Repellent Effect of Extracts and Essential Oils of Citrus limon (Rutaceae) and Melissa officinalis (Labiatae) Against Main Malaria Vector, Anopheles stephensi (Diptera: Culicidae)

Abstract

Repellet effect of extracts and essential oils of Citrus limon (L.) Burm.F., (lemon) and Melissa officinalis, (balm) were evaluated against Anopheles stephensi in laboratory on animal and human and compared with synthetic repellent, N,N- diethyl-3-methylbenzamide (Deet) as a standard. Results of statistical analysis revealed significant differences between oils and extracts (P< 0.05) against the tested species, thus oils were more effective than extracts. There was no significant difference between Deet and lemon oil, whereas the difference between lemon and melissa oils was significant. Relative efficacy of lemon oil to Deet was 0.88 whereas it was 0.71 for melissa oil. The results were found marginally superior in repellency for animals than human. Due to advantages of botanic compounds to synthetic compounds we recommend lemon essential oil as an effective alternative to Deet with potential as a means of personal protection against mosquito vectors of disease.

Full text

MA Oshaghi et al: Repellent Effect…
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Repellent Effect of Extracts and Essential Oils of Citrus limon
(Rutaceae) and Melissa officinalis (Labiatae) Against Main
Malaria Vector, Anopheles stephensi (Diptera: Culicidae)
*MA Oshaghi1, R Ghalandari 2, H Vatandoost 1, M Shayeghi1, M Kamali-nejad 2, H Tourabi-Khaledi 2,
M Abolhassani1, M Hashemzadeh1
1Dept. of Medical Entomology, School of Public Health & Institute of Health Research , Tehran University of
Medical Sciences, Iran
2 School of Pharmacy, Shaheed Beheshti University of Medical & Health Sciences, Tehran, Iran
Abstract
Repellet effect of extracts and essential oils of Citrus limon (L.) Burm.F., (lemon) and Melissa officinalis, (balm) were
evaluated against Anopheles stephensi in laboratory on animal and human and compared with synthetic repellent, N,N-
diethyl-3-methylbenzamide (Deet) as a standard. Results of statistical analysis revealed significant differences between oils
and extracts (P< 0.05) against the tested species, thus oils were more effective than extracts. There was no significant
difference between Deet and lemon oil, whereas the difference between lemon and melissa oils was significant. Relative
efficacy of lemon oil to Deet was 0.88 whereas it was 0.71 for melissa oil. The results were found marginally superior in
repellency for animals than human. Due to advantages of botanic compounds to synthetic compounds we recommend
lemon essential oil as an effective alternative to Deet with potential as a means of personal protection against mosquito
vectors of disease.
Keywords: Repellent, Anopheles stephensi, Citrus limon, Melissa officinalis
Introduction
The repellent properties of plants to mosquitoes
and other pest insects were well known before
the advent of synthetic chemicals (1). However,
the most commonly used insect repellents are
synthetic chemicals that mostly have contained
Deet (N, N-diethyl-3-methylbenzamide) in their
formulations. Although Deet is an effective
repellent against a broad spectrum of insects,
however there are disadvantages associated
with the use of Deet, which stem principally
from its activity as a solvent of paints,
varnishes, and some plastic and synthetic
fabrics. There have also been concerns over
toxicity of Deet (2-4). A variety of botanical
substances have been evaluated for their
repellency against mosquitoes (5). In ancient
medicine Lemon citrus (Citrus limon Burm)
and melissa (Melissa officinalis L.) have long
been used as natural insect repellents in Iran
(personal observation) and the world (5-6).
These plants are cultivated widely in tropical,
subtropical, and temperate parts of the world
especially in parts of Iran and neighbouring
countries where malaria is endemic. In this
paper we report the repellent effects of extracts
and essential oils of lemon citrus and melissa in
comparison with a solution formulation of Deet
against An. stephensi Liston (Diptera:
Culicidae). This species is the main vector of
malaria in the Middle East, including Iran and
Indian sub continent.
Materials and Methods
Mosquitoes The mosquitoes of An. stephensi
obtained from a well-established laboratory
*Corresponding author: Tel: +98 21 6112606, E-mail: oshaghi@ lycos.com
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colony from School of Public Health & Institute
of Health Research, Tehran University of
Medical Sciences. Mosquitoes were reared and
maintained at 27±3o C and 80±10% Relative
Humidity (RH) under a 12:12 (L:D) photoperiod.
Larvae were reared on a diet of floating catfish
food. The adults were maintained in screen cages
on 10% sucrose solution but 24 hours before
experiments the sucrose solution was removed
from the cages. Repellency was tested against 3-
5-day-old, blood-starved mosquitoes, and for
each test 35 mosquitoes were used.
Repellent In this paper we compared the
efficacy of alcoholic extracts and oils of M.
officinalis and C. limon. They were identified
based on plant taxonomy keys by botanist of
Agriculture Department, Tehran University.
Mellisa cultivated in the farm of Karaj, Dept. of
Agriculture, affiliated to Tehran University and
its leaves were used to prepare extracts and oils.
Lemon fruits were purchased from the market.
Lemon oil was obtained from fresh skin and
extracts from whole dried fruit. The method of
maceration was used to prepare extracts from the
plants. They converted to powder and then 100 g
of powder was macerated with 1 litre ethanol.
After deletion of extra alcohol, 14 g and 12 g
lemon and mellisa extracts respectively obtained.
Essential oils were obtained from the plants using
the method explained earlier (7). Distillations
were performed by adding adequate double
distilled water to 100 g of the plants in Clevenger
apparatus. Using this protocol we were able to
produce 2 ml lemon oil and 0.3 ml mellisa oil.
The extract or oil products were obtained as
required and subsequently diluted to 20%
concentration. To prepare different
concentrations, the products were further
diluted using alcohol as diluent. Extract and
essential oil solutions were formulated on a
volume- volume basis at a concentration of 3%
and 1%, respectively. The compounds were
applied as 4-ml aliquots of ethanol solution and
were spread evenly over the animal skin or hands
of volunteer as explained previously (8, 9). After
a period of 30 min, one treated subject at a time
was exposed to 35 adult female mosquitoes for
30 minutes. Fifteen percent Deet in ethanol
solution was used as a standard to evaluate the
candidate compounds against the mosquito
species.
Experiments Two series of experiments were
carried out in laboratory condition. In the first
series, the extracts and essential oils were tested
on animal guinea pigs. The animals were
laboratory reared albino males with average 400-
450 g weight. A 4×6 cm of animal abdomen hairs
was removed then washed and cleaned by
ethanol. Treatments were 4 ml of either the
extracts containing 0.12 and 0.04 g of active
ingredients of extracts and oils respectively, Deet
(standard), or ethanol (control). After treatment,
the animal was bound on top of the cage in which
the treated position was exposed to mosquitoes
for thirty minutes. Each test was repeated three
times replacing new mosquitoes and new
animal, and number of bites through the fabrics
was recorded. The compounds, which revealed
significant efficacy, were selected for the second
series of tests. The second series of tests were
performed on the skin of volunteers. Based on
low repellent efficacy of extracts, only oils
were tested in second experiment. For these
evaluations, hands of volunteers from wrist to
tips of fingers were again treated with 4 ml of
either of the selected compounds (i.e. oils of
lemon and melissa), Deet, or ethanol and
exposed to mosquitoes in the cage. Twelve
subjects participated for three replicates, thus for
each replicate four subjects were treated by either
of mentioned compounds. To count number of
bites, all mosquitoes were aspirated from the
cage, anaesthetised with carbon dioxide and
crushed on paper to determine the number of
individuals that obtained blood meal. The number
of bites in treated and control tests in each trial
was recorded and the mean percentage protection
from bites was calculated. The percentage
protection, defined as the average number of bites
received by subjects in each trial relative to that

MA Oshaghi et al: Repellent Effect…
49
of control, was calculated as (control-
treatment)/control × 100. To compare the
repellent efficacy of the compounds we used a
paired t-test to determine if there were significant
differences at 5% level between the candidates
and between the candidates and the standard.
Results
Results of laboratory study on animals
comparing lemon extract, lemon oil, melissa
extract, melissa oil, and Deet against caged
population of A. stephensi are presented in table
1. Results showed that oils were significantly
more effective than extracts. Repellent efficacy
of both oils in animal tests was as similar as
Deet. Although Deet with 97% effectiveness
was more effective than lemon (92.70%) and
melissa (92.67%) oil, however, these
differences were not significant. The lemon
extract with the rate of 56% and melissa extract
with the rate of 66% were significantly less
protective than oils. Results of tests on human
skin comparing oils products and Deet are
summarised in table 2. Mean repellent efficacy
of Deet (80.5%) against A. stephensi was higher
than Melissa and Lemon oils (60.00 and
71.16% respectively), but the difference was
not significant in case of lemon oils (71.16%).
Melissa oil with 60% protective rate was
significantly less protective than lemon oil and
Deet (P<0.05).
Table 1: Relative repellent effectiveness of 3% extracts and 1% oils of lemon citrus and melissa against laboratory
mosquitoes of Anopheles stephensi on guinea-pigs in the laboratory
Repellent I
Replicate
II
Replicate
III
Replicate
Mean (%) ± SE Ratio to
Deet
Lemon extract 29.41 58.8 62.15 56.12 ± 18.01 0.58**
Lemon oil 95.90 90.00 92.00 92.70 ± 3.0 0.96
Melissa extract 71.42 83.3 74.2 76.31 ± 6.21 0.79**
Melissa oil 93.50 84.50 100 92.67 ± 7.78 0.95
Deet 97.00 100 94.00
97.00 ± 3.0 1
Control 0 0 0 0 0
* t- test of paired test data at 5% level of confidence, same letters indicate no significant difference
Table 2: Relative repellent effectiveness of 1% melissa and lemon citrus essential oils against laboratory mosquitoes of
Anopheles stephensi on hands of human skin
Repellent I
Replicate
II
Replicate
III
Replicate
Mean (%) ± SE Ratio to
Deet*
Melissa oil 63.50 59.50 57.00 60.00 ± 3.27 0.74**
Lemon oil 71.00 69.50 73.00 71.16 ± 1.76 0.88
DEET 82.00 80.00 79.50 80.50 ± 1.32 1
Control 0 0 0 0 0
* t- test of paired test data at 5% level of confidence, same letters indicate no significant difference
Discussion
The present study clearly showed that the
application of lemon citrus repellent
compounds gave acceptable percentage biting
protection against A. stephensi which is less
than but not significant with the percentage
protection seen in Deet under similar
conditions. No adverse effects on the skin or
other parts of the body of the human volunteers
were observed during the study period and
through one month after application. This study
showed that oils provide better protection than

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50
extracts; however it is known that extracts are
more stable than oils and could be kept for
longer time than oils. Secondly oils are more
prone to irritate human skin, and finally
extracts are less expensive to produce than oils.
Therefore it is recommended to improve the
existing methods to provide better quality
extracts. There are many reports indicated that
low concentrations of repellent materials are
attractant to mosquitoes (10-11). On the other
hand, many materials that are normally thought
as attractants have been reported to be repellent
at high concentrations (12-13). In the present
study, because of time limitation, a 3%
concentration of extracts were used but and it
would be worth to evaluate higher
concentrations of extracts. Also it is
recommended to test repellent efficacy of
individual components of the extracts to
identify the most effective ingredient.
Moreover these compounds have to be tested in
field condition to evaluate their efficacy and
effective durability. Between-species
differences in sensitivity to repellents have
been widely documented (14-15). In this work,
we have tested the repellents against only one
species and do not know if these compounds
are protective against other mosquito species or
medically important insects. However studies
on the efficacy of lemon citrus oils against
sandflies of Luzomyia youngi showed that this
compound gave higher protection than Deet
and Citronella (6). Also another study showed
that the essential oils of six plants, mainly
Citrus species, were highly toxic against insects
of Liposcelis bostrychopia (16). The repellents
of lemon citrus and melissa have a lemon smell
and unlike Deet, do not have undesirable
dissolving properties. Citronelol, the most
prevalent component alcohol, and linalool
which are parts of main active ingredients of
lemon and melissa in the distillate have been
among the main active ingredients of other
botanical repellents such as citrosa and
eucalyptus (17-21). It is known that different
cultivars may contain different components
with different characters at different places.
There are some reports on the local Iranian
cultivar components (22-27), however, it is
highly recommended to do further experiments
to identify which part or parts of these
components have got the most repellent or toxic
effect against insects. There are several studies
on the larvicidal activities of Iranian herbs
against A. stephensi. In these experiments
authors tested different essential oils of plants
against A.stephensi larvae and calculated LC50
values. They concluded that in some extent
these herbal extracts can be used for malaria
vectors control (28).
It is concluded that because of the simplicity of
production, cheapness, and protective activity,
we recommend lemon essential oil as an
effective alternative to Deet with potential as a
means of personal protection against mosquito
vectors of disease.
Acknowledgements
The authors would like to appreciate the kind
cooperation of Eng. M.R Abai and his
colleagues from School of Public Health &
Institute of Health Research, Tehran University
of Medical Sciences for their kind collaboration
in this study.
References
1. Curtis CF, Lines JD, Baolin L, Renz A
(1989). Natural and synthetic repellents.
In: Appropriate technology in vector
control. Ed. CF Curtis. CRC Press.
Boca Raton, FL, pp. 75-92.
2. Anonymous (2002). Diethyl-m-toluamide
(DEET) [134-62-3] Insect Repellent:
Review of the toxicology literature for
the topical insect repellent Diethyl-m-
toluamide (DEET). Scientific evaluation
and assessment. Department of Health
Toxicology Unit at Imperial College,
www.doh.gov.uk/pdfs/reviewofdeet.
pdf

MA Oshaghi et al: Repellent Effect…
51
3. Rotland EH, Jan JE, Rigg JM (1985). Toxic
encephalopathy in a child after brief
exposure to insect repellents. Can Med
Assoc J, 132: 155-56.
4. Liu WK, Wong MH, Miu YL (1987). Toxic
effects of mosquito coil (a mosquito
repellent) smoke on rats I. Properties of
the mosquito coil and its smoke.
Toxicological Letter, 39: 223-30.
5. Sukumar K, Perich MJ, Boobar LR (1991).
Botanical derivatives in mosquito
control: a review. J Am Mosq Control
Assoc, 7: 210-37.
6. Rojas E, Scorza JV (1991). The use of
lemon essential oil as a sandfly
repellent. Trans Roy Soc Trop Med Hyg,
85: 803.
7. Tyler E, Brady LR, Robbers JE (1988).
Volatiles oils, In: Pharmacognosy. Eds,
Lea & Febiger. 9th Ed. Philadelphia, pp.
105-107.
8. Buescher MD, Rutledge LC, Writz RA,
Nelson JH (1985). Laboratory repellent
tests against Rhodnius prolixus. J Med
Ent, 22: 49-53.
9. Klun JA, Debboun M (2000). A new model
for quantitative evaluation of repellents
efficacy using human subjects. J Med
Ent, 37: 177- 81.
10. Sabitov EA (1985). Assessment of the
effectiveness of the unrestricted use of
DTEA repellent and prevention of
zoonotic cutaneous leishmaniasis. Med
Parasit Bolezni, 5: 74-8, [In Russian].
11. Meher ZA, Rutledge LC, Buescher MD,
Gupta K, Raj K, Zakaria MM (1990).
Attraction of mosquitoes to diethyl
methylbenzamide and ethyl hexanediol.
J Am Mosq Control Assoc, 6: 469-75.
12. Kramer WL, Hwang Y, Mulla MS (1980).
Oviposition repellents of mosquitoes:
negative responses elicited by lower
aliphatic acids. J Chem Ecol, 6: 415-24.
13. Gillies MT (1980). The role of carbon
dioxide in host finding by mosquitoes
(Diptera: Culicidae): a review. Bull
Entomol Res, 70: 525-32.
14. Robert LL, Hallan JA, Seeley DC, Roberts
LW, Wirtz RA (1991). Comparative
sensitivities of four Anopheles (Diptera:
Culicidae) to five repellents. J Med Ent,
28: 417-20.
15. Rutledge LC, Moussa MA, Lowe CA,
Sofield RK (1978). Comparative
sensitivity of mosquito species and
strains to the repellent diethyl
toluamide. J Med Ent, 14: 536-41.
16. Wang JJ, Tsai JH, Ding W, Zhao ZM, Li
LS (2002). Toxic effect of six plant oils
alone and in the combination with
controlled atmosphere on Liposcelis
bostrychophila (Psocoptera:
Liposcelididae). J Econ. Ent. 94(5):
1296-1301.
17. Morton J (1987). Lemon. In: Fruits of
warm climates. Ed. JF Morton. Miami,
FL, pp. 160- 68.
18. Bruneton J (1995). Pharmacognosy,
Phytochemistry, Medicinal Plants.
Paris, Lavoisier Publishing.
19. Leung AY, Foster S (1996). Encyclopedia
of Common Natural Ingredients Used in
Food, Drugs, and Cosmetics, 2nd ed.
New York: John Wiley & Sons, Inc.
20. Matsuda BM, Surgeoner GA, Heal JD,
Tucker AO, Maciarello MJ (1996).
Essential oil analysis and field
evaluation of the sitrosa plant
“Pelargonium citrosum” as a repellent
against populations of Aedes
mosquitoes. J Am Mosq Control Assoc,
12: 69-74.
21. Trigg JK (1996). Evaluation of eucalyptus-
based repellent against Anopheles spp.
in Tanzania. J Am Mosq Control Assoc,
12: 243-46.
22. Vahidi A (1965). Components and
important characters of sour lemon.
Thesis, Tabriz University of Medical
Sciences, p 66.

Iranian J Publ Health, Vol. 32, No. 4, pp.47-52, 2003
52
23. Raji-Darageh A (1966). Chemical studies
on lemon. Thesis, Tabriz University of
Medical Sciences, p 92.
24. Asgharian B (1995). Botanical and
cytochemical investigation on wild
Melissa officinalis growing in north of
Iran and comparing the results with the
standard samples. Thesis, Esfahan
University of Medical Sciences, p 67.
25. Faghih N (1997) Investigation on
pharmacognosy of Melissa officinalis.
Thesis, Tehran University of Medical
Sciences, p 154.
26. Ali-madad M (1996). Investigation on
components of essential oils of Melissa
officinalis. Thesis, Tehran University of
Medical Sciences, p 231.
27. Badihi-Ghazvini F (1988) Investigation on
essential oils and phytochemistry of
Melissa officinalis. Thesis, Tehran
University of Medical Sciences, p 87.
28. Hadjiakhondi A, Aghel N, Zamanizadeh-
Nadgar N, Vatandoost H (2000).
Chemical and biological study of
Mentha specata L. essential oil from
Iran. Daru, 8(1-2): 19-21.

MA Oshaghi et al: Repellent Effect…
53