Modulation of ROS production in human leukocytes by ganglioside micelles

Recent studies have reported that exogenous gangliosides, the sialic acid-containing glycosphingolipids, are able to modulate many cellular functions. We examined the effect of micelles of mono- and trisialoganglioside GM1 and GT1b on the production of reactive oxygen species by stimulated human polymorphonuclear neutrophils using different spectroscopic methods. The results indicated that exogenous gangliosides did not influence extracellular superoxide anion (O2.-) generation by polymorphonuclear neutrophils activated by receptor-dependent formyl-methionyl-leucyl-phenylalanine. However, when neutrophils were stimulated by receptor-bypassing phorbol 12-myristate 13-acetate (PMA), gangliosides above their critical micellar concentrations prolonged the lag time preceding the production in a concentration-dependent way, without affecting total extracellular O2.- generation detected by superoxide dismutase-inhibitable cytochrome c reduction. The effect of ganglioside GT1b (100 µM) on the increase in lag time was shown to be significant by means of both superoxide dismutase-inhibitable cytochrome c reduction assay and electron paramagnetic resonance spectroscopy (P < 0.0001 and P < 0.005, respectively). The observed phenomena can be attributed to the ability of ganglioside micelles attached to the cell surface to slow down PMA uptake, thus increasing the diffusion barrier and consequently delaying membrane events responsible for PMA-stimulated O2.- production.

An HPLC-UV method for the measurement of permeability of marker drugs in the Caco-2 cell assay

The Caco-2 cell line has been used as a model to predict the in vitro permeability of the human intestinal barrier. The predictive potential of the assay relies on an appropriate in-house validation of the method. The objective of the present study was to develop a single HPLC-UV method for the identification and quantitation of marker drugs and to determine the suitability of the Caco-2 cell permeability assay. A simple chromatographic method was developed for the simultaneous determination of both passively (propranolol, carbamazepine, acyclovir, and hydrochlorothiazide) and actively transported drugs (vinblastine and verapamil). Separation was achieved on a C18 column with step-gradient elution (acetonitrile and aqueous solution of ammonium acetate, pH 3.0) at a flow rate of 1.0 mL/min and UV detection at 275 nm during the total run time of 35 min. The method was validated and found to be specific, linear, precise, and accurate. This chromatographic system can be readily used on a routine basis and its utilization can be extended to other permeability models. The results obtained in the Caco-2 bi-directional transport experiments confirmed the validity of the assay, given that high and low permeability profiles were identified, and P-glycoprotein functionality was established.

Acute exposure to lead increases myocardial contractility independent of hypertension development

We studied the effects of the acute administration of small doses of lead over time on hemodynamic parameters in anesthetized rats to determine if myocardial contractility changes are dependent or not on the development of hypertension. Male Wistar rats received 320 µg/kg lead acetateiv once, and their hemodynamic parameters were measured for 2 h. Cardiac contractility was evaluated in vitro using left ventricular papillary muscles as were Na+,K+-ATPase and myosin Ca2+-ATPase activities. Lead increased left- (control: 112 ± 3.7 vs lead: 129 ± 3.2 mmHg) and right-ventricular systolic pressures (control: 28 ± 1.2vs lead: 34 ± 1.2 mmHg) significantly without modifying heart rate. Papillary muscles were exposed to 8 µM lead acetate and evaluated 60 min later. Isometric contractions increased (control: 0.546 ± 0.07 vs lead: 0.608 ± 0.06 g/mg) and time to peak tension decreased (control: 268 ± 13vs lead: 227 ± 5.58 ms), but relaxation time was unchanged. Post-pause potentiation was similar between groups (n = 6 per group), suggesting no change in sarcoplasmic reticulum activity, evaluated indirectly by this protocol. After 1-h exposure to lead acetate, the papillary muscles became hyperactive in response to a β-adrenergic agonist (10 µM isoproterenol). In addition, post-rest contractions decreased, suggesting a reduction in sarcolemmal calcium influx. The heart samples treated with 8 µM lead acetate presented increased Na+,K+-ATPase (approximately 140%, P < 0.05 for control vs lead) and myosin ATPase (approximately 30%, P < 0.05 for control vs lead) activity. Our results indicated that acute exposure to low lead concentrations produces direct positive inotropic and lusitropic effects on myocardial contractility and increases the right and left ventricular systolic pressure, thus potentially contributing to the early development of hypertension.

Inhibition of PKC-dependent extracellular Ca2+ entry contributes to the depression of contractile activity in long-term pressure-overloaded endothelium-denuded rat aortas

We examined the contractile responsiveness of rat thoracic aortas under pressure overload after long-term suprarenal abdominal aortic coarctation (lt-Srac). Endothelium-dependent angiotensin II (ANG II) type 2 receptor (AT2R)-mediated depression of contractions to ANG II has been reported in short-term (1 week) pressure-overloaded rat aortas. Contractility was evaluated in the aortic rings of rats subjected to lt-Srac or sham surgery (Sham) for 8 weeks. ANG I and II levels and AT2R protein expression in the aortas of lt-Srac and Sham rats were also evaluated. lt-Srac attenuated the contractions of ANG II and phenylephrine in the aortas in an endothelium-independent manner. However, lt-Srac did not influence the transient contractions induced in endothelium-denuded aortic rings by ANG II, phenylephrine, or caffeine in Ca2+-free medium or the subsequent tonic constrictions induced by the addition of Ca2+ in the absence of agonists. Thus, the contractions induced by Ca2+ release from intracellular stores and Ca2+ influx through stored-operated channels were not inhibited in the aortas of lt-Srac rats. Potassium-elicited contractions in endothelium-denuded aortic rings of lt-Srac rats remained unaltered compared with control tissues. Consequently, the contractile depression observed in aortic tissues of lt-Srac rats cannot be explained by direct inhibition of voltage-operated Ca2+ channels. Interestingly, 12-O-tetradecanoylphorbol-13-acetate-induced contractions in endothelium-denuded aortic rings of lt-Srac rats were depressed in the presence but not in the absence of extracellular Ca2+. Neither levels of angiotensins nor of AT2R were modified in the aortas after lt-Srac. The results suggest that, in rat thoracic aortas, lt-Srac selectively inhibited protein kinase C-mediated activation of contraction that is dependent on extracellular Ca2+ entry.

Assessment of indicators of vitamin A status in non-cirrhotic chronic hepatitis C patients

Subjects with chronic liver disease are susceptible to hypovitaminosis A due to several factors. Therefore, identifying patients with vitamin deficiency and a requirement for vitamin supplementation is important. Most studies assessing vitamin A in the context of hepatic disorders are conducted using cirrhotic patients. A cross-sectional study was conducted in 43 non-cirrhotic patients with chronic hepatitis C to evaluate markers of vitamin A status represented by serum retinol, liver retinol, and serum retinol-binding protein levels. We also performed the relative dose-response test, which provides an indirect estimate of hepatic vitamin A reserves. These vitamin A indicators were assessed according to the stage of liver fibrosis using the METAVIR score and the body mass index. The sample study was predominantly composed of male subjects (63%) with mild liver fibrosis (F1). The relative dose-response test was <20% in all subjects, indicating vitamin A sufficiency. Overweight or obese patients had higher serum retinol levels than those with a normal body mass index (2.6 and 1.9 µmol/L, respectively; P<0.01). Subjects with moderate liver fibrosis (F2) showed lower levels of serum retinol (1.9 vs 2.5 µmol/L, P=0.01) and retinol-binding protein levels compared with those with mild fibrosis (F1) (46.3 vs 67.7 µg/mL, P<0.01). These results suggested an effect of being overweight on serum retinol levels. Furthermore, more advanced stages of liver fibrosis were related to a decrease in serum vitamin A levels.

Evaluation of total carotenoids, alpha- and beta-carotene in carrots (Daucus carota L.) during home processing

This study aims to analyze the influence of dehydration and different preparation methods during home processing related toalpha-carotene, beta-carotene and total carotenoids stability in carrots. Vitamin A values were evaluated after different treatments. Thus, carrots were submitted to steam cooking, water cooking with and without pressure, moist/dry cooking and conventional dehydration. Determination of alpha- and beta-carotenes was made by High-Performance Liquid Chromatography (HPLC) (conditions were developed by us) using spectrophotometric detection visible-UV at 470 nm; a RP-18 column and methanol: acetonitrile: ethyl acetate (80: 10: 10) as mobile phase. Total carotenoids quantification was made by 449 nm spectrophotometer. The retention of the analyzed carotenoids ranged from 60.13 to 85.64%. Water cooking without pressure promoted higher retention levels of alpha- and beta-carotene and vitamin A values, while water cooking with pressure promoted higher retention levels of total carotenoids. Dehydration promoted the highest carotenoid losses. The results showed that, among the routinely utilized methods under domestic condition, cooking without pressure, if performed under controlled time and temperature, is the best method as it reduces losses in the amount of alpha- and beta-carotene, the main carotenoids present in the carrots. Despite the significant carotenoid losses, carrots prepared through domestic methods, remain a rich source of provitamin A.

Atividade antioxidante do beta-caroteno e da vitamina A. Estudo comparativo com antioxidante sintético

Foi avaliada a atividade antioxidante da vitamina A na forma de acetato de retinol e de seu principal precursor, o beta-caroteno, adicionados a um sistema de óleo de soja previamente sensibilizado à oxidação. Os parâmetros utilizados como grau de atividade oxidativa foram: índice de peróxidos, teores de malonaldeído durante os intervalos de 24 a 72 horas, e perfil dos ácidos linoléico e linolênico após 144 horas de oxidação. Pelos resultados pode-se verificar que o retinol apresentou atividade antioxidante superior ao beta-caroteno. As determinações das atividades antioxidantes foram comparadas à do butilhidroxitolueno (BHT). A eficiência antioxidante da vitamina A e do beta-caroteno foram proporcionais às suas resistências à decomposição no sistema oxidativo. O acetato de retinol, a exemplo do BHT, mostrou uma rápida interação com os radicais ativos, pois já no início de sua adição ao óleo de soja, reduziu o nível da oxidação em relação ao respectivo controle.

CAROTENÓIDES DE PIMENTÕES AMARELOS (Capsicum annuum, L.). CARACTERIZAÇÃO E VERIFICAÇÃO DE MUDANÇAS COM O COZIMENTO

Três cultivares de pimentões amarelos (Capsicum annuum, L.), Amador Híbrido F1, Zarco Híbrido F1 e Sunboy Híbrido F1, cultivados no Estado de São Paulo, foram analisados com o objetivo de se determinar os teores de carotenóides presentes, avaliar o valor de vitamina A e verificar a perda desta atividade após o cozimento. O procedimento analítico consistiu de saponificação com KOH aquoso 10%, antes da extração com solvente orgânico e separação por coluna cromatográfica. Os três cultivares apresentaram o mesmo perfil de carotenóides, sendo nove identificados: fitoflueno, alfa-caroteno, beta-caroteno, cis-zeta-caroteno, 5,6,5’,6’-diepóxi-beta-caroteno, 5,6,5’,6’-diepóxi-criptoxantina, beta-criptoxantina, luteína e violaxantina. O alfa e o beta-caroteno foram os principais carotenóides que contribuíram com a atividade pró-vitamínica A. Os teores de vitamina A, expressos em equivalentes de retinol/100g variaram entre 23,17 e 48,70 para as amostras cruas. Como os carotenóides são sensíveis ao calor, após o cozimento durante 10 minutos os valores de vitamina A foram menores, variando entre 18,22 e 36,27, com reduções entre 21,08 e 30,80%.

Novos fatores de conversão de carotenóides provitamínicos A

Este trabalho aborda os cálculos dos novos fatores de conversão de carotenóides provitamínicos A, propostos pelo Institute of Medicine, e agentes que afetam a biodisponibilidade de carotenóides em alimentos. Adicionalmente, revisa estudos que determinaram o conteúdo de carotenóides provitamínicos A em frutas e hortaliças no Brasil, para aplicar os novos fatores de conversão no cálculo do valor de vitamina A desses alimentos. O valor de vitamina A caiu pela metade ao se utilizarem os novos fatores, o que mostra que estes valores vêm sendo superestimados nas tabelas de composição química de alimentos. Paralelamente aos estudos demonstrando a baixa biodisponibilidade de carotenóides provenientes de vegetais, há estudos que apresentam o impacto positivo de vegetais e frutas ricos em carotenóides na concentração de retinol sérico. Medidas a curto e médio prazo, como a suplementação medicamentosa e a fortificação de alimentos, devem ser tomadas para conter a deficiência de vitamina A. A longo prazo, pode-se focalizar o consumo dos alimentos, como frutas e vegetais processados, que contêm carotenóides mais biodisponíveis. Enquanto os fatores que afetam a biodisponibilidade de carotenóides não estão bem definidos, as tabelas de composição devem trazer o conteúdo de carotenóides e não apenas o valor de vitamina A dos alimentos.

A method for fast determination of epigallocatechin gallate (EGCG), epicatechin (EC), catechin (C) and caffeine (CAF) in green tea using HPLC

Tea has been considered a medicine and a healthy beverage since ancient times, but recently it has received a great deal of attention because of its antioxidant properties. Green tea polyphenols have demonstrated to be an effective chemopreventive agent. Recently, investigators have found that EGCG, one of the green tea catechins, could have anti-HIV effects when bound to CD4 receptor. Many factors can constitute important influences on the composition of tea, such as species, season, age of the leaf, climate, and horticultural practices (soil, water, minerals, fertilizers). This paper presents an HPLC analytical methodology development, using column RP-18 and mobile phase composed by water, acetonitrile, methanol, ethyl acetate, glacial acetic acid (89:6:1:3:1 v/v/v/v/v) for simultaneous determination and quantification of caffeine (CAF), catechin (C), epicatechin (EC) and epigallocatechin gallate (EGCG) in samples of Camellia sinensis (green tea) grown in Brazil and harvested in spring, in summer and in autumn, in comparison to Brazilian black tea, to samples of Japanese and Chinese green tea and to two standardized dry extracts of green tea. The method has been statistically evaluated and has proved to be adequate to qualitative and quantitative determination of the samples.

Avaliação química e sensorial de ovos obtidos por diferentes tratamentos

Os ovos estão entre os produtos mais importantes economicamente no país, e sua maneira de produção apresenta diferentes opções. A pesquisa teve por objetivo avaliar, química e sensorialmente, ovos obtidos pela forma tradicional, semi-orgânica e orgânica. Foram coletados 144 ovos convencionais brancos e vermelhos, semi-orgânicos e orgânicos, nos locais de produção. As análises químicas de composição centesimal e minerais foram realizadas. O teor de retinol, alfa e beta caroteno foi determinado por cromatografia líquida de alta eficiência. Foram realizados teste hedônico e teste de ordenação. O delineamento experimental foi em blocos completos casualizados. Foi realizado teste de Tukey (p < 0,05) e qui-quadrado. Para as análises químicas não houve diferença quanto aos teores de umidade, cinzas, carboidratos e ferro, porém houve diferença (p < 0,05) para proteínas, lipídios, cálcio e magnésio. Para o retinol não houve diferença significativa e não foram detectados alfa e beta caroteno. Para o teor de colesterol houve diferença significativa, mas para a análise sensorial não. Portanto, o manejo influencia tanto no teor de proteína e lipídeos quanto no cálcio e magnésio, assim como no teor de colesterol das gemas, porém, não altera os teores de vitamina A e as características sensoriais.

Processamento e avaliação da vida-de-prateleira do tucumã (Astrocaryum aculeatum Meyer) desidratado e pulverizado

O presente estudo teve como objetivo processar frutos de tucumã por desidratação e pulverização e avaliar sua vida-de-prateleira em diferentes tipos de embalagens e temperaturas de armazenagem. A parte comestível do fruto foi desidratada, triturada e passada em tamisador para padronização do diâmetro dos grânulos, embalada em três diferentes tipos de embalagens (plástico polietileno, laminado e lata de aço) e armazenada em diferentes temperaturas (4 e 24 °C). Os frutos in natura e desidratados foram analisados quanto a umidade, pH, acidez, açúcares totais e redutores, proteínas, lipídios, cinzas, carboidratos, energia, β-caroteno e equivalente de retinol. O tucumã desidratado e pulverizado foi analisado a cada 30 dias durante 150 dias de armazenagem quanto aos índices de peróxido, de acidez e de iodo e também quanto aos parâmetros microbiológicos. Os constituintes que se destacaram, tanto no fruto in natura quanto no desidratado e pulverizado, foram: lipídios, carboidratos e, conseqüentemente, energia e β-caroteno. Em relação à vida-de-prateleira, todos os tratamentos apresentaram boa estabilidade química e microbiológica durante os 150 dias de armazenamento. Conclui-se que o tucumã desidratado e pulverizado, independente do tipo de embalagem e temperatura de armazenamento, pode ser estocado e consumido por até 150 dias, além de seu potencial nutricional como fonte de energia e β-caroteno.

Chemical characterization and antimicrobial activity of essential oils of salvia L. species

In this work, the essential oils of S. officinalis, S. sclarea, S. lavandulifolia and S. triloba were chemically analyzed by gas chromatography coupled to a mass spectrometry detector (GC/MSD), and their antimicrobial activity was tested against 10 microorganisms using the disk diffusion method and the Minimum Inhibitory Concentration (MIC) technique. The following major compounds were identified in the essential oils: α - and β-thujone, camphor and 1,8-cineole, except in S. sclarea, where linalool, linalyl acetate and α-terpineol were the major constituents. The antimicrobial activity showed significant differences (p < 0.05) only when obtained by the MIC method. Gram-positive microorganisms presented larger sensitivity for the essential oils. The lowest MIC was observed when Staphylococcus aureus was exposed to 2.31 mg.mL-1 of S. lavandulifolia essential oil, while the highest MIC value was obtained when Shigella flexneri was exposed to 9.25 mg.mL-1 of the same essential oil, thus demonstrating that this essential oil may be effective as a bacteriostatic agent against Gram-positive microorganisms.

Inhibitory effect of the essential oil from Cinnamomum zeylanicum Blume leaves on some food-related bacteria

Cinnamomum zeylanicum Blume, Lauraceae, has long been known for having many biological properties. This study aimed to identify the constituents of the essential oil from C. zeylanicum leaves using GC-MS and to assess its inhibitory effect on Salmonella enterica, Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa based on MIC and MBC determination and kill-time study. Eugenol (73.27%) was the most prevalent compound in the essential oil followed by trans-β-cariophyllene (5.38%), linalool (3.31%), and alcohol cinamic acetate (2.53%). The results showed an interesting antibacterial activity of the oil with MIC ranging from 1.25 to 10 µL.mL-1. MBC values were in the range of 20 - 80 µL.mL-1. A concentration of 10 and 40 µL.mL-1 of the essential oil caused a fast and steady decrease in viable cell count (2 to 5 log cycles) of all assayed strains along 24 hours. A concentration of 40 µL.mL-1 of the oil provided a total elimination of the initial inocula of S. aureus after 2 hours. These results show the possibility of regarding the essential oil from C. zeylanicum leaves as alternative sources of antimicrobial compounds to be applied in food conservation systems.

Changes in guava (Psidium guajava L. var. Paluma) nectar volatile compounds concentration due to thermal processing and storage

Guava nectars were formulated for approximately 10, 12, or 14 ºBrix, with 40% guava pulp. Sodium benzoate, 500 mg.kg-1 was used as preservative. The Brix value was adjusted with saturated sucrose syrup. The guava nectar was pasteurized (85 ºC/42 seconds) in tubular heat exchanger and then hot filled in 500 mL white glass bottles. The products were stored either at room temperature (25 ± 5 ºC) or refrigerated (5 ± 2 ºC) under fluorescent light exposure and analyzed on the day after processing (time zero) and also 40, 80, and 120 days of storage. Eight compounds were identified and quantified by Gas Chromatography (GC) -Mass Spectrometry (MS): hexanal, (E)-hex-2-enal, 1-hexenol, (Z)-hex-3-enol, (Z)-hex-3-enyl acetate, phenyl-3-propyl acetate, cinnamyl acetate, and acetic acid. There was no significant effect of thermal treatment on the volatile compound concentrations, except for a significant decrease (p = 0.0001) in hexanal and (Z)-hex-3-enyl acetate (p = 0.0029). As for the storage time, there was a much greater decrease in the esters contents, such as (Z)-hex-3-enyl and phenyl-3-propyl acetates. Cinnamyl acetate had the greatest decrease over storage time. Refrigeration was better than room temperature for guava nectar volatile compounds stability over storage time, mainly for esters compounds, which are important for the product aroma and flavor