Improving the anthelmintic effectiveness of Benzyl 4-Aminochalcone with polysaccharides nanoemulsions

Improving the anthelmintic effectiveness of Benzyl 4-Aminochalcone with polysaccharides nanoemulsions | 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 Improving the anthelmintic effectiveness of Benzyl 4-Aminochalcone with polysaccharides nanoemulsions Matheus Luiggi Freitas Barbosa, Andreza Pereira Braga, Karin Vitoria Maia Mendonça Ferreira, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5662121/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 Small ruminant farming in developing countries is hindered by gastrointestinal nematodes, such as Haemonchus contortus causing anemia, leading to weight loss and reduced productivity. The excessive use of synthetic anthelmintics has driven resistance. Therefore, natural compounds are being investigated as alternative control strategies. Hence, the present study investigates the anthelmintic activity and cytotoxicity of Benzyl 4-Aminochalcone (B4AM) and its nanoemulsions. Three nanoemulsions (sodium alginate-coated B4AM (CNAlg), gum arabic-coated B4AM (CNGA) and uncoated B4AM (UN)) were prepared and characterized physicochemically. B4AM and nanoemulsions were evaluated in the egg hatch test (EHT) using H. contortus and their cytotoxicity was evaluated on murine fibroblasts. The release kinetics of B4AM, CNAlg and UN were studied. The CNAlg and CNGA showed superior visual stability compared to UN. The CNAlg, CNGA and UN presented, respectively, 267.64, 234.3 and 144.56 nm (particle size); -56.64, -58.26 and − 46.06 mV (zeta potential); 0.751, 0.782 and 0.049 (polydispersity index). Encapsulation efficiencies were 87.5% (CNAlg), 43.8% (CNGA), and 69.87% (UN). In the EHT, CNAlg and UN were more effective. The effective concentrations to inhibit 50% (EC50) of eggs ranging from 0.02 to 0.10 mg/mL. SEM revealed more pronounced changes in eggs treated with the UN. No cytotoxicity was observed (IC50 > 120 µg/mL). Kinetics presented a faster release for UN followed by CNAlg and B4AM. CNAlg improved B4AM solubilization, stability and anti-helminthic efficacy, with ovicidal effect comparable to the pure chalcone. Nanoemulsions showed promise for ovicidal control, but further studies are needed to assess their effectiveness in other life stages. Chalcones biopolymers drug delivery nanotechnology Haemonchus spp. cytotoxicity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Small ruminant farming provides income, nutrition, and acts as a safety net for households in numerous developing nations [1]. However, the presence of gastrointestinal nematode infections presents considerable global challenges [2]. Haemonchus contortus , a highly pathogenic gastrointestinal nematode, feeds on blood in sheep and goats, causing severe anemia, weight loss, decreased productivity and sometimes death [3]. The method more used for controlling gastrointestinal nematodes is based on administration of synthetic anthelmintic. Nevertheless, the uncontrolled administration of these drugs has caused anthelmintic resistance [4]. Hence, the attention has shifted towards studying natural compounds as alternative control methods [5]. Chalcones, which are aromatic ketones, serve as precursors for flavonoids and isoflavones. They are made up of two benzene rings connected by an α,β-unsaturated system [6]. These substances can be found in abundance in plants of the Leguminosae, Compositae and Moraceae families [7] and are usually obtained naturally or synthesized using the Claisen-Schmidt reaction [8]. Studies have proven the antioxidant [9], antitumor [10], antifungal [11], antibacterial [12] and antiparasitic [13] effects of chalcones. The anthelmintic activity of these secondary metabolites has only been minimally researched, with a focus on their effectiveness against H. contortus larvae [14,15,16]. The therapeutic usefulness of chalcones is hindered by their poor water solubility [17]. Nanoemulsions have been utilized to address this challenge by safeguarding drugs, enhancing their bioavailability and providing controlled release [18]. Biopolymers like sodium alginate (Alg) and gum arabic (GA) show potential as encapsulation materials for hydrophobic substances [19,20]. The effectiveness of bioactive compounds against H. contortus was enhanced by encapsulating with Alg and GA [21,22]. Irfan et al. [23] reported various biological properties of aminochalcones. However, no anthelmintic activity of these compounds has been described, nor has nanoencapsulation been investigated for anthelmintic purposes, making the evaluation of B4AM promising. Consequently, the objectives of this research included formulating three nanoemulsions with B4AM compound, assessing their physicochemical properties, analyzing release kinetics, evaluating efficacy against H. contortus using the egg hatching test (EHT), analyzing potential morphological alterations in the eggshell using scanning electron microscopy (SEM) and verifying cytotoxicity. Materials and Methods Ethical approval Animal studies were approved by the Ethics Committee of the State University of Ceará (protocol number. 018265831-2023). Materials The chalcone (E)-1-(4-(dimethylamino)phenyl)but-2-en-1-one or B4AM (189.12 g/mol) was given by the Organic Synthesis Lab at State University of Vale do Acaraú, Sobral, Brazil. B4AM was synthesized and its structure characterized through spectroscopic techniques [24]. The reaction was kept under mechanical stirring at room temperature, and after 30 minutes, the mixture was filtered, and the solid was washed with distilled water. Recrystallization of the compound was carried out with ethanol as the solvent. Commercially sourced Tween 80 (Vetec®, Brazil), dimethylsulfoxide - DMSO (Sigma-Aldrich®, USA), Alginate (Alg), Gum Arabic GA (Dinâmica®, Brazil), and canola oil were used. Preparation of nanoemulsions The dilution of B4AM was achieved by adding 3% DMSO to distilled water. Three nanoemulsions were prepared, one uncoated (UN), one coated with polymeric solution of Alg (CNAlg) and other coated with polymeric solution of GA (CNGA). The nanoemulsions underwent a high-energy emulsification process using the Ultronique QR500 ultrasonic tip sonicator (Brazil) at a frequency of 20 kHz and power of 350W for 2 min. It was mixed 3 mg of B4AM, 100 μL DMSO, 100 μL Tween 80 and 100 μL canola oil in 100 ml of 1% polymeric solution. Nanoemulsion without polymer matrix was prepared by using the same constituents, but with distilled water substituting the polymer solution. Characterizations of nanoemulsions The physical stability of the nanoemulsions (CNAlg; CNGA and UN) was evaluated at 7, 14, 21, 28, 35, 42 and 49 days. For this procedure, approximately 10 mL of each of the nanoemulsions were kept in closed test tubes, without exposure to light, at 27°C. Periodic observations were implemented to detect any visual signs of instability. Creaming and sedimentation were measured over time with the aid of a caliper. The creaminess index (CI) and sedimentation index (SI) were determined by relating the height in centimeters of the precipitates formed in the test tube (Ha) with the total height of the sample (Ht) after the mentioned preparation periods, calculated according to equations (1) and (2), adapted from Mwangi et al. [25]: CI (%) =Ha/Ht×100 Equation 1 SI (%) =Ha/Ht×100 Equation 2 Dynamic light scattering with the Malvern Zetasizer equipment was used to determine the droplet size, polydispersity index and zeta potential of CNAlg, CNGA and UN after 15 days. The nanoemulsions were dispersed in distilled water, forming a concentration of 1% (v/v) in water and left stirring for 12 h to ensure total dispersion of the matrix in aqueous medium. Scanning electron microscopy (SEM) was used to visualize the morphology of the nanoemulsions droplets. 20 μL of each nanoemulsion were placed in rounded metal structures and dehydrated at 50°C for approximately 1 h in a drying oven. Then the samples were observed using a microscope from FEI Company® (USA) at an acceleration voltage of 15 kV. The magnification was set at 150.000× Encapsulated chalcone content (%EE) The encapsulation efficiency (EE%) of CNAlg, CNGA and UN were assessed by adding 1 mL of each emulsion to 4 mL of 96% ethanol and allowing it to stand for 24 h. Once the phases were separated, 1 mL of the ethanolic phase was isolated and diluted with 96% ethanol (3 mL) for measurement on the Kazuaki Spectrophotometer (Genesys 6 UV-Vis) at 390 nm. The absorbance in the calibration curve was adjusted by performing additional dilutions, up to a factor of 100x, to maintain linearity with concentration. The chalcone content in the nanoemulsions was determined through a calibration curve, prepared in triplicate in a standard solution of 150 mg/L in 96% ethanol. Additional dilutions were made to obtain concentrations of 75 to 1 mg/L. The calibration curve for B4AM is represented, respectively, by Equation (3): y=0.673×+ 0.0343 →R2=0:09953 Equation 3 The accuracy of the %EE determination may be affected due to high dilution, leading to fluctuations. Duplicate tests were performed, and Equation (4) was used to determine the value of %EE: %EE = Determined chalcone content/Total chalcone content Equation 4 Anthelmintic activity To recover H. contortus eggs, feces were collected of sheep harboring a monospecific infection and processed according to the technique described by Hubert and Kerboeuf [26]. The H. contortus isolate used - Kokstad (KOK) - is resistant to benzimidazoles, levamisole, and macrocyclic lactones [27,28] and was provided by the Institut National de Recherche pour l'Agriculture, l'Alimentation et l'Environnement (INRAE), Nouzilly, France. The egg hatch test ( EHT) was performed according to Coles et al. [29]. Briefly, 250 μL of an egg’s suspension containing approximately 100 fresh eggs was incubated for 48 h at 27°C with 250 μL of four formulations (B4AM; CNAlg; CNGA and UN) at concentrations of 0.15 to 0.009375 mg/mL. Drops of Lugol's solution were added to stop egg hatching, and eggs and first-stage larvae (L1) were counted under a light microscope. This test was performed with controls: 3% Tween, 1% Alg and 1% GA were the negative controls and 0.1 mg/mL thiabendazole (Sigma-Aldrich ® , USA) was the positive control. Three repetitions were performed with five replications for each treatment and control. The eggs of H. contortus exposed to the highest concentrations of four formulations were analyzed by SEM. At least 10 eggs presented the same patterns of change to be considered. Samples of 10 μL containing eggs were deposited on aluminum stubs with double-sided carbon adhesive tape and observed. This methodology was adapted from Zarza-Albarrán et al. [30]. Microscope from FEI Company® (USA) at an acceleration voltage of 15 kV was used. The magnification was set at 8.000×. Cytotoxicity The cytotoxicity test was performed using cell cultures of murine fibroblasts (L929 cell line) as a model. Cell toxicity was quantified by the ability of living cells to reduce the yellow dye 3-(4,5-dimethyl-2-thiozolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) to a purple formazan product [31]. For the experiments, L929 cells were plated in 96-well plates (0.7 x 105 cells/well). The compounds (B4AM, CNAlg, CNGA, UN) were tested at 0.46 to 120 μg/mL and the plates were incubated for 24 h. This assay was performed with controls: 3% Tween, 1% Alg and 1% GA were the negative controls and bleomycin (0.39 to 100 μg/mL) was used as positive control. Thereafter, the plates were centrifuged and fresh medium (150 μL) containing MTT at 0.5 mg/mL was placed in wells. After 3 h, the formazan product was dissolved in 150 µL of DMSO, and absorbance was measured using a multi-plate reader (Spectra Count, Packard, Ontario, Canada). The drug effect was quantified as the percentage of absorbance of the reduced dye at 595 nm. Three repetitions were performed for each treatment and control. In vitro release kinetic profile The release kinetics of B4AM, CNAlg and UN were conducted using a dialysis system. For each sample, 60 mg of formulations were introduced into cellulose acetate membranes (14 kDa pores) and dialyzed against 60 mL of phosphate buffer solution (PBS) and 1% (v/v) Tween 80 at pH 7 for 50 h. Three aliquots of 2 mL were removed every three hours and analyzed by spectrophotometry in the Genesys 10S UV-Vis apparatus (Thermo Fisher Scientific, New York, USA). Thus, the concentration of B4AM present in the medium was calculated using a calibration curve in PBS at pH 7, resulting in equation 5: Abs=0.0028conc-0.0368;R2=0.994 Equation 5 The release mechanisms of B4AM from nanoemulsions were evaluated using zero-order, first-order, Higuchi and Korsmeyer-Peppas mathematical models [32]. Statistical analysis In the EHT, efficacy was determined according to the following equation 6: number of eggs/(number of eggs + number of L1)]×100 Equation 6 The percentage of hatched larvae was analyzed by one-way analysis of variance (ANOVA), followed by multiple comparisons using the Tukey test, with the aid of GraphPad Prism® 8.0 software. The effective concentration to inhibit 50% of egg hatching (EC 50 ) was determined by the probit method, using the SPSS 23.0 program for Windows. Results Characterizations of nanoemulsions The stability indices of nanoemulsions are demonstrated in Figure 1. The CNAlg showed signs of instability (CI = 4%) on day 1 and (CI = 8%) day 21. However, this index returned to 4% on the last observation (day 49). Sedimentation for CNAlg was noted only on day 21 (SI = 1%) and decreased to null (day 28). CNGA presented stability (CI = 2% and SI = 1%) throughout the observation period. UN CI ranging from 14% (day 14) to 8% (day 49). On day 7, the UN SI was 6%, decreased to 1% on days 14-42 and reached 4% on the last day of analysis. Table 1 displays the results for particle size, zeta potential and PDI of emulsions. Nanometric particle sizes were observed. The average particle sizes for CNAlg, CNGA and UN were 267.64, 234.3 and 144.56 nm, respectively . The PDI revealed moderate homogeneity for UN, and some degree of heterogeneity for emulsions coated with polymers (CNAlg and CNGA). The encapsulation efficiency values were 87.5% ± 0.77, 43.8 ± 0.58 and 69.87% ± 0.58 for CNAlg, CNGA and UN, respectively. Figure 2 displays the SEM images of dried film nanoemulsions at a magnification of 150.000×. CNAlg, CNGA and UN (Figure 2a, 2b and 2c, respectively) exhibit nanodroplets with size below 100 nm. UN exhibited smaller domains than CNAlg and CNGA, which might be a result of the lack of polymer coating, potentially impacting the droplets size of emulsions. Anthelmintic activity The efficacy of chalcone formulations in the EHT is demonstrated in figure 3. B4AM and their nanoemulsions showed ovicidal activity on H. contortus . The highest concentration tested (0.15 mg/ml) inhibited more than 94% of egg hatching. These results did not differ statistically from the positive control (TBZ). The negative controls (3% Tween, 1% Alg and 1% GA) have no demonstrated effect on larvae hatching. The ovicidal effect of formulations was dose dependent. The EC 50 values were 0.04 mg/mL for B4AM and CNAlg, 0.10 mg/mL for CNGA and 0.02 mg/mL for UN. Figure 4 illustrates the H. contortus eggs after treatment with the formulations and controls, highlighting morphological damage in the eggs exposed to the four treatments: B4AM (Figure 4a), CNAlg (Figure 4b), CNGA (Figure 4c) and UN (Figure 4d). Figure (4e) presents the negative control (3% Tween), characterized by intact morphology and a smooth eggshell surface. Similarly, the eggs treated with polysaccharides (Figures 4f and 4g) also exhibit preserved morphology. In contrast, (Figure 4h) illustrates the alteration in the morphology of the egg caused by the positive control (TBZ). Cytotoxicity The maximum inhibitory concentration (IC 50 ) of both B4AM chalcone and the nanoemulsions was found to be greater than 120 μg/mL, indicating no cytotoxicity after 24 hours. The nanoemulsions had no toxicity because the B4AM and the biopolymers are non-toxic. In vitro release kinetic profile The in vitro release profile of the nanoemulsions and the B4AM diluted in DMSO, conducted at pH 7.0, is depicted in Figure 5. After 10 hours, the UN nanoemulsion released 92.22% of B4AM, reaching 96.66% after 30 hours. The CNAlg nanoemulsion released 17.77% after 10 hours, increasing to 34.44% after 30 hours. In comparison, free B4AM exhibited a release of less than 14% after 10 hours and 25.55% after 30 hours. This limited release suggests that B4AM has low solubility in aqueous environments. On the other hand, the use of nanoemulsions significantly enhances the release of the compound due to the controlled and prolonged release mechanism provided by these formulations. Table 2 presents the kinetic constant and the correlation coefficient for the studied kinetic models. The Higuchi model provided the best fit for B4AM and CNAlg, while the Zero-order model was the best fit for UN, with a high correlation coefficient (R²) in both cases. Discussion The study involved the use of nanoemulsions to enclose an aminochalcone, which were subsequently analyzed and assessed for its impact on egg hatching of H. contortus and cytotoxicity. Among the three nanoemulsions created the %EE was highest with the Alg matrix (>80%). The UN nanoemulsion experienced a small drop in %EE, almost reaching 70%. Conversely, the %EE for CNGA was under 45%. The interaction of a substance's hydrophilic and lipophilic components is identified as the hydrophilic-lipophilic balance (HLB). For the stabilization of both aqueous and oily phases, it is essential for the values to range from 8 to 18, showcasing the surfactant's hydrophilic traits during nanoemulsion formation [33]. Tween 80 is an emulsifier that has an HLB value of 15 [34]. Due to its hydrophilic nature, it rapidly shifts to the aqueous phase following the addition of the organic phase [35]. The higher %EE observed in CNAlg could result from the Alg and Tween 80 interaction, affecting the lipid droplets' surface charge [36]. Abreu et al. [37] reported a comparable value when encapsulating B4AM with Alg matrix. The authors explained that this outcome might be due to the molecules having a stronger attraction to the oil phase in the droplets than to the aqueous phase of Alg during preparation, as supported by Winter et al. [38]. The lack of a polymeric matrix led to a decreased EE% for UN in comparison to CNAlg, although it remained higher than CNGA. Gum arabic, being a higher molecular weight and a branched polysaccharide, tends to present higher hydrodynamic ratio, which may imply in the droplet’s stability as a coating agent and in the viscosity of the nanoemulsions [39]. Hence, EE% can be influenced by both the surfactant amount and the matrix. The nanoemulsions exhibited suitable particle sizes, ranging from 144.56 to 267.64 nm. Gago et al. [40] reported smaller particle sizes in nanoemulsions containing Alg with lemongrass essential oil, carnauba wax alone, or a combination of both, with values between 24.2 nm and 179.13 nm. In contrast to the observed zeta potential values (from -16.0 to -31.73 mV), our values, ranging from -46 mV to -58.3 mV, demonstrated superior stability. By increasing electrostatic repulsion, higher zeta potential values can strengthen the stability of colloidal dispersions and avoid particle aggregation [41]. Hence, the nanoemulsions created in our investigation demonstrate increased stability when compared to the mentioned study. To further support this information, Li et al. [42] executed nanoencapsulation methods aimed at enhancing the stability of curcumin in liposomes. In this study, liposomes stabilized with GA, or Alg were prepared at concentrations ranging from 0 to 2% (w/v). The evaluation of zeta potential revealed that it varied with increasing amounts of GA or Alg. At a concentration of 1%, which is the same concentration used in our study, the zeta potential of the isolated liposomes (without GA or Alg), which was initially -5 mV, increased to -10 mV. Regarding droplet size, previous studies have reported seemingly contradictory results on the effect of Alg concentration on droplet size in emulsions. Hosseini et al. [43] investigated the interaction between β-lactoglobulin and Alg before and after sonication using isothermal titration calorimetry (ITC). Their findings revealed that increasing the concentration of Alg resulted in an increase in droplet diameter. In contrast, Salvia-Trujillo et al. [44] examined fish oil-in-water nanoemulsions stabilized with Tween 80 and combined with Alg at various concentrations. They reported that increasing the Alg concentration from 0.5% to 1% resulted in a reduction in droplet size. These findings suggest that lower concentrations promote droplet flocculation and coalescence, whereas higher concentrations provide greater protection against coalescence. This discrepancy may be attributed to differences in the synthesis methods of nanoemulsions. Bortoluzzi et al. [45] prepared a nanoemulsion of Mentha villosa Huds. essential oil using Tween 80 as a surfactant, without the addition of a polymeric matrix. The sample was initially subjected to high-pressure homogenization using an IKA digital Ultra-Turrax T25 (Staufen, Germany) and subsequently processed with an APLAB 10 high-pressure homogenizer (Artepeças, São Paulo, Brazil). This nanoemulsion exhibited a larger particle diameter (164 nm) compared to the UN nanoemulsion in the present study (144.56 nm), which was also prepared without a polymeric matrix. Studies indicate that the combination of primary homogenization with high-energy methods, such as microfluidization, significantly reduces particle size [46] (Linares et al., 2018). However, the application of a single high-energy homogenization technique (ultrahomogenizer) in the present study yielded particles of slightly smaller size compared to those obtained by Bortoluzzi et al. [45], who employed two homogenization cycles. Particle distribution uniformity is indicated by the PDI, which has values between 0.0 and 1.0 [47]. UN exhibited the lowest index among all nanoemulsions tested, reaching a value of 0.489. This outcome consistently highlighted the crucial role of tween 80, echoing Tsichlis et al. [48] research on using tween 80 and span 80 in developing nanoparticles for quercetin incorporation. It was observed that a lower polydispersity index in the emulsion containing Tween 80. The authors reported that nanosystems with Span 80 required stabilization with phytantriol, unlike Tween 80, which was able to stabilize the systems independently. The stability of nanoemulsions during storage can be compromised by phenomena such as creaming and sedimentation [49]. Abreu et al. [12] encapsulated B4AM with an Alg matrix, and the CI was 4% after 28 days of storage, continuing to show satisfactory indices. These data are similar to those found in our study, which, even with a chalcone concentration twice as high and an observation period extended to 49 days (CI = 2%), continued to maintain satisfactory indices. This confirms that Alg serves as an effective encapsulating matrix due to its electrostatic stabilization capacity within the nanoemulsion, provided by the carboxylate (–COO) and hydroxyl (–OH) groups in its structure, as reported by Artiga-Artigas et al. [50]. The sedimentation values for CNAlg were also satisfactory, presenting sedimentation index (SI) of 1% on day 21, with no signs of instability by the final evaluation day. In contrast, CNGA showed an IC of 2% throughout the entire observation period. The instability results were significantly greater for the formulation lacking a biopolymer matrix (UN) compared to those that included matrices. The pronounced formation of creaming observed can be attributed to the lower density of the nanoemulsion droplets relative to the surrounding liquid, which facilitates the upward migration of the particles [51]. For the evaluation of anthelmintic activity, EHT was chosen due to its recognition in the literature as a well-established screening method for natural products [52]. Additionally, the development of an anthelmintic compound with ovicidal effect is highly relevant, as it can prevent the development of the nematode H. contortus until the infective larval stage (L3). Efficacy on eggs can also result in a reduction in pasture contamination and livestock infections, contributing to sustainable helminth control strategies [53]. The ovicidal efficacy of the B4AM probably can be attributed to chemical structure, in particular the presence of the highly reactive amine group (H₂N) linked to the aromatic ring. Nitrogen is an electronegative element that can induce a destabilization of the aromatic ring and facilitate its penetration into the H. contortus eggshell. Additionally, the double bonds present in the compound structure act as active sites, contributing to the increase in chemical reactivity and enhancing the ovicidal effect, something that generally does not occur with structures of compounds with longer chains. In the study by Kozlowska et al. [54], the effect of the structure of the 18 amino-chalcones on biological activity was revealed, where the presence of an amino group in the meta position with the addition of the aromatic ring in the compound increased the hydrophobicity facilitating the chalcone penetration. Regarding the nanoemulsions CNAlg and UN, they exhibited the highest efficacy, in contrast to CNGA, which, besides failing to present satisfactory results in physicochemical characterization, also did not enhance the ovicidal effect. The ovicidal effect of the polymer-free formulation (UN) was superior (EC50 = 0.02 mg/mL) compared to the other formulations in the study. A similar outcome was observed by Aguiar et al. [22], who demonstrated that a biopolymer-free nanoemulsion containing carvone exhibited greater ovicidal efficacy against H. contortus eggs compared with emulsions that utilized Alg. Additionally, a reduced particle size was noted, which likely facilitated improved penetration into the eggshell, thereby promoting a more effective ovicidal effect. The presence of the sodium alginate matrix as an encapsulating agent in the polymeric system of the CNAlg may have influenced the controlled release process in the aqueous medium of EHT, prolonging the release of the encapsulated chalcone, 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 [55]. The images of H. contortus eggs, obtained through SEM following treatment with the formulations, revealed alterations suggesting the potential mechanisms of action. Chalcones typically exhibit a crystalline morphology [56] and deposits of this crystalline structure were observed on the H. contortus eggshell (figure 4a). This finding corroborates the study by Zarza-Albarrán et al. [30], who, when evaluating organic fractions of galloyl flavonoids from Acacia farnesiana on H. contortus eggs, reported the presence of granular structures attached to the eggshell. For CNAlg (figure 4b), although the oval morphology was not entirely compromised, the SEM microphotographs revealed cracks that may have weakened the eggshell, hindering hatching. This effect may be associated with the viscosity of Alg, which likely resulted in a slower release of B4AM. In the case of CNGA (figure 4c), residues of gum arabic were more prominently observed in the microphotographs. Conversely, the UN (figure 4d) formulation exhibited more pronounced damage to the eggshell morphology, highlighting its greater efficacy in the EHT. Cytotoxicity refers to the potential of a substance to induce cellular damage or death, assessed through various methods, such as the MTT assay [57]. B4AM exhibited no toxicity. The non-toxicity of chalcones is widely documented in the literature. Studies show that administering high doses of chalcones to animals results in little or no toxicity [58-60]. There are also toxicity reports indicating the safety of both natural and synthetic chalcones in mice and plants [61,62]. In vitro assays have demonstrated no or limited cytotoxic effects at medium to high micromolar concentrations, depending on the type of chalcone substituent and the tested cell line [63-65]. Turani et al. [66] (2024) evaluated methoxylated chalcones in the adult stage of the model nematode Caenorhabditis elegans and found that, in cytotoxicity tests with human embryonic kidney cells (HEK-293), no toxicity was detected. Nascimento et al. [67] evaluated various emulsification techniques to optimize the properties of chalcone nanoemulsions with antifungal potential. They observed that the chalcone (1E,4E)-1,5-bis (4-methoxyphenyl) penta-1,4-dien-3-one (DB 4 OCH 3 ) exhibited an IC50 above 150 µg/mL, indicating toxicological safety in the MTT assay. However, significant differences in IC50 values were noted between the nanoformulations and the free form of DB 4 OCH 3 . The authors suggest that these variations may be attributed to nanoparticle size and the increase in specific surface area, which enhance interactions with cellular components. In our study, we observed no cytotoxicity from B4AM or any of the tested nanoemulsions. These findings underscore the importance of optimizing the nanoparticulate system to reduce toxicity compared to the free compound, thereby improving cell culture conditions in the MTT assay. Notably, we used only the ultrasonic device to standardize the emulsification method in this study. Over a 50-hour period, the release kinetics described the release of chalcone from the three formulations with the best efficacy in the EHT. The results indicate that nanoemulsions (CNAlg and UN) serve as effective vehicles for enhancing drug solubility, facilitating homogeneous dispersion, and significantly increasing drug release. This is evidenced by the in vitro release profile of the UN and CNAlg nanoemulsions. The UN nanoemulsion released over 96% of chalcone after 30 hours, whereas CNAlg achieved a release exceeding 34% in the same period. In comparison, free B4AM exhibited less than 26% release. The kinetic analysis revealed that the Higuchi model was the most suitable to describe the release profile of CNAlg and B4AM suggesting that the release is controlled by diffusion through the matrix and that the drug concentration is initially uniform, decreasing over time. UN fit well in the zero-order release, assuming a constant release rate, regardless of how much drug remains in the system. Emulsification enhanced the solubility of B4AM, enabling a faster release of chalcone. In comparison, free B4AM, due to its low solubility in water, was unable to release more chalcone. It was discovered that incorporating Alg as a matrix was crucial for regulating the release of B4AM in the EHT's aqueous setting. Similar results were reported by Nascimento et al. [67], who encapsulated chalcone DB4OCH3 and observed controlled release with the use of an Alg matrix. UN released the chalcone more rapidly than the free form. Therefore, emulsification increased the solubility of the chalcone and enabled a more efficient release in a shorter time interval, probably because B4AM reached its solubility limit in water, no longer releasing the chalcone. Additionally, the small droplet size of the nanoemulsions provided a large surface area, which enhanced the dissolution rate of the active ingredient, releasing the drug consistently and in a controlled manner over time. Therefore, emulsification enhanced the solubility of chalcone, allowing for a more efficient release over a shorter period, which was not observed in the free B4AM formulation, likely due to reaching its solubility limit in water, preventing further drug release. Conclusion In conclusion, CNAlg, in addition to enhancing the physicochemical properties of B4AM, demonstrated the ability to improve chalcone solubilization and exhibited significant anti-helminthic efficacy. The ovicidal effect in the presence of Alg was comparable to that of the pure drug, with the added benefits of increased stability and prolonged action. The UN nanoemulsion showed excellent efficacy against eggs, superior to that of free B4AM, suggesting only minor adjustments in concentration and application intervals may be needed. These findings indicate that the use of nanoemulsions, with or without polymeric matrices, offers promising strategies for ovicidal control, with application possibilities tailored to the desired action profile. However, it is necessary to evaluate its performance in other stages of the life cycle of H. contortus , aiming at a more comprehensive understanding of their potential as new anthelmintic agents. Declarations Acknowledgements Mr. Barbosa has received a doctoral research scholarship from Ceará Foundation for Support of Scientific and Technological Development (FUNCAP). The authors would like to thank the Central Analítica-UFC/CT-INFRA/MCT-SISANO/Pró Equipamentos. Funding sources Hélcio Silva dos Santos received financial support from CNPq-PQ (Grant 306008/2022-0) and FUNCAPUNIVERSAL (Grant UNI-0210-00337.01.00/23). Flavia Oliveira Monteiro da Silva Abreu received financial support from CNPq (Grant 406522/2021-9-1). Data availability Data will be made available on request. References McKune S, Serra R, Touré A (2021) Gender and intersectional analysis of livestock vaccine value chains in Kaffrine, Senegal. PLoS ONE 16(7):e0252045. doi: 10.1371/journal.pone.0252045 Chagas ACS, Tupy O, Santos IB, Esteves SN, et al. (2022) Economic impact of gastrointestinal nematodes in Morada Nova sheep in Brazil. Rev Bras Parasitol Vet 31(3): e008722. doi: 10.1590/S1984-29612022044 Parvin S, Dey AR, Shohana NN, et al. (2024) Haemonchus contortus , an obligatory haematophagus worm infection in small ruminants: Population genetics and genetic diversity. Saudi J Biol Sci 31(8):104030. doi: 10.1016/j.sjbs.2024.104030 Antonopoulos A, Higgins O, Doyle SR, et al. (2024) Real-time single-base specific detection of the Haemonchus contortus S168T variant associated with levamisole resistance using loop-primer endonuclease cleavage loop-mediated isothermal amplification. Mol Cell Probes 73:101946. doi: 10.1016/j.mcp.2023.101946 Pava LDA, Flores-Jiménez NG, Cuéllar-Ordaz JA, et al. (2024) Exploring alternative anthelmintic compounds: Impact of peruvin, hentriacontane/1-nonacosanol and their synergistic effect on the health of Meriones unguiculatus infected with Haemonchus contortus . Vet Parasitol 332:110303. doi: 10.1016/j.vetpar.2024.110303 Fu Y, Liu D, Zeng H, et al. (2020) New chalcone derivatives: synthesis, antiviral activity and mechanism of action. RSC Adv 10(41):24483-24490. doi: 10.1039/D0RA03684F Dan W, Dai J (2020) Recent developments of chalcones as potential antibacterial agents in medicinal chemistry. Eur J Med Chem 187:111980. doi: 10.1016/j.ejmech.2019.111980 Rocha JE, Freitas TS, Xavier JC, et al. (2021) Antibacterial and antibiotic modifying activity, ADMET study and molecular docking of synthetic chalcone (E)-1-(2-hydroxyphenyl)-3-(2,4-dimethoxy-3-methylphenyl)prop-2-en-1-one in strains of Staphylococcus aureus carrying NorA and MepA efflux pumps. Biomed Pharmacother 140:111768. doi: 10.1016/j.biopha.2021.111768 Okolo EN, Ugwu DI, Ezema BE, et al. (2021) New chalcone derivatives as potential antimicrobial and antioxidant agent. Sci Rep 11:21781. doi: 10.1038/s41598-021-01292-5 Zhu M, Wang J, Xie J, et al. (2018) Design, synthesis, and evaluation of chalcone analogues incorporate α,β-Unsaturated ketone functionality as anti-lung cancer agents via evoking ROS to induce pyroptosis. Eur J Med Chem 157:1395-1405. doi: 10.1016/j.ejmech.2018.08.072 Zhou Q, Tang X, Chen S, et al. (2022) Design, Synthesis, and Antifungal Activity of Novel Chalcone Derivatives Containing a Piperazine Fragment. Agric Environ Chem 70(4): 1029–1036. doi: 10.1021/acs.jafc.1c05933 Abreu FOMS, Holanda T, Nascimento JF, et al. (2024) Evaluation of the antibacterial and antifungal capacity of nanoemulsions loaded with synthetic chalcone derivatives Di-benzyl cinnamaldehyde and Benzyl 4-aminochalcone. Renew Mater 12(2):285–304. doi: 10.32604/jrm.2023.043919 Bezerra LL, Almeida-Neto WQ, Marinho MM, et al. (2023) Synthesis of aminochalcones and in silico evaluation of their antiparasitic potential against Leishmania . J Biomol Struct Dyn 41(13):6434-6441. doi: 10.1080/07391102.2022.2103030 Ouattara M, Sissouma D, Koné MW, et al. (2011) Synthesis and anthelmintic activity of some hybrid Benzimidazolyl-chalcone derivatives. Trop J Pharm Res 10(6):767-775. doi: 10.4314/tjpr.v10i6.10 Sissouma D, Ouattara M, Koné MW, et al. (2011) Synthesis and in vitro nematicidal activity of new chalcones vectorised by imidazopyridine. Afr J Pharm Pharmacol 5(18):2086-2093. doi: 10.5897/AJPP11.550 Vázquez-Bravo J, Aguilar-Marcelino L, Castañeda-Ramírez GS, et al. (2020) In vitro nematicidal activity of two ferrocenyl chalcones against larvae of Haemonchus contortus (L3) and Nacobbus aberrans (J2). J Helminthol 94:e190. doi: 10.1017/S0022149X2000070X Nikolic I, Lunter DJ, Randjelovic D, et al. (2018) Curcumin-loaded low-energy nanoemulsions as a prototype of multifunctional vehicles for different administration routes: Physicochemical and in vitro peculiarities important for dermal application. Int J Pharm 550(1-2):333-346. doi: 10.1016/j.ijpharm.2018.08.060 Preeti, Sambhakar S, Malik R, et al. (2023) Nanoemulsion: An emerging novel technology for improving the bioavailability of drugs. Scientifica 2023(1). doi: 10.1155/2023/6640103 Rhein-Knudsen N, Ale MT, Ajalloueian F, et al. (2017) Characterization of alginates from Ghanaian brown seaweeds: Sargassum spp. and Padina spp. Food Hydrocoll 71:236-244. doi: 10.1016/j.foodhyd.2017.05.016 Raj V, Kim Y, Kim YG, et al. (2022) Chitosan-gum arabic embedded alizarin nanocarriers inhibit biofilm formation of multispecies microorganisms. Carbohydr Polym 284:118959. doi: 10.1016/j.carbpol.2021.118959 André WPP, Junior JRP, Cavalcante GS, et al. (2020) Anthelmintic activity of nanoencapsulated carvacryl acetate against gastrointestinal nematodes of sheep and its toxicity in rodents. Rev Bras Parasitol Vet 29(1):e013119. doi: 10.1590/S1984-29612019098 Aguiar AARM, Araújo-Filho JV, Pinheiro HN, et al. (2022) In vitro anthelmintic activity of an R-carvone nanoemulsions towards multiresistant Haemonchus contortus . Parasitoloy 149(12):1631-1641. doi: 10.1017/S0031182022001135 Irfan R., Mousavi S., Meshari A, et al. (2020) A Comprehensive Review of Aminochalcones. Molecules 25(22):5381. doi: 10.3390/molecules25225381 Romeu MC, Freire PTC, Ayala AP, et al (2022) Synthesis, crystal structure, ATR-FTIR, FT-Raman and UV spectra, structural and spectroscopic analysis of (3E)‐4‐[4‐(dimethylamine)phenyl]but‐3‐en‐2‐one. J Mol Struct 1264:133222. doi: 10.1016/j.molstruc.2022.133222 Mwangi WW, Ho KW, Tey BT, et al. (2016) Effects of environmental factors on the physical stability of pickering-emulsions stabilized by chitosan particles. Food Hydrocoll 60:543–550. doi: 10.1016/j.foodhyd.2016.04.023 Hubert J, Kerboeuf D. (1992) A microlarval development assay for the detection of anthelmintic resistance in sheep nematodes. Vet Rec 130(20):442–448. doi:10.1136/vr.130.20.442 Neveu C, Charvet C, Fauvin A, et al. (2007) Identification of levamisole resistance markers in the parasitic nematode Haemonchus contortus using a cDNA-AFLP approach. Parasitology 134(8):1105–1110. doi: 10.1017/S0031182007000030 Fauvin A, Charvet C, Issouf M, et al. (2010) cDNA-AFLP analysis in levamisole-resistant Haemonchus contortus reveals alternative splicing in a nicotinic acetylcholine receptor subunit. Mol Biochem Parasitol 170(2):105–107. doi: 10.1016/j.molbiopara.2009.11.007 Coles GC, Jackson F, Pomroy WE, et al. (2006) The detection of anthelmintic resistance in nematodes of veterinary importance. Vet Parasitol 136(3–4):167–185. doi: 10.1016/j.vetpar.2005.11.019 Zarza-Albarrán MA, Olmedo-Juárez A, Rojo-Rubio R, et al. (2020) Galloyl flavonoids from Acacia farnesiana pods possess potent anthelmintic activity against Haemonchus contortus eggs and infective larvae. J Ethnopharmacol. 249:112402. doi: 10.1016/j.jep.2019.112402 Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assay. J Immunol Methods 65(1-2):55-63. doi: 10.1016/0022-1759(83)90303-4 Costa P (2002) In vitro evaluation of the lyoequivalence of pharmaceutical formulations. Braz J Pharm Sci 38(2):141–153. doi:10.1590/S1516-93322002000200003 Rai VK, Mishra N, Yadav KS, et al. (2018) Nanoemulsion as pharmaceutical carrier for dermal and transdermal drug delivery: Formulation development, stability issues, basic considerations and applications. J Control Release 270:203–225. doi: 10.1016/j.jconrel.2017.11.049 Hong IK, Kim SI, Lee SB (2018) Effects of HLB value on oil-in-water emulsions: Droplet size, rheological behavior, zeta-potential, and creaming index. J Ind Eng Chem 67:123–131. doi: 10.1016/j.jiec.2018.06.022 Guttoff M, Saberi AH, McClements (2015) DJ Formation of vitamin D nanoemulsion-based delivery systems by spontaneous emulsification: Factors affecting particle size and stability. Food Chem 171:117–122. doi: 10.1016/j.foodchem.2014.08.087 Salvia-Trujillo L, Rojas-Graü MA, Soliva-Fortuny R, et al. (2013) Effect of processing parameters on physicochemical characteristics of microfluidized lemongrass essential oil-alginate nanoemulsions. Food Hydrocoll 30(1):401–407. doi: 10.1016/j.foodhyd.2012.07.004 Abreu FOMS, Holanda T, Nascimento JF, et al. (2024) Evaluation of the antibacterial and antifungal capacity of nanoemulsions loaded with synthetic chalcone derivatives Di-Benzyl Cinnamaldehyde and Benzyl 4-Aminochalcone. J Renew Mater 12(2):285–304. doi: 10.32604/jrm.2023.043919 Winter E, Pizzol CD, Locatelli C, et al. (2014) In vitro and in vivo effects of free and chalcones-loaded nanoemulsions: Insights and challenges in targeted cancer Chemotherapies. Int J Environ Res Public Health 11(10): 10016-10035. doi: 10.3390/ijerph111010016 Abreu FOMS, Nascimento JF, Pinheiro HN, et al. (2024) Formulation of nanoemulsions enriched with chalcone-based compounds: formulation process, physical stability and antimicrobial effect. Polym Bull 81:7367-7391. doi: 10.1007/s00289-023-05069w Gago C, Guerreiro A, Souza M, et al. (2024) Effectiveness of Sodium Alginate and Carnauba Wax Nanoemulsions with Lemongrass Essential Oil on the quality of ‘Hass’ Avocado Fruit from early, middle, and late harvest season during prolonged cold storage. Sci Hortic 333:113237. doi: 10.1016/j.scienta.2024.113237 Honary S, Zahir F (2013) Effect of Zeta Potential on the Properties of Nano-Drug Delivery Systems - A Review (Part 2). Trop J Pharm Res 12(2):265-273. doi: 10.4314/tjpr.v12i2.20 Liu Y, Wei ZC, Deng YY, et al. (2020) Comparison of the Effects of Different Food-Grade Emulsifiers on the Properties and Stability of a Casein-Maltodextrin-Soybean Oil Compound Emulsion. Molecules 25(3):458. doi: 10.3390/molecules25030458 Hosseini SMH, Emam-Djomeh Z, Razavi SH, et al. (2013) β-Lactoglobulin–sodium alginate interaction as affected by polysaccharide depolymerization using high intensity ultrasound. Food Hydrocoll 32(2):235-244. doi: 10.1016/j.foodhyd.2013.01.002 Salvia-Trujillo L, Decker EA, McClements DJ (2016) Influence of an anionic polysaccharide on the physical and oxidative stability of omega-3 nanoemulsions: Antioxidant effects of alginate. Food Hydrocoll 52:690-698. doi: 10.1016/j.foodhyd.2015.07.035 Bortoluzzi BB, Buzatti A, Chaaban A, et al. (2021) Mentha villosa Hubs., M. x piperita and their bioactives against gastrointestinal nematodes of ruminants and the potential as drug enhancers. Vet Parasitol 289:109317. doi: 10.1016/j.vetpar.2020.109317 Llinares R, Santos J, Trujillo-Cayado LA, et al. (2018) Enhancing rosemary oil-in-water microfluidized nanoemulsion properties through formulation optimization by response surface methodology. LWT 97:370-375. doi: 10.1016/j.lwt.2018.07.033 Iskandar B, Mei HC, Liu TW, et al. (2024) Evaluating the effects of surfactant types on the properties and stability of oil-in-water Rhodiola rosea nanoemulsion. Colloids Surf B Biointerfaces 234:113692. doi: 10.1016/j.colsurfb.2023.113692 Tsichlis I, Manou AP, Manolopoulou V, et al. (2023) Development of Liposomal and Liquid Crystalline Lipidic Nanoparticles with Non-Ionic Surfactants for Quercetin Incorporation. Materials. 16:5509. doi: 10.3390/ma16165509 Hassanshahian M, Saadatfar A, Masoumipour F (2020) Formulation and characterization of nanoemulsion from Alhagi maurorum essential oil and study of its antimicrobial, antibiofilm, and plasmid curing activity against antibiotic-resistant pathogenic bacteria. J Environ Health Sci Eng 18:1015-1027. doi: 10.1007/s40201-020-00523-7 Artiga-Artigas M, Fani AA, Martín-Belloso O (2017) Effect of sodium alginate incorporation procedure on the physicochemical properties of nanoemulsions. Food Hydrocoll 70:191-2000. doi: 10.1016/j.foodhyd.2017.04.006 McClements DJ (2010) Emulsion Design to Improve the Delivery of Functional Lipophilic Components. Annu Rev Food Sci Technol 1:241-269. doi: 10.1146/annurev.food.080708.100722 Barbosa MLF, Ribeiro WLC, Filho JVA, et al. (2023) In vitro anthelmintic activity of Lippia alba essential oil chemotypes against Haemonchus contortus . Exp Parasitol 244: 108439. doi: 10.1016/j.exppara.2022.108439 Santos FO, Lima HGL, Santos NSS, et al. (2017) In vitro anthelmintic and cytotoxicity activities the Digitaria insularis (Poaceae). Vet Parasitol 245:48-54. doi: 10.1016/j.vetpar.2017.08.007 Kozłowska J, Potaniec B, Baczyńska D, et al (2019) Synthesis and Biological Evaluation of Novel Aminochalcones as Potential Anticancer and Antimicrobial Agents. Molecules 24(22):4129. doi: 10.3390/molecules24224129 Dash M., Chiellini F, Ottenbrite RM, et al. (2011) Chitosan—A versatile semi-synthetic polymer in biomedical applications. Prog Polym Sci 36(8):981-1014. doi: 10.1016/j.progpolymsci.2011.02.001 Arshad MN, Al-Dies AAM., Asiri AM, et al. (2017) Synthesis, crystal structures, spectroscopic and nonlinear optical properties of chalcone derivatives: A combined experimental and theoretical study. J Mol Struct 1141:142-156. doi: 10.1016/j.molstruc.2017.03.090 Adan A, Kiraz Y, Baran Y. (2016) Cell Proliferation and Cytotoxicity Assays. Curr Pharm Biotechnol 17:1213-1221. doi: 10.2174/1389201017666160808160513 Morris M, Zhang S. (2006) Flavonoid–drug interactions. Effects of flavonoids on ABC transporters. Life Sci 78(18):2116-2130. doi: 10.1016/j.lfs.2005.12.003 Bandgar BP, Gawande SS, Bodade RG, et al. (2010) Synthesis and biological evaluation of simple methoxylated chalcones as anticancer, anti-inflammatory and antioxidant agents. Bioorg Med Chem 18:1364-1370. doi: 10.1016/j.bmc.2009.11.066Abstract Zampini IC, Villena J, Salva S, et al. (2012) Potentiality of standardized extract and isolated flavonoids from Zuccagnia punctata for the treatment of respiratory infections by Streptococcus pneumoniae : In vitro and in vivo studies. Ethnopharmacol 140(2):287- 292. doi: 10.1016/j.jep.2012.01.019 González JA, Braun AE. (1998) Effect of (E)-Chalcone on Potato-Cyst Nematodes ( Globodera pallida and G. rostochiensis ). J Agric Food Chem 46(3):1163-1165. doi: 10.1021/jf9706686 Cancino K, Castro I, Yauri C. (2021) Toxicity assessment of synthetic chalcones with antileishmanial potential in BALB/c mice. Rev Peru Med Exp Salud Pública 38:424-433. doi: 10.17843/rpmesp.2021.383.6937 Forejtníková H, Lunerová K, Kubínová R. (2005) Chemoprotective and toxic potentials of synthetic and natural chalcones and dihydrochalcones in vitro . Toxicology 208(1):81-93. doi: 10.1016/j.tox.2004.11.011 Mai CW, Yaeghoobi M, Abd-Rahman N, et al. (2014) Chalcones with electron-withdrawing and electron-donating substituents: anticancer activity against TRAIL resistant cancer cells, structureeactivity relationship analysis and regulation of apoptotic. Eur J Med Chem 77:378-387. doi: 10.1016/j.ejmech.2014.03.002 Dong N, Liu X, Zhao T, et al. (2018) Apoptosis-inducing effects and growth inhibitory of a novel chalcone, in human hepatic cancer cells and lung cancer cells. Biomed Pharmacother 105:195-203. doi: 10.1016/j.biopha.2018.05.126 Turani O, Castro MJ, Vazzana J, et al. (2024) Potent Anthelmintic Activity of Chalcones Synthesized by an Effective Green Approach. ChemMedChem 19(13): e202400071. doi: 10.1002/cmdc.202400071 Nascimento JF, Abreu FOMS, Holanda T, et al. (2024) Evaluation of Emulsification Techniques to Optimize the Properties of Chalcone Nanoemulsions for Antifungal Applications. Pharmaceuticals 17(11):1442. doi: 10.3390/ph171114 Tables Table 1. Particle size, zeta potential, polydispersity index and encapsulation efficiency of nanoemulsions CNAlg, CNGA and UN with B4AM. Nanoemulsions Particle size (nm) Zeta potential (mV) Polydispersity index Encapsulation efficiency (%) CNAlg 267.64 ± 3.05 -56.64 ± 2.81 0.751 ± 0.095 87.5 ± 0.77 CNGA 234.3 ± 6.58 -58.28 ± 2.81 0.782 ± 0.065 43.8 ± 0.58 UN 144.56 ± 1.05 -46.06 ± 0.95 0.489 ± 0.045 69.87 ± 0.58 CNAlg: Sodium alginate-coated B4AM; CNGA: Gum arabic-coated B4AM; UN: Uncoated B4AM Table 2. Kinetic constant (K) and the correlation coefficient (R2) for the kinetic models studied. Formulations Zero-Order First-Order Higuchi Korsmeyer–Peppas R 2 K 0 (h −1 ) R 2 K t (h −1 ) R 2 K H (h −1/2 ) R 2 Ka (h −n ) B4AM 0.9534 0.0102 0.8086 0.0003 0.9755 0.6374 0.9709 0.5388 CNAlg 0.9085 0.013 0.8236 0.0004 0.9508 0.8139 0.9412 0.6492 UN 0.9461 0.4444 0.3125 7E-05 0.5988 0.8656 0.7235 0.1737 B4AM: Benzyl 4-Aminochalcone; CNAlg: Sodium alginate-coated B4AM; UN: Uncoated B4AM Additional Declarations No competing interests reported. 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. 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13:23:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5662121/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5662121/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":71851232,"identity":"66820d67-708b-4cee-909e-a6bd8acc66b6","added_by":"auto","created_at":"2024-12-19 07:25:34","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":102332,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Creaming and (B) sedimentation index as a function of the period of analysis for CNAlg (sodium alginate coated B4AM), CNGA (gum arabic coated B4AM) and UN (uncoated B4AM).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5662121/v1/7c22f4df91b89c9cafe513ee.png"},{"id":71851284,"identity":"efe07dba-d4c2-49ed-bad2-2d8e0e6b9de6","added_by":"auto","created_at":"2024-12-19 07:25:45","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":336572,"visible":true,"origin":"","legend":"\u003cp\u003eScanning electron microscopy for nanoemulsions at 150.000×: (a) CNAlg (sodium alginate coated B4AM), (b) CNGA (gum arabic coated B4AM) and (c) UN (uncoated B4AM).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5662121/v1/e25c401b9a960dc6ba879fbb.png"},{"id":71851363,"identity":"27052af6-bb3b-4126-a9cc-3090e9b3ce71","added_by":"auto","created_at":"2024-12-19 07:25:55","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":125788,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of B4AM (A), CNAlg (B), CNGA (C) and UN (D) on the hatching of \u003cem\u003eHaemonchus contortus\u003c/em\u003e larvae. *The number of different asterisks indicates statistical differences (p\u0026lt;0.05). B4AM (Benzyl 4-Aminochalcone); CNAlg (sodium alginate coated Benzyl 4-Aminochalcone); CNGA (gum arabic coated Benzyl 4-Aminochalcone); UN (uncoated without a polymeric matrix); Thiabendazole (TBZ) was used as positive control; Sodium alginate (1%), gum arabic (1%) and Tween 80 (3%) were used as negative controls.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5662121/v1/008145d47855f72c6092778a.png"},{"id":71851238,"identity":"0a5af082-5397-43d3-95ef-c1e1ced7e87b","added_by":"auto","created_at":"2024-12-19 07:25:38","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":401961,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eHaemonchus contortus\u003c/em\u003e eggs observed in a scanning electron microscopy treated with B4AM (a); CNAlg (b); CNGA (c); UN (d); Negative control - 3% Tween (e); Negative control - 1% Alg (f); Negative control - 1% GA (g); Positive control - Thiabendazole (h). The red arrows show the main changes observed.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5662121/v1/79b5a206c3ec509c9c8dd2e1.png"},{"id":71851219,"identity":"6d5d2a15-0fd6-4906-801b-0399224b5e91","added_by":"auto","created_at":"2024-12-19 07:25:22","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":69929,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e controlled release profile of CNAlg, UN and free chalcone B4AM.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5662121/v1/e3eaa141df71b45b39d34af0.png"},{"id":83811257,"identity":"0d7e9a66-ae72-4699-b30d-4f6d3ef18a0f","added_by":"auto","created_at":"2025-06-03 07:02:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1923986,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5662121/v1/1825b3b5-d25f-48c7-a525-a2defd052e9c.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Improving the anthelmintic effectiveness of Benzyl 4-Aminochalcone with polysaccharides nanoemulsions","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSmall ruminant farming provides income, nutrition, and acts as a safety net for households in numerous developing nations [1]. However, the presence of gastrointestinal nematode infections presents considerable global challenges [2]. \u003cem\u003eHaemonchus contortus\u003c/em\u003e, a highly pathogenic gastrointestinal nematode, feeds on blood in sheep and goats, causing severe anemia, weight loss, decreased productivity and sometimes death [3].\u003c/p\u003e \u003cp\u003eThe method more used for controlling gastrointestinal nematodes is based on administration of synthetic anthelmintic. Nevertheless, the uncontrolled administration of these drugs has caused anthelmintic resistance [4]. Hence, the attention has shifted towards studying natural compounds as alternative control methods [5].\u003c/p\u003e \u003cp\u003eChalcones, which are aromatic ketones, serve as precursors for flavonoids and isoflavones. They are made up of two benzene rings connected by an α,β-unsaturated system [6]. These substances can be found in abundance in plants of the Leguminosae, Compositae and Moraceae families [7] and are usually obtained naturally or synthesized using the Claisen-Schmidt reaction [8]. Studies have proven the antioxidant [9], antitumor [10], antifungal [11], antibacterial [12] and antiparasitic [13] effects of chalcones. The anthelmintic activity of these secondary metabolites has only been minimally researched, with a focus on their effectiveness against \u003cem\u003eH. contortus\u003c/em\u003e larvae [14,15,16].\u003c/p\u003e \u003cp\u003eThe therapeutic usefulness of chalcones is hindered by their poor water solubility [17]. Nanoemulsions have been utilized to address this challenge by safeguarding drugs, enhancing their bioavailability and providing controlled release [18]. Biopolymers like sodium alginate (Alg) and gum arabic (GA) show potential as encapsulation materials for hydrophobic substances [19,20]. The effectiveness of bioactive compounds against \u003cem\u003eH. contortus\u003c/em\u003e was enhanced by encapsulating with Alg and GA [21,22]. Irfan et al. [23] reported various biological properties of aminochalcones. However, no anthelmintic activity of these compounds has been described, nor has nanoencapsulation been investigated for anthelmintic purposes, making the evaluation of B4AM promising. Consequently, the objectives of this research included formulating three nanoemulsions with B4AM compound, assessing their physicochemical properties, analyzing release kinetics, evaluating efficacy against \u003cem\u003eH. contortus\u003c/em\u003e using the egg hatching test (EHT), analyzing potential morphological alterations in the eggshell using scanning electron microscopy (SEM) and verifying cytotoxicity.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnimal studies were approved by the Ethics Committee of the State University of Cear\u0026aacute; (protocol number. 018265831-2023).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaterials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe chalcone (E)-1-(4-(dimethylamino)phenyl)but-2-en-1-one or B4AM (189.12 g/mol) was given by the Organic Synthesis Lab at State University of Vale do Acara\u0026uacute;, Sobral, Brazil. B4AM was synthesized\u0026nbsp;and its structure characterized through spectroscopic techniques [24]. The reaction was kept under mechanical stirring at room temperature, and after 30 minutes, the mixture was filtered, and the solid was washed with distilled water. Recrystallization of the compound was carried out with ethanol as the solvent. Commercially sourced Tween 80 (Vetec\u0026reg;, Brazil), dimethylsulfoxide - DMSO (Sigma-Aldrich\u0026reg;, USA), Alginate (Alg), Gum Arabic GA (Din\u0026acirc;mica\u0026reg;, Brazil), and canola oil were used.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of nanoemulsions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe dilution of B4AM was achieved by adding 3% DMSO to distilled water. Three nanoemulsions were prepared, one uncoated (UN), one coated with polymeric solution of Alg (CNAlg) and other coated with polymeric solution of GA (CNGA). The nanoemulsions underwent a high-energy emulsification process using the Ultronique QR500 ultrasonic tip sonicator (Brazil) at a frequency of 20 kHz and power of 350W for 2 min. It was mixed 3 mg of B4AM, 100 \u0026mu;L DMSO, 100 \u0026mu;L Tween 80 and 100 \u0026mu;L canola oil in 100 ml of 1% polymeric solution. Nanoemulsion without polymer matrix was prepared by using the same constituents, but with distilled water substituting the polymer solution.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCharacterizations of nanoemulsions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe physical stability of the nanoemulsions (CNAlg; CNGA and UN) was evaluated at 7, 14, 21, 28, 35, 42 and 49 days. For this procedure, approximately 10 mL of each of the nanoemulsions were kept in closed test tubes, without exposure to light, at 27\u0026deg;C. Periodic observations were implemented to detect any visual signs of instability. Creaming and sedimentation were measured over time with the aid of a caliper. The creaminess index (CI) and sedimentation index (SI) were determined by relating the height in centimeters of the precipitates formed in the test tube (Ha) with the total height of the sample (Ht) after the mentioned preparation periods, calculated according to equations (1) and (2), adapted from Mwangi et al. [25]:\u003c/p\u003e\n\u003cp\u003eCI (%) =Ha/Ht\u0026times;100 \u003cstrong\u003eEquation 1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSI (%) =Ha/Ht\u0026times;100\u003cstrong\u003e\u0026nbsp;Equation 2\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDynamic light scattering with the Malvern Zetasizer equipment was used to determine the droplet size, polydispersity index and zeta potential of CNAlg, CNGA and UN after 15 days. The nanoemulsions were dispersed in distilled water, forming a concentration of 1% (v/v) in water and left stirring for 12 h to ensure total dispersion of the matrix in aqueous medium.\u003c/p\u003e\n\u003cp\u003eScanning electron microscopy (SEM) was used to visualize the morphology of the nanoemulsions droplets. 20 \u0026mu;L of each nanoemulsion were placed in rounded metal structures and dehydrated at 50\u0026deg;C for approximately 1 h in a drying oven. Then the samples were observed using a microscope from FEI Company\u0026reg; (USA) at an acceleration voltage of 15 kV. The magnification was set at 150.000\u0026times;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEncapsulated chalcone content (%EE)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe encapsulation efficiency (EE%) of CNAlg, CNGA and UN were assessed by adding 1 mL of each emulsion to 4 mL of 96% ethanol and allowing it to stand for 24 h. Once the phases were separated, 1 mL of the ethanolic phase was isolated and diluted with 96% ethanol (3 mL) for measurement on the Kazuaki Spectrophotometer (Genesys 6 UV-Vis) at 390 nm. The absorbance in the calibration curve was adjusted by performing additional dilutions, up to a factor of 100x, to maintain linearity with concentration. The chalcone content in the nanoemulsions was determined through a calibration curve, prepared in triplicate in a standard solution of 150 mg/L in 96% ethanol. Additional dilutions were made to obtain concentrations of 75 to 1 mg/L. The calibration curve for B4AM is represented, respectively, by Equation (3):\u003c/p\u003e\n\u003cp\u003ey=0.673\u0026times;+ 0.0343 \u0026rarr;R2=0:09953 \u003cstrong\u003eEquation 3\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe accuracy of the %EE determination may be affected due to high dilution, leading to fluctuations. Duplicate tests were performed, and Equation (4) was used to determine the value of %EE:\u003c/p\u003e\n\u003cp\u003e%EE = Determined chalcone content/Total chalcone content\u003cstrong\u003e\u0026nbsp;Equation 4\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnthelmintic activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo recover \u003cem\u003eH. contortus\u003c/em\u003e eggs, feces were collected of sheep harboring a monospecific infection and processed according to the technique described by Hubert and Kerboeuf [26]. The \u003cem\u003eH. contortus\u003c/em\u003e isolate used - Kokstad (KOK) - is resistant to benzimidazoles, levamisole, and macrocyclic lactones [27,28] and was provided by the Institut National de Recherche pour l\u0026apos;Agriculture, l\u0026apos;Alimentation et l\u0026apos;Environnement (INRAE), Nouzilly, France.\u003c/p\u003e\n\u003cp\u003eThe egg hatch test\u003cstrong\u003e\u0026nbsp;(\u003c/strong\u003eEHT) was performed according to Coles et al. [29]. Briefly, 250 \u0026mu;L of an egg\u0026rsquo;s suspension containing approximately 100 fresh eggs was incubated for 48 h at 27\u0026deg;C with 250 \u0026mu;L of four formulations (B4AM; CNAlg; CNGA and UN) at concentrations of 0.15 to 0.009375 mg/mL. Drops of Lugol\u0026apos;s solution were added to stop egg hatching, and eggs and first-stage larvae (L1) were counted under a light microscope. This test was performed with controls: 3% Tween, 1% Alg and 1% GA were the negative controls and 0.1 mg/mL thiabendazole (Sigma-Aldrich\u003csup\u003e\u0026reg;\u003c/sup\u003e, USA) was the positive control. Three repetitions were performed with five replications for each treatment and control.\u003c/p\u003e\n\u003cp\u003eThe eggs of \u003cem\u003eH. contortus\u003c/em\u003e exposed to the highest concentrations of four formulations were analyzed by SEM. At least 10 eggs presented the same patterns of change to be considered. Samples of 10 \u0026mu;L containing eggs were deposited on aluminum stubs with double-sided carbon adhesive tape and observed. This methodology was adapted from Zarza-Albarr\u0026aacute;n et al. [30]. Microscope from FEI Company\u0026reg; (USA) at an acceleration voltage of 15 kV was used. The magnification was set at 8.000\u0026times;.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCytotoxicity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe cytotoxicity test was performed using cell cultures of murine fibroblasts (L929 cell line) as a model. Cell toxicity was quantified by the ability of living cells to reduce the yellow dye 3-(4,5-dimethyl-2-thiozolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) to a purple formazan product [31]. For the experiments, L929 cells were plated in 96-well plates (0.7 x 105 cells/well). The compounds (B4AM, CNAlg, CNGA, UN) were tested at 0.46 to 120 \u0026mu;g/mL and the plates were incubated for 24 h. This assay was performed with controls: 3% Tween, 1% Alg and 1% GA were the negative controls and bleomycin (0.39 to 100 \u0026mu;g/mL) was used as positive control. Thereafter, the plates were centrifuged and fresh medium (150 \u0026mu;L) containing MTT at 0.5 mg/mL was placed in wells. After 3 h, the formazan product was dissolved in 150 \u0026micro;L of DMSO, and absorbance was measured using a multi-plate reader (Spectra Count, Packard, Ontario, Canada). The drug effect was quantified as the percentage of absorbance of the reduced dye at 595 nm. Three repetitions were performed for each treatment and control.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn vitro\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;release kinetic profile\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe release kinetics of B4AM, CNAlg and UN were conducted using a dialysis system. For each sample, 60 mg of formulations were introduced into cellulose acetate membranes (14 kDa pores) and dialyzed against 60 mL of phosphate buffer solution (PBS) and 1% (v/v) Tween 80 at pH 7 for 50 h. Three aliquots of 2 mL were removed every three hours and analyzed by spectrophotometry in the Genesys 10S UV-Vis apparatus (Thermo Fisher Scientific, New York, USA). Thus, the concentration of B4AM present in the medium was calculated using a calibration curve in PBS at pH 7, resulting in equation 5:\u003c/p\u003e\n\u003cp\u003eAbs=0.0028conc-0.0368;R2=0.994 \u003cstrong\u003eEquation 5\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe release mechanisms of B4AM from nanoemulsions were evaluated using zero-order, first-order, Higuchi and Korsmeyer-Peppas mathematical models [32].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the EHT, efficacy was determined according to the following equation 6:\u003c/p\u003e\n\u003cp\u003enumber of eggs/(number of eggs + number of L1)]\u0026times;100 \u0026nbsp;\u003cstrong\u003eEquation 6\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe percentage of hatched larvae was analyzed by one-way analysis of variance (ANOVA), followed by multiple comparisons using the Tukey test, with the aid of GraphPad Prism\u0026reg; 8.0 software. The effective concentration to inhibit 50% of egg hatching (EC\u003csub\u003e50\u003c/sub\u003e) was determined by the probit method, using the SPSS 23.0 program for Windows.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eCharacterizations of nanoemulsions\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eThe stability indices of nanoemulsions are demonstrated in Figure 1. The CNAlg showed signs of instability (CI = 4%) on day 1 and (CI = 8%) day 21. However, this index returned to 4% on the last observation (day 49). Sedimentation for CNAlg was noted only on day 21 (SI = 1%) and decreased to null (day 28). CNGA presented stability (CI = 2% and SI = 1%) throughout the observation period.\u003ca href=\"https://d.docs.live.net/126e3b8748905dc7/Documentos/Documentos%20-%20Doutorado/Artigos%20e%20documentos%20para%20estudo/Artigo%20t%C3%A9cnico%201%20-%2008.10.2024%20NOVO.docx#_msocom_2\"\u003e\u0026nbsp;\u003c/a\u003eUN CI ranging from 14% (day 14) to 8% (day 49). On day 7, the UN SI was 6%, decreased to 1% on days 14-42 and reached 4% on the last day of analysis.\u003c/p\u003e\u003cp\u003eTable 1 displays the results for particle size, zeta potential and PDI of emulsions. Nanometric particle sizes were observed. The average particle sizes for CNAlg, CNGA and UN were 267.64, 234.3 and 144.56 nm, respectively\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003eThe PDI revealed moderate homogeneity for UN, and some degree of heterogeneity for emulsions coated with polymers (CNAlg and CNGA). The encapsulation efficiency values were 87.5% ± 0.77, 43.8 ± 0.58 and 69.87% ± 0.58 for CNAlg, CNGA and UN, respectively.\u003c/p\u003e\u003cp\u003eFigure 2 displays the SEM images of dried film nanoemulsions at a magnification of 150.000×. CNAlg, CNGA and UN (Figure 2a, 2b and 2c, respectively) exhibit nanodroplets with size below 100 nm. \u0026nbsp;UN exhibited smaller domains than CNAlg and CNGA, which might be a result of the lack of polymer coating, potentially impacting the droplets size of emulsions.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eAnthelmintic activity\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eThe efficacy of chalcone formulations in the EHT is demonstrated in figure 3. B4AM and their nanoemulsions showed ovicidal activity on \u003cem\u003eH. contortus\u003c/em\u003e. The highest concentration tested (0.15 mg/ml) inhibited more than 94% of egg hatching. These results did not differ statistically from the positive control (TBZ). The negative controls (3% Tween, 1% Alg and 1% GA) have no demonstrated effect on larvae hatching. The ovicidal effect of formulations was dose dependent. The EC\u003csub\u003e50\u003c/sub\u003e values were 0.04 mg/mL for B4AM and CNAlg, 0.10 mg/mL for CNGA and 0.02 mg/mL for UN.\u003c/p\u003e\u003cp\u003eFigure 4 illustrates the \u003cem\u003eH. contortus\u003c/em\u003e eggs after treatment with the formulations and controls, highlighting morphological damage in the eggs exposed to the four treatments: B4AM (Figure 4a), CNAlg (Figure 4b), CNGA (Figure 4c) and UN (Figure 4d). Figure (4e) presents the negative control (3% Tween), characterized by intact morphology and a smooth eggshell surface. Similarly, the eggs treated with polysaccharides (Figures 4f and 4g) also exhibit preserved morphology. In contrast, (Figure 4h) illustrates the alteration in the morphology of the egg caused by the positive control (TBZ).\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eCytotoxicity\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eThe maximum inhibitory concentration (IC\u003csub\u003e50\u003c/sub\u003e) of both B4AM chalcone and the nanoemulsions was found to be greater than 120 μg/mL, indicating no cytotoxicity after 24 hours. The nanoemulsions had no toxicity because the B4AM and the biopolymers are non-toxic.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn vitro\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;release kinetic profile\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eThe \u003cem\u003ein vitro\u003c/em\u003e release profile of the nanoemulsions and the B4AM diluted in DMSO, conducted at pH 7.0, is depicted in Figure 5. After 10 hours, the UN nanoemulsion released 92.22% of B4AM, reaching 96.66% after 30 hours. The CNAlg nanoemulsion released 17.77% after 10 hours, increasing to 34.44% after 30 hours. In comparison, free B4AM exhibited a release of less than 14% after 10 hours and 25.55% after 30 hours. This limited release suggests that B4AM has low solubility in aqueous environments. On the other hand, the use of nanoemulsions significantly enhances the release of the compound due to the controlled and prolonged release mechanism provided by these formulations.\u003c/p\u003e\u003cp\u003eTable 2 presents the kinetic constant and the correlation coefficient for the studied kinetic models. The Higuchi model provided the best fit for B4AM and CNAlg, while the Zero-order model was the best fit for UN, with a high correlation coefficient (R²) in both cases.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe study involved the use of nanoemulsions to enclose an aminochalcone, which were subsequently analyzed and assessed for its impact on egg hatching of \u003cem\u003eH. contortus\u003c/em\u003e and cytotoxicity. Among the three nanoemulsions created the %EE was highest with the Alg matrix (\u0026gt;80%). The UN nanoemulsion experienced a small drop in %EE, almost reaching 70%. Conversely, the %EE for CNGA was under 45%. The interaction of a substance's hydrophilic and lipophilic components is identified as the hydrophilic-lipophilic balance (HLB). For the stabilization of both aqueous and oily phases, it is essential for the values to range from 8 to 18, showcasing the surfactant's hydrophilic traits during nanoemulsion formation [33]. Tween 80 is an emulsifier that has an HLB value of 15 [34]. Due to its hydrophilic nature, it rapidly shifts to the aqueous phase following the addition of the organic phase [35]. The higher %EE observed in CNAlg could result from the Alg and Tween 80 interaction, affecting the lipid droplets' surface charge [36]. Abreu et al. [37] reported a comparable value when encapsulating B4AM with Alg matrix. The authors explained that this outcome might be due to the molecules having a stronger attraction to the oil phase in the droplets than to the aqueous phase of Alg during preparation, as supported by Winter et al. [38]. The lack of a polymeric matrix led to a decreased EE% for UN in comparison to CNAlg, although it remained higher than CNGA.\u0026nbsp;Gum arabic, being a higher molecular weight and a branched polysaccharide, tends to present higher hydrodynamic ratio, which may imply in the droplet’s stability as a coating agent and in the viscosity of the nanoemulsions [39]. Hence, EE% can be influenced by both the surfactant amount and the matrix.\u003c/p\u003e\n\u003cp\u003eThe nanoemulsions exhibited suitable particle sizes, ranging from 144.56 to 267.64 nm. Gago et al. [40] reported smaller particle sizes in nanoemulsions containing Alg with lemongrass essential oil, carnauba wax alone, or a combination of both, with values between 24.2 nm and 179.13 nm. In contrast to the observed zeta potential values (from -16.0 to -31.73 mV), our values, ranging from -46 mV to -58.3 mV, demonstrated superior stability. By increasing electrostatic repulsion, higher zeta potential values can strengthen the stability of colloidal dispersions and avoid particle aggregation [41]. Hence, the nanoemulsions created in our investigation demonstrate increased stability when compared to the mentioned study. To further support this information, Li et al. [42] executed nanoencapsulation methods aimed at enhancing the stability of curcumin in liposomes. In this study, liposomes stabilized with GA, or Alg were prepared at concentrations ranging from 0 to 2% (w/v). The evaluation of zeta potential revealed that it varied with increasing amounts of GA or Alg. At a concentration of 1%, which is the same concentration used in our study, the zeta potential of the isolated liposomes (without GA or Alg), which was initially -5 mV, increased to -10 mV.\u003c/p\u003e\n\u003cp\u003eRegarding droplet size, previous studies have reported seemingly contradictory results on the effect of Alg concentration on droplet size in emulsions. Hosseini et al. [43] investigated the interaction between β-lactoglobulin and Alg before and after sonication using isothermal titration calorimetry (ITC). Their findings revealed that increasing the concentration of Alg resulted in an increase in droplet diameter. In contrast, Salvia-Trujillo et al. [44] examined fish oil-in-water nanoemulsions stabilized with Tween 80 and combined with Alg at various concentrations. They reported that increasing the Alg concentration from 0.5% to 1% resulted in a reduction in droplet size. These findings suggest that lower concentrations promote droplet flocculation and coalescence, whereas higher concentrations provide greater protection against coalescence. This discrepancy may be attributed to differences in the synthesis methods of nanoemulsions. Bortoluzzi et al. [45] prepared a nanoemulsion of \u003cem\u003eMentha villosa\u003c/em\u003e Huds. essential oil using Tween 80 as a surfactant, without the addition of a polymeric matrix. The sample was initially subjected to high-pressure homogenization using an IKA digital Ultra-Turrax T25 (Staufen, Germany) and subsequently processed with an APLAB 10 high-pressure homogenizer (Artepeças, São Paulo, Brazil). This nanoemulsion exhibited a larger particle diameter (164 nm) compared to the UN nanoemulsion in the present study (144.56 nm), which was also prepared without a polymeric matrix. Studies indicate that the combination of primary homogenization with high-energy methods, such as microfluidization, significantly reduces particle size [46] (Linares et al., 2018). However, the application of a single high-energy homogenization technique (ultrahomogenizer) in the present study yielded particles of slightly smaller size compared to those obtained by Bortoluzzi et al. [45], who employed two homogenization cycles.\u003c/p\u003e\n\u003cp\u003eParticle distribution uniformity is indicated by the PDI, which has values between 0.0 and 1.0 [47]. UN exhibited the lowest index among all nanoemulsions tested, reaching a value of 0.489. This outcome consistently highlighted the crucial role of tween 80, echoing Tsichlis et al. [48] research on using tween 80 and span 80 in developing nanoparticles for quercetin incorporation. It was observed that a lower polydispersity index in the emulsion containing Tween 80. The authors reported that nanosystems with Span 80 required stabilization with phytantriol, unlike Tween 80, which was able to stabilize the systems independently.\u003c/p\u003e\n\u003cp\u003eThe stability of nanoemulsions during storage can be compromised by phenomena such as creaming and sedimentation [49]. Abreu et al. [12] encapsulated B4AM with an Alg matrix, and the CI was 4% after 28 days of storage, continuing to show satisfactory indices. These data are similar to those found in our study, which, even with a chalcone concentration twice as high and an observation period extended to 49 days (CI = 2%), continued to maintain satisfactory indices. This confirms that Alg serves as an effective encapsulating matrix due to its electrostatic stabilization capacity within the nanoemulsion, provided by the carboxylate (–COO) and hydroxyl (–OH) groups in its structure, as reported by Artiga-Artigas et al. [50]. The sedimentation values for CNAlg were also satisfactory, presenting sedimentation index (SI) of 1% on day 21, with no signs of instability by the final evaluation day. In contrast, CNGA showed an IC of 2% throughout the entire observation period. The instability results were significantly greater for the formulation lacking a biopolymer matrix (UN) compared to those that included matrices. The pronounced formation of creaming observed can be attributed to the lower density of the nanoemulsion droplets relative to the surrounding liquid, which facilitates the upward migration of the particles [51].\u003c/p\u003e\n\u003cp\u003eFor the evaluation of anthelmintic activity, EHT was chosen due to its recognition in the literature as a well-established screening method for natural products [52]. Additionally, the development of an anthelmintic compound with ovicidal effect is highly relevant, as it can prevent the development of the nematode \u003cem\u003eH. contortus\u003c/em\u003e until the infective larval stage (L3). Efficacy on eggs can also result in a reduction in pasture contamination and livestock infections, contributing to sustainable helminth control strategies [53]. The ovicidal efficacy of the B4AM probably can be attributed to chemical structure, in particular the presence of the highly reactive amine group (H₂N) linked to the aromatic ring. Nitrogen is an electronegative element that can induce a destabilization of the aromatic ring and facilitate its penetration into the \u003cem\u003eH. contortus\u003c/em\u003e eggshell. Additionally, the double bonds present in the compound structure act as active sites, contributing to the increase in chemical reactivity and enhancing the ovicidal effect, something that generally does not occur with structures of compounds with longer chains. In the study by Kozlowska et al. [54], the effect of the structure of the 18 amino-chalcones on biological activity was revealed, where the presence of an amino group in the meta position with the addition of the aromatic ring in the compound increased the hydrophobicity facilitating the chalcone penetration. Regarding the nanoemulsions CNAlg and UN, they exhibited the highest efficacy, in contrast to CNGA, which, besides failing to present satisfactory results in physicochemical characterization, also did not enhance the ovicidal effect. The ovicidal effect of the polymer-free formulation (UN) was superior (EC50 = 0.02 mg/mL) compared to the other formulations in the study. A similar outcome was observed by Aguiar et al. [22], who demonstrated that a biopolymer-free nanoemulsion containing carvone exhibited greater ovicidal efficacy against \u003cem\u003eH. contortus\u003c/em\u003e eggs compared with emulsions that utilized Alg. Additionally, a reduced particle size was noted, which likely facilitated improved penetration into the eggshell, thereby promoting a more effective ovicidal effect. The presence of the sodium alginate matrix as an encapsulating agent in the polymeric system of the CNAlg may have influenced the controlled release process in the aqueous medium of EHT, prolonging the release of the encapsulated chalcone, 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 [55].\u003c/p\u003e\n\u003cp\u003eThe images of \u003cem\u003eH. contortus\u003c/em\u003e eggs, obtained through SEM following treatment with the formulations, revealed alterations suggesting the potential mechanisms of action. Chalcones typically exhibit a crystalline morphology [56] and deposits of this crystalline structure were observed on the \u003cem\u003eH. contortus\u003c/em\u003e eggshell (figure 4a). This finding corroborates the study by Zarza-Albarrán et al. [30], who, when evaluating organic fractions of galloyl flavonoids from \u003cem\u003eAcacia farnesiana\u003c/em\u003e on \u003cem\u003eH. contortus\u003c/em\u003e eggs, reported the presence of granular structures attached to the eggshell. For CNAlg (figure 4b), although the oval morphology was not entirely compromised, the SEM microphotographs revealed cracks that may have weakened the eggshell, hindering hatching. This effect may be associated with the viscosity of Alg, which likely resulted in a slower release of B4AM. In the case of CNGA (figure 4c), residues of gum arabic were more prominently observed in the microphotographs. Conversely, the UN (figure 4d) formulation exhibited more pronounced damage to the eggshell morphology, highlighting its greater efficacy in the EHT.\u003c/p\u003e\n\u003cp\u003eCytotoxicity refers\u0026nbsp;to the potential of a substance to induce cellular damage or death, assessed through various methods, such as the MTT assay [57]. B4AM exhibited no toxicity. The non-toxicity of chalcones is widely documented in the literature. Studies show that administering high doses of chalcones to animals results in little or no toxicity [58-60]. There are also toxicity reports indicating the safety of both natural and synthetic chalcones in mice and plants [61,62]. \u003cem\u003eIn vitro\u003c/em\u003e assays have demonstrated no or limited cytotoxic effects at medium to high micromolar concentrations, depending on the type of chalcone substituent and the tested cell line [63-65]. Turani et al. [66] (2024) evaluated methoxylated chalcones in the adult stage of the model nematode \u003cem\u003eCaenorhabditis elegans\u003c/em\u003e and found that, in cytotoxicity tests with human embryonic kidney cells (HEK-293), no toxicity was detected. Nascimento et al. [67] evaluated various emulsification techniques to optimize the properties of chalcone nanoemulsions with antifungal potential. They observed that the chalcone (1E,4E)-1,5-bis (4-methoxyphenyl) penta-1,4-dien-3-one (DB\u003csub\u003e4\u003c/sub\u003eOCH\u003csub\u003e3\u003c/sub\u003e) exhibited an IC50 above 150 µg/mL, indicating toxicological safety in the MTT assay. However, significant differences in IC50 values were noted between the nanoformulations and the free form of DB\u003csub\u003e4\u003c/sub\u003eOCH\u003csub\u003e3\u003c/sub\u003e. The authors suggest that these variations may be attributed to nanoparticle size and the increase in specific surface area, which enhance interactions with cellular components.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn our study, we observed no cytotoxicity from B4AM or any of the tested nanoemulsions. These findings underscore the importance of optimizing the nanoparticulate system to reduce toxicity compared to the free compound, thereby improving cell culture conditions in the MTT assay. Notably, we used only the ultrasonic device to standardize the emulsification method in this study.\u003c/p\u003e\n\u003cp\u003eOver a 50-hour period, the release kinetics described the release of chalcone from the three formulations with the best efficacy in the EHT. The results indicate that nanoemulsions (CNAlg and UN) serve as effective vehicles for enhancing drug solubility, facilitating homogeneous dispersion, and significantly increasing drug release. This is evidenced by the \u003cem\u003ein vitro\u003c/em\u003e release profile of the UN and CNAlg nanoemulsions. The UN nanoemulsion released over 96% of chalcone after 30 hours, whereas CNAlg achieved a release exceeding 34% in the same period. In comparison, free B4AM exhibited less than 26% release. The kinetic analysis revealed that the Higuchi model was the most suitable to describe the release profile of CNAlg and B4AM suggesting that the release is controlled by diffusion through the matrix and that the drug concentration is initially uniform, decreasing over time. UN fit well in the zero-order release, assuming a constant release rate, regardless of how much drug remains in the system. Emulsification enhanced the solubility of B4AM, enabling a faster release of chalcone. In comparison, free B4AM, due to its low solubility in water, was unable to release more chalcone. It was discovered that incorporating Alg as a matrix was crucial for regulating the release of B4AM in the EHT's aqueous setting. Similar results were reported by Nascimento et al. [67], who encapsulated chalcone DB4OCH3 and observed controlled release with the use of an Alg matrix. UN released the chalcone more rapidly than the free form. Therefore, emulsification increased the solubility of the chalcone and enabled a more efficient release in a shorter time interval, probably because B4AM reached its solubility limit in water, no longer releasing the chalcone. Additionally, the small droplet size of the nanoemulsions provided a large surface area, which enhanced the dissolution rate of the active ingredient, releasing the drug consistently and in a controlled manner over time. Therefore, emulsification enhanced the solubility of chalcone, allowing for a more efficient release over a shorter period, which was not observed in the free B4AM formulation, likely due to reaching its solubility limit in water, preventing further drug release.\u0026nbsp;\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, CNAlg, in addition to enhancing the physicochemical properties of B4AM, demonstrated the ability to improve chalcone solubilization and exhibited significant anti-helminthic efficacy. The ovicidal effect in the presence of Alg was comparable to that of the pure drug, with the added benefits of increased stability and prolonged action. The UN nanoemulsion showed excellent efficacy against eggs, superior to that of free B4AM, suggesting only minor adjustments in concentration and application intervals may be needed. These findings indicate that the use of nanoemulsions, with or without polymeric matrices, offers promising strategies for ovicidal control, with application possibilities tailored to the desired action profile. However, it is necessary to evaluate its performance in other stages of the life cycle of \u003cem\u003eH. contortus\u003c/em\u003e, aiming at a more comprehensive understanding of their potential as new anthelmintic agents.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMr. Barbosa has received a doctoral research scholarship from Ceará Foundation for Support of Scientific and Technological Development (FUNCAP). The authors would like to thank the Central Analítica-UFC/CT-INFRA/MCT-SISANO/Pró Equipamentos.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding sources\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHélcio Silva dos Santos received financial support from CNPq-PQ (Grant 306008/2022-0) and FUNCAPUNIVERSAL (Grant UNI-0210-00337.01.00/23). Flavia Oliveira Monteiro da Silva Abreu received financial support from CNPq (Grant 406522/2021-9-1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available on request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMcKune S, Serra R, Tour\u0026eacute; A (2021) Gender and intersectional analysis of livestock vaccine value chains in Kaffrine, Senegal. PLoS ONE 16(7):e0252045. doi: 10.1371/journal.pone.0252045\u003c/li\u003e\n\u003cli\u003eChagas ACS, Tupy O, Santos IB, Esteves SN, et al. (2022) Economic impact of gastrointestinal nematodes in Morada Nova sheep in Brazil. Rev Bras Parasitol Vet 31(3): e008722. doi: 10.1590/S1984-29612022044\u003c/li\u003e\n\u003cli\u003eParvin S, Dey AR, Shohana NN, et al. (2024) \u003cem\u003eHaemonchus contortus\u003c/em\u003e, an obligatory haematophagus worm infection in small ruminants: Population genetics and genetic diversity. Saudi J Biol Sci 31(8):104030. doi: 10.1016/j.sjbs.2024.104030\u003c/li\u003e\n\u003cli\u003eAntonopoulos A, Higgins O, Doyle SR, et al. (2024) Real-time single-base specific detection of the \u003cem\u003eHaemonchus contortus\u003c/em\u003e S168T variant associated with levamisole resistance using loop-primer endonuclease cleavage loop-mediated isothermal amplification. Mol Cell Probes 73:101946. doi: 10.1016/j.mcp.2023.101946\u003c/li\u003e\n\u003cli\u003ePava LDA, Flores-Jim\u0026eacute;nez NG, Cu\u0026eacute;llar-Ordaz JA, et al. (2024) Exploring alternative anthelmintic compounds: Impact of peruvin, hentriacontane/1-nonacosanol and their synergistic effect on the health of \u003cem\u003eMeriones unguiculatus\u003c/em\u003e infected with \u003cem\u003eHaemonchus contortus\u003c/em\u003e. Vet Parasitol 332:110303. doi: 10.1016/j.vetpar.2024.110303\u003c/li\u003e\n\u003cli\u003eFu Y, Liu D, Zeng H, et al. (2020) New chalcone derivatives: synthesis, antiviral activity and mechanism of action. RSC Adv 10(41):24483-24490. doi: 10.1039/D0RA03684F\u003c/li\u003e\n\u003cli\u003eDan W, Dai J (2020) Recent developments of chalcones as potential antibacterial agents in medicinal chemistry. Eur J Med Chem 187:111980. doi: 10.1016/j.ejmech.2019.111980\u003c/li\u003e\n\u003cli\u003eRocha JE, Freitas TS, Xavier JC, et al. (2021) Antibacterial and antibiotic modifying activity, ADMET study and molecular docking of synthetic chalcone (E)-1-(2-hydroxyphenyl)-3-(2,4-dimethoxy-3-methylphenyl)prop-2-en-1-one in strains of \u003cem\u003eStaphylococcus aureus\u003c/em\u003e carrying NorA and MepA efflux pumps. Biomed Pharmacother 140:111768. doi: 10.1016/j.biopha.2021.111768\u003c/li\u003e\n\u003cli\u003eOkolo EN, Ugwu DI, Ezema BE, et al. (2021) New chalcone derivatives as potential antimicrobial and antioxidant agent. Sci Rep 11:21781. doi: 10.1038/s41598-021-01292-5\u003c/li\u003e\n\u003cli\u003eZhu M, Wang J, Xie J, et al. (2018) Design, synthesis, and evaluation of chalcone analogues incorporate \u0026alpha;,\u0026beta;-Unsaturated ketone functionality as anti-lung cancer agents via evoking ROS to induce pyroptosis. Eur J Med Chem 157:1395-1405. doi: 10.1016/j.ejmech.2018.08.072\u003c/li\u003e\n\u003cli\u003eZhou Q, Tang X, Chen S, et al. (2022) Design, Synthesis, and Antifungal Activity of Novel Chalcone Derivatives Containing a Piperazine Fragment. Agric Environ Chem 70(4): 1029\u0026ndash;1036. doi: 10.1021/acs.jafc.1c05933\u003c/li\u003e\n\u003cli\u003eAbreu FOMS, Holanda T, Nascimento JF, et al. (2024) Evaluation of the antibacterial and antifungal capacity of nanoemulsions loaded with synthetic chalcone derivatives Di-benzyl cinnamaldehyde and Benzyl 4-aminochalcone. Renew Mater 12(2):285\u0026ndash;304. doi: 10.32604/jrm.2023.043919\u003c/li\u003e\n\u003cli\u003eBezerra LL, Almeida-Neto WQ, Marinho MM, et al. (2023) Synthesis of aminochalcones and \u003cem\u003ein silico\u003c/em\u003e evaluation of their antiparasitic potential against \u003cem\u003eLeishmania\u003c/em\u003e. J Biomol Struct Dyn 41(13):6434-6441. doi: 10.1080/07391102.2022.2103030\u003c/li\u003e\n\u003cli\u003eOuattara M, Sissouma D, Kon\u0026eacute; MW, et al. (2011) Synthesis and anthelmintic activity of some hybrid Benzimidazolyl-chalcone derivatives. Trop J Pharm Res 10(6):767-775. doi: 10.4314/tjpr.v10i6.10\u003c/li\u003e\n\u003cli\u003eSissouma D, Ouattara M, Kon\u0026eacute; MW, et al. (2011) Synthesis and \u003cem\u003ein vitro\u003c/em\u003e nematicidal activity of new chalcones vectorised by imidazopyridine. Afr J Pharm Pharmacol 5(18):2086-2093. doi: 10.5897/AJPP11.550\u003c/li\u003e\n\u003cli\u003eV\u0026aacute;zquez-Bravo J, Aguilar-Marcelino L, Casta\u0026ntilde;eda-Ram\u0026iacute;rez GS, et al. (2020) \u003cem\u003eIn vitro\u003c/em\u003e nematicidal activity of two ferrocenyl chalcones against larvae of \u003cem\u003eHaemonchus contortus\u003c/em\u003e (L3) and \u003cem\u003eNacobbus aberrans\u003c/em\u003e (J2). J Helminthol 94:e190. doi: 10.1017/S0022149X2000070X\u003c/li\u003e\n\u003cli\u003eNikolic I, Lunter DJ, Randjelovic D, et al. (2018) Curcumin-loaded low-energy nanoemulsions as a prototype of multifunctional vehicles for different administration routes: Physicochemical and \u003cem\u003ein vitro\u003c/em\u003e peculiarities important for dermal application. Int J Pharm 550(1-2):333-346. doi: 10.1016/j.ijpharm.2018.08.060\u003c/li\u003e\n\u003cli\u003ePreeti, Sambhakar S, Malik R, et al. (2023) Nanoemulsion: An emerging novel technology for improving the bioavailability of drugs. Scientifica 2023(1). doi: 10.1155/2023/6640103\u003c/li\u003e\n\u003cli\u003eRhein-Knudsen N, Ale MT, Ajalloueian F, et al. (2017) Characterization of alginates from Ghanaian brown seaweeds: \u003cem\u003eSargassum\u003c/em\u003e spp. and \u003cem\u003ePadina\u003c/em\u003e spp. Food Hydrocoll 71:236-244. doi: 10.1016/j.foodhyd.2017.05.016\u003c/li\u003e\n\u003cli\u003eRaj V, Kim Y, Kim YG, et al. (2022) Chitosan-gum arabic embedded alizarin nanocarriers inhibit biofilm formation of multispecies microorganisms. Carbohydr Polym 284:118959. doi: 10.1016/j.carbpol.2021.118959\u003c/li\u003e\n\u003cli\u003eAndr\u0026eacute; WPP, Junior JRP, Cavalcante GS, et al. (2020) Anthelmintic activity of nanoencapsulated carvacryl acetate against gastrointestinal nematodes of sheep and its toxicity in rodents. Rev Bras Parasitol Vet 29(1):e013119. doi: 10.1590/S1984-29612019098\u003c/li\u003e\n\u003cli\u003eAguiar AARM, Ara\u0026uacute;jo-Filho JV, Pinheiro HN, et al. (2022) \u003cem\u003eIn vitro\u003c/em\u003e anthelmintic activity of an R-carvone nanoemulsions towards multiresistant \u003cem\u003eHaemonchus contortus\u003c/em\u003e. Parasitoloy 149(12):1631-1641. doi: 10.1017/S0031182022001135\u003c/li\u003e\n\u003cli\u003eIrfan R., Mousavi S., Meshari A, et al. (2020) A Comprehensive Review of Aminochalcones. Molecules 25(22):5381. doi: 10.3390/molecules25225381 \u003c/li\u003e\n\u003cli\u003eRomeu MC, Freire PTC, Ayala AP, et al (2022) Synthesis, crystal structure, ATR-FTIR, FT-Raman and UV spectra, structural and spectroscopic analysis of (3E)‐4‐[4‐(dimethylamine)phenyl]but‐3‐en‐2‐one. J Mol Struct 1264:133222. doi: 10.1016/j.molstruc.2022.133222\u003c/li\u003e\n\u003cli\u003eMwangi WW, Ho KW, Tey BT, et al. (2016) Effects of environmental factors on the physical stability of pickering-emulsions stabilized by chitosan particles. Food Hydrocoll 60:543\u0026ndash;550. doi: 10.1016/j.foodhyd.2016.04.023\u003c/li\u003e\n\u003cli\u003eHubert J, Kerboeuf D. (1992) A microlarval development assay for the detection of anthelmintic resistance in sheep nematodes. Vet Rec 130(20):442\u0026ndash;448. doi:10.1136/vr.130.20.442\u003c/li\u003e\n\u003cli\u003eNeveu C, Charvet C, Fauvin A, et al. (2007) Identification of levamisole resistance markers in the parasitic nematode \u003cem\u003eHaemonchus contortus\u003c/em\u003e using a cDNA-AFLP approach. Parasitology 134(8):1105\u0026ndash;1110. doi: 10.1017/S0031182007000030\u003c/li\u003e\n\u003cli\u003eFauvin A, Charvet C, Issouf M, et al. (2010) cDNA-AFLP analysis in levamisole-resistant \u003cem\u003eHaemonchus contortus\u003c/em\u003e reveals alternative splicing in a nicotinic acetylcholine receptor subunit. Mol Biochem Parasitol 170(2):105\u0026ndash;107. doi: 10.1016/j.molbiopara.2009.11.007\u003c/li\u003e\n\u003cli\u003eColes GC, Jackson F, Pomroy WE, et al. (2006) The detection of anthelmintic resistance in nematodes of veterinary importance. Vet Parasitol 136(3\u0026ndash;4):167\u0026ndash;185. doi: 10.1016/j.vetpar.2005.11.019\u003c/li\u003e\n\u003cli\u003eZarza-Albarr\u0026aacute;n MA, Olmedo-Ju\u0026aacute;rez A, Rojo-Rubio R, et al. (2020) Galloyl flavonoids from \u003cem\u003eAcacia farnesiana\u003c/em\u003e pods possess potent anthelmintic activity against \u003cem\u003eHaemonchus contortus\u003c/em\u003e eggs and infective larvae. J Ethnopharmacol. 249:112402. doi: 10.1016/j.jep.2019.112402\u003c/li\u003e\n\u003cli\u003eMosmann T (1983) Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assay. J Immunol Methods 65(1-2):55-63. doi: 10.1016/0022-1759(83)90303-4\u003c/li\u003e\n\u003cli\u003eCosta P (2002) \u003cem\u003eIn vitro\u003c/em\u003e evaluation of the lyoequivalence of pharmaceutical formulations. Braz J Pharm Sci 38(2):141\u0026ndash;153. doi:10.1590/S1516-93322002000200003\u003c/li\u003e\n\u003cli\u003eRai VK, Mishra N, Yadav KS, et al. (2018) Nanoemulsion as pharmaceutical carrier for dermal and transdermal drug delivery: Formulation development, stability issues, basic considerations and applications. J Control Release 270:203\u0026ndash;225. doi: 10.1016/j.jconrel.2017.11.049\u003c/li\u003e\n\u003cli\u003eHong IK, Kim SI, Lee SB (2018) Effects of HLB value on oil-in-water emulsions: Droplet size, rheological behavior, zeta-potential, and creaming index. J Ind Eng Chem 67:123\u0026ndash;131. doi: 10.1016/j.jiec.2018.06.022\u003c/li\u003e\n\u003cli\u003eGuttoff M, Saberi AH, McClements (2015) DJ Formation of vitamin D nanoemulsion-based delivery systems by spontaneous emulsification: Factors affecting particle size and stability. Food Chem 171:117\u0026ndash;122. doi: 10.1016/j.foodchem.2014.08.087 \u003c/li\u003e\n\u003cli\u003eSalvia-Trujillo L, Rojas-Gra\u0026uuml; MA, Soliva-Fortuny R, et al. (2013) Effect of processing parameters on physicochemical characteristics of microfluidized lemongrass essential oil-alginate nanoemulsions. Food Hydrocoll 30(1):401\u0026ndash;407. doi: 10.1016/j.foodhyd.2012.07.004\u003c/li\u003e\n\u003cli\u003eAbreu FOMS, Holanda T, Nascimento JF, et al. (2024) Evaluation of the antibacterial and antifungal capacity of nanoemulsions loaded with synthetic chalcone derivatives Di-Benzyl Cinnamaldehyde and Benzyl 4-Aminochalcone. J Renew Mater 12(2):285\u0026ndash;304. doi: 10.32604/jrm.2023.043919\u003c/li\u003e\n\u003cli\u003eWinter E, Pizzol CD, Locatelli C, et al. (2014) \u003cem\u003eIn vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e effects of free and chalcones-loaded nanoemulsions: Insights and challenges in targeted cancer Chemotherapies. Int J Environ Res Public Health 11(10): 10016-10035. doi: 10.3390/ijerph111010016\u003c/li\u003e\n\u003cli\u003eAbreu FOMS, Nascimento JF, Pinheiro HN, et al. (2024) Formulation of nanoemulsions enriched with chalcone-based compounds: formulation process, physical stability and antimicrobial effect. Polym Bull 81:7367-7391. doi: 10.1007/s00289-023-05069w\u003c/li\u003e\n\u003cli\u003eGago C, Guerreiro A, Souza M, et al. (2024) Effectiveness of Sodium Alginate and Carnauba Wax Nanoemulsions with Lemongrass Essential Oil on the quality of \u0026lsquo;Hass\u0026rsquo; Avocado Fruit from early, middle, and late harvest season during prolonged cold storage. Sci Hortic 333:113237. doi: 10.1016/j.scienta.2024.113237\u003c/li\u003e\n\u003cli\u003eHonary S, Zahir F (2013) Effect of Zeta Potential on the Properties of Nano-Drug Delivery Systems - A Review (Part 2). Trop J Pharm Res 12(2):265-273. doi: 10.4314/tjpr.v12i2.20\u003c/li\u003e\n\u003cli\u003eLiu Y, Wei ZC, Deng YY, et al. (2020) Comparison of the Effects of Different Food-Grade Emulsifiers on the Properties and Stability of a Casein-Maltodextrin-Soybean Oil Compound Emulsion. Molecules 25(3):458. doi: 10.3390/molecules25030458\u003c/li\u003e\n\u003cli\u003eHosseini SMH, Emam-Djomeh Z, Razavi SH, et al. (2013) \u0026beta;-Lactoglobulin\u0026ndash;sodium alginate interaction as affected by polysaccharide depolymerization using high intensity ultrasound. Food Hydrocoll 32(2):235-244. doi: 10.1016/j.foodhyd.2013.01.002\u003c/li\u003e\n\u003cli\u003eSalvia-Trujillo L, Decker EA, McClements DJ (2016) Influence of an anionic polysaccharide on the physical and oxidative stability of omega-3 nanoemulsions: Antioxidant effects of alginate. Food Hydrocoll 52:690-698. doi: 10.1016/j.foodhyd.2015.07.035\u003c/li\u003e\n\u003cli\u003eBortoluzzi BB, Buzatti A, Chaaban A, et al. (2021) \u003cem\u003eMentha villosa\u003c/em\u003e Hubs., \u003cem\u003eM.\u003c/em\u003e x \u003cem\u003epiperita\u003c/em\u003e and their bioactives against gastrointestinal nematodes of ruminants and the potential as drug enhancers. Vet Parasitol 289:109317. doi: 10.1016/j.vetpar.2020.109317\u003c/li\u003e\n\u003cli\u003eLlinares R, Santos J, Trujillo-Cayado LA, et al. (2018) Enhancing rosemary oil-in-water microfluidized nanoemulsion properties through formulation optimization by response surface methodology. LWT 97:370-375. doi: 10.1016/j.lwt.2018.07.033\u003c/li\u003e\n\u003cli\u003eIskandar B, Mei HC, Liu TW, et al. (2024) Evaluating the effects of surfactant types on the properties and stability of oil-in-water Rhodiola rosea nanoemulsion. Colloids Surf B Biointerfaces 234:113692. doi: 10.1016/j.colsurfb.2023.113692\u003c/li\u003e\n\u003cli\u003eTsichlis I, Manou AP, Manolopoulou V, et al. (2023) Development of Liposomal and Liquid Crystalline Lipidic Nanoparticles with Non-Ionic Surfactants for Quercetin Incorporation. Materials. 16:5509. doi: 10.3390/ma16165509\u003c/li\u003e\n\u003cli\u003eHassanshahian M, Saadatfar A, Masoumipour F (2020) Formulation and characterization of nanoemulsion from \u003cem\u003eAlhagi maurorum\u003c/em\u003e essential oil and study of its antimicrobial, antibiofilm, and plasmid curing activity against antibiotic-resistant pathogenic bacteria. J Environ Health Sci Eng 18:1015-1027. doi: 10.1007/s40201-020-00523-7\u003c/li\u003e\n\u003cli\u003eArtiga-Artigas M, Fani AA, Mart\u0026iacute;n-Belloso O (2017) Effect of sodium alginate incorporation procedure on the physicochemical properties of nanoemulsions. Food Hydrocoll 70:191-2000. doi: 10.1016/j.foodhyd.2017.04.006\u003c/li\u003e\n\u003cli\u003eMcClements DJ (2010) Emulsion Design to Improve the Delivery of Functional Lipophilic Components. Annu Rev Food Sci Technol 1:241-269. doi: 10.1146/annurev.food.080708.100722\u003c/li\u003e\n\u003cli\u003eBarbosa MLF, Ribeiro WLC, Filho JVA, et al. (2023) \u003cem\u003eIn vitro\u003c/em\u003e anthelmintic activity of \u003cem\u003eLippia alba\u003c/em\u003e essential oil chemotypes against \u003cem\u003eHaemonchus contortus\u003c/em\u003e. Exp Parasitol 244: 108439. doi: 10.1016/j.exppara.2022.108439\u003c/li\u003e\n\u003cli\u003eSantos FO, Lima HGL, Santos NSS, et al. (2017) \u003cem\u003eIn vitro\u003c/em\u003e anthelmintic and cytotoxicity activities the \u003cem\u003eDigitaria insularis\u003c/em\u003e (Poaceae). Vet Parasitol 245:48-54. doi: 10.1016/j.vetpar.2017.08.007\u003c/li\u003e\n\u003cli\u003eKozłowska J, Potaniec B, Baczyńska D, et al (2019) Synthesis and Biological Evaluation of Novel Aminochalcones as Potential Anticancer and Antimicrobial Agents. Molecules 24(22):4129. doi: 10.3390/molecules24224129\u003c/li\u003e\n\u003cli\u003eDash M., Chiellini F, Ottenbrite RM, et al. (2011) Chitosan\u0026mdash;A versatile semi-synthetic polymer in biomedical applications. Prog Polym Sci 36(8):981-1014. doi: 10.1016/j.progpolymsci.2011.02.001\u003c/li\u003e\n\u003cli\u003eArshad MN, Al-Dies AAM., Asiri AM, et al. (2017) Synthesis, crystal structures, spectroscopic and nonlinear optical properties of chalcone derivatives: A combined experimental and theoretical study. J Mol Struct 1141:142-156. doi: 10.1016/j.molstruc.2017.03.090\u003c/li\u003e\n\u003cli\u003eAdan A, Kiraz Y, Baran Y. (2016) Cell Proliferation and Cytotoxicity Assays. Curr Pharm Biotechnol 17:1213-1221. doi: 10.2174/1389201017666160808160513\u003c/li\u003e\n\u003cli\u003eMorris M, Zhang S. (2006) Flavonoid\u0026ndash;drug interactions. Effects of flavonoids on ABC transporters. Life Sci 78(18):2116-2130. doi: 10.1016/j.lfs.2005.12.003\u003c/li\u003e\n\u003cli\u003eBandgar BP, Gawande SS, Bodade RG, et al. (2010) Synthesis and biological evaluation of simple methoxylated chalcones as anticancer, anti-inflammatory and antioxidant agents. Bioorg Med Chem 18:1364-1370. doi: 10.1016/j.bmc.2009.11.066Abstract\u003c/li\u003e\n\u003cli\u003eZampini IC, Villena J, Salva S, et al. (2012) Potentiality of standardized extract and isolated flavonoids from \u003cem\u003eZuccagnia punctata\u003c/em\u003e for the treatment of respiratory infections by \u003cem\u003eStreptococcus pneumoniae\u003c/em\u003e: \u003cem\u003eIn vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e studies. Ethnopharmacol 140(2):287- 292. doi: 10.1016/j.jep.2012.01.019\u003c/li\u003e\n\u003cli\u003eGonz\u0026aacute;lez JA, Braun AE. (1998) Effect of (E)-Chalcone on Potato-Cyst Nematodes (\u003cem\u003eGlobodera pallida\u003c/em\u003e and \u003cem\u003eG. rostochiensis\u003c/em\u003e). J Agric Food Chem 46(3):1163-1165. doi: 10.1021/jf9706686\u003c/li\u003e\n\u003cli\u003eCancino K, Castro I, Yauri C. (2021) Toxicity assessment of synthetic chalcones with antileishmanial potential in BALB/c mice. Rev Peru Med Exp Salud P\u0026uacute;blica 38:424-433. doi: 10.17843/rpmesp.2021.383.6937\u003c/li\u003e\n\u003cli\u003eForejtn\u0026iacute;kov\u0026aacute; H, Lunerov\u0026aacute; K, Kub\u0026iacute;nov\u0026aacute; R. (2005) Chemoprotective and toxic potentials of synthetic and natural chalcones and dihydrochalcones \u003cem\u003ein vitro\u003c/em\u003e. Toxicology 208(1):81-93. doi: 10.1016/j.tox.2004.11.011\u003c/li\u003e\n\u003cli\u003eMai CW, Yaeghoobi M, Abd-Rahman N, et al. (2014) Chalcones with electron-withdrawing and electron-donating substituents: anticancer activity against TRAIL resistant cancer cells, structureeactivity relationship analysis and regulation of apoptotic. Eur J Med Chem 77:378-387. doi: 10.1016/j.ejmech.2014.03.002\u003c/li\u003e\n\u003cli\u003eDong N, Liu X, Zhao T, et al. (2018) Apoptosis-inducing effects and growth inhibitory of a novel chalcone, in human hepatic cancer cells and lung cancer cells. Biomed Pharmacother 105:195-203. doi: 10.1016/j.biopha.2018.05.126\u003c/li\u003e\n\u003cli\u003eTurani O, Castro MJ, Vazzana J, et al. (2024) Potent Anthelmintic Activity of Chalcones Synthesized by an Effective Green Approach. ChemMedChem 19(13): e202400071. doi: 10.1002/cmdc.202400071\u003c/li\u003e\n\u003cli\u003eNascimento JF, Abreu FOMS, Holanda T, et al. (2024) Evaluation of Emulsification Techniques to Optimize the Properties of Chalcone Nanoemulsions for Antifungal Applications. Pharmaceuticals 17(11):1442. doi: 10.3390/ph171114\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1. Particle size, zeta potential, polydispersity index and encapsulation efficiency of nanoemulsions CNAlg, CNGA and UN with B4AM.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"567\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNanoemulsions\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eParticle size (nm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eZeta potential (mV)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePolydispersity index\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eEncapsulation efficiency (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCNAlg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e267.64 \u0026plusmn; 3.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e-56.64 \u0026plusmn; 2.81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.751 \u0026plusmn; 0.095\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e87.5 \u0026plusmn; 0.77\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCNGA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e234.3 \u0026plusmn; 6.58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e-58.28 \u0026plusmn; 2.81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.782 \u0026plusmn; 0.065\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e43.8 \u0026plusmn; 0.58\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eUN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e144.56 \u0026plusmn; 1.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e-46.06 \u0026plusmn; 0.95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.489 \u0026plusmn; 0.045\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e69.87 \u0026plusmn; 0.58\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eCNAlg: Sodium alginate-coated B4AM; CNGA: Gum arabic-coated B4AM; UN: Uncoated B4AM\u003c/p\u003e\n\u003cp\u003eTable 2. Kinetic constant (K) and the correlation coefficient (R2) for the kinetic models studied.\u003c/p\u003e\n\u003ctable border=\"1\" cellpadding=\"0\" width=\"595\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eFormulations\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eZero-Order\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eFirst-Order\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eHiguchi\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eKorsmeyer\u0026ndash;Peppas\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eR\u003csup\u003e2\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eK\u003csub\u003e0\u003c/sub\u003e (h\u003csup\u003e\u0026minus;1\u003c/sup\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eR\u003csup\u003e2\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eK\u003csub\u003et\u003c/sub\u003e (h\u003csup\u003e\u0026minus;1\u003c/sup\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eR\u003csup\u003e2\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eK\u003csub\u003eH\u003c/sub\u003e (h\u003csup\u003e\u0026minus;1/2\u003c/sup\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eR\u003csup\u003e2\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eKa\u0026nbsp;(h\u003csup\u003e\u0026minus;n\u003c/sup\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eB4AM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.9534\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.0102\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.8086\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.0003\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.9755\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.6374\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.9709\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.5388\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eCNAlg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.9085\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.013\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.8236\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.0004\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.9508\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.8139\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.9412\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.6492\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eUN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.9461\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.4444\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.3125\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e7E-05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.5988\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.8656\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.7235\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.1737\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eB4AM: Benzyl 4-Aminochalcone; CNAlg: Sodium alginate-coated B4AM; UN: Uncoated B4AM\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","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":"Chalcones, biopolymers, drug delivery, nanotechnology, Haemonchus spp., cytotoxicity","lastPublishedDoi":"10.21203/rs.3.rs-5662121/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5662121/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSmall ruminant farming in developing countries is hindered by gastrointestinal nematodes, such as \u003cem\u003eHaemonchus contortus\u003c/em\u003e causing anemia, leading to weight loss and reduced productivity. The excessive use of synthetic anthelmintics has driven resistance. Therefore, natural compounds are being investigated as alternative control strategies. Hence, the present study investigates the anthelmintic activity and cytotoxicity of Benzyl 4-Aminochalcone (B4AM) and its nanoemulsions. Three nanoemulsions (sodium alginate-coated B4AM (CNAlg), gum arabic-coated B4AM (CNGA) and uncoated B4AM (UN)) were prepared and characterized physicochemically. B4AM and nanoemulsions were evaluated in the egg hatch test (EHT) using \u003cem\u003eH. contortus\u003c/em\u003e and their cytotoxicity was evaluated on murine fibroblasts. The release kinetics of B4AM, CNAlg and UN were studied. The CNAlg and CNGA showed superior visual stability compared to UN. The CNAlg, CNGA and UN presented, respectively, 267.64, 234.3 and 144.56 nm (particle size); -56.64, -58.26 and \u0026minus;\u0026thinsp;46.06 mV (zeta potential); 0.751, 0.782 and 0.049 (polydispersity index). Encapsulation efficiencies were 87.5% (CNAlg), 43.8% (CNGA), and 69.87% (UN). In the EHT, CNAlg and UN were more effective. The effective concentrations to inhibit 50% (EC50) of eggs ranging from 0.02 to 0.10 mg/mL. SEM revealed more pronounced changes in eggs treated with the UN. No cytotoxicity was observed (IC50\u0026thinsp;\u0026gt;\u0026thinsp;120 \u0026micro;g/mL). Kinetics presented a faster release for UN followed by CNAlg and B4AM. CNAlg improved B4AM solubilization, stability and anti-helminthic efficacy, with ovicidal effect comparable to the pure chalcone. Nanoemulsions showed promise for ovicidal control, but further studies are needed to assess their effectiveness in other life stages.\u003c/p\u003e","manuscriptTitle":"Improving the anthelmintic effectiveness of Benzyl 4-Aminochalcone with polysaccharides nanoemulsions","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-19 07:25:07","doi":"10.21203/rs.3.rs-5662121/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":"96e978c1-98e2-4002-9a3f-63fbcc5f313a","owner":[],"postedDate":"December 19th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-06-03T06:54:01+00:00","versionOfRecord":[],"versionCreatedAt":"2024-12-19 07:25:07","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5662121","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5662121","identity":"rs-5662121","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}