Fungi of the genus Paracoccidioides are responsible for paracoccidioidomycosis. The occurrence of drug toxicity and relapse in this disease justify the development of new antifungal agents. Compounds extracted from fungal extract have showing antifungal activity. Extracts of 78 fungi isolated from rocks of the Atacama Desert were tested in a microdilution assay against Paracoccidioides brasiliensis Pb18. Approximately 18% (5) of the extracts showed minimum inhibitory concentration (MIC) values≤ 125.0 µg/mL. Among these, extract from the fungus UFMGCB 8030 demonstrated the best results, with an MIC of 15.6 µg/mL. This isolate was identified as Aspergillus felis (by macro and micromorphologies, and internal transcribed spacer, β-tubulin, and ribosomal polymerase II gene analyses) and was grown in five different culture media and extracted with various solvents to optimise its antifungal activity. Potato dextrose agar culture and dichloromethane extraction resulted in an MIC of 1.9 µg/mL against P. brasiliensis and did not show cytotoxicity at the concentrations tested in normal mammalian cell (Vero). This extract was subjected to bioassay-guided fractionation using analytical C18RP-high-performance liquid chromatography (HPLC) and an antifungal assay using P. brasiliensis. Analysis of the active fractions by HPLC-high resolution mass spectrometry allowed us to identify the antifungal agents present in the A. felis extracts cytochalasins. These results reveal the potential of A. felis as a producer of bioactive compounds with antifungal activity.

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.

Annona cornifolia A. St. -Hil. é uma pequena árvore anual perene encontrada no cerrado brasileiro; seu fruto verde é usado popularmente no tratamento de úlceras. As acetogeninas isoladas a partir das sementes de Annona cornifolia anteriormente apresentaram atividade antioxidante. Continuando as nossas investigações sobre as atividades biológicas das acetogeninas, quatro misturas binárias e dez acetogeninas bis-tetra-hidrofurânicas adjacentes puras foram avaliadas quanto às atividades: citotóxica (sobre três linhagens de células tumorais humanas), antifúngica (sobre Paracoccidioides brasiliensis), tripanocida (sobre Trypanosoma cruzi) e leishmanicida (sobre Leishmania amazonensis). As acetogeninas apresentaram atividade citotóxica, confirmando seu uso potencial na terapia anticâncer. Quanto às atividades leishmanicida e tripanocida, foi observada uma inibição de 87% das formas amastigotas de L. amazonensis e 100% de formas amastigotas e tripomastigotas de T. cruzi, quando testadas a uma concentração de 20 µg mL–1. Além disso, seis acetogeninas mostraram maior atividade sobre todas as três cepas testadas de P. brasiliensis que o trimetoprim-sulfametoxazol, um medicamento usado no tratamento da paracoccidioidomicose. Assim, as acetogeninas podem ser uma alternativa no tratamento de uma série de doenças que têm um enorme impacto em milhões de pessoas no mundo. Este trabalho relata pela primeira as atividades antifúngica, leishmanicida e tripanocida para estas acetogeninas.

Annona cornifolia A. St. -Hil. is a small annual perennial tree found in the Brazilian savannah; their green fruit is popularly used in the treatment of ulcers. The acetogenins isolated from the seeds of Annona cornifolia previously showed to possess antioxidant activity. In continuation of our investigations on the biological activities of acetogenins, four binary mixtures and ten pure adjacent bis-tetrahydrofuran annonaceous acetogenins were evaluated: the cytotoxic (against three human tumor cell lines), antifungal (against Paracoccidioides brasiliensis), trypanocidal (against Trypanosoma cruzi) and leishmanicidal (against Leishmania amazonensis) activities. Acetogenins presented cytotoxic activity confirming their potential use in anti-cancer therapy. Regarding leishmanicidal and trypanocidal activities, an inhibition of 87% of L. amazonensis amastigotes and 100% of T. cruzi amastigotes and trypomastigotes was observed, when tested at the concentration of 20 µg mL–1. Moreover, six acetogenins showed more activity against all the three tested isolates of P. brasiliensis than trimethoprim-sulfamethoxazole, a drug used for treating paracoccidioidomycosis. Thus, acetogenins may be an alternative in treating a number of diseases that have a huge impact on millions of people worldwide. This paper reports for the first time the antifungal, leishmanicidal and trypanocidal activities for these acetogenins.

Many phenolic compounds such as xanthones, quinones and coumarins have been isolated from Kielmeyera species; however the presence of flavonoids have been showed in other genera in the Calophylleae tribe as Caraipa, Mesua and Calophyllum. Six known glycosidic flavonoids: quercetin 3-β-O-galactopyranoside (1), quercetin 3-β-O-glucopyranoside (2), quercetin 3-O-α-rhamnoside (3), luteolin 6-C-β-glucopyranoside (4), isovitexin (5), kaempferol 3-O-α-rhamnoside (6) and one triterpene, lupenone (7) were isolated, for the first time, from organic crude extract of Kielmeyera variabilis Mart. & Zucc., Calophyllaceae, leaves. The crude organic extract from K. variabilis leaves exhibited 95% of leishmanidal activity at 20 µg/mL on amastigote-like form of Leishmania (Leishmania) amazonensis in vitro model and only compound 3 showed 40-45% of growth inhibition at concentration ranging from 0.78 to 20 µg/mL. In addition, quercetin 3-O-α-rhamnoside (quercitrin) was found to be the major metabolite. Our results and previous reports suggest that synergistic effects of flavonoid glycosides are the cause of significant leishmanidal activity of the crude organic extract from K. variabilis leaves.

Em nosso trabalho anterior (Fabri et al. 2009), mostramos que diferentes extratos de Mitracarpus frigidus apresentaram atividades antibacteriana, antifúngica e leishmanicida significativas. Com o objetivo de aprofundar o conhecimento sobre essa espécie, esse trabalho objetiva identificar os constituintes químicos e avaliar as atividades biológicas in vitro de seu óleo essencial. Dessa forma, o óleo essencial das partes aéreas de M.frigidus foi obtido por hidrodestilação e analisado por CG/EM. Entre os compostos detectados, 11 foram identificados, sendo linalol e o acetato de eugenol os componentes majoritários. O óleo essencial de M. frigidus exibiu moderada atividade antibacteriana contra Staphyloccocus aureus, Bacillus cereus, Pseudomonas aeruginosa e Enterobacter cloacae (CIM 250 µg/mL). Por outro lado, apresentou forte efeito antifúngico contra Cryptoccocus neoformans (CIM 8 µg/mL) e Candida albicans (CIM 63 µg/mL). Expressiva atividade contra as formas promastigostas de Leishmania major e Leishmania amazonensis com valores CI50 de 47, 2 e 89, 7 µg/mL, respectivamente, também foi observada. Além disso, a atividade antioxidante foi investigada através do ensaio com o radical DPPH e este apresentou uma significativa atividade com CI50 de 38 µg/mL. A citotoxidade contra Artemia salina foi moderada com CL50 de 88 µg/mL. Os resultados aqui apresentados são o primeiro relato sobre a composição química e propriedades biológicas do óleo essencial M. frigidus.

In our previous work (Fabri et al. 2009), we showed that different extracts of Mitracarpus frigidus had significant antibacterial, antifungal and leishmanicidal activities. In order to increase our knowledge about this species, this work assesses the chemical composition and the in vitro biological activity of its essential oil. Thus, the essential oil obtained by hydrodistillation of the aerial parts of M. frigidus was analyzed by GC/MS. Among several compounds detected, 11 were identified, being linalool and eugenol acetate the major components. The essential oil exhibited a moderate antibacterial effect against Staphyloccocus aureus, Bacillus cereus, Pseudomonas aeruginosa and Enterobacter cloacae (MIC 250 µg/mL). On the other hand, it showed a strong antifungal effect against Cryptoccocus neoformans (MIC 8 µg/mL) and Candida albicans (MIC 63 µg/mL). Expressive activity against L. major and L. amazonensis promastigote forms with IC50 values of 47.2 and 89.7 µg/mL, respectively, were also observed. In addition, the antioxidant activity was investigated through DPPH radical-scavenging and showed a significative activity with IC50 of 38 µg/mL. The cytotoxicity against Artemia salina was moderate with LC50 of 88 µg/mL. The results presented here are the first report on the chemical composition and biological properties of M. frigidus essential oil.

Blepharocalyx salicifolius (Kunth) O. Berg, Myrtaceae, is an endemic species that occurs at Southern America. This species was studied to intend to isolation of the active compounds that could be used in vitro model against leishmaniosis, tumoral cell and paracoccidioidomycosis. After Gel Permeation Chromatography, the ethanolic extract from leaves yielded sixteen fractions. Five compounds were isolated and assayed, showing activity against tumoral cells, from 3.33 to 12.83 µg.mL-1; Leishmania (Leishmania) amazonensis from 2.19 to 20.80 µg.mL-1 and Paracoccidioides brasiliensis from 3.10 to 12.5 µg.mL-1.

Foi realizada a comparação entre dois métodos de extração dos compostos voláteis obtidos de seis espécies de Verbenaceae, coletadas na Serra do Cipó, Minas Gerais, Brasil. Os óleos essenciais e as frações hexanica obtidos das folhas de duas espécies de Lantana e quatro espécies de Lippia, coletadas em duas diferentes estações do ano, foram analisados por CG/EM. Grande número de constituintes foi identificado nas amostras oriundas dos dois métodos de extração e o componente majoritário para a maior parte das espécies foi o β-cariofileno, seguido pelo germacreno D, biciclogermacreno e α-pineno. Para os dois gêneros estudados, foram observadas pequenas diferenças na composição do óleo essencial e fração hexânica. Esses resultados sugerem que a análise da fração hexânica pode ser usada para identificar os componentes voláteis majoritários dessas espécies, além de ser uma técnica alternativa para a análise dos compostos voláteis presentes no óleo essencial, uma vez que ambos mostraram composição similar.

A comparison between two extraction approaches of volatiles compounds from six species of Verbenaceae collected at Serra do Cipó, Minas Gerais, Brazil was done. The essential oil and hexanic fraction of leaves from two Lantana and four Lippia species collected in two different seasons were analyzed by GC/MS. Among various identified compounds from both extraction methods the majority of species showed major amounts of β-caryophyllene followed by germacrene D, bicyclogermacrene and α-pinene. Few differences were observed between the composition of essential oil and the hexanic fraction regarding the two studied genera. These results suggest that the analysis of hexanic fraction can be used, as an alternative way, to analyze the volatile compounds of the essential oil.

One hundred and twenty-one isolates of endophytic fungi were recovered from leaves of the bioactive Brazilian plant species Ageratum myriadenia, Palicourea tetraphylla, Piptadenia adiantoides, and Trixis vauthieri. All fungal isolates were cultivated in liquid media and crude extracts were obtained with ethyl acetate. The crude extracts were tested in bioassay panels using Leishmania amazonensis, Trypanosoma cruzi, the enzyme trypanothione reductase (TryR) from Trypanosoma cruzi, and three human cancer cell lines. Thirty-three extracts (27.2%) exhibited at least one biological activity. Seventeen extracts (14%) were cytotoxic against one or more human cancer cell line with the IC50 values ranged of >0.2 to 25 µg/mL. Twenty-four extracts (19.8%) inhibited the activity of TryR, and three showed ability to inhibit the growth of T. cruzi above 60% and their IC50 values ranged among 1 to 10 µg/mL. Eleven extracts (9%) were able to inhibit the growth of L. amazonensis and showed with IC50 values ranged among 4.6 to 24.4 µg/mL. The endophytic fungi were identified as belonging to the genera Alternaria, Arthrinium, Cochliobolus, Colletotrichum, Penicillium, Fusarium, and Gibberella. An interesting result was obtained for the bioactive isolates UFMGCB 508, 537, 899 and 903, which were related to fungi associated with medicinal plants native to Asia, Australia, Africa, and Polynesia. These results indicate that bioactive plants living in Brazilian ecosystems are a potential host of endophytic fungi able to produce bioactive prototype molecules for drug development against neglected tropical diseases.

Trypanosoma cruzi is a protozoan parasite that causes a severe disease (Chagas'disease) in Central and South America. The currently available chemotherapeutic agents against this disease are still inadequate. The enzyme trypanothione reductase (TR) is considered a validated molecular target for the development of new drugs against this parasite. In this regard, a series of arylfurans based on 2,5-bis-(4-acetamidophenyl)furan was synthesized and tested for their in vitro inhibitory activity against TR. Molecular modeling studies of putative enzyme-inhibitor complexes revealed a possible mechanism of interaction. From synthesized compounds, a benzylaminofuran derivative was found to be more active than the lead compound.

Uma nova substância, denominada habenariosídeo {[(2R)-2-[(2,3,4,6-tetra-O-acetil-β-D-glicopiranosil)oxi]-2-(2-metilpropil)-1,4-dioxo-1,4-butanodiil]bis(oximetileno-4,1-fenileno) bis-β-D-glicopiranosídeo}, foi isolada das frações polares do extrato etanólico de Habenaria petalodes Lindl. (Orchidaceae), juntamente com duas substâncias conhecidas e estruturalmente relacionadas, a loroglossina e a militarina. Os flavonóides: isoquercitrina, isorramnetina 3-O-β-D-glicopiranosídeo e isorramnetina 3,7-di-O-β-D-glicopiranosídeo, também foram isolados. As estruturas do habenariosídeo e das demais substâncias foram determinadas pela análise dos dados de EM, IV, UV e RMN mono e bidimensional e comparação com os dados publicados na literatura.

Habenarioside, a new natural product identified as [(2R)-2-[(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)oxy]-2-(2-methylpropyl)-1,4-dioxo-1,4-butanediyl]bis(oxymethylene-4,1-phenylene) bis-β-D-glucopyranoside, along with two known related metabolites, loroglossin and militarin, were isolated from the ethanol extract of the whole plant Habenaria petalodes Lindl. (Orchidaceae). The flavonoids isoquercitrin, isorhamnetin 3-O-β-D-glucopyranoside, and isorhamnetin 3,7-di-O-β-D-glucopyranoside were also isolated. The structures of all compounds were established by analysis of their MS, IR, UV, 1D and 2D NMR spectra, and comparison with published data.

O Laboratório de Química de Produtos Naturais (LQPN) possui uma coleção ex situ de pequenas quantidades de extratos obtidos de componentes da biodiversidade para fins de bioprospecção. Esta coleção conta atualmente com cerca de 4000 extratos de mais de 1000 espécies distintas. Os extratos são usados na identificação de novos compostos bioativos que possam servir para o desenvolvimento de novas drogas contra as doenças negligenciadas como leishmanioses, doença de Chagas, malária e tuberculose. Após serem submetidos aos ensaios biológicos, os extratos que apresentaram atividade precisam ser preparados em uma quantidade maior a partir de recoletas dos vegetais, para permitir o isolamento dos seus componentes ativos. Neste ponto, o desenvolvimento de metodologias padronizadas que permitam comparar a composição dos extratos recém obtidos com a dos extratos originais são importantes para confirmação da identidade dos mesmos. Avaliou-se a metodologia de Micro-Extração em Fase Sólida, seguida de análise por Cromatografia Gasosa e Espectrometria de Massa (MEFS-CG-EM). Foi usado o software AMDIS (Automatic Mass Spectral Deconvolution and Identification System) para armazenar e comparar os perfis gerados (fingerprint). Quarenta e seis amostras foram analisadas, onde foi possível inferir sobre os constituintes de cada amostra e traçar um perfil de composição e de componentes comuns. Foram analisados nove grupos de amostras, coletadas em diferentes períodos onde se estudou as variações sazonais ocorridas entre elas. Os resultados mostraram a viabilidade do uso desta ferramenta para monitorar a composição de extratos, permitindo avaliar alterações químicas durante a estocagem, a comparação entre extratos oriundos de coletas distintas, e na ocorrência de alguns componentes em diferentes extratos.

The Laboratory of Chemistry of Natural Products has an ex situ collection of extracts from organisms of the biodiversity aiming at bioprospecting. Nowadays the collection has about 4000 extracts from 1000 different species. Extracts are used to identify new bioactive compounds that could be useful for developing new drugs against neglected diseases like leishmaniosis, Chagas disease, malaria and tuberculosis. After biologic assays, the bioactive extracts need to be prepared in larger quantity to allow isolation and characterization of the bioactive component. At this time, it is important to not only confirm the bioactivity of new extract but also check if its composition is similar to the old one. It was evaluated the ability of Solid Phase Microextraction and Gas Chromatography-Mass Spectrometry analysis (SPME-GC-MS). It was used the AMDIS (Automatic Mass Spectral Deconvolution and Identification System) software as tools to collect and to compare the chromatographic profiles of each extract (fingerprint). Forty six samples were analyzed, it was possible to infer from the composition of each sample and common compounds. Nine groups of samples, collected at different time, were analyzed and seasonal modifications between then could be elucidated. The results showed that this methodology can be used to monitor the composition of extracts, allowing to monitor chemical changes that may occur during storage periods and to investigate the occurrence of a determined component in different extracts.

The natural lignans veraguensin and grandisin have been reported to be active against Trypanosoma cruzi bloodstream forms. Aiming at the total synthesis of these and related compounds, we prepared three 2-arylfurans and eight 2,5-diarylfurans. They were evaluated for their potential as T. cruzi trypanothione reductase (TR) inhibitors as well against the parasite's intracellular (amastigote) and bloodstream (trypomastigote) forms. Compound 12 was the most effective against TR with an IC50 of 48.5 µM while 7 and 14 were active against amastigotes, inhibiting the parasite development by 60% at 20 µg/ml (59 and 90 µM, respectively). On the other hand, none of the compounds was significantly active against the parasite bloodstream forms even at 250 µg/ml (0.6-1.5 mM).

The enzyme trypanothione reductase is a recognised drug target in trypanosomatids and has been used in the search of new compounds with potential activity against diseases such as leishmaniasis, Chagas disease and African trypanosomiasis. 8-Methoxy-naphtho [2,3-b] thiophen-4,9-quinone was selected in a screening of natural and synthetic compounds using an in vitro assay with the recombinant enzyme from Trypanosoma cruzi. Its mode of inhibition fits a non-competitive model with respect to the substrate (trypanothione) and to the co-factor (NADPH), with Ki-values of 5 and 3.6 µM, respectively. When tested against human glutathione reductase, this compound did not display any significant inhibition at 100 µM, indicating a good selectivity against the parasite enzyme.

Three hundred and thirteen extracts from 136 Brazilian plant species belonging to 36 families were tested for their suppressive activity on phytohemaglutinin (PHA) stimulated proliferation of human peripheral blood mononuclear cells (PBMC). The proliferation was evaluated by the amount of [³H]-thymidine incorporated by the cells. Twenty extracts inhibited or strongly reduced the proliferation in a dose-dependent manner at doses between 10 and 100 µg/ml. Three of these extracts appeared to be non-toxic to lymphocytes, according to the trypan blue permeability assay and visual inspection using optical microscopy. Bioassay-guided fractionation of Alomia myriadenia extract showed that myriadenolide, a labdane diterpene known to occur in this species, could account for the observed activity of the crude extract. Using a similar protocol, an active fraction of the extract from Gaylussacia brasiliensis was obtained. Analysis of the ¹H and13C NMR spectra of this fraction indicates the presence of an acetylated triterpene whose characterization is underway. The extract of Himatanthus obovatus is currently under investigation.