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Repellent Activities of Essential Oils and Monoterpenes Against Culex Pipiens Pallens

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Repellent Activities of Essential Oils and Monoterpenes Against Culex Pipiens Pallens Journal of the American Mosquito Control Association, 18(4):348-351,2OO2 Copyright @ 2OOZby the American Mosquito Control Association,Inc. REPELLENT ACTIVITIES OF ESSENTIAL OILS AND MONOTERPENES AGAINST CULEX PIPIENS PALLENS WON-SIK CHOI,I BYEOUNG-SOO PARK,I SAE-KWANG KU? AND SUNG-EUN LEE14 ABSTRACT. Essential oils of Eulcalyptus globulus, Lovender fficinalis, Rosemarinus fficinalis, andThymus vulgaris were examined for their repellent activities against Culex pipiens pallens. A114 essential oils effectively repelled adult mosquitoes on hairless mice. Essential oil of T. vulgaris (thyme) had potent repellent activity within the tested materials, with a protection rate of 91Vo at a concentration of 0.057o topical treatment. Thyme essential oil significantly extended the duration of protection until 3 bites by mosquitoes. With gas chromatog- raphy-mass spectrometry analysis, thyme essential oil was a rich source of 5 monoterpenes, including in de- scending order thymol, p-cymene, carvacrol, linalool, and o-terpinene. These 5 monoterpenes also were assessed to determine their repellent activities to the mosquitoes. o-Terpinene had a potent repellent activity with a protection rate of 97Eo at a concentration of 0.057o topical treatment. Additionally, carvacrol and thymol showed an equivalent level of repellency. A spray-type solution containing 2Vo a-terpinene was tested for its repellent activity against Cx. pipiens. This solution showed stronger repellent activity than the currently used repellent N,N-diethyl-z-methylbenzamide (deet). KEY WORDS Thymus vulgaris, essential oil, Culex pipiens, repellents, d-terpinene INTRODUCTION yl-thio)ethyl)guanidine (eudragit El00; a histamine The control of mosquito vectors is becoming dif- antagonist); polyvinyl-pyrrolidone (povidone); and ficult because of resistance to insecticides (Chandre polyethylene glycol 100 (PEG 100) were kindly pro- et al. 1998, Penilla et al. 1998). As agenrs of chem- vided by the Dong-Wha Co. (Seoul, Korea). Four ical control, the use of repellents can be effective essential o|ls, Eucalyptus globulus (eucalyptus), l^av- for protecting humans from mosquitoes. One way ender officinalls (lavender), Rosemarinus fficinalis to prevent mosquito bites is to apply the repellent (rosemary), and Thymus vulgaris (thyme), were pur- N,N-diethyl-m-methylbenzamide (deet) to the skin. chased from the Nature Co. (Sydney, Australia). However, toxic reactions to deet have been found Five monoterpenes, carvacrol, p-cymene, linalool, ct- in some circumstances and age groups, although terpinene, and thymol, were purchased from Sigma these reactions do not seem to present serious prob- Chemical Co. (St. Louis, MO). lems for deet use (Mody et al. 1989, Clem et al. Animals and husbandry: All experiments were 1993). Nevertheless, there is a need to find alter- conducted in accordance with the Korean Food and natives to the currently used repellents. Drug Administration (KFDA) guidelines (98-116) Phytochemicals derived from various botanical in this study. All experimental animals were treated sources have provided numerous compounds with according to U.S. Environmental Protection Agen- potential use as repellents (Sukumar et al. 1991, cy guidelines. Watanabe et al. 1995). Essential oils and monoter- Mice: Twenty-five male SKH-I HRBR hairless penes are used as fragrances in cosmetics, food ad- mice (7 wk old, 26-38 g, Charles River Laborato- ditives, household products, medicine, and insecti- ries, Wilmington, MA) were used after acclimati- cides. Some of these generally are recognized as zation for 13 days. Animals were allocated 5 per safe by the U.S. Food and Drug Administration (FDA). polycarbonate cage in a temperature- and humidity- controlled room. The daily light cycle was 12:12 h In this paper, we report the mosquito repellency light:dark, and feed (Samyang Co., Seoul, Korea) of Thymus vulgaris (thyme) essential oil and its pri- mary constituents against Culex pipiens pallens Co- and water were supplied continuously. Animals quillett, by using the mouse bioassay system (Rut- were identified by marking the tail with a marking ledge et al. 1994). pen. Insects: Five hundred female mosquitoes (Cx. pipiens pallens F,, adults) were acquired from MATERIALS AND METHODS KFDA for use in this study. Mosquitoes were C hemical s: N, N-Diethyl-m-methylbenzamide; 2- reared at 27 + 2"C and 75-8OVo relative humidity -methyl-3-(2-(5-methyl-imidazol-4-yl-meth- cyano- 1 in a 12:12 h light:dark cycle and fed an 87o sugar solution in water. For each test, IOO 6- to 7-day- I Department of Lif'e Science, Soonchunhyang Univer- old nulliparous females were placed into a wire sity, Asan, Chungnam 337-745, Republic of Korea. screen cage measuring 30 X 30 X 3O cm. The sugar 'Pharmacology and Toxicology Lab, Dong-Wha Phar- solution pad was removed from this cage at least maceutical Co., Anyang, Kyounggido 430-010, Republic of Korea. 12 h before testing. 3 Department of Agricultural Chemistry, Kyungpook Bioassay: Bioassays were conducted according National University, Taegu 7O2-7O1, Republic of Korea. to the method of Rutledge et al. (1994). Test ma- a To whom corresoondence should be addressed. terials (0.057o in 0.1,Voethanol) were deposited on 348 DeceNasen2002 RepeLLnN-r AcrrvrrlEs oF ESSENTTALOrls ern MoNorprrexss 349 Thble 1. Spray formulation of test repellent topically Table 2. Repellent activities of essential oils (EO) applied to hairless mice. topically applied on hairless mice against Culex pipiens pallens Coquillett. Raw materials (7o) Content (g) Protec- Duration a-terpinene (2.OVo) 0.5 tion rate of protec- EudragitE100 (5.07o) 1.25 Tested EO Total bitesr (Vo) tion (min)' Povidone (2.07o) 0.5 PEG4OO(O.2Vo) 0.05 Control 77.2 + 1.64a3 77.8 + l.92ab Ethanol (90.8Vo) 22.7 Eucalyptus 5.2 + O.84b 70 33.2 + 4.O9ac Lavender 6.0 + 1.00b 65 31.0 + 3-67ac Rosemary 4.O + 1.71b 77 47.0 + 8-28ac Thymus 1.6 + 1.34c 9l 65.4 + l2.2bc rTotal bites were measured at I h after exposure to mosquitoes. 5 hairless mice and application of O.lVo ethanol 2 Mice were considered protected until 3 bites were recorded. served as a control. Treatments were applied with 3 Means within a column followed by a different letter ae sig- absorbent (sterile) cotton over the whole body to nificantly different (P < 0.05; Scheffe's test [SAS 1995]). point of runoff. After drying, treated mice were transferred to a mosquito cage containing 100 fe- RESULTS male mosquitoes. During each test, the cage was supported I 0 cm above the surface of the worktable Four essential oils (0.57o in O.lVa ethanol) pre- to permit air circulation. The number of mosquitoes vented mosquito biting in comparison with controls biting each mouse was recorded at 5-min intervals (Table 2). Numbers biting control mice at 5 min for I h. Total bites for I h on each mouse were after exposure to mosquitoes were zero, but after I -t calculated at end of the test. Tests were replicated h total numbers of bites were 17.2 1.64. On the 5 times. Total numbers of 5 tests were converted to other hand, total bites by mosquitoes at I h after mean + standard deviation. Mean totals were con- exposure to mice treated with eucalyptus, lavender, verted to percentage of the totals with the following rosemary, and thyme werc 5.2, 6.0, 4.O, and 1.6, ef- equation: respectively. Thyme essential oil was the most fective repellent among the test materials and pro- protection rate (Vo) vided 9l%o protection from mosquito bites. Thyme oil also showed 3.7-fold stronger protection time - : tl (total biting of treatment (65.4 min) than the control (17.8 min) until 3 bites analysis, thyme es- + total biting of control)l x 100. by mosquitoes. With GC-MS sential oil was a rich source of 5 monoterpenes, linalool, In addition, mouse skin tests for each active mono- including thymol, p-cymene, carvacrol, These 5 pri- terpene were examined to minimize the high vola- and a-terpinene, in descending order. oil were as- tility of the commercial formulae, as described in mary components of thyme essential repellent against mosquitoes. Thble l. The experimental procedures were exactly sessed for activities Two principal monoterpenes, ct-terpinene and thy- same as described as above. The control was an mol, had potent repellent activities with 96 and identical treatment, but without any mosquito-re- 977o protection, respectively, from mosquito bites pellent compound in the spray solution. Riferan-S@ when compared with deet, which provided 89Vo (a commercial deet product available as a spray; protection (Table 3). Shin-Shin Pharmaceutical Co., Seoul, Korea) was A spray-type solution (Table 4) was developed used as a positive control. Two percent o.-terpinene to minimize volatilization and to prolong the re- in a spray solution described in Table 1 was tested pellency of cr-terpinene to mosquitoes. The spray- and compared with the control. type solution containing 27o a-terpinene resulted in Tests for the determination of duration of protec- 8O.3Vorepellency after 3 h of exposure, whereas the tion were conducted according to the methods de- repellency of the control was 66.67o. The solution (1993). scribed by Frances et al. Duration of pro- also had 1.5-fold and 6-fold higher protection time tection was the time (minutes) until 3 bites were than the control and the positive-control Riferan-S, recorded. Tests were replicated 5 times. Total num- respectively (Fig. 1). bers of 5 tests were converted to mean + standard deviation. Identification of constituents in essential oils was DISCUSSION determined with electron-impact mass spectra ob- Application of thyme essential oil to experimen- tained by gas chromatography-mass spectrometry tal mice resulted in a 9lVo protection rate against (GC-MS) as described by Lee et al.
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