RESUMO
The objective of the present study was to Standardize a Polymerase Chain Reaction (PCR) protocol for the authentication of bovine and buffalo milk, and to detect the presence of Salmonella spp. and Listeria monocytogenes. For this, the target DNA was extracted, mixed, and subjected to a PCR assay. Milk samples were defrauded and experimentally contaminated with microorganisms to assess the detection of target DNA at different times of cultivation, bacterial titers, and concentration of genetic material. In addition, the protocol was tested with DNA extracted directly from food, without a pre-enrichment step. The proposed quadruplex PCR showed good accuracy in identifying target DNA sequences. It was possible to simultaneously identify all DNA sequences at the time of inoculation (0h), when the samples were contaminated with 2 CFU/250mL and with 6h of culture when the initial inoculum was 1 CFU/250mL. It was also possible to directly detect DNA sequences from the food when it was inoculated with 3 CFU/mL bacteria. Thus, the proposed methodology showed satisfactory performance, optimization of the analysis time, and a potential for the detection of microorganisms at low titers, which can be used for the detection of fraud and contamination.
O objetivo do presente estudo foi padronizar um protocolo de reação em cadeia da polimerase (PCR) para a autenticação de leite bovino e bubalino e a detecção da presença de Salmonella spp. e Listeria monocytogenes. Para isso, o DNA-alvo foi extraído, misturado e submetido ao ensaio de PCR. Amostras de leite foram fraudadas e contaminadas experimentalmente com os micro-organismos, para se avaliar a detecção do DNA-alvo em diferentes tempos de cultivo, os títulos bacterianos e a concentração de material genético. Além disso, o protocolo foi testado com DNA extraído diretamente do alimento, sem a etapa de pré-enriquecimento. A PCR quadriplex proposta mostrou boa precisão na identificação de sequências de DNA-alvo. Foi possível identificar simultaneamente todas as sequências de DNA no momento da inoculação (0h), quando as amostras estavam contaminadas com 2 UFC/250mL, e com seis horas de cultura, quando o inóculo inicial foi de 1 UFC/250mL. Também foi possível detectar diretamente as sequências de DNA do alimento quando este foi inoculado com 3 UFC/mL de bactérias. Dessa forma, a metodologia proposta apresentou desempenho satisfatório, otimização do tempo de análise e potencial para detecção de micro-organismos em baixos títulos, podendo ser utilizada para detecção de fraude e contaminação.
Assuntos
Animais , Bovinos , Salmonella/isolamento & purificação , Búfalos , Leite/microbiologia , Fraude/prevenção & controle , Listeria monocytogenes/isolamento & purificação , Inocuidade dos Alimentos/métodos , Reação em Cadeia da Polimerase Multiplex/veterináriaRESUMO
The purpose of this study was to develop extemporaneous liquid pharmaceutical formulations from commercial tablets containing spironolactone and to assess their stability for use in children or adults with difficulty in swallowing. The content and stability of spironolactone in the tablets, as well as in water, 1.5% carboxymethylcellulose (CMC) or simple syrup dispersions were determined by high performance liquid chromatography (HPLC) analysis on a C18 silica column (250 mm ? 4.6 mm ? 5 ?m), with a mobile phase of methanol:water (75:25 v/v), flowing at 1 mL/min, and UV detection at 240 nm. The extemporaneous formulations were tested over a 35-day period at 8, 27, and 40 ºC. Drug content in the aqueous dispersion was far lower than expected, with significant fluctuations at all temperatures, owing to rapid sedimentation. The content proved adequate in aqueous 1.5% CMC dispersion at 27 ºC, with undesirable variations at the other temperatures. The syrup-based dispersion remained stable at all three temperatures, with suitable drug content and no significant variability. No degradation products were observed in any of the formulations. The syrup-based dispersion is easy to prepare, self-preserving, stable, palatable, offering satisfactory drug content per dose, and can therefore be recommended as an extemporaneous formulation for enhancing treatment adherence and effectiveness...
O objetivo desse trabalho foi desenvolver e avaliar a estabilidade de formas farmacêuticas líquidas extemporâneas, a partir de amostras comerciais (comprimidos), contendo espironolactona, para que possam ser empregadas em pacientes pediátricos ou adultos com dificuldade de deglutição. A metodologia empregada para a análise do teor e da estabilidade do fármaco espironolactona nos comprimidos e nas dispersões utilizando água, carboximetilcelulose (CMC) 1,5% e xarope simples foi a Cromatografia Líquida de Alta Eficiência (CLAE), utilizando coluna de sílica C18 (250 mm x 4,6 mm x 5 μm), fluxo de 1 mL/min, comprimento de onda 240 nm e fase móvel metanol:água (75:25 v/v). As formulações extemporâneas foram analisadas durante 35 dias nas temperaturas de 8, 27 e 40 ºC. A dispersão aquosa apresentou teor muito abaixo do esperado, com variações significativas em todas as temperaturas, devido à rápida sedimentação. A dispersão aquosa de CMC 1,5% apresentou teor adequado na temperatura de 27 ºC com variações indesejadas nas demais temperaturas. A dispersão de xarope simples apresentou-se estável nas três temperaturas, com teor adequado e sem variações significativas. Não foi observado produto de degradação em nenhuma das formulações propostas. Por ser de fácil preparação, autoconservante, estável e de sabor agradável, a dispersão de xarope simples é a formulação extemporânea recomendada, pois garante teor satisfatório por dose e, portanto, favorece aumento à adesão e à eficácia do tratamento...
Assuntos
Humanos , Espironolactona , Química Farmacêutica , Cromatografia Líquida de Alta Pressão/métodos , Estabilidade de Medicamentos , ComprimidosRESUMO
Endothelins (ETs) are vasoactive peptides evolutionary well conserved that exert their effects through two specific receptors (ET(A) and ET(B)) widely distributed in all vertebrates. In snakes, the presence and function of endothelins and their receptors are still scarcely described. We have recently demonstrated the presence of ET(A) and ET(B2) receptors in the snake Bothrops jararaca (Bj). In the present work we showed that distinctively from Bj, the vascular contraction induced by endothelin in Oxyrhopus guibei (Og) snake is mediated only by ET(A) receptors. Selective ET(B) agonists (SRTX-c and IRL(1620)) and antagonists (IRL(1038) and BQ(788)) were ineffective in Og preparations of isolated aorta. We also showed that ET-1 response on Og arterial blood pressure was monophasic hypertensive as opposed to biphasic (hypotension followed by hypertension) in Bj. Furthermore, we characterized the relaxing properties of endothelin receptor ET(B1) in pre-contracted aorta preparations. We showed that IRL(1620) induced relaxation of pre-contracted Bj aorta but was ineffective in relaxing Og preparations. IRL(1620) relaxing effect on Bj aorta was abolished by l-NAME, indicating involvement of NO release, and was reduced by selective ET(B) antagonists. Our findings suggest that Og snake has a more primitive spectrum of ET receptors (only ET(A) receptor) than Bj (presence of ET(A), ET(B1) and ET(B2) receptors).
Assuntos
Aorta/metabolismo , Bothrops/metabolismo , Colubridae/metabolismo , Endotelinas/metabolismo , Receptor de Endotelina A/metabolismo , Receptor de Endotelina B/metabolismo , Vasoconstrição , Vasodilatação , Animais , Aorta/efeitos dos fármacos , Pressão Sanguínea/efeitos dos fármacos , Relação Dose-Resposta a Droga , Endotelinas/farmacologia , Inibidores Enzimáticos/farmacologia , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/metabolismo , Oligopeptídeos/farmacologia , Fragmentos de Peptídeos/farmacologia , Peptídeos/farmacologia , Piperidinas/farmacologia , Receptor de Endotelina A/efeitos dos fármacos , Receptor de Endotelina B/efeitos dos fármacos , Vasoconstrição/efeitos dos fármacos , Vasodilatação/efeitos dos fármacos , Venenos de VíborasRESUMO
Cardiovascular function is affected by many mechanisms, including the autonomic system, the kallikrein-kinin system (KKS), the renin-angiotensin system (RAS) and the endothelin system. The function of these systems seems to be fairly well preserved throughout the vertebrate scale, but evolution required several adaptations. Snakes are particularly interesting for studies related to the cardiovascular function because of their elongated shape, their wide variation in size and length, and because they had to adapt to extremely different habitats and gravitational influences. To keep the normal cardiovascular control the snakes developed anatomical and functional adaptations and interesting structural peculiarities are found in their autonomic, KKS, RAS and endothelin systems. Our laboratory has characterized some biochemical, pharmacological and physiological properties of these systems in South American snakes. This review compares the components and function of these systems in snakes and other vertebrates, and focuses on differences found in snakes, related with receptor or ligand structure and/or function in autonomic system, RAS and KKS, absence of components in KKS and the intriguing identity between a venom and a plasma component in the endothelin system.
Assuntos
Animais , Serpentes/anatomia & histologia , Serpentes/crescimento & desenvolvimento , Serpentes/fisiologia , Serpentes/imunologiaRESUMO
Endothelins (ETs) and sarafotoxins (SRTXs) are active isopeptides that have very similar structures and functions. All isoforms interact with two specific G-protein-coupled receptors, ET(A) and ET(B). To characterize functional vascular ET receptors in the poisonous snake, Bothrops jararaca, cumulative concentration-response curves to ETs and SRTXs were performed in isolated aortic rings, in the absence and presence of selective ET receptor antagonists. Vascular expression of ET receptor messenger RNA (mRNA) was evaluated by reverse transcriptase (RT) polymerase chain reaction (PCR) analysis, and a fragment of the ET(A) receptor was cloned and sequenced. In vivo, ET-1 induced a dose-dependent biphasic response on anesthetized B. jararaca snakes. In vitro, ET-1, SRTX-b, ET-3, SRTX-c, and IRL-1620 induced concentration-dependent vasoconstriction, with a potency order suggesting the presence of typical ET(A) receptors. BQ-123, a selective ET(A) antagonist, inhibited contractions induced by ET-1 and SRTX-b with expected negative log of the dissociation constant, K(B), (pK(B)) values for mixed ET(A)/ET(B) receptor populations. The nonselective ET(A)/ET(B) receptors antagonist, PD-142893, produced similar inhibition. The ET(B) antagonist, IRL-1038, potentiated contractile responses to SRTX-c. ET-1 and SRTX-c responses were also potentiated when aortic rings were pretreated with N(omega)-nitro-L-arginine methyl ester (L-NAME) plus indomethacin. Processing of the B. jararaca aortic first-strand complementary DNA, by RT-PCR with primers designed from the Gallus gallus ET(A) receptor sequence, enabled isolation, purification, cloning, and sequencing of a single band. The partial sequence of the B. jararaca ET(A) receptor showed a very high sequence similarity with ET(A) receptor sequences from chicken, rat, human, and Xenopus. In conclusion, vascular responses to SRTXs/ETs in the B. jararaca aorta are mediated predominantly, but not exclusively, by typical ET(A) receptors.