In vitro evaluation of 2-(1H-pyrazol-1-yl)-1,3,4-thiadiazole derivatives against replicative and infective stages of Trypanosoma cruzi.

Current treatment of Chagas disease (CD) is based on two substances, nifurtimox (NT) and benzonidazole (BZ), both considered unsatisfactory mainly due to their low activities and high toxicity profile. One of the main challenges faced in CD management concerns the identification of new drugs active in the acute and chronic phases and with good pharmacokinetic profiles. In this work, we studied the bioactivity of twenty 2-(1H-pyrazol-1-yl)-1,3,4-thiadiazole derivatives against Trypanosoma cruzi epimastigotes and trypomastigotes. We identified seven derivatives with promising activity against epimastigote forms with IC50 values ranging from 6 µM to 44 µM. Most of the compounds showed no significant toxicity against murine macrophages. Our initial investigation on the mechanism of action indicates that this series of compounds may exert their anti-parasitic effect, inducing cell membrane damage. The results in trypomastigotes showed that one derivative, PDAN 78, satisfactorily inhibited metabolic alteration at all concentrations. Moreover, we used molecular modeling to understand how tridimensional and structural aspects might influence the observed bioactivities. Finally, we also used in silico approaches to assess the potential pharmacokinetic and toxicological properties of the most active compounds. Our initial results indicate that this molecular scaffold might be a valuable prototype for novel and safe trypanocidal compounds.

A New Strategy for Mapping Epitopes of LACK and PEPCK Proteins of Leishmania amazonensis Specific for Major Histocompatibility Complex Class I.

Leishmaniasis represents a complex of diseases with a broad clinical spectrum and epidemiological diversity, considered a major public health problem. Although there is treatment, there are still no vaccines for cutaneous leishmaniasis. Because Leishmania spp. is an intracellular protozoan with several escape mechanisms, a vaccine must provoke cellular and humoral immune responses. Previously, we identified the Leishmania homolog of receptors for activated C kinase (LACK) and phosphoenolpyruvate carboxykinase (PEPCK) proteins as strong immunogens and candidates for the development of a vaccine strategy. The present work focuses on the in silico prediction and characterization of antigenic epitopes that might interact with mice or human major histocompatibility complex class I. After immunogenicity prediction on the Immune Epitope Database (IEDB) and the Database of MHC Ligands and Peptide Motifs (SYFPEITHI), 26 peptides were selected for interaction assays with infected mouse lymphocytes by flow cytometry and ELISpot. This strategy identified nine antigenic peptides (pL1-H2, pPL3-H2, pL10-HLA, pP13-H2, pP14-H2, pP15-H2, pP16-H2, pP17-H2, pP18-H2, pP26-HLA), which are strong candidates for developing a peptide vaccine against leishmaniasis.

Synthesis of 3-aryl-4-(N-aryl)aminocoumarins via photoredox arylation and the evaluation of their biological activity.

A new series of 3-aryl-4-(N-aryl)aminocoumarins was synthesized in two steps starting from the natural product 4-hydroxycoumarin using the photoredox catalysis for the key step. These conditions reactions allowed to make CC bonds is up to 95% yields in mild conditions, easy operation, in an environmentally benign way, and are compatible with several patterns of substitution. The biological activity of the new compounds was tested in vitro against MCF-7, MDA-MB-231, and CCD-1072Sk cancer cell lines, as soon as to promastigotes and intracellular amastigotes of Leishmania amazonensis. Compounds 17d, 17s and 17x showed activity against promastigote forms (IC50 = 5.96 ± 3.210, 9.05 ± 2.855 and 5.65 ± 2.078 µM respectively), and compound 17x presented the best activity against L. amazonensis amastigote intracellular form (IC50 = 9.6 ± 1.148 µM), no BALB/c peritoneal macrophage cytotoxicity at assayed concentrations (CC50 > 600 µM), and high selectivity to parasites over the mammalian cells (Selectivity Index > 62.2). There was no expressive activity for the cancer cell lines. Single crystal X-ray diffraction analysis was employed for structural elucidation of compounds 17a and 17s. In silico analyses of physicochemical, pharmacokinetic, and toxicological properties suggest that compound 17x is a potential candidate for anti-leishmaniasis drugs.

ATRvD1 Attenuates Renal Tubulointerstitial Injury Induced by Albumin Overload in Sepsis-Surviving Mice.

Novel strategies for the prevention and treatment of sepsis-associated acute kidney injury and its long-term outcomes have been required and remain a challenge in critical care medicine. Therapeutic strategies using lipid mediators, such as aspirin-triggered resolvin D1 (ATRvD1), can contribute to the resolution of acute and chronic inflammation. In this study, we examined the potential effect of ATRvD1 on long-term kidney dysfunction after severe sepsis. Fifteen days after cecal ligation and puncture (CLP), sepsis-surviving BALB/c mice were subjected to a tubulointerstitial injury through intraperitoneal injections of bovine serum albumin (BSA) for 7 days, called the subclinical acute kidney injury (subAKI) animal model. ATRvD1 treatment was performed right before BSA injections. On day 22 after CLP, the urinary protein/creatinine ratio (UPC), histologic parameters, fibrosis, cellular infiltration, apoptosis, inflammatory markers levels, and mRNA expression were determined. ATRvD1 treatment mitigated tubulointerstitial injury by reducing proteinuria excretion, the UPC ratio, the glomerular cell number, and extracellular matrix deposition. Pro-fibrotic markers, such as transforming growth factor ß (TGFß), type 3 collagen, and metalloproteinase (MMP)-3 and -9 were reduced after ATRvD1 administration. Post-septic mice treated with ATRvD1 were protected from the recruitment of IBA1+ cells. The interleukin-1ß (IL-1ß) levels were increased in the subAKI animal model, being attenuated by ATRvD1. Tumor necrosis factor-α (TNF-α), IL-10, and IL-4 mRNA expression were increased in the kidney of BSA-challenged post-septic mice, and it was also reduced after ATRvD1. These results suggest that ATRvD1 protects the kidney against a second insult such as BSA-induced tubulointerstitial injury and fibrosis by suppressing inflammatory and pro-fibrotic mediators in renal dysfunction after sepsis.

Antiprotozoal and Antibacterial Activity of Ravenelin, a Xanthone Isolated from the Endophytic Fungus Exserohilum rostratum.

The natural compound ravenelin was isolated from the biomass extracts of Exserohilum rostratum fungus, and its antimicrobial, antiplasmodial, and trypanocidal activities were evaluated. Ravenelin was isolated by column chromatography and HPLC and identified by NMR and MS. The susceptibility of Gram-positive and Gram-negative bacteria strains to ravenelin was determined by microbroth dilution assay. Cytotoxicity was evaluated in hepatocarcinoma cells (HepG2) and BALB/c peritoneal macrophages by using MTT. SYBR Green I-based assay was used in the asexual stages of Plasmodium falciparum. Trypanocidal activity was tested against the epimastigote and intracellular amastigote forms of Trypanosoma cruzi. Ravenelin was active against Gram-positive bacteria strains, with emphasis on Bacillus subtilis (MIC value of 7.5 µM). Ravenelin's antiparasitic activities were assessed against both the epimastigote (IC50 value of 5 ± 1 µM) and the intracellular amastigote forms of T. cruzi (IC50 value of 9 ± 2 µM), as well as against P. falciparum (IC50 value of 3.4 ± 0.4 µM). Ravenelin showed low cytotoxic effects on both HepG2 (CC50 > 50 µM) and peritoneal macrophage (CC50 = 185 ± 1 µM) cells with attractive selectivity for the parasites (SI values > 15). These findings indicate that ravenelin is a natural compound with both antibacterial and antiparasitic activities, and considerable selectivity indexes. Therefore, ravenelin is an attractive candidate for hit-to-lead development.

Synthesis of new N,S-acetal analogs derived from juglone with cytotoxic activity against Trypanossoma cruzi.

A series of 11 new N,S-acetal juglone derivatives were synthesized and evaluated against T. cruzi epimastigote forms. These compounds were obtained in good to moderate yields using a microwave irradiation protocol. Among all compounds, two N,S-acetal analogs, showed significant trypanocidal activity. Notably, one compound 11g exhibited selectivity index 10-fold higher than the reference drug benznidazole for epimastigote. The compound 11h was more effective for amastigote forms. Both prototypes exhibited S.I. higher than the benznidazole description. Thus, both compounds proving to be useful candidate molecules to further studies in infected animals.

Marine-Derived Penicillium purpurogenum Reduces Tumor Size and Ameliorates Inflammation in an Erlich Mice Model.

BACKGROUND: This study addresses the antitumoral properties of Penicillium purpurogenum isolated from a polluted lagoon in Northeastern Brazil. METHODS: Ethyl Acetate Extracellular Extract (EAE) was used. The metabolites were studied using direct infusion mass spectrometry. The solid Ehrlich tumor model was used for antitumor activity. Female Swiss mice were divided into groups (n = 10/group) as follows: The negative control (CTL-), treated with a phosphate buffered solution; the positive control (CTL+), treated with cyclophosphamide (25 mg/kg); extract treatments at doses of 4, 20, and 100 mg/kg; animals without tumors or treatments (Sham); and animals without tumors treated with an intermediate dose (EAE20). All treatments were performed intraperitoneally, daily, for 15 days. Subsequently, the animals were euthanized, and the tumor, lymphoid organs, and serum were used for immunological, histological, and biochemical parameter evaluations. RESULTS: The extract was rich in meroterpenoids. All doses significantly reduced tumor size, and the 20 and 100 mg/kg doses reduced tumor-associated inflammation and tumor necrosis. The extract also reduced the cellular infiltration of lymphoid organs and circulating TNF-α levels. The extract did not induce weight loss or renal and hepatic toxic changes. CONCLUSIONS: These results indicate that P. purpurogenum exhibits immunomodulatory and antitumor properties in vivo. Thus, fungal fermentation is a valid biotechnological approach to the production of antitumor agents.

1,4-Disubstituted-1,2,3-Triazole Compounds Induce Ultrastructural Alterations in Leishmania amazonensis Promastigote: An in Vitro Antileishmanial and in Silico Pharmacokinetic Study.

The current standard treatment for leishmaniasis has remained the same for over 100 years, despite inducing several adverse effects and increasing cases of resistance. In this study we evaluated the in vitro antileishmanial activity of 1,4-disubstituted-1,2,3 triazole compounds and carried out in silico predictive study of their pharmacokinetic and toxicity properties. Ten compounds were analyzed, with compound 6 notably presenting IC50: 14.64 ± 4.392 µM against promastigotes, IC50: 17.78 ± 3.257 µM against intracellular amastigotes, CC50: 547.88 ± 3.256 µM against BALB/c peritoneal macrophages, and 30.81-fold selectivity for the parasite over the cells. It also resulted in a remarkable decrease in all the parameters of in vitro infection. Ultrastructural analysis revealed lipid corpuscles, a nucleus with discontinuity of the nuclear membrane, a change in nuclear chromatin, and kinetoplast swelling with breakdown of the mitochondrial cristae and electron-density loss induced by 1,4-disubstituted-1,2,3-triazole treatment. In addition, compound 6 enhanced 2.3-fold the nitrite levels in the Leishmania-stimulated macrophages. In silico pharmacokinetic prediction of compound 6 revealed that it is not recommended for topical formulation cutaneous leishmaniasis treatment, however the other properties exhibited results that were similar or even better than miltefosine, making it a good candidate for further in vivo studies against Leishmania parasites.

The Influence of Anthocyanidin Profile on Antileishmanial Activity of Arrabidaea chica Morphotypes.

Arrabidaea chica Verlot (crajiru) is a plant used in folk medicine as an astringent, anti-inflammatory, wound healing and to treat fungal and viral diseases such as measles chickenpox and herpes. Arrabidaea chica has several morphotypes recognized but little is known about its chemical variability. In the present study the anthocyanidin profile of A. chica morphotypes collected in two seasons (summer and winter) have been examined and their activity against Leishmania infection compared. High-performance liquid chromatography coupled to a diode-array detector (HPLC-DAD-UV) and by tandem mass spectrometry with electrospray ionization (ESI-MS/MS) were used for anthocyanidin separation and identification. Antileishmanial activity was measured against promastigote forms of Leishmania amazonensis. Multivariate analysis, principal component analysis (PCA) and Pearson's correlation were performed to classify morphotypes accordingly to their anthocyanidin profile. The presence of 6,7,3',4'-tetrahydroxy-5-methoxyflavylium (3'-hydroxy-carajurone) (1), carajurone (2), 6,7,3'-trihydroxy-5,4'-dimethoxy-flavylium (3'-hydroxy-carajurin) (3) and carajurin (4), and three unidentified anthocyanidins were detected. Two different groups were recognized: group I containing 3'-hydroxy-carajurone; and group II with high content of carajurin. Among anthocyanidins identified in the extracts, only carajurin showed significant statistical correlation (p = 0.030) with activity against L. amazonensis. Carajurin could thus be considered as a pharmacological marker for the antileishmanial potential of the species.

A Triazole Hybrid of Neolignans as a Potential Antileishmanial Agent by Triggering Mitochondrial Dysfunction.

In the search for new compounds with antileishmanial activity, we synthesized a triazole hybrid analogue of the neolignans grandisin and machilin G (LASQUIM 25), which was previously found highly active against both promastigotes and intracellular amastigote forms of Leishmania amazonensis. In this work, we investigated the leishmanicidal effects of LASQUIM 25 to identify the mechanisms involved in the cell death of L. amazonensis promastigotes. Transmission electron microscopy (TEM) analysis showed marked effects of LASQUIM 25 (IC50 = 7.2 µM) on the morphology of promastigote forms, notably on mitochondria. The direct action of the triazole derivative on the parasite was noticed over time from 2 h to 48 h, and cells displayed several ultrastructural alterations characteristic of apoptotic cells. Also, flow cytometric analysis (FACS) after TMRE staining detected changes in mitochondrial membrane potential after LASQUIM 25 treatment (64.83% labeling versus 83.38% labeling in nontreated cells). On the other hand, FACS after PI staining in 24 h-treatment showed a slight alteration in the integrity of the cell membrane, a necrotic event (16.76% necrotic cells versus 3.19% staining in live parasites). An abnormal secretion of lipids was observed, suggesting an exocytic activity. Another striking finding was the presence of autophagy-related lysosome-like vacuoles, suggesting an autophagic cell death that may arise as consequence of mitochondrial stress. Taken together, these results suggest that LASQUIM 25 leishmanicidal mechanisms involve some degree of mitochondrial dysregulation, already evidenced by the treatment with the IC50 of this compound. This effect may be due to the presence of a methylenedioxy group originated from machilin G, whose toxicity has been associated with the capacity to generate electrophilic intermediates.

Endlicheria bracteolata (Meisn.) Essential Oil as a Weapon Against Leishmania amazonensis: In Vitro Assay.

The difficulties encountered and the numerous side effects present in the treatment of cutaneous leishmaniasis have encouraged the research for new compounds that can complement or replace existing treatment. The growing scientific interest in the study of plants, which are already used in folk remedies, has led our group to test Endlicheria bracteolata essential oil against Leishmania amazonensis. Several species of the Lauraceae family, or their compounds, have relevant antiprotozoal activities Therefore, the biological potential on L. amazonensis forms from the essential oil of Endlicheria bracteolata leaves was verified for the first time in that work. The antileishmanial activity was evaluated against promastigotes and intracellular amastigotes, and cytotoxicity were performed with J774.G8, which were incubated with different concentrations of E. bracteolata essential oil. Transmission electron microscopy and flow cytometry were performed with E. bracteolata essential oil IC50. Promastigote forms showed E. bracteolata essential oil IC50 of 7.945 ± 1.285 µg/mL (24 h) and 6.186 ± 1.226 µg/mL (48 h), while for intracellular amastigote forms it was 3.546 ± 1.184 µg/mL (24 h). The CC50 was 15.14 ± 0.090 µg/mL showing that E. bracteolata essential oil is less toxic to macrophages than to parasites. Transmission electron microscopy showed that E. bracteolata essential oil treatment is capable of inducing mitochondrial damage to promastigote and intracellular amastigote forms, while flow cytometry showed ΔÑ°m disruption in treated parasites. These results could bring about new possibilities to develop products based on E. bracteolata essential oil to treat cutaneous leishmaniasis, especially for people who cannot receive the conventional therapy.

Main lesions in the central nervous system of dogs due to Leishmania infantum infection.

BACKGROUND: Canine visceral leishmaniasis (CVL) is endemic in São Luís Maranhão/Brazil and it leads a varied clinical picture, including neurological signs. RESULTS: Histopathological evaluation showed that 14 dogs exhibited pathological alterations in at least one of the analyzed areas. Of these, mononuclear inflammatory reaction was the most frequent, although other lesions, such as hemorrhage, chromatolysis and gliosis were also observed. The presence of L. infantum amastigotes was confirmed in eight dogs, identified in four regions: telencephalon, hippocampus, thalamus and caudal colliculus, but only one presented neurological signs. Polymerase chain reaction results detected the DNA of the parasite in 11 samples from seven dogs. The positive areas were the telencephalon, thalamus, hippocampus, cerebellum, caudal and rostral colliculus. CONCLUSION: These results reveal that during canine visceral leishmaniasis, the central nervous system may display some alterations, without necessarily exhibiting clinical neurological manifestations. In addition, the L. infantum parasite has the ability to cross the blood brain barrier and penetrate the central nervous system.

N-acetyl-cysteine inhibits liver oxidative stress markers in BALB/c mice infected with Leishmania amazonensis.

BACKGROUND: Leishmaniasis is a parasitosis caused by several species of the genus Leishmania. These parasites present high resistance against oxidative stress generated by inflammatory cells. OBJECTIVES: To investigate oxidative stress and molecular inflammatory markers in BALB/c mice infected with L. amazonensis and the effect of antioxidant treatment on these parameters. METHODS: Four months after infection, oxidative and inflammatory parameters of liver, kidneys, spleen, heart and lungs from BALB/c mice were assessed. FINDINGS: In liver, L. amazonensis caused thiol oxidation and nitrotyrosine formation; SOD activity and SOD2 protein content were increased while SOD1 protein content decreased. The content of the cytokines IL-1ß, IL-6, TNF-α, and the receptor of advanced glycation endproducts (RAGE) increased in liver. Treatment with the antioxidant N-acetyl-cysteine (20 mg/kg b.w) for five days inhibited oxidative stress parameters. MAIN CONCLUSIONS: L. amazonensis induces significant alterations in the redox status of liver but not in other organs. Acute antioxidant treatment alleviates oxidative stress in liver, but it had no effect on pro-inflammatory markers. These results indicate that the pathobiology of leishmaniasis is not restricted to the cutaneous manifestations and open perspectives for the development of new therapeutic approaches to the disease, especially for liver function.

Morinda citrifolia Linn. fruit (Noni) juice induces an increase in NO production and death of Leishmania amazonensis amastigotes in peritoneal macrophages from BALB/c.

Leishmaniasis is a complex disease that is considered a serious public health problem. Due to the absence of an effective vaccine and debilitating chemotherapy better therapies are urgently needed. This situation has stimulated the search for alternative treatments such as the use of herbal medicines. Several studies conducted with Morinda citrifolia Linn. have shown various biological activities such as antitumor, immunomodulation and antileishmanial activity, however its mechanisms of action are still unknown. This study aimed to analyze the activity of M. citrifolia fruit juice against Leishmania amazonensis and its action on peritoneal macrophages from BALB/c infected with L. amazonensis. Activity against the promastigote forms showed IC50 at 275.3 µg/mL. Transmission electron microscopy was used to evaluate the ultrastructural alterations in the promastigotes treated with the juice and the results showed cytoplasmic vacuolization, lipid inclusion and increased activity of exocytosis. The juice treatment presented an IC50 at 208.4 µg/mL against intracellular amastigotes and led to an increased nitrite production in infected and non-infected macrophages. When macrophages were pre-treated with iNOS inhibitors, aminoguanidine or 1400W, the intracellular amastigotes increased, demonstrating the important role of NO production in M. citrifolia fruit activity. In conclusion, our results reveal that treatment with M. citrifolia fruit juice can increase NO production in peritoneal macrophages and this ability has an important role in the killing of L. amazonensis intracellular amastigotes.

Increased tau phosphorylation and receptor for advanced glycation endproducts (RAGE) in the brain of mice infected with Leishmania amazonensis.

Leishmaniasis is a parasitosis caused by several species of the genus Leishmania, an obligate intramacrophagic parasite. Although neurologic symptoms have been observed in human cases of leishmaniasis, the manifestation of neurodegenerative processes is poorly studied. The aim of the present work was to investigate if peripheral infection of BALB/c mice with Leishmania amazonensis affects tau phosphorylation and RAGE protein content in the brain, which represent biochemical markers of neurodegenerative processes observed in diseases with a pro-inflammatory component, including Alzheimer's disease and Down syndrome. Four months after a single right hind footpad subcutaneous injection of L. amazonensis, the brain cortex of BALB/c mice was isolated. Western blot analysis indicated an increase in tau phosphorylation (Ser(396)) and RAGE immunocontent in infected animals. Brain tissue TNF-α, IL-1ß, and IL-6 levels were not different from control animals; however, increased protein carbonylation, decreased IFN-γ levels and impairment in antioxidant defenses were detected. Systemic antioxidant treatment (NAC 20mg/kg, i.p.) inhibited tau phosphorylation and recovered IFN-γ levels. These data, altogether, indicate an association between impaired redox state, tau phosphorylation and RAGE up-regulation in the brain cortex of animals infected with L. amazonensis. In this context, it is possible that neurologic symptoms associated to chronic leishmaniasis are associated to disruptions in the homeostasis of CNS proteins, such as tau and RAGE, as consequence of oxidative stress. This is the first demonstration of alterations in biochemical parameters of neurodegeneration in an experimental model of Leishmania infection.

In vitro evaluation of (-)α-bisabolol as a promising agent against Leishmania amazonensis.

Current treatments for leishmaniasis present some difficulties due to their toxicity, the use of the intravenous route for administration and therapy duration, which may lead to treatment discontinuation. The aim of this study is to investigate new treatment alternatives to improve patients well being. Therefore, we evaluated the inhibitory effect of (-)α-bisabolol, a sesquiterpene alcohol found in various essential oils of different plant species, against the promastigotes and intracellular amastigotes forms of Leishmania amazonensis, as well as the cytotoxic, morphological and ultrastructural alterations of treated cells. Promastigotes forms of L. amazonensis were incubated with (-)α-bisabolol to determine the antileishmanial activity of this compound. The cytotoxicity effect was evaluated by testing against J774.G8 cells. After these tests, the infected and uninfected cells with L. amazonensis were used to determine if the (-)α-bisabolol was able to kill intracellular parasites and to cause some morphological changes in the cells. The (-)α-bisabolol compound showed significant antileishmanial activity against promastigotes with a 50% effective concentration of 8.07 µg/ml (24 h) and 4.26 µg/ml (48 h). Against intracellular amastigotes the IC50 (inhibitory concentration) of (-)α-bisabolol (24 h) was 4.15 µg/ml. The (-)α-bisabolol also showed a cytotoxic effect against the macrophage strain J774.G8. The value of 50% cytotoxic concentration was 14.82 µg/ml showing that (-)α-bisabolol is less toxic to macrophages than to the parasite. Ultrastructural studies of treated promastigotes and amastigotes showed several alterations, such as loss of cytoplasmic organelles, including the nucleus, and the presence of lipid inclusions. This study showed that (-)α-bisabolol has promising antileishmanial properties, as it can act against the promastigote forms and is able to penetrate the cell, and is also active against the amastigote forms. About 69% of the promastigotes forms suffered mitochondrial membrane damage after treatment with IC50 of (-)α-bisabolol, suggesting inhibition of the metabolic activity of parasites. These results open new prospects for research that can contribute to the development of products based on essential oils or isolated compounds from plants for the treatment of cutaneous leishmaniasis.

Morphometric changes in C57BL/6 mice retina infected by Toxoplasma gondii ME 49 strain.

This study evaluated the morphometric implications in C57BL/6 mouse retina infected by Toxoplasma gondii, ME 49 strain. Twenty C57BL/6 female mice were divided into group 1 (n=8, intraperitoneally infected with 30 cysts of T. gondii ME 49 strain) and group 2 (n=12 non-infected controls). The eyes were enucleated on the 60th day after infection, fixed and processed for light microscopy. Changes in retinal thickness and in the perimeter/area ratio (P/A) of the retinal layers were analyzed by digital morphometry. We considered that P/A was the measurement of retinal architecture distortion induced by toxoplasmosis. This study considered the ganglion cells and nerve fiber layers as a monolayer, thus six layers of retina were evaluated: photoreceptors (PRL), outer nuclear (ONL), outer plexiform (OPL), inner nuclear (INL), inner plexiform (IPL) and ganglion cells/nerve fiber monolayer (GNL). Histological analysis of infected mouse retina showed inflammatory infiltrate, necrosis, glial reaction and distortion of the retina architecture. It also presented increased thickness (167.8±24.9µm versus 121.1±15.4µm, in controls) and increased retinal thickness within the retinitis foci (187.7±16.6µm versus 147.9±12.2µm out of the retinitis foci). A statistically significant difference in P/A was observed between infected and uninfected mouse retinas. The same was observed in PRL, OPL, INL and GNL. Retinal morphometry may be used to demonstrate differences between infected and uninfected mouse retinas.

Leishmanicidal and immunomodulatory activity of Terminalia catappa in Leishmania amazonensisin vitro infection.

Leishmaniases are infectious-parasitic diseases that impact public health around the world. Antileishmanial drugs presented toxicity and increase in parasitic resistance. Studies with natural products show an alternative to this effect, and several metabolites have demonstrated potential in the treatment of various diseases. Terminalia catappa is a plant species with promising pharmaceutical properties. The objective of this work was to evaluate the therapeutic potential of extracts and fractions of T. catappa on Leishmania amazonensis and investigate the immunomodulatory mechanisms associated with its action. In anti-Leishmania assays, the ethyl acetate fraction exhibited activity against promastigotes (IC50 86.07 ± 1.09 µg/mL) and low cytotoxicity (CC50 517.70 ± 1.68 µg/mL). The ethyl acetate fraction also inhibited the intracellular parasite (IC50 25.74 ± 1.08 µg/mL) with a selectivity index of 20.11. Treatment with T. catappa ethyl acetate fraction did not alter nitrite production by peritoneal macrophages stimulated with L. amazonensis, although there was a decrease in unstimulated macrophages treated at 50 µg/mL (p = 0.0048). The T. catappa ethyl acetate fraction at 100 µg/mL increased TNF-α levels (p = 0.0238) and downregulated HO-1 (p = 0.0030) and ferritin (p = 0.0002) gene expression in L. amazonensis-stimulated macrophages. Additionally, the total flavonoid and ellagic acid content for ethyl acetate fraction was 13.41 ± 1.86 mg QE/g and 79.25 mg/g, respectively. In conclusion, the T. catappa ethyl acetate fraction showed leishmanicidal activity against different forms of L. amazonensis and displayed immunomodulatory mechanisms, including TNF-α production and expression of pro and antioxidant genes.

In Vitro Antioxidant and Antitrypanosomal Activities of Extract and Fractions of Terminalia catappa.

Chagas disease is a severe infectious and parasitic disease caused by the protozoan Trypanosoma cruzi and considered a public health problem. Chemotherapeutics are still the main means of control and treatment of the disease, however with some limitations. As an alternative treatment, plants have been pointed out due to their proven pharmacological properties. Many studies carried out with Terminalia catappa have shown several biological activities, but its effect against T. cruzi is still unknown. The objective of this work is to evaluate the therapeutic potential of extracts and fractions obtained from T. catappa on the parasite T. cruzi, in addition to analyzing its antioxidant activity. T. catappa ethyl acetate fraction were produced and submitted the chemical characterization by Liquid Chromatography Coupled to Mass Spectrometry (LC-MS). From all T. catappa extracts and fractions evaluated, the ethyl acetate and the aqueous fraction displayed the best antioxidant activity by the 2,2-diphenyl-1-picryl-hydrazyl (DPPH) radical scavenging method (IC50 of 7.77 ± 1.61 and 5.26 ± 1.26 µg/mL respectively), and by ferric ion reducing (FRAP) method (687.61 ± 0.26 and 1009.32 ± 0.13 µM of Trolox equivalent/mg extract, respectively). The ethyl acetate fraction showed remarkable T. cruzi inhibitory activity with IC50 of 8.86 ± 1.13, 24.91 ± 1.15 and 85.01 ± 1.21 µg/mL against epimastigotes, trypomastigotes and intracellular amastigotes, respectively, and showed no cytotoxicity for Vero cells (CC50 > 1000 µg/mL). The treatment of epimastigotes with the ethyl acetate fraction led to drastic ultrastructural changes such as the loss of cytoplasm organelles, cell disorganization, nucleus damage and the loss of integrity of the parasite. This effect could be due to secondary compounds present in this extract, such as luteolin, kaempferol, quercetin, ellagic acid and derivatives. The ethyl acetate fraction obtained from T. catappa leaves can be an effective alternative in the treatment and control of Chagas disease, and material for further investigations.

Effects of Passovia ovata Mistletoe on Pro-Inflammatory Markers In Vitro and In Vivo.

New agents that can suppress inflammatory responses are being sought, since chronic inflammation is associated with several pathologies. This work aims to elucidate phytochemicals from the hydroethanolic extract of mistletoe Passovia ovata (POH) and its anti-inflammatory potential. POH is submitted to HPLC-UV, qualitative analysis of chemical constituents, and flavonoid quantification. Cytotoxicity is evaluated in RAW 264.7 macrophages by MTT. LPS-stimulated RAW 264.7 cells are treated with POH and, after 48 h, the nitrite and cytokine levels are quantified. BALB/c mice are treated by gavage with POH and stimulated with λ-carrageenan to induce paw oedema or peritonitis. POH yield is 25% with anthraquinones, tannins, anthocyanins, anthocyanidins, flavonols, catechins and flavanones present and flavonoid content of 4.44 ± 0.157 mg QE/g dry weight. POH exhibits low cytotoxicity and significantly reduced (p < 0.01) nitrite, IL-1ß, IL-6, and TNF-α quantification at 500 µg/mL. POH at 500 mg/kg prevents paw edema increase and also reduces inflammatory infiltrate and mast cells in the footpad. In the peritonitis model, POH does not influence cytokines levels or cell counts. Overall, POH demonstrates a high concentration of flavonoids and prominent effects in the reduction in pro-inflammatory markers in vitro and in the inhibition of paw oedema.