Secondary metabolites produced by endophytic fungi: novel antifungal activity of fumiquinone B

Fungi are present in the most diverse environments including the interior of plant tissues, living as endophytes without causing apparent damage. These endophytes are producers of secondary metabolites, also known as natural products, such as fungicides. Here, we evaluated the ethyl acetate fractions obtained from endophytic fungiisolated from plants in the genus Begonia. The fractions were submitted to inhibitorytest against the plant pathogens Diaporthe phaseolorum and Colletotrichum gloeosporioides. From the 88 ethyl acetate fractions evaluated, 14.7 % inhibited C. gloeosporioidesand 11.3 %inhibited D. phaseolorum. One fungal isolate displaying an active fraction was selected for chemical investigation. The fungus identified as Neopestalotiopsissp., produced a compound that was active against D. phaseolorum, with a MIC of 312 μg mL-1(1,695.3 μM). The compound was identified by mass spectrometry and 1H NMR as the known compound fumiquinone B. The results highlight that the endophytes are capable of producing compounds that may be used to control plant pathogens. The compound fumiquinone B is reported for the first time as an antifungal agent against D. phaseolorum, a relevant plant pathogen worldwide. This is also the first report of the production of fumiquinone B by the genus Neopestalotiopsis.

Synthetic acylsugars and their effects on the control of arthropod pests / Acilaçúcares sintéticos e seu efeito no controle de artrópodes-praga

ABSTRACT One of the main problems facing agriculture is the loss of production as a result of the attack of agricultural pests. Alternative ways to work around this problem are being sought. There are substances called acylsugars that are naturally produced by the wild tomato species S. pennellii and affect arthropod pests. The objectives of this work were to synthesize two acylsugars and assess the biological effect of these on the arthropod pests Bemissia tabaci and Tetranycus urticae. The syntheses were performed via the reactions of glucose and sucrose (saccharose) with acetic anhydride using sodium acetate as the catalyst. The products of these reactions were sucrose octa-acetate and glucose penta-acetate, the structures of which were confirmed by spectroscopic techniques. In a resistance test against the mite, a linear correlation between the concentration of the synthesized substances, and the dislocation of the mite was obtained. A delay in the hatching of the arthropod eggs was observed, causing a mortality rate of approximately 95% in the 1st instar larvae of mites that was confirmed in adults. In the biological tests with Bemisia tabaci, there was a low rate of hatching and emergence, and the effect on the nymphs was the deformation of the emergent adults.

RESUMO Um dos grandes problemas que a agricultura enfrenta é a perda de produção causada pelo ataque de pragas agrícolas. Assim, buscam-se maneiras alternativas de contornar esse problema. Dentre esses, encontram-se substâncias, denominadas de acilaçúcars, que são produzidas naturalmente por espécies selvagens do tomate S. pennellii e que apresentam efeito sobre artrópodes-praga. Os objetivos desse trabalho foram sintetizar dois compostos de acilaçúcares e avaliar o efeito biológico destas sobre os artrópodes-praga Bemissia tabaci e Tetranycus urticae. A síntese foi feita via as reações de glicose e sacarose com anidrido acético, utilizando acetato de sódio como catalisador. Os produtos dessas reações foram o octa-acetato de sacarose e o penta-acetato de glicose, cujas estruturas foram confirmadas por técnicas espectroscópicas. No teste de resistência do ácaro, foi possível obter um ajuste linear entre a concentração das substâncias sintetizadas e o deslocamento do ácaro. Ambas as substâncias obtidas apresentaram um atraso na eclosão dos ovos do artrópode, ocasionando uma mortalidade de aproximadamente 95% em ácaros de 1º ínstar que foram confirmadas em adultos. Nos testes biológicos com Bemisia tabaci, verificou-se uma baixa taxa de eclosão e emergência, sendo que o efeito sobre as ninfas foi a má formação para adultos emergidos.

Physicochemical characteristics of pollen collected by Amazonian stingless bees / Características físico-químicas do pólen coletado por abelhas amazônicas sem ferrão

ABSTRACT: The purpose of this study was to determine the physicochemical characteristics of pollen collected by the Amazonian stingless bees Melipona seminigra and Melipona interrupta, in order to verify whether their characteristics meet the physicochemical requirements established by the Brazilian Technical Regulation for Identity and Quality of Bee Pollen. Physicochemical analyses were performed through official analytical methods. Results of pollen analyses collected by M. seminigra and M. interrupta were respectively as follows: moisture: 53.39 and 37.12%; protein: 37.63 and 24.00%; lipids: 10.81 and 6.47%; ash: 4.03 and 2.74%; crude fiber: 9.30 and 13.65%; carbohydrates: 25.66 and 44.27%; energy: 350.47 and 331.33kcal%; pH: 3.70 and 3.34; total solids: 46.60 and 62.87%, and water activity: 0.91 and 0.85. The percentages of moisture and pH in pollen collected by both studied bees are not in agreement with the Technical Regulation for bee pollen. Since some characteristics, which are inherent to the Melipona pollen, were not in conform to the current Regulation, we recommend that further studies should be conducted to better characterize it, and correct the Regulation, if necessary.

RESUMO: O objetivo deste estudo foi determinar as características físico-químicas do pólen coletado pelas abelhas amazônicas sem ferrão, Melipona seminigra e Melipona interrupta, para verificar se suas características atendem aos requisitos físico-químicos estabelecidos no Regulamento Técnico Brasileiro de Identidade e Qualidade de Pólen Apícola. As análises físico-químicas foram realizadas através de métodos analíticos oficiais. Os resultados das análises do pólen coletado por M. seminigra e M. interrupta foram, respectivamente: umidade: 53,39 e 37,12%; proteínas: 37,63 e 24,00%; lipídeos: 10,81 e 6,47%; cinzas: 4,03 e 2,74%; fibra bruta: 9,30 e 13,65%; carboidratos: 25,66 e 44,27%; energia: 350,47 e 331,33kcal%; pH: 3,70 e 3,34; sólidos totais: 46,60 e 62,87%; e atividade de água: 0,91 e 0,85. O percentual de umidade e o pH do pólen coletado por ambas as abelhas estudadas está em desacordo com o Regulamento Técnico para pólen apícola. Recomendamos que sejam realizados mais estudos para melhor caracterizar este produto e atualizar o Regulamento vigente, se necessário, pois algumas características que não atendem ao Regulamento são inerentes ao pólen de Melipona.

Alkaloids and triterpene from Almeidea coerulea (Nees and Mart.) a. St.-Hil. and anti-leishmanial activity

The dichloromethane extract of Almeidea coerulea stems yielded the (11-hydroxyrutaecarpine alkaloid reported for the first time from this species) and the triterpene (28-hydroxy-28, 29-dihydrolupeol). The dictamine, skimianine, sitosterol and stigmasterol were also isolated from methanol extract. Extracellular forms of Leishmania amazonensis (promastigotes) was tested with dichloromethane extract and 28-hydroxy-28, 29-dihydrolupeol with showed anti-leishmanial activity above 0.1 mg/mL and 75µg/mL (inhibited 50 percent promastigote growth), respectively.

Resíduos madeireiros do alburno de pau-rainha (Brosimum rubescens): investigação de metabólitos secundários e alguns aspectos tecnológicos / Residues of sapwood of "pau-rainha" (Brosimum rubescens): investigation of secondary metabolites and some technological aspects

Estudos fitoquímicos prévios com resíduos do cerne de pau-rainha (Brosimum rubescens), identificaram um alto teor de xantiletina, uma cumarina com potencial biológico. Dando prosseguimento aos estudos com serragens desta espécie, este estudo relata a densidade básica bem como o isolamento e identificação do triterpeno 3ß-acetoxi-olean-12-eno-28-al e do ß- sitosterol nos extratos hexânico e metanólico do alburno da planta. A estrutura do triterpeno foi determinada com base nos espectros de RMN em 1D (¹H e 13C) e 2D (HSQC e HMBC) além de comparação com dados da literatura. A densidade básica encontrada para o alburno foi de 0,58 g cm-3 que, embora seja inferior a do cerne, poderá ser utilizada na confecção de vários produtos, inclusive em técnicas de marchetaria.

Previous phytochemical studies on residues of pau-rainha's heartwood (Brosimum rubescens) showed a high content of xanthyletin, a coumarin with biological potential. Continuing our studies with sawdust of this species, this work relates the basic density, isolation and identification of the triterpene 3ß-acetoxy-olean-12-ene-28-al and ß-sitosterol in n-hexane and MeOH extracts of the plant´s sapwood. The structure of the triterpene was determined on the basis of NMR spectra in 1D (¹H and 13C) and 2D (HSQC and HMBC) and comparison with literature data. The basic density found for sapwood was 0.58 g cm-3, that even so is inferior the one of heartwood, could be used in the confection of some products, also in marquetry techniques.

Proposta de aproveitamento em resíduos de pau-rainha (Brosimum rubescens) descartados pelo setor madeireiro / Proposal of profite in residues of \"pau-rainha\" (Brosimum rubescens) discarded for the lumber sector

A indústria da madeira do estado de Amazonas (Brasil) contribui com a produção de uma quantidade grande de resíduos. Este trabalho visa indicar o uso final para espécie florestal pau-rainha (Brosimum rubescens Taubert, Moraceae). Os resíduos descartados durante o processamento mecânico da madeira foram utilizados na confecção de artefatos como: instrumentos musicais e artigos decorativos gerando resíduos menores (serragens). Foram obtidos extratos das serragens do cerne e alburno por maceração com hexano e metanol. O teor extrativo no cerne foi 19,87 por cento e a porcentagem (com relação a serragem) de xantiletina (2,2-dimetilcromeno cumarina) obtida foi 2,35 por cento. Não foi detectada a cumarina nos extratos do alburno. A xantiletina é reportada pelas atividades antiplaquetária, antifúngica e herbicida e alguns derivados possuem atividade em linhagens de células leucêmicas. A proposta de uso final adequado dos resíduos de pau-rainha para confecção de artefatos tem uma grande importância social e a busca de metabólitos secundários é bastante promissora pois estes podem ser transformados em novos produtos.

Timber industry of Amazonas state (Brazil) contribute with the production of great amount of residues. This paper aims to indicating end-uses for this forest species pau-rainha (Brosimum rubescens Taubert, Moraceae). The residues discharged during the mechanical processing in timber production were used as manufactured goods such as: musical instruments and decorative articles generating less wastes in sawmills. They were carried sawdust of the heartwood and sapwood and extraction by maceration with hexane and methanol. The heartwood extractive yield was 19.87 percent and content of xanthyletin (2,2-dimethylchromene coumarin) was 2.35 percent with basis dry mass. It was not detected the coumarin in extracts of sapwood. Xanthyletin is reported as antiplatelet, antifungal and herbicide and some its derivatives have a leukaemic cells lineage activities. The proposal of adequate end-uses of "pau-rainha" as manufacture-goods is a great social benefit and the search of secondary metabolites is quite promising it can be transformed into novel products.

Síntese e atividade biológica do derivado 6-formil-oxamniquina / Synthesis and biological activity of 6-formyl-oxamniquine derivative

A esquistossomose, uma importante doença no Brasil, é causada por trematódeo pertencente ao gênero Schistosoma, atingindo milhões de pessoas, numa das maiores regiões endêmicas dessa doença em todo globo. O principal objetivo desse trabalho foi sintetizar o derivado 6-formil-oxamniquina e avaliar sua atividade biológica. O derivado 6-formil- oxamniquina foi obtido por oxidação da oxamniquina com dióxido de manganês empregando diclorometano como solvente, à temperatura ambiente, por 24 horas. Sua obtenção foi confirmada por espectrometria na região de infravermelho e espectroscopia de RMN 13C e ÕH, apresentando atividade similar quando comparada à oxamniquina comercial (Mansil®).

Schistosomiasis, an important disease in Brazil, is caused by a trematode of the genus Schistosoma, reaching millions of person in one of the most endemic region of this disease in the whole globe. The main goal of this work was to syntetize the 6-formyl-oxamniquine derivative and evaluate its biological activity. The 6-formyl-oxamniquine derivative was obtained by the oxidation of oxamniquine with MnO2, applying CH2Cl2 as solvent at room temperature for 24 hours. The obtaintion of 6-formyl-oxamniquine derivative compound was confirmed by IR spectroscopy and 13C NMR and ÕH NMR, presenting similar activity when compared to the commercial oxamniquine (Mansil®).

Diterpenoids from Copaifera reticulata Ducke with larvicidal activity against Aedes aegypti (L. ) (Diptera, Culicidae) / Diterpenos de Copaifera reticulata Ducke com atividade larvicida contra Aedes aegypti (L. ) (Diptera, Culicidae)

The objective of this study was to evaluate the larvicidal activity of diterpenoids obtained from the oil-resin of Copaifera reticulata against Aedes aegypti larvae, the principal vector of dengue and urban yellow fever. Four diterpenes were obtained from oil-resin extraction with organic solvents and subsequent chromatographic and spectroscopic procedures allowed to isolation and identification of these compounds as 3-b-acetoxylabdan-8(17)-13-dien-15-oic acid (1), alepterolic acid (2), 3-b-hidroxylabdan-8(17)-en-15-oic acid (3), and ent-agatic acid (4). Each compound was previously dissolved in dimethylsulphoxide, and distilled water was added to obtain the desired concentrations. Twenty larvae of third instars were placed into plastic beckers, containing the solution test (25 mL), in a five repetitions scheme, and their mortality, indicated by torpor and darkening of the cephalic capsule, was recorded after 48h. Probit analyses were used to determine lethal concentrations (LC50 and LC90) and their respective 95 percent confidence intervals. This study showed that only diterpenoids 1 and 2 exhibited larvicidal properties with LC50 of 0.8 ppm and 87.3 ppm, respectively, revealing the former as the most toxic compound against third instars of Ae. aegypti. Therefore, this compound seems to be an interesting source for new metabolite to be exploited.

O objetivo deste trabalho foi avaliar a atividade larvicida de diterpenos isolados do óleo-resina de Copaifera reticulata sobre Aedes aegypti, principal vetor de dengue e febre amarela urbana. Quatro diterpenóides foram obtidos a partir da extração do óleo-resina com solventes orgânicos e, subseqüentes procedimentos cromatográficos e espectroscópicos permitiram o isolamento e a identificação desses compostos como ácido 3-b-acetoxylabdan-8(17)-13-dien-15-óico (1), ácido alepterólico (2), ácido 3-b-hidroxylabdan-8(17)-en-15-óico (3) e ácido ent-agático (4). Cada um desses compostos foi previamente solubilizado em dimetilsulfóxido, acrescentando-se água, até se obterem as concentrações desejadas. Em cada bioensaio foram utilizadas 20 larvas de 3° estádio de Ae. aegypti colocadas em 25 mL da solução-teste. Foram feitas cinco repetições, e a mortalidade avaliada 48 h após a exposição, indicada pela ausência de movimentos e escurecimento da cápsula cefálica. Os dados obtidos da mortalidade x concentração (ppm) foram analisados, em gráfico de Probit para avaliar as concentrações letais (CL50 e CL90). Este estudo revelou que os diterpenóides 1 e 2 mostraram atividade larvicida com CL50 de 0,8 e 87,3 ppm, respectivamente, sendo o diterpeno 1 o composto mais promissor a ser usado como larvicida para o controle de Ae. aegypti.

Identificação de bergenina e carotenóides no fruto de uchi (Endopleura uchi, Humiriaceae) / Identification of bergenin and carotenoids in the uchi (Endopleura uchi, Humiriaceae) fruit

O extrato obtido em metanol da polpa de frutos Endopleura uchi foi submetido a fracionamento utilizando-se técnicas cromatográficas convencionais levando ao isolamento de bergenina. Na análise da composição dos carotenóides foi evidenciada a predominância de beta-caroteno (16,57 mig.g-1). Os isômeros do beta-caroteno foram detectados, trans-beta-caroteno (89,3 por cento), 13-cis-beta-caroteno (8 por cento) e 9-cis-beta-caroteno (3 por cento). Considerando a importância do papel nutricional dos carotenóides e a bioatividade do glicosídeo bergenina, esse estudo sugeriu o potencial desse fruto como alimento funcional.

The extract obtained in methanol from the fruits pulp of Endopleura uchi was fractionated using conventional chromatographic tecniques to isolate bergenin. In the analysis of the composition of carotenoids was evidenced the predominance of beta-carotene (16.57 mug.g-1). The isomers of beta-carotene: trans-beta-carotene (89.3 percent), 13-cis-beta-carotene (8 percent) and 9-cis-beta-carotene (3 percent) were detected. Considering the importance of the nutritional role of carotenoids and the bioactivity of the bergenina, this study suggested the potential of this fruit as functional food.

Constituintes químicos de Almeidea coerulea (Nees & Mart.) A. St.-Hil. Rutaceae / Chemical constituents of Almeidea coerulea (Nees & Mart.) A. St.-Hil. Rutaceae

Do extrato diclorometano do caule de Almeidea coerulea (Nees & Mart.) A. St.-Hil. foi isolado um triterpeno o 20-hidroxi-diidrolupeol (1), e do extrato metanólico foram isolados os alcalóides dictamina (2) e eskimianina (3), além de uma mistura dos esteróides sitosterol (4) e estigmasterol (5). A identificção substâncias foi realizada pela análise dos espectros de Massas, RMN ¹H, RMN 13C, HSQC, HMBC, e por comparação com dados da literatura.

From the dichloromethane extract of Almeidea coerulea stem one triterpene, 20-hydroxy-dihydrolupeol was isolated (1) and from the methanol extract the known alkaloids dictamnine (2) and skimmianine (3), as well the steroids sitosterol (4) and stigmasterol (5). The identification of these compounds was performed on the basis of spectroscopic analyses (MS, ¹H NMR, 13C NMR, HSQC and HMBC) as well as comparison with literature data.

Analysis of the molecular association of rifampicin with hydroxypropyl-ß-cyclodextrin

Inclusion complexes of rifampicin (RP) were prepared with hydroxypropyl-ß-cyclodextrin (HPßCD). The aqueous solubility of RP increased linearly with cyclodextrin concentration in all range of the solubility diagram. The data was analyzed using the framework of Higuchi and Connors. The stability constant (K) values for RP/HPßCD complex at pH 6.9 were 18 and 120-125 M-1 for ionic strength 0.01 and 0.18M, respectively. The analysis of the chemical shift data of 1H and 15N for free RP and RP/HPßCD inclusion complex reveal that only peaks of the side chain related to the piperazine ring of RP change substantially, probably due to interaction of this region of RP molecule with the hydrophobic core of HPßCD...

Antibacterial xanthones from Kielmeyera variabilis mart. (Clusiaceae)

The bioassay-guided fractionation of stems from Kielmeyera variabilis, traditionally used in Brazilian folk medicine, yielded assiguxanthone-B (1), kielcorin (4), 2,5-dihydroxybenzoic acid (3), and a mixture of xanthones containing assiguxanthone-B (1) and 1,3,5,6-tetrahydroxy-2-prenylxanthone (2) (1:1 w/w). The xanthone mixture inhibited Staphylococcus aureus and Bacillus subtilis at a concentration of 6.25 µg/ml. When tested alone, the minimal inhibitory concentration of assiguxanthone-B was 25 µg/ml against B. subtilis. Kielcorin and 2,5-dihydroxybenzoic acid were inactive against both strains. None of the fractions was active against Escherichia coli or Pseudomonas aeruginosa. Viable cells of S. aureus were reduced by a 1-3 log CFU/ml within 12 h after exposure of one to eight times the MIC of the xanthone mixture. It is not known whether the tetrahydroxy-2-prenylxanthone or other components of the xanthone mixture are responsible for the main antibacterial activity or whether additive or synergistic action is involved