**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**

**Denerieth Ximena Espinel-Mesa <sup>1</sup> , Clara Isabel González Rugeles 2, Julio César Mantilla Hernández 3, Elena E. Stashenko <sup>4</sup> , Carlos Andrés Villegas-Lanau 5, John Jaime Quimbaya Ramírez <sup>1</sup> and Liliana Torcoroma García Sánchez 1,\***


**Abstract:** In the late phase of *Trypanosoma cruzi* infection, parasite persistence and an exaggerated immune response accompanied by oxidative stress play a crucial role in the genesis of Chronic Chagasic Cardiomyopathy (CCC). Current treatments (Benznidazole (BNZ) and Nifurtimox) can effect only the elimination of the parasite, but are ineffective for late stage treatment and for preventing heart damage and disease progression. In vivo trypanocidal and cardioprotective activity has been reported for *Lippia alba* essential oils (EOs), ascribed to their two major terpenes, limonene and caryophyllene oxide. To investigate the role of antioxidant and immunomodulatory mechanisms behind these properties, chronic-*T. cruzi*-infected rats were treated with oral synergistic mixtures of the aforementioned EOs. For this purpose, the EOs were optimized through limonene-enrichment fractioning and by the addition of exogenous caryophyllene oxide (LIMOX) and used alone or in combined therapy with subtherapeutic doses of BNZ (LIMOXBNZ). Clinical, toxicity, inflammatory, oxidative, and parasitological (qPCR) parameters were assessed in cardiac tissue. These therapies demonstrated meaningful antioxidant and immunomodulatory activity on markers involved in CCC pathogenesis (IFN-γ, TNF-α, IL-4, IL-10, and iNOS), which could explain their significant trypanocidal properties and their noteworthy role in preventing, and even reversing, the progression of cardiac damage in chronic Chagas disease.

**Keywords:** chronic Chagas disease; *Trypanosoma cruzi*; antioxidant; immunomodulation; immunohistochemistry; essential oils; *Lippia alba*; limonene; caryophyllene oxide
