Comparative toxicity of cinnamon oil, cinnamaldehyde and their nano-emulsions against Culex pipiens (L.) larvae with biochemical and docking studies

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Introduction
The mosquitoes are at the center of worldwide entomological research due to their importance as vectors of several viral and parasitic diseases affecting about 700 million people worldwide (Li et al., 2022;Wilder-Smith et al., 2017).According to the World Health Organization (WHO), more than 80 % of the world's population lives in areas at risk of at least one vector-borne disease that causes more than 700.000 deaths annually (WHO 2017).Culex is a predominant mosquito that mainly thrives in tropical and sub-tropical areas, breeds in dirty waters like congested drains and impaired septic tanks near human dwellings.In Egypt, Culex species can transmit a nematode worm (Wuchereria bancrofti), responsible for the filarial disease (Holder et al., 1999).Filariasis is the fastest spreading insect-borne disease to humans worldwide; 146 million cases are reported worldwide every year.About 3492 species of mosquito are recorded; 100 species of them are vectors and can transmit many diseases to humans and mammals (Ghosh et al., 2012).Once limited to tropical and subtropical zones, numerous vector-borne diseases have emerged in temperate areas because of climate change.Hence, temperate countries may be the most threatened by the emergence and re-emergence of vector-borne diseases (Karuppusamy et al., 2021;Rocklöv and Dubrow, 2020).
programs target the larval stage in their breeding sites with larvicides because they are most concentrated, immobile, sensitive, and accessible at this stage.At the same time, the use of adulticides only reduces the adult population temporarily (Rehman et al., 2014).Moreover, the insecticide application is in a defined area, decreasing environmental contamination.Pyrethroids and organophosphates are the main groups used to control mosquitos in Egypt and worldwide.Organophosphate insecticides were used outdoor as larvicides, while pyrethroids were used indoors as adulticides (Zahran et al., 2017).However, synthetic insecticides to control mosquitoes have created resistance among target species, adverse effects on non-target organisms, and a secondary impact on the environment and public health.
These drawbacks have prompted researchers to explore new insectselective products with low toxicological risks, environmentally safer, target-specific, and cost-effective insecticides as an alternative vector control tool (Alsaraf et al., 2021;Zeghib et al., 2020).Therefore, enormous efforts have been made to explore the efficiency of plant-based products against mosquitoes (Wangai et al., 2020;Zahran et al., 2017;Zeghib et al., 2020).Plant-based insecticides have bioactivity, biodegradability, and human and environmental safety compared to traditional insecticides.They also have various novel modes of action, which is crucial in combating insect resistance (Norris et al., 2018).Insecticidal properties of natural bioactive compounds derived from plants, especially essential oils, are suitable against mosquito larvae (El Gohary et al., 2021).These essential oils are complex natural mixtures of volatile, semi-volatile organic compounds, odorous, and secondary metabolites, principally mono-and sesquiterpenes, which are considered among the best alternatives for the control of disease vectors (Alsaraf et al., 2021;Taktak and Badawy, 2019).Monoterpenes are secondary plant metabolites with insecticidal, nematicidal, acaricidal, and larvicidal properties (El-Sabrout et al., 2020;Kweka et al., 2016).The physical and chemical properties of monoterpenes, such as chemical instability, volatility, and low water solubility, rendered them unsuitable for widespread use against Culex larvae (Moretti et al., 2002).As a result, incorporating essential oils (EOs) and monoterpenes into NEs could improve the system to be more effective than bulk substances.In addition, NEs provide several benefits, such as a substantial increase in water solubility, dissolution rate, dispersion uniformity, and significant bioavailability after application (El Gohary et al., 2021).
It is generally understood that insects exposed to these products may encounter toxic compounds that have relatively nonspecific effects on a wide range of molecular targets.Among the targets are proteins (enzymes, receptors, signaling molecules, ion-channels, structural proteins), nucleic acids, biomembranes, and other cell components (Rattan, 2010).As a result, insect physiology is impacted in several ways, including the nervous system malfunctioning, which is the most important one.Rattan (2010) reviewed several physiological effects of plant secondary metabolites on insects, including inhibition of acetylcholinesterase (AChE), blockage of the gamma-aminobutyric acid (GABA)-gated chloride channel, disruption of sodium and potassium ion exchange, and inhibition of cellular respiration (Rattan, 2010).In particular, inhibition of AChE activity is significant since it is responsible for terminating nerve impulse transmission through the synaptic pathway (Badawy et al., 2018;Rattan, 2010).
Therefore, the main objective of this study was to prepare NEs of cinnamon EO and cinnamaldehyde as larvicidal agents against C. pipiens.Droplet size distribution, polydispersity index (PDI), viscosity, pH, stability, and surface morphology of the produced NEs were examined.The larvicidal effect against C. pipiens of the technical grades (T) was compared to the NEs.The in vitro biochemical studies were also investigated on AChE, adenosine triphosphatase (ATPase), and GABA transaminase (GABA-T).In addition, molecular docking of cinnamaldehyde and a reference larvicide temephos was examined with some enzymes.

C. pipiens mosquito rearing
A colony of a susceptible strain of C. pipiens culture was reared in the High Institute of Public Health insectary, Alexandria University, Alexandria, Egypt.The larvae were feeding on biscuits until pupated in shallow trays with 2-3 liters of dechlorinated water.In adult cages (30 x 30 x 30 cm), male adults were fed on 30% sucrose solution, while females were fed on pigeon blood four times a week.The use of a live pigeon in our research was approved by the High Institute of Public Health's Ethics committee and confirmed by Alexandria University under reference number 481.The egg rafts were moved from adult cages to white trays containing dechlorinated water for egg hatching.

NEs preparation
The cinnamon oil NE was prepared from 10% active ingredient as an organic phase with a polar phase mixture of Tween 80 (10%) and water (80%).While NE of cinnamaldehyde was prepared as follows: active ingredient was dissolved in DMSO to prepare the organic phase, Tween 80 dissolved in distilled water at 50 °C to prepare the polar phase.Considering the concentrations of the active ingredient, DMSO and Tween 80 were 2.5%, 5%, and 2.5%, respectively, in the prepared total volume (Abdelrasoul et al., 2020).The organic phase was dropped into the polar phase by stirring at room temperature for 30 min at 4000 rpm to form the coarse emulsion.Using a highenergy ultrasonic procedure, the ultrasonic probe was used to transform the coarse emulsion into a NE (Figure 1).The ultrasonication process was carried out for 5 min at a rate of 9 cycles per second and a power of 15 kHz for cinnamon oil and 5 kHz for cinnamaldehyde (Munawiroh et al., 2017).

Droplet size and polydispersity index (PDI)
At room temperature, droplet size and PDI of the NE were determined by Dynamic Light Scattering (DLS) technique with a Zetasizer NanoZSlaser diffractometer (Malvern Instruments Ltd, Worcestershire, UK) working at 633 nm and equipped with a backscatter detector (173˚).At a fixed 90° angle, the droplet size (nm) was characterized by distribution curves in intensity (%).The NEs were 500-fold diluted in ultrapure water and sonicated for 5 min at 9 cycles/sec and 5 kHz power before being measured to prevent multiple scattering effects.The droplet size was expressed as a mean diameter in nanometer (Mibielli et al., 2021;Silva et al., 2012).

Viscosity and pH measurements
The dynamic viscosity was determined without further dilution using a digital viscometer (a Rotary Myr VR 3000) with an L2 spindle spinning at 200 rpm at 25 °C.The viscosity was measured three times, and the results were expressed in mPa.s.The pH values of the prepared NE were determined using a digital pH meter (Crison pH Meter Basic 20, EU) (Badawy et al., 2017;Drais and Hussein, 2015).

Stability tests
Three samples from each formulation were centrifuged for 30 min at 5000 rpm, and phase separation, creaming, and cracking were observed.The NE should have enough stability without phase separation.Other thermodynamic stability tests were performed on stable formulations.Three samples of each NE were held at 25 and 40 °C then subjected to a heating-cooling test.The thermodynamic stability examinations were done by storing the NE at -21 °C for 24 h and then at 21 °C until melting for 24 h.A transparent tube was filled with 25 ml of each freshly prepared NE for three months at 25 °C as a storage period, the transition from steady-state to creaming and coalescence was investigated (Badawy et al., 2017;Drais and Hussein, 2015).

Zeta potential (ζ)
Using the Zetasizer NanoZSlaser (Malvern Instruments, Worcestershire, UK) at 25 °C, the zeta potential of NEs was investigated (Silva et al., 2012).Zeta potential was measured by transferring the samples to electrophoretic cells, then applying an electrical potential ± 150 mV.The formulations were 200-fold diluted in ultrapure water and sonicated for 5 min at 9 cycles/sec and 5kHz power before being analyzed to avoid multiple scattering effects (Smoluchowski, 1916).

TEM
TEM (JEOL JEM-1400 Plus transmission electron microscope, USA, Inc.) equipped with 20 mM aperture at 20 Kv was used to examine the surface morphology and topology of prepared NEs.Bright-field imaging with increasing magnification and diffraction modes was chosen.The sample from each NE was diluted with distilled water (1:100) and added to 200-mesh form war-coated copper TEM sample holders (EM Sciences, Hatfi eld, PA, Japan).

Toxicity assay against C. pipiens larvae
The larval bioassay was performed on C. pipiens larvae to compare the effect of cinnamon oil and cinnamaldehyde and their NEs by dipping method (WHO, 2005).Tested compounds and their NEs were tested against third instar larvae of C. pipiens to obtain the LC50 values.The technical cinnamaldehyde and pure cinnamon oil were dissolved in Tween 80/DMSO (0.05%) then dissolved in dechlorinated water.In comparison, their NEs were dissolved directly in de-chlorinated water.After preliminary screening for all products, different stock solutions for each compound were prepared to determine the toxicity.The tested concentrations of cinnamaldehyde were between 50 to 600 mg/l.In contrast, the tested concentrations of cinnamon oil were between 20 to 300 mg/l.The acute toxicity of technical temephos (90%) was also evaluated by mixing different concentrations (2, 4, 6, 8, 10, 20, 30, and 40 µg/l) prepared in DMSO with dechlorinated tap water.In a control experiment, DMSO and Tween 80 were mixed with dechlorinated water.Twenty C. pipiens larvae were put into plastic cups containing 100 ml of de-chlorinated water.Mortality caused by the compounds was recorded as larval mortalities percentage of dead versus live larvae numbers after 24 and 48 h.The 50% lethal concentration (LC50) values of the compounds were calculated using probit analysis (Finney, 1971).

Preparation of enzyme homogenates
After 24 h of exposure to LC50 values of tested compounds, surviving larvae were homogenized in 10 mM NaCl (1%, w/v) Triton X-100 and 40 mM sodium phosphate buffer (pH 7.4) at 4 °C to assess ATPase, AChE, and GABA enzyme activities.The homogenate was centrifuged at 4 °C for 20 min at 5000 rpm.The supernatant was either used right away for an enzymatic assay or processed at -20 °C for later use (Taktak and Badawy, 2019).

Total protein assay
According to Lowry et al. (1951) method, total protein in crude enzyme was determined using BSA for the standard curve.Protein extract (200 µl) was added to 1900 µl alkaline copper reagent [48 ml of sodium carbonate 2% (w/v) in 0.1 N sodium hydroxide + 1ml sodium-potassium tartrate 1% (w/v) + 1 ml copper sulfate 0.5% (w/v)] and immediately mixed with 200 µl Folin-Ciocalteu phenol reagent.After 30 min of incubation at 25 °C, the absorbance was recorded at 600 nm.By comparing the protein content of the measured samples to the BSA standard curve, the protein content of the samples was calculated.

Acetylcholinesterase (AChE) assay
AChE activity was determined by Ellman colorimetric method (Ellman et al., 1961).The reaction mixture contained 10 µl ATChI (0.075 M), 1340 µl phosphate buffer (pH 8), 100 µl of the crude enzyme, and 50 µl DTNB (0.01 M).Reaction mixture free of crude enzyme sample was checked as a blank, while a mixture containing non-treated larval extract was also studied as a control.Using a Unico 1200 Spectrophotometer (Laxco Inc, USA), the absorbance was measured at 412 nm after 10 minutes of incubation at 37 °C.The enzyme activity was expressed as OD412.mgprotein -1 .min - .

Adenosine triphosphatase (ATPase) assay
The Koch method was used to measure the activity of ATPase (Koch, 1969).The reaction mixture was prepared with 400 mM Na + , 20 mM K + , 5 mM Mg + , and 5 mM ATP, and 200 l of the crude enzyme was transferred.After that, the volume was completed to 950 µl with tris-HCl buffer (pH 7.4).The reaction was stopped with 200 µl of TCA after 10 min of incubation at 37 °C.10 ml of a fresh color reagent containing 5 g of ferrous sulfate dissolved in a solution of ammonium molybdate and sulfuric acid (10 N) was added to the reaction mixture.A reaction mixture free of crude enzyme sample was examined as a blank.As a control, a reaction mixture containing untreated larval extract was tested.The enzyme activity was measured according to the absorbance of the developed color at 740 nm and expressed as OD740.mgprotein -1 .min - (Kessler et al., 2014).

Gamma aminobutyric acid transaminase (GABA-T) assay
The activity of GABA-T was assayed according to Pandey and Singh (1985) with minor modifications.Enzyme extract (100 μl) was added to 1450 μl of 50 mM Tris-HCl (pH 8.5), 100 μl of 2 mM αketoglutarate, 100 μl of 2-mercaptoethanol (20 mM), and 20 μl of β-NAD (1.1 mM).The reaction was started by adding 200 μl of GABA (3 mM) to the mixture.Finally, 30 μl of 1% triton-x 100 were added to the previous mixture.Samples including the blank and control samples were incubated at 25 to 30 °C for 30 min.The absorbance was measured at 340 nm, and the enzyme activity was presented as OD340.mgprotein -1 .min - .

Molecular docking
Using molecular docking studies, digital values were used to determine the effectiveness of cinnamaldehyde and temephos against various enzyme targets.The crystal structures of AChE (PDB: 5X61), ATPase (PDB: 4BYG), and GABA-T (PDB: 1SF2) were obtained from protein data bank (PDB) (http://www.rcsb.org/pdb/home/home.do).The selection of AChE PDB was based on the model for C. pipiens AChE1 that was built using the template protein of malaria mosquito vector Anopheles gambiae (PDB: 5X61, Chain B), with 99% query coverage and 92.36% similarity (Rao et al., 2021).3D structure of the tested enzymes was visualized using Molecular Operating Environment (MOE) 2014.13 software (Chemical Computing Group Inc, Montreal, Quebec, Canada) and examined for missing atoms, bonds, and contacts (Chemical Computing Group, 2008).The tested compounds were converted to 3D, and the energy was minimized by the MMFF94 function (Halgren, 1999).A triangle-matching algorithm was chosen to dock the compounds into the active sites of the enzymes.The contributions of hydrophobic, ionic, hydrogenated, and Van der Waals interactions were used to quantify the free energy of binding.A ligand was considered adequate for a minimum docking score value (or interaction energy calculation) of an enzyme-ligand complex.

Statistical analysis
Statistical analysis was performed using the IBM SPSS software version 25.0 (SPSS, Chicago, IL, USA) (IBM, 2017).Mortality percentages were calculated for each treatment and corrected using Abbott's equation (Abbott, 1925).Means and standard error (SE) were obtained from three independent replications for each treatment.According to the probit analysis, the log dose-response lines (LdP line) were used to determine the LC50 values (Finney, 1971).The least-square regression analysis was used to determine the 95% confidence limits.Analysis of variance (ANOVA) of the biochemical data was conducted and means property values were separated (p ≤ 0.05) with Student-Newman-Keuls (SNK).

GC/MS analysis of cinnamon oil
The GC/MS chromatogram of the oil is shown in Figure 2. The chemical composition of cinnamon oil was done using GC/MS analysis.A chromatogram of C. verum EO constituents showed that the oil is a mixture of 12 different components, representing 99.40% of the total oil (Table 1).The identified chemical compounds belong to olefinic hydrocarbons, monoterpenes, sesquiterpenes, and other essential phytochemicals.The major components detected in the oil, according to retention time and their relative abundances, were -pinene (1.03%), β-cymene (1.01%), 1,8-cineole (6.12%), linalool (2.02%), (E)-cinnamaldehyde (80.09%), eugenol (3.18%), methyl linoleate (1.06%) and (E)-cinnamyl acetate (2.56%).However, other constituents, including β-pinene, (-)-camphor, β-terpinyl acetate, and caryophyllene, were less abundant with a percentage of lower than 1%.The GC/MS analysis results of the cinnamon EO showed that cinnamaldehyde was the major component of this essential oil.According to studies carried out by several investigators, the major component of cinnamon EO was cinnamaldehyde in the range of 44-97% (Adinew, 2014;Wang et al., 2009;Yu et al., 2020).These findings agree with the current study since the concentration of cinnamaldehyde was 80.09%.On the contrary, the results obtained by Ghosh et al. (2013) proved that eugenol was found to be the main component of cinnamon oil (C.zeylanicum) with 59.918% of the total peak area.In conclusion, most C. verum oil components were similar to those reported in this oil chemistry.

Droplet size and polydispersity index (PDI)
Table 2 presented the droplet size and PDI data of produced NEs.There is a significant difference between the droplet size values for cinnamon oil and cinnamaldehyde, which were 95.67 and 174.59 nm, respectively.In general, the scale of 10-500 nm corresponding to the average droplet size of O/W NEs (Izquierdo et al., 2002;Kabri et al., 2011).This finding showed that all compounds were successfully prepared at the nanometric scale.In agreement with our results, the findings obtained by Abdelrasoul et al. (2018) reported that the mean particle size of nano-sized cinnamaldehyde was 128.07 nm.While the particle size detected by Mibielli et al. (2021) in the carvacrol, NE was 354.20 nm.Under room temperature and accelerated stability assessment, no phase separation, creaming, or sedimentation were observed (Balaji et al., 2017;Qin et al., 2017).The tiny droplets in a NE contribute to its long-term physical stability, which is why this sort of formulation is often known as "approaching thermodynamic stability".(Izquierdo et al., 2002;Solans et al., 2005).NEs have several advantages over traditional emulsions due to the small size of the droplets: higher optical visibility, more excellent stability against droplet aggregation and gravitational separation, and increased bioactivity of encapsulated components (McClements, 2012).For these reasons, NEs have emerged as an alternative drug, pesticide carriers, and other vital applications (Feng et al., 2018;Tang et al., 2013).
The PDI of the prepared NEs were 0.33 and 0.58 for cinnamon and cinnamaldehyde, respectively (Table 2).The PDI reflects the distribution, homogeny, and stability of particle size in the emulsion.These findings indicated that the size distribution of all NEs was relatively narrow.In agreement with our results, the PDI value of cinnamaldehyde NE prepared by Abdelrasoul et al. (2018) was 0.322.While the PDI value of carvacrol NE formulated by Mibielli et al. (2021) was 0.104.The PDI, which ranges from zero to one, is a dimensionless estimate of the width of the size distribution measured from the cumulate analysis (Kentish et al., 2008).A lower PDI value indicates the presence of a uniform droplet size distribution (homogeneous system), while a PDI value closer to one indicates the presence of a large variety of droplet sizes (heterogeneity of the system) (Tyagi et al., 2012).

Zeta potential
Table 2 and Figure 3 show that the NEs revealed negative zeta potential (-30.30 and -21.20 for cinnamon and cinnamaldehyde, respectively).This finding implies a sufficiently high negative surface charge for droplet-droplet repulsion and, as a result, improved NE stability (Bruxel et al., 2012).The zeta potential is a better way to optimize sample stability and save time consumed in shelf-life tests.It is considered a potent indicator of NE stability, resist flocculation and aggregation for more extended periods, and is associated with to surface potential of the droplets (Benita and Levy, 1993).The charge acquired by a particle or molecule in a given medium is its zeta potential.It arises from the surface charge, concentration, and types of ions in the solution.Since particles of similar charge will repel each other, those with high charges will resist flocculation and aggregation for more prolonged periods making such samples more stable.This finding means that the stability can be modified by altering the pH, the ionic concentration, the type of ions, and additives such as surfactants and polyelectrolytes (Mahdi et al., 2011).

Viscosity and pH measurements
The viscosity values of the NEs were 7.03 and 6.93 for cinnamon and cinnamaldehyde, respectively (Table 2).The pH values were 6.11 and 6.08 for cinnamon and cinnamaldehyde, respectively (Table 2).As mentioned previously, the pH range of a NE has a significant impact on its stability.The surface charge of the globule changes as the pH value changes, affecting their stability during storage.A rise in globule surface charge facilitates electrostatic repulsion and decreases NE flocculation and dissolution (Pengon et al., 2018).

Thermodynamic stability studies
An accelerated storage testing was carried out to predict the longterm physical stability of the NEs (Badawy et al., 2017).The recorded data of visual stability, centrifugation, and heating-cooling stability of NEs showed that all prepared NEs were stable at 5000 rpm of centrifugation, at 25 °C for up to 3 months, and under heating-cooling cycle test.

TEM
The morphological study of the structure of NE droplets is carried out using TEM.TEM micrographs of NEs were presented in Figure 4, showing their spherical form and typical NE appearance at magnification 30.000x.

Larvicidal efficacy of tested compounds and their NEs
After 24 and 48 h of exposure, the larvicidal activity of cinnamon oil and technical (T) cinnamaldehyde was compared with their NEs and temephos against C. pipiens.Table 3 presented the LC50 values with 95% confidence limits and other statistical parameters.Cinnamaldehyde T and NE were more effective (LC50 = 70.44-94.46mg/l) than the EO products (LC50 = 112.34-154.08 mg/l).Cinnamaldehyde NE gave the LC50 value of 72.90 mg/l more than the T form (94.46 mg/l) after 24 h of the experiment.At the same time, this compound gave LC50 values 70.44 and 84.91 mg/l after 48 h of exposure for NE and T, respectively.NE form of EO gave an LC50 value of 123.13 mg/l, which was more effective than nonformulated EO (LC50 = 154.08mg/l) after 24 h of exposure.It can be observed that the larvicidal behavior of the NE was the best.Temephos as a reference insecticide against mosquito larvae exhibited the LC50 of 0.012 and 0.009 mg/l after 24 and 48 h, respectively..65 a ±1.01 --EO: Essential oil, T: Technical, NE: Nanoemulsion, OD: Optical density, SE: Standard error, AChE: Acetylcholinesterase, ATPase: Adenosine triphosphatase, GABA-T: Gammaaminobutyric acid transaminase.Values in the column with different letters are significantly different at p ≤ 0.05 using one-way analysis of variance (ANOVA) followed by the Student-Newman-Keuls (SNK) test.
Based on the literature review, a natural product with LC50 < 100 mg/l could be a promising candidate for mosquito control (Dias and Moraes, 2014;Kharoubi et al., 2021;Ríos et al., 2017).In our earlier study, twenty-one monoterpenes from different chemical groups were evaluated against C. pipiens larvae, and the results indicated that (±)-β-citronellol and cinnamyl acetate had the highest mortality (LC50 = 5.520 and 5.603 mg/l, respectively) (Taktak and Badawy, 2019).In addition, our results are approximately similar to the data obtained by Pavela (2015), who proved that the LC50 values of pcymene and linalool against C. quinquefasciatus larvae were 21 and 247 mg/l, respectively (Pavela, 2015).Moreover, Michaelakis et al. (2014) proved that the LC50 value of linalool was more than 200 mg/l against C. pipiens larvae (Michaelakis et al., 2014).In addition, the data observed for cinnamon and eucalyptus oils showed a 100% larvicidal effect at 1000 mg/l and a 100% knockdown effect at 10% against C. quinquefasciatus (Manimaran et al., 2012).
In recent years, nanotechnology in pest management has been increasing, and one of the most important goals is to prepare nano pesticides (Jesser et al., 2020).Essential oils or monoterpenes in nanometric emulsions enhanced mosquitocidal activity (Nenaah et al., 2021).For example, the NE of neem oil with the hydrodynamic size of 30.12 nm and PDI of 0.262 exhibited noteworthy larvicidal activity against C. quinquefasciatus (LC50 = 11.75 mg/l after 24 h) (Mishra et al., 2018).Currently, extensive efforts are being made to study the potential toxic behavior of prevalent nano pesticides in mosquitoes and other pest ecosystems (Kumar et al., 2020;Mishra et al., 2018).However, limited experimental studies have been carried out to shed light on the toxicity profile of these nanometric pesticides towards the non-target species prevalent in the target ecosystem (Côa et al., 2020;Taktak et al., 2021).

Biochemical studies
To explain some biochemical actions of the tested compounds on C. pipiens larvae, the effect of the LC50 values of these compounds on the activity of AChE, ATPase, and GABA-T extracted from larvae after 24 h of the treatment was studied.Generally, the activities of AChE and GABA-T were inhibited after treatment, while ATPase activity was increased (Table 4).The activity of enzymes in untreated larvae was 0.93, 1.07, and 0.11 for AChE, ATPase, and GABA-T, respectively.Cinnamaldehyde NE inhibited the AChE enzyme by 60.59%, while the unformulated form decreased the activity by 30.83% compared to the untreated larvae.At the same time, the effectiveness of cinnamon NE (46.40% inhibition) was more than the pure oil (17.26% inhibition) on AChE activity.NE of cinnamon oil caused 75.42% activation of ATPase enzyme compared to its pure oil (28.56% activation).In contrast, cinnamaldehyde NE caused more effect (172.56% activation) than the T form (120.53%).All tested compounds, at the level of the LC50, proved an activation of GABA-T activity.Cinnamaldehyde NE was the most active product, which caused 271.23% activation for 93.14,and 46.16% activation for technical cinnamaldehyde, cinnamon NE, and cinnamon EO, respectively.According to some authors, the inhibition of acetylcholinesterase by terpenes has no relationship with their larvicidal properties (Cai et al., 2018;Seo et al., 2015).Miyazawa and Yamafuji (2005) proved that bicyclic hydrocarbons with allylic methyl group uncompetitive inhibitors for AChE.In contrast, bicyclic alcohols and ketones exhibited weak inhibition.Inhibition of AChE by α-pinene, terpineol, linalool, β-myrcene, nerol, and geraniol was found at high concentrations (Zarrad et al., 2017).In another study, fenchone, Scarvone, and linalool, followed by estragole, produced a higher inhibition on AChE, but (E)-anethole was not active (López and Pascual-Villalobos, 2010).The effects of twenty-one monoterpenes on AChE, acid and alkaline phosphatases, ATPase, and GABA-T were investigated (Taktak and Badawy, 2019).The authors proved that all compounds proved a significant inhibition of all tested enzymes except ATPase.Monoterpenes such as thymol, linalool, menthol, camphor, carvone, and borneol have been discovered to bind to ionotropic GABA receptors in insects (Tong, 2010).Glycolysis produces ATP in the insect body, both in the cell wall and cytoplasm.Consequently, essential oils and their constituents affect the intracellular and extracellular ATP balance (Faleiro, 2011).In vivo activation of ATPase at these points may be due to changes in ATP balance as a result of the influence of essential oils and their constituents on cell membranes.

Molecular docking
Table 5 and Figure 5 show the docking results of cinnamaldehyde with AChE (PDB: 5X61), ATPase (PDB: 4BYG), and GABA-T (PDB: 1SF2), respectively.The data presented as docking scores and different interactions include H-bonds, Van der Waals, and hydrophobic.The binding affinity values of cinnamaldehyde were -4.32, -2.62, and -4.06 kcal/mol on the active sites of AChE, ATPase, and GABA-T, respectively.It can be noted that the highest binding affinity was found with AChE and GABA-T.However, temephos as a reference insecticide exhibited binding affinity values -7.41, -4.53, and -3.06 kcal/mol on the active sites of AChE, ATPase, and GABA-T, respectively (Table 5).
Figure 5A shows the recognized binding modes and molecular orientations (2D and 3D) of cinnamaldehyde with AChE.Seventeen amino acids surround the compound through Van der Waals interactions.The oxygen atom formed two HBA interactions with the amino acids Cys A447 and Cys B447.No hydrophobic interactions were observed.Cinnamaldehyde exhibited low binding affinity with high docking energy toward the active sites of the ATPase (ΔG = -2.62 kcal/mol).It was interacted with ATPase by Van der Waals through ten amino acids (Ala B486, Glu A488, Glu B488, Gly A497, Gly B497, His B503, His B515, and Val B496).
Cinnamaldehyde did not show hydrogen bonds to amino acids or hydrophobic interactions with ATPase in the active pockets.The binding confirmation of the cinnamaldehyde with ATPase is shown in Figure 5B.This compound is surrounded by fourteen amino acids through Van der Waals interactions in GABA-T (Figure 5C).The O1 atom formed one HBA with the amino acid Lys C329 via hydrogen bonding interaction (3.19 Å).Left are the 2D interaction diagrams, and right are the complex structures in 3D.
Figure 6 shows the recognized binding modes and molecular orientations (2D and 3D) of temephos with the target enzymes.The insecticide bonded with AChE Van der Waals, hydrogen bonding, and hydrophobic interactions.Sulfur atom in the thiophosphate group interacted with four amino acids, Cys A447-S1, Cys A447-S1, Ile A446-S1, and Ile A446-S1 through HBA (Figure 6A).No hydrophobic interactions were found.The binding confirmation with ATPase is shown in Figure 6B.The sulfur atom in the thiophosphate group formed two HBAs with the amino acids Gly B300 and Ser B301 with 3.39 and 3.46 Å, respectively.The insecticide is surrounded by fourteen amino acids through Van der Waals interaction in GABA-T (Figure 6C).The sulfur atom in the thiophosphate group formed two HBDs with the amino acids Arg A222 and Arg B222 with 3.39 and 3.46 Å, respectively.Moreover, the sulfur atom of sulfanylphenoxy moiety formed two HBAs with the amino acids Pro A399 and Pro B399 with 3.39 and 3.46 Å, respectively.No hydrophobic interactions were found.Because the mechanism of larvicidal activity of monoterpenes is not wholly understood, molecular docking is a method to predict and understand molecular recognition.It detects the predominant binding mode and binding affinity between the protein and ligand and gives a 3D structural explanation of the interaction in several biological processes (Gumede et al., 2012;Lie et al., 2011).This tool was used to elucidate the biological activities of some larvicides with their pharmacokinetic properties (Kumar et al., 2018;Zeng et al., 2016).The docking study revealed that cinnamaldehyde has low binding energy and a high affinity for the functional pocket of the enzyme targets and can bind to the active sites of those enzymes.

Conclusions
Natural pesticides derived from natural resources are urgently needed to control mosquitoes without causing harm to beneficial organisms or the environment.Therefore, NEs of cinnamon oil and cinnamaldehyde were prepared and characterized as alternatives to synthetic mosquitocides.The NEs enhanced the mosquitocidal activity against C. pipiens larvae and displayed a remarkable effect.As a result, cinnamaldehyde had more larvicidal activity than cinnamon oil 24 h after exposure.Furthermore, the action of the NEs was higher than the unformulated products on the target enzymes (AChE, ATPase, and GABA-T).Additionally, the molecular docking study demonstrated that cinnamaldehyde had a greater affinity for AChE and GABA-T than ATPase.These results indicate that cinnamon oil and cinnamaldehyde NEs have an overall significant effect on C. pipiens larvae and might prove helpful as mosquitocide agents.
Reviewed by: Yahyea Baktiar Laskar: Assam University, Silchar, INDIA Kemal Buyukguzel: Zonguldak Bulent Ecevit University, Zonguldak, TURKEY Publisher's Note: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers.Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.
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Figure 1 .
Figure 1.Schematic illustration of NEs preparation

Figure 2 .
Figure 2. GC/MS chromatogram of cinnamon essential oil

Figure 3 .
Figure 3. Zeta potential distribution graph of NEs of cinnamon oil (A) and cinnamaldehyde (B)

Table 1 .
The chemical constituents of the cinnamon EO isolated from C. verum by GC/MS

Table 2 .
The visual appearance, droplet size, PDI, zeta potential, dynamic viscosity, and pH of the prepared NEs

Table 3 .
Larvicidal activity of cinnamon EO, its main component cinnamaldehyde, and their NEs against C. pipiens Essential oil, T: Technical, NE: Nanoemulsion, a The concentration causing 50% mortality, b Slope of the concentration-mortality regression line, c Intercept of the regression line, d Chi-squared value, SE: Standard error

Table 4 .
The activity of cinnamon EO, its main component cinnamaldehyde, and their NEs on AChE, ATPase, and GABA-T activity in C. pipiens larvae after 24 h of the treatment with LC50 of each treatment

Table 5 .
Molecular docking, binding scores, and binding interactions of cinnamaldehyde and temephos within the active sites of AChE (PDB ID: 5X61), ATPase (PDB ID: 4BYG), and GABA-T (PDB ID: 1SF2) The root means square deviation of the pose, in Å, from the original ligand.This field is present if the site definition is identical to the ligand definition.