Mosquito Repellent Activities of Ocimum Volatile Oils

O. CHOKECHAIJAROENPORN1, N. BUNYAPRAPHATSARA1and S. KONGCHUENSIN2

1 Medicinal Plant Inform ation Center, Faculty of Pharmacy, Mahidol University, Sri-ayuthaya Road, Bang• kok 10400, Thailand 2 Facult y of Science, Chulalongkorn University, Phyath ai Road, Bangkok 10330, Thail and.

Summary

Essential oils obtained from Ocimum americanum, O. basilicum, O. basilicum fa. citratum, O. gratissimum and O. tenuiflorum, were tested for mosquito repellent and larvicidal activities. All the oils exhibited both activities. O. basilicum showed the strongest larvicidal activity (ECso =81, EC90 =113 ppm ), while O. gratissimum exhibited the longest dur ation of action for mos• quito repellent activity (more than two hours). Gas chro matographic analysis indicated the pres• ence of camphor, caryoph yllene oxide, cineole, methyleugenol, limonen e, myrcene, and thymol, all kno wn insect repellents.

Key wo rds: mosquito repellent activity; larvicidal activity; Ocimum americanum; O. basilicum; O. basilicum fa. citratum; O. gratissimum; O. tenuiflorum.

Introduction Materials and Methods Plant Materials Haemorrhagic fever transmitted by Aedes aegypti is still one of the leading diseases in Thailand. The best way to di• Ocimum basilicum L. (Horapha a) (BK 19437) minish the incidence of this disease is to prevent children Ocimum basilicum L. fa. citratum Bach (Maeng Lak) (BK from being bitten by mosquitoes. Dur ing our search for me• 61602) dicinal plants possibly useful as insect repellents, we found Ocimum gratissimum L. (Ho raphaa Chang) (BK 59726) interesting information on Ocimum americanum. The peo• Ocimum americanum L. (Maeng ka-saeng) (BK 61603) ple in Prachuabkirikhan pro vince wear young shoots of this Ocimum tenuiflorum L. (Kaphrao) (BK 48492) plant behind their ears to repel small insects while working Th ese Ocimum spp. belong to the Lab iatae (Lamiaceae). in the fields. Thi s finding prompted us to carry out studies All the plants were cultivated at Salaya Medicinal Plant on the mosquito repellent and larvicidal activities of Oci• Gard en, Faculty of Pharm acy, Mahidol University, and mum species grown in Thailand . There are reports on the were identified by Associate Professor Busban Na-Songkla, insecticidal (Ma nsour et aI., 1986) and juvenile hormone Department of Botany, Faculty of Science, Chulalongkorn activities (Bowers & Nishida, 1980) of Ocimum oils. There University. The herbarium specimens were deposited at the arc also reports on the insect repellent activity of thymol, Bangkok Herbarium, Botany and Weed Sciences Section, carvacrol (Ma nsour et aI., 1986), camphor (Masui & Ko• Department of Agriculture, Ministry of Agriculture and chi, 1974), caryo phyllene oxide (Hubert & Wiemer, 1985), Coo pera tives. All the plants were planted in May, 1990 and and cineole (Klocke et aI., 1987; Verma, 1981; Verma and collected in the morning in August, 1990. Meloan, 1981), comp ounds found in Ocimum oils. There• fore, in this communication the attempt to identify these An imals and Insects compounds in Ocimum oils by GC and GC-MS analyses and the investigation of their insect repellent activities are Adult, female mosquitoes (Aedes aegypti (L.), Culicidae) reported. and their larvae were obtained from The Medical Science 136 O. Chokechaijaroenporn et al.

Department, The Ministry of Public Health. Five week old, 5890GC) and GC-MS (Jeol JMS-DX 300). Gas chroma• male mice (strain ICR) obtained from National Laboratory tography was equipped with FID and split type injector. A Animal Center, Mahidol University, were used. capillary column with 0.32 mm internal diameter and 25 m long containing 10% carbowax 20 M, was em• Volatile Oils ployed. The carrier gas was helium with a flow rate of 1 ml/min and the auxiliary gas was nitrogen with a flow All the volatile oils were obtained by steam distillation of rate of 29 mllmin. Temperatures were: injector 150°C, de• the aerial parts of Ocimum spp. The steam distillation of tector 260 °C, column at 50°C for 3 min, programmed to volatile oil was done by using a set of steam distillation 200°C at 5°C/min, and then maintained at 20°C for 3 equipment made of stainless steel with a 10 kg capacity. min. These column temperatures were programed for all The steam pressure was set at 2-3 bar throughout the distil• the oils except that of O. americanum, which was per• lation. formed under the following conditions: the column tem• The following reference compounds were obtained from perature was set up at 50°C for 15 min, programmed to Sigma Co.: borneol, camphene, camphor, a-caryophyllene, 110 °C with 10°Clmin, maintained at 110°C for 15 min, cineole, citral, eugenol, d-limonene, linalool, methylchavi• programmed to 200 °C with 10°C/min, and finally main• col, methyleugenol, ~-myrcene, y-terpinene and thymol. tained at 200°C for 3 min. The identification was done by comparison of the retention time with reference com• Determination of Larvicidal Activities pounds and GC-MS. Various concentrations of the volatile oil of each plant species were freshly prepared by dissolving the oil in dis• Results tilled water and adjusting the final volume to 100 ml (each concentration was prepared in triplicate ). Ten stage 4 mos• The essential oils were obtained from Ocimum basilicum, quito larvae were put into each of the solutions and after 24 O. gratissimum, O. tenuiflorum, O. americanum and O. hours the larvae were observed and the percentage of lar• basilicum fa. citratrum in yields of 1.07%, 1.03%,0.97%, val mortality was calculated and averaged. The EC and so 1.27% and 1.00%, respectively. EC values of each sample were calculated from the semi• 90 All the Ocimum oils exhibited larvicidal activity against logarithmic curve which was plotted between percentage of the mosquito larvae. The EC and EC values for the oils larval mortality and the concentrations of the volatile oils. so 90 of O. basilicum, O. basilicum fa. citratrum, O. american• urn, O. gratissimum and O. tenuiflorum are shown in Table Determination ofInsect Repellent Activities 1 and Fig. 1. Mice used as the insect attractors were immobilized with Table 1. The EC and EC values of Ocimum oils on the mortal• a clip and put into a plastic bag with a cut hole of 2 x 5 em. so 90 ity of mosquito larvae. The hole in the plastic bag was adjusted to be aligned with the abdomen of a mouse. This area was used as the obser• Plant Oils ECso (ppm) EC90 (ppm) vation area for the determination of insect repellent activity O. basilicum 81 113 of the volatile oils. O. basilicum fa. citratum 161 283 The essential oil of each plant species was freshly diluted O. americanum 168 279 to 10 percent with acetone and 0.1 ml of each solution was O. gratissimum 138 184 O. tenuiflorum 133 240 separately applied to each observed area on the mouse. Each solution was tested in triplicate and acetone at the same di• lution was used as a control sample. Fifteen minutes after All the Ocimum oils except that of O. basilicum exhibit• application, the mouse was put into the cage containing 250 ed complete repellent activity. The duration of action of O. mosquitoes. The observation was done every 15 seconds for gratissimum, O. basilicum, O. basilicum fa. citratum, O. 3 minutes by counting the number of mosquitoes attacking americanurn and O. tenuiflorum volatile oils were 135, 75, the observed area; the average number of mosquito attacks 75, 15 and 105 minutes, respectively (Fig.2). per 15 second period was recorded. The observation was Gas chromatographic and GC-MS analyses showed the done at 30 minute intervals after the first observation peri• presence of (active) compounds possibly responsible for in• od (:3 min) for 2 hours. The percentage of mosquito attacks sect repellent activities observed, i.e., camphor, caryophyl• was calculated and averaged for each observation. lene oxide, cineole, d-limonene, methyleugenol, myrcene, and thymol. Compounds found which were possibly re• Gas chromatographic Analyses ofthe Volatile Oils sponsible for the observed larvicidal activity were eugenol and methylchavicol (Table2). Qualitative analyses of the volatile oils were performed by means of gas chromatography (Hewlett Packard Mosquito Repellent Activities of Ocimum Volatile Oils 137

Table 2. Compounds found in Ocimum oils. Discussion Compounds Identifi- I II III IV V cation The investigation of the larvicidal activities, which was performed by using stage 4 mosquito larvae, showed that borneol 1 + + + + all the Ocimum oils exhibited larvicidal activity in a dose• ~-cadinene 2 + camphene 1 + dependent manner. The effectiveness order based on the camphor" 1 + + + +a + EC90s of the plant oils was o. basilicum > o. gratissimum 4-carene 2 + > o. tenuiflorum > o. basilicum fa. citratum =o. ameri• u-caryophyllene 1 + + + + canum (Fig. 1). )-~-caryophyllene (- 2 + + + The results of the study of mosquito repellent activities caryophyllene oxide" 2 + + were as follows: o. gratissimum volatile oil exhibited the (-)-cedrenol 2 + cineole" 1 + strongest activity, while that of o. americanum exhibited citraI 1 +a the least activity. The volatile oil of o. gratissimum showed ~-cubebene 2 + + complete repellence for a period of two hours. The volatile ~-elemene 2 oils of o. basilicum fa. citratum and o. tenuiflorum also +a eugenol" 1 + + completely repelled the mosquitoes, but their duration of d-Iirnonene" 1 + action was shorter, their activities only lasting for over one linalool 1 + + + + + methylchavicol" 1 + +a + + + hour. The volatile oil of o. basilicum exhibited almost com• methyleugenol'' 1 + + +a plete repellent activity with a duration of action of over one ~-myrceneb 1 + hour. That of o. americanum only exhibited complete re• myrtenol 2 + pellent activity for 15 minutes. Therefore o. gratissimum ~-ocimene 2 + + + volatile oil is the most potent mosquito repellent oil for po• u-selinene 2 + + tential commercial development. ~-selinene 2 + y-terpinene 1 + The known insecticidal compounds found in the volatile 4-terpinenol 2 + + + + oils of some Ocimum species, are methylcinnamate, terpinolene 2 + methylchavicol (Bowers and Nishida, 1980) and eugenol thymol" 1 + (Sharma and Saxena, 1974). Other chemicals which have Identification: 1 =comparison with known compounds, 2 =GC• been reported to have insect repellent activity are thymol, MS carvacrol (Mansour et aI., 1986), camphor (Masui and Ko• I =O. gratissimum, II =O. basilicum, III =O. basilicum fa. citra• chi, 1974), caryophyllene oxide (Hubert and Wiemer, tum, IV = o. americanum, V = o. tenuiflorum. 1985), cineole, limonene, and myrcene (Nutting et aI., a major constituents, b compound known as insect repellent, = = 1974). Therefore gas chromatographic analyses were per• c =compound known as insecticide. formed in an attempt to find any correlation between the observed activities and the constituents of the oils. The re• sults showed that O. basilicum oil, which contained meth-

o 0. basilicum L. 0 0. basilicum L. var. citratum []] O. americanumL. o 0. gratissimum L. • 0. tenuiflorum L. 350

300

250

200 ee, e, 150

Fig. 1. EDsoand ED90 values of plant oilson the mortality of mosquito larvae. The EDso 100 - and ED90 values of each oil were calculated from the semilogarithmic curve which was 50 - plotted betweenpercentage of larval mortal• ity and the concentrationsof the volatileoils. At least five concentrations were tested in triplicate for each oil. 138 O. Chokechaijaroenporn et al. a 1.2 b • O. gratissimum L. • O. tenuiflorum L. ~ ...... 0.8 8 Control ~ 8 Control .:.: .:.: ...... 0.8 ~ 0.6 ~ i 0.6 ~ 0.4 0 ::I .-=::I 0.4 C' C' til ~ 0.2 0 ~ 0.2 '" o'*------l_-_--"'''' "'''' "'''' ....."'''' -_"'''' 0 "'''' "'''' "'''' "'''' IS ~ ~ 1M 1~ IS 45 75 105 135 Observing time (min.) Observing time (min. )

O. americanum L. 1.4 c • d • 0. basilicum L...... 8 Control 0.8 8 Control ~ 1.2 ~ .:.:... .:.:... 0.6 ~ 0.8 ~ ~ ~ 0.4 0 0 '6 - .-=::I C' til C' 0.2 0 s ~ 0.2 ~ 0 0 "'''' 15 45 75 105 135 15 45 75 105 135 Observing time (min. ) Observing time (min.)

1.2 e • O. basilicum L. f. citratum 8 Control ~...... 1 .:.: ~ 0.8 ~ 0 0.6 ~ C' til 0.4 0 ~ 0.2 0 '" "'''' "'''' '" 15 45 75 105 135 Observing time (min.)

Fig. 2. Mosquito repellent activities of Ocimum oils. Each percentage of mosquito attacks on the observed area was calculated from 12 countings every 15 seconds for 3 minutes (see methods). Each oil was triplicately tested. (* and ** indicate significant differences from the controls with p < 0.05 and < 0.01, respectively).

ylchavicol as its major component, exhibited the strongest the oils, it was expected to be at least partly responsible for activity. Therefore, methylchavicol may be one of the active the mosquito-repellent activities observed. However, O. compounds. Eugenol may also be responsible for larvicidal americanum, which contained camphor as its major com• activity because it was found as a major component of O. ponent, did not show stro ng mosquito-repellent activity. gratissimum, which also exhibited potent larvicidal activity. Therefore camphor possessed only weak activity against In add ition, eugenol was also found in the volatile oils of O. mosquitoes. However, camphor may be active against basilicum fa. citratum and O. tenuiflorum. other insects as evidenced by its folkloric use as an insect Since camphor, a known insect repellent, was found in all repellent by Thais. Besides camphor, caryophyllene oxide Mosquito Repellent Activities of Ocimum Volatile Oils 139 was another potentially active compound found in O. gra• References tissimum, O. basilicum fa. citratum and O. tenuiflorum. Bowers, W.S., Nishida, R.: Juvocimenes: potent juvenile hormone Methyleugenol, a commercial insect repellent, was found as mimics from sweet basil. Science; 209: 1030-1032, 1980. the major component in O. tenuiflorum oil and as a minor Hubert, T.D., Wiemer, Q. E: Anti-repellent terpenoids from Me• component in O. basilicum and O. basilicum fa. citratum lampodium divaricatum. Phytochemistry; 24 (6): 1197-1198, oils. Myrcene, another insect repellent, was found only as a 1985. Klocke, J.A., Darlington, M. V., Balandrin, M. E: 1,8-Cineole (eu• minor component in O. americanum, a weakly repellent calyptol), a mosquito feeding and ovipositional repellent from oil. volatile oil of Hemizonia fitchii (Asteraceae). J. Chem. Ecol.; 13 In conclusion, Ocimum oils possess potent mosquito-re• (12): 2131-2141,1987. pellent activities, with camphor, caryophyllene oxide, Mansour, E, Ravid, D., Putievsky, E.: Studies of the effects of es• methyleugenol and myrcene as possible active constituents. sential oils isolated from 14 species of Labiatae on the carmine The variation in activity observed depends on the types and spider mite, Tetranychus cinnabaricus. Pbytoparasitica; 14: 137-142,1986. amounts of active constituents found in each oil. The two Masui, K., Kochi, H.: Camphor and tricyclodecane in deodorants most potent repellent oils were those of O. gratissimum and and insect repelling compositions. Japan Kokai 74: 100239. O. tenuiflorum. The oil of O. gratissimum contained only 1974, Through Chem. Abstr. 84: 165913 F (1976). one known insect repellent compound, but it exhibited Nutting, W.1., Blum, M. S., Fales, H. M.: Behaviour of the North strong activity which must be due to other components. American termite, Tenuirostritermes tenuirostris, with special The oil of O. tenuiflorum, another potent mosquito repel• reference to the soldier frontal gland secretion, its chemical com• lent contained a high content of methyleugenol, a commer• position, and its use in defense. Psyche; 81: 166, 1974, Through Chem Abstr 82: 1955H (1975). cial repellent. Although the oil of O. basilicum fa. citratum Sharma, R. N., Saxena, K. N.: Orientation and developmental in• did not contain known repellents as major components, it hibition in the housefly by certain terpenoids. J. Med. Entomol.; did contain three known repellents in smaller quantities, 11 (5): 617-621, 1974. camphor, caryophyllene oxide, and methyleugenol, which Verma, M. M.: The isolation and identification of a cockroach re• may contribute to the repellent activity observed. The oil of pellent in bay leaves and a fluorescence method for determina• O. basilicum, containing camphor and methyleugenol as tion of protein in wheat. Diss. Abstr. Int. B; 41: 4514,1981. Verma, M., Meloan, C. E.: A natural cockroach repellent in bay minor components, did not show complete repellent activ• leaves. Am. Lab.; 13 (10): 64-69, 1981. ity. In terms of commercial potency, all the Ocimum spp. are commonly found in Thailand. Therefore further stud• ies on humans and product development may lead to a low• Address ering of the incidence of haemorrhagic fever and the devel• opment of new commercial mosquito repellent products. In N. Bunyapraphatsara, Ph. D., Medicinal Plant Information addition to mosquitoes, studies on other pest insects are Center, Faculty of Pharmacy, Mahidol University, Sri-ayu• currently being carried out. thaya Road, Bangkok 10400, Thailand