In the present investigation we evaluate methods for the isolation and growth of marine-derived fungal strains in artificial media for the production of secondary metabolites. Inoculation of marine macroorganisms fragments in Petri dishes proved to be the most convenient procedure for the isolation of the largest number of strains. Among the growth media used, 3% malt extract showed the best result for strains isolation and growth, and yielded the largest number of strains from marine macroorganisms. The percentage of strains isolated using each of the growth media which yielded cytotoxic and/or antibiotic extracts was in the range of 23-35%, regardless of the growth media used. Further investigation of extracts obtained from different marine-derived fungal strains yielded several bioactive secondary metabolites, among which (E)-4-methoxy-5-(3-methoxybut-1-enyl)-6-methyl-2H-pyran-2-one is a new metabolite isolated from the Penicillium paxilli strain Ma(G)K.

Destruxinas (Dtx) são ciclodepsipetídeos produzidos por fungos entomopatogênicos, que são utilizados como controle biológico em insetos pragas em diferentes agriculturas. A presente investigação reporta uma nova abordagem para análises de destruxinas produzidas por uma linhagem do fungo Beauveria felina, utilizando-se de LC-PDA-ELSD-MS. Em comparação com os métodos anteriores, a nova abordagem utiliza-se de uma limpeza prévia da amostra em cartuchos C18 que removem efetivamente os constituintes do meio de cultura. Além disso, o uso dos solventes MeCN/MeOH 50:50, (v/v) como eluentes mais fortes no sistema de gradiente em 0,1% de H2O demonstrou fornecer a melhor resolução dos picos cromatográficos. Detecções simultâneas usando arranjo de fotodiodos (PDA), detector de espalhamento de luz evaporativa (ELSD) e espectrometria de massas (MS) indicaram praticamente uma resposta idêntica de todos os detectores na análise das destruxinas. Cinco amostras obtidas da cultura de B. felina foram analisadas, e indicaram a presença de 20 destruxinas conhecidas e de 6 ciclodepsipetídeos ainda não reportados. Considerando a redução do uso do MeCN, e a eficácia do ELSD como detector para destruxina, o método prova que pode ser de grande valia e de baixo custo operacional para controle de qualidade nas análises de destruxinas produzidas por linhagens de fungos.

Destruxins (Dtx) are cyclodepsipeptides produced by enthomopathogenic fungi, which are used in biological control of different agricultural insect plagues. The present investigation reports a new approach for analysis of destruxins produced by the fungal strain Beauveria felina, using LC-PDA-ELSD-MS. Compared to previous methods, the new approach uses a clean-up on C18 cartridges, which effectively removes growth media constituents. Moreover, the use of 50:50 (v/v) MeCN/MeOH as the strongest eluting solvent in a gradient system over 0.1% H2O proved to give a better resolution of chromatographic peaks. Simultaneous detection using photodiode array (PDA), evaporative light scattering detector (ELSD) and mass spectrometry (MS) indicated a practically identical response of all detectors for destruxins analysis. Five samples obtained from the culture media of B. felina were analysed, and indicated the presence of twenty known destruxins and six yet unreported cyclodepsipeptides. Considering the reduced use of MeCN, and the effectiveness of ELSD as a detector for destruxins, the method proved to be valuable and cost-effective for quality control analysis of destruxin-producing fungal strains.

Extracts obtained from 57 marine-derived fungal strains were analyzed by HPLC-PDA, TLC and ¹H NMR. The analyses showed that the growth conditions affected the chemical profile of crude extracts. Furthermore, the majority of fungal strains which produced either bioactive of chemically distinctive crude extracts have been isolated from sediments or marine algae. The chemical investigation of the antimycobacterial and cytotoxic crude extract obtained from two strains of the fungus Beauveria felina have yielded cyclodepsipeptides related to destruxins. The present approach constitutes a valuable tool for the selection of fungal strains that produce chemically interesting or biologically active secondary metabolites.

No presente estudo apresentamos resultados da triagem biológica realizada com 349 extratos obtidos de esponjas marinhas, ascídias, briozoários e octocorais do Brasil, em testes contra 16 linhagens de bactérias comuns e resistentes à antibióticos, uma levedura (Candida albicans), Mycobacterium tuberculosis H37Rv, três linhagens de células tumorais MCF-7 (mama), B16 (melanoma murínico) e HCT8 (cólon), e de inibição da enzima adenina fosforribosil transferase de Leishmania tarentolae (L-APRT). Menos de 15% dos extratos de esponja marinhas apresentaram atividade antibacteriana, contra linhagens resistentes ou não a antibióticos. Quase 40% dos extratos de esponjas marinhas apresentaram atividade antimicobacteriana contra Mycobacterium tuberculosis H37Rv. Foi observada citotoxicidade para 18% dos extratos de esponjas marinhas. Finalmente, menos de 3% dos extratos de esponjas apresentaram atividade inibitória da enzima L-APRT. Menos de 10% dos extratos de ascídias apresentaram atividade antibacteriana. Mais de 25% dos extratos de ascídias apresentaram atividade contra M. tuberculosis e as três linhagens de células tumorais. Somente dois extratos obtidos da ascídia Polysyncraton sp. coletada em duas diferentes épocas (1995 e 1997) apresentaram atividade contra L-APRT. Menos de 2% dos extratos de briozoários e octocorais apresentaram atividade antibacteriana, mas uma alta percentagem de extratos destes animais apresentaram atividades citotóxica (11% e 30%, respectivamente) e antimicobacteriana (60%). O extrato de somente uma espécie de briozoário, Bugula sp., apresentou atividade inibitória da enzima L-APRT. A análise taxonômica de algumas espécies de invertebrados que forneceram alguns dos extratos mais ativos, indicou a ocorrência de atividade biológica em espécies pertencentes a grupos taxonômicos que já foram anteriormente investigados do ponto de vista químico. Estes incluem esponjas marinhas pertencentes aos gêneros Aaptos, Aplysina, Callyspongia, Haliclona, Niphates, Cliona, Darwinella, Dysidea, Ircinia, Monanchora e Mycale, ascídias dos gêneros Didemnum, Aplidium, Botrylloides, Clavelina, Polysyncraton e Symplegma, o briozoário Bugula sp. e octocorais dos gêneros Carijoa e Lophogorgia. A subseqüente investigação química de alguns dos extratos ativos levou ao isolamento de vários metabólitos secundários biologicamente ativos. Os resultados obtidos estão de acordo com resultados obtidos em programas de triagem de atividade biológica de extratos brutos de invertebrados marinhos anteriormente reportados, que apresentaram altas percentagens de extratos bioativos oriundos de Porifera, Ascidiacea, Cnidária e Bryozoa.

Herein we present the results of a screening with 349 crude extracts of Brazilian marine sponges, ascidians, bryozoans and octocorals, against 16 strains of susceptible and antibiotic-resistant bacteria, one yeast (Candida albicans), Mycobacterium tuberculosis H37Rv, three cancer cell lines MCF-7 (breast), B16 (murine melanoma ) and HCT8 (colon), and Leishmania tarentolae adenine phosphoribosyl transferase (L-APRT) enzyme. Less than 15% of marine sponge crude extracts displayed antibacterial activity, both against susceptible and antibiotic-resistant bacteria. Up to 40% of marine sponge crude extracts displayed antimycobacterial activity against M. tuberculosis H37Rv. Cytotoxicity was observed for 18% of marine sponge crude extracts. Finally, less than 3% of sponge extracts inhibited L-APRT. Less than 10% of ascidian crude extracts displayed antibacterial activity. More than 25% of ascidian crude extracts were active against M. tuberculosis and the three cancer cell lines. Only two crude extracts from the ascidian Polysyncraton sp. collected in different seasons (1995 and 1997) displayed activity against L-APRT. Less than 2% of bryozoan and octocoral crude extracts presented antibacterial activity, but a high percentage of crude extracts from bryozoan and octororal displayed cytotoxic (11% and 30%, respectively) and antimycobacterial (60%) activities. The extract of only one species of bryozoan, Bugula sp., presented inhibitory activity against L-APRT. Overall, the crude extracts of marine invertebrates herein investigated presented a high level of cytotoxic and antimycobacterial activities, a lower level of antibacterial activity and only a small number of crude extracts inhibited L-APRT. Taxonomic analysis of some of the more potently active crude extracts showed the occurrence of biological activity in taxa that have been previously chemically investigated. These include marine sponges belonging to genera Aaptos, Aplysina, Callyspongia, Haliclona, Niphates, Cliona, Darwinella, Dysidea, Ircinia, Monanchora and Mycale, ascidians of the genera Didemnum, Aplidium, Botrylloides, Clavelina, Polysyncraton and Symplegma, the bryozoan Bugula sp. and octocorals of the genera Carijoa and Lophogorgia. The subsequent chemical investigation of some of the active extracts led to the isolation of several new biologically active secondary metabolites. Our results are in agreement with previous screening programs carried out abroad, that showed a high percentage of bioactive extracts from Porifera, Ascidiacea, Cnidaria and Bryozoa.

Dois derivados cumarínicos, o ester metílico do ácido 4-esculetinocarboxílico (1) e o éster etílico do ácido 4-esculetinocarboxílico (2), foram isolados da esponja marinha Axinella cf. corrugata. A determinação estrutural dos compostos isolados foi realizada pela análise de seus dados espectroscópicos e pela síntese do composto 2. Estes são os primeiros derivados cumarínicos isolados de uma esponja marinha. O éster etílico 2 apresentou atividade in vitro contra a SARS 3Cl-protease e de inibição de células Vero infectadas com o SARS coronavírus, em concentrações que não provocaram citotoxicidade.

Two coumarin derivatives, esculetin-4-carboxylic acid methyl ester (1) and esculetin-4-carboxylic acid ethyl ester (2), have been isolated from the marine sponge Axinella cf. corrugata. Structure determination included analysis of spectroscopic data and total synthesis of compound 2. These are the first coumarin derivatives isolated from a marine sponge. The ethyl ester 2 was found to be an in vitro inhibitor of SARS 3CL-protease and an effective inhibitor of SARS-CoV replication in Vero cells at non-cytotoxic concentrations.

Two new marine metabolites, 3Z, 6Z, 9Z-dodecatrien-1-ol (1) from the ascidian Botrylloides giganteum and 4H-pyran-2ol acetate from the sponge Ircinia felix (4) are herein reported. The known bromotyrosine compounds, 2-(3,5-dibromo-4-methoxyphenyl)-N,N,N-dimethylethanammonium (2) and 2,6-dibromo-4-(2-(trimethylammonium)ethyl)phenol (3), have been isolated from the sponge Verongula gigantea. Serotonin (5) is reported for the first time from the sponge Cliona delitrix, and tambjamines A (15) and D (16) isolated as their respective salts from the nudibranch Tambja eliora. Only tambjamine D presented cytotoxicity against CEM (IC50 12.2 µg/mL) and HL60 (IC50 13.2 µg/mL) human leukemya cells, MCF-7 breast cancer cells (IC50 13.2 µg/mL), colon HCT-8 cancer cells (IC50 10.1 µg/mL) and murine melanoma B16 cancer cells (IC50 6.7 µg/mL).