Made available in DSpace on 2014-08-20T14:38:03Z (GMT). No. of bitstreams: 1 dissertacao_cristina_nunes.pdf: 1093744 bytes, checksum: 0c42a534dae5175850fd939832f086a8 (MD5) Previous issue date: 2011-02-28 / Currently, the drug industry looks for new drugs based on natural products, for the production of drugs more efficient, for which the microorganisms did not show resistance to both humans and animals. A natural product that has been the subject of intense pharmacological and chemical studies by scientists for the control of diseases is propolis, a resinous substance produced by honeybees from exudates collected from different parts of the plant, which has been used for centuries in popular medicine due to its therapeutic properties. Chemical studies revealed the complex chemical composition, identifying in some cases more than 300 components including various bioactive phenolic compounds responsible for the virucidal action. This work initially describes the standardization of an ethanol extract of green propolis (EEPV), where the chemicals were identified by high performance liquid chromatography (HPLC), phytochemical characterization by thin layer chromatography (TLC), soluble solids, content of phenolics and flavonoids and antioxidant activity by 2.2 diphenil picryl hydrazyl (DPPH). The EEPV was also evaluated in vitro and in vivo for their capacity lentogenic virucidal against a strain of the virus of Newcastle disease (NDV) at two different temperatures (22 and 37 ° C), 5 incubation periods (0, 1, 2, 4 and 8 hours) of NDV in five different concentrations of EEPV (4000μg/dose, 400μg/dose, 40μg/dose, and 4μg/dose 0μg/dose). The EEPV standard is within the standards required by the MAP, with high levels of phenolics and flavonoids (12.93 and 6.05% respectively) as shown by HPLC, which identified high concentrations of phenolic acids (p-coumaric acid, hydroxycinnamic acid diprenyl , cinâmino acid derivatives), which are assigned the antibacterial, antioxidant, antiviral and virucidal. This extract showed dose-dependent virucidal activity (4000μg/dose e 400μg/dose) and time of incubation with the virus (2 hour). The inhibitory activity of EEPV against the strain of NDV lentogenic found in the present study suggests the use of this extract as an alternative to fight the infection by this virus. / Atualmente, a indústria farmacêutica busca novos medicamentos com base em produtos naturais, visando à produção de fármacos mais eficientes, para os quais os microrganismos não apresentem resistência, tanto para humanos quanto para animais. Um dos produtos naturais que tem sido objeto de intensos estudos farmacológicos e químicos por cientistas para o controle de enfermidades é a própolis, uma substância resinosa produzida por abelhas melíferas a partir de exsudatos coletados em diferentes partes das plantas, que tem sido utilizada durante séculos na medicina popular devido as suas propriedades terapêuticas. Estudos químicos revelaram a complexa composição da própolis, identificando em alguns casos mais de 300 componentes, incluindo vários compostos bioativos fenólicos responsáveis pela ação virucida. Este trabalho inicialmente descreve a padronização de um extrato etanólico de própolis verde (EEPV), onde foram identificados os compostos químicos por cromatografia liquida de alta eficiência (CLAE), caracterização fitoquímica por cromatografia em camada delgada (CCD), teor de sólidos solúveis, teor de fenóis e flavonóides totais e atividade antioxidante por 2,2 diphenil picril hidrazil(DPPH). O EEPV foi avaliado também in vitro e in vivo, quanto a sua capacidade virucida contra uma cepa lentogênica do vírus da doença de Newcastle (NDV) em duas temperaturas distintas (22 e 37°C), 5 períodos de incubação (0, 1, 2, 4 e 8 horas) do NDV em 5 concentrações de EEPV distintos (4000μg/dose, 400μg/dose, 40μg/dose, 4μg/dose e 0μg/dose). O EEPV padronizado está dentro dos padrões requisitados pelo MAPA, com altos níveis de fenóis e flavonóides totais (12.93 e 6,05% respectivamente), comprovado por CLAE, o qual identificou altas concentrações de ácidos fenólicos (ácido p-cumárico, ácido diprenil hidroxicinâmico, derivados do ácido cinâmino), os quais são atribuídos as propriedades antibacteriana, antioxidante, antiviral e virucida. Este extrato apresentou atividade virucida dependente da dose (4000μg/dose, 400μg/dose) e do tempo de incubação com o vírus (2 horas). A atividade inibitória do EEPV contra a cepa lentogênica de NDV, encontrada no presente estudo sugere a utilização deste extrato como uma alternativa no combate a infecções por este vírus.

Doctor of Philosophy / Department of Horticulture, Forestry, and Recreation Resources / Channa B. Rajashekar / Studies were conducted to examine the role of antioxidants in adaptation of lettuce (Lactuca sativa L.) to unprotected environmental conditions and various environmental stresses. Antioxidants, in addition to being a plant defense mechanism, are phytochemicals that provide significant health-promoting and nutritive value in human diet. Various approaches involving mild environmental stresses and cultural and management practices have been used to enhance the quality of this commonly consumed leafy vegetable by improving its phytochemical and health-promoting attributes. Lettuce plants grown in protective environments adapt to unprotective environmental conditions by activating antioxidant genes such as phenylalanine ammonia-lyase (PAL), gamma-tocopherol methyl transferase (gamma-TMT), and L-galactose dehydrogenase (L-GalDH) involved in the biosynthesis of phenolic compounds,alpha-tocopherol and ascorbic acid, respectively. Mild environmental stresses such as heat shock, chilling, water stress and high light also activate these genes leading to the accumulation of secondary metabolites and phytochemicals without any adverse effect on biomass accumulation. The phytochemicals included chlorogenic acid, chicoric acid, caffeic acid, quercetin-3-O-glucoside and luteolin-7-O-glucoside. However, under field conditions, application of drought stress did not produce consistent results with regard to the phytochemical composition of lettuce. Plants grown in open field have higher phenolic content and higher antioxidant capacity than those grown in high tunnel. However, these plants also had less biomass accumulation. Many factors such as plant age, variety, fertilization, transplanting shock affected the phytochemical composition of lettuce. The red leaf variety 'Red Sails' had higher antioxidant capacity than 'Baronet' and younger plants had the highest phytochemical content. With regard to the management practices, there was no significant difference in phytochemical composition between organically and conventionally managed crops. Also, low fertility favored the accumulation of phytochemicals and increased the antioxidant capacity. Thus, the results show that mild environmental stresses along with appropriate cultural and management practices can enhance the quality of lettuce by improving their phytochemical composition.

This diploma thesis deals with comparison of the properties of chokeberry spreads of different origins, composition and different production technologies. The theoretical part contains a brief description of the chokeberry – Aronia melanocarpa, an overview of the most important substances contained in the chokeberry and examples of the use of aronia, not only in the food industry. Furthermore, the legislative requirements for the production of spreads and the production technology itself are described. In this thesis is also described a new patented technology of spread production using HTD technology. Another part of the theoretical part is a literature review of the determination of anthocyanins in chokeberry by HPLC method. The experimental part describes the laboratory procedures used for determination of dry matter, carbohydrates by HPLC with ELSD detector, anthocyanin dyes, total phenolic compounds and antioxidant activity. Another part of the work was a questionnaire survey and sensory analysis of selected chokeberry spreads. The panel members prefered the Lowicz chokeberry spread, for which was determined 29,39% dry matter, 15,15 mg.g-1 anthocyanins, 9,20 mg.g-1 total phenolic compounds, 158,42 mg.g-1 fructose and 151,26 mg.g-1 glucose and the antioxidant activity was determined to be 5,94 mg.g-1.

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Current fruit-zone management recommendation in the eastern US aims for 1-2 basal shoot leaf layers after fruit set to limit fungal disease and sunburn incidence, and prevent extreme heating of grapes. The goal of this work was to assess if fruit-zone leaf removal to an uncommonly greater extent, and/or at an earlier phenological stage, would favorably alter yield components or fruit composition in three popularly grown, red-fruited, Bordeaux varieties – Cabernet franc, Petit Verdot, and Cabernet Sauvignon. Pre-bloom leaf removal to various extents reduced crop yield by 41-78% when compared to no leaf removal across seasons and varieties. Pre-bloom leaf removal implementation in consecutive seasons tended to further reduce crop yield components compared to implementation in the first year. Pre-bloom leaf removal tended to reduce cluster compactness and bunch rot incidence when compared to post-fruit set and no leaf removal. Basal leaf removal to the greatest extents inconsistently reduced soluble solids and titratable acidity across varieties and seasons. Pre-bloom and post-fruit set leaf removal to the greatest extent consistently increased total grape phenolics and anthocyanins compared to no leaf removal in Cabernet Sauvignon, but inconsistently increased total grape phenolics compared to no leaf removal in Cabernet franc and Petit Verdot. Basal leaf removal to the greatest extents tended to increase the synthesis and degradation of carotenoids more consistently than no leaf removal, and this was particularly true for zeaxanthin. Petit Verdot and Cabernet franc wine color and aroma were inconsistently distinguishable between leaf removal treatments, and color intensity was rated higher in wines made with fruit from pre-bloom leaf removal compared to modest post fruit-set leaf removal plots. Waiting until after fruit set to remove fruit-zone leaves maintained crop yield and offered comparable improvements in fruit composition to pre-bloom leaf removal. Pre-bloom leaf removal of no more than four leaves is recommended to limit crop yield reduction, and modestly improve fruit composition. This work showed that fruit-zone leaf removal does not need to be conservative in the eastern US, particularly because the climate does not appear to be detrimental to fruit composition, and open fruit-zones reduce grape fungal disease incidence. / Ph. D.

This thesis focuses on mechanisms and processes underlying spatial patterns of resistance and virulence and on local adaptations in plant–parasite interactions. The model system used comprises the plant host Salix triandra, the pathogenic rust fungus Melampsora amygdalinae, the leaf beetle Gonioctena linnaeana, and the galler Pontania triandrae. In this work, I (1) emphasize the most important factors determining the outcome of a plant–pathogen interaction, and the types of systems in which local adaptations can be expected, (2) examine the resistance structures of different populations of S. triandra, and whether the leaf beetle G. linnaeana responds to the local conditions of the populations of S. triandra in Sweden, and (3) address whether the distribution of parasites on S. triandra can be explained by the plant content of secondary metabolites. A review of several studies of the subject leads to the conclusion that adaptation of pathogens to their local hosts is more likely to be found in systems in which the pathogen is host-specific, non-systemic, and has a larger dispersal range and evolutionary potential than its host does. Furthermore, the scale of the study must be adjusted to that of the pathogen’s local population distribution. In addition, the temporary nature of host–pathogen interactions influences the importance of sample size, and too-small sample sizes can lower the chance of finding local adaptations, even though they may have evolved in a given system. The results of an inoculation experiment using material from physically isolated natural populations of S. triandra and M. amygdalinae confirm the importance of previous conclusions. Spatial variation in the resistance structure of S. triandra also has effects on the insect herbivore G. linnaeana, which has responded by adapting to the local hosts. However, local differences in secondary chemistry affect different parasites in different ways, and while P. triandrae is attracted by high levels of phenolic compounds, including tannins, M. amygdalinae and G. linnaeana are more rarely found on plant individuals with high concentrations of tannins. In addition, brood deposition by adult females of G. linnaeana and the performance of larvae are positively affected by luteolin-7-glucoside and an additional unidentified flavonoid, whereas they are negatively affected by the presence of (+)-catechin and high levels of tannins. Our results also show that plants traits that provide resistance to one type of parasite do not necessarily provide resistance to others. This indicates that different natural enemies potentially assert divergent selection pressure on S. triandra phenotypes which can be important for maintaining phenotypic variation in plant species.

Cette étude examine les effets bénéfiques des marcs de raisins obtenus après vinification de différents cépages caractéristiques de la Vallée du Rhône, de leurs compositions phénoliques à leurs effets in vivo. Les raisins et leurs marcs respectifs des cépages Grenache (provenant de deux endroits différents [GRE1] et [GRE2]), Syrah (provenant de deux endroits différents [SYR1] et [SYR2]), Carignan (CAR), Mourvèdre (MOU), Counoise (COU) et Alicante (ALI) ont été étudiés. La comparaison des extraits de raisins et de leurs marcs respectifs montre que les marcs représentent une source importante d’antioxydants phénoliques malgré le processus de vinification. Les pépins et pellicules de marcs renferment des quantités appréciables de flavan-3-ols et anthocyanes. La distribution qualitative et quantitative des polyphenols dans les marcs de raisin présentent des différences significatives au travers des variétés et millésimes allant de 15% à 70% de polyphénols extraits. Les pépins de Grenache (GRE1), Syrah (SYR1) et les pellicules de Syrah (SYR1), Carignan et Alicante sont les fractions les plus intéressantes dues a la présence d’importantes quantités de flavan-3-ols (monomères, dimères et trimère) jusqu’à 8.7 mg/g MS et d’anthocyanes (glycosylées, acétylées et coumaroylées jusqu’à 17.40 mg/g MS, 1.57 mg/g MS et 2.38 mg/g MS respectivement). L’analyse des extraits aqueux (EAQ) et hydro-alcoolique 70% (EA70) indique que les pépins de Carignan et Syrah (SYR1) et les pellicules de Carignan et d’Alicante contiennent les plus fort taux en composés phénoliques et activités antioxydantes. L’efficacité in vivo sur l’hypertension de certains extraits a été évaluée utilisant un modèle de rat SHR. Les résultats des expériences in vivo démontrent que certains extraits administrés seuls ou en association avec le vérapamil possèdent un effet anti-hypertenseur. Cette capacité a été mise en évidence une fois que les extraits de pépins de marcs de GRE1 (EA70) et SYR1 (EA70) et de pellicules de marcs d’ALI (EA70) ont été administrés seuls ou lorsque les extraits de pépins de marcs GRE1 (EA70) et SYR1 (EAQ) et les pellicules de marcs d’ALI (EA70) et SYR2 (EAQ) ont été administrés en association avec le vérapamil. Cette étude met en évidence la biodisponibilité des extraits de pépins et de pellicules de marcs des rats SHR incluant à la fois le métabolisme de phase II et de la microflore intestinale. Les extraits de marcs administrés seuls et en association au vérapamil sont absorbés en tant que métabolites de phase II dérivant du métabolisme des monomères de flavan-3-ols. La détection de métabolites microbiens dérivés de flavan-3-ols, d’hydroxyphényl-γ-valérolactones sous leurs formes glucuronidés et sulfatés confirme l'absorption des métabolites dérivés des flavan-3-ols monomères et polymères et des conjugaisons supplémentaires dans le foie. De nombreux métabolites issus de la dégradation microbienne des hydroxyvalerolactones ont également été détectés. L’excrétion urinaire de ces métabolites représente une plus grande proportion de polyphénols ingérés comparé à ceux issus de métabolisme de phase II des monomères de flavan-3-ols, indiquant un rôle important des bactéries intestinales dans le métabolisme des molécules hautement polymérisées. Ces métabolites peuvent avoir exercé leurs effets biologiques lors de leur passage dans la circulation sanguine. Cette étude constitue une première étape de valorisation de marcs de raisins après vinification, en tant qu’activateur de vérapamil, un médicament couramment utilisé contre l’hypertension. Des quantités suffisantes de composés phénoliques subsistent dans les marcs, en particulier en terme de flavan-3-ols et d’anthocyanes, pour exercer des effets antihypertenseurs. En effet, selon le type et la composition des extraits, il est possible de moduler les effets antihypertenseurs en amplifiant ou en diminuant l’absorption des polyphénols et/ou du vérapamil. / This study investigated the beneficial potential effects of grape pomaces obtained after winemaking of different Mediterranean grape varieties from crude materials to their in vivo effectiveness. Grapes and their respective grape pomaces from six different V. vinifera L. cultivar were studied namely Grenache (from two different locations [GRE1 and GRE2]), Syrah (from two different locations [SYR1 and SYR2]), Carignan (CAR), Mourvèdre (MOU), Counoise (COU) and Alicante (ALI) grape varieties from the Rhône Valley. The comparison of several wine industry by-products with their respective grapes provided evidence that pomace remaining at the end of the winemaking process can be very rich sources of antioxidants. The quantitative and qualitative distribution of polyphenols by HPLC-PDA-Fluo-MS in grape pomaces showed significant differences through varieties and vintages varying from 15% to 70% of polyphenols extracted. Seeds from Grenache (GRE1), Syrah (SYR1) and skins from Syrah (SYR1), Carignan and Alicante were of particular interest because of their higher polyphenol contents in terms of flavan-3-ols (monomers, dimers and trimers) up to 8.7 mg/g DW and anthocyanins (glycosides, acetylated and coumaroylated derivatives up to 17.40, 1.57 and 2.38 mg/g DW, respectively). The investigation of aqueous and hydro-alcoholic 70% extracts of seeds from Carignan and Syrah (SYR1) and skins from Carignan and Alicante was carried out as they contained high levels of total phenols and antioxidant activity. Several extracts, were tested in order to evaluate their in vivo biological effects on hypertension using a spontaneously hypertensive rat (SHR) model. A series of different grape pomace extracts were tested in association with verapamil. All in vivo experiments demonstrated that some grape pomace extracts administrated with or without co-ingestion with verapamil possessed an anti-hypertensive activity. This was evident with GRE1 (EA70) seed pomace extract, SYR1 (EA70) seed pomace extract, ALI (EA70) skin pomace extract administrated alone and with GRE1 (EA70) seed pomace extract, SYR1 (EAQ) seed pomace extract, ALI (EA70) skin pomace extract and SYR2 (EAQ) skin pomace extract administrated in association with verapamil. Grape pomace extracts with or without co-ingestion with verapamil were absorb as phase II metabolites mainly including glucuronide, O-methyl glucuronide, sulfate, and O-methyl sulfate derivatives of (epi)catechin which arise from the metabolism of monomeric flavan-3-ols. The detection by HPLC-PDA-Fluo-MSn and GC-MS of microbial-derived metabolites of flavan-3-ols, hydroxyphenyl-γ-valerolactones in their glucuronide and sulfate forms confirmed the absorption of metabolites derived from both monomeric and polymeric flavan-3-ols from grape pomace extracts and subsequent post-absorption conjugation. Numerous metabolites derived from further microbial degradation of hydroxyvalerolactones were also detected. The urinary excretion of these metabolites accounted for a larger proportion of the total polyphenol ingested than phase II metabolites of monomeric flavan-3-ols, indicating the important role of intestinal bacteria in the metabolism of polymerized procyanidins. All these metabolites may have exerted biological effects during the period in which they circulated in the bloodstream. This study constitutes the first step of assessing grape pomace as an enhancer of the verapamil, an anti-hypertensive drug. Substantial levels of polyphenols, especially flavan-3-ols, procyanidins and anthocyanins, remain in pomace after the winemaking process in quantities sufficient to exert anti-hypertensive effects. In addition, according to the extract used and its composition, it is feasible to modulate anti-hypertensive effects by amplifying or decreasing polyphenols and/or verapamil absorption.

<div>Shifts in the human diet to more refined foods and ingredients have contributed to the rise in metabolic disease rates associated with long-term consumption of foods causing swift rises in blood glucose response. Foods which result in a more moderate blood glucose curve are considered healthier by increasing satiety and reducing oxidative stress. Sorghum products contain naturally slowly digested starch. The matrix of sorghum porridges contains kafirin protein bodies which cross link around gelatinizing starch molecules, while similar nascent matrices in other cereals aggregate and collapse. The 3-deoxyanthocyanidin pigments unique to sorghum may be accountable for the difference in matrix stability. The density of the starch entrapped in the matrices is thought to partially inhibit α-amylase access to the starch, reducing overall starch digestion and thereby mitigating glucose response. The purpose of this work was to increase our understanding of how phenolic compounds in sorghum interact with endosperm proteins to create a stable matrix, and to explore if the knowledge might be translated to other starchy cereal products. In the first study, phenolic extracts from flours (sorghum, corn masa, white rice) were characterized for phenolic content, antioxidant activity, phenolic components, and their ability to interact with a model protein system (ovalbumin) in order to examine protein polymerization. In the second study, specific phenolic compounds in sorghums (<i>p</i>-coumaric, sinapic, and gallic acids; (+)-catechin; and apigeninidin, a 3-deoxyanthocyanidin found in sorghums) were interacted in the model protein system at different concentrations to observe extent and type of protein polymerization, and promising compounds subjected to fluorescence quenching spectroscopy to examine the nature of the interactions. The final study explored the effects of apigeninidin addition to a yellow corn flour and naturally present anthocyanin (blue corn) on starch digestion and microstructure of porridges by utilizing an <i>in vitro</i> α-amylase assay and confocal microscopy. </div><div>The slow digestion of starch in cooked sorghum products can be attributed to the 3-deoxyanthocyanidin compounds present in the grain participating in sulfhydryl-disulfide interchanges which results in extensive kafirin cross-linking surrounding starch granules. While other phenolic and redox-active components may affect matrix formation and stability, 3-deoxyanthocyanidins appear to have the most direct influence, and their ability to modify food protein matrices appears to have a direct result on starch digestion <i>in vitro</i>.</div>