*3.3. Cardioprotective Activity*

Following the completion of the treatment regimens (at 98 d.a.i.), cardiac effects were evaluated by echocardiography, recording the changes in the cardiac silhouette (maximum length or ML and maximum diameter or MD) at the third ultrasound analysis. Over this time interval, the results obtained showed no significant variations in either parameter for animals belonging to the negative control group. In contrast, animals infected and left untreated (positive control) and those infected and treated with BNZ (reference group) exhibited a significant increase in MD (*p* = 0.0156 and *p* = 0.0050, respectively). In fact, the animals assigned to these two groups were the only ones which demonstrated effects upon clinical parameters; exhibiting decay, dehydration, piloerection, and brown discharge in the conjunctiva (a stress indicator). In contrast, a reversion of both cardiac measures (ML and MD) to values similar to those found in uninfected subjects (negative control, *p* > 0.05) were evidenced in those infected animals which received the LIMOX and LIMOXBNZ therapies (with statistically significant differences when compared to the positive control, *p* < 0.05) (Figure 1).


**Figure 1.** Echocardiography of Wistar rats infected with *Trypanosoma cruzi*. (**a**) Cardiac ultrasound maximum length start and end of treatment; (**b**) Cardiac ultrasound maximum diameter start and end of treatment; (**c**) Cardiac silhouette measurements via ultrasound (maximum length or ML and maximum diameter or MD) at the first (I), second (II), and third (III) echocardiography: (I) one day prior to infection; (II) at the start date of the therapies; and (III) at the end date of the treatments; *p* # compared with negative control measure from ultrasound II; *p* & compared with negative control measure from ultrasound III; \* statistically significant difference at *p* < 0.05; Negative: untreated and uninfected animals; Positive: untreated and infected animals; LIMOX: infected animals treated with a mixture of an essential oil fraction of *L. alba* carvone chemotype enriched in limonene (68.9 mg/kg/day) and with added caryophyllene oxide (Sigma-Aldrich) (70 mg/kg/day); LIMOXBNZ: infected animals treated with LIMOX plus benznidazole (7.9 mg/kg/day); BNZ: infected animals treated with benznidazole (100 mg/kg/day). Data are representative of six independent experiments and values are expressed in mean ± SEM.

Post mortem macroscopic and microscopic (histopathology) heart observations are presented in Table 3 and Figure 2. The successful induction of the experimental cardiomyopathy model was confirmed by bulging and dilated forms, especially in the left ventricles, of the hearts in the positive control group (observed in 100% of the animals). Consistently, in these animals, histopathology evidenced: multiple foci of inflammatory infiltrate with cell diversity (predominantly lymphocytes, plasma cells, and histiocytes); damage to the myocardial tissue, the atria-ventricle junction sites, and the neurons of the cardiac plexus [10,15].

**Figure 2.** Macro and microscopic findings in the hearts of Wistar rats infected with *Trypanosoma cruzi*. Histological images taken using a 40× objective on Hematoxylin and Eosin stained tissue sections. Negative: untreated, uninfected animals; Positive: untreated, infected animals; LIMOX: infected animals treated with an essential oil fraction of *Lippia alba* carvone chemotype enriched in limonene (68.9 mg/kg/day) and with added caryophyllene oxide (Sigma-Aldrich) (70 mg/kg/day); LIMOXBNZ: infected animals treated with LIMOX and benznidazole (7.9 mg/kg/day); BNZ: infected animals treated with benznidazole (100 mg/kg/day). (**A**–**C**): Normal heart tissue. (**D**). Large focal inflammatory infiltrate in myocardium with lymphocytes, histiocytes, and plasma cells. (**E**). Aggregate

of cardiac plexus neurons, surrounded by lymphocytic infiltrate. (**F**). Multifocal infiltrate with minimal angiogenesis and loose, elongated, and sinusoidal fibers with fibrotic process. (**G**). Small histiocytic inflammatory infiltrate. (**H**). Apparent reparative process. (**I**). Lymphocytic inflammatory infiltrate with minimal fibrosis. (**J**). Loose and elongated fibers, with histiocytic lymphocyte inflammatory infiltrate. (**K**). Mild lymphohistiocytic infiltrate. (**L**). Diffuse linear inflammatory infiltrate, with minimal fibrosis. (**M**) Focal of inflammatory infiltrate in plasma lymphocytoid, loose and elongated fibers. (**N**). Foci of lymphocytic inflammatory infiltrate; loose, elongated fibers. (**O**). Focal of inflammatory infiltrate of lymphocytes and plasma cells; dilated, loose, and elongated fibers with a sinuous appearance; with fibrosis. Figures are representative of six independent experiments.


**Table 3.** Macroscopic and microscopic findings in the hearts of Wistar rats after receiving treatments.

RW: Relative weight; SEM: standard error of the mean; ̇: mean; Negative: untreated, uninfected animals; hLIMOX-Control: not infected animals treated with higher doses of an essential oil fraction of *Lippia alba* carvone chemotype enriched in limonene (170 mg/kg/day) and with added caryophyllene oxide (Sigma-Aldrich) (70 mg/kg/day); Positive: untreated, infected animals; LIMOX: infected animals treated with an essential oil fraction of *L. alba* carvone chemotype enriched in limonene (68.9 mg/kg/day) and with added caryophyllene oxide (Sigma-Aldrich) (70 mg/kg/day); LIMOXBNZ: infected animals treated with LIMOX and benznidazole (7.9 mg/kg/day); BNZ: infected animals treated with benznidazole (100 mg/kg/day). Data are representative of six independent experiments and values are expressed in mean ± SEM.

> In infected animals submitted to the array of treatments, the best performance in terms of protection against cardiac damage induced by the *T. cruzi* infection was observed in LIMOX treated animals, followed by LIMOXBNZ (Table 3 and Figure 2). In contrast, among the experimental groups, major effects upon the heart structure were apparent in the BNZ group, whose animals presented macroscopic and microscopic features very similar to infected and untreated rats (positive control).
