Desenvolvimento de uma formulação pour on da associação do óleo essencial de Citrus aurantium var amara L. com ivermectina para o controle de nematódeos gastrointestinais

Abstract

Gastrointestinal nematodes (GIN) significantly impact the livestock economy by compromising cattle health and are traditionally controlled with synthetic anthelmintics. However, the indiscriminate use of these drugs has led to increased parasite resistance, making it necessary to develop new strategies to optimize their efficacy. The essential oil of Citrus aurantium var. amara (OECa) may have a synergistic effect against parasites and enhance the transdermal permeation of drugs. Thus, this study evaluated the anthelmintic efficacy of the combination of OECa with ivermectin (IVM), both in vitro and in vivo, against GIN in naturally infected cattle. The study included the analysis of OECa by gas chromatography-mass spectrometry (GC-MS). Anthelmintic activity was evaluated in vitro using the larval development test (LDT), exposing L1 larvae to different concentrations of the oil, ivermectin, and their combination. For the in vivo tests, a pour- on formulation containing essential oil and ivermectin was developed, using vegetable sunflower oil as an excipient. The formulation's stability was assessed at different temperatures. Finally, anthelmintic efficacy was analyzed in naturally infected cattle through the fecal egg count reduction test (FECRT). Statistical analyses were performed by calculating means and standard deviations. Chemical analysis identified limonene (59.94%) and carvone (4.93%) as the main constituents of the oil. The in vitro LDT assay showed that OECa had a median inhibitory concentration (IC50) of 0.554 ± 0.006 mg/mL, while ivermectin alone had an IC50 of 2.432 ± 0.053 ng/mL. The combination of the compounds demonstrated synergy, with a Synergy Score (SS) of 14.71 and a Combination Sensitivity Score (CSS) of 65.58. A pour-on formulation combining OECa with IVM was developed using vegetable sunflower oil as an excipient, remaining stable for three months at different temperatures (5 °C, 25 °C, and 37 °C), without phase separation or degradation, and with a constant pH (5.0). In the in vivo bioassay, the formulation containing the combination of OECa and IVM reduced egg excretion in feces by 40.60%. However, when evaluating different nematode genera, OECa alone was highly effective against Haemonchus spp. (93%, 95% CI: 90–96), while its combination with ivermectin resulted in an 88% reduction (95% CI: 84–93). For Cooperia spp., the combination showed lower efficacy (20%, 95% CI: 14–27) compared to ivermectin alone (40%, 95% CI: 29–51). Similarly, against Oesophagostomum spp., the combination had a modest effect (9%, 95% CI: 6–11), while IVM alone was more effective (51%, 95% CI: 48–55). The results indicate that OECa has promising anthelmintic activity, especially against Haemonchus spp., and may act synergistically with ivermectin. However, its in vivo efficacy against other genera was limited. Further research is needed to optimize future formulations and explore its potential in anthelmintic therapy.