Vernonia brasiliana (L.) Druce induces ultrastructural changes and apoptosis-like death of Leishmania infantum promastigotes.

The present study aimed to evaluate the antileishmanial effect, the mechanisms of action and the association with miltefosine of Vernonia brasiliana essential oil against Leishmania infantum promastigotes. This essential oil was obtained by hydrodistillation and its chemical composition was determined by gas chromatography-mass spectrometry (GC-MS). The antileishmanial activity against L. infantum promastigotes and cytotoxicity on DH82 cells were evaluated by MTT colorimetric assay. Ultrastructural alterations were evaluated by transmission electron microscopy. Changes in mitochondrial membrane potential, in the production of reactive oxygen species, and analysis of apoptotic events were determined by flow cytometry. The association between the essential oil and miltefosine was evaluated using the modified isobologram method. The most abundant component of the essential oil was ß-caryophyllene (21.47 %). Anti-Leishmania assays indicated an IC50 of 39.01 ±â€¯1.080 µg/mL for promastigote forms after 72 h of treatment. The cytotoxic concentration for DH82 cells was 63.13 ±â€¯1.211 µg/mL after 24 h of treatment. The effect against L. infantum was proven through the ultrastructural changes caused by the oil, such as kinetoplast and mitochondrial swelling, vesicles in the flagellar pocket, discontinuity of the nuclear membrane, nuclear fragmentation and condensation, and loss of organelles. It was observed that the oil leads to a decrease in the mitochondrial membrane potential (35.10 %, p = 0.0031), increased reactive oxygen species production, and cell death by late apoptosis (17.60 %, p = 0.020). The combination of the essential oil and miltefosine exhibited an antagonistic effect. This study evidences the antileishmanial action of V. brasiliana essential oil against L. infantum promastigotes.

An enquiry into antileishmanial activity and quantitative analysis of polyhydroxylated steroidal saponins from Solanum paniculatum L. leaves.

Solanum paniculatum L. is species whose fruits are widely consumed in Brazil as a tonic beverage with higher content of steroidal saponins. In this work, we developed an analytical method for the quantification of the eight saponins present in the 70 % ethanol extract from the leaves using ultra-high-performance liquid chromatography coupled to mass spectrometry (UHPLC-MS). Besides, the eight spirostanic saponins were screened for in vitro antileishmanial activity against promastigote and amastigote forms of Leishmania (L.) amazonensis. Substances 1, 2 and 3 were found to be the most active compounds, with inhibitory concentration (IC50) values of 8.51 ± 4.38, 10.75 ± 6.85 and 10.45 ± 4.21 µM, respectively, against promastigote forms and effective concentration (EC50) values of >25, 17.73 ± 0.99 and 19.57 ± 0.84 µM, respectively, against amastigote forms. The cytotoxic test with compounds 1-3 evidenced low toxicity in murine macrophage cells, with values above 50 µM at concentration lower than 25 µM. These findings show that saponins 1-3 should be evaluated in further studies for the treatment of cutaneous leishmaniasis.

Mode of action of a formulation containing hydrazones and saponins against leishmania spp. Role in mitochondria, proteases and reinfection process.

Toxicity and poor adherence to treatment that favors the generation of resistance in the Leishmania parasites highlight the need to develop better alternatives. Here, we evaluated the in vitro effectiveness of hydrazone derived from chromanes 2-(2,3-dihydro-4H-1-benzothiopyran-4-ylidene) hydrazide (TC1) and 2-(2,3-dihydro-4H-1-benzopyran-4-ylidene) hydrazide (TC2) and the mixture of triterpene saponin hederagenin-3-O-(3,4-O-diacetyl-ß-D-xylopyranosyl-(1à3)-a-L- rhamnopyranosyl-(1à2)-a-L-arabinofuranoside, hederagenin-3-O-(3,4-O-diacetyl-a-L- arabinopyranosyl-(1à3)-a-L-rhamnopyranosyl-(1à2)-a-L-arabinofuranoside and, hederagenin-3-O-(4-O-acetyl-ß-D-xylopyranosyl-(1à3)-a-L-rhamnopyranosyl-(1à2)-a-L-arabinofuranoside from Sapindus saponaria (SS) on L. braziliensis and L. pifanoi. Mixtures of TC1 or TC2 with saponin were formulated for topical application and the therapeutic effectiveness was evaluated in the model for cutaneous leishmaniasis (CL) in golden hamster. The mode of action of these compounds was tested on various parasite processes and ultrastructural parasite modifications. TC1, TC2 and SS showed moderate cytotoxicity when tested independently but toxicity was improved when tested in combination. The compounds were more active against intracellular Leishmania amastigotes. In vivo studies showed that combinations of TC1 or TC2 with SS in 1:1 ratio (w/w) cured 100% of hamsters with no signs associated with toxicity. The compounds did cause changes in the mitochondrial activity of the parasite with a decrease in ATP levels and depolarization of membrane potential and overproduction of reactive oxygen species; nevertheless, these effects were not related to alterations in membrane permeability. The phagolysosome ultrastructure was also affected impacting the survival of Leishmania but the function of the lysosome nor the pH inside the phagolysosome did not change. Lastly, there was a protease inhibition which was directly related to the decrease in the ability of Leishmania to infect and multiply inside the macrophage. The results suggest that the combination of TC1 and TC2 with SS in a 1:1 ratio is capable of curing CL in hamsters. This effect may be due to the ability of these compounds to affect parasite survival and the ability to infect new cells.

(±)-trans-2-phenyl-2,3-dihydrobenzofurans as leishmanicidal agents: Synthesis, in vitro evaluation and SAR analysis.

Leishmaniasis, a neglected tropical disease caused by parasites of the genus Leishmania, causes a serious burden of disease around the world, represents a threat to the life of millions of people, and therefore is a major public health problem. More effective and non-toxic new treatments are required, especially for visceral leishmaniasis, the most severe form of the disease. On the backdrop that dihydrobenzofurans have previously shown antileishmanial activity, we present here the synthesis of a set of seventy trans-2-phenyl-2,3-dihydrobenzofurans and evaluation of their in vitro activity against Leishmania donovani as well as a discussion of structure-activity relationships. Compounds 8m-o and 8r displayed the highest potency (IC50 < 2 µmol/L) and interesting selectivity of the antileishmanial activity over cytotoxicity against mammalian cells (SI > 4.6). Nonetheless, structural optimization as further requirement was inferred from the high clearance of the most potent compound (8m) observed during determination in vitro of its metabolic stability. On the other hand, chiral separation of 8m and subsequent biological evaluation of its enantiomers demonstrated no effect of chirality on activity and cytotoxicity. Holistic analysis of in silico ADME-like properties and ligand efficiency metrics by a simple scoring function estimating druglikeness highlighted compounds 16c, 18 and 23 as promising candidates for further development. Overall, the potential of trans-2-phenyl-2,3-dihydrobenzofurans as leishmanicidal agents was confirmed.

An ex vivo multiparametric flow cytometry assay using human whole blood to simultaneously measure cytotoxicity and leishmanicidal activities.

Therapeutic options for the treatment of leishmaniasis are insufficient and need improvements owing to their low efficiency and high toxicity as well as the emergence of resistant strains. The limited number of new drugs for neglected diseases and lack of innovation in your development are still challenges. In this context, the process of discovery and development of biological assays play a pivotal role for the identification of bioactive compounds. The assays currently used for screening of drugs with cytotoxic activity against Leishmania parasites, include different processes that utilize intact parasite (free or intracellular) or specific enzymes of metabolism as a target cell. These assays allow the screening of large numbers of samples followed by more detailed secondary confirmatory assays to confirm the observed activity and assess their toxicity. In the present study, we described the development of a new functional and more complete assay that enables simultaneous assessment of potential anti-Leishmania compounds through evaluation of internalization of fluorescein-labeled L. braziliensis promastigotes by human peripheral blood monocytes and their cytotoxicity by flow cytometry. We standardized the conditions for parasite labeling to achieve better phagocytosis analysis by setting the ratio of number of parasites per cell as 1 to 2, at incubation time of 6h. The cytotoxicity assessment was performed by the quantification of cells undergoing early/late apoptosis and necrosis using a double labelling platform employing 7AAD for late apoptosis and necrosis analysis and Annexin-V for early apoptosis evaluation. Hemolysis analysis was an additional parameter to test cytotoxicity. Two drugs used on clinic (Amphotericin B and Glucantime®) were used to validate the proposed methodology, and the assay was able to detect their known leishmanicidal activity and immunotoxicity properties. This new predictive assay will contribute to the development of translational medicine strategies in drug discovery for neglected diseases such as leishmaniasis.

Investigation of pyrimidine nucleoside analogues as chemical probes to assess compound effects on the proliferation of Trypanosoma cruzi intracellular parasites.

Trypanosoma cruzi parasites utilise de novo pyrimidine biosynthesis to produce DNA and survive within mammalian host cells. This pathway can be hijacked to assess the replication of intracellular parasites with the exogenous addition of a DNA specific probe. To identify suitable probe compounds for this application, a collection of pyrimidine nucleoside analogues was assessed for incorporation into T. cruzi intracellular amastigote DNA using image-based technology and script-based analysis. Associated mammalian cell toxicity of these compounds was also determined against both the parasite host cells (3T3 cells) and HEK293 cells. Incorporation of 5-ethynyl-2'-deoxyuridine (EdU) into parasite DNA was the most effective of the probes tested, with minimal growth inhibition observed following either two or four hours EdU exposure. EdU was subsequently utilised as a DNA probe, followed by visualisation with click chemistry to a fluorescent azide, to assess the impact of drugs and compounds with previously demonstrated activity against T. cruzi parasites, on parasite replication. The inhibitory profiles of these molecules highlight the benefit of this approach for identifying surviving parasites post-treatment in vitro and classifying compounds as either fast or slow-acting. F-ara-EdU resulted in <50% activity observed against T. cruzi amastigotes following 48 hours incubation, at 73 µM. Collectively, this supports the further development of pyrimidine nucleosides as chemical probes to investigate replication of the parasite T. cruzi.

Protective Cellular Immune Response Induction for Cutaneous Leishmaniasis by a New Immunochemotherapy Schedule.

The palladacycle complex DPPE 1.2 was previously shown to inhibit Leishmania (Leishmania) amazonensis infection in vitro and in vivo. The present study aimed to evaluate the effect of DPPE 1.2 associated with a recombinant cysteine proteinase, rLdccys1, and the adjuvant Propionibacterium acnes on L. (L.) amazonensis infection in two mouse strains, BALB/c, and C57BL/6. Treatment with this association potentiated the leishmanicidal effect of DPPE 1.2 resulting in a reduction of parasite load in both strains of mice which was higher compared to that found in groups treated with either DPPE 1.2 alone or associated with P. acnes or rLdccys1. The reduction of parasite load in both mice strains was followed by immunomodulation mediated by an increase of memory CD4+ and CD8+ T lymphocytes, IFN-γ levels and reduction of active TGF-ß in treated animals. No infection relapse was observed 1 month after the end of treatment in mice which received DPPE 1.2 associated with rLdccys1 or rLdccys1 plus P. acnes in comparison to that exhibited by animals treated with DPPE 1.2 alone. Evaluation of serum levels of AST, ALT, urea, and creatinine showed no alterations among treated groups, indicating that this treatment schedule did not induce hepato or nephrotoxicity. These data indicate the potential use of this association as a therapeutic alternative for cutaneous leishmaniasis caused by L. (L) amazonensis.

Drug screening of food and drug administration-approved compounds against Babesia bovis in vitro.

Babesia (B.) bovis is one of the main etiological agents of bovine babesiosis, causes serious economic losses to the cattle industry. Control of bovine babesiosis has been hindered by the limited treatment selection for B. bovis, thus, new options are urgently needed. We explored the drug library and unbiasedly screened 640 food and drug administration (FDA) approved drug compounds for their inhibitory activities against B. bovis in vitro. The initial screening identified 13 potentially effective compounds. Four potent compounds, namely mycophenolic acid (MPA), pentamidine (PTD), doxorubicin hydrochloride (DBH) and vorinostat (SAHA) exhibited the lowest IC50 and then selected for further evaluation of their in vitro efficacies using viability, combination inhibitory and cytotoxicity assays. The half-maximal inhibitory concentration (IC50) values of MPA, PTD, DBH, SAHA were 11.38 ± 1.66, 13.12 ± 4.29, 1.79 ± 0.15 and 45.18 ± 7.37 µM, respectively. Of note, DBH exhibited IC50 lower than that calculated for the commonly used antibabesial drug, diminazene aceturate (DA). The viability result revealed the ability of MPA, PTD, DBH, SAHA to prevent the regrowth of treated parasite at 4 × and 2 × of IC50. Antagonistic interactions against B. bovis were observed after treatment with either MPA, PTD, DBH or SAHA in combination with DA. Our findings indicate the richness of FDA approved compounds by novel potent antibabesial candidates and the identified potent compounds especially DBH might be used for the treatment of animal babesiosis caused by B. bovis.

In Vitro Screening to Identify Anti-Toxoplasma Compounds and In Silico Modeling for Bioactivities and Toxicity.

Toxoplasmosis, which affects more than a billion people worldwide, is a common parasitic infection caused by the obligate intracellular parasite, Toxoplasmagondii. Current treatment strategies have several limitations, including unwanted side effects and poor efficacy. Therefore, newer therapies are needed for toxoplasmosis. Drug repurposing and screening of a vast array of natural and/or synthetic compounds is a viable option for antiparasitic drug discovery. In this study, we screened 62 compounds comprising natural products (NPs) and FDA-approved (FDA) drugs, to identify the hit compounds that suppress the growth of T. gondii. To determine the parasite inhibitory potential of the compounds, host mammalian cells were infected with a transgenic T. gondii strain, and the viability of the parasite was evaluated by luminescence. Of the 62 compounds, tubericidin, sulfuretin, peruvoside, resveratrol, narasin and diacetoxyscirpenol of the natural product isolates, as well as bortezonib, 10-Hydroxycamtothecin, mebendazole, niflumic acid, clindamycin HCl, mecamylamine, chloroquine, mitomycin C, fenbendazole, daunorubicin, atropine, and cerivastatin of FDA molecules were identified as "hits" with ≥ 40 percent anti-parasite action. Additionally, mitomycin C, radicicol, naringenin, gitoxigenin, menadione, botulin, genistin, homobutein, and gelsemin HCl of the natural product isolates, as well as lomofungin, cyclocytidine, prazosin HCl, cerivastatin, camptothecin, flufenamic acid, atropine, daunorubicin, and fenbendazole of the FDA compounds exhibited cytotoxic activity, reducing the host viability by ≥ 30 percent. Our findings not only support the prospects of drug repurposing, but also indicate that screening a vast array of molecules may provide viable sources of alternative therapies for parasitic infection.

Efficacy of lapachol on treatment of cutaneous and visceral leishmaniasis.

Leishmaniasis is one of the most important neglected diseases worldwide. It is a life-threatening disease and causes significant morbidity, long-term disability, and early death. Treatment involves disease control or use of intervention measures, although the currently used drugs require long-lasting therapy, and display toxicity and reduced efficacy. The use of natural products isolated from plants, such as lapachol, an abundant naphthoquinone naturally occurring in South American Handroanthus species (Tabebuia, Bignoniaceae), is a promising option for the treatment of leishmaniasis. In this study, we investigated the leishmanicidal activity of lapachol in vitro and in vivo against Leishmania infantum and L. amazonensis, causative agents of visceral and cutaneous leishmaniasis, respectively. Low cytotoxicity in HepG2 cells (3405.8 ±â€¯261.33 µM), good anti-Leishmania activity, and favorable selectivity indexes (SI) against promastigotes of both L. amazonensis (IC50 = 79.84 ±â€¯9.10 µM, SI = 42.65) and L. infantum (IC50 = 135.79 ±â€¯33.04 µM, SI = 25.08) were observed. Furthermore, anti-Leishmania activity assays performed on intracellular amastigotes showed good activity for lapachol (IC50 = 191.95 µM for L. amazonensis and 171.26 µM for L. infantum). Flow cytometric analysis demonstrated that the cytotoxic effect of lapachol in Leishmania promastigotes was caused by apoptosis-like death. Interestingly, the in vitro leishmanicidal effect of lapachol was confirmed in vivo in murine models of visceral and cutaneous leishmaniasis, as lapachol (25 mg/kg oral route for 24 h over 10 days) was able to significantly reduce the parasitic load in skin lesions, liver, and spleen, similar to amphotericin B, the reference drug. These results reinforce the therapeutic potential of lapachol, which warrants further investigations as an anti-leishmaniasis therapeutic.

Discovery of benzimidazole-based Leishmania mexicana cysteine protease CPB2.8ΔCTE inhibitors as potential therapeutics for leishmaniasis.

Chemotherapy is currently the only effective approach to treat all forms of leishmaniasis. However, its effectiveness is severely limited due to high toxicity, long treatment length, drug resistance, or inadequate mode of administration. As a consequence, there is a need to identify new molecular scaffolds and targets as potential therapeutics for the treatment of this disease. We report a small series of 1,2-substituted-1H-benzo[d]imidazole derivatives (9a-d) showing affinity in the submicromolar range (Ki  = 0.15-0.69 µM) toward Leishmania mexicanaCPB2.8ΔCTE, one of the more promising targets for antileishmanial drug design. The compounds confirmed activity in vitro against intracellular amastigotes of Leishmania infantum with the best result being obtained with derivative 9d (IC50  = 6.8 µM), although with some degree of cytotoxicity (CC50  = 8.0 µM on PMM and CC50  = 32.0 µM on MCR-5). In silico molecular docking studies and ADME-Tox properties prediction were performed to validate the hypothesis of the interaction with the intended target and to assess the drug-likeness of these derivatives.

Anti-plasmodial activities of Combretum molle (Combretaceae) [Zwoo] seed extract in Swiss albino mice.

OBJECTIVE: Objective of the study was to evaluate in vivo anti-plasmodial activities of Combretum molle seed extract. METHODS: As a standard protocol, initially the acute toxicity of the plant seed extract was checked following single administration of crude seed extract of the plant at doses 500, 1000 and 2000 mg/kg. This was followed by evaluation of anti-plasmodial activity of crude seed extract of the plant following a 4 days suppressive test. RESULTS: In acute toxicity study sign of toxicity was not observed. Also physical and behavioural changes were not detected. The crude seed extract of C. molle showed, 63.5% parasite suppression in mice infected with Plasmodium berghei ANKA (PbA) murine parasite and treated with 250 mg/kg of seed extract of C. molle. Relative survival time of mice treated with 250 mg/kg showed significantly longer survival than the negative control, while lower than mice treated with the standard drug, chloroquine. The plant seed extract on day-4 post-infection showed significant (P < 0.05) protection against body weight reduction, high body temperature and hemolysis of RBC at relatively lower doses. At optimum dose the crude extract of C. molle seed has good chemo-suppressive activity against PbA parasite and improved some clinical symptoms of malaria in mice.

Embryonic toxico-pathological effects of meglumine antimoniate using a chick embryo model.

Leishmaniasis is one of the diverse and neglected tropical diseases. Embryo-toxicity of drugs has always been a major concern. Chick embryo is a preclinical model relevant in the assessment of adverse effects of drugs. The current study aimed to assess embryonic histopathological disorders and amniotic fluid biochemical changes following meglumine antimoniate treatment. The alteration of vascular branching pattern in the chick's extra-embryonic membrane and exploration of molecular cues to early embryonic vasculogenesis and angiogenesis were also quantified. Embryonated chicken eggs were treated with 75 or 150 mg/kg of meglumine antimoniate. Embryo malformations, growth retardation and haemorrhages on the external body surfaces were accompanied by histopathological lesions in the brain, kidney, liver and heart in a dose-dependent manner. Significant rise occurred in the biochemical indices of alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase and amylase in the amniotic fluid. Quantification of the extra-embryonic membrane vasculature showed that the anti-angiogenic and anti-vasculogenic effects of the drug were revealed by a significant decrease in fractal dimension value and mean capillary area. The relative expression levels of vascular endothelial growth factor A and vascular endothelial growth factor receptor 2 mRNA also significantly reduced. Concerns of a probable teratogenicity of meglumine antimoniate were established by data presented in this study. It is concluded that tissue lesions, amniotic fluid disturbance, altered early extra-embryonic vascular development and gene expression as well as the consecutive cascade of events, might eventually lead to developmental defects in embryo following meglumine antimoniate treatment. Therefore, the use of meglumine antimoniate during pregnancy should be considered as potentially embryo-toxic. Hence, physicians should be aware of such teratogenic effects and limit the use of this drug during the growing period of the fetus, particularly in rural communities. Further pharmaceutical investigations are crucial for planning future strategies.

Kostchyienones A and B, new antiplasmodial and cytotoxicity of limonoids from the roots of Pseudocedrela kotschyi (Schweinf.) Harms.

Two new limonoids, kostchyienones A (1) and B (2), along with 12 known compounds 3-14 were isolated from the roots of Pseudocedrela kostchyi. Compound (7) was isolated for the first time from a natural source. Their structures were elucidated on the basis of spectroscopic evidence. Compounds 1-6 and 13-14 gave IC50 values ranging from 0.75 to 5.62 µg/mL for antiplasmodial activity against chloroquine-sensitive (Pf3D7) and chloroquine-resistant (PfINDO) strains of Plasmodium falciparum. Compound 5 showed moderate potential cytotoxicity against the HEK239T cell line with an IC50 value of 22.2±0.89 µg/mL. The antiplasmodial efficacy of the isolated compounds supports the medicinal value of this plant and its potential to provide novel antimalarial drugs.

Plants of Brazilian restingas with tripanocide activity against Trypanosoma cruzi strains.

Chagas disease is caused by the Trypanosoma cruzi affecting millions of people, and widespread throughout Latin America. This disease exhibits a problematic chemotherapy. Benznidazole, which is the drug currently used as standard treatment, lamentably evokes several adverse reactions. Among other options, natural products have been tested to discover a novel therapeutic drug for this disease. A lot of plants from the Brazilian flora did not contain studies about their biological effects. Restinga de Jurubatiba from Brazil is a sandbank ecosystem poorly studied in relation to plant biological activity. Thus, three plant species from Restinga de Jurubatiba were tested against in vitro antiprotozoal activity. Among six extracts obtained from leaves and stem parts and 2 essential oils derived from leave parts, only 3 extracts inhibited epimastigote proliferation. Substances present in the extracts with activity were isolated (quercetin, myricetin, and ursolic acid), and evaluated in relation to antiprotozoal activity against epimastigote Y and Dm28 Trypanosoma cruzi strains. All isolated substances were effective to reduce protozoal proliferation. Essentially, quercetin and myricetin did not cause mammalian cell toxicity. In summary, myricetin and quercetin molecule can be used as a scaffold to develop new effective drugs against Chagas's disease.

Acanthamoebicidal activity of periglaucine A and betulinic acid from Pericampylus glaucus (Lam.) Merr. in vitro.

Acanthamoeba species are pathogenic protozoa which account for amoebic keratitis, conjunctivitis and granulomatous amoebic encephalitis. These amoebae form cysts which resist drugs and more effective acanthamoebicidal agents are needed. Medicinal plants could be useful in improving the current treatment strategies for Acanthamoeba infections. In the present study, we examined the amoebicidal effects of Pericampylus glaucus (Lam.) Merr., a medicinal plant used for the treatment of conjunctivitis in Malaysia. Pathogenic Acanthamoeba triangularis were isolated from environmental water samples and treated with different concentrations of fractions obtained from Pericampylus glaucus (Lam.) Merr. as well as main constituents for 24-72 h. Chlorhexidine was used as a reference drug. Ethanol fraction of stem showed significant (p < 0.05) inhibition of trophozoites survival. Betulinic acid and periglaucine A from this plant at 100 µg/mL inhibited more than 70% survival of both cysts and trophozoites. The calculated therapeutic index for betulinic acid and periglaucine A was 170 and 1.5 for trophozoites stage and 3.75 and 8.5 for cysts stage. The observed amoebicidal efficacies indicate the beneficial aspects of this plant in the treatment of Acanthamoeba infection. Periglaucine A could also be of value for the treatment of Acanthamoeba infection.

Piqueria trinervia as a source of metabolites against Giardia intestinalis.

CONTEXT: Piqueria trinervia Cav. (Asteraceae) is a plant species with a long history in traditional medicine to cure diarrhoea and other digestive disorders. OBJECTIVE: The study investigates the antigiardial activity of piquerol, trinervinol, red oil and two fractions (F1 and F2) from P. trinervia. MATERIALS AND METHODS: P. trinervia was collected in the Ajusco in Mexico City. Aerial parts were ground and mixed with water to obtain the extract, which was treated with dichloromethane to isolate piquerol and trinervinol (P & T). Remnants were the red oil, fractions 1 and 2 (RO, F1 & F2). Trophozoites of Giardia intestinalis were treated with P, T, RO, F1 and F2 at different concentrations (0.78-200 µg/mL) for 48 h. Antigiardial activity was measured using the methylene blue reduction, and the cytotoxicity assayed on human fibroblasts and Vero cells by reduction of tetrazolium salts. RESULTS: Trinervinol and piquerol showed antigiardial activity with an IC50 = 2.03 and 2.42 µg/mL, and IC90 = 13.03 and 8.74 µg/mL, respectively. The concentrations of trinervinol (CC50 = 590 µg/mL) and piquerol (CC50 = 501 µg/mL) were not cytotoxic to human fibroblasts. CONCLUSIONS: Compounds from P. trinervia showed antigiardial activity; to enhance this activity, piquerol and trinervinol can be chemically modified.

Cytotoxicity and anti-Leishmania amazonensis activity of Citrus sinensis leaf extracts.

CONTEXT: Leishmania amazonensis is the main agent of diffuse cutaneous leishmaniasis, a disease characterized by lesional polymorphism and the commitment of skin surface. Previous reports demonstrated that the Citrus genus possess antimicrobial activity. OBJECTIVE: This study evaluated the anti-L. amazonensis activity of Citrus sinensis (L.) Osbeck (Rutaceae) extracts. MATERIALS AND METHODS: Citrus sinensis dried leaves were subjected to maceration with hexane (CH), ethyl acetate (CEA), dichloromethane/ethanol (CD/Et - 1:1) or ethanol/water (CEt/W - 7:3). Leishmania amazonensis promastigotes were treated with C. sinensis extracts (1-525 µg/mL) for 120 h at 27 °C. Ultrastructure alterations of treated parasites were evaluated by transmission electron microscopy. Cytotoxicity of the extracts was assessed on RAW 264.7 and J774.G8 macrophages after 48-h treatment at 37 °C using the tetrazolium assay. In addition, Leishmania-infected macrophages were treated with CH and CD/Et (10-80 µg/mL). RESULTS: CH, CD/Et and CEA displayed antileishmanial activity with 50% inhibitory activity (IC50) of 25.91 ± 4.87, 54.23 ± 3.78 and 62.74 ± 5.04 µg/mL, respectively. Parasites treated with CD/Et (131.2 µg/mL) presented severe alterations including mitochondrial swelling, lipid body formation and intense cytoplasmic vacuolization. CH and CD/Et demonstrated cytotoxic effects similar to that of amphotericin B in the anti-amastigote assays (SI of 2.16, 1.98 and 1.35, respectively). Triterpene amyrins were the main substances in CH and CD/Et extracts. In addition, 80 µg/mL of CD/Et reduced the number of intracellular amastigotes and the percentage of infected macrophages in 63% and 36%, respectively. CONCLUSION: The results presented here highlight C. sinensis as a promising source of antileishmanial agents.

New iminodibenzyl derivatives with anti-leishmanial activity.

Leishmaniasis is an infection caused by protozoa of the genus Leishmania and transmitted by sandflies. Current treatments are expensive and time-consuming, involving Sb(V)-based compounds, lipossomal amphotericin B and miltefosine. Recent studies suggest that inhibition of trypanothione reductase (TR) could be a specific target in the development of new drugs because it is essential and exclusive to trypanosomatids. This work presents the synthesis and characterization of new iminodibenzyl derivatives (dado) with ethylenediamine (ea), ethanolamine (en) and diethylenetriamine (dien) and their copper(II) complexes. Computational methods indicated that the complexes were highly lipophilic. Pro-oxidant activity assays by oxidation of the dihydrorhodamine (DHR) fluorimetric probe showed that [Cu(dado-ea)]2+ has the highest rate of oxidation, independent of H2O2 concentration. The toxicity to L. amazonensis promastigotes and RAW 264,7 macrophages was assessed, showing that dado-en was the most active new compound. Complexation to copper did not have an appreciable effect on the toxicity of the compounds.

Insight into the mechanism of action of temporin-SHa, a new broad-spectrum antiparasitic and antibacterial agent.

Antimicrobial peptides (AMPs) are promising drugs to kill resistant pathogens. In contrast to bacteria, protozoan parasites, such as Leishmania, were little studied. Therefore, the antiparasitic mechanism of AMPs is still unclear. In this study, we sought to get further insight into this mechanism by focusing our attention on temporin-SHa (SHa), a small broad-spectrum AMP previously shown to be active against Leishmania infantum. To improve activity, we designed analogs of SHa and compared the antibacterial and antiparasitic mechanisms. [K3]SHa emerged as a highly potent compound active against a wide range of bacteria, yeasts/fungi, and trypanosomatids (Leishmania and Trypanosoma), with leishmanicidal intramacrophagic activity and efficiency toward antibiotic-resistant strains of S. aureus and antimony-resistant L. infantum. Multipassage resistance selection demonstrated that temporins-SH, particularly [K3]SHa, are not prone to induce resistance in Escherichia coli. Analysis of the mode of action revealed that bacterial and parasite killing occur through a similar membranolytic mechanism involving rapid membrane permeabilization and depolarization. This was confirmed by high-resolution imaging (atomic force microscopy and field emission gun-scanning electron microscopy). Multiple combined techniques (nuclear magnetic resonance, surface plasmon resonance, differential scanning calorimetry) allowed us to detail peptide-membrane interactions. [K3]SHa was shown to interact selectively with anionic model membranes with a 4-fold higher affinity (KD = 3 x 10-8 M) than SHa. The amphipathic α-helical peptide inserts in-plane in the hydrophobic lipid bilayer and disrupts the acyl chain packing via a detergent-like effect. Interestingly, cellular events, such as mitochondrial membrane depolarization or DNA fragmentation, were observed in L. infantum promastigotes after exposure to SHa and [K3]SHa at concentrations above IC50. Our results indicate that these temporins exert leishmanicidal activity via a primary membranolytic mechanism but can also trigger apoptotis-like death. The many assets demonstrated for [K3]SHa make this small analog an attractive template to develop new antibacterial/antiparasitic drugs.