Avaliação anti-inflamatória e antipeçonhenta das espécies Hancornia speciosa e Mimosa tenuiflora em modelos experimentais de inflamação e envenenamento induzidos por Bothrops jararaca e Tityus serrulatus

Accidents caused by venomous animals represents a significant and serious public health problem in certain regions of Brazil, as well as in other parts of the world by the frequency with which they occur and the mortality they cause. The use of plant extracts as an antidote for poisoning cases is an ancient practice used in many communities that have no access to antivenom. Medicinal plants represent an important source of obtaining bioactive compounds able to assist directly in the treatment of poisoning or indirectly supplementing serum therapy currently used. The aim of this study was to evaluate the effect of extracts, fractions and isolated compounds from M. tenuiflora and H. speciosa in the inflammatory process induced by carrageenan and the venom of B. jararaca and T. serrulatus. The results showed that both M. tenuiflora and H. speciosa were capable of inhibiting cell migration and cytokines levels in peritonitis induced by carrageenin and venom of T. serrulatus. In poisoning by B. jararaca model, mice treated with the plants in studies decreased the leukocyte influx into the peritoneal cavity. Finally the M. tenuiflora and H. speciosa had antiphlogistic activity, reducing edema formation and exerted inhibitory action of leukocyte migration in local inflammation induced by the venom of B. jararaca. Through of Thin Layer Chromatography (TLC) analysis was possible identified the presence of flavonoids ,saponins and/or terpenes in aqueous extract of M. tenuiflora. By High Performance Liquid Chromatography analysis, it was possible to identify the presence of rutin and chlorogenic acid in aqueous extract of H. speciosa. We conclude that the administration of extracts, fractions and isolated compounds of H. speciosa and M. tenuiflora resulted in inhibition of the inflammatory process in different experimental models. This study demonstrates for the first time the effect of M. tenuiflora and H. speciosa in inhibition of the inflammation caused by B. jararaca and T. serrulatus venom.

Tensoativo derivado do ácido cítrico: síntese e aplicação em fluidos de perfuração de emulsão inversa

This work describes the synthesis and study of the application of a new surfactant (Triester Lipophilic – TEL) obtained by citric acid with octanol. It is reaction was followed by thin layer chromatography (TLC) and after purification the product was characterized by proton and 13 – carbon nuclear magnetic resonance spectroscopy ( H and 13C NMR), thermogravimetric analysis (TGA) and surface tension analysis of oil-in-water emulsions. The TEL performance as surfactant in ester, n-paraffin and biodiesel based drilling fluids on the 70/30 and 60/40 water- oil rations (WOR) was evaluated by comparative tests of two commercial products used in the fields. These drilling fluids were aged in roller oven at 200 0 F during 16 h. The rheological and electric stability measurements were carried out at 135 ºF, the phase separation was evaluated after seven days at rest and the filtrate volume of drilling fluids was determined at high temperature and high pressure. The rheological behavior of the drilling fluids was evaluated by the flow curves. The results showed that the drilling fluids studied here presented Binghamian behavior as well as the used in the oil fields. The laboratory tests showed that the TEL reduced the filtrate volume and promoted the enhance of the thermal and mechanical stabilities.

Inibição dos efeitos locais induzidos pelas peçonhas das serpentes Bothrops erythromelas e Bothrops jararaca pelo extrato aquoso das folhas de Jatropha mollissima (Pohl) Bail

Snakebites are a serious public health problem in tropical and subtropical countries and Bothrops genus is responsible for the accidents in Brazil and throughout Latin America (90% of cases). The local effects (pain, edema, hemorrhage and myonecrosis) and systemic (cardiovascular alterations, shock and blood clotting disorders) caused by the venom of Bothrops are due to the numerous protein and non-protein components, which are part of the constitution of the poison. The only form of therapy is scientifically validated antivenom serum therapy which, however, is not effective with respect to local effects produced, risk of immunological reactions, high cost and difficult access in some regions. Thus, the search for new alternatives to serum therapy becomes important, and in this context, many medicinal plants have been highlighted by the popular use as antiophidic. Among these plants, we can mention the species Jatropha mollissima (Euphorbiaceae) which has popular use in traditional medicine as antiophidic, anti-inflammatory, antimicrobial and antipyretic. Therefore, this study aims to evaluate the neutralizing potential of local effects induced by the venom of Bothrops erythromelas and Bothrops jararaca with the aqueous extract of the leaves of J. mollissima. The leaf extracts were prepared by decoction, fractionated (by liquid-liquid partition) and characterized by thin layer chromatography (TLC) and High Performance Liquid Chromatography (HPLC). Antiophidic activity of the extract was evaluated in model of paw edema, peritonitis, bleeding and myotoxicity induced by venoms of B. jararaca and B. erythromelas. In all models, the extract was evaluated by intraperitoneal route at the doses of 50, 100 and 200 mg/kg, administered 30 minutes prior to injection of the venom (pretreatment protocol). Stains suggestive of the presence of flavonoids: apigenin, luteolin, orientin, isoorientin, vitexin and vitexin-2-O-rhamnoside were detected in the extract by co-CCD. By means of HPLC were identified isoorientin, orientin, vitexin and isovitexin. All tested doses of J. mollissima extract reduced the paw edema induced by the venom with intensity similar to dexamethasone. The aqueous extract of J. mollissima leaves on all evaluated doses, inhibited cell migration induced by B. jararaca and B. erythromelas promoting inhibition of recruitment of mononuclear cells and the polymorphonuclear cells. Local bleeding induced by B. jararaca venom was significantly inhibited by the extract. Both venoms were inhibited by the extract in myotoxic activity. These results indicate that the aqueous extract of J. mollissima leaves have snakebite potential, particularly with respect to local effects, which may justify the use of this plant in traditional medicine and complementary therapy as anti-venom serum.

Inibição dos efeitos locais induzidos pelas peçonhas das serpentes Bothrops erythromelas e Bothrops jararaca pelo extrato aquoso das folhas de Jatropha mollissima (Pohl) Bail

Snakebites are a serious public health problem in tropical and subtropical countries and Bothrops genus is responsible for the accidents in Brazil and throughout Latin America (90% of cases). The local effects (pain, edema, hemorrhage and myonecrosis) and systemic (cardiovascular alterations, shock and blood clotting disorders) caused by the venom of Bothrops are due to the numerous protein and non-protein components, which are part of the constitution of the poison. The only form of therapy is scientifically validated antivenom serum therapy which, however, is not effective with respect to local effects produced, risk of immunological reactions, high cost and difficult access in some regions. Thus, the search for new alternatives to serum therapy becomes important, and in this context, many medicinal plants have been highlighted by the popular use as antiophidic. Among these plants, we can mention the species Jatropha mollissima (Euphorbiaceae) which has popular use in traditional medicine as antiophidic, anti-inflammatory, antimicrobial and antipyretic. Therefore, this study aims to evaluate the neutralizing potential of local effects induced by the venom of Bothrops erythromelas and Bothrops jararaca with the aqueous extract of the leaves of J. mollissima. The leaf extracts were prepared by decoction, fractionated (by liquid-liquid partition) and characterized by thin layer chromatography (TLC) and High Performance Liquid Chromatography (HPLC). Antiophidic activity of the extract was evaluated in model of paw edema, peritonitis, bleeding and myotoxicity induced by venoms of B. jararaca and B. erythromelas. In all models, the extract was evaluated by intraperitoneal route at the doses of 50, 100 and 200 mg/kg, administered 30 minutes prior to injection of the venom (pretreatment protocol). Stains suggestive of the presence of flavonoids: apigenin, luteolin, orientin, isoorientin, vitexin and vitexin-2-O-rhamnoside were detected in the extract by co-CCD. By means of HPLC were identified isoorientin, orientin, vitexin and isovitexin. All tested doses of J. mollissima extract reduced the paw edema induced by the venom with intensity similar to dexamethasone. The aqueous extract of J. mollissima leaves on all evaluated doses, inhibited cell migration induced by B. jararaca and B. erythromelas promoting inhibition of recruitment of mononuclear cells and the polymorphonuclear cells. Local bleeding induced by B. jararaca venom was significantly inhibited by the extract. Both venoms were inhibited by the extract in myotoxic activity. These results indicate that the aqueous extract of J. mollissima leaves have snakebite potential, particularly with respect to local effects, which may justify the use of this plant in traditional medicine and complementary therapy as anti-venom serum.

Estudo fitoquímico e biológico das folhas de Banisteriopsis argyrophylla (A. Juss.) B. Gates (Malpighiaceae)

With the increasing fungi resistance compared with existing drugs on the market and the side effects reported by some compounds with antioxidant properties and enzymatic inhibitors, in particular against α-amylase and α-glucosidase, the discovery of new compounds with biological potential, becomes a need. In this context, natural products can be an important source for the discovery of new active molecular architectures. Then, this study aimed to evaluate the antioxidant activity, the enzymatic inhibitory activity of α-amylase and α-glucosidase, the antifungal and cytotoxic activities of ethanolic extract (EE) the leaves of Banisteriopsis argyrophylla (Malpighiaceae) and their fractions, obtained by liquid-liquid extraction using solvents of increasing polarity. The antioxidant activity was evaluated by the free radical DPPH scavenging method (2,2-diphenyl-1-picrylhydrazyl) and the ethyl acetate fractions (FAE) and n-butanol (FB) were the most active, confirmed by the peak current and the oxidation potential obtained by differential pulse voltammetry (DPV). The inhibitory activity of the α-amylase and α-glucosidase was analyzed considering the reactions between substrates α-(2-chloro-4-nitrophenyl)-β-1,4-galactopiranosilmaltoside (Gal-α-G2-CNP) and 4-nitrophenyl-α-D-glucopyranoside (p-NPG), respectively. Initially, it was found that the EE showed considerable activity against α-amylase (EC50 = 2.89±0.1 μg m L–1) compared to the acarbose used as positive control (EC50 = 0.08±0.1 μg mL–1) and that did not showed promising activity against the α-glucosidase. After this observation we evaluated the inhibitory activity of α-amylase fractions, with FAE (EC50 = 2.33±0.1 μg mL–1) and FB (EC50 = 2.57 ± 0.1 μg mL–1) showing the best inhibitions. The antifungal activity was evaluated against Candida species, and the FAE had better antifungal potential (MIC's between 93.75 and 11.72 μg mL–1) compared with amphotericin as positive standard (MIC = 1.00 and 2.00 μg L–1 for C. parapsilosis and C. krusei used as controls, respectively). The EE (CC50 = 360.00 ± 12 μg mL–1) and fractions (CC50's> 270.00 μg mL–1) were considerably less toxic to Vero cells than the cisplatin used as positive control (CC50 = 7.01 ± 0 6 μg mL–1). The FAE showed the best results for the activities studied, this fraction was submitted to ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS)), and the following flavonoids have been identified: (±)-catechin, quercetin-3-O-β-D-Glc/ quercetin-3-O-β-D-Gal, quercetin-3-O-β-L-Ara, quercetin-3-O-β-D-Xyl, quercetin-3-O-α-L-Rha, kaempferol-3-O-α-L-Rha, quercetin-3-O-(2''-galoil)-α-L-Rha, quercetin-3-O-(3''-galoil)-α-L-Rha and kaempferol-3-O-(3''-galoil)-α-L-Rha,. FAE was submitted to column chromatography using C18 phase, and (±)-catechin was isolated (FAE-A1, 73 mg) and three fractions consisting of a mixture of flavonoids were obtained (FAE-A2, FAE-A3 and FAE-A4). These compounds were identified by thin layer chromatography (TLC) and (–)-ESI-MS. The (±)-catechin fraction showed an MIC = 2.83 μg ml–1 in assay using C. glabrata, with amphotericin as positive control. The fractions FAE-A2, FAE-A3, FAE-A4, showed less antifungal potential in tested concentrations. The identified flavonoids are described in the literature, regarding their antioxidant capacity and (±)-catechin, quercetin-3-O-Rha and kaempferol-3-O-Rha are described as α-amylase inhibitors. Thus, B. argyrophylla is an important species that produces compounds with antioxidant potential that can be related to the traditional use as anti-inflammatory and also has antifungal compounds and inhibitors of α-amylase. Therefore, these leaves are promising resources for the production of new drugs.

The cellular lipids of Romboutsia.

We have examined the lipids of three isolates, Romboutsia lituseburensis, Romboutsia ilealis, and Romboutsia sp. strain FRIFI, of the newly described genus Romboutsia by two-dimensional thin-layer chromatography (2D-TLC) and by liquid chromatography/mass spectrometry (LC/MS). We have found three phospholipids, phosphatidylglycerol (PG), cardiolipin and phosphatidic acid in all three species. A fourth phospholipid, lysyl-PG, was found in R. lituseburensis and strain FRIFI. Polyprenyl-phosphates were identified in the lipid extracts of all three species. Three glycolipids, mono-, di- and tri-hexosyldiacylglycerol, were common to all three species. An additional glycolipid, tetrahexosyl-diacylglycerol was identified in strain FRIFI. Acylated trihexosyldiacylglycerol and acyl-tetrahexosydiacylglycerol were also found in R. ilealis and strain FRIFI. Remarkably, no alk-1-enyl ether lipids (plasmalogens) were present in Romboutsia as distinct from bacteria of the related genus Clostridium in which these ether lipids are common. We have compared the lipidome of Romboutsia with that recently described for Clostridium difficile, which has plasmalogens, no lysyl-PG, and no tetrahexosyl-diacylglycerol. According to 16S rRNA gene sequencing, Romboutsia spp. and C. difficile are closely related (>95% sequence identity).

From traditional resource to global commodities: a comparison of Rhodiola species using NMR spectroscopy - metabolomics and HPTLC

The fast developing international trade of products based on traditional knowledge and their value chains has become an important aspect of the ethnopharmacological debate. The structure and diversity of value chains and their impact on the phytochemical composition of herbal medicinal products has been overlooked in the debate about quality problems in transnational trade. Different government policies and regulations governing trade in herbal medicinal products impact on such value chains. Medicinal Rhodiola species, including Rhodiola rosea L. and Rhodiola crenulata (Hook.f. & Thomson) H.Ohba, have been used widely in Europe and Asia as traditional herbal medicines with numerous claims for their therapeutic effects. Faced with resource depletion and environment destruction, R. rosea and R. crenulata are becoming endangered, making them more economically valuable to collectors and middlemen, and also increasing the risk of adulteration and low quality. We compare the phytochemical differences among Rhodiola raw materials available on the market to provide a practical method for Rhodiola authentication and the detection of potential adulterant compounds. Samples were collected from Europe and Asia and nuclear magnetic resonance spectroscopy coupled with multivariate analysis software and high performance thin layer chromatography techniques were used to analyse the samples. A method was developed to quantify the amount of adulterant species contained within mixtures. We compared the phytochemical composition of collected Rhodiola samples to authenticated samples. Rosavin and rosarin were mainly present in R. rosea whereas crenulatin was only present in R. crenulata. 30% of the Rhodiola samples purchased from the Chinese market were adulterated by other Rhodiola spp. Moreover, 7 % of the raw-material samples were not labelled satifactorily. The utilisation of both 1H-NMR and HPTLC methods provided an integrated analysis of the phytochemical differences and novel identification method for R. rosea and R. crenulata. Using 1H-NMR spectroscopy it was possible to quantify the presence of R. crenulata in admixtures with R. rosea. This quantitative technique could be used in the future to assess a variety of herbal drugs and products. This project also highlights the need to further study the links between producers and consumers in national and trans-national trade.

Perturbations in Blood Phosphatidylcholine and Sphingomyelin Fatty Acid Composition in a Sample Population of Cigarette Smokers

Docosahexaenoic (DHA) and arachidonic acids (AA) are polyunsaturated fatty acids (PUFAs), major components of brain tissue and neural systems, and the precursors of a number of biologically active metabolites with functions in inflammation resolution, neuroprotection and other actions. As PUFAs are highly susceptible to peroxidation, we hypothesised whether cigarette smokers would present altered PUFAs levels in plasma and erythrocyte phospholipids. Adult males from Indian, Sri-Lankan or Bangladeshi genetic backgrounds who reported smoking between 20 and 60 cigarettes per week were recruited. The control group consisted of matched non-smokers. A blood sample was taken, plasma and erythrocyte total lipids were extracted, phospholipids were separated by thin layer chromatography, and the fatty acid content analysed by gas chromatography. In smokers, dihomo-gamma-linolenic acid, the AA precursor, was significantly reduced in plasma and erythrocyte phosphatidylcholine. AA and DHA were significantly reduced in erythrocyte sphingomyelin. Relatively short term smoking has affected the fatty acid composition of plasma and erythrocyte phospholipids with functions in neural tissue composition, cell signalling, cell growth, intracellular trafficking, neuroprotection and inflammation, in a relatively young population. As lipid peroxidation is pivotal in the pathogenesis of atherosclerosis and neurodegenerative diseases such as Alzheimer disease, early effects of smoking may be relevant for the development of such conditions.

Produção de lipidios funcionais por ação de lipase fúngica

A modificação estrutural de óleos e gorduras é uma das principais áreas de interesse de pesquisa em diferentes setores industriais. No caso da indústria de alimentos, a interesterificação é empregada para melhorar propriedades nutricionais e funcionais, em que se obtêm compostos diferentes dos que lhes deram origem. As lipases microbianas são os biocatalisadores mais utilizados industrialmente, por serem mais estáveis, específicas e com propriedades bem mais diversificadas que as lipases de outras fontes. Este trabalho objetivou, primeiramente, a caracterização da gordura da pele de frango (GPF) e sua comparação com óleo de soja, como referência, visando a utilização de GPF em reações de interesterificação. Para isto foram caracterizados quanto aos índices de rancidez hidrolítica e oxidativa, bem como de matéria insaponificável, índices de saponificação, refração e iodo. Foi realizado ainda o fracionamento e perfil de ácidos graxos destes lipídios e suas frações, com o cálculo de seus índices nutricionais. Foi verificado que a GPF apresentou qualidade satisfatória devido aos baixos índices de acidez (0,65 g ácido oleico.100 g -1 ), peróxido (2,14 meq.kg-1 ), p-anisidina (0,70 unidades de absorvância.g-1 ), além de fonte de ácidos graxos mono-insaturados (40%), sendo fonte promissora para estudos de interesterificação. Em um segundo momento o objetivo foi produzir lipídios modificados ricos em ácidos graxos essenciais a partir da gordura da pele de frango e ácidos graxos ramificados, utilizando lipase sn-1,3 específica e interesterificação do tipo acidólise. Foram estudados os fatores concentração de enzima, adição de água, proporção de substratos e tempo, segundo um planejamento experimental fatorial completo 2 4 . As separações analíticas foram executadas em placas de cromatografia de camada delgada, sendo as frações posteriormente extraídas, ressuspensas e injetadas no cromatógrafo a gás. Foi verificado que a adição de água ao meio reacional apresentou efeito significativo (p<0,05) para todos ácidos graxos avaliados dos triacilgliceróis, sendo que para o ácido essencial linoleico (C18:2) o efeito do tempo de reação também foi significativo, sendo verificado que quanto maior o tempo de reação, menor a quantidade de água a ser adicionada. Em um terceiro momento, o objetivo foi produzir éster fenólico a partir do DHCA, além de realizar reações de transesterificação deste éster com tricaprilina. Para a reação de transesterificação, foi utilizado um delineamento composto central rotacional (DCCR) variando a quantidade de enzima, tempo de reação e temperatura sobre a resposta (%) dos reagentes consumidos. A lipase Novozym® 435 de Candida antarctica foi utilizada como catalisador de todas reações. Foi verificado que a maior produção de éster (50%) ocorreu em oito dias. Nas reações de transesterificação, as relações molares em que houve maior consumo do éster produzido foram 1:5 e 1:10, sendo obtidos 21,1% e 29,6% de residual de dihidrocafeato de octila, respectivamente em 24 h. Foi observado que em altas temperaturas e tempo superior a 26 h, houve o menor residual de dihidrocafeato de octila (18,2%). Foram identificados três diferentes compostos fenólicos, contendo em sua estrutura dihidrocafeato de octila e ácido caprílico.

Ocratoxina A e citrinina em arroz: um estudo de metodologia para determinação simultânea de ocorrência

A ocratoxina A e a citrinina são micotoxinas encontrados naturalmente em alimentos e rações animais. A avaliação do risco do consumo de alimentos contaminados por esses compostos deve ser estimada a partir de dados confiáveis que reflitam a verdadeira concentração destas toxinas em diferentes alimentos ou insumos, especialmente se ingeridos com freqüência. Isto gera a necessidade de métodos analíticos precisos que sejam rápidos sem desconsiderar as etapas clássicas de avaliação de traços, amostragem representativa, extração, limpeza, concentração, separação de formas químicas, detecção, confirmação de identidade e quantificação. Para as micotoxinas em geral as técnicas cromatográficas são as mais aplicadas e relatadas em vista da diversidade estrutural destes compostos. Neste trabalho foi realizada uma otimização de extração utilizando o método QuEChERS modificado em comparação com os métodos Soares e Rodrigues Amaya (1989), Tanaka (2001) e Ultrassom (Palma et. al., 2007) empregando diferentes técnicas cromatográficas com detectores distintos para análise destas micotoxinas simultaneamente. Foram analisadas 38 amostras de arroz cultivadas e armazenadas em campos experimentais de Cachoeirinha na região Sul do Brasil. O uso dos sistemas CCD, HPLC-DAD e LC-MS proporcionou especificidade, precisão e sensibilidade, de modo que os limites de detecção e quantificação, obtivessem valores inferiores ao limite máximo estabelecido por órgãos reguladores internacionais (5 µg Kg-1 para ocratoxina A). Os limites de detecção encontrados para citrinina e ocratoxina A em camada delgada foram 4,7 e 6 vezes maior que para cromatografia líquida de alta eficiência acoplada a detector de arranjo de diodos, 14 e 300 vezes maior que cromatografia líquida acoplada a detector de massas. Os limites de quantificação das duas micotoxinas ficaram dentro do exigido pela legislação européia para OTA de 5 µg Kg-1 para HPLC-DAD e LC-MS. Na cromatografia de camada delgada esse valor ficou 4 vezes acima do estabelecido para ocratoxina A. A ocorrência de ocratoxina A e citrinina foi verificada em 16 % das amostras estando os teores variando entre 3 e 560 µg Kg-1, sugerindo possível exposição crônica a estas micotoxinas caso as amostras sejam consumidas.

Casca de arroz como adsorvente para extração de micotoxinas em cebola

A produção mundial de arroz chega a 80 milhões de toneladas ao ano, considerando que as cascas representam 20% deste valor, anualmente são geradas cerca de 1.162.000 toneladas desse rejeito. Há alguns anos, esse material era descartado no ambiente, atualmente as leis de proteção ambiental, demandaram na preocupação com resíduos de casca de arroz (FOLETO, 2005). Segundo Mayer (2006) a casca leva aproximadamente 5 anos para se decompor e exala um volume elevado de metano, um dos gases responsáveis pelo efeito estufa. Visando proteger a integridade do meio ambiente, estão sendo buscadas alternativas para reduzir os impactos ambientais do descarte e recuperar os investimentos na cultura do grão. Devido seu alto teor de silício a casca de arroz, é matéria-prima de grande interesse para aplicação em vários ramos: indústria eletrônica, cerâmica e na agricultura e também pode ser utilizada como fonte energética e ser aplicadas como adsorvente, em análises químicas (FOLETO, 2005; ROSA, 2009). Neste trabalho o objetivo foi padronizar método para a determinação das aflatoxinas B1, B2, G1, G2 e ocratoxiana A em cebola, empregando a técnica de extração, Dispersão da Matriz em Fase Sólida (MSPD), tendo a casca de arroz como adsorvente, de forma a possibilitar a determinação dos contaminantes empregando cromatografia de camada delgada de alta eficiência (HPTLC) e/ou cromatografia líquida de alta eficiência acoplada a detector de fluorescência (HPLC-FD). A cebola (Allium cepa L.) foi a matriz escolhida devido sua importância econômica, ela é a terceira hortaliça mais importante economicamente no Brasil, depois do tomate e da batata. O país está entre os dez maiores produtores do mundo, sendo que na safra de 2010 a produção foi de 1.548.146 toneladas. Dentre os estados que se destacam pela sua produção estão: Santa Catarina, São Paulo e Rio Grande do Sul. No entanto, a cebola assim como os demais alimentos é suscetível à contaminação fúngica e se entre a microbiota estiverem espécies toxigênicas pode ocorrer à produção de micotoxinas. O método foi validado avaliando-se curva analítica, linearidade, limites de detecção e quantificação, precisão (repetitividade e precisão intermediária) e exatidão (recuperação) para cada tipo de determinação cromatográfica. Para HPTLC, os limites de detecção variaram entre 0,33-5µg Kg-1 e os de quantificação entre 1-15µg Kg-1 Para o método HPLC-FD os limites de detecção variaram entre 0,003– 0,26 µg Kg-1 e os de quantificação 0,03 – 2,6 µg Kg-1 . As recuperações para o método HPTLC variaram entre 76- 95% e para HPLC-FD variaram entre 72-88%. O método desenvolvido foi aplicado para verificar a ocorrência de micotoxinas em 14 amostras de cebola. A contaminação com aflatoxinas foi verificada em 43% das amostras analisadas. O nível máximo encontrado foi de 90 µg Kg-1 para aflatoxina B2 em uma amostra de cebola crioula, com o defeito de mancha negra.

Molecular and biochemical mechanisms involved in the toxicity of a marine cyanobacteria compound on cancer cell lines

In recent years marine biotechnology has revealed a crucial role in the future of bioindustry. Among the many marine resources, cyanobacteria have shown great potential in the production of bioactive compounds with diverse applicability. The pharmacological potential of these organisms has been one of the most explored areas in particular its antibacterial, antifungal and anticancer potential. This work was based on the assessment of potential anticancer compound E13010 F 5.4 isolated from marine cyanobacteria strain Synechocystis salina LEGE 06099. Thus the aim of this work was to explore molecular and biochemical mechanisms underlying the bioactivity detected in human cancer cells, specifically in lines RKO colon carcinoma and HT-29. The isolation of the compound was performed from biomass obtained by large-scale culture. To obtain the compound fractionation was carried and confirmation and isolation performed by Nuclear Magnetic Resonance (NMR), Thin Layer Chromatography (TLC) and High-Performance Liquid Chromatography (HPLC). Cell viability assays were performed based on reduction of 3- (4,5-dimetiltiaziol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) to assess the cytotoxic potential of the compound. From the battery of cell lines RKO (colon carcinoma), HT-29 (colorectal adenocarcinoma), MG-63 (osteosarcoma) and T47D (breast carcinoma) the cell lines RKO and HT-29 were selected for elucidation of mechanisms of cytotoxicity. For the elucidation of the mechanisms involved in cytotoxicity the cell lines RKO and HT29 were exposed to the compound. A genomic approach based in the mRNA expression of genes involved in apoptosis and cell cycle by Real-Time PCR and a proteomic approach based on the separation of proteins by two-dimensional electrophoresis (2DGE) was performed. For mRNA expression were selected the genes RPL8, HPRT1, VDAC, SHMT2, CCNE, CCNB1, P21CIP, BCL-2 and BAD and for proteomics isoelectric focussing between 3 – 10 and molecular weight of 19 – 117 kDa separated by polyacrylamide gels (2DGE). The MTT results confirmed the reduction of the cell viability. The RT-PCR results for the expression of genes studied were not yet fully elucidative. For the cell line RKO there was a significant reduction in the expression of the gene P21CIP, and a tendency for reduction in the BAD gene expression and for increased expression of gene CCNB1, pointing to an effort for cell proliferation. In HT-29 cell line, there was a tendency for increase in the expression of P21CIP and BAD, which may explain the reduction in cell viability. The 2DGE results indicate proteomic patterns with differentially altered spots in the treated and control cells with both qualitative and quantitative differences, and differences in response between the RKO and HT-29 cell lines.

Estudo de metodologias de controle de qualidade do Mo-99 utilizado no preparo de geradores de Mo-99/Tc-99m

Dissertação (Mestrado em Tecnologia Nuclear)

Efecto antiinflamatorio de extractos metanólicos de plantas de Azuay y Loja (Ecuador) a través del modelo de peces cebra

Los peces cebra son utilizados como modelo biológico para screening primario de extractos de plantas con potencial bioactividad, aprovechando sus similitudes: genética, fisiológica y respuesta farmacológica con los mamíferos. En el estudio se empleó este modelo para valorar la actividad antiinflamatoria de 36 extractos metanólicos de plantas medicinales utilizadas en las provincias de Azuay y Loja (Ecuador). Parte del material vegetal fue recolectado con el aporte de una hierbatera de etnia Saraguro. Los extractos fueron preparados por percolación y su toxicidad fue evaluada en peces cebra en concentraciones variables de 400 a 3,125 μg/ml, determinándose la máxima concentración tolerada para cada uno de ´estos. La actividad antiinflamatoria se evaluó a través del ensayo de migración leucocitaria inducida por lipopolisacárido de Sallmonella typhi. Los extractos de: Cestrum aff. peruvianum, Galinsoga parviflora, Galium sp., Oenothera tetraptera, Peperomia aff. galioides , Passiflora ampullaceae y Ambrosia arborescens, correspondientes al 18,92% de los analizados, mostraron un potencial antiinflamatorio comparable con indometacina y dexametasona; siendo el extracto metanólico de Cestrum aff. peruvianum el más relevante a 50 g/ml. El análisis fitoquímico básico de los extractos se realizó por cromatografía de capa fina, evidenciándose la presencia de saponinas y terpenoidoes como compuestos principales en la mayoría de los extractos.