**1. Introduction**

Chagas disease is a parasitosis caused by *Trypanosoma cruzi* that affects approximately 10 million people worldwide, of whom around 30% will eventually develop organomegaly of the digestive tract or heart [1]. Dilated cardiomyopathy, or Chagas heart disease (CHD), is the most relevant manifestation of this infection during its chronic phase [1], making it the most prevalent cardiac infection in the world, and causing a significant public health

**Citation:** Espinel-Mesa, D.X.; González Rugeles, C.I.; Mantilla Hernández, J.C.; Stashenko, E.E.; Villegas-Lanau, C.A.; Quimbaya Ramírez, J.J.; García Sánchez, L.T. Immunomodulation and Antioxidant Activities as Possible Trypanocidal and Cardioprotective Mechanisms of Major Terpenes from *Lippia alba* Essential Oils in an Experimental Model of Chronic Chagas Disease. *Antioxidants* **2021**, *10*, 1851. https:// doi.org/10.3390/antiox10111851

Academic Editor: Serge Ankri

Received: 16 October 2021 Accepted: 15 November 2021 Published: 22 November 2021

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problem in Latin American countries where it is endemic [2]. Because of the silent course of this parasitosis and the late appearance of its symptoms (usually 8–10 years after acquiring the parasite, and without any other pathognomonic signs), CHD can be confused with other etiologies, delaying timely diagnosis [3]. These factors combined have contributed to CHD being presently considered a neglected and high-cost disease, with an average cost of care for chronic cases estimated at USD \$44,955 per person [4].

Progression of the disease is characterized by the persistence of the parasite in smooth muscle, and is associated with an exacerbated immune response and a permanent oxidative environment. Research indicates that these conditions together contribute to tissue, neurological, and microvasculature injury; with a consequent deterioration of contractile capacity and dilation of muscle tissue; which in turn can culminate in an eventual loss of the organ's physiological function and even result in death [5]. Current treatments available for therapeutic intervention against this infection are based on the use of two nitro-heterocyclic compounds: nitrofuran Nifurtimox (NFX) and nitroimidazole Benznidazole (BNZ). These compounds, well-established for more than 50 years as the conventional therapy against *T. cruzi* infection, have been found to exhibit limited trypanocidal activity (between 50–80% in the acute phase, and 8–20% in the chronic) [6], with high toxicity due to non-selective oxidative damage.

In general terms, patients assigned therapies based on these drugs demonstrate low treatment adherence due to the presence of multiple adverse side effects (mainly severe anorexia, digestive intolerance, and neurological disorders), as well as long treatment times; all characteristics of therapeutic regimens with high rates of treatment abandonment [7]. In response, regulatory entities such as the US Food and Drug Administration (FDA) have not approved the use of NFX in human therapy, thus limiting the available prophylactic and therapeutic options to BNZ, alone, for all clinical phases of Chagas disease.

Recent studies have reported interesting in vitro trypanocidal, antioxidant, and immunomodulatory activity for fractions derived from *Lippia alba* (mill.) N.E. Brown essential oils (EOs) rich in terpenes such as limonene, citral, and caryophyllene oxide [8,9]. In addition, trypanocidal and cardioprotective qualities have also been ascribed to these oils in animals with chronic *T. cruzi* infection [10]. The aim of the present work is to assess, in an animal model of chronic Chagas disease, potential immunomodulatory and antioxidant properties as possible mechanisms for the trypanocidal and cardioprotective activity observed for these compounds. In order to accomplish this, synergistic mixtures were generated by combining a limonene-rich fraction of *L. alba* EOs with added exogenous caryophyllene oxide (LIMOX); or by the interaction of this LIMOX with subtherapeutic doses of BNZ (LIMOXBNZ). These experimental therapies were used in a daily, oral scheme of 31 doses on a murine model (Wistar rats) infected with chronic *T. cruzi*, in which CCC had been induced.

The effect of the treatments was determined through evaluation of the clinical progression of heart disease (biochemical and morphological parameters), the trypanocidal efficacy (qPCR), and by immunohistochemical analysis of the cytokine profiles relevant to the immune response against the parasite (TNF-α, IFN-γ, IL-10, and IL-4). Likewise, a marker of oxidative stress (iNOS) was also measured. The phytotherapeutics studied showed promising data for a therapy that could be used as an adjuvant to current treatments (BNZ). Such a novel therapy would be based on standardized production technologies, with Good Agricultural Practices and environmentally sustainable EOs extracted from *L. alba*, a wild shrub from the Colombian Andean Region.
