GC-MS Profile, Antioxidant Activity, and In Silico Study of the Essential Oil from Schinus molle L. Leaves in the Presence of Mosquito Juvenile Hormone-Binding Protein (mJHBP) from Aedes aegypti

Schinus molle is a medicinal plant used as an anti-inflammatory and for rheumatic pain in the traditional medicine of Peru. On the other hand, Aedes aegypti is the main vector of several tropical diseases and the transmitter of yellow fever, chikungunya, malaria, dengue, and Zika virus. In this study, the aim was to investigate the antioxidant activity in vitro and the insecticidal activity in silico, in the presence of the mosquito juvenile hormone-binding protein (mJHBP) from Aedes aegypti, of the essential oil from S. molle leaves. The volatile phytochemicals were analyzed by gas chromatography-mass spectrometry (GC-MS), and the profile antioxidants were examined by DPPH, ABTS, and FRAP assays. The evaluation in silico was carried out on mJHBP (PDB: 5V13) with an insecticidal approach. The results revealed that EO presented as the main volatile components to alpha-phellandrene (32.68%), D-limonene (12.59%), and beta-phellandrene (12.24%). The antioxidant activity showed values for DPPH = 11.42 ± 0.08 μmol ET/g, ABTS = 134.88 ± 4.37 μmol ET/g, and FRAP = 65.16 ± 1.46 μmol ET/g. Regarding the insecticidal approach in silico, alpha-muurolene and gamma-cadinene had the best biding energy on mJHBP (ΔG = −9.7 kcal/mol), followed by beta-cadinene (ΔG = −9.5 kcal/mol). Additionally, the volatile components did not reveal antioxidant activity, and its potential insecticidal effect would be acting on mJHBP from A. aegypti.

female promoting the ovary maturation before blood feeding; additionally, it is involved with the nutritional state and its relationship with blood meal-dependent reproductive development [17]. In recent years, a class of insecticides named JH analogues have been designed to disrupt this endocrine process and affect the normal development in mosquitoes. A synthetic JH analogue is pyriproxyfen, which is a phenyl carbonyl derivative, and its main effect is to produce an imbalance in the mosquito hormonal system, inhibiting the embryogenesis, adult metamorphosis, and development of the adult mosquito [18].
Although the insecticidal activity of S. molle EO was evaluated in several species such as Trogoderma granarium, Tribolium castaneum [19], Sitophilus oryzae [20], Haematobia irritans [21], Ctenocephalides felis felis [22], and Gonipterus platensis [23], currently, there is not scientific literature on A. aegypti. Thus, we investigated the potential insecticidal effect in silico of the volatile components of S. molle EO to find any responsible molecule as a JH analogue, which might combat the presence of mosquitoes in tropical regions and reduce the prevalence of diseases transmitted by them. The aim in this study was (1) to determine the total volatile component of the EO from S. molle leaves by gas chromatography-mass spectrometry (GC-MS), (2) to evaluate the antioxidant activity using the DPPH, ABTS, and FRAP methods, and (3) to determine the insecticidal activity using a virtual screening of the EO from S. molle leaves on the mosquito juvenile hormone-binding protein from Aedes aegypti.

Plant Material.
A quantity of 4700 g of Schinus molle (leaves) cultivated in Tinguiña, Ica Region, Peru (406 m.a.s.l.) in December 2020 was received. Leaves were cleaned and peeled to be incorporated in a Clevenger equipment and to obtain essential oil by hydrodistillation for 2 h [24]. The essential oil was separated by decantation; then, anhydrous Na 2 SO 4 was added to eliminate any water drops. Finally, the EO was stored in a sealed amber vial until further use.

Identification of Volatile Compounds by Gas
Chromatography-Mass Spectrometry (GC-MS). Volatile chemicals were determined with a GC-MS system (Agilent  Technologies 7890 Gas Detector and Agilent Technologies   2 BioMed Research International 5975C Mass Spectrometer Detector, Santa Clara, CA, USA). Then, 20 μL of EO was mixed with 1.0 mL of dichloromethane. Next, 1.0 μL of the working solution was injected into the equipment in splitless mode (split: 20 : 1). The EO was run on a J&W 122-1545.67659 DB-5 ms column, (60 m × 250 μm × 0:25 μm) (Agilent Technologies, Santa Clara, CA, USA). The working conditions were as follows: the temperature program was 40°C, starting with increments of 5°C/min up to 180°C, followed by increases of 2.5°C/min up to 200°C for 5 min, and finally 10°C/min up to 300°C, remaining for 3 min. The helium flow rate was at 1 mL/ min. Volatile components' identification was based on a comparison of relative retention indices (RIs) and mass spectra data with the NIST20 library data and the published literature [25]. Each RI was calculated compared with a homologous series of n-alkanes C9-C25 (C9, BHD purity 99%; C10-C25, Fluka purity 99%). The relative amount (expressed as a percentage) of each compound identified in the EO was calculated by comparing the area of the corresponding peak in the chromatogram with the total area of identified peaks.

Determination of the Antioxidant Capacity by the Free
Radical 2,2-Diphenyl-1-picrylhydrazyl (DPPH). A 150 μL of EO (10 mg/mL) was mixed with 2850 μL of a methanolic solution of DPPH (20 mg/L) with an absorbance adjusted to 1:1 ± 0:02 nm. After mixing, it was incubated in the dark for 30 minutes, and the absorbance reading was carried out at 515 nm. The standard curve was elaborated with Trolox at concentrations of 0 to 800 μmol/mL. The Trolox equivalent antioxidant capacity (TEAC) was expressed as μmol TE/g of essential oil [26]. To calculate the half inhibitory concentration (IC50), a linear regression method was used based on the different concentrations of the EO. Method  of  the  Radical  2,2′-Azinobis-(3ethylbenzothiazoline)-6-sulfonic Acid (ABTS .+ ). To carry out the antioxidant activity using the ABTS radical, a solution was prepared using the mixing of 10 mL of ABTS (4.06 mg/mL) and 10 mL of potassium persulfate (0.7 mg/ mL), both reacted for 16 hours. Then, 150 μL of the EO (5 mg/mL, diluted with methanol) was mixed with 2850 μL of ABTS radical and incubated for 7 min, the same procedure was used with Trolox standard ranging between 0 and 400 μmol/mL. The absorbance was read to 0:7 ± 0:02 at a wavelength of 734 nm. The Trolox equivalent antioxidant capacity (TEAC) was expressed as μmol TE/g of essential oil [27]. To calculate the half inhibitory concentration (IC 50 ), a linear regression method was used based on the different concentrations of the EO.

Determination of Antioxidant Capacity by the Ferric
Reducing/Antioxidant Power (FRAP). In the determination of FRAP of EO, a reagent battery was made using 25 mL of acetate buffer pH 3.6; 2.5 mL of 20 mM TPTZ was dissolved in 40 mM HCl and 2.5 mL of 20 mM ferric chloride hexahydrate. The mixing of this reagents constituted the FRAP solution which reacted with the EO at different concentrations as well as Trolox standard. 150 μL of EO (1 mg/mL) was mixed with 2850 μL of FRAP reagent and reacted for 4 min at room temperature. The absorbance reading was carried out at 593 nm. A standard curve was prepared with Trolox (50-800 μM). The results were expressed as μmol equivalents of Trolox per gram of essential oil (μmol TE/g EO) [28]. To calculate the half inhibitory concentration (IC 50 ), the linear regression method was used based on the different concentrations of the EO.         The binding cavity residues comprised mainly  TYR33, LEU37, TRP50, AL51, TRP53, TYR64, SER69,  TYR129, TYR133, ILE140, PHE269, TRP278,

Molecular Dynamics Simulation (MD).
This evaluation was carried on the dock complexes for α-phellandrene (the most abundant in GC-MS analysis) and α-muurolene (the most active in molecular docking analysis) with the mosquito juvenile hormone-binding protein (mJHBP) using the Desmond 2020.1 from Schrödinger, LLC. The reproducibility was completed using three replicates for each MD run. The OPLS-2005 force field and explicit solvent model with the SPC water molecules were used in this system. To neutralize and simulate the physiological conditions, the charge Na+ ions and 0.15 M NaCl were added, respectively. The system was equilibrated using constant-temperature and constant-volume ensemble (NVT) for 100 ps. This was followed by a short run equilibration and minimization using constant-temperature and constant-pressure ensemble (NPT) for 12 ps and was set up using the Nose-Hoover chain coupling scheme with temperature 27°C, the relaxation time of 1.0 ps, pressure 1 bar, and a time of 2 fs. The Martyna-Tuckerman-Klein chain coupling scheme barostat method was used for pressure control using a relaxation time of 2 ps. The particle mesh Ewald method was used for calculating long-range electrostatic interactions with a radius of 9 Å for the coulomb interactions. Bonded forces were calculated using a RESPA integrator with a time step of 2 fs for each trajectory. The root mean square deviation (RMSD), radius of gyration (Rg), root mean square fluctuation (RMSF), and solvent accessible surface area (SAS area) were calculated to monitor the stability of the MD simulation.
2.9. Statistical Analysis. In the analysis of the antioxidant profile of essential oil from S. molle, the IC 50 values were estimated by linear regression statistics. A Spearman's Rho coefficient was calculated to establish a correlation

Results and Discussion
3.1. Chemical Profile of the Essential Oil of S. molle. The obtained EO of fresh leaves showed a light-yellow color, a density of 0:873 ± 0:02 g/mL at 20°C, and an extraction yield of 0.73% during 2 hours of distillation. The profiles of volatile constituents of S. molle essential oil were analyzed by GC-MS and are presented in Figure 1 and Table 1. Regarding the chemical profile, the EO showed 34 compounds (Figure 1), two of which are of unknown structures (compounds 27 and 29), which accounted for 99.7% of the total composition. Those unknown chemical structures were classified as oxygenated sesquiterpenes with the following formula C 15 H 26 O and a molecular weight of 222.37 g/mol. The analysis identified alphaphellandrene as the main volatile chemical with 32.68%, followed by D-limonene (12.59%) and beta-phellandrene (12.24%). According to Figure 1, the retention time at 17.80 min corresponded to the major component in the EO (compound 7).

Antioxidant
Profile of S. molle Essential Oil. S. molle EO exhibited a low antioxidant activity, as is shown in Table 2. On the other hand, there was a significant difference between the different methods used in the antioxidant activity (P = 0:0004). Other reports have shown different values; according to Eryigit et al., EO showed a TEAC ðABTSÞ = 4:7 ± 1:2 mM Trolox of S. molle grown in Turkey [36]. EO from Portugal reported an inhibition value of 4.8% at 16 mg/ mL and was obtained by hydrodistillation. Although alphaphellandrene, myrcene, limonene, and beta-phellandrene were the main components in this EO, its low antioxidant activity could be associated with the low ability of monoterpene hydrocarbons for DPPH scavenging activity. However, in other methods, such as beta-carotene bleaching, the antioxidant activity was high and might be justified by the presence of the monoterpenes α and β-phellandrene, α-pinene, sabinene, limonene, β-myrcene, and others [10]. In this study, the IC 50 against DPPH was 41.84 μg/mL, which was higher compared to the findings of the EO from southeast Portugal [10]. In another study, S. molle from Egypt showed an IC 50 at DPPH of 172.45 μg/mL and a high percentage of oxygenated sesquiterpene (17.73%) [37]. According to Table 2, the best effect was found in the FRAP assay, followed by ABTS and DPPH. It is known that DPPH and ABTS methods are based on electron and H atom transfer reaction, while the FRAP method is based on electron transfer reaction. Thus, the volatile phytochemicals from S. molle EO could be acted by electron transfer to reduce the free radicals.
3.3. Molecular Docking of the Essential Oil from S. molle in the Presence of the Mosquito Juvenile Hormone-Binding Protein (mJHBP) from Aedes aegypti. The virtual screening of the 32 chemical components, additionally JH3 and the pyriproxyfen (synthetic insecticide), was carried out in order to understand the interaction profile of various volatile compounds present in S. molle EO leaves with mJHBP from A. aegypti. Out of 32 specific compounds found abundantly in chromatography, gamma-cadinene and alpha-muurolene displayed a lower binding energy (ΔG) of -9.7 kcal/mol and predicted inhibitory concentration (Ki) of 0.134 μM (Table 3). Although the major component (alpha-phellandrene) had a high binding free energy (ΔG = −7 kcal/mol), compared to alpha-muurolene, the total components might be synergizing the insecticidal effect.
The principal residues of mJHBP, TYR133, TRP53, TYR33, and PHE144 were involved in Pi-Sigma bond formation with alpha-muurolene ( Figure 2). We observed that TYR133 was the main residue of mJHBP in which the molecules of the EO established a Pi-sigma, Van der Walls, and Pi-alkyl bonds (Supplementary Materials S1-S32). Interestingly, elixene, germacrene D, beta-cadinene, gamma-muurolene, germacrene-D, humulene, and gamma-eludesmol exhibited activity and binding energies close to those of the known insecticide pyriproxyfen and the cocrystallized ligand JH3. Alpha-phellandrene, beta-caryophyllene, beta-elemene, alpha-pinene, beta-phellandrene, beta-pinene, elemol, etc. have exhibited significant binding and inhibition of the mosquito juvenile hormone-binding protein. Therefore, from the docking study, it can be predicted that the molecules from this essential oil have great potential as inhibitors of the mosquito juvenile hormone-binding protein. The interactions of JH3 in this study are according to the results of Ramos et al., which the epoxy group forms a conventional hydrogen bond with the phenolic hydroxyl of Tyr-129, and other interactions showed on the isoprenoid chain were Val65,Val68, Pro55, Phe144, Tyr64, Tyr33, Ala281, Trp53, and Phe269 [18]. 13 BioMed Research International Furthermore, the low binding free energy (ΔG > −9 kcal/mol) of some volatile components was evidenced in those interactions with the Tyr133, Trp53, Tyr33, and Phe144 residues as shown in Supplementary Materials Figures S1-S32. These could infer that the insecticidal activity is related to these residues mentioned above.
3.4. Molecular Dynamics of the Phytoconstituents of the Essential Oil from S. molle. Molecular dynamics and simulation (MD) studies were carried out in order to determine the stability and convergence of mJHBP+α-phellandrene and mJHBP+α-muurolene complexes. Each simulation of 100 ns displayed stable conformation comparing the root mean square deviation (RMSD) values. The Cα backbone of mJHBP bound to α-phellandrene exhibited a deviation of 0.5 Å (Figure 3(a)), while mJHBP bound to α-muurolene exhibited a deviation of 1.0 Å (Figure 3(f)). RMSD plots are within the acceptable range signifying the stability of mJHBP in the ligand-bound state before and after simulation, and it can also be suggested that ligand α-phellandrene and αmuurolene-bound mJHBP are quite stable in complex due to the higher affinity of the ligand. The plots for root mean square fluctuations (mMSF) displayed a significant spike in fluctuation (3.5 Å) at amino acid residue 220 in mJHBP, while the rest of the residues fluctuated less during the entire 100 ns simulation (Figures 3(b) and 3(g)) indicating the stable amino acid conformations during the simulation time. Therefore, from the RMSF plots, it can be suggested that the structures of mJHBP were stable during simulation in α-phellandreneand α-muurolenebound conformations. The radius of gyration is the measure of compactness of the protein. In this study, the mJHBP Cα backbone displayed a lowering of the radius of gyration (Rg) from 19.6 Å to 19.4 Å, and the lowering of the Rg indicates the compactness of the complex (Figure 3(c)). On the other hand, the lowering of Rg was observed for the mJHBP+α-muurolene complex till 35 ns, and a later increment of the peak was observed. This indicates the less stable conformation of mJHBP+α-muurolene as compared to mJHBP+α-phellandrene. However, the stable Rg peak confirmed the significant compactness of the protein in the α-muurolene-bound state (Figure 3(h)). The overall quality analysis from RMSD and Rg suggests that α-phellandrene and α-muurolene bound to the protein targets posthumously in the binding cavities and played a significant role in stability of the proteins. Solvent accessible surface area provides the information about the compactness of protein complex with the ligand. The lowering of SASA in the case of α-phellandrene and α-muurolene bound to mJHBP as compared to the unbound state signifies the achievement of stable converged structures due to the high compactness of both the systems (Figures 3(d) and 3(i)). The interaction plots of both αphellandrene and α-muurolene bound to mJHBP displayed no involvement of conventional hydrogen bonds, while hydrophobic interactions played a major role in ligand stabilization at the binding cavity of mJHBP (Figures 3(e) and 3(j)).

Conclusions
The essential oil of S. molle leaves presented 34 volatile constituents determined by gas chromatography-mass spectrometry (GC-MS). Alpha-phellandrene was the major component, which represented 32.68% of the total composition; furthermore, beta-phellandrene and D-limonene were the following more abundant metabolites with 12.24% and 12.59%, respectively. Additionally, the antioxidant activity was evaluated in order to determine the antioxidant profile of EO using the three methods of DPPH, ABTS, and FRAP. According to the results, the EO showed better affinity and a good effect on FRAP assay and IC 50 equivalent to 1:50 ± 0:02 mg/mL. However, this study revealed that S. molle EO is not a good antioxidant. Regarding the insecticidal activity in silico based on a virtual screening on mosquito juvenile hormone-binding protein, several volatile compounds were active against this target such as alpha-muurolene and gamma-cadinene, being similar to pyriproxyfen, which is a synthetic insecticide analogue of JH. The molecular dynamics carried out for alpha muurolene (the best result in molecular docking) and alpha-phellandrene (the most abundant molecule) were very stable during 100 ns of evaluation. In the future, S. molle EO might be used as bioinsecticide on A aegypti, but an in vitro and in vivo assay has to be evaluated to validate our findings.

Data Availability
The data used to support the findings of this study are included within the supplementary information file.

Conflicts of Interest
The authors declare no conflicts of interest.