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Efficacy of Thai herbal essential oils as green repellent against mosquito vectors
Acta Tropica 142 (2015) 127–130
Contents lists available at ScienceDirect
Acta Tropica journal homepage: www.elsevier.com/locate/actatropica
Short Communication
Efficacy of Thai herbal essential oils as green repellent against mosquito vectors Mayura Soonwera a , Siriporn Phasomkusolsil b,∗ a b
Plant Production Technology Section, Faculty of Agricultural Technology, King Mongkut’s Institute of Technology Ladkrabang, Bangkok 10520, Thailand Department of Entomology, US Army Medical Component, Armed Forces Research Institute of Medical Sciences, Bangkok 10400, Thailand
a r t i c l e
i n f o
Article history: Received 14 August 2014 Received in revised form 18 November 2014 Accepted 21 November 2014 Available online 28 November 2014 Keywords: Repellency Cananga odorata oil Cymbopogon citratus oil Aedes aegypti Culex quinquefasciatus
a b s t r a c t Repellency activity of Thai essential oils derived from ylang ylang (Cananga odorata (Lamk.) Hook.f. & Thomson: Annonaceae) and lemongrass (Cymbopogon citratus (DC.) Stapf: Poaceae) were tested against two mosquito vectors, Aedes aegypti (L.) and Culex quinquefasciatus (Say). There were compared with two chemical repellents (DEET 20% w/w; Sketolene Shield® and IR3535, ethyl butylacetylaminopropionate 12.5% w/w; Johnson’s Baby Clear Lotion Anti-Mosquito® ). Each herbal repellent was applied in three diluents; coconut oil, soybean oil and olive oil at 0.33 l/cm2 on the forearm of volunteers. All herbal repellent exhibited higher repellent activity than IR3535 12.5% w/w, but lower repellent activity than DEET 20% w/w. The C. odorata oil in coconut oil exhibited excellent activity with 98.9% protection from bites of A. aegypti for 88.7 ± 10.4 min. In addition, C. citratus in olive oil showed excellent activity with 98.8% protection from bites of C. quinquefasciatus for 170.0 ± 9.0 min. While, DEET 20% w/w gave protection for 155.0 ± 7.1–182.0 ± 12.2 min and 98.5% protection from bites of two mosquito species. However, all herbal repellent provided lower repellency activity (97.4–98.9% protection for 10.5–88.7 min) against A. aegypti than C. quinquefasciatus (98.3–99.2% protection for 60–170 min). Our data exhibited that C. odorata oil and C. citratus oil are suitable to be used as green repellents for mosquito control, which are safe for humans, domestic animals and environmental friendly. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Repellents, clothing, netting and other personal protection measures can help prevent mosquito bites and the diseases mosquitoes carry. Especially, insect repellents are effective and safe when properly used. Repellent repels mosquitoes, it reduces the chances of being bitten. In this light, repellent use offers individuals added protection against mosquito-borne diseases. In the community, it reduces the transmission of mosquito-borne diseases (Gul et al., 2013). N,N-diethyl-m-toluamide (DEET) seems to be most effective and is the best studied insect repellent currently available to the general public. DEET is designed for direct application to human skin to repel insects, rather than kill them. Currently, DEET is formulated in aerosols, pump sprays, lotions, creams, liquids, sticks, roll-ons and impregnated towelettes, with concentrations ranging from 5% to 100%. However, there are concerns about the potential toxic effects of DEET, especially when used by children. Children who absorb high amounts of DEET through insect repellents have
∗ Corresponding author. Tel.: +66 089 124 6455. E-mail address: [email protected] (S. Phasomkusolsil). http://dx.doi.org/10.1016/j.actatropica.2014.11.010 0001-706X/© 2014 Elsevier B.V. All rights reserved.
developed seizures, slurred speech, hypotension and bradycardia (Clem et al., 1993). Mosquito repellents based on chemicals has a remarkable safety profile, but they are toxic against the skin and nervous system like rashes, swelling, eye irritation, and worse problems, though unusual including brain swelling in children, anaphylactic shock, and low blood pressure (Shasany et al., 2000; Phal et al., 2012). To overcome these problems, it is necessary to search for alternative methods of vector control. The failure of chemical insecticides to control the insect and growing public concern for safe food and a healthy environment have catalyzed the search for more environmentally benign control methods for the management of the vectors (Amerasan et al., 2012). Essential oil has been the active principle of most important herbal remedies since ancient times. Insecticides of plant origin have been extensively used on agricultural pests, and to a very limited extent, against insect’s vectors of public health importance, which deserve careful and thorough screening. The use of plant extracts for insect control has several appealing features, as these are generally more biodegradable, less hazardous, and rich storehouse of chemicals of diverse biological activity (Nath et al., 2006). In Thailand, several Thai herbs act as natural insect repellent, including ylang ylang (Cananga odorata) and lemongrass
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M. Soonwera, S. Phasomkusolsil / Acta Tropica 142 (2015) 127–130
(Cymbopogon citratus) that expressed high degree of repellency against insect (Caballero-Gallardo et al., 2011; Adeniran and Fabiyi, 2012). The present investigation aims to assess the repellent properties of the essential oils of ylang ylang and lemongrass which mixed in the natural oils by six formulations against medically important mosquitoes of dengue vector (Aedes aegypti) and filarial vector (Culex quinquefasciatus). Additionally, the study utilized natural oils; soybean oil, coconut oil and olive oil used locally for cooking and cosmetic purposes to formulate the volatile oil into lotions to improve its acceptability and preclude any adverse effect that could emanate from use of synthetic bases. 2. Materials and methods 2.1. Mosquito cultures and rearing conditions A. aegypti and C. quinquefasciatus lab bred mosquitoes were used in this study. They were maintained in the laboratory of the Entomology and Environment Program, Plant Production Technology Section, Faculty of Agricultural Technology, King Mongkut’s Institute of Technology Ladkrabang (KMITL), Bangkok. Adult mosquitoes were reared at 25 ± 2 ◦ C, and a relative humidity of 80 ± 10% with a photoperiod of 12 h light followed by 12 h dark (12L:12D); adults were fed with 5% multivitamin solution. Prior to testing, 5- to 7-day-old female mosquitoes were starved by providing them with only water for 12 h. 2.2. Preparation of herbal essential oils In this paper, the efficacy of essential oils of ylang ylang (C. odorata (Lamk.) Hook.f. & Thomson) and lemongrass (C. citratus DC. Stapf) were compared. Flowers of ylang ylang (C. odorata) and stems of lemongrass (C. citratus) were collected and essential oils from plant parts were extracted by water distillation method (Charles and Simo, 1990). These essential oils were then prepared at 0.33 l/cm2 in three diluents; coconut oil, soybean oil and olive oil. 2.3. Chemical repellent (1) DEET, 20% w/w; Sketolene Shield® is a commercial chemical repellent in Thailand. (2) IR3535, 12.5% w/w Ethyl butylacetylaminopropionate; Johnson’s Baby Clear Lotion Anti-Mosquito® , is a commercial chemical repellent in Thailand. 2.4. Human volunteers Five adult volunteers of both sexes, 25–45 years old, weight 50–70 kg, who had no history of allergic reaction to arthropod bites were recruited. Before signing an informed consent form, the volunteers were interviewed and instructed on the methodology, probable discomforts to subjects and remedial arrangements. 2.5. Laboratory repellent bioassay Repellency of C. odorata and C. citratus oils and their formulations were evaluated against A. aegypti and C. quinquefasciatus under laboratory conditions using the methods which followed the guidelines by World Health Organization (2009). To compare the repellency of essential oils with that of the standard repellent, DEET 20% w/w and IR3535 12.5% w/w were tested. Due to different biting behavior, the tests against A. aegypti was carried out from 0800 am to 0400 pm, whereas those against C. quinquefasciatus was conducted between 0400 pm and 1200 pm because
Table 1 Repellency activities of two herbal essential oils which diluted in three diluents at 0.33 l/cm2 (coconut oil, soybean oil and olive oil) against Aedes aegypti. Herbal essential oils
Protection time (min)a
Ylang ylang oil + coconut oil Ylang ylang oil + soybean oil Ylang ylang oil + olive oil Lemongrass oil + coconut oil Lemongrass oil + soybean oil Lemongrass oil + olive oil DEET 20% (w/w) (Sketolene Shield® ) IR3535 12.5% (w/w) (Johnson’s Baby Clear Lotion® )
88.7 ± 10.4b 10.50 ± 2.1c
1.1 2.4
98.9 97.6
85.5 ± 12.0b 85.5 ± 10.5b
1.4 1.1
98.6 98.9
72.0 ± 12.4b
2.6
97.4
60.0 ± 12.5b 155.0 ± 7.1a
1.2 1.5
98.8 98.5
21.0
79.0
3.0 ± 0c
% Biting
% Protection
a Means in each column against each mosquito species followed by the difference letters are significantly different (P < 0.05, by one-way ANOVA and Duncan’s Multiple Range Test).
A. aegypti is a day-biter but C. quinquefasciatus is a night-biter (Govindarajan, 2011). Two hundred and fifty nonblood-fed starved female mosquitoes were randomly selected and placed in an experimental cage (30 cm × 30 cm × 30 cm) and left to acclimatize for 1 h. After cleaning with distilled water, each forearm of the volunteer was wrapped in a plastic sleeve attached with double-sided tape, and a cutout was aligned with a 3 cm × 10 cm area on the ventral portion of the forearm. Therefore, only a restricted zone of the skin was exposed to the mosquitoes. However, before the start of each exposure, the bare hand, used as control area (no treatment) of each volunteer, was exposed for up to 30 s. If at least two mosquitoes landed on the test area of the control arm, the arm was shaken off before imbibing any blood and withdrawn from the cage and the repellency test was then continued. This was done to ensure that the mosquitoes were still active. An amount of 0.1 ml of test repellent was applied to the marked area of one forearm of each volunteer. Subsequently, the test arm was introduced into the cage for 3 min. The total number of mosquitoes biting on the treatment was recorded. If no mosquito bite occurred within 3 min, the forearm was then taken out and the test was repeated every 30-min interval. The experiment was completed after two mosquitoes had bitten. The study period was carried out every 30 min until at least 2 mosquitoes bit during the 3 min study period, at which time the study was stopped. The protection time was the time from repellent application until the study was stopped. On each day, only one repellent preparation was tested to assure that residual material has disappeared from the skin before the next test (Curtis and Hill, 1988). 2.6. Statistical analysis The mean protection time was used as a standard repellency measure of the test samples against A. aegypti and C. quinquefasciatus in the laboratory. Differences in significance were analyzed by one-way analysis of variance (ANOVA) and Duncan’s multiple comparisons by SPSS for Windows (version 16.0). 3. Results Each of plant products was applied onto the forearm and they were evaluated. The results for plant oils repellency against A. aegypti were summarized in Table 1. Ylang ylang oil containing coconut oil provided longer lasting complete repellency (88.7 min) while the ylang ylang oil containing olive oil and lemongrass oil containing coconut oil which gave complete protection for
M. Soonwera, S. Phasomkusolsil / Acta Tropica 142 (2015) 127–130 Table 2 Repellency activities of two herbal essential oils which diluted in three diluents at 0.33 l/cm2 against Culex quinquefasciatus. Herbal essential oils
Protection time (min)a
Ylang ylang oil + coconut oil Ylang ylang oil + soybean oil Ylang ylang oil + olive oil Lemongrass oil + coconut oil Lemongrass oil + soybean oil Lemongrass oil + olive oil DEET 20% (w/w) (Sketolene Shield® ) IR3535 12.5% (w/w) (Johnson’s Baby Clear Lotion® )
126.7 ± 5.8b 60.0 ± 0d
0.8 1.2
99.2 98.8
85.5 ± 12.0c 115.5 ± 10.4b
1.4 1.1
98.6 98.9
84.0 ± 25.1c
1.7
98.3
170.0 ± 9.0a 182.0 ± 12.2a
1.2 1.5
98.8 98.5
24.2
75.8
3.0 ± 0c
% Biting
% Protection
a Means in each column against each mosquito species followed by the difference letters are significantly different (P < 0.05, by one-way ANOVA and Duncan’s Multiple Range Test).
85.5 min. In addition, both products of lemongrass oil containing soybean oil and olive oil gave complete repellency for 72 min and 60 min, respectively. However, there were not significant differences in the protection time among the repellents against A. aegypti (P > 0.05). WHO standard (2009) determines the repellency time against A. aegypti mosquitoes should be more than 2 h; none of the repellents met this requirement. DEET, which is the best-known synthetic insect repellent, at 20% concentration of active ingredient, provided the longest-lasting complete protection for 155 min or more than 2 h after application. Unfortunately, IR3535, which is the commercial insect repellent, at 12.5% concentration of active ingredient, showed no repellency against A. aegypti (3 min). The results of repellency tests on six different formulations against C. quinquefasciatus were shown in Table 2. The essential oil of lemongrass oil in olive oil provided the longest lasting complete repellency for 170 min after application. Compared against the standard mosquito repellent, DEET, lemongrass oil in olive oil performed equally well in protection time against C. quinquefasciatus (P > 0.05). However, IR3535-based repellent showed no repellency against mosquitoes (3 min after application). Furthermore, ylang ylang oil and lemongrass oil in coconut oil provided moderate protection time against C. quinquefasciatus, for 126.7 and 115.5 min, respectively. Moreover, the degree repellency was obtained from ylang ylang oil in olive oil (85.5 min), followed by lemongrass oil in soybean oil (84 min) and ylang ylang oil in soybean oil (60 min). There were significant overall among the efficacy of the essential oils against C. quinquefasciatus (P < 0.05). The percentage of repellency for C. quinquefasciatus ranged between 98% and 99% but reached only 75.8% for IR3535. 4. Discussion In this study, the standard repellent (DEET) has also been found to be effective. There was a significant different between the repellent activity of the standard and the test samples. Therefore, essential oils from C. citratus and C. odorata were investigated the repellency activity against A. aegypti and C. quinquefasciatus. The use of plant of the C. citratus or lemongrass as insect repellent is widespread throughout the world and the formulations tested represent the range of concentration which is commercially available. However, further investigation of formulation of C. odorata (ylang ylang) is still lack of information. In this study, we focused on the differences between repellent effect and feed deterrent effect. Because the protection times were determined as the time form material application until the first two bites. The
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percentage of repellency depended on the total number of landing and biting mosquitoes in treated and control areas. If the protection time of a compound was long while the percentage of repellency was high, this compound exhibited both high efficiency to repel mosquito as well as feed deterrent activity. If the protection time was low and also the percentage of repellency was high, then this compound was more a feed deterrent than a repellent. Conversely, if the protection time was long but the percentage of repellency was low, then the compound was more a repellent than a feed deterrent (Amer and Mehlhorn, 2006). Therefore, in this study shows that the protection time of ylang ylang oil in coconut oil and soybean oil and lemongrass in coconut oil were 85.5–88.7 min, but the repellency increased to 98.6–98.9%. This shows that these oils are only a feed deterrent and no repellent. Meanwhile, the protection period for lemongrass oil in olive oil against C. quinquefasciatus was 170 min, in addition, the protection time of ylang ylang oil and lemongrass oil in coconut oil were 126.7 and 115.5 min, while the repellency were up to 98%. Thus, these repellents showed the attributes of a repellent and a feed deterrent. The use of essential oils as commercial repellent products needs a formulation to fix the aromatic materials onto the skin for as long as possible. The present study compares a mixture of essential oil within the base material of plant products as coconut oil, soybean oil and olive oil. Some researchers evaluated that the repellent properties of mixture of essential oil in three base materials. Bite Blocker, a commercial preparation containing glycerin, lecithin, vanillin, coconut oil, geranium, and 2% soybean oil can achieve similar repellency to DEET, providing 7.2 h mean protection time against a dengue vector and nuisance biting mosquitoes in one study (Barnard and Xue, 2004). Oparaocha et al. (2010) examined the mosquito-repellent and mosquitocidal activities of the 30% (v/v) of Ocimum gratissimum in olive oil base exhibiting highest average percentage repellencies of 97.2%, 95.7% and 96.3% at World Bank Estate, Ihitte and Umuekunne centres, respectively. Das et al. (2003) investigated the repellent properties of Zanthoxylum limonella, Citrus aurantifolia and petroleum ether extract of Z. limonella (fruits) as repellent against Aedes albopictus in mustard and coconut oil base under laboratory conditions. However, the results showed that repellents in mustard oil afforded longer protection time against the bites of A. albopictus mosquitoes than those in coconut oil. Moreover, the natural oils which used in this experiment, there were also of great nutritional value and as well as therapeutic values. Coconut oil is an indispensable ingredient in cosmetics, as well as cooking. It possesses healing properties far beyond that of any other dietary oil. In traditional medicine, coconut oil is used to treat wide variety of health problem including the following, allergic conditions, bronchial asthma, gonorrhea, skin infections and malnutrition (Agarwal and Lakshmi, 2013). Meanwhile, soybean oil is a rich source of vitamin E. Vitamin E is essential to protect the body fat from oxidation and to scavenge the free radicals and therefore helps to prevent their potential effect upon chronic diseases such as coronary heart diseases and cancer (Lu and Liu, 2002). In addition, olive oil is a rich source of valuable nutrients and bioactives of medicinal and therapeutic interest. Olive fruit contains appreciable concentration, 1–3% of fresh pulp weight, of hydrophilic (phenolic acids, phenolic alchohols, flavonoids and secoiridoids) and lipophilic (cresols) phenolic compounds that are known to possess multiple biological activities such as antioxidant, anticarcinogenic, antiinflammatory, antimicrobial, antihypertensive, antidyslipidemic, cardiotonic, laxative, and antiplatelet. Olive oil is widely used for food preparations (as salad oil, cooking oil, in frying and pasta sauces), in cosmetics and the pharmaceutical industry (Gavriilidou and Boskou, 1991). Thus, the as well as the plants are abundant, the local people could be taught how to prepare the lotions.
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Currently, there is no effective vaccine against dengue, and due to insecticide resistance. Despite this fact, protection from mosquito bites could be achieved by avoiding physical contact with mosquitoes using insect repellents, and there are many commercially available repellents on the market. Many factors play a role in how effective any repellent is, including the frequency, dosage and uniformity of application, the number and species of the organisms attempting to bite, the user’s inherent attractiveness to blood-sucking arthropods, and the overall activity level of the potential host (Schreck, 1995). While the plants mentioned in this article have been shown to have mosquito-repelling properties, but requires reapplication to be effective over several hours. For the period the test was conducted and even several hours after the experiment, no skin irritation or dermatitis was observed on the treated skin of the human subject after application of the repellent treatments. An insect repellent of plant origin ought to be welldefined and harmless to human and other non-target organisms. Therefore, use of these botanical derivatives in mosquito control instead of synthetic insecticides could reduce the cost and environment effects. Acknowledgments The authors are highly grateful to Faculty of Agricultural Technology, King Mongkut’s Institute of Technology Ladkrabang (KMITL), Bangkok, Thailand for providing financial assistance to carry out this study. Grateful thanks are due to the volunteers from Plant Production Technology Section, Faculty of Agricultural Technology, KMITL for their assistance in repellent tests. Thanks are extended to plant taxonomist of Faculty of Agricultural Technology, KMITL for herbal identification. The standard disclaimer that the findings/conclusions reflect the efforts and opinions of the authors and not of the US Army or AFRIMS or the Department of Entomology. References Adeniran, O.I., Fabiyi, E., 2012. A cream formulation of an effective mosquito repellent: a topical product from lemongrass oil (Cymbopogon citratus) Stapf. J. Nat. Product Plant Resour. 2, 322–327.
Agarwal, R., Lakshmi, T., 2013. Essential oils in dentistry – an update. Int. J. PharmTech Res. 5, 1804–1807. Amer, A., Mehlhorn, H., 2006. Repellency effect of forty-one essential oils against Aedes, Anopheles, and Culex mosquitoes. J. Parasitol. Res. 99, 478–490. Amerasan, D., Murugan, K., Kovendan, K., Kumar, P.M., Pannerselvam, C., Subramaniam, J., William, S.J., Hwang, J.S., 2012. Adulticidal and repellent properties of Cassia tora Linn. (Family: Caesalpinaceae) against Culex quinquefasciatus, Aedes aegypti, and Anopheles stephensi. J. Parasitol. Res. 111, 1953–1964. Barnard, D.R., Xue, R.D., 2004. Laboratory evaluation of mosquito repellents against Aedes albopictus, Culex nigripalpus, and Ochierotatus triseriatus (Diptera: Culicidae). J. Med. Entomol. 41, 726–730. Caballero-Gallardo, K., Olivero-Verbel, J., Stashenko, E.E., 2011. Repellent activity of essential oils and some of their individual constituents against Tribolium castaneum Herbst. J. Agric. Food Chem. 59, 1690–1696. Charles, D.J., Simo, J.E., 1990. Comparison of extraction methods for the rapid determination of essential oil content and composition of basil. J. Am. Soc. Hortic. Sci. 115, 458–462. Clem, J.R., Havemann, D.F., Raebel, M.A., 1993. Insect repellent (N,N-diethyl-mtoluamide) cardiovascular toxicity in an adult. Ann. Pharmacother. 27, 289–293. Curtis, C.F., Hill, N., 1988. Comparison of methods of repelling mosquitoes. Entomol. Exp. Appl. 49, 175–179. Das, N.G., Baruah, I., Talukdar, P.K., Das, S.C., 2003. Evaluation of botanicals as repellents against mosquitoes. J. Vector Borne Dis. 40, 49–53. Gavriilidou, V., Boskou, D., 1991. Chemical interesterification of olive oil-tristearin blends for margarines. Int. J. Food Sci. Technol. 26, 451–456. Govindarajan, M., 2011. Ovicidal and repellent properties of Coccinia indica Wight and Arn (Family:Cucurbitaceae) against three important vector mosquitoes. Eur. Rev. Med. Pharmacol. Sci. 15, 1010–1019. Gul, S., Ibrahim, S., Wasif, N., Zafar, A., Syed, R., 2013. Mosquito repellents: killing mosquitoes or yourselves. J. Sci. Innov. Res. 2, 1052–1057. Lu, C., Liu, Y., 2002. Interaction of lipoic acid radical cations with vitamins C and E analogue and hydroxycinnamic acid derivatives. Arch. Biochem. Biophys. 406, 78–84. Nath, D.R., Bhuyan, M., Goswami, S., 2006. Botanicals as mosquito Larvicides. Def. Sci. J. 56, 507–511. Oparaocha, E.T., Iwu, I., Ahanakuc, J.E., 2010. Preliminary study on mosquito repellent and mosquitocidal activities of Ocimum gratissimum (L.) grown in eastern Nigeria. J. Vector Borne Dis. 47, 45–50. Phal, D., Patil, S., Naik, R., 2012. Concentration of d-trans allethrin in air after complete smoldering of mosquito repellent coil manufactured using different fillers. Int. J. Biol. Pharm. Allied Sci. 1, 1312–1321. Schreck, C.E., 1995. Protection from blood-feeding arthropods. In: Wilderness Medicine: Management of Wilderness and Environmental Emergencies, 3rd ed. Mosby, St. Louis. Shasany, A.K., Lal, R.K., Patra, N.K., Darokar, M.P., Garg, A., Kumar, S., Khanuja, S.P.S., 2000. Phenotypic and RAPD diversity among Cymbopogon winterianus Jowitt accessions in relation to Cymbopogon nardus Rendle. Gene. Resour. Crop Evol. 47, 553–559. World Health Organization, 2009. Guidelines for Efficacy Testing of Mosquito Repellents for Human Skin. WHO/HTM/NTD/WHOPES/2009.4. World Health Organization.
Contents lists available at ScienceDirect
Acta Tropica journal homepage: www.elsevier.com/locate/actatropica
Short Communication
Efficacy of Thai herbal essential oils as green repellent against mosquito vectors Mayura Soonwera a , Siriporn Phasomkusolsil b,∗ a b
Plant Production Technology Section, Faculty of Agricultural Technology, King Mongkut’s Institute of Technology Ladkrabang, Bangkok 10520, Thailand Department of Entomology, US Army Medical Component, Armed Forces Research Institute of Medical Sciences, Bangkok 10400, Thailand
a r t i c l e
i n f o
Article history: Received 14 August 2014 Received in revised form 18 November 2014 Accepted 21 November 2014 Available online 28 November 2014 Keywords: Repellency Cananga odorata oil Cymbopogon citratus oil Aedes aegypti Culex quinquefasciatus
a b s t r a c t Repellency activity of Thai essential oils derived from ylang ylang (Cananga odorata (Lamk.) Hook.f. & Thomson: Annonaceae) and lemongrass (Cymbopogon citratus (DC.) Stapf: Poaceae) were tested against two mosquito vectors, Aedes aegypti (L.) and Culex quinquefasciatus (Say). There were compared with two chemical repellents (DEET 20% w/w; Sketolene Shield® and IR3535, ethyl butylacetylaminopropionate 12.5% w/w; Johnson’s Baby Clear Lotion Anti-Mosquito® ). Each herbal repellent was applied in three diluents; coconut oil, soybean oil and olive oil at 0.33 l/cm2 on the forearm of volunteers. All herbal repellent exhibited higher repellent activity than IR3535 12.5% w/w, but lower repellent activity than DEET 20% w/w. The C. odorata oil in coconut oil exhibited excellent activity with 98.9% protection from bites of A. aegypti for 88.7 ± 10.4 min. In addition, C. citratus in olive oil showed excellent activity with 98.8% protection from bites of C. quinquefasciatus for 170.0 ± 9.0 min. While, DEET 20% w/w gave protection for 155.0 ± 7.1–182.0 ± 12.2 min and 98.5% protection from bites of two mosquito species. However, all herbal repellent provided lower repellency activity (97.4–98.9% protection for 10.5–88.7 min) against A. aegypti than C. quinquefasciatus (98.3–99.2% protection for 60–170 min). Our data exhibited that C. odorata oil and C. citratus oil are suitable to be used as green repellents for mosquito control, which are safe for humans, domestic animals and environmental friendly. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Repellents, clothing, netting and other personal protection measures can help prevent mosquito bites and the diseases mosquitoes carry. Especially, insect repellents are effective and safe when properly used. Repellent repels mosquitoes, it reduces the chances of being bitten. In this light, repellent use offers individuals added protection against mosquito-borne diseases. In the community, it reduces the transmission of mosquito-borne diseases (Gul et al., 2013). N,N-diethyl-m-toluamide (DEET) seems to be most effective and is the best studied insect repellent currently available to the general public. DEET is designed for direct application to human skin to repel insects, rather than kill them. Currently, DEET is formulated in aerosols, pump sprays, lotions, creams, liquids, sticks, roll-ons and impregnated towelettes, with concentrations ranging from 5% to 100%. However, there are concerns about the potential toxic effects of DEET, especially when used by children. Children who absorb high amounts of DEET through insect repellents have
∗ Corresponding author. Tel.: +66 089 124 6455. E-mail address: [email protected] (S. Phasomkusolsil). http://dx.doi.org/10.1016/j.actatropica.2014.11.010 0001-706X/© 2014 Elsevier B.V. All rights reserved.
developed seizures, slurred speech, hypotension and bradycardia (Clem et al., 1993). Mosquito repellents based on chemicals has a remarkable safety profile, but they are toxic against the skin and nervous system like rashes, swelling, eye irritation, and worse problems, though unusual including brain swelling in children, anaphylactic shock, and low blood pressure (Shasany et al., 2000; Phal et al., 2012). To overcome these problems, it is necessary to search for alternative methods of vector control. The failure of chemical insecticides to control the insect and growing public concern for safe food and a healthy environment have catalyzed the search for more environmentally benign control methods for the management of the vectors (Amerasan et al., 2012). Essential oil has been the active principle of most important herbal remedies since ancient times. Insecticides of plant origin have been extensively used on agricultural pests, and to a very limited extent, against insect’s vectors of public health importance, which deserve careful and thorough screening. The use of plant extracts for insect control has several appealing features, as these are generally more biodegradable, less hazardous, and rich storehouse of chemicals of diverse biological activity (Nath et al., 2006). In Thailand, several Thai herbs act as natural insect repellent, including ylang ylang (Cananga odorata) and lemongrass
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(Cymbopogon citratus) that expressed high degree of repellency against insect (Caballero-Gallardo et al., 2011; Adeniran and Fabiyi, 2012). The present investigation aims to assess the repellent properties of the essential oils of ylang ylang and lemongrass which mixed in the natural oils by six formulations against medically important mosquitoes of dengue vector (Aedes aegypti) and filarial vector (Culex quinquefasciatus). Additionally, the study utilized natural oils; soybean oil, coconut oil and olive oil used locally for cooking and cosmetic purposes to formulate the volatile oil into lotions to improve its acceptability and preclude any adverse effect that could emanate from use of synthetic bases. 2. Materials and methods 2.1. Mosquito cultures and rearing conditions A. aegypti and C. quinquefasciatus lab bred mosquitoes were used in this study. They were maintained in the laboratory of the Entomology and Environment Program, Plant Production Technology Section, Faculty of Agricultural Technology, King Mongkut’s Institute of Technology Ladkrabang (KMITL), Bangkok. Adult mosquitoes were reared at 25 ± 2 ◦ C, and a relative humidity of 80 ± 10% with a photoperiod of 12 h light followed by 12 h dark (12L:12D); adults were fed with 5% multivitamin solution. Prior to testing, 5- to 7-day-old female mosquitoes were starved by providing them with only water for 12 h. 2.2. Preparation of herbal essential oils In this paper, the efficacy of essential oils of ylang ylang (C. odorata (Lamk.) Hook.f. & Thomson) and lemongrass (C. citratus DC. Stapf) were compared. Flowers of ylang ylang (C. odorata) and stems of lemongrass (C. citratus) were collected and essential oils from plant parts were extracted by water distillation method (Charles and Simo, 1990). These essential oils were then prepared at 0.33 l/cm2 in three diluents; coconut oil, soybean oil and olive oil. 2.3. Chemical repellent (1) DEET, 20% w/w; Sketolene Shield® is a commercial chemical repellent in Thailand. (2) IR3535, 12.5% w/w Ethyl butylacetylaminopropionate; Johnson’s Baby Clear Lotion Anti-Mosquito® , is a commercial chemical repellent in Thailand. 2.4. Human volunteers Five adult volunteers of both sexes, 25–45 years old, weight 50–70 kg, who had no history of allergic reaction to arthropod bites were recruited. Before signing an informed consent form, the volunteers were interviewed and instructed on the methodology, probable discomforts to subjects and remedial arrangements. 2.5. Laboratory repellent bioassay Repellency of C. odorata and C. citratus oils and their formulations were evaluated against A. aegypti and C. quinquefasciatus under laboratory conditions using the methods which followed the guidelines by World Health Organization (2009). To compare the repellency of essential oils with that of the standard repellent, DEET 20% w/w and IR3535 12.5% w/w were tested. Due to different biting behavior, the tests against A. aegypti was carried out from 0800 am to 0400 pm, whereas those against C. quinquefasciatus was conducted between 0400 pm and 1200 pm because
Table 1 Repellency activities of two herbal essential oils which diluted in three diluents at 0.33 l/cm2 (coconut oil, soybean oil and olive oil) against Aedes aegypti. Herbal essential oils
Protection time (min)a
Ylang ylang oil + coconut oil Ylang ylang oil + soybean oil Ylang ylang oil + olive oil Lemongrass oil + coconut oil Lemongrass oil + soybean oil Lemongrass oil + olive oil DEET 20% (w/w) (Sketolene Shield® ) IR3535 12.5% (w/w) (Johnson’s Baby Clear Lotion® )
88.7 ± 10.4b 10.50 ± 2.1c
1.1 2.4
98.9 97.6
85.5 ± 12.0b 85.5 ± 10.5b
1.4 1.1
98.6 98.9
72.0 ± 12.4b
2.6
97.4
60.0 ± 12.5b 155.0 ± 7.1a
1.2 1.5
98.8 98.5
21.0
79.0
3.0 ± 0c
% Biting
% Protection
a Means in each column against each mosquito species followed by the difference letters are significantly different (P < 0.05, by one-way ANOVA and Duncan’s Multiple Range Test).
A. aegypti is a day-biter but C. quinquefasciatus is a night-biter (Govindarajan, 2011). Two hundred and fifty nonblood-fed starved female mosquitoes were randomly selected and placed in an experimental cage (30 cm × 30 cm × 30 cm) and left to acclimatize for 1 h. After cleaning with distilled water, each forearm of the volunteer was wrapped in a plastic sleeve attached with double-sided tape, and a cutout was aligned with a 3 cm × 10 cm area on the ventral portion of the forearm. Therefore, only a restricted zone of the skin was exposed to the mosquitoes. However, before the start of each exposure, the bare hand, used as control area (no treatment) of each volunteer, was exposed for up to 30 s. If at least two mosquitoes landed on the test area of the control arm, the arm was shaken off before imbibing any blood and withdrawn from the cage and the repellency test was then continued. This was done to ensure that the mosquitoes were still active. An amount of 0.1 ml of test repellent was applied to the marked area of one forearm of each volunteer. Subsequently, the test arm was introduced into the cage for 3 min. The total number of mosquitoes biting on the treatment was recorded. If no mosquito bite occurred within 3 min, the forearm was then taken out and the test was repeated every 30-min interval. The experiment was completed after two mosquitoes had bitten. The study period was carried out every 30 min until at least 2 mosquitoes bit during the 3 min study period, at which time the study was stopped. The protection time was the time from repellent application until the study was stopped. On each day, only one repellent preparation was tested to assure that residual material has disappeared from the skin before the next test (Curtis and Hill, 1988). 2.6. Statistical analysis The mean protection time was used as a standard repellency measure of the test samples against A. aegypti and C. quinquefasciatus in the laboratory. Differences in significance were analyzed by one-way analysis of variance (ANOVA) and Duncan’s multiple comparisons by SPSS for Windows (version 16.0). 3. Results Each of plant products was applied onto the forearm and they were evaluated. The results for plant oils repellency against A. aegypti were summarized in Table 1. Ylang ylang oil containing coconut oil provided longer lasting complete repellency (88.7 min) while the ylang ylang oil containing olive oil and lemongrass oil containing coconut oil which gave complete protection for
M. Soonwera, S. Phasomkusolsil / Acta Tropica 142 (2015) 127–130 Table 2 Repellency activities of two herbal essential oils which diluted in three diluents at 0.33 l/cm2 against Culex quinquefasciatus. Herbal essential oils
Protection time (min)a
Ylang ylang oil + coconut oil Ylang ylang oil + soybean oil Ylang ylang oil + olive oil Lemongrass oil + coconut oil Lemongrass oil + soybean oil Lemongrass oil + olive oil DEET 20% (w/w) (Sketolene Shield® ) IR3535 12.5% (w/w) (Johnson’s Baby Clear Lotion® )
126.7 ± 5.8b 60.0 ± 0d
0.8 1.2
99.2 98.8
85.5 ± 12.0c 115.5 ± 10.4b
1.4 1.1
98.6 98.9
84.0 ± 25.1c
1.7
98.3
170.0 ± 9.0a 182.0 ± 12.2a
1.2 1.5
98.8 98.5
24.2
75.8
3.0 ± 0c
% Biting
% Protection
a Means in each column against each mosquito species followed by the difference letters are significantly different (P < 0.05, by one-way ANOVA and Duncan’s Multiple Range Test).
85.5 min. In addition, both products of lemongrass oil containing soybean oil and olive oil gave complete repellency for 72 min and 60 min, respectively. However, there were not significant differences in the protection time among the repellents against A. aegypti (P > 0.05). WHO standard (2009) determines the repellency time against A. aegypti mosquitoes should be more than 2 h; none of the repellents met this requirement. DEET, which is the best-known synthetic insect repellent, at 20% concentration of active ingredient, provided the longest-lasting complete protection for 155 min or more than 2 h after application. Unfortunately, IR3535, which is the commercial insect repellent, at 12.5% concentration of active ingredient, showed no repellency against A. aegypti (3 min). The results of repellency tests on six different formulations against C. quinquefasciatus were shown in Table 2. The essential oil of lemongrass oil in olive oil provided the longest lasting complete repellency for 170 min after application. Compared against the standard mosquito repellent, DEET, lemongrass oil in olive oil performed equally well in protection time against C. quinquefasciatus (P > 0.05). However, IR3535-based repellent showed no repellency against mosquitoes (3 min after application). Furthermore, ylang ylang oil and lemongrass oil in coconut oil provided moderate protection time against C. quinquefasciatus, for 126.7 and 115.5 min, respectively. Moreover, the degree repellency was obtained from ylang ylang oil in olive oil (85.5 min), followed by lemongrass oil in soybean oil (84 min) and ylang ylang oil in soybean oil (60 min). There were significant overall among the efficacy of the essential oils against C. quinquefasciatus (P < 0.05). The percentage of repellency for C. quinquefasciatus ranged between 98% and 99% but reached only 75.8% for IR3535. 4. Discussion In this study, the standard repellent (DEET) has also been found to be effective. There was a significant different between the repellent activity of the standard and the test samples. Therefore, essential oils from C. citratus and C. odorata were investigated the repellency activity against A. aegypti and C. quinquefasciatus. The use of plant of the C. citratus or lemongrass as insect repellent is widespread throughout the world and the formulations tested represent the range of concentration which is commercially available. However, further investigation of formulation of C. odorata (ylang ylang) is still lack of information. In this study, we focused on the differences between repellent effect and feed deterrent effect. Because the protection times were determined as the time form material application until the first two bites. The
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percentage of repellency depended on the total number of landing and biting mosquitoes in treated and control areas. If the protection time of a compound was long while the percentage of repellency was high, this compound exhibited both high efficiency to repel mosquito as well as feed deterrent activity. If the protection time was low and also the percentage of repellency was high, then this compound was more a feed deterrent than a repellent. Conversely, if the protection time was long but the percentage of repellency was low, then the compound was more a repellent than a feed deterrent (Amer and Mehlhorn, 2006). Therefore, in this study shows that the protection time of ylang ylang oil in coconut oil and soybean oil and lemongrass in coconut oil were 85.5–88.7 min, but the repellency increased to 98.6–98.9%. This shows that these oils are only a feed deterrent and no repellent. Meanwhile, the protection period for lemongrass oil in olive oil against C. quinquefasciatus was 170 min, in addition, the protection time of ylang ylang oil and lemongrass oil in coconut oil were 126.7 and 115.5 min, while the repellency were up to 98%. Thus, these repellents showed the attributes of a repellent and a feed deterrent. The use of essential oils as commercial repellent products needs a formulation to fix the aromatic materials onto the skin for as long as possible. The present study compares a mixture of essential oil within the base material of plant products as coconut oil, soybean oil and olive oil. Some researchers evaluated that the repellent properties of mixture of essential oil in three base materials. Bite Blocker, a commercial preparation containing glycerin, lecithin, vanillin, coconut oil, geranium, and 2% soybean oil can achieve similar repellency to DEET, providing 7.2 h mean protection time against a dengue vector and nuisance biting mosquitoes in one study (Barnard and Xue, 2004). Oparaocha et al. (2010) examined the mosquito-repellent and mosquitocidal activities of the 30% (v/v) of Ocimum gratissimum in olive oil base exhibiting highest average percentage repellencies of 97.2%, 95.7% and 96.3% at World Bank Estate, Ihitte and Umuekunne centres, respectively. Das et al. (2003) investigated the repellent properties of Zanthoxylum limonella, Citrus aurantifolia and petroleum ether extract of Z. limonella (fruits) as repellent against Aedes albopictus in mustard and coconut oil base under laboratory conditions. However, the results showed that repellents in mustard oil afforded longer protection time against the bites of A. albopictus mosquitoes than those in coconut oil. Moreover, the natural oils which used in this experiment, there were also of great nutritional value and as well as therapeutic values. Coconut oil is an indispensable ingredient in cosmetics, as well as cooking. It possesses healing properties far beyond that of any other dietary oil. In traditional medicine, coconut oil is used to treat wide variety of health problem including the following, allergic conditions, bronchial asthma, gonorrhea, skin infections and malnutrition (Agarwal and Lakshmi, 2013). Meanwhile, soybean oil is a rich source of vitamin E. Vitamin E is essential to protect the body fat from oxidation and to scavenge the free radicals and therefore helps to prevent their potential effect upon chronic diseases such as coronary heart diseases and cancer (Lu and Liu, 2002). In addition, olive oil is a rich source of valuable nutrients and bioactives of medicinal and therapeutic interest. Olive fruit contains appreciable concentration, 1–3% of fresh pulp weight, of hydrophilic (phenolic acids, phenolic alchohols, flavonoids and secoiridoids) and lipophilic (cresols) phenolic compounds that are known to possess multiple biological activities such as antioxidant, anticarcinogenic, antiinflammatory, antimicrobial, antihypertensive, antidyslipidemic, cardiotonic, laxative, and antiplatelet. Olive oil is widely used for food preparations (as salad oil, cooking oil, in frying and pasta sauces), in cosmetics and the pharmaceutical industry (Gavriilidou and Boskou, 1991). Thus, the as well as the plants are abundant, the local people could be taught how to prepare the lotions.
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Currently, there is no effective vaccine against dengue, and due to insecticide resistance. Despite this fact, protection from mosquito bites could be achieved by avoiding physical contact with mosquitoes using insect repellents, and there are many commercially available repellents on the market. Many factors play a role in how effective any repellent is, including the frequency, dosage and uniformity of application, the number and species of the organisms attempting to bite, the user’s inherent attractiveness to blood-sucking arthropods, and the overall activity level of the potential host (Schreck, 1995). While the plants mentioned in this article have been shown to have mosquito-repelling properties, but requires reapplication to be effective over several hours. For the period the test was conducted and even several hours after the experiment, no skin irritation or dermatitis was observed on the treated skin of the human subject after application of the repellent treatments. An insect repellent of plant origin ought to be welldefined and harmless to human and other non-target organisms. Therefore, use of these botanical derivatives in mosquito control instead of synthetic insecticides could reduce the cost and environment effects. Acknowledgments The authors are highly grateful to Faculty of Agricultural Technology, King Mongkut’s Institute of Technology Ladkrabang (KMITL), Bangkok, Thailand for providing financial assistance to carry out this study. Grateful thanks are due to the volunteers from Plant Production Technology Section, Faculty of Agricultural Technology, KMITL for their assistance in repellent tests. Thanks are extended to plant taxonomist of Faculty of Agricultural Technology, KMITL for herbal identification. The standard disclaimer that the findings/conclusions reflect the efforts and opinions of the authors and not of the US Army or AFRIMS or the Department of Entomology. References Adeniran, O.I., Fabiyi, E., 2012. A cream formulation of an effective mosquito repellent: a topical product from lemongrass oil (Cymbopogon citratus) Stapf. J. Nat. Product Plant Resour. 2, 322–327.
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