Larvicidal evaluation of the Origanum majorana L. Essential Oil against the larvae of the Aedes aegypti mosquito

This study evaluated the larvicidal activity of O. majorana essential oil, identified the chemical composition, evaluated the antimicrobial, cytotoxic and antioxidant potential. The larvicidal activity was evaluated against larvae of the third stage of Aedes aegypti, whereas the chemical composition was identified by gas chromatography coupled to mass spectrometer, the antimicrobial activity was carried out against the bacteria Pseudomonas aeruginosa, Escherichia coli and Staphylococcus auereus, the antioxidant activity was evaluated from of 2.2-diphenyl-1-picryl-hydrazila sequestration and Artemia salina cytotoxicity. Regarding to the results, the larvicidal activity showed that O. majorana essential oil caused high mortality in A. aegypti larvae. In the chromatographic analysis, the main component found in O. majorana essential oil was pulegone (57.05%), followed by the other components verbenone (16.92%), trans-p-menthan-2-one (8.57%), iso-menthone (5.58%), piperitone (2.83%), 3-octanol (2.35%) and isopulegol (1.47%). The antimicrobial activity showed that E. coli and P. aeruginosa bacteria were more sensitive to oil than S. aureus, which was resistant at all concentrations. Essential oil did not present antioxidant activity, but it has high cytotoxic activity against A. salina.

The A. aegypti mosquito (Linnaeus, 1762) is a vector of viruses that cause diseases known as dengue, chikungunya, and zika [5]. It has holometabolic development, with egg, larva, pupa and adult phases. Because it is a mosquito highly adapted to the urban environment, its most common breeding sites are artificial containers that accumulate water, such as bottles, tires, cans, and pots [6].
Among the control policies adopted in Brazil, the mechanical control is carried out by Agents to Combat Endemics (ACE), with the participation of the population, aiming at the protection, destruction or adequate allocation of potent breeding sites. The intensive collaboration of the population is crucial to hinder the proliferation and installation of the mosquito. In addition, it reinforces the need for adequate sanitary conditions in the cities, eliminating stocks of water that allow eggs to hatch. An important strategy is the promotion of educational actions during home visits made regularly by the health agents [7].
The spread and flow of various serotypes of the dengue virus over the years also have a significant influence on epidemics, as well as an increase in cases diagnosed for the most severe form of the disease. These factors demonstrate the importance of introducing preventive measures in order to reduce dengue rates [8].
Origanum majorana L. belongs to the Lamiaceae family, and it contains several terpenoids, which are isolated from aerial parts of the Origanum plant and exhibit antimicrobial, antiviral and antioxidant properties, without toxic effects [9,10]. In folk medicine, O. majorana is used for cramp, depression, migraine and nerve headaches [9].
The antimicrobial and antioxidant properties of many spices and their essential oils have been known for a long time, but only in recent years have consumers given proper attention to the use of these substances [11]. Because many plants are toxic to mosquitoes, the mixture of essential oils may represent an efficient outlet for this problem, compared to the A. aegypti mosquito [12].
In the literature, there are no reports on larvicidal activity against A. aegypti and cytotoxicity against A. salina, and few studies have been reported on the antioxidant and antimicrobial effects of the essential oils of this species. Therefore, the objective of this study was to evaluate the larvicidal activity against A. aegypti, to determine the chemical composition, to evaluate the antimicrobial activity against E. coli, P. aeruginosa and S. aureus bacteria, to determine the antioxidant potential through the sequestration of DPPH and cytotoxicity against A. salina of O. majorana essential oil.

Plant Material
The leaves of O. majorana were collected in the district of Fazendinha (00 "36'955" S and 51 "11'03'77" W) in the Municipality of Macapá, Amapá. Five samples of the plant species were deposited at the Amapaense Herbarium (HAMAB) of the Institute of Scientific Research and Technology of Amapá (IEPA).

Obtaining Essential Oil
The essential oil (EO) was obtained by the hydrodistillation process using the Clevenger type apparatus, 131 g of O. majorana dried leaves were dried at 45 °C for a period of 2 h [13]. The EO was kept under refrigeration (4ºC).

Identification of the Chemical Composition by Gas Chromatography Coupled to Mass
Spectrometer (GC-MS).
The EO analysis was performed by Gas Chromatography coupled to the Mass Spectrometer (GC-MS) of the Museu Paraense Emílio Goeldi. The Shimadzu equipment, CGEM-SHIMADZU QP 5000 was used. A fused silica capillary column (OPTIMA®-5-0.25 μm) was used. It has 30 m of length and 0.25 mm of internal diameter and nitrogen as carrier gas. The operating conditions of the gas chromatograph were: internal column pressure 67.5 kPa, division ratio 1:20, gas flow at column 1.2 mL/min (210 °C), injector temperature 260 °C, temperature detector or interface (GC-MS) of 280 °C. The initial column temperature was 50°C, followed by an increase from 6 °C/min to 260 °C kept constant for 30 min. The mass spectrometer was programmed to perform readings at intervals of 29-400 Da, 0.5 s with ionization energy of 70 eV.
The identification of the chemical compounds present in the EO was made from the comparisons of the Indices of Retention (IR) and Kovats (IK) of the homologous series of n-alkanes (C8-C26) and the literature [14]. Identification was also made by In the bioassay, 18 beakers of 100 mL of glass were organized into six groups.
The mother solution was distributed as follows: in group I, it was added 10 mL, in group II it was added 8 mL, in the group III it was added 6 mL, in group IV it was added 4 mL and in group V it was added 2 mL. In group VI, 80 mL of positive control solution was added.
Then, about 80% of distilled water were added to each beaker from the total volume and plus 25 A. aegypti larvae. Group I was 100 μg.mL -1 , group II was 80 μg.mL -1 , group III was 60 μg.mL -1 , group IV was 40 μg.mL -1 group V had a total of 20 μg.mL -1 of test solution. After 24 and 48 h, the number of dead larvae was counted, it is considered as dead all those that were unable to reach the surface.

Larvicidal Statistical Analysis
The experiment was carried out in triplicate. The larval mortality efficiency data were calculated in percentages using the Abbott formula and later tabulated in Microsoft For each microorganism, the stock culture was stored in BHI medium (Brain Heart Infusion) with 20% glycerol and stored at -80 °C. An aliquot of 50 μL of this culture was inoculated into 5 mL of sterile BHI broth medium and incubated for 24 h at 37 °C.

Determination of Minimum Inhibitory Concentration (MIC) and Minimum
Bactericidal Concentration (MBC).
The MIC and MBC were determined using the microplate dilution technique (96 wells) according to the protocol established by Icls [16], with adaptations.
Bacteria were initially reactivated from the stock cultures, kept in BHI broth, for 18 h at 37 °C. Subsequently, bacterial growth was prepared in 0.9% saline inoculum for each microorganism, adjusted to the McFarland 0.5 scale, then diluted in BHI and tested at 2 x 10 6 UFC.mL -1 concentration.
In determining the MIC, the EO was diluted in Dimethylsulfoxide (2% DMSO).
Each well of the plate was initially filled with 0.1 mL of 0.9% NaCl, except for the first column, which was filled with 0.2 mL of the EO at the concentration of 2000 μg.mL -1 .
Subsequently, base two serial dilutions were performed in the ratio of 1:2 to 1:128 dilution in a final volume of 0.1 mL. After this process, 0.1 mL of cells (2 x 106 CFU.mL -1 ) added in each well related to the second preceding item, resulting in a final volume of 0.2 mL.
Control of culture medium, control of EO, and negative control (DMSO 2%) were performed. And for the positive control, amoxicillin (50 μg.mL -1 ) was used. After incubation of the microplates in an incubator at 37ºC for 24 hours, the plates were read in ELISA reader (DO630nm). Significant differences between the groups were verified using the One-way ANOVA test with Bonferroni post-test. The data were considered statistically significant when p <0.001.

Antioxidant Activity
The antioxidant quantitative test was based on the methodology recommended by Sousa et al. [17], Lopes-Lutz et al. [18] and Andrade et al. [19] by the use of 2. The A. salina cytotoxicity assay was based on the technique of Araújo et al. [20] and Lôbo et al. [21] with adaptations. An aqueous solution of artificial sea salt was prepared (35 gL -1 ) at pH 9.0 for incubation of 45 mg of A. salina eggs, which were placed in the dark for 24 h for the larvae to hatch (nauplii), then the nauplii were exposed to artificial light in 24 h, period to reach the stage methanuplii. The stock solution was prepared to contain 0.06 g of EO, 1.  Notes: Gas Trap: Helium (He); initial temperature 60 ° C; initial time 1.0 min; the column temperature increased 3 ° C / min. at 240 ° C, maintained at this temperature for 30 min.

Larvicidal Activity
The percentage of dead A. aegypti larvae is shown in

Antimicrobial Activity
The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) that were identified for O. majorana EO can be verified in Fig. 2.
The results show that gram-negative bacteria were more sensitive to EO presenting MIC = 31.25 μg.mL -1 compared to the negative control. The MBC for E. coli was at the concentration of 500 μg.mL -1 and for P. aeruginona was at the concentration of 1000 μg.mL -1 in relation to the negative control (amoxicillin). While the S. aureus bacterium did not present MIC and neither MBC.

DISCUSSION
This result corroborates with other studies that have shown that environmental factors may affect certain chemical compounds, while in others they have no influence on their production [22,23].
Lima et al. [24], reports that piperitone has three organic functions in its chemical structure and it can be used for the synthesis of other compounds. Piperitone is derived from the metabolic pathway for the formation of piperitenone oxide, in which cispulegone is also, derived [25].
Macêdo et al. [26] observe that the variations of the active components of the plant are important parameters to correlate the activities, such as antibacterial and insecticide.
In addition, a number of biotic factors such as plant/ microorganism Stoppacher et al. [27], plants/ insects Kessler and Baldwin [28] plant interactions, age and stage of development. As well as abiotic factors such as luminosity Takshak and Agrawal [29], temperature, precipitation, nutrition, time and harvest time Bitu et al. [30], they may present correlations with each other, acting together, and they may exert a joint influence on chemical variability and yield of essential oil [26].
The results of the larvicidal activity of this study show that O. majorana EO is active against A. aegypti larvae. A fact that Komalamisra et al. [31], Magalhães et al. [32] and Dias et al. [33], classified with the values of the minimum lethal concentration that eliminates 50% of the population (LC50) as a criterion for the activity. Because if LC50 <50 μg.mL -1 , the product is considered very active, if 50 <LC50 <100 μg.mL -1 the product is considered active, and when LC50> 750 μg.mL -1 the product is considered inactive.
There were no reports of studies on the larvicidal activity of O. majorana essential oil against A. aegypti larvae.
According to Cantrell et al. [34], larvicidal compounds act by absorption through the cuticle, via the respiratory tract, and/or enter by ingestion via the gastrointestinal tract.
Once inside the larva, the substances may reach the site of action or may cause systemic effects by diffusion in different tissues [35].
Studies on the insecticidal effect of Mentha spp. reported that menthol, mentona, pulegone and carvone help to clarify the mechanisms of action on insects [36]. Previous studies indicate that limonene, camphene, and verbenone have insecticidal insect activity [37].
Some EOs are known to cause dissuasive or anti-eating behavior in insects suggesting a neurotoxic action Satyan et al. [38], while some act as growth-regulating insects through analogous effects or antagonistic endogenous hormones. In the present study, it was found that even short-term exposure of larvae to lethal doses can dramatically increase their mortality over time and thereby reduce the total number of viable adults, leading to a possible reduction in total populations [39].
In relation to the microbiological activity, it was possible to verify that gramnegative bacteria were more sensitive to O. majorana EO than gram-positive bacteria.
According to Rosato et al. [40], the antibacterial activity in gram-negative bacteria occurs due to the high percentage of oxygenated monoterpenes present in the EO and consequently the synergism between these components. On the other hand, bacteria can also respond to adverse conditions in a transient way, through so-called stress tolerance responses. Bacterial stress tolerance responses include structural and physiological modifications in the cell, and complex genetic regulatory machines mediate them [41].
In the study by Duru et al. [42] pulegone showed high antimicrobial activity, particularly against Candida, albicans and Salmonella typhimurium. Pulegone is classified as a monoterpene, in the same way as carvone. It can be obtained from a variety of plants [43,44]. Menthone is a common volatile compound in Lamiaceae, which may also be active against a large number of bacteria, such as E. coli and Enterococcus faecalis [45,46]. Some studies have argued that monoterpenes can cross cell membranes and interact with intracellular sites critical for antibacterial activity [47].
However, reports of non-adaptation or cross-adaptation of bacteria to sublethal concentrations of major constituents of essential oils have also been reported [48]. Crossresistance can occur when different antimicrobial agents attack the same target in the cell, reach common route of access to the respective targets or initiate a common pathway for cell death, ie, the resistance mechanism is the same for more than one antibacterial agent [49].
Many antioxidants derived from natural products demonstrate neuroprotective activity in vitro and/or in vivo models such as flavonoid phenolic compounds [50].
The percentage of antioxidant activity of the essential oil showed a high IC50 = 16.83 μg.mL -1 , whereas ascorbic acid presented 16.71 μg.mL -1 [51]. According to Rodrigues [52] the higher the consumption of DPPH for a smaller sample will be its IC50 and the greater its antioxidant capacity.
According to Beatović et al. [53], the antioxidant capacity of EO is related to its main compounds. However, this study did not present antioxidant activity. The importance concerning the performance of antioxidants depends on the factors types of free radicals formed; where and how these radicals are generated; analysis and methods for identifying damage, and ideal doses for protection [54].
A. salina is a microcrustacean used in fish feed, and it is widely used in toxicological studies because of the low cost and easy cultivation. Several studies have attempted to correlate toxicity on A. salina with antifungal, virucidal, antimicrobial, trypanosomicidal and parasiticidal activities. Lethality assays are performed in toxicological tests and the median lethal concentration (LC50), which indicates death in half of a sample, can be obtained [55].
The lethal concentration of mortality against the A. salina larvae of this assay showed moderate cytotoxic activity. In order to evaluate the cytotoxicity of a given sample, it is possible to elucidate the cytotoxic effect of the cytotoxic mechanism and the mechanism of action of different compounds during their interaction with the tissues [57].