New limonoids from Hortia oreadica and unexpected coumarin from H. superba using chromatography over cleaning Sephadex with sodium hypochlorite.

Previous investigations of H. oreadica reported the presence of a wide spectrum of complex limonoids and dihydrocinnamic acids. Our interest in the Rutaceae motivated a reinvestigation of H. oreadica, H. brasiliana and H. superba searching for other secondary metabolites present in substantial amounts for taxonomic analysis. In a continuation of the investigation of the H. oreadica, three new limonoids have now been isolated 9α-hydroxyhortiolide A, 11ß-hydroxyhortiolide C and 1(S*)-acetoxy-7(R*)-hydroxy-7-deoxoinchangin. All the isolated compounds from the Hortia species reinforce its position in the Rutaceae. With regard to limonoids the genus produces highly specialized compounds, whose structural variations do not occur in any other member of the Rutaceae, thus, it is evident from limonoid data that Hortia takes an isolated position within the family. In addition, H. superba afforded the unexpected coumarin 5-chloro-8-methoxy-psoralen, which may not be a genuine natural product. Solid-state cross-polarisation/magic-angle-spinning 13C nuclear magnetic resonance, X-Ray fluorescence and Field-emission gun scanning electron microscopy experiments show that the Sephadex LH-20 was modified after treatment with NaOCl, suggesting that when xanthotoxin (8-methoxy-psoralen) was extracted from cleaning of the gel column, chlorination of the aromatic system occurred.

Evaluation of the leaching of florfenicol from coated medicated fish feed into water.

Florfenicol is one of the most-used antimicrobial agents in global fish farming. Nevertheless, in most countries, its use is not conducted in accordance with good practices. The aim of this work was to evaluate the leaching of florfenicol from coated fish feed into the water. Analytical methods were developed and validated for the quantitation of florfenicol in medicated feed and water by UHPLC-MS/MS. Florfenicol residues in the water were quantified after 5- and 15-min exposures of the medicated feed in the water at 22 and 28 °C and at pH 4.5 and 8.0. The influence of pellet size and three coating agents (vegetable oil, carboxymethylcellulose, and low-methoxylated pectin) on the leaching of the drug was also assessed. Pellet size, coating agent, water temperature, and time of exposure significantly (p < 0.05) affected florfenicol leaching, while water pH did not interfere with the leaching. Coating with vegetable oil was the most efficient method to reduce florfenicol leaching, while coating with carboxymethylcellulose presented the highest leaching (approximately 60% after 15 min at 28 °C). Thus, the coating agent has a significant effect on the florfenicol leaching rate and, consequently, on the necessary dose of the drug to be administered. Moreover, it is worth mentioning that higher florfenicol leaching will pose a greater risk to environmental health, specifically in terms of the development of bacteria resistant to florfenicol. Additional studies are needed with other polymers and veterinary drugs used in medicated feed for fish farming.

Rapid identification of bovine mastitis pathogens by MALDI-TOF Mass Spectrometry / Rápida identificação de agentes causadores de mastite por espectrometria de massas MALDI-TOF

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been shown to be an alternative method for identification of bacteria via their protein profile spectra, being able to identify bacteria at the genus, species and even at subspecies level. With the aim of large-scale identification of pathogens causing mastitis by this platform, a total of 305 isolates of bacteria identified from cows with subclinical mastitis were analyzed by conventional microbiological culture (MC) as well as by MALDI-TOF MS coupled with Biotyper data processing. Approximately 89% of the identifications performed by MALDI-TOF MS were consistent with results obtained by MC. From the remaining isolates (11%), 6.3% of isolates were classified as misidentified (discordance for both genus and species level), and 4.7% showed identification agreement at the genus level but not at the species level, being classified as unidentified at species level. The disagreement results were mostly associated with identification of Streptococcus and Enterococcus species probably due to the narrow phenotypic similarity between these two genera. These disagreement results suggest that biochemical assays might be prone to identification errors and, MALDI-TOF MS therefore may be an alternative to overcome incorrect species-specific identification. Standard microbiological methods for bovine mastitis diagnosis are time consuming, laborious and prone to errors for some bacteria genera. In our study, we showed that MALDI-TOF MS coupled with Biotyper may be an alternative method for large-scale identification of bacteria isolated from milk samples compared to classical microbiological routine protocols.(AU)

A espectrometria de massas (MALDI-TOF MS) tem mostrado ser um método alternativo para a identificação de bactérias, sendo capaz de identificar as bactérias causadoras de mastite em gênero, espécie ou até mesmo subespécie. Com o objetivo de identificar os patógenos causadores de mastite em grande-escala por esta plataforma, um total de 305 isolados bacterianos oriundos de vacas com mastite subclínica foram analisados pela cultura microbiológica convencional (CM) e pela MALDI-TOF MS acoplada ao software Biotyper. Aproximadamente 89% das identificações realizadas pela MALDI-TOF MS foram consistentes com os resultados obtidos pela CM. Do restante de isolados bacterianos (11%), 6,3% foram classificados como identificação errônea (discordância de gênero e espécie), e 4,7% apresentaram concordância de gênero, mas discordância da espécie. Os resultados que apresentaram divergência estavam mais associados com a identificação das espécies de Streptococcus spp. e Enterococcus spp. devido à similaridade fenotípica entre os dois gêneros. Estes resultados divergentes sugerem que os ensaios bioquímicos podem ser propensos a erros de identificação, por isso a MALDI-TOF MS pode ser considerada um método alternativo para superar os erros de identificação da CM. A cultura microbiológica padrão e os ensaios bioquímicos utilizados na identificação de agentes causadores de mastite são demorados, trabalhosos e propensos a erros quando utilizados na identificação em nível de espécie. No presente estudo, demonstramos que a MALDI-TOF MS acoplada ao software Biotyper pode ser considerada um método alternativo de identificação de bactérias causadoras de mastite em grande-escala quando comparado com a cultura microbiológica convencional.(AU)

Microorganisms in cryopreserved semen and culture media used in the in vitro production (IVP) of bovine embryos identified by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS).

Commercial cattle breeders produce their own herd offspring for the dairy and beef market using artificial insemination. The procedure involves sanitary risks associated with the collection and commercialization of the germplasm, and the in vitro production and transfer of the bovine embryos must be monitored by strict health surveillance. To avoid the spreading of infectious diseases, one must rely on using controlled and monitored germplasm, media, and reagents that are guaranteed free of pathogens. In this article, we investigated the use of a new mass spectrometric approach for fast and accurate identification of bacteria and fungi in bovine semen and in culture media employed in the embryo in vitro production process. The microorganisms isolated from samples obtained in a commercial bovine embryo IVP setting were identified in a few minutes by their conserved peptide/protein profile, obtained applying matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), matched against a commercial database. The successful microorganisms MS identification has been confirmed by DNA amplification and sequencing. Therefore, the MS technique seems to offer a powerful tool for rapid and accurate microorganism identification in semen and culture media samples.

Cyclopropane- and spirolimonoids and related compounds from Hortia oreadica.

The dichloromethane extract from taproots of Hortia oreadica afforded six limonoids, these are 9,11-dehydro-12α-acetoxyhortiolide A, hortiolide C, 11α-acetoxy-15-deoxy-6-hydroxyhortiolide C, hortiolide D, hortiolide E, 12ß-hydroxyhortiolide E, in addition to the known limonoid, guyanin. The dichloromethane extract from stems of H. oreadica also afforded two limonoids 9,11-dehydro-12α-hydroxyhortiolide A and 6-hydroxyhortiolide C. As a result of this study and literature data, Hortia has been shown to produce highly specialized limonoids that are similar to those from the Flindersia (Flindersioideae). The taxonomy of Hortia has been debatable, with most authors placing it in the Toddalioideae. Considering the complexity of the isolated limonoids, Hortia does not show any close affinity to the genera of Toddalioideae. That is, the limonoids appear to be of little value in resolving the taxonomic situation of Hortia.