Research Journal # 11

Valerie Fay D.L. Gabertan November 20,2019

8-Adelfa Group # 5

Lemongrass oil as an alternative insect repellent

Abstract

Background: Safety concerns over the use of chemical-based pesticides are provoking intense studies and application
of bio-pesticides in disease control and agriculture. Aim: This qualitative study was aimed at testing the repellent
efficacy; and suitability of Cymbopogon citratus (Lemongrass) for the development of natural product-based
mosquito repellent. Method: Cymbopogon oil (75ml) was extracted from 200g of fresh Lemongrass leaves using
Soxhlet method with n-Hexane as solvent. Human volunteers were topically treated with 2ml and 6ml of
Cymbopogon oil applied on exposed areas of the body at night; and observed for 4hrs 30mins. Direct test on
mosquitoes in captivity was conducted using a 2-chambered insect box treated with 4 ml of oil in one chamber.
Results: Human subjects were free from mosquito bites for 3 hours. Mosquitoes in captivity exhibited active
movement away from oil-treated chamber of the box within the first minute of application; 43% repellency and 100%
mortality were recorded after 18 minutes. Conclusion: Findings suggest that Cymbopogon citratus extracts could be
used for the production of potent chemical-free and safe mosquito repellent and insecticide for vector control in the
fight against malaria and other mosquito-vectored diseases. However further studies and qualitative analyses are
necessary to determine suitable concentrations for human topical application.
Introduction

Mosquitoes have been implicated in the transmission of very debilitating and deadly diseases to humans; such as
malaria, filariasis, dengue fever and yellow fever. Little wonder that the quest for the control of this arthropod vector
have remained a major focus in disease control / eradication programmes; which could be traced back to the earliest
global malaria eradication effort of residual insecticidal campaign [1]. Apart from the use of insecticides for residual
house spraying and other basic strategies targeted at the vector, the fight against mosquito-borne infections also
engages preventive measures to avoid or reduce vector-man contact, such as avoiding places of high mosquito
densities, wearing protective clothing outdoors, use of bed and window nets, and use of mosquito repellents, etc.
These formed part of the Global Malaria Control Strategy [2–5]. These have today come to constitute the
components of integrated vector control.
Mosquito control has been known among people of many cultures around the globe. A variety of local mosquito
control practices have been documented in the past few decades; such as the use of traditional fumigants made from
natural products as source of deterrent smoke, e.g. herb smoke in Burundi, [6]; burning of cassava chips and herbs in
Ghana [7]; burning of bark of the tree Daniella oliveri in Gambia, [8]; and other such fumigants in Sri Lanka [9]. The
use of a paste (Thanaka) derived from Limonia acidissima (the wood apple tree) for skin application as repellent had
been reported in Thailand [10][8]. In some parts of Nigeria deterrent smoke from palm kernel shells and dried orange
peels serve the purpose. These indicate the long existing use of natural products to control mosquitoes, earlier
before the current trend in bio-pesticide use. There is evidence that the use of biological control agents against
mosquitoes grew in the beginning of the 20th Century, but reduced with the discovery of the insecticidal properties
of Dichlorodiphenyl trichloroethane (DDT) in 1939 [11]. However, the gradual development of resistance by many
species of insects, suggested association with human cancers [4] and other public health problems [12], the use of
DDT dwindled, even as it was banned in the USA in 1973[13].

Commercially available chemical-based insecticides and repellents are known to contain harmful and poisonous
compounds which could pose threats to human health and toxicity to non-target organisms [14]. The repellent
efficacy of the chemical N,N-diethyl-3-methylbenzamide (DEET) over other products made it the compound of choice
in pesticides [15]. However, concerns of its safety have not ceased to be considered. The effects on human health,
aquatic species and the environment have been reviewed [16],[17],[18]. The seeming renewed focus on natural
productbased repellents sets to replace hazardous chemical-based products with safe alternatives; hence the
concept of green pesticides [19].
Studies have demonstrated the insecticidal effect of plant products, which make them suitable as safe pest control
products. Essential oils from a variety of plant species have been used as very effective pest repellents; such as
Cymbopogon nardus, Cymbopogon excavatus, Azaradicta indica, Eucalyptus maculata etc. [11]. Etta and others
reported the insecticidal property of Ocimum gratissimum extract on wild-caught adult Anopheles mosquitoes [20]
Cymbopogon citratus (Lemon grass) oil has been known as effective insect repellent; its effectiveness in mosquito
repellence has also been documented [21],[22]. This study was aimed at testing the mosquito repellent property of
Cymbopogon citratus on human volunteers and mosquitoes in captivity, to determine its suitability as natural
product-based mosquito repellent.

Methods

Extraction of Lemongrass Oil

Fresh leaves of Cymbopogon. citratus (lemon grass) were sliced and ground. Two hundredmgrams (200g) of ground
leaves were placed in a porous thimble, for oil extraction using the Soxhlet extraction method, with n-Hexane
solvent. About 75ml of oil, weighing 36.4g, was extracted and stored in the refrigerator at 4oC

Collection of Mosquito Specimens

Mosquitoes were collected in an open area using a sieve net and safely transferred into an insect box. This was
repeated severally to obtain the desired number of mosquito samples. The mosquitoes were taken to the Biology
laboratory, Department of Biological Sciences, Cross River University of Technology, Calabar, and identified as
Anopheles species by an entomologist before the test.

Test of Repellent Activity of C. citratus on Human Volunteers

The test was conducted at Cross River University of Technology, Calabar Campus Hostel, Hall 2. Five student
volunteers participated in the study; 2 of them applied 2ml and 2 applied 6ml of lemon grass oil topically on the legs,
arms and head (including the ear), while 1 did not apply oil and was used as control. All volunteers were awake and
reading throughout the duration of the experiment in a hostel room where relative abundance of mosquitoes was
previously established. The experiment was observed for 4 hours thirty minutes (from 22.00 hours to 02:30 hours).
Results recorded included individual observations by each volunteer and the researchers; with respect to mosquito
landing attempts/bites, place of bites, number of bites and time interval between bites.

Test for repellent activity of C. citratus on mosquitoes in captivity

The laboratory experiment was conducted using an insect box designed with a surface area of 2756cm2 ; with an
open surface covered with net. The box had two chambers, A (lemon grass oiltreated) and B (untreated), with an
opening linking both. A petri dish containing 6ml of Cymbopogon oil was placed in chamber A; and thirty (30) freshly
caught, live mosquitoes were carefully transferred into same chamber of the box. Timed observations of the reaction
of mosquitoes were recorded.

Ethical considerations

Ethical approval for the study was obtained from the Chief Medical Director of the Cross River University of
Technology, through the Dean, Student Affairs Division of the University. Informed consent was obtained from all
volunteers that participated in the research.

Results

Repellent Activity of C. citratus on Human Volunteers

The test volunteers experienced zero mosquito landing attempts and bites until three hours, while the control
volunteer had mosquito landings and bites within 2 minutes of the experiment. One volunteer who applied 2mls of C.
citratus oil had a bite at 3 hours into the experiment (01.00 hours), while one who used 6ml of oil experienced a bite
only 1 hour 30 minutes later at 02:30; that is 4 hours 30 minutes from start of experiment. Of 30 mosquito landing
attempts/bites experienced during the study, 21(70%) were on the control subject; 8(27%) on the volunteer with 2ml
of oil and 1(3%) on the volunteer with 6ml of oil (Table 1).
Repellent activity of lemon grass oil on mosquitoes in captivity

Mosquitoes were observed to fly restlessly exhibiting frantic attempts to escape from the test box; and 13 (43%) flew
away from the oil-treated section (A) into the untreated section (B) of the insect box within the first minute of
exposure. However, 17 (57%) mosquitoes were knocked down, among which those that got in direct contact with the
oil died within few minutes; but by the 18th minute, 100% mortality was recorded in section A (Table 2).

Discussion

The study with human volunteers provided a real case situation for confirmatory observations on the repellent
activity of lemon grass oil against mosquitoes. Mosquitoes usually make surreptitious landings on exposed skin to
feed; reason why the wearing of protective clothing stands as a good measure of preventing man-vector contact, a
primary method of malaria prevention [23]. Application of lemongrass oil prevented landing attempts and bites by
mosquitoes; and this verifies already documented evidence that C. citratus extract is a potent mosquito repellent [21,
[22].
However, the interval of protection is a matter of concern; in this study, lemon grass oil provided anti-mosquito
protection for 3 hours after which landing attempts and bites were experienced. This observation would corroborate
previous records of volatility of plants essential oils [11]; hence the duration of repellence might be undoubtedly
connected with the concentration of oil. Using different volumes (2ml and 6ml) of the oil gave a suggestive result to
consider that the intensity of oil could have relevance with its retention time on the skin and hence interval of
protection. Only one bite was experienced by the volunteer who applied 6ml of oil and at 4 hours thirty minutes of
experiment; by which time there were already 5 landing attempts/bites on volunteer with 2ml of oil. While the
difference in volume is not equal to difference in concentration, it is assumable that at a higher level the essential oil
would persist longer. This could also be explained from the result of laboratory test with mosquitoes in captivity.
Mosquitoes which escaped from the oil-treated chamber of the insect box survived, while those that remained
longer within the influence of the volatile oil were knocked down and eventually killed after 18 minutes. It is obvious
that all the mosquitoes would have flown away from the source of discomfort (oil) if they were not in captivity.

Further, quantitative, studies would beam on the issue of interval of protection and provide useful guide for
commercial production of lemon grass oil repellent for topical application against insect vectors. It would also
provide reliable information for use of the extract in local mosquito prevention and control practices. Considering the
high volatility, commercial formulation of plant-based repellents from essential oils would have to use fixatives to
maintain longevity of their action [24].

The study with live mosquitoes also confirmed the insecticidal property of lemon grass oil, [25], [26], [27]. Assumed
to be safer, compared with chemical based pesticides [11], [24], lemon grass oil would serve as a very effective
natural product-based repellent and insecticide.
 Conclusion

This study reports the repellent and insecticidal properties of Lemon grass oil against malaria mosquitoes in the study
area; and contributes to the growing literature on safe bio-pesticides for disease control. While commercial
formulations should be expected, local use of this and other plants essential oil is recommendable to prevent man-
vector contact in local communities where transmission rates are high but healthcare interventions are lacking. While
this study did not consider chemical composition and characterization of C. citratus essential oil, indeed, in-depth and
continuous studies in the area of health and environmental safety of bio-pesticides are of immediate necessity in the
midst of current global awareness for natural products.

Acknowledgement

We thank the management and student volunteers of Hall 2 male hostel, Cross River University of Technology,
Calabar for cooperation and participation in the study, respectively.

Recommendation

1) Usage of another material or ingredient that can help increase the effectiveness of the oil. 2) Prolong the
process of letting stay the lemongrass stalks inside the jar full of oil before putting it in a different container. 3)
Trying a different method in creating the oil. 4) Test the product in different surroundings (e.g. public parks with
numerous bushes and trees).

References

[1] World Health Organization (1957). Malaria: Sixth report of the expert committee. Technical Report Series, No.
123, WHO, Geneva, Switzerland.[2] World Health Organization (1993a). A global strategy for malaria control. WHO,
Geneva, Switzerland. [3] World Health Organization (1993b). Implementation of the global malaria control strategy.
WHO, Geneva, Switzerland. [4] World Health Organization (1995).Vector control for malaria and other mosquitoe-
borne diseases. WHO Technical Report Series 857, WHO, Geneva, Switzerland. [5] Eldridge, B. (2000). Mosquitoes:
Home and landscape, UC Pest management guidelines; the Regents of the University of California, www-
ipm.uc.davis.edu. [6] Baribwira, C., Kanyange, L. and Barutwanayo, M. (1997). The management of malaria and its
vector: knowledge and practices in households in urban areas of Bujumbura (Burundi), Malar. Infect. Dis. Africa, 3:13-
19. [7] Ahorlu, C. K., Dunyo, S. K., Afari, E. A., Koram, K. A. and Nkrumah, F. K. (1997). Malariarelated beliefs and
behavior in Southern Ghana: Implications for treatment, prevention and control. Trop. Med. Int. Hlt, 2(5): 488-499.
[8] Greenwood, B. (1999). What can the residents of malaria endemic countries do to protect themselves against
malaria? Parassitologia, 41: 295-299. [9] W. Van de Hoek, W., Konradsen, F., Dijkstra, D. S. Amerasinghe, P. H. and
Amerasinghe, F. P. (1998). Risk factors for malaria: A micro-epidemiological study in a village in Sri Lanka. Trans. R.
Soc. Trop. Med. Hyg., 92; 265-269. 10] Lindsay, S. W.,Ewald, J. A.,Samung, Y.,Apiwathnasorn,C. and Nosten, F.(1998).
Thanaka (Limonia acidissima) and DEET (Dimethyl benzamide) mixture as a mosquito repellent for use by Karen
women. Med. Vet. Entomology, 12: 295-301. [11] Bhupen, K., Somi, B. and Sharma, A. K. (2013). Plant essential oils as
mosquito repellent: A Review. International Journal of Research and Development in Pharmacy and Life Sciences.
3(1): 741-747. [12] L. Lopez-Carrillo, L., Torres-Arreola, L., Torres-Sanchez, L., Espinosa-Torres, F., Jimenez, C.,
Cebrian, M., Waliszewski, S. and Saldate, O. (1996). Is DDT use a public health problem in Mexico? Env. Hlth.
Perspectives, 104(6): 584-588. [13] Harrison, K. (1997). DDT a banned insecticide,
www.chem.ox.ac.uk/mom/ddt/ddt.html. [14] Mandavgane, S. A., Pattalwar, V. V., and Kalambe, A. R. (2005).
Development of cow dung based herbal mosquito repellent. Natural Project Radiance. 4(4): 270-273. [15] Fradin, M.
S. and Day, F. J. (2002). Comparative efficacy of insect repellents against mosquito
bites. New England J. Medicine, 347(1): 13-18. [16] Environmental health Association of Nova Scotia (2003). The
hazards of DEET. www.environmentalhealth.ca. Accessed December, 2016. [17] Fishel, F. (2003). Pesticides and the
environment. University of Missouri extension, www.extension.mission.edu. Accessed December, 2016.
[18] National Pesticide Information Center (2008). DEET General fact sheet, www.npic.orst.edu.
Accessed December, 2016. [19] Bakkali, F., Averbeck, S., Averbeck, D. and Idaomar, M. (2008). Biological effects of
essential oils. Food and Chemical Toxicology, 46(2): 446-475. [20] Etta, H. E., Ukpong, I. G., Iboh, C. I. and Momoh, E.
N. (2016). Preliminary investigations of the insecticidal effect of Ocimum Gratissimum (L) on adult Anopheles
mosquitoes

Glossary

Anopheles
Anopheles (/əˈnɒfɪliːz/) is a genus of mosquito first described and named by J. W. Meigen in 1818. About 460 species
are recognised; while over 100 can transmit human malaria, only 30–40 commonly transmit parasites of the genus
Plasmodium, which cause malaria in humans in endemic areas.

Arthropod vector

other than insects and arachnids, which transmit infective organisms from one host to another or from an inanimate
reservoir to an animate host.

Azaradicta indica

commonly known as neem, nimtree or Indian lilac, is a tree in the mahogany family Meliaceae

Cymbopogon citratus

Cymbopogon citratus, Stapf (Lemon grass) is a widely used herb in tropical countries, especially in Southeast Asia

Cymbopogon excavatus,

Cymbopogon excavatus (Hochst.) Stapf ex Burtt Davy. Back to results. Group. Monocot. Family. Poaceae. Genus.
Cymbopogon. Species.

Cymbopogon nardus

Cymbopogon nardus, common name citronella grass, is a perennial aromatic plant from the family Poaceae,
originating in tropical Asia.

Dengue fever

Dengue (DENG-gey) fever is a mosquito-borne disease that occurs in tropical and subtropical areas of the world. Mild
dengue fever causes a high fever, rash, and muscle and joint pain

Dichlorodiphenyl

sulfone is rapidly absorbed from the gut and metabolized by a saturable process.

Eucalyptus maculata

commonly known as spotted gum, is an endemic Australian tree.

Filariasis

Filariasis is a parasitic disease caused by an infection with roundworms of the Filarioidea type

Kernel shells

are the shell fractions left after the nut has been removed after crushing in the Palm Oil mill.

Limonia acidissima

is a large tree growing to 9 metres (30 ft) tall, with rough, spiny bark. The leaves are pinnate, with 5-7 leaflets, each
leaflet 25–35 mm long and 10–20 mm broad, with a citrus-scent when crushed. The flowers are white and have five
petals.

Malaria

Malaria is a mosquito-borne infectious disease that affects humans and other animals. Malaria causes symptoms that
typically include fever, tiredness, vomiting, and headaches. In severe cases it can cause yellow skin, seizures, coma, or
death.
Ocimum gratissimum

also known as clove basil, African basil, and in Hawaii as wild basil, is a species of Ocimum. It is native to Africa,
Madagascar, southern Asia, and the Bismarck Archipelago, and naturalized in Polynesia, Hawaii, Mexico, Panama,
West Indies, Brazil, and Bolivia.

Oliveri

Oliveri (Sicilian: Uliveri) is a comune (municipality) in the Province of Messina in the Italian region Sicily, located about
150 kilometres (93 mi) east of Palermo and about 45 kilometres (28 mi) west of Messina. As of 31 December 2004, it
had a population of 2,076 and an area of 10.3 square kilometres (4.0 sq mi).[3]

Trichloroethane

The organic compound 1,1,1-trichloroethane, also known as methyl chloroform, is a chloroalkane. This colourless,
sweet-smelling liquid was once produced industrially in large quantities for use as a solvent.

Yellow fever

Yellow fever is a disease caused by a virus that is spread through mosquito bites.