Betania Barros Cota

Antifungal activity of extracts of some plants used in Brazilian traditional medicine against the pathogenic fungus Paracoccidioides brasiliensis

Paracoccidioidomycosis (PCM) is a systemic granulomatous disease caused by Paracoccidioides brasiliensis Almeida (Onygenales) that requires 1–2 years of treatment. In the absence of drug therapy, the disease is usually fatal, highlighting the need for the identification of safer, novel, and more effective antifungal compounds. With this need in mind, several plants employed in Brazilian traditional medicine were assayed on P. brasiliensis and murine macrophages. Extracts were prepared from 10 plant species: Inga spp. Mill. (Leguminosae), Schinus terebinthifolius Raddi (Anacardiaceae), Punica granatum L. (Punicaceae), Alternanthera brasiliana Kuntze (Amaranthaceae), Piper regnellii CDC. (Piperaceae), P. abutiloides Kunth (Piperaceae), Herissantia crispa L. Briz. (Malvaceae), Rubus urticaefolius Poir (Rosaceae), Rumex acetosa L. (Polygonaceae), and Baccharis dracunculifolia DC. (Asteraceae). Hexane fractions from hydroalcoholic extracts of Piper regnellii and Baccharis dracunculifolia were the most active against the fungus, displaying minimum inhibitory concentration (MIC) values of 7.8 μg/mL and 7.8–30 μg/mL, respectively. Additionally, neither of the extracts exhibited any apparent cytotoxic effects on murine macrophages at 20 μg/mL. Analyses of these fractions using gas chromatography-mass spectrometry (GC-MS) showed that the major components of B. dracunculifolia were ethyl hydrocinnamate (14.35%) and spathulenol (16.02%), while the major components of the hexane fraction of Piper regnellii were 1-methoxy-4-(1-propenyl) benzene (21.94%) and apiol (21.29%). The activities of these fractions against P. brasiliensis without evidence of cytotoxicity to macrophages justify their investigation as a potential source of new chemical agents for the treatment of PCM.

Leishmanicidal Metabolites from Cochliobolus sp., an Endophytic Fungus Isolated from Piptadenia adiantoides (Fabaceae)

Protozoan parasites belonging to genera Leishmania and Trypanosoma are the etiological agents of severe neglected tropical diseases (NTDs) that cause enormous social and economic impact in many countries of tropical and sub-tropical areas of the world. In our screening program for new drug leads from natural sources, we found that the crude extract of the endophytic fungus Cochliobolus sp. (UFMGCB-555) could kill 90% of the amastigote-like forms of Leishmania amazonensis and inhibit by 100% Ellmans reagent reduction in the trypanothione reductase (TryR) assay, when tested at 20 µg mL−1. UFMGCB-555 was isolated from the plant Piptadenia adiantoides J.F. Macbr (Fabaceae) and identified based on the sequence of the internally transcribed spacer (ITS) regions of its ribosomal DNA. The chromatographic fractionation of the extract was guided by the TryR assay and resulted in the isolation of cochlioquinone A and isocochlioquinone A. Both compounds were active in the assay with L. amazonensis, disclosing EC50 values (effective concentrations required to kill 50% of the parasite) of 1.7 µM (95% confidence interval = 1.6 to 1.9 µM) and 4.1 µM (95% confidence interval = 3.6 to 4.7 µM), respectively. These compounds were not active against three human cancer cell lines (MCF-7, TK-10, and UACC-62), indicating some degree of selectivity towards the parasites. These results suggest that cochlioquinones are attractive lead compounds that deserve further investigation aiming at developing new drugs to treat leishmaniasis. The findings also reinforce the role of endophytic fungi as an important source of compounds with potential to enter the pipeline for drug development against NTDs.

Altenusin, a biphenyl isolated from the endophytic fungus Alternaria sp., inhibits trypanothione reductase from Trypanosoma cruzi

Parasitic protozoan species belonging to the genera Trypanosoma and Leishmania are the etiological agents of several diseases in tropical areas of the world, for which there is an urgent need for effective and affordable treatment. In this regard, we are screening the Brazilian biodiversity, especially its flora and mycota, for natural products that could serve as leads for drug development against these diseases. Trypanothione reductase (TR) is an enzyme involved in the protection of Trypanosoma and Leishmania species against oxidative stress, and is considered to be a validated drug target. The endophytic fungus Alternaria sp. (UFMGCB55) was isolated from the plant Trixis vauthieri DC (Asteraceae), known to contain trypanocidal compounds. The organic extract of the culture of Alternaria sp. was able to inhibit TR by 99%, when tested at 20 microg mL(-1). Fractionation of the extract identified altenusin, a biphenyl derivative with an IC50 value of 4.3+/-0.3 microM in the TR assay. This compound is the first in its class to have shown TR inhibitory activity, opening new perspectives for the design of more effective derivatives that could serve as drug leads for new chemotherapeutic agents to treat trypanosomiasis and leishmaniasis.

Activity of compounds isolated from Baccharis dracunculifolia D.C. (Asteraceae) against Paracoccidioides brasiliensis

Paracoccidioidomycosis is a prevalent systemic mycosis in Latin America which requires prolonged treatment with highly toxic antifungals. Baccharis dracunculifolia is a medicinal plant in Brazil that is a candidate in the search for new drugs. Fractions of the hexanic extracts were obtained using chromatographic procedures and assessed using an antifungal assay with Paracoccidioides brasiliensis (Pb18), tumor cell lines and amastigote forms of Leishmania, L. amazonensis. Four compounds were isolated, i.e., ursolic acid (1), methyl linolenate (2), caryophyllene oxide (3), and trans-nerolidol (4). Compounds 2, 3 and 4 displayed antifungal activity against four isolates of Paracocci dioides with MIC values ranging from 3.9-250 μg/ml. Only caryophyllene oxide showed differences in the MIC values against Pb18 when the medium was supplemented with ergosterol, which suggested that the compound interacts with ergosterol. Ursolic acid was active in the cytotoxic assays and showed leishmanicidal activity. Scanning electron microscopy demonstrated that compounds 2, 3 and 4 decreased the cell size and produced an irregular cell wall surface on P. brasiliensis cells. The present results showed the biological activities of the isolated compounds and revealed that these compounds may affect the cell surface and growth of P. brasiliensis isolates.

New cassane diterpenes from Caesalpinia echinata.

An investigation of the ethanolic extract from stems of Caesalpinia echinata Lam (Leguminosae-Caesalpinioideae) led to the isolation of five new cassane diterpenes along with known lambertianic acid. Their structures were determined based on spectroscopic methods. A preliminary study on leishmanicidal activity demonstrated that compounds 1, 2 and 6 were found to inhibit the growth of amastigote-like forms of Leishmania amazonensis without affecting mononuclear cells obtained from human peripheral blood.

Antifungal activity of altenusin isolated from the endophytic fungus Alternaria sp. against the pathogenic fungus Paracoccidioides brasiliensis

BACKGROUND Altenusin is a biphenyl derivative isolated from different species of fungi, which presents several biological activities. AIMS We report the antifungal activity of the altenusin isolated from the endophytic fungus Alternaria sp., against clinical isolates of Paracoccidioides brasiliensis, and its action on cell walls of P. brasiliensis and the nonpathogenic yeast Schizosaccharomyces pombe. METHODS In vitro antifungal activity of altenusin was evaluated using the broth microdilution method against 11 strains of P. brasiliensis and one strain of S. pombe. The effects of the altenusin on the cell wall were estimated using the sorbitol protection assay. RESULTS The altenusin presented strong activity against P. brasiliensis with MIC values ranging between 1.9 and 31.2 μg/ml, and 62.5 μg/ml for S. pombe. Our results demonstrated that the MIC values for altenusin were increased for P. brasiliensis Pb18 and for S. pombe when the medium was supplemented with sorbitol. Additionally, S. pombe cells treated with altenusin were more rounded in shape than untreated cells. CONCLUSIONS Altenusin showed activity against clinical strains of P. brasiliensis at the concentration tested, and this compound probably affects fungal cell walls. These findings suggest that altenusin could act through the inhibition of cell wall synthesis or assembly in P. brasiliensis and S. pombe, and could be considered as a lead compound for the design of new antifungals.

Antifungal activity of trichothecenes from Fusarium sp. against clinical isolates of Paracoccidioides brasiliensis

Paracoccidioidomycosis (PCM), a human mycosis caused by the dimorphic fungus Paracoccidioides brasiliensis, is a serious public health problem in several countries of Latin America. In our search we found that the crude extract of the endophytic fungus UFMGCB 551 was able to inhibit several clinical strains of P. brasiliensis, and was also active in the bioautographic assay against Cladosporium sphaerospermum. The endophytic fungus UFMGCB 551 was isolated from the plant Piptadenia adiantoides J.F. Macbr (Fabaceae). The fungus was identified as Fusarium sp. based on its macro‐ and micro‐morphology, and on the sequence of the internally transcribed spacer regions (ITS) of its rRNA gene. The chromatographic fractionation of the fungal extract was guided by the bioautographic assay to afford three known trichothecene mycotoxins: T2‐toxin (1) and a mixture of 8‐n‐butyrylneosolaniol (2) and 8‐isobutyrylsolaniol (3). The minimal inhibitory concentrations (MIC) of the these compounds against eleven clinical strains of P. brasiliensis were evaluated and found to be in the range between 75 and 640 nmol l−1 for 1 and 160–640 nmol l−1 for the mixture of 2 and 3.

Flavonoids from leaves of Mauritia flexuosa

The chromatographic fractionation of the Mauritia flexuosa L. f., Arecaceae, leaves extract, a plant known by the name of buriti palm tree, resulted in the isolation of six flavonoids: tricin-7-O-rutinoside, apigenin-6-C-arabinoside, 8-C-glucoside (isoschaftoside), kaempferol-3-O-rutinoside (nicotii¬‚orine), quercetin-3-O-rutinoside (rutin), luteolin-8-C-glucoside (orientin) and luteolin-6-C-glucoside (isoorientin). The flavonoids were found out and previously reported as constituents of the Arecaceae family plants, but the occurrence of C-glucoside flavonoids, in the species being analyzed, is described for the first time on this study. The structural elucidations of all of the isolated compounds were performed by means of the comparison of their spectral data (1H and 13C NMR, UV and ESI-MS) with those ones of the literature.

A potent trypanocidal component from the fungus Lentinus strigosus inhibits trypanothione reductase and modulates PBMC proliferation

The fungus Lentinus strigosus (Pegler 1983) (Polyporaceae, basidiomycete) was selected in a screen for inhibitory activity on Trypanosoma cruzi trypanothione reductase (TR). The crude extract of L. strigosus was able to completely inhibit TR at 20 microg/ml. Two triquinane sesquiterpenoids (dihydrohypnophilin and hypnophilin), in addition to two panepoxydol derivatives (neopanepoxydol and panepoxydone), were isolated using a bioassay-guided fractionation protocol. Hypnophilin and panepoxydone displayed IC50 values of 0.8 and 38.9 microM in the TR assay, respectively, while the other two compounds were inactive. The activity of hypnophilin was confirmed in a secondary assay with the intracellular amastigote forms of T. cruzi, in which it presented an IC50 value of 2.5 micro M. Quantitative flow cytometry experiments demonstrated that hypnophilin at 4 microM also reduced the proliferation of human peripheral blood monocluear cells (PBMC) stimulated with phytohemaglutinin, without any apparent interference on the viability of lymphocytes and monocytes. As the host immune response plays a pivotal role in the adverse events triggered by antigen release during treatment with trypanocidal drugs, the ability of hypnophilin to kill the intracellular forms of T. cruzi while modulating human PBMC proliferation suggests that this terpenoid may be a promising prototype for the development of new chemotherapeutical agents for Chagas disease.

Bioactive endophytic fungi isolated from Caesalpinia echinata Lam. (Brazilwood) and identification of beauvericin as a trypanocidal metabolite from Fusarium sp.

Aiming to identify new sources of bioactive secondary metabolites, we isolated 82 endophytic fungi from stems and barks of the native Brazilian tree Caesalpinia echinata Lam. (Fabaceae). We tested their ethyl acetate extracts in several in vitro assays. The organic extracts from three isolates showed antibacterial activity against Staphylococcus aureus and Escherichia coli [minimal inhibitory concentration (MIC) 32-64 μg/mL]. One isolate inhibited the growth of Salmonella typhimurium (MIC 64 μg/mL) and two isolates inhibited the growth of Klebsiella oxytoca (MIC 64 μg/mL), Candida albicans and Candida tropicalis (MIC 64-128 μg/mL). Fourteen extracts at a concentration of 20 μg/mL showed antitumour activities against human breast cancer and human renal cancer cells, while two isolates showed anti-tumour activities against human melanoma cancer cells. Six extracts were able to reduce the proliferation of human peripheral blood mononuclear cells, indicating some degree of selective toxicity. Four isolates were able to inhibit Leishmania (Leishmania) amazonensis and one isolate inhibited Trypanosoma cruzi by at least 40% at 20 μg/mL. The trypanocidal extract obtained from Fusarium sp. [KF611679] culture was subjected to bioguided fractionation, which revealed beauvericin as the compound responsible for the observed toxicity of Fusarium sp. to T. cruzi. This depsipeptide showed a half maximal inhibitory concentration of 1.9 μg/mL (2.43 μM) in a T. cruzi cellular culture assay.