The Maillard reaction is a network of different non-enzymatic reactions between carbonyl groups of reducing sugars and amino groups from amino acids, peptides, or proteins, which progresses in three major stages and originates a very heterogeneous mixture of reaction products. It is also known as non-enzymatic browning, due to the brown macromolecular pigments formed in the final stage of the reaction. The chemistry underlying the Maillard reaction is complex. It encloses not only one reaction pathway, but a whole network of various transformations. As virtually all foods contain both proteins and carbohydrates, Maillard reaction products are present in the daily diet in considerable amounts. The endogenous formation of Maillard reaction products, especially related to ageing and diabetes, aroused intense discussions about the health consequences of the “glycation”, the term that describes the in vivo reaction corresponding to the Maillard reaction in foods. Melanoidins are the final brown products of the Maillard reaction. They are responsible for the color formed during the heat processing of foods like coffee, bread, malt, and beef. Melanoidins are high molecular weight polydisperse polymers containing nitrogen. Their structure is largely unknown. Coffee melanoidins, which are object of the present study, contain thermally transformed polysaccharides, proteins, and phenolic compounds. Since the mechanisms involved on the formation of these macromolecules, and the chemical transformations which take place during the heat treatment are not completely elucidated, key structural features were analyzed. Especially the incorporation of chlorogenic acids in the melanoidin skeleton was object of attention of the present work. Another major aim of this work was to investigate the influence of the Maillard reaction on the inhibitory potential of food components against zinc metalloproteases. The studied enzymes were three human matrix metalloproteases (MMP-1, -2 and -9), which are able to degrade matrix proteins and participate in many physiological processes, including tissue turnover and repair, but also constitute important targets in malignant and degenerative diseases. A microbial collagenase from Chlostridium histolyticum was chosen due to its subtract similarity to MMPs. Furthermore, Angiotensin Converting Enzyme (ACE), which plays a central role in cardiovascular pathologies such as hypertension and cardiac hypertrophy, was investigated. As a prototypical Maillard reaction product, coffee melanoidin was adopted. Due to the roast dependent inhibitory activity of the coffee melanoidin fractions against matrix metalloproteases, the functionalization caused by the non-enzymatic browning was closer investigated. Na-carboxyalkylated derivatives of a sequence of relevant peptides were synthesized, in a variation of the process-induced formation of Nε-carboxymethyllysine, a major advanced glycation end-product (AGE). The inhibitory activity against zinc metalloproteases of the sequence of selected peptides and their Na-carboxymethyl- (CM-) and Na-carboxyethyl- (CE-) derivates was investigated.

Spécialisée dans la production d’actifs végétaux, l’entreprise PAT développe une activité innovante de recherche qui consiste à produire ou à modifier par voie enzymatique, des molécules présentes naturellement dans les plantes. L’espèce Ipomoea batatas contient de nombreux dérivés d’acide caféoyl-quinique dont majoritairement du 3,5-dicaféoyl-quinique (3,5-DCQ), une molécule antioxydante très recherchée en cosmétique. Cependant, la voie de biosynthèse est assez méconnue pour pouvoir exploiter les gènes d’intérêt par des approches d’ingénierie métabolique en vue d’augmenter la production. Les travaux réalisés dans le cadre de cette thèse ont porté sur l’identification et la caractérisation fonctionnelle d’une hydroxycinnamoyl transférase (HCT) en vue d’augmenter la production de 3,5-DCQ soit en micro-organismes soit dans des plantes recombinantes. Pour réaliser ces travaux, une banque de données RNAseq a été générée permettant ainsi d'accéder à des séquences codantes. Ces séquences ont été analysées par alignement avec des séquences codant pour des hydroxycinnamoyl transférases (HCT, HQT) impliquées dans la synthèse d’un précurseur potentiel, l’acide chlorogénique. Un premier tri a été mené en utilisant des approches d’expression différentielle de gènes. La fonction des gènes a été étudiée par des approches d’expression hétérologue en système bactérien et de transgénèse végétale. La production des métabolites cibles a été analysée dans des plantes transgéniques et dans des cultures cellulaires. Sur ces plantes, nous avons réalisé des tests de résistance à des pathogènes fongiques. Nous avons identifié une HCT qui partage 85% d’identité avec une HCT de café impliquée dans la synthèse de 3,5-DCQ. Cette activité a pu être démontrée in vitro pour l’HCT d’ipomée. De plus, l’expression du gène codant pour l’HCT conduit à une surproduction de 3,5-DCQ dans les cultures cellulaires de tabacs exprimant les HCT. Cette molécule inhibe la croissance de Botrytis cinerea et de Phytophthora parasitica. Compte tenu des teneurs en 3,5-DCQ très faibles par rapport à la plante d’origine, ces résultats suggèrent l’implication d’autres gènes dans cette voie de biosynthèse. L’activité antifongique du 3,5-DCQ pourrait être exploitée pour des applications agrochimiques / Specialized in the production of plant actives, the company PAT develops an innovate research activity that consists of producing or modifying by enzymatic pathway, molecules naturally present in plants. The species Ipomoea batatas contains numerous caffeoylquinic acid derivatives, predominantly 3,5-dicaffeoylquinic acid (3,5-DCQ), an antioxydant molecule arousing interest in cosmetics. However, biosynthesis pathway of this molecule is poorly established in order to exploit the genes of interest by metabolic engineering approaches to increase the production. The work realized in the frame of this PhD concerns the identification and functional characterization of a hydroxycinnamoyl transferase (HCT) in order to increase the production of 3,5-DCQ either in microorganisms or in recombinant plants. To perform this work, an RNAseq databank was generated allowing to access to coding sequences. These sequences were analysed by alignment of sequences encoding for hydroxycinnamoyl transferases (HCT, HQT) involved in the biosynthesis of a potential precursor, chlorogenic acid. A first screening was performed by utilizing an approach of differential expression of genes. The function of genes was studied by heterologous expression in bacterial systems and by plant transgenesis approaches. The production of target metabolites was analyzed in transgenic plants and cell cultures. On these plants, we conducted tests of resistance to fungal pathogens. We identified an HCT that shares 85% of identity with a HCT isolated from coffee previously characterized in 3,5-DCQ biosynthesis. This activity was shown in vitro for HCT of Ipomee. Moreover, the expression of target gene led to an overproduction of 3,5-DCQ in cell cultures of tobacco expressing HCT. This molecule inhibits growth of Botrytis cinerea and Phytophthora parasitica. Giving that amounts of 3,5-DCQ are very low compared to the plant of origin, these results suggest the involvement of other genes in this biosynthesis pathway. Antifungal activity of 3,5-DCQ could be exploited for agrochimic applications

The growing world demand for renewable energy production in substitution of fossil fuels has given great prominence in the culture of sugarcane (Saccharum sp.). This has been considered the most efficient crop for energy production, as Brazil being the world's largest producer, with a production of 655.6 million tons in 2015/2016 harvest in an area of 8.5 million hectares. One of the major obstacles to the production of sugarcane is still the attack by pest and estimates that about 10% of the crop losses are caused by insects, being a sugarcane borer (Diatraea Saccharalis) the most importante pest. The plants, during its evolution, to reduce the damages caused by the attack of pests, they have developed a series of defense mechanisms, among them, physical barriers, proteins and toxic metabolites and volatile metabolic flags. Many works have been developed to decipher the mechanisms of defense of sugarcane to the attack of herbivorous insects, an attempt of genetic breeding programs and information biotechnology for the development of more resistant plants, however, many of the mechanisms still remain to be clarified. These works are mostly restricted to the study of genes and proteins and global studies of transcript and proteome analyzes of the plant. The current work proposes to perform an analysis of the metabolism of two varieties of sugarcane (RB92579 and SP791011) through direct and indirect extraction methods in response to herbivory by Diatraea saccharalis using NMR spectroscopy and multivariate statistical analysis from data by principal component analysis (PCA) and least squares analysis of discrimination (OPLS-DA). In the metabolomic analysis of sugarcane of the variety RB92579 was studied using the direct extraction method (leaves) in the 4 herbivory times (24, 48, 72 and 96 hours), only after 48, 72 and 96 hours of stress by herbivory, it was possible to get differences between the control groups and herbivore groups, which the greater timing of response was obtained in 72 hours, that indicated the increase of the primary metabolites asparagine, aspartic acid, dimethylamine, glutamic acid , isoleucine, leucine, malic acid, tyrosine and phenylalanine. The variety of sugarcane SP791011, the method of indirect extraction of the leaves of the plant was applied in times 24, 48 and 72 hours by stress induced by caterpillars of Diatraea saccharalis and, coincidentally, showed in the time 72 hours a greater number of herbivory response metabolites, which caused a depletion of aconitic acid, formic acid, asparagine, alanine and elevation of acetic acid and chlorogenic acid. Despite their different metabolic profiles in response to herbivory, the elucidated metabolites suggest the metabolic pathway of shikimic acid to produce phenylpropanoids due to the increase of tyrosine, phenylalanine in the leaves of the sugarcane variety RB92579, and increase of chlorogenic acid in the leaves of the cane. In addition to the herbivory test, it was carried out a biological assay with chlorogenic acid inserted in the artificial diet of Diatraea saccharalis caterpillars, which demonstrated a decrease in the development time of the pupae comparing with pupae of Caterpillars Control, which caused an outbreak of moths with deformations at all concentrations of chlorogenic acid used in the bioassay. These results may help in the development of sugarcane varieties more resistant to the attack by Diatraea Saccharalis. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A crescente demanda mundial para a produção de energias renováveis em substituição aos combustíveis fósseis tem dado grande destaque à cultura da cana-de-açúcar (Saccharum sp.). Esta tem sido considerada a cultura mais eficiente para a produção de energia, tendo o Brasil como o maior produtor mundial, com uma produção de 655,6 milhões de toneladas na safra 2015/2016 em uma área de 8,5 milhões de hectares. Um dos grandes entraves à produção de cana-de-açúcar ainda é o ataque de pragas e doenças e estima-se que cerca de 10% das perdas para esta cultura sejam ocasionadas por insetos, sendo a broca da cana-de-açúcar (Diatraea saccharalis) a praga mais importante. As plantas, durante a evolução, para reduzir os danos causados pelo ataque de pragas, têm desenvolvido uma série de mecanismos de defesa, dentre eles, barreiras físicas, proteínas, metabólitos tóxicos e metabólitos voláteis sinalizadores. Muitos trabalhos têm sido desenvolvidos para decifrar os mecanismos de defesa da cana-de-açúcar ao ataque de insetos herbívoros, na tentativa de subsidiar programas de melhoramento genético e a biotecnologia de informação para o desenvolvimento de plantas mais resistentes, porém, muitos destes mecanismos ainda permanecem a ser esclarecidos. Estes trabalhos, em sua maioria, estão restritos ao estudo de genes e proteínas e a estudos globais de análises dos transcritos e do proteoma da planta. O presente trabalho propõe realizar a análise do metaboloma de duas variedades de cana-de-açúcar (RB92579 e SP791011) através dos métodos de extração direto e indireto na resposta a herbivoria por Diatraea saccharalis, utilizando espectroscopia de RMN, e processamento e estatística dos dados pelos métodos de análise de componentes principais (PCA) e análise dos mínimos quadrados parciais-análise discriminante (OPLS-DA). A análise metabolômica da cana-de-açúcar da variedade RB92579 foi realizada pelo método de extração direta (folhas) nos 4 tempos de herbivoria (24, 48, 72 e 96 horas), sendo que, apenas após 48, 72 e 96 horas sob herbivoria, foi possível haver diferenças entre os grupos controle e grupo herbivoria, no qual o tempo em que se obteve maior resposta da planta foi no tempo 72 horas, que indicou o aumento dos metabólitos primários asparagina, ácido aspártico, dimetilamina, ácido glutâmico, isoleucina, leucina, ácido málico, tirosina e fenilalanina. Já com relação a variedade de cana-de-açúcar SP 1011, aplicou-se o método de extração indireto das folhas da planta nos tempos 24, 48 e 72 horas sob estresse induzido pelas lagartas de Diatraea saccharalis e, coincidentemente, apresentou no tempo 72 horas o maior número de metabólitos de resposta a herbivoria, o que causou numa redução dos níveis de ácido cis e trans aconítico, ácido fórmico, asparagina, alanina e no aumento dos níveis de ácido acético e ácido clorogênico. Apesar de fornecerem perfis metabólicos diferentes em resposta a herbivoria, os metabólitos elucidados sugerem a via metabólica do ácido chiquímico devido ao aumento de tirosina, fenilalanina nas folhas da cana-de-açúcar da variedade RB92579, e aumento do ácido clorogênico nas folhas da cana-de-açúcar da variedade SP791011. Além do ensaio de herbivoria, foi realizado um ensaio biológico com o ácido clorogênico inserido na dieta artificial das lagartas de Diatraea saccharalis, que demonstrou uma diminuição do tempo de desenvolvimento das pupas em comparação com as pupas de lagartas controle, que provocou a eclosão das mariposas com deformações em todas as concentrações de ácido clorogênico usadas no bioensaio. Esses resultados podem ajudar no desenvolvimento de variedades de cana-de-açúcar mais resistentes ao ataque da Diatraea Saccharalis.

Introdução: A incidencia e prevalencia do Diabetes Mellitus aumentam a cada ano, alcancando proporcoes epidemicas. A infusao aquosa de erva mate apresenta consideraveis teores de acidos clorogenicos, que alem de atuarem como antioxidantes, podem diminuir a producao e absorcao de glicose, conforme indicado na literatura. Objetivos: Avaliar a influencia da ingestao de infusao de erva mate sobre parametros bioquimicos relacionados ao diabetes mellitus e o metabolismo de glicose. Metodologia: Ratos Wistar (n=41) foram divididos em: nao diabeticos controle (NDC, n=10); nao diabeticos erva mate (NDE, n=10); diabeticos controle (DC, n=11) e diabeticos erva mate (DE, n=10). O diabetes foi induzido por aloxana. Os animais receberam extrato de erva mate (1 g/kg) ou solucao fisiologica por gavagem durante 28 dias, com agua e racao comercial ad libitum. Seus tecidos foram submetidos as analises (glicemia, insulinemia, colesterol total, atividade da enzima glicose-6-fosfatase hepatica e expressao genica do cotransportador intestinal de Na+/glicose SGLT1, sendo este responsavel pela maior parte da absorcao de glicose no lumen). Os dados foram analisados por analise de variancia com dois fatores (ANOVA 2-way) com ou sem medidas repetidas, de acordo com a variavel. O nivel de significancia adotado foi 5%. Resultados: Nao houve diferenca significativa entre os grupos controle (NDC e DC) e os grupos que ingeriram erva mate (NDE e DE) para a glicemia, insulinemia, colesterol total e atividade da glicose-6-fosfatase hepatica. No entanto, a expressao genica 7 dos transportadores de glicose SGLT1 foi significantemente menor nos animais que receberam erva mate, tanto no duodeno (p=0,007) quanto no jejuno (p<0,001) Conclusão: A ingestao da erva mate nao modificou significativamente os parametros bioquimicos estudados dos animais sob intervencao relativamente ao controle. No entanto, foi observada diferenca significativa quanto a expressao do transportador de glicose SGLT1, sugerindo que compostos bioativos da erva mate sao capazes de reduzir a absorcao da glicose. / Introduction: The incidence and prevalence of Diabetes Mellitus are increasing, reaching epidemic proportions. Yerba mate infusions are rich in polyphenols, especially chlorogenic acids, that are known to have antioxidant properties. Evidences suggest that dietary polyphenols could also play a role in glucose metabolism and absorption. Objective: The aim of this study was to evaluate if yerba mate extract could have antidiabetic properties in alloxaninduced diabetic Wistar rats. Research design and methods: Wistar rats (n=41) were divided in four groups: non diabetic control (NDC, n=10); non diabetic yerba mate (NDM, n=10); diabetic control (DC, n=11) and diabetic yerba mate (DM, n=10). Diabetes was induced by alloxan (38 mg/kg bw). The mate group animals received yerba mate extract diluted in saline solution in a 1g extract/kg bw dose for 28 days, controls received saline solution only. The following biochemical parameters were measured: serum glucose, insulin and total cholesterol; hepatic glucose-6-fosfatase activity and gene expression of the intestinal Na+/glucose cotransporter SGLT1. Results: There were no significant differences in serum glucose, insulin, total cholesterol and hepatic glucose-6-phosphatase activity between the groups that ingested yerba mate extract (NDM and DM) and the controls (NDC and DC). However, the intestinal SGLT1 gene expression was significantly lower in animals that received yerba mate both in upper (p=0.007) and middle small intestine (p<0,001). Conclusion: These results indicate that bioactive 9 compounds present in yerba mate might be capable to decrease intestinal glucose absorption, by decreasing SGLT1 expression.

As leishmanioses são antropozoonoses com amplo problema de saúde pública, que acometem aproximadamente 12 milhões de indivíduos em todo o mundo e causam cerca de 60 mil mortes por ano. No Brasil, a leishmaniose tegumentar (LT) apresenta coeficiente médio de detecção de 16 casos por 100 mil habitantes, onde em notificações no estado do Maranhão apresenta um dos maiores índices de incidência. Os fármacos utilizados atualmente apresentam eficácia limitada e algumas vezes significante toxicidade e efeitos adversos, sendo necessária a busca por novos tratamentos. Um dos meios para obtenção deste propósito é obter extratos vegetais que proporcionem a inativação da enzima arginase presente no parasito e responsável pela produção de poliaminas essenciais a sua sobrevivência. A escolha do material vegetal foi baseada através de práticas medicinais de populações onde a planta Stachytarpheta cayennensis é utilizada como analgésica, antiinflamatória e no tratamento de úlceras causadas por Leishmania. A planta foi coletada e feita a identificação botânica, e a droga foi utilizada para preparo de dois extratos por decocção e maceração em etanol 50%. Ambos foram fracionados com n-butanol e utilizados em testes de inibição da arginase. A fração butanólica do chá (BUF) foi a que apresentou menor número de constituintes. A análise do BUF por ressonância magnética nuclear indicou a presença de uma mistura de 7:3 de verbascosídeo/isoverbascosideo. Em seguida foi realizado o teste de inibição enzimática com a mesma fração e com os compostos estruturalmente relacionado com o verbascosídeo: ácido cafeico (IC50 = 1.5 &micro;M), ácido clorogênico (IC50 = 3.4 &micro;M), e ácido rosmarínico (IC50 = 1.5 &micro;M). O teste de inibição na forma promastigota do parasito com a fração (BUF-CHÁ) em 72h de tratamento apresentou EC50 de 51 &micro;g/mL. A partir dos resultados encontrados faz-se necessário mais experimentos com a planta, como a realização de ensaios com amastigotas de Leishmania e teste de toxicidade celular. Conclusão: S. cayennensis pode ser uma promissora fonte de novos fármacos para leishmaniose. / Stachytarpheta cayennensis is a plant traditionally used to treat tegumentary Leishmaniasis and as an anti-inflammatory. The aim of this study was to evaluate the mechanisms of action of extracts from S. cayennensis on the arginase enzyme of the trypanosome parasite Leishmania (Leishmania) amazonensis. S. cayennensis was collected in Formosa da Serra do Maranhão, Brazil. Crude water extract was fractionated with n-butanol and fractions were tested against arginase enzymes collected from L. (L.) amazonensis. The most potent arginase inhibitor fraction was then tested against L. (L.) amazonensis promastigotes in axenic culture. To verify arginase inhibition in L. (L.) amazonensis, promastigote cultures were supplemented with L-arginine (substrate) and L-ornithine, a product of hydrolysis of L-arginine by the arginase enzyme. Butanolic fraction (BUF) is a potent L. (L.) amazonensis arginase inhibitor (IC50 = 5 ± 1 &micro;g/mL). BUF showed an IC50 of 51 &micro;g/mL against L. (L.) amazonensis promastigotas. In addition, caffeic acid and two acids containing caffeoyl moiety were tested against arginase showing IC50 1.5-3.4 &micro;M. The inhibition of arginase by BUF in the promastigote cultures was demonstrated by adding L-ornithine, which enhances parasite growth. In conclusion, verbascoside present in S. cayennensis extracts (BUF) target the arginase enzyme of L. (L.) amazonensis, resulting in the death of the parasites.

The Maillard reaction is a network of different non-enzymatic reactions between carbonyl groups of reducing sugars and amino groups from amino acids, peptides, or proteins, which progresses in three major stages and originates a very heterogeneous mixture of reaction products. It is also known as non-enzymatic browning, due to the brown macromolecular pigments formed in the final stage of the reaction. The chemistry underlying the Maillard reaction is complex. It encloses not only one reaction pathway, but a whole network of various transformations. As virtually all foods contain both proteins and carbohydrates, Maillard reaction products are present in the daily diet in considerable amounts. The endogenous formation of Maillard reaction products, especially related to ageing and diabetes, aroused intense discussions about the health consequences of the “glycation”, the term that describes the in vivo reaction corresponding to the Maillard reaction in foods. Melanoidins are the final brown products of the Maillard reaction. They are responsible for the color formed during the heat processing of foods like coffee, bread, malt, and beef. Melanoidins are high molecular weight polydisperse polymers containing nitrogen. Their structure is largely unknown. Coffee melanoidins, which are object of the present study, contain thermally transformed polysaccharides, proteins, and phenolic compounds. Since the mechanisms involved on the formation of these macromolecules, and the chemical transformations which take place during the heat treatment are not completely elucidated, key structural features were analyzed. Especially the incorporation of chlorogenic acids in the melanoidin skeleton was object of attention of the present work. Another major aim of this work was to investigate the influence of the Maillard reaction on the inhibitory potential of food components against zinc metalloproteases. The studied enzymes were three human matrix metalloproteases (MMP-1, -2 and -9), which are able to degrade matrix proteins and participate in many physiological processes, including tissue turnover and repair, but also constitute important targets in malignant and degenerative diseases. A microbial collagenase from Chlostridium histolyticum was chosen due to its subtract similarity to MMPs. Furthermore, Angiotensin Converting Enzyme (ACE), which plays a central role in cardiovascular pathologies such as hypertension and cardiac hypertrophy, was investigated. As a prototypical Maillard reaction product, coffee melanoidin was adopted. Due to the roast dependent inhibitory activity of the coffee melanoidin fractions against matrix metalloproteases, the functionalization caused by the non-enzymatic browning was closer investigated. Na-carboxyalkylated derivatives of a sequence of relevant peptides were synthesized, in a variation of the process-induced formation of Nε-carboxymethyllysine, a major advanced glycation end-product (AGE). The inhibitory activity against zinc metalloproteases of the sequence of selected peptides and their Na-carboxymethyl- (CM-) and Na-carboxyethyl- (CE-) derivates was investigated.:LIST OF CONTENTS I LIST OF TABLES IV LIST OF FIGURES V LIST OF ABBREVIATIONS VII 1 INTRODUCTION 1 2 BACKGROUND 3 2.1 Maillard reaction in food 3 2.1.1 Melanoidins 8 2.2 Coffee 11 2.2.1 General aspects 11 2.2.1.1 Coffee production 12 2.2.1.2 General chemical composition 14 2.2.1.3 Coffee and health 20 2.2.2 Coffee melanoidins 24 2.2.2.1 Chemistry of coffee melanoidins 24 2.2.2.2 Properties of coffee melanoidins 29 2.3 Zinc metallopeptidases 32 2.3.1 Matrix metalloproteinases (MMPs) 33 2.3.1.1 Functions of MMPs 35 2.3.1.2 Structure of MMPs 37 2.3.1.3 Inhibition of MMPs 39 2.3.2 Clostridium histolyticum collagenase (ChC) 43 2.3.2.1 Functions of ChC 43 2.3.2.2 Structure of ChC 43 2.3.2.3 Inhibition of ChC 44 2.3.3 Agiotensin converting enzyme (ACE) 45 2.3.3.1 Functions of ACE 45 2.3.3.2 Structure of ACE 46 2.3.3.3 Inhibition of ACE 48 3 EXPERIMENTAL SECTION 50 3.1 Chemicals, materials and equipment 50 3.1.1 Chemicals 50 3.1.2 Material 52 3.1.3 Equipment 52 3.1.4 Solutions 54 3.2 Synthesis of Nα-carboxyalkylated peptides 55 3.2.1 Nα-carboxyalkylation of GP, LL, IA, GA, GL, AP, IP and IPP by reductive alkylation 55 3.2.2 Nα-carboxyalkylation of IW using sodium cyanoborohydride 56 3.3 Purification 57 3.3.1 Ion Exchange Chromatographic purification 57 3.3.1.1 Spotting test 58 3.3.2 HPLC purification of CM-IW 58 3.3.3 Overview of the synthesis and elution conditions 59 3.4 Characterization of carboxyalkylated peptides 61 3.4.1 Mass spectrometry 61 3.4.2 Elemental Analysis 61 3.4.3 Analytical characteristics of carboxyalkylated peptides 62 3.5 Preparation of coffee fractions 65 3.5.1 Roasting conditions 65 3.5.2 Fractionation of coffee samples: Isolation of coffee melanoidins 67 3.6 Structural studies 69 3.6.1 Estimation of the molecular weight 69 3.6.2 C/N ratio 70 3.6.3 Amino acid analysis 70 3.6.3.1 Acid hydrolysis 70 3.6.3.2 General amino acid analysis 70 3.6.3.3 Lysinoalanine 71 3.6.3.4 Pentosidine 72 3.6.4 Total phenols 74 3.6.5 Raman spectroscopy 74 3.7 Study on inhibition of zinc metalloproteases 75 3.7.1 Inhibition of ACE 75 3.7.1.1 General enzymatic assay 75 3.7.1.2 Quantification 78 3.7.2 Inhibition of MMP-1, -2 and -9 79 3.7.2.1 General enzymatic assay 80 3.7.2.2 Effect of zinc addition on the inhibition of MMP-1 by melanoidins 81 3.7.3 Inhibition of ChC 82 3.7.3.1 General enzymatic assay 82 3.7.3.2 Quantification 84 3.7.4 Calculation of IC50 84 4 RESULTS AND DISCUSSION 86 4.1 Coffee melanoidins 86 4.1.1 Isolation of coffee fractions 86 4.2 Inhibition of zinc-dependent peptidases by coffee fractions 89 4.2.1 Inhibition of MMPs 89 4.2.2 Inhibition of other zinc metalloproteases 98 4.2.3 General considerations 99 4.3 Structural studies on coffee melanoidins 101 4.3.1 Gel permeation chromatography 102 4.3.2 Elemental analysis: C/N ratio 113 4.3.3 Amino acid analysis 116 4.3.4 Total phenolics 120 4.3.5 Correlation between total phenols content and C/N ratio in coffee melanoidins 123 4.3.6 Raman spectroscopy 124 4.4 Derivatization of peptides 129 4.4.1 Nα-carboxyalkylation of peptides by reductive alkylation 130 4.5 Preliminary investigations on the inhibitory potential of Nα-carboxyalkyl derivatives of peptides against metalloproteases 133 4.5.1 Inhibition against ACE 134 4.5.2 Inhibition against other zinc metalloproteases 138 5 SUMMARY 141 6 REFERENCES 145 LIST OF PUBLICATIONS AND CONFERENCE CONTRIBUTIONS 168 AKNOWLEDGMENTS 169 ERKLÄRUNG 170

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Au cours de ce travail de thèse nous avons étudié l’influence des apports en azote nitrique sur le contenu en polyphénols chez la tomate. Plusieurs dispositifs de culture hors sols ont été utilisés (hydroponie, laine de roche, NFT). Nous avons quantifié les principaux composés phénoliques de la tomate, à savoir : l’acide chlorogénique, la rutine, le kaempférol rutinoside dans les parties végétatives de la plante (limbe, tige et racine), ainsi que des dérivés de l’acide caféique et la naringénine chalcone dans les fruits. Ces composés ont été analysés par chromatographie liquide à haute performance. Nous avons observé que les limbes étaient le compartiment le plus sensible aux conditions de nutrition. Nous y avons observé une multiplication par deux des concentrations en polyphénols lors d’une diminution des apports en azote de 15 à 0.05mM NO3-. Nous avons mis en évidence, en établissant des courbes de réponses, que ces augmentations s’effectuaient de façon nettement plus importante dans des conditions de nutrition azotée limitante pour la croissance de la plante. Dix à 20 jours de carence ou de limitation semblent nécessaires pour obtenir une modification du contenu en polyphénols à l’échelle de la plante. Dans les fruits les concentrations en polyphénols ont été modifiées par le niveau de fertilisation azotée, mais nous n’avons pas mis en évidence d’augmentations très significatives. L’impact d’autres facteurs, comme les conditions climatiques (lumière et température) a également été pris en compte. Nos résultats semblent indiquer que les conditions climatiques régissent le contenu en polyphénols de manière plus importante que la nutrition azotée / During my PhD we studied the effects of nitrate supply on tomato polyphenolics content. Several cultural systems were used (hydroponic, rockwool culture, NFT). We quantified the main tomato phenolic compounds: chlorogenic acid, rutine, kaempferol rutinoside in vegetative parts (leaf, stem, root), together with some caffeic acid derivates and naringenine chalcone in fruits. These compounds were analyses by High Performance Liquid Chromatography. We noticed that leaves were the more responsive compartment of the plant, to the nutrition conditions. We observed a two-fold increase in polyphenolics concentrations when nitrate supply decrease from 15 to 0.05mM. We found, by drawing response curves, that this increase was more important when nitrate supply limited plant growth. Ten to 20 days seem necessary to observe a modification, at the plant level, of polyphenolics content. In tomato fruits, polyphenolics concentrations were modified by the nitrate supply, but we do not observed very significant increases. The effects of other environmental factors, such as climate conditions (light, temperature) were studied. Our results seem to indicate that climate is more important than nitrogen nutrition for the determination of the polyphenolic compounds concentrations

Made available in DSpace on 2015-03-26T13:30:53Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1175860 bytes, checksum: 8d49f98b8b8a4ab64b4d0d84434f5297 (MD5) Previous issue date: 2007-02-14 / Universidade Federal de Viçosa / The green cochineal Coccus viridis (green) (Hemipetera: Coccidae) causes problems in sapling plants of Coffea arabica and in parts of the canopy under low luminosity. Plant fertilization with nutrients like nitrogen and potassium may influence the survival, the development, the growth, the reproduction and the behavior of insects. The impact of the application of nitrogen and potassium doses may have direct effects (via nutrients on leaves) and indirect (on phytochemists) on C. viridis. Another impact of nitrogen and potassium fertilization on the interaction of C. viridis on coffee trees is on the tolerance of the plants to the losses caused by this insect-pest. So far, this work aimed to study the relation among nitrogen and potassium doses given to the plants, concentration of foliar phytochemical compounds and attack of C. viridis and also to determine the losses incurred to plants of C. Arabica by this insect. This research was conducted in a green house. Deficient, normal and excessive nitrogen and potassium fertilizations were used. Each treatment was composed of two plants (infested and uninfested). Nymphs and adults were counted every week. The phytochemical and nutrient levels on leaves were determined for infested plants, and dry material of roots, stem, leaves and overall for uninfested plants. The analysis conducted were those of Pearson´s correlation, path and multiple linear regressions. Raised the nitrogen levels in the nutritive solution, the intensity of nymph and adult attacks of C. viridis were observed throughout the experiment period. A direct impact of those nutrients was observed through the increasing levels of nitrogen on leaves. On the other hand, the indirect effect is due to the decreasing of caffeine levels, chlorogenic acid and cafeic acid on leaves that may act as alomones on C. viridis. It was also observed that plants when fertilized with larger doses of nitrogen and potassium presented smaller loss of foliar, stem and total dry material as well as a smaller diameter reduction when attacked by that pest. / A cochonilha verde Coccus viridis (Green) (Hemiptera: Coccidae) causa problemas em plantas jovens de Coffea arabica e em partes do dossel com baixa luminosidade. A adubação das plantas com nutrientes como o nitrogênio e o potássio pode influenciar a sobrevivência, o desenvolvimento, o crescimento, a reprodução e o comportamento dos insetos. O impacto da aplicação de doses do nitrogênio e do potássio pode ter efeitos diretos (via nutrientes na folha) e indiretos (sobre os fitoquímicos) sobre C. viridis. Outro impacto da adubação nitrogenada e potássica sobre a interação de C. viridis no cafeeiro é na tolerância das plantas às perdas causadas por este inseto-praga. Assim este trabalho teve por objetivo estudar as relações entre doses de nitrogênio e de potássio fornecidas às plantas, concentração de compostos fitoquímicos foliares e ataque de C. viridis, e ainda, determinar as perdas em vigor causadas por este inseto a plantas de C. arabica. Esta pesquisa foi conduzida em casa de vegetação. Utilizaram-se adubações de nitrogênio e de potássio em deficiência, normal e excessiva. Cada tratamento foi composto por duas plantas (infestada e não infestada). Semanalmente, contaram-se os números de adultos e de ninfas nas plantas. Foram determinados os teores dos fitoquímicos e nutrientes nas folhas para plantas infestadas e matéria seca das raízes, caule, folhas e total para plantas não infestadas. Realizou-se análise de correlação de Pearson, análise de trilha e regressão linear múltipla. Verificou-se que com a elevação dos teores de nitrogênio na solução nutritiva ocorreu aumento da intensidade de ataque de ninfas e de adultos de C. viridis ao cafeeiro ao longo do tempo. Verificou-se que estes nutrientes têm impacto direto através do aumento dos teores de nitrogênio nas folhas. Já o efeito indireto deve-se à redução dos teores de cafeína, ácido clorogênico e ácido cafeico nas folhas, os quais atuam possivelmente como alomônios sobre C. viridis. Observou-se que plantas adubadas com maiores doses de nitrogênio e de potássio tiveram menores perdas de matéria seca total, foliar, caule e menor redução do diâmetro quando atacadas pela praga.

The thesis deals with the content of selected phenolic compounds in some species of the genus Amaranthus, in black elderberry (Sambucus nigra L.) and buckwheat (Fagopyrum esculentum M.). Phenolic compounds are a group of natural compounds exclusively vegetable character. Flavonoids represent only one group of phenolic compounds. Flavonoids show many positive biological effects, in particular act as antioxidants. Natural flavonoids may cause to prevent from coronary- heard diseases and other diseases associated with older age. In recent years the increased attention is paid to flavonoid investigation due to its biological effects. For the determination of phenolic substances there were used two independent analytical methods. There are the high-performance liquid chromatography (HPLC) and micellar electrokinetic capillary chromatography (MECC). The MECC method was used for determination rutin and free quercetin. The highest content of rutin was found in leaves of buckwheat (76,400 mg/kg of dry weight) and the lowest content of rutin was determined in buckwheat hulls. The highest content of rutin was observed in teas from buckwheat leaves and inflorescence. This amount of rutin corresponds with rutin content in more than two pills of Ascorutin (the most favourite flavonoid medicament in the Czech Republic) The HPLC method was used for quantitative determination of phenolic acids. The content of free quercetin was monitored in all samples. No free quercetin was found both in plant material and in samples of teas. The ethanolic extract from the elderberry inflorescence didn´t contain any free quercetin. Free quercetin wasn?t found in any further samples of teas, which were prepared by described methods.