Mosquito Repellency of Essential Oils

In this study we evaluated and reported repellent effects of essential oils from Thai plants against 4 mosquito vectors: Aedes aegypti, Ae. albopictus, Anopheles dirus and Culex quinquefasciatus under laboratory conditions using human volunteers. The essential oils were extracted from 18 plant species, belonging to 11 families, and the oils were then prepared as 10% solution in absolute ethanol with additives. Two chemical repellents, deet and IR3535, were also prepared in the same formulation as the essential oil repellents and tested for repellency as controls. The essential oils were also evaluated for oviposition deterrent effects against Ae. aegypti under laboratory conditions. The results show night-biting mosquitoes (An. dirus and Cx. quinquefasciatus) and Ae. albopictus were more sensitive to all the essential oils (repellency 4.5 8 hours) than was Ae. aegypti (repellency 0.3 2.8 hours), whereas deet and IR3535 provided excellent repellency against all four mosquito species (repellency 6.78 hours). All essential oils exhibited oviposition deterrent activity against Ae. aegypti with various degrees of repellency ranging from 16.6 to 94.7%, whereas deet and IR3535 had no repellency. The present study demonstrates the potential for using essential oils as mosquito repellents and oviposition deterrents. These findings may lead to new and more effective strategies for protection from and control of mosquitoes. SOUTHEAST ASIAN J TROP MED PUBLIC HEALTH 916 Vol 37 No. 5 September 2006 spread by mosquito vectors when they visit and seek leisure away from their home country. Although the most common mosquito repellents currently available on the market containing deet (N,N-diethyl-3-methylbenzamide) have shown excellent protection from mosquito bites (Yap, 1986; Walker et al, 1996; Thavara et al, 2001) and other biting insects (Coleman et al, 1993), there were reports of toxicity problems after application of deet range from mild effects, such as contact urticaria (Maibach and Johnson, 1975) and skin eruption (Reuveni and Yagupsky, 1982), to severe reactions, such as toxic encephalopathy (Zadikoff, 1979; Roland et al, 1985; Edwards and Johnson, 1987). To overcome these adverse effects, attempts to find and develop repellents derived from plant extracts have been made by many researchers. In Thailand, some plant extracts, such as basil (Chokechaijaroenporn et al, 1994), galanga (Choochote et al, 1999), turmeric (Tawatsin et al, 2001), aromatic turmeric (Pitasawat et al, 2003), celery (Choochote et al, 2004; Tuetun et al, 2004) and clove (Trongtokit et al, 2004) have been investigated for repellent activity against various mosquito species under laboratory and field conditions. The development and use of locally available plants showing repellent activity avails an alternative strategy for the control or minimization of mosquitoborne diseases, especially in developing countries. In the present study, we evaluated and report on the repellent effects of essential oils extracted from 18 species of Thai plants against four mosquito vectors: Aedes aegypti (L.), Ae. albopictus (Skuse), Anopheles dirus Peyton & Harrison, and Culex quinquefasciatus Say under laboratory conditions. Comparison of repellency over different exposure periods was also carried out to standardize repellent testing methods. In addition, we evaluated the oviposition deterrent activity of each repellent composition against Ae. aegypti under laboratory conditions. MATERIALS AND METHODS Plant species Eighteen plant species belonging to 11 families were selected for this study because most of them are known or used traditionally as mosquito repellents by Thai people. They were Eleutherococcus trifoliatus (L.) (Phak paem), Schefflera leucantha R. Vig. (Hanuman prasankai), Ocimum sanctum L. (Holy basil), Vitex trifolia L. (Khon thi so), Litsea cubeba (Lour.) Pers. (Ta khrai ton), Manglietia garrettii Craib (Montha doi), Aglaia odorata Lour. (Prayong), Myristica fragans Houtt. (Nutmeg tree), Melaleuca cajuputi Powell (Cajuput tree), Psidium guajava L. (Guava), Piper betle L. (Betel pepper), Piper nigrum L. (Black pepper), Murraya paniculata (L.) Jack (Orange jasmine), Houttuynia cordata Thunb. (Fishwort), Zingiber officinale Roscoe (Ginger), Alpinia galanga (L.) Wild (Galanga), Curcuma longa L. (Turmeric), and Hedychium coronarium J. Konig (White ginger). Extraction of essential oils Essential oils were extracted from each plant by steam distillation. One to two kilograms of fresh plant material (by particular part of each plant, see Table 1) were cut into small pieces and placed in a distillation flask with approximately five times as much water, and 10 glass beads. The distillation chamber was heated in a liquid paraffin bath at about 120oC until the distillation was completed. The distillate was collected in a separate funnel in which the aqueous portion was separated from the essential oil (oily phase). The aqueous phase (lower layer) was slowly drawn off until only the oil layer remained. This procedure was repeated until at least 5 ml of essential oil was collected. Each essential oil was kept in a screwed-cap glass vial at 4oC until it was tested for mosquito repellency and ovipositional deterrent activity. Analysis of chemical constituents All essential oils were analyzed for chemiMOSQUITO REPELLENCY OF ESSENTIAL OILS Vol 37 No. 5 September 2006 917 cal constituents employing the Gas Chromatography / Mass Spectroscopy (GC/MS) assay. Briefly, the essential oil (50 μl) was diluted with 1.5 ml of hexane and CH2Cl2 (1:1) to a final concentration of 3.33% v/v. The diluted sample (0.1 μl) was then injected into the column (DBTM-1ms, 30 m x 0.25 mm x 0.25 μm, 100% dimethylpolysiloxane) for analysis with a GC-MS instrument (QP2010, Shimatzu). The operation conditions were as follows: the injection temperature was 200oC. Helium was used as a carrier gas and the purge flow rate was 3 ml/minute. The pressure was 69.4 kPa and the split ratio was 1:100. The chemical constituents of each essential oil were obtained by searching each peak and comparing with data from the National Institute of Science and Technology (NIST) library spectra. The relative amounts of the individual chemical components of each essential oil were computed from the GC peak areas (%). Preparation of repellents for testing The essential oils were formulated as 10% lotion in absolute ethanol and additives (vanillin, propylene glycol and polyethylene glycol). For comparison with standard repellents, two chemical repellents, N,N-diethyl-3-methylbenzamide (deet) and ethyl butylactylamino-propionate (IR3535), were formulated as 10% lotion similar to the essential oil repellents. All formulated repellents were placed in screw-cap vials and kept at room temperature before testing. Test mosquitoes The mosquitoes used in this study were laboratory-reared female mosquitoes (age 4-5 days) Aedes aegypti, Ae. albopictus, Anopheles dirus, and Culex quinquefasciatus. These were reared according to the standard protocol of the National Institute of Health, Thailand, and maintained at the insectary of the institute. Repellent test The repellency of essential oils and standard repellents was assessed in the laboratory using a human-bait technique (Tawatsin et al, 2001). Ethical clearance was approved by the Ethics Committee, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand (TM-IRB004/2005). Six volunteers (age 25-61 years) participated in the laboratory tests. The testing period lasted up to eight hours, depending on the efficacy of repellent. The timing of the tests depended on whether the target mosquitoes were dayor night-biters. Ae. aegypti and Ae. albopictus were tested during the daytime from 0900 to 1700, while An. dirus and Cx. quinquefasciatus were tested during the night from 1900 to 0300. Evaluations were carried out in a 6x6x3 m room, at 25-29oC with relative humidity of 6080%. An area of 3x10 cm on each forearm of the six human volunteers was marked out with a permanent marker. Each test repellent formulation (0.1 ml) was applied to the marked area of one forearm of each volunteer while the other forearm was treated with 0.1 ml of solution base (without active ingredient) as a control. Before the start of each exposure period, the bare hand of the test person, used as control area for each volunteer, was exposed for up to 10 seconds in a mosquito cage (30x30x30 cm), containing 250 hostseeking female mosquitoes (4-5 days old). If at least two mosquitoes landed on or bit the hand, the repellency test was then continued. This was done to ensure that the mosquitoes were host seeking. Then each volunteer put the test forearm and hand covered by a paper sleeve with a hole corresponding to the marked area into the mosquito cage for the first three minutes of each half-hour interval. The number of mosquitoes biting the treated area of each volunteer was recorded each minute (at 1, 2 and 3 minutes) of each 3minute exposure. To determine the duration of protection for each repellent, the exposures continued until at least two bites occurred in a given exposure period, or until a bite in the previous exposure period was followed by a confirmatory second bite in the following exSOUTHEAST ASIAN J TROP MED PUBLIC HEALTH 918 Vol 37 No. 5 September 2006 posure period. The time between application of the test repellent and the second successive bite was recorded as the protection time. Ovipositional deterrent test Ovipositional deterrent activity of essential oils and standard repellents were studied for gravid Ae. aegypti under laboratory conditions at room temperature. Two black plastic cups (300 ml in capacity) were filled with 200 ml de-ionized water. One cup was a control and the other cup was treated with essential oil (undiluted) or standard chemical repellent (deet or IR3535) at dosage of 20 μl/cup. The final concentration of the treated material (essential oil or chemical repellent) in each treated cup was 0.01%. Each cup was fitted inside with a white filter-paper sheet (7x28 cm) for deposition of mosquito eggs. The paper was located in each cup so as the lower half of the paper was submerged in water. The cups were placed in a mosquito cage (30x30x30 cm) containing 50 gravid female mosquitoes for 48 hours then, the eggs laid in each cup were counted after removal of the oviposition paper. Each test repellent was tested in six cages. The percentage of repellency for each essential oil and standard repellent was calculated by Xue et al (2001) as follows: C-T Repellency (%) = x 100 C where C stands for the number of mosquito eggs collected from the control cup and T denotes the number of mosquito eggs collected from the treated cup. Data analysis The mean protection time was used as a standard measure of repellency for the essential oils, deet and IR3535 against the four mosquito species. Comparison of repellency for each test repellent derived from the different exposure per iods and oviposi t ion deterrency against gravid Ae. aegypti were carried out employing the one-way analysis of variance (ANOVA) with Duncan’s multiple range test. All differences were considered significant at p ≤ 0.05.