Cláudio Galuppo Diniz

In vitro antioxidant potential of medicinal plant extracts and their activities against oral bacteria based on Brazilian folk medicine

BACKGROUND AND OBJECTIVES This study aims to determine antibacterial activities of Cocos nucifera (husk fiber), Ziziphus joazeiro (inner bark), Caesalpinia pyramidalis (leaves), aqueous extracts and Aristolochia cymbifera (rhizomes) alcoholic extract against Prevotella intermedia, Porphyromonas gingivalis, Fusobacterium nucleatum, Streptococcus mutans and Lactobacillus casei. The antioxidant activity and acute toxicity of these extracts were also evaluated. MATERIAL AND METHODS The plant extracts antibacterial activity was evaluated in vitro and the minimal inhibitory concentration (MIC) was determined by the broth micro-dilution assay. The bacterial killing kinetic was also evaluated for all extracts. In addition, the antibacterial effect of the extracts was tested in vitro on artificial oral biofilms. The acute toxicity of each extract was determined in according to Lorke [Lorke D. A new approach to practical acute toxicity testing. Arch Toxicol 1983;54:275-87] and the antioxidant activity was evaluated by DPPH photometric assay [Mensor LL, Menezes FS, Leitão GG, Reis AS, Santos TC, Coube CS, et al. Screening of Brazilian plants extract for antioxidant activity by the use of DPPH free radical method. Phytother Res 2001;15:127-30]. RESULTS MIC and the bactericidal concentrations were identical, for each evaluated extract. However, microbes of artificial biofilms were less sensitive to the extracts than the planktonic strains. A. cymbifera extract induced the highest bactericidal effect against all tested bacteria, followed by C. nucifera, Z. joazeiro and C. pyramidalis extracts, respectively. All extracts showed good antioxidant potential, being C. nucifera and C. pyramidalis aqueous extracts the most active ones. CONCLUSION In conclusion, all oral bacteria tested (planktonic or in artificial biofilms) were more susceptible to, and rapidly killed in presence of A. cymbifera, C. pyramidalis and C. nucifera than Z. joazeiro extracts, respectively. Thus, these extracts may be of great interest for future studies about treatment of oral diseases, considering their potent antioxidant activity and low toxicity.

Antigenic, microbicidal and antiparasitic properties of an l-amino acid oxidase isolated from Bothrops jararaca snake venom.

Venoms from the bee Apis mellifera, the caterpillar Lonomia achelous, the spiders Lycosa sp. and Phoneutria nigriventer, the scorpions Tityus bahiensis and Tityus serrulatus, and the snakes Bothrops alternatus, Bothrops jararaca, Bothrops jararacussu, Bothrops moojeni, Bothrops neuwiedi, Crotalus durissus terrificus, and Lachesis muta were assayed (800mug/mL) for activity against Staphylococcus aureus. Venoms from B. jararaca and B. jararacussu showed the highest S. aureus growth inhibition and also against other Gram-positive and Gram-negative bacteria. To characterize the microbicidal component(s) produced by B. jararaca, venom was fractionated through gel exclusion chromatography. The high molecular weight, anti-S. aureus P1 fraction was further resolved by anion exchange chromatography through Mono Q columns using a 0-0.5M NaCl gradient. Bactericidal Mono Q fractions P5 and P6 showed significant LAAO activity using l-leucine as substrate. These fractions were pooled and subjected to Heparin affinity chromatography, which rendered a single LAAO activity peak. The anti-S. aureus activity was abolished by catalase, suggesting that the effect is dependent on H(2)O(2) production. SDS-PAGE of isolated LAAO indicated the presence of three isoforms since deglycosylation with a recombinant N-glycanase rendered a single 38.2 kDa component. B. jararaca LAAO specific activity was 142.7 U/mg, based on the oxidation of l-leucine. The correlation between in vivo neutralization of lethal toxicity (ED(50)) and levels of horse therapeutic antibodies anti-LAAO measured by ELISA was investigated to predict the potency of Brazilian antibothropic antivenoms. Six horses were hyperimmunized with Bothrops venoms (50% from B. jararaca and 12.5% each from B. alternatus, B. jararacussu, B. neuwiedii and B. moojeni). To set up an indirect ELISA, B. jararaca LAAO and crude venom were used as antigens. Correlation coefficients (r) between ED(50) and ELISA antibody titers against B. jararaca venom and LAAO were 0.846 (p<0.001) and 0.747 (p<0.001), respectively. The hemolytic and leishmanicidal (anti-Leishmania amazonensis) activity of LAAO was also determined.

Enhanced pathogenicity of susceptible strains of the Bacteroides fragilis group subjected to low doses of metronidazole

Different concentrations of metronidazole are used widely to treat protozoan and fungal infections. As an antibacterial drug, metronidazole is mainly used against anaerobes, of which the Bacteroides fragilis group is the most important in terms of the frequency of recovery and antimicrobial resistance patterns. The objective of this study was to investigate (1) in vivo metronidazole-induced modifications in the B. fragilis group reflected by altered virulence, and (2) the interference of metronidazole in cellular viability of these samples when subjected in vitro to human polymorphonuclear leukocytes (PMNs). Strains adapted to low metronidazole concentrations were observed to be more virulent, as demonstrated experimentally in mice by weight loss, quantitative evidence of tissue damage, hemorrhage and anatomopathology of spleen, liver and small intestine samples. A significant increase (P < 0.05) in mean bacterial viability rate of about 2.62-fold was observed for all the drug-adapted strains after contact with human PMNs. However, the level of this phenomenon was quite different among the tested species. These results draw attention to the risk that prolonged therapy, even with low concentrations of metronidazole, may affect the pathogenicity of Bacteroides strains, producing changes in host-bacteria relationships.

Use of 2-D electrophoresis and ESI mass spectrometry techniques to characterize Fusobacterium nucleatum proteins up-regulated after oxidative stress.

The genus Fusobacterium belongs to the Fusobacteriaceae family and is a Gram-negative obligate anaerobic bacterium found in the human oral microbiota. Even that Fusobacterium nucleatum cannot grow under aerobic conditions, they may exhibit aerotolerance as an adaptive response which could figure as an important virulence factor, during the stages of infection, when these anaerobes are shifted to aerobic conditions. In this regard, little is known about bacterial oxidative stress adaptive response and the influence of this adaptation on the host-bacteria relationship. We aimed to use both techniques 2-DE and Electrospray Ionization Mass Spectrometry (ESI-MS) to characterize proteins in F. nucleatum, after oxidative stress. We related three different proteins which were up-regulated by oxidative stress. As its genome is already sequenced, these proteins were found in data base search, by homology. Thus, by using techniques as ESI-Q/TOF-MS, in addition to 2-DE, the opportunity exists to gain a more holistic view of the bacterial proteome of human pathogens, to achieve a better understanding of species diversity and to elucidate the role of specific proteins in disease. This work represents one of the first studies using genetic and physiological approaches to understand the phenomenon of oxidative stress in F. nucleatum.

Determinação do perfil protéico da membrana externa da Leptospira interrogans sorovariedade Hardjoprajitno

The protein profile of the outer membrane of Leptospira interrogans serovar Hardjoprajitno was determined by two-dimensional gel electrophoresis. The outer membrane was extracted with Triton x 114 and the proteins were precipitated with acetone. The images were analyzed for the determination of the molecular weight of the detected proteins. Thirty-five spots for the proteins that are predominant in the outer membrane of this Leptospira were observed and five proteins were found in higher quantities: 22.54KDa (LipL22), 30/26KDa (LipL32), 34.41KDa (PME34) (2), 42.75KDa (LipL41), and 58.59KDa (LipL63).