Resveratrol and Curcumin for Chagas Disease Treatment—A Systematic Review

Chagas disease (CD) is a neglected protozoan infection caused by Trypanosoma cruzi, which affects about 7 million people worldwide. There are two available drugs in therapeutics, however, they lack effectiveness for the chronic stage—characterized mainly by cardiac (i.e., cardiomyopathy) and digestive manifestations (i.e., megaesophagus, megacolon). Due to the involvement of the immuno-inflammatory pathways in the disease’s progress, compounds exhibiting antioxidant and anti-inflammatory activity seem to be effective for controlling some clinical manifestations, mainly in the chronic phase. Resveratrol (RVT) and curcumin (CUR) are natural compounds with potent antioxidant and anti-inflammatory properties and their cardioprotective effect have been proposed to have benefits to treat CD. Such effects could decrease or block the progression of the disease’s severity. The purpose of this systematic review is to analyze the effectiveness of RVT and CUR in animal and clinical research for the treatment of CD. The study was performed according to PRISMA guidelines and it was registered on PROSPERO (CDR42021293495). The results did not find any clinical study, and the animal research was analyzed according to the SYRCLES risk of bias tools and ARRIVE 2.0 guidelines. We found 9 eligible reports in this study. We also discuss the potential RVT and CUR derivatives for the treatment of CD as well.


Introduction
Carlos Chagas was the researcher who first described the Chagas Disease (CD) in 1909. CD, also named American Trypanosomiasis, is an infectious disease caused by the flagellated protozoan Tryparnosoma cruzi (T. cruzi). Until the year of 2021, no effective and safe treatment focusing on the cure was described, and this neglected disease is now considered a great Health Problem, mainly for some developing countries [1][2][3].
According to the WHO (2022), there are about 6-7 million people infected with T. cruzi, worldwide and 75 million are at risk of getting sick [3]. Although most severe cases occur in Latin America, globalization has contributed to the spread of the disease worldwide. Thus, it is not uncommon to find reported cases in developed countries, such as the USA, Canada, and Japan, bringing for those countries concerns that were previously in infected mongrel dogs, even though the parasitemia was not reduced, a protective effect on the left ventricle ejection fraction, diastolic end diameter, and mass index was shown. Moreover, an increase in the expression of the IL-10 messenger RNA was observed, whereas the proinflammatory cytokine IFN-γ was detected only in infected and untreated animals [30]. The improvement of the cardiac condition lead to a clinical trial of atorvastatin (NCT04984616) [31] and other anti-inflammatory nutrients, such as selenium (NCT00875173) [32] and omega-3 (NCT01863576) [33], demonstrating a new therapeutic focus in the treatment of Chagas disease.
The cardioprotective effect of RVT may also involve the increase of nitric oxide (NO) production by the increase of NO synthase (eNOS) expression in endothelium cells. This is caused by the overexpression of SIRT-1 [70][71][72][73], which prevents the uncoupling of eNOS, leading to decreasing of superoxide (ROS) production under pathological conditions [73]. In addition, the cardioprotective activity of RVT is associated with the modulation of the composition of gut microbiota which can alter the profile of the host metabolite involved in cardiovascular health [74] and can modulate the biological circadian rhythm [75,76].
Curcumin (CUR) is a natural polyphenol [1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6heptadiene-3,5-dione] found in the rhizome of Curcuma longa (turmeric) and in others Curcuma spp. The compound presents several biological activities, including antioxidant and anti-inflammatory, by inhibiting ROS, NF-κβ, TNF-α, and inflammatory cytokines [77,78]. CUR interferes in epigenetic pathways through the inhibition of p300/CREBspecific acetyltransferase, which leads to the repression of the acetylation of histone/nonhistone proteins [79]. CUR, as well as RVT, is considered a pan inhibitor, acting on several therapeutic targets, which can explain its effectiveness in a large range of diseases, mainly in their chronic phase [80,81]. It has been shown to prevent and reverse cardiac hypertrophy and failure in animal models [82,83].
Based on the cardioprotective, antioxidant, and anti-inflammatory effect of RVT and CUR, both of these natural compounds have been tested against T.cruzi activity and the protection of cardiomyocytes from damage. As the several attempts to treat CCC with trypanocide drugs have produced inconsistent results, despite reductions in parasite load, the purpose of this work was to search clinical and animal studies of RVT and CUR for the treatment of CD. We set out to answer the following review questions:  Are RVT and CUR trypanocide agents?  Are there any RVT and CUR benefits in in vivo-infected animals with T cruzi which can support the clinical study?
The cardioprotective effect of RVT may also involve the increase of nitric oxide (NO) production by the increase of NO synthase (eNOS) expression in endothelium cells. This is caused by the overexpression of SIRT-1 [70][71][72][73], which prevents the uncoupling of eNOS, leading to decreasing of superoxide (ROS) production under pathological conditions [73]. In addition, the cardioprotective activity of RVT is associated with the modulation of the composition of gut microbiota which can alter the profile of the host metabolite involved in cardiovascular health [74] and can modulate the biological circadian rhythm [75,76].
Curcumin (CUR) is a natural polyphenol [1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6heptadiene-3,5-dione] found in the rhizome of Curcuma longa (turmeric) and in others Curcuma spp. The compound presents several biological activities, including antioxidant and anti-inflammatory, by inhibiting ROS, NF-κβ, TNF-α, and inflammatory cytokines [77,78]. CUR interferes in epigenetic pathways through the inhibition of p300/CREBspecific acetyltransferase, which leads to the repression of the acetylation of histone/nonhistone proteins [79]. CUR, as well as RVT, is considered a pan inhibitor, acting on several therapeutic targets, which can explain its effectiveness in a large range of diseases, mainly in their chronic phase [80,81]. It has been shown to prevent and reverse cardiac hypertrophy and failure in animal models [82,83].
Based on the cardioprotective, antioxidant, and anti-inflammatory effect of RVT and CUR, both of these natural compounds have been tested against T.cruzi activity and the protection of cardiomyocytes from damage. As the several attempts to treat CCC with trypanocide drugs have produced inconsistent results, despite reductions in parasite load, the purpose of this work was to search clinical and animal studies of RVT and CUR for the treatment of CD. We set out to answer the following review questions:  Are RVT and CUR trypanocide agents?  Are there any RVT and CUR benefits in in vivo-infected animals with T cruzi which can support the clinical study?
Are there any RVT and CUR benefits in in vivo-infected animals with T. cruzi which can support the clinical study?
In addition, this work aimed to review derivatives of RVT and CUR as potential antichagasic compounds.

Literature Search
We have searched for the animal and human studies based on literature published until 30 October 2021 in databanks Pubmed/Medline, Embase, Lilacs, Cochrane Library, and Clinical.trials.gov, using Medical Subject Healing (MESH) terms for Chagas disease, Trypanosoma cruzi, resveratrol, curcumin, antioxidant, and anti-inflammatory. The review was performed according to PRISMA guidelines [84] and was registered in PROSPERO (CDR42021293495). In addition, this review includes potential resveratrol and curcumin derivatives proposed for the treatment of CD.
Inclusion criteria: All animal research (male or female, strains of T. cruzi, and stage of the disease), clinical trials, case reports, all stages of CD with the intervention of resveratrol and/or curcumin (oral or i.p administration, before or after infection in any time). Only English language was included in the study.
Exclusion criteria: No RVT or CUR intervention, in vitro, ex vivo RVT or CUR intervention, in silico and genetic experiments, comments, editorials, posters, letters, notes, and reviews were excluded.
Data extraction: Three independent groups of two authors extracted information from the selected articles and, if needed, the differences were resolved by another author. The extracted data included type of studies, and important results and references. The primary outcome was the decrease of parasitemia, and the second was the improvement of heart and digestive function and anti-inflammatory activity.
Primary outcome: parasitemia (blood or organs parasite load) and/or animal survival. Second outcome: effects on the organs (heart, brain, liver, esophagus, colon, and others). Inflammation and/or oxidative markers.
Data and bias analysis: Joanna Brigs Appraisal Critical and Cochrane bias guidelines, and SYRCLE's risk of bias [85] and ARRIVE 2.0 guideline tools [86,87] for animal studies were used and classified for quality as green (good), yellow (fair), and red (bad). The risk of bias was classified as yes, no, and unclear. If yes, it was classified as low, fair, or high risk.

Literature Search and Study Selection
The literature search in Pubmed/Medline, Embase, Lilacs, Cochrane Library, and Clinical.trials.gov, using the MESH terms anti-inflammatory, antioxidant, Chagas Disease, and Trypanosoma cruzi, found 2180 reports, including 9 clinical trials. The advanced search choosing more specific terms such as resveratrol, curcumin, Chagas disease, and Trypanosoma cruzi found 114 reports, of which 63 were excluded due to duplicity and 38 were excluded for having no RVT or CUR intervention or no infection with the parasite, and 4 were excluded because two were reviews and other two showed only in vitro and ex vivo experiments. After exclusion and re-searching in February 2022, 9 records were included in the study, as shown in the flowchart in Figure 1. Tables 1 and 2 show the results of the selected reports. No clinical study was found and almost all included records were conducted with no randomization or blindness. This is not an experimental practice in animal models. However, it is an important point to evaluate avoiding discrepancies in assessing the methodological quality and bias [85]. In view of this, the works without animal randomization and blindness were considered as half orange in color classification of bias/quality.        Primary outcome: RVT has no effect on parasitemia over 8 dpi. Secondary outcome:  brains (cerebral cortex) at 8 dpi: RVT decrease ROS levels compared to control (p < 0.05).  RVT alone or in combination with BZN did not affect lipid peroxidation (TBARS levels) in infected animals (p < 0.05) Primary outcome: RVT has no effect on parasitemia over 8 dpi. Secondary outcome:  brains (cerebral cortex) at 8 dpi: RVT decrease ROS levels compared to control (p < 0.05).  RVT alone or in combination with BZN did not affect lipid peroxidation (TBARS levels) in infected animals (p < 0.05) cardiomyopathy at 60 dpi  RVT: short term treatment (20 h) did not present benefits on cardiac function  RVT did not significantly alter the number of invading inflammatory cells infiltrating the heart, heart vascularization, or collagen content Primary outcome: at 7 dpi: RVT has no influence on parasitemia (trypomastigote forms) Secondary outcomes:  RVT did not revert lipid peroxidation caused by the infection and did not modulate the oxidative stress nor exert effects in antioxidant enzymes  RVT decrease SOD activity in infected animals  RVT reverses the lower GST in infected animals  RVT can downregulate inflammatory response stimulating the expression of NOx Q: quality were classified as green (good), yellow (fair), and red (bad) according to ARRIVE 2.0 guideline tools [82,83]. B: The risk of bias was classified as yes, no, and unclear. If yes, it was classified as low (green), fair (orange), or high (red) risk according to SYRCLE's risk of bias [81]. Half orange: no randomization and blinding. Primary outcome: at 7 dpi: RVT has no influence on parasitemia (trypomastigote forms) Secondary outcomes:  RVT did not revert lipid peroxidation caused by the infection and did not modulate the oxidative stress nor exert effects in antioxidant enzymes  RVT decrease SOD activity in infected animals  RVT reverses the lower GST in infected animals  RVT can downregulate inflammatory response stimulating the expression of NOx Q: quality were classified as green (good), yellow (fair), and red (bad) according to ARRIVE 2.0 guideline tools [82,83]. B: The risk of bias was classified as yes, no, and unclear. If yes, it was classified as low (green), fair (orange), or high (red) risk according to SYRCLE's risk of bias [81]. Half orange: no randomization and blinding.    RVT reverses the lower GST in infected animals  RVT can downregulate inflammatory response stimulating the expression of NOx Q: quality were classified as green (good), yellow (fair), and red (bad) according to ARRIVE 2.0 guideline tools [82,83]. B: The risk of bias was classified as yes, no, and unclear. If yes, it was classified as low (green), fair (orange), or high (red) risk according to SYRCLE's risk of bias [81]. Half orange: no randomization and blinding.  RVT reverses the lower GST in infected animals  RVT can downregulate inflammatory response stimulating the expression of NOx Q: quality were classified as green (good), yellow (fair), and red (bad) according to ARRIVE 2.0 guideline tools [82,83]. B: The risk of bias was classified as yes, no, and unclear. If yes, it was classified as low (green), fair (orange), or high (red) risk according to SYRCLE's risk of bias [81]. Half orange: no randomization and blinding.      • significant reduction of inflammation of myocardial arteries: observed by the significant decrease of the inflammatory cell infiltration of heart vessels (histologically analyzed and scored), vascular permeability, and IL-6 and TNF-α mRNA levels in total heart extracts by CUR. Primary outcome: at 130 dpi survival: heart tissue parasitemia: T. cruzi load was 4.39-fold (p < 0.01) higher in the myocardium from mice administered with PBS than in those treated with BZ Cur therapy had no significant effect on cardiac parasitism and did not limit BZ parasiticidal activity. Secondary outcome: at 130 dpi  CK activity: Chronic Chagas mice receiving BZ or CUR showed lower (p < 0.01) CK activity than that recorded in untreated animals.  BZN + CUR + 16-fold decrease in circulating CK values (p < 0.001 vs. untreated infected mice).  CUR reduce circulating ANP levels alone or in combination with BZN. BZN alone does not revert ANP serum elevation  CUR reduce intensity of long-term inflammation in myocardium (IL-1β, TNF-α, IL-6, and CCL5) but not by BZN (exception for TNF-α)  BZN is unable to impair leukocyte influx  CUR + BZN enhances cardioprotective effect Q: quality were classified as green (good), yellow (fair), and red (bad) according to ARRIVE 2.0 guideline tools [82,83]. B: The risk of bias was classified as yes, no, and unclear. If yes, it was classified as low (green), fair (orange), or high (red) risk according to SYRCLE's risk of bias [81]. Half orange: no randomization and blinding. Primary outcome: parasitemia 14 dpi: CUR very little effect on parasitemia profile compared to non-treated animals Secondary outcome:  significant reduction of inflammation of myocardial arteries: observed by the significant decrease of the inflammatory cell infiltration of heart vessels (histologically analyzed and scored), vascular permeability, and IL-6 and TNF-α mRNA levels in total heart extracts by CUR.
Hernandez et al. 2021 [96] C57BL/6 mice female and male mice (eight weeks old) 10,000 Brazil strain (DTU I, routinely maintained by serial subinoculation in C3HeJ mice at threeweek intervals) Nano formulated Cur preparations (size range, 250-300 nm) contained 0.15 mg CUR per mg of polymer, suspended in an aqueous solution of 1% wt/vol sodium carboxymethylcellulose and administered orally by gavage (0.15 mL) once a day. Treatment for 30 consecutive days, starting on day 60 of infection.
Primary outcome: at 130 dpi survival: heart tissue parasitemia: T. cruzi load was 4.39-fold (p < 0.01) higher in the myocardium from mice administered with PBS than in those treated with BZ Cur therapy had no significant effect on cardiac parasitism and did not limit BZ parasiticidal activity. Secondary outcome: at 130 dpi  CK activity: Chronic Chagas mice receiving BZ or CUR showed lower (p < 0.01) CK activity than that recorded in untreated animals.  BZN + CUR + 16-fold decrease in circulating CK values (p < 0.001 vs. untreated infected mice).  CUR reduce circulating ANP levels alone or in combination with BZN. BZN alone does not revert ANP serum elevation  CUR reduce intensity of long-term inflammation in myocardium (IL-1β, TNF-α, IL-6, and CCL5) but not by BZN (exception for TNF-α)  BZN is unable to impair leukocyte influx  CUR + BZN enhances cardioprotective effect Q: quality were classified as green (good), yellow (fair), and red (bad) according to ARRIVE 2.0 guideline tools [82,83]. B: The risk of bias was classified as yes, no, and unclear. If yes, it was classified as low (green), fair (orange), or high (red) risk according to SYRCLE's risk of bias [81]. Half orange: no randomization and blinding.

Discussion
Natural products have been used in Medicinal Chemistry as hits and/or leads to be optimized with the aim to discover new agents for many different diseases [97]. This occurs also with neglected tropical diseases, such as Chagas disease [1]. Therefore, biodiversity, that is mainly strictly related to medicinal plants, has been a source of compounds that could turn into drug candidates [98]. Searching for a mechanism of RVT action, in 2012, Vera and colleagues [99] performed docking with arginine kinase, a possible target from T. cruzi, using 24 polyphenolic compounds and 18 arginine analogues downloaded from the ZINC database. From those compounds, RVT was chosen for additional tests, considering its ligand efficiency for the binding site of arginine kinase. This compound inhibits 50% of the recombinant arginine kinase activity at the concentration of 325 µM. The IC 50 observed in T. cruzi trypomastigotes-infected CHOK1 cells was 77 µM. Despite its low activity, this compound showed to be promising due to its lack of toxicity and its accessibility, as it is commercially available with a low price. In addition, the selectivity of its target, as it is not found in mammalian hosts, led to its possible use against T. cruzi.
Despite RVT showing trypanocide in vitro activity, based on the results, the RVT failed to decrease T. cruzi parasitemia in the acute phase of Chagas disease in vivo. We found four reports involving treatment with RVT in mice infected with T. cruzi and only three have parasitemia data [88][89][90][91]. Two of them were the same group of research and showed no significant parasitemia load difference at 8 days post-infection (dpi) with the RVT, 100 mg/kg via gavage, or the control [90,91]. Even though RVT has no trypanocidal activity in vivo in the acute phase, it was shown to decrease ROS in the brain [90] and liver [91], protecting those organs against the inflammatory aggression which was promoted as a defense response from the infection by the parasite.
Vilar et al. [88] established the chronic parasitemia with the Colombian I strain of T. cruzi (60 dpi) and started a 30-day treatment with 15 mg/kg (ip) or 40 mg/kg (per os) of RVT. At 90 dpi, they showed a decrease of around 90% of the heart tissue parasitemia observed, as detected by quantitative PCR, without altering the number of inflammatory cells infiltrating the heart, heart vascularization, or collagen content. They showed that infected mice presented normal ECG profiles after RVT treatment, 15/47 (31%), while the vehicle (VEH) presented cardiac alterations such as sinus arrhythmia, and atrial and/or atrioventricular conduction disorders in 48/48 (100%, p < 0.0001). RVT also restored the ejection fraction showing an improvement in the stroke volume and cardiac output compared to the VEH. In addition, RVT activated AMPK phosphorylation and reduced oxidative stress in the heart. These results were not observed in a shorter time of treatment (20 h) or with a lower dose (5 mg/kg).
In contrast, Wan and co-workers [89] reported that RVT-treated infected animals (90-111 dpi) exhibited a moderate (up to 20%) improvement in the end systolic volume (ESV), stroke volume (SV), and cardiac output (CO) induced by the parasite infection and statistically insignificant improvement in the ejection fraction (EF) and fractional shortening (FS). In addition, RVT exhibited modest control of the left ventricular mass and no improvement in the inter-ventricular septum (IVS), LV posterior wall (LVPW) thickness, or LV area. These results could have occurred because the bias of the treatment chosen by the authors (20 mg/mL in drinking water) did not measure the amount of water drunk by the animal during the time of treatment, making it difficult to control the right concentration given to the animal. In addition, the solubility of RVT in water is very low (0.03 mg/mL) [100]. It was not considered by the authors that the concentration used was probably insoluble and, with the time, the RVT could be precipitated, suggesting an insufficient dose of RVT and a low response. The authors also tested a small SIRT agonist molecule (SIRT1720) which was not RVT-structure related and showed a better response compared to RVT. However, the administration route was different and, because the experiment was not blind, the possibility of bias in this experiment could be increased. CUR, (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione (commonly called diferuloylmethane), the other natural bioactive compound, is also a natural phenolic an-tioxidant, free radical scavenger, acting on the release of superoxide radicals, on nitric oxide in immune cells, and as an inhibitor of lipoperoxidation [101]. Therefore, it is estimated that this phenolic compound acts on modulating signaling molecules, transcription factors, besides some important enzymes, such as protein kinases and protein reductases related to cardiovascular diseases [101].
Five reports were included involving CUR treatment and most of them carried out in vivo and in vitro experiments [92][93][94][95][96]. We collected only the in vivo results. The most common problem found was the quality of the CUR from 65% up to 80% purity by High Performance Liquid Chromatography (HPLC). CUR solution preparation was different or not informed by the authors.
In 2012, Nagajyothi et al. [92] tested the inhibitory effect of CUR on the parasite invasion. The results showed that CUR can decrease the heart parasitemia load compared to the control at 23 dpi and increase the survival rate (100% against 60% in non-treated animals). The authors reported 35 days of CUR treatment (100 mg/kg, orally) and 23 days of infection, suggesting 8 days of pretreatment or a mistake, that was not solved, as we tried to contact the authors with no success. The experiments also showed very good results in decreasing inflammatory markers (heart and liver), suggesting the protection of infection damage. The CUR used was presented as having a >65% purity and there was no information about the vehicle used.
Novaes et al. [93] performed two different experiments: first, the CUR (100 mg/kg) alone and in association with benznidazol (BZN, 50 and 100 mg/kg), the currently used drug; the second, the same groups with 3 immunosuppression cycles with cyclophosphamide to evaluate infection recrudescence or cure. The results showed that CUR can decrease parasitemia (decrease of parasitemia with BZN100 + CUR > BZN50 + CUR > BZN 100 > BZN 50 >> CUR compared to untreated animals). CUR improved survival, decreasing 25% of mortality (at 24 dpi) against 58.33% of untreated animals after 20 days of treatment. The negative parasitemia animals were selected to perform the recrudescence experiment. Recrudescence after immunosuppression occurred in CUR (75%), BZN50 (66,67%), BZN 100 (25%), but was not observed when CUR was associated to BZN (with both doses, 50 and 100 mg/kg). This methodology is very important to show real antichagasic efficacy on the parasitemia load after treatment, as the immunosuppressor can be reactive, dormant, or in a non/low-replicating (latent) form during the intracellular cycle of T. cruzi, improving the quality of the work. The authors also reported a significant reduction (38%) of macrophage infiltration and a large decrease of the heart and liver inflammation markers, such as TNF-α (8000 ↓), at 20 dpi. The purity of the CUR used was not informed and it was suspended in an aqueous solution of 1% carboxymetylcellulose.
The Hernandez's group conducted the other three researches involving CUR [90][91][92]. In the 2016 [94] and 2018 [95] experiments, CUR presented a purity ≥ 94% for curcuminoids and ≥80% for curcumin (by HPLC), dissolved in corn oil. In the first publication [94], Hernandez et al. treated infected mice (T. cruzi RA strain) with different concentrations of CUR (25,50, and 100 mg/kg) for 35 days. The results showed 100% survival with 100 mg/kg (but not 25 or 50 mg/kg) and 100 mg/kg BZN against 55% of untreated infected mice after 35 dpi. Despite this, the cardiac parasitemia burden was not modified with CUR (all doses) at 21 dpi compared to the non-treated animals observed by quantitative PCR analysis. The second outcome, despite the parasitism, a significant inflammatory process of attenuation was shown in the heart tissue with CUR 100 treatment, analyzed by leukocyte infiltration, cyclooxygenase-2 (COX-2), microsomal prostaglandin E synthase-1 (mPGES-1), and B-type natriuretic peptide (BNP) mRNA expression to a normal (non-infected animal) level. The antichagasic BZN showed almost a 100% decrease on heart parasitism at 21 dpi. However, the inflammation was not attenuated and showed similar results to the infected and non-treated animals.
In 2018, Hernandez et al. [95] performed the infection of the mice with the T. cruzi Tulahuen strain for 14 days. The treatment with CUR (100 mg/kg) or BZN (100 mg/kg) showed that CUR had no effect on decreasing the bloodstream parasite burden, while BZN presented an antiparasitic effect. Even though CUR had no antiparasitic effect, 100% survival was shown after 14 days of infection and there was a significant reduction in the inflammation of the myocardial arteries, decreasing the inflammatory cell infiltration of the heart vessels (histologically analyzed and scored), vascular permeability, and IL-6 and TNF-α mRNA levels in the total heart extracts, compared to BZN and non-treated animals. The histological analysis of the heart was double-blind, conducted of randomized slices.
Based on the poor solubility of CUR that hinders the bioavailability and the lack of drugs for the chronic phase of CD, Hernandez et al., in 2021 [96], conducted the experiment in the chronic phase with 200 mg/kg of CUR nanoparticules (nano-CUR) for 30 days, starting at 60 dpi, comparing with BZN in a suboptimal dose (25 mg/kg), at 130 dpi. The survival level was not reported and the parasitemia load was detected only in the myocardium, which was shown to be 4.39-fold higher in infected animals and nano-CUR, compared to those treated by BZN, suggesting that nano-CUR has no antiparasitic activity. However, lower CK (creatine kinase) activity was observed, which was not reverted by the current therapeutic drug, BZN. CK is also known as creatine phosphokinase (CPK), a very important protein marker for myocardium damage [102]. The effect of CUR in combination with BZN decreases the level of CK circulating by about 16-fold compared to untreated animals. The authors found an intense reduction of the long-term inflammation in the myocardium, observed by downloading the inflammatory marker levels (IL-1β, TNF-α, IL-6, and CCL5, and heart histopathological analysis with CUR, but not BZN, with the exception of TNF-α). The tissue collagen deposition was decreased in CUR, and was not changed with BZN, but it was more effective when used in combination with the latter drug. These results are very consistent in showing the cardiac benefits of CUR plus BZN [96].

Limitations of the Study
The limitation of the study was the low number of experimental studies in vivo (9 reports), performed with different experimental strains, animals, phases of the disease, period of treatment, and doses, making the comparison difficult. None of works have been executed with randomization/blinding and were considered as half orange. Due to the low solubility of both of the compounds, we found problems with the RVT concentrations and vehicles used for improving CUR that was not uniform. Additionally, the purity of CUR varied with the experiment (found by the informed CAS number), from ≥65% CUR (no total curcuminoids were informed) to ≥94% of curcuminoids and ≥80% of CUR, that can increase the bias of the study.

RVT and CUR Derivatives
The absorption, distribution, metabolism, and excretion (ADME) properties are one of the most important barriers that both RVT and CUR found decreased their effectivity. In spite of their lipophilic nature, they showed a low bioavailability due to poor absorption, a fast metabolism, and elimination [103,104]. Several derivatives were synthetized to improve RSV and CUR physicochemical (poor solubility) and pharmacokinetics (poor bioavailability) properties. Most of the compounds were studied for anticancer activity. The methoxylated, hydroxylated, and halogenated RSV derivatives were obtained and showed beneficial biological effects and a potential increased oral bioavailability [99]. None of the compounds were tested for Chaga's disease, however, some of them were reported to be beneficial for cardiovascular diseases (Figure 2), such as pteorstilbene (1), piceatannol (2), dihydroxystilbene (3), or DHS and a tetramethoxylated derivative (4), DMS 212 or TMS.
Pterostilbene (1) is a natural dimethoxylated analogue of RVT (trans-3,5-dimethoxy-4 -hidroxystilbene) which is reported to have similar activities, including cardioprotective activity [105] and decreasing cardiac oxidative stress [106]. Pterostilbene is 80% bioavailable against 20% of RVT and at concentrations of 1 and 3 uM it prevents myocardiac hypoxia/reperfusion (H/R) or ischemia-reperfusion (IR), and induces H 9c2 apoptosis, against the dose of 20 uM observed by RVT, suggesting it might be more potent than RVT [107]. A docking experiment revealed that pterostilbene interacts with Cys482 and Arg 466 of the active pocket of SIRT-1,2 and its effect is abolished by pretreatment with the SIRT-1 antagonist, the splitomicin [108]. Pterostilbene (1) is a natural dimethoxylated analogue of RVT (trans-3,5-dimethoxy-4′-hidroxystilbene) which is reported to have similar activities, including cardioprotective activity [105] and decreasing cardiac oxidative stress [106]. Pterostilbene is 80% bioavailable against 20% of RVT and at concentrations of 1 and 3 uM it prevents myocardiac hypoxia/reperfusion (H/R) or ischemia-reperfusion (IR), and induces H 9c2 apoptosis, against the dose of 20 uM observed by RVT, suggesting it might be more potent than RVT [107]. A docking experiment revealed that pterostilbene interacts with Cys482 and Arg 466 of the active pocket of SIRT-1,2 and its effect is abolished by pretreatment with the SIRT-1 antagonist, the splitomicin [108].
Piceatannol (2), is a metabolite of RVT from cytochrome P4501B1 which has similar RVT activity [109], including various cardioprotective effects [110] such as the prevention of cardiac arrythmia, ischemia/reperfusion (I/H) injury in rats, delaying sodium ion current inactivation, showing to be more potent than RVT, strengthening the effective refractor period elongating the action potential in the cardiomyocytes [111]. The study by Wang et al. (2019) showed that (2) can protect the heart tissue from peroxidative injury by the upregulation of PI3K-Akt-eNOS (phosphoinositide 3 kinase-protein kinaseendothelial nitric oxide synthase) signaling [112]. Coppa et al. (2011) reported the possible cardioprotective activity mechanism of the DHS (3), a dihydroxylate derivative of RVT. They found that (3) can inhibit the secretion of endothelin-1 mRNA expression, a vascular tension regulator, and decrease the endothelin-converting enzyme-1mRNA levels, a protein involved in the proteolytic processing of endothelin-1, suggesting that the cardioprotective element is independent of their antioxidant activity [113].
A tetrahydroxylmethylated derivative of RVT (4) named DMS 212 or TMS (trans-3,4,5,4′-tetramethoxystilbene) is a more soluble derivative, presenting faster absorption than RVT because of the presence of one more hydroxy group [105]. Liu et al. [114] showed that TMS can prevent cardiovascular diseases by remodeling H/R induced in pulmonary Piceatannol (2), is a metabolite of RVT from cytochrome P4501B1 which has similar RVT activity [109], including various cardioprotective effects [110] such as the prevention of cardiac arrythmia, ischemia/reperfusion (I/H) injury in rats, delaying sodium ion current inactivation, showing to be more potent than RVT, strengthening the effective refractor period elongating the action potential in the cardiomyocytes [111]. The study by Wang et al. (2019) showed that (2) can protect the heart tissue from peroxidative injury by the upregulation of PI3K-Akt-eNOS (phosphoinositide 3 kinase-protein kinase-endothelial nitric oxide synthase) signaling [112]. Coppa et al. (2011) reported the possible cardioprotective activity mechanism of the DHS (3), a dihydroxylate derivative of RVT. They found that (3) can inhibit the secretion of endothelin-1 mRNA expression, a vascular tension regulator, and decrease the endothelin-converting enzyme-1mRNA levels, a protein involved in the proteolytic processing of endothelin-1, suggesting that the cardioprotective element is independent of their antioxidant activity [113].
A tetrahydroxylmethylated derivative of RVT (4) named DMS 212 or TMS (trans-3,4,5,4 -tetramethoxystilbene) is a more soluble derivative, presenting faster absorption than RVT because of the presence of one more hydroxy group [105]. Liu et al. [114] showed that TMS can prevent cardiovascular diseases by remodeling H/R induced in pulmonary hypertensive rats through the inhibition of NOX/VPO1 pathway-mediated oxidative stress and the inflammatory reaction.
hypertensive rats through the inhibition of NOX/VPO1 pathway-mediated oxidative stress and the inflammatory reaction.

Final Remarks
In summary, the limitation of the study was the low number of experimental research in vivo (9 reports), performed with different experimental strains, animals, phases of the disease, periods of treatment, and doses, which made the conclusion difficult. Most of the works have not been performed with randomization/blinding and were considered as half orange. Due to the low solubility of RVT, we found problems with the concentrations and, with CUR, the problem of different purities, which can increase the bias in the study. The studies showed that RVT did not present antiparasitic activity in the acute or chronic phase of Chagas disease in mice. Despite only four studies being found, they showed beneficial effects for the heart, liver, and brain of the infected mice. The reports focusing on CUR showed antiparasitic activity, however, it was not superior to BZN, the current therapeutic drug. However, when used in combination, CUR enhanced the antiparasitic  (5), demethoxycurcumin or DMC (6), and bis-demethoxycurcumin or BMC (7); synthetic CUR derivative 4a (8), 4e (9).

Final Remarks
In summary, the limitation of the study was the low number of experimental research in vivo (9 reports), performed with different experimental strains, animals, phases of the disease, periods of treatment, and doses, which made the conclusion difficult. Most of the works have not been performed with randomization/blinding and were considered as half orange. Due to the low solubility of RVT, we found problems with the concentrations and, with CUR, the problem of different purities, which can increase the bias in the study. The studies showed that RVT did not present antiparasitic activity in the acute or chronic phase of Chagas disease in mice. Despite only four studies being found, they showed beneficial effects for the heart, liver, and brain of the infected mice. The reports focusing on CUR showed antiparasitic activity, however, it was not superior to BZN, the current therapeutic drug. However, when used in combination, CUR enhanced the antiparasitic activity of BZN, a result which was observed in a recrudescence experiment. According to the medicinal chemistry point of view, the design and synthesis of a series of RVT and CUR analogues could bring light into their structure-activity relationship (SAR) as trypanocide, providing the means to optimize the structure of this hit and further lead to obtain a drug candidate with a higher efficiency and better pharmacokinetic properties, maintaining the prototype's lack of toxicity as well. It is worth noting that most of the derivatives found in the literature were designed for cancer and chronic diseases. We found two new derivatives of RVT with good activity in heart damage in the T. cruzi infection and two related to CUR which was more potent against T. cruzi in vitro. Despite the low reports in T. cruzi-infected animals, we support that both RVT and CUR can be tested as adjuvants in the treatment of CD in a clinical trial with the aim to decrease inflammatory processes of the disease infection's progression, decreasing heart damage. CUR can also be tested in combination with BZN to block the parasite's life cycle. We also point to a concern about the correct formulation to guarantee the right concentration without bias.