Synthesis and characterization of solid 2-methoxycinnamylidenepyruvic acid

The 2-methoxycinnamylidenepyruvic acid (2-MeO-HCP) was synthesized and characterized for nuclear magnetic resonance (¹H and 13C NMR), mass spectrometry (MS), Infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC). The application of DSC for purity determination is well documented in literature and is used in the analysis of pure organic compounds. The molecular geometry and vibrational frequencies of 2-MeO-HCP have been calculated.

Cuticular and Suberin Polymers of Edible Plants. Analysis by Gas Chromatographic-Mass Spectrometric and Solid State Spectroscopic Methods

Cutin and suberin are structural and protective polymers of plant surfaces. The epidermal cells of the aerial parts of plants are covered with an extracellular cuticular layer, which consists of polyester cutin, highly resistant cutan, cuticular waxes and polysaccharides which link the layer to the epidermal cells. A similar protective layer is formed by a polyaromatic-polyaliphatic biopolymer suberin, which is present particularly in the cell walls of the phellem layer of periderm of the underground parts of plants (e.g. roots and tubers) and the bark of trees. In addition, suberization is also a major factor in wound healing and wound periderm formation regardless of the plants’ tissue. Knowledge of the composition and functions of cuticular and suberin polymers is important for understanding the physiological properties for the plants and for nutritional quality when these plants are consumed as foods. The aims of the practical work were to assess the chemical composition of cuticular polymers of several northern berries and seeds and suberin of two varieties of potatoes. Cutin and suberin were studied as isolated polymers and further after depolymerization as soluble monomers and solid residues. Chemical and enzymatic depolymerization techniques were compared and a new chemical depolymerization method was developed. Gas chromatographic analysis with mass spectrometric detection (GC-MS) was used to assess the monomer compositions. Polymer investigations were conducted with solid state carbon-13 cross polarization magic angle spinning nuclear magnetic resonance spectroscopy (13C CP-MAS NMR), Fourier transform infrared spectroscopy (FTIR) and microscopic analysis. Furthermore, the development of suberin over one year of post-harvest storage was investigated and the cuticular layers from berries grown in the North and South of Finland were compared. The results show that the amounts of isolated cuticular layers and cutin monomers, as well as monomeric compositions vary greatly between the berries. The monomer composition of seeds was found to differ from the corresponding berry peel monomers. The berry cutin monomers were composed mostly of long-chain aliphatic ω-hydroxy acids, with various mid-chain functionalities (double-bonds, epoxy, hydroxy and keto groups). Substituted α,ω-diacids predominated over ω-hydroxy acids in potato suberin monomers and slight differences were found between the varieties. The newly-developed closed tube chemical method was found to be suitable for cutin and suberin analysis and preferred over the solvent-consuming and laborious reflux method. Enzymatic hydrolysis with cutinase was less effective than chemical methanolysis and showed specificity towards α,ω-diacid bonds. According to ₁₃C CP-MAS NMR and FTIR, the depolymerization residues contained significant amounts of aromatic structures, polysaccharides and possible cutan-type aliphatic moieties. Cultivation location seems to have effect on cuticular composition. The materials studied contained significant amounts of different types of biopolymers that could be utilized for several purposes with or without further processing. The importance of the so-called waste material from industrial processes of berries and potatoes as a source of either dietary fiber or specialty chemicals should be further investigated in detail. The evident impact of cuticular and suberin polymers, among other fiber components, on human health should be investigated in clinical trials. These by-product materials may be used as value-added fiber fractions in the food industry and as raw materials for specialty chemicals such as lubricants and emulsifiers, or as building blocks for novel polymers.

Interação entre temperatura do solo, profundidade de semeadura e tratamento de sementes com fungicida na emergência de plantas de milho

O trabalho avaliou a interação entre temperaturas do solo (13ºC, 15ºC, 17ºC e 19ºC), profundidades de semeadura (2,5; 5,0 e 7,5 cm) e tratamento de semente com fungicida (captan + tiabendazole) na emergência de plantas de milho. Utilizaram-se sementes do híbrido AS 1565 com 22% de incidência de Fusarium verticillioides e solo de lavoura naturalmente infestado. O ensaio foi conduzido em câmaras climatizadas por 25 dias, com contagem diária da emergência. Houve diferença significativa entre sementes tratadas e não tratadas, sendo que quanto menor a temperatura do solo e maior a profundidade de semeadura, maior a resposta ao tratamento de sementes na emergência de plantas. A estabilidade de emergência ocorreu aos 23, 18, 12 e 12 dias após a semeadura respectivamente para 13ºC, 15ºC, 17ºC e 19ºC, mostrando redução na velocidade de emergência nas temperaturas baixas.

Conservative nutrient use by big-leaf mahogany (Swietenia macrophylla king) planted under contrasting environmental conditions

We analyzed the nutritional composition and isotope ratios (C and N) of big-leaf mahogany (Swietenia macrophylla King) leaves in plantations established on contrasting soils and climates in Central America (State of Quintana Roo, Yucatán, México) and South America (State of Pará, Brazil). The objective was to determine the adaptability of this species to large differences in nutrient availability and rainfall regimes. Nutrient concentrations of leaves and soils were determined spectrophotometrically, and isotope ratios were measured using mass spectrometric techniques.In Pará soils were sandier, and acidic, receiving above 2000 mm of rain, whereas in Quintana Roo soils were predominantly clayey, with neutral to alkaline pH due to the underlying calcareous substrate, with about 1300 mm of rain. Leaf area/weight ratio was similar for both sites, but leaves from Quintana Roo were significantly smaller. Average N and K concentrations of adult leaves were similar, whereas Ca concentration was only slightly lower in Pará in spite of large differences in Ca availability. Leaves from this site had slightly higher P and lower Al concentrations. Differences in water use efficiency as measured by the natural abundance of 13C were negligible, the main effect of lower rainfall in Quintana Roo seemed to be a reduction in leaf area. The N isotope signature (δ15N) was more positive in Pará than in Quintana Roo, suggesting higher denitrification rates in the former. Results reveal a calciotrophic behavior and a remarkable capacity of mahogany to compensate for large differences in soil texture and nutrient availability.

Sheep poisoning by Panicum dichotomiflorum in northeastern Brazil

Different species of Panicum, including P. dichotomiflorum,have been reported as a cause of photosensitization in sheep, horses, cattle and goats. An outbreak of hepatogenous photosensitization occurred in 3 flocks of hair sheep in the Brazilian semiarid region. Eighty one out of 365 sheep were affected and 39 died. The main affected animals were nursing lambs and sheep younger than one year old. Donkeys, goats and cattle grazing in the same pasture were not affected. Clinical signs were edema of the head, followed by dermatitis, mainly in the face, ears, and croup, ocular discharge, corneal opacity with blindness, and redness of the coronary band and hoof. At necropsy of one affected lamb the liver was yellowish. Upon histologic examination scattered necrotic hepatocytes were observed in the liver and focal areas of necrosis of myocytes appeared in the heart. Samples of P. dicotomiflorum were analyzed by TLC and those containing saponins were isolated by HPLC using RP-C18 column and eluted with a mixture of MeOH and H2O. The isolated compounds were submitted to ¹H and 13C NMR spectroscopy. Reactions were positive to furostanol saponins with the same Rf of the standard protodioscin (0.21) and methylprotodioscin (0.32). The spectroscopic results indicated a mixture of (25R)- and (25S)-protodioscin isomers in a proportion of 3:1, and methylprotodioscin.

Extraction of polymeric galactoglucomannans from spruce wood by pressurized hot water

The major type of non-cellulosic polysaccharides (hemicelluloses) in softwoods, the partly acetylated galactoglucomannans (GGMs), which comprise about 15% of spruce wood, have attracted growing interest because of their potential to become high-value products with applications in many areas. The main objective of this work was to explore the possibilities to extract galactoglucomannans in native, polymeric form in high yield from spruce wood with pressurised hot-water, and to obtain a deeper understanding of the process chemistry involved. Spruce (Picea abies) chips and ground wood particles were extracted using an accelerated solvent extractor (ASE) in the temperature range 160 – 180°C. Detailed chemical analyses were done on both the water extracts and the wood residues. As much as 80 – 90% of the GGMs in spruce wood, i.e. about 13% based on the original wood, could be extracted from ground spruce wood with pure water at 170 – 180°C with an extraction time of 60 min. GGMs comprised about 75% of the extracted carbohydrates and about 60% of the total dissolved solids. Other substances in the water extracts were xylans, arabinogalactans, pectins, lignin and acetic acid. The yields from chips were only about 60% of that from ground wood. Both the GGMs and other non-cellulosic polysaccharides were extensively hydrolysed at severe extraction conditions when pH dropped to the level of 3.5. Addition of sodium bicarbonate increased the yields of polymeric GGMs at low additions, 2.5 – 5 mM, where the end pH remained around 3.9. However, at higher addition levels the yields decreased, mainly because the acetyl groups in GGMs were split off, leading to a low solubility of GGMs. Extraction with buffered water in the pH range 3.8 – 4.4 gave similar yields as with plain water, but gave a higher yield of polymeric GGMs. Moreover, at these pH levels the hydrolysis of acetyl groups in GGMs was significantly inhibited. It was concluded that hot-water extraction of polymeric GGMs in good yields (up to 8% of wood) demands appropriate control of pH, in a narrow range about 4. These results were supported by a study of hydrolysis of GGM at constant pH in the range of 3.8 – 4.2 where a kinetic model for degradation of GGM was developed. The influence of wood particle size on hot-water extraction was studied with particles in the range of 0.1 – 2 mm. The smallest particles (< 0.1 mm) gave 20 – 40% higher total yield than the coarsest particles (1.25 – 2 mm). The difference was greatest at short extraction times. The results indicated that extraction of GGMs and other polysaccharides is limited mainly by the mass transfer in the fibre wall, and for coarse wood particles also in the wood matrix. Spruce sapwood, heartwood and thermomechnical pulp were also compared, but only small differences in yields and composition of extracts were found. Two methods for isolation and purification of polymeric GGMs, i.e. membrane filtration and precipitation in ethanol-water, were compared. Filtration through a series of membranes with different pore sizes separated GGMs of different molar masses, from polymers to oligomers. Polysaccharides with molar mass higher than 4 kDa were precipitated in ethanol-water. GGMs comprised about 80% of the precipitated polysaccharides. Other polysaccharides were mainly arabinoglucuronoxylans and pectins. The ethanol-precipitated GGMs were by 13C NMR spectroscopy verified to be very similar to GGMs extracted from spruce wood in low yield at a much lower temperature, 90°C. The obtained large body of experimental data could be utilised for further kinetic and economic calculations to optimise technical hot-water extractionof softwoods.

Efeitos teratogênicos de Prosopis juliflora em ratos e análise da toxicidade das vagens

Objetivou-se com este estudo determinar os possíveis efeitos teratogênicos de Prosopis juliflora, verificar se existe perda da toxicidade entre vagens armazenadas e recém-coletadas e determinar se existe diferença de toxicidade entre as vagens coletadas em diferentes localidades. Trinta ratas prenhes da linhagem Wistar foram separadas, aleatoriamente, em cinco grupos: um controle (G1) e quatro experimentais (G2, G3, G4 e G5). Os animais dos grupos G2 e G3 foram alimentados com ração contendo 70% de vagens de P. juliflora recém-coletadas em dois municípios diferentes. Os grupos G4 e G5 foram alimentados com ração preparada com vagens das mesmas procedências, porém armazenadas por um período de 6 meses. O grupo controle recebeu ração sem vagens de P. juliflora. No grupo controle o número de malformações por ninhada (1,16 ± 0,98) foi significativamente menor do que os dos grupos experimentais (14,00 ± 2,96, 6,16 ± 2,22, 7,66 ± 2,94 e 4,66 ± 1,63 para os grupos G2, G3, G4 e G5, respectivamente) indicando que a planta é teratogênica. Não foram observadas diferenças significativas na frequência de malformações e no número de fetos nascidos entre os grupos que receberam vagens de diferentes localidades. No entanto, o número de malformações nos grupos que receberam as vagens recém-colhidas (65,80 ± 21,20 a, 67,59 ± 18,10 a), foi significativamente maior que o observado nas ratas que receberam as vagens após o armazenamento (35,74±10,10b, 21,85±5,13c) sugerindo que o efeito teratogênico da planta diminui durante o armazenamento. Conclui-se que as vagens de P. juliflora são teratogênicas para ratas Wistar e que a fetoxicidade das mesmas diminui com o armazenamento.

Análise comparativa dos efeitos alelopáticos das substâncias químicas titonina e titonina acetilada

Este trabalho teve por objetivo estabelecer as variações na atividade alelopática da substância química titonina, em função da acetilação de sua molécula. Bioensaios de germinação (25 ºC de temperatura constante e fotoperíodo de 12 horas) e de desenvolvimento da radícula e do hipocótilo (25 ºC de temperatura constante e fotoperíodo de 24 horas) foram desenvolvidos. Como planta receptora, utilizou-se a planta daninha Mimosa pudica (malícia). Análise de espectros RMN ¹H e 13C e técnicas de RMN bidimensionais foram realizadas na molécula acetilada. O processo de acetilação produziu a molécula 3'-acetil-7,4'-dimetoxiflavona, que diferiu da molécula original, identificada como 3'-hidroxi-7,4'-dimetoxiflavona. A estrutura da titonina-Ac foi confirmada pelos espectros de RMN ¹H, 13C, DEPT, COSY e HETCOR. A titonina foi acetilada com anidrido acético em piridina. A análise comparativa da atividade alelopática das duas substâncias revelou que titonina-Ac apresentou maior potencial para inibir tanto a germinação das sementes como o desenvolvimento da radícula e do hipocótilo da planta daninha malícia. A intensidade dos efeitos alelopáticos das duas substâncias esteve positivamente associada à concentração. O conjunto das informações obtidas permite sugerir a possibilidade de se aumentar a atividade alelopática de uma substância química sem comprometer suas peculiaridades biológicas desejáveis à natureza e aos interesses da sociedade.

Potencial alelopático de duas neolignanas isoladas de folhas de Virola surinamensis (Myristicaceae)

Este trabalho teve por objetivos isolar, identificar e caracterizar a atividade alelopática de substâncias químicas presentes nas folhas de Virola surinamensis. O processo de isolamento e identificação das substâncias químicas envolveu o uso de solventes orgânicos e de Ressonância Magnética Nuclear (RMN ¹H, RMN 13C e RMN 13C-DEPT), espectro de COSY e de HETCOR. A avaliação da atividade alelopática foi realizada em bioensaios de germinação de sementes, em condições de 25 ºC de temperatura constante e fotoperíodo de 12 horas, e de desenvolvimento da radícula e do hipocótilo, com 25 ºC de temperatura constante e fotoperíodo de 24 horas, empregando-se concentrações variando de 1,0 a 8,0 mg L-1. Como plantas receptoras, foram utilizadas as espécies daninhas Mimosa pudica, Senna obtusifolia e Senna occidentalis. Foram isoladas e identificadas duas neolignanas: a surinamensina e a virolina. A tendência geral observada nos resultados foi de aumento da intensidade dos efeitos alelopáticos inibitórios em função do aumento da concentração, com inibições máximas obtidas, sempre, na concentração de 8,0 mg L-1. A surinamensina apresentou maior potencial para inibir a germinação e o desenvolvimento da radícula e do hipocótilo do que a virolina, independentemente da espécie receptora e do fator da planta analisado. Considerando-se as intensidades dos efeitos promovidos sobre os três fatores das plantas, o desenvolvimento da radícula e o do hipocótilo foram mais intensamente inibidos pelas duas substâncias do que a germinação das sementes. À exceção dos efeitos verificados sobre o desenvolvimento do hipocótilo, malícia foi a espécie de maior sensibilidade aos efeitos alelopáticos das duas neolignanas, enquanto mata-pasto foi aquela que evidenciou inibições de menor magnitude.

Variation in nitrogen use strategies and photosynthetic pathways among vascular epiphytes in the Brazilian Central Amazon

The variation in nitrogen use strategies and photosynthetic pathways among vascular epiphyte families was addressed in a white-sand vegetation in the Brazilian Central Amazon. Foliar nitrogen and carbon concentrations and their isotopic composition (δ15N and δ13C, respectively) were measured in epiphytes (Araceae, Bromeliaceae and Orchidaceae) and their host trees. The host tree Aldina heterophylla had higher foliar N concentration and lower C:N ratio (2.1 ± 0.06% and 23.6 ± 0.8) than its dwellers. Tree foliar δ15N differed only from that of the orchids. Comparing the epiphyte families, the aroids had the highest foliar N concentration and lowest C:N ratios (1.4 ± 0.1% and 34.9 ± 4.2, respectively). The orchids had more negative foliar δ15N values (-3.5 ± 0.2‰) than the aroids (-1.9 ± 0.7‰) and the bromeliads (-1.1 ± 0.6‰). Within each family, aroid and orchid taxa differed in relation to foliar N concentrations and C:N ratios, whereas no internal variation was detected within bromeliads. The differences in foliar δ15N observed herein seem to be related to the differential reliance on the available N sources for epiphytes, as well as to the microhabitat quality within the canopy. In relation to epiphyte foliar δ13C, the majority of epiphytes use the water-conserving CAM-pathway (δ13C values around -17‰), commonly associated with plants that live under limited and intermittent water supply. Only the aroids and one orchid taxon indicated the use of C3-pathway (δ13C values around -30‰).

The secondary alcohol and aglycone metabolites of doxorubicin alter metabolism of human erythrocytes

Anthracyclines, a class of antitumor drugs widely used for the treatment of solid and hematological malignancies, cause a cumulative dose-dependent cardiac toxicity whose biochemical basis is unclear. Recent studies of the role of the metabolites of anthracyclines, i.e., the alcohol metabolite doxorubicinol and aglycone metabolites, have suggested new hypotheses about the mechanisms of anthracycline cardiotoxicity. In the present study, human red blood cells were used as a cell model. Exposure (1 h at 37ºC) of intact human red blood cells to doxorubicinol (40 µM) and to aglycone derivatives of doxorubicin (40 µM) induced, compared with untreated red cells: i) a ~2-fold stimulation of the pentose phosphate pathway (PPP) and ii) a marked inhibition of the red cell antioxidant enzymes, glutathione peroxidase (~20%) and superoxide dismutase (~60%). In contrast to doxorubicin-derived metabolites, doxorubicin itself induced a slighter PPP stimulation (~35%) and this metabolic event was not associated with any alteration in glutathione reductase, glutathione peroxidase, catalase or superoxide dismutase activity. Furthermore, the interaction of hemoglobin with doxorubicin and its metabolites induced a significant increase (~22%) in oxygen affinity compared with hemoglobin incubated without drugs. On the basis of the results obtained in the present study, a new hypothesis, involving doxorubicinol and aglycone metabolites, has been proposed to clarify the mechanisms responsible for the doxorubicin-induced red blood cell toxicity.

Pressure-assisted cold denaturation of hen egg white lysozyme: the influence of co-solvents probed by hydrogen exchange nuclear magnetic resonance

COSY proton nuclear magnetic resonance was used to measure the exchange rates of amide protons of hen egg white lysozyme (HEWL) in the pressure-assisted cold-denatured state and in the heat-denatured state. After dissolving lysozyme in deuterium oxide buffer, labile protons exchange for deuterons in such a way that exposed protons are substituted rapidly, whereas "protected" protons within structured parts of the protein are substituted slowly. The exchange rates k obs were determined for HEWL under heat treatment (80ºC) and under high pressure conditions at low temperature (3.75 kbar, -13ºC). Moreover, the influence of co-solvents (sorbitol, urea) on the exchange rate was examined under pressure-assisted cold denaturation conditions, and the corresponding protection factors, P, were determined. The exchange kinetics upon heat treatment was found to be a two-step process with initial slow exchange followed by a fast one, showing residual protection in the slow-exchange state and P-factors in the random-coil-like range for the final temperature-denatured state. Addition of sorbitol (500 mM) led to an increase of P-factors for the pressure-assisted cold denatured state, but not for the heat-denatured state. The presence of 2 M urea resulted in a drastic decrease of the P-factors of the pressure-assisted cold denatured state. For both types of co-solvents, the effect they exert appears to be cooperative, i.e., no particular regions within the protein can be identified with significantly diverse changes of P-factors.

Protein turnover, amino acid requirements and recommendations for athletes and active populations

Skeletal muscle is the major deposit of protein molecules. As for any cell or tissue, total muscle protein reflects a dynamic turnover between net protein synthesis and degradation. Noninvasive and invasive techniques have been applied to determine amino acid catabolism and muscle protein building at rest, during exercise and during the recovery period after a single experiment or training sessions. Stable isotopic tracers (13C-lysine, 15N-glycine, ²H5-phenylalanine) and arteriovenous differences have been used in studies of skeletal muscle and collagen tissues under resting and exercise conditions. There are different fractional synthesis rates in skeletal muscle and tendon tissues, but there is no major difference between collagen and myofibrillar protein synthesis. Strenuous exercise provokes increased proteolysis and decreased protein synthesis, the opposite occurring during the recovery period. Individuals who exercise respond differently when resistance and endurance types of contractions are compared. Endurance exercise induces a greater oxidative capacity (enzymes) compared to resistance exercise, which induces fiber hypertrophy (myofibrils). Nitrogen balance (difference between protein intake and protein degradation) for athletes is usually balanced when the intake of protein reaches 1.2 g·kg-1·day-1 compared to 0.8 g·kg-1·day-1 in resting individuals. Muscular activities promote a cascade of signals leading to the stimulation of eukaryotic initiation of myofibrillar protein synthesis. As suggested in several publications, a bolus of 15-20 g protein (from skimmed milk or whey proteins) and carbohydrate (± 30 g maltodextrine) drinks is needed immediately after stopping exercise to stimulate muscle protein and tendon collagen turnover within 1 h.

Análise da adulteração de méis por açúcares comerciais utilizando-se a composição isotópica de carbono

O mel é um alimento apreciado por seu sabor característico e pelo seu considerável valor nutritivo. Devido a oferta do produto ser menor que a procura, seu preço é relativamente alto, incentivando sua adulteração. Geralmente, a adulteração do mel é feita através da adição de açúcares comerciais, derivados de cana-de-açúcar e milho. Como essas plantas têm uma composição isotópica do carbono distinta das plantas utilizadas pelas abelhas como fonte de néctar (flores silvestres, citros e eucaliptos), é possível utilizar a composição isotópica do carbono de amostras de mel para se avaliar a adulteração desse produto por açúcares comerciais oriundos da cana e do milho. Foram analisadas amostras de plantas pertencentes ao ciclo fotos-sintético C3, subprodutos de plantas C4 (açúcares comerciais) e 61 amostras de mel obtidas no mercado. As plantas C3 analisadas apresentaram valores de d13C de -28,9±1,1‰ (n=8), enquanto os açúcares apresentaram valores de -11,1±0,7‰ (n=3). Das 61 amostras de mel analisadas, cerca de 8% (5 amostras) tiveram sinais claros de adulteração. A amostra de número 34 teve um valor igual a -12,9‰, indicando que açúcar puro de cana-de-açúcar ou milho estaria sendo comercializado como mel. As amostras 13, 14, 33 e 54 apresentaram valores iguais a -21,0; -19,9; -21,9 e -17,6‰, respectivamente. Esses valores indicam também adição de açúcares de cana-de-açúcar ou milho, no entanto em menor proporção. A metodologia testada neste trabalho foi aprovada como um método simples, confiável e complementar aos métodos químicos e físicos convencionais visando detectar adulteração de mel.

Carbon isotope analysis in apple nectar beverages

The aims of this study were to use the isotope analysis method to quantify the carbon of C3 photosynthetic cycle in commercial apple nectars and to determine the legal limit to identify the beverages that do not conform to the safety standards established by the Brazilian Ministry of Agriculture, Livestock and Food Supply. These beverages (apple nectars) were produced in the laboratory according to the Brazilian legislation. Adulterated nectars were also produced with an amount of pulp juice below the permitted threshold limit value. The δ13C values of the apple nectars and their fractions (pulp and purified sugar) were measured to quantify the C3 source percentage. In order to demonstrate the existence of adulteration, the values found were compared to the limit values established by the Brazilian Law. All commercial apple nectars analyzed were within the legal limits, which enabled to identify the nectars that were in conformity with the Brazilian Law. The isotopic methodology developed proved efficient to quantify the carbon of C3 origin in commercial apple nectars.