A eficiência de antocianinas como inibidores de corrosão da liga de alumínio 2024-T3 foi avaliada através de medidas eletroquímicas como a espectroscopia de impedância eletroquímica e polarizações anódica e catódica. Soluções de NaCl 0,025 mol.L-1 contendo diferentes concentrações (800, 1000, 2000, 3000, 5000 e 7200 ppm) de antocianinas extraídas da uva foram preparadas para analisar o efeito da presença destas substâncias quando em contato com a liga de alumínio 2024-T3. Medidas de espectroscopia de impedância eletroquímica apresentaram uma diminuição sobre a dispersão dos pontos nas regiões de baixas frequências com o acréscimo do tempo de contato das antocianinas com a superfície da liga de alumínio 2024-T3 de 1 hora para 3 dias de imersão. Além disso, o comportamento indutivo nestas regiões decresceu após 3 dias de imersão com uma elevação considerável da resistência à polarização e da eficiência de inibição em 1000 ppm de antocianinas. Sob polarizações anódica e catódica, também foi verificado um melhoramento generalizado das propriedades anticorrosivas como potencial de corrosão, corrente de corrosão e eficiência de proteção contra a corrosão, para a concentração de 1000 ppm de antocianinas. Análises de microscopia eletrônica de varredura, microscopia de força atômica e espectroscopia por dispersão de energia, confirmam a adsorção de antocianinas na superfície da liga, assim como medidas de absorbância após 3 dias de imersão. / The efficiency of anthocyanins as corrosion inhibitor of the 2024-T3 aluminum alloy was evaluated by electrochemical measurements such as electrochemical impedance spectroscopy and anodic and cathodic polarizations. NaCl solutions 0.025 mol.L-1 containing different concentrations (800, 1000, 2000, 3000, 5000 and 7200 ppm) of anthocyanins extracted from grape were prepared to examine the effect of the presence of these substances when in contact with the 2024-T3 aluminum alloy. Electrochemical impedance spectroscopy measurements showed a decrease on the dispersion of points in the lower frequency regions with an increase of contact time of anthocyanins with the surface of the 2024-T3 aluminum alloy from 1 hour to 3 days of immersion. In addition, the inductive behavior in these regions decreased after 3 days of immersion with a considerable increase of the polarization resistance and inhibition efficiency of 1000 ppm of anthocyanins. Under cathodic and anodic polarizations, it was noted a general improvement in the anticorrosive properties such as corrosion potential, corrosion current and efficiency of protection against corrosion for the concentration of 1000 ppm of anthocyanins. Analyses of scanning electron microscopy, atomic force microscopy and energy dispersive spectroscopy confirm anthocyanins adsorption on to the surface alloy, as well as absorbance measurements in a spectrophotometer after 3 days of immersion.

A cada ano a indústria de vinhos descarta uma alta quantidade de resíduos líquidos e sólidos, constituído em grande parte por bagaço de uva. Este resíduo pode ser considerado um subproduto da indústria e apresenta quantidades significativas de antocianinas, as quais podem apresentar diversas aplicações. Apesar da extração de antocianinas ser um passo importante na recuperação de pigmentos em matrizes vegetais não existe um método de extração padrão na literatura. Dessa forma, existem estudos tanto em relação ao uso de métodos tradicionais quanto emergentes, sendo estes últimos normalmente mais favoráveis ao Meio Ambiente. Neste estudo foi usado o método de extração enzimática, técnica emergente, que se baseia em alterações da parede celular de matrizes alimentares para exposição dos materiais intracelulares. Assim, o objetivo geral do trabalho foi realizar a extração enzimática de antocianinas presentes em casca de uva para posterior aplicação como corante alimentício em quefir e bebida carbonatada. Foram encontrados diferentes temperaturas e porcentagens de preparado enzimático ótimos dependendo do cultivar analisado. Após aperfeiçoamento foi selecionado o uso de casca de Cabernet Sauvignon, a uma temperatura de 40 oC e 0,25 % de Pectinex Ultra Color®. O corante natural produzido foi aplicado em bebida carbonatada e quefir, ambos sob análises de tempo de meia-vida de antocianinas e parâmetros físico-químicos durante 16 dias de armazenagem. Dentre os resultados obtidos foi destacado que o quefir com adição do corante manteve características semelhantes ao encontrado na literatura para quefir natural. Em análises em bebida carbonatada, houve maior estabilidade de antocianinas nas amostras armazenadas sem presença de luz. A aplicação do extrato antociânico foi favorável em ambas matrizes alimentares, sendo recomendado estudos futuros visando o aumento do tempo de meia vida da estabilidade das antocianinas. / Every year, wine industry discards a high amount of liquid and solid waste, consisting largely of grape pomace. This residue can be considered a by-product of the industry and presents significant quantity of anthocyanins, which can provide several applications. Despite the extraction of anthocyanins be an important step on the recovery of pigments in vegetables, there is not a standard extraction method in the literature. In this way, there are studies regarding the use of traditional and emerging methods, which the latter usually being more environmentally friendly. In this study, controlled enzymatic extraction method was used, an emerging technique that is based on alterations of the cell wall of alimentary matrices for exposure of the intracellular materials. Thus, the aim of this study was to perform the enzymatic extraction of anthocyanins present in grape skins for subsequent application as a food dye in kefir and carbonated beverage. Eight different samples of grape pomace were used to improvement the extraction process. Preliminary responses of the study defined that low extraction times are required for the enzymatic extraction process, where the maximum ones being found when the process was conducted for thirty minutes using Pectinex Ultra Color®. In addition, different temperatures and percentages of enzyme preparation were found depending on the variety analyzed. After improvement, the use of Carbenet Sauvignon skin was selected, at a temperature of 40 ° C and 0.25% of enzymatic preparation. The natural dye produced was applied in carbonated beverage and kefir, both under analysis of the half-life of anthocyanins and physical- chemical parameters during 16 days of storage. Among the results obtained, it was pointed out that kefir with dye addition maintained similar characteristics to that found in the literature for natural kefir. In analyzes in carbonated beverage, there was greater stability of anthocyanins when the samples were stored without light. The application of the anthocyanin extract was favorable in both food matrices, and future studies are recommended aiming to increase the half-life of the stability of anthocyanins.

A produção do fruto do palmiteiro juçara tem sido bastante estimulada na região da Mata Atlântica. Este fruto possui elevados teores de compostos bioativos, como antocianinas e outros compostos fenólicos. Devido ao recente estímulo ao consumo deste fruto e ao impacto de compostos bioativos da dieta sobre a estabilidade genômica, os objetivos deste trabalho foram investigar os efeitos citotóxico, genotóxico e antigenotóxico da polpa do fruto da juçara em ratos Wistar, usando o teste do micronúcleo e o ensaio do cometa. Ratos Wistar machos receberam por gavagem água ou a polpa liofilizada do fruto da juçara reidratada com água (125, 250 ou 500 mg/kg p.c) durante 14 dias. No último dia de tratamento (14º dia), salina (NaCl, 0,9 %) ou metil-metanosulfonato (MMS, 40 mg/kg p.c.) foram administrados por via intraperitoneal, 24 horas antes da eutanásia. Foram coletados fígado e rins para a análise de glutationa reduzida (GSH), catalase (CAT), substâncias reativas ao ácido tiobarbitúrico (TBARS) e para a realização do ensaio do cometa. Medula óssea e sangue periférico foram usados para o teste do micronúcleo. Os resultados mostraram que o tratamento com todas as doses da polpa do fruto da juçara não alterou os parâmetros de estresse oxidativo avaliados em rins e fígado de ratos Wistar. Além disso, não foi constatado efeito genotóxico nestes órgãos, nas três doses testadas da polpa do fruto da juçara. Em todas as doses avaliadas, a polpa do fruto da juçara não apresentou efeito mutagênico nem citotóxico em células da medula óssea e do sangue periférico de ratos Wistar. Por outro lado, a polpa do fruto do palmiteiro juçara apresentou efeito antigenotóxico frente aos danos induzidos pelo MMS em células hepáticas nas doses de 125 e 500 mg/kg p.c. A polpa deste fruto, nestas mesmas doses, também apresentou efeito antimutagênico em células do sangue periférico de ratos Wistar. A polpa liofilizada do fruto da juçara apresentou alta concentração de antocianinas, sendo majoritárias a cianidina-3-glicosídeo e cianidina-3-rutinosídeo. A presença destes compostos bioativos neste fruto pode estar relacionada aos efeitos protetores frente aos danos induzidos pelo MMS, verificados neste presente estudo. Como conclusão, o consumo da polpa do fruto do palmiteiro juçara, nas doses avaliadas, não induziu genotoxicidade e mutagenicidade em ratos Wistar. Além disso, a administração da polpa do fruto da juçara em associação com o MMS reduziu a genotoxicidade e mutagenicidade induzidas por este agente indutor de danos ao DNA. / Juçara palm fruit\'s production has been intensely promoted in the Atlantic Forest region. This fruit has high content of bioactive compounds, including anthocyanins and other phenolic compounds. Due to the recent stimulus to the consumption of this fruit and the impact of dietary bioactive compounds on the genomic stability, this study aimed to evaluate the cytotoxic, genotoxic and antigenotoxic effects of juçara fruit pulp in Wistar rats, using the comet assay and the micronucleus test. Male Wistar rats received water or lyophilized juçara fruit pulp rehydrated with water (125, 250 or 500 mg/kg b.w.) by gavage for 14 days. On the last day of treatment (day 14th), saline (NaCl, 0,9 %) or methyl methanesulfonate (MMS, 40 mg/kg b.w.) were administered intraperitoneally 24 hours before euthanasia. Liver and kidneys were collected for analysis of reduced glutathione (GSH), catalase (CAT), thiobarbituric acid reactive substances (TBARS) and to perform the comet assay. Bone marrow and peripheral blood were used for the micronucleus test. Results showed that treatment with all doses of juçara fruit pulp did not alter the oxidative stress parameters evaluated in kidneys and liver of Wistar rats. Furthermore, no genotoxic effects were observed in these organs, at all doses evaluated. At all tested doses, juçara fruit pulp also showed no mutagenic or cytotoxic effects on cells of bone marrow and peripheral blood of rats. On the other hand, juçara fruit pulp showed antigenotoxic effect at doses of 125 and 500 mg/kg b.w. against MMS-induced DNA damage in liver cells. At these same doses, juçara fruit pulp also exhibited antimutagenic effect on peripheral blood cells of Wistar rats. Juçara fruit pulp exhibited a high concentration of anthocyanins, with cyanidin-3-glucoside and cyanidin-3-rutinoside as major compounds. These bioactive compounds present in this fruit may be related to the protective effects against MMS-induced DNA damage, demonstrated in the present study. In conclusion,juçara palm fruit\'s consumption, at tested doses, did not induce genotoxicity and mutagenicity in Wistar rats. Furthermore, juçara fruit pulp associated with MMS, a DNA-damaging agent, decreased the genotoxicity and mutagenicity induced by this DNA-damaging agent.

Diversos estudos buscam identificar novas moléculas com ações regulatórias sobre a via de sinalização da insulina e consequentemente na homeostase da glicose. Assim, este trabalho visa avaliar o potencial de extratos de frutos no controle da homeostase glicêmica. Os frutos avaliados foram o morango (cv. Toianoca, Camarosa, Oso Grande e Camino Real), a amora-preta e a framboesa vermelha, em dois tempos de amostragem, sendo considerado como tempo A1 e tempo A2. As amostras foram caracterizadas quanto ao seu conteúdo de fenólicos totais, conteúdo de antocianinas monoméricas, capacidade antioxidante, avaliada pelos métodos DPPH e ORAC, ácido elágico total, capacidade de inibição da alfa-glicosidase e captação de glicose e lipólise em tecido adiposo de camundongos (ensaio explante). Dentre os frutos, no primeiro tempo de amostragem, a amora-preta e o morango, cv Oso Grande, foram os que apresentaram maior conteúdo de fenólicos totais (62,36 e 34,89 mg AG/g, respectivamente) no entanto não foram mantidos esses valores no segundo tempo de amostragem, com concentração 30% e 60% inferior, respectivamente; e maior concentração de antocianinas monoméricas (45,33 mg/g e 3,09 mg/g, respectivamente). Em relação a inibição da enzima alfa-glicosidase, avaliado em extrato metanólico, o fruto framboesa vermelha e o morango cv. Camino Real foram as que apresentaram alto potencial inibitório nos dois tempos de amostragem (IC50 0,47 mg FT e IC50 0,57 mg FT para framboesa vermelha e IC50 0,50 mg FT e IC50 0,46 mg FT para Camino Real). Quando avaliado os extratos enriquecidos em fenólicos, o valor de IC50 com maior potencial dentre os frutos avaliados foi da amora-preta, nos dois tempos A1 e A2 (0,0023 mg FT e 0,0021 mg FT, respectivamente). Para captação de glicose em tecido adiposo explate, ao utilizar a insulina para estimular a captação de glicose juntamente com o tratamento (extrato), esse estimulo foi efetivo no aumento da captação de glicose somente com as amostras cv. Camino Real e cv. Oso Grande. Isso pode ser explicado pela alta correlação encontrada de antocianinas identificadas no fruto, como pelargonidina-3-O-glicosídeo. Por outro lado, somente a amora-preta A1 aumentou a lipólise em condição basal, mas nenhuma fruta foi eficiente para reduzir a lipólise em condição estimulada pelo isoproterenol. Sendo assim, frutas vermelhas podem ser boas fontes de compostos bioativos, principalmente antocianinas, as quais podem ter corroborado positivamente com os resultados. / Several studies seek to identify new molecules with regulatory actions on the insulin signaling pathway and consequently on glucose homeostasis. Thus, this work aims to evaluate the potential of fruit extracts in the control of glycemic homeostasis. The fruits evaluated were strawberry (cv. Toianoca, Camarosa, Oso Grande and Camino Real), blackberry and red raspberry, in two sampling times, being considered as time A1 and time A2. Fruits were evaluated for total phenolic, monomeric anthocyanins and contents, antioxidant capacity, evaluated by DPPH and ORAC methods, alpha-glycosidase inhibition capacity and glucose uptake and lipolysis in adipose tissue of mice (explant assay). Among the fruits, in the first sampling period, blackberry and strawberry, cv. Oso Grande, showed the highest total phenolic content (62.36 and 34.89 mg AG / g, respectively), with a decrease of the 30% and 60%, respectively, in the second sampling time; and higher monomeric anthocyanins concentration (45.33 mg/g and 3.09 mg/g, respectively). The methanolic extracts of raspberry and the strawberry cv Camino Real (A1 and A2) presented the highest alpha-glucosidase inhibitory potential. Otherwise, the enriched-polyphenol extract of blackberry (A1 and A2) presented the highest potential among the evaluated fruits. Adipose tissue treated with strawberry cv. Camino Real and cv. Oso Grande was effective in increasing glucose uptake stimulated by insulin. This can be explained by the high correlation with the anthocyanins pelargonidin-3-O-glycoside identified in this fruit. In addition, blackberry A1 was the only sample to increase the lipolysis in basal condition, but all other fruits were not effective to decrease lipolysis in stimulated condition. Thus, berries could be a good sources of bioactive compounds to maintain the glucose homeostasis.

FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico / Atualmente, ocorre um crescimento mundial da preferÃncia dos consumidores pelos alimentos que contÃm ingredientes naturais. Em relaÃÃo aos pigmentos, ainda hà certa dificuldade na utilizaÃÃo de corantes naturais na indÃstria alimentÃcia, pois estes apresentam menor estabilidade que os corantes sintÃticos. Com o objetivo de aumentar a estabilidade e a utilizaÃÃo dos corantes naturais como ingredientes alimentÃcios, aplicou-se aos mesmos, a tÃcnica de microencapsulaÃÃo por atomizaÃÃo. Os corantes utilizados foram bixina, pigmento lipossolÃvel do urucum, fruto do urucuzeiro (Bixa orellana L.) e antocianinas, pigmentos hidrossolÃveis do aÃaÃ, fruto do aÃaizeiro (Euterpe oleracea Mart.), com os agentes encapsulantes goma arÃbica, goma do cajueiro e maltodextrina, em diferentes formulaÃÃes. Testes de umidade, solubilidade e cristalinidade foram executados, bem como a microscopia eletrÃnica, para observaÃÃo da morfologia das microcÃpsulas. AtravÃs da colorimetria, os pigmentos naturais microencapsulados foram estudados em diferentes apresentaÃÃes, ou seja, dissolvidos em soluÃÃo-tampÃo pH 4,0 e na forma de pÃs. Na referida soluÃÃo-tampÃo utilizou-se tambÃm o extrato puro de antocianinas como testemunha. Verificou-se entÃo, a estabilidade ao longo do tempo das formulaÃÃes descritas, em temperatura ambiente de 25ÂC  1ÂC na presenÃa (450h) e ausÃncia (1400h) da luz. A degradaÃÃo dos pigmentos microencapsulados ocorreu mais rapidamente na luz. A goma do cajueiro mostrou-se como bom encapsulante e sendo um abundante recurso natural do Cearà poderà ser utilizado como substituto da goma arÃbica, cujo custo de importaÃÃo à elevado. / Nowadays, there is a world increase in the consumerâs preference for food that contains natural ingredients. As it comes to pigments, there is still a difficulty in the utilization of natural pigments on the food industry, because these have less stability than the synthetic ones. With the objective of increasing the stability and the utilization of natural pigments as food ingredients, they were submitted to microencapsulation by atomization. The utilized pigments were bixin, a liposoluble pigment from urucum (Bixa orellana L.) and anthocyanins, water soluble pigments from aÃaà (Euterpe oleracea Mart.), combined with encapsulating agents (arabic gum, cashew tree gum and maltodextrin), in different formulations. Moisture, solubility and crystallinity tests were executed, as well as scanning electronic microscopy, in order to observe the microcapsules morphology. The microencapsulated natural pigments were studied by calorimetry in different presentations, that is, they were dissolved in a pH 4,0 buffer-solution and as powders. In the referred buffer-solution, a pure anthocyanin extract was used as a control. Then, the stability of the described formulations were evaluated through time, at room temperature of 23ÂC  1ÂC, with and without light exposure. The microencapsulated natural pigments degeneration occurred faster under light. The cashew tree gum proved to be a good encapsulating agent and, since it is an abundant natural resource in CearÃ, it can be utilized to replace arabic gum, whose importation cost is high.

As antocianinas compreendem o maior conjunto de pigmentos solúveis em água do reino vegetal. Responsáveis pelas cores vermelha, azul e roxa da maioria das frutas e flores, estas pertencem ao grupo dos flavonóides (polifenois). A cor apresentada por uma determinada antocianina depende do padrão de substituição, presença ou não de íons metálicos ou de outras moléculas capazes de estabilizar a cor, co-pigmentos, do pH local do meio e da natureza do microambiente em que a antocianina se encontra. Neste projeto, realizou-se um estudo de especiação e das propriedades antioxidantes de uma série de compostos análogos às antocianinas: os sais de 3\' -hidroxiflavílio (3\'-HF), 4\'- hidroxiflavílio (4\'-HF), 7,4-dihidroxiflavílio (7,4\'-DHF), 3\',4\'-dihidroxiflavílio (3\',4\'-DHF), 5,7-dihidroxi-4-metilflavílio (5,7-DH-4-MF), 7-hidroxi-4-metilflavílio (7-H-4-MF) e 7- metoxi-4-metilflavílio (7-metox-4-MF). No estudo de especiação, foram determinados o pK imediatamente após a preparação da amostra e o pKfinal após 1 dia. Para 3\'-HF, os valores do pK e pKfinal foram 2,88±0,05 e 1,94±0,15; para 4\'-HF, 4,56±0,10 e 4,23±0,20. Para os flavílios com dois grupos hidroxila ionizáveis, as constantes pK1, pK2, pK1final e pK2final para 7,4\'-DHF foram 4,12±0,07, 7,85±0,01, 3,29±0,01 e 7,40±0,03; para 3\',4\'-DHF, 3,98±0,10, 8,80±0,40, 3,70±0,01 e 7,01±0,22. Para o 5,7-DH-4-MF, que não sofre hidratação devido à presença do grupo metílico na posição 4, o pK1 e pK2 foram 4,87±0,01 e 7,97±0,01. Para a determinação da atividade antioxidante, foi utilizado o método de ABTS em que o poder antioxidante é obtida comparando-se a descoloração do radical 2,2\'-azino-bis(3- etilbenzoiltiazolina-6-sulfônico) (ABTS+), a 734 nm, pela amostra com a do composto padrão Trolox (ácido 6-hidroxi-2,5,7,8-tetrametilcroman-2-carboxílico), sendo expressa como a TEAC (Trolox Equivalent Antioxidant Capacity). A cinética da reação com os flavílios é bifásica, com uma fase lenta devido à atividade antioxidante residual dos subprodutos da reação. Utilizou-se a primeira derivada das curvas cinéticas para identificar o fim da fase rápida e, portanto, o tempo ótimo para a determinação do valor da TEAC. Os monohidroxiflavílios (3\'-HF e 4\'-HF) e o 7,4\'-DHF não apresentaram poder antioxidante em solução aquosa a pH 2,1 (H3PO4/H2PO4-). Por outro lado, o grupo catecol presente no anel B do 3\',4\'-DHF e o grupo resorcinol no anel A do 5,7-DH-4-MF conferem propriedades antioxidantes, mesmo em pH baixo. Também apresentaram atividade antioxidante a pH 2,1 o 7-H-4-MF e o 7-metox-4-MF. Em pHs acima do pK, a formação de cis- e trans-chalconas, devido à hidratação, tautomerização e isomerização dos flavílios, resultou num aumento do valor da TEAC. / Anthocyanins are the major water-soluble pigments of the plant kingdom. Responsible of the red, blue and purple colors of the majority of fruit and flowers, they belong to the group of flavonoids (polyphenols). The color presented by an anthocyanin depends on the pattern of substitution, the presence or not of metal ions or other molecules capable to stabilize the color (co-pigments), the local pH of the solution and the nature of the microenvironment of the anthocyanin. In this project, a study was made of the speciation and the antioxidant properties of a series of compounds analogous to anthocyanins: 3\'-hydroxyflavylium (3\'-HF), 4\'- hydroxyflavylium (4\'-HF), 7,4\'-dihydroxyflavylium (7,4\'-DHF), 3\',4\'-dihydroxyflavylium (3\',4\'-DHF), 5,7-dihydroxy-4-methylflavylium (5,7-DH-4-MF), 7-hydroxy-4- methylflavylium (7-H-4-MF) and 7-methoxy-4-methylflavylium (7-Methox-4-MF). In the study of the speciation, the pK is determined immediately after preparation of the samples and the pKfinal after 1 day. For 3\'-HF, the values of pK and pKfinal were 2.88±0.05 and 1.94±0.15; for 4\'-HF, 4.56±0.10 and 4.23±0.20. For the flavyliums with two ionizable hydroxyl groups, the constants pK1, pK2, pK1final and pK2final for 7,4\'-DHF were 4.12±0.07, 7.85±0.01, 3.29±0.01 and 7.40±0.03; for 3\',4\'-DHF, they were 3.98±0.10, 8.80±0.40, 3.70±0.01 and 7.01±0.22. For 5,7-DH-4-MF, which does not hydrate due to the presence of the methyl group at position 4, pK1 and pK2 were 4.87±0.01 and 7.97±0.01. For the determination of the antioxidant activity, the ABTS method was employed, in which the antioxidant power is obtained by comparing the rate of bleaching of the 2,2\'-azinobis( 3-ethylbenzothiazoline-6-sulfonate) radical (ABTS+), at 734 nm, by the sample with that of the standard compound Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), the result being expressed as the TEAC (Trolox Equivalent Antioxidant Capacity). The reaction kinetics with the flavyliums are biphasic, with the slower phase due to the residual antioxidant activity of the reaction subproducts. The first derivative of the kinetic curves was employed to determine the end of the faster phase and thus the optimum time for measuring the TEAC value. The monohydroxyflavyliums (3\'-HF and 4\'-HF) and 7,4\'DHF showed no antioxidant power in aqueous solution at pH 2.1 (H3PO4/H2PO4-). However, the catechol group in ring B of 3\',4\'-DHF and the resorcinol group in ring A of 5,7-DH-4-MF conferred antioxidant properties even at low pH. In addition, 7-H-4-MF and 7-Methox-4-MF also showed antioxidant activity at pH 2.1. At pH above the pK, the formation of the cis- and trans-chalcones via hydration, tautomerization and isomerization resulted in an increase in the value of the TEAC.

Plants inherently produce polyphenols (i.e., antioxidants) as a response to reduce oxidative stress caused by abusive environmental pre- and postharvest conditions. These antioxidants, as well as vitamin C, are present in considerable levels in strawberries; however, excessive oxidative stress brought on by improper postharvest handling conditions can reduce the levels of antioxidants in the fruit and shorten the shelf-life of strawberries. Nevertheless, it may be possible to utilize strawberry’s naturally occurring polyphenols to reduce postharvest stress and extend their shelf life. The polyphenolic profile has been previously investigated in several strawberry cultivars, however no studies have determined the unique polyphenolic profiles of three important Florida strawberry cultivars (‘Florida Radiance’, Sweet Sensation® ‘Florida 127’and ‘Florida Beauty’) at harvest and during cold storage. Therefore, in order to better understand the distribution of individual polyphenols within these cultivars and their impact on postharvest shelf-life, this study examined the polyphenolic profiles throughout 7 days of cold storage (1 °C) using an HPLC-DAD. The activity of phenylalanine ammonia lyase (PAL), a key enzyme in the biosynthesis of polyphenols, and polyphenol oxidase (PPO), the enzyme responsible for polyphenol degradation, were also examined during cold storage to understand their possible influences on postharvest synthesis or degradation of polyphenols. This study revealed that the polyphenolic profile of strawberry fruit was genotype dependent; however, pelargonidin 3-glucoside was consistently the anthocyanin found in higher concentrations in the fruit regardless of the cultivar. Apart from the anthocyanins, the flavonols showed the most variation among the three cultivars. PAL was slightly induced during strawberry postharvest storage suggesting that a stress response occurred during cold storage while PPO showed variable induction patterns across all three cultivars most likely due to their different polyphenol profiles. Analysis of the distribution of polyphenols in the cortex and pith of strawberries showed that polyphenols were mostly concentrated in the cortex of the fruit and that the concentration of individual polyphenol in each fruit tissue varied by cultivar. These results indicate that the oxidative stress response varies in each of the strawberry cultivars studied contributing to their unique polyphenolic profile. Results from this study can ultimately help to identify the polyphenols and enzymes related to superior postharvest quality in future studies.

In this thesis, the main concern has been to improve the reliability of different parts of the analytical workflow (Paper I, II, IV &V). Additionally, one of the resulting optimized methods was used in a real application (Paper III). Paper I-II concerned the extraction of acrylamide (AA) from foods. In Paper I different parameters such as sample particle size, extraction solvent, extraction time and extraction temperature were optimized, leading to a method that showed good agreement with the assigned AA levels of several proficiency test samples. Later, after the publication of the paper, the method showed good performance in a collaborative trial validation, in terms of trueness, repeatabil­ity and reproducibility figures. It was labeled “undoubtedly fit for the purpose”. In Paper II, it was shown that the ‘extra’ amounts of AA obtained during extraction of foods with an alkaline aqueous solution was not due to improved extractability of AA from the food matrix. Strongly alkaline conditions seemed to rather induce net formation of AA from water-soluble precursors formed during thermolysis. This phenomenon should therefore be regarded as an extraction artifact. Paper III was an application of the optimized method from Paper I, where it was used to study the reduction of AA in potato chips (crisps) by using pre-treatments and frying at reduced pressure. There were significant reductions in AA, down to below the limit of quantification (5 µg/kg) for the method. Paper IV-V concerned analysis of anthocyanins (AC) in red onion. In Paper IV, a new separation method using capillary electrophoresis was developed, and its rapidness combined with an acidic background electrolyte helped in preventing AC degradation. Furthermore, its alternative separation mechanism is a complement to that of the more commonly used liquid chromatography technique. In Paper V, simultaneous extraction and degradation of anthocyanins from red onion was studied in a static batch reactor at 110ºC. The extraction and degradation kinetics were successfully separated, and an ideal theoretical extraction curve was constructed by compensating mathematically for degradation effects, showing that more anthocyanins, 21 to 36% depending on different species, could be extracted if no degradation occurred. The results give important information about the different kinetics competing during an extraction procedure, and also show that quantitative extraction is not to recommend in the batch system used in the study.

<p>Determining the genetic basis of adaptation has become a central focus of evolutionary biology, and the incorporation of increasingly sophisticated analytical tools from molecular biology has made identifying causal genes a practical reality. The work presented herein addresses the effects of pleiotropic constraint on evolutionary change at the level of individual genes and genetic networks. In the first chapter, I combine molecular phylogenetic analyses and direct assays of enzymatic function to determine the evolutionary processes following a gene duplication in the anthocyanin pathway. My results show that, prior to duplication, the DFR gene was constrained from functional improvement by its multiple enzymatic roles. Following duplication, this constraint was released and adaptive evolution proceeded along both paralog lineages. In the second chapter, I determine the molecular genetic basis of a flower color transition that is associated with change in pollinator attraction in morning glories. A regulatory change in a branching gene in the flavonoid biosynthetic pathway restricted flux down the cyanidin-producing branch, conferring nearly exclusive production of red pelargonidin pigment in flowers. I further demonstrate that this regulatory change was restricted to floral tissue, and that ancestral pathway flux predominates in vegetative tissues. I propose that deleterious pleiotropic effects prevented evolutionary change via enzymatic changes in the pathway due to the numerous essential products downstream of this branching point. Together, these two results show that evolutionary change may be constrained by the molecular genetic context in which prospective adaptive mutations occur.</p> / Dissertation

The influence of genotype (selection), location, and year on antioxidant activity (AOA), total phenolics (TP), total carotenoids (TC), phenolic and carotenoid composition was studied using specialty (colored) potatoes (Solanum tuberosum L.) from the Texas Potato Variety Development Program, grown at two Texas locations (McCook and Dalhart), and in two years (2003 and 2004). Chlorogenic acid, gallic acid, catechin, caffeic acid, and malvidin-3-(p-coumaryl rutinoside)–5-galactoside were the major phenolics, and lutein and violaxanthin were the major carotenoids identified. The AOA, TP, and TC and phenolic composition differed significantly with genotype, location and year. However, genotypic effects were larger than location and year effects. Selection CO112F2-2 was high in all the measured parameters and also stable across locations and years, suggesting that this selection could be used as a parent in breeding varieties with improved health benefits. The AOA, TP and chlorogenic acid content were highly significantly correlated with one another. The effects of whole specialty potato extracts, fractions and individual compounds on LNCaP (androgen-dependent) and PC-3 (androgen-independent) prostate cancer cells were also investigated. Ethanol extract of the selection CO112F2-2 (5 µg chlorogenic acid eq/ml), the anthocyanin fraction (AF; 5 µg chlorogenic acid eq/ml), gallic acid and chaconine showed potent anti-proliferative properties and increased the cyclin-dependent kinase inhibitor p27 levels in LNCaP and PC-3 cells. Induction of apoptosis was cell context dependent and associated with JNK (c-Jun NH2-terminal Kinase) and Erk (extracellular signal regulated kinase) activation. Cell death pathways, induced by potato extract and the AF, were associated with Erk and JNK activation, and these kinases activated caspase-independent apoptosis through nuclear translocation of endonuclease G (endo G) and apoptosis-inducing factor (AIF) in both cell lines. Induction of caspase-dependent apoptosis was also kinase-dependent but was observed only in LNCaP cells. Kinase inhibitors reversed this nuclear translocation of endo G and AIF. This is the first report showing that the cytotoxic activities of potato extract/AF in cancer cells were due to activation of caspase-independent apoptosis.