Toxoplasma gondii, the etiologic agent of toxoplasmosis, is an obligate intracellular protozoan parasite belonging to the phylum Apicomplexa that has a worldwide distribution and can invade and replicate in all nucleated cells of warm-blooded animals (Matta et al., 2021). Sexual propagation of T. gondii occurs exclusively in intestinal epithelial cells of definitive hosts (species of the Felidae family). The infected host contaminates the environment with feces carrying millions of oocysts that sporulate and become infective in the environment, spreading the infection to a high number of animal species (Kaushik et al., 2014). In the intermediate host, such as humans, the parasite permeates between a replicative state called tachyzoite and a semi-dormant state known as bradyzoite (Matta et al., 2021). This last form presents asynchronous and slow growth, configuring one of the greatest challenges for the treatment of T. gondii infection (Kaushik et al., 2014).

It is estimated that chronic infection by T. gondii affects about one-third of the world's human population and, in specific regions, such as some parts of Africa and Europe, the seroprevalence can reach 90% and 60% of the population, respectively (Molan et al., 2019; Montoya and Liesenfeld, 2004). Although subclinical in most cases, the chronic infection can be reactivated in immunocompromised individuals, leading to a high rate of morbidity and mortality, especially for HIV-infected people with progression to AIDS (Andreani et al., 2012). In spite of the fact that it is typically asymptomatic, the acute infection can cause eye diseases, cervical lymphadenopathy and pneumonia that may lead to death (Demar et al., 2007). Furthermore, latent infections can be locally reactivated in the retina in immunocompetent individuals, causing damage to the eye and a consequent decrease in work power and economic loss (Dunay et al., 2018).

The immune response to T. gondii infection involves, primarily, the activation of type 1 Innate Lymphoid Cells (ILCs), promoting the production of interferon (IFN)-γ and tumor necrosis factor (TNF)-α (Klose and Artis, 2016; Klose et al., 2014). Also, the production of pro-inflammatory cytokines acts on the activation of the adaptive immune response mediated by TCD4 and TCD8 lymphocytes, promoting the release of inflammatory mediators such as nitric oxide (NO), which favors the establishment of a toxic environment for T. gondii (Buzoni-Gatel et al., 2006; Melo et al., 2011; Miller et al., 2009). Additionally, cytokines such as IL-6, IL-8, IL-10 and macrophage migration inhibitory factor (MIF) proved to be important regulators of T. gondii infection (Barbosa et al., 2015; Denney et al., 1999; Zhao et al., 2013).

Currently, the treatment for toxoplasmosis comprises the use of the drug spiramycin, as well as the association of pyrimethamine with sulfadiazine (Khan and Khan, 2018). The use of these gold-standard drugs is not able to eliminate the parasitic infection, and it has been associated with severe side effects in long-term use, such as skin irritation, nausea or vomiting, leukopenia, fever, thrombocytopenia and diarrhea, thus leading to a discontinuation of the treatment in one-third of patients (Dannemann et al., 1992; Katlama et al., 1996; Porter and Sande, 1992; Silva et al., 2019). Despite the existence of alternative therapies such as atovaquone and azithromycin, these still lack clinical data regarding intolerance by patients (Jacobson et al., 2001; Torres et al., 1997). Henceforth, the search for alternative approaches for the treatment of toxoplasmosis that are accessible, effective and safe is mandatory (Alday and Doggett, 2017).

Medicinal plants have been used for centuries to treat numerous diseases (Al Nasr et al., 2016). Plants of the genus Copaifera (Leguminosae family), popularly known as “copaíba”, “copaibeira”, “pau-de-óleo”, “palo-de-balsamo”, are acclaimed for their medicinal properties that include purgative, anticarcinogenic, anti-inflammatory, antibiotic, antiviral, antiparasitic, anesthetic, bactericidal, insecticidal and healing activities (Veiga Junior and Pinto, 2002). Copaifera oleoresin (OR) is a liquid composed of a non-volatile portion (solid phase or resin) comprising mainly diterpene acids, and a volatile oil part consisting of a mixture of sesquiterpenes (Arruda et al., 2019).

Due to their broad pharmacological properties, the ORs are used in Brazilian folk medicine to treat microbial diseases, thus highlighting the genus Copaifera as a promising new source of anti-infective agents (Alves et al., 2020; Furtado et al., 2018; Leandro et al., 2016). Although several published studies have reported the use of the ORs and their isolated molecules to treat infectious diseases, especially Chagas disease, leishmaniosis and malaria (da Trindade et al., 2018), knowledge about a possible anti-Toxoplasma action is still scarce. In this scenario, our research group demonstrated in a pioneering way that ORs and leaf hydroalcoholic extract from Copaifera spp. reduced T. gondii growth in in vitro and ex vivo models at maternal-fetal interface (Martínez et al., 2023; Teixeira et al., 2020). To gain insights into the underlying mechanisms related to the anti-T. gondii effect of these natural compounds, the present study aimed to investigate the activity of four Copaifera ORs (C. duckei, C. paupera, C. pubiflora and C. reticulata), as well as two isolated diterpene acids, ent-kaurenoic acid [ent-kaur-16-en-19-oic acid] and the ent-polyalthic acid [ent-15,16-epoxy-8(17),13(16),14-labdatrien-19-oic acid] regarding their antiparasitic and immune-modulatory activities during T. gondii infection in a distinct experimental model.