Studies on the Phytochemical Profile of Ocimum basilicum var. minimum (L.) Alef. Essential Oil, Its Larvicidal Activity and In Silico Interaction with Acetylcholinesterase against Aedes aegypti (Diptera: Culicidae)

Aedes aegypti L. (Diptera: Culicidae) is an important transmitter of diseases in tropical countries and controlling the larvae of this mosquito helps to reduce cases of diseases such as dengue, zika and chikungunya. Thus, the present study aimed to evaluate the larvicidal potential of the essential oil (EO) of Ocimum basilicum var. minimum (L.) Alef. The EO was extracted by stem distillation and the chemical composition was characterized by gas chromatography coupled with mass spectrometry (GC/MS and GC-FID). The larvicidal activity of EO was evaluated against third instar Ae. aegypti following World Health Organization (WHO) standard protocol and the interaction of the major compounds with the acetylcholinesterase (AChE) was evaluated by molecular docking. The predominant class was oxygenated monoterpenes with a concentration of 81.69% and the major compounds were limonene (9.5%), 1,8-cineole (14.23%), linalool (24.51%) and methyl chavicol (37.41%). The O. basilicum var. minimum EO showed unprecedented activity against third instar Ae. aegypti larvae at a dose-dependent relationship with LC50 of 69.91 (µg/mL) and LC90 of 200.62 (µg/mL), and the major compounds were able to interact with AChE in the Molecular Docking assay, indicating an ecological alternative for mosquito larvae control.


Introduction
Aedes aegypti L. (Diptera: Culicidae) is the vector responsible for the transmission of infectious diseases such as dengue, yellow fever, zika and chikungunya and has caused serious public health problems, especially in tropical regions [1]; these problems are related to the accelerated rate of proliferation of this mosquito, which has strong resistance to insecticides and commercial repellents. Thus, controlling the proliferation of this vector remains the main tool to eradicate or reduce the harmful effects of Ae. aegypti [2].
Natural products from plants are promising in controlling this proliferation due to their biological properties, as well as essential oils, which are defined as volatile substances

Essential Oil Yield
The essential oil (EO) of O. basilicum var. minimum leaves showed a yield of 1.57% (v/w); this yield was higher than that found [19] in essential oils extracted from the dry leaves of O. basilicum var. minimum by hydrodistillation, which showed yields of 1.20% (v/w), 1.06% (v/w) and 1.0% (v/w), respectively, in the years 2005, 2007 and 2008, and lower when compared to the same study in the years 2003, 2004 and 2006, which showed yields of 1.93% (v/w), 1.83% (v/w) and 1.78% (v/w). In another study carried out by Safari Dolatabad et al. [20], the essential oil of O. basilicum var. minimum showed a yield of 0.5% (v/w), much lower than that found in this study. Table 1 presents the data related to the chemical composition obtained by GC-MS in ascending order of retention rates for each constituent, with 24 constituents identified in the EO of O. basilicum var. minimum, representing 99.65% of the oil composition. The chemical profile of the EO was characterized by the major compounds methyl chavicol (37.41%), linalool (24.51%), 1,8-cineole (14.23%) and limonene (9.50%). The levels of methyl chavicol and linalool were higher than that found by [21] in a study carried out in South Africa with the essential oil of O. basilicum var. minimum, with methyl chavicol contents of 34.3% and linalool of 17.8%. Linalool (25.6%) has also characterized the essential oil of O. basilicum var. minimum, as well as the compound geranyl acetate (45.6%). The chemical composition of the EO of this species in collections carried out in Iran, during the years 2003 to 2008, demonstrated the strong presence of linalool with concentrations ranging from 40.2% to 88.34%, followed by 1,8-cineole (1.46% to 8.87%) and eugenol (0.28% to 7.23%) [19]. Ion-chromatogram of the essential oil is shown in Figure 1. chemical profile of the EO was characterized by the major compounds methyl chavicol (37.41%), linalool (24.51%), 1,8-cineole (14.23%) and limonene (9.50%). The levels of methyl chavicol and linalool were higher than that found by [21] in a study carried out in South Africa with the essential oil of O. basilicum var. minimum, with methyl chavicol contents of 34.3% and linalool of 17.8%. Linalool (25.6%) has also characterized the essential oil of O. basilicum var. minimum, as well as the compound geranyl acetate (45.6%). The chemical composition of the EO of this species in collections carried out in Iran, during the years 2003 to 2008, demonstrated the strong presence of linalool with concentrations ranging from 40.2% to 88.34%, followed by 1,8-cineole (1.46% to 8.87%) and eugenol (0.28% to 7.23%) [19]. Ion-chromatogram of the essential oil is shown in Figure 1.    chemical profile of the EO was characterized by the major compounds methyl chavicol (37.41%), linalool (24.51%), 1,8-cineole (14.23%) and limonene (9.50%). The levels of methyl chavicol and linalool were higher than that found by [21] [19]. Ion-chromatogram of the essential oil is shown in Figure 1.   [19]. Ion-chromatogram of the essential oil is shown in Figure 1.  chemical profile of the EO was characterized by the major compounds methyl chavicol (37.41%), linalool (24.51%), 1,8-cineole (14.23%) and limonene (9.50%). The levels of methyl chavicol and linalool were higher than that found by [21] [19]. Ion-chromatogram of the essential oil is shown in Figure 1.  chemical profile of the EO was characterized by the major compounds methyl chavicol (37.41%), linalool (24.51%), 1,8-cineole (14.23%) and limonene (9.50%). The levels of methyl chavicol and linalool were higher than that found by [21] in a study carried out in South Africa with the essential oil of O. basilicum var.  [19]. Ion-chromatogram of the essential oil is shown in Figure 1.  chemical profile of the EO was characterized by the major compounds methyl chavicol (37.41%), linalool (24.51%), 1,8-cineole (14.23%) and limonene (9.50%). The levels of methyl chavicol and linalool were higher than that found by [21] in a study carried out in South Africa with the essential oil of O. basilicum var.  [19]. Ion-chromatogram of the essential oil is shown in Figure 1.  chemical profile of the EO was characterized by the major compounds methyl chavicol (37.41%), linalool (24.51%), 1,8-cineole (14.23%) and limonene (9.50%). The levels of methyl chavicol and linalool were higher than that found by [21] in a study carried out in South Africa with the essential oil of O. basilicum var.  [19]. Ion-chromatogram of the essential oil is shown in Figure 1.   chemical profile of the EO was characterized by the major compounds methyl chavicol (37.41%), linalool (24.51%), 1,8-cineole (14.23%) and limonene (9.50%). The levels of methyl chavicol and linalool were higher than that found by [21] in a study carried out in South Africa with the essential oil of O. basilicum var.  [19]. Ion-chromatogram of the essential oil is shown in Figure 1.   [23], O. gratissimum and O. tenuiflorum [24]. Furthermore, studies report antioxidant activities that may be related to the presence of linalool and methyl chavicol [25]. Linalool is described as having cytotoxic activities against HeLa, HEp-2 and NIH 3T3 type cancer cells [26], and antibacterial activities against Listeria monocytogenes, Enterobacter aerogenes, Escherichia coli and Pseudomonas aeruginosa [27]. Methyl chavicol is reported for antimicrobial action in essential oils and  [23], O. gratissimum and O. tenuiflorum [24]. Furthermore, studies report antioxidant activities that may be related to the presence of linalool and methyl chavicol [25]. Linalool is described as having cytotoxic activities against HeLa, HEp-2 and NIH 3T3 type cancer cells [26], and antibacterial activities against Listeria monocytogenes, Enterobacter aerogenes, Escherichia coli and Pseudomonas aeruginosa [27]. Methyl chavicol is reported for antimicrobial action in essential oils and  [23], O. gratissimum and O. tenuiflorum [24]. Furthermore, studies report antioxidant activities that may be related to the presence of linalool and methyl chavicol [25]. Linalool is described as having cytotoxic activities against HeLa, HEp-2 and NIH 3T3 type cancer cells [26], and antibacterial activities against Listeria monocytogenes, Enterobacter aerogenes, Escherichia coli and Pseudomonas aeruginosa [27]. Methyl chavicol is reported for antimicrobial action in essential oils and  [23], O. gratissimum and O. tenuiflorum [24]. Furthermore, studies report antioxidant activities that may be related to the presence of linalool and methyl chavicol [25]. Linalool is described as having cytotoxic activities against HeLa, HEp-2 and NIH 3T3 type cancer cells [26], and antibacterial activities against Listeria monocytogenes, Enterobacter aerogenes, Escherichia coli and Pseudomonas aeruginosa [27]. Methyl chavicol is reported for antimicrobial action in essential oils and  [23], O. gratissimum and O. tenuiflorum [24]. Furthermore, studies report antioxidant activities that may be related to the presence of linalool and methyl chavicol [25]. Linalool is described as having cytotoxic activities against HeLa, HEp-2 and NIH 3T3 type cancer cells [26], and antibacterial activities against Listeria monocytogenes, Enterobacter aerogenes, Escherichia coli and Pseudomonas aeruginosa [27]. Methyl chavicol is reported for antimicrobial action in essential oils and  [23], O. gratissimum and O. tenuiflorum [24]. Furthermore, studies report antioxidant activities that may be related to the presence of linalool and methyl chavicol [25]. Linalool is described as having cytotoxic activities against HeLa, HEp-2 and NIH 3T3 type cancer cells [26], and antibacterial activities against Listeria monocytogenes, Enterobacter aerogenes, Escherichia coli and Pseudomonas aeruginosa [27]. Methyl chavicol is reported for antimicrobial action in essential oils and  [24]. Furthermore, studies report antioxidant activities that may be related to the presence of linalool and methyl chavicol [25]. Linalool is described as having cytotoxic activities against HeLa, HEp-2 and NIH 3T3 type cancer cells [26], and antibacterial activi-ties against Listeria monocytogenes, Enterobacter aerogenes, Escherichia coli and Pseudomonas aeruginosa [27]. Methyl chavicol is reported for antimicrobial action in essential oils and against phytopathogenic agents such as Brenneria nigrifluens [28]. Studies in silico have indicated that this compound has antilipase biological action and may be a promising molecular target for the treatment of diseases related to oxidative damage [29].

Chemical Composition
The monoterpenic compounds 1,8-cineole and limonene showed significant levels in the composition of the essential oil of the species under study; these contents were higher than those found in the essential oil of a sample collected in Gabon, Africa, which were 0.7% and 0.2%, respectively. Other species of the genus Ocimum of the aforementioned study were also characterized by the respective compounds, as described in the essential oil from O. basilicum, O. gratissimum, O. americanum and O. lamiifolium [21]. There are reports showing that 1,8-cineole has biological properties with antibacterial, antifungal, anesthetic and allelopathic potential [30].
Limonene naturally presents two enantiomeric forms: R-(+)-and S-(−)-. Among these forms, the terpene R-(+)-limonene is the most common found in nature; this compound has shown potential in activities with fumigant and repellent action, electroencephalographic, cytotoxicity against tumor cells and antimicrobial activities against bacteria [31,32].

Larvicidal Activity
Mortality data for third instar Ae. aegypti larvae exposed to different concentrations of O. basilicum var. minimum essential oil. demonstrate the efficacy of the larvicidal action on mosquito larvae at a dose-dependent relationship with a LC 50  There was no significant difference in mortality between the control groups of larvae exposed to water and 2% DMSO, indicating that there is no influence of the diluent on the mortality observed in the solutions of EO (p < 0.05).
The World Health Organization (WHO) does not establish a criterion for evaluating the larvicidal potential of natural products, but some authors consider LC 50 values between 50 and 100 µg/mL as active [33], moderately active [34] or with significant activity [35], framing the EO of O. basilicum var. minimum in these categories (LC 50 = 69.91 µg/mL); this activity can be attributed mainly to the major compounds present in the EO, or even the synergism between them and the other components. For instance, methyl chavicol and limonene have already been shown to be highly active against third instar larvae of Ae. aegypti with LC 50 values of 46.40 µg/mL (CI = 42.50-50.00 µg/mL) and 13.0 µg/mL (CI = 10.50-16.70 µg/mL), respectively [36].
These results are in line with those obtained for most species of the Lamiaceae family that represent 10.5% of the active oils (LC 50 < 100 µg/mL) against third instar larvae of Ae. aegypti, behind only the Myrtaceae family with 13.5% [37]   The larvicidal activity observed for the EO was lower when compared to synthetic larvicides [43]; this lower efficiency and the higher cost compared to synthetic compounds is found for the vast majority of plant derivatives and makes their use much lower than conventional larvicides [37]. suHowever, the increasing use of synthetic larvicides has led to an increase in the resistance of these organisms [44]. Thus, the EO of O. basilicum var. minimum becomes a relevant alternative to combat A. aegypti larvae or even to obtain highly active substances (LC 50 < 50.00 µg/mL) such as methyl chavicol and limonene.

Evaluation of the Interaction of EO Compounds with AChE
Molecular modeling approaches have been successfully used to evaluate the interaction of major volatile compounds with molecular targets of pharmacological interest [45][46][47]. We used AChE as a molecular target because this enzyme is a promising molecular target for essential oils with larvicidal activity [9]. Before starting the docking, MD simulations and free energy analyzes, the crystallographic ligand was redocked. Redocking was performed to assess whether the software was capable of simulating the experimental binding mode found in the crystallographic of the AChE-tacrine derivative complex 9-(3iodobenzylamino)-1,2,3,4-tetrahydroacridine. According to the literature, for the docking protocol used to be considered satisfactory, the RMSD found between the crystallographic ligand and the redocked ligand must be equal to or less than 2 angstroms [48][49][50]. The results of this study showed an RMSD value equal to 1.8 angstroms; thus, our docking protocol is suitable to evaluate the way of interaction of biomolecules with the AChE binding cavity. Figure 2 shows the overlap between crystallographic and redocked ligands. The larvicidal activity observed for the EO was lower when compared to synthetic larvicides [43]; this lower efficiency and the higher cost compared to synthetic compounds is found for the vast majority of plant derivatives and makes their use much lower than conventional larvicides [37]. suHowever, the increasing use of synthetic larvicides has led to an increase in the resistance of these organisms [44]. Thus, the EO of O. basilicum var. minimum becomes a relevant alternative to combat A. aegypti larvae or even to obtain highly active substances (LC50 < 50.00 µg/mL) such as methyl chavicol and limonene.

Evaluation of the Interaction of EO Compounds with AChE
Molecular modeling approaches have been successfully used to evaluate the interaction of major volatile compounds with molecular targets of pharmacological interest [45][46][47]. We used AChE as a molecular target because this enzyme is a promising molecular target for essential oils with larvicidal activity [9]. Before starting the docking, MD simulations and free energy analyzes, the crystallographic ligand was redocked. Redocking was performed to assess whether the software was capable of simulating the experimental binding mode found in the crystallographic of the AChE-tacrine derivative complex 9-(3iodobenzylamino)-1,2,3,4-tetrahydroacridine. According to the literature, for the docking protocol used to be considered satisfactory, the RMSD found between the crystallographic ligand and the redocked ligand must be equal to or less than 2 angstroms [48][49][50]. The results of this study showed an RMSD value equal to 1.8 angstroms; thus, our docking protocol is suitable to evaluate the way of interaction of biomolecules with the AChE binding cavity. Figure 2 shows the overlap between crystallographic and redocked ligands. Docking analyzes have been used to evaluate the molecular binding mode of compounds present in EO. Through these analyzes, it is possible to evaluate the interactions established between the EO compounds and the molecular target under study. In Figure  3, it is possible to visualize the interactions and the chemical nature of the bonds formed between the compounds present in the EO and the AChE. Docking analyzes have been used to evaluate the molecular binding mode of compounds present in EO. Through these analyzes, it is possible to evaluate the interactions established between the EO compounds and the molecular target under study. In Figure 3, it is possible to visualize the interactions and the chemical nature of the bonds formed between the compounds present in the EO and the AChE. ol. Sci. 2022, 23, x FOR PEER REVIEW 8 of 16 In the enzyme binding pocket, all ligands remained interacting throughout the simulation. During the 100 ns of MD simulations, the ligands remained bound to the enzyme and exhibited a low fluctuation profile, as can be seen in Figure 4A-D. In the enzyme binding pocket, all ligands remained interacting throughout the simulation. During the 100 ns of MD simulations, the ligands remained bound to the enzyme and exhibited a low fluctuation profile, as can be seen in Figure 4A-D.
Methyl chavicol interacted with the amino acid residues of Trp83 and Tyr370 through stacked π-π interactions formed mainly by the benzene ring of the molecule. Linalool remained bound to the active site of the protein, forming mainly alkyl-type interactions with residues of Trp427, Leu479, Tyr370, Trp83, Tyr71 and 374 and a hydrogen bond with Glu80. The 1,8-cineole interacted with Trp472, Trp83 and Tyr370 through alkyl and πalkyl hydrophobic interactions and with the residues of Try374 and Tyr71 π-σ interactions. Limonene has formed van der Waals interactions with Asn84, Glu80, Gly79, His480, Gly481 and Tyr73 and alkyl or π-alkyl interactions with Tyr71, Tyr374, Trp83, Tyr370, Trp472 and Leu479.
As  Methyl chavicol interacted with the amino acid residues of Trp83 and Tyr370 through stacked π-π interactions formed mainly by the benzene ring of the molecule. Linalool remained bound to the active site of the protein, forming mainly alkyl-type interactions with residues of Trp427, Leu479, Tyr370, Trp83, Tyr71 and 374 and a hydrogen bond with Glu80. The 1,8-cineole interacted with Trp472, Trp83 and Tyr370 through alkyl and πalkyl hydrophobic interactions and with the residues of Try374 and Tyr71 π-σ interactions. Limonene has formed van der Waals interactions with Asn84, Glu80, Gly79, His480, Gly481 and Tyr73 and alkyl or π-alkyl interactions with Tyr71, Tyr374, Trp83, Tyr370, Trp472 and Leu479. As

Plant Material
Leaves of O. basilicum var. minimum were collected in the municipality of

Plant Material
Leaves of O. basilicum var. minimum were collected in the municipality of Ananindeua, Pará, Brazil. The specimen of the sample was identified and incorporated into the collection of the João Murça Pires Herbarium (MG) of the Emílio Goeldi Museum of Pará, in the collection of Aromatic Plants of the Amazon, Belém, Pará, under the registration MG167656.

Preparation of Botanical Material
The samples of O. basilicum var. minimum leaves were dried in an oven with air circulation at a temperature of 35 • C for 5 days and then ground in a knife mill (Tecnal, model TE-631/3, Brazil). The moisture content was analyzed using an infrared moisture analyzer (ID50; GEHAKA, Duquesa de Góias, Real Parque, São Paulo, Brazil), in the temperature range from 60 to 180 • C with increments of 1 • C and bidirectional output.

Essential Oil Extraction
The essential oil (EO) from the leaves of O. basilicum var. minimum was extracted using 130 g of dried material by stem distillation with a Clevenger-type apparatus for 3 h as described by Oliveira et al. [51]. The EO obtained was dehydrated with anhydrous sodium sulfate and centrifuged for 5 min at 3000 rpm.

Essential Oil Analysis
The chemical compositions of the EO of O. basilicum var. minimum were analyzed as reported by our research group [52,53], using a Shimadzu QP-2010 (Kyoto, Japan) plus gas chromatography system equipped with an Rtx-5MS capillary column (Restek Corporation, Bellefonte, PA, USA) (30 m × 0.25 mm; 0.25 µm film thickness) coupled with a mass spectrometer (GC/MS) (Shimadzu, Kyoto, Japan) and the components were quantified using gas chromatography (CG) on a Shimadzu QP-2010 system (Kyoto, Japan), equipped with a flame ionization detector (FID). The program temperature and injection were the same operating conditions as described in the literature [54,55], except for the carrier hydrogen gas, under the same operating conditions as before. The retention index for all volatile constituents was calculated using a homologous series of n-alkanes (C 8 -C 40 ) Sigma-Aldrich (San Luis, CA, USA), according to van den Dool and Kratz [56]. The components were identified by comparison of: (i) the experimental mass spectra with those compiled in libraries, and (ii) their retention indices to those found in the literature [57].

Larvicidal Assay
The methodology adopted was the World Health Organization standard protocol [58]. To obtain the larvae, Ae. aegypti eggs were placed in a tray with 500 mL of distilled water added to 1 g of rat chow. Hatching occurred within 24 h and larvae were allowed to grow. Upon reaching the third instar, batches of 25 larvae were transferred by droppers to small disposable test cups, each containing 100 mL of water, and added appropriate volume of stock solution to obtain the desired target dosage.
The larvae were exposed to concentrations of 15.62, 31.25, 62.5, 125, 250 and 500 ppm (v/v) of the oil diluted in 2% dimethyl sulfoxide aqueous solution (DMSO 2%) for 24 h. The entire assay was performed in quadruplicate, with 25 larvae for each concentration, at a temperature of 25 • C and a photoperiod of 12 h of light followed by 12 h in the dark. Negative controls water (H 2 O) and DSMO 2% were evaluated under the same conditions as the sample.
3.6. In Silico Analysis 3.6.1. Molecular Docking The compounds methyl chavicol, linalool, 1,8-cineole and limonene were drawn in GaussView 6 and their structure were optimized via B3LYP/6-31G* using the Gaussian quantum chemistry software 16 [59]. The software Molegro Virtual Docker [60] was then used to assess how these compounds are able to interact with the acetylcholinesterase (AChE) binding cavity. For that, the crystal structure of the protein was collected in the Protein Data Bank (www.rcsb.org, accessed on 5 March 2022) and located using the PDB ID: 1QON. The MolDock Score (GRID) scoring function was used with a Grid resolution of 0.30 Å and 5 Å radius encompassing the entire connection cavity. The MolDock SE algorithm was used for the docking with a number of runs equal to 10, 1500 max interactions, and max population size equal to 50. The maximum evaluation of 300 steps, with neighbor distance factor of 1 and an energy threshold of 100, was used during the molecular docking simulation.

MD Simulations
The charges of the methyl chavicol, linalool, 1,8-cineole and limonene atoms were calculated HF/6-31G*. Ligand parameters were constructed using GAFF and the proteins were described by the ff14SB force field in all simulations. The protonation of protein residues was evaluated using PROPKA server.
Each system was solvated in an octahedron periodic box with a 12-Å cutting radius in all directions from the solute (Waterp-TIP3P). An adequate number of counterions were added to neutralize the partial charge of the systems.
The MD simulations were performed using the Amber 16 software [61]. The minimization of system energy occurred in three steps. In the first step, 2000 cycles were executed using the steepest descent method and conjugate gradient algorithm, applying a harmonic force constant of 50 kcal·mol −1 ·Å −2 to the solute. In the second step, the harmonic force constant applied to the solute was 25 kcal·mol −1 ·Å −2 and 1000 more cycles were run using the steepest descent method and conjugate gradient algorithm. In the last step, the constraints were removed and 1000 cycles were run using steepest descent method and conjugate gradient algorithm.
To increase the system temperature from 0 to 300 k, 900 ps simulations were run. Warming up was carried out in three steps. In the first step, the solute was constrained with a harmonic force constant of 25 kcal·mol −1 ·Å −2 , thus, only the solvent and counterions were free to move. In the following two steps, the harmonic force constant was removed. To balance the complexes, 2 ns simulations were run at constant temperature and without restrictions. Then, for each complex, 100 ns of MD simulation with NVT ensemble were generated.

Free Energy Calculations
The free energy calculations were performed using MM-GBSA method [62]. The free energy was calculated as follows: where ∆G bind is the free energy of the complex, resulting from the sum of the molecular mechanics energy (∆E MM ), desolvation free energy (∆G solv ), and entropy (−T∆S).
The molecular mechanics energy of the gas phase (∆E MM ) can be described by the sum of the internal energy contributions (∆E internal ); sum of the connection, angle, and dihedral energies; electrostatic contributions (∆E electrostatic ); and van der Waals terms (∆E vdW ).
The desolvation free energy (∆G solv ) is the sum of the polar (∆G GB ) and nonpolar (∆G nonpol ) contributions. The polar desolvation term was calculated using the implicit generalized born (GB) approach.

Statistical Analysis
Mortality data from replicates of each concentration were grouped and presented as mean ± standard deviation. The values of LC 50 , LC 90 and 95% confidence intervals (CI) of upper and lower confidence levels were calculated from probit regression analysis using the software GraphicPad Prism 8. Larval mortality was corrected using the formula by Abbott [63], when mortality of the control group H 2 O varied between 5-20%. A one-way ANOVA statistical test was also performed, followed by a Tukey post-test. The statistical difference was considered significant when p < 0.05.

Conclusions
Chemical characterization of O. basilicum var. minimum essential oil extracted by stem distillation revealed the presence of hydrocarbon monoterpenes, oxygenated monoterpenes, hydrocarbon sesquiterpenes, oxygenated sesquiterpenes, with methyl chavicol, linalool, 1,8-cineole and limonene as the major compounds. In addition, the essential oil of O. basilicum var. minimum showed larvicidal action against Ae. aegypti larvae and the major compounds were able to interact with the binding cavity of the target enzyme acetylcholinesterase (AChE), indicating a potential ecological alternative for the control of larvae of this mosquito.