Introduction
Gastrointestinal nematode infections (GNIs) are one of the main causes of economic losses for sheep and goats worldwide. Among nematodes, Haemonchus contortus is considered the most pathogenic, as it is a haematophagous parasite that causes anaemia, submandibular oedema and, in more severe cases, can lead to death (Wang et al., Reference Wang, Li, Zhang, Yang, Ahmad, Li, Du and Hu2017; Toscano et al., Reference Toscano, Lopes, Giraldelo and Da Silva2018). The control of H. contortus is mainly based on the use of synthetic anthelmintics. However, the intensive use of these drugs and inadequate management have resulted in the selection of resistant and multiresistant nematode populations worldwide (Jackson and Coop, Reference Jackson and Coop2000; Bartram et al., Reference Bartram, Leathwick, Taylor, Geurden and Maeder2012; Kaplan and Vidyashankar, Reference Kaplan and Vidyashankar2012; Kotze and Prichard, Reference Kotze and Prichard2016; Tuersong et al., Reference Tuersong, He, Zhu, Yang, Zhang, Ahmad, Di, Wang, Zhou, Liu, Chen and Hu2020). Thus, new control strategies are being implemented, such as the use of essential oils from plants and their bioactive compounds (Ribeiro et al., Reference Ribeiro, Camurça-Vasconcelos, Macedo, Santos, Ribeiro, Pereira, Viana, Paula and Bevilaqua2015; Ferreira et al., Reference Ferreira, Benincasa, Fachin, França, Contini, Chagas and Beleboni2016; Araújo-Filho et al., Reference Araújo-Filho, Ribeiro, André, Cavalcante, Rios, Schwinden, Rocha, Macedo, Morais, Bevilaqua and Oliveira2019).
Essential oils are complex mixtures of active substances of plant origin that exhibit a variety of properties, such as anthelmintic effects, due to the synergism of their compounds (Zhu et al., Reference Zhu, Dai, Li Yang and Qiu2013; Katiki et al., Reference Katiki, Barbieri, Araujo, Veríssimo, Louvandini and Ferreira2017). Studies evaluating the anthelmintic efficacy of compounds isolated from essential oils, such as monoterpenes e.g. carvacrol, thymol, terpinen-4-ol, limonene and carvone, have demonstrated in vitro and in vivo effects on H. contortus (André et al., Reference André, Ribeiro, Cavalcante, Santos, Macedo, Paula, Freitas, Maia, Melo and Bevilaqua2016; Grando et al., Reference Grando, De Sá, Baldissera, Oliveira, De Souza, Raffin, Santos, Domingues, Minho, Leal and Monteiro2016; Katiki et al., Reference Katiki, Barbieri, Araujo, Veríssimo, Louvandini and Ferreira2017, Reference Katiki, Araujo, Ziegelmeyer, Gomes, Gutmanis, Rodrigues, Bueno, Veríssimo, Louvandini and Ferreira2019; André et al., Reference André, Ribeiro, Oliveira, Macedo, Rondon and Bevilaqua2018; Garbin et al., Reference Garbin, Munguía, Saldaña, Deschamps, Cipriano and Molento2021; Silva et al., Reference Silva, Lifschitz, Macedo, Campos, Viana-Filho, Alcântara, Araújo, Alencar and Costa-Junior2021). Carvone is a monoterpene that can be found as the main active component of several essential oils, Mentha spicata L., Anethum graveolens L., Carum carvi L. and Lippia alba (De Sousa et al., Reference De Sousa, Nóbrega and Almeida2007; Johri, Reference Johri2011). However, one of the limiting factors for the use of this biocompound is its volatility, instability and low water solubility. Thus, an alternative to improve these characteristics is encapsulation in biopolymer matrices (Lertsutthiwong and Rojsitthisak, Reference Lertsutthiwong and Rojsitthisak2011).
Nanoemulsions containing bioactive compounds form nanostructured systems that improve physicochemical stability, modulation of release rates and bioavailability of active principles (Abreu et al., Reference Abreu, Costa, Cardial and André2020). For nanoencapsulation, biopolymers such as sodium alginate are used. It is a natural polysaccharide extracted from brown marine algae, red algae or bacteria. It has been used as an encapsulating matrix because it is a biodegradable polymer that is biocompatible, is sensitivity to pH, has low toxicity and promotes the controlled release of drugs (Wong et al., Reference Wong, Preston and Schiller2000; Prabaharan, Reference Prabaharan2015; Martins et al., Reference Martins, Poncelet, Rodrigues and Renard2017; Cheng et al., Reference Cheng, Jiang, Xu, Liu and Mao2020; Hariyadi and Islam, Reference Hariyadi and Islam2020). The composition of formulations, method and preparation conditions are intrinsically related (Lovelyn and Attama, Reference Lovelyn and Attama2011). Thus, the comparison between the preparation methods of nanoemulsions is advantageous, as it allows obtaining more stable nanoemulsions and efficient controlled release systems (Abreu et al., Reference Abreu, Costa, Cardial and André2020).
Therefore, the objective of this work was to evaluate the anthelmintic activity of R-carvone nanoemulsions produced by different homogenization methods towards H. contortus in vitro.
Materials and methods
Preparation of nanoemulsions
R-(−)-Carvone nanoemulsions (Quinarí®, Ponta Grossa, Paraná, Brazil) were prepared by the high-energy emulsification method with 2 different homogenization techniques (high-pressure homogenization and ultrasound) using the following equipment: an ultrahomogenizer ULTRA380 (Ultrastirrer, Jacareí, São Paulo, Brazil)) and a sonicator Fisherbrand™ Model 50 Sonic Dismembrator (Thermo Fisher Scientific, Waltham, Massachusetts, EUA) with or without the addition of sodium alginate (Dinâmica, Indaiatuba, São Paulo, Brazil) as a polymer matrix. For the preparation of the uncoated R-carvone nanoemulsion with sodium alginate (UNAlg-son), R-carvone and Tween 80 (Dinâmica, Indaiatuba, São Paulo, Bra) were added at a proportion of 1:15. Then, they were homogenized in a sonicator (Fisherbrand™ Model 50 Sonic Dismembrator) for 5 min at an amplitude of 30 μm. For preparation of the uncoated R-carvone nanoemulsion with sodium alginate (UNAlg-ultra), Tween 80 and R-carvone were added at a proportion of 1:2. Then, they were placed in an ultrasonic eco-sonics Q1.8/25 (Indaiatuba, São Paulo, Brazil) bath for 5 min and homogenized in an ultrahomogenizer ULTRA380 (Ultrastirrer) at 18 000 rpm for 2 min. To prepare the sodium alginate-coated R-carvone nanoemulsion (CNAlg-ultra), 4% sodium alginate, Tween 80 and R-carvone were added in proportions of 1:1:4. The solution was subjected to an ultrasonic bath (Q1.8/25, Ultronique) for 5 min and then homogenized in an ultrahomogenizer (ULTRA380, Ultrastirrer) at 22 000 rpm for 5 min.
Stability of nanoparticles
The physical stability of the emulsions homogenized in an ultrahomogenizer (UNAlg-ultra and CNAlg-ultra) was evaluated at 1, 7, 14, 21, 30, 60 and 90 days after preparation. For this procedure, ~10 mL of each of the nanoemulsions was maintained in closed test tubes, without exposure to light, at a temperature of 26°C. Periodic observation was implemented to detect any visual signs of instability, such as creaming, sedimentation or phase separation. Creaming and sedimentation were measured over time with the aid of a caliper. The creaming index (CI) and sedimentation index (SI) were determined by relating the height in centimetres of the precipitates formed in the test tube (H a) with the total height of the sample (H t) after the mentioned preparation periods, calculated according to equations (1) and (2), adapted from Mwangi et al. (Reference Mwangi, Ho, Tey and Chan2016):
Encapsulation efficiency
The encapsulation efficiency (EE) was determined by the difference between the initial amount of carvone in the nanoemulsion and the quantified free amount after ultracentrifugation (8000 rpm for 30 min) (equation (3)) using an Amicon® filter (molecular weight cutoff of 100 kDa, Millipore, UK) (Campelo et al., Reference Campelo, Melo, Arrais, Nascimento, Gramosa, Soares, Ribeiro, Silva, Júnior and Ricardo2021). In the assay, an aliquot of 1 mL of each nanoemulsion (equivalent to 1.65 mg of carvone) was centrifuged, and the absorbance was determined at 235 nm (λ max of carvone) using a Genesys 6 Spectrophotometers (Thermo Fisher Scientific, Waltham, Massachusetts, EUA). The %EE was determined from a previously prepared carvone standard curve at concentrations ranging from 2 to 18 μg mL−1 (y = 0.0678 − 0.016; R 2 = 0.9995). The maximum wavelength obtained in the spectrophotometric scan of carvone was λ = 235 nm. From the constructed standard curve, it was possible to determine the content of encapsulated carvone in the nanoemulsion as well as the EE. The experiment was carried out in triplicate, and the results are expressed as the mean ± standard error:
Physicochemical characterization of nanoemulsions
Particle size and zeta potential were determined by dynamic light scattering using the Malvern Zetasizer/Nanoseries Z590 equipment.
Detection of the main vibrational stretching modes was carried out by means of spectroscopy in the infrared region by Fourier transform (FTIR). Sample readings were performed on a Shimadzu spectrometer from 400 to 4000 cm−1 using KBr inserts.
Scanning electron microscopy (SEM) of nanoemulsions was performed after 42 days of production. A Zeiss 940A microscope was used at an accelerating voltage of 20 kV and a magnification of 100–3000×. Approximately 200 μL of nanoemulsions were placed on rounded metallic structures and dehydrated in a drying oven at 50°C for ~1 h until a thin sample film was obtained on the coin.
Haemonchus contortus isolate
The Kokstad (KOK) isolate of H. contortus multiresistant to commercial synthetic anthelmintics, benzimidazoles, levamisole and macrocyclic lactones was used (Neveu et al., Reference Neveu, Charvet, Fauvin, Cortet, Castagnone-Sereno and Cabaret2007, Reference Neveu, Charvet, Fauvin, Cortet, Beech and Cabaret2010; Fauvin et al., Reference Fauvin, Charvet, Issouf, Cortet and Neveu2010; Barrere et al., Reference Barrere, Beech, Charvet and Prichard2014). The isolate was supplied by the Institut National de Recherche pour l'Agriculture, l'Alimentation, et l'Environnement.
Egg hatch test
The egg hatch test (EHT) was performed according to the technique described by Coles et al. (Reference Coles, Jackson, Pomroy, Prichard, Von Samsonhimmelstjerna, Silvestre, Taylor and Vercruysse2006). For egg recovery, feces were collected directly from the rectal ampoule of a sheep experimentally infected with the KOK isolate and processed according to Hubert and Kerboeuf (Reference Hubert and Kerboeuf1992). Suspensions (250 μL) containing ~100 fresh eggs were incubated with the same volume of solutions from the following treatments: G1: UNAlg-son and G2: CNAlg-ultra, at concentrations ranging from 0.06 to 2.0 mg mL−1; G3: UNAlg-ultra, at concentrations ranging from 0.125 to 0.007 mg mL−1; G4: 0.025 mg mL−1 of thiabendazole (Sigma-Aldrich®) (positive control); G5: 3% Tween 80 (negative control) and G6: 4% sodium alginate polymer matrix (negative control). The suspension was incubated for 48 h at 25°C, and 5% Lugol's iodine solution was added to stop the hatching of the larvae. For each treatment and control, 5 replicates and 3 repetitions were performed.
Larval development test (LDT)
Eggs of H. contortus were incubated for 24 h to obtain L1. Solutions consisting of 500 μL of L1 (~250 larvae) and equal volumes of the following treatments: G1: UNAlg-son; G2: UNAlg-ultra and G3: CNAlg-ultra, in concentrations ranging from 0.06 to 2.0 mg mL−1; G4: 0.008 mg mL−1 of ivermectin (Sigma-Aldrich®) (positive control); G5: 3% Tween 80 (negative control) and G6: 4% sodium alginate polymer matrix (negative control), were incubated with GNI-free sheep feces for 6 days at room temperature (27°C) (Camurça-Vasconcelos et al., Reference Camurça-Vasconcelos, Bevilaqua, Morais, Maciel, Costa, Macedo, Oliveira, Braga, Silva and Vieira2007). After the incubation period, L3 were recovered following the technique described by Roberts and O'Sullivan (Reference Roberts and O'Sullivan1950) and counted using a microscope. For each treatment and control, 3 repetitions were performed with 5 replicates for each.
Adult worm motility test
The adult worm motility test (AWMT) was performed according to the method described by Hounzangbe-Adote et al. (Reference Hounzangbe-Adote, Paolini, Fouraste, Moutairou and Hoste2005). Adult females of H. contortus were collected from the abomasum of a sheep with monospecific infection. Soon after the animal was euthanized, the abomasum was removed, and the parasites were washed with saline solution. The mobile females were transferred to a 24-well plate at a density of 3 specimens per well, to which 1 mL phosphate-buffered saline (PBS) with 4% penicillin/streptomycin (Sigma-Aldrich®) was added and maintained at 37°C in a greenhouse with 5% carbon dioxide. After 1 h, 1 mL of treatment was added to the wells: G1: UNAlg-son; G2: UNAlg-ultra and G3: CNAlg-ultra, at concentrations ranging from 800 to 100 μg mL−1; G4: 100 μg mL−1 ivermectin (Sigma-Aldrich®); G5: 3% Tween 80 + PBS (negative control) and G6: 4% sodium alginate polymer matrix (negative control). The plates were maintained in the oven, and after 3, 6 and 12 h of incubation, motility was evaluated using a stereomicroscope. Eight replicates were made for each treatment and control groups.
SEM of adult H. contortus
Nematodes obtained from the AWMT were collected and subjected to SEM. Adult specimens of H. contortus were fixed in a 2.5% glutaraldehyde solution in 0.1 m sodium cacodylate buffer for a period of 72 h. After 3 washes in the same buffer, the parasites were dehydrated in a graded ethanol series (10, 20, 30, 60, 70, 80, 90 and 100%, 5 min each). The samples were dried in an EMSCOPE CPD 750, mounted in metal stubs and coated with gold–palladium for 5 min at 100 Å min−1. Possible changes in the cuticle of the treated parasites were observed with an S450 scanning electron microscope (Hitachi) at an accelerating voltage of 15 kV.
Statistical analysis
In EHT, inhibition of egg hatching was determined according to the following formula: (number of eggs/number of eggs + number of hatched larvae) × 100. In LDT, the effect was determined according to the following formula: (number of L3 in the negative control − number of L3 in the treated group/number of L3 in the negative control) × 100. The percentage of motile adults in AWMT was evaluated as the number of motile worms/total number of worms per well.
In EHT and LDT, the inhibition effects at each concentration of treatment were analysed using analysis of variance (ANOVA) and compared with the Tukey test. To analyse the differences between treatments, the data were subjected to the unpaired t test (2-tailed). In AWMT, the inhibition effects at each concentration of treatments at different times were analysed by 2-way ANOVA and compared with the Tukey test, using the SPSS 22.0 program, considering a significance level of 5% (P < 0.05).
GraphPad Prism® 6.0 software was used considering a significance level of 5% (P < 0.05). Effective concentrations to inhibit 50% (EC50) of egg hatching and larval development were calculated by linear regression using the SPSS 22.0 program.
Results
The macroscopic effects of nanoemulsion stability, such as creaming, sedimentation and phase separation were evaluated, and there was no macroscopic presence of sedimentation phenomena for UNAlg-ultra. For CNAlg-ultra, it was only possible to observe instability phenomena after 60 days, with a sedimentation rate of 6.6%. Creaming formation was observed only for CNAlg-ultra 7 days after storage. The CI values were 18.2% after 7, 14 and 21 days. After 30, 60 and 90 days, the creaming rates varied between 16.1 and 21.5%. In both nanoemulsions, no phase separation phenomenon was observed. The formulations presented nanometric and multimodal particle sizes. The particle size and zeta potential results are shown in Table 1. Samples UNAlg-ultra and UNAlg-son presented an average particle size value of 152.7 and 281.1 nm, respectively; CNAlg-ultra coated with alginate showed the highest particle size value (557.8 nm).
Table 1. Particle size, zeta potential and polydispersity index of R-carvone nanoemulsions without polymer matrix (NCS-SM and NCT-SM) and R-carvone with polymer matrix (NCT-CM)
There was an average content of 20.24 ± 1.98% and an average EE of 99.84 ± 0.01%. The FTIR spectra of the compounds carvone, Tween 80 and alginate, as well as UNAlg-ultra and CNAlg-ultra are shown in Fig. 1. Carvone presents the main peaks at 2922 cm−1, which correspond to the stretching of the methyl groups (−CH2), as well as at 1670 cm−1, corresponding to the stretching of the C=C bond, and at 1445 cm−1, due to the C–H strain. Sodium alginate showed bands at 3392 cm−1 due to the stretching of the −OH bond, at 1739 cm−1 corresponding to the stretching of the COO– bond and at 1028 cm−1 attributed to the COH stretch. Tween 80 showed the stretching of CH3 and CH2 groups at 2918 and 2860 cm−1, respectively. Furthermore, it presented an absorption band at 1735 cm−1 due to stretching of the C=O bond, at 1500 cm−1 referring to the angular deformation of the −CH2 methyl groups and at 1095 cm−1 due to stretching of the COC bond. The aqueous nanoemulsions UNAlg-ultra and CNAlg-ultra exhibited a combination of peaks from the constituent materials. The peaks at 1670 and 1336 cm−1 were attributed to C=C and −CH2 stretches of carvone, respectively. The peaks at 1500 cm−1 present in Tween were distinctly found in nanoemulsions. The nanoemulsion with sodium alginate matrix CNAlg-ultra presented a distinction in relation to the nanoemulsion without matrix (UNAlg-ultra) by the discrete presence of a shoulder at 1739 cm, which could be attributed to stretching of the COO− bond of alginate.
Fig. 1. FTIR spectra of carvone, sodium alginate, Tween 80 and uncoated (UNAlg-ultra) and sodium alginate-coated (CNAlg-ultra) R-carvone nanoemulsions homogenized in an ultrahomogenizer.
The SEM images of the nanoemulsions are shown in Fig. 2. Figure 2A–C shows the 3 types of nanoemulsions at the same magnification (50 000×). Samples UNAlg-Ultra and UNAlg-son showed similar patterns in comparison with the particle size from DLS (Dynamic Light Scattering) results (Table 1). Figure 2A (UNAlg-ultra) shows micelles with spherical morphology and homogeneously arranged in the medium in which they were dispersed. Figure 2B (UNAlg-son) shows particles with similar morphology, but with higher particle size, with uneven spacing between micelles. Figure 2C (CNAlg-ultra) shows the presence of particles with different sizes arranged in a non-uniform manner dispersed along the alginate matrix. Figure 2D–F shows in detail the presence of microspherical inclusions as small particles aggregated to the polymeric matrix network for the sample CNAlg-ultra, in addition to the formation of structures with porous characteristics in the polymeric network. Figure 2C (UNAlg-ultra) shows the presence of particles with different sizes arranged in a non-uniform manner in the dispersion medium.
Fig. 2. SEM surface images: (a) UNAlg-ultra at 50 000×; (b) UNAlg-son at 50 000×; (c) CNAlg-ultra at 50 000×. Images (d), (e) and (f) show CNAlg-ultra at different magnifications: 1000×, 5000× and 10 000×.
The mean effects of the UNAlg-son, UNAlg-ultra and CNAlg-ultra formulations obtained in the EHT on the H. contortus-resistant isolate to synthetic anthelmintics are shown in Fig. 3. At a concentration of 2 mg mL−1, UNAlg-son, UNAlg-ultra and CNAlg-ultra were 100% effective, showing statistical similarity to the positive control (P > 0.05) and a dose-dependent effect. The EC50 values of UNAlg-son, UNAlg-ultra and CNAlg-ultra were 0.19, 0.02 and 0.17 mg mL−1, respectively.
Fig. 3. Effects of UNAlg-son (A), UNAlg-ultra (B) and CNAlg-ultra on the hatching of Haemonchus contortus eggs. * Different numbers indicate statistical difference (P < 0.05). UNAlg-son, uncoated R-carvone nanoemulsion with sodium alginate; UNAlg-ultra, uncoated R-carvone nanoemulsion with sodium alginate; CNAlg-ultra, sodium alginate coated R-carvone nanoemulsion; TBZ, thiabendazole (positive control). Sodium alginate solution (4%) was used as a negative control for nanoemulsions.
The inhibitory effects of nanoemulsions on LDT are shown in Fig. 4. UNAlg-son and UNAlg-ultra inhibited larval development by 100% at a concentration of 2 mg mL−1, while for CNAlg-ultra, the inhibition was 80.23%. These results did not differ statistically from the positive control (P > 0.05) and showed a dose-dependent effect. The EC50 values in the LDT for UNAlg-son, UNAlg-ultra and CNAlg-ultra were 0.29, 0.31 and 0.95 mg mL−1, respectively.
Fig. 4. Effects of UNAlg-son (A), UNAlg-ultra (B) and CNAlg-ultra on larval development of H. contortus. *Different numbers indicate statistical difference (P < 0.05). UNAlg-son, uncoated R-carvone nanoemulsion with sodium alginate; UNAlg-ultra, uncoated R-carvone nanoemulsion with sodium alginate; CNAlg-ultra, sodium alginate coated R-carvone nanoemulsion; IVM, ivermectin (positive control). Sodium alginate solution (4%) was used as a negative control for nanoemulsions.
The effects of R-carvone nanoemulsions on the inhibition of H. contortus motility are shown in Fig. 5. UNAlg-ultra and CNAlg-ultra inhibited worm motility by 100% at a concentration of 800 μg mL−1 after 3 h of exposure, while the effectiveness of UNAlg-son was 79.16% after 12 h of exposure. These results did not differ statistically from the positive control (P > 0.05). The motility in the negative (Tween 80® + PBS) and positive controls was 100 and 0%, respectively, at 12 h post-exposure.
Fig. 5. Effects of UNAlg-son (A), UNAlg-ultra (B) and CNAlg-ultra on adult motility of H. contortus. IVM, ivermectin (positive control); Tween 80 + PBS (negative control).
SE micrographs (Fig. 6) showed changes in the buccal capsule and along the cuticle of female H. contortus after exposure to 800 μg mL−1 nanoemulsions. The alterations consisted of the wrinkling of the longitudinal cuticular ridges (Fig. 3B, E and H). There was wrinkling of the buccal capsule and deformation of the lancet (Fig. 3A and G), similar to the positive control (Fig. 3J). The longitudinal cuticular ridges, buccal capsule and lancet seemed to be well preserved in the negative control group (Fig. 3C, F, I and L).
Fig. 6. SEM images of ultrastructural changes in the cuticle and cephalic region of adult females of H. contortus: (A) and (B) UNAlg-son; (D) and (E) UNAlg-ultra; (G) and (H) CNAlg-ultra; (C) and (F) Tween 80 + PBS (negative control); (I) and (L) sodium alginate solution 4% (negative control); (J) and (K) ivermectin (positive control).
Discussion
Instability phenomena can limit the period of effective application of emulsified formulations; thus, nanoemulsions based on essential oils require the evaluation of their stability during storage (Guerra-Rosas et al., Reference Guerra-Rosas, Morales-Castro, Ochoa-Martınez, Salvia-Trujillo and Olga Martín-Belloso2016). The UNAlg-ultra and CNAlg-ultra formulations were more stable for sedimentation and creaming phenomena, respectively, as they presented a percentage of secreted volume lower than 1%, even after 90 days of preparation. The formation of creaming in UNAlg-ultra occurred because the nanoemulsion droplets had a lower density than the surrounding liquid, causing an upward movement of the particles, while the sedimentation of CNAlg-ultra might be due to the larger particle size because of the use of sodium alginate as an encapsulating matrix, in addition to droplets having a higher density than the surrounding liquid, promoting downward movement of the particles (Mcclements, Reference Mcclements2010).
The UNAlg-ultra formulation had the smallest particle size (152.7 nm), followed by UNAlg-son (281.1 nm), while the largest size was found for CNAlg-ultra (557.8 nm). UNAlg-Ultra and UNAlg-son showed similar pattern sizes in SEM images from lower to higher sizes. In evaluating the morphology of nanoemulsions by SEM, UNAlg-son presented micelles with higher particle size than UNAlg-Ultra ones, which might be due to the instability process causing a possible aggregation of the droplets and, thus, an increase in their size (Daltin, Reference Daltin2011). The formation of porous structures seen in nanoemulsions, and observed in CNAlg-ultra, is a characteristic of polymeric coating, and has been attributed to the formation of micellar vacuoles in the polymer network (Fernandes et al., Reference Fernandes, Borges and Botrel2014; Morais et al., Reference Morais, Alencar, Xavier, Oliveira, Marcelino, Barratt, Fessi, Egito and Elaissari2016).
The UNAlg-son was homogenized for 5 min, probably because a longer homogenization time could have contributed to obtaining a smaller particle size (Abismail et al., Reference Abismail, Canselier, Wilhelm, Delmas and Gourdon1999). To prepare the UNAlg-ultra, the high-pressure homogenization method (ultrahomogenizer) was used, producing a nanoemulsion with a smaller particle size due to the homogenization time (2 min) and rotation speed (18 000 rpm). The particle size decreases as the homogenization time or the rotation speed increases (Benavides et al., Reference Benavides, Cortés, Parada and Franco2016). In this case, it is evident that UNAlg-ultra sample showed the lowest particle size, where the ultra-stirrer technique showed higher efficiency in the dispersion of the oily phase in nanometric droplets in comparison with the sonication technique. Although CNAlg-ultra was prepared with a higher number of revolutions per minute (22 000 rpm) and a longer homogenization time (5 min), this nanoemulsion presented larger particles. Alginate-coated nanoemulsion showed an increased size due to the external polymer matrix which, because of the size of its molecule, promotes the formation of a larger adsorption interface. The alginate matrix involves the nanoemulsion droplets, and despite the occurrence of an increase in the particle size of the nanoemulsion (Salvia-Trujillo et al., Reference Salvia-Trujillo, Rojas-Grau, Soliva-Fortuny and Martın-Belloso2014), the external coating may prevent from further coalescence stabilizing nanodroplets size for longer periods of time.
Bortoluzzi et al. (Reference Bortoluzzi, Buzatti, Chaaban, Pritsch, Anjos, Cipriano, Deschamps and Molento2020) prepared a nanoemulsion of the essential oil of Mentha villosa Hubs (68.8% carvone) using Tween 80 as a surfactant without the addition of a polymer matrix. The sample was first subjected to high-pressure homogenization in an Ultra-Turrax T25 digital IKA (Staufen, Germany) and later in high pressure homogenizer APLAB 10 (Artepeças, São Paulo,Brazil). This nanoemulsion exhibited a larger particle diameter (164 nm) than the CNAlg-ultra nanoemulsion utilized in the present study (152.7 nm), prepared without the addition of a polymer matrix. It has been reported that the primary homogenization of the sample and subsequent use of a high-energy homogenization method, such as microfluidization, promotes a significant reduction in particle size (Santos et al., Reference Santos, Ramírez, Llinares, Muñoz and Trujillo-Cayado2018). However, using only 1 high-energy homogenization technique (ultrahomogenizer), it was possible to obtain a slightly smaller particle size compared with the results obtained by Bortoluzzi et al. (Reference Bortoluzzi, Buzatti, Chaaban, Pritsch, Anjos, Cipriano, Deschamps and Molento2020) when using 2 homogenization cycles.
The 3 nanoemulsions had negative zeta potentials, which was related to the use of Tween 80, a nonionic surfactant that can generate a negative charge on oil droplets due to the preferential adsorption of hydroxyl ions from the aqueous phase and sodium alginate, which endows it with a negative charge at neutral pH as an encapsulating matrix (Mcclements, Reference McClements2005; Machado et al., Reference Machado, Lundberg, Ribeiro, Veiga, Lindman, Miguel and Olsson2012; Gago et al., Reference Gago, Artiga-Artigas, Antunes, Faleiro, Miguel and Martín-Belloso2019). The zeta potential value of CNAlg-ultra is close to the recommended stability value, as potentials greater than +30 mV or less than −30 mV are strongly cationic and anionic, respectively, and are classified as stable (Clogston and Patri, Reference Clogston, Patri and McNeil2011).
The R-carvone nanoemulsion was evaluated for its in vitro anthelmintic effect on H. contortus eggs, larvae and adults that were multiresistant. The ovicidal effect of UNAlg-ultra was potentiated (EC50 = 0.02 mg mL−1) with homogenization in an ultrahomogenizer without a polymer matrix in comparison with UNAlg-son (EC50 = 0.18 mg mL−1) and CNAlg-ultra (EC50 = 0.19 mg mL−1). The superior results obtained for the UNAlg-ultra formulation can be explained by its smaller particle size, which possibly allowed greater penetration into the eggshell and, consequently, a better inhibitory effect on larval hatching. The effectiveness of the UNAlg-son and CNAlg-ultra formulations was similar (P > 0.05), indicating that the polymeric system did not enhance the effectiveness of R-carvone on egg hatching. The presence of the sodium alginate matrix as an encapsulating agent in the polymeric system of the UNAlg-ultra may have influenced the controlled release process in the aqueous medium of EHT, prolonging the release of the encapsulated carvone, since the diffusion of drugs retained in the nanoemulsions occurs when the water enters the polymeric system, resulting in swelling and subsequent release of encapsulated drugs (Dash et al., Reference Dash, Chiellini, Ottenbrite and Chiellini2011).
UNAlg-son (CE50 = 0.29 mg mL−1) and UNAlg-ultra (CE50 = 0.31 mg mL−1) formulations showed similar effects (P > 0.05), indicating that the type of homogenization did not influence larval development. The CNAlg-ultra (CE50 = 0.95 mg mL−1) formulation was less effective than the other formulations because the LDT uses a solid medium based on feces, hindering the release of the biocompound from the polymeric system. These results are similar to those of Ribeiro et al. (Reference Ribeiro, Camurça-Vasconcelos, Macedo, Santos, Ribeiro, Pereira, Viana, Paula and Bevilaqua2015), where the nanoemulsion (EC50 = 2.3 mg mL−1) of Eucalyptus staigeriana oil had a lower inhibitory effect than the oil in its free form (EC50 = 1.8 mg mL−1).
UNAlg-ultra and CNAlg-ultra showed a similar effect on the inhibition of adult motility (100%), differing from UNAlg-son, which only attained 16.65% of the inhibitory effect on adult H. contortus motility. This phenomenon may be indicative that high pressurization with an ultrahomogenizer increases the effectiveness of carvone. The use of monoterpenes to inhibit the motility of adult nematodes has been previously reported (André et al., Reference André, Ribeiro, Cavalcante, Santos, Macedo, Paula, Freitas, Maia, Melo and Bevilaqua2016; Araújo-Filho et al., Reference Araújo-Filho, Ribeiro, André, Cavalcante, Guerra, Muniz, Macedo, Rondon, Bevilaqua and Oliveira2018; André et al., Reference André, Paiva, Cavalcante, Ribeiro, Araújo-Filho, Cavalcanti, Morais, Oliveira, Bevilaqua and Abreu2020). The monoterpenes citronellal and thymol used in the free form completely inhibit the motility of adult H. contortus after 12 and 24 h of exposure, respectively (André et al., Reference André, Cavalcante, Ribeiro, Santos, Macedo, Paula, Morais, Melo and Bevilaqua2017; Araújo-Filho et al., Reference Araújo-Filho, Ribeiro, André, Cavalcante, Rios, Schwinden, Rocha, Macedo, Morais, Bevilaqua and Oliveira2019). We can conclude that the R-carvone nanoemulsion had a better inhibitory effect on adults, since the inhibition was 100% after 3 h of exposure. Thus, the use of encapsulation can potentiate the anthelmintic effect of bioactive compounds and ensure protection for encapsulated substances, making it a useful alternative for controlling multiresistant populations of gastrointestinal nematodes (Ribeiro et al., Reference Ribeiro, Macedo, Santos, Oliveira, Camurça-Vasconcelos, Paula and Bevilaqua2013; Ribeiro et al., Reference Ribeiro, Ribeiro, Camurça-Vasconcelos, Macedo, Santos, Paula, Aráujo-Filho, Magalhães and Bevilaqua2014).
SEM allowed the observation of damage in the mouth capsule and cuticle of females of H. contortus exposed to R-carvone nanoemulsions. Cuticle wrinkling in H. contortus caused by exposure to substances with potential anthelmintic effects, such as essential oils, extracts and isolated compounds such as monoterpenes, has been previously described (André et al., Reference André, Ribeiro, Cavalcante, Santos, Macedo, Paula, Freitas, Maia, Melo and Bevilaqua2016, Reference André, Cavalcante, Ribeiro, Santos, Macedo, Paula, Morais, Melo and Bevilaqua2017; Ribeiro et al., Reference Ribeiro, Camurça-Vasconcelos, Santos, Macedo, Ribeiro, Oliveira, Paula and Bevilaqua2017; Cavalcante et al., Reference Cavalcante, Morais, André, Araújo-Filho, Muniz, Rocha, Ribeiro, Rodrigues, Oliveira, Bevilaqua and Ramos2020). The changes observed in SEM may be related to interactions with the internal structures of nematodes causing physiological disturbances (Brunet et al., Reference Brunet, Fourquaux and Hoste2011), associated with the loss of protective properties of the cuticle, involvement in motility and metabolic changes that occur in the digestive tract of small ruminants (Martínez-Ortíz-de-Montellano et al., Reference Martínez-Ortíz-de-Montellano, Arroyo-López, Fourquaux, Torres-Costa and Hoste2013).
Some studies suggest that carvone acts as an acetylcholinesterase (AChE) inhibitor (Ryan and Byrne, Reference Ryan and Byrne1988; Jukic et al., Reference Jukic, Politeo, Maksimovic, Milos and Milos2007; López and Pascual-Villalobos, Reference López and Pascual-Villalobos2010; Kurt et al., Reference Kurt, Gazioglu, Dag, Salmas, Kaylk, Durdagi and Sonmez2017). Its possible action on AChE receptors observed in vertebrates and invertebrates causes neurotoxic lesions similar to organophosphates, which are potent anthelmintics that are highly toxic to mammals (Miller et al., Reference Miller, Baker and Farver1986; Lopez and Pascual-Villalobos, Reference López and Pascual-Villalobos2010; Ross et al., Reference Ross, McManus, Harrison and Mason2013).
Acknowledgements
The authors acknowledge Central Analítica-UFC/CT-INFRA/MCTI-SISANO/Pró-Equipamentos CAPES for the support. Ms Aguiar has received a master research scholarship from Conselho Nacional de Desenvolvimento Científico e Tecnologico (CNPq). Dra Bevilaqua has received a researcher fellowship from CNPq (310663/2016-4-5).
Author contributions
A. A. R. M. A., C. M. L. B., W. P. P. A. and L. M. B. O.: conceptualization. A. A. R. M. A., W. P. P. A., J. V. A. F., F. O. M. S. A., H. N. P., W. L. C. R.; A. C. F. L. M., M. E. N. P. R.; N. M. P. S. R.; L. O. R. and L. M. B. O.: performed experiments. A. A. R. M. A., C. M. L. B. and W. P. P. A.: data analysis, supervision and project administration. A. A. R. M. A., C. M. L. B., W. P. P. A. and L. M. B. O.: writing the manuscript. All the authors reviewed and approved the manuscript.
Financial support
This study was funded by the Brazilian funding agency Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). Mrs Aguiar received a master's research grant from the National Council for Scientific and Technological Development (130372/2020-0). Dr Bevilaqua is a CNPq fellow (310663/2016-4-5).
Conflict of interest
The authors declare that they have no conflicts of interest.
Ethical standards
This study was approved by the Ethics Committee for Animal Use of the State University of Ceará (UECE) and registered under protocol number 390620/2020.
