Potencial bioativo de sucos de uva Vitis labrusca L.: caracterização química e atividade antioxidante, influência de sementes de uva e de pectinases na composição fenólica, e bioatividade do trans-resveratrol em células humanas

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências Agrárias, Programa de Pós-Graduação em Ciência dos Alimentos, Florianópolis, 2016.As propriedades bioativas de compostos fenólicos são bastante estudadas em vinhos e uvas V. vinifera L. Entretanto, variedades V. labrusca L., amplamente cultivadas no Brasil e principalmente destinadas à produção do suco de uva, representam uma atrativa fonte de compostos fenólicos. Os objetivos deste trabalho foram avaliar a influência da adição de sementes de uva e de enzimas pectinolíticas no teor de compostos fenólicos, na atividade antioxidante in vitro e no teor de macro e microelementos de sucos de uva de diferentes variedades V. labrusca L. cultivadas na região Sul do Brasil; e avaliar suas propriedades bioativas in vitro e in vivo. Nas etapas de elaboração experimental dos sucos, os efeitos da adição de sementes de uva e de pectinases foram avaliados em sucos de uvas Bordô, Concord e Isabel. Para a avaliação do potencial bioativo, foram empregados sucos de uvas industrializados produzidos na região da Serra Gaúcha, RS, Brasil. Sucos de uva tintos e branco foram caracterizados quanto às composições fenólica e elementar e à atividade antioxidante in vitro. O efeito do consumo agudo de sucos de uva orgânico e convencional sobre a atividade antioxidante e marcadores de estresse oxidativo em indivíduos saudáveis foi avaliado através de estudo de intervenção controlado randomizado. Em estudo com células humanas, os efeitos bioativos do trans-resveratrol (RSV) foram investigados quanto a mecanismos oxidativos envolvidos na disfunção endotelial, através de cocultura de células intestinais Caco-2 e endoteliais EA.hy926. A adição de sementes de uva aumentou significativamente o teor de polifenóis totais e a atividade antioxidante nos sucos, e demonstrou correlação positiva com os minerais Ca, Mg, Na, K, Mn e Zn. A otimização da atividade pectinolítica através da metodologia de superfície de resposta gerou um aumento na extração de minerais e polifenóis nos sucos, demonstrando correlação positiva entre as variáveis. As condições otimizadas da extração de polifenóis foram estimadas pelo modelo nas concentrações enzimáticas de 1,30 e 1,57 g L-1, nas temperaturas de 46,8 e 54,8 ºC, respectivamente. A caracterização dos sucos de uva industrializados demonstrou maior concentração de polifenóis totais, antocianinas monoméricas, trans-resveratrol, Mg e Mn, e maior atividade antioxidante in vitro no suco de uva orgânico. O consumo agudo de sucos de uva V. labrusca L. promoveu reduções significativas nos níveis séricos de hidroperóxidos lipídicos (LOOH) e nasconcentrações plasmáticas de substâncias reativas ao ácido tiobarbitúrico (TBARS) em indivíduos saudáveis. O consumo dos sucos aumentou significativamente a capacidade antioxidante sérica e a atividade das enzimas catalase (CAT), glutationa peroxidase (GPx) e superóxido dismutase (SOD) em eritrócitos. As intervenções com os sucos não afetaram significativamente o estado glicêmico e os níveis de ácido úrico. A análise multivariada confirmou a influência das principais classes fenólicas nas respostas biológicas. Todavia, as diferenças químicas associadas aos sucos orgânico e convencional não foram um fator preditivo para seus efeitos in vivo. Em cultura de células, o tratamento com RSV reduziu significativamente os níveis de fator de crescimento endotelial vascular (VEGF), espécies reativas de oxigênio (ERO), interleucina 8 (IL-8) e molécula de adesão intercelular 1 (ICAM-1) induzidos pelo fator de necrose tumoral a (TNF-a), e aumentou a produção endotelial de óxido nítrico (NO). As análises por UPLC-MS/MSE revelaram a presença de RSV e quatro metabólitos conjugados. A bioatividade do RSV e seus metabólitos sobre mecanismos inflamatórios foi demonstrada pela primeira vez em cocultura. A caracterização química e os resultados in vitro e in vivo evidenciaram que os sucos V. labrusca L. são ricos em macro e microelementos e compostos fenólicos, com potencial bioativo na saúde humana.Abstract : The bioactive properties of polyphenols are extensively studied in wines and grapes from V. vinifera L. However, the V. labrusca L. varieties, widely cultivated in Brazil and mainly destined to grape juice production, represent an attractive source of phenolic compounds. The objectives of this study were to evaluate the influence of the addition of grape seeds and pectinolytic enzymes on the polyphenols content, antioxidant activity in vitro and on the macro and microelements contents in grape juices from different V. labrusca L. varieties grown in the South region of Brazil; and to evaluate their bioactive properties in vitro and in vivo. In the experimental preparation of juices, the effects of grape seed addition and pectinases were evaluated in Bordo, Concord and Isabel grape juices. Industrialized grape juices produced in the Serra Gaúcha Region, RS, Brazil, were used to investigate the bioactive potential. Characterization of the phenolic and elemental composition and in vitro antioxidant activity was performed in red and white grape juices. The effect of acute consumption of organic and conventional grape juices on antioxidant activity and oxidative stress markers in healthy subjects was evaluated through a randomized controlled intervention trial. In human cells studies, the bioactive effects of trans-resveratrol (RSV) towards oxidative mechanisms related to endothelial dysfunction were investigated through a co-culture of intestinal Caco-2 cells and endothelial EA.hy926 cells. Grape seed addition significantly increased the total phenolics and antioxidant capacity of juices, and was positively correlated with minerals Ca, Mg, Na, K, Mn e Zn. The optimization of pectinolytic activity through response surface methodology led to an increased extraction of minerals and phenolics in juices, and depicted a positive correlation among the variables. The optimized conditions for polyphenols extraction were estimated by the model at enzyme concentrations of 1.30 and 1.57 g L-1, at 46.8 and 54.8 ºC, respectively. The characterization of industrialized grape juices showed higher concentration of total phenolics, monomeric anthocyanins, trans-resveratrol, Mg and Mn, and higher antioxidant activity in vitro in the organic grape juice. The acute consumption of V. labrusca L. juices promoted significant reductions on levels of hydroperoxides (LOOH) in serum and thiobarbituric acid reactive substances (TBARS) in plasma of healthy individuals. The consumption of juices significantly augmented serum antioxidant capacity andenzymatic activities of catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD) in erythrocytes. The interventions with grape juices did not affected significantly the glycaemic status and uric acid levels. The multivariate analysis confirmed the influence of the main phenolic classes on biological responses. However, the chemical differences associated with organic and conventional juices were not a predictive factor for its effects in vivo. In cell culture, RSV treatment significantly reduced the tumor necrosis factor a (TNF-a)-induced levels of vascular endothelial growth factor (VEGF), reactive oxygen species (ROS), interleukin 8 (IL-8) and intercellular adhesion molecule 1 (ICAM-1) and increased the endothelial production of nitric oxide (NO). The UPLC-MS/MSE analysis revealed the presence of RSV and four conjugated metabolites. The bioactivity of RSV and its metabolites towards inflammatory mechanisms was demonstrated in a co-culture for the first time. The chemical characterization and the in vitro and in vivo results have demonstrated that V. labrusca L. grape juices are rich sources of macro and microelements and phenolic compounds, with bioactive potential in human health

Desenvolvimento e validação de método multirresíduo para determinação de resíduos de antimicrobianos em leite por CLAE-FL/DAD e CLAE-MS/MS

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Agrárias, Programa de Pós-Graduação em Ciência dos Alimentos, Florianópolis, 2011Antimicrobianos são extensivamente empregados na medicina veterinária para prevenção e tratamento de infecções em animais produtores de alimentos. O uso disseminado desses fármacos na prática leiteira acarreta a presença de seus resíduos no leite. Resíduos de antimicrobianos representam riscos à saúde do consumidor, podendo causar reações de hipersensibilidade e resistência microbiana. O objetivo deste trabalho foi desenvolver e validar método multirresíduo para análise de resíduos de sulfonamidas, fluorquinolonas e ceftiofur em leite, empregando a técnica de cromatografia líquida de alta eficiência com detecção por fluorescência, arranjo de diodos e espectrometria de massas. Diferentes etapas foram avaliadas quanto à extração dos antimicrobianos em leite, sendo empregadas extrações líquido-líquido e em fase sólida. O método quantitativo foi avaliado quanto aos critérios de desempenho: seletividade, linearidade e faixa de trabalho, sensibilidade, precisão, exatidão, limite de detecção, limite de quantificação, limite de decisão (CC?), capacidade de detecção (CC?) e robustez, para validação analítica segundo a Diretiva 657/2002 da Comunidade Européia. Adicionalmente, a identidade dos compostos foi confirmada por cromatografia líquida com detecção por espectrometria de massas, a partir de extração líquido-líquido em leite. De modo a avaliar o desempenho dos métodos, diferentes amostras de leite obtidas comercialmente foram analisadas pelos métodos quantitativo e confirmatório. A extração líquido-líquido com água 10% ácido tricloroacético demonstrou melhor resultado para extração dos resíduos em leite, em comparação à extração em fase sólida. O gradiente de eluição permitiu a separação cromatográfica de onze antimicrobianos, compreendo quatro fluorquinolonas (ciprofloxacino, norfloxacino, danofloxacino e enrofloxacino), seis sulfonamidas (sulfatiazol, sulfaclorpiridazina, sulfametoxazol, sulfamerazina, sulfadoxina e sulfadimetoxina) e uma cefalosporina (ceftiofur). O método foi validado em conformidade com a Legislação Européia com desempenho analítico adequado. Os coeficientes de correlação (r2) foram superiores a 0,98 para todas as curvas, com recuperações superiores a 98% para todos os analitos e limites de detecção e quantificação abaixo dos valores de limite máximo permitido para os compostos em leite. A repetibilidade e a reprodutibilidade foram inferiores ou iguais a 7,40% e 10,13%, respectivamente. Nas amostras de leite não houve detecção para os antimicrobianos. Os métodos desenvolvidos representam alternativa eficiente para análise multirresíduo em leite, com aplicabilidade confirmada pela análise de amostras reais de leite de diferentes origens.Antimicrobials are extensively used in veterinary medicine for prevention and treatment of infections in food-producing animals. The widespread use of these drugs in dairy practice leads to the presence of its residues in milk. Antimicrobial residues can pose risks to consumers health, may causing hypersensitivity reactions and microbial resistance. The objective of this work was to develop and validate multiresidue method for the analysis of sulfonamide, fluoroquinolone, and ceftiofur residues in milk, using high performance liquid chromatography with fluorescence, diode array and mass spectrometry detections. Different steps were evaluated for extracting antimicrobials in milk, by applying liquid-liquid and solid phase extractions. The quantitative method was evaluated regarding performance criteria: selectivity, linearity and working range, sensitivity, precision, accuracy, detection limit, quantification limit, decision limit (CC?), detection capability (CC?) and robustness, for analytical validation according to Directive 657/2002 of the European Community. Additionally, the analytes identities were confirmed by liquid chromatography with mass spectrometry detection, through a liquid-liquid extraction in milk. To evaluate methods performance, different milk samples obtained at local markets were analyzed by both quantitative and confirmatory methods. The liquid-liquid extraction with 10% trichloroacetic acid in water showed better results for residue extraction in milk compared to solid phase extraction. The elution gradient allowed the chromatographic separation of eleven antimicrobials, comprising four fluoroquinolones (ciprofloxacin, norfloxacin, danofloxacin and enrofloxacin), six sulfonamides (sulfathiazole, sulfachlorpyridazine, sulfamethoxazole, sulfamerazine, sulfadoxine and sulfadimethoxine) and one cephalosporin (ceftiofur). The quantitative method was validated in line with European requirements with adequate analytical performance. The correlation coefficients (r2) were greater than 0.98 for all curves, also the recoveries rates were above 98% for all analytes and the LOD and LOQ limits were lower than the maximum permitted limit for the compounds in milk. The intra-day and inter-day precisions were below or equal to 7.40% and 10.13%, respectively. Milk samples showed no detection for the antimicrobials. The developed methods comprise an efficient alternative to multiresidue analysis in milk, with applicability confirmed by analysis of real milk samples from different origins

Resveratrol improves TNF-α-induced endothelial dysfunction in a coculture model of a Caco-2 with an endothelial cell line

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Phytochemical polyphenol extraction and elemental composition of Vitis labrusca L. grape juices through optimization of pectinolytic activity

Grape juice is a natural source of polyphenols with potential health benefits. The aim of this study was to assess the optimized basis of the pectinolytic activity through a multivariate design for the bioactive enrichment of polyphenols in grape juices and to evaluate the effect of pectinases on the extraction of minerals by ICP-MS and FAAS. Grape juices were treated with pectinases EverzymA (R) Color and RapidaseA (R) Smart at different conditions of enzyme concentration (0.16-1.84 g L-1), temperature (14.8-83.6 A(0)C), and incubation time (9.6-110.5 min), and the effect on total phenolics, anthocyanin content, and antioxidant activity of juices were determined by employing a second-order central composite design in combination with response surface methodology. The different conditions showed significant variations in polyphenol levels of grape juices. For both of the enzymes, the increase of temperature and enzyme concentration led to a higher extraction of polyphenols, with a significant effect. Estimated effects depict a good correlation among dependent variables. For Rapidase, the optimized extraction of polyphenols was determined at an enzyme concentration of 1.57 g L-1 and at a temperature of 54.8 A(0)C. For Everzym, higher concentrations of polyphenols were obtained at the enzyme concentration of 1.30 g L-1 at 46.8 A(0)C. The extraction using the Rapidase enzyme significantly increased the levels of Na, K, Mg, and Fe in grape juices (p < 0.05). The pectinolytic activity increased the polyphenol and mineral concentrations in grape juices. In addition, the optimal conditions of the pectinolytic treatment comprise a feasible alternative to improve the bioactive potential of grape juices

Bioactive profiling of polyphenolics and oenological properties of red wines from Italian grapes (<i>Vitis vinifera</i> L.) cultivated in a selected subtropical region

<p>The polyphenolic and oenological properties were evaluated in red wines from Italian varieties Ancellotta, Rebo, Nebbiolo, Barbera and Teroldego cultivated in a selected subtropical region. Ancellotta wines showed the highest concentrations of phenolics, particularly flavanols, flavonols and anthocyanins 3-<i>O</i>-glycosides. Grape variety rather than vintage had stronger influence on polyphenols. Wines from vintage 2012 showed the highest antioxidant activity and highest concentrations of polyphenols, mainly Ancellotta and Teroldego wines. The wines showed similar phenolic profile regarding the presence of phenolics, whereas their concentrations varied greatly among varieties and vintages. The results demonstrated good adaptability of these varieties and their potential to produce quality wines.</p

Bioactive potential of Vitis labrusca L. grape juices from the Southern Region of Brazil: phenolic and elemental composition and effect on lipid peroxidation in healthy subjects

AbstractGrapes are rich in polyphenols with biologically active properties. Although the bioactive potential of grape constituents are frequently reported, the effects of Brazilian Vitis labrusca L. grape juices ingestion have not been demonstrated in humans. This study identified the phenolic and elemental composition of red and white grape juices and the effect of organic and conventional red grape juice consumption on lipid peroxidation in healthy individuals. Concentrations of anthocyanins, flavanols and phenolic acids and the in vitro antioxidant activity were significantly higher in the organic juice. The macro-elements K, Ca, Na and Mg were the most abundant minerals in all juices. The acute consumption of red grape juices promoted significant decrease of lipid peroxides in serum and TBARS levels in plasma. It is concluded that red V. labrusca L. grape juices produced in Southern Brazil showed lipid peroxidation inhibition abilities in healthy subjects, regardless of the cultivation system

Milk quality parameters associated with the occurrence of veterinary drug residues in bulk tank milk

Veterinary drug residues in bulk tank milk are important to all sectors of the dairy chain because they are one of the major factors which determine the safety of the final product. This study attempted to identify milk quality parameters that are associated with the occurrence of veterinary drug residues using multivariate principal component analysis (PCA). A total of 132 raw milk samples were collected from 45 dairy farms in the state of Minas Gerais - Brazil and analyzed for 42 analytes, including pyrethroids, macrocyclic lactones and antibacterials, using liquid chromatography coupled with mass spectrometry in tandem mode and gas chromatography with electron capture detection. Out of the 132 milk samples, 40 samples tested positive for at least one analyte (above the detection limit). The milk parameters associated with the antimicrobial residues by confirmatory tests were lactose and nonfat concentrations, as revealed by PCA. This analysis showed that fat and total solid concentrations, as well as the somatic cell and total bacteria counts were associated with macrocyclic lactone residues in bulk tank milk. A PCA assessing pyrethroid residues in bulk tank milk revealed that the lactose and nonfat solid concentrations and titratable acidity were inversely associated with these residues. Thus, the data analysis indicated that the veterinary drug residues were associated with certain milk quality parameters that can be used to target farms at higher risk of veterinary drug residue contamination for testing programs in combination with incentives, education and training programs to improve mammary health, milk hygiene and safety