B.P. Dias Filho

Antibacterial activity of indole alkaloids from Aspidosperma ramiflorum.

We evaluated the antibacterial activities of the crude methanol extract, fractions (I-V) obtained after acid-base extraction and pure compounds from the stem bark of Aspidosperma ramiflorum. The minimum inhibitory concentration (MIC) was determined by the microdilution technique in Mueller-Hinton broth. Inoculates were prepared in this medium from 24-h broth cultures of bacteria (10(7) CFU/mL). Microtiter plates were incubated at 37 masculineC and the MICs were recorded after 24 h of incubation. Two susceptibility endpoints were recorded for each isolate. The crude methanol extract presented moderate activity against the Gram-positive bacteria B. subtilis (MIC = 250 microg/mL) and S. aureus (MIC = 500 microg/mL), and was inactive against the Gram-negative bacteria E. coli and P. aeruginosa (MIC > 1000 microg/mL). Fractions I and II were inactive against standard strains at concentrations of < or =1000 microg/mL and fraction III displayed moderate antibacterial activity against B. subtilis (MIC = 500 microg/mL) and S. aureus (MIC = 250 microg/mL). Fraction IV showed high activity against B. subtilis and S. aureus (MIC = 15.6 microg/mL) and moderate activity against E. coli and P. aeruginosa (MIC = 250 microg/mL). Fraction V presented high activity against B. subtilis (MIC = 15.6 microg/mL) and S. aureus (MIC = 31.3 microg/mL) and was inactive against Gram-negative bacteria (MIC > 1000 microg/mL). Fractions III, IV and V were then submitted to bioassay-guided fractionation by silica gel column chromatography, yielding individual purified ramiflorines A and B. Both ramiflorines showed significant activity against S. aureus (MIC = 25 microg/mL) and E. faecalis (MIC = 50 microg/mL), with EC50 of 8 and 2.5 microg/mL for ramiflorines A and B, respectively, against S. aureus. These results are promising, showing that these compounds are biologically active against Gram-positive bacteria.

Avaliação da atividade antibacteriana e antifúngica de extratos de plantas utilizados na medicina popular

Extracts of 13 Brazilian medicinal plants were screened for their antimicrobial activity against bacteria and yeast. Of these, 10 plant extracts showed varied levels of antibacterial activity. Five of the plant extracts presented compounds with Rf values similar to the antibacterial compounds visible on bioautogram. Of these, three plants belong to the Compositae family. This may mean that the same compounds are responsible for the antibacterial activity in these plants. Anticandidal activity was detected in 9 plant extracts. The results might explain the ethnobotanical use of the studied species for the treatment of various infectious diseases.

Effects of (-) mammea A/BB isolated from Calophyllum brasiliense leaves and derivatives on mitochondrial membrane of Leishmania amazonensis.

We have previously demonstrated antileishmanial activity on Leishmania amazonensis of the natural (1-2), synthetic (7) and derivatives of coumarin (-) mammea A/BB (3-6) isolated from the dichloromethane extract of Calophyllum brasiliense leaves. The aim of the present study was to evaluate morphological and ultrastructural alterations in Leishmania amazonensis induced by these compounds. In promastigote forms, all seven compounds produced significant morphological and ultrastructural alterations, as revealed by scanning and transmission electron microscopy. The compound 5,7-dihydroxy-8-(2-methylbutanoyl)-6-(3-methylbutyl)-4-phenyl-chroman-2-one (3), the most active antileishmanial with LD₅₀ of 0.9 μM), induced cell shrinkage and a rounded appearance of the cells. Parasites incubated in the presence of compound (3) showed ultrastructural changes, such as the appearance of mitochondrial swelling with a reduction in the density of the mitochondrial matrix and the presence of vesicles inside the mitochondrion, indicating damage and significant change in this organelle; abnormal chromatin condensation, alterations in the nuclear envelope, intense atypical cytoplasmic vacuolization, and the appearance of autophagic vacuoles were also observed. In addition, the compound (3) may be acting to depolarize the mitochondrial membrane potential of the cells, leading to death of the parasite.

Further evidence of the trypanocidal action of eupomatenoid-5: Confirmation of involvement of reactive oxygen species and mitochondria owing to a reduction in trypanothione reductase activity

Our group assays natural products that are less toxic and more effective than available nitroheterocycles as promising therapeutic options for patients with Chagas disease. Our previous study reported the trypanocidal activity of eupomatenoid-5, a neolignan isolated from the leaves of Piper regnellii var. pallescens, against the three main parasitic forms of Trypanosoma cruzi. The present study further characterizes the biochemical and morphological alterations induced by this compound to elucidate the mechanisms of action involved in the cell death of T. cruzi. We show that eupomatenoid-5 induced oxidative imbalance in the three parasitic forms, especially trypomastigotes, reflected by a decrease in the activity of trypanothione reductase and increase in the formation of reactive oxygen species (ROS). A reduction of mitochondrial membrane potential was then triggered, further impairing the cell redox system through the production of more ROS and reactive nitrogen species. Altogether, these effects led to oxidative stress, reflected by lipid peroxidation and DNA fragmentation. These alterations are key events in the induction of parasite death through various pathways, including apoptosis, necrosis, and autophagy.

BACTERIAL POPULATION OF A TWO-PHASE ANAEROBIC DIGESTION PROCESS TREATING EFFLUENT OF CASSAVA STARCH FACTORY

Different groups of microorganisms in a two-phase anaerobic system were enumerated to evaluate the prevalence of specific groups and species. Total and fecal coliforms showed similar values both in acidogenic and methanogenic reactors. The fecal streptococci were 4-fold higher in the acidogenic reactor, when compared with those of the methanogenic reactor. As expected, no methane forming or sulfate reducing bacteria were found in the acidogenic reactor. The populations of methanogenic bacteria were dominated by a mixed population of straight to curved rods and multicellular filaments which strongly resembled members of the genus Methanosaeta. Seven prevalent species of facultative anaerobic gram-negative bacteria were identified as Previdencia alcalifacienciens, Providencia rettgeri, Enterobacter cloaceae, Citrobacter freundii, Klebsiella oxytoca, Proteus pennri and Yersinia enterocolitica. The species Pseudomonas aeruginosa, Stenotrophomonas maltophila and Acinotobacter iwoffi, were the most frequently isolated glucose non-fermenting gram-negative bacilli. Among these species, only P. aeruginosa was present in high number in each sample.

Estudo físico-químico, químico e biológico de extrato das cascas de Stryphnodendron polyphyllum Mart. (Leguminosae)

Two flavan-3-ols and one proanthocyanidins have been isolated from the stem bark of Stryphnodendron polyphyllum Mart., which is traditionally used in Brazil against various diseases. The structure was determined on the basis of spectroscopic data including 1-D (1H, 13C) and 2-D NMR (1H/1H COSY) and MS. The antibacterial activities of an acetone:water and semipurified extracts from the stem bark of Stryphnodendron polyphyllum Mart. were evaluated. Both the crude and semipurified extracts showed activity against Bacillus subtilis and Staphylococcus aureus. Quality control was determined using several pharmacopoeial assay.

Neolignanas e análise do óleo essencial das folhas de Piper regnellii (Miq.) C. DC. var. pallescens (C. DC.) Yunck

The essential oil of Piper regnellii (Miq.) C. DC. var. pallescens (C. DC.) Yunck Piparaceae leaves, which were collected at a tree farm named Horto de Plantas Medicinais of the Universidade Estadual de Maringa, was obtained by hydrodistillation. A preliminary analysis by GC/MS was carried out. b-mirceno (70%) was identified as the main constituent by comparing MS and 13C NMR with the literature data. Four neolignans were isolated from the leaves and identified: eupomatenoid-6, eupomatenoid-5, eupomatenoid-3 and conocarpan. Their structures were established by extensive 1H and 13C NMR, 1H x 1H - COSY, HETCOR, HMBC, gNOE and MS spectral studies.

Purification and immunocytochemical localization of neuraminidase from Tritrichomonas foetus.

Lysis of Tritrichomonas foetus with a solution of the non-ionic detergent Triton X-114 at 0 degree C, followed by low-speed centrifugation, resulted in a detergent-insoluble pellet and a detergent-soluble supernatant. The supernatant was further fractionated by phase separation at 30 degrees C into a detergent-rich phase and an aqueous phase. Neuraminidase activity was mostly located in the detergent-insoluble pellet. When the parasites were incubated with bacterial phosphatidylinositol phospholipase C (PI-PLC) prior to detergent solubilization and phase separation neuraminidase activity was predominantly recovered in aqueous phase, rather than in the pellet and detergent phase. The molecular mass determined by gel permeation in high performance liquid chromatography (HPLC) and SDS-PAGE was 80,000 Da. Indirect immunofluorescence microscopy using polyclonal antibodies raised in rabbits against the purified neuraminidase, indicated that the enzyme is exposed on the cell surface. Previous treatment of the cells with PI-PLC significantly reduced antibody binding. Incubation of cryo-sections with the antibodies followed by detection using gold-labelled anti-rabbit IgG confirmed the presence of neuraminidase in the plasma membrane enclosing the cell body and flagella and in the membrane of vesicles preferentially located at the peripheral region of the protozoan.

Microbial populations of an upflow anaerobic sludge blanket reactor treating wastewater from a gelatin industry.

The microbial populations of an upflow anaerobic sludge blanket reactor, used for treating wastewater from the gelatin industry, were studied by microbiological methods and phase-contrast and electron microscopy. Microscopy examination of the sludge showed a complex mixture of various rod-shaped and coccoid bacterial pluslong filaments and verymobile curved rods. In addition free-living anaerobic ciliates and flagellates were also observed. The trophic group population observed in decreasing order of dominance were hydrolytic and acetogenic at 10(6) and sulfate reducing and methanogenic at 10(5). The rate of methane production in anaerobic granular sludge cultivated in growth medium supplement with formate pressurized with H2:CO2 showed a significant increase in methane yield compared with theseed culture containingthe same substrate and atmosphere of N2:CO2. Similar rates of methane production were observed when the growth medium was supplemented with acetate pressurized either with H2:CO2 or N2:CO2. The number of total anaerobic bacteria at 10(7), fecal coliforms and total coliforms at 10(6), and fecal streptococci at 10(3) is based on colony counts on solid media. The four prevalent species of facultative anaerobic gram-negative bacteria that belong to the family of Enterobacteriaceae were identified as Escherichia coli, Esherichia fergusonii, Klebsiella oxytoca, and Citrobacter freundii. The species Aeromonas hydrophila, Aeromonas veronii, Acinetobacter iwoffi and Stenotrophomonas maltophila were the most frequently isolated glucose fermenting and nonfermenting gram-negative bacilli.

Effect of the marine brown alga Canistrocarpus cervicornis on promastigote forms of Leishmania (L.) amazonensis

Leishmaniasis is a disease resulting from infection by protozoan parasites of the genus Leishmania. Pentavalent antimonials, used clinically for more than 50 years, are still the first-choice drugs for the treatment of leishmaniasis, but they are toxic, require long-term treatment, and are prone to stimulate drug resistance [1]. Marine brown algae (Phaeophyceae) belonging to the order Dictyotales have emerged as an exceptionally rich source of diterpenoids, which form part of a defensive strategy against herbivores in the marine environment [2]. We have investigated the activity of crude extracts, a fraction, and an isolated compound (4R, 9S, 14S)-4α-Acetoxy-9β,14α-dihydroxydolast-1(15),7-diene of the brown alga Canistrocarpus cervicornis against promastigote forms of Leishmania amazonensis. The antiproliferative assays showed a dose-dependent effect against promastigotes with IC50 values in the range between 20.0 and 80.0µg/mL for crude extracts, 5.0µg/mL for the fraction and 2.0µg/mL for the isolated compound from C. cervicornis. We also investigated targets in the parasite by means of electron microscopy. Ultrastructural alterations were mainly observed in the mitochondrion of parasites treated with the isolated compound. Based on the current study, compounds from C. cervicornis appear to be an alternative for the development of new antiparasitic chemotherapies. However, further in vitro and in vivo studies are necessary to elucidate the mechanism of action of this compound. Acknowledgements: CNPq, FINEP, PRONEX/Fundacao Araucaria. References: [1] Croft, S.L. et al. (2006) Indian J. Med. Res. 123:399–410. [2] Garcia, D.G. et al. (2009) Phytother. Res., in press.