Electrospray Ionization Mass Spectrometry Fingerprinting Of Propolis.

Crude ethanolic extracts of propolis, a natural resin, have been directly analysed using electrospray ionization mass (ESI-MS) and tandem mass spectrometry (ESI-MS/MS) in the negative ion mode. European, North American and African samples have been analyzed, but emphasis has been given to Brazilian propolis which displays diverse and region-dependent chemical composition. ESI-MS provides characteristic fingerprint mass spectra, with propolis samples being divided into well-defined groups directly related to their geographical origins. Chemometric multivariate analysis statistically demonstrates the reliability of the ESI-MS fingerprinting method for propolis. On-line ESI-MS/MS tandem mass spectrometry of characteristic [M - H](-) ion markers provides an additional dimension of fingerprinting selectivity, while structurally characterizing the ESI-MS marker components of propolis. By comparison with standards, eight such markers have been identified: para-coumaric acid, 3-methoxy-4-hydroxycinnamaldehyde, 2,2-dimethyl-6-carboxyethenyl-2H-1-benzopyran, 3-prenyl-4-hydroxycinnamic acid, chrysin, pinocembrin, 3,5-diprenyl-4-hydroxycinnamic acid and dicaffeoylquinic acid. The negative mode ESI-MS fingerprinting method is capable of discerning distinct composition patterns to typify, to screen the sample origin and to reveal characteristic details of the more polar and acidic chemical components of propolis samples from different regions of the world.129739-4

Cell Invasion by Neisseria meningitidis Requires a Functional Interplay between the Focal Adhesion Kinase, Src and Cortactin

Entry of Neisseria meningitidis (the meningococcus) into human brain microvascular endothelial cells (HBMEC) is mediated by fibronectin or vitronectin bound to the surface protein Opc forming a bridge to the respective integrins. This interaction leads to cytoskeletal rearrangement and uptake of meningococci. In this study, we determined that the focal adhesion kinase (FAK), which directly associates with integrins, is involved in integrin-mediated internalization of N. meningitidis in HBMEC. Inhibition of FAK activity by the specific FAK inhibitor PF 573882 reduced Opc-mediated invasion of HBMEC more than 90%. Moreover, overexpression of FAK mutants that were either impaired in the kinase activity or were not capable of autophosphorylation or overexpression of the dominant-negative version of FAK (FRNK) blocked integrin-mediated internalization of N. meningitidis. Importantly, FAK-deficient fibroblasts were significantly less invaded by N. meningitidis. Furthermore, N. meningitidis induced tyrosine phosphorylation of several host proteins including the FAK/Src complex substrate cortactin. Inhibition of cortactin expression by siRNA silencing and mutation of critical amino acid residues within cortactin, that encompass Arp2/3 association and dynamin binding, significantly reduced meningococcal invasion into eukaryotic cells suggesting that both domains are critical for efficient uptake of N. meningitidis into eukaryotic cells. Together, these results indicate that N. meningitidis exploits the integrin signal pathway for its entry and that FAK mediates the transfer of signals from activated integrins to the cytoskeleton. A cooperative interplay between FAK, Src and cortactin then enables endocytosis of N. meningitidis into host cells

UHPLC–MS quantification of coumarin and chlorogenic acid in extracts of the medicinal plants known as guaco (Mikania glomerata and Mikania laevigata)

In Brazil, Mikania glomerata Spreng. and M. laevigata Sch. Bip. ex Baker, Asteraceae, known popularly as guaco, are widely used for colds and asthma. Although coumarin is adopted as the chemical marker of both species, it was not always detected in M. glomerata, for which chlorogenic acid was identified and quantified instead. The purpose of this study was to develop and validate a method to quantify both coumarin and chlorogenic acid and apply it to extracts of plants identified as M. glomerata, M. laevigata, or as guaco, to determine the pattern of composition of these two species and to observe differences between oven-dried and lyophilized leaves. A method using ultra-high resolution liquid chromatography–mass spectrometry (UHPLC–MS) in the full scan mode was validated for selectivity, matrix effect, linearity, limits of detection and quantification, precision and accuracy. The concentration of coumarin varied between species and samples, therefore these two species should not be used interchangeably. The concentration of chlorogenic acid was also determined for all samples. The UHPLC–MS method permitted the quantification of coumarin and chlorogenic acid in 16 samples of guaco and several commercial samples were possibly misidentified252105110CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP473597/2013-62008/58035-

Damage and drying modify the composition of mikania glomerata and mikania laevigata leaves

This study compared the influence of mechanical damage and drying on the chemical composition of Mikania glomerata Spreng. and Mikania laevigata Sch.Bip. ex Baker, Asteraceae, leaves. Leaves were collected 1-24 h after damage. Oven-drying at 40 degrees C and shade-drying at ambient temperature were compared to lyophilization. Samples were extracted in 70% ethanol and analyzed by ultra-high-performance liquid chromatography with mass spectrometry. Significant (p< 0.05) increases of caffeoylquinic acids were observed in damaged leaves of both species and coumarin decreased in M. laevigata, indicating stress. Although the final water content was similar, the drying method affected the leaf composition. In shade-dried leaves of M. laevigata coumarin decreased and the presence of umbelliferone was observed; caffeoylquinic acid contents increased for 288 h in in both species. Apparently, enzymes were inactivated after 6 h in oven drying, stabilizing their chemical composition, while shade drying allowed enzymatic and microbial activity to continue; illustrating the importance of post harvesting procedures on the quality of medicinal plants296793797CAPES - Coordenação de Aperfeiçoamento de Pessoal e Nível SuperiorFAPESP – Fundação de Amparo à Pesquisa Do Estado De São PauloSem informação2013/15962-

Analysis of Soluble Lignin in Sugarcane by Ultrahigh Performance Liquid Chromatography–Tandem Mass Spectrometry with a Do-It-Yourself Oligomer Database

Lignin is a polymer found in the cell wall of plants and is one of the main obstacles to the implementation of second-generation ethanol production because it confers the recalcitrance of the lignocellulosic material. The recalcitrance of biomass is affected by the amount of lignin, by its monomer composition, and the way the monomers are arranged in the plant cell wall. Analysis of lignin structure demands mass spectrometry analysis, and identification of oligomers is usually based on libraries produced by laborious protocols. A robust method to build a do-it-yourself lignin oligomer library was tested. This library can be built using commercially available enzymes, standards, and reagents and is relatively easy to accomplish. An ultrahigh performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method for the separation and characterization of monomers and oligomers was developed and was equally applicable to the synthetic lignin and to soluble lignin extracted from a sample of sugar cane

Analysis of the composition of Brazilian propolis extracts by chromatography and evaluation of their in vitro activity against gram-positive bacteria Análise da composição de extratos de própolis brasileiros por cromatografia e avaliação de sua atividade in vitro sobre bactérias

Brazilian propolis from São Paulo state was submitted to extraction using several solvents, resulting in extracts with different composition. These extracts were submitted to Thin Layer Chromatography (TLC). Bioauthographic analysis of the TLC plates identified fractions with inhibitory activity, which were then analysed by High Performance Liquid Chromatography (HPLC). In vitro assays, commonly used to evaluate the activity of propolis against Gram-positive bacteria, were compared to determine which rendered the most consistent results. The bactericidal activity of these extracts were analysed by Serial Dilution in Tubes and Agar Plate Diffusion. Serial Dilution in Tubes obtained the most consistent results, with the Minimal Bactericidal Concentration of the extracts ranging between 2.5 and 20.0 mg/mL, for the species of Gram-positive bacteria tested. The results of the Agar Plate Diffusion were directly proportional to the hydro-solubility of the extracts and did not evaluate their bactericidal activity correctly. The bactericidal activity of this sample of propolis was due to the combined effect of several components that were identified by HPLC and were best extracted using 50% ethanol as a solvent.<br>Própolis brasileira, proveniente do estado de São Paulo, foi submetida à extração usando vários solventes, resultando em extratos com diferentes composições. Estes extratos foram submetidos à Cromatografia em Camada Delgada (CCD). Análise bioautográfica das placas de CCD permitiu identificar as frações com atividade antimicrobiana, que foram então analisadas por Cromatografia Líquida de Alta Eficiência (CLAE). Ensaios in vitro freqüentemente utilizados para avaliar a atividade de própolis frente a bactérias Gram-positivas foram comparados para determinar qual renderia os resultados mais consistentes. A atividade bactericida destes extratos foi analisada por Diluição Seriada em Tubos e por testes de Difusão em Agar. O método de Diluição em Tubos permitiu obter os resultados mais consistentes e a Concentração Bactericida Mínima dos extratos variou entre 2,5 e 20,0 mg/mL, para as espécies de bactérias Gram-positivas testadas. Os resultados do método de Difusão em Agar foram diretamente proporcionais à hidrossolubilidade dos extratos, e não avaliaram a atividade bactericida corretamente. A atividade bactericida desta amostra resultou da combinação de vários componentes, identificados por CLAE, que foram extraídos preferencialmente usando etanol 50% como solvente

Den økonomiske krise og arbejderklassen. Socialistiske Økonomers Småskrifter, 2. Roskilde: Roskilde Universitetsforlag, 1977. 40 pp. Kr. 7,50. NT 77-2008

Pilocarpine, an important imidazole alkaloid, is extracted from the leaves of Pilocarpus microphyllus (Rutaceae), known in Brazil as jaborandi and used mainly for the treatment of glaucoma. Jaborandi leaves also contain other imidazole alkaloids, whose pharmacological and physiological properties are unknown, and whose biosynthetic pathways are under investigation. In the present study, a HPLC method coupled with ESI-MS(n) was developed for their qualitative and quantitative analysis. This method permits the chromatographic separation of the imidazole alkaloids found in extracts of jaborandi, as well as the MS/MS analysis of the individual compounds. Thus two samples: leaves of P. microphyllus and a paste that is left over after the industrial extraction of pilocarpine; were compared. The paste was found to contain significant amounts of pilocarpine and other imidazole alkaloids, but had a slightly different alkaloid profile than the leaf extract. The method is suitable for the routine analysis of samples containing these alkaloids, as well as for the separation and identification of known and novel alkaloids from this family, and may be applied to further studies of the biosynthetic pathway of pilocarpine in P. microphyllus

Detoxification Of Atrazine By Endophytic Streptomyces Sp. Isolated From Sugarcane And Detection Of Nontoxic Metabolite.

Atrazine is still one of the most used agricultural pesticides worldwide and it has been recognized as a major contaminant of surface and ground water. The aims of this research were to isolate an endophytic microorganism from leaves of sugarcane, evaluate its ability to degrade atrazine, and investigate the formation of metabolites. By sequencing of the 16S rRNA gene, the endophytic isolate atz2 was identified as Streptomyces sp. The reduction in atrazine concentration by Streptomyces sp. atz2 was 98 % and UHPLC-MS/MS analyses showed the appearance of an unknown metabolite observed as m/z 311. Ecotoxicity tests with an aquatic organism, Daphnia similis, confirmed that this metabolite was nontoxic. This mechanism of detoxification of atrazine is different from the ones of other free-living microorganisms that inhabit the soil or rhizosphere. The results show new aspects of atrazine detoxification, highlighting a new role of endophytic bacteria in plants.95803-80