Beni-Suef University Journal of Basic and Applied Sciences xxx (2017) xxx–xxx

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Beni-Suef University
Journal of Basic and Applied Sciences
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Full Length Article

Adulticidal and repellent activities of some botanical oils against malaria

mosquito: Anopheles gambiae (Diptera: Culicidae)
Olajide J. Afolabi ⇑, Iyabo A. Simon-Oke, Oluwadoyinsolami O. Elufisan, Mobolanle O. Oniya
Department of Biology, Federal University of Technology Akure, Ondo State, Nigeria

a r t i c l e i n f o a b s t r a c t

Article history: The study is undertaken to determine the adulticidal efficacy and repellent activity of different solvent
Received 10 April 2017 extracts of Ocimum caninum, Ocimum gratissimum, Chromolaena odorata and Datura stramonium against
Received in revised form 23 August 2017 adult Anopheles gambiae. The extracts showed adulticidal activity at LC50 (0.82–4.58 mg/l) and LC90
Accepted 19 September 2017
(2.38–8.33 mg/l). The highest mortality was observed in Ocimum gratissimum and Datura stramonium
Available online xxxx
with the LC50 and LC90 values of 2.35 mg/l, 4.75 ml/l and 0.82 mg/l, 2.38 mg/l respectively. Meanwhile,
the lowest adulticidal activity was recorded in Chromolaena odorata with the lowest LC50 (4.52 mg/l)
Keywords:
and LC90 (8.32 mg/l). The repellent efficacy was determined against the mosquitoes at five concentrations
Botanical extracts
Adulticidal
viz 1.0, 2.0, 3.0, 4.0 and 5.0 mg/l under laboratory condition (28 ± 2 °C, 75 ± 5% RH, 12L: 12D photope-
Repellence riod). The results showed complete protections of 90 min for Ocimum caninum, 120 min for Ocimum
Anopheles gambiae gratissimum, 30 min for Chromolaena odorata and 120 min for Datura stramonium at 5 mg/l of the extracts
respectively. It was observed that D. stramonium and O. gratissimum provided highest protection
(120 min) against Anopheles gambiae while the least protection (30 s) was observed in Chromolaena odor-
ata at 5 mg/l. The results of the study showed that all the crude extracts with the exception of
Chromolaena odorata were potential botanicals for confirming protection on the exposed parts of human
body against Anopheles gambiae for at least 90 min and as such can be included in integrated vector man-
agement to reduce malaria infection in endemic areas.
Ó 2017 Production and hosting by Elsevier B.V. on behalf of Beni-Suef University. This is an open access
article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction chemicals and botanicals as larvicides (Akinkurolere et al., 2011),

Mosquitoes are regarded as public enemies because of their bit-
ing annoyance, noise nuisance, sleeplessness, allergic reactions and
disease transmission during their biting and feeding activities
(Onyido et al., 2008). Some mosquitoes that bite humans routinely
act as vectors for a number of infectious diseases affecting millions
of people per year (Molavi, 2003), others that do not routinely bite
humans, but act as vectors for animal diseases, may become disas-
trous agents for zoonosis of new diseases when their habitat is dis-
turbed especially by sudden deforestation. In most mosquito
species, only the female transmits human and animal diseases such
as malaria, yellow fever, dengue haemorrhagic fever, West Nile
virus and encephalitis (Soulsby, 1982). All these diseases cause
great suffering to man and livestock. They do not only cause high
mortality and morbidity in human and animal population but also
lead to economic loss (Service, 1986).
In the past, several control measures had been directed on
different stages of mosquito, these include the use of synthetic
⇑ Corresponding author.
E-mail address: ojafolabi@futa.edu.ng (O.J. Afolabi).
pupacides and adulticides (Bagavan et al., 2009; Akinneye and
Afolabi, 2014; Afolabi, 2016). Most insecticides have been noted
to have high mammalian toxicity which limits their success in vec-
tor control. In addition, total eradication of mosquito and mosquito
borne diseases remain difficult due to large number of mosquito
species and their varied ecology (Fang, 2010). Noteworthy, among
mosquito control methods is the use of long lasting insecticide
treated nets (LLINs) which has recorded high degree of success.
Meanwhile, this method has not been able to completely eradicate
mosquitoes in endemic communities because the treated nets are
often used indoor while most people in the developing countries
such as Nigeria are always bitten outdoors by female mosquitoes
especially during dry season when the weather is hot. Therefore,
in order to completely eradicate mosquito-borne diseases, inte-
grated control methods which include outdoor protection from
mosquito bites should be intensified such methods include insect
repellents which are good control measures for both indoor and
outdoor protection against sucking and biting insects. The use of
insect repellent compounds dates back to antiquity, when various
plant oils, smokes, tars etc. were used to displace or kill insects.
Insect repellents have generally been regarded to be safe because

https://doi.org/10.1016/j.bjbas.2017.09.004
2314-8535/Ó 2017 Production and hosting by Elsevier B.V. on behalf of Beni-Suef University.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Please cite this article in press as: Afolabi, O.J., et al. Adulticidal and repellent activities of some botanical oils against malaria mosquito: Anopheles gambiae
(Diptera: Culicidae). Beni-Suef Univ. J. Basic Appl. Sci. (2017), https://doi.org/10.1016/j.bjbas.2017.09.004
2 O.J. Afolabi et al. / Beni-Suef Univ. J. Basic Appl. Sci. xxx (2017) xxx–xxx
some of these repellents can be synthesized from edible plants
which are easily available in the tropics. The aim of this study is
to explore the adulticidal and repellent activities of different
solvent extracts of Ocimum caninum, Ocimum gratissimum,
Chromolaena odorata and Datura stramonium against adult female
Anopheles gambiae.
2. Materials and methods
2.1. Plant collection
The fully developed leaves of Ocimum caninum, Ocimum gratis-
simum, Chromolaena odorata and Datura stramonium were obtained
from Ilara-Mokin, Apatapiti, Oke-Aro and Redemption Estate,
Akure, Ondo State respectively. The plants were identified by a
plant taxonomist in the Department of Crop Science and Protection
of the Federal University of Technology Akure, Ondo State, Nigeria.
2.2. Extraction of oil from plant materials
The plant materials (leaves) were washed with tap water, shade
dried, cut into small pieces and air dried for 7 days in the labora-
tory. They were further pulverized into fine powders using an
industrial electric pulverizing machine at the Department of
Animal Production and Health Laboratory of the Federal University
of Technology Akure. The powders were sieved into fine powder
and kept in an air-tight plastic containers for storage before use
at ambient temperature (28 ± 2) °C.
Hundred grams (100 g) each of the pulverized materials was
weighed into thimble and their oils were extracted using absolute
ethanol while Datura oil was extracted using petroleum ether. The
oils were extracted using soxhlet extractor. Thereafter, the thimble
was removed from the unit and the ethanol and ether were recov-
ered by redistilling the contents using rotary evaporator and then
air dried to remove any trace of solvent. The oil was kept in labeled
plastic bottles till when needed.
2.3. Insect culture
Clean transparent white bucket was filled with water and 10 g
of yeast (Bakers’ yeast) dissolved in the water which was then
placed behind Biology Laboratory, School of Science, Federal
University of Technology Akure, Ondo State, Nigeria. The water
was monitored for 3–5 days for the development of the egg/first
instar larva. These larvae were taken into the laboratory. In the lab-
oratory, the larvae were identified into species level using the mor-
phological keys (Gillies and De Meillon, 1968). The An. gambiae
larvae were separated from the mixed culture and transferred into
another plastic container containing water. The An. gambiae larvae
were nurtured for 6–7 days before they were transferred into a
cage (54
54 square cm) where they were nurtured into adults
by feeding them with Baker’s yeast. Adult mosquitoes that
emerged were provided with 10% sucrose solution and were peri-
odically blood fed on restrained 5–7 weeks old chick. These adult
mosquitoes were maintained at 28 ± 2 °C, 75 ± 5% relative humid-
ity, photoperiod of 12 h light followed by 12 h dark (12L:12D).
2.4. Adulticidal activity
Five-six day old sugar-fed mosquitoes were used for the adulti-
cidal experiment. All the plant extracts with the exception of Dat-
ura stramonium were diluted with ethanol while D. stramonium
was diluted using petroleum ether. The dilution was made into
1.0, 2.0, 3.0, 4.0 and 5.0 mg/cm2 and impregnated on filter papers
(140
120 mm). Two blank papers one soaked with ethanol and
Please cite this article in press as: Afolabi, O.J., et al. Adulticidal and repellent activities of some botanical oils against malaria mosquito: Anopheles gambiae
(Diptera: Culicidae). Beni-Suef Univ. J. Basic Appl. Sci. (2017), https://doi.org/10.1016/j.bjbas.2017.09.004
the other soaked with ether were used as controls. The bioassay
for the experiment was conducted using a standard protocol of
(WHO, 2006). Twenty (20) sucrosed–fed and blood starved mos-
quitoes were released into the tubes containing the impregnated
papers. Subsequently, adult mortality was observed every 6 h
interval for 24 h.
2.5. Repellency test
The essential oil was evaluated for their repellent activities
against Anopheles gambiae mosquito using the human-bait tech-
niques adopted from (WHO, 2009; Marimuthu and Rajamohan,
2011). First, the essential oil was diluted in ethanol and ether as
applicable and prepared to 1–5 mg/cm2 concentrations. For each
test, 100 disease free, laboratory-reared female mosquitoes emerge
from the culture medium in the laboratory cage (45
30
45 cm)
were used. Before the commencement of the experiment, volun-
teers consent was received after explaining the procedures and
benefits of the study to them. Four volunteers were used for each
test concentrations. For each test concentration volunteer’s skin
was washed with unscented soap and the tested essential oil was
applied from the elbow to the wrist covering the fingers with a
gloves. The test was carried out during the night hours (19.00–
05.00 h) when the female mosquitoes were active. The control
(washed with ethanol) and treated arms (rub with crude extracts)
were introduced simultaneously into the experimental cages, and
the sides of the cages were gently tapped to activate the mosqui-
toes. Each test concentration was repeated 5 times. The mosqui-
toes that landed on the arm and the time of landing were
recorded. Percentage repellency was calculated using the formula
(Marimuthu and Rajamohan, 2011).
% Repellency ¼ ½ðTa  TbÞ=Ta
100
where
Ta = Number of mosquitoes in the control group
Tb = Number of mosquitoes in the treated group
2.6. Statistical analysis
The LC50 and LC90 were calculated using the probit values of the
average mortality. The significance of the results was calculated
using one-way Analysis of Variance (ANOVA) and the means were
separated using Turkey’s test at 0.05% level of significance. Results
with P < 0.05 were considered to be statistically significant. All
analyses were done using SPSS 20.0 version software.
3. Results
The results of the adulticidal efficacy of Ocimum caninum, Oci-
mum gratissimum, Chromolaena odorata and Datura stramonium
against adult Anopheles gambiae mosquito as presented in Table 1
and Table 2 revealed that among the crude extracts, the highest
mortality was observed in Ocimum gratissimum and Datura stramo-
nium with the LC50 and LC90 values of 2.35 mg/l, 4.75 ml/l and
0.82 mg/l, 2.38 mg/l respectively. Meanwhile, the lowest adultici-
dal activity was recorded in Chromolaena odorata with the lowest
LC50 (4.52 mg/l) and LC90 (8.32 mg/l). The results show that the
most effective of the extracts on the adult mosquitoes is Datura
stramonium while the least effective extract is the Chromolaena
odorata. It was further observed that all the adult mosquitoes
exposed to Ocimum gratissimum and Datura stramonium extracts
were killed at 5.0 mg/l and 3.0 mg/l at 24 h of exposure respec-
tively. In contrast, not all the adult mosquitoes exposed to Chromo-
laena odorata and Ocimum caninum extracts were killed, these
 O.J. Afolabi et al. / Beni-Suef Univ. J. Basic Appl. Sci. xxx (2017) xxx–xxx 3

Table 1 extracts could only achieved 60% and 75% mortality at 24 h of

Adulticidal activity of crude extracts of Ocimum caninum and Ocimumgratissimum exposure respectively.
against Anopheles gambiae.
In addition, the extracts were also observed to show significant
Extracts Concentration (mg/l) % Mortality ± SD LC50 (mg/l) LC90 (mg/l) repellent activity against Adult An. gambiae s.l (Tables 3 and 4). All
Ocimum caninum the plant extracts gave protection against mosquito bites at vary-
Control 0.00e±0.00 3.38 7.15 ing time of exposure without any allergic reaction to the human
1.0 15.00d±0.3 subjects. Chromolaena odorata was observed to protect the exposed
2.0 35.00d±0.8
3.0 45.00c±1.2
arms for 30 min, Ocimum caninum protected the exposed arms for
4.0 60.00b±0.6 90 min, while Ocimum gratissimum and Datura stramonium pro-
5.0 75.00b±0.7 tected the exposed arms for 120 min. This shows that all these
Ocimum gratissimum extracts with the exception of Chromolaena odorata can be used
Control 0.00e±0.00 2.35 4.73 as repellent for both indoor and outdoor protection of skin against
1.0 35.00d±0.7 mosquito bites. The repellent activity of the plant extracts was
2.0 50.00c±1.2
observed to increase as the strength of the extracts increases.
3.0 60.00b±0.5
4.0 75.00b±1.7
5.0 100.00a±0.3
4. Discussion

Table 2
From the result obtained, the study showed that the essential
Adulticidal activity of crude extracts of Chromolaena odorata and Datura stramonium oils from the plant extracts used have adulticidal and repellent
against Anopheles gambiae. effects on the An. gambiae mosquitoes. The increase in the protec-
Extracts Concentration % Mortality ± SD LC50 LC90
tion time of the oil extract at higher concentrations of the extract
(mg/l) (mg/l) + SE (mg/l) + SE may be due to the increase in the concentration of the active ingre-
dient present in the extracts. Plant extracts usually contain alka-
Chromolaena odorata
Control 0.00e ± 0.00 4.52 ± 0.52 8.33 ± 1.12 loids, saponins, terpenoids and tanins which have insecticidal
1.0 10.00d ± 0.2 properties on insect vectors. The extracts were observed to show
2.0 25.00d ± 0.3 adulticidal activity at LC50 (0.82–4.58 mg/l) and LC90 (2.38–
3.0 30.00d ± 0.7
8.33 mg/l). Adulticidal activity of the plant extracts has been sup-
4.0 40.00b ± 1.2
5.0 60.00b ± 0.6
ported by the findings of (Marimuthu and Rajamohan, 2011). The
authors reported that solvent extracts of Eclipta alba and
Datura stramonium
Control 0.00e ± 0.00 0.82 ± 0.02 2.38 ± 0.05
Andrographis paniculata showed adult mortality at LC50 range
1.0 60.00b ± 0.3 (150.36–197.88 ppm), LC90 range (285.22–361.03 ppm) and LC50
2.0 75.00b ± 0.8 (130.19–174.20 ppm), LC90 (244.16–306.12 ppm) respectively.
3.0 100.00a ± 0.1 The plant extracts also showed varying degrees of repellent
4.0 100.00a ± 0.0
activities at different concentrations and time intervals. Among
5.0 100.00a ± 0.0
the four plants extracts, Datura stramonium and Ocimum gratissi-
SD = Standard deviation, SE = Standard error. mum were able to protect the skin for 120 min, while Chromolaena

Table 3
Repellent activities of different crude extracts against Anopheles gambiae.

Extracts Conc Time of post application of repellents (min)

(mg/l) 15 30 60 90 120 150 180
Ocimum caninum 1.0 100 ± 0.0 100 ± 0.0 100 ± 0.0 74.5 ± 0.1 69.4 ± 0.9 42.2 ± 0.5 40.0 ± 0.2
2.0 100 ± 0.0 100 ± 0.0 100 ± 0.0 100 ± 0.0 52.2 ± 1.2 45.6 ± 0.3 43.5 ± 0.2
3.0 100 ± 0.0 100 ± 0.0 100 ± 0.0 100 ± 0.0 54.4 ± 0.7 49.4 ± 0.6 44.7 ± 0.2
4.0 100 ± 0.0 100 ± 0.0 100 ± 0.0 100 ± 0.0 60.2 ± 0.5 50.6 ± 0.4 47.1 ± 1.3
5.0 100 ± 0.0 100 ± 0.0 100 ± 0.0 100 ± 0.0 69.4 ± 0.7 63.3 ± 0.2 56.0 ± 0.5
Ocimum gratissimum 1.0 100 ± 0.0 100 ± 0.0 100 ± 0.0 100 ± 0.0 69.3 ± 0.1 63.2 ± 0.4 57.9 ± 0.8
2.0 100 ± 0.0 100 ± 0.0 100 ± 0.0 100 ± 0.0 71.4 ± 0.5 69.4 ± 0.3 60.0 ± 1.3
3.0 100 ± 0.0 100 ± 0.0 100 ± 0.0 100 ± 0.0 100 ± 0.0 75.5 ± 0.6 73.5 ± 0.9
4.0 100 ± 0.0 100 ± 0.0 100 ± 0.0 100 ± 0.0 100 ± 0.0 84.4 ± 1.5 81.4 ± 0.2
5.0 100 ± 0.0 100 ± 0.0 100 ± 0.0 100 ± 0.0 100 ± 0.0 87.6 ± 0.8 84.5 ± 0.5

Table 4
Repellent activities of different crude extracts against Anopheles gambiae.

Extracts Conc Time of post application of repellents (min)

(mg/l) 15 30 60 90 120 150 180
Datura stramonium 1.0 100 ± 0.0 100 ± 0.0 100 ± 0.0 100 ± 0.0 89.4 ± 0.9 86.2 ± 0.5 83.0 ± 0.2
2.0 100 ± 0.0 100 ± 0.0 100 ± 0.0 100 ± 0.0 91.6 ± 1.2 87.4 ± 0.3 84.2 ± 0.2
3.0 100 ± 0.0 100 ± 0.0 100 ± 0.0 100 ± 0.0 100 ± 0.0 90.7 ± 0.6 87.6 ± 0.2
4.0 100 ± 0.0 100 ± 0.0 100 ± 0.0 100 ± 0.0 100 ± 0.0 92.8 ± 0.4 89.7 ± 1.3
5.0 100 ± 0.0 100 ± 0.0 100 ± 0.0 100 ± 0.0 100 ± 0.0 94.9 ± 0.2 90.8 ± 0.5
Chromolaena odorata 1.0 100 ± 0.0 63.3 ± 0.5 58.3 ± 0.2 50.6 ± 0.7 43.5 ± 0.2 37.5 ± 0.7 33.3 ± 0.2
2.0 100 ± 0.0 100 ± 0.0 59.8 ± 0.6 54.1 ± 0.1 46.7 ± 1.2 42.2 ± 0.3 35.3 ± 0.2
3.0 100 ± 0.0 100 ± 0.0 61.5 ± 0.1 56.9 ± 0.5 50.0 ± 0.3 45.6 ± 0.6 40.0 ± 0.2
4.0 100 ± 0.0 100 ± 0.0 64.9 ± 0.3 58.3 ± 0.2 52.2 ± 0.5 49.4 ± 0.4 44.7 ± 1.3
5.0 100 ± 0.0 100 ± 0.0 68.6 ± 0.9 63.5 ± 0.0 58.3 ± 0.7 50.6 ± 0.2 47.1 ± 0.5

Please cite this article in press as: Afolabi, O.J., et al. Adulticidal and repellent activities of some botanical oils against malaria mosquito: Anopheles gambiae
(Diptera: Culicidae). Beni-Suef Univ. J. Basic Appl. Sci. (2017), https://doi.org/10.1016/j.bjbas.2017.09.004
4 O.J. Afolabi et al. / Beni-Suef Univ. J. Basic Appl. Sci. xxx (2017) xxx–xxx
odorata and Ocimum canimum protected the skin for 30 mins and
90 min respectively. The repellent activities of botanicals against
malaria mosquitoes have also been reported by (Elango et al.,
2009). The authors reported that methanolic extracts of Aegle mar-
melos and Acacia lineata gave mean complete protection time ran-
ged from 90 to 120 min. The repellency activity of solvent
extracts of Eclipta alba and Andrographis paniculata has also been
reported to protect the skin for 150–180 min of exposure
(Marimuthu and Rajamohan, 2011). Ocimum gratissimum and Dat-
ura stramonium were able to protect the subjects for 120 min at
3–5 mg/l. Other plants in which repellency has been reported
include: Neem (Azadirachta indica), citronella grass (Ocimum basili-
cum, Ocimum americanum), Clove (Syzgium aromaticum), prickly
straggler (Solanum trilobatum), musk basil (Moschosma polysta-
chyum) (Chokechaijaroenporn et al., 1994; Suwonkerd and
Tantrarongraj, 1994; Rajkumar and Jebanesan, 2004, 2005) pepper-
mint and thyme (Thymus vulgaris) (Barnard, 1999). The results of
this study suggest that the plant extracts can be included in the
mosquito control programs for both indoor and outdoor protection
against mosquito bites.
5. Conclusion
The report has shown that the plant extracts used for this
research have good adulticidal and repellent properties against
malaria mosquitoes. The oil extracts from Datura stramonium and
Ocimum gratissimum can protect the skin for 120 min while Oci-
mum caninum can protect the skin for 90 min. Hence, the oils from
the plants can be used for both indoor and outdoor protections
against mosquitoes. The use of these potential repellents is eco-
friendly with no mammalian toxicity and as such can be included
in vector management to reduce the morbidity and mortality
caused by mosquito-borne diseases in tropical countries where
mosquito-borne diseases are endemic.
Conflicting interests
The authors declare that they have no competing interests.
Acknowledgements
The authors are grateful to department of crop protection for
the identification of the plants. We also appreciate Mrs E.T. Alade
for the extraction of the botanical oils.
Please cite this article in press as: Afolabi, O.J., et al. Adulticidal and repellent activities of some botanical oils against malaria mosquito: Anopheles gambiae
(Diptera: Culicidae). Beni-Suef Univ. J. Basic Appl. Sci. (2017), https://doi.org/10.1016/j.bjbas.2017.09.004
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