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Aromatic plant-derived essential oil: An alternative larvicide for mosquito control
Fitoterapia 78 (2007) 205 – 210 www.elsevier.com/locate/fitote
Aromatic plant-derived essential oil: An alternative larvicide for mosquito control B. Pitasawat a,⁎, D. Champakaew a , W. Choochote a , A. Jitpakdi a , U. Chaithong a , D. Kanjanapothi b , E. Rattanachanpichai a , P. Tippawangkosol a , D. Riyong a , B. Tuetun a , D. Chaiyasit a a
Department of Parasitology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand b Chulabhorn Research Institute, Chiang Mai 50200, Thailand Received 11 November 2005; accepted 8 January 2007 Available online 6 February 2007
Abstract Five aromatic plants, Carum carvi (caraway), Apium graveolens (celery), Foeniculum vulgare (fennel), Zanthoxylum limonella (mullilam) and Curcuma zedoaria (zedoary) were selected for investigating larvicidal potential against mosquito vectors. Two laboratory-reared mosquito species, Anopheles dirus, the major malaria vector in Thailand, and Aedes aegypti, the main vector of dengue and dengue hemorrhagic fever in urban areas, were used. All of the volatile oils exerted significant larvicidal activity against the two mosquito species after 24-h exposure. Essential oil from mullilam was the most effective against the larvae of A. aegypti, while A. dirus larvae showed the highest susceptibility to zedoary oil. © 2007 Elsevier B.V. All rights reserved. Keywords: Larvicidal activity; Aedes aegypti; Anopheles dirus; Carum carvi; Apium graveolens; Foeniculum vulgare; Zanthoxylum limonella; Curcuma zedoaria
1. Introduction Interest in the control of Aedes aegypti and Anopheles dirus lies in the fact that they act as vectors of dengue and dengue hemorrhagic fever, and malaria, respectively, which are serious public health problems in Thailand and many developing countries. Although some vector-transmitting diseases such as Japanese encephalitis and yellow fever have been reasonably brought under control by vaccination, no effective vaccine is available for dengue or malaria. Therefore, the only efficacious approach of minimizing the incidence of these diseases is to eradicate and control mosquito vectors mainly by application of insecticides to larval habitats, destroying unwanted containers, and educating the public [1]. During epidemics, these measures are complemented by insecticide space-spraying against adult mosquitoes. However, aerial toxicants for eradicating A. aegypti are not effective, since this species is highly
⁎ Corresponding author. Tel.: +66 53 945343; fax: +66 53 217144, +66 53 945347. E-mail address: [email protected] (B. Pitasawat). 0367-326X/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2007.01.003
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B. Pitasawat et al. / Fitoterapia 78 (2007) 205–210
domesticated and many adults rest in hidden places indoors [2]. It is acknowledged that attacking breeding places with larvicides is a potential alternative to diminish mosquito populations. The most commonly used larvicides are organophosphorus compounds such as temephos, fenthion and chlorpyrifos, which are highly active against mosquito larvae and other aquatic insects. Organophosphorate temephos is recommended as the most appropriate larvicide for the control of Aedes and Anopheles, because it has low toxicity to fish, birds, mammals and humans [3,4]. However, its toxicity to fish and other nontarget organisms as well as the environment, and the insecticide resistance of arthropods, are being increasingly reported [5–8]. Even though susceptibility of various species of mosquito larvae to plant products has been studied extensively, most plant-derived constituents have shown tendencies to exhibit slower actions and weaker effects compared with synthetic compounds. The plant-derived natural products, however, still have immense potential for the control of mosquito vectors, if they are reasonably effective and harmless to beneficial nontarget organisms and the environment. Determination of the larvicidal activity of AkseBio2, a new botanical natural product derived from a mixture of various plant extracts, revealed that this product is promising as a larvicide against Culex pipiens and could be useful in the search of new larvicidal natural compounds [9]. Recently, essential oils derived from plants have received much interest as potential bioactive agents against mosquito vectors. Many plant oils such as basil, cinnamon, citronella and thymus are promising as mosquito larvicides [10–13]. Investigation of plant oil-derived larvicides used for mosquito control is, therefore, a vital point of this research. The selection of plants used in this study focused on those belonging to similar or associated plant families reported to have potential against mosquitoes [10,14,15]. In addition, herbs used as vegetables and spices in traditional medicine were mainly considered in the search for effective and safe materials. 2. Experimental 2.1. Plants Carum carvi L. (Apiaceae), Apium graveolens L. (Apiaceae), Foeniculum vulgare Mill (Apiaceae), Zanthoxylum limonella Alston (Rutaceae) and Curcuma zedoaria Roscoe (Zingiberaceae) were obtained from commercial suppliers, Phachinai Industry or E.A.R. Samunpri, in Chiang Mai Province, Thailand. The voucher specimens were deposited in the Department of Parasitology, Faculty of Medicine, Chiang Mai University, Thailand. 2.2. Extraction and preparation Essential oils from each plant material were steam-distillated. The obtained oil was dried over anhydrous sodium sulfate, collected and kept in a light-protected bottle under refrigeration at 4 °C for later investigation. 2.3. Test mosquitoes A. dirus was obtained from the Armed Forces Research Institute of Medical Sciences (AFRIMS). A. aegypti was collected at various breeding places in Chiang Mai Province, northern Thailand. The free-mating colonies of these mosquitoes were established in the insectarium of the Department of Parasitology, Faculty of Medicine, Chiang Mai University, by following the common standard mosquito rearing technique previously described [16]. The mosquito colony was maintained continuously at 25–30 °C and 80–90% relative humidity under a photoperiod of 14:10 h (light/
Table 1 Physical and organoleptic properties, and yields of volatile oils derived from C. carvi, A. graveolens, F. vulgare, Z. limonella and C. zedoaria Plants
Voucher specimen
Appearance
Color
Odor
Density (g/ml)
% Yield (v/w)
C. carvi A. graveolens F. vulgare Z. limonella C. zedoaria
PARA-CA-001/1 PARA-AP-001/2 PARA-FO-001/1 PARA-ZA-001/1 PARA-CU-001/2
Liquid Liquid Liquid Liquid Viscous liquid
Light yellow Pale yellow Pale yellow Light green Golden yellow
Pepper-like Orange-like Pepper-like Lemon-like Cineolic-like
0.93 0.89 0.97 0.84 0.90
1.46 1.25 0.77 5.72 1.17
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dark) for over 20 generations in a laboratory free of exposure to pathogens or insecticides. Freshly molted larvae of A. dirus and A. aegypti were continuously available for the mosquito larvicidal experiments. 2.4. Determination of larvicidal activity Mosquito larvicidal assays were carried out according to the standard procedures of the WHO [17], with slight modifications. Each plant's volatile oil was diluted in dimethylsulphoxide (DMSO) to prepare a serial dilution of test dosage. Early fourth instar larvae of each mosquito species were selected, transferred in 25 ml of distilled water (25 larvae/beaker), and allowed to acclimatize for 1 h before testing. For experimental treatment, 1 ml of serial dilutions was added to 224 ml of distilled water in a 500-ml enamel bowl and shaken lightly to ensure a homogeneous test
Table 2 Larvicidal activity of volatile oils derived from C. carvi, A. graveolens, F. vulgare, Z. limonella, C. zedoaria on A. aegypti Volatile oil (ppm)
C. carvi 37.2 46.5 55.8 65.1 74.4 83.7 A. graveolens 26.7 35.6 44.5 53.4 62.3 71.2 F. vulgare 42.7 44.6 46.6 48.5 50.4 52.4 54.3 56.3 Z. limonella 8.4 12.6 16.8 21.0 25.2 29.4 33.6 C. zedoaria 18.0 22.5 27.0 31.5 36.0 40.5 45.0 a
A. aegypti % Mortality (Mean ± SE)
12.67 ± 9.29 22.67 ± 9.02 54.33 ± 8.08 73.00 ± 22.65 87.67 ± 11.59 97.00 ± 3.61 13.67 ± 15.18 41.67 ± 30.83 55.33 ± 31.09 68.00 ± 28.21 78.33 ± 25.66 96.67 ± 4.16 17.67 ± 8.08 25.33 ± 12.34 31.00 ± 14.73 37.33 ± 10.02 48.00 ± 3.00 73.00 ± 6.00 79.00 ± 2.65 88.33 ± 5.86 1.00 ± 1.00 7.33 ± 0.58 21.33 ± 4.04 39.33 ± 8.33 43.33 ± 12.01 67.33 ± 15.31 78.00 ± 5.00 4.33 ± 2.31 12.67 ± 0.58 29.00 ± 7.00 52.33 ± 14.19 63.67 ± 16.50 77.00 ± 16.09 86.67 ± 11.06
Larvicidal activity a LC50
LC95
LC99
54.62 (52.62–56.64)
90.06 (83.09–100.76)
119.21 (105.70–141.33)
42.07 (39.62–44.56)
99.12 (85.00–124.85)
160.25 (126.82–228.68)
49.32 (48.70–49.94)
62.09 (60.08–64.88)
70.64 (67.23–75.53)
24.61 (23.29–25.98)
55.81 (47.50–72.01)
88.32 (69.10–130.72)
31.87 (30.71–33.02)
55.50 (51.05–62.30)
75.75 (66.73–90.46)
The lethal concentrations with the corresponding 95% confidence intervals are shown in parenthesis.
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solution. Selected mosquito larvae were transferred in distilled water into a bowl of prepared test solution with a final surface area of 124 cm2 (25 larvae/bowl). Four replicates were run simultaneously yielding a final total of 100 larvae for each dosage, with at least five dosages providing a range of 0–100% mortality. For each test, controls and untreated sets containing a mixture of 1 ml of diluent DMSO dissolved in 249 ml of distilled water and 250 ml of distilled water, respectively, were also run for comparison. Symptoms of treated larvae were observed and recorded immediately and at time-intervals. Mortality and survival were determined after 24 h of exposure, during which time no food was offered to the larvae. The moribund and dead larvae in the four replicates were combined and expressed as the percentage mortality for each concentration. Larvae were considered dead or moribund if they were unrousable within a reasonable period of time, even when gently prodded with a needle, as described in the World Health Organization's technical report series. They might also show discoloration, unnatural positions, tremors, incoordination, or rigor. Three independent experiments for each volatile oil against each mosquito species from different rearing batches were carried out at 25–30 °C on separate days.
Table 3 Larvicidal activity of volatile oils derived from C. carvi, A. graveolens, F. vulgare, Z. limonella, C. zedoaria on A. dirus Plant oil (ppm)
C. carvi 46.5 55.8 65.1 74.4 83.7 93.0 A. graveolens 35.6 44.5 53.4 62.3 71.2 80.1 89.0 F. vulgare 33.9 34.9 35.9 36.9 37.8 38.8 Z. limonella 37.8 42.0 46.2 50.4 54.6 58.8 63.0 C. zedoaria 23.4 25.2 27.0 28.8 30.6 32.4 a
A. dirus % Mortality (Mean ± SE)
4.00 ± 3.61 11.00 ± 10.82 30.00 ± 13.53 57.67 ± 10.21 75.00 ± 5.57 91.33 ± 2.52 10.25 ± 8.34 24.00 ± 8.29 36.00 ± 13.56 50.75 ± 19.47 68.75 ± 4.92 84.25 ± 3.10 92.75 ± 3.30 25.00 ± 14.11 39.00 ± 17.06 63.67 ± 7.02 83.00 ± 5.20 88.00 ± 3.61 93.33 ± 3.06 4.00 ± 1.73 8.33 ± 0.58 13.33 ± 0.58 18.67 ± 4.73 34.00 ± 2.65 59.33 ± 8.62 74.00 ± 9.54 4.33 ± 6.66 7.00 ± 6.00 27.00 ± 11.14 46.33 ± 15.28 54.33 ± 20.50 70.67 ± 14.15
Larvicidal activity a LC50
LC95
LC99
72.28 (70.23–74.53)
104.69 (97.19–117.23)
128.87 (115.46–152.62)
59.44 (56.87–61.98)
111.69 (99.59–133.00)
159.06 (133.47–208.45)
35.27 (35.01–35.50)
38.78 (38.19–39.64)
40.90 (39.95–42.34)
57.22 (56.10–58.51)
76.23 (72.04–82.93)
89.52 (82.40–101.43)
29.69 (29.04–30.37)
40.23 (37.25–46.67)
47.70 (42.40–60.00)
The lethal concentrations with the corresponding 95% confidence intervals are shown in parenthesis.
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2.5. Statistical analysis It was essential to obtain not less than 3 mortality counts of between 10% and 90%. If the mortality in controls was between 5 and 20%, correction of the observed percentage mortality (%M) was performed by applying the Abbott's formula [18]: %M ¼
% test mortality−% control mortality 100 100−% control mortality
When the control mortality was over 20%, the tests were discarded and repeated. The average mortality percentage derived from individual tests was determined. The lethal concentrations of 50%, 95% and 99% (LC50, LC95 and LC99, respectively) as well as the corresponding 95% confidence intervals (95% CI), used to measure differences between plant samples, were determined by using a computerized log-probit analysis (Harvard Programming; Hg1, 2). 3. Results and discussion Steam distillation of C. carvi, A. graveolens, F. vulgare, Z. limonella and C. zedoaria yielded from 0.77 to 5.72% (v/w, dry weight) of volatile oils (Table 1). The highest yield of volatile oil was obtained from Z. limonella, whereas that of F. vulgare was the lowest. All volatile oils obtained were less dense than water, and their physical and organoleptic properties presented in Table 1 demonstrated differences in appearance, color and odor. The larvicidal activities of the essential oils against mosquito larvae under laboratory conditions are given in Tables 2 and 3. All plant oils exerted significant larvicidal potential against A. aegypti (Table 2) and A. dirus (Table 3), after exposure for 24 h. The lethal dosage of 50% (LC50) on A. aegypti larvae ranged between 24.61 and 54.62 ppm and on A. dirus between 29.69 and 72.28 ppm. Z. limonella oil was the most effective against A. aegypti with LC50 and LC95 values of 24.61 and 55.81 ppm, respectively. A. dirus larvae showed the highest susceptibility to C. zedoaria oil (LC50 and LC95 values: 29.69 and 40.23 ppm, respectively). After exposure to the essential oils, the treated larvae exhibited restlessness, sluggishness, tremors and convulsions followed by paralysis at the bottom of the bowl. After that, the number of toxic affected larvae increased, and some if not all of them subsequently died within 24 h. Some A. aegypti larvae, treated with fennel oil, paralyzed and sank to bottom of the container, and after a short time of exposure recovered within 4–7 h. After 24 h, however, some moribund and dead larvae were found. Finally, ethnobotanical data and chemical composition of the used plants are reported in Table 4. Chemical identification by gas chromatography coupled with mass spectrometry (GC/MS) revealed the composition of the investigated oils. Carvone was a main constituent representing 32.7% of C. carvi oil. A principal component found in essential oils that were derived from A. graveoles and Z. limonella was limonene (60% and 31%, respectively). F. vulgare oil was found to contain anethole (85.63%) as a major substance, while a high content of 1,8-cineole (18.5%), p-cymene (18.42%) and α-phellandrene (14.93%) was found in C. zedoaria oil. These compounds have been isolated Table 4 Ethnobotanical data and chemical composition of C. carvi, A. graveolens, F. vulgare, Z. limonella and C. zedoaria Plants
English name
Part used
C. carvi
Caraway Seed
Phytomedicine and pharmacology
Treatment of digestive disorders; laxative on the duodenum; antiproliferative; antiulcerogenic; antibacterial A. graveolens Celery Seed Treatment of arthritis, nervousness, hysteria, headaches, bronchitis, asthma, liver and spleen disease; hepatoprotecive; sedative; anticonvulsive; anticarcinogen F. vulgare Fennel Fruit Treatment of dyspeptic complaints, respiratory disorder, pediatric colic; promoter of menstruation; hepatoprotecive Z. limonella Mullilam Fruit Treatment of wounds; promoter of digestion; anthelminthic; gastrointestinal stimulant C. zedoaria Zedoary Rhizome Treatment of wounds; promoter of digestion; relief of flatulence and colic; antiseptic; antimicrobial activity
Chemical composition of plant oil (GC/MS)
References
Carvone (32.7%), limonene (17.4%), γ-terpinene (17.2%) D-Limonene (60%), selinene (10%), phthalides (3%)
[21–25] [26–31]
trans-Anethole (85.63%), estragole [32–37] (5.27%), D-limonene (3.8%), p-anisaldehyde (2.68%) D -Limonene (31%), terpin-4-ol [38–40] (13.94%), sabinene (9.13%) 1,8-Cineole (18.5%), p-cymene [41,42] (18.42%), α-phellandrene (14.93%)
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from many herbal oils [12,19,20] that showed mosquito larvicidal activity. The efficacy of each of these compounds and their synergistic mechanisms need to be tested, to see if any have a potential use against mosquitoes. In conclusion, this study clearly illustrated the efficacy of the investigated herbs, which encourages the development of alternative active ingredients as effective mosquito larvicides. Acknowledgements The authors are thankful to the botanist J.F. Maxwell from CMU Herbarium, Department of Biology, Faculty of Science, Chiang Mai University. Special thanks also go to the Armed Forces Research Institute of Medical Sciences (AFRIMS) for providing the test mosquito, Anopheles dirus. Acknowledgment is extended to the Faculty of Medicine Research Fund, Chiang Mai University for their financial support, and the Faculty of Medicine Endowment Fund for Research Publication for helping defray the publication cost. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16]
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Corbel V, Duchon S, Zaim M, Hougard JM. J Med Entomol 2004;41:712. Ciccia G, Coussio J, Mongelli E. J Ethnopharmacol 2000;72:185. WHO. WHO Tech Rep Ser 1973;513. Rozendaal JA. Vector control. Methods for use by individuals and communities. Geneva: WHO; 1997. Mittal PK, Adak T, Sharma VP. Indian J Malariol 1991;28:167. Brown MD, Thomas D, Kay BH. J Am Mosq Control Assoc 1998;14:463. Pinkney AE, McGowan PC, Murphy DR, Lowe TP, Sparling DW, Meredith WH. J Am Mosq Control Assoc 1999;15:321. NICC-Comunicação e Saúde., 2003. Available at: www.cpqam.fiocruz.br/nicc/comunic/jc200101.htm. Cetin H, Erler F, Yanikoglu A. Fitoterapia 2004;75:724. Mansour SA, Messeha SS, el-Gengaihi SE. J Nat Toxins 2000;9:49. Cheng SS, Chang HT, Chang ST, Tsai KH, Chen WJ. Bioresour Technol 2003;89:99. Cheng SS, Liu JY, Tsai KH, Chen WJ, Chang ST. J Agric Food Chem 2004;52:4395. Cavalcanti ESB, de Morais SM, Lima MAA, Santana EWP. Mem Inst Oswaldo Cruz 2004;99:541. Sukumar K, Perich MJ, Booba LR. J Am Mosq Control Assoc 1991;7:210. Mongelli E, Coussio J, Ciccia G. Phytother Res 2002;16:S71. Limsuwan S, Rongsriyam Y, Kerdpibule V, Apiwathnasorn C, Chiang GL, Cheong WH. Rearing techniques for mosquitos. In: Supavej S, editor. Entomology malaria and filariasis—practical entomology malaria and filariasis. Bangkok: Museum and Reference Center, Faculty of Tropical Medicine, Mahidol University; 1987. WHO, 1981a. WHO/VBC/81.807. Abbott WS. J Econ Entomol 1925;18:265. Bassole IH, Guelbeogo WM, Nebie R, Costantini C, Sagnon N, Kabore ZI, et al. Parasitologia 2003;45:23. Thomas TG, Rao S, Lal S. JPN J Infect Dis 2004;57:176. Matev M, Chakurski L, Koichev A, Angelov L. Vutr Boles 1981;20:48. Nakano Y, Matsunaga H, Saita T, Mori M, Katano M, Okabe H. Biol Pharm Bull 1998;21:257. Khayyal MT, El-Ghazaly MA, Kenawy SA, Seif-el-Nasr M, Mahran LG, Kafafi YA, et al. Arzneim-Forsch 2001;51:545. Singh G, Kapoor IP, Pandey SK, Singh UK, Singh RK. Phytother Res 2002;16:680. Sadraei H, Ghannadi A, Takei-Bavani M. Int J Aromath 2003;13:121. Satyavati GV, Raina MK. Medicinal plants of India. New Delhi: Indian Council of Medical Research; 1976. Bjeldaness LF, Kim IS. J Org Chem 1977;42:2333. Zheng GQ, Zhang J, Kenney PM, Lam LKT. Nutr Cancer 1993;19:77. Bisset NG. Herbal drugs and phytopharmaceuticals. Stuttgart: Scientific Publishers; 1994. Rafikali AM, Muraleednaran GN. J Agric Food Chem 2001;49:142. Claus R, Hoppen HO. Experientia 1979;35:1674. Albert-Puleo M. J Ethnopharmacol 1980;2:337. Reynolds JEF. Essential oils and aromatic carminatives. London: Martindale—The Extra Pharmacopeia, Royal Pharmaceutical Society; 1982. Farrell KT. Spices, condiments and seasonings. Westport, CT: AVI Publishing; 1985. Hänsel R, Keller K, Rimpler H. Foeniculum 5. Hagers handbuch der pharmazeutischen, praxis 5. New York: Springer; 1993. Özbek H, Ugras S, Dülger H, Bayram I, Tuncer I, Öztürk G, et al. Fitoterapia 2003;74:317. Dadalioglu I, Evrendilek GA. J Agric Food Chem 2004;52:8255. Kalyani GA, Aithal KS, Srivastava KK. Fitoterapia 1989;60:160. Shiva MP, Lehri A, Shiva A. Aromatic and medicinal plants. Yielding essential oil for pharmaceutical, perfumery, cosmetic industries and trade. Dehradun: International Book Distributors; 2002. Itthipanichpong C, Ruangrungsi N, Pattanaautsahakit C. J Med Assoc Thai 2002;85(Suppl 1):S344. Barbara B. Encyclopedia of spices; 2003. Available from: URL: http://www.theepicentre.com/Spices/zedoary.html. Lai EY, Chyau CC, Mau JL, Chen CC, Lai YJ, Shih CF, et al. Am J Chin Med 2004;32:281.
Aromatic plant-derived essential oil: An alternative larvicide for mosquito control B. Pitasawat a,⁎, D. Champakaew a , W. Choochote a , A. Jitpakdi a , U. Chaithong a , D. Kanjanapothi b , E. Rattanachanpichai a , P. Tippawangkosol a , D. Riyong a , B. Tuetun a , D. Chaiyasit a a
Department of Parasitology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand b Chulabhorn Research Institute, Chiang Mai 50200, Thailand Received 11 November 2005; accepted 8 January 2007 Available online 6 February 2007
Abstract Five aromatic plants, Carum carvi (caraway), Apium graveolens (celery), Foeniculum vulgare (fennel), Zanthoxylum limonella (mullilam) and Curcuma zedoaria (zedoary) were selected for investigating larvicidal potential against mosquito vectors. Two laboratory-reared mosquito species, Anopheles dirus, the major malaria vector in Thailand, and Aedes aegypti, the main vector of dengue and dengue hemorrhagic fever in urban areas, were used. All of the volatile oils exerted significant larvicidal activity against the two mosquito species after 24-h exposure. Essential oil from mullilam was the most effective against the larvae of A. aegypti, while A. dirus larvae showed the highest susceptibility to zedoary oil. © 2007 Elsevier B.V. All rights reserved. Keywords: Larvicidal activity; Aedes aegypti; Anopheles dirus; Carum carvi; Apium graveolens; Foeniculum vulgare; Zanthoxylum limonella; Curcuma zedoaria
1. Introduction Interest in the control of Aedes aegypti and Anopheles dirus lies in the fact that they act as vectors of dengue and dengue hemorrhagic fever, and malaria, respectively, which are serious public health problems in Thailand and many developing countries. Although some vector-transmitting diseases such as Japanese encephalitis and yellow fever have been reasonably brought under control by vaccination, no effective vaccine is available for dengue or malaria. Therefore, the only efficacious approach of minimizing the incidence of these diseases is to eradicate and control mosquito vectors mainly by application of insecticides to larval habitats, destroying unwanted containers, and educating the public [1]. During epidemics, these measures are complemented by insecticide space-spraying against adult mosquitoes. However, aerial toxicants for eradicating A. aegypti are not effective, since this species is highly
⁎ Corresponding author. Tel.: +66 53 945343; fax: +66 53 217144, +66 53 945347. E-mail address: [email protected] (B. Pitasawat). 0367-326X/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2007.01.003
206
B. Pitasawat et al. / Fitoterapia 78 (2007) 205–210
domesticated and many adults rest in hidden places indoors [2]. It is acknowledged that attacking breeding places with larvicides is a potential alternative to diminish mosquito populations. The most commonly used larvicides are organophosphorus compounds such as temephos, fenthion and chlorpyrifos, which are highly active against mosquito larvae and other aquatic insects. Organophosphorate temephos is recommended as the most appropriate larvicide for the control of Aedes and Anopheles, because it has low toxicity to fish, birds, mammals and humans [3,4]. However, its toxicity to fish and other nontarget organisms as well as the environment, and the insecticide resistance of arthropods, are being increasingly reported [5–8]. Even though susceptibility of various species of mosquito larvae to plant products has been studied extensively, most plant-derived constituents have shown tendencies to exhibit slower actions and weaker effects compared with synthetic compounds. The plant-derived natural products, however, still have immense potential for the control of mosquito vectors, if they are reasonably effective and harmless to beneficial nontarget organisms and the environment. Determination of the larvicidal activity of AkseBio2, a new botanical natural product derived from a mixture of various plant extracts, revealed that this product is promising as a larvicide against Culex pipiens and could be useful in the search of new larvicidal natural compounds [9]. Recently, essential oils derived from plants have received much interest as potential bioactive agents against mosquito vectors. Many plant oils such as basil, cinnamon, citronella and thymus are promising as mosquito larvicides [10–13]. Investigation of plant oil-derived larvicides used for mosquito control is, therefore, a vital point of this research. The selection of plants used in this study focused on those belonging to similar or associated plant families reported to have potential against mosquitoes [10,14,15]. In addition, herbs used as vegetables and spices in traditional medicine were mainly considered in the search for effective and safe materials. 2. Experimental 2.1. Plants Carum carvi L. (Apiaceae), Apium graveolens L. (Apiaceae), Foeniculum vulgare Mill (Apiaceae), Zanthoxylum limonella Alston (Rutaceae) and Curcuma zedoaria Roscoe (Zingiberaceae) were obtained from commercial suppliers, Phachinai Industry or E.A.R. Samunpri, in Chiang Mai Province, Thailand. The voucher specimens were deposited in the Department of Parasitology, Faculty of Medicine, Chiang Mai University, Thailand. 2.2. Extraction and preparation Essential oils from each plant material were steam-distillated. The obtained oil was dried over anhydrous sodium sulfate, collected and kept in a light-protected bottle under refrigeration at 4 °C for later investigation. 2.3. Test mosquitoes A. dirus was obtained from the Armed Forces Research Institute of Medical Sciences (AFRIMS). A. aegypti was collected at various breeding places in Chiang Mai Province, northern Thailand. The free-mating colonies of these mosquitoes were established in the insectarium of the Department of Parasitology, Faculty of Medicine, Chiang Mai University, by following the common standard mosquito rearing technique previously described [16]. The mosquito colony was maintained continuously at 25–30 °C and 80–90% relative humidity under a photoperiod of 14:10 h (light/
Table 1 Physical and organoleptic properties, and yields of volatile oils derived from C. carvi, A. graveolens, F. vulgare, Z. limonella and C. zedoaria Plants
Voucher specimen
Appearance
Color
Odor
Density (g/ml)
% Yield (v/w)
C. carvi A. graveolens F. vulgare Z. limonella C. zedoaria
PARA-CA-001/1 PARA-AP-001/2 PARA-FO-001/1 PARA-ZA-001/1 PARA-CU-001/2
Liquid Liquid Liquid Liquid Viscous liquid
Light yellow Pale yellow Pale yellow Light green Golden yellow
Pepper-like Orange-like Pepper-like Lemon-like Cineolic-like
0.93 0.89 0.97 0.84 0.90
1.46 1.25 0.77 5.72 1.17
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dark) for over 20 generations in a laboratory free of exposure to pathogens or insecticides. Freshly molted larvae of A. dirus and A. aegypti were continuously available for the mosquito larvicidal experiments. 2.4. Determination of larvicidal activity Mosquito larvicidal assays were carried out according to the standard procedures of the WHO [17], with slight modifications. Each plant's volatile oil was diluted in dimethylsulphoxide (DMSO) to prepare a serial dilution of test dosage. Early fourth instar larvae of each mosquito species were selected, transferred in 25 ml of distilled water (25 larvae/beaker), and allowed to acclimatize for 1 h before testing. For experimental treatment, 1 ml of serial dilutions was added to 224 ml of distilled water in a 500-ml enamel bowl and shaken lightly to ensure a homogeneous test
Table 2 Larvicidal activity of volatile oils derived from C. carvi, A. graveolens, F. vulgare, Z. limonella, C. zedoaria on A. aegypti Volatile oil (ppm)
C. carvi 37.2 46.5 55.8 65.1 74.4 83.7 A. graveolens 26.7 35.6 44.5 53.4 62.3 71.2 F. vulgare 42.7 44.6 46.6 48.5 50.4 52.4 54.3 56.3 Z. limonella 8.4 12.6 16.8 21.0 25.2 29.4 33.6 C. zedoaria 18.0 22.5 27.0 31.5 36.0 40.5 45.0 a
A. aegypti % Mortality (Mean ± SE)
12.67 ± 9.29 22.67 ± 9.02 54.33 ± 8.08 73.00 ± 22.65 87.67 ± 11.59 97.00 ± 3.61 13.67 ± 15.18 41.67 ± 30.83 55.33 ± 31.09 68.00 ± 28.21 78.33 ± 25.66 96.67 ± 4.16 17.67 ± 8.08 25.33 ± 12.34 31.00 ± 14.73 37.33 ± 10.02 48.00 ± 3.00 73.00 ± 6.00 79.00 ± 2.65 88.33 ± 5.86 1.00 ± 1.00 7.33 ± 0.58 21.33 ± 4.04 39.33 ± 8.33 43.33 ± 12.01 67.33 ± 15.31 78.00 ± 5.00 4.33 ± 2.31 12.67 ± 0.58 29.00 ± 7.00 52.33 ± 14.19 63.67 ± 16.50 77.00 ± 16.09 86.67 ± 11.06
Larvicidal activity a LC50
LC95
LC99
54.62 (52.62–56.64)
90.06 (83.09–100.76)
119.21 (105.70–141.33)
42.07 (39.62–44.56)
99.12 (85.00–124.85)
160.25 (126.82–228.68)
49.32 (48.70–49.94)
62.09 (60.08–64.88)
70.64 (67.23–75.53)
24.61 (23.29–25.98)
55.81 (47.50–72.01)
88.32 (69.10–130.72)
31.87 (30.71–33.02)
55.50 (51.05–62.30)
75.75 (66.73–90.46)
The lethal concentrations with the corresponding 95% confidence intervals are shown in parenthesis.
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solution. Selected mosquito larvae were transferred in distilled water into a bowl of prepared test solution with a final surface area of 124 cm2 (25 larvae/bowl). Four replicates were run simultaneously yielding a final total of 100 larvae for each dosage, with at least five dosages providing a range of 0–100% mortality. For each test, controls and untreated sets containing a mixture of 1 ml of diluent DMSO dissolved in 249 ml of distilled water and 250 ml of distilled water, respectively, were also run for comparison. Symptoms of treated larvae were observed and recorded immediately and at time-intervals. Mortality and survival were determined after 24 h of exposure, during which time no food was offered to the larvae. The moribund and dead larvae in the four replicates were combined and expressed as the percentage mortality for each concentration. Larvae were considered dead or moribund if they were unrousable within a reasonable period of time, even when gently prodded with a needle, as described in the World Health Organization's technical report series. They might also show discoloration, unnatural positions, tremors, incoordination, or rigor. Three independent experiments for each volatile oil against each mosquito species from different rearing batches were carried out at 25–30 °C on separate days.
Table 3 Larvicidal activity of volatile oils derived from C. carvi, A. graveolens, F. vulgare, Z. limonella, C. zedoaria on A. dirus Plant oil (ppm)
C. carvi 46.5 55.8 65.1 74.4 83.7 93.0 A. graveolens 35.6 44.5 53.4 62.3 71.2 80.1 89.0 F. vulgare 33.9 34.9 35.9 36.9 37.8 38.8 Z. limonella 37.8 42.0 46.2 50.4 54.6 58.8 63.0 C. zedoaria 23.4 25.2 27.0 28.8 30.6 32.4 a
A. dirus % Mortality (Mean ± SE)
4.00 ± 3.61 11.00 ± 10.82 30.00 ± 13.53 57.67 ± 10.21 75.00 ± 5.57 91.33 ± 2.52 10.25 ± 8.34 24.00 ± 8.29 36.00 ± 13.56 50.75 ± 19.47 68.75 ± 4.92 84.25 ± 3.10 92.75 ± 3.30 25.00 ± 14.11 39.00 ± 17.06 63.67 ± 7.02 83.00 ± 5.20 88.00 ± 3.61 93.33 ± 3.06 4.00 ± 1.73 8.33 ± 0.58 13.33 ± 0.58 18.67 ± 4.73 34.00 ± 2.65 59.33 ± 8.62 74.00 ± 9.54 4.33 ± 6.66 7.00 ± 6.00 27.00 ± 11.14 46.33 ± 15.28 54.33 ± 20.50 70.67 ± 14.15
Larvicidal activity a LC50
LC95
LC99
72.28 (70.23–74.53)
104.69 (97.19–117.23)
128.87 (115.46–152.62)
59.44 (56.87–61.98)
111.69 (99.59–133.00)
159.06 (133.47–208.45)
35.27 (35.01–35.50)
38.78 (38.19–39.64)
40.90 (39.95–42.34)
57.22 (56.10–58.51)
76.23 (72.04–82.93)
89.52 (82.40–101.43)
29.69 (29.04–30.37)
40.23 (37.25–46.67)
47.70 (42.40–60.00)
The lethal concentrations with the corresponding 95% confidence intervals are shown in parenthesis.
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2.5. Statistical analysis It was essential to obtain not less than 3 mortality counts of between 10% and 90%. If the mortality in controls was between 5 and 20%, correction of the observed percentage mortality (%M) was performed by applying the Abbott's formula [18]: %M ¼
% test mortality−% control mortality 100 100−% control mortality
When the control mortality was over 20%, the tests were discarded and repeated. The average mortality percentage derived from individual tests was determined. The lethal concentrations of 50%, 95% and 99% (LC50, LC95 and LC99, respectively) as well as the corresponding 95% confidence intervals (95% CI), used to measure differences between plant samples, were determined by using a computerized log-probit analysis (Harvard Programming; Hg1, 2). 3. Results and discussion Steam distillation of C. carvi, A. graveolens, F. vulgare, Z. limonella and C. zedoaria yielded from 0.77 to 5.72% (v/w, dry weight) of volatile oils (Table 1). The highest yield of volatile oil was obtained from Z. limonella, whereas that of F. vulgare was the lowest. All volatile oils obtained were less dense than water, and their physical and organoleptic properties presented in Table 1 demonstrated differences in appearance, color and odor. The larvicidal activities of the essential oils against mosquito larvae under laboratory conditions are given in Tables 2 and 3. All plant oils exerted significant larvicidal potential against A. aegypti (Table 2) and A. dirus (Table 3), after exposure for 24 h. The lethal dosage of 50% (LC50) on A. aegypti larvae ranged between 24.61 and 54.62 ppm and on A. dirus between 29.69 and 72.28 ppm. Z. limonella oil was the most effective against A. aegypti with LC50 and LC95 values of 24.61 and 55.81 ppm, respectively. A. dirus larvae showed the highest susceptibility to C. zedoaria oil (LC50 and LC95 values: 29.69 and 40.23 ppm, respectively). After exposure to the essential oils, the treated larvae exhibited restlessness, sluggishness, tremors and convulsions followed by paralysis at the bottom of the bowl. After that, the number of toxic affected larvae increased, and some if not all of them subsequently died within 24 h. Some A. aegypti larvae, treated with fennel oil, paralyzed and sank to bottom of the container, and after a short time of exposure recovered within 4–7 h. After 24 h, however, some moribund and dead larvae were found. Finally, ethnobotanical data and chemical composition of the used plants are reported in Table 4. Chemical identification by gas chromatography coupled with mass spectrometry (GC/MS) revealed the composition of the investigated oils. Carvone was a main constituent representing 32.7% of C. carvi oil. A principal component found in essential oils that were derived from A. graveoles and Z. limonella was limonene (60% and 31%, respectively). F. vulgare oil was found to contain anethole (85.63%) as a major substance, while a high content of 1,8-cineole (18.5%), p-cymene (18.42%) and α-phellandrene (14.93%) was found in C. zedoaria oil. These compounds have been isolated Table 4 Ethnobotanical data and chemical composition of C. carvi, A. graveolens, F. vulgare, Z. limonella and C. zedoaria Plants
English name
Part used
C. carvi
Caraway Seed
Phytomedicine and pharmacology
Treatment of digestive disorders; laxative on the duodenum; antiproliferative; antiulcerogenic; antibacterial A. graveolens Celery Seed Treatment of arthritis, nervousness, hysteria, headaches, bronchitis, asthma, liver and spleen disease; hepatoprotecive; sedative; anticonvulsive; anticarcinogen F. vulgare Fennel Fruit Treatment of dyspeptic complaints, respiratory disorder, pediatric colic; promoter of menstruation; hepatoprotecive Z. limonella Mullilam Fruit Treatment of wounds; promoter of digestion; anthelminthic; gastrointestinal stimulant C. zedoaria Zedoary Rhizome Treatment of wounds; promoter of digestion; relief of flatulence and colic; antiseptic; antimicrobial activity
Chemical composition of plant oil (GC/MS)
References
Carvone (32.7%), limonene (17.4%), γ-terpinene (17.2%) D-Limonene (60%), selinene (10%), phthalides (3%)
[21–25] [26–31]
trans-Anethole (85.63%), estragole [32–37] (5.27%), D-limonene (3.8%), p-anisaldehyde (2.68%) D -Limonene (31%), terpin-4-ol [38–40] (13.94%), sabinene (9.13%) 1,8-Cineole (18.5%), p-cymene [41,42] (18.42%), α-phellandrene (14.93%)
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from many herbal oils [12,19,20] that showed mosquito larvicidal activity. The efficacy of each of these compounds and their synergistic mechanisms need to be tested, to see if any have a potential use against mosquitoes. In conclusion, this study clearly illustrated the efficacy of the investigated herbs, which encourages the development of alternative active ingredients as effective mosquito larvicides. Acknowledgements The authors are thankful to the botanist J.F. Maxwell from CMU Herbarium, Department of Biology, Faculty of Science, Chiang Mai University. Special thanks also go to the Armed Forces Research Institute of Medical Sciences (AFRIMS) for providing the test mosquito, Anopheles dirus. Acknowledgment is extended to the Faculty of Medicine Research Fund, Chiang Mai University for their financial support, and the Faculty of Medicine Endowment Fund for Research Publication for helping defray the publication cost. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16]
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