Cytotoxic plant extracts towards insect cells: bioactivity and nanoencapsulation studies for application as biopesticides

molecules Article Cytotoxic Plant Extracts towards Insect Cells: Bioactivity and Nanoencapsulation Studies for Application as Biopesticides Ana I. F. Lopes 1,2, Mariana Monteiro 1,2, Ana R. L. Araújo 1, Ana Rita O. Rodrigues 2, Elisabete M. S. Castanheira 2, David M. Pereira 3, Pedro Olim 3, A. Gil Fortes 1 and M. Sameiro T. Gonçalves 1,* 1Centre of Chemistry, Department of Chemistry, Campus of Gualtar, University of Minho, 4710-057 Braga, Portugal; [email protected] (A.I.F.L.); [email protected] (M.M.); [email protected] (A.R.L.A.); [email protected] (A.G.F.) 2Centre of Physics, Department of Physics, Campus of Gualtar, University of Minho, 4710-057 Braga, Portugal; [email protected] (A.R.O.R.); [email protected] (E.M.S.C.) 3REQUIMTE/LAQV, Laboratory of Pharmacognosy, Department of Chemistry, Faculty of Pharmacy, University of Porto, R. Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal; [email protected] (D.M.P.); [email protected] (P.O.) *Correspondence: [email protected]; Tel.: +351253604372 Received: 12 November 2020; Accepted: 8 December 2020; Published: 11 December 2020   Abstract: The potential of plant extracts as bioinsecticides has been described as a promising field of agricultural development. In this work, the extracts of Punica granatum (pomegranate), Phytolacca americana (American pokeweed), Glandora prostrata (shrubby gromwell), Ulex europaeus (gorce), Tagetes patula (French marigold), Camellia japonica red (camellia), Ruta graveolens (rue or herb-of-grace) were obtained, purified, and their activity against Spodoptera frugiperda (Sf9) insect cells was investigated. From the pool of over twenty extracts obtained, comprising different polarities and vegetable materials, less polar samples were shown to be more toxic towards the insect cell line Sf9. Among these, a dichloromethane extract of R. graveolens was capable of causing a loss of viability of over 50%, exceeding the effect of the commercial insecticide chlorpyrifos. This extract elicited chromatin condensation and the fragmentation in treated cells. Nanoencapsulation assays of the cytotoxic plant extracts in soybean liposomes and chitosan nanostructures were carried out. The nanosystems exhibited sizes lower or around 200 nm, low polydispersity, and generally high encapsulation efficiencies. Release assays showed that chitosan nanoemulsions provide a fast and total extract release, while liposome-based systems are suitable for a more delayed release. These results represent a proof-of-concept for the future development of bioinsecticide nanoformulations based on the cytotoxic plant extracts. Keywords: biopesticides; bioinsecticides; Ruta graveolens; chlorpyrifos; nanoencapsulation 1. Introduction Many synthetic chemicals have been used in plant protection against agroindustrial and medicinal pests for the last 60 years [ 1 – 3 ]. This has caused a strong negative impact at different levels, and there are serious concerns about their side effects, such as the presence of residues on food and drinking water, toxicity to humans as well as to other non-target organisms, and high persistence, leading to an emergence of pest resistance. These facts have demanded for a drastic decrease in the use of chemical pesticides and opening up space for new and safer alternative strategies [ 1 ]. The use of natural compounds to control weeds has long been accepted as an environment friendly strategy, Molecules 2020,25, 5855; doi:10.3390/molecules25245855 www.mdpi.com/journal/molecules Molecules 2020,25, 5855 2 of 14 but farmers have mostly relied on toxic synthetic agrochemicals. New tools in pest management are based on biopesticides, which include semiochemicals and plant-incorporated protectants, botanical and microbial derived chemicals, and also new synthetic analogues [ 1 ]. The major problems in organic agriculture faced by farmers are weed management, crop diseases and insect controlling tools. Due to the diversity of chemicals present, natural products have become important sources for most biological activity optimization, also playing also an important role in drug discovery [ 4 ]. In recent years, research has been focused on the pesticidal efficiency of many phytocompounds, from essential oils to more complex mixtures extracted from different plants [ 5 , 6 ]. Compared to synthetic pesticides, phytochemical biopesticides present lower toxicity, are less persistent, and biodegradable [ 1 , 7 ]. Phytochemical biopesticides, such as nicotine, rotenone, pyrethrins and sabadilla alkaloids, are some examples of the first series of botanical pesticides used. However, due to either their stability or toxicity and adverse effects on human health, their use has been restricted, and despite an increasing application, these remain less than 5% of the total pesticide use [8]. Phytochemical biopesticides comprise a series of secondary metabolites that act in a synergistic way, which strongly reduces the chances for insect pests to develop resistance. Literature has covered in detail many plant species and their use as biopesticides. Punica granatum L. 1753, Phytolacca americana L. 1753, Glandora prostrata Loisel, 1844, Ulex europaeus L. 1753, Tagetes patula L. 1753, Camellia japonica L. 1753 and Ruta graveolens L. 1753 displayed a huge range of biological activities [ 3 , 9 – 19 ], including antibacterial (e.g., P. granatum) [ 9 ], antiviral (e.g., P. granatum, C. japonica) [ 10 , 11 ], antifungal (e.g., P. granatum,T. patula,C. japonica) [ 12 ], nematicide (T. patula) [ 13 ], herbicide (U. europaeus) and insecticide (e.g., T. patula and R. graveolens) [14–19]. The chemical composition and bioactivity of R. graveolens leaves were studied and tested against the maize weevil. The insecticidal and repellent properties of R. graveolens were analyzed and evaluated in contact and vapor forms, with a total of 45 compounds detected by chromatographic techniques, most of which were 2-ketones. The research carried out to date supports the evidence that R. graveolens leaves contain bioactive secondary plant metabolites, which justify its use against S.zeamais in storage pest management programs [ 20 ]. Lee et al. isolated essential oils from the flowers and leaves of R. graveolens, which were evaluated using fumigant and contact toxicity bioassays against store food pests, supporting the eco-potential of using R. graveolens essential oil and its major constituents in the management of stored pests [21,22]. Despite the reported activity against pests, several plant compounds exhibited some limitations in their application, due to their easy degradation and high volatility. Nanoencapsulation techniques arise as suitable strategies to allow for the preservation and controlled release of plant components [ 23 , 24 ]. Specifically, lipid-based nanosystems have been widely used as vehicles for bioactives in cosmetic and pharmaceutical industries [ 25 , 26 ], as well as for the encapsulation of plant extracts with insecticidal activity [ 27 , 28 ], being a promising route for a safe application of pesticides [ 29 ]. Plant extracts have also been encapsulated in cyclodextrins [ 24 ] and polymeric nanoparticles, the latter including alginate [ 30 ], gum arabic/maltodextrin [ 31 ], gelatin [ 32 ], polyvinylalcohol (PVA) [ 33 ], poly(dl-lactide-co-glycolide) (PLGA) [34] and chitosan nanostructures [35,36]. In this work, extracts from seven plant species, P. granatum (pomegranate), P. americana (American pokeweed), G. prostrata (shrubby gromwell), U. europaeus (gorce), T. patula (French marigold), C. japonica (camellia) and R. graveolens (rue or herb-of-grace), were isolated using water/ethanol and dichloromethane as solvents. The obtained extracts were evaluated as potential insecticides, through the assessment of their biological activity against Sf9 insect cell lines, compared with a commercial synthetic pesticide. Considering the insecticidal activity exhibited by the R. graveolens dichloromethane extract, encapsulation assays in chitosan nanoemulsions and lipid nanosystems were carried out, for the future development of green insecticide nanoformulations. Molecules 2020,25, 5855 3 of 14 2. Results and Discussion 2.1. Plant Extracts A series of seven plant species were selected, taking into account their production in the country, endemic characteristics, the information of literature, as well as their unexploited potential in the field of bioinsecticides. After the harvest, the parts of plants/fruits that were intended to be studied were separated, namely fruit peel in the case of P. granatum (pomegranate); berries in the case of P. americana (Americanpokeweed); flowersandleavesinthecaseofG.prostrata(shrubbygromwell),U.europaeus(gorce), R. graveolens (rue or herb-of-grace), and T. patula (French marigold)—in the former, flowers (separated from the receptacle) and leaves were extracted separately—and leaves in the case of C. japonica. The preparation of the plant material included drying under sunlight (20–25 ◦ C, 45 days) (peel of P. Granatum), the lyophilization (berries of P. americana, leaves and flowers of T. patula), or drying in an oven (40–45 ◦ C, 3 days), followed in both cases by grounding with a shredder to obtain the corresponding powder, which was passed through a sieve until a particle size ≤900 µm was obtained. Furthermore, these materials were subjected to Soxhlet extraction with solvent systems of different polarities, namely in a polar system, using a water/ethanol solution (1:1), and in a less polar one, using dichloromethane, for 9 or 4 h, respectively. The solvents were removed by evaporation under reduced pressure (dichloromethane extracts) followed by lyophilization (aqueous ethanolic extracts). In order to remove the chlorophyll content, dichloromethane extracts were passed through a Chromabond C18 column, using methanol as eluent, followed by solvent evaporation. The 22 extracts thus obtained (11 in water/ethanol and 11 in dichloromethane, Table 1) were submitted to assays of cell viability using the insect cell line Spodoptera frugiperda (Sf9), to assess their capacity as potential bioinsecticides. Table 1. Extracts of various species obtained using two different solvent systems and studied against Sf9. Entry Specie Part of the Plant Solvent Code 1Phytolacca americana Leaves DCM A1 2Phytolacca americana Berries DCM A2 3Phytolacca americana Berries Water/EtOH (1:1) D2 4Phytolacca americana Leaves Water/EtOH (1:1) D3 5Tagetes patula (red flowers) Leaves DCM (F.1-3) A3 6Tagetes patula (yellow flowers) Leaves DCM (F.1-4) A4 7Tagetes patula (orange flowers) Leaves DCM (F.1-3) A5 8Tagetes patula (red flowers) Flowers DCM A6 9Tagetes patula (red flowers) Leaves Water/EtOH (1:1) B6 10 Tagetes patula Flowers (red) Water/EtOH (1:1) C1 11 Tagetes patula (yellow flowers) Leaves Water/EtOH (1:1) C2 12 Tagetes patula (orange flowers) Leaves Water/EtOH (1:1) C3 13 Ruta graveolens Leaves DCM B1 14 Ruta graveolens Leaves Water/EtOH (1:1) D4 15 Ulex europaeus Leaves and flowers DCM B2 16 Ulex europaeus Leaves and flowers Water/EtOH (1:1) C5 17 Glandora prostrata Leaves and flowers DCM B3 18 Glandora prostrata Leaves and flowers Water/EtOH (1:1) D1 19 Punica granatum Fruit peel DCM B4 20 Punica granatum Fruit peel Water/EtOH (1:1) C4 21 Camellia japonica Leaves DCM B5 22 Camellia japonica Leaves Water/EtOH (1:1) C6 2.2. Evaluation of the Activity Against Sf9 Cells Given the high number of samples under study (22), we were interested in knowing which samples were the most toxic towards the Sf9 cell line. To this end, all extracts were evaluated at the same concentration (100 µ g/mL) for their impact in the viability of these cells after 24 h of incubation. For a clearer understanding, a heatmap of all results can be found in Figure 1(left). Considering the results, Molecules 2020,25, 5855 4 of 14 there was a clear cluster of a group of samples that were active (red) and those that were mostly inactive (blue). The most active samples were, in fact, those arising from the use of non-polar solvents, as it can be seen in clearer detail in Figure 1(right), when plotting the results from viability assays against the polarity of the extracts. This result can be explained by the fact that the use of nonpolar solvents allows the extraction of classes of natural products that are usually not extractable with highly polar solvents, such as water. As so, while polar solvents will extract molecules such as sugars, polyphenols and their glycosylated derivatives, the use of non-polar solvents widens the chemical diversity of the target molecules to include classes such as alkaloids, terpenes, coumarins, among others. Molecules 2020, 25, x FOR PEER REVIEW 4 of 15 same concentration (100 μg/mL) for their impact in the viability of these cells after 24 h of incubation. For a clearer understanding, a heatmap of all results can be found in Figure 1 (left). Considering the results, there was a clear cluster of a group of samples that were active (red) and those that were mostly inactive (blue). The most active samples were, in fact, those arising from the use of non-polar solvents, as it can be seen in clearer detail in Figure 1 (right), when plotting the results from viability assays against the polarity of the extracts. This result can be explained by the fact that the use of nonpolar solvents allows the extraction of classes of natural products that are usually not extractable with highly polar solvents, such as water. As so, while polar solvents will extract molecules such as sugars, polyphenols and their glycosylated derivatives, the use of non-polar solvents widens the chemical diversity of the target molecules to include classes such as alkaloids, terpenes, coumarins, among others. Figure 1. (Left): Results of the viability (%) of insect cells after treatment with the indicated samples. (Right): Comparison of the viability of cells treated with non-polar (A1–B5) vs. polar (B6–C4) extracts. Having reduced the number of samples under study, we were interested in knowing which species were triggering higher levels of toxicity. As seen in Figure 2, most samples reduced cell viability to the 50–75% range; however, the R. graveolens dichloromethane extract (B1, Table 1) was able to reduce the viability below 50%. For benchmarking purposes, we additionally tested the viability of Sf9 cells after exposure to the commercial synthetic insecticide chlorpyrifos. As shown in Figure 2, this molecule elicited ca. 50% of viability loss, thus being less potent than B1. For this reason, the latter was selected for subsequent studies. Extract Viability (%) Polar Apolar 0 50 100 Figure 1. ( Left ): Results of the viability (%) of insect cells after treatment with the indicated samples. ( Right ): Comparison of the viability of cells treated with non-polar ( A1 – B5 ) vs. polar ( B6 – C4 ) extracts. Having reduced the number of samples under study, we were interested in knowing which species were triggering higher levels of toxicity. As seen in Figure 2, most samples reduced cell viability to the 50–75%range; however, theR. graveolens dichloromethaneextract ( B1 ,Table 1) wasabletoreducethe viability below 50%. For benchmarking purposes, we additionally tested the viability of Sf9 cells after exposure to the commercial synthetic insecticide chlorpyrifos. As shown in Figure 2, this molecule elicited ca. 50% of viability loss, thus being less potent than B1 . For this reason, the latter was selected for subsequent studies. Molecules 2020, 25, x FOR PEER REVIEW 5 of 15 Figure 2. Viability of Sf9 cells after exposure to the indicated samples at 100 μg/mL, for 24 h. CHPY = chlorpyrifos. *** p < 0.001. Identity of samples (A1–A6 and B1–B5) as in Table 1. The UHPLC ESI-Q-TOF-MME analysis of R. graveolens extract revealed the presence of ten compounds, some of which show a molecular mass compatible with quinolin, benzopyran, and acridone derivatives (Table 2). A chromatogram of the extract is shown in the Supporting Information, Figure S1. Table 2. Mass spectrometry data and putative identification for the R. graveolens extract. The peak mentioned is the chromatogram peak number shown in the Supporting Information, Figure S2. Peak RT M+ Compound Reference 10 19.477 279 Graveoline [37] 11 20.455 259 Skimmianin [37] 11 20.455 261 4,6,7-Trimethoxy-furo[2,3-b]quinoline [38] Figure 2. Viability of Sf9 cells after exposure to the indicated samples at 100 µ g/mL, for 24 h. CHPY =chlorpyrifos. *** p<0.001. Identity of samples (A1–A6 and B1–B5) as in Table 1. Molecules 2020,25, 5855 5 of 14 The UHPLC ESI-Q-TOF-MME analysis of R. graveolens extract revealed the presence of ten compounds, some ofwhich showa molecularmass compatiblewith quinolin, benzopyran, andacridone derivatives (Table 2). A chromatogram of the extract is shown in the Supporting Information, Figure S1. Table 2. Mass spectrometry data and putative identification for the R. graveolens extract. The peak mentioned is the chromatogram peak number shown in the Supporting Information, Figure S2. Peak RT M+Compound Reference 10 19.477 279 Molecules 2020, 25, x FOR PEER REVIEW 5 of 15 Figure 2. Viability of Sf9 cells after exposure to the indicated samples at 100 μg/mL, for 24 h. CHPY = chlorpyrifos. *** p < 0.001. Identity of samples (A1–A6 and B1–B5) as in Table 1. The UHPLC ESI-Q-TOF-MME analysis of R. graveolens extract revealed the presence of ten compounds, some of which show a molecular mass compatible with quinolin, benzopyran, and acridone derivatives (Table 2). A chromatogram of the extract is shown in the Supporting Information, Figure S1. Table 2. Mass spectrometry data and putative identification for the R. graveolens extract. The peak mentioned is the chromatogram peak number shown in the Supporting Information, Figure S2. Peak RT M+ Compound Reference 10 19.477 279 Graveoline [37] 11 20.455 259 Skimmianin [37] 11 20.455 261 4,6,7-Trimethoxy-furo[2,3-b]quinoline [38] Graveoline [37] 11 20.455 259 Molecules 2020, 25, x FOR PEER REVIEW 5 of 15 Figure 2. Viability of Sf9 cells after exposure to the indicated samples at 100 μg/mL, for 24 h. CHPY = chlorpyrifos. *** p < 0.001. Identity of samples (A1–A6 and B1–B5) as in Table 1. The UHPLC ESI-Q-TOF-MME analysis of R. graveolens extract revealed the presence of ten compounds, some of which show a molecular mass compatible with quinolin, benzopyran, and acridone derivatives (Table 2). A chromatogram of the extract is shown in the Supporting Information, Figure S1. Table 2. Mass spectrometry data and putative identification for the R. graveolens extract. The peak mentioned is the chromatogram peak number shown in the Supporting Information, Figure S2. Peak RT M+ Compound Reference 10 19.477 279 Graveoline [37] 11 20.455 259 Skimmianin [37] 11 20.455 261 4,6,7-Trimethoxy-furo[2,3-b]quinoline [38] Skimmianin [37] 11 20.455 261 Molecules 2020, 25, x FOR PEER REVIEW 5 of 15 Figure 2. Viability of Sf9 cells after exposure to the indicated samples at 100 μg/mL, for 24 h. CHPY = chlorpyrifos. *** p < 0.001. Identity of samples (A1–A6 and B1–B5) as in Table 1. The UHPLC ESI-Q-TOF-MME analysis of R. graveolens extract revealed the presence of ten compounds, some of which show a molecular mass compatible with quinolin, benzopyran, and acridone derivatives (Table 2). A chromatogram of the extract is shown in the Supporting Information, Figure S1. Table 2. Mass spectrometry data and putative identification for the R. graveolens extract. The peak mentioned is the chromatogram peak number shown in the Supporting Information, Figure S2. Peak RT M+ Compound Reference 10 19.477 279 Graveoline [37] 11 20.455 259 Skimmianin [37] 11 20.455 261 4,6,7-Trimethoxy-furo[2,3-b]quinoline [38] 4,6,7-Trimethoxy-furo[2,3-b]quinoline [38] 16 23.932 216 Molecules 2020, 25, x FOR PEER REVIEW 6 of 15 16 23.932 216 8-Methoxypsoralen 5-Methoxypsoralen [39] 17 24.082 229 Pteleine [37] 18 33.006 314 Chalepin [37] 21 38.681 356 Rutamarin [37] 20 37.305 271 2-N-Nonyl-4-quinolone [38] 21 38.681 357 Gravacridonetriol [37] 2.3. Morphological Assessment In light of the pronounced impact of B1 in Sf9 cell viability, we were interested in understanding the underlying effect of the extract. To this end, the control and B1-treated cells have been imaged in order to assess putative differences in overall cell morphology and chromatin. As shown in Figure 3, B1 elicits a significant degree of chromatin condensation (blue arrows) and even fragmentation (yellow arrows), although overall cell integrity is maintained. This result suggests that B1 may be eliciting the loss of cell viability by triggering an organized process of cell death, in which morphological traits are compatible to those described here. Molecules 2020, 25, x FOR PEER REVIEW 6 of 15 16 23.932 216 8-Methoxypsoralen 5-Methoxypsoralen [39] 17 24.082 229 Pteleine [37] 18 33.006 314 Chalepin [37] 21 38.681 356 Rutamarin [37] 20 37.305 271 2-N-Nonyl-4-quinolone [38] 21 38.681 357 Gravacridonetriol [37] 2.3. Morphological Assessment In light of the pronounced impact of B1 in Sf9 cell viability, we were interested in understanding the underlying effect of the extract. To this end, the control and B1-treated cells have been imaged in order to assess putative differences in overall cell morphology and chromatin. As shown in Figure 3, B1 elicits a significant degree of chromatin condensation (blue arrows) and even fragmentation (yellow arrows), although overall cell integrity is maintained. This result suggests that B1 may be eliciting the loss of cell viability by triggering an organized process of cell death, in which morphological traits are compatible to those described here. 8-Methoxypsoralen 5-Methoxypsoralen [39] 17 24.082 229 Molecules 2020, 25, x FOR PEER REVIEW 6 of 15 16 23.932 216 8-Methoxypsoralen 5-Methoxypsoralen [39] 17 24.082 229 Pteleine [37] 18 33.006 314 Chalepin [37] 21 38.681 356 Rutamarin [37] 20 37.305 271 2-N-Nonyl-4-quinolone [38] 21 38.681 357 Gravacridonetriol [37] 2.3. Morphological Assessment In light of the pronounced impact of B1 in Sf9 cell viability, we were interested in understanding the underlying effect of the extract. To this end, the control and B1-treated cells have been imaged in order to assess putative differences in overall cell morphology and chromatin. As shown in Figure 3, B1 elicits a significant degree of chromatin condensation (blue arrows) and even fragmentation (yellow arrows), although overall cell integrity is maintained. This result suggests that B1 may be eliciting the loss of cell viability by triggering an organized process of cell death, in which morphological traits are compatible to those described here. Pteleine [37] 18 33.006 314 Molecules 2020, 25, x FOR PEER REVIEW 6 of 15 16 23.932 216 8-Methoxypsoralen 5-Methoxypsoralen [39] 17 24.082 229 Pteleine [37] 18 33.006 314 Chalepin [37] 21 38.681 356 Rutamarin [37] 20 37.305 271 2-N-Nonyl-4-quinolone [38] 21 38.681 357 Gravacridonetriol [37] 2.3. Morphological Assessment In light of the pronounced impact of B1 in Sf9 cell viability, we were interested in understanding the underlying effect of the extract. To this end, the control and B1-treated cells have been imaged in order to assess putative differences in overall cell morphology and chromatin. As shown in Figure 3, B1 elicits a significant degree of chromatin condensation (blue arrows) and even fragmentation (yellow arrows), although overall cell integrity is maintained. This result suggests that B1 may be eliciting the loss of cell viability by triggering an organized process of cell death, in which morphological traits are compatible to those described here. Chalepin [37] 21 38.681 356 Molecules 2020, 25, x FOR PEER REVIEW 6 of 15 16 23.932 216 8-Methoxypsoralen 5-Methoxypsoralen [39] 17 24.082 229 Pteleine [37] 18 33.006 314 Chalepin [37] 21 38.681 356 Rutamarin [37] 20 37.305 271 2-N-Nonyl-4-quinolone [38] 21 38.681 357 Gravacridonetriol [37] 2.3. Morphological Assessment In light of the pronounced impact of B1 in Sf9 cell viability, we were interested in understanding the underlying effect of the extract. To this end, the control and B1-treated cells have been imaged in order to assess putative differences in overall cell morphology and chromatin. As shown in Figure 3, B1 elicits a significant degree of chromatin condensation (blue arrows) and even fragmentation (yellow arrows), although overall cell integrity is maintained. This result suggests that B1 may be eliciting the loss of cell viability by triggering an organized process of cell death, in which morphological traits are compatible to those described here. Rutamarin [37] 20 37.305 271 Molecules 2020, 25, x FOR PEER REVIEW 6 of 15 16 23.932 216 8-Methoxypsoralen 5-Methoxypsoralen [39] 17 24.082 229 Pteleine [37] 18 33.006 314 Chalepin [37] 21 38.681 356 Rutamarin [37] 20 37.305 271 2-N-Nonyl-4-quinolone [38] 21 38.681 357 Gravacridonetriol [37] 2.3. Morphological Assessment In light of the pronounced impact of B1 in Sf9 cell viability, we were interested in understanding the underlying effect of the extract. To this end, the control and B1-treated cells have been imaged in order to assess putative differences in overall cell morphology and chromatin. As shown in Figure 3, B1 elicits a significant degree of chromatin condensation (blue arrows) and even fragmentation (yellow arrows), although overall cell integrity is maintained. This result suggests that B1 may be eliciting the loss of cell viability by triggering an organized process of cell death, in which morphological traits are compatible to those described here. 2-N-Nonyl-4-quinolone [38] 21 38.681 357 Molecules 2020, 25, x FOR PEER REVIEW 6 of 15 16 23.932 216 8-Methoxypsoralen 5-Methoxypsoralen [39] 17 24.082 229 Pteleine [37] 18 33.006 314 Chalepin [37] 21 38.681 356 Rutamarin [37] 20 37.305 271 2-N-Nonyl-4-quinolone [38] 21 38.681 357 Gravacridonetriol [37] 2.3. Morphological Assessment In light of the pronounced impact of B1 in Sf9 cell viability, we were interested in understanding the underlying effect of the extract. To this end, the control and B1-treated cells have been imaged in order to assess putative differences in overall cell morphology and chromatin. As shown in Figure 3, B1 elicits a significant degree of chromatin condensation (blue arrows) and even fragmentation (yellow arrows), although overall cell integrity is maintained. This result suggests that B1 may be eliciting the loss of cell viability by triggering an organized process of cell death, in which morphological traits are compatible to those described here. Gravacridonetriol [37] Molecules 2020,25, 5855 6 of 14 2.3. Morphological Assessment In light of the pronounced impact of B1 in Sf9 cell viability, we were interested in understanding the underlying effect of the extract. To this end, the control and B1 -treated cells have been imaged in order to assess putative differences in overall cell morphology and chromatin. As shown in Figure 3, B1 elicits a significant degree of chromatin condensation (blue arrows) and even fragmentation (yellow arrows), although overall cell integrity is maintained. This result suggests that B1 may be eliciting the loss of cell viability by triggering an organized process of cell death, in which morphological traits are compatible to those described here. Molecules 2020, 25, x FOR PEER REVIEW 7 of 15 Figure 3. Sf9 cells exposed to B1 (100 μg/mL) for 24 h (S Plan Fluor ELWD 40 × DIC N1 objective). Cell morphology was evaluated using 4′,6-diamidino-2-phenylindole (DAPI, chromatin) and phalloidin (actin). Yellow arrow: chromatin fragmentation; Blue arrow: chromatin condensation. (C)—control. Color blindfriendly image in Supporting Information Figure S2. 2.4. Nanoencapsulation Studies The size and size distribution of the chosen nanoencapsulation systems was determined by dynamic light scattering (DLS), Table 3. The liposomes prepared by the ethanolic injection method have a smaller size than the ones prepared by thin film hydration but exhibit a slightly higher polydispersity. The chitosan nanostructures (prepared by ionic gelation) are the encapsulation systems with the lowest hydrodynamic diameter. The sizes were maintained constant for two weeks, with no significant change in polydispersity, proving the stability of the prepared nanosystems. Table 3. Size (hydrodynamic diameter) and the polydispersity of soybean liposomes and chitosan nanostructures determined by dynamic light scattering (DLS) (SD: standard deviation from five independent measurements). System Size ± SD (nm) PDI ± SD Liposomes (ethanolic injection) 121 ± 28 0.256 ± 0.08 Liposomes (thin film hydration) 203 ± 17 0.185 ± 0.05 Chitosan nanostructures 118 ± 23 0.203 ± 0.02 Liposomes have been described as ideal bioactive delivery systems, due to their biocompatibility and their ability to carry both hydrophilic (in the aqueous lumen) and hydrophobic (in the lipid bilayer) payloads [40]. The results in Table 3 are in accordance with the ones reported in the literature for lecithin-based liposomal systems prepared by ethanolic injection [41,42] and thin film hydration [43,44], the latter being slightly larger. Chitosan nanostructures have also been described as advantageous for the encapsulation of pesticides and natural extracts [45–48]. The hydrodynamic diameter and polydispersity obtained for chitosan nanocapsules (Table 3) are also in accordance with the reported results for a similar preparation method [45]. The encapsulation efficiencies of the extract with the best insecticidal activity B1 in soybean liposomes and chitosan nanoemulsions are presented in Table 4. Figure 3. Sf9 cells exposed to B1 (100 µ g/mL) for 24 h (S Plan Fluor ELWD 40 × DIC N1 objective). Cell morphology was evaluated using 4 0 ,6-diamidino-2-phenylindole (DAPI, chromatin) and phalloidin (actin). Yellow arrow: chromatin fragmentation; Blue arrow: chromatin condensation. ( C )—control. Color blind-friendly image in Supporting Information Figure S2. 2.4. Nanoencapsulation Studies The size and size distribution of the chosen nanoencapsulation systems was determined by dynamic light scattering (DLS), Table 3. The liposomes prepared by the ethanolic injection method have a smaller size than the ones prepared by thin film hydration but exhibit a slightly higher polydispersity. The chitosan nanostructures (prepared by ionic gelation) are the encapsulation systems with the lowest hydrodynamic diameter. The sizes were maintained constant for two weeks, with no significant change in polydispersity, proving the stability of the prepared nanosystems. Table 3. Size (hydrodynamic diameter) and the polydispersity of soybean liposomes and chitosan nanostructures determined by dynamic light scattering (DLS) (SD: standard deviation from five independent measurements). System Size ±SD (nm) PDI ±SD Liposomes (ethanolic injection) 121 ±28 0.256 ±0.08 Liposomes (thin film hydration) 203 ±17 0.185 ±0.05 Chitosan nanostructures 118 ±23 0.203 ±0.02 Molecules 2020,25, 5855 7 of 14 Liposomes have been described as ideal bioactive delivery systems, due to their biocompatibility and their ability to carry both hydrophilic (in the aqueous lumen) and hydrophobic (in the lipid bilayer) payloads [ 40 ]. The results in Table 3are in accordance with the ones reported in the literature for lecithin-based liposomal systems prepared by ethanolic injection [ 41 , 42 ] and thin film hydration [ 43 , 44 ], the latter being slightly larger. Chitosan nanostructures have also been described as advantageous for the encapsulation of pesticides and natural extracts [ 45 – 48 ]. The hydrodynamic diameter and polydispersity obtained for chitosan nanocapsules (Table 3) are also in accordance with the reported results for a similar preparation method [45]. The encapsulation efficiencies of the extract with the best insecticidal activity B1 in soybean liposomes and chitosan nanoemulsions are presented in Table 4. Table 4. Encapsulation efficiencies (EE% ± SD) of the extract with the best insecticidal activity in liposomes and chitosan nanoemulsions (SD: standard deviation of three independent measurements). System Sample B1 Liposomes (ethanolic injection) 93.3 ±6 Liposomes (thin film hydration) 73.0 ±9 Chitosan nanoemulsions 94.0 ±2 The encapsulation efficiencies were generally high (above 70%), especially in liposomes prepared by the ethanolic injection method and in chitosan nanostructures. The release of the encapsulated extract was followed for 24 h at room temperature (25 ◦ C) towards phosphate buffer, pH =7.3 (Figure 4). The results evidence that chitosan nanostructures are suitable for a faster and complete release, while liposomes allow a delayed release. The difference in the release profiles obtained from liposomes prepared by the two techniques (thin film hydration and ethanolic injection) may be related with the hydrophobic character of this extract, as ethanolic injection is more advantageous for this type of bioactives [41]. Molecules 2020, 25, x FOR PEER REVIEW 8 of 15 Table 4. Encapsulation efficiencies (EE% ± SD) of the extract with the best insecticidal activity in liposomes and chitosan nanoemulsions (SD: standard deviation of three independent measurements). System Sample B1 Liposomes (ethanolic injection) 93.3 ± 6 Liposomes (thin film hydration) 73.0 ±9 Chitosan nanoemulsions 94.0 ± 2 The encapsulation efficiencies were generally high (above 70%), especially in liposomes prepared by the ethanolic injection method and in chitosan nanostructures. The release of the encapsulated extract was followed for 24 h at room temperature (25 °C) towards phosphate buffer, pH = 7.3 (Figure 4). The results evidence that chitosan nanostructures are suitable for a faster and complete release, while liposomes allow a delayed release. The difference in the release profiles obtained from liposomes prepared by the two techniques (thin film hydration and ethanolic injection) may be related with the hydrophobic character of this extract, as ethanolic injection is more advantageous for this type of bioactives [41]. The experimental release profiles were analyzed by a modified Korsmeyer–Peppas model [49] and the results are summarized in Table 5 (the fittings are shown in Supporting Information Figure S3). Figure 4. Release profiles of sample B1 from the liposomes and chitosan nanostructures. Table 5. Release parameters of the Korsmeyer–Peppas model fitted to the release profiles (R is the coefficient of determination). Nanosystem Ks n l  Liposomes (thin film hydration) 0.07 0.50 0.9 0.98 Liposomes (ethanolic injection) Liposomes (ethanolic injection) * 0.01 1.39 0 0.98 0.0045 1.74 0 0.99 Chitosan nanostructures Chitosan nanostructures * 0.04 1.07 2.13 0.98 0.0314 1.21 2.11 0.99 Considering that the nanoencapsulation systems are spherical, the n value in the range 0.43 < n < 0.85 suggests that the release profiles from liposomes prepared by thin film hydration can be mainly ascribed to diffusion and swelling, i.e., both contributions have comparable rates [49]. The samples marked with * in Table 5 were subjected to a different analysis, for which the release is initially assumed to be associated with the super case-ii transport mechanism, where erosion, relaxation and diffusion are responsible for extract release. Despite the reasonable fitting of the model to the Figure 4. Release profiles of sample B1 from the liposomes and chitosan nanostructures. The experimental release profiles were analyzed by a modified Korsmeyer–Peppas model [ 49 ] and the results are summarized in Table 5(the fittings are shown in Supporting Information Figure S3). Molecules 2020,25, 5855 8 of 14 Table 5. Release parameters of the Korsmeyer–Peppas model fitted to the release profiles (Ris the coefficient of determination). Nanosystem Ksn l R2 Liposomes (thin film hydration) 0.07 0.50 0.9 0.98 Liposomes (ethanolic injection) Liposomes (ethanolic injection) * 0.01 1.39 0 0.98 0.0045 1.74 0 0.99 Chitosan nanostructures Chitosan nanostructures * 0.04 1.07 2.13 0.98 0.0314 1.21 2.11 0.99 Considering that the nanoencapsulation systems are spherical, the nvalue in the range 0.43 <n<0.85 suggests that the release profiles from liposomes prepared by thin film hydration can be mainly ascribed to diffusion and swelling, i.e., both contributions have comparable rates [ 49 ]. The samples marked with * in Table 5were subjected to a different analysis, for which the release is initially assumed to be associated with the super case-ii transport mechanism, where erosion, relaxation and diffusion are responsible for extract release. Despite the reasonable fitting of the model to the experimental data obtained in liposomes prepared by injection and chitosan nanostructures (as implied by the coefficients of determination), it does not properly explain the data from a qualitative point of view. Therefore, the release profiles were also fitted to the Gompertz empirical function (Table 6), defined as Xt=Xmaxe−aeblog10 t where Xt and Xmax are the dissolved fraction percentages at time tand its maximum, a is a shape parameter and b is the dissolution rate per unit of time [ 50 ]. The fittings to this model are also generally good (see Supporting Information Figure S4). Although the model does not provide mechanistic information, the larger bvalues of the samples in chitosan indicate faster release kinetics, while liposomes obtained by hydration seem to be more effective in ensuring a slower release. Table 6. Release parameters obtained from the fitting of the Gompertz model to the release profiles. Nanosystem Xmax a B R2 Liposomes (thin film hydration) 49.38 3.61 1.55 0.99 Liposomes (ethanolic injection) 100 680.48 5.99 0.99 Chitosan nanostructures 100 163.23 5.34 0.97 The results obtained herein indicate that lipid-based and chitosan nanoencapsulation systems allow high encapsulation efficiencies of the extracts with insecticidal activity and provide different release rates. This represents a proof-of-concept to future developments of bioinsecticide nanoformulations using the most active extracts. 3. Experimental Section 3.1. Chemicals and Reagents Ethanol, methanol, dichloromethane, isopropanol, DMSO, acetic acid and tetrahydrofuran were purchased from Merck KGaA (Darmstadt, Germany). Chromabond C18 column was purchased from Sigma-Aldrich (St. Louis, MO, USA). Trypan blue and 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s modified eagle medium (DMEM), Hank’s balanced salt solution (HBSS), fetal bovine serum (FBS), penicillin–streptomycin solution (penicillin 5000 units/mL and streptomycin 5000 µ g/mL) and 0.25% trypsin-EDTA were obtained from GIBCO, Invitrogen™(Grand Island, NY, USA). Molecules 2020,25, 5855 9 of 14 3.2. Plant Material The seven species of plants were harvested in the North of Portugal, namely in Melgaço (42.1144 ◦ N, 8.2580 ◦ W) (P. granatum,P. americana,G. prostrata,U. europaeus,T. patula,C. japonica red) and Guimar ã es (41.4425 ◦ N, 8.2918 ◦ W) (R. graveolens), at the flowering stage in the case of G. prostrata,U. europaeus and T. patula in March 2019 (G. prostrata,U. europaeus) or October 2019 for the remaining plants. The plant material included fruit peel (P. granatum), berries (P. americana), flowers and leaves (G. prostrata, U. europaeus, and T. patula), as well as only leaves (C. japonica and R. graveolens). In the case of T. patula, the leaves and yellow, orange and red flowers (separated from the receptacle) were used in separate experiments. At least three specimens of each species were collected. The pomegranate peel (P. granatum) was dried under sunlight at 20–25 ◦ C during 45 days. The berries of P. americana, as well as the leaves and flowers of T. patula, were frozen at − 20 ◦ C until lyophilization in an Alpha 1-4 ld Plus–Christ freeze dryer (Martin Christ Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany). The leaves and flowers of the remaining species were dried in an oven at 40–45 ◦C during 3 days. After drying or lyophilization, the vegetable matter was ground with a shredder, and the resulting powder passed through a sieve until a particle size ≤ 900 µ m was obtained. The material was stored under vacuum for further use. 3.3. Plant Materials Extraction The dried or lyophilized plant materials of P. granatum,P. americana,G. prostrata,U. europaeus, T. patula,C.japonica,R. graveolens (8 g) were subjected to Soxhlet extraction with water/ethanol (1:1) (50 mL) or dichloromethane (50 mL) for 9 or 4 h, respectively. The organic solvents were removed by evaporation under reduced pressure at 40 ◦ C (Rotavapor ® R-210, BÜCHI Labortechnik AG, Flawil, Switzerland), followed by lyophilization (Alpha 1-4 ld Plus–Christ freeze dryer, Martin Christ Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany) in the case of extractions with water/ethanol (1:1). The resulting lyophilized extracts were stored under vacuum for further use. The evaporated extracts of dichloromethane were passed through a Chromabond C18 column, using methanol as eluent, for the removal of chlorophylls. After the solvent evaporation under vacuum at 35 ◦C, the resulting extracts were stored at −20 ◦C for further use. 3.4. Cell Culture Sf9 (Spodoptera frugiperda)cells werefromATCC/LGC Standards(Spain)maintained as asuspension culture and cultivated in Grace’s medium with 10% FBS and antibiotics at 28 ◦ C. Cells were used in the experiments while in the exponential phase of growth. HaCaT (human keratinocytes) cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin at 37 ◦ C, in a humidified atmosphere of 5% CO2. 3.5. Assessment of Viability For the assessment of viability, a resazurin-based method was used, as previously described [ 51 ]. Sf9 cells were plated at a density of 3 × 10 4 cells/well, incubated for 24 h and then exposed to the molecules under study for 24 h. After this period, a commercial solution of resazurin was added (1:10) and the kinetic reaction of fluorescence increase was monitored. An incubation time of 30 min was used. 3.6. Morphological Assessment For morphological studies, the cells were cultured in 96-well plates, as described above for viability experiments, in the presence of the samples under study. After incubation, the cells were washed with HBSS and fixed in 10% formalin solution for 30 min, at room temperature. DAPI (0.25 µ g/mL) and CF543 (5 U/mL) were added and the cells were stained for 25 min at room temperature and washed with HBSS, as described before [52,53].