Leaves of A. indica from Guayaquil - Ecuador (2°07'32" S, 79°50'48" W) were collected in February 2015, during the tree fIowering stage. A sample of the plant material was taken for botanical identification, being herborized in the National Herbarium of Ecuador (QCNE), keeping an herbal control (Code: CIBE019) in the Bioproducts laboratory of CIBE-ESPOL, Guayaquil, Ecuador.

The plant extract was obtained according to the procedure described by Maragathavalli et al. (2012). Fresh leaves were dried in an oven with air recirculation for 24 h at 55 °C for subsequent manual milling and sieving, the selected fraction was the one remaining on the 2-mm mesh sieve. Three successive macerations were performed with ethanol at 96% (100 g dry sample per 500 mL of solvent) under stirring at room temperature for 48 h and then filtered. The solvent of the combined maceration was removed by roto-evaporation at 40 °C, and the extracted material was kept at 4 °C until use (Heidolph, 40001). Three samples (100, 200, 300 mg) were dissolved on 100 mL of distilled water, giving an initial concentration of 1, 2, and 5 mg mL^-1.

The plant extract was analyzed by GC-MS according to the method of Umar et al. (2014) with some modifications, using Agilent Technologies gas chromatography and mass spectrometry equipment (7890A GC and 5975C XL MSD inert with triple-axis detector). The samples dissolved in distilled water were filtered or centrifuged to remove any insoluble matter. The injection of 2.0 µL of a sample (10 mg mL^-1) was performed at 250 °C with no division mode; the detector temperature was 280 °C, and the oven was equipped with an HP-5 capillary column (30 m×0.25 mm ID×0.25 µm film thickness). The GC-MS was programmed with a ramp of 70 °C for 2 min, at a speed of 5 °C min^-1 until reaching 285 °C. The carrier fIow of helium was adjusted at a speed of 1.2 mL min^-1 to have a run of 45 min. Electronic ionization was set at 70 eV (135 and 230 °C), and the data were collected in full scan mode (40-1000 amu). Lastly, the compounds were identified by comparing their retention index and the Wiley 9th mass spectrum data with the NIST 2011 MS Library.

The Chemistry Faculty of Universidad de Guayaquil provided the A. aegypti larvae. The assay was developed according to the methodology of Wandscheer et al. (2004) and Cruz-Estrada et al. (2013). Three concentrations of neem extract were prepared by diluting 1 mL of the initial extracts (1, 2, and 5 mg mL^-1) in 100 mL of water, obtaining final doses of 10 mg L^-1, 20 mg L^-1, and 50 mg L^-1; dose range was selected based on Wandscheer et al. (2004). Ten larvae (III and IV instar stage) were placed in each polyethylene plastic containers with test solutions (100 mL) at a temperature from 25 to 30 °C and 12 h photoperiod. The tests were performed thrice. Negative control (NC) was water, and positive control (PC) was Bactivec (Labiofam), bio-larvicide which active ingredient (0.6%) is Bacillus thuringiensis var. israelensis; for the assay, it was diluted to the recommended application (1% v/v). After 24, 48, and 72 h, the percentage of mortality was registered.

Assumptions of normality and homogeneity of variances were corroborated, then the data was submitted to 2 way ANOVA. Tukey's test at a 5% probability was used for the comparison of means. The statistical software use was MINITAB 16.

In Figure 2, the gas analytical chromatogram of the compounds identified by GC-MS of the ethanolic extract is shown. The significant component detected in the extract was phytol (14.24%). The presence of terpenes and fatty acids was also reported (Table 1).

Figure 2

Table 1

The presence of the terpene phytol, as the main component of the ethanolic extract of A. indica is consistent with that reported by Cruz-Estrada et al. (2013). Furthermore, previous reports suggest that phytol extracted from plants have larvicidal activity against mosquitoes (Renjana and Thoppil, 2013). Phytol presented in the leaf extract of Lantara chamber, Azadirachta indica and Ocimum gratissimum possess larvicidal properties against Aedes aegypti and Culex quinquefasciatus (Maneemegalai and Sathish, 2008; Maragathavalli et al., 2012; Pratheeba et al., 2015), and Premna latifolia extract against Aedes albopictus as observed in the studies conducted by Krishnaveni and Ramamurthy (2014). The combination of neophytadiene and phytol has been shown to have higher insecticidal activity than its components alone (Cáceres et al., 2015). Additionally, potent toxicity of 2-Furancarboxaldehyde has been shown against Drosophila melanogaster larvae (Miyazawa et al., 2003). On the other hand, linoleic acid has larvicidal activity against Aedes aegypti with LC₅₀ and LC₉₀ values of 35.39 and 96.33 ppm, respectively (Rahuman et al., 2008), so this compound in the present study could be contributing to larvicidal activity.

According to the results mentioned above may be noted that the neem tree leaves have a high amount of terpenes, which is corroborated by this study where phytol was the most abundant metabolite, a component that confers the insecticide property and possibly the responsible of the reported larvicidal action.