Дисертації з теми "Anthocyanin and antioxidant activity"

Thesis (M.S. in food science)--Washington State University, December 2008.
Title from PDF title page (viewed on Dec. 31, 2008). "School of Food Science and Human Nutrition." Includes bibliographical references.

Ce travail porte sur la synthèse chimique d’analogues simples d’anthocyanes, une classe majeure de pigments naturels solubles dans l’eau. Onze ions flavylium substitués par des groupements hydroxyl,méthoxyl et beta-D-glucopyranosyloxyl en positions 4’, 5 et 7 ont été préparés en utilisant des procédures simples. De plus, les deux principales 3-désoxyanthocyanidines du sorgho rouge, l’apigéninidine (APN) et la lutéolinidine (LTN), ont été synthétisées en une seule étape. Les propriétés physico-chimiques ainsi que l’activité antioxydante ont été étudiées pour le chlorure de 3’,4’,7-trihydroxyflavylium (P1), son 7-O-beta-D-glucoside (P2) et le chlorure de 3’,4’,5,7-tétrahydroxyflavylium (LTN). Grâce à leur noyau catéchol, ces pigments complexent rapidement FeIII, AlIII and CuI et ne se lient que faiblement à FeII tout en stimulant son autoxydation en FeIII. Suite à la complexation de CuII, les pigments subissent une oxydation. Les aglycones P1 et LTN sont des ligands modérés de l’albumine de sérum humain (HSA) et leurs chalcones ont montré une plus grande affinité pour la HSA que leurs formes colorées. Leur capacité antioxydante a été démontrée par le test de réduction du radical stable DPPH et par l’inhibition de la peroxydation lipidique induite par le fer héminique, un modèle de stress oxydant postprandial dans l’estomac. Les aglycones P1 et LTN (particulièrement, dans leur forme incolore chalcone) sont plus efficaces que le glucoside P2
This work deals with the chemical synthesis of simple analogs of anthocyanins, the main class of watersolublenatural pigments. Eleven flavylium ions with hydroxyl, methoxyl and beta-D-glucopyranosyloxylsubstituents at positions 4’, 5 and 7 have been prepared by straightforward chemical procedures.Moreover, the two main 3-deoxyanthocyanidins of red sorghum, apigeninidin (APN) and luteolinidin(LTN), have been synthesized in a one-step protocol. The physicochemical properties and antioxidantactivity are investigated for 3’,4’,7-trihydroxyflavylium chloride (P1), its 7-O-beta-D-glucoside (P2) and3’,4’,5,7-tetrahydroxyflavylium chloride (LTN). Owing to their catechol B-ring, they rapidly bind FeIII,AlIII and CuI, more weakly interact with FeII while promoting its autoxidation to FeIII. Following CuIIbinding, the pigments undergo oxidation. Aglycones P1 and LTN are moderate ligands of human serumalbumin (HSA) with chalcones having a higher affinity for HSA than the corresponding colored forms.The antioxidant activity of P1, P2 and LTN is investigated via two tests: reduction of the stable DPPHradical and inhibition of heme-induced lipid peroxidation (a model of postprandial oxidative stress inthe stomach). Aglycones P1 and LTN (especially in their colorless chalcone form) are more potent thanglucoside P2

Orientador: Adriana Zerlotti Mercadante
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: Os pigmentos naturais, tais como antocianinas e carotenóides, proporcionam cor aos alimentos, contribuindo para o seu aspecto visual, atributo este de fundamental importância na aceitação e escolha de um alimento por seus consumidores. Concomitante, estes pigmentos possuem importantes funções e ações biológicas, podendo ser considerados promotores da saúde humana. Já é reconhecida a associação entre a ingestão de frutas e vegetais e a diminuição do risco de desenvolvimento de diversas desordens crônico-degenerativas, sendo os pigmentos um dos grupos de compostos bioativos aos quais são atribuídas tais ações. Acerola e açaí são frutas tropicais que contém elevados teores de compostos bioativos, como carotenóides e antocianinas. Os pigmentos, carotenóides e antocianinas, de duas variedades de acerola (Waldy Cati 30 e Olivier) obtidas em uma plantação brasileira durante as safras de verão de 2003 e 2004 foram analisados por cromatografia líquida de alta eficiência (CLAE). As duas variedades apresentaram ß-caroteno (265,5 ¿ 1669,4 µg/100g), luteína (37,6 ¿ 100,7 µg/100g), ß-criptoxantina (16,3 ¿ 56,5 µg/100g) e a-caroteno (7,8 ¿ 59,3 µg/100g) como carotenóides majoritários. Em ambas safras, os teores de ß-caroteno, ß-criptoxantina e a-caroteno foram significativamente maiores na variedade Olivier, entretanto o teor de luteína foi maior na variedade Waldy Cati 30. As duas variedades de acerola mostraram composição similar em termos de antocianinas e a cianidina-3-ramnosídeo foi a antocianina majoritária, seguida da pelargonidina-3-ramnosídeo, da cianidina-3,5-di-ramnosídeo e da peonidina-3-ramnosídeo. O teor de antocianinas totais da variedade Waldy variou de 6,5 a 7,6 mg/100g, enquanto para a variedade Olivier os teores variaram entre 7,9 e 8,4 mg/100g, para as safras de 2003 e 2004, respectivamente. Não foi verificada diferença significativa entre os teores de antocianinas totais das diferentes variedades e entre as duas safras. Dois lotes de polpa de açaí congelado apresentaram teores de antocianinas totais variando entre 282 e 303 mg/100g, apresentando a cianidina-3-glucosídeo e a cianidina-3-rutinosídeo nas proporções médias de 13,2% e 87,5%, respectivamente. A acerola é considerada uma das melhores fontes naturais de ácido ascórbico (AA) e, por esta razão, foi determinada a influência do AA na estabilidade do extrato de antocianinas de acerola utilizando como comparação o extrato de açaí, que não possui AA. Os experimentos demonstraram que o AA presente ao nível de 276 mg /100mL no extrato antociânico de acerola foi o principal responsável pela intensa degradação destas antocianinas, pois a adição de AA à solução de antocianinas de açaí na concentração de 276 mg/100mL provocou um aumento de 22 vezes na velocidade de degradação (kobs) quando comparada ao açaí não fortificado. Embora os sistemas-modelo antociânicos das duas frutas apresentassem a mesma concentração de AA (276 mg /100mL), o valor de kobs encontrado no sistema de acerola foi 3 vezes maior do que o kobs da solução de açaí. Essa diferença pode ter ocorrido devido à concentração 10 vezes maior de flavonóides no extrato antociânico de açaí, uma vez que os teores de polifenóis totais foram semelhantes nos dois extratos. A viabilidade do emprego dos extratos antociânicos de acerola e de açaí como ingrediente funcional foi avaliada em sistema simulador de bebidas isotônicas. Em todas as condições avaliadas, a degradação das antocianinas seguiu cinética de primeira ordem. A estabilidade das antocianinas em sistema simulador de bebida isotônica foi 1,3 e 1,7 vezes menor do que nos sistemas tampão citrato-fosfato para as antocianinas de acerola e de açaí, respectivamente, a pH 2,5, na presença de luz e de oxigênio. Em todos os sistemas, a perda gradual da cor vermelha e aumento da cor amarela foi observada através do incremento do parâmetro CIE L* (luminosidade) e diminuição dos valores de C* (intensidade de cor) que podem ser resultado da degradação do cátium flavilium e formação de chalconas, respectivamente. Utilizando os radicais estáveis ABTS¿+ e DPPH¿, a atividade anti-radical dos extratos de antocianinas de acerola e de açaí com diferentes graus de purificação foi avaliada, e comparada com padrões de antocianinas e de uma antocianidina. Os extratos bruto (EB), parcialmente purificado (EPP) e purificado (EP) de acerola apresentaram maior atividade antioxidante do que os respectivos extratos de açaí. A atividade anti-radical dos extratos brutos das duas frutas diminuiu à medida que o extrato bruto foi sendo purificado e, portanto, a presença de polifenóis, flavonóides e ácido ascórbico no extrato antociânico bruto influenciou na atividade anti-radical. Os resultados obtidos com padrões confirmaram que a atividade anti-radical aumenta com o número de grupos OH no anel B das antocianinas e que a atividade antioxidante diminui à medida que o número de açúcares ligados à molecula aumenta. Foram realizados estudos sobre a reatividade de antocianinas frente ao oxigênio singlete (O2(1?g)), através da avaliação do efeito protetor do EB, EPP e EP das antocianinas de acerola e de açaí sobre o actinômetro dimetil-antraceno, utilizando azul de metileno como sensitizador. A constante de desativação física (kq) foi determinada através dos métodos de foto-oxidação sensitizada e pela detecção da fosforescência do O2(1?g), e o nível de energia do estado triplete das antocianinas de açaí foi avaliado utilizando a técnica de time resolved photoacoustics (TRP). À medida que os extratos foram sendo purificados, a capacidade de desativação do O2(1?g) foi diminuindo. Dentre os padrões avaliados, malvidina, cianidina-3-rutinosídeo e cianidina-3-glucosídeo, a malvidina foi o melhor desativador de O2(1?g), independentemente do método utilizado para cálculo do kq. Os experimentos de TRP demonstraram que o estado triplete das antocianinas de açaí apresentam tempo de vida muito curto, indicando que a desativação do O2(1?g) não depende da formação do estado triplete das antocianinas
Abstract: Natural pigments, such as anthocyanins and carotenoids, are responsible for the colors found in foods, influencing its visual aspect, which is considered the main attribute for the acceptance and preference of a food product. At the same time, these pigments show important biological functions and actions, being considered human health promoters. The association between the intake of fruits and vegetables and the decreased risk of developing several chronic-degenerated disorders is already recognized, being the pigments one of the bioactive compounds responsible for such actions. Acerola and açaí are tropical fruits that contain high levels of bioactive compounds, such as carotenoids and anthocyanins. The pigments, carotenoids and anthocyanins, of two acerola varieties (Waldy Cati 30 and Olivier) harvested from a Brazilian plantation during the 2003 and 2004 summer harvests were analyzed by high performance liquid chromatography (HPLC). Both varieties presented ß-carotene (265.5 ¿ 1669.4 µg/100g), lutein (37.6 ¿ 100.7 µg/100g), ß-cryptoxanthin (16.3 ¿ 56.5 µg/100g) and a-carotene (7.8 ¿ 59.3 µg/100g) as the major carotenoids. In both harvests, the ß-carotene, ß-cryptoxanthin and a-carotene levels were significantly higher in the Olivier variety, whereas the lutein content was higher in the Waldy Cati variety. The two acerola varieties showed similar anthocyanin composition, being cyanidin-3-rhamnoside the major anthocyanin, followed by pelargonidin-3-rhamnoside, cyanidin-3,5-di-rhamnoside and peonidin-3-rhamnoside. The Waldy variety of acerola showed total anthocyanin content of 6.5 and 7.6 mg/100g, whilst 7.9 and 8.4 mg/100g were found in the Olivier variety, for the harvests 2003 and 2004, respectively. No statically differences for the total anthocyanin content were found between varieties and between harvests. Two brand of frozen pulp of açai presented 282 to 303 mg/100 g of total anthocyanin, with cyanidin-3-glucoside and cyanidin-3-rutinoside being found in average proportions of 13.2% and 87.5%, respectively. Acerola is considered one of the best natural source of ascorbic acid (AA) and due to this fact, the influence of AA on the stability of the acerola anthocyanin extract was evaluated using as comparison the açai anthocyanin extract which does not have AA. The experiments demonstrated that the 276 mg/100mL AA level present in the acerola anthocyanin extract is the major responsible for the intense anthocyanin degradation, since the degradation rate (kobs) of the anthocyanins in the açai extract fortified with 276mg/100mL AA, was 22 times higher than the kobs found in the natural açai extract. Although the anthocyanic model-systems from both fruits had the same AA concentration (276 mg /100mL), the kobs value observed in the acerola system was 3 times higher than that found for the açai solutions. This difference can be attributed to the 10 times higher flavonoid content found in the anthocyanin extract from açai, since the total polyphenol levels were similar in the systems from both fruits. The viability to employ the anthocyanin extracts of acerola and açai as functional ingredient was evaluated in isotonic soft drink model-systems. In all conditions studied, the anthocyanin degradation followed a first order kinetics rate. The anthocyanin stability in the isotonic soft drink system was 1.3 e 1.7 times lower compared to the citrate-phosphate buffer for the acerola and açai extracts, respectively, at pH 2.5, in the presence of light and oxygen. In all systems, the gradual fading of the red color was observed by the incresead L* values CIE parameter and decreased C* values, probably resulted respectively from the degradation of the flavillium cation and formation of chalcones. Using the stable radicals ABTS¿+ and DPPH¿, the radical scavenger activities of the acerola and açai anthocyanin extracts with different purification degrees were evaluated and compared to standards of anthocyanins and anthocyanidin. The crude (EB), partially purified (EPP) and purified (EP) extracts of acerola showed higher antioxidant activity than the respective extracts from açai. Since the radical scavenger activity of both crude extracts decreased with the purification process, the presence of polyphenols, flavonoids and AA in the anthocyanic crude extracts affected the antioxidant activity. The results obtained with the standards confirmed that the antioxidant activity increases with the increased number of OH groups in the B ring of the anthocyanins and that the activity decreases with the increased numbers of glycosilated sugars. Studies concerning the reactivity of anthocyanin extracts towards singlet oxygen (O2(1?g)) were carried out using the EB, EPP and EP anthocyanin extracts from acerola and açai, dimethyl-anthracene as actinometer and methylene blue as sensitizer. The physical quenching constant (kq) was determined by the sensitized photooxidation method and by the O2(1?g) phosphorescence detection. The energy level of the excited triplet state of açai anthocyanins was determined by the time resolved photoacoustics (TRP) technique. It was observed a correlation between the increased purification degree and a decrease in the O2(1?g) quenching. Among the standards evaluated, malvidin, cyanidin-3-rutinoside and cyanidin-3-glucoside, the malvidin was the best O2(1?g) quencher, independently of the method used for kq calculation. The experiments with TRP demonstrated that the açai anthocyanin triplet state had a very short life time, indicating that the O2(1?g) quenching does not occur via formation of the anthocyanin excited triplet state
Doutorado
Mestre em Ciência de Alimentos

The diploma thesis deals with the preparation of extract and concentrate of anthocyanin dyes from aronia moldings (Aronia melanocarpa). It also determines chemical properties important for human´s health, which are important also for further possible use of extract/concentrate as natural red food coloring. A series of extractions of dried and powdered aronia moldings was carried out in the experimental part of the thesis, aiming for the optimization the conditions of the extraction. Extracts were characterized by content of total anthocyans, content of total fenolic substances and total antioxidant activity. The best laboratory conditions for extraction were determined as follows: amount of moldings 12,5 g, composition of extraction agent ethanol:water 1:1, volume of extraction mixture 60 ml and time of the extraction 11 hours. Prepared extracts were concentrated on vacuum evaporator. It has been found that the temperature of the water bath in the vacuum evaporator can not exceed 40 °C. At higher temperatures there is significant degradation of anthocyanins. Furthemore, the content of individual anthocyanic dyes in extract and concentrate, from aronia moldings, was quantified by HPLC. The major anthocyanin, cyanidine-3-galactoside, was contained in the concentrate at 923,6 ± 8,5 mg·l-1, the other specified anthocyanin, minor cyanidine-3-glucoside, was present at a concentration of 53,5 ± 0,3 mg·l-1.

O cultivo de plantas das espécies de Vitis (Vitaceae), conhecidas popularmente como videiras, representa uma das atividades econômicas mais proeminentes no Sul do Brasil. Figuram entre as espécies mais ricas em compostos polifenólicos, sendo os mesmos responsáveis por inúmeras atividades biológicas e farmacológicas importantes, dentre as quais, a antioxidante. Neste contexto, este trabalho objetivou analisar quali e quantitativamente os principais grupos polifenólicos encontrados em extratos de videiras, os antocianos e flavonóides, bem como realizar ensaio para avaliação da atividade antioxidante in vitro. Neste sentido foram desenvolvidos e validados dois métodos por cromatografia líquida de alta eficiência, com detector UV/VIS. A análise dos compostos fenólicos incluíram a utilização de duas colunas diferentes, sendo uma constituída por sílica C18 e outra RP-18 com partículas híbridas contendo ligações etano-silicone. Em ambos os métodos foram utilizados sistema de eluição em gradiente, cuja composição das fases móveis foram água e acetonitrila acificadas com ácido trifluoracético. Os métodos desenvolvidos foram validados segundo as normas vigentes e atenderam aos parâmetros de validação como especificidade, linearidade, precisão, sensibilidade, exatidão e robustez nas faixas de concentração avaliadas. O presente estudo também investigou a atividade antioxidante in vitro dos extratos de videira através do método potencial antioxidante reativo total (TRAP), o qual foi otimizado e validado segundo normas internacionais, utilizando Trolox como referência. A partir deste método também foi possível avaliar a reatividade total antioxidante (TAR). Todas as concentrações testadas apresentaram reatividade instantânea inferior ao composto de referência, entretanto, demonstraram atividade antioxidante superior ao Trolox 200 nM.
The cultivation of plants from Vitis species (Vitaceae), popularly known as grapevine, is one of the most prominent economic activities in southern Brazil. The Vitis species is one of the plants that is most rich in polyphenolic compounds, responsible for numerous important pharmacological and biological activities, such as antioxidant activity. In this context, this study aimed to analyze qualitatively and quantitatively the principal groups encountered in grapevine extracts, the anthocyans and flavonoids, and to assay their antioxidant activities in vitro. For this purpose, two methods were developed and validated using high-performance liquid chromatography, employing a UV/VIS detector. The analysis of phenolic compounds included the utilization of two different columns: a column constituted by silica C18 and other RP18 column with bridged ethane-silicon hybrid particles. In both methods, a gradient system was used, with water and acetonitrile acidified with trifluoroacetic acid as a mobile phase. The methods developed were validated according to ICH evaluating parameters such as specificity, linearity, precision, sensibility, accuracy and robustness in the concentration range evaluated. The present study also analyzed the antioxidant activity in vitro of grapevine extracts employing the Total Reactive Antioxidant Potencial (TRAP) method, which was optimized and validated according to international patterns, using Trolox as a reference substance. This method also allowed evaluation of the total instantaneous reactivity (TAR). In all concentrations, the samples presented lower instantaneous reactivity than that of the reference compound. In contrast, the samples presented higher antioxidant activity than Trolox 200 nM.

O consumo de frutas está relacionado à baixa incidência de doenças crônico-degenerativas pela variedade de compostos que possuem a interação destes e seu potencial antioxidante. O processamento a partir dos alimentos in natura é necessário, tanto para preservação quanto para disponibilizar produtos sazonais durante todo o ano, no entanto se devem saber quais suas implicações. O araçá vermelho (Psidium cattleyanum SABINE) e o guabiju (Myrcianthes Pungens) são pequenos frutos nativos do sul do Brasil. Sua composição físicoquímica, perfil de antocianinas, carotenóides e atividade antioxidante, através de dois métodos diferentes (DPPH e ABTS), foram investigados em frutos in natura, bem como o comportamento dos compostos bioativos e da atividade antioxidante em relação à secagem por ar quente a 70 °C, liofilização e congelamento. Foi também realizado um experimento com ratos Wistar para examinar o potencial antioxidante destes frutos contra a toxicidade do antitumoral cisplatina. O guabiju apresentou maior atividade antioxidante, teor de antocianinas e carotenóides do que o araçá vermelho, entretanto este fruto possui elevada quantidade de fibra. No guabiju in natura predominaram o β-caroteno e malvidina 3- glicosídeo, com 40,4 % e aproximadamente 60 % do total de carotenóides e antocianinas, respectivamente. No araçá vermelho in natura o carotenóide e a antocianina predominantes foram a β-criptoxantina, com 44,8 % e a cianidina 3-glicosídeo com 51,7 % do total. Frutos secos a 70 °C e os liofilizados tiveram maior atividade antioxidante pelo método DPPH, enquanto no ABTS o araçá vermelho seco a 70 °C e o guabiju congelado após 90 dias destacaram-se. No entanto, em relação aos compostos bioativos, nos dois frutos, a secagem por ar quente (70 °C) resultou nos menores teores de antocianinas e carotenóides. O congelamento por 30 e 90 dias aumentou as concentrações de carotenóides no araçá vermelho e guabiju, respectivamente. Para as antocianinas, os maiores teores foram encontrados no guabiju liofilizado e no araçá vermelho in natura. Em relação ao estudo com animais, os frutos nativos atuaram protegendo os animais contra os efeitos produzidos pela cisplatina, diminuindo os níveis de colesterol total (CT), LDL, LDL oxidado (LDL-ox) e os níveis de gordura no fígado.
The fruit consumption is related to the low incidence of chronic diseases by a variety of compounds that have the interaction of these and their antioxidant potential. The processing from fresh food is necessary, both for preservation and to provide seasonal products all year round, however you must know what its implications. The red guava (Psidium cattleyanum SABINE) and guabiju (Myrcianthes pungens) are small native fruits southern Brazil. Its physical and chemical composition profile of anthocyanins, carotenoids and antioxidant activity, using two different methods (DPPH and ABTS), were investigated in fresh fruit, as well as the behavior of bioactive compounds and antioxidant activity in respect to air drying warm to 70 °C, freeze-drying and freezing. It was also carried out an experiment with rats to examine the antioxidant potential of fruit against the toxicity of antitumor cisplatin. The guabiju showed higher antioxidant activity, anthocyanins and carotenoids content than the red guava, but this fruit has high fiber content. In fresh guabiju β-carotene and malvidin 3- glucoside were predominant, with 40.4 % and approximately 60 % of total carotenoids and anthocyanins, respectively. In the fresh red guava predominant carotenoid and anthocyanin were β-cryptoxanthin, with 44.8 % and cyanidin 3-glucoside with hydrochloric 51.71 % of the total. Fruits dried at 70 °C and freeze- dried had higher antioxidant activity by DPPH method, while in the ABTS red guava dried at 70 °C and frozen after 90 days guabiju stood out. However, for bioactive compounds in the two fruits, hot air drying (70 °C) resulted in lower levels of anthocyanins and carotenoids. Freeze for 30 and 90 days increased the concentrations of carotenoids in red guava and guabiju, respectively. For anthocyanins, the highest levels were found in guabiju freeze dried and fresh red guava. Regarding the study of animals, fruits native acted protecting animals against the effects of cisplatin, reducing the levels of total cholestreol (TC), LDL and oxidized LDL (ox-LDL) levels of fat in the liver.

In this thesis was determinated selected chemical and nutritional parameters in the wild elderberry and 17 cultivated varieties of elderberry. The fruits was provided by the Research and Breeding Institute of Pomology Holovousy Ltd. The theoretical part deals describes elderberry, chemical composition of elderberry, especially biological active substances and its using not only food industry. Closer attention has been focused on effect of saccharides, anthocyanins and methods of their determination, specially determination by HPLC. The experimental part describes the various methods of determination of selected chemical and nutritional parameters. Total dry matter, content of total polyphenolic compounds, monomeric anthocyanin pigment and antioxidant activity was defined in all varieties of elderberry fruits. For some varieties was defined total soluble dry matter, content of organic acids and saccharides by various methods. In the end was compared the values from different methods varieties.

Orientador: Adriana Zerlotti Mercadante
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: Considerando a importancia de carotenoides e antocianinas, tanto como pigmentos naturais como pelas suas propriedades antioxidantes, este trabalho foi desenvolvido com o objetivo de avaliar, por meio de cromatografia liquida de alta eficiencia acoplada aos detectores arranjo de diodos e espectrometro de massas (HPLC-DAD-MS/MS), a composicao de carotenoides e antocianinas em nespera, jaca e jambolao, bem como estudar a atividade antioxidante apresentada por esses pigmentos em diferentes sistemas. Em funcao da coloracao desses frutos, apenas o jambolao foi submetido a analise de antocianinas. Cinco cultivares brasileiros de nespera foram avaliados: Centenaria, Mizauto, Mizuho, Mizumo e Nectar de Cristal. De vinte e cinco carotenoides separados, vinte e tres foram identificados, sendo que os principais foram all-trans-?-caroteno (19-55 %), alltrans-?-criptoxantina (18-28 %), 5,6:5¿,6¿-diepoxi-?-criptoxantina (9-18 %) e 5,6- epoxi-?-criptoxantina (7-10 %). Os conteudos de carotenoides totais variaram entre 196 ?g/100 g (cv. Nectar de Cristal) e 3020 ?g/100 g (cv. Mizumo). Os cultivares Mizauto, Mizuho, Mizumo e Centenaria apresentaram valores de provitamina A entre 89 e 162 ?g RAE/100 g e podem ser considerados uma boa fonte desta pro-vitamina. Nos tres lotes de jaca analisados, quatorze dos dezoito carotenoides identificados foram relatados pela primeira vez, sendo que os principais foram all-trans-luteina (24-44 %), all-trans-?-caroteno (24-30 %), alltrans-neoxantina (4-19 %), 9-cis-neoxantina (4-9 %) e 9-cis-violaxantina (4-10 %). O lote A apresentou o menor conteudo de carotenoides totais (34,1 ?g/100 g) e o menor valor de pro-vitamina A (0,8 ?g RAE/100 g), enquanto nos lotes B e C, os conteudos de carotenoides totais foram de 129,0 e 150,3 ?g/100 g e os valores de pro-vitamina A foram 3,3 e 4,3 ?g RAE/100 g, respectivamente. Os frutos de jambolao apresentaram dois carotenoides principais: all-trans-luteina (43,7 %) e all-trans-?-caroteno (25,4 %). A composicao de antocianinas foi marcada pela presenca de diglucosideos de cinco das seis agliconas comumente encontradas em alimentos, sendo que as principais antocianinas foram delfinidina 3,5- diglucosideo (45 %), petunidina 3,5-diglucosideo (32 %) e malvidina 3,5- diglucosideo (15 %). Esse padrao tambem foi observado para outros flavonoides, onde os principais compostos identificados foram diglucosideos de diidromirecetina, metil-diidromirecetina e dimetil-diidromirecetina, alem de mirecetina glucosideo e um ester de galoil-glucose. A atividade antirradical ABTS?+ (2,2¿-azinobis-(3-etilbenzotiazolina-6-acido sulfonico)) do extrato funcional rico em antocianinas, obtido de frutos de jambolao, foi dependente do pH do meio, com valores de TEAC (capacidade antioxidante equivalente a Trolox) entre 4,8 ?mol Trolox/g fruta (pH 1,0) e 12,7 ?mol Trolox/g fruta (pH 5,0). Esse extrato funcional apresentou cerca de 60 % de protecao ao dimetilantraceno frente a oxidacao por oxigenio singlete, em condicoes de pH 1,0 e 3,0, sendo essa atividade superior aquela apresentada por outras frutas vermelhas. Padrao de cianidina 3-glucosideo foi incorporado em lipossomas unilamelares grandes (LUV) preparados em duas condicoes diferentes de pH (3,1 e 7,4), sendo esses sistemas submetidos a oxidacao induzida por AAPH (diidrocloreto de ?,?¿-azodiisobutiramidina) a 37 °C. Observou-se que as formas de cianidina 3-glucosideo presentes no meio com pH 7,4 (hemiacetais e/ou chalconas e bases quinonoidais), com EC50 = 9,3 ???1,3 ?mol/L (concentracao de antioxidante necessaria para obter 50 % de protecao), apresentaram uma atividade 2,6 vezes maior que as formas presentes no meio com pH 3,1 (maior proporcao de cation flavilium e hemiacetais), cujo EC50 foi de 23,6 ???0,9 ?mol/L. Por fim, padroes de carotenoides, trolox e tocoferol foram microencapsulados com dois materiais de parede diferentes, goma arabica (GA) e maltodextrina (MD), por meio da tecnica de spray-drying. Quando comparada a MD, a parede de GA formou dominios hidrofobicos mais rigidos e compactos, melhorando a solubilizacao de moleculas apolares e reduzindo a acessibilidade de moleculas de oxigenio. Devido a efeitos de compartimentalizacao das moleculas de antioxidante nas microcapsulas (MC), as constantes de desativacao total (kQ) de oxigenio singlete (1O2) foram cerca de duas ordens de magnitude menores nas solucoes de MC, quando comparadas aos valores de kQ em meio homogeneo
Abstract: Considering the importance of carotenoids and anthocyanins, as both natural pigments and antioxidants, the present study was carried out to evaluate, by high-performance liquid chromatography connected to a photodiode array and a mass spectrometry detector (HPLC-DAD-MS/MS) the composition of carotenoids and anthocyanins from loquat, jackfruit and jambolao fruits, as well as to study the antioxidant activity showed by these pigments in different systems. Due to the colour of these fruits, only jambolao was submitted to anthocyanin analysis. Five loquat cultivars from Brazil were evaluated: Centenaria, Mizauto, Mizuho, Mizumo and Nectar de Cristal. Twenty five carotenoids were separated, and twenty three of them were identified. The main carotenoids were all-trans-?-carotene (19-55 %), all-trans-?-cryptoxanthin (18-28 %), 5,6:5¿,6¿-diepoxy-?-cryptoxanthin (9-18 %) and 5,6-epoxy-?-cryptoxanthin (7-10 %). The total carotenoid contents ranged from 196 ?g/100 g (cv. Nectar de Cristal) to 3020 ?g/100 g (cv. Mizumo). Cultivars Mizauto, Mizuho, Mizumo and Centenaria showed provitamin A values between 89 and 162 ?g RAE/100 g, and can be considered good source of this provitamin. In the three analyzed batches of jackfruit, fourteen of the eighteen identified carotenoids were reported for the first time in this fruit. The major carotenoids were all-trans-lutein (24-44 %), all-trans-?-carotene (24-30 %), all-trans-neoxanthin (4-19 %), 9-cisneoxanthin (4-9 %) and 9-cis-violaxanthin (4-10 %). Batch A showed the lowest total carotenoid content (34.1 ?g/100 g) and provitamin A value (0.8 ?g RAE/100 g), whereas for batches B and C, respectively, the total carotenoid contents were 129.0 and 150.3 ?g/100 g and the provitamin A values were 3.3 and 4.3 ?g RAE/100 g. The jambolao fruits showed two main carotenoids: all-trans-lutein (43.7 %) and all-trans-?-carotene (25.4 %). The anthocyanin composition was marked by the presence of diglucosides of five among the six aglycones that are commonly found in foods. The major anthocyanins were delphinidin 3,5-diglucoside (45 %), petunidin 3,5-diglucoside (32 %) and malvidin 3,5-diglucoside (15 %). This pattern was also observed for other flavonoids, where the main identified compounds were diglucosides of dihydromyricetin, methyl-dihydromyricetin, and dimethyldihydromyricetin, in addition to myrecetin glucoside and a galoyl-glucose ester. The scavenging capacity of ABTS?+ (2,2'-azinobis(3-ethylbenzthiazoline-6-sulphonic acid)) showed by the anthocyanin-rich functional extract from jambolao fruits, was dependent on the medium pH, showing TEAC (Trolox equivalent antioxidant capacity) values between 4.8 ?mol Trolox/g fruit (pH 1.0) and 12.7 ?mol Trolox/g fruit (pH 5.0). This functional extract showed about 60 % of protection on dimethylantracene oxidation by singlet oxygen, in pH conditions of 1.0 and 3.0, being this activity higher than those showed by other red fruits. Cyanidin 3-glucoside standard was incorporated to large unilamelar liposomes (LUV) prepared at two different pH conditions (3.1 and 7.4). These systems were submitted to oxidation induced by AAPH (?,?¿-azodiisobutyramidine dihydrochloride) at 37 °C. The cyanidin 3-glucoside forms present in the medium at pH 7.4 (hemiacetals and/or chalcones and quinonoidal bases), with EC50 = 9.3 ???1.3 ?mol/L (antioxidant concentration required for 50 % protection), showed na activity 2.6 times higher than that showed by the forms present at pH 3.1 (higher proportion of flavylium cation and hemiacetals), whose EC50 was 23.6 ???0.9 ?mol/L. Finally, carotenoids, trolox and tocopherol standards were microencapsulated with two different wall materials, gum arabic (GA) and maltodextrin (MD), by the spraydrying technique. GA wall formed more rigid and compacted hydrophobic domains than those in the MD microcapsules, improving the solubilization of apolar molecules and reducing the accessibility of oxygen molecules. Due to compartmentalization effects of antioxidant molecules in the microcapsules (MC), the quenching rate constants (kQ) of singlet oxygen (1O2) were reduced almost twoorders of magnitude in MC solutions as compared with those observed in homogenous media. was also observed for other flavonoids, where the main identified compounds were diglucosides of dihydromyricetin, methyl-dihydromyricetin, and dimethyldihydromyricetin, in addition to myrecetin glucoside and a galoyl-glucose ester. The scavenging capacity of ABTS?+ (2,2'-azinobis(3-ethylbenzthiazoline-6-sulphonic acid)) showed by the anthocyanin-rich functional extract from jambolao fruits, was dependent on the medium pH, showing TEAC (Trolox equivalent antioxidant capacity) values between 4.8 ?mol Trolox/g fruit (pH 1.0) and 12.7 ?mol Trolox/g fruit (pH 5.0). This functional extract showed about 60 % of protection on dimethylantracene oxidation by singlet oxygen, in pH conditions of 1.0 and 3.0, being this activity higher than those showed by other red fruits. Cyanidin 3-glucoside standard was incorporated to large unilamelar liposomes (LUV) prepared at two different pH conditions (3.1 and 7.4). These systems were submitted to oxidation induced by AAPH (?,?¿-azodiisobutyramidine dihydrochloride) at 37 °C. The cyanidin 3-glucoside forms present in the medium at pH 7.4 (hemiacetals and/or chalcones and quinonoidal bases), with EC50 = 9.3 ???1.3 ?mol/L (antioxidant concentration required for 50 % protection), showed an activity 2.6 times higher than that showed by the forms present at pH 3.1 (higher proportion of flavylium cation and hemiacetals), whose EC50 was 23.6 ???0.9 ?mol/L. Finally, carotenoids, trolox and tocopherol standards were microencapsulated with two different wall materials, gum arabic (GA) and maltodextrin (MD), by the spraydrying technique. GA wall formed more rigid and compacted hydrophobic domains than those in the MD microcapsules, improving the solubilization of apolar molecules and reducing the accessibility of oxygen molecules. Due to compartmentalization effects of antioxidant molecules in the microcapsules (MC), the quenching rate constants (kQ) of singlet oxygen (1O2) were reduced almost twoorders of magnitude in MC solutions as compared with those observed in homogenous media
Doutorado
Doutor em Ciência de Alimentos

Il frumento duro [Triticum turgidum (L.) subsp. turgidum (L.) convar. durum (Desf.)] è una coltura di largo uso per la nutrizione umana, in particolare nel bacino del Mediterraneo. Costituisce la principale fonte di semola utilizzata per la produzione di pasta, pane di frumento duro, ma viene utilizzato anche per la produzione di cuscus e burghul. Condizioni ambientali avverse riducono la produzione e influenzano le caratteristiche qualitative della granella. Gli stress che più comunemente colpiscono le piante coltivate consistono in un eccesso o carenza di acqua, eccesso di sale o metalli pesanti nel terreno, e temperature estreme. In particolare dall’inizio del secolo si sta assistendo ad un incremento della temperatura media mondiale, ed un ulteriore aumento è previsto per i prossimi decenni. Alte temperature durante la fase riproduttiva del frumento possono determinare una riduzione del peso della granella e del numero di semi per spiga. Queste espressioni fenotipiche sono il risultato dell’alterazione del profilo dei trascritti e di variazione dei livelli di accumulo dei metaboliti nelle piante, in risposta ai cambiamenti di temperatura. Lo scopo di questa ricerca è di studiare le variazioni dei trascritti e dei metaboliti in risposta a stress termico, in due varietà di frumento duro, e di comprendere come tali variazioni possano influenzare la composizione della granella e quindi la qualità nutrizionale e nutraceutica del prodotto finale acquistato dai consumatori. Lo studio è stato condotto sulla varietà di frumento duro Primadur e su una cultivar non commercializzata T1303 (codice USDA PI 352395); la prima è una cultivar élite caratterizzata da alta resa produttiva e un alto indice di giallo (altamente correlato al contenuto di pigmenti carotenoidi), la seconda è una cultivar caratterizzata da un alto contenuto di antociani che conferiscono alla granella un colore rosso (purple-cultivar). Lo shock termico è stato imposto a 37°C, durante il periodo di riempimento della granella. La temperatura elevata è stata imposta per 5 giorni in un periodo che va dal quinto al decimo giorno dopo la fioritura. Le analisi sono state condotte su seme immaturo, prelevato 14 giorni dopo la fioritura, e seme maturo, prelevato alla fine del ciclo riproduttivo della pianta. I risultati ottenuti da seme derivante da piante sottoposte a shock termico sono stato confrontati con quelli ottenuti da piante controllo (20°C). La risposta allo stress è stata valutata con due differenti approcci. L’analisi dell’espressione genica, condotta con un approccio di sequenziamento genome–wide di nuova generazione che ha utilizzato la tecnologia Illumina, ha mostrato che un totale di 1202 geni hanno subito variazioni dei livelli di trascritto in risposta a stress. Molti dei geni sovraespressi in risposta a stress in Primadur ricadevano nelle categorie geniche note per avere un ruolo in risposta a stress, mentre tali categorie erano meno rappresentate in T1303 in cui erano sovraespressi geni codificanti per proteine di 3 riserva. Tali risultati erano in accordo con la maggiore sensibilità allo stress termico mostrata da Primadur rispetto a T1303, valutata come calo in peso della granella. L'accumulo dei metaboliti è stato valutato con gas-cromatografia associata a spettromeria di massa (GS-MS) in seme immaturo e maturo. I due genotipi hanno mostrato differente risposta allo stress termico. Il contenuto di molti dei metaboliti analizzati è risultato incrementato in ‘Primadur’, mentre un decremento è stato osservato in T1303; tuttavia per alcuni metaboliti con un ruolo chiave di risposta alle condizioni di stress (per esempio saccarosio e glicerolo) è stato osservato un incremento del contenuto per entrambi i genotipi. Per alcuni metaboliti le variazioni sono state osservate già nel seme immaturo e le stesse variazioni sono state conservate anche nel seme maturo. Questo studio ha evidenziato come un incremento di temperatura, della durata di pochi giorni nelle fasi iniziali di riempimento della granella, sia in grado di determinare cambiamenti a breve e lungo termine in grado di cambiare qualità e produzione della granella di frumento duro. In risposta a stress, il contenuto di antociani è risultato incrementato mentre quello dei carotenoidi non è variato. Solo lo sfarinato della granella di T1303, maturata dopo l’imposizione dello stress, ha mostrato un incremento della capacità antiossidante (misurata come ‘Trolox Equivalent Antioxidant Capacity’-TEAC) rispetto allo sfarinato della granella maturata in condizioni controllo. Il contenuto proteico è risultato incrementato solo in ‘T1303’, che ha mostrato un minore calo in peso della granella rispetto a Primadur, in risposta allo stress. Questo studio fornisce nuove conoscenze sugli effetti dell'adattamento allo stress termico del frumento duro sulle proprietà nutrizionali e qualitative della granella e mostra come tali proprietà possano essere differentemente alterate in due genotipi. Anche se il ‘background’ genetico dei due genotipi è differente, non può essere escluso che alcune delle differenze osservate, nella risposta a stress termico, tra i due genotipi possano essere legate al metabolismo degli antociani. L’incremento del loro accumulo in risposta a stress termico insieme al ridotto calo in peso della granella sono considerati tratti di interesse per le future attività di miglioramento genetico del frumento duro.
Durum wheat [Triticum turgidum (L.) subsp. turgidum (L.) convar. durum (Desf.)] is an important crop for human nutrition, especially in the Mediterranean area, where it is the main source of semolina for the production of pasta, traditional/typical bread, couscous, and burghul. Environmental stress can strongly limit the yield potential and affect qualitative characteristics of grain. The stress conditions that are commonly experienced by crops are extreme lack or excess of water (i.e., drought, flooding), presence of salt or contaminants (e.g., heavy metals), and temperature (i.e., cold, heat). In particular, environmental temperatures have increased since the beginning of the last century and they are predicted to further increase under the present conditions of climate change. Wheat is very sensitive to high temperatures during the reproductive phase, due to direct effects of temperature on grain numbers and dry weight. These phenotypic effects are due to molecular modifications at the different levels of gene expression, and to changes in metabolite accumulation levels. The aim of this study was to investigate the transcriptomic and metabolomic variations in response to heat stress in grains of two durum wheat genotypes, and their relationship with variation in grain composition that can affect the nutraceutical quality of the final product. The study was carried out on the durum wheat cv Primadur and T1303 (PI 352395 USDA code); the first is an elite cultivar with high grain yield and yellow index and the second one an anthocyanin-rich purple cultivar. Heat shock was imposed at 37°C from 5 up to 10 days after flowering (DAF). Immature seeds (14 DAF) and mature seeds (seeds at physiological maturity stage), collected from heat shocked and control plants (20°C), were analyzed. The response to stress was investigated with different approaches. Genome–wide gene expression analysis, carried out using Illumina HiSeq2000, showed that 1202 genes were differentially expressed in response to stress in the two genotypes. The number of up-regulated genes belonging to stress-related categories was higher in Primadur with respect to T1303, whereas the number of genes with nutrient reservoir activity was larger in T1303 compared to Primadur. These results were according to the higher sensitivity to stress of Primadur with respect to T1303, measured as grain weight loss. The profile of polar metabolites was investigated by gas chromatography-mass spectrometry (GC-MS). The effects of heat stress were genotype dependent. Although some metabolites (e.g., sucrose, glycerol) increased in response to heat stress in both genotypes, clear differences were observed. Following the heat stress, there was a general increase in most of the analyzed metabolites in Primadur, with a general decrease in T1303. Heat shock applied early during seed development produced changes that were observed in immature seeds, and also long-term effects 5 that changed the qualitative and quantitative parameters of the mature grain. The level of anthocyanins increased significantly in response to the heat stress whereas the levels of carotenoids were not affected. Only for T1303, the Trolox equivalent antioxidant capacity (TEAC) method assessed an increased antioxidant capacity of ground grains, in response to heat stress. Protein content increased only in T1303 that showed lower grain weight loss with respect to Primadur, in response to stress. Therefore, short heat-stress treatments can affect the nutritional and nutraceutical value of grain of different genotypes of durum-wheat in different ways. The present study increased the knowledge about the effect of mechanisms of adaptation to stress of wheat plants on nutritional and quality traits of grain and provides a global picture of the effects of the heat stress on metabolies and transcriptome of two different genotypes of durum wheat. Moreover, although the genetic backgrounds of two durum-wheat genotypes were different, it cannot be excluded that some of the differences observed in the responses to heat stress can be due to anthocyanin accumulation, which can be considered a trait of interest for future breeding activities for durum wheat.

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Strawberry crop (Fragaria x ananassa Duchesne) is grown in all continents, and this fruit is very popular due of its color, scent and flavor. The aim of this work was to evaluate the adaptation, yield and quality of different strawberry cultivars under the climatic conditions of Pelotas, Rio Grande do Sul state. The experiments were conducted at Embrapa Temperate Climate, Pelotas-RS from 2011 to 2012. At first, we evaluated fruit, yield, number and average fruit size of the cultivars Albion, Aromas, Camarosa, Camino Real, Portola, Monterey, San Andreas, Strawberry Festival and Palomar. The second test was conducted at the Laboratory of Food Technology, and it evaluated physical and chemical characteristics (soluble solids contents, titratable acidity, soluble solids/titratable acidity, pH, and vitamin C) and quantified the bioactive compounds (phenolic compounds, anthocyanins and antioxidant activity) of the cultivars in three harvest dates: September, October and November. The cultivars showed significant differences for date and for comparison between cultivars in the physical and chemical characteristics. Phenolic compounds and antioxidant activity were higher in September, while anthocyanins was higherin November. The third test was conducted at the Laboratory of Postharvest Physiology. We observed the behavior of cultivars under the cold storage for up to eight days, using three replicates of five fruits in a factorial scheme 8x3 (eight cultivars x three dates). The variables studied were soluble solids content, titratable acidity, soluble solids/acidity, pH, color, luminosity, mass rot and decay incidence. During this period, the cultivars maintained the quality and there was no significant loss of mass, degradation of the compounds or symptoms of illness or unpleasant odor.
O morangueiro (Fragaria x ananassa Duchesne) é cultivado em todos os continentes, e sua fruta, o morango, é muito popular devido sua coloração, aroma e sabor. Objetivou-se neste trabalho avaliar a adaptação, produção e qualidade de diferentes cultivares de morangueiro às condições edafoclimáticas de Pelotas-RS. Os ensaios foram realizados na Embrapa Clima Temperado, durante as safras de 2011/2012 e 2012/2013. No primeiro ensaio avaliou-se a produção, produtividade, número e tamanho médio de frutas das cultivares Albion, Aromas, Camarosa, Camino Real, Portola, Monterey, San Andreas, Strawberry Festival e Palomar. O delineamento experimental foi em blocos casualizados. As cvs. Strawberry Festival, Camarosa e Monterey proporcionaram a maior produtividade na safra 2011/2012, enquanto que a cultivar que se destacou na safra 2012/2013 foi Camarosa, sendo o pico de produção de todas as cultivares no mês de novembro. O segundo ensaio foi realizado no Laboratório de Tecnologia de Alimentos na safra 2012/2013. Avaliaram-se os atributos físicos e químicos (sólidos solúveis, acidez titulável, relação sólidos solúveis/acidez titulável, pH, teor de vitamina C) e quantificaram-se os compostos bioativos das cultivares (compostos fenólicos, antocianinas e atividade antioxidante) em três datas de colheita: setembro, outubro e novembro. As cultivares apresentaram diferenças significativas tanto para data, quanto para a comparação entre as cultivares nas características físicas e químicas. Os compostos fenólicos e a atividade antioxidante foram maiores no mês de setembro, enquanto que as antocianinas em novembro. O terceiro ensaio foi realizado no Laboratório de Fisiologia Pós-colheita. Utilizaram-se três repetições de cinco frutas, constituindo um esquema fatorial de 8x3 (oito cultivares x três datas). Observou-se o comportamento das cultivares diante do armazenamento em câmara fria por zero, quatro e oito dias, à temperatura de 1±0,5ºC e UR 90-95%. As variáveis estudadas foram sólidos solúveis, acidez titulável, relação sólidos solúveis/acidez, pH, coloração e luminosidade da epiderme, perda de massa e incidência de podridões. No período avaliado, as cultivares mantiveram a qualidade pois não houve perdas significativas de massa, degradação de compostos ou presença de sintomas de doenças ou odor desagradável.

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.

Presented diploma thesis is dedicated to the determination of selected chemical parameters of fruits of new currant varieties. The theoretical part is focused on the characteristics of currant, the biologically active substances of its fruits and also the utilization of current in the food industry. Further part of the theory deals with the main chemical parameters which were examined: polyphenols, anthocyanins, ascorbic acid and antioxidant activity. In the practical part there were determined selected chemical parameters in six white, twelve red and thirteen black varieties of currant. Anthocyanins and polyphenols have been determined spectrophotometrically. The content of ascorbic acid in selected varieties of currant was determined by HPLC. As a technique for the determinationof the antioxidant activity was selected the method of free radical DPPH.

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Large amounts of wine by-products are produced as a result of processing grapes in Brazil. Considering the presence of bioactive pigments in this material, the aim of this work was to investigate the antioxidant potential and stability of anthocyanins from the grape juice pomace. The grape residue (Vitis labrusca, cv. Isabella) was ceded by an industry producing of pure grape juice, located in the region São Francisco Valley, in the city of Petrolina-PE. The anthocyanin content was determined using pH-differential method. The isolated anthocyanin were identified and quantified by chromatography analysis using a RP-HPLC-DAD system. The antioxidant activity of the purified anthocyanins were evaluated for their ability to scavenge free radicals DPPH• (1,1-diphenyl-2-picrylhydrazyl) and ABTS•+ [2,2'-azino-bis (3-ethylbenzthiazoline-6sulfonic acid)], using gallic acid as standard. Purified anthocyanins were diluted in ethanol solution 95%: HCl 1.5N; 85:15 v/v (pH = 1.0) to evaluate the light stability, and distributed into test tubes, exposed and not exposed to light for a period of 840 hours (35 days) at 21°C ± 1°C, monitored by spectrophotometer readings. The effect of temperature on the stability of anthocyanins was evaluated using test tubes containing purified anthocyanins diluted into water, acidified with HCl (pH = 1.0), and subjected to temperatures of 50°C, 70°C and 90°C in thermostatic bath. Absorbance readings were made in the control sample (0h), and after 1, 3, 5 and 7 hours of heating. The absorbance data were used to calculate the rate constant (k) and half-life (t1/2) of anthocyanins through first-order kinetics equations. The average content of anthocyanins was 31.66 ± 0.63 mg. 100g-1 residue (expressed as malvidin-3-glucoside). A total of six majority different anthocyanins were identified: cyanidin-3-O-glucoside, delphinidin-3-O-glucoside, malvidin-3-O-glucoside, malvidin-3,5-O-diglucoside, pelargonidin-3-O-glucoside and peonidin-3-O-glucoside, with different concentrations. Malvidin 3-glucoside was the most abundant (61.66%) of the total observed, while malvidin-3,5-O-diglucoside (14.88%) was the second. The anthocyanin fraction showed high antioxidant potential against the radical DPPH• (EC50: 0.26 g. g DPPH•-1; TEC50: 1.37 minutes; AE: 2.79). After 5 minutes of reaction, at concentrations of 5-40 μg. mL-1, showed variations in the percentage scavenging between 48 and 94%, respectively, though there is a high correlation between anthocyanin content and antioxidant capacity (R = 0.93). Against the radical ABTS•+ anthocyanins exhibited significant action in capturing the radical with value of 199.35 uM Trolox. g-1. The half-life (t1/2) of the anthocyanin pigments, in the absence of light, was 3.320h (138 days) and in the presence of light was 505h (21 days), with the follows rate constants (k): 2.09 x 10-4 h-1 e 1.37 x 10-3 h-1, respectively, showing that presence of light contributed significantly to the instability of anthocyanins. In the study of heat stability, the rate constants (k) were: 1.84 x 10-2 h-1 (50°C); 7.82 x 10-2 h-1 (70°C); and 1.99 x 10-1 h-1 (90°C). The half-life of the samples occurred after 3.5h at 90°C and would occur after 8.9h and 37.7h for the samples at 70°C and 50°C, respectively, indicating the higher instability of anthocyanins to high temperatures. Anthocyanins from cv. Isabella grape juice residue are a promising source of natural dye which has antioxidant action with potential application in food products with an acidic pH, packaged in opaque packaging, and not subjected to severe heat treatment during processing.
Elevada quantidade de subprodutos vitivinícolas é produzida em decorrência do processamento de uvas no Brasil. Considerando a presença de pigmentos bioativos em sua composição, objetivou-se neste trabalho investigar o potencial antioxidante e a estabilidade das antocianinas do resíduo agroindustrial de suco de uva. O resíduo de uva (Vitis labrusca, cv. Isabel) foi cedido por uma indústria produtora de suco de uva integral, situada na região do Vale do São Francisco, Petrolina-PE. O teor de antocianinas totais foi determinado utilizando metodologia de pH diferencial. Para identificação e quantificação das antocianinas individuais, análises cromatográficas foram efetuadas por CLAE-DAD em fase reversa. A atividade antioxidante das antocianinas foi avaliada quanto à capacidade de sequestro dos radicais DPPH• (1,1-difenil-2-picrilhidrazil) e ABTS•+ (2,2’-azino-bis-3-etilbenzotiazolina-6-acido sulfônico), tendo o ácido gálico como composto de referência. Para avaliar a estabilidade à luz, as antocianinas foram diluídas em uma solução de etanol 95%: HCl 1,5N; 85:15 v/v (pH = 1,0) e distribuídas em tubos de ensaio, expostos à luz e ao abrigo da luz, a 21°C ± 1°C, durante um período de 840 horas (35 dias), monitorados por leituras em espectrofotômetro. O efeito da temperatura sobre a estabilidade das antocianinas foi avaliado utilizando tubos de ensaio contendo antocianinas diluídas em água acidificada com HCl (pH = 1,0) e submetidos a temperaturas de 50ºC, 70ºC e 90ºC, em banho termostatizado. Leituras de absorbância foram realizadas na amostra recém-preparada (0h) e após 1, 3, 5 e 7 horas de aquecimento. Os dados de absorbância foram utilizados para o cálculo da constante de velocidade (k) e do tempo de meia-vida (t1/2) das antocianinas por meio de equações cinéticas de 1ª ordem. O teor médio de antocianinas totais foi de 31,66 mg. 100g–1 de resíduo (expresso em malvidina-3-glicosídeo). Seis antocianinas majoritárias foram identificadas: cianidina-3-O-glicosídeo; delfinidina-3-O-glicosídeo; malvidina-3-O-glicosídeo; malvidina-3,5-O-glicosídeo; pelargonidina-3-O-glicosídeo; e peonidina-3-O-glicosídeo, em diferentes concentrações. Malvidina-3-O-glicosídeo foi a mais abundante (61,66%) seguida da malvidina-3,5-O-diglicosídeo (14,88%). A fração antociânica demonstrou elevado potencial antioxidante frente ao radical DPPH• (EC50: 0,26 g. g DPPH•-1; TEC50: 1,37 minutos; EA: 2,79). Aos 5 minutos de reação, nas concentrações de 5 a 40 μg. mL–1, apresentou variações no percentual de sequestro entre 48 e 94%, respectivamente, constatando uma alta correlação entre o teor de antocianinas e a capacidade antioxidante (R = 0,93). Frente ao radical ABTS•+ as antocianinas exibiram significativa ação na captura do radical com valor de 199,35 μM Trolox. g-1. Na ausência de luz, o tempo de meia vida dos pigmentos antociânicos foi de 3.320h (138 dias), e na presença de luz foi de 505h (21 dias), apresentando constantes de velocidade de 2,09 x 10-4 h-1 e 1,37 x 10-3 h-1, respectivamente, demonstrando que a luminosidade contribuiu para a instabilidade das antocianinas. No estudo de estabilidade ao calor, as constantes de velocidade foram: 1,84 x 10-2 h-1 (50°C); 7,82 x 10-2 h-1 (70°C); e 1,99 x 10-1 h-1 (90°C). O tempo de meia-vida ocorreu após 3,5h a temperatura de 90°C, e ocorreria após 8,9h e 37,7h, a 70°C e 50°C, respectivamente, constatando a maior instabilidade das antocianinas à altas temperaturas. As antocianinas do resíduo de suco de uva cv. Isabel são uma fonte promissora de corante natural que possui ação antioxidante, com potencial aplicação em produtos alimentícios que tenham pH ácido, sejam acondicionados em embalagens opacas, e não sejam submetidos à tratamentos térmicos severos durante o seu processamento.

Orientadores: Helena Teixeira Godoy, Elaine Conceição de Oliveira
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: A groselha do Ceilão (Dovyalis hebecarpa) é uma fruta exótica de coloração roxa quando madura. Não foram encontrados trabalhos que identifiquem os principais compostos fenólicos na espécie, ação antioxidante e efeitos biológicos. Sendo assim, além da otimização da extração, esse trabalho avaliou os principais compostos fenólicos na casca e polpa de D. hebecarpa por cromatografia líquida acoplada a espectrômetro de massas (CLAE-DAD-EM), o potencial antioxidante in vitro e o efeito sobre a resposta imunológica em camundongos. Com exceção da razão entre amostra e solvente, avaliada univariadamente, a otimização das variáveis de extração foi realizada utilizando planejamento experimental multivariado. O processo otimizado foi realizado em menor tempo (20 minutos), com menos solvente orgânico (20% de acetona) e com maior rendimento (aumento de 10% no teor total de compostos fenólicos e de 26% no teor total de antocianinas monoméricas) do que a metodologia inicial. A caracterização do perfil de antocianinas revelou que D. hebecarpa é fonte de compostos não acilados, sendo delfinidina-3-O-rutinosídeo e cianidina-3-O-rutinosídeo os majoritários. Casca e polpa possuem composição similar, no entanto, maiores concentrações são encontradas na parte externa do fruto. Amostras de dois anos consecutivos e em duas datas de amostragem foram recolhidas para avaliação de variações entre estações do ano. Considerando que a ação antioxidante se dá por diversos mecanismos, metodologias de FRAP, ABTS e ORAC foram empregadas. Os maiores resultados de atividade antioxidante foram obtidos com a metodologia de ORAC, indicando marcada atividade sequestradora de radicais peroxil dos compostos extraídos. De forma diferenciada para polpa e casca dos frutos, efeitos significativos sob os teores de antocianinas, de compostos fenólicos e capacidade antioxidante, foram observados devido à variação climática entre as datas. Adicionalmente foram avaliados parâmetros biométricos e composição nutricional. Alterações no peso e tamanho dos frutos (p<0,05), assim como, na composição nutricional (p<0,05) foram observadas. Esses resultados, possivelmente, devem-se as diferenças de disponibilidade de água e incidência solar entre as datas de amostragem. Os valores médios dos teores de compostos fenólicos e antocianinas encontradas são similares aos relatados para outras frutas vermelhas. Nos testes in vivo, camundongos C57Black6 foram divididos em 4 grupos que receberam por 5 dias uma dose diária de extrato bruto em alta, média e baixa dosagem (32µg, 16µg e 8µg equivalentes de cianidina-3-O-glucosídeo/animal, respectivamente). Para o grupo controle foi utilizada solução salina. Após 24 horas do fim do tratamento, os animais foram imunizados com OVA (ovalbumina), conforme metodologia padrão. No 14° dia, os animais foram sacrificados e os tecidos recolhidos para análise. Foram monitoradas a proliferação celular em baço e linfonodos, assim como, sua diferenciação em linfócitos T CD4+/CD8+ por citometria de fluxo. Os grupos tratados com CGCE apresentaram redução na proliferação celular em linfonodos, assim como na razão CD4+/CD8+. Sendo assim, os resultados indicam um possível efeito anti-inflamatório do extrato aquoso bruto de groselha do Ceilão, porém pesquisas adicionais de caracterização de citocinas e tratamentos prolongados são necessárias para confirmação dos resultados encontrados
Abstract: D. hebecarpa is a dark purple/red berry produced in Brazil as an exotic fruit with potential for large scale production and commercialization. Fruits are believed to contain high phenolic and anthocyanin concentration that provide color and defense to the plant. Moreover, these compounds have been extensively studied for their antioxidant activity and potential human health benefits. Thus, this work has optimized extraction prior to the skin and pulp anthocyanin profile characterization by HPLC-PDA-MS aiming to obtain the simplest and mildest conditions with maximum phenolic yield. Moreover, it was evaluated in vitro antioxidant capacity and the effect of water-based crude extract on immune system of mice. Solid-liquid ratio was determined in a linear experiment using ANOVA and Tukey (p<0.05). Acetone, ethanol and water were the extraction solvents evaluated by Simplex Lattice design. Time and acid concentration were evaluated using response surface methodology (RSM) to determine variables effect and their interactions. The optimized conditions achieved were: solid-liquid ratio of 1:120, acetone 20% with 0.35% formic acid, 20min, and no re-extraction. However, satisfactory results were obtained using just water as solvent. The optimized extraction used less organic solvent than the other conditions tested and showed higher yields than the initial ones (an increase of 10% and 26% of total phenolic compounds and total monomeric anthocyanins, respectively). The analytical HPLC chromatogram showed five major anthocyanins (Delphinidin-3-glucoside, Delphinidin-3-rutinoside, Cyanidin-3-glucoside, Cyanidin-3-rutinoside, and Petunidin-3-rutinoside) in addition to two minor pigments (Peonidin-3-rutinoside and Malvidin-3-rutinoside). Samples from two consecutive years were used for quantification purposes and antioxidant measurements. High concentration of phenolics and anthocyanins were detected in skin samples with the same anthocyanin profile of pulp part. FRAP, ABTS, and ORAC methodologies were used to evaluate antioxidant mechanisms. A strong peroxyl scavenger capacity was detected for D.hebecarpa samples by ORAC methodology which could indicate possibly health effects of fruit consumption. Significant variations of anthocyanin, total phenolic content, and antioxidant capacity were observed in pulp and skim possibly as a result of weather conditions. Concomitantly, biometric parameters and nutritional composition seems to be affected as well (p<0.05). C57Black6 mice were used for in vivo evaluation of immuno-modulatory effect of water-based Ceylon gooseberry crude extract (CGCE). Animals were treated with high, medium, and low dosages of CGCE (32µg, 16µg, and 8µg cyanidin-3-O-glucose/animal/day, respectively) during 5 days. Saline solution was used as a control group. Animals were challenged after 24 hours from the last dose using a standardized immunization protocol with OVA and CFA. After euthanasia, blood, spleen, and lymph nodes were collected and analyzed. Cell proliferation using MTT and lymphocyte profile by flown citometry was determined in spleen and lymph nodes, as well as IgG levels in blood samples. Results indicate a possible anti-inflammatory effect. Since, there was an decrease in lymph node cell proliferation response as well as in CD4+/CD8+ ratio. In summary, D. hebecarpa is a rich source of anthocyanins with concentration levels as high as reported for other berry fruits. The strong antioxidant activity and some evidences of in vivo anti-inflammatory effects suggest this fruit as source for future scientific works aiming to evaluate possible health effects
Doutorado
Ciência de Alimentos
Doutora em Ciência de Alimentos

Diploma thesis deals with the characterization of chokeberry (Aronia melanocarpa) and saskatoon berry (Amelanchier alnifolia) fruits. The theoretical part focuses on the botanical classification, chemical composition, curative effects and the use of chokeberry and saskatoon berry in the food industry. Further, the attention is also devoted to a review of methods for the determination of important antioxidants, specialized on the determination of polyphenols, anthocyanins, ascorbic acid and total antioxidant activity. The experimental part of the work deals with the determination of selected parameters in different varieties of chokeberry and serviceberry, harvested in 2012 and 2013 using spectroscopic and chromatographic techniques with an emphasis on effect of solvent (50% ethanol, destilled water, DMSO). Specifically total content of polyphenols and anthocyanins, color characteristics using the CIE L*a*b* system and the ability of the sample to reduce the iron are determined using the UV-VIS-NIR spectroscopy. Further ascorbic acid concentration in individual extracts is quantified by High performance liquid chromatography coupled with a diode array detector and the total antioxidant activity is characterized by electron paramagnetic resonance involving •DPPH and ABTS•+ assays.

Growing numbers of morbidity and mortality due to the cancer and coronary heart disease is recognized as the increasing challenge in the world. The initial stage of these diseases, early diagnosis and treatment are the main objectives of current researches. According to Lithuanian Health Information Centre morbidity and mortality due to these diseases in Lithuania exceed average of European Union. The oxidative stress occurs in cells as a result of normal physiological processes and environmental interactions, and the complex antioxidant defense system are of particular importance in protecting biological systems against oxidative damage by free radicals. Recently polyphenols and anthocyanins containing foods and preparations have attracted attention due to their health promoting benefits. Anthocyanins make one of the main groups of flavonoids, and represent polyphenolic compounds, that are found in vegetables and fruits, particularly in berries. An important effect of anthocyanins is the scavenging of oxygen-derived free radicals. They can interfere with few different free radical-producing systems, and can increase the function of the endogenous antioxidants. The aim of the research is to choose sources for the products with antioxidant activity from plants cultivated in Lithuania, evaluate quality and antioxidant activity of black currant, red currant and chokeberry products, to develop and evaluate technologies of phytopharmaceuticals with antioxidant activity... [to full text]

O Brasil é o segundo maior produtor de soja (Glycine max, L.), sendo responsável por quase 30% da colheita mundial. Mais de 80% da produção total é destinada à extração de óleo e proteína para a alimentação humana, sendo que o consumo na forma de grão e de produtos industrializados vem crescendo, como alternativa a proteínas de origem animal e devido aos potenciais benefícios à saúde. Grande parte da literatura científica refere-se à soja amarela, enquanto estudos sobre soja preta ainda são escassos. Este projeto visou estudar e comparar as características físicas e de composição química, incluindo os compostos fenólicos e atividade antioxidante da soja preta e amarela, cultivadas no Brasil em condições climáticas e ambientais similares. Fez parte do escopo do projeto avaliar o efeito do cozimento sobre os compostos polifenólicos e a atividade antioxidante. Não foi observada diferença significativa no conteúdo de nutrientes e no perfil de ácidos graxos entre a soja preta e amarela, nem nas características físicas, como massa, capacidade de hidratação e tempo de cozimento dos grãos. Porém, a soja preta apresentou teores muito mais elevados de compostos fenólicos totais (CFT) e de flavonóides em relação à soja amarela. Os teores na soja preta foram, em média, de 4,78 mg eq. ácido gálico/g e de 1,75 mg eq. catequina/g, de CFT e de flavonoides,respectivamente. A soja amarela apresentou em média 40% a menos de CFT e aproximadamente 60% a menos de flavonóides. Ao contrário da soja amarela que é desprovida de antocianinas, na soja preta foi encontrado um teor médio de 0,92 mg eq. cianidina-3-O-glicosídeo/g. Empregando a técnica de HPLC-DAD-MS/MS foram identificadas duas antocianinas, a cianidina-3-O-glicosídeo e a peonidina-3-Oglicosídeo, além da cianidina. A soja preta apresentou atividade antioxidante 70% superior à soja amarela pelo método de DPPH e 50% superior, pelo método de ORAC. O cozimento dos grãos reduziu em pelo menos 40% o teor de compostos fenólicos e a atividade antioxidante, tanto na soja preta como na amarela. Foi observada uma forte correlação entre os teores de CFT, flavonóides e a atividade antioxidante, antes e após o cozimento. Assim, o consumo de soja preta ou mesmo a produção de novos alimentos a partir desses grãos poderá oferecer ao consumidor a possibilidade de diversificar a sua dieta, introduzindo um alimento com a mesma qualidade nutricional da soja amarela e, possivelmente, maiores benefícios à saúde. Vislumbra-se a possibilidade de expansão do cultivo da soja preta, inclusive por pequenos produtores, criando um novo nicho de mercado para um alimento diferenciado.
Brazil is the second largest producer of soybean (Glycine max, L.), accounting for almost 30% of the world\'s production. Over 80% of the total production is destined to oil extraction and protein for human consumption. Soybean and soy-product consumption has been rising as an alternative to animal protein and due to its potential health benefits. Most of the scientific literature refers to yellow soybeans, while studies on black soybeans are still scarce. This project aimed to study and compare black and yellow soybeans, cultivated in Brazil in similar climatic and environmental conditions, as to their physical characteristics and chemical composition, including phenolic compounds and antioxidant activity. The effect of cooking on phenolic compounds and antioxidant activity was also investigated. No significant difference was observed between black and yellow soybeans as to nutrient content, fatty acid composition and physical characteristics, such as seed-weight, hydration capacity and cooking time. However, black soybeans had a much higher content of total phenolic compounds (TPC) and flavonoids than yellow soybeans. TPC and flavonoid contents in black soybeans were 4.78 mg gallic acid equivalents/g and 1.75 mg (+)-catechin equivalents/g, respectively. Yellow soybeans had on average 40% less TPC and approximately 60% less flavonoids. Unlike yellow soybeans which lack anthocyanins, black soybeans were found to have an average content of 0.92 mg cyanidin-3-O-glucoside equivalents/g. High performance liquid chromatography coupled to photodiode array and mass spectrometry detectors (HPLCDADMS/MS) was used to identify two anthocyanins, namely cyanidin-3-Oglucoside and peonidin-3-O-glucoside, and cyanidin. Black soybeans also showed a 70% higher DPPH-free radical scavenging activity than yellow soybeans, and a 50% higher ORAC value. Cooking reduced at least 40% of phenolic compounds and antioxidant capacity in all soybean samples, both black and yellow. High correlations between phenolic compositions and antioxidant activities were observed, before and after cooking. These results suggest that black soybean consumption or new processed food products from these crops may offer consumers an opportunity to diversify their diet with food nutritionally equivalent to yellow soybeans, but possibly with greater health benefits. The possibility of expanding black soybean cultivation, including small-scale production, may create a new market niche for a value-added food product.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES
The agribusiness processing of tropical fruits have generated waste that may have great potential, as well as having nutritional constituents, still have significant levels of bioactive compounds. Thus, this research aimed to use frozen pulp residue of grape extract and purify anthocyanins, evaluate its antioxidant capacity as well as its stability to light and heat.The residue was transferred by a processing unit (Recife - PE), whose grapes cultivar Isabel (Vitis labrusca L.) came from the city of São Vincente Ferrer - PE. The anthocyanin content was determined by differential pH method and its purification using C18 cartridges promoted the removal of sugars, acids and not anthocyanin phenolic compounds.The antioxidant activity of purified anthocyanins was evaluated by the ability to scavenge the radicals DPPH• (1,1-diphenyl-2-picrylhydrazyl) and ABTS•+ (2,2’-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid), with gallic acid as reference compound.To evaluate light fastness, anthocyanins were diluted in 95% ethanol solution: 1.5N HCl; 85:15 v/v (pH 1.0) and exposed to light and no light.To assess the effect of heat, anthocyanins were diluted in water, acidified with HCl (pH = 1.0) and subjected to temperatures of 50°C, 70°C and 90°C in thermostated bath. Absorbance readings were taken after 1, 3, 5 and 7 hours of heating. The data obtained from reading the absorbance of diluted anthocyanins were used for the calculation of the rate constant (k) and half-life (t1/2) using 1st order kinetics equations.The total anthocyanin content in grape residue was 15.56 ± 0.33 mg. 100g-1 residue. Anthocyanins purified showed good efficiency against the radical DPPH• (EC50 of 0.14 ± 0.01 g. g DPPH-1 and TEC50 < 5 minutes) and strong ability to sequester (> 80%). Against the radical ABTS•+ anthocyanins showed sequestration capacity of 241.36 ± 3.74 μmol TEAC.g-1 sample. The results showed that in the presence of light, time of half-life (t1/2) of the anthocyanin pigments was 656.87h and in the absence of light was 6.525.42h, demonstrating that the absence of light contributed significantly to the increase the stability of anthocyanins. Anthocyanins subjected to heat at temperatures of 50°C, 70°C and 90°C showed a half-life (t1/2) 159.31h; 8.77h and 3.05h, respectively, demonstrating that these pigments are degraded with increasing temperature. The results show the agroindustrial residue grape frozen pulp is a promising source of anthocyanins, which showed significant antioxidant properties, being efficient at capturing the DPPH• and ABTS•+ and can be used in foods that have acidic pH, put in opaque packaging, and are not subjected to severe heat treatment during processing.
As agroindústrias processadoras de frutas tropicais têm gerado resíduos que podem apresentar grande potencial, pois além de possuir constituintes nutritivos, ainda apresentam significantes teores de compostos bioativos. Assim, esta pesquisa teve como objetivo utilizar resíduo de polpa congelada de uva para extrair e purificar antocianinas, avaliar sua capacidade antioxidante assim como, sua estabilidade frente à luz e ao calor. O resíduo foi cedido por uma unidade processadora (Recife – PE), cujas uvas da cultivar Isabel (Vitis labrusca L.) foram provenientes da cidade de São Vicente Férrer – PE. O teor de antocianinas foi quantificado pelo método de pH diferencial e sua purificação, utilizando cartuchos C18,promoveu a remoção de açúcares, ácidos e compostos fenólicos não antociânicos. A atividade antioxidante das antocianinas purificadas foi avaliada pela capacidade de sequestrar os radicais DPPH• (1,1-difenil-2-picrilhidrazil) e ABTS•+ (2,2’-azino-bis-3-etilbenzotiazolina-6-acido sulfônico), tendo o ácido gálico como composto de referência. Para avaliar a estabilidade à luz, antocianinas foram diluídas em solução de etanol 95%:HCl 1,5N; 85:15 v/v (pH=1,0) e expostas à luz e à ausência de luz. Para avaliar o efeito do calor, antocianinas foram diluídas em água, acidificada com HCl (pH=1,0) e submetidas a temperaturas de 50ºC, 70ºC e 90ºC, em banho termostatizado. Leituras da absorbância foram efetuadas após 1, 3, 5 e 7 horas de aquecimento. Os dados obtidos na leitura da absorbância das antocianinas diluídas foram utilizados para o cálculo da constante de velocidade (k) e do tempo de meia vida (t1/2) utilizando equações cinéticas de 1ª ordem. O teor de antocianinas totais no resíduo de uva foi de 15,56 ± 0,33mg. 100g-1 de resíduo. As antocianinas purificadas apresentaram boa eficiência frente ao radical DPPH• (EC50 de 0,14 ± 0,01 g.g DPPH-1 e TEC50 < 5 minutos) e forte capacidade de sequestrar (> 80%). Frente ao radical ABTS•+ as antocianinas apresentaram capacidade de sequestro de 241,36 ± 3,74 μmol TEAC.g-1 de amostra. Os resultados obtidos evidenciaram que em presença de luz, o tempo de meia vida (t1/2) dos pigmentos antociânicos foi de 656,87h e na ausência de luz foi de 6.525,42h, demonstrando que a ausência de luz contribuiu significativamente para o aumento da estabilidade das antocianinas.As antocianinas submetidas ao calor, às temperaturas de 50ºC, 70ºC e 90ºC apresentaram de tempo de meia vida (t1/2) de 159,31h; 8,77h e 3,05h, respectivamente, demonstrando que estes pigmentos sofrem degradação com aumento da temperatura. Os resultados evidenciam o resíduo agroindustrial de polpa congelada de uva é uma fonte promissora de antocianinas, que apresentaram significativa propriedade antioxidante, mostrando-se eficientes em capturar os radicais DPPH• e ABTS•+, podendo ser utilizadas em alimentos que possuam pH ácido, acondicionados em embalagens opacas, e que não sejam submetidos à tratamentos térmicos severos durante seu processamento.

The awareness of consumers on the importance of food safety and of potential benefits of many fruit and vegetable derived products are, more and more, driving the interest of research institutes and food industries to deepen the knowledge on the quality of raw materials for fresh or processed use or to design food products enriched with nutraceutical substances. Several factors influence composition and quality of food products in pre- and post-harvest stages, such as cultivar and rootstock, agronomical techniques and storage conditions. The possibility to enhance the synthesis of some chemical compounds, in particular nutraceuticals (flavonoids, such as phenols and anthocyanins) is an important strategy in order to obtain foods with high functional activity. The overall aim of the PhD thesis is the evaluation of agronomical and postharvest factors that can influence the qualitative and nutraceutical traits of two important fruit products i.e. blood oranges and pomegranates. These fruits are characterized by a high anthocyanin content greatly appreciated by the consumers for their nutraceutical properties. The influence of several rootstocks on yield precocity and fruit quality and the effect of postharvest treatments on qualitative parameters were mainly considered in the case of blood oranges. As concerning pomegranate the investigation was focused on nutraceutical and physicochemical evolution observed in different studies regarding the comparison of international cultivars grown in Italy and Spain and the characterization of several Sicilian pomegranate accessions. On the whole, the results are interesting for their contribution to the comprehension of the many factors, from varietal choice up to fruit postharvest management, affecting the qualitative profiles of the products with a special emphasis on those compounds valuable for their nutraceutical properties.

L'Hibiscus sabdariffa (hibiscus) est une plante dont les calices comestibles, contiennent des polyphénols et des molécules d'anthocyane qui sont à la fois des composés antioxydants et responsables de leur couleur rouge attrayante pour le consommateur. Ces particularités constituent un fort potentiel santé qui répond aux demandes actuelles du marché et un atout économique pour les industries. Cependant, cette plante tropicale et saisonnière dont les calices riches en eau, sont très périssables, sensibles à l'humidité et à la chaleur, et les molécules d'intérêt (anthocyanes) sont thermosensibles et instables en milieu aqueux. Dans ce contexte, la transformation des calices en poudre permet, en plus d'optimiser le coût du transport et l'espace de stockage, de stabiliser les calices en assurant une longue durée de conservation du produit, de faciliter l'accessibilité aux biomolécules et donc à ses bienfaits pour la santé, ceci, à condition que le procédé soit maîtrisé. L'un des meilleurs moyens de préserver les propriétés nutritionnelles d'origine (teneur en anthocyanes, activité antioxydante) des calices est d'obtenir des produits peu transformés ou « minimally processed products » en combinant le séchage à l'étuve (contrôlé), le broyage et le fractionnement par tamisage. Ce procédé a été étudié dans ce travail et les propriétés physicochimiques (granulométrie, forme, structure, porosité, proportion de fibre, teneur en anthocyanes, activité antioxydante) et fonctionnelles des poudres (écoulement, reconstitution, extractibilité des biomolécules) ont été systématiquement évaluées dans un but de formulation de boisson. L'intérêt d'un tel procédé est la facilité de sa mise en œuvre, applicable à tous végétaux, la facilité d'usage de la poudre, l'amélioration de la disponibilité et l'accessibilité des biomolécules. Ce travail a permis d'identifier l'impact du séchage solaire et du broyage sur les propriétés physicochimiques des poudres, ces dernières impactant directement les propriétés fonctionnelles. La substitution du séchage solaire par le séchage contrôlé à l'étuve et un fractionnement par tamisage supplémentaire après le broyage ont permis d'améliorer les propriétés fonctionnelles des poudres. Ces propriétés singulières des poudres ainsi obtenues, ont permis de distinguer des applications qui leur sont propres
Hibiscus sabdariffa (hibiscus) is a plant with edible calyxes containing polyphenol and anthocyanin molecules that are both antioxidant compounds and responsible for their attractive red color for consumers. These particularities constitute a good health potential that meets the current market demands, and an economic potential for industries. However, this tropical and seasonal plant whose water-rich calyxes are highly perishable, sensitive to humidity and heat, and whose the interesting molecules (anthocyanins) are heat-sensitive and unstable in aqueous medium. In this context, the calyx transformation into powder allows, besides optimizing the transport cost and storage space, to stabilize the calyxes by ensuring a long product shelf-life, and to improve the biomolecule accessibility. This allows to benefit to its health assets provided that the process is controlled. One of the best ways to preserve the calyx original nutritional properties (anthocyanin content, antioxidant activity) is to obtain minimally processed products by combining controlled oven-drying, grinding and fractionation by sieving. This process was studied in this work and the powder physicochemical properties (particle size and shape, structure, porosity, fiber proportion, anthocyanin content, antioxidant activity) and functional properties (flowability, reconstitution, biomolecule extractability) were systematically evaluated for drink formulation. The interest of such a process is the ease of its implementation applicable to all plants, the powder ease of use, the improvement of the biomolecule availability and accessibility. This work allows identifying the impact of sun-drying and grinding on the powder physicochemical properties, the latter impacting the functional properties. The substitution of sun-drying by controlled oven-drying and an additional fractionation by sieving preceded by grinding allowed improving the powder functional properties. These powder functional properties were singular and allowed to distinguish specific applications for each powder type (fine or coarse powders)

Darbo tikslas: atlikti Lietuvoje registruotų mėlynių uogų pasirinktų preparatų sudėčių ir technologijos palyginamąją analizę, įvertinti kiekybiškai ir nustatyti antioksidacinį aktyvumą. Darbo uždaviniai: Ištirti Lietuvoje registruotų mėlynių uogų preparatų pasiūlą. Atlikti šių preparatų palyginamąją analizę vertinant sudėtis. Pasirinktuose preparatuose nustatyti fenolinių junginių ir antocianinų kiekį spektrofotometriniu metodu. Nustatyti pasirinktų preparatų antioksidacinį aktyvumą DPPH metodu. Tyrimo objektai ir metodai: Preparatai su mėlynių uogų ekstraktu ir mėlynių uogų milteliais. Spektrofotometrinis metodas naudotas analizei: bendras fenolinių junginių kiekis – pagal Galo rūgšties kalibracinę kreivę; bendras antocianinų kiekis – pH diferenciniu metodu; antioksidacinis aktyvumas – DPPH laisvojo radikalo inaktyvavimo kreivė. Tyrimo rezultatai ir išvados: Iki 2013 m. birželio mėn. nustatyti 47 Lietuvoje registruoti mėlynių uogų preparatai. Į 97 proc. šių preparatų sudėtį įeinančių mėlynių uogų žaliavos forma – ekstraktas. 19 proc. visų tirtųjų sudaro kompleksiniai ir 9 proc. vienkomponenčiai preparatai. Spektrofotometriškai nustačius fenolinius junginius, didžiausias jų kiekis yra ,,Mėlynių ekstraktas“, kapsulėse (,,Vitaforce“, Jungtinė Karalystė), mažiausias - ,,Visioblue strong“, kapsulėse (,,Hankintatukku Oy“, Suomija). Preparatas ,,Mėlynių uogų ekstrakto tabletės su cinku forte 0,25 g”, („Elit-farm", Ukraina) eksperimentinio tyrimo metu nedavė apčiuopiamo rezultato... [toliau žr. visą tekstą]
Purpose of the work: To make the comparable ingredients and technology analysis of selected blueberry preparations registered in Lithuania, evaluate quantitatively and determine the antioxidant activity. Tasks: to investigate the supply of blueberry preparations registered in Lithuania. To make the comparable ingredients analysis of preparations mentioned above. Determine the phenolic compounds and anthocyanins quantity using the spectrophotometry method. Measure the antioxidant activity of selected preparation using DPPH method. Research objects and methodology: Preparations with blueberry extract and blueberry powder. Spectrophotometric methods were used for analysis: total phenolic content according calibration curve of galic acid; total anthocyanins - pH differential method; antioxidant activity - DPPH free radical inhibition. Results and conclusions: Until June, 2013 y. Lithuania set 47 record Blueberry fruit preparations. There was noticed that in 97 percent composition of the preparation, blueberry raw material shape - extract. 91 percent of explored preparations are multicomponent and just 9 percent - one-component. Determined by spectrophotometry, that the highest levels of the phenolic compounds are in ”Mėlynių ekstraktas“, capsules of (“Vitaforce”, United Kingdom) and the lowest – “Visioblue strong’’, capsules of (”Hankintatukku Oy’’, Finland). The quantity of Anthocyanins per recommended daily dose in the preparation was studied by spectrophotometry method. The... [to full text]

Diploma thesis deals with determination of individual chemical parameters contained in modern varietes of gooseberries (Grossularia uva-crispa). The theoretical part is focused on the botanical characteristics of the plant, a description of selected varietes, chemical composition and food utilization fruits. The second half of the theoretical part is devoted to the characterization of selected nutritional parameters and methods for determination of total anthocyanins, phenolic compounds, vitamin C and antioxidant activity. Total of 17 varietes of gooseberries, which come from growers Vondráček and the Research and Breeding Institute of Pomology Holovousy Ltd. were analyzed in the experimental part. pH differential method was used to determination total monomeric anthocyanins. Total content of polyphenols were determined by using the Folin-Ciocalteu reagent. Next was determined content of vitamin C in all varieties by HPLC and was evaluated the total antioxidant aktivity using radical DPPH. In conclusion, individual varieties of gooseberries were compared by the contents of the determined chemical parameters.

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Blackberry (Rubus fruticosus) is considered a fruit rich in the phytochemical compounds, mainly phenolic acids and anthocyanins, which have a high potential for antioxidant and are also present L-ascorbic acid, carotenoids and tocopherols. Based on the concentration changes that these compounds undergo during the fruit ripening stages, this work aims to study the changes in the phytochemical profile of blackberry cv. Tupy, grown in temperate climates, in Pelotas, and effects on antioxidant and antimicrobial activity. Were determined five stages of ripening from green to mature through the external color of the fruit, and to confirm the differences between them, each stage was measured physical-chemical, determining total acidity (TA), total soluble solids (SS),ripening index (SS/TA) and instrumental analysis of color (hue angle). With regard to the phytochemical profile were analyzed phenolic compounds, L-ascorbic acid, carotenoids and tocopherols. It can be stated that according to the results of the different ripening stages did not affect significantly (p≤0.05) the total phenolic compounds, catechin and L-ascorbic acid. For the individual phenolic compounds during the fruit development, the derivatives of benzoic acid increased, the same behavior occurs with epicatechin, that among these is composed of higher concentration, the levels of cinnamic acid derivatives showed a different behavior, there fall of these levels. Anthocyanins increased during the fruit ripening, detaching the cyanidin-3-glucoside. The carotenoids and tocopherols decreased during ripening. One can conclude that it is possible to mount a phytochemical profile of blackberry cv. Tupy, grown in temperate climates, to differentiate their ripening stages, but it is necessary to carry out several determinations, since it is not possible to say which assessment contributed most to the differentiation of stages along the ripening. Another observation concerns the antioxidant activity increased during ripening, in vitro and in vivo, but noted that it is not possible to determine which compound has a higher antioxidant capacity in isolation, but found that there was a synergism between the phytochemical compounds present in fruit, which vary over the course of its development. Likewise, we can not say if there was an increase in antimicrobial activity over the course of ripening, and even if there was a specific secondary compound responsible for this antimicrobial activity, but that it exists, since the bacterium Salmonella enteritidis showed sensitivity to the extracts but with different answers.
Amora-preta (Rubus fruticosus) é considerada um fruto rico em compostos fitoquímicos, principalmente ácidos fenólicos e antocianinas, que possuem um elevado potencial antioxidante e também estão presentes ácido L-ascórbico, carotenóides e tocoferóis. Com base nas mudanças de concentração que esses compostos sofrem ao longo dos estádios de amadurecimento do fruto, este trabalho tem por objetivo estudar as alterações no perfil fitoquímico da amora-preta cv. Tupy, cultivada em clima temperado, na região de Pelotas, e os efeitos na capacidade antioxidante e antimicrobiana. Foram determinados cinco pontos de maturação do verde ao mais maduro, através da coloração externa do fruto, e para confirmar as diferenças entre estes, cada estádio foi avaliado físico-químicamente, determinandose acidez total titulável (AT), sólidos solúveis totais (SS), índice de maturação (SS/AT) e análise instrumental da cor (ângulo hue). Com relação ao perfil fitoquímico foram analisados os compostos fenólicos, ácido L-ascórbico, carotenóides e tocoferóis. Pode-se afirmar que de acordo com os resultados os diferentes estádios de maturação não afetaram significativamente (p≤0,05) os compostos fenólicos totais, a catequina e o ácido L-ascórbico. Para os compostos fenólicos individuais ao longo do desenvolvimento dos frutos, os derivados do ácido benzóico aumentaram, o mesmo comportamento ocorre com a epicatequina, que dentre todos é o composto de maior concentração, os teores dos derivados do ácido cinâmico apresentaram um comportamento diferenciado, houve queda destes teores. As antocianinas aumentaram ao longo da maturação do fruto, destacando a cianidina-3-glicosídeo. Os carotenóides e os tocoferóis decresceram no decorrer da maturação. Pode-se concluir que é possível montar um perfil fitoquímico da amora-preta cv. Tupy, cultivada em clima temperado, para diferenciar seus estádios de maturação, porém é necessária a realização de diversas determinações, uma vez que não é possível afirmar qual avaliação contribuiu em maior parte para a diferenciação dos estádios ao longo da maturação. Outra observação, diz respeito à atividade antioxidante que aumentou ao longo da maturação, tanto in vitro quanto in vivo, contudo foi observado que não é possível determinar qual composto tem maior capacidade antioxidante isoladamente, mas sim foi verificado que há um sinergismo entre os compostos fitoquímicos presentes no fruto, que variam ao decorrer do seu desenvolvimento. Da mesma maneira, não podemos afirmar se houve um aumento na atividade antimicrobiana ao decorrer da maturação, e nem se houve um composto secundário específico responsável por esta atividade antimicrobiana, mas sim que esta existe, uma vez que a bactéria Salmonella Enteritidis mostrou sensibilidade aos extratos, porém com diferentes respostas.

This Diploma thesis deals with the production of the concentrate from the Elderberry marc extract (Sambucus nigra L.). The basic chemical characteristics in this concentrate were determined. The theoretical part of the thesis deal with the botanic characteristic of the Elderberry and its berries. The characteristic of the phenolic compounds, mainly anhocyanins was summarized. The use of the anthocyanin pigments in the food industry, the possibilities of their extraction and the concentrate production was discussed. Thereafter the methods of the anthocyanin content and the phenolic content determination were described. In the experimental part of study, the production of the anthocyanin rich extract from Elderberry marc was optimized. The extraction of the marc by the 1:1 ethanol/water (V/V) solvent with the ratio marc/solvent 1:2 (w/V) showed the most convenient. The production of the concentrate followed. During the concentration on the rotary evaporator the pigment degradation was investigated. The degradation decreased with the decrease of the temperature. The final concentrate was prepared from the extract on the rotary evaporator by 45 °C for 15 minutes. This product had the 7times lower volume compared to the initial extract. In the final concentrate come of the chemical characteristic were determined. By liquid chromatography (HPLC) the major pigments cyanidin-3-sambubiosid and cyanidin-3-glucosid were determined. Their total content was determined to 34,9 gl-1. The total anthocyanin content was determined by pH differential method to 7,1 gl-1 (expressed as cyanidin-3-glucoside equivalent). The total phenolic content was determined to 27,1 gl-1 (expressed as gallic acid equivalent) by Folin-Ciocalteu method. By electron paramagnetic resonance the increase in antioxidant activity with the increase in concentration was found. Up to 42 aromatic compounds was determined in Elderberry marc, its extract and the final concentrate.

Diabetes mellitus—in particular, Type 2 diabetes mellitus (T2DM)—is one of the most prevalent chronic illnesses in many countries. Diabetes mellitus is regarded as an independent risk factor for cardiovascular disease (CVD). Cardiovascular disease is one of the main causes of mortality in patients with diabetes, mainly due to macrovascular complications. One of these macrovascular complications in diabetes is atherosclerosis, which involves a complicated pathophysiological process. In addition to hyperglycaemia, oxidative stress (OS) plays a significant role in the pathogenesis of diabetes and its associated risk of CVD. Platelet hyperactivity, in the presence of OS, has a major effect on the progression of atherosclerosis and thrombotic events. Aspirin (AS) is the most-used antiplatelet therapy for the prevention of thrombotic complications in people with T2DM. AS attenuates platelet hyperactivity. Although aspirin is a frequently used therapy for the inhibition of platelet hyperactivity, there is much evidence for AS non-responsiveness. The anthocyanin (AC) antioxidant has been shown to have an inhibitory effect on platelets and consequently may be used as a complement to other antiplatelet therapies to attenuate the negative effect of atherosclerosis and CVD in people with T2DM. Although many dietary intervention studies have shown that intake of AC-rich food may be negatively related to some CVD risk factors, the effect of pure AC on thrombotic markers such as platelet hyperactivity and haemostasis is yet to be explored. The main aim of the studies commenced for this thesis were to examine the effect of AC on thrombotic parameters and reveal the pathways by which AC might affect platelet hyperactivity, thereby providing individuals with T2DM with better protection against CVD. The aim of the first study (see Chapter 4) was to evaluate the in vitro effect of AC on platelet activation and aggregation. Fasting blood samples were collected from 13 screened and healthy volunteers after obtaining ethics clearance and signed informed consent. A full blood examination was conducted, and a dose-response curve was created by incubating platelets with five concentrations of AC (25–200 mg/L). Flow-cytometer assessed platelet activity by recording platelet surface marker expression of activation independent (CD41a) and dependent (P-selectin and PAC-1). Platelet aggregation studies were performed using the turbidimetric method by stimulating platelets using three different agonists: adenosine diphosphate (ADP), collagen and arachidonic acid (AA). The results of this study confirmed that AC at 50 mg/L significantly lowered platelet activation as expressed by the P-selectin surface activation marker and AA-stimulated platelet aggregation. However, a similar effect of AC was not detected when ADP or collagen was applied to induce platelet aggregation. Reduced AA-stimulated platelet aggregation by in vitro–adding of AC suggests that AC may reduce platelet hyperactivity, thus reducing the risk of vascular thrombosis and promoting cardioprotective effects. Following the results of Chapter 4, a subsequent study (see Chapter 5) was conducted to assess if AC had a comparable antithrombotic effect ex vivo. Twenty-six randomly recruited healthy (25–75-year-old) participants contributed to this study and consumed 320 mg of AC a day in the form of Medox® capsules for 28 days. This study was conducted in laboratories of the School of Medical Science at Griffith University. Fasting blood samples were collected pre- and post-intervention to perform platelet activation studies, which were done by measuring platelet surface marker expression of CD41a and P-selectin, and platelet–monocyte aggregates in ADP-stimulated platelets. Platelet aggregation studies were performed by stimulating platelets with various agonists such as ADP, collagen and AA. Full blood examination, coagulation and biochemistry profile analyses were also evaluated pre- and post-intervention. A flow-cytometry analysis showed that AC had a significant effect on the expression of P-selectin as measured by the platelet surface expression of CD62p. A significant decrease in ADP-stimulated platelet aggregation was detected in the blood of healthy individuals. These results endorse the idea that AC might reduce platelet aggregation by affecting a mechanism of platelet activation, specifically the P2Y2–P2Y12 receptor. Similarly, AC significantly reduced platelet activation, as a lesser concentration of fibrinogen and decreased mean platelet volume (MPV) were detected due to AC effects in normal participants. The results from Chapter 5 suggest that AC consumption may enhance protection against platelet hyperactivity–related thrombosis. Based on the results of Chapter 5, the aim of the next study (see Chapter 6) was to identify and elucidate any possible influence of AC on thrombotic risks in people with T2DM. This study involved patients with T2DM. Twenty-four patients with T2DM were recruited for this study, and they consumed 320 mg of AC a day in the form of Medox® capsules for 28 days. Blood pressure and anthropometric measures were taken before and after the intervention period. Fasting blood samples were collected pre- and post-intervention to perform platelet activation studies, which were done by measuring platelet surface marker expression of CD41a and P-selectin in ADP-stimulated platelets. Platelet aggregation, full blood examination, coagulation and biochemistry profile analyses were also evaluated pre- and post-intervention. The data from this study showed that AC had a probable lowering effect on collagen and ADP-induced platelet aggregation in T2DM. This clinical trial also demonstrated the reducing effect of AC on the TC level in the blood. The figures shown in Chapter 6 suggest that the ingestion of AC may mitigate the development of thrombotic risks due to platelet hyperactivity. Following the outcome of Chapter 6, a fourth study (see Chapter 7) was conducted to assess if AC was comparable to AS in lowering different thrombotic biomarkers as well as platelet activation and aggregation. Antiplatelet medications, such as AS, diminish platelet hyperactivity and aggregation and decrease the risk of thrombosis. These antiplatelet drugs inhibit platelet activation through different pathways. Antiplatelet agents are indicated for mitigating thrombosis, which is partly mediated by platelet hyperactivity. However, AS non-responsiveness and side effects have been reported. Antioxidants alleviate the development of atherosclerosis and mitigate the prognosis of CVD. Two groups of healthy participants consumed AC and AS for four weeks. They were tested before and after the intervention period for different parameters including full blood count, platelet activation and aggregation, biochemical tests of lipid profile, uric acid, glucose and C-reactive protein, and coagulation assay. This study (see Chapter 7) showed a significant decrease in platelet hyperactivity—as expressed by CD62p (P-selectin) caused by AC—in the participants, yet the effect of AS was more powerful. AC had a reducing effect on ADP and collagen-stimulated platelet aggregation, but AS applied a greater inhibitory effect on this. Alleviated platelet activation, along with reduced platelet aggregation, were also detected. Lower platelet degranulation correlates with a decrease in thrombus size, as P-selectin (which is expressed by platelets upon activation) is recognised to attract nearby white blood cells (WBCs) dynamically, thus increasing thrombus size. The outcomes from this study (see Chapter 7) suggest that AC could possibly be used to decrease platelet function. However, this study also showed AC to be less useful than AS in lowering the risk of thrombosis. The results achieved from the studies completed for this thesis demonstrate a positive relationship between the consumption of AC and a decrease in platelet activity, which may be instrumental in lowering the risk of thrombosis, thus providing better prevention against CVD. The hypothesised total antioxidant effect of AC may be responsible for reduced platelet activity, which is expected to delay or even prevent macrovascular or microvascular events in patients who suffer from elevated OS as a result of different diseases such as T2DM. Reduced MPV and lowered fibrinogen levels also suggest that ingestion of AC may have an effect in suspending the early stages of atherosclerosis. Thus, AC has the potential to alleviate thrombotic risk and probably reduce the risk of cardiovascular events.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Medical Science
Griffith Health
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Several seven-coordinate manganese complexes have been synthesised, characterised and tested for both superoxide dismutase and catalase activity. Macrocyclic ring contractions have led to a series of new seven coordinate mononuclear manganese(II) macrocycles that have potential for their use as working superoxide dismutase mimics. Numerous polynuclear seven coordinate manganese(II) macrocycles have been synthesised via Schiff base condensation. Subsequent reduction of the imine bonds has led to a variety of reduced amine analogues with varying axial ligands. The geometry has been compared about the manganese centres where possible. From the results of each complex tested for superoxide dismutase activity, the µ-chloro bridged tetranuclear complex [Mn2(C24H29N6O2)(Cl)2]2(ClO4)2 has proved to be the most efficient mimic with a calculated KMcCF value of 7.7 x 106 [M-1 s-1]. A method for measuring catalase activity has been developed, and the most efficient catalase active compound was found to be [Mn5(C24H29N6O2)2(OAc)2(ClO4)2](ClO4)2 with one molecule of complex breaking down approximately 59000 molecules of hydrogen peroxide after one minute. Catalase testing showed that a reduction of the imine bonds produced an increase in activity overall for the complexes of H2L1 (C24H29N6O2), but a decrease was observed for the reduced tripodal complexes. An increase in the number of manganese centres resulted in a rise in catalase activity. Many of the complexes tested for catalase activity showed an induction period prior to the activity being observed. This may suggest that the complexes undergo a change in structure, or that there is a rearrangement occurring before catalase activity may be observed. The results that are presented indicate that the axial ligands have an effect on the rate of catalase activity and the observed induction period. Of the molecules that were tested for both superoxide dismutase and catalase activity, the pentanuclear complex [Mn5(C24H29N6O2)2(OAc)2(ClO4)2](ClO4)2 showed high activity for both analyses. This may be due to the extra manganese centre within the complex and the axial ligands that are present when compared with other tetranuclear complexes. The complex [Mn5(HL1)(OAc)2(ClO4)2](ClO4)2 may prove to be a good candidate for a working superoxide dismutase mimic. Ring contracted complexes show high rates of superoxide dismutase activity but possess limited catalase activity. Attempts have been made to produce a direct method of measuring superoxide dismutase activity using a stop-flow technique to complement the results using the indirect NBT (Nitro blue Tetrazoleum) method. This was carried out by analysing low concentration solutions of both complex and superoxide on a millisecond timescale. Progress has been made for this method with preliminary results being obtained.

In industry, antioxidants are added to prevent changes that affect quality due to oxidation, such as the development of off flavors, unacceptable odors and discoloration. New resources that are familiar to consumers and may work as antioxidants should be studied. The changes in antioxidant activity were determined for four freeze-dried native wild berry powder species in Utah, including skunkbush (Rhus trilobata), chokecherry (Prunus virginiana), river hawthorn (Crataegus douglasii) and silver buffaloberry (Shepherdia argentea), during cold storage (-20 °C) for 6 months. The total antioxidant activity was stable in most of the berry powders during the six months of frozen storage. Overall the river hawthorn and buffaloberry contained the highest antioxidant activity values, which indicated that they would be more efficient for use as food antioxidants. The effectiveness of two different levels of native Utah river hawthorn and buffaloberry freeze-dried powders in maintaining the quality of ground poultry and ground beef was studied. Meat patties mixed with berry powders were held at 4 °C and analyzed every three days for two weeks. The river hawthorn berry and silver buffaloberry delayed the undesirable changes in flavor or color caused by the oxygen present in the air in both chicken and beef ground patties. However, the plant pigments in the silver buffaloberry increased the redness of chicken patties. Neither berry significantly changed the color of ground beef patties or changed the microbial growth in ground meats. Overall, both river hawthorn and buffaloberry are suitable for using in ground beef product as antioxidants. Milk mineral and sodium tripolyphosphate are antioxidants that could bind free metal ions into insoluble or inactive forms. They can delay the changes of the meat pigment and maintain color in meat products. The purpose of the third study was to examine the effect antioxidants on meat pigment.pH describes the amounts of acid in food. Slowed color changes in the poultry meat pigment with antioxidants was seen both at the pH of living poultry muscle (7.2) and the pH of raw poultry meat (pH 6.0). This suggested that addition of metal-binding antioxidants like milk mineral or sodium tripolyphosphate could help preserve poultry meat pigment and maintain the color of poultry products.

Mestrado em Biologia Aplicada - Biologia Molecular Celular
It is generally accepted that the principal roles of metallothioneins (MTs) lie in the detoxification of toxic metals and regulation of the metabolism of essential trace metals. However, there is increasing evidence that it can act as a free radical scavenger. Although the great number of studies on the antioxidant activity of MTs, the effective physiological role of this protein is still unclear. In order to understand the role of MTs in the protection against metal contamination and oxidative stress, the bivalve Cerastoderma edule was used to evaluate the response of MTs in both situations. Cadmium, a widely reported MT inducer, was used to simulate metal contamination whereas H2O2, an oxidizing compound, was used to simulate oxidative stress. In the first approach, cockles were exposed to a range of Cd and H2O2 concentrations and MTs and TBARS were quantified. Results showed that both treatments induced MT synthesis, confirming the involvement of MTs in metal contamination and oxidative stress. Indeed, the use of MTs as biomarkers for metal pollution was questioned due to the similar synthesis of MT in the two highest concentration used. At last, one concentration of Cd (10 μM) and of H2O2 (20 μM) were selected and cockles were exposed again. TBARS concentration and the intracellular amount of H2O2 were determined. Metal-MT complexes in the two conditions and control were isolated by size exclusion chromatography, and the binding of Zn and Cd to MTs and other cytosolic proteins was evaluated. Furthermore, MTs were quantified and their content in each treatment and control was compared to the amount of Zn associated to them. Results showed that the >H2O2 treatment induced high levels of oxidative stress, demonstrated by the high lipid peroxidation and intracellular concentration of H2O2. Data also indicated that Cd was mainly associated with MTs pool in the Cd treatment, confirming that the protective role of MTs in metal contamination in this bivalve species was due to the binding of MTs to Cd ions. Additionally, the percentage of Zn bound to MTs decreased in the H2O2 treatment, indicating Zn release in oxidative stress. Also, MTs molecules were not as metalated as in the control, confirming Zn release from MTs in oxidative stress and indicating that MTs were needed for demands other than Zn distribution. Further studies on the redox status of MTs are needed to determine the redox status of MTs in the oxidative stress, and understand if, in this bivalve, MTs are acting as ROS scavengers.
Os papéis geralmente associados às metalotioninas (MTs) resumem-se á desintoxicação de metais tóxicos e à regulação do metabolismo dos metais essenciais. No entanto, existem evidências cada vez mais acentuadas de que as MTs atuam na proteção contra o stresse oxidativo. Apesar do grande número de estudos que se focam na actividade antioxidante das MTs, o papel fisiológico efetivo destas proteínas não foi ainda clarificado. A fim de compreender o papel das MTs no stress oxidativo e na proteção contra o efeito dos metais, o bivalve Cerastoderma edule foi selecionado neste estudo para avaliar a resposta das MTs em ambas as situações. O cádmio, um forte indutor das MTs foi usado para causar contaminação metálica enquanto o H2O2, sendo um composto oxidante, foi usado para provocar stresse oxidativo. Numa primeira abordagem, os berbigões foram expostos a uma gama de concentrações de Cd e H2O2 e as MTs e os TBARS foram quantificados. Os resultados mostraram que apenas o H2O2 provocou peroxidação lipídica no berbigão e que ambos os tratamentos induziram a síntese de MTs, confirmando o envolvimento destas na contaminação metálica e no stresse oxidativo. De facto, a utilização das MTs como biomarcadores de poluição metálica foi neste estudo questionada devido á síntese de quantidades semelhantes de MTs nas duas concentrações mais elevadas de Cd e H2O2. Numa segunda abordagem, os berbigões foram novamente expostos a uma concentração selecionada para cada tratamento (10 μM de Cd e 20 μM de H2O2). A concentração dos TBARS e a quantidade intracelular de H2O2 foram determinados. Os complexos metal-MT em ambas as condições e no controlo foram isolados por cromatografia de exclusão molecular e a ligação entre os iões de Zn e Cd e as MTs e outras proteínas citosólicas foi avaliada. Para além disso, As MTs foram quantificadas em cada tratamento e no controlo, sendo o seu conteúdo comparado com a quantidade de Zn ligado. Os dados indicaram que o tratamento com H2O2 induziu elevados níveis de stresse oxidativo, demonstrado pela elevada peroxidação lipídica e pela grande concentração intracelular de H2O2. Relativamente aos resultados da cromatografia, os iões de Cd estavam principalmente ligados às MTs no tratamento com Cd, confirmando o efeito protector das MTs na contaminação metálica nesta espécie de bivalve. Adicionalmente, a percentagem de Zn ligado às MTs diminuiu no tratamento com H2O2, indicando que o stresse oxidativo impõe a libertação de Zn por parte das MTs. Em jeito de confirmação, as MTs estavam menos metaladas no tratamento com H2O2 do que no controlo. Seriam necessários estudos complementares para perceber se neste bivalve as MTs actuam como eliminadoras de ROS.

Reactive Oxygen Species (ROS) produced during normal aerobic metabolism, if not promptly removed by the detoxification mechanisms of the cells, can easily react with cell components, especially lipids, producing secondary cytotoxic molecules called reactive carbonyl species (RCSs). RCSs exhibit significant chemical reactivity and can cause protein modification and dysfunction. One of the most important ReSs is 4-hydroxinonenal (HNE), an unsaturated aldehyde that has been strongly linked to Alzheimer'S disease (AD). A new series of dipeptide histidyl hydrazide analogues of carnosine was prepared, with the aim of producing molecules with enhanced HNE scavenging activity. These compounds were demonstrated to scavenge HNE and to protect SH-SY5Y cells from HNE-induced tOxlcity and were superior in action to carnosine. The synthesis of an analogue of the best compound of the series containing caffeic acid, resulted in the generation of a new molecule exhibiting complete retention of HNE-scavenging activity and also possessing free Radical Scavenging Activity (RSA). Mitochondria are the major sites of high levels of oxidative stress and the targeting of a reactive carbonyl scavenger directly to these organelles would result in extinguishing the primary source of RCS, thus arresting any consequent cellular damage. In the present work, the possibility of specifying the cellular localization of histidyl hydrazide was investigated using previously described mitochondria penetrating peptides (MPPs) and new examples of such sequences I modified to be more resistant to protease degradation. The ligation of histidyl hydrazide to a previously reported MPP was successful in targeting the former to the mitochondria of HeLa cells. Finally. a particular chemical ligation approach was investigated for the development of a system of linking a common peptide vector and a variety of cargo molecules of choice through facile in situ coupling, without requiring the de novo synthesis of a new chemical conjugate in each instance.

Certain components of flaxseed are known to exhibit antioxidant activity. The objective of this research was to study the antioxidant activity of flaxseed proteins and protein hydrolysates. Proteins and their hydrolysates were prepared from defatted, non-defatted, and demucilaged flaxseed with and without dialysis, and the antioxidant activities were examined by DPPH scavenging assay, reducing power assay, and metal ion chelating assay. The degree of hydrolysis (DH) of the proteins using bacterial protease was higher than using trypsin. Using bacterial protease, higher DH was observed for demucilaged and defatted flaxseed protein/dialysis (DDFPD, 30% DH), defatted flaxseed protein/dialysis (DFPD, 28% DH), and non-defatted flaxseed proteins/dialysis with 14% DH, compared to flaxseed protein/non-dialysis which were DDFPND 28% DH, DFPND 25% DH, and NDFPND with 12% DH. Proteins from dialysis treatment as well as their hydrolysates showed positive effect on antioxidant activity. DDFPD hydrolysates using bacterial protease showed higher DPPH radical scavenging activity (73.23%) and reducing power activity (0.15) at the concentration of 2.5 mg/ml as well as Fe2+ chelating ability (75%) at of concentration 1 mg/ml. SDS-PAGE and native-PAGE results of non-hydrolyzed samples showed no change in electrophoretic behavior as a result of the treatments; a major band corresponding to MW 48 KDa and three minor bands with MW of 16, 23, and 34 KDa. SDS-PAGE of DDFPNDH and NDFPNDH hydrolysates obtained using trypsin showed one resistant band.
Certaines composantes des graines de lin sont reconnues pour avoir des effets antioxydants. L'objectif du projet de recherche ci-présent est d'étudier les effets antioxydants des protéines des graines de lin et de leurs hydrolysâtes. Les protéines et leurs hydrolysâtes ont été préparé à partir de graines de lin dégraissées ou natures et démucilaginées, avec et sans dialyse. Leurs effets antioxydants ont été examinés avec le test de scannage DPPH, le test du pouvoir de réduction, et le test des ions métalliques chélates. Le degré d'hydrolyse (DH) des protéines était plus élevé avec l'utilisation d'une protéase bactériale qu'avec l'utilisation de trypsine. En utilisant la protéase bactériale, un DH plus élevé a été observé avec les graines démucilaginées et dégraissées avec dialyse/protéine (DDFPD, 30% DH), les graines dégraissées avec dialyse/protéine (DFPD, 28% DH), et les graines nature avec dialyse/protéines (14% DH), comparé aux graines protéine/sans dialyse qui ont eu comme résultat DDFPND 28%, DFPND 25% DH, et NDFPND avec 12% DH. Les protéines et leurs hydrolysâtes du traitement avec dialyse ont montré qu'il y avait bel et bien un effet antioxydant. Les hydrolysâtes, du test DDFPD et de l'utilisation du protéase bactériale, ont montré un niveau d'activité de radicaux libres plus élevé (DPPH 73.23%) ainsi qu'un pouvoir de réduction de (0.15) à une concentration de 2.5 mg/ml, de même qu'une habilité au Fe2+ de 75% à la concentration de 1mg/ml. Les résultats de SDS-PAGE et PAGE-original des échantillons non hydrolysés montrent qu'il n'y a aucun changement dans le comportement électrophorétique résultant du traitement: une bande majeure qui correspond au MW 48 KDa et trois bandes mineures avec un MW de 16, 23, et 34 KDa. Dans les échantillons d'hydrolysâtes SDS-PAGE du DDFPNDH et les hydrolysâtes du NDFPNDH obtenus en utilisant la trypsine ont montré une bande résistante.

Mn-salophen complex with superoxide-scavenging activity was prepared from manganese(III) acetate dihydrate and salophen in ethanol. Visible absorption spectrum of the red-brown solution exhibited a broad absorption band at 430 - 450 nm with two shoulders between 500 and 600 nm which were absent with either salophen or manganic acetate alone. Titration of salophen with manganese(III) was consistent with a 1:1 Mn to salophen stoichiometry of the complex based on changes in the absorbance at 500 nm or of superoxide scavenging activity. The SOD-like activity of the complex in the xanthine-xanthine oxidase/cytochrome c assay was 1450 units/mg salophen. The SOD activity of the complex was suppressed 50% in the presence of EDTA (1 mM), but was not altered in the presence of bovine serum albumin (1 mg/ml) or crude protein extract of E. coli QC779 sodA sodB (1 mg/ml). E. coli QC779 sodA sodB grew scantily after an 8 hour lag phase in aerobic M63 glucose minimal medium.
Ph. D.

Free radicals are unstable and reactive molecules, with one ore more unpaired electrons in the outer orbit and adverse effects on plant, animal, and human cells. In particular free radicals are responsible of lipids peroxidation, proteins oxidative damages, and DNA cleavage, resulting in increased risk of mutation. Antioxidants represent an important defence against these injuries. Vegetable derived foods are known as important sources of dietary antioxidants such as phenols, anthocyanin, tocopherols, tocotrienols, carotenoids, retinol precursors, and ascorbate. With this background, the aims of this Ph.D. project were: i) to develop and validate analytical methods for quantification of tocopherols, thiols, and total antioxidant activity (TAA) of milk; ii) to describe the phenotypic variation of thiols in milk of dairy and dual-purpose cow breeds; iii) to describe the phenotypic variation of TAA in milk of cow, buffalo, goat, and sheep; iv) to evaluate the feasibility of mid infrared spectroscopy to predict TAA of cow milk; v) to evaluate the effect of skimming and heat treatments on milk tocopherols and TAA. In the 1st Chapter, a simplified saponification protocol, followed by cheap HPLC method based on methanol elution and sensible fluorescence detection was developed and validated for the quantification of α-tocopherol and γ-tocopherol, in different types of commercial fluid milk. Chromatograms showed two analytical peaks, corresponding to α-T and γ-T. The method was able to detect the adverse effects of skimming and UHT treatment on α-T and γ-T concentrations. The proposed method could be usefully adopted in future for large scale studies, aiming to investigate phenotypic variation of tocopherols in milk. The 2nd Chapter represent a first contribution to the characterization of low molecular weight thiols in milk of different cattle breeds. Thiols were extracted from the soluble fraction of milk, and following a derivatization protocol they were separated by reverse phase HPLC and detected fluorimetrically. Six thiol species were detected and two of them, glutathione (GSH) and cysteine-glycine (Cys-Gly), were identified and quantified. The average concentration of Cys-Gly in milk was greater than that of GSH, and milk from dual-purpose breeds was richer in thiols than milk from dairy cows. The 3rd Chapter deals with the development and the validation of a robust and fast spectrophotometric method for the determination of TAA of different types of milk. The method was linear, and highly repeatable and reproducible. Preservative added on raw milk had negligible effects on TAA measurement. The greatest TAA was measured on skimmed pasteurised milk, followed by skimmed UHT milk, raw milk, whole pasteurised milk, and whole UHT milk. The 4th Chapter is the first contribution to the phenotypic characterization of TAA of bovine milk. This phenotype exhibited an exploitable variability, similar to that of other quality traits. Favourable phenotypic correlations of TAA with fat, protein, and casein percentages were observed, as well as with somatic cell score. Total antioxidant activity of milk increased across lactation. Mid-infrared prediction models developed to predict milk TAA were not enough accurate for analytical purposes. The 5th Chapter deals with the phenotypic characterization of TAA of buffalo, goat, and sheep milk. Sheep milk showed the greatest TAA. This is probably due to its relatively high content in fat, protein, and casein percentages, that are known as compounds contributing to milk antioxidant capacity. Accordingly, buffalo and goat milk had lower TAA as well as lower fat, protein and casein percentages. Milk TAA was unfavourably correlated with milk yield, but the relationship was significant only for buffalo, whereas protein and casein percentages were positively correlated with TAA of goat milk.
I radicali liberi sono molecole reattive e instabili, con uno o più elettroni spaiati nell'orbitale esterno, con possibili effetti negative a livello di cellule vegetali e animali. In particolare, i radicali liberi sono responsabili della perossidazione dei lipidi, dell'ossidazione delle proteine e del danneggiamento del DNS, che causa un aumento del rischio di mutazioni. Gli antiossidanti rappresentano un'importante difesa contro questi danni. Gli alimenti di origine vegetale sono noti come risorse primarie di antiossidanti, come i fenoli, gli antociani, i tocoferoli, i carotenoidi, i precursori del retinolo e l'ascorbato. In questo contesto, gli scopi della presente tesi di dottorato sono: i) lo sviluppo e la validazione di metodi analitici per la quantificazione dei tocoferoli, dei tioli e della capacità antiossidante totale nel latte; ii) la descrizione della variazione fenotipica dei tioli in vacche da latte e a duplice attitudine; iii) la descrizione della variabilità della capacità antiossidante totale (TAA) nel latte di vacca, di bufala, pecora e capra; iv) la valutazione della capacità del medio infrarosso nella predizione della TAA del latte vaccino; v) la valutazione dell'effetto della scrematura e del trattamento al calore sulla concentrazione in tocoferoli e sulla TAA.

The objective of this research was to study the antioxidant activity of soybean protein hydrolysates (SPH) and chickpea protein hydrolysates (CPH) at different concentrations, and to measure the antioxidant activity of fractions collected from the RP-HPLC analysis of SPH and CPH. Protein hydrolysates were prepared by the proteolytic enzyme trypsin. The hydrolysates obtained were subjected to DPPH (1, 1-diphenyl-2 picrylhydrazyl) radical scavenging assay. The SPH and CPH at concentration of 2.5-10 mg/ml showed antioxidant activity of 16.5-32 % and 3.4-26.8 %. SPH and CPH were fractionated by using RP-HPLC on C18 column. The antioxidant activity of four SPH and CPH fractions (F I, F II, F III, and F IV) was measured by using DPPH radical scavenging assay. For SPH, antioxidant activity of F III (47.7 %) was higher than other fractions at protein concentration of 1 mg/mL and for CPH; F II showed maximum antioxidant activity 27.9 % at protein concentration 1 mg/mL. The results from the SDS-PAGE confirmed the hydrolysis of protein samples. The second part of the study was to measure the impact of high pressure processing (HPP) on the degree of hydrolysis and antioxidant activity of proteins. High pressure processing (HPP) of isolated soybean protein (ISP) and isolated chickpea protein (ICP) was done at 400 MPa and 600 MPa for 5 min and 10 min. The degree of hydrolysis of isolated soybean protein and isolated chickpea protein treated with high pressure processing and with trypsin hydrolysis showed continuous increase from 12.4 to 24.9 % for SPH and 13.6 to 26.2 % for CPH. The DPPH radical scavenging assay showed a more than two fold increase in antioxidant activity of SPH and CPH: 67 % as compared to the 32 % of SPH without HPP and 56.6 % as compared to the 26.8 % of CPH without HPP at concentration 10 mg/mL. These results show that HPP increased the degree of hydrolysis and antioxidant activity of protein hydrolysates.
Le but principal de cette recherche constituait l'analyse du potentiel antioxydant, à diverses concentrations, d'hydrolysats de protéine de soya (HPS) et d'hydrolysats de protéine de pois chiche (HPP). Les hydrolysats de protéine ont été isolés à l'aide de l'enzyme protéolytique trypsine. Les HPS et HPP démontraient respectivement un potentiel antioxydant de 16.5 à 32% et 3.4 à 26.8 % lorsque présents à des concentrations de 2.5 à 10 mg/mL. L'utilisation d'une colonne C18 a permis de séparer, par CLHP-PI, les HPS et HPP en quatre fractions (F I, F II, F III, et F IV) qui furent dosées avec du DPPH (1,1-diphényl-2-picrylhydrazyle) afin de comparer leur pouvoir de scavenging sur les radicaux. Pour les HPS, le potentiel antioxydant de F III (47.7 %) était supérieur à celui des autres échantillons alors que pour les HPP, 27.9 % (F II) était le seuil maximal. Dans les deux cas, les hydrolysats étaient concentrés à 1mg/mL. L'hydrolyse des échantillons de protéine a été confirmée par SDS-page. La deuxième partie de l'étude visait à mesurer l'impact de la pascalisation sur le degré d'hydrolyse et le potentiel antioxydant des protéines. Des isolats de protéine de soya (IPS) et de protéine de pois chiche (IPP) ont été traités à haute pression (400 MPa et 600 MPa) pendant 5 et 10 min. Le degré d'hydrolyse des IPS et IPP soumis à la pascalisation et à la trypsin ont démontré une augmentation constante allant de 12.4 à 24.9 % pour les isolats de protéine de soya et de 13.6 à 26.2 % pour les isolats de protéine de pois chiche. L'analyse au DPPH du pouvoir d'épuration des radicaux a montré que le potentiel antioxydant des hydrolysats a plus que doublé, passant de 32 à 67 % pour les HPS et de 26.8 à 56.6 % pour les HPP, lorsqu'ils étaient traités par hautes pressions. Cela démontre que la pascalisation améliore le degré d'hydrolyse et le potentiel antioxydant des hydrolysats de protéines.

The methanolic crude extract of aerial parts of the plant Scrophularia nodosa was shown to have potent DPPH radical scavenging activity (IC50 = 48.75 ± 7.00 μg/ml). Activity-guided fractionation resulted in the isolation of three principal antioxidant compounds; acteoside, angoroside C and angoroside A. Acteoside (yield = 1.21%, IC50 = 15.2 μM) appeared to be the most abundant and most antioxidant-active. The potent antioxidant activity is in support of the traditional use of the plant for wound healing and anti-inflammatory conditions. The methanolic extract of aerial parts of Tanacetum vulgare has potent DPPH radical scavenging activity (IC50 = 37.00 ± 1.20 μg/ml). Activity-guided fractionation on the methanolic extract of T. vulgare resulted in the isolation of 3,5-di-caffeoylquinic acid (3,5-DCQA), axillarin and luteolin. 3,5-DCQA appeared to be the most abundant and most antioxidant-active compound (yield = 7.28%, IC50 = 9.7 μM). The potent antioxidant activity is in support of the traditional use of the herb for fever, rheumatic conditions and anti-inflammatory conditions. The methanolic crude extract of Cassia auriculata and its fractions were shown to have potent scavenging activity on DPPH, hydroxyl and hydroperoxide radicals, moderate superoxide radical scavenging activity and potent ion(III) reducing power. The activity-directed studies resulted in the isolation of kaempferol-3-0-β-D-rutinoside, kaempferol, luteolin, quercetin and unknown antioxidant inactive compound. The previously reported pharmacological aspects of the above flavonoids and flavonoid glycosides (anti-carcinogenic, anti-inflammatory, hyperglycaemic, antidiabetic) along with the shown antioxidant behaviour explain the traditional medicinal values of the plant. Cassia alata L crude extract and its fractions showed potent radical scavenging activity against formation of lipid peroxide radicals. The activity directed isolations resulted in the isolation of kaempferol along with p-hydroxybenzoic acid. These may contribute towards the traditional medicinal values of the plant as an antidiabetic, anti-microbial and for skin diseases.

The objectives of the studies presented in this thesis were to identify and quantify the major phenolic components of red wine and to assess the contribution of individual compounds to the total antioxidant activity. Red wines were analysed for their phenolic content and antioxidant activity using a range of complementary techniques including HPLC-tandem mass spectrometry, preparative HPLC and HPLC with an on-line antioxidant detection system. HPLC-MS2 revealed the presence of a number of flavoids and phenolic compounds of which 19 were identified, with gallic acid, the flavin-3-ols and anthocyanins being the most abundant. Preparative HPLC was used in an effort to isolate the antioxidant components in red wine and 60 aliquots were collected. Each wine fraction was analysed for total phenolics, catechins and anthocyanins, while antioxidant activity was determined by electron spin resonance spectroscopy (ESR). The preparative HPLC step did into completely separate the compounds in red wine, nonetheless increasing antioxidant activity was highly and significantly associated with total phenolics (r = 0.816, P < 0.001) and total catechins (r = 0.188, p = 0.151). HPLC with an on-line antioxidant detection system was subsequently used to separate and identify red wine phenolics. The findings from this study indicate that gallic acid, (+)-catechin, (-)-epicatechin, and procyanidin dimersB1 and B2 were the major in vitro antioxidants identified in red wine. Collectively, the flavin-3-ols contributed > 50% of the total antioxidant capacity of each wine, while gallic acid contributed between 24-44%. The flavonols and anthocyanins were minor antioxidant components in red wines. By combining HPLC, MS2 and on-line assessment of antioxidant activity, the major phenolic compounds present in red wine were identified, together with their direct contribution to the total antioxidant activity.

Peptides derived from food proteins after enzymatic treatment and/or processing, are known to be bioactive in both biological and food systems; for this reason fish muscle peptides were investigated for their antihypertensive and antioxidant activity. Atlantic mackerel muscle proteins were hydrolysed with pepsin and pancreatine and the resultant hydrolysate was sequentially fractionated on 2 kDa membrane ultrafilters and further by gel filtration, ion exchange and high performance liquid chromatography and the resultant peptide fraction contained the amino acids histidine, proline, tyrosine, methionine, leucine, tryptophan and lysine. For ACE inhibitory activity, the peptide fraction (MFPH-V-JPA) had an inhibitory concentration (IC50) of 0.15 mg/ml and showed competitive inhibition for ACE with an inhibition constant (Ki) of 0.32 mg/ml. In terms of antioxidant activity, the HPLC isolated peptide fraction (LC1-Z) contained the amino acids serine, histidine, tyrosine, phenylalanine, tryptophan and lysine. It inhibited the formation of both peroxides and malonaldehyde in a linoleic acid model emulsion in a dose dependent manner with lipid oxidation inhibitory concentration (IC50) of 1.80 mg/ml. At concentration of 8 mg/ml, the inhibition of linoleic acid oxidation was more than that of 0.01 % butylated hydroxytoluene (BHT) and trolox (p < 0.001). The mechanism of antioxidant activity of the peptide (LC1-Z) was by carbon centered radical scavenging (5.34 %), hydroxyly radical scavenging (IC50 value of 1.60 mg/ml), metal chelating (5.72 %) and reducing ability. In caco-2 cells, 1 mg/ml of the peptide (LC1-Z) was not toxic to the cells seeded at 2 x 104 cells/well. Proxidant tBHP (2.5 mM) reduced cell viability significantly (79.3 %) but this increased to 94.7 % in the presence of the peptide or trolox. The peptide (1 mg/ml) also reduced TBARS formation (33.18 mug/ml) in cells compared to cells treated with tBHP alone (38.18 mug/ml). The activity of caspases-3 and -7, was higher in caco-2 cells treated with tBHP only (157.5 +/- 7.99 %) compared with those treated with the peptide (25.7 + 3.92 %). Morphological modification of the caco-2 cells treated with tBHP was evident as the cells appeared detached from the flask surface compared to those treated with the peptide (LC1-Z) which were healthy and attached to the flask surface. In Ea.hy 926 cells, reactive oxygen species were reduced by 26 % and 39 % in the lucigenin-chemiluminscence and flourescence methods respectively in cells treated with the peptide. In a caco-2 cell monolayer, transepithelial transport of the peptide was observed in both directions with a basolateral to apical apparent permeability of 0.95 +/- 0.12 cm-1 and apical to basolateral flux of 0.74 + 0.20 cm-1. HPLC chromatograms of the buffer solution taken from the apical and basolateral side showed the presence of the peptide in both sides i.e. 11.5% for apical to basolateral flux and 12.2 % for basolateral to apical. These results demonstrate that peptides with antihypertensive and antioxidant activity can be derived from Atlantic mackerel muscle proteins, with potential for neutraceutical applications.

碩士
國立屏東科技大學
食品科學系
92
The purple variety of napiergrass (Pennisetum purpureum), with the common name of “medicine sugarcane”, possesses higher antioxidant capacity, which was accredited to its anthocyanin contents. At present, there are no related researches available in Taiwan regarding purple Napiergrass. This research was aimed to study the characteristics and processing stability of this anthocyanin. Since sucrose and vitamin C were reasoned to affect the stability of this pigment, a model system was used to investigate the changes of color quality, patterns of anthocyanin and antioxidant capacity as being heated under different sucrose or vitamin C concentration. The results showed that there were five kinds of anthocyanin with possible acyl groups in the purple napiergrass. Preservation at 5℃ revealed that pH 1 and 2 were the most stable environments for the pigment, whereas pH 2 exhibited the best heat stability after being heat treated at 90℃ for 12 hours. Heating the pH 3 model system at 90℃ showed that appropriate concentrations of sucrose and vitamin C facilitated the hyperchromic effects, which could favor the heat stability of pigment. The best pigment retention was found with vitamin C of 4 mg/100 ml which prolonged for 12 hours. The conservation durations of anthocyanin were shortened with increasing amounts of sucrose addition. 20% of sucrose accompanied with by 4 mg/100 ml of vitamin C would reinforce the anthocyanin retention. Monomeric ACN increased before heating but polymeric ACN enhanced after heating, with the presence of sucrose or vitamin C. Monomeric ACN was found being highest for concurrent additions of 20% sucrose and 4 mg/100 ml of vitamin C, yet the addition of either one would promote the formation of monomeric ACN. Correlation analysis revealed a significantly negative correlation (-0.99) between the degradation index (DI) and monomeric ACN, which implied the preservation ability of pigment by monomeric ACN. As to the antioxidant capacity of the anthocyanins extract of purple napiergrass, the DPPH scavenging ability before heating presented no significant differences regardless of treatments, yet both the FRAP reducing power and TEAC total antioxidant capacity rapidly increased after heating with sucrose additions. FRAP reducing power augmented by two folds with sucrose alone or accompanied by vitamin C after being heated for 24 hours. Vitamin C alone in contrast reduced the antioxidant capacity during heating. The correlation coefficient between FRAP reducing power and monomeric ACN was determined to be 0.95 before heating. Heating the solutions with the presence of sucrose resulted in the large enhancement of FRAP yet decreasing monomeric ACN. It was hypothesized as the effect of sucrose browning reaction. The proof of existence for ascorbic acid-anthocyanin complex could not be deduced. The intensification of maximum absorption peaks for anthocyanin with various metal ions could be attributed to the copigmentation effects. The formation and the optimum environments for sustaining the complex would need to be investigated further.

碩士
國立宜蘭大學
園藝學系碩士班
104
Recently, there has been an increased interest in plant pigment and phenolic compounds of fruits, due to their antioxidant capacity. Other than that, as consumers are more wary of the usage of synthetic colour additives in food products, researchers are looking into the potential use of natural pigments such as anthocyanin, and also the methods to better retain this beneficial pigments. Thermal processing is the main method used in jam making, therefore the impact of this process on the bioactive compounds is important in order to assess the antioxidant capacity and colour quality changes over processing and storage of food product. The first part of this study aimed to determine the effect of processing and storage on colour, total phenolics, anthocyanin, carotenoid and antioxidant capacity of various fruit jams viz. passion fruit, kumquat, pineapple and dried roselle. While the second part of this study is to evaluate the effect of processing and storage of mix pineapple and roselle jams on their color, total phenolics, anthocyanin, carotenoid and antioxidant capacities. Roselle was added into pineapple pulp as a natural acidifier in order to meet the gelatinization requirement of pectin and increase antioxidative effect. The results of present study showed that jam processing caused significant changes in bioactive compounds of fruits, as well as their antioxidant capacity and colour properties. Long term storage of kumquat and roselle jam caused significant changes in total phenolics, total anthocyanin, total carotenoid and DPPH radical scavenging activity. While only significant reduction of total anthocyanin was found in passion fruit jam after storage. In addition, total phenolics, total anthocyanins and antioxidant capacity of pineapple showed significant reduction after 5 months of storage at room temperature. The addition of roselle pulp into pineapple jam increased the total anthocyanin compounds, when compared to only single pineapple jam. Total anthocyanins reduced significantly in all mix jam samples. In comparison of bioactive compound retention in different jam pH, pH 3.0 of pineapple and roselle mix jam was able to retain the highest percentage of total anthocyanin, total carotenoid and antioxidant capacity. It can also be observed that after storage in room temperature for 5 months, the presence of brown pigments increased as a result of anthocyanin degradation. Fruit jams also lost their bright colour. Despite the reduction in valuable bioactive compounds, jam processing is able to retain them during storage.

碩士
臺灣大學
食品科技研究所
98
This study investigated the cytoprotection effect of mulberry juice (MJ) anthothyanin on neuronal glial cell. The results showed that freeze dried mulberry juice powder (MJP) contained 19 mg /g anthocyanin. After purified with solid phase extraction device, anthocyanin content in freeze dried anthocyanin extract from mulberry juice (AEMJ) increased to 243 mg/g. The HPLC analysis revealed that cyanindin-3-glucoside and cyanidin-3-rutinoside are the major anthocyanin in AEMJ. The results of in vitro antioxidant test revealed that the DPPH free radical scavenging ability of MJP achieved to 81% at a concentration of 4 mg/ml. The DPPH free radical scavenging ability of 1 mg/ml AEMJ achieved to 90%, higher than that of catechin at the same concentration. The reducing power of MJP increased with the treated concentration. The absorption value was about 1.58 with the treated concentration of 2 mg/ml. The reducing power of AEMJ, similar to catechin, achieved to 2 at a concentration of 0.25 mg/ml. The rat glial tumor C6 cell was used for investigating the cytoprotection ability of MJP or AEMJ under hypoxia condition induced with Na2S2O4. The cell viability was 60.98 % for hypoxia rat glial tumor C6 cell control group at Na2S2O4 concentration of 5 mM. The cell viability of hypoxia C6 cells was 89.74% when pre-incubated with MJP contains 25 μg /ml anthocyanin. The cell viability of hypoxia C6 cells was 92.21% when pre-incubated with AEMJ contains 50 μg /ml anthocyanin. The intercellular antioxidant glutathione of hypoxia C6 cells increased, however, the superoxide dismutase activity of hypoxia C6 cells decreased, when pre-cultured with AEMJ. The results indicated that both MJP and AEMJ possess cytoprotection ability on hypoxia rat C6 glial cells.

8. Abstract The fruit of the European elder, Sambucus nigra L., is used in therapy as a drug Sambuci fructus for its diaphoretic, diuretic and laxative effects. Cultivars of European elder are being studied for the use in the food industry and pharmacy. The fruit contains especially large amount of anthocyanins and flavonoids that are known for their antioxidant activity. The aim of this work is to measure the antioxidant activity of the drug Sambuci fructus and of the fruit of the European elder cultivars Samdal, Mammut, Sambo, Sambu, Sampo, Bohatka, Haschberg, Weihenstephan, Allesö, and Samyl. An extract containing anthocyanins was obtained from fruit by a process based on extraction of frozen material with acidified water at high temperature, followed by separation with polymer non-ionogenic polystyrene-divinylbenzene sorbent. The content of anthocyanins in the extracts was determined spectrophotometrically using a modified pH-differential method. The absorbance was measured along the range of 350 - 600 nm. The content of the anthocyanins was quantified in units of mg of cyanidin 3-glucoside in 1 ml of extract. The content ranged from 0.33 - 2.76 mg cyanidin 3-glucoside/ml of extract. The highest content was in the extract from the fruit of the cultivar Samdal and the lowest was of the cultivar Mammut....

Phytochemicals found in grains complement those found in fruits and vegetables. These phytochemicals, though minor compounds, contribute to the antioxidant properties which are related to the health benefits associated with the consumption of whole grain. In this thesis project, nine barley genotypes of diverse origin namely CI2230 from Nepal, CI1248 from Israel, 3 Peruvian genotypes; Peru 3, Peru 16 and Peru 35, Hokuto Hadaka from Japan, EX116; a cross between Moroccan and Canadian genotype, EX83; a cross between two Canadian genotypes and EX127; a cross between Canadian and German genotypes were studied. The genotypes were categorized based on appearance into purple, black and yellow grains. Phenolic acids and flavonoids were identified and quantified in these diverse genotypes using HPLC-ESI-MS analysis. The main classes of dietary flavonoids studied in the barleys were anthocyanins and flavan-3-ols. Phenolic acids were identified and quantified (p-coumaric, ferulic, sinapic, caffeic, vanillic). Three ferulic acid dehydrodimers (8-0-4’ DFA, 8-5’ benzofuran form and 5-5’ DFA) were also identified. The most abundant dimeric flavan-3-ols were procyanidins B3 and prodelphinidin B3. The monomeric unit, (+)-catechin, was the most abundant while catechin glucoside (m/z 451) was also identified. Among the Peruvian genotypes, Peru 16 and Peru 35 exhibited relatively high levels of total PA content. Total phenolic content and antioxidant activities of methanolic, acetone and alkali hydrolyzed extracts of the nine barley genotypes was determined by the Folin- Ciocalteau assay, DPPH radical scavenging assay and oxygen radical absorbance capacity (ORAC assay).The acetone extract exhibited the highest antioxidant capacity using all the methods of analysis. Furthermore, dark colored grains were found to exhibit higher contents of phenolic compounds. The phenolic acids, PAs and anthocyanins identified and quantified had significant contribution to the overall antioxidant capacity of the barley whole grain. Four hull-less genotypes namely CI2230, EX127, CI1248 and Peru 35 were further partially sprouted to establish the effects of sprouting on phenolic acid composition. Partial sprouting was observed to significantly increase the soluble conjugated phenolic acids. The barley genotypes studied were found to contain different quantities of phytochemicals and had high proanthocyanidin content thereby rendering them as alternative sources of antioxidants. Barley sprouts present a possible novel food ingredient with improved properties such as phenolic acid composition and other benefits such as easier incorporation into food products under development.

Content of free- (FPC) and bound- phenolics (BPC) significantly (p<0.05) increased during mixing, fermenting and baking. Bread crust and crumb contained the highest FPC and BPC, respectively. Antioxidant activities (AOA) followed the trends of their respective phenolic contents. HPLC analysis demonstrated that different phenolic acids showed various responses to the bread-making process. Total anthocyanin content (TAC) was significantly (p<0.05) reduced through mixing and baking, but fermentation elevated the levels. Anthocyanin extract of purple wheat exerted higher AOA than those of common wheat. Digested purple wheat extracts after in-vitro digestion demonstrated significantly (p<0.05) higher AOA than common wheat. During in-vitro testing, extracts exhibited concentration-dependent effects, while the use of different cell lines exhibited varying levels of cellular antioxidant and pro-oxidant properties. Purple wheat demonstrated higher cytoprotectivity and cellular AOA than those of common wheat. Our findings suggest that purple wheat has the potential to act as functional food in bakery products.