Antioxidant essential oil–based nanoemulsions associated with levothyroxine: Physicochemical characterization and toxicity assessment in zebrafish

Antioxidant essential oil–based nanoemulsions associated with levothyroxine: Physicochemical characterization and toxicity assessment in zebrafish | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Antioxidant essential oil–based nanoemulsions associated with levothyroxine: Physicochemical characterization and toxicity assessment in zebrafish Thamiris Pinheiro Santos, Paulo Eduardo da Silva Bastos, Matheus Victor Viana de Melo, and 12 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9568099/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract This study aimed to develop nanoemulsions containing levothyroxine associated with essential oils with antioxidant activity and to evaluate their toxicity in zebrafish ( Danio rerio ). The essential oils of Piper aduncum , Citrus sinensis , and Carum carvi were characterized by qualitative and quantitative gas chromatography. The oil/water nanoemulsions were prepared using a high-energy emulsification method, and the effect of time and stirring speed was studied by experimental design. The mean size, polydispersity index (PDI), and zeta potential were evaluated as response variables. Antioxidant activity was also analyzed. Toxicity tests were conducted on zebrafish based on the OECD 236 guidelines, and the response variables were mortality, teratogenic effects, and behavioral tests. GC-MS analysis of C. carvi , C. sinensis , and P. aduncum identified 6, 16, and 13 compounds, with carbon (60.44 ± 0.80%), limonene (90.13 ± 1.11%), and dilapiol (78.41 ± 0.98%) being the major constituents. Nanoemulsions were successfully developed, with a mean size ranging from 218.5 to 489.2 nm, PDI of 0.113 to 0.478, and zeta potential of -12.5 to -39.1 mV. All oils exhibited antioxidant activity, with EC 50 values ranging from 204.3 to 288.5 µg/mL for DPPH and 156.6 to 194.9 µg/mL for ABTS. Toxicity tests revealed that all oils exhibited toxic effects with animals exposed to all nanoemulsions showing teratogenic effects, mostly at the larval stage. Only the C. carvi nanoemulsion affected the behavioral tests. In summary, nanoemulsions showed strong antioxidant activity and were well-characterized, but toxicity in zebrafish suggests safety concerns that require further research before therapeutic use. Chemical Biology Nanoscience Toxicology Carum carvi Citrus sinensis Piper aduncum Levothyroxine Danio rerio Figures Figure 1 Figure 2 1. Introduction Essential oils (EOs) are products of spices used since antiquity due to their medicinal properties. Wide acceptance by consumers requires the natural product to show safety and efficacy (Bilia et al., 2014 ). EOs are volatile liquids, being soluble in lipid and organic solvents, and synthesized by all plant organs (flowers, leaves, and bark) (Bilia et al., 2014 ; Pavoni et al., 2020 ). EO constituents are secondary plant metabolites (Aziz et al., 2019 ), including monoterpenes, sesquiterpenes, and phenylpropanoids. Citrus is the genus that includes the world's major fruit-cultivated species, Citrus sinensis (sweet orange), and Brazil is the world’s main producer of this fruit (Rezende et al., 2020 ). According to the Food and Agriculture Organization of the United Nations (FAO), in Brazil, the total production of this fruit was 16.713.534 tons in 2018. Antibacterial, antifungal, and antioxidant properties are reported for C. sinensis essential oil (Frassinetti et al., 2011 ). Popular uses include skin health and promoting detoxification, and pharmacological actions include reduced anxiety, agitation, stress, challenging behaviors, and insomnia (Ali et al., 2015 ). Piper is a genus of the Piperaceae family, widely distributed in subtropical regions. Piper aduncum is popularly known as long pepper, and monkey pepper among other names (Pohlit et al., 2006 ; Silva et al., 2019a ). In Brazil, it is used as a bactericide, leishmanicide, fungicide, antioxidant, anticancer, larvicide, insecticide, antiplatelet (anticoagulant), molluscicide, and antiviral (Pohlit et al., 2006 ; Silva et al., 2019a ). Carum carvi , also known as caraway and black zeera, is a plant from the Apiaceae family, grown in Northern Africa, western Asia, and Europe. Recently, it was introduced in other regions of the world like Russia, Iran, Iraq, Indonesia, and North America. Its potential biological effects include antioxidant, anti-inflammatory, anti-diabetic, anti-obesity, anti-spasmodic, anti-asthmatic, anti-microbial, anti-hemolytic, anti-foaming, and galactagogue (Obeid et al., 2020 ; Trifan et al., 2016 ). In addition, the essential oil of Carum Carvi is a potent hypothyroidism treatment (Obeid et al., 2020 ). In hypothyroidism, there is an accelerated production of free radicals and low availability of antioxidants that eliminate free radicals (Mancini et al., 2016 ). As a result of this imbalance, oxidative stress arises, which is implicated in the pathophysiological mechanism of hypothyroidism and other diseases (Mancini et al., 2016 ). In subclinical hypothyroidism patients, symptoms like memory deficit, despondency, depression, and anxiety are frequently reported (Almeida et al., 2007 ). Levothyroxine monotherapy is the standard treatment for patients with hypothyroidism. Patients are biochemically well-controlled with levothyroxine, but a substantial proportion of patients have persistent complaints, like symptoms of depression and impaired mental well-being. This raised the question of whether treatment with this synthetic hormone is sufficient for all patients or whether alternative therapies can be adopted to minimize these adverse effects (Chaker et al., 2017 ). The formulation of nanoemulsions (NEs) containing levothyroxine associated with essential oils with antioxidant activity may be a good alternative and overcome these limitations. Nanotechnology applied to the pharmaceutical industry is an opportunity for new treatment approaches that overcome the obstacles of conventional therapies (Rojas-Aguirre et al., 2016 ). Therefore, controlled release systems have been widely investigated to enable delayed and sustained drug release, controlling the site of drug release and reducing the amount of drug required for treatment (Singh et al., 2017 ). Among the controlled drug release systems, NEs are widely reported in the pharmaceutical research literature. NEs are produced by the mixture of two immiscible liquids (oil and water are usually used), stabilized by the addition of a surfactant suitable for the compounds used, which decreases the surface tension between the immiscible liquids (Singh et al., 2017 ). Sometimes, a combination of surfactants is necessary to ensure the formation and stabilization of NEs (McClements, 2012 ). NEs offer better bioavailability and drug release due to vesicle size, which is usually < 500 nm, giving emulsions a clear or hazy appearance (Aziz et al., 2019 ; Singh et al., 2017 ). Emulsification processes are commonly used for essential oils to solubilize and stabilize volatile compounds, which are easily degraded by exposure to environmental factors like heat, light, oxygen, and humidity (Aziz et al., 2019 ; Pavoni et al., 2020 ), allowing essential oils to be used in drug development. Emulsification of the C. sinensis (Velmurugan et al., 2017 )d aduncum (Silva et al., 2019a ) essential oils are described in the literature. In silico , in vitro , and in vivo methods are used to evaluate the toxicity of NEs because the nanoscale can change their toxicity. In silico and in vitro methods are beneficial for the preliminary toxicological assessment of NEs. However, animal tests are still needed. Zebrafish have become a well-established animal model for the toxicological and pharmacological screening of new drugs. Zebrafish can also reveal toxic mechanisms of discovery candidates as NEs and provide 3 Rs (replacement, reduction, and refinement) value to animal research (Cassar et al., 2020 ). In addition, animal model research can reveal the benefit/risk of new products, and nanotechnological products present a new dimension to the pharmacological/toxicological profile as compared with traditional drugs (Pensado-Lopez et al., 2021 ). Using zebrafish to evaluate the toxicity of new drugs at the pre-clinical stage allows easier and more rapid decisions on the viability of candidates and information about modifying these new drugs to produce safer and more effective alternatives (Cassar et al., 2020 ; Jayasinghe and Jayawardena, 2019 ). Zebrafish embryos are an effective model to study the toxicological effects of a broad spectrum of herbal medicines (Jayasinghe and Jayawardena, 2019 ). First, zebrafish are externally fertilized, and embryos/larvae are optically transparent during early developmental stages, allowing direct, noninvasive observations (Cassar et al., 2020 ). The zebrafish genome is 87% conserved with human genes (Jayasinghe and Jayawardena, 2019 ), and the zebrafish shares 82% of human disease genes (Cassar et al., 2020 ), but also behavioral similarities. Thyroid receptor proteins have 91% identity with human thyroid receptors (MacRae and Peterson, 2015 ). This conservation allows an effective comparison of this animal model with the human condition, including hypothyroidism. These advantages can be exploited to evaluate drug toxicity using D. rerio in pre-clinical studies to develop safer drugs (Jayasinghe and Jayawardena, 2019 ). In the present study, NEs of essential oils associated with levothyroxine were produced and characterized, and antioxidant activity was analyzed. Also, the toxicity of these produced NEs was evaluated in the zebrafish ( Danio rerio ) model. Toxic effects on fish development were tested using mortality, teratogenic effects, and animal behavior assays (thigmotaxis and touch sensitivity). These experiments provide insights into the potential utility of these NEs. 2. Material and methods 2.1. Collection of plant material Fresh leaves of the P. aduncum (8°01'10.1"S 34°56'50.4" W) were collected in the Mata de Dois Irmãos , in Recife , Pernambuco , Brazil. Leaf collections for specimens were performed at around 9:00 am in the dry season, in February 2019. The plants were identified by botanist Dra. Ângela Maria de Miranda Freitas ( Universidade Federal Rural de Pernambuco ). Voucher specimens were mounted and deposited in the Sérgio Tavares Herbarium of the UFRPE under numbers HST 18177. Fruits of the C. sinensis were collected in Sítio Cigarra , in the municipality of Santana do Mundaú , Alagoas-AL, in May 2019. Fruits collections for specimens were performed at around 11:00 am in the dry season. The plant was identified by Dra. Suzene Izídio da Silva from the Biology Department of the Universidade Federal Rural de Pernambuco (UFRPE). Voucher specimens were deposited in the Vasconcelos Sobrinho Herbarium of the UFRPE, under the number: 48735, C. sinensis Osbeck var. pear ). The oil of the Carum carvi was acquired from a local supplier. 2.2. Isolation, chemical analysis, identification, and quantification of the essential oil The essential oils from fresh leaves of Piper aduncum , and shells of the fruit of Citrus sinensis (100 g) were separately isolated using a modified Clevenger-type apparatus and hydrodistillation for 2h. The oil layers were separated and dried over anhydrous sodium sulfate, stored in hermetically sealed glass containers, and kept at a low temperature (-5 ºC) until analysis. Total oil yields were expressed as percentages (g/100 g of fresh plant material). All experiments were carried out in triplicate. For the Carum carvi oil was acquired from a local supplier (LASZLO, Brazil). Quantitative GC analysis was carried out using a PerkinElmer Clarus 500 GC apparatus equipped with a flame ionization detector (FID) and a non-polar DB-5 fused silica capillary column (30 m x 0.25 mm x 0.25 µm film thickness) (J & W Scientific). The oven temperature was programmed from 60 to 240°C at a rate of 3°C/min. Injector and detector temperatures were 260°C. Hydrogen was used as the carrier gas at a flow rate of 1 mL/min in split mode (1:30). The injection volume was 0.5 µL of diluted solution (1/100) of oil in n-hexane. The amount of each compound was calculated from GC-FID peak areas in the order of DB-5 column elution and expressed as a relative percentage of the total area of the chromatograms. Analyses were carried out in triplicate. The qualitative Gas Chromatography-Mass Spectrometry (GC-MS) analysis was carried out using a Varian 220-MS IT GC system with a mass selective detector, a mass spectrometer in EI 70 eV with a scan-interval of 0.5 s, and fragments from 40 to 550 Da. fitted with the same column and temperature program as that for the GC-FID experiments, with the following parameters: carrier gas = helium; flow rate = 1 mL/min; split mode (1:30); injected volume = 1 µL of diluted solution (1/100) of oil in n-hexane. Identification of the components was based on GC-MS retention indices with reference to a homologous series of C8-C40 n-alkanes calculated using the Van der Dool and Kratz equation (van Den Dool and Dec. Kratz, 1963 ) and by computer matching against the mass spectral library of the GC-MS data system (NIST 14 and WILEY 11th) and co-injection with authentic standards as well as other published mass spectra (Adams, 2017 ). Area percentages were obtained from the GC-FID response without the use of an internal standard or correction factors. 2.3. Preparation of levothyroxine stock solution Commercial levothyroxine (L-T 4 ) (composition: L-T 4 (37,5 µg), mannitol, maize starch, microcrystalline cellulose, butyl-hydroxyanisole, and magnesium stearate) was used in the experiments. The stock solution of L-T 4 was prepared by dissolving in distilled water, obtaining the final nominal concentration of 250 µg/L, which was stored under refrigeration (4˚C) until further use. 2.4. Preparation of nanoemulsions The oil/water (O/W) NEs were prepared using a high-energy emulsification method (dos Santos Magnabosco et al., 2022 ) by magnetic stirring from an oily phase (OP) produced from a mixture of 10–20% (w/w) of individual essential oils ( Carum carvi , Citrus sinensis , or Piper aduncum ), 10–20% (w/w) of sorbitan monooleate (Tween 80) and/or 10% polysorbate 80 (Span 80). The OP was first homogenized using a magnetic stirrer for 30 min at 22 g at room temperature (25 ± 2 ºC). Simultaneously in another container, water phase (WP) was prepared with or without (empty) levothyroxine diluted in 43.75 mL of phosphate buffer 100 mM, pH 7.4. Finally, 70% of the WP was dripped (flow of 3.5 mL/min) using a burette into the OP under magnetic stirring according to Duarte et al. ( 2015 ). The prepared NEs were designated by the initials of each essential oil: Carum carvi – CC; Citrus sinensis – CS; and Piper aduncum - PA. 2.5. Experimental designs A two-level 3 x 2 2 full experimental design was carried out to study the effect of time and stirring speed in the preparations of NEs. Factors (stirring speed and stirring time) of each formulation that mainly influenced the physicochemical parameters were evaluated at two levels and a central point (Table 1 ) in authentic triplicate. The mean size, polydispersity index (PDI), and zeta potential (ζ mV) of NEs were used as the response variables of the design study. Table 1 Two-level 3 x 2 2 full experimental designs of nanoemulsions of each essential oil associated with levothyroxine. Run Stirring speed ( g ) Stirring time (h) Essential oil (g) Tween 80 (g) Span 80 (g) Phosphate-buffered + Levothyroxine (250 µg/L) 1 10 g 1 h 0.5 ( Carum carvi ) 1.0 0.0 3.5 2 39 g 1 h 0.5 ( Carum carvi ) 1.0 0.0 3.5 3 10 g 2 h 0.5 ( Carum carvi ) 1.0 0.0 3.5 4 39 g 2 h 0.5 ( Carum carvi ) 1.0 0.0 3.5 5 (CP) 22 g 1 h 30 min 0.5 ( Carum carvi ) 1.0 0.0 3.5 6 10 g 1 h 1.0 ( Citrus sinensis ) 1.0 0.0 3.0 7 39 g 1 h 1.0 ( Citrus sinensis ) 1.0 0.0 3.0 8 10 g 2 h 1.0 ( Citrus sinensis ) 1.0 0.0 3.0 9 39 g 2 h 1.0 ( Citrus sinensis ) 1.0 0.0 3.0 10 (CP) 22 g 1 h 30 min 1.0 ( Citrus sinensis ) 1.0 0.0 3.0 11 10 g 1 h 0.5 ( Piper aduncum ) 0.5 0.5 3.5 12 39 g 1 h 0.5 ( Piper aduncum ) 0.5 0.5 3.5 13 10 g 2 h 0.5 ( Piper aduncum ) 0.5 0.5 3.5 14 39 g 2 h 0.5 ( Piper aduncum ) 0.5 0.5 3.5 15 (CP) 22 g 1 h 30 min 0.5 ( Piper aduncum ) 0.5 0.5 3.5 Legend: Runs 1 to 5 are Carum carvi samples with a central point (CP – run 5); 6 to 10 are Citrus sinensis samples with a central point (CP – run 10); 11 to 15 are Piper aduncum samples with a central point (CP – run 15). 2.6. Physicochemical characterization The NEs were diluted (50x) in double-distilled water before analysis to reduce the turbidity before microscopic analysis. The average size (nm), polydispersity index (PDI), and zeta potential (mV) of the oil vesicles were evaluated using the standard photon correlation spectroscopy (PCS) technique fixed at 90º to 25 ºC using a Zetasizer Nano ZS (Malvern, UK) (Cadena et al., 2013 ; dos Santos Magnabosco et al., 2022 ). The analysis was performed at Laboratórios Associados em Rede de Nanotecnologia (LARnano) of the Universidade Federal de Pernambuco (UFPE). The data were measured in triplicate. 2.7. Antioxidant Activity of nanoemulsions Antioxidant activity was analyzed by DPPH˙ and ABTS˙ + radicals. The experiments were performed at Laboratório de Produtos Naturais Bioativos (LPNBio) of the Universidade Federal Rural de Pernambuco (UFRPE). The antioxidant activity of the NEs was tested against the free radical DPPH following the methodology of Araujo et al. ( 2022 ). Stock solutions were prepared from the extracts and methanol fraction at several concentrations (0.1 to 5.0 mg/mL). Through preliminary analysis, appropriate quantities of stock solutions of the samples and 450 µL of the solution of DPPH ˙ (23.6 mg/mL in EtOH) were transferred to 0.5 mL Eppendorf tubes, and the sample was diluted with EtOH following homogenization. Samples were sonicated for 30 min and the amount of DPPH ˙ was measured in a 96-well plate using a UV-vis spectrophotometer (Biochrom EZ Read 2000) at 517 nm. Ascorbic acid was used as a positive control. The determination of the antioxidant activity in the NEs to detect the radical cation ABTS ˙ + was carried out following the methodology described by Araujo et al. ( 2022 ), in a UV-vis spectrophotometer (Biochrom EZ Read 2000) using Trolox as the standard compound. The starting concentrations of the solutions of the samples were 0.1–1.0 mg/mL, with the addition of 450 µL of the radical ABTS ˙ + solution to give final concentrations of 2.5–100.0 µg/mL samples. Samples were protected from light and sonicated for 6 min. The absorbance of the samples and the positive control in a 96-well microplate were measured at a wavelength of 734 nm. In the two tests each concentration was tested in triplicate, and the percentage of free radical scavenging activity of DPPH˙ or ABTS ˙ + was calculated by the equation: %SA = 100 x (Abs control – Abs sample ) / Abs control , where Abs control is the absorbance of the control containing only the ethanol solution of DPPH˙ or ABTS + and Abs sample is the absorbance of the radical in the presence of the sample or standard ascorbic acid. Antiradical efficiency was established using linear regression analysis and the 95% confidence interval (p < 0.05) obtained using the statistical program GraphPad Prism 5.0. 2.8. Fish husbandry, crossing, and toxicity test Toxicity tests were performed using zebrafish ( Danio rerio ; WT strain; 7 months old) as an animal model. The experiments were carried out at Laboratório de Ecofisiologia e Comportamento Animal (LECA) – Universidade Federal Rural de Pernambuco (UFRPE). Adult zebrafish used in the experiments were obtained from a commercial supplier and housed in quarantine to detect or confirm the absence of pathogens or diseases maintained according to OECD 236 guidelines (OECD, 2025 ). The protocols used in this study were approved by the Ethics Committee in the use of animals of the same University, protocol number 2008150721. Adult male/female zebrafish were isolated in spawning aquariums (Alesco Zebclean) with a 2:1 ratio (Westerfield, 2000 ). For fish crossing, dividers between males and females were removed, and they were allowed to breed for 40 min. The embryos were collected and washed with distilled water in Petri dishes. Healthy embryos (normal development of the blastula) were used to form experimental groups. The experiments were conducted with 5 x 15 animals per experimental group (a total of 825 animals with replicates and repetitions). The assay was based on the OECD guidelines on the Fish Embryo Acute Toxicity (FET) Test (OECD, 2025 ) and Silva et al. ( 2019b ). The toxicity test started at 1-hour post-fertilization (hpf). Embryos were maintained in sterile polystyrene test chambers (80 mL) and maintained in an incubator with a light-dark cycle of 14:10 h, controlled temperature of 26 ± 1˚C, and pH of 7.5 ± 0.5. Daily water renewal was performed to maintain the NEs levels. The study used a control group and experimental groups that were described in Table 2 with two dilution rates where 4444x and 3200x to evaluate toxicity. These dilution ratios were chosen based on studies showing most of the animals survived at 96 hpf. The final nominal concentration of L-T 4 in all experimental groups was the same as described above (item 2.3). The exposure to NEs started at 1-h post-fertilization (hpf) being renewed daily until 96 hpf, according to OECD 236 (2025). Any residue generated by these compounds during the experiments was treated by the advanced oxidative process (AOP) in a reactor using photo-oxidation UV (16w) and H 2 O 2 (Hansen and Andersen, 2012 ; Silva et al., 2019b ). Table 2 The dilution ratio of the chemical compounds used in the experimental groups for the acute toxicity test. Experimental groups Abbreviations Dilution ratio Control C - Levothyroxine L-T 4 320x Empty (E) Carum carvi (CC) nanoemulsion (0.5 g oil, 1.0 g tween, 3.5 g of phosphate buffer) diluted ECC 3200x Carum carvi (CC) nanoemulsion (0.5 g oil, 1.0 g tween, 3.5 g of phosphate buffer with levothyroxine) diluted CCN44 4444x Carum carvi (CC) nanoemulsion (0.5 g oil, 1.0 g tween, 3.5 g phosphate buffer with levothyroxine) diluted CCN32 3200x Empty (E) Citrus sinensis (CS) nanoemulsion (1.0 g oil, 1.0 g tween, 3.0 g phosphate buffer) diluted ECS 3200x Citrus sinensis (CS) nanoemulsion (1.0 g oil, 1.0 g tween, 3.0 g phosphate buffer with levothyroxine) diluted CSN44 4444x Citrus sinensis (CS) nanoemulsion (1.0 g oil, 1.0 g tween, 3.0 g phosphate buffer with levothyroxine) diluted CSN32 3200x Empty Piper aduncum (PA) nanoemulsion (0.5 g oil, 0.5 g tween, 0.5 g spam, 3.5 g of phosphate buffer) diluted EPA 3200x Piper aduncum (PA) nanoemulsion (0.5 g oil, 0.5 g tween, 0.5 g spam, 3.5 g of phosphate buffer with levothyroxine) diluted PAN44 4444x Piper aduncum (PA) nanoemulsion (0.5 g oil, 0.5 g tween, 0.5 g spam, 3.5 g of phosphate buffer with levothyroxine) diluted PAN32 3200x 2.9. Effects of levothyroxine and nanoemulsions on zebrafish embryonic development Mortality data were observed at 24, 48, 72, and 96 hours post-fertilization (hpf). The determination of embryo lethality was assayed according to OECD 236 (2025). Determination of mortality as coagulation, tail not detached, no somite formation, absence of heartbeat, and lack of hatching. Teratogenic effects were evaluated according to OECD 236 (2025), Silva et al. ( 2019b ), and Cadena et al. ( 2020 ). The teratogenic effects studied were spine deformation, tail deformation, yolk sac edema, and pericardial edema. The effects were identified in images of lateral and dorsal views in vivo . The images were captured using a Hayear Mod. HY-2307 digital camera microscope and S-EYE 1.4.2.474 software. We consider larvae affected when at least, mortality or one teratogenic effect was observed under a microscope (Cadena et al., 2020 ). These results were presented as a percentage of affected animals per experimental group. 2.10. Effects of levothyroxine and nanoemulsions on zebrafish animal behavior Behavioral tests were used as a method to study the effects of NEs s in zebrafish ( Danio rerio ). The thigmotaxis (TH Test) is the tendency of an animal to move toward the edge or periphery of plates that are introduced. Therefore, this animal avoids the center of this environment. This parameter evaluates the anxiety like-behavior in zebrafish (Basnet et al., 2019 ). The thigmotaxis test was evaluated at 144 hpf, according to Cadena et al. ( 2020 ). The larvae were left at 10 min of acclimatization in 48-well plates (48 larvae per experimental group) in a light environment, and if larvae remained near the wall of plates, the response was recorded. The touch sensitivity test (TS Test) was performed according to Cadena et al. ( 2020 ). This test evaluated the larval response to mechanical stimuli. At 144 hpf, the larvae were touched on the tail or head with a pipette tip. The response was recorded if the larva exhibited swimming behaviors (slow or fast), that is, an escape response. 2.11. Statistical analysis All data were shown as mean ± SD. ANOVA was used to analyze data, and the means were compared by Tukey's test (p < 0.05). Statistical analyses were carried out using the Origin Pro Academic 2015 (Origin Lab. Northampton, MA USA) and GraphPad Prism 5.0. 3. Results 3.1. Isolation, chemical analysis, identification, and quantification of the essential oil The chemical composition of essential oils from the leaves of P. aduncum , C.carvi , and C. sinensis is shown in Table 3 . Hydrodistillation of P. aduncum leaves provided yellowish oil. The extraction of the oil revealed a yield of 2.52 ± 0.10%. Moreover, for C. sinensis hydrodistillation provided a colorless citrus-scented oil. The extraction of the oil revealed a yield of 1.4 ± 0.13%. The GC-MS analysis of P. aduncum oils allowed the identification of 13 compounds, representing a total of 98.53 ± 1.01% of the oil. The oil revealed the presence of compounds belonging to the class of monoterpenes (4.03 ± 0.05%), sesquiterpenes (16.09 ± 0.10%), and phenylpropanoid (78.41 ± 0.98%). The phenylpropanoid dilapiol (78.41 ± 0.98%) was characterized as a major constituent in the oil. For C. carvi oil the GC-MS analysis allowed the identification of 6 compounds, representing a total of 98.37 ± 0.82% of the oil. The oil revealed a predominance of compounds belonging to the monoterpene class (98.37 ± 0.82%), having as main constituent carbon (60.44 ± 0.80%) followed by limonene (34.14 ± 0.64%). Finally, C. sinensis , the analysis by GC-MS allowed the identification of 16 compounds, representing a total of 97.47 ± 1.22% of the oil. The oil revealed the presence of compounds belonging to the class of monoterpenes (96.39 ± 1.08%) and sesquiterpenes (1.09 ± 0.01%). For this oil, limonene (90.13 ± 1.11%) was characterized as a major constituent in the oil. Table 3 Chemical composition of essential oils of Piper aduncum, Carum carvi , and Citrus sinensis. Compounds RI a RI b P. aduncum C. carvi C. sinensis Method of identification α -Thujene 921 924 - - 0.16 ± 0.00 RI, MS α -Pinene 928 932 - 0.33 ± 0.02 1.25 ± 0.07 RI, MS, CI Sabinene 966 969 - - 0.95 ± 0.03 RI, MS β -Pinene 971 974 0.11 ± 0.00 0.13 ± 0.01 1.87 ± 0.10 RI, MS, CI iso-Sylvestrene 1005 1007 0.45 ± 0.02 - - RI, MS p -Cymene 1018 1020 - 2.14 ± 0.08 - RI, MS, CI Limonene 1023 1024 - 34.14 ± 0.64 90.13 ± 1.11 RI, MS, CI β -Phellandrene 1025 1025 - - 0.11 ± 0.00 RI, MS, CI (Z)- β -Ocimene 1031 1032 - - 0.28 ± 0.01 RI, MS ( E )- β -Ocimene 1040 1044 1.01 ± 0.05 - - RI, MS γ -Terpinene 1055 1054 - - 0.35 ± 0.02 RI, MS Terpinolene 1088 1086 0.13 ± 0.00 - 0.38 ± 0.01 RI, MS Linalool 1094 1095 - - 0.22 ± 0.01 RI, MS, CI cis - β -Terpineol 1143 1140 - - 0.36 ± 0.01 RI, MS Citronellal 1145 1148 - - 0.11 ± 0.00 RI, MS Terpinen-4-ol 1174 1174 0.50 ± 0.01 - - RI, MS, CI ( E )-Isocitral 1175 1177 - - 0.22 ± 0.03 RI, MS Dill ether 1180 1184 - 1.19 ± 0.10 - RI, MS α -Terpineol 1189 1186 1.83 ± 0.03 - - RI, MS, CI δ -Elemene 1340 1335 0.75 ± 0.04 - - RI, MS Carvone 1235 1239 - 60.44 ± 0.80 - RI, MS β -Caryophyllene 1415 1417 3.20 ± 0.09 - 0.08 ± 0.00 RI, MS, CI α - trans -Bergamotene 1431 1432 - - 0.11 ± 0.01 RI, MS α -Humulene 1456 1452 8.32 ± 0.34 - RI, MS, CI γ-Gurjunene 1474 1475 - - 0.9 ± 0.00 RI, MS γ -Muurolene 1477 1478 0.55 ± 0.02 - - RI, MS γ -Himachalene 1488 1481 1.93 ± 0.06 - - RI, MS Germacrene B 1555 1559 1.34 ± 0.09 - - RI, MS Dillapiole 1620 1620 78.41 ± 0.98 - - RI, MS, CI Total 98.53 ± 1.01 98.37 ± 0.82 97.47 ± 1.22 Monoterpenes 4.03 ± 0.05 98.37 ± 0.82 96.39 ± 1.12 Sesquiterpenes 16.09 ± 0.10 - 1.09 ± 0.01 Phenylpropanoids 78.41 ± 0,98 - - Legend: RI a = Retention indices calculated from retention times in relation to those of a series C 8 -C 40 of n-alkanes on a 30m DB-5 capillary column. RI b = Retention indices from literature. SD = Standard deviation. RI = retention indices, MS = mass spectroscopy, and CI = Co-injection with authentic compounds. See supplementary material for chromatograms. 3.2. Physicochemical characterization of nanoemulsions The results of the physicochemical characterization of NEs are shown in Table 4 (A and B). Our data revealed significant results in the zeta potential for the C. carvi NE suggesting that the higher agitation speed promotes more negative zeta potential. For the C. sinensis NE, a significant result was observed only in the size of the vesicles, demonstrating that the higher stirring speed reduces the size of the vesicles. In addition, a significant result in PDI values observed in the P. aduncum NEs suggested that the shorter stirring time induces the smaller PDI values. Finally, the C. carvi nanoemulsions were produced using a high stirring speed and short stirring time (1129 g /1 h), C. sinensis NE was produced using a low stirring speed, and short stirring time (282 g /1 h) and P. aduncum NEs produced using low stirring speed and long stirring time (282 g /2 h) were chosen for the next experiments. Our choice was based on the best physicochemical parameters for industrial production. Table 4 Effect of variables stirring speed ( g ) and stirring time (h) in the physicochemical parameters of nanoemulsions of each essential oil associated with levothyroxine. Bold values indicate significant results by the Tukey test (p < 0.05). A) Size, polydispersity index (PDI), and zeta potential data for each nanoemulsion Emulsion formulations (g/h) Mean size (nm) PDI ζ (mV) Carum carvi 282 g / 1 h 384.7 ± 4.9 0.333 -12.5 ± 0.6 Carum carvi 1129 g / 1 h 277.1 ± 2.5 0.304 -22.7 ± 0.4 Carum carvi 282 g / 2 h 408.6 ± 7.8 0.409 -15.1 ± 0.4 Carum carvi 1129 g / 2 h 259.0 ± 12.0 0.323 -14.6 ± 0.4 Carum carvi 635 g / 1 h 30 min 218.5 ± 4.4 0.315 -12.8 ± 0.5 Carum carvi 635 g / 1 h 30 min 223.1 ± 3.3 0.317 -13.3 ± 0.9 Carum carvi 635 g / 1 h 30 min 225.8 ± 6.3 0.257 -14.6 ± 0.6 Citrus sinensis 282 g / 1 h 287.3 ± 20.5 0.226 -21.3 ± 2.6 Citrus sinensis 1129 g / 1 h 401.9 ± 45.5 0.113 -18.5 ± 0.3 Citrus sinensis 282 g / 2 h 325.3 ± 3.7 0.424 -16.9 ± 0.7 Citrus sinensis 1129 g / 2 h 391.8 ± 35.7 0.223 -20.9 ± 1.6 Citrus sinensis 635 g / 1 h 30 min 200.2 ± 7.4 0.339 -12.9 ± 0.3 Citrus sinensis 635 g / 1 h 30 min 235.2 ± 6.0 0.460 -16.0 ± 0.3 Citrus sinensis 635 g / 1 h 30 min 224.9 ± 10.2 0.235 -18.5 ± 2.0 Piper aduncum 282 g / 1 h 489.1 ± 78.1 0.234 -32.2 ± 0.3 Piper aduncum 1129 g / 1 h 445.9 ± 298.0 0.299 -36.8 ± 0.3 Piper aduncum 282 g / 2 h 326.9 ± 15.7 0.406 -30.6 ± 1.3 Piper aduncum 1129 g / 2 h 401.7 ± 21.1 0.478 -39.1 ± 0.4 Piper aduncum 635 g / 1 h 30 min 631.3 ± 118.2 0.302 -36.1 ± 1.3 Piper aduncum 635 g / 1 h 30 min 426.4 ± 42.7 0.343 -31.3 ± 1.6 Piper aduncum 635 g / 1 h 30 min 489.2 ± 57.4 0.265 -35.7 ± 0.6 B. ANOVA of 3 x 2 2 full experimental design Carum carvi Size Sum of squares Df Mean square F P Stirring speed 16537.96 1 16537.96 2.393784 0.219558 Stirring time 8.41 1 8.41 0.001217 0.974359 PDI Sum of squares Df Mean square F P Stirring speed 0.003306 1 0.003306 1.011704 0.420410 Stirring time 0.002256 1 0.002256 0.690407 0.493425 Zeta Sum of squares Df Mean square F P Stirring speed 77.4400 1 77.44000 89.6988 0.010965 Stirring time 1.4400 1 1.44000 1.6680 0.325658 Citrus sinensis Size Sum of squares Df Mean square F P Stirring speed 8226.49 1 8226.49 25.42729 0.037150 Stirring time 198.81 1 198.81 0.61450 0.515195 PDI Sum of squares Df Mean square F P Stirring speed 0.024649 1 0.24649 1.943876 0.297943 Stirring time 0.023716 1 0.023716 1.870298 0.304843 Zeta Sum of squares Df Mean square F P Stirring speed 0.36000 1 0.36000 0.045743 0.850467 Stirring time 1.00000 1 1.00000 0.127065 0.755588 Piper aduncum Size Sum of squares Df Mean square F P Stirring speed 249.64 1 249.64 0.022653 0.894171 Stirring time 10650.24 1 10650.24 0.966443 0.429218 PDI Sum of squares Df Mean square F P Stirring speed 0.004692 1 0.004692 3.08228 0.221235 Stirring time 0.030800 1 0.030800 20.23226 0.046039 Zeta Sum of squares Df Mean square F P Stirring speed 42.90250 1 42.90250 6.048285 0.133109 Stirring time 0.12250 1 0.12250 0.017270 0.907475 3.3. Antioxidant activity of nanoemulsions (DPPH˙ and ABTS˙ + ) The antioxidant activity of NEs from the leaves of P. aduncum , C. carvi , and C. sinensis associated with levothyroxine is presented in Table 5 . According to our results, all NEs showed antioxidant activity. In the DPPH test, the highest antioxidant activity was observed for C. carvi NE (EC 50 = 204.3 µg/mL). In the test with the radical ABTS, the P. aduncum NE (EC 50 = 156.6 µg/mL) showed the best antioxidant activity. Table 5 Antioxidant activity of the essential oil of species of Carum carvi , Citrus sinensis , and Piper aduncum . Legend: Ascorbic acid and TROLOX (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) were used as reference antioxidants. Chemicals DPPH ABTS EC 50 µg/mL (Confidence interval) EC 50 µg/mL (Confidence interval) C. carvi 204.3 (199.4-208.2) 194.9 (190.4-199.4) C. sinensis 288.5 (280.8-295.1) 191.5 (186.7-196.3) P. aduncum 266.5 (261.4–272.0) 156.6 (151.4-161.8) Ascorbic acid 1.6 (1.4–1.8) - TROLOX - 4.1 (3.7–5.8) 3.4. Effects of levothyroxine and nanoemulsions on zebrafish embryonic development Toxicity tests were carried out with zebrafish embryos and larvae to evaluate the toxicity of the NEs. The results are shown in Fig. 1 from the zebrafish embryo and larval stages exposed to different dilution ratios of L-T 4 and NEs of each essential oil. Mortality was the only significant effect observed at 24 hpf in the PAN32 group. Teratogenic effects were observed in embryos still in the chorion. However, toxic effects were most observed in the 72 hpf larvae stage for the following groups: ECS, CSN32, EPA, PAN44, and PAN32, with P. aduncum NE being the most toxic. While at 96 hpf, significant teratogenic effects were observed in groups exposed to all NEs. Yolk sac edema is the most common toxic effect presented in zebrafish larvae, followed by pericardial edema. 3.5. Effects of levothyroxine and nanoemulsions on zebrafish animal behavior The results of the thigmotaxis test (TH test) and touch sensitivity response (TS test) are shown in Fig. 2 A and 2 B. The data in the TH test represents the number of animals that move to the edge or periphery of plates. We did not observe toxic effects in this test when larvae were exposed to L-T 4 or any of the NEs (Fig. 2 A). TS test was also performed by the mechanical stimuli to evaluate if L-T 4 or NEs exposure affects the swimming activity of the larvae. Our results (Fig. 2 B) show that this parameter was affected only in the larvae exposed to empty C. carvi NE (ECC). 4. Discussion Essential oils have been used by our ancestors as therapeutic agents for the treatment of various diseases. Their chemical constituents have high volatility. As a result, nanoencapsulation of these oils has been proposed as a drug delivery system since this formulation technique is capable of improving the solubility, stability, and bioavailability of the compounds. NEs are being studied in recent years using formulations produced from essential oils (Pavoni et al., 2020 ). In the present study, GC-MS and GC-FID identified 16 chemical constituents in Citrus sinensis essential oil. Limonene was found to be the main constituent at 90.13%. Rezende et al. ( 2020 ) also found that essential oil from C. sinensis had a high limonene content (91.4%), which corroborates our data. In addition, traces of α- and β-pinene (1.25 ± 0.07, 1.87 ± 0.10, respectively), and phellandrene are present. Also, there is aldehyde (E)-isocitral (0.22 ± 0.03), which provides the characteristic odor present in this essential oil, as noted by Ali et al. ( 2015 ). Piper aduncum essential oil was identified with 13 chemical constituents, and dillapiole was the main constituent (78.41%), corroborating data in studies reported by Pino et al. ( 2004 ) and Maia et al. ( 1998 ). Finally, Carum carvi essential oil showed 6 constituents. The main ones are carvone (60.44 ± 0.80) and limonene (34.14 ± 0.64). This dataset corroborates the findings by Obeid et al. ( 2020 ) and Trifan et al. ( 2016 ). Increased therapeutic potential of the essential oil is possible through nanoencapsulation (Aziz et al., 2019 ). The emulsification technique was successfully used to develop the three NEs from essential oils in our study. The formulation of NEs was affected by the stirring speed and stirring time. Our formulations were characterized as NEs since their vesicle sizes were less than 300 nm (Singh et al., 2017 ). It is preferable that the polydispersion index (PDI), a measure that characterizes the homogeneity of the vesicle size, have values below 0.2 (Danaei et al., 2018 ), which indicates a high degree of size homogeneity (Caddeo et al., 2008 ). PDI values can vary more or less depending on the technique used to produce NEs. The results obtained in our study correspond to PDI values varying between 0.3 and 0.4, which were obtained in our best NEs, demonstrating the homogeneity of the formulations. Additionally, the zeta potential (ζ - mv) parameter measures the surface charge and electrostatic parameters of vesicles (Grisham and Nanda, 2020 ). Our data revealed that all formulations presented high negative potential, indicating the stability of the NEs by repulsion forces between the vesicles and showing the absence of aggregations of the vesicles. The use of surfactants with intermediate high hydrophilic-lipophilic balance (HLB) (generally between 11 and 16), such as tween, is the best choice to produce cheaper NEs by dispersing the oily phase in the aqueous phase. In addition, non-ionic surfactants such as Span are important due to their better toxicological safety for oral ingestion (Pavoni et al., 2020 ). According to Aziz et al. ( 2019 ), a higher value HLB surfactant is needed to produce smaller vesicles in the NEs, which was also used in our studies. NEs containing C. carvi , C. sinensis , and P. aduncum essential oils showed good antioxidant activity, according to our values by EC 50 (concentration of sample required to scavenge 50% of free radicals). This data confirms findings by Trifan et al. ( 2016 ), Rodríguez et al. ( 2013 ), and Frassinetti et al. ( 2011 ) for C. carvi , P. aduncum , and C. sinensis , respectively. These results can be explained because oils have a large percentage of oxygenated compounds in their compositions, such as limonene, carvone, and dilapiol, besides phenylpropanoid that is present in their structure methoxy and methylenedioxy groups. Furthermore, we cannot rule out the presence of other oxygenated compounds found in lower percentages than the cited ones. Our findings are important to developing products that reduce the toxicity of monotherapy levothyroxine treatment (Chaker et al., 2017 ), and we suggest a combination of therapeutics and nanoemultions can be effective. Keeping this in mind, this study focused on the development of the NEs produced from essential oils, and their characterization and toxicity evaluation. We used the zebrafish ( Danio rerio ) to evaluate the toxicity. The ethical consideration of the use of lower vertebrates such as zebrafish to evaluate the toxicity of NE was taken into account (Jayasinghe and Jayawardena, 2019 ). Our results revealed that the animals exposed to all NEs presented teratogenic effects. Independent of the type of essential oils used, despite P. aduncum NE producing more toxicity, similar teratogenic effects were observed for each NE. This could indicate that the same chemical compound used in the formulation of nanoemulsions has related toxicity effects. We did not observe significant teratogenic effects in the L-T 4 solution. However, all NEs were produced with surfactants (tween 80 and span 80). This idea is supported by Yang et al. ( 2015 ), who found that tween 80 induced anaphylactoid reactions in zebrafish. Ginzburg et al. ( 2018 ) investigated the influence of surfactants on nanoparticle biocompatibility by studying mixtures of ligand-stabilized gold nanoparticles and polysorbate 80 (span 80) in embryonic zebrafish and observed that the mixtures produced synergistic toxicity at concentrations where the individual components were benign. The acute toxicity of span 80 was previously studied in zebrafish (Sun et al., 2017 ). This study revealed that span 80 has high mortality rate on zebrafish. This indicates that pharmaceutical excipients, including mixtures such as span 80, have potential risks when the safety of these new nanotechnological drugs were evaluated in zebrafish. Additionally, tween 80 also could be toxic to zebrafish (Ali et al., 2011 ). In our study, we saw more toxic effects in the larval stage and the absence of chorion may explain this effect. The chorion functions to protect the embryo against chemical exposure, for example (Kim and Tanguay, 2014 ), and when it ruptures, the fish are more readily exposed to toxins. Finally, our results indicate these NEs affected morphology rather than behavioral endpoints. This may be because the animals chosen for tests did not show any morphological defects, and probably these animals were more resistant to or less affected by toxic compounds present in NEs. Further research is necessary to reduce adverse effects before the use of these NEs. 5. Conclusion In this study, antioxidant essential oil–based nanoemulsions associated with levothyroxine were successfully developed using a high‑energy emulsification approach and optimized by factorial experimental design. The formulations showed suitable physicochemical characteristics, including nanometric mean size, acceptable polydispersity index, and negative zeta potential, indicating good colloidal stability. All nanoemulsions exhibited relevant antioxidant activity, confirming the contribution of the essential oils and supporting their potential to mitigate oxidative stress associated with hypothyroidism. Despite these promising properties, in vivo toxicity assessment using the zebrafish model revealed significant teratogenic effects, particularly during the larval stage, regardless of the essential oil used. Behavioral alterations were limited and formulation‑dependent, with effects observed mainly for the Carum carvi nanoemulsion. The absence of toxicity in levothyroxine alone and the occurrence of toxic effects in empty nanoemulsions suggest that surfactants employed in the formulations likely play a major role in the observed toxicity. Further studies focusing on reformulation strategies, surfactant replacement and long‑term toxicity are necessary before these nanoemulsions can be considered viable therapeutic alternatives for hypothyroidism. Declarations Competing Interests The research reported in this manuscript is part of a patent (BR102020025513-4) in the course of the process. Funding The authors would like to thank the Universidade Federal Rural de Pernambuco , Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – CAPES for the scholarship to Thamiris P. Santos and fellowship for Pabyton G. Cadena, Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq for the scholarship to Paulo E. S. Bastos and productivity grant to Pabyton G. Cadena (306947/2020-0). The authors also thank Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq (Grant 423038/2018-4) and Pró-Reitoria de Pesquisa e Pós-Graduação of UFRPE – PRPPG (Grant PRPPG 05/2018) for financial support. Ethics approval The protocols used in this study were approved by the Ethics Committee in the use of animals of the Universidade Federal Rural de Pernambuco - Brazil, protocol number 091/2018. Consent to participate Not applicable. Consent for publication Not applicable. Availability of data and material The data and data and material used in this study are available from the corresponding author on request. Declaration of generative AI in scientific writing During the preparation of this work the author(s) used Copilot in order to correct the language of the manuscript. Author Contributions Details of each author with their contribution in this paper: Conceptualization, methodology, validation, formal analysis, Investigation, data curation, writing - original draft, writing - review and editing, and visualization were performed by Thamiris Pinheiro Santos. Investigation and data Curation also were performed by Paulo Eduardo da Silva Bastos. Authors Matheus Victor Viana de Melo, Tiago Queiroz da Mota Bittencourt, Renata Meireles Oliveira Padilha, Renatta Priscilla Ferreira Silva, Niely Priscila Correia da Silva, Carolina Alves de Araujo, Marcilio Martins de Moraes, Cláudio Augusto Gomes da Câmara and Amanda Rodrigues dos Santos Magnabosco performed investigation. Authors Jadson Freitas da Silva and André Lucas Corrêa de Andrade performed the investigation and formal analysis. Co-supervision, project administration, writing - original draft, writing - review & editing were performed by Marilia Ribeiro Sales Cadena, Samara da Silva Gomes and Yuri Mateus Lima de Albuquerque. Conceptualization, methodology, validation, formal analysis, resources, data curation, writing - original draft, writing - review and editing, visualization, supervision, project administration, and funding acquisition were performed by Pabyton Gonçalves Cadena. All authors read and approved of the final manuscript. Acknowledgments The authors would like to thank the Laboratório de Ecofisiologia Comportamento Animal – LECA for the inspiration. References Adams RP (2017) Identification of essential oil components by gas chromatography/ mass spectrometry. Allured Publishing Corporation, Carol Stream, Illinois Ali B, Al-Wabel NA, Shams S, Ahamad A, Khan SA, Anwar F (2015) Essential oils used in aromatherapy: A systemic review. 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University of Oregon, Eugene, OR Yang R, Lao QC, Yu HP, Zhang Y, Liu HC, Luan L, Sun HM, Li CQ (2015) Tween-80 and impurity induce anaphylactoid reaction in zebrafish. J Appl Toxicol 35:295–301 Additional Declarations The authors declare no competing interests. Supplementary Files Supplementarymaterial.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9568099","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":631907966,"identity":"bf3fd697-d8bf-42b8-a4b9-66eacf2408a6","order_by":0,"name":"Thamiris Pinheiro Santos","email":"","orcid":"https://orcid.org/0000-0003-1878-4088","institution":"Universidade Federal de Pernambuco","correspondingAuthor":false,"prefix":"","firstName":"Thamiris","middleName":"Pinheiro","lastName":"Santos","suffix":""},{"id":631908319,"identity":"517e7cac-9790-450a-bfae-2e7076e442a8","order_by":1,"name":"Paulo Eduardo da Silva Bastos","email":"","orcid":"","institution":"Universidade Federal Rural de Pernambuco","correspondingAuthor":false,"prefix":"","firstName":"Paulo","middleName":"Eduardo da Silva","lastName":"Bastos","suffix":""},{"id":631911965,"identity":"11edb4e4-f459-44a6-b445-3b6867871349","order_by":2,"name":"Matheus Victor Viana de Melo","email":"","orcid":"https://orcid.org/0000-0002-4349-8318","institution":"Universidade Federal Rural de Pernambuco","correspondingAuthor":false,"prefix":"","firstName":"Matheus","middleName":"Victor Viana","lastName":"de Melo","suffix":""},{"id":632039628,"identity":"68be0d73-79e7-4a34-9e26-589e3f388529","order_by":3,"name":"André Lucas Corrêa de Andrade","email":"","orcid":"https://orcid.org/0000-0002-8339-8338","institution":"Universidade Federal de Pernambuco","correspondingAuthor":false,"prefix":"","firstName":"André","middleName":"Lucas Corrêa","lastName":"de Andrade","suffix":""},{"id":632039629,"identity":"7c90e86d-28e3-4a24-8c46-e625642001aa","order_by":4,"name":"Tiago Queiroz da Mota Bittencourt","email":"","orcid":"https://orcid.org/0000-0002-5900-4861","institution":"Universidade Federal Rural de Pernambuco","correspondingAuthor":false,"prefix":"","firstName":"Tiago","middleName":"Queiroz da Mota","lastName":"Bittencourt","suffix":""},{"id":632039688,"identity":"574179a3-aecb-417b-b58e-1a3d600823d2","order_by":5,"name":"Renata Meireles Oliveira Padilha","email":"","orcid":"https://orcid.org/0000-0002-3069-4053","institution":"Universidade Federal Rural de Pernambuco","correspondingAuthor":false,"prefix":"","firstName":"Renata","middleName":"Meireles Oliveira","lastName":"Padilha","suffix":""},{"id":632039797,"identity":"8605c651-0191-4ac9-b130-b4010630339c","order_by":6,"name":"Renatta Priscilla Ferreira Silva","email":"","orcid":"https://orcid.org/0000-0002-6713-7023","institution":"Universidade Federal de Pernambuco","correspondingAuthor":false,"prefix":"","firstName":"Renatta","middleName":"Priscilla Ferreira","lastName":"Silva","suffix":""},{"id":632039891,"identity":"47937047-3b93-492a-bc5c-589133c0b5f9","order_by":7,"name":"Niely Priscila Correia da Silva","email":"","orcid":"https://orcid.org/0000-0002-7908-458X","institution":"Universidade Federal Rural de Pernambuco","correspondingAuthor":false,"prefix":"","firstName":"Niely","middleName":"Priscila Correia da","lastName":"Silva","suffix":""},{"id":632061858,"identity":"022b3059-931b-4413-bb98-8706f7c5191e","order_by":8,"name":"Carolina Alves de Araujo","email":"","orcid":"","institution":"Universidade Federal Rural de Pernambuco","correspondingAuthor":false,"prefix":"","firstName":"Carolina","middleName":"Alves","lastName":"de Araujo","suffix":""},{"id":632062335,"identity":"31950370-6e76-4846-8e15-24f0d5ceade1","order_by":9,"name":"Marcilio Martins de Moraes","email":"","orcid":"","institution":"Universidade Federal Rural de Pernambuco","correspondingAuthor":false,"prefix":"","firstName":"Marcilio","middleName":"Martins","lastName":"de Moraes","suffix":""},{"id":632062400,"identity":"6abf44ae-2e61-44b4-b038-16ebe0433f51","order_by":10,"name":"Samara da Silva Gomes","email":"","orcid":"https://orcid.org/0000-0003-3902-4483","institution":"Universidade Federal Rural de Pernambuco","correspondingAuthor":false,"prefix":"","firstName":"Samara","middleName":"da Silva","lastName":"Gomes","suffix":""},{"id":632062620,"identity":"044a58a9-11f2-451b-ac83-5a77f5d619ca","order_by":11,"name":"Yuri Mateus Lima de Albuquerque","email":"","orcid":"https://orcid.org/0000-0002-0580-9920","institution":"Universidade Federal Rural de Pernambuco","correspondingAuthor":false,"prefix":"","firstName":"Yuri","middleName":"Mateus Lima","lastName":"de Albuquerque","suffix":""},{"id":632062791,"identity":"3f329df1-193e-4eda-b96f-e16227cdb186","order_by":12,"name":"Cláudio Augusto Gomes da Câmara","email":"","orcid":"https://orcid.org/0000-0001-8508-1230","institution":"Universidade Federal Rural de Pernambuco","correspondingAuthor":false,"prefix":"","firstName":"Cláudio","middleName":"Augusto Gomes da","lastName":"Câmara","suffix":""},{"id":632062792,"identity":"18e725c5-a138-4e59-ade0-3ffe56f62cfd","order_by":13,"name":"Marilia Ribeiro Sales Cadena","email":"","orcid":"https://orcid.org/0000-0002-2620-9158","institution":"Universidade Federal Rural de Pernambuco","correspondingAuthor":false,"prefix":"","firstName":"Marilia","middleName":"Ribeiro Sales","lastName":"Cadena","suffix":""},{"id":631908320,"identity":"85fa45fb-cc7f-4f01-b182-baa64987fad0","order_by":14,"name":"Pabyton Gonçalves Cadena","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/klEQVRIiWNgGAWjYFACHjjLAIhtGNgbgBRjAwEtBxBa0hh4DpCo5TBhLebsZ49Jf6i4w8Dffnjj44Ka84k90mcPPmDccQ+nFsuevDSJA2eeMUicSSs2nnHsdmIPX16yAeOZYpxaDA7kmEkcbDsMdFWOmTQP2+3E/Tw8ZhKMbQm4tZx/A9XC/8b8N8+/c4k9PDzmP/BquQGzRSLHjJm37QBIixkDPi2WM94lW5w5c5hH4sazYmnevmTjHh6+ZInEM7i1mPPnHrxRUXFYjr8/eeNnnm92sj08vAc/fNyBx2EMDCwSDMhpAMzGrQGshfkDmhgPVpWjYBSMglEwcgEAnyFV2skGPBEAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0003-2297-5872","institution":"Universidade Federal Rural de Pernambuco","correspondingAuthor":true,"prefix":"","firstName":"Pabyton","middleName":"Gonçalves","lastName":"Cadena","suffix":""}],"badges":[],"createdAt":"2026-04-29 16:04:12","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":true,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":true},"doi":"10.21203/rs.3.rs-9568099/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9568099/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108419604,"identity":"a49ff2b5-2f77-49c7-a1e8-05ff8981cf8a","added_by":"auto","created_at":"2026-05-04 12:21:05","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":58913,"visible":true,"origin":"","legend":"\u003cp\u003ePercentage of affected animals after 24, 48, 72, and 96 h (post-fertilization) in toxicity test. The reduction in the percentage of affected animals was considered significant when * = p \u0026lt; 0.05. Legend: L-T\u003csub\u003e4\u003c/sub\u003e - levothyroxine; ECC - Empty \u003cem\u003eC. carvi\u003c/em\u003e nanoemulsion; CCN44 - \u003cem\u003eC. carvi\u003c/em\u003e nanoemulsion (4444x dilution rate); CCN32 - \u003cem\u003eC. carvi\u003c/em\u003e nanoemulsion (3200x dilution rate); ECS - Empty \u003cem\u003eC. sinensis\u003c/em\u003e nanoemulsion; CSN44 - \u003cem\u003eC. sinensis\u003c/em\u003e nanoemulsion (4444x dilution rate); CSN32 - \u003cem\u003eC. sinensis\u003c/em\u003e nanoemulsion (3200x dilution rate); EPA - Empty \u003cem\u003eP. aduncum \u003c/em\u003enanoemulsion; PAN44 - \u003cem\u003eP. aduncum\u003c/em\u003e nanoemulsion (4444x dilution rate); PAN32 - \u003cem\u003eP. aduncum \u003c/em\u003enanoemulsion (3200x dilution rate).\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-9568099/v1/c92c5d31cd9989916692e98e.png"},{"id":108493802,"identity":"ab8623ac-9e6a-4e04-b30b-687b709fe405","added_by":"auto","created_at":"2026-05-05 10:01:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":88165,"visible":true,"origin":"","legend":"\u003cp\u003eThe thigmotaxis test (A) showed that the empty nanoemulsion and levothyroxine nanoemulsion did not affect the thigmotaxis response, but the empty \u003cem\u003eC. carvi\u003c/em\u003e nanoemulsions affected the touch sensitivity response (B). The response for each group was compared to the control group by one-way ANOVA followed by Tukey’s test (thigmotaxis (F(10, 732) = 1.15, p \u0026lt; 0.05); touch sensitivity response (F(10, 732) = 2.81, p \u0026lt; 0.05). * = p \u0026lt; 0.05. Legend: L-T\u003csub\u003e4\u003c/sub\u003e - levothyroxine; ECC - Empty \u003cem\u003eC. carvi\u003c/em\u003e nanoemulsion; CCN44 - \u003cem\u003eC. carvi\u003c/em\u003e nanoemulsion (4444x dilution rate); CCN32 - \u003cem\u003eC. carvi\u003c/em\u003e nanoemulsion (3200x dilution rate); ECS - Empty \u003cem\u003eC. sinensis\u003c/em\u003e nanoemulsion; CSN44 - \u003cem\u003eC. sinensis\u003c/em\u003e nanoemulsion (4444x dilution rate); CSN32 - \u003cem\u003eC. sinensis\u003c/em\u003e nanoemulsion (3200x dilution rate); EPA - Empty \u003cem\u003eP. aduncum \u003c/em\u003enanoemulsion; PAN44 - \u003cem\u003eP. aduncum\u003c/em\u003e nanoemulsion (4444x dilution rate); PAN32 - \u003cem\u003eP. aduncum \u003c/em\u003enanoemulsion (3200x dilution rate).\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-9568099/v1/ac48eee099c8e02ce96efafd.png"},{"id":108804683,"identity":"f0fc30e4-fba1-4395-a254-d6a11495c2fb","added_by":"auto","created_at":"2026-05-08 15:22:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1093598,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9568099/v1/f7cd729a-5ef9-4176-b4d6-2294dc6def80.pdf"},{"id":108493553,"identity":"ab2c8e4d-7e7f-4a61-a44e-eb2267fc655e","added_by":"auto","created_at":"2026-05-05 10:00:55","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":573440,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-9568099/v1/344e269c6d4439ea0f5ef871.docx"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eAntioxidant essential oil–based nanoemulsions associated with levothyroxine: Physicochemical characterization and toxicity assessment in zebrafish\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eEssential oils (EOs) are products of spices used since antiquity due to their medicinal properties. Wide acceptance by consumers requires the natural product to show safety and efficacy (Bilia et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). EOs are volatile liquids, being soluble in lipid and organic solvents, and synthesized by all plant organs (flowers, leaves, and bark) (Bilia et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Pavoni et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). EO constituents are secondary plant metabolites (Aziz et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), including monoterpenes, sesquiterpenes, and phenylpropanoids.\u003c/p\u003e \u003cp\u003e \u003cem\u003eCitrus\u003c/em\u003e is the genus that includes the world's major fruit-cultivated species, \u003cem\u003eCitrus sinensis\u003c/em\u003e (sweet orange), and Brazil is the world\u0026rsquo;s main producer of this fruit (Rezende et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). According to the Food and Agriculture Organization of the United Nations (FAO), in Brazil, the total production of this fruit was 16.713.534 tons in 2018. Antibacterial, antifungal, and antioxidant properties are reported for \u003cem\u003eC. sinensis\u003c/em\u003e essential oil (Frassinetti et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Popular uses include skin health and promoting detoxification, and pharmacological actions include reduced anxiety, agitation, stress, challenging behaviors, and insomnia (Ali et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003ePiper\u003c/em\u003e is a genus of the Piperaceae family, widely distributed in subtropical regions. \u003cem\u003ePiper aduncum\u003c/em\u003e is popularly known as long pepper, and \u003cem\u003emonkey pepper\u003c/em\u003e among other names (Pohlit et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Silva et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2019a\u003c/span\u003e). In Brazil, it is used as a bactericide, leishmanicide, fungicide, antioxidant, anticancer, larvicide, insecticide, antiplatelet (anticoagulant), molluscicide, and antiviral (Pohlit et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Silva et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2019a\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eCarum carvi\u003c/em\u003e, also known as caraway and black zeera, is a plant from the Apiaceae family, grown in Northern Africa, western Asia, and Europe. Recently, it was introduced in other regions of the world like Russia, Iran, Iraq, Indonesia, and North America. Its potential biological effects include antioxidant, anti-inflammatory, anti-diabetic, anti-obesity, anti-spasmodic, anti-asthmatic, anti-microbial, anti-hemolytic, anti-foaming, and galactagogue (Obeid et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Trifan et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In addition, the essential oil of \u003cem\u003eCarum Carvi\u003c/em\u003e is a potent hypothyroidism treatment (Obeid et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn hypothyroidism, there is an accelerated production of free radicals and low availability of antioxidants that eliminate free radicals (Mancini et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). As a result of this imbalance, oxidative stress arises, which is implicated in the pathophysiological mechanism of hypothyroidism and other diseases (Mancini et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In subclinical hypothyroidism patients, symptoms like memory deficit, despondency, depression, and anxiety are frequently reported (Almeida et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Levothyroxine monotherapy is the standard treatment for patients with hypothyroidism. Patients are biochemically well-controlled with levothyroxine, but a substantial proportion of patients have persistent complaints, like symptoms of depression and impaired mental well-being. This raised the question of whether treatment with this synthetic hormone is sufficient for all patients or whether alternative therapies can be adopted to minimize these adverse effects (Chaker et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The formulation of nanoemulsions (NEs) containing levothyroxine associated with essential oils with antioxidant activity may be a good alternative and overcome these limitations.\u003c/p\u003e \u003cp\u003eNanotechnology applied to the pharmaceutical industry is an opportunity for new treatment approaches that overcome the obstacles of conventional therapies (Rojas-Aguirre et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Therefore, controlled release systems have been widely investigated to enable delayed and sustained drug release, controlling the site of drug release and reducing the amount of drug required for treatment (Singh et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Among the controlled drug release systems, NEs are widely reported in the pharmaceutical research literature. NEs are produced by the mixture of two immiscible liquids (oil and water are usually used), stabilized by the addition of a surfactant suitable for the compounds used, which decreases the surface tension between the immiscible liquids (Singh et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Sometimes, a combination of surfactants is necessary to ensure the formation and stabilization of NEs (McClements, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). NEs offer better bioavailability and drug release due to vesicle size, which is usually\u0026thinsp;\u0026lt;\u0026thinsp;500 nm, giving emulsions a clear or hazy appearance (Aziz et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Singh et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eEmulsification processes are commonly used for essential oils to solubilize and stabilize volatile compounds, which are easily degraded by exposure to environmental factors like heat, light, oxygen, and humidity (Aziz et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Pavoni et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), allowing essential oils to be used in drug development. Emulsification of the \u003cem\u003eC. sinensis\u003c/em\u003e (Velmurugan et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2017\u003c/span\u003e)d \u003cem\u003eaduncum\u003c/em\u003e (Silva et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2019a\u003c/span\u003e) essential oils are described in the literature.\u003c/p\u003e \u003cp\u003e \u003cem\u003eIn silico\u003c/em\u003e, \u003cem\u003ein vitro\u003c/em\u003e, and \u003cem\u003ein vivo\u003c/em\u003e methods are used to evaluate the toxicity of NEs because the nanoscale can change their toxicity. \u003cem\u003eIn silico\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e methods are beneficial for the preliminary toxicological assessment of NEs. However, animal tests are still needed. Zebrafish have become a well-established animal model for the toxicological and pharmacological screening of new drugs. Zebrafish can also reveal toxic mechanisms of discovery candidates as NEs and provide 3 Rs (replacement, reduction, and refinement) value to animal research (Cassar et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In addition, animal model research can reveal the benefit/risk of new products, and nanotechnological products present a new dimension to the pharmacological/toxicological profile as compared with traditional drugs (Pensado-Lopez et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Using zebrafish to evaluate the toxicity of new drugs at the pre-clinical stage allows easier and more rapid decisions on the viability of candidates and information about modifying these new drugs to produce safer and more effective alternatives (Cassar et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Jayasinghe and Jayawardena, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eZebrafish embryos are an effective model to study the toxicological effects of a broad spectrum of herbal medicines (Jayasinghe and Jayawardena, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). First, zebrafish are externally fertilized, and embryos/larvae are optically transparent during early developmental stages, allowing direct, noninvasive observations (Cassar et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The zebrafish genome is 87% conserved with human genes (Jayasinghe and Jayawardena, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), and the zebrafish shares 82% of human disease genes (Cassar et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), but also behavioral similarities. Thyroid receptor proteins have 91% identity with human thyroid receptors (MacRae and Peterson, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). This conservation allows an effective comparison of this animal model with the human condition, including hypothyroidism. These advantages can be exploited to evaluate drug toxicity using \u003cem\u003eD. rerio\u003c/em\u003e in pre-clinical studies to develop safer drugs (Jayasinghe and Jayawardena, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn the present study, NEs of essential oils associated with levothyroxine were produced and characterized, and antioxidant activity was analyzed. Also, the toxicity of these produced NEs was evaluated in the zebrafish (\u003cem\u003eDanio rerio\u003c/em\u003e) model. Toxic effects on fish development were tested using mortality, teratogenic effects, and animal behavior assays (thigmotaxis and touch sensitivity). These experiments provide insights into the potential utility of these NEs.\u003c/p\u003e"},{"header":"2. Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Collection of plant material\u003c/h2\u003e \u003cp\u003eFresh leaves of the \u003cem\u003eP. aduncum\u003c/em\u003e (8\u0026deg;01'10.1\"S 34\u0026deg;56'50.4\" W) were collected in the \u003cem\u003eMata de Dois Irm\u0026atilde;os\u003c/em\u003e, in \u003cem\u003eRecife\u003c/em\u003e, \u003cem\u003ePernambuco\u003c/em\u003e, Brazil. Leaf collections for specimens were performed at around 9:00 am in the dry season, in February 2019. The plants were identified by botanist \u003cem\u003eDra. \u0026Acirc;ngela Maria de Miranda Freitas\u003c/em\u003e (\u003cem\u003eUniversidade Federal Rural de Pernambuco\u003c/em\u003e). Voucher specimens were mounted and deposited in the \u003cem\u003eS\u0026eacute;rgio Tavares\u003c/em\u003e Herbarium of the UFRPE under numbers HST 18177. Fruits of the \u003cem\u003eC. sinensis\u003c/em\u003e were collected in \u003cem\u003eS\u0026iacute;tio Cigarra\u003c/em\u003e, in the municipality of \u003cem\u003eSantana do Munda\u0026uacute;\u003c/em\u003e, Alagoas-AL, in May 2019. Fruits collections for specimens were performed at around 11:00 am in the dry season. The plant was identified by \u003cem\u003eDra. Suzene Iz\u0026iacute;dio da Silva\u003c/em\u003e from the Biology Department of the \u003cem\u003eUniversidade Federal Rural de Pernambuco\u003c/em\u003e (UFRPE). Voucher specimens were deposited in the \u003cem\u003eVasconcelos Sobrinho\u003c/em\u003e Herbarium of the UFRPE, under the number: 48735, \u003cem\u003eC. sinensis\u003c/em\u003e Osbeck var. \u003cem\u003epear\u003c/em\u003e). The oil of the \u003cem\u003eCarum carvi\u003c/em\u003e was acquired from a local supplier.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Isolation, chemical analysis, identification, and quantification of the essential oil\u003c/h2\u003e \u003cp\u003eThe essential oils from fresh leaves of \u003cem\u003ePiper aduncum\u003c/em\u003e, and shells of the fruit of \u003cem\u003eCitrus sinensis\u003c/em\u003e (100 g) were separately isolated using a modified Clevenger-type apparatus and hydrodistillation for 2h. The oil layers were separated and dried over anhydrous sodium sulfate, stored in hermetically sealed glass containers, and kept at a low temperature (-5 \u0026ordm;C) until analysis. Total oil yields were expressed as percentages (g/100 g of fresh plant material). All experiments were carried out in triplicate. For the \u003cem\u003eCarum carvi\u003c/em\u003e oil was acquired from a local supplier (LASZLO, Brazil). Quantitative GC analysis was carried out using a PerkinElmer Clarus 500 GC apparatus equipped with a flame ionization detector (FID) and a non-polar DB-5 fused silica capillary column (30 m x 0.25 mm x 0.25 \u0026micro;m film thickness) (J \u0026amp; W Scientific). The oven temperature was programmed from 60 to 240\u0026deg;C at a rate of 3\u0026deg;C/min. Injector and detector temperatures were 260\u0026deg;C. Hydrogen was used as the carrier gas at a flow rate of 1 mL/min in split mode (1:30). The injection volume was 0.5 \u0026micro;L of diluted solution (1/100) of oil in n-hexane. The amount of each compound was calculated from GC-FID peak areas in the order of DB-5 column elution and expressed as a relative percentage of the total area of the chromatograms. Analyses were carried out in triplicate. The qualitative Gas Chromatography-Mass Spectrometry (GC-MS) analysis was carried out using a Varian 220-MS IT GC system with a mass selective detector, a mass spectrometer in EI 70 eV with a scan-interval of 0.5 s, and fragments from 40 to 550 Da. fitted with the same column and temperature program as that for the GC-FID experiments, with the following parameters: carrier gas\u0026thinsp;=\u0026thinsp;helium; flow rate\u0026thinsp;=\u0026thinsp;1 mL/min; split mode (1:30); injected volume\u0026thinsp;=\u0026thinsp;1 \u0026micro;L of diluted solution (1/100) of oil in n-hexane. Identification of the components was based on GC-MS retention indices with reference to a homologous series of C8-C40 n-alkanes calculated using the Van der Dool and Kratz equation (van Den Dool and Dec. Kratz, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1963\u003c/span\u003e) and by computer matching against the mass spectral library of the GC-MS data system (NIST 14 and WILEY 11th) and co-injection with authentic standards as well as other published mass spectra (Adams, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Area percentages were obtained from the GC-FID response without the use of an internal standard or correction factors.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Preparation of levothyroxine stock solution\u003c/h2\u003e \u003cp\u003eCommercial levothyroxine (L-T\u003csub\u003e4\u003c/sub\u003e) (composition: L-T\u003csub\u003e4\u003c/sub\u003e (37,5 \u0026micro;g), mannitol, maize starch, microcrystalline cellulose, butyl-hydroxyanisole, and magnesium stearate) was used in the experiments. The stock solution of L-T\u003csub\u003e4\u003c/sub\u003e was prepared by dissolving in distilled water, obtaining the final nominal concentration of 250 \u0026micro;g/L, which was stored under refrigeration (4˚C) until further use.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Preparation of nanoemulsions\u003c/h2\u003e \u003cp\u003eThe oil/water (O/W) NEs were prepared using a high-energy emulsification method (dos Santos Magnabosco et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) by magnetic stirring from an oily phase (OP) produced from a mixture of 10\u0026ndash;20% (w/w) of individual essential oils (\u003cem\u003eCarum carvi\u003c/em\u003e, \u003cem\u003eCitrus sinensis\u003c/em\u003e, or \u003cem\u003ePiper aduncum\u003c/em\u003e), 10\u0026ndash;20% (w/w) of sorbitan monooleate (Tween 80) and/or 10% polysorbate 80 (Span 80). The OP was first homogenized using a magnetic stirrer for 30 min at 22 \u003cem\u003eg\u003c/em\u003e at room temperature (25\u0026thinsp;\u0026plusmn;\u0026thinsp;2 \u0026ordm;C). Simultaneously in another container, water phase (WP) was prepared with or without (empty) levothyroxine diluted in 43.75 mL of phosphate buffer 100 mM, pH 7.4. Finally, 70% of the WP was dripped (flow of 3.5 mL/min) using a burette into the OP under magnetic stirring according to Duarte et al. (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The prepared NEs were designated by the initials of each essential oil: \u003cem\u003eCarum carvi\u003c/em\u003e \u0026ndash; CC; \u003cem\u003eCitrus sinensis\u003c/em\u003e \u0026ndash; CS; and \u003cem\u003ePiper aduncum\u003c/em\u003e - PA.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Experimental designs\u003c/h2\u003e \u003cp\u003eA two-level 3 x 2\u003csup\u003e2\u003c/sup\u003e full experimental design was carried out to study the effect of time and stirring speed in the preparations of NEs. Factors (stirring speed and stirring time) of each formulation that mainly influenced the physicochemical parameters were evaluated at two levels and a central point (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) in authentic triplicate. The mean size, polydispersity index (PDI), and zeta potential (ζ mV) of NEs were used as the response variables of the design study.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eTwo-level 3 x 2\u003csup\u003e2\u003c/sup\u003e full experimental designs of nanoemulsions of each essential oil associated with levothyroxine.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRun\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eStirring speed (\u003cem\u003eg\u003c/em\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eStirring time (h)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEssential oil\u003c/p\u003e \u003cp\u003e(g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTween 80 (g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSpan 80 (g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePhosphate-buffered\u0026thinsp;+\u0026thinsp;Levothyroxine (250 \u0026micro;g/L)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10 \u003cem\u003eg\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1 h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.5 (\u003cem\u003eCarum carvi\u003c/em\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e3.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e39 \u003cem\u003eg\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1 h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.5 (\u003cem\u003eCarum carvi\u003c/em\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e3.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10 \u003cem\u003eg\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2 h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.5 (\u003cem\u003eCarum carvi\u003c/em\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e3.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e39 \u003cem\u003eg\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2 h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.5 (\u003cem\u003eCarum carvi\u003c/em\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e3.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5 (CP)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e22 \u003cem\u003eg\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1 h 30 min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.5 (\u003cem\u003eCarum carvi\u003c/em\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e3.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10 \u003cem\u003eg\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1 h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.0 (\u003cem\u003eCitrus sinensis\u003c/em\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e3.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e39 \u003cem\u003eg\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1 h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.0 (\u003cem\u003eCitrus sinensis\u003c/em\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e3.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10 \u003cem\u003eg\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2 h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.0 (\u003cem\u003eCitrus sinensis\u003c/em\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e3.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e39 \u003cem\u003eg\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2 h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.0 (\u003cem\u003eCitrus sinensis\u003c/em\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e3.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10 (CP)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e22 \u003cem\u003eg\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1 h 30 min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.0 (\u003cem\u003eCitrus sinensis\u003c/em\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e3.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10 \u003cem\u003eg\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1 h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.5 (\u003cem\u003ePiper aduncum\u003c/em\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e3.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e39 \u003cem\u003eg\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1 h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.5 (\u003cem\u003ePiper aduncum\u003c/em\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e3.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10 \u003cem\u003eg\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2 h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.5 (\u003cem\u003ePiper aduncum\u003c/em\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e3.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e39 \u003cem\u003eg\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2 h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.5 (\u003cem\u003ePiper aduncum\u003c/em\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e3.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e15 (CP)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e22 \u003cem\u003eg\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1 h 30 min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.5 (\u003cem\u003ePiper aduncum\u003c/em\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e3.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eLegend: Runs 1 to 5 are \u003cem\u003eCarum carvi\u003c/em\u003e samples with a central point (CP \u0026ndash; run 5); 6 to 10 are \u003cem\u003eCitrus sinensis\u003c/em\u003e samples with a central point (CP \u0026ndash; run 10); 11 to 15 are \u003cem\u003ePiper aduncum\u003c/em\u003e samples with a central point (CP \u0026ndash; run 15).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Physicochemical characterization\u003c/h2\u003e \u003cp\u003eThe NEs were diluted (50x) in double-distilled water before analysis to reduce the turbidity before microscopic analysis. The average size (nm), polydispersity index (PDI), and zeta potential (mV) of the oil vesicles were evaluated using the standard photon correlation spectroscopy (PCS) technique fixed at 90\u0026ordm; to 25 \u0026ordm;C using a Zetasizer Nano ZS (Malvern, UK) (Cadena et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; dos Santos Magnabosco et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The analysis was performed at \u003cem\u003eLaborat\u0026oacute;rios Associados em Rede de Nanotecnologia\u003c/em\u003e (LARnano) of the \u003cem\u003eUniversidade Federal de Pernambuco\u003c/em\u003e (UFPE). The data were measured in triplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Antioxidant Activity of nanoemulsions\u003c/h2\u003e \u003cp\u003eAntioxidant activity was analyzed by DPPH˙ and ABTS˙\u003csup\u003e+\u003c/sup\u003e radicals. The experiments were performed at \u003cem\u003eLaborat\u0026oacute;rio de Produtos Naturais Bioativos\u003c/em\u003e (LPNBio) of the \u003cem\u003eUniversidade Federal Rural de Pernambuco\u003c/em\u003e (UFRPE). The antioxidant activity of the NEs was tested against the free radical DPPH following the methodology of Araujo et al. (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Stock solutions were prepared from the extracts and methanol fraction at several concentrations (0.1 to 5.0 mg/mL). Through preliminary analysis, appropriate quantities of stock solutions of the samples and 450 \u0026micro;L of the solution of DPPH\u003cb\u003e˙\u003c/b\u003e (23.6 mg/mL in EtOH) were transferred to 0.5 mL Eppendorf tubes, and the sample was diluted with EtOH following homogenization. Samples were sonicated for 30 min and the amount of DPPH\u003cb\u003e˙\u003c/b\u003e was measured in a 96-well plate using a UV-vis spectrophotometer (Biochrom EZ Read 2000) at 517 nm. Ascorbic acid was used as a positive control. The determination of the antioxidant activity in the NEs to detect the radical cation ABTS\u003cb\u003e˙\u003c/b\u003e\u003csup\u003e\u003cb\u003e+\u003c/b\u003e\u003c/sup\u003e was carried out following the methodology described by Araujo et al. (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), in a UV-vis spectrophotometer (Biochrom EZ Read 2000) using Trolox as the standard compound. The starting concentrations of the solutions of the samples were 0.1\u0026ndash;1.0 mg/mL, with the addition of 450 \u0026micro;L of the radical ABTS\u003cb\u003e˙\u003c/b\u003e\u003csup\u003e\u003cb\u003e+\u003c/b\u003e\u003c/sup\u003e solution to give final concentrations of 2.5\u0026ndash;100.0 \u0026micro;g/mL samples. Samples were protected from light and sonicated for 6 min. The absorbance of the samples and the positive control in a 96-well microplate were measured at a wavelength of 734 nm. In the two tests each concentration was tested in triplicate, and the percentage of free radical scavenging activity of DPPH˙ or ABTS\u003cb\u003e˙\u003c/b\u003e\u003csup\u003e\u003cb\u003e+\u003c/b\u003e\u003c/sup\u003e was calculated by the equation: %SA\u0026thinsp;=\u0026thinsp;100 x (Abs\u003csub\u003econtrol\u003c/sub\u003e \u0026ndash; Abs\u003csub\u003esample\u003c/sub\u003e) / Abs\u003csub\u003econtrol\u003c/sub\u003e, where Abs\u003csub\u003econtrol\u003c/sub\u003e is the absorbance of the control containing only the ethanol solution of DPPH˙ or ABTS\u003csup\u003e\u003cb\u003e+\u003c/b\u003e\u003c/sup\u003e and Abs\u003csub\u003esample\u003c/sub\u003e is the absorbance of the radical in the presence of the sample or standard ascorbic acid. Antiradical efficiency was established using linear regression analysis and the 95% confidence interval (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) obtained using the statistical program GraphPad Prism 5.0.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Fish husbandry, crossing, and toxicity test\u003c/h2\u003e \u003cp\u003eToxicity tests were performed using zebrafish (\u003cem\u003eDanio rerio\u003c/em\u003e; WT strain; 7 months old) as an animal model. The experiments were carried out at \u003cem\u003eLaborat\u0026oacute;rio de Ecofisiologia e Comportamento Animal\u003c/em\u003e (LECA) \u0026ndash; \u003cem\u003eUniversidade Federal Rural de Pernambuco\u003c/em\u003e (UFRPE). Adult zebrafish used in the experiments were obtained from a commercial supplier and housed in quarantine to detect or confirm the absence of pathogens or diseases maintained according to OECD 236 guidelines (OECD, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The protocols used in this study were approved by the Ethics Committee in the use of animals of the same University, protocol number 2008150721. Adult male/female zebrafish were isolated in spawning aquariums (Alesco Zebclean) with a 2:1 ratio (Westerfield, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). For fish crossing, dividers between males and females were removed, and they were allowed to breed for 40 min. The embryos were collected and washed with distilled water in Petri dishes. Healthy embryos (normal development of the blastula) were used to form experimental groups.\u003c/p\u003e \u003cp\u003eThe experiments were conducted with 5 x 15 animals per experimental group (a total of 825 animals with replicates and repetitions). The assay was based on the OECD guidelines on the Fish Embryo Acute Toxicity (FET) Test (OECD, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) and Silva et al. (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2019b\u003c/span\u003e). The toxicity test started at 1-hour post-fertilization (hpf). Embryos were maintained in sterile polystyrene test chambers (80 mL) and maintained in an incubator with a light-dark cycle of 14:10 h, controlled temperature of 26\u0026thinsp;\u0026plusmn;\u0026thinsp;1˚C, and pH of 7.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5. Daily water renewal was performed to maintain the NEs levels. The study used a control group and experimental groups that were described in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e with two dilution rates where 4444x and 3200x to evaluate toxicity. These dilution ratios were chosen based on studies showing most of the animals survived at 96 hpf. The final nominal concentration of L-T\u003csub\u003e4\u003c/sub\u003e in all experimental groups was the same as described above (item 2.3). The exposure to NEs started at 1-h post-fertilization (hpf) being renewed daily until 96 hpf, according to OECD 236 (2025). Any residue generated by these compounds during the experiments was treated by the advanced oxidative process (AOP) in a reactor using photo-oxidation UV (16w) and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (Hansen and Andersen, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Silva et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2019b\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe dilution ratio of the chemical compounds used in the experimental groups for the acute toxicity test.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eExperimental groups\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAbbreviations\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eDilution ratio\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLevothyroxine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eL-T\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e320x\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEmpty (E) \u003cem\u003eCarum carvi\u003c/em\u003e (CC) nanoemulsion (0.5 g oil, 1.0 g tween, 3.5 g of phosphate buffer) diluted\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eECC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3200x\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCarum carvi\u003c/em\u003e (CC) nanoemulsion (0.5 g oil, 1.0 g tween, 3.5 g of phosphate buffer with levothyroxine) diluted\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCCN44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4444x\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCarum carvi\u003c/em\u003e (CC) nanoemulsion (0.5 g oil, 1.0 g tween, 3.5 g phosphate buffer with levothyroxine) diluted\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCCN32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3200x\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEmpty (E) \u003cem\u003eCitrus sinensis\u003c/em\u003e (CS) nanoemulsion (1.0 g oil, 1.0 g tween, 3.0 g phosphate buffer) diluted\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eECS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3200x\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCitrus sinensis\u003c/em\u003e (CS) nanoemulsion (1.0 g oil, 1.0 g tween, 3.0 g phosphate buffer with levothyroxine) diluted\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCSN44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4444x\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCitrus sinensis\u003c/em\u003e (CS) nanoemulsion (1.0 g oil, 1.0 g tween, 3.0 g phosphate buffer with levothyroxine) diluted\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCSN32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3200x\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEmpty \u003cem\u003ePiper aduncum\u003c/em\u003e (PA) nanoemulsion (0.5 g oil, 0.5 g tween, 0.5 g spam, 3.5 g of phosphate buffer) diluted\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEPA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3200x\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePiper aduncum\u003c/em\u003e (PA) nanoemulsion (0.5 g oil, 0.5 g tween, 0.5 g spam, 3.5 g of phosphate buffer with levothyroxine) diluted\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePAN44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4444x\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePiper aduncum\u003c/em\u003e (PA) nanoemulsion (0.5 g oil, 0.5 g tween, 0.5 g spam, 3.5 g of phosphate buffer with levothyroxine) diluted\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePAN32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3200x\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Effects of levothyroxine and nanoemulsions on zebrafish embryonic development\u003c/h2\u003e \u003cp\u003eMortality data were observed at 24, 48, 72, and 96 hours post-fertilization (hpf). The determination of embryo lethality was assayed according to OECD 236 (2025). Determination of mortality as coagulation, tail not detached, no somite formation, absence of heartbeat, and lack of hatching. Teratogenic effects were evaluated according to OECD 236 (2025), Silva et al. (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2019b\u003c/span\u003e), and Cadena et al. (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The teratogenic effects studied were spine deformation, tail deformation, yolk sac edema, and pericardial edema. The effects were identified in images of lateral and dorsal views \u003cem\u003ein vivo\u003c/em\u003e. The images were captured using a Hayear Mod. HY-2307 digital camera microscope and S-EYE 1.4.2.474 software. We consider larvae affected when at least, mortality or one teratogenic effect was observed under a microscope (Cadena et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). These results were presented as a percentage of affected animals per experimental group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10. Effects of levothyroxine and nanoemulsions on zebrafish animal behavior\u003c/h2\u003e \u003cp\u003eBehavioral tests were used as a method to study the effects of NEs s in zebrafish (\u003cem\u003eDanio rerio\u003c/em\u003e). The thigmotaxis (TH Test) is the tendency of an animal to move toward the edge or periphery of plates that are introduced. Therefore, this animal avoids the center of this environment. This parameter evaluates the anxiety like-behavior in zebrafish (Basnet et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The thigmotaxis test was evaluated at 144 hpf, according to Cadena et al. (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The larvae were left at 10 min of acclimatization in 48-well plates (48 larvae per experimental group) in a light environment, and if larvae remained near the wall of plates, the response was recorded. The touch sensitivity test (TS Test) was performed according to Cadena et al. (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). This test evaluated the larval response to mechanical stimuli. At 144 hpf, the larvae were touched on the tail or head with a pipette tip. The response was recorded if the larva exhibited swimming behaviors (slow or fast), that is, an escape response.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11. Statistical analysis\u003c/h2\u003e \u003cp\u003eAll data were shown as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. ANOVA was used to analyze data, and the means were compared by Tukey's test (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Statistical analyses were carried out using the Origin Pro Academic 2015 (Origin Lab. Northampton, MA USA) and GraphPad Prism 5.0.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Isolation, chemical analysis, identification, and quantification of the essential oil\u003c/h2\u003e \u003cp\u003eThe chemical composition of essential oils from the leaves of \u003cem\u003eP. aduncum\u003c/em\u003e, \u003cem\u003eC.carvi\u003c/em\u003e, and \u003cem\u003eC. sinensis\u003c/em\u003e is shown in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Hydrodistillation of \u003cem\u003eP. aduncum\u003c/em\u003e leaves provided yellowish oil. The extraction of the oil revealed a yield of 2.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10%. Moreover, for \u003cem\u003eC. sinensis\u003c/em\u003e hydrodistillation provided a colorless citrus-scented oil. The extraction of the oil revealed a yield of 1.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13%. The GC-MS analysis of \u003cem\u003eP. aduncum\u003c/em\u003e oils allowed the identification of 13 compounds, representing a total of 98.53\u0026thinsp;\u0026plusmn;\u0026thinsp;1.01% of the oil. The oil revealed the presence of compounds belonging to the class of monoterpenes (4.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05%), sesquiterpenes (16.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10%), and phenylpropanoid (78.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.98%). The phenylpropanoid dilapiol (78.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.98%) was characterized as a major constituent in the oil. For \u003cem\u003eC. carvi\u003c/em\u003e oil the GC-MS analysis allowed the identification of 6 compounds, representing a total of 98.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.82% of the oil. The oil revealed a predominance of compounds belonging to the monoterpene class (98.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.82%), having as main constituent carbon (60.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.80%) followed by limonene (34.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.64%). Finally, \u003cem\u003eC. sinensis\u003c/em\u003e, the analysis by GC-MS allowed the identification of 16 compounds, representing a total of 97.47\u0026thinsp;\u0026plusmn;\u0026thinsp;1.22% of the oil. The oil revealed the presence of compounds belonging to the class of monoterpenes (96.39\u0026thinsp;\u0026plusmn;\u0026thinsp;1.08%) and sesquiterpenes (1.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01%). For this oil, limonene (90.13\u0026thinsp;\u0026plusmn;\u0026thinsp;1.11%) was characterized as a major constituent in the oil.\u003c/p\u003e \u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eChemical composition of essential oils of \u003cem\u003ePiper aduncum, Carum carvi\u003c/em\u003e, and \u003cem\u003eCitrus sinensis.\u003c/em\u003e\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCompounds\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRI\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eRI\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eP. aduncum\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cem\u003eC. carvi\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cem\u003eC. sinensis\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eMethod of identification\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eα\u003c/em\u003e-Thujene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e921\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e924\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eRI, MS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eα\u003c/em\u003e-Pinene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e928\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e932\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eRI, MS, CI\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSabinene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e966\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e969\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eRI, MS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eβ\u003c/em\u003e-Pinene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e971\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e974\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eRI, MS, CI\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eiso-Sylvestrene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1005\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1007\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eRI, MS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003ep\u003c/em\u003e-Cymene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1018\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1020\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eRI, MS, CI\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLimonene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1023\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1024\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e34.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.64\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e90.13\u0026thinsp;\u0026plusmn;\u0026thinsp;1.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eRI, MS, CI\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eβ\u003c/em\u003e-Phellandrene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1025\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1025\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eRI, MS, CI\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e(Z)-\u003cem\u003eβ\u003c/em\u003e-Ocimene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1031\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1032\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eRI, MS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e(\u003cem\u003eE\u003c/em\u003e)-\u003cem\u003eβ\u003c/em\u003e-Ocimene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1040\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1044\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eRI, MS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eγ\u003c/em\u003e-Terpinene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1055\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1054\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eRI, MS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTerpinolene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1088\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1086\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eRI, MS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLinalool\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1094\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1095\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eRI, MS, CI\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003ecis\u003c/em\u003e-\u003cem\u003eβ\u003c/em\u003e-Terpineol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1143\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1140\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eRI, MS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCitronellal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1145\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1148\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eRI, MS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTerpinen-4-ol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1174\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1174\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eRI, MS, CI\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e(\u003cem\u003eE\u003c/em\u003e)-Isocitral\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1175\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1177\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eRI, MS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDill ether\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1180\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1184\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eRI, MS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eα\u003c/em\u003e-Terpineol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1189\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1186\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eRI, MS, CI\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eδ\u003c/em\u003e-Elemene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1340\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1335\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eRI, MS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCarvone\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1235\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1239\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e60.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eRI, MS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eβ\u003c/em\u003e-Caryophyllene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1415\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1417\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eRI, MS, CI\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eα\u003c/em\u003e-\u003cem\u003etrans\u003c/em\u003e-Bergamotene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1431\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1432\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eRI, MS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eα\u003c/em\u003e-Humulene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1456\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1452\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e8.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eRI, MS, CI\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eγ-Gurjunene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1474\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1475\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eRI, MS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eγ\u003c/em\u003e-Muurolene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1477\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1478\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eRI, MS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eγ\u003c/em\u003e-Himachalene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1488\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1481\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eRI, MS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGermacrene B\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1555\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1559\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eRI, MS\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDillapiole\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1620\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1620\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e78.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.98\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eRI, MS, CI\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTotal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e98.53\u0026thinsp;\u0026plusmn;\u0026thinsp;1.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e98.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.82\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e97.47\u0026thinsp;\u0026plusmn;\u0026thinsp;1.22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMonoterpenes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e98.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.82\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e96.39\u0026thinsp;\u0026plusmn;\u0026thinsp;1.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSesquiterpenes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e16.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePhenylpropanoids\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e78.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0,98\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"7\"\u003eLegend: RI\u003csup\u003ea\u003c/sup\u003e = Retention indices calculated from retention times in relation to those of a series C\u003csub\u003e8\u003c/sub\u003e-C\u003csub\u003e40\u003c/sub\u003e of n-alkanes on a 30m DB-5 capillary column. RI\u003csup\u003eb\u003c/sup\u003e = Retention indices from literature. SD\u0026thinsp;=\u0026thinsp;Standard deviation. RI\u0026thinsp;=\u0026thinsp;retention indices, MS\u0026thinsp;=\u0026thinsp;mass spectroscopy, and CI\u0026thinsp;=\u0026thinsp;Co-injection with authentic compounds. See supplementary material for chromatograms.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Physicochemical characterization of nanoemulsions\u003c/h2\u003e \u003cp\u003eThe results of the physicochemical characterization of NEs are shown in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e (A and B). Our data revealed significant results in the zeta potential for the \u003cem\u003eC. carvi\u003c/em\u003e NE suggesting that the higher agitation speed promotes more negative zeta potential. For the \u003cem\u003eC. sinensis\u003c/em\u003e NE, a significant result was observed only in the size of the vesicles, demonstrating that the higher stirring speed reduces the size of the vesicles. In addition, a significant result in PDI values observed in the \u003cem\u003eP. aduncum\u003c/em\u003e NEs suggested that the shorter stirring time induces the smaller PDI values. Finally, the \u003cem\u003eC. carvi\u003c/em\u003e nanoemulsions were produced using a high stirring speed and short stirring time (1129 \u003cem\u003eg\u003c/em\u003e/1 h), \u003cem\u003eC. sinensis\u003c/em\u003e NE was produced using a low stirring speed, and short stirring time (282 \u003cem\u003eg\u003c/em\u003e/1 h) and \u003cem\u003eP. aduncum\u003c/em\u003e NEs produced using low stirring speed and long stirring time (282 \u003cem\u003eg\u003c/em\u003e/2 h) were chosen for the next experiments. Our choice was based on the best physicochemical parameters for industrial production.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of variables stirring speed (\u003cem\u003eg\u003c/em\u003e) and stirring time (h) in the physicochemical parameters of nanoemulsions of each essential oil associated with levothyroxine. Bold values indicate significant results by the Tukey test (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"8\" nameend=\"c8\" namest=\"c1\"\u003e \u003cp\u003eA) Size, polydispersity index (PDI), and zeta potential data for each nanoemulsion\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"1\" nameend=\"c9\" namest=\"c9\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEmulsion formulations (g/h)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e\u003cem\u003eMean size (nm)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e\u003cem\u003ePDI\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e\u003cem\u003eζ (mV)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c9\" namest=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCarum carvi\u003c/em\u003e 282 \u003cem\u003eg\u003c/em\u003e / 1 h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e384.7\u0026thinsp;\u0026plusmn;\u0026thinsp;4.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e0.333\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e-12.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c9\" namest=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCarum carvi\u003c/em\u003e 1129 \u003cem\u003eg\u003c/em\u003e / 1 h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e277.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e0.304\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e-22.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c9\" namest=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCarum carvi\u003c/em\u003e 282 \u003cem\u003eg\u003c/em\u003e / 2 h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e408.6\u0026thinsp;\u0026plusmn;\u0026thinsp;7.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e0.409\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e-15.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c9\" namest=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCarum carvi\u003c/em\u003e 1129 g / 2 h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e259.0\u0026thinsp;\u0026plusmn;\u0026thinsp;12.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e0.323\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e-14.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c9\" namest=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCarum carvi\u003c/em\u003e 635 g / 1 h 30 min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e218.5\u0026thinsp;\u0026plusmn;\u0026thinsp;4.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e0.315\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e-12.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c9\" namest=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCarum carvi\u003c/em\u003e 635 g / 1 h 30 min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e223.1\u0026thinsp;\u0026plusmn;\u0026thinsp;3.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e0.317\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e-13.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c9\" namest=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCarum carvi\u003c/em\u003e 635 g / 1 h 30 min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e225.8\u0026thinsp;\u0026plusmn;\u0026thinsp;6.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e0.257\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e-14.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c9\" namest=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCitrus sinensis\u003c/em\u003e 282 \u003cem\u003eg\u003c/em\u003e / 1 h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e287.3\u0026thinsp;\u0026plusmn;\u0026thinsp;20.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e0.226\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e-21.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c9\" namest=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCitrus sinensis\u003c/em\u003e 1129 g / 1 h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e401.9\u0026thinsp;\u0026plusmn;\u0026thinsp;45.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e0.113\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e-18.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c9\" namest=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCitrus sinensis\u003c/em\u003e 282 \u003cem\u003eg\u003c/em\u003e / 2 h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e325.3\u0026thinsp;\u0026plusmn;\u0026thinsp;3.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e0.424\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e-16.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c9\" namest=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCitrus sinensis\u003c/em\u003e 1129 g / 2 h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e391.8\u0026thinsp;\u0026plusmn;\u0026thinsp;35.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e0.223\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e-20.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c9\" namest=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCitrus sinensis\u003c/em\u003e 635 g / 1 h 30 min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e200.2\u0026thinsp;\u0026plusmn;\u0026thinsp;7.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e0.339\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e-12.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c9\" namest=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCitrus sinensis\u003c/em\u003e 635 g / 1 h 30 min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e235.2\u0026thinsp;\u0026plusmn;\u0026thinsp;6.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e0.460\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e-16.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c9\" namest=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCitrus sinensis\u003c/em\u003e 635 g / 1 h 30 min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e224.9\u0026thinsp;\u0026plusmn;\u0026thinsp;10.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e0.235\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e-18.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c9\" namest=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePiper aduncum\u003c/em\u003e 282 \u003cem\u003eg\u003c/em\u003e / 1 h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e489.1\u0026thinsp;\u0026plusmn;\u0026thinsp;78.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e0.234\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e-32.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c9\" namest=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePiper aduncum\u003c/em\u003e 1129 g / 1 h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e445.9\u0026thinsp;\u0026plusmn;\u0026thinsp;298.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e0.299\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e-36.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c9\" namest=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePiper aduncum\u003c/em\u003e 282 \u003cem\u003eg\u003c/em\u003e / 2 h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e326.9\u0026thinsp;\u0026plusmn;\u0026thinsp;15.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e0.406\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e-30.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c9\" namest=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePiper aduncum\u003c/em\u003e 1129 g / 2 h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e401.7\u0026thinsp;\u0026plusmn;\u0026thinsp;21.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e0.478\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e-39.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c9\" namest=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePiper aduncum\u003c/em\u003e 635 g / 1 h 30 min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e631.3\u0026thinsp;\u0026plusmn;\u0026thinsp;118.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e0.302\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e-36.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c9\" namest=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePiper aduncum\u003c/em\u003e 635 g / 1 h 30 min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e426.4\u0026thinsp;\u0026plusmn;\u0026thinsp;42.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e0.343\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e-31.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c9\" namest=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePiper aduncum\u003c/em\u003e 635 g / 1 h 30 min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e489.2\u0026thinsp;\u0026plusmn;\u0026thinsp;57.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e0.265\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e-35.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c9\" namest=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"9\" nameend=\"c9\" namest=\"c1\"\u003e \u003cp\u003eB. ANOVA of 3 x 2\u003csup\u003e2\u003c/sup\u003e full experimental design\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"9\" nameend=\"c9\" namest=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCarum carvi\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSize\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eSum of squares\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eDf\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e\u003cem\u003eMean square\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e\u003cem\u003eF\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStirring speed\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16537.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e16537.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e2.393784\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e0.219558\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStirring time\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e8.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e0.001217\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e0.974359\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePDI\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eSum of squares\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eDf\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e\u003cem\u003eMean square\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e\u003cem\u003eF\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStirring speed\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.003306\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e0.003306\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e1.011704\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e0.420410\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStirring time\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.002256\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e0.002256\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e0.690407\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e0.493425\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eZeta\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eSum of squares\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eDf\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e\u003cem\u003eMean square\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e\u003cem\u003eF\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStirring speed\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e77.4400\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e\u003cb\u003e77.44000\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e\u003cb\u003e89.6988\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e\u003cb\u003e0.010965\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStirring time\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.4400\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e1.44000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e1.6680\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e0.325658\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"9\" nameend=\"c9\" namest=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCitrus sinensis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSize\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eSum of squares\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eDf\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e\u003cem\u003eMean square\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e\u003cem\u003eF\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStirring speed\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e8226.49\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e\u003cb\u003e8226.49\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e\u003cb\u003e25.42729\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e\u003cb\u003e0.037150\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStirring time\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e198.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e198.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e0.61450\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e0.515195\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePDI\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eSum of squares\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eDf\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e\u003cem\u003eMean square\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e\u003cem\u003eF\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStirring speed\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.024649\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e0.24649\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e1.943876\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e0.297943\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStirring time\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.023716\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e0.023716\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e1.870298\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e0.304843\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eZeta\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eSum of squares\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eDf\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e\u003cem\u003eMean square\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e\u003cem\u003eF\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStirring speed\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.36000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e0.36000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e0.045743\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e0.850467\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStirring time\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.00000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e1.00000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e0.127065\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e0.755588\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"9\" nameend=\"c9\" namest=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePiper aduncum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSize\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSum of squares\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDf\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eMean square\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStirring speed\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e249.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e249.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e0.022653\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e0.894171\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStirring time\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10650.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e10650.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e0.966443\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e0.429218\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePDI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSum of squares\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDf\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eMean square\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStirring speed\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.004692\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e0.004692\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e3.08228\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e0.221235\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStirring time\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e0.030800\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e\u003cb\u003e0.030800\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e\u003cb\u003e20.23226\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e\u003cb\u003e0.046039\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZeta\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSum of squares\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDf\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eMean square\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStirring speed\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e42.90250\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e42.90250\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e6.048285\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e0.133109\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStirring time\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.12250\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e0.12250\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e0.017270\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e0.907475\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Antioxidant activity of nanoemulsions (DPPH˙ and ABTS˙\u003csup\u003e+\u003c/sup\u003e)\u003c/h2\u003e \u003cp\u003eThe antioxidant activity of NEs from the leaves of \u003cem\u003eP. aduncum\u003c/em\u003e, \u003cem\u003eC. carvi\u003c/em\u003e, and \u003cem\u003eC. sinensis\u003c/em\u003e associated with levothyroxine is presented in Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. According to our results, all NEs showed antioxidant activity. In the DPPH test, the highest antioxidant activity was observed for \u003cem\u003eC. carvi\u003c/em\u003e NE (EC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;204.3 \u0026micro;g/mL). In the test with the radical ABTS, the \u003cem\u003eP. aduncum\u003c/em\u003e NE (EC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;156.6 \u0026micro;g/mL) showed the best antioxidant activity.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAntioxidant activity of the essential oil of species of \u003cem\u003eCarum carvi\u003c/em\u003e, \u003cem\u003eCitrus sinensis\u003c/em\u003e, and \u003cem\u003ePiper aduncum\u003c/em\u003e. Legend: Ascorbic acid and TROLOX (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) were used as reference antioxidants.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eChemicals\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eDPPH\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eABTS\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eEC\u003c/em\u003e\u003csub\u003e\u003cem\u003e50\u003c/em\u003e\u003c/sub\u003e \u003cem\u003e\u0026micro;g/mL (Confidence interval)\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eEC\u003c/em\u003e\u003csub\u003e\u003cem\u003e50\u003c/em\u003e\u003c/sub\u003e \u003cem\u003e\u0026micro;g/mL (Confidence interval)\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eC. carvi\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e204.3 (199.4-208.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e194.9 (190.4-199.4)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eC. sinensis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e288.5 (280.8-295.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e191.5 (186.7-196.3)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP. aduncum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e266.5 (261.4\u0026ndash;272.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e156.6 (151.4-161.8)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAscorbic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.6 (1.4\u0026ndash;1.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTROLOX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.1 (3.7\u0026ndash;5.8)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\u003cp\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Effects of levothyroxine and nanoemulsions on zebrafish embryonic development\u003c/h2\u003e \u003cp\u003eToxicity tests were carried out with zebrafish embryos and larvae to evaluate the toxicity of the NEs. The results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003e from the zebrafish embryo and larval stages exposed to different dilution ratios of L-T\u003csub\u003e4\u003c/sub\u003e and NEs of each essential oil. Mortality was the only significant effect observed at 24 hpf in the PAN32 group. Teratogenic effects were observed in embryos still in the chorion. However, toxic effects were most observed in the 72 hpf larvae stage for the following groups: ECS, CSN32, EPA, PAN44, and PAN32, with \u003cem\u003eP. aduncum\u003c/em\u003e NE being the most toxic. While at 96 hpf, significant teratogenic effects were observed in groups exposed to all NEs. Yolk sac edema is the most common toxic effect presented in zebrafish larvae, followed by pericardial edema.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Effects of levothyroxine and nanoemulsions on zebrafish animal behavior\u003c/h2\u003e \u003cp\u003eThe results of the thigmotaxis test (TH test) and touch sensitivity response (TS test) are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eB. The data in the TH test represents the number of animals that move to the edge or periphery of plates. We did not observe toxic effects in this test when larvae were exposed to L-T\u003csub\u003e4\u003c/sub\u003e or any of the NEs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). TS test was also performed by the mechanical stimuli to evaluate if L-T\u003csub\u003e4\u003c/sub\u003e or NEs exposure affects the swimming activity of the larvae. Our results (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) show that this parameter was affected only in the larvae exposed to empty \u003cem\u003eC. carvi\u003c/em\u003e NE (ECC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eEssential oils have been used by our ancestors as therapeutic agents for the treatment of various diseases. Their chemical constituents have high volatility. As a result, nanoencapsulation of these oils has been proposed as a drug delivery system since this formulation technique is capable of improving the solubility, stability, and bioavailability of the compounds. NEs are being studied in recent years using formulations produced from essential oils (Pavoni et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In the present study, GC-MS and GC-FID identified 16 chemical constituents in \u003cem\u003eCitrus sinensis\u003c/em\u003e essential oil. Limonene was found to be the main constituent at 90.13%. Rezende et al. (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) also found that essential oil from \u003cem\u003eC. sinensis\u003c/em\u003e had a high limonene content (91.4%), which corroborates our data. In addition, traces of α- and β-pinene (1.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07, 1.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10, respectively), and phellandrene are present. Also, there is aldehyde (E)-isocitral (0.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03), which provides the characteristic odor present in this essential oil, as noted by Ali et al. (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). \u003cem\u003ePiper aduncum\u003c/em\u003e essential oil was identified with 13 chemical constituents, and dillapiole was the main constituent (78.41%), corroborating data in studies reported by Pino et al. (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2004\u003c/span\u003e) and Maia et al. (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). Finally, \u003cem\u003eCarum carvi\u003c/em\u003e essential oil showed 6 constituents. The main ones are carvone (60.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.80) and limonene (34.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.64). This dataset corroborates the findings by Obeid et al. (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) and Trifan et al. (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIncreased therapeutic potential of the essential oil is possible through nanoencapsulation (Aziz et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The emulsification technique was successfully used to develop the three NEs from essential oils in our study. The formulation of NEs was affected by the stirring speed and stirring time. Our formulations were characterized as NEs since their vesicle sizes were less than 300 nm (Singh et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). It is preferable that the polydispersion index (PDI), a measure that characterizes the homogeneity of the vesicle size, have values below 0.2 (Danaei et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), which indicates a high degree of size homogeneity (Caddeo et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). PDI values can vary more or less depending on the technique used to produce NEs. The results obtained in our study correspond to PDI values varying between 0.3 and 0.4, which were obtained in our best NEs, demonstrating the homogeneity of the formulations. Additionally, the zeta potential (ζ - mv) parameter measures the surface charge and electrostatic parameters of vesicles (Grisham and Nanda, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Our data revealed that all formulations presented high negative potential, indicating the stability of the NEs by repulsion forces between the vesicles and showing the absence of aggregations of the vesicles.\u003c/p\u003e \u003cp\u003eThe use of surfactants with intermediate high hydrophilic-lipophilic balance (HLB) (generally between 11 and 16), such as tween, is the best choice to produce cheaper NEs by dispersing the oily phase in the aqueous phase. In addition, non-ionic surfactants such as Span are important due to their better toxicological safety for oral ingestion (Pavoni et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). According to Aziz et al. (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), a higher value HLB surfactant is needed to produce smaller vesicles in the NEs, which was also used in our studies.\u003c/p\u003e \u003cp\u003eNEs containing \u003cem\u003eC. carvi\u003c/em\u003e, \u003cem\u003eC. sinensis\u003c/em\u003e, and \u003cem\u003eP. aduncum\u003c/em\u003e essential oils showed good antioxidant activity, according to our values by EC\u003csub\u003e50\u003c/sub\u003e (concentration of sample required to scavenge 50% of free radicals). This data confirms findings by Trifan et al. (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), Rodr\u0026iacute;guez et al. (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), and Frassinetti et al. (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) for \u003cem\u003eC. carvi\u003c/em\u003e, \u003cem\u003eP. aduncum\u003c/em\u003e, and \u003cem\u003eC. sinensis\u003c/em\u003e, respectively. These results can be explained because oils have a large percentage of oxygenated compounds in their compositions, such as limonene, carvone, and dilapiol, besides phenylpropanoid that is present in their structure methoxy and methylenedioxy groups. Furthermore, we cannot rule out the presence of other oxygenated compounds found in lower percentages than the cited ones. Our findings are important to developing products that reduce the toxicity of monotherapy levothyroxine treatment (Chaker et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), and we suggest a combination of therapeutics and nanoemultions can be effective.\u003c/p\u003e \u003cp\u003eKeeping this in mind, this study focused on the development of the NEs produced from essential oils, and their characterization and toxicity evaluation. We used the zebrafish (\u003cem\u003eDanio rerio\u003c/em\u003e) to evaluate the toxicity. The ethical consideration of the use of lower vertebrates such as zebrafish to evaluate the toxicity of NE was taken into account (Jayasinghe and Jayawardena, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Our results revealed that the animals exposed to all NEs presented teratogenic effects. Independent of the type of essential oils used, despite \u003cem\u003eP. aduncum\u003c/em\u003e NE producing more toxicity, similar teratogenic effects were observed for each NE. This could indicate that the same chemical compound used in the formulation of nanoemulsions has related toxicity effects. We did not observe significant teratogenic effects in the L-T\u003csub\u003e4\u003c/sub\u003e solution. However, all NEs were produced with surfactants (tween 80 and span 80). This idea is supported by Yang et al. (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), who found that tween 80 induced anaphylactoid reactions in zebrafish. Ginzburg et al. (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) investigated the influence of surfactants on nanoparticle biocompatibility by studying mixtures of ligand-stabilized gold nanoparticles and polysorbate 80 (span 80) in embryonic zebrafish and observed that the mixtures produced synergistic toxicity at concentrations where the individual components were benign.\u003c/p\u003e \u003cp\u003eThe acute toxicity of span 80 was previously studied in zebrafish (Sun et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). This study revealed that span 80 has high mortality rate on zebrafish. This indicates that pharmaceutical excipients, including mixtures such as span 80, have potential risks when the safety of these new nanotechnological drugs were evaluated in zebrafish. Additionally, tween 80 also could be toxic to zebrafish (Ali et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). In our study, we saw more toxic effects in the larval stage and the absence of chorion may explain this effect. The chorion functions to protect the embryo against chemical exposure, for example (Kim and Tanguay, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), and when it ruptures, the fish are more readily exposed to toxins. Finally, our results indicate these NEs affected morphology rather than behavioral endpoints. This may be because the animals chosen for tests did not show any morphological defects, and probably these animals were more resistant to or less affected by toxic compounds present in NEs. Further research is necessary to reduce adverse effects before the use of these NEs.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn this study, antioxidant essential oil\u0026ndash;based nanoemulsions associated with levothyroxine were successfully developed using a high‑energy emulsification approach and optimized by factorial experimental design. The formulations showed suitable physicochemical characteristics, including nanometric mean size, acceptable polydispersity index, and negative zeta potential, indicating good colloidal stability. All nanoemulsions exhibited relevant antioxidant activity, confirming the contribution of the essential oils and supporting their potential to mitigate oxidative stress associated with hypothyroidism. Despite these promising properties, \u003cem\u003ein vivo\u003c/em\u003e toxicity assessment using the zebrafish model revealed significant teratogenic effects, particularly during the larval stage, regardless of the essential oil used. Behavioral alterations were limited and formulation‑dependent, with effects observed mainly for the \u003cem\u003eCarum carvi\u003c/em\u003e nanoemulsion. The absence of toxicity in levothyroxine alone and the occurrence of toxic effects in empty nanoemulsions suggest that surfactants employed in the formulations likely play a major role in the observed toxicity. Further studies focusing on reformulation strategies, surfactant replacement and long‑term toxicity are necessary before these nanoemulsions can be considered viable therapeutic alternatives for hypothyroidism.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompeting Interests\u003c/h2\u003e\n\u003cp\u003eThe research reported in this manuscript is part of a patent (BR102020025513-4) in the course of the process.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThe authors would like to thank the \u003cem\u003eUniversidade Federal Rural de Pernambuco\u003c/em\u003e, \u003cem\u003eCoordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior\u003c/em\u003e \u0026ndash; CAPES for the scholarship to Thamiris P. Santos and fellowship for Pabyton G. Cadena, Conselho Nacional de Desenvolvimento Cient\u0026iacute;fico e Tecnol\u0026oacute;gico \u0026ndash; CNPq for the scholarship to Paulo E. S. Bastos and productivity grant to Pabyton G. Cadena (306947/2020-0). The authors also thank \u003cem\u003eConselho Nacional de Desenvolvimento Cient\u0026iacute;fico e Tecnol\u0026oacute;gico\u003c/em\u003e \u0026ndash; CNPq (Grant 423038/2018-4) and \u003cem\u003ePr\u0026oacute;-Reitoria de Pesquisa e P\u0026oacute;s-Gradua\u0026ccedil;\u0026atilde;o\u003c/em\u003e of UFRPE \u0026ndash; PRPPG (Grant PRPPG 05/2018) for financial support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe protocols used in this study were approved by the Ethics Committee in the use of animals of the \u003cem\u003eUniversidade Federal Rural de Pernambuco\u003c/em\u003e - Brazil, protocol number 091/2018.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data and data and material used in this study are available from the corresponding author on request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of generative AI in scientific writing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDuring the preparation of this work the author(s) used Copilot in order to correct the language of the manuscript.\u003c/p\u003e\n\u003ch2\u003eAuthor Contributions\u003c/h2\u003e\n\u003cp\u003eDetails of each author with their contribution in this paper: Conceptualization, methodology, validation, formal analysis, Investigation, data curation, writing - original draft, writing - review and editing, and visualization were performed by Thamiris Pinheiro Santos. Investigation and data Curation also were performed by Paulo Eduardo da Silva Bastos. Authors Matheus Victor Viana de Melo, Tiago Queiroz da Mota Bittencourt, Renata Meireles Oliveira Padilha, Renatta Priscilla Ferreira Silva, Niely Priscila Correia da Silva, Carolina Alves de Araujo, Marcilio Martins de Moraes, Cl\u0026aacute;udio Augusto Gomes da C\u0026acirc;mara and Amanda Rodrigues dos Santos Magnabosco performed investigation. Authors Jadson Freitas da Silva and Andr\u0026eacute; Lucas Corr\u0026ecirc;a de Andrade performed the investigation and formal analysis. Co-supervision, project administration, writing - original draft, writing - review \u0026amp; editing were performed by Marilia Ribeiro Sales Cadena, Samara da Silva Gomes and Yuri Mateus Lima de Albuquerque. Conceptualization, methodology, validation, formal analysis, resources, data curation, writing - original draft, writing - review and editing, visualization, supervision, project administration, and funding acquisition were performed by Pabyton Gon\u0026ccedil;alves Cadena. All authors read and approved of the final manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgments\u003c/h2\u003e\n\u003cp\u003eThe authors would like to thank the \u003cem\u003eLaborat\u0026oacute;rio de Ecofisiologia Comportamento Animal \u0026ndash; LECA\u003c/em\u003e for the inspiration.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAdams RP (2017) Identification of essential oil components by gas chromatography/ mass spectrometry. Allured Publishing Corporation, Carol Stream, Illinois\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAli B, Al-Wabel NA, Shams S, Ahamad A, Khan SA, Anwar F (2015) Essential oils used in aromatherapy: A systemic review. Asian Pac J Trop Biomed 5:601\u0026ndash;611\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAli S, van Mil HG, Richardson MK (2011) Large-scale assessment of the zebrafish embryo as a possible predictive model in toxicity testing. PLoS ONE 6:e21076\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlmeida C, Brasil MA, Costa AJL, Reis FAA, Reuters V, Teixeira P, Ferreira M, Marques AM, Melo BA, Teixeira LBBM, Buescu A, Vaisman M (2007) Subclinical hypothyroidism: psychiatric disorders and symptoms. 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Soft Matter 8:1719\u0026ndash;1729\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eObeid HA, Mshimesh BAR, Jasim GA (2020) Study on the Thyrotropic Activity Of Hydroalcoholic Caraway Seeds Extract In Female Rats With Hypothyroid- Induction Model. Biochem Cell Archives 20:3933\u0026ndash;3943\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOECD (2025) Education at a Glance 2013: OECD Indicators. OECD Publishing\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePavoni L, Perinelli DR, Bonacucina G, Cespi M, Palmieri GF (2020) An Overview of Micro- and Nanoemulsions as Vehicles for Essential Oils: Formulation, Preparation and Stability. 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Surf Interfaces 9:124\u0026ndash;132\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWesterfield M (2000) The zebrafish book: A guide for the laboratory use of zebrafish (Danio rerio). University of Oregon, Eugene, OR\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang R, Lao QC, Yu HP, Zhang Y, Liu HC, Luan L, Sun HM, Li CQ (2015) Tween-80 and impurity induce anaphylactoid reaction in zebrafish. J Appl Toxicol 35:295\u0026ndash;301\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[{"identity":"b1a47d10-c25e-46c6-ad7d-b5775d414228","identifier":"10.13039/501100002322","name":"Coordenação de Aperfeiçoamento de Pessoal de Nível Superior","awardNumber":"Postgraduate scholarships","order_by":0},{"identity":"3a893375-569e-4a6f-b34e-173e8adb3686","identifier":"10.13039/501100003593","name":"Conselho Nacional de Desenvolvimento Científico e Tecnológico","awardNumber":"306947/2020-0","order_by":1},{"identity":"f68f955e-3e20-4bd3-a1cb-b8f3ca0b01e6","identifier":"10.13039/501100003593","name":"Conselho Nacional de Desenvolvimento Científico e Tecnológico","awardNumber":"423038/2018-4","order_by":2}],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Federal Rural University of Pernambuco","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Carum carvi, Citrus sinensis, Piper aduncum, Levothyroxine, Danio rerio","lastPublishedDoi":"10.21203/rs.3.rs-9568099/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9568099/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study aimed to develop nanoemulsions containing levothyroxine associated with essential oils with antioxidant activity and to evaluate their toxicity in zebrafish (\u003cem\u003eDanio rerio\u003c/em\u003e). The essential oils of \u003cem\u003ePiper aduncum\u003c/em\u003e, \u003cem\u003eCitrus sinensis\u003c/em\u003e, and \u003cem\u003eCarum carvi\u003c/em\u003e were characterized by qualitative and quantitative gas chromatography. The oil/water nanoemulsions were prepared using a high-energy emulsification method, and the effect of time and stirring speed was studied by experimental design. The mean size, polydispersity index (PDI), and zeta potential were evaluated as response variables. Antioxidant activity was also analyzed. Toxicity tests were conducted on zebrafish based on the OECD 236 guidelines, and the response variables were mortality, teratogenic effects, and behavioral tests. GC-MS analysis of \u003cem\u003eC. carvi\u003c/em\u003e, \u003cem\u003eC. sinensis\u003c/em\u003e, and \u003cem\u003eP. aduncum\u003c/em\u003e identified 6, 16, and 13 compounds, with carbon (60.44 ± 0.80%), limonene (90.13 ± 1.11%), and dilapiol (78.41 ± 0.98%) being the major constituents. Nanoemulsions were successfully developed, with a mean size ranging from 218.5 to 489.2 nm, PDI of 0.113 to 0.478, and zeta potential of -12.5 to -39.1 mV. All oils exhibited antioxidant activity, with EC\u003csub\u003e50\u003c/sub\u003e values ranging from 204.3 to 288.5 µg/mL for DPPH and 156.6 to 194.9 µg/mL for ABTS. Toxicity tests revealed that all oils exhibited toxic effects with animals exposed to all nanoemulsions showing teratogenic effects, mostly at the larval stage. Only the \u003cem\u003eC. carvi\u003c/em\u003e nanoemulsion affected the behavioral tests. In summary, nanoemulsions showed strong antioxidant activity and were well-characterized, but toxicity in zebrafish suggests safety concerns that require further research before therapeutic use.\u003c/p\u003e","manuscriptTitle":"Antioxidant essential oil–based nanoemulsions associated with levothyroxine: Physicochemical characterization and toxicity assessment in zebrafish","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-04 12:21:01","doi":"10.21203/rs.3.rs-9568099/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"607b3f5d-4849-4ea9-a17f-8526722d3805","owner":[],"postedDate":"May 4th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":67276087,"name":"Chemical Biology"},{"id":67276088,"name":"Nanoscience"},{"id":67276089,"name":"Toxicology"}],"tags":[],"updatedAt":"2026-05-04T12:21:01+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-04 12:21:01","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9568099","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9568099","identity":"rs-9568099","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}