Blood shizonticidal activities of phenazines and naphthoquinoidal compounds against Plasmodium falciparum in vitro and in mice malaria studies

Due to the recent advances of atovaquone, a naphthoquinone, through clinical trials as treatment for malarial infection, 19 quinone derivatives with previously reported structures were also evaluated for blood schizonticide activity against the malaria parasite Plasmodium falciparum. These compounds include 2-hydroxy-3-methylamino naphthoquinones (2-9), lapachol (10), nor-lapachol (11), iso-lapachol (12), phthiocol (13) and phenazines (12-20). Their cytotoxicities were also evaluated against human hepatoma and normal monkey kidney cell lines. Compounds 2 and 5 showed the highest activity against P. falciparum chloroquine-resistant blood-stage parasites (clone W2), indicated by their low inhibitory concentration for 50% (IC50) of parasite growth. The therapeutic potential of the active compounds was evaluated according to the selectivity index, which is a ratio of the cytotoxicity minimum lethal dose which eliminates 50% of cells and the in vitro IC50. Naphthoquinones 2 and 5, with activities similar to the reference antimalarial chloroquine, were also active against malaria in mice and suppressed parasitaemia by more than 60% in contrast to compound 11 which was inactive. Based on their in vitro and in vivo activities, compounds 2 and 5 are considered promising molecules for antimalarial treatment and warrant further study.

The efficacy of 2-nitrovinylfuran derivatives againstLeishmania in vitro and in vivo

Despite recent advances in the treatment of some forms of leishmaniasis, the available drugs are still far from ideal due to inefficacy, parasite resistance, toxicity and cost. The wide-spectrum antimicrobial activity of 2-nitrovinylfuran compounds has been described, as has their activity against Trichomonas vaginalis and other protozoa. Thus, the aim of this study was to test the antileishmanial activities of six 2-nitrovinylfurans in vitro and in a murine model of leishmaniasis. Minimum parasiticide concentration (MPC) and 50% inhibitory concentration (IC50) values for these compounds against the promastigotes of Leishmania amazonensis, Leishmania infantum and Leishmania braziliensis were determined, as were the efficacies of two selected compounds in an experimental model of cutaneous leishmaniasis (CL) caused by L. amazonensis in BALB/c mice. All of the compounds were active against the promastigotes of the three Leishmania species tested. IC50 and MPC values were in the ranges of 0.8-4.7 µM and 1.7-32 µM, respectively. The compounds 2-bromo-5-(2-bromo-2-nitrovinyl)-furan (furvina) and 2-bromo-5-(2-methyl-2-nitrovinyl)-furan (UC245) also reduced lesion growth in vivo at a magnitude comparable to or higher than that achieved by amphotericin B treatment. The results demonstrate the potential of this class of compounds as antileishmanial agents and support the clinical testing of Dermofural(r) (a furvina-containing antifungal ointment) for the treatment of CL.

In vitro activity of 1,3-bisaryloxypropanamines against Trypanosoma cruzi-infected L929 cultures

We describe herein the antitrypanosomal activity of 20 novel 1,3-bis(aryloxy)propan-2-amine derivatives. Compounds 2, 4, 6, 12, 15, 16 and 19 were significantly active against amastigote and trypomastigote forms, with half maximal inhibitory concentrationvalues in the range of 6-18 µM. In the cytotoxicity tests against L929 cells, only compound 4 presented selectivity index value above 10, indicating low toxicity.

Aspidosperma (Apocynaceae) plant cytotoxicity and activity towards malaria parasites. Part II: experimental studies withAspidosperma ramiflorum in vivo and in vitro

Several species of Aspidosperma plants are used to treat diseases in the tropics, including Aspidosperma ramiflorum, which acts against leishmaniasis, an activity that is experimentally confirmed. The species, known as guatambu-yellow, yellowperoba, coffee-peroba andmatiambu, grows in the Atlantic Forest of Brazil in the South to the Southeast regions. Through a guided biofractionation of A. ramiflorum extracts, the plant activity against Plasmodium falciparum was evaluated in vitro for toxicity towards human hepatoma G2 cells, normal monkey kidney cells and nonimmortalised human monocytes isolated from peripheral blood. Six of the seven extracts tested were active at low doses (half-maximal drug inhibitory concentration < 3.8 µg/mL); the aqueous extract was inactive. Overall, the plant extracts and the purified compounds displayed low toxicity in vitro. A nonsoluble extract fraction and one purified alkaloid isositsirikine (compound 5) displayed high selectivity indexes (SI) (= 56 and 113, respectively), whereas compounds 2 and 3 were toxic (SI < 10). The structure, activity and low toxicity of isositsirikine in vitro are described here for the first time in A. ramiflorum, but only the neutral and precipitate plant fractions were tested for activity, which caused up to 53% parasitaemia inhibition of Plasmodium bergheiin mice with blood-induced malaria. This plant species is likely to be useful in the further development of an antimalarial drug, but its pharmacological evaluation is still required.

Tolerância de estirpes e isolados de Bradyrhizobium e de Azorhizobium a zinco, cádmio e cobre "in vitro"

Com o objetivo de avaliar a tolerância a metais pesados "in vitro" de estirpes inoculantes (I), isolados de solos contaminados com metais (ISC) e de solos não contaminados (ISNC) de Bradyrhizobium, simbiontes de Enterolobium contortisiliquum (tamboril) e de Acacia mangium (acácia) e de Azorhizobium, simbiontes de Sesbania virgata (sesbânia), foram realizados dois experimentos. No primeiro, dez estirpes e, ou, isolados para cada espécie vegetal foram testados em meio YMA modificado por adição de tampões biológicos (HEPES e MES), suplementado com diferentes concentrações de Cu, Cd e Zn. Cobre e Cd foram testados em concentrações de 0 a 40 mg L-1, para ambos os gêneros, enquanto Zn variou de 0 a 1.000 mg L-1, para Bradyrhizobium, e de 0 a 500 mg L-1, para Azorhizobium. O crescimento de rizóbio nas diferentes concentrações de metais foi avaliado com atribuição de valores para os padrões observados (0 a 5). Os ISC de ambos os gêneros foram mais tolerantes, mas Bradyrhizobium tolerou Zn (800 mg L-1) até duas vezes e Cu (40 mg L-1) até oito vezes mais que Azorhizobium. No segundo experimento, estirpes e isolados tolerantes (T), sensíveis (S) e de tolerância média (TM) a metais selecionados em meio YMA modificado foram estudados em soluções aquosas com diferentes concentrações de Cu (0 a 0,01 mg L-1), Cd e Zn (0 a 1,0 mg L-1). A avaliação do número de células viáveis em soluções de metais foi feita por contagem das unidades formadoras de colônia em 0, 24, 48, 72 e 96 h de incubação, pelo método das diluições sucessivas e inoculação em YMA. Embora as soluções de metais tenham sido mais discriminatórias quanto a tolerância a metais que o meio YMA, estes dois métodos mostraram que: (a) Azorhizobium foi mais sensível que Bradyrhizobium, (b) os ISC de ambos os gêneros foram mais tolerantes do que os ISNC e (c) a ordem de toxidez dos metais foi Cu > Cd > Zn.

Manganês e germinação de esporos de fungos micorrízicos arbusculares in vitro

A alta disponibilidade de íons metálicos no solo, dentre eles o Mn2+, pode inibir os fungos micorrízicos arbusculares (FMA), retardando a germinação dos esporos e, conseqüentemente, a formação de micorriza, o que reduz a eficiência simbiótica. O objetivo deste trabalho foi avaliar o efeito do íon Mn2+ sobre a germinação de esporos de seis espécies de FMA dos gêneros Glomus, Acaulospora, Gigaspora e Scutellospora em experimento in vitro Em substrato constituído por areia lavada, adicionaram-se 15; 30 e 75 mg kg-1 de Mn2+, na forma de MnCl2.4H2O, mantendo-se um controle sem adição de Mn. Acondicionaram-se os esporos em envelopes membranosos, introduzindo-os entre duas camadas de areia com diferentes níveis de Mn, em placas de Petri. Após 30 dias de incubação, avaliou-se a germinação dos esporos. Houve decréscimo médio de 32, 49 e 75 % na germinação dos esporos, à medida que se aumentaram as doses de Mn, em comparação ao controle. A germinação de esporos do gênero Glomus foi totalmente inibida na maior dose. O gênero Acaulospora sofreu decréscimos de até 50 % já na dose de 15 mg kg-1 de Mn2+, enquanto os gêneros Scutellospora e Gigaspora apresentaram os maiores índices de germinação de esporos, tendo havido tolerância no caso de Gigaspora, mesmo na maior dose de Mn2+, e estímulo à germinação no caso de Scutellospora até à dose 30 mg kg-1 de Mn2+.

Indução in vitro da germinação de sementes de Oncidium flexuosum (Orchidaceae) por fungos micorrízicos rizoctonióides

A germinação de sementes e o desenvolvimento de protocórmios de Oncidium flexuosum (Orchidaceae) induzidos simbioticamente são descritos pela primeira vez. As sementes de O. flexuosum foram inoculadas com dez fungos micorrízicos rizoctonióides, previamente isolados de micorrizas de dez espécies de orquídeas neotropicais do Brasil, incluindo O. flexuosum. Foram utilizados um isolado pertencente à espécie Epulorhiza repens, dois pertencentes à Epulorhiza epiphytica, seis de Ceratorhiza spp. e um de Rhizoctonia sp. Sementes inoculadas com o isolado M2 de Ceratorhiza sp., originalmente isolado do sistema radicular de O. flexuosum em habitat natural, promoveu a germinação das sementes em sete dias e em, aproximadamente, 30 % das plântulas, houve formação de folhas após 50 dias de incubação, apresentando pelotons em algumas células do protocórmio e das radicelas. Os demais isolados promoveram a germinação das sementes; entretanto, não promoveram um desenvolvimento ótimo dos protocórmios. Sementes incubadas na ausência de fungos micorrízicos não germinaram. A especificidade e a alta dependência de O. flexuosum pela associação micorrízica ficaram claras. Aspectos relativos à especificidade, anatomia da interação fungo-planta e a importância da seleção de estirpes fúngicas, previamente ao uso de fungos micorrízicos para o cultivo simbiótico a partir de sementes de O. flexuosum são discutidos.

Molecular Identification of Trichoderma spp. in Garlic and Onion Fields and In Vitro Antagonism Trials on Sclerotium cepivorum

ABSTRACT Trichoderma species are non-pathogenic microorganisms that protect against fungal diseases and contribute to increased crop yields. However, not all Trichoderma species have the same effects on crop or a pathogen, whereby the characterization and identification of strains at the species level is the first step in the use of a microorganism. The aim of this study was the identification – at species level – of five strains of Trichoderma isolated from soil samples obtained from garlic and onion fields located in Costa Rica, through the analysis of the ITS1, 5.8S, and ITS2 ribosomal RNA regions; as well as the determination of their individual antagonistic ability over S. cepivorum Berkeley. In order to distinguish the strains, the amplified products were analyzed using MEGA v6.0 software, calculating the genetic distances through the Tamura-Nei model and building the phylogenetic tree using the Maximum Likelihood method. We established that the evaluated strains belonged to the species T. harzianum and T. asperellum; however it was not possible to identify one of the analyzed strains based on the species criterion. To evaluate their antagonistic ability, the dual culture technique, Bell’s scale, and the percentage inhibition of radial growth (PIRG) were used, evidencing that one of the T. asperellum isolates presented the best yields under standard, solid fermentation conditions.