(-)-5-Demethoxygrandisin B a New Lignan from Virola surinamensis (Rol.) Warb. Leaves: Evaluation of the Leishmanicidal Activity by In Vitro and In Silico Approaches.

Leishmaniasis is a complex disease caused by infection with different Leishmania parasites. The number of medications used for its treatment is still limited and the discovery of new drugs is a valuable approach. In this context, here we describe the in vitro leishmanicidal activity and the in silico interaction between trypanothione reductase (TryR) and (-)-5-demethoxygrandisin B from the leaves of Virola surinamensis (Rol.) Warb. The compound (-)-5-demethoxygrandisin B was isolated from V. surinamensis leaves, a plant found in the Brazilian Amazon, and it was characterized as (7R,8S,7'R,8'S)-3,4,5,3',4'-pentamethoxy-7,7'-epoxylignan. In vitro antileishmanial activity was examined against Leishmania amazonensis, covering both promastigote and intracellular amastigote phases. Cytotoxicity and nitrite production were gauged using BALB/c peritoneal macrophages. Moreover, transmission electron microscopy was applied to probe ultrastructural alterations, and flow cytometry assessed the shifts in the mitochondrial membrane potential. In silico methods such as molecular docking and molecular dynamics assessed the interaction between the most stable configuration of (-)-5-demethoxygrandisin B and TryR from L. infantum (PDB ID 2JK6). As a result, the (-)-5-demethoxygrandisin B was active against promastigote (IC50 7.0 µM) and intracellular amastigote (IC50 26.04 µM) forms of L. amazonensis, with acceptable selectivity indexes. (-)-5-demethoxygrandisin B caused ultrastructural changes in promastigotes, including mitochondrial swelling, altered kDNA patterns, vacuoles, vesicular structures, autophagosomes, and enlarged flagellar pockets. It reduced the mitochondria membrane potential and formed bonds with important residues in the TryR enzyme. The molecular dynamics simulations showed stability and favorable interaction with TryR. The compound targets L. amazonensis mitochondria via TryR enzyme inhibition.

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.

Antitumor Effect of Açaí (Euterpe oleracea Mart.) Seed Extract in LNCaP Cells and in the Solid Ehrlich Carcinoma Model.

Euterpe oleracea (açaí) fruit has approximately 15% pulp, which is partly edible and commercialized, and 85% seeds. Although açaí seeds are rich in catechins-polyphenolic compounds with antioxidant, anti-inflammatory, and antitumor effects-almost 935,000 tons/year of seeds are discarded as industrial waste. This work evaluated the antitumor properties of E. oleracea in vitro and in vivo in a solid Ehrlich tumor in mice. The seed extract presented 86.26 ± 0.189 mg of catechin/g of extract. The palm and pulp extracts did not exhibit in vitro antitumor activity, while the fruit and seed extracts showed cytotoxic effects on the LNCaP prostate cancer cell line, inducing mitochondrial and nuclear alterations. Oral treatments were performed daily at 100, 200, and 400 mg/kg of E. oleracea seed extract. The tumor development and histology were evaluated, along with immunological and toxicological parameters. Treatment at 400 mg/kg reduced the tumor size, nuclear pleomorphism, and mitosis figures, increasing tumor necrosis. Treated groups showed cellularity of lymphoid organs comparable to the untreated group, suggesting less infiltration in the lymph node and spleen and preservation of the bone marrow. The highest doses reduced IL-6 and induced IFN-γ, suggesting antitumor and immunomodulatory effects. Thus, açaí seeds can be an important source of compounds with antitumor and immunoprotective properties.

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.

Monomethylsulochrin isolated from biomass extract of Aspergillus sp. against Leishmania amazonensis: In vitro biological evaluation and molecular docking.

Leishmaniasis represents a serious world health problem, with 1 billion people being exposed to infection and a broad spectrum of clinical manifestations with a potentially fatal outcome. Based on the limitations observed in the treatment of leishmaniasis, such as high cost, significant adverse effects, and the potential for drug resistance, the aim of the present study was to evaluate the leishmanicidal activity of the compounds pseurotin A and monomethylsulochrin isolated from the biomass extract of Aspergillus sp. The chromatographic profiles of the extract were determined by high-performance liquid chromatography coupled with a diode-array UV-Vis detector (HPLC-DAD-UV), and the molecular identification of the pseurotin A and monomethylsulochrin were carried out by electrospray ionization mass spectrometry in tandem (LC-ESI-MS-MS) and nuclear magnetic resonance (NMR). Antileishmanial activity was assayed against promastigote and intracellular amastigote of Leishmania amazonensis. As a control, cytotoxicity assays were performed in non-infected BALB/c peritoneal macrophages. Ultrastructural alterations in parasites were evaluated by transmission electron microscopy. Changes in mitochondrial membrane potential were determined by flow cytometry. Only monomethylsulochrin inhibited the promastigote growth (IC50 18.04 ± 1.11 µM), with cytotoxicity to peritoneal macrophages (CC50 5.09 91.63 ± 1.28 µM). Activity against intracellular amastigote forms (IC50 5.09 ± 1.06 µM) revealed an increase in antileishmanial activity when compared with promastigotes. In addition to a statistically significant reduction in the evaluated infection parameters, monomethylsulochrin altered the ultrastructure of the promastigote forms with atypical vacuoles, electron-dense corpuscles in the cytoplasm, changes at the mitochondria outer membrane and abnormal disposition around the kinetoplast. It was showed that monomethylsulochrin leads to a decrease in the mitochondrial membrane potential (25.9%, p = 0.0286). Molecular modeling studies revealed that monomethylsulochrin can act as inhibitor of sterol 14-alpha-demethylase (CYP51), a therapeutic target for human trypanosomiasis and leishmaniasis. Assessed for its drug likeness, monomethylsulochrin follows the Lipinski Rule of five and Ghose, Veber, Egan, and Muegge criteria. Furthermore, monomethylsulochrin can be used as a reference in the development of novel and therapeutically useful antileishmanial agents.

Amentoflavone isolated from Selaginella sellowii Hieron induces mitochondrial dysfunction in Leishmania amazonensis promastigotes.

Leishmaniasis chemotherapy is a bottleneck in disease treatment. Although available, chemotherapy is limited, toxic, painful, and does not lead to parasite clearance, with parasite resistance also being reported. Therefore, new therapeutic options are being investigated, such as plant-derived anti-parasitic compounds. Amentoflavone is the most common biflavonoid in the Selaginella genus, and its antileishmanial activity has already been described on Leishmania amazonensis intracellular amastigotes but its direct action on the parasite is controversial. In this work we demonstrate that amentoflavone is active on L. amazonensis promastigotes (IC50 = 28.5 ± 2.0 µM) and amastigotes. Transmission electron microscopy of amentoflavone-treated promastigotes showed myelin-like figures, autophagosomes as well as enlarged mitochondria. Treated parasites also presented multiple lipid droplets and altered basal body organization. Similarly, intracellular amastigotes presented swollen mitochondria, membrane fragments in the lumen of the flagellar pocket as well as autophagic vacuoles. Flow cytometric analysis after TMRE staining showed that amentoflavone strongly decreased mitochondrial membrane potential. In silico analysis shows that amentoflavone physic-chemical, drug-likeness and bioavailability characteristics suggest it might be suitable for oral administration. We concluded that amentoflavone presents a direct effect on L. amazonensis parasites, causing mitochondrial dysfunction and parasite killing. Therefore, all results point for the potential of amentoflavone as a promising candidate for conducting advanced studies for the development of drugs against leishmaniasis.

Nitric Oxide Induction in Peritoneal Macrophages by a 1,2,3-Triazole Derivative Improves Its Efficacy upon Leishmania amazonensis In Vitro Infection.

1,2,3-Triazole is one of the most flexible chemical scaffolds broadly used in various fields. Here, we report the antileishmanial activity of 1,2,3-triazole derivatives, the ultrastructural alterations induced by their treatment, and the nitric oxide (NO) modulation effect on their efficacy against Leishmania amazonensis in vitro infection. After the screening of eleven compounds, compound 4 exhibited better results against L. amazonensis promastigotes (IC50 = 15.52 ± 3.782 µM) and intracellular amastigotes (IC50 = 4.10 ± 1.136 µM), 50% cytotoxicity concentration at 84.01 ± 3.064 µM against BALB/c peritoneal macrophages, and 20.49-fold selectivity for the parasite over the cells. Compound 4 induced ultrastructural mitochondrial alterations and lipid inclusions in L. amazonensis promastigotes, upregulated tumor necrosis factor α, interleukin (IL)-1ß, IL-6, IL-12, and IL-10 messenger RNA expressions, and enhanced the NO production, verified by nitrite (p = 0.0095) and inducible nitric oxide synthase expression (p = 0.0049) quantification, which played an important role in its activity against intramacrophagic L. amazonensis. In silico prediction in association with antileishmanial activity results showed compound 4 as a hit compound with promising potential for further studies of new leishmaniasis treatment options.

Antileishmanial Activity of Flavones-Rich Fraction From Arrabidaea chica Verlot (Bignoniaceae).

Acknowledging the need of identifying new compounds for the treatment of leishmaniasis, this study aimed to evaluate, from in vitro trials, the activity of flavones from Arrabidaea chica against L. amazonensis. The chromatographic profiles of the hydroethanolic extract and a flavone-rich fraction (ACFF) from A. chica were determined by high-performance liquid chromatography coupled with a diode-array UV-Vis detector (HPLC-DAD-UV) and electrospray ionization mass spectrometry in tandem (LC-ESI-MS-MS). The flavones luteolin (1) and apigenin (2), isolated from chromatographic techniques and identified by Nuclear Magnetic Resonance of 1H and 13C, were also quantified in ACFF, showing 190.7 mg/g and apigenin 12.4 mg/g, respectively. The other flavones were identified by comparing their spectroscopic data with those of the literature. The in vitro activity was assayed against promastigotes and intramacrophagic amastigote forms of L. amazonensis. Cytotoxicity tests were performed with peritoneal macrophages of BALB/c mice. Nitrite quantification was performed with Griess reagent. Ultrastructural investigations were obtained by transmission electron microscopy. Anti-Leishmania assays indicated that the IC50 values for ACFF, apigenin, and luteolin were obtained at 40.42 ± 0.10 and 31.51 ± 1.13 µg/mL against promastigotes, respectively. ACFF and luteolin have concentration-dependent cytotoxicity. ACFF and luteolin also inhibited the intra-macrophagic parasite (IC50 3.575 ± 1.13 and 11.78 ± 1.24 µg/mL, respectively), with a selectivity index of 11.44 for ACFF. Promastigotes exposed to ACFF and luteolin exhibited ultrastructural changes, such as intense cytoplasm vacuolization and mitochondrial swelling. These findings data evidence the antileishmanial action of flavone-rich fractions of A. chica against L. amazonensis, encouraging further studies.

Amentoflavone as an Ally in the Treatment of Cutaneous Leishmaniasis: Analysis of Its Antioxidant/Prooxidant Mechanisms.

Treatment of leishmaniasis is a challenging subject. Although available, chemotherapy is limited, presenting toxicity and adverse effects. New drugs with antileishmanial activity are being investigated, such as antiparasitic compounds derived from plants. In this work, we investigated the antileishmanial activity of the biflavonoid amentoflavone on the protozoan Leishmania amazonensis. Although the antileishmanial activity of amentoflavone has already been reported in vitro, the mechanisms involved in the parasite death, as well as its action in vivo, remain unknown. Amentoflavone demonstrated activity on intracellular amastigotes in macrophages obtained from BALB/c mice (IC50 2.3 ± 0.93 µM). No cytotoxicity was observed and the selectivity index was estimated as greater than 10. Using BALB/c mice infected with L. amazonensis we verified the effect of an intralesional treatment with amentoflavone (0.05 mg/kg/dose, in a total of 5 doses every 4 days). Parasite quantification demonstrated that amentoflavone reduced the parasite load in treated footpads (46.3% reduction by limiting dilution assay and 56.5% reduction by Real Time Polymerase Chain Reaction). Amentoflavone decreased the nitric oxide production in peritoneal macrophages obtained from treated animals. The treatment also increased the expression of ferritin and decreased iNOS expression at the site of infection. Furthemore, it increased the production of ROS in peritoneal macrophages infected in vitro. The increase of ROS in vitro, associated with the reduction of NO and iNOS expression in vivo, points to the antioxidant/prooxidant potential of amentoflavone, which may play an important role in the balance between inflammatory and anti-inflammatory patterns at the infection site. Taken together these results suggest that amentoflavone has the potential to be used in the treatment of cutaneous leishmaniasis, working as an ally in the control and development of the lesion.

Host Genetics Background Influence in the Intragastric Trypanosoma cruzi Infection.

Background: Considering the complexity of the factors involved in the immunopathology of Chagas disease, which influence the Chagas' disease pathogenesis, anti-T. cruzi immune response, and chemotherapy outcome, further studies are needed to improve our understanding about these relationships. On this way, in this article we analyzed the host genetic influence on hematological, histopathological and immunological aspects after T. cruzi infection. Methods: BALB/c and A mice were intragastrically infected with T. cruzi SC2005 strain, isolated from a patient of an outbreak of Chagas disease. Parameters such as parasite load, survival rates, cytokines production, macrophages, T and B cell frequencies, and histopathology analysis were carried out. Results: BALB/c mice presented higher parasitemia and mortality rates than A mice. Both mouse lineages exhibited hematological alterations suggestive of microcytic hypochromic anemia and histopathological alterations in stomach, heart and liver. The increase of CD8+ T cells, in heart, liver and blood, and the increase of CD19+ B cells, in liver, associated with a high level of proinflammatory cytokines (IL-6, TNF-α, IFN-γ), confer a resistance profile to the host. Although BALB/c animals exhibited the same findings observed in A mice, the response to infection occurred later, after a considerable parasitemia increase. By developing an early response to the infection, A mice were found to be less susceptible to T. cruzi SC2005 infection. Conclusions: Host genetics background shaping the response to infection. The early development of a cytotoxic cellular response profile with the production of proinflammatory cytokines is important to lead a less severe manifestation of Chagas disease.

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.

In vitro and in vivo leishmanicidal activity of a ruthenium nitrosyl complex against Leishmania (Viannia) braziliensis.

Leishmaniasis is a parasitic disease caused by protozoa of the genus Leishmania. There are many complications presented by the current treatment, as high toxicity, high cost and parasite resistance, making the development of new therapeutic agents indispensable. The present study aims to evaluate the leishmanicidal potential of ruthenium nitrosyl complex cis-[Ru(bpy)2(SO3)(NO)](PF6) against Leishmania (Viannia) braziliensis. The effect of this metal complex on parasite-host interaction was evaluated by in vitro efficacy test in dermal fibrobast cells in the presence of different concentrations (1, 10, 50 and 100 µM) and by in vivo efficacy tests performed in the presence of two different concentrations of complex (100 µg/kg/day or 300 µg/kg/day) evaluating its effect on the size of the lesion and the number of parasites present in the draining lymph nodes in hamsters. Even at the lowest concentration of 1 µM of ruthenium complex, it was observed a significant decrease of the infected cells, after 24 h exposure in vitro, with total reduction at 50 µM of the ruthenium complex. In the in vivo cutaneous infection model, administration of daily doses of 300 µg/kg/day of complex reduced significantly lesion size by 51% (p < 0.05), with a 99.9% elimination of the parasites found in the lymph nodes (p < 0.001). The results suggest a promising leishmanicidal effect by that ruthenium nitrosyl complex against L. (V.) braziliensis.

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.

Infection of retinal epithelial cells with L. amazonensis impacts in extracellular matrix proteins.

One of the manifestations of leishmaniases is eye injuries which main characteristics are the injury of the anterior chamber of the eye and the resistance to specific treatments. The retinal pigment epithelial (RPE) cells participate in pathogen-induced intraocular inflammatory processes. We investigated Leishmania amazonensis-RPE cells relationship and its impact in laminin and fibronectin production. Using RPE cell (ARPE-19), we demonstrated that L. amazonensis adhere to these cells in the first hour of infection, whereas parasite internalization was only observed after 6 h. Seventy-two hours after infection, vacuoles with parasites debris were observed intracellularly, and no parasite were observed intra- or extracellularly at the 96 h, suggesting that Leishmania can infect ARPE-19 cells although this cells are able to clear the infection. Fibronectin and laminin were associated with L. amazonensis-ARPE-19 interaction. Confocal analysis showed no substantial alterations in fibronectin presence in ARPE-19-infected or ARPE-19-noninfected cells, whereas laminin levels increased three times 10 h after L. amazonensis infection. After this time, laminin levels decreased in infected cells. These results suggest that L. amazonensis-ARPE-19 infection induces increased production of laminin in the beginning of infection which may facilitate parasite-host cell interactions.

Trypanosoma cruzi and myoid cells from seminiferous tubules: interaction and relation with fibrous components of extracellular matrix in experimental Chagas' disease.

The main transmission route of Trypanosoma cruzi is by triatomine bugs. However, T. cruzi is also transmitted through blood transfusion, organ transplantation, ingestion of contaminated food or fluids, or is congenital. Sexual transmission, although suggested since the discovery of Chagas' disease, has remained unproven. Sexual transmission would require T. cruzi to be located at the testes and ovaries. Here we investigated whether T. cruzi is present in the gonads of mice infected with 10(4) T. cruzi trypomastigotes from the CL strain. Fourteen days after experimental infection, histopathological examination showed alterations in the extracellular matrix of the lamina propria of the seminiferous tubules. Furthermore, amastigotes were present in seminiferous tubules, within myoid cells, and in the adjacencies of the basal compartment. These results indicate that T. cruzi is able to reach seminiferous tubule lumen, thus suggesting that Chagas' disease could potentially be transmitted through sexual intercourse. Complementary studies are required to demonstrate that Chagas' disease can be transmitted by coitus.