and positioned over the
collagen-covered feeding solution. The K&D mod-
ule containing 5 female mosquitoes per cell was
positioned over treated organdy and the trap doors
were opened to expose the females to the treatment.
The number of mosquitoes biting through the
treated organdy in each cell was recorded after a
3-min exposure. Sets of 5 replications each with 5
females per treatment were conducted on 2–3
different days, using a newly treated organdy and
a new batch of mosquitoes in each replication.
Proportion not biting (PNB) was calculated using
the following formula:
DECEMBER 2018 273
REPELLENCY OF CARROT SEED ESSENTIAL OIL AND CAROTOL AGAINST MOSQUITOES
PNB ¼1Total number of females biting
Total number of females
:
In vitro A&K repellent bioassay
Bioassays were conducted using the A&K bioas-
say system developed by Ali et al. (2017b) for
quantitative evaluation of repellency against mosqui-
toes. Briefly, the bioassay system consists of a 30 3
30 330-cm collapsible aluminum cage having 1
panel of clear transparent acrylic sheet with 120 3
35-mm slit through which the blood box containing a
removable feeding device was attached. The top of
the blood box had a sliding door used to expose
female mosquitoes to treatment during the bioassay.
Rectangular areas of either 3 34cmor437.5 cm
were marked on the collagen sheet that matched the
measurement of the rectangular liquid reservoirs.
Treated collagen was secured on the feeding
reservoir containing the feeding solution using a thin
layer of grease. The feeding device was then pushed
inside the blood box and the sliding door was opened
to expose the insects to the treatment. The number of
females biting through the treated collagen during a
1-min exposure was recorded.
In vivo cloth patch bioassay
Cloth patch bioassays were conducted by using an
in vivo bioassay system described by Katritzky et al.
(2010) and Ali et al. (2017a). A piece of muslin cloth
measuring 8 313 cm with a 4 37.5-cm marked area
in its center was used. Approximately 2.5 37-cm
pieces of cardboard were stapled onto the sides of the
muslin cloth to secure the treated cloth on the plastic
sleeve by using an adhesive tape. Each volunteer’s
hand was covered with a soft-embossed long-cuff
poly glove and a powder-free latex glove (Diamond
Grip; Microflex Corporation, Reno, NV) followed by
a knee-high stocking (L’eggs Everyday Knee Highs;
Hanes, Winston-Salem, NC). A polyvinyl sleeve with
437.5-cm opening cut halfway between the wrist
and the elbow was placed around the arm with
Velcro strips. The treatment was applied to the
marked area by using a pipette, and the treated
muslin cloth was secured on the plastic sleeve. The
arm was inserted into the cage, and data were
recorded after 1 min of exposure. This opening
permitted attractive odors from the skin surface to
emanate out and attract mosquitoes.
In vivo direct skin application bioassay
Direct skin application bioassays were conducted
using a powder-free latex glove (Diamond Grip;
Microflex Corporation). An opening of 3 34 cm was
cut through the glove to fit on the dorsal surface of
the hand. After wearing the glove, a wristband was
used to prevent the biting on the hand near the border
of the glove. Marked skin surface was treated with
the test compound in a volume of 50 ll of ethanol.
The treated hand was inserted into the cage and data
on biting were recorded after 1 min of exposure.
Statistical analysis
Data on the PNB were analyzed using SAS Proc
ANOVA (SAS Institute, Inc., Cary, NC), and means
were separated using Ryan–Einot–Gabriel–Welsch
multiple range test. Means and standard errors of
MED values were calculated using SAS Proc Means
(SAS Institute, Inc., Cary, NC) or Microsoft Excel
2010 (Microsoft Corporation, Redmond, WA).
RESULTS
In the initial screening, the essential oil with PNB
value of 0.84 provided biting deterrent activity
similar to deet (PNB ¼0.85) in K&D bioassays.
Therefore, we selected carrot seed essential oil for
further investigation.
The GC/FID and GC/MS analysis of carrot seed
essential oil revealed the presence of 47 compounds,
mainly mono- and sesquiterpenes (Table 1). Carotol
was .75% (w/w), followed by muurolene (4.86%),
(Z)-b-farnesene (2.9%), and diepicedrene (1.1%). Of
the 7 fractions of carrot seed oil (6.0 g) obtained
through silica gel CC, fraction 3 appeared to be the
most active fraction. The GC/FID and GC/MS
analysis indicated that fraction 3 contained .95%
of carotol (Fig. 1), which was isolated as a colorless
oil. The identity of carotol was confirmed by NMR
analysis and by comparing its spectroscopic data to
those reported by Jasicka-Misiak et al. (2004).
In vitro K&D biting bioassay
Data on the biting deterrent activity of the carrot
seed essential oil, fractions, and carotol against
different species of mosquitoes are given in Table
2. Carrot seed essential oil at 10 lg/cm
2
showed a
biting deterrent activity similar to deet at 4.8 lg/cm
2
.
The results indicated that the biting deterrent activity
of fraction 3 with a PNB value of 0.72 was similar to
that of deet. Carotol, which was the major compound
in this fraction, resulted in a PNB value of 0.9 at 10
lg/cm
2
and was similar to deet (PNB ¼0.85) at 4.8
lg/cm
2
. However, the biting deterrent activity of
carotol at 5 lg/cm
2
, with PNB value of 0.66, was
slightly lower than deet. Carrot seed essential oil and
carotol were also tested against An. quadrimaculatus.
Results from carrot seed essential oil and carotol with
PNB values of 0.84 and 0.78 at 10 lg/cm
2
showed
activity similar to deet. The biting deterrence of
carotol at 5.6 lg/cm
2
, with PNB value of 0.72, was
also similar to deet at 4.8 lg/cm
2
.
In vitro A&K repellency bioassay
In A&K bioassay with treated surface area of 30
cm
2
, MED of deet, carrot essential oil, and carotol
against Ae. aegypti was 25 lg/cm
2
(Table 3). The
274 VOL. 34, NO.4
JOURNAL OF THE AMERICAN MOSQUITO CONTROL ASSOCIATION
mixture of deet with either the essential oil or carotol
showed MED value of 12.5 lg/cm
2
(6.25 þ6.25 ¼
12.5 lg/cm
2
), which is half the dose of individual
compounds. Amounts of the individual components
in the mixture were one-fourth of the MED value of
individual compounds, which indicated a synergistic
activity. In residual repellent activity bioassay, the
essential oil and carotol gave activity similar to deet
at 46.9 lg/cm
2
up to 120 min posttreatment (Table
4). At 23.4 lg/cm
2
, deet showed full activity up to
120 min, whereas the carrot seed essential oil and the
carotol crossed the MED limit after 60 min
posttreatment. At 11.7 lg/cm
2
, deet was within range
of MED value up to 30 min posttreatment, whereas
the essential oil and carotol failed at this dose.
In vivo cloth patch repellent bioassay
Data on the repellent activity of carrot seed
essential oil and carotol are given in Table 5. The
MED value of carrot seed essential oil and carotol
was higher (25 lg/cm
2
) than deet (12.5 lg/cm
2
)
against Ae. aegypti. The MED value of deet and
carotol was 12.5 lg/cm
2
against Ae. albopictus,
whereas the MED value of carotol was lower than
Ae. aegypti (25 lg/cm
2
). The MED value of deet and
carotol (6.25 lg/cm
2
) against An. quadrimaculatus
was lower than Ae. aegypti (deet ¼12.5 and carotol ¼
25 lg/cm
2
, respectively) or Ae. albopictus (MED ¼
12.5 lg/cm
2
).
In cloth patch bioassay carrot seed essential oil and
carotol showed residual repellency similar to deet up
to 120 min posttreatment at the dosages of 50 and 25
lg/cm
2
(Table 6) against Ae. aegypti, whereas only
deet showed activity at 12.5 lg/cm
2
.
Table 1. Major components of carrot seed essential oil from gas chromatography analysis.
Component Area (%) Rt (min) m/z Class
a-pinene 0.82 7.8 136 Monoterpene
2(10)-pinene 0.46 9.4 136 Monoterpene
b-pinene 0.40 9.8 136 Monoterpene
Limonene 0.75 11.4 136 Monoterpene
Anisole 0.37 23.4 148 Monoterpene
Muurolene 4.86 27.5 204 Sesquiterpene
Cryophyllene 0.5 29.4 204 Sesquiterpene
(z)-b-farnesene 2.9 33.2 204 Sesquiterpene
Diepicedrene 1.1 35.6 222 Sesquiterpene
Carotol 75.6 37.56 222 Sesquiterpene
Daucol 2.0 38.9 238 Sesquiterpene
Total area 89.47
Fig. 1. Chemical structure of carotol.
Table 2. Biting deterrent activity of deet (N,N-Diethyl-3-
methylbenzamide), carrot seed essential oil, and carotol
against different species of mosquitoes in Klun and
Debboun bioassay.
Treatment N
1
PNB 6SEM
2
Aedes aegypti 75 0.85 60.04 A
Experiment no. 1
Deet 4.8 lg/cm
2
Carrot seed essential
oil 10 lg/cm
2
75 0.84 60.05 A
Ethanol 75 0.36 60.06 B
Experiment no. 2
(Fractions [F])
25 0.84 60.04 AB
Deet 4.8 lg/cm
2
Carrot seed essential
oil 10 lg/cm
2
25 0.88 60.05 A
F-1 10 lg/cm
2
25 0.64 60.04 BC
F-2 10 lg/cm
2
25 0.60 60.06 BC
F-3 10 lg/cm
2
25 0.72 60.05 ABC
F-4 10 lg/cm
2
25 0.52 60.08 CD
F-5 10 lg/cm
2
25 0.48 60.05 CD
F-6 10 lg/cm
2
25 0.52 60.05 CD
F-7 10 lg/cm
2
25 0.60 60.06 BC
Ethanol 25 0.32 60.05 D
Experiment no. 3 75 0.85 60.04 A
Deet 4.8 lg/cm
2
Carotol 10 lg/cm
2
75 0.90 60.03 A
Carotol 5 lg/cm
2
75 0.66 60.03 B
Ethanol 75 0.29 60.05 C
Anopheles quadrimaculatus
Deet 4.8 lg/cm
2
50 0.84 60.03 A
Carrot seed oil 10 lg/cm
2
50 0.84 60.04 A
Carotol 10 lg/cm
2
50 0.78 60.04 A
Carotol 5.6 lg/cm
2
50 0.72 60.04 A
Ethanol 50 0.1860.02 B
1
N, number of females tested.
2
PNB, mean proportion of females not biting; SEM, standard
error of the mean. Means within a column in an experiment not
followed by the same letter are significantly different (Ryan–Einot–
Gabriel–Welsch multiple range test P0.05).
DECEMBER 2018 275
REPELLENCY OF CARROT SEED ESSENTIAL OIL AND CAROTOL AGAINST MOSQUITOES
In vivo direct skin application bioassay
In direct skin application bioassay, both the
essential oil and carotol at 8%, 12.5%, 25%, and
50% application rates were active and reached to the
MED values at 1.5, 1.5, 2, and 2.5 h postapplication,
respectively (Table 7).
Data on the repellent activity of mixtures of deet
with carrot seed essential oil and carotol against Ae.
aegypti are shown in Table 8. These mixtures of deet
with the essential oil and carotol showed very
promising results. All the mixtures were active,
showing a substantial increase in residual repellent
activity at lower dosages when compared with
various treatments used alone. Deet at 1% rate of
application crossed the MED level at 1 h postappli-
cation and with the addition of 1% carrot essential oil
or carotol residual activity increased by 100% against
Ae. aegypti. There was a 33% increase in residual
activity when the activity of 1 þ1¼2% mixtures was
compared with 2% of deet alone. Addition of 2%
essential oil or carotol in 2% deet increased the
residual repellent activity by 100% (1.5 h), whereas
this increase was 25% when the mixture was
compared with the MED value of 4% deet. Mixture
of 4% of the essential oil or carotol with 4% deet
increased residual activity by 175% (3.5 h) when
compared with the MED value of 4% deet, whereas
the residual repellent activity of this mixture was
similar to 8% application rate of deet. Mixture of
8.3% of the essential oil or carotol with 4.2% deet
increased the residual activity by 225–250% (4.5–5
h), 30–40% (1.5–2 h), and 0%, when compared with
4%, 8%, and 12.5% application rate of deet,
respectively.
DISCUSSION
Many researchers have reported the composition
of carrot oil. Ozcan and Chalchat (2007) reported
Table 3. Repellant activity of deet (N,N-Diethyl-3-methylbenzamide) and combinations with carrot oil (C) and carotol
(F) against Aedes aegypti in an in vitro Ali and Khan (A&K) bioassay. Values show the percentage (mean 6SEM) of
females biting out of 200 in the cage.
1
Product
2
N
3
Dose (lg/cm
2
)
50 25 12.5 6.25
Compound
Deet 15 0 0.53 60.08 .1.1
C 15 0 0.23 60.08 .1.1
F 15 0 0.43 60.08 .1.1
25 þ25 ¼50 12.5 þ12.5 ¼25 6.25 þ6.25 ¼12.5 3.12 þ3.13 ¼6.25
Mixture
Deet þC 15 0 0.13 60.06 0.57 60.07 .1
Deet þF 15 0 0.1 60.05 0.63 60.08 .1
1
Minimum effective dose is 1% biting, which was 2 females out of 200 in the cage.
2
The data are from A&K bioassay using 30-cm
2
treated surface area.
3
N, number of replications.
Table 4. Residual repellent activity of deet (N,N-Diethyl-3-methylbenzamide), carrot seed essential oil, and carotol
against Aedes aegypti females at different dosages in an in vitro Ali and Khan (A&K) bioassay. Values show the
percentage (mean 6SEM) of females biting out of 200 in the cage.
1
Compound by dose N
2
Time after treatment (min)
0 30 60 90 120
46.9 lg/cm
2
Deet 15 00000
Carotol 15 0 0 0 0.03 60.03 0.03 60.03
Carrot seed essential oil 15 0 0.07 60.05 0.13 60.06 0.33 60.06 0.8 60.1
23.4 lg/cm
2
Deet 15 0 0.0 60.0 0.0 60.0 0.03 60.03 0.43 60.07
Carotol 15 0 0.4 60.05 0.57 60.08 .1.1
Carrot seed essential oil 15 0.03 60.03 0.16 60.06 0.43 60.07 .1.1
11.7 lg/cm
2
Deet 15 0.26 60.07 0.53 60.08 .1.1.1
Carotol 15 0.1 60.05 .1.1.1.1
Carrot seed essential oil 15 0.87 60.05 .1.1.1.1
1
Minimum effective dose (MED) is 1% biting, which was 2 females out of 200 in the cage. Data are from A&K bioassay using 12-cm
2
treated surface area. Residual repellent activity based on MED is 46.9 lg/cm
2
in all treatment up to 120 min, whereas at 23.4 lg/cm
2
carotol and carrot seed essential oil crossed MED levels after 60 min posttreatment. Ethanol was regularly tested at the beginning and after
every 5 replications as solvent control. The bioassays were continued only if the ethanol treatment failed (feeding 1%).
2
N, number of replications.
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Table 5. Repellent activity of deet (N,N-Diethyl-3-methylbenzamide), carrot seed essential oil, and carotol against 3
different species of mosquitoes in an in vivo ‘‘cloth patch’’ bioassay. Values show the percentage (mean 6SEM) of
females biting out of 500 in the cage.
1
Compound N
2
Dose (lg/cm
2
)
50 25 12.5 6.25 3.125
Aedes aegypti
Deet 25 0.02 60.01 0.14 60.03 0.5 60.04 .1—
Carotol 25 0.13 60.03 0.53 60.04 .1——
Carrot seed oil 25 0.24 60.03 0.57 60.03 .1——
Aedes albopictus
Deet 5 — 0 0.23 60.05 .1—
Carotol 5 — 0.16 60.06 0.65 60.05 .1—
Anopheles quadrimaculatus
Deet 5 — 0 0.08 60.05 0.32 60.12 .1
Carotol 5 — 0 0.12 60.08 0.36 60.12 .1
1
Minimum effective dose (MED) is 1% biting, which was 5 females out of 500. The MED in deet was 12.5 lg/cm
2
whereas comparable
MED of carotol and the carrot seed essential oil was 25 lg/cm
2
. Data are based on 25 replications for 50 and 25 lg/cm
2
treatments,
respectively. Ethanol was used as solvent control, which showed heavy landing and biting in this bioassay (feeding 1%).
2
N, number of replications.
Table 6. Residual repellent activity of deet (N,N-Diethyl-3-methylbenzamide), carrot seed essential oil, and carotol
against Aedes aegypti females in an in vivo ‘‘cloth patch’’ assay. Values show the percentage (mean 6SEM) of females
biting out of 500 in the cage.
1
Compound Dose (lg/cm
2
)N
2
Time after treatment (min)
0 306090120
Deet 50 5 0.04 60.04 0.08 60.05 0.16 60.04 0.28 60.05 0.28 60.05
Carotol 50 5 0.2 60.06 0.44 60.04 0.64 60.04 0.68 60.08 0.8 60.06
Carrot seed oil 50 5 0.2 60.06 0.28 60.05 0.44 60.09 0.6 60.06 0.72 60.05
Deet 25 5 0.08 60.05 0.16 60.04 0.36 60.04 0.48 60.05 0.48 60.05
Carotol 25 5 0.4 60.06 0.44 60.04 0.56 60.07 0.72 60.05 0.76 60.04
Carrot seed oil 25 5 0.52 60.05 0.6 60.0 0.64 60.04 0.72 60.05 0.92 60.5
Deet 12.5 5 0.52 60.1 .1.1.1.1
Carotol 12.5 5 .1
Carrot seed oil 12.5 5 .1
1
Minimum effective dose (MED) is 1% biting, which was 5 females out of 500 in the cage. Residual repellency based on MED is 50
and 25 lg/cm
2
in all treatments up to 120 min, whereas deet was also active at 0 min at 12.5 lg/cm
2
. Ethanol was regularly tested at the
beginning and after every 5 replications as solvent control. The bioassays were continued only if the ethanol treatment failed (feeding
1%).
2
N, number of replications.
Table 7. Residual activity of carrot oil (C) and carotol (F) in direct skin application bioassay against Aedes aegypti.
Values show the percentage (mean 6SEM) of females biting out of 500 in the cage.
1
Time (h)
Dose (% age)
8 12.5 25 50
CFCF C F C F
0 0 0 0 0.13 60.07 0 0 0 0
0.5 0.13 60.13 0.4 60.12 0 0.07 60.07 0 0 0 0
1 0.6 60.0 0.6 60.12 0.6 60.28 0.27 60.26 0.27 60.13 0.67 60.07 0 0.2 60.12
1.5 .1.1.1.1 0.93 60.07 .1 0.33 60.07 0.47 60.13
2.1.1.1.1.1.1 0.73 60.18 0.73 60.27
2.5 .1.1.1.1.1.1.1.1
1
Minimum effective dose is 1% biting, which was 5 females out of 500 in the cage. The data are the means of 3 replications done on
different days. Ethanol was regularly tested at the beginning and after every 5 replications as solvent control. The bioassays were continued
only if the ethanol treatment failed (feeding 1%).
DECEMBER 2018 277
REPELLENCY OF CARROT SEED ESSENTIAL OIL AND CAROTOL AGAINST MOSQUITOES
Table 8. Residual activity of deet (N,N-Diethyl-3-methylbenzamide) and combinations with carrot essential oil (C) and carotol (F) against Aedes aegypti in direct skin
application bioassay. Values show the percentage (mean 6SEM) of females biting out of 500 in the cage.
1
Time
(h)
Dose (% age)
1.0 (1 þ1¼2) 2.0 (2 þ2¼4) 4.0 (4 þ4¼8) 8.0 (4.2 þ8.3 ¼12.5) 12.5
Deet Deet þC Deet þF Deet Deet þC Deet þF Deet Deet þC Deet þF Deet Deet þC Deet þF Deet
0 0.3 60.2000000000000
0.5 0.33 60.07 0 0 0.07 60.07 0 0 0 0 0 0 0 0 0
1.1 0.27 60.26 0.4 60.12 0.27 60.13 0.2 60.000000000
1.5 .1 0.53 60.29 .1.1 0.33 60.13 0.13 60.13 0.13 60.13 0 0 0 0 0 0
2.1.1.1.1 0.53 60.18 0.33 60.13 0.73 60.18 0 0 0 0 0 0
2.5 .1.1.1.1 0.6 60.12 0.4 60.0 .1 0 0 0 0.07 60.07 0.07 60.07 0
3.1.1.1.1.1.1.1 0 0 0 0.07 60.07 0.13 60.07 0
3.5 .1.1.1.1.1.1.1 0.07 60.07 0.27 60.07 0.13 60.13 0.07 60.07 0.27 60.07 0
4.1.1.1.1.1.1.1 0.2 60.12 0.4 60.12 0.0 60.0 0.07 60.07 0.33 60.07 0
4.5 .1.1.1.1.1.1.1 0.27 60.18 0.53 60.07 0.4 60.23 0.13 60.07 0.33 60.13 0
5.1.1.1.1.1.1.1 0.47 60.13 .1 0.73 60.3 0.2 60.12 0.47 60.07 0
5.5 .1.1.1.1.1.1.1 0.73 60.18 .1.1 0.27 60.07 0.73 60.18 0
6.1.1.1.1.1.1.1.1.1.1 0.33 60.07 0.87 60.13 0.13 60.07
6.5 .1.1.1.1.1.1.1.1.1.1 0.53 60.13 0.93 60.07 0.27 60.27
7.1.1.1.1.1.1.1.1.1.1 0.6 60.2 .1 0.53 60.24
7.5 .1.1.1.1.1.1.1.1.1.1.1.1.1
1
Minimum effective dose is 1% biting, which was 5 females out of 500 in the cage. The data are the means of 3 replications done on different days. Ethanol was regularly tested at the beginning and
after every 5 replications as solvent control. The bioassays were continued only if the ethanol treatment failed (feeding 1%) in this bioassay.
278 VOL. 34, NO.4
JOURNAL OF THE AMERICAN MOSQUITO CONTROL ASSOCIATION
carotol (66.8%), daucene (7.84%), (Z,Z)-a-faenesene
(5.86%), germacrene D (2.34%), trans-a-bergamo-
tene (2.41%), and b-selenene (2.2%) as the major
contents of the carrot seed essential oil, while the
seed oil contained carotol (30.6%), daucol (12.6%),
and copaenol (0.62%) as main constituents. Jasicka-
Misiak et al. (2004) reported 38.6% carotol in carrot
seed oil purchased from Augustus Oils Ltd. London,
with b-caryophyllene (10.66%), caryophyllene oxide
(4.34%), a-pinene (3.94%), and farnesene (3.35%) as
the other major contents. Mazzoni et al. (1999)
reported carotol as the major content (73.1%, 69.7%,
and 36.1%) in 3 commercial carrot seed essential oil
samples. The published data suggested that the
quantity of the major constituents of the carrot seed
essential oil can vary. This variation could have been
due to the variety, plant parts used, harvest timings,
geographical location as affected by the climatic
factors, genetic origin, and the way the samples were
prepared (Merghache et al. 2008).
The K&D bioassay is widely used as a tool to test
the biting deterrence of compounds against mosqui-
toes where deet is used as a positive control. As such,
many studies have reported data on deet whereas
biting deterrent activity of the carrot seed essential
oil and carotol is reported for the 1st time in the
present study. In most of the published studies, the
PNB value of deet is .0.8. Data on the biting
deterrent activity of deet in this study were similar to
the published literature (Klun et al. 2005, Ali et al.
2012).
The A&K bioassay is a newly developed bioassay
and data on deet are limited, whereas repellent
activity of carrot seed essential oil and carotol against
mosquitoes has not been reported in the literature.
Data from this study corroborate the findings of Ali et
al. (2017a), who reported an MED value of 18.7 lg/
cm
2
in a similar experimental setup.
In vivo cloth patch bioassay is widely used to test
repellency against mosquitoes where deet is used as a
positive control. As such, many studies have reported
the repellency of deet against different species of
mosquitoes, whereas data on repellent activity of
carrot seed essential oil and carotol are reported for
the 1st time. The MED reported in the literature
ranges from 6 to 23 lg/cm
2
as reported by Ali et al.
(2017a). Data from the cloth patch bioassay on deet
(MED ¼12.5 lg/cm
2
) in this study corroborates the
findings of Ali et al. (2017a) and Katritzky et al.
(2010), reporting MED values of 11.1 and 9 lg/cm
2
for Ae. aegypti, respectively.
Residual activity of deet (7 h) at 12.5% application
rates of deet in direct skin application bioassay
corroborates the findings of Logan et al. (2010), who
reported 100% protection for 6 h against Ae. aegypti
in arm in cage repellency trials. Witting-Bissinger et
al. (2008) reported .96% protection for 6 h at a dose
of 15%. Repellent activity of carrot seed essential oil
and carotol is reported for the 1st time. In this study,
the residual activity of carrot seed essential oil and
carotol lasted 1 h at a dose of 8% (Table 7) and deet
at 4% lasted for 2 h (Table 8). Residual activity of the
mixture of these natural products with deet at 8.3 þ
4.2%, respectively, synergistically increased from 1 h
to 6.5 h.
The carrot seed essential oil and carotol showed
excellent biting deterrent activity in all the bioassays.
The data on the mixtures of deet with the carrot seed
essential oil and carotol indicated a substantial
increase in residual repellency compared with the
essential oil, carotol, or deet alone. Data on
repellency of these natural products or their mixtures
with deet in direct skin application are very
promising, indicating good potential of these natural
products to be developed as effective repellents. This
is the 1st report on the biting deterrent and repellent
activity of carrot seed essential oil and carotol against
mosquitoes. Synergy of these natural products in
mixtures is a unique characteristic that makes them
strong candidates for development as commercial
repellents against mosquitoes alone or in blends.
Carrot seed essential oil is on the US Food and Drug
Administration’s Generally Recognized as Safe list
and is used commercially in cosmetic formulations.
A major commercial advantage of carrot seed
essential oil is that it is safe for skin use,
biodegradable, and commercially available. Carotol,
which is the major part (.75%) of the carrot seed
essential oil, can be economically extracted. Further
studies will be needed to test these natural products
under field conditions.
ACKNOWLEDGMENTS
This study was supported in part by USDA/ARS
grant No. 58-6066-6-043. We thank James J. Becnel
and Dan Kline, Mosquito and Fly Research Unit,
Center for Medical, Agricultural and Veterinary
Entomology, USDA-ARS, Gainesville, for supplying
mosquito eggs. We thank N. Tabanca, C. Cantrell,
and S. Duke, USDA-ARS, for their valuable
suggestions during the course of this study. We
thank J. F. Parcher, NCNPR, The University of
Mississippi for his critical review of the manuscript.
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