Larvicidal Activity of essential oils from Brazilian plants against Aedes aegypti L.

CONTROL

Larvicidal Activity of essential oils from Brazilian plants against Aedes aegypti L.

Eveline Solon Barreira Cavalcanti; Selene Maia de Morais¹; Michele Ashley A Lima; Eddie William Pinho Santana^II

^ICurso de Química do Centro de Ciências e Tecnologia

^IICurso de Medicina do Centro de Ciências da Saúde, Universidade Estadual do Ceará, Av. Paranjana 1700, Campus do Itaperi, 60740-000 Fortaleza, CE, Brasil

ABSTRACT

Aedes aegypti L. is the major vector of dengue fever, an endemic disease in Brazil. In an effort to find effective and affordable ways to control this mosquito, the larvicidal activities of essential oils from nine plants widely found in the Northeast of Brazil were analyzed by measurement of their LC₅₀. The essential oils were extracted by steam distillation and their chemical composition determined by GL-chromatography coupled to mass spectroscopy. The essential oils from Cymbopogon citratus and Lippia sidoides, reported in the literature to have larvicidal properties against A. aegypti, were used for activity comparison. The results show that Ocimum americanum and Ocimum gratissimum have LC₅₀ of 67 ppm and 60 ppm respectively, compared to 63 ppm for L. sidoides and 69 ppm for C. citratus. These results suggest a potential utilization of the essential oil of these two Ocimum species for the control of A. aegypti.

Key words: larvicidal activity - mosquito control - Aedes aegypti - essential oils - dengue

Dengue fever is endemic over large areas of tropics and subtropics. Outbreaks of dengue have repeatedly occurred in Brazil over the last 10 years. The etiological agent is an arbovirus and the major vector is the Aedes aegypti mosquito, which is found in 3600 Brazilian municipalities (Cives 2002). While most patients are asymptomatic, reinfection with different serotypes of dengue viruses may lead to hemorrhagic fever with high mortality. During outbreaks, public health authorities in Brazil have standardized the use of aerolized pyrethroid insecticides that can cause allergies. This measure only partially controls the mosquito population since it eliminates the adult flying insects but does not eliminate the breeding places. In these breeding sites, the larvicide used is usually the organophosphorate Temephos, although very slightly toxic may cause headaches, loss of memory, and irritability (NICC 2003).

Secondary metabolites of plants, many of them produced by the plant for its protection against microorganisms and predator insects are natural candidates for the discovery of new products to combat A. aegypti. Several studies have on focused natural products for controlling Aedes mosquitoes as insecticides and larvicides, but with varied results (Consoli et al. 1988, Perich et al. 1995, Jayaprakasha et al. 1997, Sathiyamoorthy et al. 1997, Chariandy et al. 1999, Pizarro et al. 1999, Rahuman et al. 2000, Markouk et al. 2000, Ciccia et al. 2000, Tsao et al. 2002). The repellency to A. aegypti of essential oils from orange peel (Ezeonu 2001), thyme and clove (Barnard 1999) and components of essential oils such as eugenol, cineole, and citronellal (Hummelbrunner & Isman 2001) was determined in laboratory tests. Studies with Lippia sidoides (Carvalho et al. 2003) and Cymbopogon citratus (Sukumar et al. 1991) essentials oils suggest that they are promising as larvicides against A. aegypti.

In the present study essential oils of nine plants commonly found in Northeastern Brazil were tested against third instar A. aegypti larvae in a search for effective and affordable natural products to be used in the control of dengue.

MATERIALS AND METHODS

Plant material - Citrus fruits such as lemon and orange were purchased in local markets and other plants were collected in the Medicinal Plants Garden of the Federal University of Ceará (UFC), Brazil. Taxonomic identification of plants was performed by botanists of the Prisco Bezerra Herbarium, Department of Biology, UFC, where voucher specimens are deposited. Eight plants commonly found in the Northeast of Brazil (Alpinia zerumbet, Citrus limonia, Citrus sinensis, Syzygium jambolana, Ocimum americanum, Ocimum gratissimum, Hyptis suaveolens) were evaluated in addition to C. citratus and L. sidoides, which were used for comparison. The essential oils were extracted by steam distillation in a Clevenger-type apparatus (Craveiro et al. 1976). The oils were extracted from the aerial parts (leaves and branches) of the plants and for the Citrus species fruit peels were used.

Essential oil analysis - The oils were analyzed using a Hewlett-Packard 5971 GC/MS instrument employing the following conditions: column: Dimethylpolysiloxane DB-1 coated fused silica capillary column (30 m x 0.25 mm); carrier gas: He (1 ml/min); injector temperature: 250°C; detector temperature: 200°C; column temperature: 35–180ºC at 4ºC/min then 180–250ºC at 10ºC/min; mass spectra: electron impact 70 eV. The identification of the constituents was performed by computer library search, retention indices and visual interpretation of the mass spectra (Craveiro et al. 1984, Adams 2001). The identified constituents are listed in their order of elution from a non-polar column in Table I.

Larvicidal bioassay - Larvae of A. aegypti were collected from a mosquito colony maintained at Nuend (Núcleo de Controle de Endemias Transmissíveis por Vetores Secretaria de Saúde do Estado do Ceará). For the assay, the essential oil of the plants was placed in a 50 ml beaker and DMSO (0.3 ml) was used to solubilize the oil in water (19.7 ml) that contained 50 larvae (third instar). With each experiment, a set of controls using 1% DMSO and untreated sets of larvae in tap water, were also run for comparison. Mortality was recorded after 24 h of exposure during which no nutritional supplement was added.

The experiments were carried out 28 ± 2ºC. Each test comprised of three replicates with four concentrations (500, 250, 100, 50 ppm). Data were evaluated through regression analysis. From the regression line, the LC₅₀ values were read representing the lethal concentration for 50% larval mortality of A. aegypti.

RESULTS AND DISCUSSION

The results from the A. aegypti larvicidal assay using nine different plants, common in the Northeast of Brazil, are shown in Table II. The most active essential oils against third instar larvae of A. aegypti were those of O. gratissimun (LC₅₀ 60 ppm), O. americanum (LC₅₀ 67 ppm), L. sidoides (LC₅₀ 63 ppm),and C. citratus (LC₅₀ 69 ppm). Skumar et al. (1991) reported that C. citratus causes significant growth inhibition and mortality in later developmental stages of A. aegypti. The analysis of the essential oil of this plant from the state of Ceará, showed that its major components are geranial (60.3%) and neral (39.7%). L. sidoides essential oil and its main constituent thymol were shown to be very active against A. aegypti larvae (Carvalho et al. 2003). Previous studies of O. americanum showed that solvent extracts from the whole plant have ovipositional deterrence against A. aegypti (Sukumar et al. 1991). Matos (2000) reported that O. gratissimum essential oil displays antifungal (Aspergillus and Trichoderma) and antibacterial (Staphylococcus) activities. Both eugenol and O. gratissimum oil presented anthelmintic activity against Haemonchus contortus, the main nematode of ovines and caprines in Northeastern Brazil (Pessoa et al. 2002). The citrus oils, although they have insecticidal activities (Ezeonu et al. 2001), and H. suaveolens, that is used as mosquito repellent (Palsson & Jaenson 1999), were not effective in the larvicidal test. Supavarn et al. (1974) tested 36 vegetable extracts on A. aegypti and found that 11.1% were capable of producing mortality at a concentration of 500 ppm but only 2.8% produced the same effect at a concentration of 100 ppm. In conclusion, the essential oils of O. americanum and O. gratissimun were shown to be as potent as L. sidoides and C. citratus in the larvicidal activity against A. aegypti and caused 100% mortality at a concentration of 100 ppm. These results are very promising in creating new effective and affordable approaches to the control of Aedes mosquito and, thus, of dengue fever.

ACKNOWLEDGEMENTS

To Dr Afrânio G Fernandes from Geography Course of State University of Ceará for authenticating the plant material, to Dr Afrânio A Craveiro and Mrs Olga Ramos from Technological and Scientific Park of Ceará for the gas chromatography/mass spectrometry analysis of essential oils.

Received 13 February 2004

Accepted 21 June 2004

Financial support: Fundação Cearense de Apoio ao Desenvol-vimento Científico e Tecnológico, CNPq for Pibic scholarship

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Publication Dates

• Publication in this collection
  03 Nov 2004
• Date of issue
  Aug 2004

History

• Accepted
  21 June 2004
• Received
  13 Feb 2004