**Preface to "Insecticide, Acaricide, Repellent and Antimicrobial Development"**

Nowadays, arthropod pest and vector species are still managed though the use of synthetic insecticides and acaricides. However, in an Integrated Pest/Vector Management framework, substantial efforts are directed to design and validate environmentally sustainable and selective products with multiple modes of action, which make the develop of insecticide and acaricide resistance unlikely. A similar scenario applies to antimicrobials.

Further, bites from bloodsucker insects and mites can be avoided using repellents. In this scenario, discovering novel and effective products to repel mosquitoes, ticks, tabanids and kissing bugs, just to cite some hot-topic examples, is a challenge for public health. Natural products represent a huge source of highly effective active ingredients to be used for repellent purposes.

From this perspective, the present book, a reprint of the *Molecules* Special Issue "Insecticide, Acaricide, Repellent and Antimicrobial Development", is dedicated to the development of effective and eco-friendly insecticides, acaricides, repellents, and antimicrobials, including products of natural origin.

> **Giovanni Benelli** *Editor*

### *Editorial* **Insecticide, Acaricide, Repellent and Antimicrobial Development**

**Giovanni Benelli**

Department of Agriculture, Food and Environment, University of Pisa, via del Borghetto 80, 56124 Pisa, Italy; giovanni.benelli@unipi.it; Tel.: +39-0502216141

The quick spread of invasive arthropod species worldwide, sometimes boosted by global warming and urbanization [1–4], outlines again the need for effective and timely pest and vector management tools [5]. However, most of them rely on the use of synthetic insecticides and acaricides. This represents a major problem, since synthetic molecules often rely on a single mechanism of action, making resistance development quick and hard to deal with [6,7]. Similarly, fast resistance development to widely used antimicrobials has been detected in a wide number of microbial pathogens and parasites [8,9]. The massive, often inappropriate, employ of synthetic pesticides also leads to serious non-target effects on human health and the environment [10].

Further, bites from bloodsucker insects and mites can be avoided using repellents. In this scenario, discovering novel and effective products to repel mosquitoes, ticks and tabanids, just to cite some hot examples, is a challenge for public health [11–14]. Natural products represent a huge source of highly effective active ingredients to be used for repellent purposes (e.g., *Eucalyptus citriodora* and the related molecule *p*-menthane-3,8 diol) [15].

In this framework, the present Special Issue is dedicated to the development of effective and eco-friendly insecticides, acaricides, repellents and antimicrobials, including products of natural origin (e.g., plant extracts, essential oils, selected bacterial and fungal metabolites). Research efforts shedding light on the modes of action, behavioural modifications and non-target effects of the above-mentioned natural products have been welcomed. It has been recommended to the authors to include a positive control in the experiments [16], as well as detailed information on the chemical composition of the tested products [17]. Both original research and reviews have been included in the Special Issue.

Herein, contributions on the following topics have been included:


Finally, the Special Issue ends with two reviews. The first summarized current knowledge on the use of diatomaceous earths in crop protection, stored product, and urban pest control, presenting a number of challenges for future research [31]. The second one highlights current prospects and challenges about the use of plant-borne products as pesticides for agricultural purposes [32].

**Citation:** Benelli, G. Insecticide, Acaricide, Repellent and Antimicrobial Development. *Molecules* **2022**, *27*, 386. https://doi.org/10.3390/molecules 27020386

Received: 29 December 2021 Accepted: 4 January 2022 Published: 8 January 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the author. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

In conclusion, despite the relevant research efforts undertaken in this field for discovering new insecticides, acaricides and repellents of natural origin, the road to their large-scale use in the real world appears long and windy, complicated by costly and complex authorization requirements [33], and with limited commercialization outcomes [34]. In this scenario, I sincerely hope that the present Special Issue will be useful in inspiring future research and even extension efforts on the topic, particularly among young researchers.

**Funding:** This article received no external funding.

**Acknowledgments:** I am grateful to Ashley Chen and Carey Yuan for their assistance in preparing this Special Issue for *Molecules*.

**Conflicts of Interest:** The author declares no conflict of interest.

### **References**


### *Article* **Amino Alcohols from Eugenol as Potential Semisynthetic Insecticides: Chemical, Biological, and Computational Insights**

**Renato B. Pereira 1,†, Nuno F. S. Pinto 2,†, Maria José G. Fernandes 2, Tatiana F. Vieira 3,4, Ana Rita O. Rodrigues 5, David M. Pereira 1, Sérgio F. Sousa 3,4, Elisabete M. S. Castanheira 5, A. Gil Fortes <sup>2</sup> and M. Sameiro T. Gonçalves 2,\***


**Abstract:** A series of β-amino alcohols were prepared by the reaction of eugenol epoxide with aliphatic and aromatic amine nucleophiles. The synthesized compounds were fully characterized and evaluated as potential insecticides through the assessment of their biological activity against *Sf9* insect cells, compared with a commercial synthetic pesticide (chlorpyrifos, CHPY). Three derivatives bearing a terminal benzene ring, either substituted or unsubstituted, were identified as the most potent molecules, two of them displaying higher toxicity to insect cells than CHPY. In addition, the most promising molecules were able to increase the activity of serine proteases (caspases) pivotal to apoptosis and were more toxic to insect cells than human cells. Structure-based inverted virtual screening and molecular dynamics simulations demonstrate that these molecules likely target acetylcholinesterase and/or the insect odorant-binding proteins and are able to form stable complexes with these proteins. Encapsulation assays in liposomes of DMPG and DPPC/DMPG (1:1) were performed for the most active compound, and high encapsulation efficiencies were obtained. A thermosensitive formulation was achieved with the compound release being more efficient at higher temperatures.

**Keywords:** eugenol derivatives; amino alcohols; semisynthetic insecticides; biopesticides; bioinsecticides; phenylpropanoids; *Spodoptera frugiperda*

### **1. Introduction**

The use of synthetic pesticides for decades to manage pest control in crops has resulted in an accumulation of various residues with adverse effects on many organisms and potential negative impact in human health. At the same time, crop destruction by pests, mainly by insects, is one of the main problems responsible for losses in agricultural production. Pesticides from natural sources are an effective alternative to synthetic pesticides and are becoming more important for pest management in agriculture and also public health. In this respect, plants offer a wide variety of secondary metabolites with efficacy against insects [1,2]. In recent years, essential oils (EOs) became an important natural source of pesticides. Many EOs present insecticidal, repellent, fumigant, and antifeedant activities against a wide variety of insects [3,4]. Essential oil components and their derivatives are

**Citation:** Pereira, R.B.; Pinto, N.F.S.; Fernandes, M.J.G.; Vieira, T.F.; Rodrigues, A.R.O.; Pereira, D.M.; Sousa, S.F.; Castanheira, E.M.S.; Fortes, A.G.; Gonçalves, M.S.T. Amino Alcohols from Eugenol as Potential Semisynthetic Insecticides: Chemical, Biological, and Computational Insights. *Molecules* **2021**, *26*, 6616. https://doi.org/ 10.3390/molecules26216616

Academic Editor: Giovanni Benelli

Received: 17 September 2021 Accepted: 26 October 2021 Published: 31 October 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

considered to be an alternative way of insect control. In particular, phenylpropanoids, one of the main constituents of some EOs, have proved to present efficacy against insects [4]. Eugenol is a phenylpropanoid and a major constituent of clove essential oil with many applications in pharmaceutical, food, agricultural, and cosmetics industries [5], and it has been shown to be biologically active as antioxidant [5,6], antiviral [7] anti-inflammatory [8] and antimicrobial [9]. Enan [10] showed that eugenol mimicked octopamine in increasing intracellular calcium levels in cloned cells from the brain of *Periplaneta americana* and *Drosophila melanogaster*, and this was also found to be mediated via octopamine receptors. Structural changes of eugenol are known to be a useful strategy in order to improve biological activity and to obtain new analogues with reduced side effects [11].

Further, epoxides are important intermediates in pharmaceutical and agrochemical industries. The three-membered heterocyclic ring is strained and susceptible to attack by a range of nucleophiles, including nitrogen (e.g., ammonia, amines, azides), oxygen (e.g., water, alcohols, phenols, acids), and sulfur (thiol)-containing compounds, leading to bifunctional molecules of great industrial value. The β-amino alcohols are used in the synthesis of β-blockers, insecticidal agents, and oxazolines, as well as chiral ligands in asymmetric synthesis [12–17]. β-Amino alcohol functionality is found in many biologically active compounds, being an important pharmacophore [14,18], and *N*-substituted β-amino alcohols are important building blocks in the preparation of added-value chemicals [16,19]. Salbutamol and propranolol are on the World Health Organization List of Essential Medicines and represent the most important examples of therapeutic agents having this structural feature [20].

Following our research interests in plant-inspired alternatives to synthetic pesticides [21,22], and specifically the study in which some eugenol derivatives have shown potential as biopesticides [22], in the present work, eugenol was converted to the corresponding epoxide with *m*-chloroperoxybenzoic acid (*m*-CPBA) in dichloromethane (DCM) and further reacted with a series of amine nucleophiles to afford the corresponding βamino alcohols. The β-amino alcohols were purified by column chromatography and fully characterized by 1H and 13C NMR spectroscopy and HRMS (high-resolution mass spectrometry). The obtained compounds were evaluated as potential insecticides through the assessment of their biological activity against the *Sf9* insect cell lines compared with chlorpyrifos, which is a commercial synthetic pesticide. In addition, computational studies were performed to identify the most likely protein targets responsible for the observed insecticide activity of these molecules through the application of structure-based inverted virtual screening protocol combined with molecular dynamics simulations and free energy calculations. Nanoencapsulation of the most promising compound considering its insecticidal activity was performed in liposomes of DMPG (dimyristoylphosphatidylglycerol) and DPPC/DMPG (dipalmitoylphosphatidylcholine/dimyristoylphosphatidylglycerol) (1:1), aiming at obtaining thermosensitive formulations. DMPG has a gel to liquid–crystalline phase transition temperature (Tm) at 23 ◦C, while for DPPC, it is 41 ◦C [23]. The increase in membrane fluidity upon phase transition is expected to promote an enhanced release of the encapsulated compounds, providing a triggered release by temperature above Tm of the formulation.

### **2. Results and Discussion**

### *2.1. Synthesis*

Eugenol **1** is easily obtained by hydrodistillation from clove, and is known for its various biological activities, as mentioned above, namely insecticidal. Following our recent interests in finding new biopesticides [22], the present work describes a strategy consisting of structural changes of eugenol in an attempt to obtain semisynthetic alternatives with improved insecticidal activity. Eugenol epoxide **2** was prepared from eugenol through reaction with *m*-CPBA in DCM using a known procedure [11,24] in 48% yield. Then, the epoxide was further reacted at 50 ◦C with a series of aliphatic and aromatic amine nucleophiles in ethanol/water as solvent [25], which is followed

by column chromatography purification on silica gel using dichloromethane/methanol, mixtures of increasing polarity (**3b**, **3d–f**) as the eluent, or by evaporation of solvents under reduced pressure (**3a** and **3c**) to afford the corresponding β-amino alcohol derivatives **3a**–**f** as oil materials. Thus, the reaction of 2-methoxy-4-(oxiran-2-ylmethyl)phenol **2** with 2-methylpropan-2-amine, octan-2-amine, piperidine, aniline, 3-methoxyaniline, and 4-cyanoaniline gave 4-(3-(*tert*-butylamino)-2-hydroxypropyl)-2-methoxyphenol **3a**, 4-(2-hydroxy-3-(octan-2-ylamino)propyl)-2-methoxyphenol **3b**, (4-(2-hydroxy-3-(piperidin-1-yl)-propyl)-2-methoxyphenol **3c**, 4-(2-hydroxy-3-(methyl(phenyl)amino)propyl)- 2 methoxyphenol **3d**, 4-(2-hydroxy-3-((3-methoxyphenyl)amino)propyl)-2-methoxy- phenol **3e**, and 4-(2-hydroxy-3-(4-hydroxy-3-methoxyphenyl)propyl)amino)benzonitrile **3f**, respectively, in 9–97% yield, and they were fully characterized by 1H and 13C NMR spectroscopy and HRMS (Scheme 1).

**Scheme 1.** Synthesis of eugenol amino alcohols **3a**–**f**.

The main 1H-NMR features of compounds **3a**–**f** are the signals for protons of the CHOH, CH2N, and OCH3 groups. The CH2N protons show up as two distinct signals as doublets of doublets or multiplets (δ 3.45–2.53 ppm); the CHOH proton displays as a multiplet in all compounds (δ 4.91–3.89 ppm); and the OCH3 group shows up as a singlet (δ 3.89–3.81 ppm). The *tert*-butyl group in **3a** shows up as a singlet (δ 1.16 ppm), while in **3b** and **3c**, the *N*-alkyl chain corresponds to a series of multiplets (δ 2.88–0.88 ppm). The 13C main features are the CH2N (δ 41.24–41.10 ppm), CHOH carbon (δ 71.1–67.01 ppm), OCH3 carbon (δ 55.93–55.72 ppm), and the additional OCH3 in **3e** (δ 54.96 ppm). In addition, the *tert*-butyl carbons in **3a** shows up (δ 26.79 and 24.58 ppm), while in **3b**, the methyl terminal is highlighted (δ 13.98 ppm).

### *2.2. Toxicity Assessment in Insect Cells*

All molecules obtained were evaluated for their impact in the viability of the *Sf9* cells at 100 <sup>μ</sup>g/mL (i.e., **<sup>1</sup>**—6.09 × <sup>10</sup>−<sup>4</sup> M; **<sup>2</sup>**—5.55 × <sup>10</sup>−<sup>4</sup> M; **3a**—3.95 × <sup>10</sup>−<sup>4</sup> M; **3b**— 3.23 × <sup>10</sup>−<sup>4</sup> M; **3c**—3.77 × <sup>10</sup>−<sup>4</sup> M; **3d**—3.66 × <sup>10</sup>−<sup>4</sup> M; **3e**—3.30 × <sup>10</sup>−<sup>4</sup> M; **3f**— 3.35 × <sup>10</sup>−<sup>4</sup> M;

**CHPY**– 2.85 × <sup>10</sup>−<sup>4</sup> M) by the means of a resazurin-based method. For benchmarking purposes, the insecticide chlorpyrifos was used at the same concentration. As shown in Figure 1, the only molecule devoid of toxicity was **3c**, which incidentally was also the only one bearing a piperidine ring. A second group of molecules, which elicited residual toxicity (under 25% of viability loss), was **1**, **2**, **3a**, and **3b**. Eugenol **1** was the starting material, and the results show that the replacement of the terminal methylene group by the epoxide had no effect upon the biological activity of the molecule. Finally, the most potent molecules were **3d**, **3e**, and **3f**, which caused losses of 40%, 30%, and 50% viability in insect cells, respectively. These three molecules were also the only ones bearing a benzene ring next to the nitrogen atom. Considering the unsubstituted ring, **3d**, its methoxylation resulted in decreased potency, while the presence of the cyanide group increased it. In light of these results, we decided to advance our studies solely with the two most potent molecules, **3d** and **3f**, as they were more potent than the benchmark used, chlorpyrifos.

**Figure 1.** Viability of the *Sf9* cells after incubation with the presented molecules (100 μg/mL), medium (control), or the reference insecticide chlorpyrifos (CHPY, 100 μg/mL). Cells were incubated for 24 h, after which viability was evaluated. \*\* *p* < 0.01, \*\*\* *p* < 0.001.

### *2.3. Amino Alcohols 3d and 3f Activate Caspase-Like Proteases in the Sf9 Cells*

After establishing the toxicity of the selected molecules toward insect cells, we investigated the mechanism of action behind this effect. In fact, the loss of viability could be a consequence of an array of different biological processes, from necrosis to cell cycle arrest and apoptosis, among others. Necrosis is a process of uncontrolled cell death that encompasses the destruction of cell membranes, with consequent leakage of cytoplasmic content to the surrounding tissues; for this reason, it is usually avoided in biological contexts [26]. To assess the potential unfolding of this event, we assessed the levels of leaked lactate dehydrogenase (LDH) in cells incubated with the selected molecules. Being a cytoplasmic enzyme, the finding of extracellular LDH is widely used as a marker of necrosis. As shown in Figure 2A, the incubation of cells with a lysis solution (LS) resulted in a three to four-fold increase in extracellular LDH. Conversely, the incubation of cells with **3d** and **3f** had no detectable impact in LDH levels in culture media. In light of this, we concluded that the impact of these molecules in the viability of the *Sf9* cells was not a consequence of an ongoing necrotic process. Next, we assessed if a process of organized cell death, such as apoptosis, could be taking place. Given the pivotal role of cysteine-aspartic proteases in most forms of apoptosis, we investigated the impact of **3d** and **3f** in the insect equivalent of mammal caspases, in this case DRACE, using a proluminescent substrate of this target. As shown in Figure 2B, both **3d** and **3f** significantly increased the caspase-like activity in treated cells, the latter having a more pronounced effect. This result suggests that both **3d** and **3f** elicit their cytotoxic effect toward the *Sf9* cells by triggering an organized process of cell death with the involvement of cysteine-aspartic proteases.

**Figure 2.** (**A**) LDH activity found in the culture media of the *Sf9* cells treated with compounds **3d** and **3f** (100 μg/mL) for 24 h. Lysis solution (LS) was used as positive control to generate a maximum LDH release. (**B**) Caspase-like activity of the *Sf9* cells after incubation with compounds **3d** and **3f** (100 μg/mL) for 24 h. Results are normalized for DNA content. \* *p* < 0.05, \*\*\* *p* < 0.001.

### *2.4. Amino Alcohols 3d and 3f Are More Toxic to Insect Cells Than Human Cells*

Up to this point, we had already identified two molecules that presented higher potency than the commercial insecticide chlorpyrifos and that were shown the be nonnecrotic and pro-apoptotic. In addition to these traits reported herein, it is also important that prospective new insecticides present some degree of selectivity, specifically low toxicity to human cells. To this end, we assessed the impact of the **3d** and **3f** in 2D models of human cells. We chose human keratinocytes (HaCaT cell line), as one of the most relevant routes of human contact with pesticides is usually via the skin, where keratinocytes are the first population of living cells in the skin. As shown in Figure 3, both molecules elicited a weak loss of viability, around 20%. Importantly, both molecules were less toxic than the benchmark chlorpyrifos and, relevantly, they were less toxic to human cells than insect cells.

**Figure 3.** Viability of HaCaT cells exposed to compounds **3d** and **3f** (100 μg/mL), medium (control), or the reference insecticide chlorpyrifos (CHPY, 100 μg/mL). Cells were incubated for 24 h, after which viability was evaluated. \*\*\* *p* < 0.001.

These results are promising and pave the way for further developments in the field, as the chemical diversity obtained allows drawing some structure–activity relationships, as addressed above.

### *2.5. Inverted Virtual Screening Results*

After identifying the most promising molecules and the biological processes involved in their cytotoxic effect, we were interested in shedding light on the possible molecular targets. To this end, an array of computational methods was used.

Table 1 presents the average scores obtained for compounds **3d** and **3f** for each potential target with each scoring function. Regarding the different scoring functions, it is important to mention that they are based on different metrics and scales. The score for all the GOLD scoring functions is dimensionless, and the higher the score, the better the binding affinity. The Vina scoring function, on the other hand, uses a metric that approximates that of binding free energies, so a more negative value means better affinity. The PDB structure with the best score was selected for each potential target, and they were ranked from the best target to worst, according to the predictions of the different docking programs/scoring functions.



Globally, considering the results obtained with the different scoring functions, the odorant binding proteins class (OBP) and acetylcholinesterase (AChE) are the protein targets with the highest affinity toward compounds **3d** and **3f**. This tendency was quite clear with all the different scoring functions evaluated, which further strengthens our results.

### *2.6. Molecular Dynamics Simulations and Free Energy Calculations Results*

To validate the inverted screening results, we evaluate the protein flexibility and characterize the molecular interactions formed, and molecular dynamics simulations were performed for the complexes formed with compound **3d** and compound **3f** and the two groups of targets predicted at the inverted VS stage: OBP and AChE. Structures with the best score from each group were selected (3K1E for OBP and 1QON for AChE). The stability of AChE: compound **3d**, AChE: compound **3f**, OBP: compound **3d**, and OBP: compound **3f** complexes was evaluated using RMSD calculations for the Cα atoms of each complex and for the ligands, Solvent-Accessible Surface Area (SASA) analysis, and hydrogen bonding analysis.

All systems and ligands presented relatively low RMSD values, as seen in Table 2 (and Figure S1), showing that the target–ligand complexes are well equilibrated and that the eugenol derivatives evaluated maintain their binding conformation predicted from the docking.

**Table 2.** Average protein and ligand RMSD values (Å), ligand RMSD (Å), average ligand SASA (Å), percentage of potential ligand SASA buried, and average number of ligand–target hydrogen bonds obtained from the MD simulations. ΔG binding energy determined using MM/GBSA and per-residue decomposition, which were calculated for the last 90 ns of the simulation.


When analyzing the percentage of potential SASA area buried for compound **3d** and compound **3f** when complexed with AChE and OBP, it can be seen that the two molecules remain tightly bound to the two targets evaluated and well protected from the solvent with average buried areas over 90% (Table 2). A small decrease was noticed for compound **3d** bound to AChE, in relation to the initial configuration predicted from docking, with an average buried area oscillating between 80 and 90%. These results demonstrate that compounds **3d** and **3f** remain well bound to the two targets evaluated, even after 100 ns. In particular, the eugenol derivatives evaluated in complex with OBP remain very well protected from the solvent throughout time.

Hydrogen bonding analysis is important to understand the stability of the interactions between the targets and ligands throughout time. The results presented in Table 2 show that both ligands maintain a stable hydrogen bonding profile with the targets evaluated, maintaining between one and three hydrogen bonds with AChE and one and four hydrogen bonds with OBP. Globally, the profile observed shows that compounds **3d** and **3f** establish more hydrogen bonds with OBP and with AChE.

Table 2 summarizes the results discussed so far and presents the values for the Gibbs binding free energy calculated using MM/GBSA. The analysis of the residue contribution to the eugenol derivatives' binding free energy to the two protein targets evaluated highlights the interaction profile of compounds **3d** and **3f** against AChE and OBP, showing the most important amino acid residues involved in ligand stabilization.

AChE is a serine hydrolase, and it is a very common target for pesticides as it is an enzyme vital for the regulation of acetylcholine in several organisms, from insects to mammals. Since this is an enzyme transversal to many species, the use of anticholinesterase insecticides can cause serious health and environmental problems. In addition, there are reports of insect resistance due to mutation of the AChE gene [27].

For AChE, compound **3d** binding is stabilized mostly by residues Trp83 (−2.4 ± 0.8), Tyr370 (−1.3 ± 0.4), and His480 (−1.3 ± 0.6) through non-polar interactions. For compound **3f**, the residues contributing more toward AChE binding are Tyr370 (−2.4 ± 0.1), Tyr372 (−2.5 ± 0.8), and Trp83 (−1.9 ± 0.4), with non-polar interactions playing an important role and π–π stacking with Trp83. Figure 4 represents the average structure of the dominant cluster of the AChE-eugenol derivatives complexes obtained from the analysis of the MD trajectory, illustrating the binding pocket and main interactions formed.

**Figure 4.** Compound **3d** (cyan licorice) and compound **3f** (pink licorice) interaction map with AChE. The most important residues for the interaction are highlighted in green. Blue arrow indicates π–π stacking with the ring of Trp-83. Red lines represent hydrogen bonding.

For the sake of warranting the potential off-target effect that could pose a toxicity risk, human AChE was also analyzed, the docking scores (Table S1) being inferior to the ones obtained for insect AChE, hence suggesting that the eugenol derivatives evaluated favor binding to the insect AChE considered over that of human AChE. When comparing the sequence of amino acids between insect and human AChE, there is only 53–54% sequence identity, even though their 3D structures are very similar. The active-site gorge in the insect enzyme is narrower, and the amino acid residues are different. Moreover, the residues in the opening of the gorge are also different [27], which might explain the difference in affinity of the eugenol derivatives.

The results show that the most stable complexes are OBP–compound **3d** and OBP– compound **3f**, with binding free energy values of −31.7 ± 0.2 and −41.6 ± 0.2 kcal/mol, respectively. This is consistent with the results presented so far and indicates that eugenol derivatives have indeed a high affinity toward OBP. In fact, there is a structure deposited in the PDB of a bee OBP14 from bound to eugenol [28].

OBP are a large and diverse family of insect proteins. They are involved in the transport of hydrophobic odorant and pheromone molecules toward the olfactory receptors. They are abundant in the insect family and different in structure but carry out similar roles. In the *Drosophila melanogaster*, there are 52 different types of OBPs alone. Even though diverse in number and sequence, they present some common features. They are small, have six conserved cysteine residues joined by three disulfide bridges, and have six alpha-helical domains [29–32].

Compounds **3d** and **3f** have a higher molecular weight than eugenol (273.33 g/mol, 298.34 g/mol, respectively, versus 164.20 g/mol), but they are also lipophilic and if volatile, they can in fact be capable of binding OBP. The precise mechanism of action still needs to be further validated.

For OBP1, compound **3d** binding is stabilized mostly by residues Leu67 (−2.5 ± 0.5), Trp105 (−2.1 ± 0.4), and Ala79 (−1.7 ± 0.5), through van der Waals interactions. For compound **3f**, the residues contributing more toward OBP binding are Met75 (−2.9 ± 0.4) and Phe114 (−1.8 ± 0.8) through van der Waals interactions and Trp105 (−2.5 ± 0.4) through a hydrogen bond with the backbone. Figure 5 illustrates the average structure of the dominant cluster of the OBP1 binding pocket and main interactions formed between compound **3d**-OBP1 and compound **3f**-OBP1, respectively.

**Figure 5.** Compound **3d** (cyan licorice) and compound **3f** (pink licorice) interaction map with OBP1. The most important residues are highlighted in green.

### *2.7. Nanoencapsulation and Release Assays*

The most active compound against the *Sf9* cells, compound **3f**, was encapsulated in liposomal systems of the phospholipids DMPG (100%) and DMPG/DPPC (1:1). The size (hydrodynamic diameter), polydispersity index, and zeta potential of the compoundloaded liposomes were determined by Dynamic and Electrophoretic Light Scattering (Table 3). These properties can affect the bulk properties, performance, processability, and stability of a nanoformulation. Particularly, the surface charge highly influences the stability of the liposomes, and zeta potential values more negative than −30 mV or more positive than +30 mV are considered optimal values for good stabilization of a nanodispersion [33]. In view of this fact, a negatively charged lipid, phosphatidylglycerol, was chosen for the liposomal formulation. Specifically, the phospholipid DMPG has a phase transition temperature (23 ◦C) near room temperature [23], allowing obtaining an enhanced release at summer temperatures (around or above 30 ◦C), where the lipid is in the fluid (liquid-crystalline) phase. However, the relatively short hydrocarbon chain of DMPG

and the tendency to form leaky vesicles [34] can hamper a high encapsulation efficiency of compound **3f**. Therefore, the DPPC/DPPG (1:1) formulation was also tested.

**Table 3.** Hydrodynamic diameter (Dh), polydispersity index (PDI), and zeta (ζ) potential of DMPG (100%) and DMPG:DPPC (1:1) liposomes (SD from three independent measurements).


The values in Table 3 show that both liposome formulations are small in size, with hydrodynamic diameters around 200 nm, presenting also a low polydispersity. A PDI value below 0.3 is considered to be acceptable, indicating a homogenous population of phospholipid vesicles [35]. The zeta potential values indicate a highly negative surface charge, anticipating a low aggregation (due to the electrostatic repulsion) and high colloidal stability.

The compound **3f** is a fluorescent molecule (Figure 6) in several solvents and in liposomes. This is a great advantage for the determination of the encapsulation efficiency and drug release, due to the high sensitivity (and selectivity) of fluorescence spectroscopy.

**Figure 6.** Normalized absorption and fluorescence emission (excitation at 290 nm) spectra of **3f** solution (2 <sup>×</sup> <sup>10</sup>−<sup>5</sup> M for absorption and 5 <sup>×</sup> <sup>10</sup>−<sup>6</sup> M for emission).

The encapsulation efficiency (EE%) of compound **3f** in liposomes was determined by fluorescence measurements (Table 4). In these assays, it was kept in mind that the compound is active against the *Sf9* cells at a concentration of 100 <sup>μ</sup>g/mL (3.35 × <sup>10</sup>−<sup>4</sup> M) and, therefore, at least this concentration must be encapsulated. The encapsulation efficiencies are high, the system DPPC/DMPG being the most advantageous for **3f** encapsulation. Nevertheless, the EE% values show that both formulations are able to encapsulate **3f** at concentrations that may guarantee an insecticidal activity (if compound release is effective).


**Table 4.** Encapsulation efficiency (EE%) of compound **3f** in DMPG (100%) and DMPG/DPPC (50:50) liposomes and concentration of encapsulated compound.

Compound release from both liposomal formulations was studied at 20 ◦C and 35 ◦C, to investigate the temperature dependence of the release profile (Figure 7). The experimental data were analyzed with the Weibull model (Table S2 and Figure S4 in Supporting Information) and the cumulative concentration released was compared in terms of liposome formulation and temperature.

**Figure 7.** Cumulative release of compound **3f** at 20 ◦C (black squares) and 35 ◦C (red dots) from liposomes of DMPG (100%) (**left**) and DMPG:DPPC (**right**).

An enhanced release of **3f** was observed from liposomes of DMPG, reaching a cumulative release of 62% in 24 h at 35 ◦C, while, at 20 ◦C, a 36% release was attained. This is a result of the higher membrane fluidity at 35 ◦C (above transition temperature) for DMPG liposomes, which provide a thermosensitive formulation. Moreover, at both temperatures, the compound released is higher than 100 μg/mL in 24 h. The rigidity of DPPC at temperatures below its gel to liquid-crystalline phase transition (41 ◦C) justifies the much lower compound release from DPPC/DMPG liposomes, with 14% and 16% of released compound at 20 ◦C and 35 ◦C, respectively, not displaying a significant sensitivity to temperature.

The parameter *b* of the Weibull model can be related to the release mechanism [36]. If *b* ≤ 0.75, the release is due to a Fickian diffusion, which is the case for DMPG liposomes at 20 ◦C (Table S2). If *b* > 1, a complex release mechanism takes place, with multiple mechanisms contributions, which is verified in the other cases.

### **3. Materials and Methods**

### *3.1. Chemicals and Reagents*

Dichloromethane, ethanol, methanol, ethyl acetate, light petroleum, and *m*-chloroperbenzoic acid were purchased from Fisher Scientific (Geel, Belgium). The anhydrous magnesium sulfate was PanReac Applichem (Barcelona, Spain) products. Chloroform-d was produced by Eurisotop (Cambridge, England). Thin-layer chromatography (TLC) analyses were carried out on 0.25 mm thick, precoated silica plates (Merck Fertigplatten Kieselgel 60F254, Germany), and spots were visualized under UVlight. Chromatography on silica gel was carried out on Merck Kieselgel (230–240 mesh).

### *3.2. Analytical Instruments*

NMR spectra were obtained on a Bruker Avance III (Bruker Corporation, Billerica, MA, USA) at an operating frequency of 400 MHz for 1H NMR and 100.6 MHz for 13C NMR using the solvent peak as internal reference at 25 ◦C. All chemical shifts are given in ppm using δ Me4Si = 0 ppm as reference, and J values are given in hertz. Assignments were made by comparison of chemical shifts, peak multiplicities, and J values and were supported by spin decoupling-double resonance and bidimensional heteronuclear correlation techniques. High-resolution mass spectrometry analyses were performed at the "CACTI—Unidade de Masas e Proteómica, at University of Santiago de Compostela", Spain.

### *3.3. Synthesis of 2-methoxy-4-(oxiran-2-ylmethyl)phenol 2*

A solution of eugenol **1** (0.500 g 3.0 mmol; 1 equiv) dissolved in dichloromethane (18 mL) was added dropwise to a suspension of 70% *m*-chloroperbenzoic acid (0.750 g; 4.3 mmol; 1 equiv) in dichloromethane (10 mL) at 0 ◦C. After stirring for 1 h, *m*-chloroperbenzoic acid was again added (1 equiv), and the reaction mixture was stirred for another 24 h at room temperature. A 10% aqueous solution of sodium sulfate (2 × 20 mL) was added, and the resulting solution was washed with 5% aqueous solution of sodium hydrogen carbonate (2 × 20 mL). The organic phase was dried with anhydrous magnesium sulfate, and the solvent was evaporated to afford the compound **2** as a dark yellow oil (0.239 g; 48%). Rf = 0.27 (DCM). 1H-NMR δ<sup>H</sup> (CDCl3, 400 MHz): 6.87 (d, 1H, J = 8 Hz, H-6), 6.73–6.78 (m, 2H, H-3 and H-5), 5.54 (s, 1H, OH), 3.90 (s, 3H, OCH3), 3.12–3.16 (m, 1H, CH oxirane), 2.79–2.82 (m, 3H, CH2Ph and CH2 oxirane), 2.55 (q, J = 2.8 Hz, 1H, CH2 oxirane) ppm. 13C-NMR δ<sup>C</sup> (CDCl3, 100.6 MHz): 146.46 (C-2), 144.39 (C-1), 129.03 (C-4), 121.64 (C-5), 114.32 (C-6), 111.54 (C-3), 55.90 (OCH3), 52.67 (CH oxirane), 46.79 (CH2 oxirane), 38.37 (CH2Ph) ppm.

### *3.4. Synthesis of Amino Alcohols 3a–f*

3.4.1. Synthesis of 4-(3-(tert-butylamino)-2-hydroxypropyl)-2-methoxyphenol **3a**

To a suspension of 2-methoxy-4-(oxiran-2-ilmethyl)phenol **2** (0.163 g; 0.90 mmol; 1 equiv) in H2O/EtOH 2:1 (2 mL) was added 2-methylpropan-2-amine (0.325 g; 4.44 mmol), and the resulting mixture was heated at 50 C for 5 h. The solvents and the amine were evaporated under reduced pressure to afford a compound **3a** as an orange oil (0.097 g; 0.38 mmol; 42%), Rf = 0.30 (MeOH/DCM 1:9). 1H-NMR δ<sup>H</sup> (CDCl3, 400 MHz): 6.78 (d, J = 8.0 Hz, 1H, Ar-H), 6.74 (d, J = 1.6 Hz, 1H, Ar-H), 6.45 (dd, J = 8.4 Hz, 2.0 Hz 1H, Ar-H), 4.04–3.98 (m, 1H, CH2CH(OH)), 3.81 (s, 3H, OCH3), 3.45 (s, 1H, CH2NH), 2.81 (dd, J = 12.0 Hz, 2.4 Hz, 1H, CH2NH), 2.77–2.62 (m, 2H, CH2CH(OH)), 1.16 (s, 9H, *t*-Bu) ppm. 13C-NMR δ<sup>C</sup> (CDCl3, 100.6 MHz): 146.63 (Ar-C),144.34 (Ar-C), 121.83 (Ar-C), 114.4 (Ar-C), 112.13 (Ar-C), 69.37 (CH2CH(OH)), 55.8 (OCH3), 47.06 (CH2), 41.25 (CH2), 26.79 (3×CH3), 24.58 (C(CH3)) ppm. HRMS (ESI-TOF): calcd for C14H24NO3 [M <sup>+</sup> + H]: 254.1751; found 254.1753.

### 3.4.2. Synthesis of 4-(2-hydroxy-3-(octan-2-ylamino)propyl)-2-methoxyphenol **3b**

To a suspension of 2-methoxy-4-(oxiran-2-ilmethyl)phenol **2** (0.163 g; 0.90 mmol; 1 equiv) in H2O/EtOH 2:1 (2 mL) was added octan-2-amine (0.502 g; 3.89 mmol) and the resulting mixture was heated at 50 ◦C for 4 h. Then, water (2 mL) was added, and the resulting mixture extracted with EtOAc (2 mL), The organic phase was collected, dried with anhydrous MgSO4, and the solvent evaporated to afford an oil (0.202 g), which was subjected to column chromatography using DCM/MeOH as eluent of increasing polarity to give the compound **3b** as a brown oil (0.165 g; 0.53 mmol; 59%). Rf = 0.45 (MeOH/DCM 10:90). 1H-NMR δ<sup>H</sup> (CDCl3, 400 MHz): 6.78 (d, J = 8.0 Hz, 1H, Ar-H), 6.72 (ls, 1H, Ar-H), 6.48 (d, J = 8.0 Hz, 1H, Ar-H), 3.92–3.89 (m, 1H, CH2CH(OH)), 3.81 (s, 3H, OCH3), 2.84–2.53 (m, 6H, 2×CH2 and CH2NH), 1.49 (m, 1H, NHCHCH3), 1.24 (m, 10H, 5×CH2), 1.07 (m, 3H, NHCHCH3), 0.88 (m, 3H, CH2CH2CH3) ppm. 13C-NMR δ<sup>C</sup> (CDCl3, 100.6 MHz): 146.69 (Ar-Cq),144.36 (Ar-Cq), 129.59 (Ar-Cq), 121.76 (Ar-C), 114.55 (Ar-C), 111.99 (Ar-C), 70.25

(CH), 69.93 (CH), 55.72 (OCH3), 51.48 (CH2), 41.32 (CH2), 35.61 (CH2), 31.68 (CH2), 29.21 (CH2), 25.77 (CH2), 22.51 (CH2), 19.17 (CH3), 13.98 (CH3) ppm.

### 3.4.3. Synthesis of 4-(2-hydroxy-3-(piperidin-1-yl)propyl)-2-methoxyphenol **3c**

To a suspension of 2-methoxy-4-(oxiran-2-ilmethyl)phenol **2** (0.1 g; 0.56 mmol; 1 equiv) in H2O/EtOH 2:1 (2 mL) was added piperidine (0.047 g; 0.56 mmol), and the resulting mixture was heated at 50 ◦C for 5 h. The solvent was evaporated under reduced pressure to afford compound **3c** as a brown oil (0.142 g; 0.54 mmol; 97%). Rf = 0.35 (MeOH/DCM 10:90). 1H-NMR δ<sup>H</sup> (CDCl3, 400 MHz): 6.83 (d, J = 8.4 Hz, 1 H, Ar-H), 6.77 (d, J = 2.0 Hz, 1H, Ar-H), 6.67 (dd, J = 8.0 Hz, 2.0 Hz, 1H, Ar-H), 4.18–4.12 (m, 1H, CH2CH(OH)), 3.89 (s, 3H, OCH3), 2.88–2.81 (m, 4H, CH2 and CH2NH), 2.65–2.52 (m, 4H, 2×CH2), 1.82–1.70 (m, 4H, 2×CH2) 1.58–1.47 (m, 4H, 2×CH2) ppm. 13C-NMR <sup>δ</sup><sup>C</sup> (CDCl3, 100.6 MHz): 146.52 (Ar-Cq), 144.31 (Ar-Cq), 129.52 (Ar-Cq), 121.80 (Ar-C), 114.27 (Ar-C), 111.80 (Ar-C), 67.01 (CH), 63.93 (CH2), 55.93 (OCH3), 54.76 (CH2), 41.30 (CH2), 24.26 (CH2), 23.05 (CH2) ppm. HRMS (ESI-TOF): calcd for C15H24NO3 [M <sup>+</sup> +H]: 266.1751, found 266.1752.

### 3.4.4. Synthesis of 4-(2-hydroxy-3-(phenylamino)propyl)-2-methoxyphenol **3d**

To a suspension of 2-methoxy-4-(oxiran-2-ilmethyl)phenol **2** (1 equiv) in H2O/EtOH 2:1 (2 mL) was added aniline (0.4 mL; 3.9 equiv), and the resulting mixture was heated at 50 ◦C for 5.5 h. Then, water (2 mL) was added, and the resulting mixture was extracted with EtOAc (2 mL). The organic phase was collected, dried with anhydrous MgSO4, and the solvent was evaporated to afford an oil (0.284 g), which was subjected to column chromatography using DCM/MeOH as eluent of increasing polarity to give compound **3d** as a yellow oil (0.095 g; 0.35 mmol; 36%). Rf = 0.7 (MeOH/DCM 5:95). 1H-NMR δ<sup>H</sup> (CDCl3, 400 MHz): 7.19 (t, J = 7.2 Hz, 2 H, Ar-H), 6.88 (d, J = 8 Hz, 1H, Ar-H), 6.75 (m, 3H, Ar-H), 6.65 (d, J = 8 Hz, 2H, Ar-H), 4.09–4.02 (m, 1H, CH2CH(OH)), 3.87 (s, 3H, OCH3), 3.31 (dd, J = 12.4 Hz, 7.2 Hz, 1H, CH2NH), 3.10 (dd, J = 12.4 and 8 Hz, 1H, CH2NH), 2.83 (dd, J = 14 Hz and 5.2 Hz, 1H, CH2CH(OH)), 2.75 (dd, J = 14 and 8 Hz, 1H, CH2CH(OH)) ppm. 13C-NMR δ<sup>C</sup> (CDCl3, 100.6 MHz): 147.9 (Ar-C), 146.6 (Ar-C), 144.4 (Ar-C), 129.4 (Ar-C), 129.3 (Ar-C), 122.0 (Ar-C), 118.1 (Ar-C), 114.5 (Ar-C), 113.5 (Ar-C), 111.8 (Ar-C), 71.1 (CH), 55.9 (OCH3), 49.5 (CH2), 41.2 (CH2) ppm HRMS (ESI-TOF): calcd for C16H20NO3 [M<sup>+</sup> +H]: 274.1438; found 274.1430.

### 3.4.5. Synthesis of 4-(2-hydroxy-3-((3-methoxyphenyl)amino)propyl)-2-methoxyphenol **3e**

To a suspension of 2-methoxy-4-(oxiran-2-ilmethyl)phenol **2** (0.162 g; 1 equiv) in H2O/EtOH 2:1 (2 mL) was added 3-methoxyaniline (0.544 mg; 4.42 mmol), and the resulting mixture was heated at 50 ◦C for 4 h. Then, water (2 mL) was added, and the resulting mixture was extracted with DCM (2 mL). The organic phase was collected, dried with anhydrous MgSO4, and the solvent was evaporated to afford an oil (0.579 g), which was subjected to column chromatography using light petroleum/EtOAc as an eluent of increasing polarity to give compound **3e** as a brown oil (0.098 g; 0.32 mmol; 36%). Rf = 0.35 (ethyl acetate/light petroleum 1:1). 1H-NMR δ<sup>H</sup> (CDCl3, 400 MHz): 7.08 (t, J = 8.0 Hz, 1H, Ar-H), 6.86 (d, J = 8 Hz, 1H, Ar-H), 6.73–6.71 (m, 1H, Ar-H), 6.30 (dd, J = 8 Hz, 1.6 Hz, 1H, Ar-H), 6.24 (dd, J = 8 Hz, 2.4 Hz, 1H, Ar-H), 6.20–6.16 (m, 1H, Ar-H), 4.05–3.99 (m, 1H, CH2CH(OH)), 3.84 (s, 3H, OCH3), 3.76 (s, 3H, OCH3), 3.27 (dd, J = 13.2 Hz, 3.6 Hz, 1H, CH2NH), 3.05 (dd, J = 12.8 Hz, 7.6 Hz, 1H, CH2NH), 2.83–2.70 (m, 2H, CH2CH(OH)) ppm. 13C-NMR <sup>δ</sup><sup>C</sup> (CDCl3, 100.6 MHz): 160.69 (Ar-Cq), 149.42 (Ar-Cq), 146.57 (Ar-Cq), 144.27 (Ar-Cq), 129.99 (Ar-Cq), 129.91 (Ar-CH), 121.85 (Ar-CH), 114.48 (Ar-CH), 111.81 (Ar-CH), 106.35 (Ar-CH), 102.95 (Ar-CH), 102.88 (Ar-CH), 71.07 (CH), 55.76 (OCH3), 54.96 (OCH3), 49.32 (CH2), 41.08 (CH2) ppm. HRMS (ESI-TOF): calcd for C14H24NO3 [M+ + H]: 304.1543, found 304.1547.

3.4.6. Synthesis of 4-(2-hydroxy-3-(4-hydroxy-3methoxyphenyl)propyl)amino)benzonitrile **3f**

To a suspension of 2-methoxy-4-(oxiran-2-ilmethyl)phenol **2** (0.162 g; 0.90 mmol; 1 equiv) in H2O/EtOH 2:1 (2 mL) was added 4-aminobenzonitrile (0.528 mg; 4.47 mmol), and the resulting mixture was heated at 50 ◦C for 37 h. Then, water (2 mL) was added, and the resulting mixture was extracted with EtOAc (2 mL), The organic phase was collected, dried with anhydrous MgSO4, and the solvent was evaporated to afford an oil (0.265 g), which was subjected to column chromatography using DCM/MeOH as an eluent of increasing polarity to give compound **3f** as a dark yellow oil (0.025 g; 0.08 mmol; 9%). Rf = 0.45 (MeOH/DCM 5:95). 1H-NMR δ<sup>H</sup> (CDCl3, 400 MHz): 7.39 (d, J = 8.8 Hz, 2H, Ar-H), 6.87 (d, J = 8.4 Hz, 1H, Ar-H), 6.72–6.69 (m, 2H, Ar-H), 6.58–6.50 (m, 2H, Ar-H), 4.07–4.01 (m, 1H, CH2CH(OH)), 3.85 (s, 3H, OCH3), 3.31 (dd, J = 12.8 Hz, 7.2 Hz, 1H, CH2NHPhe), 3.12 (dd, J = 12.8 Hz and 7.4 Hz, 1H, CH2NHPhe), 2.82 (dd, J = 13.6 Hz, 5.2 Hz, 1H, CH2CH(OH)), 2.73 (dd, J = 13.6 Hz and 8 Hz, 1H, CH2CH(OH)) ppm. 13C-NMR δ<sup>C</sup> (CDCl3, 100.6 MHz): 151.20 (Ar-Cq), 146.68 (Ar-Cq), 144.53 (Ar-Cq), 136.33 (Ar-Cq), 133.65 (Ar-CH), 128.83 (Ar-Cq), 121.88 (Ar-CH), 121.33 (Ar-CH), 114.62 (Ar-CH), 112.58 (Ar-CH), 111.72 (Ar-CH), 98.91 (Ar-CH), 70.97 (Ar-CH), 55.87 (OCH3), 48.10 (CH2), 41.20 (CH2) ppm.

### *3.5. Cell Culture*

Insect cells (*Sf9*, *Spodoptera frugiperda*) cells were maintained as a suspension culture and cultivated in Grace's medium with 10% FBS and 1% penicillin/streptomycin, at 28 ◦C with agitation. Cells were used in experiments while in the exponential phase of growth. On the other hand, HaCaT (human keratinocytes) cells were culture in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin at 37 ◦C in a humidified atmosphere of 5% CO2.

### *3.6. Viability Assessment*

For the assessment of viability, a resazurin-based method was used. The *Sf9* and HaCaT cells were plated at a density of 3.0 × 104 and 1.5 × <sup>10</sup><sup>4</sup> cells/well, respectively, incubated for 24 h, and then exposed to the molecules under study (at 100 μg/mL in Grace's medium) for 24 h. After this period, a commercial solution of resazurin was added (1:10), and the kinetic reaction of fluorescence increase was monitored at 560/590 nm. For HaCaT and the *Sf9* cells, 30 and 60 min of incubation were used, respectively.

### *3.7. LDH Assay*

*The Sf9* cells were cultured at the same density described above for the viability assessment. To assess the release of the stable cytosolic enzyme lactate dehydrogenase (LDH) into the media, 24 h after the incubation of cells with the molecules under study (at 100 μg/mL in Grace's medium), 50 μL of culture media were removed to a 96-well plate. The LDH released was determined using a CytoTox 96® assay kit (Promega; Madison, WI, USA) according to the manufacturer's instructions. A lysis solution (LS) was used as a positive control to generate a maximum LDH release (45 min). Absorbances were measured at 490 nm in a Multiskan GO plate reader (Thermo Fisher Scientific; Waltham, MA, USA), and results correspond to the fold increase of absorbance in treated vs. untreated cells of four independent experiments performed in duplicate.

### *3.8. Caspase-Like Activity*

The *Sf9* cells were plated at the same density described for viability studies and exposed to the molecules under study for the designated time. Generally, the same method described before by some of us was used [37]; however, it has been adapted toward insect cells, as previously reported [21,22]. After the incubation period, caspase-3/7 substrate was added to wells, and cells were incubated for 20 min at 22 ◦C. The luminescent signal was measured in a microplate reader (Cytation™ 3, BioTek, Winooski, VT, USA), and three independent experiments were performed in duplicate. Then, to normalize the results, DNA quantification was performed in a triplicate pool using a Qubit™ 1X dsDNA HS Assay Kit according to a previously described procedure [38] and manufacturer's instructions.

### *3.9. Statistical Analysis*

For biological assays, the Shapiro–Wilks normality test was performed in the data to ensure that it followed a normal distribution. Comparison between the means of controls and each experimental condition was performed using one-way ANOVA. Outliers were identified by the Grubbs' test. Data were expressed as the mean ± standard deviation (SD) of at least three independent experiments. GraphPad Prism 7.0 software was used, and values were considered statistically significant with a *p* < 0.05.

### *3.10. Molecular Docking and Inverted Virtual Screening Studies*

To identify possible molecular targets of the amino alcohols derived from eugenol, an inverted virtual screening protocol was applied. A search on Scopus was performed for papers describing virtual screening (VS) studies involving targets and molecules with insecticidal activity using the keywords: "virtual screening" and "pesticides". The selection criteria were relevance of the target and year of publication. In the 18 studies found, 23 PDB structures were identified and downloaded, enabling the creation of a structural database of putative insecticide targets. These are listed in Table S3.

The 23 PBD structures were prepared for docking starting using the Autodock Vina plugin for Pymol [39]. Crystallographic waters were removed. Then, the crystallographic ligands were saved in separate files and used as reference for active site coordinates as well as for validation in the re-docking steps. In the absence of ligands, the active site coordinates were based on the most important residues described in the literature. Redocking was used to evaluate the ability of the docking software to reproduce the geometry and orientation of the crystallographic pose, as well as the quality of the docking protocol, and to optimize the docking protocol.

The docking programs/scoring functions used were GOLD [40] (PLP, ASP, Chem-Score, and GoldScore scoring functions), and AutoDock Vina [41]. With each docking program/scoring function, the protocol was optimized for each protein target, to minimize the RMSD in the docking predictions of the reference ligand in re-docking, by comparison with the crystallographic structure of the corresponding complex.

The optimized parameters for each program/scoring function were as follows: Vinadocking box position, docking box dimension, exhaustiveness; GOLD (PLP, ASP, Chem-Score, GoldScore)-binding pocket center, docking region radius, search efficiency, number of runs. The final optimized conditions were used for the subsequent stages. Structures for the two eugenol amino alcohol derivatives with the highest insecticide activity were prepared for docking using Datawarrior [42] and OpenBabel [43] and were docked into each structure with the optimized protocol with all the five scoring functions. A ranked list of most likely targets was prepared based on the average scores obtained for each target with the different scoring functions.

### *3.11. Molecular Dynamics Simulations and Free Energy Calculations*

Molecular dynamics simulations were performed using the Amber18 software (University of California, San Francisco, USA) for the two compounds identified from the experimental studies to have the highest insecticide activity (compounds **3d** and **3f**), which are bound to the two most promising targets identified from the inverted virtual screening study (odorant binding protein 1–3KIE and acetylcholinesterase-1QON). Since 1QON presented a gap in the structure, a homology model was generated using SWISS-MODEL [44]. A total of 1466 templates were found to match the original sequence, but only the top 50 were used to build the model (Figure S5 in Supplementary Information).

Models for the MD simulations were prepared starting from the pose predicted for these complexes in the docking experiments during the inverted virtual screening

protocol with GOLD/PLP and treated with the Leap module of AMBER [45]. The protein targets were described with the ff14SB force field [46], while the eugenol derivatives were parameterized using ANTECHAMBER, with RESP HF/6-31G (d) charges calculated with Gaussian16 [47] and the General Amber Force Field (GAFF) [48]. The overall charge on the system was neutralized through the addition of counter-ions (Na<sup>+</sup> or Cl−), and the systems were placed in TIP3P water boxes with a minimum distance of 12 Å between the protein surface and the side of the box.

In order to remove the clashes, the systems were submitted to four consecutive minimizations stages, which were followed by an equilibration and production. In the first four minimization stages, the procedure was applied to (1) water molecules (2500 steps); (2) hydrogens atoms (2500 steps); (3) side chains of all the amino acid residues (2500 steps); and (4) the full system (10,000 steps). After the complete minimization, the systems were equilibrated by a procedure, which was divided into two stages: in the first stage, NVT ensemble, the systems were gradually heated to 298 K using a Langevin thermostat at constant volume (50 ps); in the second stage, the density of the systems was further equilibrated at 298 K (subsequent 50 ps). Finally, the productions runs were performed during 100 ns. Production was executed with an NPT ensemble at constant temperature (298 K, Langevin thermostat) and pressure (1 bar, Berendsen barostat), with periodic boundary conditions. An integration time of 2.0 fs using the SHAKE algorithm was used to constrain all covalent bonds involving hydrogen atoms. The nonbonded interactions were cut off at 10 Å throughout the entire molecular simulation procedure. The final trajectories were analyzed in terms of RMSD to obtain confirmation that both systems were well equilibrated after the initial 10 ns. The last 90 ns of the simulation were considered for hydrogen bonding analysis, and cluster analysis of the conformations was generated. This overall procedure has been previously used with success in the treatment of several biomolecular systems [49–57].

The Molecular Mechanics-Generalized Born Surface Area (MM-GBSA) method [27] was applied to estimate the binding free energies of compounds **3d** and **3f** to the odorant binding protein 1 and to acetylcholinesterase, considering a salt concentration of 0.100 mol.dm<sup>−</sup>3. In addition, the energy decomposition method was employed to estimate the contribution of all the amino acid residues for each of these binding free energies. From each MD trajectory, a total of 1400 conformations taken from the last 70 ns of simulation were considered for the MM-GBSA calculations.

### *3.12. Nanoencapsulation Studies*

The most active compound against the *Sf9* cells, compound **3f**, was encapsulated in liposomes composed of the phospholipids 1,2-dimyristoyl-*sn*-glycero-3-phospho- (1 -*rac*glycerol) (sodium salt) (DMPG) and 1,2-dipalmitoyl-*sn*-glycero-3-phosphocholine (DPPC), which are either composed of DMPG (100%) or DPPC/DMPG (1:1). The liposomes (2 mM total lipid concentration) were prepared by the ethanolic injection method [58] above the transition temperature of each lipid, as previously described [21]. The compound (at an initial concentration of 6.7 × <sup>10</sup>−<sup>4</sup> M) was encapsulated by co-injection with the ethanolic lipid solution. The size, polydispersity, and zeta potential of compound-loaded liposomes were measured in a Litesizer 500 Dynamic Light Scattering apparatus from Anton Paar (Anton Paar GmbH, Graz, Austria).

The encapsulation efficiency (EE%) was determined as previously reported [21] and calculated through the Equation (1):

$$EE\% = \frac{(\mathbb{C}\_{total\ compound} - \mathbb{C}\_{free\ compound})}{\mathbb{C}\_{total\ compound}} \times 100\tag{1}$$

using a calibration curve of fluorescence intensity vs. concentration (C), taking advantage of the fluorescence emission of the compound.

The compound release was followed during 24 h at 20 ◦C and 35 ◦C, below and above the phase transition temperature of DMPG. The Weibull model (a distribution function) was used to study the transport mechanism involved in the compound release [36], being expressed in terms of the compound fraction accumulated (*m*) in solution at time *t* (Equation (2)):

$$m = 1 - \exp\left[\frac{-\left(t - T\_i\right)^b}{a}\right] \tag{2}$$

where *a* is a scale parameter that defines the timescale of the process, *Ti* represents the latency time of the release process (often being zero), and *b* is a formal parameter that characterizes the type of curve (*b* = 1 is exponential; *b* > 1 is sigmoid, with ascendant curvature delimited by an inflection point; and *b* < 1 is parabolic, displaying high initial slope and a consistent exponential character).

### **4. Conclusions**

A series of β-amino alcohols were prepared by reaction of eugenol epoxide with various aliphatic and aromatic amines. The obtained eugenol derivatives were subjected to biological activity evaluation in the *Sf9* cell line, in comparison with the corresponding precursors, in order to evaluate their application as potential natural based insecticides.

We identified that the three derivatives bearing a terminal benzene ring, either substituted or unsubstituted, were those showing higher potency, in some cases higher than the benchmark used. We further clarified that the molecules were eliciting their effect by triggering organized cell death, and they were selective for insect cells.

Inverted virtual screening studies with five independent methods suggest that these molecules display their insecticide activity most likely by targeting the insect acetylcholinesterase and/or the insect odorant binding proteins. Molecular dynamics simulations and free energy calculations confirm that these two molecules bind strongly to both targets forming very stable complexes with well-defined molecular interactions that are maintained through time.

Nanoencapsulation studies allow obtaining very reasonable encapsulation efficiencies and a controlled release. Liposomes of DMPG provide a temperature-sensitive compound release, which is more effective than the DPPC/DMPG (1:1) formulation.

**Supplementary Materials:** The following are available online, Figure S1: Protein and ligand RMSD (Å) of the AChE and OBP–ligand complexes, Figure S2: Percentage of the potential solvent accessible surface area of the ligands that is buried by the protein targets evaluated, Figure S3: Number of ligand–target hydrogen bonds formed during the simulations for compound **3d** and **3f** when complexed with AchE and OBP, Figure S4: Fitting of the release profiles to the Weibull model, Figure S5: Homology model built for 1QON, Table S1: Docking scores for compounds **3d** and **3f** in complex with human and insect AChE, Table S2: Parameters of the Weibull model for the release of compound **3f** from liposomes and corresponding coefficients of determination (R2),Table S3: List of targets selected for the inverted virtual screening study.

**Author Contributions:** Conceptualization, M.S.T.G.; A.G.F.; D.M.P.; S.F.S. and E.M.S.C.; methodology, A.G.F.; M.S.T.G.; D.M.P.; S.F.S. and E.M.S.C.; validation, A.G.F.; M.S.T.G.; D.M.P.; S.F.S. and E.M.S.C.; investigation, N.F.S.P.; R.B.P.; T.F.V.; M.J.G.F. and A.R.O.R.; writing—original draft preparation, A.G.F.; M.S.T.G.; D.M.P.; S.F.S.; E.M.S.C. and R.B.P.; writing—review and editing, M.S.T.G.; A.G.F.; D.M.P.; S.F.S.; E.M.S.C. and R.B.P.; supervision, A.G.F.; M.S.T.G.; D.M.P.; S.F.S. and E.M.S.C.; project administration, M.S.T.G. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by project PTDC/ASP-AGR/30154/2017 (POCI-01-0145-FEDER-030154) of the COMPETE 2020 program, co-financed by the FEDER and the European Union. The authors acknowledge also the Foundation for Science and Technology (FCT, Portugal) and FEDER-COMPETE-QREN-EU for financial support to the research centers CQ-UM (UID/QUI/00686/2020), CF-UM-UP (UIDB/04650/2020) and REQUIMTE (UIDB/50006/2020). The NMR spectrometer Bruker Avance III 400 is part of the National NMR Network and was purchased within the framework of the National Program for Scientific Re-equipment, contract REDE/1517/RMN/2005 with funds from POCI 2010 (FEDER) and FCT.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

**Sample Availability:** Not applicable.

### **References**


### *Article* **Toxicological Stability of** *Ocimum basilicum* **Essential Oil and Its Major Components in the Control of** *Sitophilus zeamais*

**Eridiane da Silva Moura 1, Lêda Rita D'Antonino Faroni 1,\*, Fernanda Fernandes Heleno 1,2 and Alessandra Aparecida Zinato Rodrigues 1,3**


**Abstract:** Essential oils (EOs) are widely recognized as efficient and safe alternatives for controlling pest insects in foods. However, there is a lack of studies evaluating the toxicological stability of botanical insecticides in stored grains in order to establish criteria of use and ensure your efficiency. The objective of this work was to evaluate the toxicological stability of basil essential oil (*O. basilicum*) and its linalool and estragole components for *Sitophilus zeamais* (Motschulsky) adults in corn grains by fumigation. The identification of the chemical compounds of the essential oil was performed with a gas chromatograph coupled to a mass selective detector. Mortality of insects was assessed after 24 h exposure. After storage for six (EO) and two months (linalool and estragole) under different conditions of temperature (5, 20, and 35 ◦C) and light (with and without exposure to light), its toxicological stability was evaluated. Studies revealed that the essential oil of *O. basilicum* and its main components exhibited insecticidal potential against adults of *S. zeamais*. For greater toxicological stability, suitable storage conditions for them include absence of light and temperatures equal to or less than 20 ◦C.

**Keywords:** storage; monoterpenes; bioinsecticide; insect pest; toxicity

### **1. Introduction**

Essential oils (EOs) are classified as secondary metabolites produced by various parts of the plant such as seeds, stems, leaves, and flowers. They are mixtures of volatile, natural substances, characterized by strong odor and, in most cases, have lipophilic constitution [1]. As they are composed of volatile terpenoids such as monoterpenes (C10) and sesquiterpenes (C15) and phenylpropenes (derived from the phenyl group junction (aromatic ring) and a three-carbon side chain (propyl group) [2], which usually originate from various biosynthesis pathways [3], there are a wide variety of possible applications of essential oils [4]. Among the current applications of EOs is their use as an alternative to synthetic insecticides, as EOs have great biocidal potential, presenting insect toxicity [5].

EOs and their compounds are believed to have a higher barrier to pest resistance and lower risk to human health and environmental contamination compared to conventional insecticides [6]. Among the essential oils with insecticidal activity is the essential oil of *Ocimum basilicum*, aromatic and medicinal plant of the Lamiaceae family [7], composed mainly of linalool and estragole [8,9].

The toxicity of *O. basilcium* essential oil has already been proven for *Acanthoscelides obtectus* (Coleoptera: Chrysomelidae) [10], *Rhyzopertha dominica* (Coleoptera: Bostrichidae) [11], *Sitophilus zeamais* (Coleoptera: Curculionidae) [12], *Tribolium castaneum* (Coleoptera: Tenebrionidae) [13], *Zabrotes subfasciatus* (Coleoptera: Chrysomelidae) [14], *Anopheles funestus* (Diptera: Culicidae) [15] and *Sitophilus Oryzae* (Coleoptera: Curculionidae) [16]. Because this essential

**Citation:** Moura, E.d.S.; Faroni, L.R.D.; Heleno, F.F.; Rodrigues, A.A.Z. Toxicological Stability of *Ocimum basilicum* Essential Oil and Its Major Components in the Control of *Sitophilus zeamais*. *Molecules* **2021**, *26*, 6483. https://doi.org/10.3390/ molecules26216483

Academic Editor: Giovanni Benelli

Received: 29 September 2021 Accepted: 25 October 2021 Published: 27 October 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

oil is mainly composed of linalool and estragole, its toxicity to stored grain pest insects can be explained by the action of mixed inhibition of the enzyme acetylcholinesterase (AChE) caused by such compounds, especially when they are applied by fumigation [17,18].

Linalool, the most well-known monoterpene of *O. basilicum* essential oil, is present in the essential oil of various medicinal plants, mainly of the Lamiaceae family [19]. It is insect repellent [20], inhibits the reproduction of *Acanthoscelides obtectus* (Say) (Coleoptera: Chrysomelidae) [21] and exhibits larvicidal activity against *Culex quinquefasciatus* and *Aedes stephensi* larvae (Diptera: Culicidae) [22,23].

Estragole, a volatile monoterpenoid ether found in numerous plants [24]. This component and its biotransformation products have toxic potential because they are genotoxic, mutagenic or carcinogenic [25]. Nevertheless, it was considered safe (GRAS—Generally Recognized As Safe) by FEMA (Flavor and Extract Manufacturer's Association, 2008) as it does not pose a risk to human health in small quantities (0.6 mg kg−<sup>1</sup> day−1). Estragole inhibits the growth of *Aedes aegipyti* (Diptera: Culicidae) larvae and has antiparasitic and antihelmintic actions [26]. This compound has reported potential insecticide for *Oryzaephilus surinamensis* (Coleoptera: Silvanidae), *Lasioderma serricorne* (Coleoptera: Anobiidae), *Liposcelis bostrychophila* (Psocoptera: Liposcelididae) and *Tribolium castaneum* (Coleoptera: Tenebrionidae) [27–31].

Given that *O. basilicum* EO and its major components linalool and estragole are toxic to stored grain pest insects and knowing that once deprived of the protective compartmentalization of the plant, essential oil constituents are especially prone to oxidative damage, chemical transformations, or polymerization through enzymatic or chemically triggered reactions by external factors such as temperature and light [32]. the objective of this work was to determine the toxicological stability of *O. basilicum* EO and its linalool and estragole components on fumigation *Sitophilus zeamais* in corn grains, after storage as a function of temperature and luminosity for a period of six (EO) and two months (linalool and estragol).

### **2. Material and Methods**

### *2.1. Insect Colony*

The insects were raised on maize grains with water content of 12.1% (wet basis) under constant conditions of temperature (25 ± 2 ◦C), relative humidity (70 ± 5%) and scotophase 24 h. For the creation were used 3 L glass vials, closed with perforated plastic lid and internally coated with organza to allow gas exchange.

### *2.2. Essential Oil*

The essential oil used in the research was acquired through the company Mundo dos Óleos (Brasília, DF, Brazil). 100% pure and natural oil extracted from *O. basilicum* leaves by steam distillation, obtained from selected raw material, to preserve the main properties of each extracted element, as well as enhance its flavor, color, and aroma characteristics. All the essential oil used in the research was acquired on the same date, thus belonging to the same manufacturing batch, in order to avoid interference in the research due to compositional variability.

### *2.3. Essential Oil Analysis*

The analysis of the chemical composition of the essential oil was performed at the Department of Chemistry of the Federal University of Viçosa in Viçosa, Minas Gerais, Brazil. *O. basilicum* essential oil was analyzed by mass spectrometry coupled gas chromatography (GC-MS) on a QP2010 model equipment (Shimadzu, Tokyo, Japan) under the following conditions: fused silica capillary column (30 m in length) and 0.25 mm internal diameter) with RTX®-5MS stationary phase (0.25 μm film thickness) and helium as a carrier gas with a flow rate of 1.0 mL/min. Injector temperature of 220 ◦C, the initial column temperature was 60 ◦C, with programming to increase by 2 ◦C until reaching a temperature of 200 ◦C, and 5 ◦C until reaching a maximum temperature of 250 ◦C. Mass spectra were obtained by electron impact at 70 eV, with 29 to 400 (*m*/*z*) scan. 1 μL of the prepared oil solution was

injected at a concentration of 10 mg mL−<sup>1</sup> with a split ratio of 1:20. The main constituents were identified and quantified by their retention index (IR) relative to the hydrocarbon standard (C7–C30) (99%, Supelco, Bellefonte, PA, USA) and confirmed by comparing the mass spectrum of the compounds with the NIST 14 spectrotheque.

### *2.4. Exposure to Temperature and Light Radiation*

For the evaluation of the effect of temperature on the stability of the essential oil and its major compounds, clear glass containers wrapped in foil and properly sealed, 20 mL of *O. basilicum* essential oil and 1 mL of each compound were under different temperature conditions. The flasks were divided into three lots and packaged for six months for EO and two months for linalool and estragole, in the following environments: refrigerator at 5.0 ± 1 ◦C (low temperature); in an incubator chamber (model 347, CD, Fanem, São Paulo, SP, Brazil) at a temperature of 20 ± 2 ◦C (average temperature) and an incubator chamber at a temperature of 35 ± 2 ◦C (high temperature).

For the evaluation of light stability, clear glass vials containing the essential oil and its linalool and estragole compounds were kept in a B.O.D. (model 347 CD, Fanem, São Paulo, SP, Brazil) at a temperature of 20 ± 2 ◦C and subjected to light from cold white lamps (100 W each) (Philips, São Paulo, SP, Brazil) for six months for EO and two months for linalool and estragole.

### *2.5. Toxicological Stability*

The fumigation bioassays were performed in 0.8 L (8 cm diameter × 15 cm high) glass vials with 50 non-sexed *S. zeamais* adults, in four replications. The concentrations of *O. basilicum* essential oil stored under different conditions ranged from 8 to 40 μL L−<sup>1</sup> of air. Working solutions of the essential oil were prepared with toluene solvent (Sigma-Aldrich, 99.9%, Baden-Württemberg, Germany) and applied with a microsyringe (Hamilton, Reno, NV, USA) on 4 mm diameter paper filter discs. 4 cm placed in Petri dishes (6.5 cm diameter). Petri dishes were covered with organza type tissue and placed at the base of the flasks. Pure solvent (toluene) was used as a control. The vials were sealed with a screw-on metal cap and sealed with parafilm (PM996, American, NV, USA) after insect distribution to prevent oil vapor leakage during the exposure period. The flasks were kept in an incubator chamber at a temperature of 27 ± 2 ◦C for 24 h. After this period, dead and living insects were counted. Corrected mortality was calculated by Abbott's formula [33].

Pure linalool and estragole were purchased from Sigma-Aldrich (Burlington, MA, USA). Toxicity assays were performed at concentrations ranging from 8 to 40 μL L<sup>−</sup>1. Each filter paper disc (4.4 cm) was treated with 25 μL of toluene diluted linalool and estragole solution and placed in a Petri dish (6.5 cm in diameter), covered with organza and inserted into the base of glass pots with a capacity of 0.8 L. A total of 50 non-sexed adults were placed by pot to expose the insects to the fumigant activity of the compounds for 24 h. Each treatment consisted of four repetitions. As a control 25 μL of pure toluene was used.

### *2.6. Statistical Analysis*

Toxicity data were subjected to probit analysis using SAS software (SAS Institute, Cary, NC, USA), generating concentration-mortality curves. Mortality data were submitted to ANOVA and Tukey test with Statistica 8 software (StatSoft Inc., Tulsa, OK, USA).

### **3. Results and Discussion**

### *3.1. Essential Oil Composition*

The relative chemical composition of the essential oil compounds of *O. basilicum* leaves were performed by GC-MS. The major constituents were identified by their retention index (RI) relative to a homologous series of n-alkanes and confirmed by comparing the mass spectrum of the compounds with the NIST 14 spectrotheque. Chromatographic analysis showed that estragole (H2C=CHCH2C6H4OCH3) and linalool ((CH3) 2C=CHCH2CH2C

(CH3)(OH)CH=CH2) were the major components of *O. basilicum* essential oil (Figure 1), representing 85% and 12% of the identified compounds, respectively.

**Figure 1.** Chromatogram of *Ocimum basilicum* essential oil (10 mg mL−<sup>1</sup> in toluene).

These compounds are responsible for most of the composition of this essential oil [8,9]. Generally, linalool and estragole are the major components of *O. basilicum* EO, but factors such as soil type, altitude, temperature, insolation period, cultivation, drying conditions and storage influence its composition [31], explaining variations in the amount of the compounds.

### *3.2. Toxicological Stability of Essential Oil*

The Probit model was adequate for the concentration-mortality data of all fumigation treatments, based on the low χ<sup>2</sup> value and the high *p* value obtained from the *O. basilicum* essential oil curves stored under different conditions and their components. linalool and estragole over *S. zeamais*. For the untreated essential oil, the values of χ<sup>2</sup> = 0.14 and *p* = 0.98 were obtained. Lethal concentrations to cause 50 and 95% insect mortality (LC50 and LC95) were 25.4 μL L−<sup>1</sup> and 178.4 μL L−<sup>1</sup> of air, respectively (Table 1). The slope of the curve was (1.94 ± 0.37), which indicates genetic homogeneity among individuals of the *S. zeamais* population.

**Table 1.** Lethal concentrations of *Ocimum basilicum* essential oil stored under different conditions and their major components for fumigation *Sitophilus zeamais*.


LC = Lethal Concentration (μL L−<sup>1</sup> of air); FI = Fiducial Interval; MSE <sup>1</sup> = Mean square error; χ<sup>2</sup> = Chi square; *p* = Probability; df = degrees of freedom; EO = Essential oil.

> *O. basilicum* EO was lethal to *S. zeamais* by fumigation [34], but the effectiveness of essential oils depends on factors such as dose or concentration, insect species, application surface, penetration pathway, method of application and composition of oil, season, ecological conditions, method and extraction time, plant part and storage conditions [35,36]. Both the untreated EO and the EO stored under different temperatures (5 and 20 ◦C) and exposed to light for a period of six months had lower LC50 when applied by fumigation.

> The LC50 value of untreated *O. basilicum* essential oil when applied by fumigation (25.4 μL L−<sup>1</sup> air) was lower than that of *Minthostachys verticillata* (28.2 μL L−<sup>1</sup> air) and *Eucalyptus globulus* essential oil (335.7 μL L−<sup>1</sup> of air) [37] and higher than *Melaleuca al-*

*ternifolia* essential oil (7.7 μL L−<sup>1</sup> of air) for *S. zeamais* [38]. The fumigant activity of *O. basilicum* EO on *S. zeamais* can be explained by the fact that monoterpenoids inhibit the acetylcholinestrase (AChE) nerve conduction enzyme [37]. In addition, studies have shown that essential oils can significantly inhibit the activity of two detoxifying enzymes in *S. zeamais*, glutathione S-transferase (GST) and carboxylesterase (CarE), as well as negatively regulating differentially expressed genes (DEGs) in response to fumigation [37].

*O. basilicum* EO caused higher mortality of *S. zeamais* when compared to the negative control, showing that it has higher fumigant activity (Figure 2). The *O. basilicum* EO stored at 5 to 20 ◦C and without storage, differed statistically from each other in only three concentrations (8; 16 and 40 μL L−<sup>1</sup> of air), indicating that temperatures up to 20 ◦C do not interfere significantly on the toxicological stability of EO for *S. zeamais* adults when stored for six months. When comparing the EO without storage and EO stored at 35 ◦C and in light exposure, there was a statistical difference in all concentrations (Figure 2). The EO stored at 35 ◦C was more stable than the EO stored in light exposure, causing higher mortality of *S. zeamais* adults, which shows that light exposure decreases the toxicity of *O. basilicum* EO on *S. zeamais*.

**Figure 2.** Toxicological stability by fumigation of *Ocimum basilicum* essential oil stored under different conditions and their major components for *Sitophilus zeamais*. Means followed by the same letter in the column do not differ at 5% probability by Tukey test.

When comparing the untreated EO and the EO stored at 35 ◦C in the fumigation applications, there was a statistical difference in all concentrations (Figures 2 and 3). This indicates that storage at high temperatures for a period of six months affects its toxicological stability on *S. zeamais* adults. The temperature plays a crucial role in the degradation process of essential oils, which directly affects their stability. This decisively influences the stability of the essential oil in several respects [38]. Generally, chemical reactions accelerate with increasing heat due to temperature dependence of the reaction rate, as expressed by the Arrhenius equation [39]. Based on this, Van't Hoff's law states that a temperature increase of 10 ◦C doubles chemical reaction rates, a ratio that can be consulted to predict stability at different temperatures [40].

**Figure 3.** Toxicological stability by fumigation of linalool stored under different conditions for *Sitophilus zeamais*. Means followed by the same letter in the column do not differ at 5% probability by Tukey test.

Increasing temperature advances the self-oxidation and decomposition processes of hydroperoxides, as heat can contribute to free radical formation [41]. Essential oils vary in their susceptibility to self-oxidation at different storage temperatures. In general, monitoring of volatile plant extracts and essential oil composition demonstrates that stability decreases with prolonged storage time, as well as a temperature rise from 0 to 28 ◦C [42], 4 to 25 ◦C [43] and 23 to 38 ◦C [44].

There was a statistical difference between the untreated EO and the EO stored in light exposure at all concentrations (Figures 2 and 3). This indicates that six-month storage in light exposure affects its toxicological stability in *S. zeamais* adults. This is possibly due to the presence of ultraviolet light (UV) and visible light (Vis) being responsible for accelerating the self-oxidation processes in the essential oils, triggering what results in free radical formation [44]. Auto-oxidation involves a succession of chemical reactions that alter the initial composition of the oil, leading to the production of low molecular weight compounds and oxidized polymers, as well as the destruction of important fatty acids and the formation of other compounds, compromising their stability [42].

Comparison of EO stored at 35 ◦C and in light exposure shows that the toxicological stability of *O. basilicum* EO over *S. zeamais* was most affected by storage in light exposure. The light is much more important than temperature in the oxidation of essential oils [42], although the effect of light on oil oxidation is lessened with increasing temperature [45]. The effect of sunlight for 2 h caused degradation of the quality of ginger oil, while it remained stable when stored in the dark for the same period of time [46].

Processing and storage of oils in exposure to light can lead to the generation of a wide range of undesirable compounds, some of which are harmful to health because of their high toxicity, thereby altering their stability [47]. Among the components of essential oils, monoterpenes have been shown to degrade rapidly under the influence of visible light [48]. The same study also showed that there were transformation reactions in marjoram oil during storage under visible light, which led to the formation of several unidentified elements and smaller components.

### *3.3. Toxicological Stability of Linalool and Estragole*

The Probit model was adequate for concentration-mortality data, based on the low χ<sup>2</sup> values and the high *p* values obtained on the linalool and estragole curve stored under different conditions over *S. zeamais*. For untreated linalool, the values of χ<sup>2</sup> = 5.57 and *p* = 0.34 were obtained. Lethal concentrations to cause 50% and 95% insect mortality (LC50 and LC95) were 34.6 μL L−<sup>1</sup> and 330.3 μL L−<sup>1</sup> of air, respectively (Table 2). The slope of the curve was (1.67 ± 0.22), which indicates genetic homogeneity among individuals of the *S. zeamais* population.



LC = Lethal Concentration (μL L−<sup>1</sup> of air); FI = Fiducial Interval; MSE <sup>1</sup> = Mean square error; χ<sup>2</sup> = Chi square; *p* = Probability; df = degrees of freedom.

> Linalool caused higher mortality of *S. zeamais* adults when compared to the negative control, showing that it has higher fumigant activity (Figure 3). Studies have shown that essential oil components are able to inhibit cellular respiration enzymes, nervous system enzymes such as acetylcholinesterase (AChE), and detoxification system enzymes such as P450 and esterase [49], which weakens the insecticide metabolism in insects. Linalol acts together with other compounds in the cholinergic system of insects, promoting the rapid breakdown of the nervous system [50]. Linalool stored at 5 to 20 ◦C and without storage differed statistically from each other in only two concentrations (36 and 40 μL L−<sup>1</sup> of air), indicating that temperatures up to 20 ◦C do not significantly affect its toxicological stability for adults of *S. zeamais* during storage for two months. When comparing non-stored linalool and linalool stored at 35 ◦C and in light exposure, there was a statistical difference in all concentrations except one (8 μL L−<sup>1</sup> of air) (Figure 3). Linalool stored at 35 ◦C was more stable than linalool stored in light exposure, causing higher mortality of *S. zeamais* adults, which shows that light exposure is more detrimental to linalool stability than temperature increase.

> For untreated estragole, the values of χ<sup>2</sup> = 5.48 and *p* = 0.35 were obtained. Lethal concentrations to cause 50% and 95% insect mortality (LC50 and LC95) were 38.13 μL L−<sup>1</sup> and 314.01 <sup>μ</sup>L L−<sup>1</sup> of air, respectively (Table 3). The slope of the curve was (1.79 ± 0.22), which indicates genetic homogeneity among individuals of the *S. zeamais* population.



LC = Lethal Concentration (μL L−<sup>1</sup> of air); FI = Fiducial Interval; MSE <sup>1</sup> = Mean square error; χ<sup>2</sup> = Chi square; *p* = Probability; df = degrees of freedom.

> Estragole caused higher mortality of *S. zeamais* adults when compared to the negative control, showing that it has higher fumigant activity (Figure 4). Estragole stored at 5 to 20 ◦C and without storage differed statistically from only one concentration (32 μL L−<sup>1</sup> of air), indicating that temperatures up to 20 ◦C do not significantly affect its toxicological stability for adults of *S. zeamais* during storage for two months. Comparing estragole

without storage and linalool stored at 35 ◦C and in light exposure, there was a statistical difference in all concentrations, indicating that the increase of storage temperature decreases the toxicity of estragol for adults of *S. zeamais* (Figure 4). Estragole stored at 35 ◦C differed statistically from estragole stored in light exposure by only one concentration (16 μL L−<sup>1</sup> of air), which shows that both treatments decrease the toxicity of estragole for *S. zeamais* adults to the same extent.

**Figure 4.** Toxicological stability by estragole fumigation stored under different conditions for *Sitophilus zeamais*. Means followed by the same letter in the column do not differ at 5% probability by Tukey test.

Linalool LC50 and LC95 increased from 34.56 to 132.4 μL L−<sup>1</sup> of air and from 330.3 to 495.6 μL L−<sup>1</sup> of air respectively when stored at 35 ◦C for two months. The same occurred with estragol LC50 and LC95 which increased from 38.1 to 56.5 μL L−<sup>1</sup> of air and from 314.0 to 395.2 μL L−<sup>1</sup> of air, respectively. This shows that storage at 35 ◦C decreases the toxicity of this compound for *S. zeamais* adults. This can be explained by the fact that temperature directly influences the stability of volatile compounds [41]. Generally, chemical reactions accelerate with increasing heat due to temperature dependence of the reaction rate, as expressed by the Arrhenius equation [42]. Terpenoids, especially terpenes and aldehydes, are known to be susceptible to rearrangement processes at elevated temperatures. Terpenic conversion reactions by heating have been reported for both isolated compounds [51,52] and for essential oils [53]. Mircene, for example, suffered degradation as it was exposed to higher storage temperatures for 120 days. The initial percentage of mircene fell from 17.38% (6 ◦C) to 3.99% at 37.5 ◦C [54].

In this work, the comparison between linalool stored at 35 ◦C and stored at 20 ◦C in light exposure shows that it was more toxicologically stable on *S. zeamais* when exposed to 35 ◦C, indicating that light exposure is more detrimental to the stability of linalool than increase in temperature. This is because some monoterpenes are more thermodynamically stable while others demonstrate rapid degradation under the influence of visible light [50]. Linalol, for example, is 5.9 kJ mol <sup>−</sup><sup>1</sup> more stable than geraniol [55].

In contrast to linalool, the toxicological stability of estragole on *S. zeamais* was more affected by increased storage temperature (35 ◦C) than by exposure to light. This can occur due to the evaporation process of low boiling compounds, mainly hydrocarbons and sesquiterpenes [56].

There was no significant difference between linalol and estragole stored at 5 and 20 ◦C, which shows that they can be stored in this temperature range for two months without decreasing their toxicological stability on *S. zeamais*. Three temperatures (4 ◦C in a cold room, −20 ◦C in a freezer and 25 ◦C at room temperature) were used to assess the stability of *Thymus daenensis* essential oil for three months [57]. The results indicated that at room temperature, the amounts of thymol and carvacrol increased considerably by 26.6% and 23% after 3 months, respectively. The increase in thymol and carvacrol by storage at room temperature represents an increase in oil quality index. In addition, oil compositions exhibited the smallest changes and maintained primary quality when stored at low temperatures, particularly at 20 ◦C [57].

### **4. Conclusions**

The essential oil of *O. basilicum* and its linalool and estragole components components exhibited insecticidal potential against *S. zeamais* adults in corn grains by fumigation. Increasing temperature (35 ◦C) and exposure to light during storage negatively affects the stability of *O. basilicum* EO, reducing its toxicity against *S. zeamais*. Aiming at the higher toxicity of *O. basilicum* EO to *S. zeamais*, the storage conditions suitable for it are at temperatures of maximum 20 ◦C and without exposure to light.

**Author Contributions:** E.d.S.M. performed the experiments, collected the data and wrote the manuscript. L.R.D.F. conceived the idea, supervised the work and obtained funding for the research. F.F.H. analyzed the data and participated in the writing of the manuscript. A.A.Z.R. participated in the CG-MS analysis of the essential oil end edited the final version of the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Brazil (CAPES)—Finance Code 001. The APC was funded by "Graduate Program in Agricultural Engineering" of Universidade Federal de Viçosa (UFV)—PPGEA-UFV.

**Acknowledgments:** The authors are grateful to the Department of Chemistry and Department of Agricultural Engineering of the Federal University of Viçosa (Brazil) for support and research facilities. The authors would like to thank the Brazilian Agencies: Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). Also, this study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior–Brazil (CAPES)–Finance Code 001.

**Conflicts of Interest:** The authors declare that they have no conflict of interest.

**Sample Availability:** Samples of the compounds are not available from the authors.

### **References**


### *Article Zuccagnia punctata* **Cav. Essential Oil into Poly(**ε**-caprolactone) Matrices as a Sustainable and Environmentally Friendly Strategy Biorepellent against** *Triatoma infestans* **(Klug) (Hemiptera, Reduviidae)**

**Sandra López 1, Alejandro Tapia 1,\*, Julio Zygadlo 2, Raúl Stariolo 3, Gustavo A. Abraham 4and Pablo R. Cortez Tornello 4,\***


**Abstract:** The main strategies against *Triatoma infestans* (primary vector responsible for the Chagas disease transmission) are the elimination or reduction of its abundance in homes through the application of insecticides or repellents with residual power, and environmental management through the improvement of housing. The use of plant-derived compounds as a source of therapeutic agents (i.e., essential oils from aromatic plants and their components) is a valuable alternative to conventional insecticides and repellents. Essential oil-based insect repellents are environmentally friendly and provide reliable personal protection against the bites of mosquitoes and other blood-sucking insects. This study investigates, for the first time to our knowledge, the potential repellent activity of *Zuccagnia punctata* essential oil (ZEO) and poly(ε-caprolactone) matrices loaded with ZEO (ZEOP) prepared by solvent casting. The analysis of its essential oil from aerial parts by GC–FID and GC-MS, MS allowed the identification of 25 constituents representing 99.5% of the composition. The main components of the oil were identified as (−)-5,6-dehydrocamphor (62.4%), alpha-pinene (9.1%), thuja-2, 4 (10)-diene (4.6%) and dihydroeugenol (4.5%). ZEOP matrices were homogeneous and opaque, with thickness of 800 ± 140 μm and encapsulation efficiency values above 98%. ZEO and ZEOP at the lowest dose (0.5% wt./wt., 96 h) showed a repellency of 33 and 73% respectively, while at the highest dose (1% wt./wt., 96 h) exhibited a repellent activity of 40 and 66 %, respectively. On the other hand, until 72 h, ZEO showed a strong repellent activity against *T. infestans* (88% repellency average; Class V) to both concentrations, compared with positive control N-N diethyl-3-methylbenzamide (DEET). The essential oils from the Andean flora have shown an excellent repellent activity, highlighting the repellent activity of *Zuccagnia punctata*. The effectiveness of ZEO was extended by its incorporation in polymeric systems and could have a potential home or peridomiciliary use, which might help prevent, or at least reduce, Chagas' disease transmission.

**Keywords:** Chagas' disease transmission; triatomines; peridomiciliary use; Argentina

### **1. Introduction**

Chagas disease is an extraordinarily complex zoonosis that is present throughout the territory of South America, Central America, and Mexico, and continues to represent a serious threat to the health of the countries of the region. This illness affects 6 to 7 million people around the world [1]. The main strategies used to interrupt vector transmission of

**Citation:** López, S.; Tapia, A.; Zygadlo, J.; Stariolo, R.; Abraham, G.A.; Cortez Tornello, P.R. *Zuccagnia punctata* Cav. Essential Oil into Poly(ε-caprolactone) Matrices as a Sustainable and Environmentally Friendly Strategy Biorepellent against *Triatoma infestans* (Klug) (Hemiptera, Reduviidae). *Molecules* **2021**, *26*, 4056. https://doi.org/10.3390/ molecules26134056

Academic Editor: Giovanni Benelli

Received: 19 May 2021 Accepted: 28 June 2021 Published: 2 July 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

*Trypanosoma cruzi* by *Triatoma infestans* (primary responsible for the Chagas disease transmission) are the elimination or reduction of its abundance in homes through the application of insecticides with residual power, and environmental management the improvement of housing [1].

Pyrethroid insecticides have been used for over 20 years to control the vectors of Chagas disease in Argentina and other Latin American countries [2]. From them, deltamethrin has been intensively used for the chemical control of *T. infestans* "vinchucas" and in general has displayed an effect as a highly effective triatomicide [3]. In 2002, the health authorities for the control of vectors in Argentina reported failures in the chemical control of *T. infestans* associated with different levels of resistance to pyrethroids [4–6]. This fact could be caused by the rapid degradation of the active compound, as shown in several studies [7].

To overcome these problems, active agents have been incorporated in polymeric systems allowing their protection and sustained release. Polymer-based systems have shown a longer lasting effect than traditional suspension concentrate formulations, both under experimental and field conditions [8]. Nanofibrous mats containing citriodiol as biorepellent against *Aedes aegypti* mosquitoes and its incorporation into a layered fabric were recently studied [9]. Monolithic and core-enriched nanofibrous mats with repellent activity were successfully obtained, and core-enriched mats displayed a 100% of repellency for 34 days. Moreover, compounds of botanical origin such as the essential oils from aromatic plants and their components provide an alternative to conventional insecticides and repellents. Essential oil-based insect repellents are environmentally friendly and provide dependable personal protection against the bites of mosquitoes and other bloodsucking insects [10]. On the other hand, the essential oils have the advantage of being agents of low toxicity in mammals, little residual life in the environment, and fewer requirements imposed by the legal framework, because they enjoy social acceptance due to the widespread use of aromatic species [11].

The essential oils from the Andean flora growing in the province of San Juan located in the center-west of the Argentine have shown an excellent repellent activity repellents to *Triatoma infestans* (Klug) (Hemiptera, Reduviidae), the vector of Chagas disease, since they constitute a rich source of bioactive compounds that are biodegradable into nontoxic products [12–14], effect that could be enhanced by incorporating them into polymeric systems, which are considered a suitable strategy for time and distribution-controlled repellent delivery [13]. The use of repellents against *T. infestans* vectors might help prevent, or at least reduce, Chagas' disease transmission. The resinous species including the genus *Larrea* in Argentina (*Larrea ameghinoi*, *L. cuneifolia*, *L. divaricata,* and *L. nitida*), vernacular name "jarillas" and *Zuccagnia punctata*, commonly called "jarilla macho" are used extensively in traditional medicine in Argentina Andean communities for the treatment of injuries and bruises, and a good disinfectant of wounds, repellent of insects, for roof construction in rural areas and as a vegetable fuel for cooking food. [15,16].

On the other hand, poly(ε-caprolactone) (PCL) is a well-known aliphatic biocompatible polyester with a glass transition temperature at −60 ◦C and melting temperature between 59–64 ◦C. Its semicrystalline structure and hydrophobic character allow PCL to exhibit a long degradation time under humidity or physiological conditions of around 2 years. This is an attractive property for long-term applications in bioactive agent delivery [17–20]. There are reports from Peres et al. in which they propose the encapsulation of essential oil of fruit and leaves of *Xylopia aromatica* in PCL nanoparticles [21]. The nanoencapsulation of these bioactive compounds promotes their protection from environmental degradation and prolongs their biological activity. De Ávila et al. reported the preparation of PCL microparticles with encapsulated citronella oil through an emulsion technique followed by solvent evaporation [22]. Akolade et al. reported the microencapsulation of eucalyptol in poly(ethylene glycol) and PCL using particles from gas-saturated solutions [23]. Unalan et al. reported the fabrication and characterization of various concentrations of peppermint essential oil (PEP) loaded on PCL electrospun fiber mats for wound healing applications, where PEP was intended to impart antibacterial activity to the fibers [24].

This study investigated for the first time the potential repellent activity of *Zuccagnia punctata* essential oil and its incorporation in PCL matrices for increasing the duration of the repellent activity.

### **2. Results and Discussion**

### *2.1. Essential Oil Composition, Yield, and Spectroscopy Characterization*

The essential oil yield was 0.25% (v/wt.); δ25: 0.96 g/mL. Regarding the chemical profile, a total of 25 compounds amounting 99.5% of the oil were identified according [25–27]. The main constituents are showed in Table 1 and include (−)-5,6-dehydrocamphor (62.4%), alpha-pinene (9.1%), thuja-2,4(10)-diene (4.6%), terpinen-4-ol (4.4%), verbenone (3.1%) and dihydroeugenol (4.5%). The monoterpenes represented the main portion of the oil accounting for 89.2% with a high percentage of oxygenated monoterpenes (69.9%). Hydrocarbon sesquiterpenes accounted for 4.7 %. Among them, the most abundant was epi-beta-santalene (2.1%). Epi-alpha-cadinol was the oxygenated sesquiterpenoid detected (0.7%).


**Table 1.** Chemical composition of the *Zuccagnia punctata* essential oil.

Constituents listed in order of increasing retention indices (RI). Unidentified components less than 0.1% are not reported. Temperature-programmed RI referred to n-alkanes, determined on a HP-5MS capillary column. Percentage values less than 0.1% are denoted as t (traces). Method of identification of minor constituents: 1corresponds to comparison of

GC-MS data and RI with those of the volatile oil ADAMS, Wiley and NBS computer mass libraries, 2corresponds to comparison of GC-MS data and RI with those of authentic samples.

The chemical composition as well as the antifungal activity of the *Zuccagnia punctata* essential oil collected in the province of San Juan have been previously reported [27], standing out the presence of (−)-5,6-dehydrocamphor (56.5%), linalool (14.5%) and cislinalool oxide THF (3.4%). The chemical composition of the essential oil reported here shows also that the main component is (−)-5,6-dehydrocamphor (62.4%), with some differences in minor components. The chemical composition is genetically determined (intrinsic factors) and on the other hand, environmental conditions (extrinsic factors) may be responsible for significant variations in the chemical composition of plants [28]. Essential oils can qualitatively and quantitatively change their chemical composition due to climatic factors, the composition of the soil, the plant organ, age, seasonality, and the phase of the circadian cycle [29–31].

### *2.2. Morphological Characterization*

The Figure 1 shows a disc of 10 mm cut from the ZEOP matrix and the SEM image of ZEOP 1% sample. ZEOP matrices were homogeneous and opaque with thickness of 800 ± 140 μm, as measured with a low force caliper. The SEM micrograph exhibited a characteristic morphology of PCL matrices prepared by solvent casting. The surface porosity could favor the evaporation of essential oils from the polymer matrix regions with dispersed oil.

**Figure 1.** Morphological characterization, (**A**) Optical image of ZEOP 1% disc 10 mm, (**B**) SEM micrograph of ZEOP 1% (1000×).

### *2.3. Thermal Properties and Crystallinity*

The Figure 2 shows the DSC thermograms. PCL pellets exhibited a characteristic thermogram with a melting temperature of 65.6 ◦C and crystallinity of 59.3%. PCL matrices showed a decrease in the melting point (Tm = 62 ◦C) and slight increase in crystallinity (63.8%) which can be attributed to the matrix formation during solvent casting. The incorporation of ZEO led to a decrease in Tm values with the increase in the oil content (ZEOP 0.5%, 61.4 ◦C and ZEOP 1%, 59.0 ◦C). This phenomenon agrees with the decrease in the crystallinity degree (ZEOP 0.5%, 61%, and ZEOP 1%, 59.6%), and it could be ascribed to the incorporation of oil, which makes difficult the crystallization process during the solvent evaporation. The ZEO thermogram did not show thermal events in the explored temperature by subheadings. It should provide a concise and precise description of the experimental results, their interpretation, as well as the experimental conclusions that can be drawn.

**Figure 2.** Differential scanning calorimetry thermograms corresponding to PCL, PCL matrix (PCLm), ZEO, ZEOP 0.5 % and ZEOP 1% formulations.

TGA curves of ZEO are shown in Figure 3. The results indicated that the thermal behavior of *Zuccagnia punctata* is simple and present only one thermal process. A continuous weight loss starting at 62.6 ◦C and continued until 358.2 ◦C. These values indicate that ZEO decomposition with temperature begins above room temperature, and therefore it is stable at the temperature of use of ZEOP matrices.

**Figure 3.** Thermal stability of ZEO: thermogravimetric and first-derivative of TGA curves.

### *2.4. ZEO Loading Capacity and Encapsulation Efficiency*

Table 2 shows the ZEO content in ZEOP matrices per mass unit of sample (Mc), loading capacity (LC), and encapsulation efficiency (EE). LC values of ZEOP 0.5% (0.48%), and ZEOP 1% (0.97%) matrices showed that ZEO content were slightly lower than the amount present in the polymeric solution. On the other hand, EE values showed encapsulation of ZEO above 98%. These values were consistent with those reported by Peres et al. [21] for PCL nanoparticles containing the essential oils of *Xylopia* (95%), and higher than the obtained values for PCL microparticles loaded eucalyptol (77 %) [23] and PCL fibers with peppermint oil (37 %) [24]. These results indicate that PCL is a suitable polymer for ZEO encapsulation.


**Table 2.** ZEO content incorporated in ZEOP matrices per mass unit of sample (Mc), loading capacity (LC), and encapsulation efficiency (EE).

*2.5. Repellent Activity against Triatoma Infestans Nymphs*

The results of the assay of repellency of the ZEO and ZEOP are shown in Tables 3 and 4.

**Table 3.** Repellent activity of ZEO, and ZEOP against *T. infestans* nymphs fifth instars (mean ± SD, *n* = 5) at 0.5% (wt./wt.).


1) Average value of repellency in the three times. 2) Repellence class according to scale: Class 0 (0.01 to 0.01%), Class I (0.1 to 20%), Class II (20.1 to 40%), Class III (40.1 to 60%), Class IV (60.1 to 80%), and Class V (80.1 to 100). a,b indicate significant difference at the 0.05 level according to Tukey test. 3) Blank control acetone. 4) Positive control DEET at 0.5% (wt./v).

**Table 4.** Repellent activity of ZEO, and ZEOP against *T. infestans* nymphs fifth instars, the vector of Chagas disease (mean ± SD, *n* = 5) at 1.0% (wt./wt.).


1) Average value of repellency in the three times. 2) Repellence class according to scale: Class 0 (0.01 to 0.01%), Class I (0.1 to 20%), Class II (20.1 to 40%), Class III (40.1 to 60%), Class IV (60.1 to 80%), and Class V (80.1 to 100%). a,b,c indicate significant difference at the 0.05 level according to Tukey test. 3) Blank control acetone. 4) Positive control DEET at 0.5% (wt./v).

> The ZEO showed excellent repellent properties on *T. infestans* between 1 and 72 h, for the two concentrations of the oil tested (Tables 3 and 4). The percentage of repellence did not change significantly with time (no effect within subjects, *p* > 0.05), and no significant relationship was observed between time points and oil treatment (*p* > 0.05). The essential oil was Class V, which is the one with the highest repellency according to the methodology used; the mean values obtained were 88.3 and 88.8%, for concentrations of 0.5 and 1% (wt./wt.), respectively. For both concentrations, the repellent activity decays between 72 and 96 h until a Class II repellent activity. This short-term action regarding the duration of the effect may be what limits the use of insect repellent products based on essential oils, according to previous reports, it may be related to the rapid volatilization and short time of

action [32]. On the other hand, significant differences in average percentage of repellence were observed between the ZEO treatment and blank control (effects between subjects, *p* < 0.05).

In a previous report, the chemical composition, anti-insect, and antimicrobial activity of *Baccharis darwinii* essential oil from Argentina, Patagonia were reported. The major components with recognized anti-insect and antimicrobial activity were identified, including limonene (47.1%), thymol (8.1%) and, 4-terpinelol (6.4%). The in vitro evaluation of the anti-insect properties showed promising insecticidal activity against *Ceratitis capitata* (LD50 19.9–31.0 g/fly for males and females respectively at 72 h) and repellent activity against *T. infestans* (average repellence 92%, Class V) [33]. The potential of the Andean medicinal flora of Argentina as a source of essential oils with repellent activity has been reported in the last decade, together with the chemical profile of volatile compounds [12–14].

Regarding ZEOP, the repellent activity showed a low activity during the first 24 h (Class III) and it was growing until 96 h (Class IV) to both concentrations assayed (Tables 3 and 4). On the other hand, significant differences in average percentage of repellence were observed between the ZEOP treatment and blank control (effects between subjects, *p* < 0.05).

Nanoproducts developed using natural products have been highlighted as ecologically and economically sustainable alternatives for effective control of crop pest and other vectors of human incidence such as mosquitoes and triatomines. The strong activity of limonene and β-pinene against *Tribolium castaneum* has been informed; however, the high volatility and hydrophobicity hinder the use of these monoterpenes as a large-scale pest control agent [34].Recently, has been reported the nano-emulsification of monoterpenes and essential oil allowed their incorporation into an aqueous matrix without losing its repellent activities [34], which gives support to the results reported here.The controlled release systems for repellents comprise polymer micro/nanocapsules, micro/solid lipid nanoparticles, nanoemulsions/microemulsions, liposomes/niosomes, nanostructured hydrogels and cyclodextrins [35]. There are many formulations based on micro and nanocapsules containing DEET and essential oils to increase repellent action time duration and decrease permeation and consequently, systemic toxicity [36]. Limonene essential oil successfully encapsulated in microcapsules of chitosan showed a slow and prolonged liberation profile by volatilization [36]. The ZEOP has also shown a slow and prolonged release during 96 h.

The oil from *Z. punctata*, one of the endemic resinous species in Argentina that is extensively used in the traditional medicine of Argentina and Andean people for various purposes, has shown significant potential as a biorepellent against the vector of Chagas disease. Repellent activity is prolonged significantly if the oil is supported in a polymeric system.

### **3. Materials and Methods**

### *3.1. Chemicals*

All solvents used were of analytical grade. Chloroform was purchased from Fisher (Walham, MA, USA); acetone and methanol (MeOH) grade UHPLC from J.T. Baker (Phillipsburg, NJ, USA) and dichloromethane (DCM) from Aldrich Chemical Co. (St. Louis, MO, USA). Poly(ε-caprolactone) (PCL, Mw 80000 g/mol) and *N,N*-diethyl-3-methylbenzamide (DEET) were purchased from Aldrich Chemical Co. (USA). Ultra-pure water (<5 μg/L) was obtained from a purification system Arium 61316-RO plus Arium 611 UV (Sartorius, Göttingen, Germany).

### *3.2. Plant Material*

The aerial parts of *Zuccagnia punctata* Cav. (Fabaceae, Caesalpinoideae) were collected in January 2018, on Iglesia district, province of San Juan (Argentina) at an altitude of 1800 m above sea level. The species has been previously identified by Dr Gloria Barboza, IMBIV (Instituto Multidisciplinario de Biología Vegetal, Facultad de Ciencias Exactas, Físicas y Naturales, Universidad Nacional de Córdoba, Argentina). A voucher specimen has been previously deposited at the herbarium of the Botanic Museum of Córdoba (CORD 1125).

### *3.3. Essential Oil Extraction and Chemical Analysis*

Fresh aerial parts (500 g) were subjected to hydrodistillation for 2 h using a Clevenger type apparatus. The yields were averaged over two experiments and calculated according to dry weight of plant material. Essential oils (ZEO) were stored at −1 ◦C in airtight micro-tubes prior to chemical analysis. Qualitative data were determined by GC–FID and GC-MS. Gas chromatography-mass spectrometry analyses were carried out on a Hewlett-Packard 5890 II gas chromatograph coupled to a Hewlett-Packard 5989 B mass spectrometer, using a methyl silicone HP-5MS (crosslinked 5% PH ME Siloxane) capillary column (30 m × 0.25 mm), film thickness 0.25 μm. Samples were analyzed using the following GC-MS conditions: oven temperature program: 50–250 ◦C at 3 ◦C/min, carrier gas: helium, 1.5 mL/min; injection temperature: 250 ◦C, FID detector temperature: 300 ◦C; split mode ratio of 1:60. Additional parameters in the mass spectrometer unit: ion source temperature of 250 ◦C; ionizing voltage of 70 eV; scan range from *m/z* 35 to *m/z* 300. The identification of components was performed with the use of the volatile oil ADAMS library together with retention indices of reference compounds and built-in Wiley and NBS peak matching library search systems. Quantitative percentage composition was determined from the GC peak areas without correction factors [25–27].

### *3.4. Preparation of Zuccagnia punctata Essential Oil Loaded Polymeric Systems*

PCL solutions of 10 wt./v % were prepared by dissolving PCL pellets in a 5 mL of DCM:MeOH solvent mixture (50:50 by volume) under magnetic stirring. For the preparation of polymeric matrices of PCL containing *Zuccagnia punctata* essential oil (ZEOP), 0.5 and 1 % wt./wt. of ZEO with respect to PCL were added to the solution. The selected solvent mixture allowed the complete dissolution of ZEO and PCL.

ZEOP were prepared by solution casting onto a Petri dish (4.6 mm in diameter) and dried in a fume hood at room temperature for 24 h. Samples were subsequently vacuum dried to remove residual solvent. Disc samples of 10 mm were cut and stored at room temperature under vacuum until use.

### *3.5. PCL, ZEO, and ZEOP Matrices Characterization*

The morphology of matrices was examined by scanning electron microscopy (SEM, JEOL JSM6460 LV, Peabody, MA, USA) operated at 15 kV. Samples were sputter-coated with gold during 15 min in a chamber evacuated to 500 mTorr (Sputter coater, Desk II, Denton Vacuum, Moorestown, NJ, USA). Thermal properties of PCL pellets, ZEO, ZEOP and PCL matrices were determined by differential scanning calorimetry (DSC, TA instrument, Model Q-2000, New Castle, DE, USA). Scans were carried out at a heating rate of 10 ◦C/min. Glass transition temperature was taken as the onset of the transition. The degree of crystallinity of PCL (Xc) was calculated as:

### Xc (%) = (ΔHm experimental/ΔHmtheoretical) × 100 (1)

where the theoretical melting heat (ΔHm) for pure high molecular weight PCL was taken as 148.05 J/g [37]. Thermogravimetric Analysis (TGA) was conducted to study the thermal stability of ZEO. TGA data were obtained using a thermogravimetric analyzer (TA instrument, Model Q-500, New Castle, DE, USA). A sample of 5–10 mg was accurately weighed in an aluminum pan and the measurement was conducted at heating rate of 10 ◦C/min under nitrogen purging.

*Zuccagnia punctata* essential oil content was determined by ultraviolet-visible spectroscopy using an Agilent 8453 spectrometer (Santa Clara, CA, USA) equipped with a diode array system. A predetermined amount of sample was dissolved in DCM:MeOH (1:1 by volume), and quantification was carried out observing the absorption band at λ = 280 nm. At least three measurements were performed.

The loading capacity (LC) was calculated from the ratio between the ZEO mass in the sample (mZ) and the polymer mass (mP) in ZEOP matrix.

$$\text{L.C.}\left(\%\right) = \left(\text{mZ}\right) / \left(\text{mP}\right) \times 100\tag{2}$$

The encapsulation efficiency (EE) was calculated as:

$$\text{EE (\%)} = \text{(mZf/mPCLf)/(mZi/mPCLi)} \times 100\tag{3}$$

where mZfis the mass of ZEO encapsulated, mZi the initial mass of ZEO, and mPCLf and mPCLi correspond to the final and initial mass of PCL, respectively.

### *3.6. Repellent Activity against Triatoma infestans Nymphs Fifth Instars*

The bioassays were carried out according to [13,14]. *Triatoma infestans* nymphs fifth instar were provided by Servicio Nacional de Chagas (Córdoba, Argentina) and were used one day after receipt.

Filter paper discs (9 cm in diameter) divided by halves were used. One half was treated with 0.5 mL of acetone solutions of the essential oils (0.5% and 1% wt./wt.) while the other half remained untreated. As control, circular white filter papers divided in two halves, one treated with 0.5 mL of acetone and the other untreated, were used. After solvent evaporation, filter paper discs were placed covering the floor of a Petri dish. Five starved nymphs of *T. infestans* (fifth instar) were released in the center of each Petri dish and maintained under controlled conditions of temperature 24 ± 2 ◦C, 50 ± 5% RH and photoperiod of 16 h L/8h D. Experiments were performed by quintuplicate. The same procedure was carried out with the polymer discs containing the essential oil at the same concentrations (0.5% and 1% wt./wt.). Insect distribution was recorded at 1, 24, 72, and 96 h of treatment. *N,N*-diethyl-3-methylbenzamide (DEET) was used as positive control at 0.5% (wt./v) and acetone as blank control.

Data were transformed into repellency percentage (RP %) as:

$$\text{RP}\,\%=\text{(Nc}-\text{50)}\times\text{2}\tag{4}$$

Nc corresponds to the percentage of nymphs in the blank half.

Positive values show repellence while negative values show attraction. Mean values were categorized according to the following scale: Class 0 (>0.01 to <0.1), I (0.1 to 20), II (20.1 to 40); III (40.1 to 60); IV (60.1 to 80), V (80.1 to 100) according to Talukder et al. [38]. Data were analyzed by repeated measures ANOVA to determine the overall significance of the repellence means between the time points and the effect of oil treatment as a factor between subjects. Data were analyzed with the statistical software SPSS 15.0 (SPSS Inc.).

### **4. Conclusions**

Essential oil from *Zuccagnia punctata* Cav. (Caesalpinieae) growing in the province of San Juan, located in the center-west of the Argentine, may be a potential alternative repellent to *T. infestans* (Klug) (Hemiptera, Reduviidae), the vector of Chagas disease. This oil biorepellent constitutes a rich source of sustainable, bioactive, and biodegradable compounds, especially a high content of oxygenated monoterpenes, such as (−)-5,6-dehydrocamphor.

Polymeric matrices of PCL loaded with different amounts of *Zuccagnia punctata* were prepared and characterized. Essential oil content on polymeric matrices showed encapsulation efficiencies higher than 98%, and it is thermally stable. The essential oils from the Andean flora have shown an excellent repellent activity, highlighting the repellent activity of the essential oil of the medicinal species *Zuccagnia punctata*. The effectiveness of ZEO was extended by its incorporation in polymeric systems and could have a potential home or peridomiciliary use, which might help prevent, or at least reduce, Chagas' disease transmission.

**Author Contributions:** S.L., A.T., J.Z., G.A.A. and P.R.C.T. conceived and designed the experiments; S.L., A.T., R.S., obtained *Z. punctata* essential oil, physical-chemical parameters, and performed the repellence assays. J.Z. analyzed the date of GC-MS, G.A.A. and P.R.C.T. obtained and analyzed the poly(ε-caprolactone) matrices loaded with *Z. punctata* essential oil. All authors have read and agreed to the published version of the manuscript.

**Funding:** G.A.A. and P.R.C.T. received financial support from the National Scientific and Technical Research Council of Argentina (grant PIP 0153), and the Argentinean Agency for Scientific and Technological Promotion (grant PICT 2018/02334). J.Z. received financial support from SECyT-Universidad Nacional de Córdoba. This research was funded by CICITCA-Universidad Nacional de San Juan (S.L. and A.T.), Argentina.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** G.A.A.; J.Z. and P.R.C.T. are researchers from CONICET, Argentina.

**Conflicts of Interest:** The authors declare no conflict of interest.

**Sample Availability:** Samples of the *Zuccagnia punctata* essential oil are available from the authors.

### **References**


### *Article*

## **Developing a** *Hazomalania voyronii* **Essential Oil Nanoemulsion for the Eco-Friendly Management of** *Tribolium confusum, Tribolium castaneum* **and** *Tenebrio molitor* **Larvae and Adults on Stored Wheat**

**Nickolas G. Kavallieratos 1,\*, Erifili P. Nika 1, Anna Skourti 1, Nikoletta Ntalli 2, Maria C. Boukouvala 1, Catherine T. Ntalaka 1, Filippo Maggi 3, Rianasoambolanoro Rakotosaona 4,5, Marco Cespi 3, Diego Romano Perinelli 3, Angelo Canale 6, Giulia Bonacucina <sup>3</sup> and Giovanni Benelli <sup>6</sup>**


**Abstract:** Most insecticides commonly used in storage facilities are synthetic, an issue that generates concerns about food safety and public health. Therefore, the development of eco-friendly pest management tools is urgently needed. In the present study, a 6% (*w/w*) *Hazomalania voyronii* essential oil-based nanoemulsion (HvNE) was developed and evaluated for managing *Tribolium confusum*, *T. castaneum*, and *Tenebrio molitor*, as an eco-friendly wheat protectant. Larval and adult mortality was evaluated after 4, 8, and 16 h, and 1, 2, 3, 4, 5, 6, and 7 days, testing two HvNE concentrations (500 ppm and 1000 ppm). *T. confusum* and *T. castaneum* adults and *T. molitor* larvae were tolerant to both concentrations of the HvNE, reaching 13.0%, 18.7%, and 10.3% mortality, respectively, at 1000 ppm after 7 days of exposure. However, testing HvNE at 1000 ppm, the mortality of *T. confusum* and *T. castaneum* larvae and *T. molitor* adults 7 days post-exposure reached 92.1%, 97.4%, and 100.0%, respectively. Overall, the HvNE can be considered as an effective adulticide or larvicide, depending on the target species. Our results highlight the potential of *H*. *voyronii* essential oil for developing green nanoinsecticides to be used in real-world conditions against key stored-product pests.

**Keywords:** botanical-based insecticide; cereals; green grain protectant; essential oil nanoformulation; stored-product beetles; Tenebrionidae

### **1. Introduction**

The confused flour beetle, *Tribolium confusum* (Jacquelin du Val) (Coleoptera: Tenebrionidae), is a cosmopolitan secondary stored-product pest of high economic importance [1]. It has been reported to infest 119 different commodities [2]. The contamination of infested stored products by body fragments and toxins (e.g., methyl-1,4-benzoquinone, ethyl-1,4 benzoquinone, methoxybenzoquinone) may have a negative impact on consumers [3,4]. Due to its ability to infest processed commodities, it is usually found in mills, bakeries,

**Citation:** Kavallieratos, N.G.; Nika, E.P.; Skourti, A.; Ntalli, N.; Boukouvala, M.C.; Ntalaka, C.T.; Maggi, F.; Rakotosaona, R.; Cespi, M.; Perinelli, D.R.; et al. Developing a *Hazomalania voyronii* Essential Oil Nanoemulsion for the Eco-Friendly Management of *Tribolium confusum, Tribolium castaneum* and *Tenebrio molitor* Larvae and Adults on Stored Wheat. *Molecules* **2021**, *26*, 1812. https://doi.org/10.3390/molecules 26061812

Academic Editor: Vincenzo De Feo

Received: 28 February 2021 Accepted: 18 March 2021 Published: 23 March 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

pet shops, and storage units [2]. Recently, Kavallieratos et al. [5] reported that the larval and pupal development period is a complex phenomenon depending on the geographical origin of *T. confusum* and the type of infested commodity.

The red flour beetle, *Tribolium castaneum* (Herbst) (Coleoptera: Tenebrionidae), is a key secondary pest of stored products that is globally distributed [1]. It has been reported to infest 246 different commodities, an issue that makes it one of the most polyphagous stored-product insect pests [2]. It can be routinely found in mills, storage units, pet stores, and retail stores [2]. As in the case of *T. confusum*, *T. castaneum* produces quinones that cause skin irritation [6]. Skourti et al. [7,8] recently outlined that different levels of temperature and different types of commodities, chiefly alter the immature developmental period.

The yellow mealworm beetle, *Tenebrio molitor* L. (Coleoptera: Tenebrionidae), is one of the biggest stored-product insects. It is categorized as a secondary pest and a scavenger [1]. It infests fewer commodities (i.e., at least 46) compared to the other two species [2]. *Tenebrio molitor* can be easily found in flour mills, storage units, and food shops [2]. Apart from being a pest of stored products, it is reared as pet food for birds, fish, and reptiles [1,9,10], and–more recently–widely considered as a promising food for humans, while it degrades polystyrene and plastic waste [11]. *Tenebrio molitor* contaminates the commodities with quinones, but it is not as severe as the contamination by the species belonging to the *Tribolium* genus [10]. Anyway, it can cause allergic reactions in humans [6]. It can complete the biological cycle in only 30 days, while as an adult, it can survive up to two years [1,3].

It is well known that tenebrionids can develop resistance to several insecticides [12–14]. Therefore, new insecticidal formulations are necessary to be developed [15–18]. Among natural products with efficacy against stored-product insects, essential oils (EOs) and their main constituents have been revealed to be promising [18–20]. However, in real-world conditions, EOs need to be encapsulated in micro- and nanoformulations to enhance their persistence and physio-chemical stability while maintaining their biological properties [21,22]. Nanoemulsions (NEs) can be considered one of the most promising ways for the encapsulation and formulation of EOs. Nanoemulsions are kinetically stable oil droplets in water systems with a surfactant-to-oil ratio (SOR) ranging from 1 to 2 [23], and a droplet diameter <100 nm. Thanks to the reduced size of the droplets of the internal phase and the consequent increment of the surface area, NEs allow a better interaction of the encapsulated compounds into the target site [23] by overcoming the EO's poor physicochemical stability and solubility issues [24]. This strategy can boost the applicability of EOs as natural insecticides [25–29].

Therefore, EO-based NEs are alternative solutions for pest management for a wide spectrum of insects of public health importance, and crop pests as well. For example, Ghosh et al. [30] treated 3rd instar larvae of *Aedes aegypti* L. (Diptera: Culicidae) with different NE concentrations from *Ocimum basilicum* L. (Lamiaceae) EO. Larval mortality was completely suppressed even after 15 min of exposure at 200 mL of 10-fold diluted NE. Duarte et al. [31] tested the larvicidal efficacy of 5% (*w*/*w*) NE of *Rosmarinus officinalis* L. (Lamiaceae) EO against the 4th instar larvae of *Ae. aegypti*. Mortality levels reached 80% after 24 h and 90% after 48 h at 250 ppm. Recently, Benelli et al. [27] proposed a 6% (*w*/*w*) NE of *Carlina acaulis* L. (Compositae) root EO against the European grapevine moth, *Lobesia botrana* (Denis and Schiffermüller) (Lepidoptera: Tortricidae), reaching 50% and 90% mortality of 1st instar larvae with 9.04 and 17.70 μL/mL, respectively. In a further recent study, Pavela et al. [32] evaluated NEs based on *C. acaulis* EO and found that less than 1200 μL/L caused 90% mortality to 3rd instar larvae of *Culex quinquefasciatus* Say (Diptera: Culicidae).

However, there is little published research on the utilization of NEs based on EOs as grain protectants [16,33]. For instance, Hashem et al. [19] used an NE of *Pimpinella anisum* L. (Apiaceae) EO for the management *T. castaneum* adults, over a wide spectrum of NE concentrations, on cracked wheat kernels. Nevertheless, there are no data on the efficacy of *Hazomalania voyronii* (Jum.) Capuron (Hernandiaceae) EO-based NE against *T. confusum, T. castaneum*, and *T. molitor.* However, the bioactivity of the pure EO of *H. voyronii* has

been recently studied [20], showing that the raw *H*. *voyronii* EO exerted a rather limited toxicity as a grain protectant against important stored-product beetles. Even on selected species and instars (e.g., *Trogoderma granarium* Everts (Coleoptera: Dermestidae) adults), mortality rates reached about 79% after 7 days of exposure at 1000 ppm. In this framework, one may hypothesize that a way to boost the efficacy of this EO may be to develop highly stable EO-based nanoformulations [23]. As a model EO prototype, here we used the one obtained from *H. voyronii*, a traditional Malagasy plant (e.g., it is used to heal wounds, the drinkable bark decoction of stems is used for the treatment of malaria) with documented insecticidal efficacy [34]. Perilla aldehyde, the major compound of the *H. voyronii* EO, is used as a flavouring component to baked foods, sweets, meat products, dressing for salads, sauces, salted vegetables, and beverages [35]. Furthermore, perilla aldehyde is a "generally recognized as safe" (GRAS) substance [36].

To validate the hypothesis formulated above, the objective of the present study was the development of a 6% (*w*/*w*) *H. voyronii* EO-based NE for the effective and eco-friendly management of larvae and adults of three major stored-product beetles (i.e., *T. confusum, T. castaneum*, and *T. molitor).* To assess the applied potential to protect stored grains, the effectiveness of this NE as a grain protectant was investigated in small environments mimicking real wheat storage conditions.

### **2. Results**

### *2.1. Development and Characterization of H. voyronii EO-Based NE*

After a preliminary screening, the quantitative composition of the *H*. *voyronii* NE was selected as follows: 6% (*w*/*w*) of the EO phase was emulsified in the aqueous medium containing 4% (*w*/*w*) of surfactant (Polysorbate 80). A high-energy method (i.e., high pressure homogenization) was employed at the pressure of 130 MPa to obtain *H. voyronii* EO-based NE characterized by oil droplets with a size in the nanometric range. From DLS analysis, in fact, the sample showed a monomodal size distribution with a mean diameter (Z-average) of 53.54 ± 0.20 nm and a polydispersity index of 0.340 ± 0.013 after preparation (Figure 1). The absence of oil droplets with a diameter above 1 μm confirms the formation of a true NE. Indeed, the sample appeared homogenous upon observation by optical microscope.

**Figure 1.** Size distribution (d.nm) of the prepared *Hazomalania voyronii*-based nanoemulsion (6% *w*/*w*) as obtained from dynamic light scattering.

### *2.2. Insecticidal Efficacy*

When the insecticidal efficacy of the 6% (*w*/*w*) *H*. *voyronii* NE was evaluated, between exposure intervals, all main effects were significant, while the associate interaction was not significant (Table 1). Within exposure intervals, the main effect as well as the interaction exposure x insect species-stage were significant, while the interactions exposure x NE concentration and exposure x NE concentration x insect species-stage were not significant (Table 1).

**Table 1.** Evaluation of the insecticidal activity of a 6% (*w*/*w*) *Hazomalania voyronii* essential oil nanoemulsion: MANOVA parameters about the main effects and associated interactions leading to the observed mortality rates on *Tribolium castaneum, Tribolium confusum*, and *Tenebrio molitor* adults and larvae, between and within exposure intervals (error *df* = 96).


Concerning *T. castaneum* adults, the mortality caused by *H. voyronii* EO-based NE was 0.0% until the 1st day post-treatment, then reached 2.2% after 2 days of exposure for both tested NE concentrations (Table 2). The mortality remained low and did not exceed 12.5% and 18.7% at 500 ppm and 1000 ppm, respectively, after 7 days of exposure. Although the mortality of *T. castaneum* larvae did not exceed 10% at 500 ppm and 12.6% at 1000 ppm 16 h post-exposure, it reached 84.1% at 500 ppm and 97.4% at 1000 ppm, after 7 days of exposure.

Regarding *T. confusum* adults, the mortality remained at low levels (Table 3). After 4 days of exposure at 500 ppm, the mortality was 3.3%, and after 7 days it reached 10.3%. The mortality at 1000 ppm was not significantly higher than the one at 500 ppm, after 2 and 7 days of exposure reaching 3.3% and 13.0%, respectively. As far as *T. confusum* larval mortality is concerned, 5 days of exposure to the two NE concentrations led to significantly higher mortality at 1000 ppm than 500 ppm (i.e., 45.0% and 26.4%, respectively). The *H. voyronii* NE killed 59.3% of larvae at 500 ppm, and 92.1% of larvae at 1000 ppm, 7 days post-exposure.

Mortality of *T. molitor* adults was <90% after the 7th day of exposure (94.8%) at 500 ppm, while at 1000 ppm after the 6th day of exposure it was 93.5% (Table 4). Complete mortality of this life stage was achieved testing 1000 ppm of *H. voyronii* NE after 7 days post-exposure. No mortality of *T. molitor* larvae was noted testing 500 ppm and 1000 ppm, 2 days and 1 day post-exposure, respectively. At the end of the experimental period, the overall larval mortality did not exceed 5.8% at 500 ppm, and 10.3% at 1000 ppm.


*t* *p*



 0.33

 0.26

 0.14

 0.03

 0.12

 0.07

 0.05

 <0.01

 <0.01

−1

−1.2

−1.6

−2.3

−1.7

−1.9

−2.2

−7.6

−8.7


**Table 4.** Mean (%) mortality ± SE of *Tenebrio molitor* adults and larvae after 4 h, 8 h, 16 h, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, and 7 days of exposure to wheat treated

*p*


 -

 -

 -

 0.07

 0.26

 0.31

 0.58

 1.00

 0.52

### **3. Discussion**

Our results indicate that the NE-based on *H. voyronii* EO (6% *w*/*w*) is effective against *T. castaneum, T. confusum*, and *T. molitor*. This nanosystem led to high mortality levels on different life stages of the tested species, even at 500 ppm. Concerning *T. molitor* adults, the mortality reached 100% at 1000 ppm, and 94.8% at 500 ppm, after 7 days of exposure. The mortality of larvae was very low, even at the highest tested concentration (1000 ppm), reaching 10.3% after 7 days of exposure. Earlier research has revealed that the adult stage is the most susceptible life stage of *T. molitor*. For example, Kavallieratos et al. [37] found that when 0.504 ppm deltamethrin, 5 ppm pirimiphos-methyl, 1000 ppm silicoSec (which is a diatomaceous earth (DE)), and 1 ppm spinosad were applied on stored wheat as grain protectants, high adult mortality rates were achieved (92.2%, 100%, 100%, and 94,4%, respectively), while they caused moderate larval mortality (10.0%, 71.1%, 43.3%, and 28.9%, respectively). Apart from the wheat, barley and maize have also been tested on the same formulations as grain protectants, and the results followed the same pattern, with larvae being more tolerant than adults. In addition, testing the efficacy of 5 ppm pirimiphos-methyl on barley in a wide range of temperature and relative humidity levels, a greater mortality of *T. molitor* adults was found, if compared to the larvae [38].

In an earlier study, the raw EO of *H. voyronii* showed relevant efficacy on *T. granarium* adults, but not on the larvae [20]. The raw EO caused 78.9% adult mortality at 1000 ppm, but only 15.6% at 500 ppm after 7 days of exposure. Larvae were more tolerant and the mortality rates at 500 ppm and 1000 ppm after 7 days of exposure did not exceed 4.4% and 15.6%, respectively. Although the raw *H. voyronii* EO does not allow an adequate control of *T. molitor* and *T. granarium* larvae, the suppression of the adult stage of both pests is a very important finding, as adults are the vehicle of reproduction [20].

In contrast, the results of the present study outlined that the adults of *T. castaneum* and *T. confusum* remained practically unaffected by being exposed to the *H. voyronii* EO-based NE, given that the highest adult mortality was 18.7% for *T. castaneum* at 1000 ppm and 13.0% at 1000 ppm for *T. confusum,* while the larvae had a mortality of 97.4% for *T. castaneum* at 1000 ppm and 92.1% at 1000 ppm for *T. confusum* after 7 days of exposure. Previous research on *T. castaneum* has reported that the adults are tolerant to several insecticides. Fumigation studies on the species of four EOs extracted from the plants *Lantana camara* L. (Verbenaceae), *Cymbopogon nardus* (L.) Rendle (Poaceae), *Cinnamomum zeylanicum* Blume (Lauraceae), and *Trachyspermum ammi* (L.) Sprague (Apiaceae) showed that for all the tested EOs and exposure times, *T. castaneum* adults needed relatively high EO volumes (e.g., 14.56, 37.52, 4.40, and 14.86 μL, respectively, for each EO after 72 h of exposure) than the larvae (e.g., 5.00, 4.13, 2.48, and 2.73 μL, respectively, for each EO after 72 h of exposure) [39]. When the d-strain of *T. castaneum* was treated with 1 mg/L and the r-strain with 2 mg/L phosphine (PH3), after 4 h of treatment, adults have been found more tolerant than the larvae. For both d- and r-strains of *T. castaneum,* after 24 h of exposure to <10 mg/L carbonyl sulphide (COS), the larvae were less tolerant than adults [40]. Earlier, Arthur [41] reported that larvae were less tolerant than the adults. Regarding contact toxicity, Deb and Kumar [42] reported that the *Artemisia annua* L. (Asteraceae) EO had greater larvicidal than adulticidal efficacy against *T*. *castaneum*. Mujeeb and Shakoori [43] suggested the larval stage as the preferable life stage to apply pirimiphos-methyl, outlining that this is the most susceptible stage. Adults of *T. confusum* were also more tolerant than the larvae. The natural insecticide silicoSec attained 100% larval mortality after 7 days of exposure, but when tested on adults it did not exceed 85% after 14 days of exposure [44]. Spinetoram and spinosad [45] had high larval mortality reaching 98.9% in a mixture of the two insecticides after 14 days on treated wheat kernels, while the adult mortality reached 67.8% for the same mixture after 14 days on treated wheat kernels. Similarly, studying the effectiveness of eight pyrrole derivatives, a better larvicide than adulticide action has been detected [46–48].

EO constituents have a wide spectrum of effectiveness on adults and larvae that could be partially explained by their different mode of actions [49]. For instance, perilla aldehyde, the main component of *H. voyronii* EO, showed insecticidal and inhibitory effects on the enzyme acetylcholinesterase (AChE) in *Drosophila suzukii* (Matsumura) (Diptera: Drosophilidae) [50], and it has been argued that its insecticidal activity could be related to the presence of exocyclic and endocyclic double bonds in the chemical structure [51]. Furthermore, 1,8-cineole and limonene, the other two main constituents of the *H*. *voyronii* EO, showed insecticidal efficacy against a wide spectrum of insects, including mosquitoes (e.g., *Culex pipiens* L. (Diptera: Culicidae), *Cx. quinquefasciatus*, *Ae. aegypti, Ae. albopictus* (Skuse) (Diptera: Culicidae)), houseflies (*Musca domestica* L. (Diptera: Muscidae)), and stored-product beetles (*Sitophilus granarius* (L.) (Coleoptera: Curculinonidae)) [52–55].

The significant toxicity of the EO-based NE developed here against *T. castaneum* and *T. confusum* larvae is important, since targeting the adults is crucial to achieve a major reduction of the overall population. Storage units can host several species in different developmental stages existing simultaneously [56–59]. Therefore, insecticides based on natural products such as EO-based NEs, which can manage a broad spectrum of storedproduct insects, are highly desirable [16,33]. Our results showed that the *H. voyronii* EO-based NE is toxic against three tenebrionid species. Similarly, Hashem et al. [19] tested four different concentrations of a *P. anisum* EO-based NE against *T. castaneum* adults, showing that 7.5% and 10% *v*/*v* killed 51.2% and 74.3% of the exposed individuals, in comparison to the control after 9 days of exposure, respectively; 12 days post-exposure, the overall mortality rates reached 54.7% at 7.5% *v*/*v* and 81.3% at 10% v/v.

Our findings indicate that among the different species tested here, all belonging to the Tenebrionidae family, there is a wide variability of the performance of *H. voyronii* EO-based NE as a grain protectant. However, the level of efficacy depends on the life stage of the target species. A crucial factor for elevated effectiveness of insecticides is the timing of their application [7,60]. Therefore, the knowledge of the species and the life stage that infests grain commodities could maximize the management strategy if the *H. voyronii* EO-based NE is selected as a component of a management strategy against *T. castaneum, T. confusum*, and *T. molitor*.

### **4. Materials and Methods**

### *4.1. Essential Oil*

The dry bark of *H. voyronii* was obtained from trees growing in Kirindy Forest, Madagascar (coordinates: S 20◦28 15.002"; E 44◦17 56.06"; 62 m a.s.l.) in February, 2018. Then, it was subjected to hydrodistillation for 3 h using a Clevenger-type apparatus. The chemical composition was recently reported in our recent study by Benelli et al. [34]. The major components were oxygenated monoterpenes, namely perilla aldehyde (43%), 1,8-cineole (33.2%), and limonene (13%).

### *4.2. Insects*

*Tribolium confusum* and *T. castaneum* were cultured on wheat flour and 5% brewer's yeast, and *T. molitor* on a combination of oat bran and potato slices for additional moisture [61], at 30 ◦C, 65% relative humidity, and continuous darkness [37,62,63]. The founding colonies were acquired from storage facilities in Greece. The two *Tribolium* species were maintained in the Laboratory of Agricultural Zoology and Entomology, Agricultural University of Athens, since 2003, and *T. molitor* since 2014. For the tests, unsexed adults <2 weeks old, and larvae that were 3rd–4th instar old (for *T. confusum* and *T. castaneum*) and 10–14 mm long (for *T. molitor*) were used [37,62].

### *4.3. Commodity*

Hard wheat, *Triticum durum* Desf. (var. Claudio) (Poales: Poaceae) clean and free of infestations and pesticides was used in the experiments. Prior to the bioassays, moisture content of the wheat kernels was 12.4%, calibrated by a moisture meter (mini GAC plus, Dickey-John Europe S.A.S., Colombes, France).

### *4.4. Development and Characterization of H. voyronii EO-Based Nanoemulsion*

*Hazomalania voyronii* EO NE was obtained through a high-energy method, by using a high-pressure homogenizer according to the procedure reported by Cappellani et al. [64]. A 6% (*w*/*w*) of EO was added dropwise to 4% (*w*/*w*) Polysorbate 80 (Sigma-Aldrich) aqueous solution under high-speed stirring (Ultraturrax T25 basic, IKAfi Werke GmbH & Co.KG, Staufen, Germany) for 5 min at 9500 rpm. The obtained emulsion was then homogenized with a French Pressure Cell Press (American Instrument Company, Aminco, MY, USA) for four cycles at a pressure of 130 MPa. Visual inspection of the formulation was performed by using a polarizing optical microscope (MT9000, Meiji Techno Co. Ltd., Chikumazawa, Miyoshi machi, Iruma-gun Saitama, Japan) equipped with a 3-megapixel CMOS camera (Invenio 3S, DeltaPix, Smørum, Denmark) to assess NEs formation. Dynamic light scattering (DLS) analyses were then carried out to determine lipophilic internal phase droplets size using a Zetasizer Nano S (Malvern Instruments, Worcestershire, UK) equipped with backscattered light detector working at 173◦. A sample of 1 mL was analysed at 25 ◦C, following a temperature equilibration time of 180 s. The analyses were performed in triplicate.

### *4.5. Insecticidal Assays*

The NE based on *H. voyronii* EO (6% *w*/*w*) was tested at the concentrations of 500 μL/kg wheat (=500 ppm) and 1000 μL/kg wheat (=1000 ppm). The test concentrations were selected based on preliminary tests on the three tenebrionid species. Test solutions were prepared in water at the final volume of 750 μL, and sprays were performed on plates, where 0.25 kg wheat lots were laid out [20]. Additional lots of 0.25 kg wheat treated with (i) water and (ii) carrier control (4% *w*/*w* surfactant dispersed in water) served as controls. The spraying of the wheat lots was conducted by an AG-4 airbrush (Mecafer S.A., Valence, France) on different trays. Controls were sprayed using different AG-4 airbrushes. Then treated lots were transferred to 1-L glass containers and were shaken for 10 min to equally distribute the NE on the total quantity of wheat. The same procedure was followed for controls. From each treated lot or controls, three samples of 10 g each were obtained with different scoops. A Precisa XB3200D compact balance (Alpha Analytical Instruments, Gerakas, Greece) was used to weigh the samples on a filter paper that was unique for each sample. Then, samples were placed in glass vials (7.5 cm × 12.5 cm diameter and height). The caps of the vials had a 1.5 cm diameter circular opening in the middle, covered with gauze, to sufficiently aerate the content of the vials. The upper inner necks of the vials were covered by polytetrafluoroethylene (60 wt % dispersion in water) (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany), to assure that the beetles would remain in the vials. Thereafter, 10 larvae or adults of each tenebrionid species were separately transferred into the vials. The containers remained in incubators set to 30 ◦C and 65% RH for the whole experimental period. Mortality rates' evaluation was accomplished by an Olympus stereomicroscope (SZX9, Bacacos S.A., Athens, Greece) at 57x total magnification after 4, 8, and 16 h and 1, 2, 3, 4, 5, 6, and 7 days of exposure, by gently nudging each individual insect using a fine brush (Cotman 111 No 000, Winsor and Newton, London, UK) to detect any movement. For each concentration of the NE and control, different brushes were used. The above procedure was repeated three times with new insects, wheat, and vials.

### *4.6. Data Analysis*

Control mortality on wheat treated with water was low (<5%) for all insect species and life stages tested, therefore no correction was necessary. In contrast, the control mortality on wheat treated with the carrier control 4% *w*/*w*surfactant dispersed in water, was >5% for all tested species and life stages, ranging up to 17.8%. Therefore, mortality values were corrected by the Abbott formula (i.e., (1 − insect population in treated unit after treatment/insect population in control unit after treatment) × 100) [65]. Before conducting analysis, the mortality data were log(*x* + 1) transformed to normalize variance [66,67]. Statistical analyses were conducted by following the repeated-measures model [68]. Exposure

interval was the repeated factor, and mortality was the response variable. The main effects were the concentration and insect species/developmental stage. The associated interactions of the main effects were considered in the analysis. All analyses were conducted using JMP v.14 software [69]. Means were separated using the Tukey–Kramer (HSD) test at 0.05 of significance [70]. The two-tailed *t*-test at *n*-2 *df* and 0.05 significance [71] was used to compare the two tested concentrations of *H. voyronii* EO-based NE at each species or life stage.

### **5. Conclusions**

To eco-friendly manage several important insect pests of stored products, a combination of the *H. voyronii* EO-based NE with other natural insecticides (e.g., DEs) could potentially provide an enhanced level of protection for stored durable commodities against multi species infections. For example, the use of DE or natural zeolite as grain protectants caused 100% mortality on *T. castaneum* adults after 14 or 21 days of exposure, respectively [72]. A combination of *H. voyronii* EO-based NE with DE or natural zeolite could enhance the likelihood of the management of *T. castaneum, T. confusum,* and *T. molitor,* regardless of their being adults or larvae, an issue that merits further investigation. Furthermore, Athanassiou et al. [73] found that the mortality of *S. oryzae* was significantly higher when silica gel was combined with the EO of *Juniperus oxycedrus* L. ssp. *oxycedrus* (Pinales: Cupressaceae) compared to the silica gel alone. In this scenario, additional experimental efforts are necessary to shed light on the impact of *H. voyronii* EO-based NE as a multi-species killing agent.

**Author Contributions:** Conceptualization, N.G.K., F.M., and G.B. (Giovanni Benelli); methodology, N.G.K., N.N., F.M., G.B. (Giulia Bonacucina) and G.B. (Giovanni Benelli); software N.G.K., E.P.N., and G.B. (Giulia Bonacucina); validation, N.G.K., E.P.N., N.N., F.M., G.B. (Giulia Bonacucina), and G.B. (Giovanni Benelli); formal analysis, N.G.K., E.P.N., and G.B. (Giulia Bonacucina); investigation, N.G.K., E.P.N., A.S., N.N., M.C.B., C.T.N., F.M., R.R., M.C., D.R.P., A.C., G.B. (Giulia Bonacucina), and G.B. (Giovanni Benelli); resources, N.G.K. and G.B. (Giovanni Benelli); data curation, N.G.K., E.P.N., A.S., and G.B. (Giulia Bonacucina); writing—original draft preparation, N.G.K., E.P.N., F.M., G.B. (Giulia Bonacucina), and G.B. (Giovanni Benelli); writing—review and editing, N.G.K., E.P.N., A.S., N.N., M.C.B., C.T.N., F.M., R.R., M.C., D.R.P., A.C., G.B. (Giulia Bonacucina), and G.B. (Giovanni Benelli); visualization, N.G.K., E.P.N., F.M., G.B. (Giulia Bonacucina), and G.B. (Giovanni Benelli); supervision N.G.K., F.M., and G.B. (Giovanni Benelli); funding acquisition, N.G.K. and G.B. (Giovanni Benelli). All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was partially funded by the 34.0401 Project (Special Account for Research Funds of the Agricultural University of Athens).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** All data are within this manuscript.

**Conflicts of Interest:** The authors declare no competing interests.

**Sample Availability:** Not available.

### **References**


### *Article* **Plumbagin, a Potent Naphthoquinone from** *Nepenthes* **Plants with Growth Inhibiting and Larvicidal Activities**

**Asifur Rahman-Soad 1, Alberto Dávila-Lara 1, Christian Paetz <sup>2</sup> and Axel Mithöfer 1,\***


**Abstract:** Some plant species are less susceptible to herbivore infestation than others. The reason for this is often unknown in detail but is very likely due to an efficient composition of secondary plant metabolites. Strikingly, carnivorous plants of the genus *Nepenthes* show extremely less herbivory both in the field and in green house. In order to identify the basis for the efficient defense against herbivorous insects in *Nepenthes,* we performed bioassays using larvae of the generalist lepidopteran herbivore, *Spodoptera littoralis.* Larvae fed with different tissues from *Nepenthes x ventrata* grew significantly less when feeding on a diet containing leaf tissue compared with pitcher-trap tissue. As dominating metabolite in *Nepenthes* tissues, we identified a naphthoquinone, plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone). When plumbagin was added at different concentrations to the diet of *S. littoralis* larvae, an EC50 value for larval growth inhibition was determined with 226.5 μg g−<sup>1</sup> diet. To further determine the concentration causing higher larval mortality, sweet potato leaf discs were covered with increasing plumbagin concentrations in no-choice-assays; a higher mortality of the larvae was found beyond 60 μg plumbagin per leaf, corresponding to 750 μg g−1. Plantderived insecticides have long been proposed as alternatives for pest management; plumbagin and derivatives might be such promising environmentally friendly candidates.

**Keywords:** naphthoquinones; plumbagin; *Spodoptera littoralis*; insect growth inhibition; carnivorous plants; *Nepenthes*

### **1. Introduction**

*Nepenthes* is a tropical plant genus occurring mainly in Southeast Asia. Plants of this genus are carnivorous. They attract, catch, and digest insect prey in order to get additional nutrients, primarily, nitrogen and phosphate [1,2]. Therefore, *Nepenthes* species developed a pitfall trap (Figure 1), called pitcher, where insect prey falls inside due to a slippery surface and drown in a digestive fluid [1,2]. As in many other carnivorous plants, also the genus *Nepenthes* harbors a large chemical diversity; currently, several secondary metabolites are isolated for pharmaceutical, biotechnological, and ethnobotanical use [3,4]. Especially, *Nepenthes* species are well known in traditional medicine. Multiple reports are in the literature describing curative effects of *Nepenthes* extracts on diseases, e.g., on hypertension, cough, fever, urinary system infections [5], malaria [6,7], pain, asthma [7], *Staphylococcus* infection [8], celiac disease [9], and oral cancer cells [10].

However, up to now, most of the chemical analysis in *Nepenthes* has been done for the digestive pitcher fluid. Here, metabolites with antimicrobial properties have been found, e.g., naphthoquinones (NQ; droserone, 5–*O*–methyl droserone in *N. khasiana* [11]; plumbagin, 7-methyl-juglone in *N. ventricosa* [12]). Thus, it is hypothesized that such compounds mediate protection against microbes and preserve prey during digestion [11–14]. NQ derivatives are also described for tissues of various *Nepenthes* species including the pitchers [12,15–17]. In particular, plumbagin is of broad pharmaceutical interest because

**Citation:** Rahman-Soad, A.; Dávila-Lara, A.; Paetz, C.; Mithöfer, A. Plumbagin, a Potent Naphthoquinone from *Nepenthes* Plants with Growth Inhibiting and Larvicidal Activities. *Molecules* **2021**, *26*, 825. https://doi.org/10.3390/ molecules26040825

Academic Editor: Giovanni Benelli Received: 30 December 2020 Accepted: 1 February 2021 Published: 5 February 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

it is a candidate that may be used in therapies against various cancers or chronic diseases [18–21]. In addition to NQ, carotenoids, flavonoids, sterols, and triterpenes are described for *Nepenthes* leaves [1,22,23]. Recently, an untargeted metabolomics approach was performed in *N. x ventrata* comparing secondary metabolites of leaves and pitcher tissue before and after prey catches [24]. In that study, about 2000 compounds (MS/MS events) were detected in the two tissues showing enormous metabolome diversity, which was even higher in leaves. Strikingly, the tissue specificity of chemical compounds could significantly discriminate pitchers from leaves. Besides many yet unknown compounds, the common constituents were phenolics, flavonoids, and NQ [24]. These data suggest that the metabolite composition of the tissues can point to their function. In addition, the metabolite composition may represent mechanisms that promote the evolution of plant carnivory as well as enable the plants to cope with environmental challenges [14].

**Figure 1.** *Nepenthes x ventrata*. A natural hybrid of *N. ventricosa* and *N. alata*. Copyright © A. Rahman-Soad.

(A)biotic challenges include the attack of herbivorous insects. Interestingly, there are only a very few observations and studies published concerning the attack of insects on tissues of carnivorous pitcher plants. Recently, lepidopteran herbivory was described for some species of the new world pitcher plant *Sarracenia* [25,26]. There is only one investigation showing that *N. bicalcarata* plants are attacked by an insect, the weevil *Alcidodes spec*. [27]. Another study shows that in *N. gracilis* red pitchers experience less herbivory than green ones [28]. To the best of our knowledge, no other studies have been published yet that focus on herbivore damage in *Nepenthes*. Obviously, the carnivorous syndrome obtained much more attention. However, herbivory on *Nepenthes* tissue is obviously rare. The reason for this is not known but it is unlikely that all herbivores are caught and digested. Instead, *Nepenthes* very likely has an efficient setting of defensive chemistry, which is not unusual in many plants [29]. In order to address this hypothesis and gain more insight in the ecological relevance of *Nepenthes* metabolites, we performed bioassays to study the effect of tissue of *N. x ventrata,* a robust natural hybrid of *N. alata* and *N. ventricosa*, on the feeding behavior and larval development of the generalist insect herbivore *Spodoptera littoralis*.

### **2. Results and Discussion**

### *2.1. Effect of Nepenthes x ventrata Tissue on Insect Larvae Growth*

The observation that *Nepenthes* plants are rarely infested by insect herbivores forced us to study this phenomenon. Therefore, freshly harvested tissues from *N. x ventrata* leaves and pitchers were added to an artificial diet and fed to larvae of the generalist herbivore *Spodoptera littoralis*. As can be seen in Figure 2A, starting at day 4 to 5, the presence of leaf but not pitcher tissue significantly affected the performance of the larvae, which gained less weight. At this point, it might be worth to mention that recently in *N. x ventrata* [24] and before in *N. khasiana* [15], the concentration of a NQ, very likely plumbagin, was determined to be significantly higher in leaves compared with pitchers, which may explain the result found in Figure 2A. We also could support these results by comparing plumbagin content in pitcher vs. leaf; by quantitative NMR analysis, we found a 5.2-fold higher plumbagin concentration in leaf compared with pitcher tissue (650 and 125 μg g−<sup>1</sup> FW, respectively). Although significant, the growth inhibition effect was not very pronounced. Thus, the feeding experiment was repeated with dried leaf tissue in order to add more plant material to the diet, knowing that the water content of *N. x ventrata* tissue is about 90% [24]. Here, the effect of the plant tissue was more distinct (Figure 2B). Both quantities of leaf tissue, 10% and 15% (*w*/*w*), showed clear impairment on the growth and weight of the feeding *S. littoralis* larvae already at day 2. Starting from day 3 on, there was also a significant difference between the larvae feeding on either 10% or 15% of *Nepenthes* tissue that was included in the diet (Figure 2B).

**Figure 2.** Performance of *Spodoptera littoralis* larvae feeding on artificial diet containing (**A**) fresh leaf powder of *Nepenthes x ventrata* leaf and pitcher (30% (*w*/*w*)) or (**B**) dried *N. x ventrata* leaf powder (10 and 15% (*w*/*w*)) Larvae were weighed every day for 7 days. Mean (± SE) labelled with different letters indicate significant difference (*p* < 0.05); two-way ANOVA, Šidák's multiple comparisons test; *n* = 15.

### *2.2. Plumbagin in Nepenthes x ventrata Tissue*

In many carnivorous plants belonging to the order Nepenthales [14], a *sensu stricto* sister group to Caryophyllales [30] and including the plant families Droseraceae and Nepenthaceae, the presence of NQ has been described [31]. This includes species such as *Aldrovanda vesiculosus*, *Dionaea muscipula*, *Drosophyllum lusitanicum,* as well as the genera *Drosera* and *Nepenthes* [31]. Among their secondary compounds, in particular, plumbagin is slightly volatile; thus, its presence in plant tissue is often indicated by spontaneous sublimation, thereby staining the tissue surface or plastic material used for storage. We observed this effect with both leaf and pitcher tissue (Figure 3) stored in plastic vials. In order to proof its identity, a part of the compound was removed from the wall of the plastic vial by extraction with dichloromethane.

**Figure 3.** (**A**) Tissues of dry *Nepenthes x ventrata* leaf and pitcher stored for 6 months in a plastic tube. Sublimed compounds cover the dry material with a yellowish color (left) in comparison with freshly cut tissue (right). (**B**) Plastic tubes that stored the different tissue types for 6 months. New tubes do not show any color.

After evaporation of the solvent, the residue was used for NMR analysis. In parallel, leaf extracts from *N. x ventrata* were analyzed by 1H-NMR as well (Figure 4). When compared with a reference, it could be confirmed that the sublimed volatile compound was indeed plumbagin, and this compound could also be proven in leaf material (Figure 4).

**Figure 4.** *Cont*.

**Figure 4.** 1H-NMR spectra in DMSO-*d6*. (**A**) Plumbagin (see insert) extracted from *Nepenthes x ventrata* leaves and (**B**) a plumbagin reference. (**C**) Details of 1H NMR spectra of a plumbagin reference and (**D**) the volatile exudate emitted by *N. x ventrata* pitcher material. Asterisks (\*) indicate the presence of 4-tert-butylcatechol, a polymerization inhibitor probably extracted from the plastic material, and hashes (#) account for an unidentified impurity. The intensity of the aromatic range in (**C**) was increased as indicated by the factor.

These results raised the question of the function of plumbagin and other NQ in carnivorous plants and in *Nepenthes*. In general, NQ are highly bioactive compounds. Besides pharmacological properties against malaria, various cancers, inflammation, and much more [6,19,32–34], they have allelopathic effects as shown for the walnut trees (*Juglans* spp.) releasing the phytotoxin juglone (5-hydroxy-1,4-naphthalenedione) [35,36]. Many defenserelated properties are associated with NQ, among them are activities against numerous microbes including human- and phytopathogenic parasites, bacteria, and fungi [31–33]. That means, the NQ might protect the plants from pathogen infection. In addition, for *N. khasiana,* it could be shown that droserone and its derivative 5–*O*–droserone provided antimicrobial protection in the pitcher fluid of [11,37]. Buch and coworkers identified plumbagin and 7-methyl-juglone in the pitcher fluid of *N. ventricosa* [12]. These results suggest a role for NQ in the pitcher fluid in order to control the microbiome in the digestive fluid, together with, e.g., pathogenesis-related proteins such as PR-1 [13,37].

### *2.3. Growth-Inhibiting and Larvicidal Activities of Plumbagin*

Besides the hypothesis that NQ are involved in defense against microbial infection, there are several studies showing that these compounds can also affect insects [31–33,38–43]. We, therefore, performed feeding experiments with plumbagin-supplemented artificial diet and measured the weight of *S. littoralis* larvae every day. Knowing that the amount of plumbagin in *Nepenthes* leaves is about 0.05% of fresh weight [15], we covered a concentration range between 100 and 900 μg g−1, representing 0.01–0.09% fresh mass, respectively. As shown in Figure 5, with increasing plumbagin concentrations, the larvae gained less weight. Based on these data the EC50 value was calculated indicating the plumbagin concentration necessary for 50% growth inhibition (weight gain), which was determined as 226.5 μg g−<sup>1</sup> diet. For some lepidopteran species such as *Spodoptera litura*, *Achaea janata*, and *Trichoplusia ni*, it already has been shown that plumbagin affects the feeding behavior [38–41]. However, in those experiments, the focus of the analysis was on the level of feeding-avoidance rather than on the larval growth.

**Figure 5.** (**A**) Performance of *Spodoptera littoralis* larvae feeding on artificial diet containing various concentrations of plumbagin. Larvae were weighed every day for 7 days. Mean (±SE), *n* = 15. (**B**) Determination of EC50 value based on the data obtained in (**A**). EC50 was calculated with 226.5 and 1.2 μmol g−<sup>1</sup> diet, respectively.

In contrast to most other bioassays that analyzed the antifeeding activity of plumbagin, here, the compound of interest was included in the food, not painted on leaves of various plant species. Nevertheless, in order to determine the mortality rate of larvae feeding on plumbagin, we also carried out an experiment using the approach with plumbagin-painted leaves. Therefore, a sweet potato cultivar (Tainong 66) that is known to be susceptible to herbivores and does not induce strong defense response upon attack was selected [44]. In first experiments, we observed that *S. littoralis* larvae even preferred cannibalism than feeding on those leaves. As a consequence, only individualized larvae were used. Up to a plumbagin concentration of 60 μg−<sup>1</sup> leaf (13.3 μg cm−2, 750 μg g−<sup>1</sup> leaf) no larvicidal effect was determined for the period analyzed (Figure 6A). With 90 μg−<sup>1</sup> leaf (20 μg cm<sup>−</sup>2; 1.125 mg g−<sup>1</sup> leaf) dead larvae could be found at the end of day 4 and the survival rate drop to 50% at the end of day 5. At 120 μg−<sup>1</sup> leaf (26.7 μg cm−2; 1.5 mg g−<sup>1</sup> leaf), dead larvae were detected at day 3 and until the end of day 7, all larvae have died (Figure 6A). For *T. ni* feeding on plumbagin-covered cabbage leaves, an antifeeding effect was also determined in the low microgram per square centimeter range [41]. It also can be seen that the larvae avoided feeding on the leaves covered with high concentrations of plumbagin (Figure 6B,C). With respect to the results shown in Figure 5, it seems that larval growth is heavily affected at higher plumbagin concentrations of around 700 μg plumbagin g−<sup>1</sup> diet. However, the larvae were affected in growth but still survived at all concentrations tested (up to 900 μg g<sup>−</sup>1). The plumbagin concentrations used in the no-choice assay also showed no mortality up to 750 μg g−<sup>1</sup> leaf tissue. Only at the used concentration of 1.125 μg g−<sup>1</sup> leaf, we found the first larvae dying. This suggests that there might be a threshold of about 1 mg g−<sup>1</sup> food before the *S. littoralis* larvae begin to die. The experiment is somehow comparable with a recent study by Hu and colleagues [42]. They investigated the mortality of *Pieris rapae* and *Helicoverpa armigera* feeding on cabbage leaves dipped into solutions with different concentrations of plumbagin and juglone, respectively. For plumbagin, IC50 values of 11 μg mL−<sup>1</sup> (*P. rapae*) and 30 μg mL−<sup>1</sup> (*H. armigera*) were calculated [42]. However, these data are hard to rank as it is not known how much of the compounds of interest was finally on or in the leaf disc. Nevertheless, for all the latter assays, it is difficult to discriminate whether the larvae really die either because of the ingested compounds or of hunger as they consequently avoid feeding. Other studies used topical assays where the compound was added directly onto the insect's (e.g., *S. litura*, *A. janata*, and *Musa domestica*) body to investigate the toxicity of compounds [38,43]. This approach is worth to carry out but not qualified for studies on activities of compounds that are incorporated during herbivory.

**Figure 6.** (**A**) Survival rate of *Spodoptera littoralis* larvae feeding on *Ipomoea batatas* (sweet potato) leaf discs painted with various concentrations of plumbagin (*n* = 6). (**B**) Representative leaf discs at the end of the feeding period of day three. Leaf disks were renewed every day. (**C**) Leaf areas consumed by *S. littoralis* larvae (indicated in green) at day 3 depending on the applied plumbagin concentration.

However, the mode of action of NQ is not completely known. In general, NQ are redoxactive compounds that can generate oxidative stress [33]; moreover, there are hints for specific inhibition of enzymes and, hence, processes involved in insect development mainly the molting process in insects, e.g., the enzymes phenoloxidase [30], chitin synthetase [45], or ecdysone 20-monooxygenase [46]. The interaction with molting hormone pools is discussed as well [47]. Another study showed that in *Anopheles stephensi,* the level of certain enzymes such as esterases and SOD was decreased significantly in the presence of plumbagin, which also was active as repellent against *A. stephensi* at a concentration of 100 μg mL<sup>−</sup>1. Further histological investigations showed that muscles, midgut, and hindgut were the most affected tissues [48]. However, most studies suggest that, most likely, the insecticidal activity of plumbagin is based on the inhibition of ecdysis. This also includes a certain specificity against insects compared with neurotoxic insecticidal compounds.

Botanical or plant-derived insecticides have long been touted as environmentally friendly alternatives to synthetic insecticides for pest and disease management [3]; NQ combine the advantage of both low toxicity, compared with conventional pesticides, and restricted environmental contamination and, thus, might be promising candidates for an ecological agriculture.

### **3. Materials and Methods**

### *3.1. Insects and Plants*

*Spodoptera littoralis* Boisd. (Lepidoptera: Noctuidae) were hatched from eggs kindly provided by Syngenta Crop Protection (Stein, Switzerland) and reared on artificial diet (500 g hackled beans, 9 g ascorbic acid, 9 g 4-ethylbenzoic acid, 9 g vitamin E Mazola oil mixture (7.1%), 4 mL formaldehyde, 1.2 L water, 1 g-sitosterol, 1 g leucine, 10 g AIN-76 vitamin mixture, and 200 mL (7.5%) agar-water solution) at 23–25 ◦C with a 14 h photoperiod. Sweet potato (*Ipomoea batatas* Lam. cv Tainong 66) scions were grown as described [40] under a 16/8 h light/dark regime at 28/25 ◦C, respectively, and 70% relative humidity. *Nepenthes x ventrata* (*N. alata x N. ventricosa* hybrid) plants were grown at 21–23 ◦C, 50–60% relative humidity, and a 16/8 h light/dark photoperiod. Pitcher and

the associated leaf tissues were harvested at the time when the pitchers were just opened, directly frozen in liquid nitrogen and ground with mortar and pestle. Material was used directly (fresh) or freeze-dried before use.

### *3.2. Feeding Assays*

For feeding assays, second to third instar larvae of *S. littoralis* were used. Ground fresh or dried plant material (leaves and pitcher) from *N. x ventrata* was added to the artificial diet with the indicated quantities (*w*/*w*). Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone, C11H8O3; Fischer Scientific, Schwerte, Germany) was dissolved in acetone and added to the diet. Controls were prepared in the same way without plumbagin. At all the time, it was made sure that acetone was evaporated. For these feeding assays, 15 independent repeats were done. No-choice leaf disks feeding assays according to [34] were further performed on sweet potato. Therefore, leaf discs of 24 mm in diameter were punched out with a cork borer put directly on wet filter paper in a petri dish (5.5 cm diameter). Plumbagin was solved as described before and diluted to the required concentration with 2.5% (*w*/*v*) PEG 2000 (Sigma-Aldrich, Taufkirchen, Germany). That solution was added onto the surface of the discs at the concentrations indicated. For the no-choice assays, 6 independent repeats were performed.

Every day fresh diet or leaf discs were provided. All assays were performed with individual larvae to avoid cannibalism. Larvae were reared for the indicated periods on the particular diets and weighed at the given time.

### *3.3. Isolation of Plumbagin from Nepenthes x ventrata Leaves*

Freshly harvested *N. x ventrata* leaves (7.3 g) were immediately frozen in liquid N2 and freeze-dried. Dried tissue was ground and extracted with 100 mL dichloromethane (DCM) for 15 min by stirring in Erlenmeyer flasks. After precipitation for 20 min, the clear supernatant (50 mL) was collected and another 50 mL DCM was added to the remaining material for re-extraction, which was repeated six times. Collected supernatants were filtered, combined, and DCM was removed using a rotary evaporator. The dried extract (9.3 mg) was dissolved in 2 mL DCM transferred into a HPLC vial and dried again under N2 stream. For the whole procedure, only glassware was used. The NQ in the extract was identified by means of NMR spectroscopy by comparing spectral data with those of an authentic standard (plumbagin).

*N. x ventrata* leaf material was kept in 50 mL polypropylene tubes at room temperature over 6 months during which the NQ sublimed (Figure 4), leaving a yellowish stained plastic material. Absorbed compounds were extracted from closed tubes with DCM (10 mL) for 3 days at room temperature. The extract was transferred into a glass vial and evaporated using N2 gas. The residue was reconstituted with DMSO-*d*<sup>6</sup> and subjected to NMR analysis.

Identity of the sublimed and extracted plumbagin was confirmed by 1H-NMR spectroscopy. NMR spectra were measured on a Bruker Avance III HD spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany) equipped with a cryoplatform and a TCI 1.7 mm Micro-CryoProbe. Spectra were referenced to the residual solvent signal for DMSO-*d*<sup>6</sup> at δH 2.50. Spectrometer control and data processing was accomplished using Bruker TopSpin 3.6.1, and standard pulse programs as implemented in Bruker TopSpin 3.6.1 were used.

For a quantitative comparison of 1H NMR spectra of extracts of *N. x ventrata* leaf and pitcher tissue, the spectral intensity was adjusted to equal solvent signal areas. The areas of signals accounting for plumbagin (range: δH 8.00–7.00) were determined and used for calculation based on the respective areas of a plumbagin standard. For preparation of the experiment, 729 mg (FW) of each tissue was ground in liquid N2 and extracted with 20 mL of dichloromethane in closed vessels at room temperature with shaking. Extracts were filtered through Chromabond PTS phase separation cartridges (Macherey-Nagel, Düren, Germany) and the flow-through was evaporated with N2 gas at room temperature within 30 min. Afterwards, the residue was reconstituted with 1.2 mL DMSO-*d*6 and subjected to 1H-NMR spectroscopy.

### *3.4. Statistical Analysis*

Statistical calculations were performed using GraphPad Prism version 9.0.0 in all cases. Details are indicated in the particular figure legends. For EC50 analysis, the total response was normalized to run between 0% and 100% using control data. For growth experiments, larvae were picked randomly from a large population and all experiments were conducted out under highly standardized conditions to avoid investigator-included bias.

### **4. Conclusions**

Naphthoquinones are known metabolites in several plant species. Among these are various carnivorous plants including the pitcher plant *Nepenthes*. Plumbagin is a prominent NQ in *Nepenthes x ventrata* and it was detected by 1H-NMR in tissues in different concentrations (100 and 650 μg g−<sup>1</sup> fresh weight in pitcher and leaf, respectively). Plumbagin has known antimicrobial activities and is of pharmaceutical interest. Now, in different feeding assays with *Spodoptera littoralis* larvae the anti-feeding, growth-inhibiting and larvicidal activity of plumbagin or plumbagin-containing tissues was demonstrated at naturally occurring concentrations. Plumbagin as well as other NQ might become alternative compounds as natural insecticides in agriculture.

**Author Contributions:** A.R.-S., A.D.-L., and A.M. conceived the study and experiments. A.R.-S., A.D.-L., and C.P. performed the experiments and analyzed data. A.R.-S., A.D.-L., C.P., and A.M. discussed the data and wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** A.D.-L. was supported by a PhD fellowship from the DAAD (German Academic Exchange Service).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** All data generated or analyzed during this study are included in the main text.

**Acknowledgments:** We thank the greenhouse team of the MPI for cultivating the plants, Syngenta for providing *Spodoptera littoralis* and Andrea Lehr for rearing larvae.

**Conflicts of Interest:** The authors declare no conflict of interest.

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