Chemical Composition and Larvicidal Activity against Aedes aegypti Larvae of Essential Oils from Four Guarea Species

The essential oils of four Guarea species collected at Manaus (Amazonas, Brazil) were obtained by hydrodistillation and analyzed by GC-MS. Except for one diterpene detected, the compounds identified in the essential oils were hydrocarbons and oxygenated sesquiterpenes. The major sesquiterpenes were α-santalene (26.26%) and α-copaene (14.61%) from G. convergens branches; caryophyllene epoxide (40.91%) and humulene epoxide II (14.43%) from G. humaitensis branches; cis-caryophyllene (33.37%) and α-trans-bergamotene (11.88%) from G. scabra leaves; caryophyllene epoxide (36.54%) in leaves and spathulenol (14.34%) in branches from G. silvatica. The diterpene kaurene (15.61%) was found in G. silvatica leaves. Larvicidal activity assay of essential oils against third-instar Aedes aegypti larvae revealed that at higher concentrations (500 and 250 μg/mL), all the essential oils caused 100% mortality after 24 h of exposure. The most active essential oils were those of G. humaitensis branches (LC50 48.6 μg/mL), G. scabra leaves (LC50 98.6 μg/mL) and G. silvatica (LC50 117.9 μg/mL). The differences in the toxicity of essential oils of Guarea species on A. aegypti are due to qualitative and quantitative variations of the components, therefore the larvicidal effect may be due to higher amount of the sesquiterpenes with caryophyllane skeleton.

quantitative variations of the components, therefore the larvicidal effect may be due to higher amount of the sesquiterpenes with caryophyllane skeleton. Keywords: Guarea convergens; Guarea humaitensis; Guarea scabra; Guarea silvatica; Meliaceae

Introduction
Dengue is a viral disease that has major public health consequences in many parts of the world. The principal vector of dengue fever, including the haemorrhagic form, is the mosquito Aedes aegypti L. (Diptera, Culicidae) [1]. Presently, mosquito control primarily depends worldwide on continued applications of conventional toxic synthetic insecticides to which resistance has been reported in many areas where it is widely used [2][3][4]. This has stimulated the investigation of natural insecticides as an alternative control, focused on plant-derived compounds, including volatile chemical constituents (essential oils), as potentially bioactive substances against mosquito larvae.
In nature, many essential oils play an important role in protecting plants against pathogens and also against herbivores by reducing their appetite for such plants. They also may attract some insects to favour the dispersion of pollens and seeds, or act as repellent for other undesirable insects [5]. In addition, some of them have been shown to be effective in the control of mosquito larvae responsible for the transmission of dengue fever. Essential oils with larvicidal activity against third-instar of A. aegypti have been extracted from plants of the Myrtaceae [6], Piperaceae [7], Poaceae [8], Lamiaceae [8][9][10], Rutaceae [8,9,11,12], Verbenaceae [8,9], Apiaceae and Zingiberaceae [11]. There are however no published reports on larvicidal activity of Meliaceae essential oils. In this study we analyzed the constituents of essential oils from species of this family Guarea convergens, G. humaitensis, G. scabra, G. silvatica and studied their larvicidal activity against A. aegypti.
Despite the fact that the genus Guarea includes more than 50 species, few studies on the essential oils of its species have been published, namely, the Brazilian species G. guidonia [13][14][15], G. macrophylla ssp tuberculata [16][17][18] and the African species G. cedrata [19], whose dominant compounds differ from those found in the present study.

Larvicidal activity of essential oils
The five essential oils obtained were investigated for their larvicidal activities against third-instar A. aegypti larvae. At higher concentrations (500 and 250 μg/mL), all the essential oils exhibited 100% mortality after 24 h of exposure; the most active were those Guarea humaitensis branches (LC 50 48.6 μg/mL), G. scabra leaves (LC 50 98.6 μg/mL) and G. silvatica leaves (LC 50 117.9 μg/mL), in accordance with Table 3. The differences in the toxicity of essential oils of Guarea species against A. aegypti were due to both qualitative and quantitative variations of the components, therefore the larvicidal effect may be due to higher amount of sesquiterpenes with caryophyllane skeletons: caryophyllene epoxide (40.91%, GHb and 36.54%, GSil), and cis-caryophyllene (33.37%, GSl). Several studies have shown that sesquiterpenoids possess significant larvicidal activity against A. aegypti. Cheng et al. [20] reported the results of screening essential oils and suggested that oils presenting LC 50 values > 100 ppm should not be considered active, whereas those with LC 50 values < 50 ppm could be regarded as highly active. The test using known compounds conducted by Cheng et al., [21] in fouth-instar mosquito larvae A. albopictus included caryophyllene epoxide, which displayed a LC 50 value of 65.6 μg/mL at 24 h of exposure.

Plant material and oil distillation
The samples of G. convergens, G. humaitensis, G. scabra and G. silvatica were collected in November, 2006 at the Forest Reserve Adolfo Ducke, Km 26 from Manaus, Amazonas, Brazil from individual previously marked (trees no. 4451-09, 4705-09, 3095-09 and 2091-09, respectively) and identified by Dr. Terence D. Pennington during the "Flora da Reserva Ducke" project Instituto Nacional de Pesquisas da Amazônia (INPA) [22]. The leaves and branches were dried in an air conditioned room at 25 °C for seven days, milled and submitted to hydrodistillation in a Clevenger-type apparatus for 4 hours. The oils were dried in anhydrous sodium sulphate, filtered, stored in amber glass bottles in a refrigerator (4 °C) for investigation of chemical constituents and larvicidal activity.

Gas chromatography-mass spectrometry (GC/MS)
Analyses of volatile constituents were performed on a Shimadzu QP5000 instrument, equipped with a DB-5 fused silica capillary column (J&W Scientific -Serial N 0 8766726; 5% phenylmethylsiloxane; 30 m × 0.25 mm × 0.25 μm). The electron impact technique (70 eV) was used with the injector temperature at 240 °C and the detector at 230 °C. The carrier gas was helium at the working rate of 1.0 mL/min. The column temperature was initially 60°C and then was gradually increased at the rate of 3 °C/min up to 240 °C. Compounds were identified by comparing their mass spectrum to those of the database of the GC-MS (NIST 62.lib), literature [23] and retention indices [24].

Larvicidal bioassay
Larvae of Aedes aegypti were obtained from a permanent colony, maintained at temperature of 23-27 °C and relative humidity of 50-70% in the Laboratório de Vetores de Malária e Dengue of the Instituto Nacional de Pesquisas da Amazônia. The essential oils were dissolved in DMSO (20 mg/mL) and aliquots of the stock solution in appropriate amounts for different concentrations (500, 250, 100, 50 and 25 μg/mL) to the final volume of 5 mL were transferred to plastic cups containing distilled water and food. Then, thirty third-instar larvae of A. aegypti were placed in each cup. After 24 hours, the number of dead larvae was counted and the lethal percentage calculated. Each experiment was performed in triplicate and a control test was run in parallel using 5 mL of distilled water and 125 μL of DMSO solution.

Statistical analysis
Data were evaluated through Probit analysis (POLO PC program) to determine the LC 50 and LC 90 , representing the concentrations in μg/mL that caused 50 and 90% mortality along with 95% confidence intervals [25]. The 95% confidence intervals, values and degrees of freedom of the χ 2 goodness of fit tests, and regression equations were recorded. Essential oils from G. silvatica (leaves and branches) does not constitute a confidence interval because the data does not apply to the Probit model, because the value of χ 2 calc. > χ 2 tab (7.8, P = 0.05 and three degrees of freedom). That is, determining the value estimate of the confidence interval is calculated when it is ≤ 0.5 which did not occur in the case of these oils.

Conclusions
This is the first report on the composition and larvicidal activity against Aedes aegypti from essential oils of Guarea convergens, G. humaitensis, G. scabra and G. silvatica. The results obtained from the larvicidal test indicated that the essential oils containing sesquiterpenes with caryophyllane skeletons as major compounds were the most active, suggesting that caryophyllene epoxide and ciscaryophyllene are probably the active principles responsible for the observed A. aegypti larvicidal action, therefore further studies for the mode of the actions these constituents are necessary. These results can be used in the research for natural biodegradable larvicidal, compounds, besides contributing for to the knowledge of the aromatic flora of the Biological Preserves of the Amazonian forest.