GC-MS Chemical Characterization and In Vitro Evaluation of Antioxidant and Toxic Effects Using Drosophila melanogaster Model of the Essential Oil of Lantana montevidensis (Spreng) Briq.

Background and objectives: Natural products such as essential oils with antioxidant potential can reduce the level of oxidative stress and prevent the oxidation of biomolecules. In the present study, we investigated the antioxidant potential of Lantana montevidensis leaf essential oil (EOLM) in chemical and biological models using Drosophila melanogaster. Materials and methods: in addition, the chemical components of the oil were identified and quantified by gas chromatography coupled to mass spectrometry (GC-MS), and the percentage compositions were obtained from electronic integration measurements using flame ionization detection (FID). Results: our results demonstrated that EOLM is rich in terpenes with Germacrene-D (31.27%) and β-caryophyllene (28.15%) as the major components. EOLM (0.12–0.48 g/mL) was ineffective in scavenging DPPH radical, and chelating Fe(II), but showed reducing activity at 0.24 g/mL and 0.48 g/mL. In in vivo studies, exposure of D. melanogaster to EOLM (0.12–0.48 g/mL) for 5 h resulted in 10% mortality; no change in oxidative stress parameters such as total thiol, non-protein thiol, and malondialdehyde contents, in comparison to control (p > 0.05). Conclusions: taken together, our results indicate EOLM may not be toxic at the concentrations tested, and thus may not be suitable for the development of new botanical insecticides, such as fumigants or spray-type control agents against Drosophila melanogaster.


Introduction
Reactive species are known to cause damage to cellular membranes (lipid peroxidation), DNA, proteins, and mitochondria, and have been found to be involved in the pathophysiology of a wide range a glass Pasteur pipette, after which the yield was calculated. The obtained essential oil was treated with anhydrous sodium sulphate (Na2SO4) and stored at −4 °C until the chemical analyzes were carried out. The essential oil showed a yield of 0.19%.

Composition and Identification of the Constituents of the Essential Oil
After obtaining the essential oil, it was submitted to GC/MS analysis according the procedure described in the literature [33].

Fe 2+ Chelating Activity of L. montevidensis Leaf Essential Oil
The chelating capacity of essential oil from the leaves of L. montevidensis was determined according to the modified methods of Dinis et al. [35] and Kamdem et al. [36]. To summarize, the schedule for the evaluation of Fe 2+ chelation or oxidation by the oil is presented in (Figure 1). To further investigate the reductive potential of the essential oil, the same reaction mixture as described above but using FeCl3(110 µM) instead of FeSO4(110 µM) in the reaction mixture and was determined using a modified method of Kamdem et al. [36]. To further investigate the reductive potential of the essential oil, the same reaction mixture as described above but using FeCl 3 (110 µM) instead of FeSO 4 (110 µM) in the reaction mixture and was determined using a modified method of Kamdem et al. [36].

Biological Assays with Drosophila Melanogaster
Fumigant Toxicity Different concentrations of the essential oil of L. montevidensis (EOLM) (0, 0.12, 0.24, and 0.48 g/mL) were prepared by dissolving it in 4% DMSO. The filter paper fragment was treated with 300 µL of different concentrations of EOLM and placed at the bottom of the flask. Then, 50 adult flies (males and females) of 3-5 days were exposed to the oil. The top of the flask was sealed with adhesive foam to prevent evaporation of the essential oil. The control group was exposed to the filter paper soaked with 4% DMSO alone. The flasks were maintained in a light/dark cycle of 12 h, 25 • C ± 1 • C, and 60% of relative humidity. The experiment was performed in triplicate, and the number of death flies was counted after 30 min, 1 h, 2 h, 3 h, 4 h, and 5 h.

Determination of Total Thiol and Non-Protein Thiols (NPSH)
Twenty flies from each group were homogenized in 1 mL of 0.1 M potassium phosphate buffer, pH 7.4, at a ratio of 1:10 (1 mg of flies for 10 µL), and then centrifuged at 10,000 rpm for 10 min. For the determination of total thiols, 50 µL of the obtained supernatant was added to 190 µL of 0.1 M potassium phosphate buffer (pH 7.4), and then 10 µL of 5 mM DTNB (5,5 -Dithiobis(2-nitrobenzoic acid) was added to the mixture. The reaction mixture was incubated for 30 min at room temperature (protected from light), and the absorbance was measured at 405 nm using microplate reader. Glutathione (GSH) was used as standard, and the results were expressed as ηmol GSH/g of tissue. For the measurement of NPSH level, the obtained supernatant was missed with equal volume of 10% trichloroacetic acid (TCA) and centrifuged for 3 min at 10,000 rpm. The clear supernatant was used for NPSH determination as described for the total thiol.

Assessment of Cell Viability
Cell viability was assessed with MTT [3-(4,5-Dimethylthiazol, 2-yl)-2,5-diphenyl-2 -tetrazolium bromide], according to the method described by Mosman [37]. A volume of 20 µL of the supernatant from treated and untreated flies was added to 170 µL of potassium phosphate buffer (0.1 M, pH 7.4), followed by the addition of 10 µL of 1 mg/mL MTT prepared in ethanol. The reaction medium was incubated for 120 min, and then 150 µL of the mixture was pipetted and added to 50 µL of DMSO. After 10 min of incubation at room temperature, the readings were carried out at 492 nm and 630 nm, respectively, in an ELISA microplate reader.

Determination of Iron Levels
The iron (II) ions content was determined by measuring the intensity of the orange complexe formed with 1,10-phenanhroline and free Fe 2+ in the supernatant of control and treated groups with EOLM. The free Fe 2+ content was determined using a modified method of Kamdem et al. [36] and Klimaczewski et al. [38]. Briefly, ten microliters (10 µL) of 1,10-Phenanthroline (0.25%) was added to the reaction mixture containing 110 µL of saline solution (0.9% NaCl), 60 µL of 0.1 M Tris-HCl (pH 7.4), and 20 µL of the supernatant, and then incubated for 60 min at room temperature. Iron(II) sulfate was used to construct the standard curve. Absorbance was measured after incubation at 492 nm using microplate reader, and the results were expressed in ηmol of Fe (II)/g tissue.

Measurement of Malondialdehyde (MDA)
Thiobarbituric acid reactive substances (TBARS) were measured to determine lipid peroxidation products as a measure of oxidative stress. Ten flies per group, in triplicate, were homogenized and centrifuged at 10,000 rpm for 10 min. Briefly, the reaction mixture containing 100 µL of the supernatant, 100 µL of 10% trichloroacetic acid (TCA), and 100 µL of 0.75% of 2-thiobarbituric acid (TBA, prepared in 0.1 M HCl) was incubated at 95 • C for 1 h. After cooling, they were centrifuged at 10,000 rpm for 10 min, and the absorbance was measured at 405 nm using 250 µL of the reaction mixture [39]. MDA used for the standard curve was obtained by hydrolysis of 1,1,3,3-tetramethoxypropane (TMP). The results were expressed as ηmol MDA (malondialdehyde)/g of tissue.

Statistical Analysis
Results were expressed as mean ± standard error of the mean (SEM). Statistical analysis was performed using one-way (ANOVA), with multiple comparisons of Bonferroni, in order to detect significant differences between controls and treatments, and two-way, for chelating and reducing power. The probability of p < 0.05 was considered statistically significant.
All protocols were approved by the Commission of Ethics in Research in Animals (CEUA) of the Regional University of Cariri (00029/2017.1) on 23 October 2017.

Scavenging Activity of the Essential Oil from L. montevidensis on DPPH Radical
The effect of essential oil of L. montevidensis and ascorbic acid on DPPH reduction is shown in Figure 3. Ascorbic acid exhibited DPPH radical scavenging activity in a concentration-dependent manner ( Figure 3), with IC50 value of 0.042 g/mL. However, L. montevidensis leaf essential oil did not exhibit DPPH radical scavenging activity at all the concentrations tested ( Figure 3).

Scavenging Activity of the Essential Oil from L. montevidensis on DPPH Radical
The effect of essential oil of L. montevidensis and ascorbic acid on DPPH reduction is shown in Figure 3. Ascorbic acid exhibited DPPH radical scavenging activity in a concentration-dependent manner ( Figure 3), with IC 50 value of 0.042 g/mL. However, L. montevidensis leaf essential oil did not exhibit DPPH radical scavenging activity at all the concentrations tested ( Figure 3). From the results, it is possible to state that sesquiterpenes (e.g., germacrene D and β-caryophyllene) are the major groups of compounds found in EOLM (Table 1, Figure 2).

Scavenging Activity of the Essential Oil from L. montevidensis on DPPH Radical
The effect of essential oil of L. montevidensis and ascorbic acid on DPPH reduction is shown in Figure 3. Ascorbic acid exhibited DPPH radical scavenging activity in a concentration-dependent manner ( Figure 3), with IC50 value of 0.042 g/mL. However, L. montevidensis leaf essential oil did not exhibit DPPH radical scavenging activity at all the concentrations tested ( Figure 3).

Fe 2+ Chelation or Oxidation Potential of L. montevidensis Leaf Essential Oil
In Fe 2+ chelating assay, the rate of the reduction in the absorbance of the orange complex formed by the interaction of Fe 2+ and ortho-phenanthroline allows estimation of a possible chelator. Surprisingly, the incubation of L. montevidensis leaf essential oil (0.24 and 0.48 g/mL) with Fe 2+ significantly increased the absorbance of the complex Fe 2+ orthophenanthroline formed, when compared with that of Fe 2+ alone ( Figure 4). The absorbance remained unchanged after 20 min of incubation, but dramatically raised after addition of the reducing agent (ascorbic acid (Figure 4)). This finding may suggest that the leaf essential oil of L. montevidensis is oxidizing Fe 2+ in the reaction medium.
Surprisingly, the incubation of L. montevidensis leaf essential oil (0.24 and 0.48 g/mL) with Fe significantly increased the absorbance of the complex Fe 2+ orthophenanthroline formed, when compared with that of Fe 2+ alone (Figure 4). The absorbance remained unchanged after 20 min of incubation, but dramatically raised after addition of the reducing agent (ascorbic acid (Figure 4)). This finding may suggest that the leaf essential oil of L. montevidensis is oxidizing Fe 2+ in the reaction medium. After 20 min incubation of Fe 2+ with EOLM, ascorbic acid (AA) was added to the reaction medium to confirm whether or not the increase in absorbance in the presence of the oil was attributed to Fe 2+ . Absorbance did not change at 5, 10, and 20 min after addition of AA, suggesting that EOLM directly stimulated the oxidation of Fe 2+ to Fe 3+ during the incubation periods (prior to AA addition) (Figure 4).

Fe3+ Reducing Properties of L. montevidensis Leaf Essential Oil
The Fe 3+ reducing properties of L. montevidensis is shown in Figure 5. Similar to that observed with Fe 2+ , the incubation of the essential oil of L. montevidensis with Fe 3+ in the presence of ortho-phenanthroline resulted in a significant increase in the absorbance in a dose-dependent manner (p < 0.05) in comparison to that of Fe 3+ alone. However, the absorbance of the complex formed with Fe 3+ and ortho-phenanthroline was lower than that obtained with Fe 3+ and ortho-phenanthroline ( Figure 5). The addition of ascorbic acid to the reaction medium dramatically increased the absorbance of the sample ( After 20 min incubation of Fe 2+ with EOLM, ascorbic acid (AA) was added to the reaction medium to confirm whether or not the increase in absorbance in the presence of the oil was attributed to Fe 2+ . Absorbance did not change at 5, 10, and 20 min after addition of AA, suggesting that EOLM directly stimulated the oxidation of Fe 2+ to Fe 3+ during the incubation periods (prior to AA addition) (Figure 4).

Fe3+ Reducing Properties of L. montevidensis Leaf Essential Oil
The Fe 3+ reducing properties of L. montevidensis is shown in Figure 5. Similar to that observed with Fe 2+ , the incubation of the essential oil of L. montevidensis with Fe 3+ in the presence of ortho-phenanthroline resulted in a significant increase in the absorbance in a dose-dependent manner (p < 0.05) in comparison to that of Fe 3+ alone. However, the absorbance of the complex formed with Fe 3+ and ortho-phenanthroline was lower than that obtained with Fe 3+ and ortho-phenanthroline ( Figure 5). The addition of ascorbic acid to the reaction medium dramatically increased the absorbance of the sample (

Fumigant Activity of Leaf Essential Oil L. montevidensis
The fumigant toxicity of the essential oil extracted from leaves of EOLM was investigated in Drosophila melanogaster. Exposure of flies to EOLM at all the concentrations tested (0.12, 0.24 and 0.48 g/mL) for up to 5 h, caused mortality below 10% (data not shown).

Quantification of Total Thiol and Non-Protein Thiols (GSH and NPSH)
Considering that antioxidant activity has been reported for L. montevidensis [9], we investigated the effects of EOLM on the indirect biomarker of oxidative stress, total thiol. Additionally, the levels of GSH, an antioxidant found in the intracellular medium, that acts to defend the cell from oxidative

Fumigant Activity of Leaf Essential Oil L. montevidensis
The fumigant toxicity of the essential oil extracted from leaves of EOLM was investigated in Drosophila melanogaster. Exposure of flies to EOLM at all the concentrations tested (0.12, 0.24 and 0.48 g/mL) for up to 5 h, caused mortality below 10% (data not shown).

Quantification of Total Thiol and Non-Protein Thiols (GSH and NPSH)
Considering that antioxidant activity has been reported for L. montevidensis [9], we investigated the effects of EOLM on the indirect biomarker of oxidative stress, total thiol. Additionally, the levels of GSH, an antioxidant found in the intracellular medium, that acts to defend the cell from oxidative stress, were estimated in homogenates of D. melanogaster. The results presented in the Figure 6A,B revealed that the treatment with EOLM (0.12-0.48 g/mL) did not significantly alter the levels of total thiol ( Figure 6A) and non-protein thiol (NPSH) (Figure 6B), when compared with the control (p < 0.05).
The fumigant toxicity of the essential oil extracted from leaves of EOLM was investigated in Drosophila melanogaster. Exposure of flies to EOLM at all the concentrations tested (0.12, 0.24 and 0.48 g/mL) for up to 5 h, caused mortality below 10% (data not shown).

Quantification of Total Thiol and Non-Protein Thiols (GSH and NPSH)
Considering that antioxidant activity has been reported for L. montevidensis [9], we investigated the effects of EOLM on the indirect biomarker of oxidative stress, total thiol. Additionally, the levels of GSH, an antioxidant found in the intracellular medium, that acts to defend the cell from oxidative stress, were estimated in homogenates of D. melanogaster. The results presented in the Figure 6A and 6B revealed that the treatment with EOLM (0.12-0.48 g/mL) did not significantly alter the levels of total thiol ( Figure 6A) and non-protein thiol (NPSH) (Figure 6B), when compared with the control (p < 0.05).

Effect of EOLM on Fe 2+ Content
To investigate possible oxidative damage, after exposure of D. melanogaster to different concentrations of EOLM, the levels of free Fe 2+ ions were measured. As shown in Figure 8, exposure of flies to EOLM (0.12-0.48 g/mL) did not cause significant change in total iron content compared with the control group (p < 0.05).

Effect of EOLM on Fe 2+ Content
To investigate possible oxidative damage, after exposure of D. melanogaster to different concentrations of EOLM, the levels of free Fe 2+ ions were measured. As shown in Figure 8, exposure

Effect of EOLM on Fe 2+ Content
To investigate possible oxidative damage, after exposure of D. melanogaster to different concentrations of EOLM, the levels of free Fe 2+ ions were measured. As shown in Figure 8, exposure of flies to EOLM (0.12-0.48 g/mL) did not cause significant change in total iron content compared with the control group (p < 0.05).

Determination of Lipid Peroxidation
MDA, one of the well-known side products of LPO, is used as an index of lipid damage. For this reason, we measured the effect of EOLM on MDA levels following exposure. As shown in Figure 9, Concentrations (g/mL)

Determination of Lipid Peroxidation
MDA, one of the well-known side products of LPO, is used as an index of lipid damage. For this reason, we measured the effect of EOLM on MDA levels following exposure. As shown in Figure 9, the EOLM at different concentrations tested did not alter MDA level in comparison with the control (p > 0.05).

Discussion
The use of in vivo alternative models in order to perform toxicological tests is increasing [15,40]. In this scenario, several model organisms have been used to identify the pharmacological properties of plants material or active components. Drosophila melanogaster is one of them, and has been used for more than 110 years to study the biological effects of compounds and the underlying pathophysiology of numerous diseases, including Alzheimer's and Parkinson's diseases [31,41,42].
Medicinal plants have been widely used for the prevention and/or treatment of various diseases, on the basis that they do not present genotoxic risks when consumed for long time [43]. In the current study, the antioxidant activity of Lantana montevidensis leaf essential oil was evaluated in vitro, while its potential toxic effect was investigated in vivo using D. melanogaster. In general, the essential oil of the leaves of L. montevidensis appeared to be rich in terpenes, (mainly, monoterpenes and sesquiterpenes), which can justify its strong odor [44]. EOLM showed only small differences in composition compared to data reported in the literature [24,25]. For instance, in the study carried out

Discussion
The use of in vivo alternative models in order to perform toxicological tests is increasing [15,40]. In this scenario, several model organisms have been used to identify the pharmacological properties of plants material or active components. Drosophila melanogaster is one of them, and has been used for more than 110 years to study the biological effects of compounds and the underlying pathophysiology of numerous diseases, including Alzheimer's and Parkinson's diseases [31,41,42].
Medicinal plants have been widely used for the prevention and/or treatment of various diseases, on the basis that they do not present genotoxic risks when consumed for long time [43]. In the current study, the antioxidant activity of Lantana montevidensis leaf essential oil was evaluated in vitro, while its potential toxic effect was investigated in vivo using D. melanogaster. In general, the essential oil of the leaves of L. montevidensis appeared to be rich in terpenes, (mainly, monoterpenes and sesquiterpenes), which can justify its strong odor [44]. EOLM showed only small differences in composition compared to data reported in the literature [24,25]. For instance, in the study carried out by Bezerra et al. [45], the chemical composition of the essential oil of the leaves of Lantana montevidensis was β-caryophyllene (34.96%), germacrene D (25.49%), and bicyclogermacrene (9.48%), while in the study by Sousa et al. [24], it was β-caryophyllene (31.5%), germacreno D (27.5%), and bicyclerecyroreno (13.9%). Such differences can be attributed to intrinsic and/or extrinsic factors such as environmental conditions and time and place of leaf collection [46][47][48]. β-caryophyllene (28.15%), one of the major component of L. montevidensis leaf essential oil, is a sesquiterpene present in essential oils of several plants families. Plants species that have this constituent exhibit a variety of pharmacological activities such as anti-inflammatory, antinociceptive, and antioxidant, among others [49][50][51]. d-germacrene (31.27%), the major component found in L. montevidensis leaf essential oil, is a common compound in plants, being considered a precursor for the biosynthesis of many sesquiterpenes [52]. DPPH is a stable radical, exhibiting a maximum absorbance at 517 nm. Such absorption by the action of antioxidants is taken as a measure of antioxidant activity [53]. It has widespread use in the assessment of radical elimination [54,55]. Here, the essential oil of L. montevidensis showed very low DPPH scavenging activity, and Fe(II) chelating activity, but demonstrated higher reducing power. The study carried out by Barros et al. [9] demonstrated the antioxidant activity of the aqueous and ethanolic extracts of L. montevidensis against DPPH radical, and its capacity to inhibit lipid peroxidation in rat brain. Our results are in agreement with that of Hossain and Shah [40], who found low antioxidant activity for other essential oils. Consistent with this, essential oils rich in monoterpenes (e.g., from Cupressus sempervirens, Phyllostachys nigra, Eucalyptus globulus, and Psidium guayava) have been shown to be ineffective in scavenging DPPH radical [14].
Plant essential oils (EOs) have been tested against a wide range of arthropod pests, with promising results. EOs have been reported to possess high efficacy, multiple mechanisms of action, low toxicity in non-target vertebrates, and the potential to act as reducing agents and stabilizers for the synthesis of nanopesticides [56]. Therefore, the reducing power of EOLM observed in this study could justify its potential use as a natural reducing agent.
It is widely recognized that reactive oxygen species (ROS) or reactive nitrogen species (RNS) induce oxidative stress by causing significant damage to cell structure directly or indirectly, leading to a number of diseases [57][58][59]. The major targets of ROS during oxidative stress are thought to be DNA, RNA, proteins, and lipids. Different reactive species have different degrees of reactivity with the cellular components, and the availability of free iron in the form of Fe 2+ is considered to be of utmost importance in ROS toxicity due to its participation in the Fenton reaction that drives the formation of hydroxyl radicals [60]. Although free Fe 2+ ions are known to exert vital functions in the organism at low concentrations [61], high level of free Fe 2+ ions has been detected in several neurodegenerative diseases such as Alzheimer's disease [62]. Therefore, compounds able to chelate Fe 2+ to render it unavailable or less available for the participation in the Fenton reaction are of particular interest. Unfortunately, EOLM did not exhibit Fe 2+ chelating activity.
Glutathione (GSH) is an essential compound in the maintenance of cellular homeostasis because of its reducing properties [63]. Protein thiol groups are inherently very reactive, and can be oxidized by reactive species. The balance between oxidized and free thiols is important not only for the maintenance of protein and enzymatic functions, but also for cellular redox balance [64]. The results obtained in this study showed that EOLM at all the concentrations tested did not alter the total thiol and NPSH levels and the MDA content of flies homogenates, indicating that EOLM does not cause oxidative stress since the total thiol depletion is associated with increased lipid peroxidation [65]. Thus, it is possible to presume that the beneficial effects of EOLM may be attributed at least, in part, to its reducing ability. In spite of the fact exposure of flies to EOLM (0.12-0.48 g/mL) did not affect MDA content (an index of lipid peroxidation), there is report showing the potential of L. montevidensis to decrease LPO in egg phospholipid [25], and to exert antioxidant activity in vitro [9,25].

Conclusions
In conclusion, the present study demonstrated that Lantana montevidensis leaf essential oil was ineffective in scavenging the DPPH radical, but exhibited reducing activity in vitro. Exposure of Drosophila melanogaster to L. montevidensis leaf essential oil (0.12-0.48 g/mL) for 5 h did not significantly alter markers of oxidative, as evidenced by there being no change in total thiol, GSH, and MDA contents.