Anti-Leishmania compounds can be screened using Leishmania spp. expressing red fluorescence (tdTomato).

The main challenges associated with leishmaniasis chemotherapy are drug toxicity, the possible emergence of resistant parasites, and a limited choice of therapeutic agents. Therefore, new drugs and assays to screen and detect novel active compounds against leishmaniasis are urgently needed. We thus validated Leishmania braziliensis (Lb) and Leishmania infantum (Li) that constitutively express the tandem tomato red fluorescent protein (tdTomato) as a model for large-scale screens of anti-Leishmania compounds. Confocal microscopy of Lb and Li::tdTomato revealed red fluorescence distributed throughout the entire parasite, including the flagellum, and flow cytometry confirmed that the parasites emitted intense fluorescence. We evaluated the infectivity of cloned promastigotes and amastigotes constitutively expressing tdTomato, their growth profiles in THP-1 macrophages, and susceptibility to trivalent antimony, amphotericin, and miltefosine in vitro. The phenotypes of mutant and wild-type parasites were similar, indicating that the constitutive expression of tdTomato did not interfere with the evaluated parameters. We applied our validated model to a repositioning strategy and assessed the susceptibility of the parasites to eight commercially available drugs. We also screened 32 natural plant and fungal extracts and 10 pure substances to reveal new active compounds. The infectivity and Glucantime treatment efficacy of BALB/c mice and golden hamsters infected with Lb and Li::tdTomato mutant lines, respectively, were very similar compared to animals infected with wild-type parasites. Standardizing our methodology would offer more rapid, less expensive, and easier assays to screen of compounds against L. braziliensis and L. infantum in vitro and in vivo. Our method could also enhance the discovery of active compounds for treating leishmaniasis.

Thiosemicarbazone of lapachol acts on cell membrane in Paracoccidioides brasiliensis.

Paracoccidioidomycosis (PCM) is the most prevalent systemic mycosis in Latin American countries. Amphotericin B, sulfonamides, and azoles may be used in the treatment of PCM. However, the high toxicity, prolonged course of treatment, and significant frequency of disease relapse compromise their use. Therefore, there is a need to seek new therapeutic options. We conducted tests with thiosemicarbazone of lapachol (TSC-lap) to determine the antifungal activity and phenotypic effects against several isolates of Paracoccidioides spp. In addition, we evaluated the toxicity against murine macrophages and the ability to enhance phagocytosis. Further, we verified that TSC-lap was active against yeasts but did not show any interaction with the drugs tested. The TSC-lap showed no toxicity at the concentration of 40 µg/ml in macrophages, and at 15.6 µg/ml it could increase the phagocytic index. We observed that this compound induced in vitro ultrastructural changes manifested as withered and broken cells beyond a disorganized cytoplasm with accumulation of granules. We did not observe indications of activity in the cell wall, although membrane damages were noted. We observed alterations in the membrane permeability, culminating in a significant increase in K+ efflux and a gradual loss of the cellular content with increase in the concentration of TSC-lap. In addition, we showed a significant reduction of ergosterol amount in the Pb18 membrane. These data reinforce the possible mechanism of action of this compound to be closely associated with ergosterol biosynthesis and reaffirms the antifungal potential of TSC-lap against Paracoccidioides spp.

Leishmanicidal compounds of Nectria pseudotrichia, an endophytic fungus isolated from the plant Caesalpinia echinata (Brazilwood)

BACKGROUND In a screen of extracts from plants and fungi to detect antileishmanial activity, we found that the ethyl acetate extract of the fungus Nectria pseudotrichia, isolated from the tree Caesalpinia echinata (Brazilwood), is a promising source of bioactive compounds. OBJECTIVES The aims of this study were to isolate and determine the chemical structures of the compounds responsible for the antileishmanial activity of the organic extract from N. pseudotrichia. METHODS Compounds were isolated by chromatographic fractionation using semi-preparative high-performance liquid chromatography, and their chemical structures were determined by analytical and spectral data and by comparison with published data. The antileishmanial activity of the isolated compounds was evaluated in intracellular amastigote forms of Leishmania (Viannia) braziliensis expressing firefly luciferase as reporter gene, and cytotoxicity was determined in Vero and THP-1 mammalian cell lines by MTT assay. FINDINGS Fractionation of the extract yielded seven compounds: 10-acetyl trichoderonic acid A (1), 6′-acetoxy-piliformic acid (2), 5′,6′-dehydropiliformic acid (3), piliformic acid (4), hydroheptelidic acid (5), xylaric acid D (6), and cytochalasin D (7). Compounds 1, 2 and 3 are reported here for the first time. Compounds 1, 2, and 5 were more active, with IC50 values of 21.4, 28.3, and 24.8 µM, respectively, and showed low toxicity to Vero and THP-1 cells. MAIN CONCLUSIONS N. pseudotrichia produces secondary metabolites that are more toxic to intracellular amastigote forms of L. (V.) braziliensis than to mammalian cells.

Leishmanicidal compounds of Nectria pseudotrichia, an endophytic fungus isolated from the plant Caesalpinia echinata (Brazilwood).

BACKGROUND In a screen of extracts from plants and fungi to detect antileishmanial activity, we found that the ethyl acetate extract of the fungus Nectria pseudotrichia, isolated from the tree Caesalpinia echinata (Brazilwood), is a promising source of bioactive compounds. OBJECTIVES The aims of this study were to isolate and determine the chemical structures of the compounds responsible for the antileishmanial activity of the organic extract from N. pseudotrichia. METHODS Compounds were isolated by chromatographic fractionation using semi-preparative high-performance liquid chromatography, and their chemical structures were determined by analytical and spectral data and by comparison with published data. The antileishmanial activity of the isolated compounds was evaluated in intracellular amastigote forms of Leishmania (Viannia) braziliensis expressing firefly luciferase as reporter gene, and cytotoxicity was determined in Vero and THP-1 mammalian cell lines by MTT assay. FINDINGS Fractionation of the extract yielded seven compounds: 10-acetyl trichoderonic acid A (1), 6'-acetoxy-piliformic acid (2), 5',6'-dehydropiliformic acid (3), piliformic acid (4), hydroheptelidic acid (5), xylaric acid D (6), and cytochalasin D (7). Compounds 1, 2 and 3 are reported here for the first time. Compounds 1, 2, and 5 were more active, with IC50 values of 21.4, 28.3, and 24.8 µM, respectively, and showed low toxicity to Vero and THP-1 cells. MAIN CONCLUSIONS N. pseudotrichia produces secondary metabolites that are more toxic to intracellular amastigote forms of L. (V.) braziliensis than to mammalian cells.

The antimicrobial activity of lapachol and its thiosemicarbazone and semicarbazone derivatives.

Lapachol was chemically modified to obtain its thiosemicarbazone and semicarbazone derivatives. These compounds were tested for antimicrobial activity against several bacteria and fungi by the broth microdilution method. The thiosemicarbazone and semicarbazone derivatives of lapachol exhibited antimicrobial activity against the bacteria Enterococcus faecalis and Staphylococcus aureus with minimal inhibitory concentrations (MICs) of 0.05 and 0.10 µmol/mL, respectively. The thiosemicarbazone and semicarbazone derivatives were also active against the pathogenic yeast Cryptococcus gattii (MICs of 0.10 and 0.20 µmol/mL, respectively). In addition, the lapachol thiosemicarbazone derivative was active against 11 clinical isolates of Paracoccidioides brasiliensis, with MICs ranging from 0.01-0.10 µmol/mL. The lapachol-derived thiosemicarbazone was not cytotoxic to normal cells at the concentrations that were active against fungi and bacteria. We synthesised, for the first time, thiosemicarbazone and semicarbazone derivatives of lapachol. The MICs for the lapachol-derived thiosemicarbazone against S. aureus, E. faecalis, C. gattii and several isolates of P. brasiliensis indicated that this compound has the potential to be developed into novel drugs to treat infections caused these microbes.

The antimicrobial activity of lapachol and its thiosemicarbazone and semicarbazone derivatives

Lapachol was chemically modified to obtain its thiosemicarbazone and semicarbazone derivatives. These compounds were tested for antimicrobial activity against several bacteria and fungi by the broth microdilution method. The thiosemicarbazone and semicarbazone derivatives of lapachol exhibited antimicrobial activity against the bacteria Enterococcus faecalis and Staphylococcus aureus with minimal inhibitory concentrations (MICs) of 0.05 and 0.10 µmol/mL, respectively. The thiosemicarbazone and semicarbazone derivatives were also active against the pathogenic yeast Cryptococcus gattii (MICs of 0.10 and 0.20 µmol/mL, respectively). In addition, the lapachol thiosemicarbazone derivative was active against 11 clinical isolates of Paracoccidioides brasiliensis, with MICs ranging from 0.01-0.10 µmol/mL. The lapachol-derived thiosemicarbazone was not cytotoxic to normal cells at the concentrations that were active against fungi and bacteria. We synthesised, for the first time, thiosemicarbazone and semicarbazone derivatives of lapachol. The MICs for the lapachol-derived thiosemicarbazone against S. aureus, E. faecalis, C. gattii and several isolates of P. brasiliensis indicated that this compound has the potential to be developed into novel drugs to treat infections caused these microbes.

Leishmanicidal activities of the extract from Blepharocalyx salicifolius (Kunth) O. Berg, Myrtaceae / Atividade leishmanicida do extrato de Blepharocalyx salicifolius (Kunth) O. Berg, Myrtaceae

Crude ethanolic extracts from Blepharocalyx salicifolius (Kunth) O. Berg, Myrtaceae, was fractioned by Gel Permeation Chromatography, using SephadexTM LH-20 gel. Sixteen fractions were obtained and were supplied to cytotoxicity in vitro assay against Leishmania (L.) amazonensis amastigota cells. It was observed eight cytotoxic fractions against Leishmania (L.) amazonensis amastigota cells at range of 19 to 29 µg.mL-1. Two of them were not citotoxic against human peripheral blood mononuclear cell, with a great potential to isolation of more selective leishmanicidal substances.

O extrato etanólico bruto de Blepharocalyx salicifolius (Kunth) O. Berg, Myrtaceae, foi fracionado por meio de Cromatografia de Permeação em Gel, utilizando-se SephadexTM LH-20. Dezesseis frações foram obtidas e foram submetidas ao ensaio de citotoxicidade in vitro contra células amastigotas de Leishmania (Leishmania) amazonensis. Verificou-se atividade citocida contra células amastigotas de Leishmania (L.) amazonensis em oito frações, a uma concentração de 19 a 29 µg.mL-1. Duas destas frações apresentaram baixa toxicidade para células mononucleares de sangue periférico humano, com grande potencial de isolamento de substâncias leishmanicidas mais seletivas.

Polygodial, the fungitoxic component from the Brazilian medicinal plant Polygonum punctatum

Polygonum punctatum (Polygonaceae) is an herb known in some regions of Brazil as "erva-de-bicho" and is used to treat intestinal disorders. The dichloromethane extract of the aerial parts of this plant showed strong activity in a bioautographic assay with the fungus Cladosporium sphaerospermum. The bioassay-guided chemical fractionation of this extract afforded the sesquiterpene dialdehyde polygodial as the active constituent. The presence of this compound with antibiotic, anti-inflammatory and anti-hyperalgesic properties in "erva-de-bicho" may account for the effects attributed by folk medicine to this plant species

Biological screening of Brazilian medicinal plants

In this study, we screened sixty medicinal plant species from the Brazilian savanna ("cerrado") that could contain useful compounds for the control of tropical diseases. The plant selection was based on existing ethnobotanic information and interviews with local healers. Plant extracts were screened for: (a) molluscicidal activity against Biomphalaria glabrata, (b) toxicity to brine shrimp (Artemia salina L.), (c) antifungal activity in the bioautographic assay with Cladosporium sphaerospermum and (d) antibacterial activity in the agar diffusion assay against Staphylococcus aureus, Escherichia coli, Bacillus cereus and Pseudomonas aeruginosa. Forty-two species afforded extracts that showed some degree of activity in one or more of these bioassays.