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Coffee contains many antioxidants including purpurogallin, which is a hydrophobic phenolic antioxidant that is difficult to measure with reported methods. A method combining solid-phase extraction and liquid chromatography–mass spectrometry was developed to detect and quantify purpurogallin in brewed beverages, including coffee. For beverage preparation, water extraction was adopted for improved correlation with moka pot brewing. Purpurogallin was detected in all commercial coffee samples, and its content in ground coffees ranged from 455-630 ng/g dry weight. Purpurogallin was only detected in two English breakfast tea samples (335-360 ng/g dry weight) and was not detected in any cocoa sample. Antioxidant activity, total phenolic content, and phenolic profile of coffees with different degrees of roasting were determined and analyzed. The developed methodology was then further improved, and coffees with different roasting degrees were analyzed for their antioxidant activity, total phenolic content, and phenolic profile. The antioxidant activity ranged from 63.9-92.0 mg Trolox equivalents per gram of coffee (dry weight), and the total phenolic content ranged from 36.0-57.7 mg gallic acid equivalents per gram of coffee (dry weight). However, the total phenolic content was not correlated with the roasting degree (p > 0.05). When the roasting degree increases, chlorogenic acid decreases drastically, but shikimic acid, caffeic acid, gallic acid, pyrogallol, and purpurogallin increase correspondingly. The results suggest that purpurogallin is a common antioxidant in roasted coffees, and an increase in roasting degree will not only lead to dramatic breakdown of chlorogenic acid, but also promote significant formation of other phenolic compounds that can provide antioxidant activity.

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.

Cecropia obtusa é popularmente utilizada na Amazônia para vários fins medicinais, e possui atividade antioxidante comprovada por diferentes metodologias. Neste trabalho objetivou-se caracterizar o extrato; avaliar o potencial fotoquimioprotetor; separar os constituintes por CLAE; identificar os constituintes majoritários e desenvolver formulações tópicas. O conteúdo de polifenóis e flavonoides totais foi de 371,5 mg EGA/g e 66,68 mg EQ/g, respectivamente, e os IC50 para os radicais DPPHo, O2 o- e 1O2 foram, respectivamente1,83, 0,34 e 0,55 ?g/mL, respectivamente. Para o radical OHo, 125 ?g/mL de extrato foi capaz de inibir a formação do radical em 35,48%. O extrato demonstrou fotoestabilidade e capacidade de absorver a radiação UVB e UVA-II. O FPS calculado pela metodologia de Mansur e estimado pelo experimento de fotoproteção em cultura de células foi de 16. O método analítico cromatográfico apresentou boa resolução e separação, sendo linear para o intervalo de 5 a 125 ?g/mL e apresentando coeficientes de variação que variaram de 3,12% a 4,51%. Através da espectrometria de massas foi possível sugerir a presença do ácido clorogênico e três flavonas-C-heterosídicas (duas luteolinas-C-heterosídicas e uma apigenina-C-heterosídicas). Em cultura de células HaCaT submetidas à radiação UVA, o extrato diminuiu a produção de espécies reativas do oxigênio, protegeu contra o processo de peroxidação lipídica e contra a depleção da glutationa reduzida induzidos pela radiação, além de proteger contra a diminuição da atividade das enzimas catalase e superóxido dismutase. Em cultura de fibroblastos humanos submetidos à radiação UVA/UVB, o tratamento com o extrato forneceu maior proteção do que o ácido clorogênico contra o aumento da proteína carbonilada. O extrato foi capaz de retornar o conteúdo de ácido hialurônico ao nível basal enquanto o tratamento com ácido clorogênico foi capaz de aumentar acima do nível basal. Em relação ao conteúdo de colágeno, tanto o extrato quanto o ácido clorogênico foram capazes de retornar os valores ao nível basal da célula. O extrato também foi capaz de diminuir a atividade da enzima metaloproteinase enquanto o ácido clorogênico não exibiu efeito. Em relação às formulações desenvolvidas, as mudanças mais significativas nos parâmetros de estabilidade avaliados foram para as formulações incubadas a 40?C: o decréscimo da atividade antioxidante em aproximadamente 40% entre 0 e 2 meses, a diminuição do pH abaixo do limite de estabilidade do agente de viscosidade e, para a reologia, o aumento da área de histerese entre 4 e 6 meses. A formulação com oleato de isodecila dobrou a quantidade de extrato penetrada na pele em 12 e 6 horas, em comparação com as outras duas formulações, além de ter mantido a atividade de absorção da radiação. O extrato apresentou atividade fotoquimioprotetora e a formulação promoveu a penetração do extrato e manteve a atividade de absorção da radiação, sugerindo a incorporação do extrato de C. obtusa em formulações contendo filtros, visando aumentar o espectro de absorção e promover um efeito atenuador do estresse oxidativo na pele. / Cecropia obtusa is commonly used in Amazon with different medicinal purposes and the antioxidant activity of this specie has been proved by different methodologies. The aim of this work was: to characterize the purified extract of C. obtusa; to evaluate the photochemoprotective effect in cell culture; develop an HPLC methodology to separate the extract\'s compounds; suggest the identification of the major compounds, and develop a stable formulation that would be able to penetrate on the skin to exert the antioxidant effect, besides absorbing the radiation. The contents of total polyphenols and flavonoids were 371.5 mg GAE/g and 66.68 mg QE/g, respectively, and the IC50 for the radicals DPPHo, O2 o- and 1O2 were respectively 1.83, 0.34 and 0.55 ?g/mL. For the OHo radical, 125 ?g/mL of extract was able to inhibit the radical formation by 35.48%. The extract was photo stable and exhibited the ability to absorb UVB and UVA-II radiation. The SPF calculated by the Mansur methodology and confirmed by the photoprotection experiment in cell culture was 16. The HPLC method exhibited good resolution and separation, being linear from 5 to 125 ?g/mL and presenting coefficients of variation ranging from 3.12% to 4.51%. By employing mass spectrometry, it was possible to suggest the presence of chlorogenic acid and three C-heterosidic flavones (two Cheterosidic luteolins and one C-heterosidic apigenin). In HaCaT culture exposed to UVA radiation, the extract decreased the production of reactive oxygen species, protected against the lipid peroxidation process and against the depletion of the reduced glutathione. In addition, extract also protected against the decrease in the activity of the enzymes catalase and superoxide dismutase. In human fibroblasts exposed to UVA/UVB radiation, the treatment with the extract provided higher protection than chlorogenic acid against the increase in the production of carbonyl protein. The extract was able to return the content of hyaluronic acid to the basal level while the treatment with chlorogenic acid was able to increase above the basal level. Regarding the collagen content, both the extract and the chlorogenic acid were able to return the values to the basal level of the cell. The extract was also able to decrease metalloproteinase activity, while chlorogenic acid had no effect. Regarding the developed formulations, the most significant changes in the stability parameters were for the formulations incubated at 40°C: the decrease in the antioxidant activity, which was, approximately, 40% between 0 and 2 months, and the increase of hysteresis area and decrease of the pH below the limit for the viscosity agent, between 4 and 6 months. The formulation with isodecyl oleate doubled the amount of extract penetrated into the skin in 12 and 6 hours compared with the other two formulations. The extract exhibited photochemoprotective effect, and the formulation penetrated on the skin, besides absorbing the UV radiation, which suggests the incorporation of C. obtusa extract in formulations containing filters, aiming to increase the absorption spectrum and to attenuate the oxidative stress in the skin.

O principal resíduo da indústria de café solúvel é a borra de café, gerada após a extração de sólidos solúveis com água, sendo usualmente queimada em caldeiras para geração de energia para a própria indústria. Entretanto, este co-produto pode conter de 15 a 20 % de óleo, componente de grande interesse na indústria alimentícia. Em paralelo, na indústria de produção de óleos vegetais o solvente frequentemente utilizado é o hexano. Contudo, por este ser um derivado de combustíveis fósseis, alternativas para sua substituição por solventes mais amigáveis ao meio ambiente, que podem ser obtidos por via biotecnológica, estão sendo estudadas. Deste modo, o objetivo principal desta dissertação de mestrado foi a viabilização técnica do emprego de solventes alcoólicos no processo de extração de óleo de borra de café proveniente da indústria de processamento de café solúvel. Foram realizadas extrações sólido-líquido em um estágio para estudar a influência das variáveis de processo temperatura (60 a 90 °C), tipo de solvente (etanol, ET ou isopropanol, IPA) e a hidratação do solvente (absoluto ou azeotrópico) nas características das fases extrato e rafinado, em termos de rendimento de extração de óleo, de ácidos clorogênicos (ACG), de carboidratos totais, teor de proteínas e índice de solubilidade de nitrogênio (ISN) da fase rafinado. Pré-tratamento enzimático ao processo de extração também foi realizado para investigar a sua atuação sobre o rendimento de extração de óleo e ACG, além do ISN das fases rafinado obtidas na temperatura de 70 °C. De forma geral, pode-se inferir que a temperatura favorece a extração de compostos lipídicos, mas a hidratação do solvente prejudica a extração destes compostos pelo aumento da polaridade do solvente. Do mesmo modo, como o ET é mais polar que o IPA, o primeiro solvente proporcionou a obtenção de menores rendimentos de extração de óleo. A temperatura de processo também influenciou a extração de ACG, a qual foi beneficiada a temperaturas mais baixas pelo aumento da polaridade dos solventes utilizados. De tal forma, que a 60 °C, nos experimentos utilizando etanol azeotrópico obteve-se os menores rendimentos de extração de óleo, porém maior rendimento de extração de ACG. O pré-tratamento enzimático apresentou diferenças significativas nas características das fases extrato e rafinado. No entanto, somente os experimentos com etanol absoluto resultaram em rendimentos de extração de óleo economicamente viáveis. De fato, será relevante um estudo mais aprofundado das variáveis de pré-tratamento enzimático para obter resultados mais expressivos em relação à extração de compostos lipídicos. Diante dos dados experimentais obtidos, conclui-se que é tecnicamente viável o emprego de solventes alcoólicos no processo de extração de óleo de borra de café. Entretanto, nota-se que as condições de processo devem ser avaliadas minuciosamente com o objetivo de se obter altos rendimentos de extração de óleo com maior teor de ACG. / Spent coffee grounds are the main residues of the soluble coffee industry, which are generated after the extraction of soluble solids with water. They are generally burned in boilers to generate energy for the coffee industry. However, this residue may contain 15 to 20 % oil, a component of interest to the food industry. Hexane is the most common solvent used in the vegetable oil industry; however, as it is a fossil fuel byproduct, biotechnology alternatives that replace it by more environmental-friendly solvents are being studied. Therefore, the main objective of this study was to determine technical viability of alcoholic solvents in oil extraction of spent coffee grounds. Solid-liquid extractions were carried out in one stage in order to analyzed the influence of the variables of the process (temperature, 60 to 90 °C; type of solvent, ethanol, ET or isopropanol, IPA; and solvent hydration level, absolute or azeotropic degree) in the characteristics of extract and raffinate phases, in terms of yield of oil extraction, chlorogenic acids (CGA), total carbohydrates, protein content, and nitrogen solubility index (NSI) of the raffinate phase. Enzymatic pre-treatment in the extraction process was also carried out to assess its effect on the oil and CGA yields, as well as NSI in the raffinate phase obtained at 70 °C. In general, it could be inferred that temperature favored lipid extraction, but solvent hydration level negatively affected extraction due to the increased polarity of the solvent. Similarly, as ET is more polar than IPA, the former solvent enabled lower yield of oil. The temperature of the process also influenced CGA extraction, which was favored by lower temperatures and increased solvent polarity. Therefore, at 60 °C, in the trials using azeotropic ethanol, oil extraction yield was lower, and CGA yield was greater. Enzymatic pre-treatment showed significant differences in the characteristics of the extract and raffinate phases. However, only the trials with absolute ethanol led to economically viable oil yields. In fact, further studies on pre-treatment variables of the enzymatic treatment should be carried out for more relevant results in relation to lipid compound extraction. Given the experimental data obtained here, it was concluded that the use of alcoholic solvents in oil extraction of spent coffee grounds is technically viable. However, the conditions of the process should be carefully analyzed for high yields of oil with greater levels of CGA to be obtained.

Smallanthus sonchifolius (Poepp. & Endl.) H. Robinson (Asteraceae), conhecida popularmente como yacón, é um erva utilizada na medicina popular para o tratamento do diabetes. A ação hipoglicemiante do extrato aquoso das folhas do yacón foi comprovada em estudos recentes realizados por outros autores em animais diabéticos. Análises fitoquímicas preliminares revelaram que a espécie é rica em lactonas sesquiterpênicas (LST) e em derivados dos ácidos clorogênicos (ACG). Ambas as classes de substâncias possuem inúmeras atividades biológicas, como ação antioxidante, anti-inflamatória, e inibidora de enzimas que podem contribuir no quadro de melhora do estado diabético. Contudo, ainda não está comprovado se as atividades biológicas da espécie são resultantes da ação dos ACG ou das LST. Adicionalmente, análises toxicológicas do consumo das folhas do yacón ainda não foram realizadas. Assim, o objetivo deste trabalho foi avaliar três extratos preparados com folhas do yacón, com o intuito de analisar o papel das diferentes classes químicas no desenvolvimento das atividades biológicas descritas para a espécie, bem como de seus potenciais efeitos tóxicos. Os extratos obtidos foram: o extrato aquoso (EA), no qual foram detectados tanto ACG (2,0 e 1,3 µg/mL dos picos mais intensos), quanto LST (99,7 e 319,0 µg/mL dos picos mais intensos); extrato de lavagem foliar (ELF), rico em LST (1257,4 e 1997,7 µg/mL das majoritárias) e o extrato polar (EP), rico em ACG (9,9 e 9,6 µg/mL dos picos mais intensos), mas sem LST. Os estudos toxicológicos demonstraram que o EP não causou efeitos tóxicos significativos em ratos, enquanto alterações em parâmetros bioquímicos específicos no sangue (creatinina 7,0 mg/dL, glicose 212,0 mg/dL, albumina 2,8 g/dL) de ratos tratados com o EA (10, 50 e 100 mg/kg) e ELF (10 e 100 mg/kg) apontaram para dano renal, confirmado por análises histológicas dos rins. Todos os extratos apresentaram atividade anti-edematogênica in vivo, em especial no modelo de edema induzido por óleo de cróton (EA: 25,9% inibição do edema a 0,5 mg/orelha; EP: 42,7% de inibição a 0,25 mg/orelha, ELF: 44,1% de inibição a 0,25 mg/orelha). O ELF demonstrou os melhores resultados na inibição da migração de neutrófilos, na indução da IL-10, na inibição do NO, TNF- e PGE2, quando comparado aos demais extratos. Todos os extratos inibiram de forma estatisticamente similar a atividade da -amilase, enquanto o EA e o EP foram mais eficazes na redução dos níveis glicêmicos após a administração oral de glicose (controle glicêmico: 201,8±5,2, EA: 169,0±4,9, EP: 176,7,3±7,3, ELF: 219,1±7,6 mg/dL). Com os dados obtidos neste trabalho pode-se concluir que o consumo do chá das folhas do yacón por períodos prolongados pode ser tóxico e seu uso não deve ser recomendado na medicina popular. As LST parecem ser as substâncias responsáveis por essa toxicidade. Tanto as LST, quanto os ACG contribuem para atividade anti-inflamatória, embora as LST parecem ter efeitos mais pronunciados. Contudo, a atividade anti-diabética da espécie parece estar relacionada principalmente à presença ao ACG e não à LST, sendo necessários estudos mais detalhados com ACG puros para verificar seu uso potencial no tratamento do diabetes. / Smallanthus sonchifolius (Poepp. & Endl.) H. Robinson (Asteraceae), known as yacón is a herb used in folk medicine to treat diabetes. The hypoglycaemic effect of the aqueous extract from yacón leaves was demonstrated in recent studies in diabetic animal models. Phytochemical analyzes have demonstrated that this species is rich in chorogenic acids (CGAs) and sesquiterpene lactones (STLs). Both classes of compounds have many biological activities such as antioxidant, anti-inflammatory and inhibit enzymes that can contribute in the improvement of the diabetic state. However, it is not yet established whether the biological activities of this species are due to the action of the CGAs or STLs. In addition, toxicological analysis of the consumption of yacón leaves has not yet been performed. Thus, the purpose of this study was to evaluate three extracts prepared from leaves of yacón, with the aim to analyze the role of different chemical classes in the development of the biological activities described for this species, as well as their potential toxic effects. The following extracts were obtained: aqueous extract (AE), were detected CGAs (2.0 and 1.3 mg / mL of the more intense peaks), and STLs (99.7 and 319 mg / mL of most intense peaks); leaf rinse extract (LRE), rich in STLs (1257.4 and 1997.7 mg / mL of major peaks), and the polar extract (PE) rich in CGAs (9.9 and 9.6 mg / mL of the more intense peaks), but without STLs. The toxicological studies in animals showed that the PE did not cause significant toxic effects, while changes in specific biochemical parameters in blood (creatinine 7.0 mg / dL, glucose 212.0 mg / dL, albumin 2.8 g / dL) of rats treated with AE (10, 50 and 100 mg / kg) and LRE (10 and 100 mg / kg) indicated kidney damage, which was confirmed by histological analysis of kidneys. All extracts showed anti-oedematogenics activity in vivo, particularly in the model of oedema induced by croton oil (AE: 25.9% of oedema inhibition at 0.5 mg / ear, PE: 42.7% of inhibition at 0.25 mg / ear, LRE: 44.1% inhibition at 0.25 mg / ear). The LRE showed the best results in the inhibition of neutrophil migration, induction of IL-10, as well as NO, TNF- and PGE2 inhibition, when compared to other extracts. All extracts inhibited in a statistically similar way the activity of -amylase, while the AE and the PE were more effective in reducing blood glucose levels after oral glucose adminstration (glycaemic control: 201.8 ± 5.2; AE: 169, 0 ± 4.9; PE: 7.3 ± 176,7,3; LRE: 219.1 ± 7.6 mg / dL). The data obtained in this work suggested that the consumption of tea from yacón leaves for prolonged periods can be toxic and their use should not be recommended in folk medicine. The STLs seem to be the substances responsible for this toxicity. Both STLs and CGAs contribute to the anti-inflammatory activity, although the STLs seem to have more pronounced effects. However, the anti-diabetic activity of the yacón leaves seems to be related to the presence of the CGAs and not to LST. More detailed studies with pure CGAs are required in order to determine their potential use in treatment of diabetes.

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

Les acides chlorogéniques (CGAs) représentent une famille d'esters formés d'un dérivé de l'acide cinnamique conjugué à l'acide quinique ou shikimique. Ces métabolites secondaires produits par la voie des phénylpropanoides sont largement répandus chez les végétaux terrestres et sont une source majeure d'antioxydants alimentaires. Les esters hydroxycinnamoyl-CoA sont les précurseurs des CGAs et d'autres composés phénoliques tels que les lignines. Ces intermédiaires activés sont synthétisés à partir d'un acide hydroxycinnamique et du coenzyme A par la 4-coumarate CoA ligase (4CL) appartenant à la superfamille des enzymes formant des adénylates. Nicotiana tabacum 4CL2 a été utilisée pour la production d'esters et sa structure a été résolue par remplacement moléculaire. Deux gènes codant pour des hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyltransférases chez Coffea canephora ont été clonés. CcHCT et CcHQT, qui appartiennent à la superfamille des acyltransférases acyl-CoA-dépendantes, ont été surexprimées dans E. coli et purifiées à homogénéité. L'analyse par diffraction aux rayons X de cristaux de CcHCT a permis de déterminer sa structure par remplacement moléculaire. Un modèle a été dérivé par homologie de séquence pour CcHQT afin de proposer les déterminants de la préférence pour l'acide quinique ou shikimique. Des modélisations moléculaires ont été réalisées afin d'identifier les résidus potentiellement impliqués dans les intéractions enzyme-substrat. L'analyse par chromatographie liquide haute performance des réactions enzymatiques ont montré que ces enzymes sont capables de synthétiser l'acide 5-O-caféoylquinique mais aussi le diester 3,5-O-dicaffeoylquinique, qui est un composé majeur du grain de café avant mûrissement. La production de variants par mutagenèse dirigée a permis l'identification de résidus importants pour la catalyse des réactions de mono- et de diacylation. L'approche combinée de la biologie structurale et de l'enzymologie s'avère particulièrement utile pour mieux comprendre le rôle de HCT et HQT. / Chlorogenic acids (CGAs) represent a family of esters formed between a cinnamic acid derivative and quinic or shikimic acid. CGAs are secondary metabolites produced via the phenylpropanoid pathway by higher plants and are a major source of dietary antioxidants. Hydroxycinnamoyl-CoA esters are the precursors for CGAs and other phenolic compounds such as lignins. These activated intermediates are synthesized from a hydroxycinnamic acid and coenzyme A by 4-coumarate CoA ligase (4CL), which belongs to the adenylate-forming enzyme superfamily. Nicotiana tabacum 4CL2 was used to produce hydroxycinnamoyl-CoA esters and its structure was solved by molecular replacement. Two genes encoding hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyltransferases from Coffea canephora were cloned. CcHCT and CcHQT, which belong to the acyl-CoA-dependent acyltransferase superfamily, were overexpressed in E. coli and purified to homogeneity. X-ray diffraction analysis of CcHCT crystals resulted in a structural solution by molecular replacement. A homology model was derived for CcHQT in order to propose some determinants of the preference for quinic or shikimic acid. Docking experiments were carried out in order to identify potential residues involved in enzyme-substrate interactions. High performance liquid chromatography analysis of enzymatic reactions showed that these enzymes are capable of synthesizing 5-O-caffeoylquinic acid but also the diester 3,5-O-dicaffeoylquinic acid, which is a major component of the coffee grain before ripening. The production of variants by site-directed mutagenesis enabled the identification of residues important for catalysis of the mono- and diacyltransfer reactions. The combined approach of structural biology and enzymology provides molecular insights into the role of HCT and HQT in CGA biosynthesis.