Dissertations / Theses on the topic 'Polyphenol'

Increasing evidence has suggested that dietary consumption may have the potential to influence mental health. However, few experimental studies have examined the effect of polyphenol-rich foods on psychological health. Furthermore, minimal studies exist on consumer attitudes towards polyphenol-rich foods. Thus, the main aims of this thesis were to investigate the effect of polyphenol-rich foods (FV, berries and dark chocolate) on psychological health, and to examine the behaviours, attitudes and knowledge consumers have with regards to the consumption of these foods. Firstly, results from a systematic review suggested that current evidence surrounding fruit and vegetables (FV) and psychological well-being is inconclusive. The review highlighted the need for future randomised controlled trials to investigate the relationship further. Secondly, a randomised controlled trial (PPhIT) showed mixed findings with regards to the effect of an eight week polyphenol-rich dietary pattern (FV, berries and dark chocolate) on psychological health. Improvements were observed for certain outcomes, including depressed mood and mental health (quality of life), but not for others (e.g. self-esteem and body-image). The dietary intervention led to significant increases in nutritional biomarkers, indicating good participant compliance. Overall, participants showed favourable attitudes towards the polyphenol-rich diet. Whilst a number of barriers towards the study diet foods were detected at baseline, the intervention significantly reduced some of these (e.g. ease, willingness, awareness). Similarly, a second ReT (n=30 adults), highlighted various barriers and facilitators towards FV consumption. However, in contrast to PPhIT, the four week intervention did not significantly modify these. This study also detected a lack of knowledge regarding what constitutes a portion of FV. The inconsistent findings from this thesis surrounding the effect of polyphenol-rich foods on psychological health suggest further research is warranted. Future research on the capacity for dietary interventions to reduce barriers towards polyphenol-rich foods may also be of value.

Plant polyphenols are reported to have bioactive properties, which may be used for protection against diseases. Therefore, the aim of this research was to investigate the bioactive activities of a pomegranate tannin polyphenol compound, punicalagin. In particular, the antioxidant, antihypertensive and anticancer mechanisms were investigated. Punicalagin was found III pomegranate husk but not in pomegranate juice when analysed by HPLC and LC-MS. Antioxidant mechanisms involved hydrogen peroxide scavenging, ferrous chelating and reducing ability. Higher hydrogen peroxide scavenging activity was achieved by 0.1 mg/ml from both punicalagin and pomegranate juice when compared with butylated hydroxytoluene (BHT) or trolox (p :S0.05). Punicalagin and pomegranate juice exhibited ferrous chelating ability significantly lower than Ethylenediaminetetraacetic acid. Cell toxicity induced by tert-butylhydroperoxide (3 mM) was significantly inhibited by punicalagin (5 and 10 !!M) in Caco-2 cells; these results were confinned by cell morphology. Punicalagin protection was achieved by inhibiting cellular reactive oxygen species (ROS) as well as malondialdehyde levels. Glutathione level was significantly increased in stressed cells pretreated with both concentration of punicalagin, indicating good antioxidant activity for punicalagin. Punicalagin (1-60 !!M) increased nitric oxide production in endothelial cells (EA.hy926) through decreased ROS levels and increased endothelial nitric oxide synthase enzyme (eNOS) activation. Activation of eNOS enzyme was achieved by an II - increase of cellular calcium concentration. At the same examined concentration of punicalagin (1-60 f-tM), the activity of angiotensin converting enzyme (ACE) was significantly inhibited. The dual action of punicalagin as nitric oxide synthase inducer and ACE inhibitor showed antihypertensive effect. Punicalagin (50 and 75 f-tM) showed toxic effects on the colon cancer cell line (Caco-2) but not on a normal colon cell line (HCEC); both results were confinned by morphological studies. In the presence of punicalagin, cytoplasmic ROS production decreased, indicating antioxidant activity whereas superoxide radicals released from mitochondria increased due to mitochondrial dysfunction. Annexin V and caspase family (caspase 9, 8 and 3) activation confinned that cell death occurred via apoptosis pathway by both concentrations of punicalagin. The cell cycle was attested by punicalagin in the G liS-phase at the concentrations tested. The above findings indicating that punicalagin has antioxidant, antihypertensive and anticarcinogenic activity.

Mushroom polyphenol oxidase (EC 1.14.18.1) was investigated to determine its potential for application as a biocatalyst in the synthesis of o-quinones, in organic medium. In order to determine the kinetic properties of the biocatalyst, a system was devised which comprised an immobilised polyphenol oxidase extract, functioning in chloroform. The system was hydrated by the addition of buffer. A simple method for the consistent measurement of reaction rates in this heterogenous system was designed and used to obtain detailed enzyme kinetic data relating to optimisation of reaction conditions and substrate specificity. The aqueous content of the system was optimised using p-cresol as a substrate. A crude, immobilised extract of Agaricus bisporus was used to hydroxylate and oxidise a range of selected p-substituted phenolic substrates, yielding, as the sale products, o-quinones. These products were efficiently reduced to catechols by extracting the reaction mixtures with aqueous ascorbic acid solution. The biocatalytic system was also successfully utilised to produce L-DOPA, the drug used to treat Parkinson's disease, from L-acetyl tyrosine ethyl ester (ATEE). Michaelis-Menten kinetics were used to obtain apparent Km and V values with respect to the selected phenolic substrates, and the kinetic parameters obtained were found to correlate well with the steric requirements of the substrates and with their hydrophobicity. In the course of the investigation, a novel ¹H NMR method was used to facilitate measurement of the UV molar absorption coefficients of the o-quinones in reaction mixtures, thus avoiding the necessity to isolate these unstable, water-sensitive products. The biocatalytic system was extended to a continuous process, in which the immobilised enzyme was shown to function successfully in the chloroform medium for several hours, with high conversion rates. Modifications, involving partial purification and the addition of a surfactant, were investigated to determine their effect on the kinetic parameters. The results obtained using partially purified enzyme indicated that the removal of extraneous protein and/or melanoid material lead to a reduced capacity for conversion of sterically demanding substrates. The addition of the anionic detergent, sodium dodecyl sulphate (SOS), enhanced the ability of the biocatalyst to bind and oxidise sterically demanding substrates. These effects are attributed to changes in the polar state of groups within the protein binding pocket, which result in altered flexibility and hydrophobicity. Computer modelling of several biomimetic dinuclear copper complexes also indicated the importance of flexibility for effective biocatalysis. Novel binuclear copper (II complexes, containing a flexible biphenyl spacer and imidazole or benzimidazole donors, were prepared and analysed using NMR, UV, AA and cyclic voltammetric techniques. The complexes were also shown, in a detailed kinetic study, to mimic the catecholase activity of polyphenol oxidase by oxidising 3,5-di-tertbutylcatechol, and to catalyse the coupling of the phenolic substrate 2,4-di-tert-butylphenol. However, the complexes were apparently too flexible to react with smaller substrates. These biomimetic complexes provided valuable insights into the nature of the dinuclear copper binding site.

Due to the negative environmental impact of the Olive Mill Wastewaters (OMW), research is done in order to treat and valorize them. In this work, different OMW from different Italian regions (Liguria and Puglia) and harvests (from 2012 to 2014) were tested in order to recover polyphenols (PCs), molecules with a high added value because of their beneficial properties. The solid phase used for PCs recovery was the resin Amberlite XAD16; the desorption solvent was acidified ethanol. An HPLC method for total PCs content quantification was developed using a C18 column. A new, repeatable and reliable column packing method was developed. The packing quality was evaluated with step-change fluid dynamic analysis tests using NaCl 0.04M as tracer. Also, to avoid clogging problems in the packed columns an OMW pre-treatment was designed, capable to remove 98% of the solids. Several breakthrough tests were performed to evaluate the influence of linear velocity and column length (0.52m and 2.0m). A repeatability test was performed in order to evaluate the stability of the process. The process was modeled using a plug flow with axial dispersion model with solid-liquid mass transfer; implemented in COMSOL3.5a. The desorption curves were obtained with subsequent solvent regeneration. Antioxidant activity tests were performed with the desorption product using the ABTS method. On the basis of economic considerations, two new ion-exchange resins were tested (IRA958Cl and IRA 67Cl). IRA958Cl showed the best performance. Two breakthrough tests at different linear velocities were conducted with this resin. In order to recover specific high added value molecules (tyrosol and hydroxytyrosol) from the actual OMW, experiments were performed in collaboration with the Fachhoschule Nordwestschweiz (FHNW) using a Cyclodextrin-based polyurethane polymer, synthetized by the FHNW research group. Then, in order to increase the purity of tyrosol in the desorption fractions several organic solvents were tested.

Due to the negative environmental impact of the Olive Mill Wastewaters (OMW), research is done in order to treat and valorize them. In this work, different OMW from different Italian regions (Liguria and Puglia) and harvests (from 2012 to 2014) were tested in order to recover polyphenols (PCs), molecules with a high added value because of their beneficial properties. The solid phase used for PCs recovery was the resin Amberlite XAD16; the desorption solvent was acidified ethanol. An HPLC method for total PCs content quantification was developed using a C18 column. A new, repeatable and reliable column packing method was developed. The packing quality was evaluated with step-change fluid dynamic analysis tests using NaCl 0.04M as tracer. Also, to avoid clogging problems in the packed columns an OMW pre-treatment was designed, capable to remove 98% of the solids. Several breakthrough tests were performed to evaluate the influence of linear velocity and column length (0.52m and 2.0m). A repeatability test was performed in order to evaluate the stability of the process. The process was modeled using a plug flow with axial dispersion model with solid-liquid mass transfer; implemented in COMSOL3.5a. The desorption curves were obtained with subsequent solvent regeneration. Antioxidant activity tests were performed with the desorption product using the ABTS method. On the basis of economic considerations, two new ion-exchange resins were tested (IRA958Cl and IRA 67Cl). IRA958Cl showed the best performance. Two breakthrough tests at different linear velocities were conducted with this resin. In order to recover specific high added value molecules (tyrosol and hydroxytyrosol) from the actual OMW, experiments were performed in collaboration with the Fachhoschule Nordwestschweiz (FHNW) using a Cyclodextrin-based polyurethane polymer, synthetized by the FHNW research group. Then, in order to increase the purity of tyrosol in the desorption fractions several organic solvents were tested.

L’obesitat és un problema de salut en augment i suposa un risc per al desenvolupament de malalties cròniques. Les estratègies per reduir i prevenir l’obesitat no han sigut satisfactòries el que fa necessari el desenvolupament d’alternatives terapèutiques. Nombrosos estudis en animals i humans demostren que els polifenols tenen propietats protectores en front a trastorns metabòlics per la qual cosa aquests compostos bioactius poden ser útils per a reduir l’obesitat i malalties metabòliques associades. La leptina és una hormona encarregada de la regulació del balanç energètic al sistema nerviós central on activa les neurones POMC i inhibeix les AgRP produint sacietat i promovent la despesa energètica. No obstant això, l’acció de la leptina en l’obesitat es troba afectada. L’objectiu principal d’aquesta tesi és identificar polifenols que millorin la sensibilitat a la leptina en situacions d’obesitat i que tingui com a resultat la pèrdua de pes. En aquesta tesi demostrem com el consum crònic d’un extracte de pinyol de raïm ric en proantocianidines millora la senyalització de la leptina a través de l’augment de l’expressió gènica del neuropèptid POMC i redueix la ingesta energètica sense mostrar canvis al pes corporal. A més, s’ha investigat el potencial d’altres polifenols amb efectes complementaris a les proantocianidines per tal d’estimular la pèrdua de pes. Els resultats presentats mostren que el resveratrol és efectiu reduint el pes i el greix corporal i la hiperleptinèmia en animals obesos, actuant com a agent sensibilitzador de la leptina. D’altra banda, es demostra el potencial de fruites estacionals riques en polifenols en la modulació de la senyalització de la leptina en condicions normals i d’obesitat. Finalment, s’explica el rol d’una nova diana per modular l’activitat neuronal de les neurones AgRP. Els resultats d’aquesta recerca aporten nous coneixements pel disseny d’aliments funcionals que combinin diferents compostos bioactius amb el potencial de poder ser utilitzats com a teràpia anti-obesitat.
La obesidad es un problema de salud en aumento y supone un riesgo para el desarrollo de enfermedades crónicas. Las estrategias para reducir y prevenir la obesidad no han sido satisfactorias lo que hace necesario el desarrollo de alternativas terapéuticas. Numerosos estudios en animales y humanos demuestran que los polifenoles tienen propiedades protectoras frente a trastornos metabólicos por lo que estos compuestos bioactivos pueden ser útiles para reducir la obesidad y enfermedades metabólicas asociadas. La leptina es una hormona encargada de la regulación del balance energético en el sistema nervioso central donde activa las neuronas POMC e inhibe las AgRP produciendo saciedad y promoviendo el gasto energético. Sin embargo, la acción de la leptina en la obesidad se encuentra afectada. El objetivo principal de esta tesis es identificar polifenoles que mejoren la sensibilidad a la leptina en situaciones de obesidad y que tenga como resultado la pérdida de peso. En esta tesis demostramos como el consumo crónico de un extracto de pepita de uva rico en proantocianidinas mejora la señalización de la leptina a través del aumento de la expresión génica del neuropéptido POMC y reduce la ingesta energética sin mostrar cambios en el peso corporal. Además, se ha investigado el potencial de otros polifenoles con efectos complementarios a las proantocianidinas para estimular la pérdida de peso. Los resultados presentados muestran que el resveratrol es efectivo reduciendo el peso y la grasa corporal y la hiperleptinémia en animales obesos, actuando como agente sensibilizador de la leptina. Por otra parte, se demuestra el potencial de frutas estacionales ricas en polifenoles en la modulación de la señalización de la leptina en condiciones normales y de obesidad. Finalmente, se explica el rol de una nueva diana para modular la actividad neuronal de las neuronas AgRP. Los resultados de esta investigación aportan nuevos conocimientos para el diseño de alimentos funcionales que combinen diferentes compuestos bioactivos con el potencial de poder ser utilizados como terapia anti-obesidad.
Obesity is an increasing health problem and a major risk factor for a number of chronic diseases. Up to now, strategies to reduce and prevent obesity were unsuccessful. Therefore, novel approaches to treat obesity need to be developed. In this sense, several animal and human studies demonstrate that polyphenols protect against metabolic disorders including diabetes and cardiovascular disease. Thus, polyphenols emerge as bioactive compounds useful to reduce obesity and its associated metabolic diseases. Energy balance is regulated by leptin in the central nervous system, particularly in the hypothalamus where it activates POMC and inhibits AgRP neurons to produce satiety and promote energy expenditure. However, leptin action appears to be suppressed in obesity which is reflected by increased appetite and reduced energy expenditure. The aim of this thesis was to identify polyphenols that improve leptin sensitivity under obesogenic environments, which could ultimately result in a loss of body weight. We show that a chronic intake of a grape seed proanthocyanidin extract improves leptin signaling by increasing POMC gene expression and reduces food intake without decreasing body weight in obese animals. Furthermore, we investigated other polyphenols that could complement the effects of proanthocyanidins by enhancing body weight loss. Our results show that high doses of resveratrol effectively reduce body weight, fat mass and correct hyperleptinemia in obese animals acting as a leptin sensitizer compound. Additionally, we demonstrate the potential of seasonal fruits rich in polyphenols to modulate hypothalamic leptin signaling and downstream effectors in normal conditions and during obesity. Finally, the role of a novel target to modulate AgRP neurons activity is explained. The outcome of this research provides insights into the design of functional foods that combine bioactive compounds which could potentially be used as anti-obesity therapy.

The potential of using mushroom polyphenol oxidase (EC 1.14.18.1) as a biocatalyst for the biotransformation of phenols to produce catechols in an aqueous medium was investigated. Polyphenol oxidase is characterised by two distinct reactions i.e., the ortho-hydroxylation of phenols to catechols (cresolase activity) and the subsequent oxidation of catechols to orthoquinones (catecholase activity). In order to facilitate the development of a process to produce catechols, the accumulation of catechol as a true intermediate product released in the reaction system needed to be investigated, as its release had been disputed due to the oxidation of catechols to o-quinones. Using LC-MS, catechol products were successfully identified as true intermediate products formed during biocatalytic reactions in water.

This thesis explores the utilization of physico-chemical interactions between natural polyphenols and biocompatible polymers to design novel materials and complex colloidal dispersions and emulsions. These usually unwanted interactions and the resulting insoluble complexes are important in the creation of structures that could potentially be used in the design of novel foodstuffs. The theme of this thesis is therefore the interactions of polyphenols with polymers and emulsion stability and properties. The results are as follows. Chapter 3 presents a study on the interaction of polymer-polyphenol and the fabrication of new colloidal particles using the self-association of polymer-polyphenol complexes. The phase diagram of the physical appearance of mixtures of polyvinylpyrrolidone (PVP) with catechins and tannic acid has been explored and we have characterized the different structures by using a combination of light microscopy, UV-Vis and IR spectroscopy and dynamic light scattering techniques. The molar ratio between polymers and polyphenols has been found to play an important role in defining the structure of the complexes and their stability. By further optimizing the reaction conditions it is possible to obtain various structures resulting in colloids, microgels, and macroscopically gelled interfaces, which find applications in emulsion stabilization as explored in the following chapters. In Chapter 4 emulsification techniques and consideration on emulsion stability and formulation are investigated. Novel biodegradable surfactant free emulsions stabilized by non-covalently associated micro-gel particles made from PVP and TA have been successfully prepared. The strong interaction between PVP and TA, driven by hydrophobic interactions and hydrogen bonding between phenolic and pyrrolidinone rings, leads to formation of microgels during the emulsification process. The molar ratio between TA and PVP was found to play an important role in determining the mechanism of emulsion stabilisation. At low molar ratio a shell of PVP-TA microgels is formed and over time wrinkles have been observed on these shells, while at high molar ratio [TA]/[PVP] we observe Pickering emulsions stabilised by microgel particles and the continuous phase is a dispersion of microgel particles. The structure and stability of the systems have been investigated by light scattering, confocal and light transmission microscopy. The presence of TA leads to halochromism of the emulsions at high pH and the absence of surfactants makes these emulsions particularly desirable in terms of biodegradability. Chapter 5 focuses on the rheological properties of emulsions stabilised by gel particles and on the relationship between oil volume fraction and rheological behaviour. The rheological properties of emulsions and complex fluids are of pivotal importance in relation to their technical applications. These properties depend strongly on the emulsions composition, oil-phase volume fraction, microscopic droplet structure, and interfacial interactions. Our rheological tests showed that PVP-TA particles stabilised emulsions behave like flocculated dispersions and polymeric gels confirming the qualitative observation on emulsion gelation observed in the experiments carried out in the previous chapter. Chapter 6 introduces a new technique, microfluidics. Microfluidics devices were designed and built with the purpose of making monodisperse emulsions stabilized by PVP-TA complexes. The emulsions produced with microfluidics devices were found to be monodisperse rather than the polydisperse ones resulting from bulk emulsification processes. The basic theory behind the effect of microfluidic scaling affects the hydrodynamics is outlined. Experimental details on the design process and on how to use photo-lithography and soft-lithography to build microfluidics devices are given. Finally we have demonstrated how our microfluidics devices can be successfully used to make monodisperse emulsions stabilised by PVP-TA and double emulsions (oil in water in oil emulsions).

Los compuestos fenólicos son fitoquímicos ampliamente distribuidos en nuestra dieta debido a la ingesta de productos vegetales –principalmente frutas y verduras, derivados del cacao, te, café y vino tinto-. Varios efectos beneficiosos han sido relacionados con la ingesta de compuestos fenólicos, como por ejemplo la reducción del riesgo de sufrir enfermedades cardiovasculares o neurodegenerativas, la prevención de enfermedades relacionadas con el estrés oxidativo, o la reducción de algunos canceres. Pero menos información existe sobre los mecanismos de acción de estos en el cuerpo y sobre que compuestos alcanzan las dianas metabólicas. La presente tesis ha estado centrada en la evaluación de la digestibilidad y bioaccesibilidad de los compuestos fenólicos de la dieta durante el proceso de digestión y en el estudio de la absorción, el metabolismo y la distribución en tejidos de los compuestos fenólicos y de sus metabolitos. Para completar los objetivos citados, (i) se estudió la digestibilidad y bioaccesibilidad de los compuestos fenólicos de la dieta mediante el uso de un método de digestión in vitro; (ii) se desarrolló un método in vitro de fermentación colónica y se aplicó dicho método a compuestos fenólicos de la dieta; (iii) se desarrollaron metodologías analíticas para la cuantificación de compuestos fenólicos y sus metabolitos en muestras biológicas y finalmente (iv) se realizaron estudios in vivo, usando rata Wistar como modelo animal, para evaluar la absorción, el metabolismo y la distribución en tejidos de los compuestos fenólicos (principalmente procianidinas y compuestos fenólicos del aceite de oliva) y sus metabolitos.Como resultados principales de la parte experimental, se demostró que las procianidinas de bajo grado de polimerización (dimer y trímero) fueron estables durante el proceso de digestión. Se caracterizaron las rutas de formación de los productos de fermentación colónica de varios compuestos fenólicos, identificando a los ácidos fenólicos hidroxilados como los principales productos de fermentación de los flavonoides. Paralelamente, se desarrollaron y validaron dos métodos cromatográficos que combinaban la extracción en fase sólida con la ultra cromatografía líquida de alta resolución acoplada a la espectrometría de mases en tándem (UPCL-MS/MS) para la determinación de compuestos fenólicos y sus metabolitos en muestras biológicas. Dichos métodos fueron utilizados para analizar los compuestos fenólicos y sus metabolitos en las muestras biológicas que fueron obtenidas como resultado de los estudios de ingesta aguda e ingesta crónica de extractos ricos en compuestos fenólicos y de alimentos ricos en compuestos fenólicos en ratas Wistar. De los estudios in vivo se estableció que los metabolitos fenólicos fueron ampliamente distribuidos alcanzando prácticamente todos los órganos del cuerpo y que la presencia de metabolitos de procianidinas en plasma y su distribución en tejidos fue modulada por la composición de la matriz alimentaria que vehiculizaba a los compuestos fenólicos. Además, se demostró, mediante la comparación de un producto rico y un producto enriquecido en compuestos fenólicos, que la naturaleza de los compuestos fenólicos puede modificar significativamente parámetros farmacocinéticos de los metabolitos fenólicos. Adicionalmente, se demostró, mediante la exposición de los animales durante el embarazo y la lactancia a una dieta rica en fructosa y grasas saturadas, que la dieta pueden modular la capacidad de glucuronidación del hígado sobre los flavonoides en la generación parental y en la generación hija. No obstante, se ha de tener en cuenta que los resultados obtenidos en estudios con animales y en estudios in vitro representan el paso previo a los estudios clínicos y que los resultados obtenidos pueden no ser directamente extrapolables a humanos. Por lo que, los estudios clínicos representan el objetivo final de todo estudio nutricional relacionado con alimentos funcionales.
Phenolic compounds are phytochemicals widely distributed in our diet due to the intake of plant-derived products -e.g. fruits and vegetables, cocoa derivatives, tea, coffee or red wine-. Several beneficial effects have been established related to the intake of dietary phenolic compounds, such as the reduction of the risk of cardiovascular and neurodegenerative diseases, prevention of illnesses related with oxidative stress, or the reduction of some cancers, among others. But less is known about how phenolic compounds act in the body after their ingestion and which phenolic metabolites reach the metabolic targets. This thesis was focus on the evaluation of the bioaccessibility and digestibility of dietary phenolic compounds during the digestion process; and on the study of the absorption, metabolism and tissue distribution of phenolic compounds and their metabolites. To complete with these objectives, (i) the evaluation of the bioaccessibility and digestibility of dietary phenolic compounds by the use of an in vitro digestion system; (ii) the development of an in vitro colonic fermentation model and the evaluation of the colonic fermentation of dietary phenolic compounds (ii) the development of analytical methodologies to quantify polyphenols and their metabolites in biological samples (plasma and tissues) and (iii) the study of the absorption, metabolism and tissue distribution of dietary phenolic compounds (mainly procyanidins and olive oil phenolics) and their metabolites in vivo using rats as an animal model were performed. As a result of the experimental work developed within this dissertation, the stability during the digestion process of dimer and trimerprocyanidins was demonstrated. The colonic metabolic pathways of individual phenolic compounds were successfully established detecting hydroxylated phenolic acids as the main colonic metabolites of flavonoids. Parallel, two chromatographic methods combining off-line solid-phase extraction and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) were successfully validated for the determination of phenolic compounds and their metabolites in biological samples, which were used to detect and quantify polyphenols and their metabolites in the biological samples obtained as a result of the in vivo acute and chronic studies performed using phenolic extract and polyphenol-rich foods as a polyphenol sources. Derived from the in vivo studies were established that, phenolic metabolites were widely distributed through the body reaching practically all the organs; procyanidin plasmatic bioavailability and tissue distribution were modulated according to the food matrix composition and the intake of phenols by a naturally rich-phenol or by a phenol-enriched food could vary the pharmacokinetic parameters of the phenolic metabolites. Additionally environmental factors, such as the diet, may modulate the hepatic glucuronidation capacity toward flavonoids in the parental and offspring rats, as was demonstrated with an in utero exposure of a high fructose and saturated fat diet.Nonetheless, data from animal and in vitro experiments represent the previous step to the human clinical studies because it may not be readily extrapolatable to humans. So, human clinical studies should be the colophon of all the nutritional studies related with functional foods.

It has been proposed that polyphenol-rich foods have a role in disease prevention and are associated with health benefits due to their antioxidant, anti-inflammatory, prebiotic, and antibacterial properties. However, associated health benefits depend on their intake, metabolism, and bioavailability. The metabolism and the bioavailability of polyphenols have been studied in young adults and show substantial variability. As the majority of polyphenols are metabolised in the colon, this may result in different bioactive microbial metabolites in the large intestine where they may have an impact on the risk of colorectal cancer (CRC). This variability could be due to: 1) dietary habits including intake polyphenol-rich foods; 2) ethnic-specific colonic microbiota; and 3) ageing and its effect on colonic physiology. Little is known about the impact of ethnicity, ageing, and the risk of CRC on polyphenol metabolism. Therefore, this thesis aimed to investigate the effect of the factors that could have an impact on the colonic metabolism of dietary polyphenols in a human feeding study measuring the biomarkers of polyphenol metabolism, colonic fermentation, and gut health; and an in-vitro faecal fermentation study measuring the colonic metabolites of quercetin-3-O-rutinoside (rutin). The first aim of this thesis (Chapter 3) was to examine the effect of ethnicity (Europeans versus Indians) on polyphenol metabolism. The findings of this study suggest that ethnicity could have a role on the colonic metabolism of polyphenols which could be due to the differences in disease incidence between countries such as the lowest risk of CRC in India among the world. The Indian group excreted less urinary phenolic acid after the high-polyphenol diet compared to the Europeans; however, Indians were more capable and faster in metabolizing rutin in the in-vitro model. This could be due to the differences in: 1. Genetics and its effect on gastrointestinal tract absorption. 2. Gut microbiota, as Indians have a significantly higher level of Bifidobacterium. 3. Gut environment, in particular the colonic pH and SCFA could have an influence as the colonic pH was lower in the Indian group. 4. Cultural daily diet between groups, as Indians significantly consumed a high amount of onions, tomatoes, chillies, spices, curry-based products, and yoghurt. These food types are high in polyphenols, fibre, and probiotics. The second study of this thesis aimed to investigate the effect of ageing on polyphenol metabolism. The results suggest another factor, ageing, which could influence the colonic metabolism of polyphenols. The older group excreted less urinary phenolic acid and some of the acid was not detected in certain of the participants’ urine compared to the younger group. However, the sum of the phenolic acid that formed after the faecal fermentation of rutin was not significantly different between the groups. This could suggest different reasons behind these variations. First, the lack of absorption of some phenolic acids by the older group as ageing was shown to decrease the colonic absorption. Secondly, the effect of ageing on gut microbiota composition and function. Thirdly, changes in dietary habits and physical activity may be influenced by ageing. Thus, this may suggest that older people can have fewer benefits of polyphenol metabolites which could be associated with an increase in risk for age-related diseases including CRC. As the risk of CRC is different between countries and increases with age, the supportive findings of the first and second study suggest that ethnicity and ageing could have a role on the metabolism of polyphenols so this raises the questions whether a low intake of polyphenols can be one of the factors that may lead to CRC, or whether polyphenols can reduce the risk of CRC due to their colonic health benefits. Therefore, the last study examined the metabolism of polyphenols on patients who are at risk of CRC (history of polyps). No significant differences were observed between the healthy control and polypectomy groups in terms of the sum urinary phenolic acid excretion and phenolic acid formation in the faecal fluids. However, some phenolic acids were not detected in all of the urine samples of the polypectomy group as well as one acid in the faecal fermentation fluids, while some of the acids were not detected in few participants in the healthy group. No hard conclusion can be made from this study due to the small sample size. However, this study gives us an idea that there could be differences if a larger sample size were used. Therefore, more studies are needed to determine the effect of CRC risk as being one of the factors that can influence the metabolism of polyphenols. In conclusion, the work of this thesis showed that ethnicity, ageing, and gut health are likely some of the key factors that could contribute to the variations in polyphenol metabolism which were observed previously by many in-vivo and in-vitro studies. These variations could result in bioavailability variation and consequential differences in the biological activity of polyphenol metabolites leading to differences in health and optimal health among individuals.

Dietary polyphenols have been positively correlated to the reduced risk of several chronic diseases, including Alzheimer' disease (AD). Grape (Vitis vinifera) seed contains 5-8 % polyphenols and annually, 2.5 million tonnes of grape seed is generated in the juice and wine industry. Grape seed extract (GSE) and its compounds gallic acid (GA), resveratrol (RSV) and epigallocatechin gallate (EGCG) have been shown to attenuate AD aetiological features including oxidative stress, protein aggregation, and mitochondrial dysfunction in cell and animal models. The main objectives of this work were to optimize the methods for grape seed polyphenol extraction and assess their antioxidant properties. Polyphenols from grape seeds were extracted using ethanol, methanol, and acetone as solvents. Total phenolic content (TPC), and total flavonoid content (TFC) of GSEs were determined by Folin-Ciocalteu’s and AlCl3 colorimetric methods. Free radical scavenging activities were measured using standard antioxidant assays (DPPH and ABTS) and levels of GA, EGCG, and RSV were determined by high performance liquid chromatography (HPLC). Findings from this study showed the highest TFC levels in acetone extraction that was consistent with previous studies. Ethanol showed improved extraction of phenolic compounds compared to acetone and better than methanol in overall polyphenol extraction. Ethanol extracted GSEs exhibited high free radical scavenging capacity measured using ABTS and DPPH demonstrating its potent antioxidant activity. Ethanol extracted GSEs displayed the highest ABTS scavenging ability as compared to acetone and methanol extractions. Ethanol extracted GSEs showed high free polyphenols content but low level of bound polyphenols. The TFC, TPC and radical scavenging properties of the free polyphenol fraction was significantly higher than those of the bound polyphenol fraction from GSE. Thus, potential therapeutic effects of GSE may be attributed to the free polyphenol fraction. GA and EGCG were detected in both unbound and bound phenolic extracts. However, RSV was only detected in bound polyphenol extract. Overall, the findings presented here have unravelled new insights into the polyphenol content of GSEs, solubility, efficiency of different extraction conditions and more importantly generated new knowledge that will be critical for developing industrial processes for developing GSE as a commercial food product for alleviating AD.

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

Polyphenol oxidases are enzymes that catalyze the oxidation of certain phenolic substrates to quinones in the presence of molecular oxygen. Polyphenol oxidases are widely used in several applications. In food industry, they are used for enhancement of flavor in coffee, tea and cocoa production, and determination of food quality. In medicine, they have several uses in treatments of Parkinson&rsquo
s disease, phenlyketonurea and leukemia. In wastewater treatment, they are used for the removal of phenolic pollutants from wastewaters. In pharmaceutical industry, differentiation of morphine from codeine is possible by means of polyphenol oxidase immobilized electrodes. In this study, a thermophilic fungus, Thermomyces lanuginosus was evaluated in terms of poyphenol oxidase production. The effect of different nutrient sources, inducers and fermentation parameters on enzyme production were investigated and maximum PPO activity of 97 U/ml was observed in bioreactor experiments at 50°
C, 400 rpm and pH 8.0 in a fermentation medium containing 1.4% yeast extract, 0.3% MgSO4, 1% KH2PO4, 0.003% CuSO4, 0.032% gallic acid. Type of polyphenol oxidase produced by Thermomyces lanuginosus was determined as laccase. For biochemical characterization studies, the enzyme was enriched by electrophoresis. Temperature and pH optima for the enzyme were determined as 60°
C and 8.0, respectively. Enzyme retained 67% activity after 1 h incubation at 80°
C and retained 87% of its activity after 1 hour of incubation at pH 9.0 at room temperature. The enzyme obeys Michealis-Menten kinetics with Km and Vmax values being 5 mg /ml catechol and 38 U/ml, respectively. Molecular weight of the enzyme was determined as 29 kDa and isoelectric point of enzyme was found to be approximately 6.0.

"This thesis is organized in five parts: introduction, three chapters of experimental work and a general discussion. In the introduction section, major topics necessary for a broad comprehension of this thesis is described. Major aspects about neurodegenerative diseases and (poly)phenols importance in the disease prevention are present. State of art of major cellular models used for studying (poly)phenols role in Parkinson’s disease (PD) is also described. Emphasis to the knowledge gaps of (poly)phenols potential to act inside the brain was considered such as their ability to cross and/or interact with the blood-brain barrier (BBB). Finally, a thesis rational and major work objectives are concluding the introductory section. Part of this section was included in a review published in Current Neuropharmacology.(...)"
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Non-communicable disease (NCD) represent a modern global health challenge, with decreased life expectancy and quality of life, and a greater increase in health costs. Overweight and obesity are recognized as key risk factors for NCD, and the major link between these states is low-grade inflammation (LGI). Adipose tissues are now established as major contributors of pro-inflammatory molecules. One driver for low-grade inflammation is endotoxaemia (characterised by an increase in circulating endotoxin, or lipopolysaccharide (LPS)). Endotoxin is released in the gut lumen as part of the cell wall of Gram-negative bacteria, and endotoxaemia has been associated with high-fat high-calorie intake, one of the factors contributing to obesity. Thus high-fat high-calorie intake, endotoxaemia, LGI, obesity and NCD may all be linked and dietary approaches, such as functional foods with anti-inflammatory properties, may modulate this interaction. Curcuminoids (polyphenols found in the turmeric root) have anti-inflammatory and anti-bacterial effects and are potential dietary agents for preventing LGI and NCD. Little is known about the potential preventive action of curcuminoids on LGI. Therefore, this thesis aims to investigate the potential for curcuminoids to reduce endotoxaemia and LGI in apparently healthy overweight and obese people. A systematic review of the evidence for the anti-inflammatory effect of curcuminoids in healthy people and people at risk of NCD was performed. Nine papers describing five intervention studies published in this context have been assessed. Promising effects were seen in reducing CRP level (mean difference -0.71 mg/dl; 95% CI: -1.31 to -0.12; p < 0.05) and increasing adiponectin level (mean difference 5.12 ng/ml; 95% CI: 3.78 to 6.45; p < 0.01). However, there was insufficient data to make a clear conclusion that curcuminoids reduce LGI in those groups, thereby justifying the need for further work on the topic. In the second study, the phenolics and curcuminoids content in fresh turmeric root, turmeric powder, and four types of supplements were characterised to choose the supplement for a human intervention. Amongst all turmeric extracts assessed, the supplement BCM-95® (Dolcas-Biotech, Landing, NJ, USA) had the highest total phenolics (7.11 vs 1.94 mg GAE/g in average) and curcumin content (191.75 vs 13.7 mg/g in average). Therefore, BCM-95® was used in the later studies. The fate and effect of curcuminoids in the human gut were investigated further using established in vitro models for digestion and fermentation. Phenolic compounds were released after gastric and intestinal digestion. The presence of oil during digestion increased the release of phenolics from turmeric extracts. The presence of curcuminoids in a 24 h fermentation by faecal bacteria in vitro did not affect the production of short-chain fatty acids from Raftiline, which was used as the substrate for fermentation. The third study investigated the effect of curcuminoids on the intestinal permeability and endotoxin translocation using Caco-2 cell monolayers intestinal model. Administration of 115 nM of curcuminoids (in BCM-95®) for 3 day (3 h each day) unexpectedly resulted in increased monolayer permeability (by 30%) and endotoxin translocation (by 6-fold). The final study was a double-blind, placebo-controlled, crossover trial of curcuminoids supplementation (BCM-95®) for 21 days in healthy overweight and obese adults (n =14). Due to ongoing work in other parts of this project, the treatment code was not broken at the time of submission. In conclusion, there is very little published data on curcuminoids and LGI, however the Caco-2 cell monolayers study in this thesis suggest that turmeric may impair gut permeability under the experimental conditions tested. The effect may be dependent on the type of curcuminoids, the dose, and the associated oils. From the digestion study, it would appear that it is better to consume supplements associated with oil in order to optimize the release of phenolics. Although we cannot determine which of the two treatments in the human study was the active compound, the study was feasible, no side effects were reported and there was a difference between treatments. The relevance of this will be known when the code is broken.

Polyphenol oxidases are important enzymes because of their role in food spoilage, oxidizing monophenols into o-diphenols and/or diphenols into the corresponding o-quinones. The resulting compounds are unstable and can rapidly form brown colored compounds, called melanins. Polyphenol oxidases, (PPOs) have been purified from several sources, particularly from fruits and vegetables. However, successful purification of PPO to homogeneity from plant sources has always been difficult.
The purification procedure of PPOs from cassava tuber consisted of (1) the preparation of cassava acetone powder; (2) the buffer extraction of the acetone powder to obtain a crude extract, followed by one of two possible purification procedures. The first consisted of ammonium sulfate fractionation, ion exchange chromatography on Mono-Q and gel filtration on Superdex G75 to yield two isoenzymes, PPO1 and PP02 having molecular weights of 71.8 +/- 6.0 and 69.6 +/- 1.5 kDa, respectively. The second purification procedure involved hydrophobic interaction chromatography (HIC) on phenyl-sepharose CL-4B followed by gel filtration on Superdex G75 to yield a single active PPO fraction of 68.3 +/- 2.8 kDa molecular weight.
The two isoenzymes obtained by ion exchange chromatography exhibited pH optima of 6.5 (PPO1) and 6.8 (PPO2) and were stable in the pH range of 7.5 to 10.0. These two isoenzymes had a temperature optimum of 30--40°C. PPO2 retained 65% of its original activity after heating at 50°C for 10 min whereas PPO1 was completely inactivated by the same treatment. The PPO fraction obtained by HIC purification exhibited a pH optimum of 7.5 with catechol and D,L-dopa as substrates and was stable in the pH range 4 to 8. Its temperature optima, were 20 and 30°C respectively with D,L-dopa and catechol as substrates and this PPO fraction was able to retain 80% of its original activity after heating at 50°C for 10 min. Unstable enzymes were obtained by the ion exchange chromatography purification procedure suggesting that artifacts were created.
Kinetic studies performed with the PPO fraction obtained by the HIC purification showed that catechol had the highest catalytic efficiency ratio. The Km values were 28.1, 5.27 and 3.72 mM for catechol, catechin and D,L-dopa, respectively. The PPO from the HIC purification procedure was inhibited by benzoic acid and p-coumaric acid and inactivated by diethyldithiocarbamate but not by EDTA. L-Cysteine, ascorbic acid and its derivatives (erythorbic acid and sodium erythorbate) were also inhibitors of the oxidation of catechol, catechin and D,L-dopa.

Ce travaille de thèse concerne la synthèse de dérivés de polyphénols de type flavanol, ellagitannins C-glucosidique et procyanidine, modifiés avec un espaceur comportant une biotine terminale. Cette biotine terminale a permis d’immobiliser ces polyphénols modifiés sur des surfaces SPR, permettant ainsi l’étude d’interactions polyphénol-protéine en temps réel. Ainsi, la topoisomerase II alpha et l'actine fibrilaire ont montré une plus grande affinité pour les polyphenols de type ellagitannins que pour ceux de type flavanol. Nous avons également pu montrer que d’autres protéines (BSA, myoglobine, actine globulaire, streptavidine, collagen type I) n’avaient pas d’interaction avec les flavanols et les ellagitannins
This work concerns the synthesis of derivatives of polyphenols of the type flavanol, C-glucosidic ellagitannin and procyanidin, which are modified to bear a spacer ending with a biotin unit. This biotin ending unit allowed to immobilize these modified polyphenols on to SPR surfaces, which allowed the study of polyphenol-protein interactions in real time. The proteins topoisomerase II alpha and fibrilar actin showed a higher affinity for the polyphenols of the type ellagitannins than for those of the type flavanol. It was also showed that other proteins (BSA, myoglobin, globular actin, streptavidin, collagen type I) did not interact with either the flavanols or the ellagitannins

Ce travail concerne l'extraction d'enzymes de la famille des polyphénol oxydases à partir de champignons, leur caractérisation biochimique et leur immobilisation dans des matrices solides. Ces enzymes ont tout d'abord été extraites du champignon de Paris (Agaricus bisporus) puis partiellement purifiées. Une étude de leur activité enzymatique, de leur domaine de stabilité et de leur comportement thermique a été effectuée, ainsi que l'identification d'inhibiteurs. Cette approche a été étendue à la polyphénol oxydase de la truffe de désert (Terfezia leonis Tul.). Ces deux enzymes ont ensuite été piégées dans des gels de silice pour le dosage de la dopamine par un biocapteur optique et dans un gel d'alginate pour la dégradation du phénol
This work is devoted to the extraction of enzymes belonging to the polyphenol oxidase family from mushrooms, their biochemical characterization and their immobilization in solid hosts. These enzymes were first extracted from Paris mushrooms (Agaricus bisporus) and partially purified. A study of their enzymatic activity, stability conditions and thermal behavior was performed, together with the identification of inhibitors. A similar approach was applied to polyphenol oxidase extracted from desert truffle (Terfezia leonis Tul.). These enzymes were then trapped in silica gels for dopamine determination using an optical biosensor and in an alginate gel for phenol degradation

Tea and coffee are rich in polyphenols with a variety of biological activities. Polyphenols found in tea are predominantly flavonoids, of which up to 15% are present as free or esterified gallic acid. Coffee polyphenols are almost wholly comprised of chlorogenic acids. Many of the demonstrated activities of polyphenols are consistent with favourable effects on the risk of chronic diseases. In investigating the relationships between intake and exposure to such compounds and chronic disease-related endpoints, it is important to be able to identify biomarkers that are specific to the compounds of interest. 4-O-methyl gallic acid (4OMGA) and isoferulic acid have been identified as potential biomarkers of intake and exposure to polyphenols derived from tea and coffee, respectively. 4OMGA is derived from gallic acid in tea, and isoferulic acid from chlorogenic acid in coffee. The major objectives of the research which is the subject of this thesis were (1) to establish a dose-response relationship of 24h urinary excretions of 4OMGA and isoferulic acid following ingestions of black tea and coffee of different strengths, and (2) to explore relationships of tea and coffee intake with 24h urinary excretion of 4OMGA and isoferulic acid in human populations. It was found that there was rapid excretion of both 4OMGA and isoferulic acid in the first 6h after tea and coffee ingestion, respectively. Approximately 60 80% of the ingested dose was excreted during the first 6h after ingestion. Urinary excretion of 4OMGA and isoferulic acid was directly related to the dose of tea and coffee, respectively. That is, higher intake resulted in increased urinary excretion of the metabolites. The relationships of 24h urinary excretion of 4OMGA and isoferulic acid with long-term usual (111 participants) and contemporary recorded current (344 participants) tea and coffee intake were assessed. 4OMGA was strongly related to usual (r = 0.50, P < 0.001) and current (r = 0.57, P < 0.001) tea intake. Isoferulic acid was less strongly, but significantly associated with usual (r = 0.26, P = 0.008) and current (r = 0.18, P < 0.001) coffee intake. Overall, the results are consistent with the proposal that 4OMGA is a good biomarker for black tea derived polyphenol intake and exposure, but isoferulic acid may have only limited use as a biomarker for coffee-derived polyphenol exposure.

The latest generation of fighter aircraft utilizes a 270Vdc power system [1]. Such high voltage DC power systems are difficult to protect with conventional circuit breakers because the current does not automatically go to zero twice per cycle during a fault like it does in an AC power system and thus arcing of the contacts is a problem. Solid state power controllers (SSPCs) are the solid state equivalent of a circuit breaker that do not arc and which can respond more rapidly to a fault than a mechanical breaker [2]. Present SSPCs are limited to lower voltages and currents by the available power semiconductors [8,9]. This dissertation presents design and experimental results for a SSPC that utilizes SiC power JFETs for the SSPC power switch to extend SSPC capability to higher voltages and currents in a space that is smaller than what is practically achievable with a Si power switch. The research started with the thermal analysis of the SSPCs power switch, which will guide the development of a SiC JFET multi-chip power module to be fabricated by Solid State Devices Inc. (SSDI) using JFETs from SiCED and/or Semisouth LLC. Multiple multi-chip power modules will be paralleled to make the SSPC switch. Fabricated devices were evaluated thermally both statically and dynamically and electrically both statically and dynamically. In addition to the SiC module research a detailed design of the high voltage SSPC control circuit capable of operating at 200andamp;ordm;C was completed including detailed analysis, modeling and simulations, detailed schematic diagrams and detailed drawings. Finally breadboards of selected control circuits were fabricated and tested to verify simulation results. Methods for testing SiC JFET devices under transient thermal conditions unique to the SSPC application was also developed.

Anthocyanins are polyphenolic plant pigments that are responsible for much of the attractive colour displays found in many flowers, fruit and vegetables. Anthocyanins are divided into different classes based on the number of hydroxyl groups on their phenyl B-ring and subsequent side chain modifications. It has been shown in our laboratory that the introduction of the regulatory genes Delila and Rosea1 activates the biosynthetic pathway leading to accumulation of trihydroxylated anthocyanins in tomatoes. Tomato DFR substrate specificity for trihydroxylated precursors prevents the formation of other anthocyanin classes. By inhibiting the activity of F3’5’H, blocking biosynthesis of trihydroxylated anthocyanins, and introducing a non-specific DFR from A. majus, I developed two transgenic tomato lines that produced mono- and dihydroxylated anthocyanins. My work demonstrated that tomatoes could be enriched with different classes of anthocyanins using a metabolic engineering approach. Dietary consumption of plant secondary metabolites such as anthocyanins and other polyphenols are linked to a reduced risk of developing chronic, non-communicable diseases such as cancer, type 2 diabetes and cardiovascular diseases. High-anthocyanin tomato extracts induced cell death, cell cycle alteration and apoptosis in the human breast cancer cell lines, MCF-7 and MDA-MB-231, whilst WT tomato extracts exerted no biological effects. Purified anthocyanins and polyphenolic compounds had no or little effect on the metabolic status of breast cancer cells, suggesting that anthocyanins exert their biological effects in synergy with other components in the food matrix. Polyphenolic compounds may act as chemosensitizers for the treatment of tumours with chemotherapeutic agents. Co-treatment of breast cancer cells with the chemotherapeutic agents, doxorubicin or roscovitine, and high-anthocyanin or -resveratrol tomato extracts showed that dietary polyphenols might potentiate the effects of pharmacological agents in vitro. This work demonstrated that the health benefits of dietary plants can be significantly improved through nutritional enrichment with plant bioactives using metabolic engineering.

Tableau d'honneur de la FÉSP
L'obésité et son large spectre de maladies associées ont atteint des proportions pandémiques inquiétantes, soulignant la nécessité d’identifier des stratégies alternatives afin de lutter contre ce problème. À ce titre, les régimes riches en fruits et légumes représentent des déterminants bien établis d'une incidence plus faible de ces désordres métabolique. Grandement soutenus par des évidences épidémiologiques reliant les régimes riches en polyphénols et un meilleur état de santé, des efforts considérables ont été déployés afin d’étudier les bienfaits de ces métabolites secondaires des plantes. Malgré tout, les mécanismes par lesquels ces phytoéléments améliorent la santé métabolique demeurent encore mal compris, ce qui en justifie une étude plus approfondie. D’autre part, de plus en plus d’évidences indiquent que les bactéries intestinales exercent un important contrôle sur des aspects clés du métabolisme, et on comprend aujourd’hui que plusieurs phytoéléments de baies ont une biodisponibilité limitée, atteignant ainsi le colon qui abrite la plus vaste part du microbiote intestinal. Le travail présenté dans cette thèse vise donc à étudier l’impact de phytoéléments de baies sur le syndrome métabolique de souris soumises à une diète obèsogène et d’en comprendre le rôle du microbiote intestinal dans ces effets. En traitant quotidiennement ces animaux avec des extraits riches en polyphénols d'une gamme de baies aux compositions polyphénoliques variées, nous avons montré que les extraits les plus bioactifs (c.- à-d., canneberge, cloudberry, alpine bearberry, lingonberry et camu camu) partagent la capacité de diminuer l'inflammation intestinale, l’entotoxémie métabolique, la stéatose hépatique et la résistance à l'insuline. L'analyse des populations microbiennes fécales par séquençage du gène 16S ARNr a révélé que l'état métabolique amélioré lié à l'administration de ces extraits était associé à un remodelage draconien du microbiote intestinal, marqué par une expansion d'Akkermansia muciniphila. Cette bactérie intestinale est fortement associée à un faible niveau d’adiposité chez l’humain et son administration à des souris obèses a été montrée suffisante pour renverser le syndrome métabolique. Par ailleurs, les résultats présentés dans cette thèse suggèrent que les polymères de polyphénols, à savoir les proanthocyanidines et les ellagitannins, pourraient bien être des iv molécules clés dans les effets bénéfiques observés, ouvrant la voie à plus de recherche en ce sens. L’ingestion régulière de ces polyphénols par la consommation de canneberges, de cloudberry, d'alpine bearberry, de lingonberry et de camu camu représentent donc une stratégie efficace pour la prévention de désordres métaboliques associés à l’obésité. Cet ouvrage ouvre ainsi à de nouveaux concepts mécanistiques, ciblant l’axe intestin-foie et le microbiote intestinal pour expliquer les effets bénéfiques des polyphénols sur la santé métabolique.
Obesity and its wide spectrum of associated diseases have reached worrisome pandemic proportions, underscoring the need for alternative strategies to fight this problem. Plant-rich diets are well-established determinants of a lower incidence of obesity-related diseases, and fruits are important components of these diets. Supported by strong epidemiological evidence linking polyphenol-rich diets and better health status, research has been focused on the potential health effects of these plant secondary metabolites, albeit the mechanisms by which these poorly bioavailable phytonutrients improve metabolic health remains are not yet fully understood. Since there is compelling evidence for a relationship between host metabolic control and the gut microbiota, the work presented in this thesis aimed to investigate the impact of polyphenol-rich berry extracts on features of the metabolic syndrome in diet-induced obese mice. The work presented in this thesis also focuses on the relationship between putative gut microbial alterations driven by dietary polyphenols and its relevance to host metabolism. By daily treating dietinduced obese mice with polyphenol-rich extracts of a wide range of berries (with varied polyphenolic concentration and composition) we demonstrated that the most bioactive extracts (i.e., cranberry, cloudberry, alpine bearberry, lingonberry and camu camu) shared in common the ability to dampen intestinal inflammation and bacterial lipopolysaccharide leakage to systemic circulation, findings associated with reduced hepatic steatosis and improved insulin resistance. 16S rRNA genebased analysis of fecal DNA revealed that the improved metabolic status linked to the administration of these polyphenolic extracts was associated with a drastic gut microbial remodeling, marked by a consistent bloom of Akkermansia muciniphila. This gut bacterium is strongly associated with leanness in humans and its administration to obese mice reversed features of the metabolic syndrome. The findings presented in this thesis suggest that polymers of polyphenols, namely proanthocyanidins and ellagitannins, may have a superior impact on the gut-liver homeostasis, supporting further research on these particular classes of phenolic phytonutrients. While bringing evidence that substantiate the regular consumption of sources of proanthocyanidins and ellagitannins as a strategy to prevent prevalent chronic diseases associated with obesity, this work provides novel mechanistic insights pointing to the gut-liver axis and the gut microbiota as primary targets of dietary polyphenols in order to improve metabolic health.

The enzymatic browning and melanogenesis are associated respectively with the most of qualitative and economical losses during post-harvest processing in agro-food industry and human skin disorders in cosmetic field. The main responsible is tyrosinase or polyphenol oxidase (PPO, EC 1.14.18.1), a copper-containing oxidoreductase that catalyses two different enzymatic reactions involving polyphenolic substrates and oxygen and producing dark pigments. Recently, the research of new eco-friendly systems for controlling PPO activity is focused on innovative non-thermal technologies and bioactive compounds to replace the conventional thermal treatments and traditional additives. All of these have critical points related not only to organoleptic and nutritional qualities of agro-food products and stability in cosmetic formulations but also to human health after topical, oral or parenteral exposure. In this regards, the goal of this study is to evaluate, by in vitro and in vivo assays, the anti-browning performances of a UV-A LED technology (first contribution) and two natural extracts obtained from agro-food by-products such as citrus hydrosols (CIHs; second contribution) and agricultural wastes like vineyard pruning residues (VPRs; third contribution). In the first contribution, after fixing the optimal operational conditions of a UV LED illuminator prototype (2.43·10-3 Wm-2 irradiance) in accordance with number of LED diodes, voltage, and distance from sample, the UV-A light (390 nm) treatment at 25 °C over increasing time periods up to 60 min was applied on fresh-cut apples (Golden Delicious, Granny Smith, and Fuji) and pears (Abate Fétel and Decana). The total colour change (ΔE) and its percent reduction (%RΔE) were measured using a colorimeter and the greatest performances were observed in apples with higher %RΔE values than pears (58.3% vs. 25.5% on average after 60 min exposure, respectively). Moreover electrophoretic and zymographic techniques on the commercial mushroom tyrosinase (TYR) and PPO extracted from irradiated Golden Delicious apple slices confirmed the inhibitory effects of UV-A light on PPO activity. The anti-browning effectiveness of UV-A LED technology was related to irradiance, exposure time, and fruit type and cultivar. In the second contribution, three kinds of hydrosols, which have been obtained by subjecting citron, lemon, and orange peels to steam distillation (CH, LH, and, OH respectively), were spectrophotometrically assessed for anti-TYR activity in the presence of (+)–epicatechin and L-DOPA as the model phenolic substrates of plant enzymatic browning and human skin melanogenesis, respectively. All of the CIHs showed a mixed-type inhibition at varying levels in the 21.8–68.9 % range, depending on substrate type and concentration. The gas chromatography analysis (GC) of their terpene contents indicated that some known TYR inhibitors including myrcene, sabinene, geraniol and citral were present in CIHs. The third contribution investigate the anti-browning and antioxidant potentials of some grape juices obtained by cold-pressing the berries collected from the VPRs of Barbera (B) and Merlot (M) cultivars during 2013 (1) and 2014 (2) seasons. Among the VPRs, Merlot wastes spectrophotometrically exhibited a greater uncompetitive inhibition towards TYR activity than those of Barbera (68.2% and 67.8% for M1 and M2, respectively; 56.3% and 58.8% for B1 and B2, respectively), in the presence of catechol substrate, as confirmed also by gel diffusion assay. The zymographic techniques on the isoforms isolated from TYR and some plant PPOs (Fuji and Golden Delicious apples; Abate Fétel pears; Bintje potatoes) as well as in vivo trials on several fresh-cut fruits (Fuji, Golden Delicious, Granny Smith apples; Abate and Decana pears) vegetables (Bintje potatoes; eggplants), and dried apple slices (Golden Delicious) demonstrated that the inhibitory performances were related mainly to enzyme source. In this regards, this chemical treatment with VPRs was not effective on pear PPO. However, the VPRs showed not only anti-browning but also whitening and antioxidant capacities that were associated mainly with their high organic acids and epigallocatechin gallate (EGCG) contents detected by HPLC analysis. Overall this research confirms that the inhibitory effectiveness is a function of PPO source and inhibitor type and dose. The UV-A LED technology, CIHs, and VPRs are eco-friendly, safe, and inexpensive systems for effectively inhibiting PPO activity, thus preserving the enzymatic browning in agro-food and cosmetic industries. Moreover, these natural extracts, whose anti-browning performances depends mainly on their bioactive compounds contents, suggest a possible recycling use with high value added of these agro-food by-products.
L’imbrunimento enzimatico dei prodotti agro-alimentari in post-raccolta e le problematiche associate alla melanogenesi nel settore cosmetico comportano consistenti perdite qualitative ed economiche. Il principale responsabile di entrambi questi fenomeni è la tirosinasi o polifenol ossidasi (PPO, EC 1.14.18.1), una ossido riduttasi contenente un ione rame all’interno del sito attivo, che catalizza due differenti reazioni enzimatiche di ossidazione di substrati polifenolici e quindi rende possibile la successiva formazione di composti scuri. Negli ultimi anni, lo studio di nuovi sistemi ecocompatibili per il controllo dell’attività enzimatica si è focalizzato sulle tecnologie non-termiche e sugli inibitori di origine naturale da proporre in alternativa ai convenzionali trattamenti termici ed ai tradizionali additivi chimici. Un impulso alla ricerca in questa direzione è stato dato dalla dimostrazione del loro impatto negativo non solo sulla qualità organolettica e nutrizionale dei prodotti agro-alimentari e sulla stabilità delle formulazioni cosmetiche, ma anche sulla sicurezza in seguito ad ingestione o contatto. Partendo da questi presupposti il progetto di ricerca alla base di questa tesi di dottorato vuole valutare, attraverso saggi in vitro e in vivo, l’efficacia anti-imbrunimento di tre possibili sistemi alternativi: una tecnologia UV-A basata su fonte di luce a LED (primo contributo) e due estratti naturali ottenuti da sottoprodotti agro-industriali, gli idrosol degli agrumi (CHIs; secondo contributo) e gli scarti di potatura del vigneto (VPRs; terzo contributo). Nel primo contributo, il trattamento basato su luce UV-A, alla lunghezza d’onda di 390 nm, è stato applicato, a temperature ambiente, in intervalli fino un’ora complessiva, su fette di mela (Golden Delicious, Granny Smith, Fuji) e pera (Abate Fétel, Decana), utilizzando un prototipo di illuminatore a LED, dove alcuni parametrici fisici, quali numero di diodi, voltaggio e distanza dal campione, sono stati impostati in modo tale da garantire il massimo irraggiamento (2.43·10-3 Wm-2). La variazione totale di colore (ΔE) e la sua riduzione percentuale (%RΔE) sono state misurate utilizzando un colorimetro; le mele trattate mostravano una maggiore percentuale di riduzione del colore rispetto alle pere (rispettivamente 58.3% e 25.5% in media, dopo un irraggiamento di 60 minuti). Le ottime potenzialità inibitorie del trattamento con luce UV-A nei confronti dell’attività PPO sono state confermate anche dalle prove elettroforetiche e zimografiche eseguite su una tirosinasi commerciale di origine fungina (TYR) e sulla PPO estratta dalle fette di mela Golden Delicious dopo l’irraggiamento. Sulla base dei risultati ottenuti, l’efficacia anti-imbrunimento di questa tecnologia non termica, basata su luce UV-A con fonte a LED dipende non solo da tempo e intensità di irraggiamento, ma anche da tipo e cultivar di frutti utilizzati. Nel secondo contributo, l’inibizione tirosinasica da parte di tre diversi tipi d’idrosol, co-prodotti durante distillazione in corrente di vapore delle bucce di cedro, arancia e limone (CH, LH, OH, rispettivamente), è stata determinata spettrofotometricamente, utilizzando (+)–epicatechina e L-DOPA come substrati fenolici rappresentanti, rispettivamente, l’imbrunimento enzimatico delle piante e la melanogenesi della pelle. Tutti gli idrosol di agrumi testati mostravano un’inibizione enzimatica di tipo misto (tra 21.8 e 68.9 %), in base al tipo e alla concentrazione di substrato fenolico utilizzato. L’analisi gas cromatografica (GC) degli idrosol di agrumi ha permesso di individuare tra i terpeni alcuni noti inibitori dell’enzima TYR, quali mircene, sabinene, geraniolo e citrale. Il terzo contributo esamina le potenzialità anti-imbrunimento e antiossidante di alcuni centrifugati di bacche d’uva provenienti dagli scarti di potatura del vigneto di due diverse cultivar, Barbera (B) e Merlot (M), durante le stagioni di vendemmia dell’anno 2013 (1) e 2014 (2). Tra gli scarti di diradamento, quelli di Merlot inibivano maggiormente l’attività dell’enzima commerciale TYR, quantificata allo spettrofotometro in presenza del substrato catecolo, rispetto a quelli di Barbera (68.2% e 67.8% per M1 e M2, rispettivamente; 56.3% and 58.8% per B1 e B2, rispettivamente) mostrando un’inibizione di tipo acompetitiva; i risultati spettrofotometrici sono stati confermati anche dai test su piastra. Le tecniche zimografiche applicate sulle isoforme enzimatiche isolate da TYR e da alcune PPO vegetali (mele Fuji e Golden Delicious; pere Abate Féte; patate Bintje) così come le prove in vivo, condotte su diverse fette di frutta (mele Fuji, Golden Delicious e Granny Smith; pere Abate e Decana) verdura (patate Bintje; melanzane) e su fette essiccate di mela Golden Delicious, hanno dimostrato che il grado d’inibizione dipende principalmente dall’origine dell’enzima. Infatti, questo trattamento chimico non si è rivelato efficace nei confronti della PPO di pera. Tuttavia, lo studio effettuato sugli scarti di potatura di vigneto ha messo in luce le loro potenzialità non solo come agenti anti-imbrunimento, ma anche come sbiancanti e antiossidanti; le loro molteplici proprietà possono essere correlate al loro alto contenuto in acidi organici ed epigallocatechin gallato (EGCG). Nel complesso, questa ricerca dimostra come l’efficacia inibitoria sia legata principalmente non solo all’origine della PPO, ma anche alla dose e al tipo di inibitore applicato. La tecnologia UV-A con fonte a LED, gli idrosol di agrumi e gli scarti di potatura del vigneto rappresentano sistemi sicuri, economici ed a basso impatto ambientale per controllare l’imbrunimento enzimatico nel settore agro-alimentare e cosmetico. Inoltre, questi estratti naturali, ricchi in composti bioattivi con forti proprietà inibitorie, suggeriscono un possibile impiego alternativo che potrebbe conferire un interessante valore aggiunto a questi sottoprodotti della filiera agro-industriale.

Flavonoids, and phenolics in general, are very interesting compounds, because of their reactivity, and their properties, with the consequent importance on health and food quality. They are a group of very similar compounds, but little differences are enough to change their role (as between anthocyanidins and flavanols). The situation is still more complicated when we consider polymers, or interactions, with the HPLC limitation on their evaluation. Many works have been done concerning these molecules, and in spite of their importance, a number of questions are still open for the researcher imagination. In my PhD thesis, I studied different aspects of flavonoid behavior in grape and wine: • concerning to anthocyanins, I made experiments related to: • evaluation of anthocyanins extractability by simulated maceration process: the influence of bunch exposure • copigmentation and anticopigmentation in grape extracts study by spectrophotometry and post column reaction HPLC Studying tannins, my experiments were related to: • preliminary study: grape tannins accumulation • seed phenolics oxidation: development of a new ripening index • proanthocyanidins polymerization: a different hypothesis

Polyphenol oxidase (PPO) affects both kinds of matters that are nowadays at the forefront of the quality concept: those aspects related to health and those regarding organoleptic perception of food. In this Thesis, PPO activity has been characterized in different fruits and situations, and mathematical models to describe melanin formation from monophenolic and o diphenolic substrates have been developed. In addition, potential toxicity of these melanins on the pancreatic proteases carboxypeptidase A, carboxypeptidase B and trypsin has been studied. The second part of the Thesis covers PPO inactivation by innovative technologies, and the side effects that they cause on different parameters. Ultraviolet-visible irradiation has been modeled, and PPO inactivation by this method has been assessed in model solutions and in apple, pear and grape juices. Moreover, the effects of must irradiation on the quality of wine have also been assessed. In addition, high-hydrostatic pressure effectiveness in inactivating apple PPO was tested, as well as its effects on juices color.
La polifenol oxidasa (PPO) afecta a ambdós tipus de qüestions que són actualment al capdavant del concepte de qualitat: els aspectes relacionats amb la salut i els relatius a la percepció organolèptica dels aliments. En aquesta Tesi, l'activitat de la PPO s'ha caracteritzat en diferents fruites i situacions, i s'han desenvolupat models matemàtics per descriure la formació de melanina a partir de substrats monofenòlics i o difenòlics. A més, s'ha estudiat la toxicitat potencial d'aquestes melanines sobre les proteases pancreàtiques carboxipeptidasa A, carboxipeptidasa B i tripsina. La segona part de la Tesi tracta la inactivació de la PPO per tecnologies innovadores, i els efectes secundaris que causen en diferents paràmetres. La irradiació ultraviolada-visible ha estat modelitzada, i la inactivació de la PPO per aquest mètode s'ha avaluat en solucions model i en sucs de poma, pera i raïm. A més, els efectes de la irradiació del most sobre la qualitat del vi també han estat avaluats. En darrer lloc, s'estudià l'eficàcia de l'alta pressió hidrostàtica en la inactivació de la PPO de poma, així com els seus efectes sobre el color dels sucs.
La polifenol oxidasa (PPO) afecta a ambos tipos de cuestiones que se encuentran actualmente al frente del concepto de calidad: los aspectos relacionados con la salud y los relativos a la percepción organoléptica de los alimentos. En esta Tesis, la actividad de la PPO se ha caracterizado en diferentes frutas y situaciones, y se han desarrollado modelos matemáticos para describir la formación de melanina a partir de sustratos monofenólicos y o difenólicos. Además, se ha estudiado la toxicidad potencial de estas melaninas sobre las proteasas pancreáticas carboxipeptidasa A, carboxipeptidasa B y tripsina. La segunda parte de la Tesis trata la inactivación de la PPO por tecnologías innovadoras, y los efectos secundarios que causan en diferentes parámetros. La irradiación ultravioleta-visible ha sido modelizada, y la inactivación de la PPO por este método se ha evaluado en soluciones modelo y en zumos de manzana, pera y uva. Además, los efectos de la irradiación del mosto sobre la calidad del vino también han sido evaluados. Por último, se estudió la eficacia de la alta presión hidrostática en la inactivación de la PPO de manzana, así como sus efectos sobre el color de los zumos.

Polyphenol oxidases (PPOs) from several plant species, including wheat, have been implicated in the undesirable brown discoloration of food products. Wheat (Triticum aestivum L.) represents an interesting system to advance our understanding of plant PPO function for two important reasons, namely (1) the large size an complexity of its (allohexaploid) genome, and (2) its economic importance. Prior to this study, the molecular and biochemical properties of wheat PPOs were largely unknown. To remedy this situation, we have performed several BLAST searches of expressed sequence tag (EST) databases, using a known wheat PPO sequence as a search tool. This study suggested the presence of at least six PPO genes in hexaploid wheat, falling into two different phylogenetic clusters of three genes each. Presence of a wheat PPO multigene family was confirmed by Southern blotting. A combination of biochemical (enzyme purification and mass spectrometric analysis) and molecular (Northern) approaches confirmed that members of one cluster are not expressed in the developing kernels and senescing flag leaves, while regulation of one or several members of the other gene cluster controls PPO activity in these tissues. Our data, including immunoblotting and enzyme activity studies, further indicated that wheat PPOs are synthesized as inactive precursor (early kernel development) which are proteolytically processed and activated as the kernels mature. Activation of PPO precursor proteins was also demonstrated in vitro, in presence of purified trypsin. In these experiments, PPO activity increased during the first four hours and remained stable thereafter, indicating that the protein domains responsible for catalytic activity are quite stable. Research performed as a part of this dissertation has also demonstrated that wheat PPO activity is influenced by strong anionic detergents such as SDS and N-lauroylsarcosine. The corresponding experiments indicated that these detergents influenced both enzyme extraction and activity, at least in high-PPO wheat varieties. This work also has practical aspects, making PPO assays (as used by breeders for germplasm screening) more robust. In conclusion, as a result of this dissertation, wheat PPOs have emerged as a fascinating example of a plant multigene family with complex transcriptional and posttranscriptional regulation.

Cellular membranes are important targets for many membrane-active peptides and drug compounds. Here we are interested in deciphering how lipid membranes are perturbed by several membrane-active molecules, including the transmembrane domain of the influenza M2 protein (M2TM), aggregates formed by a synthetic polyglutamine peptide, and three polyphenol compounds (i.e., tamoxifen, genistein, and verapamil). We employ phase-separated ternary lipid model membranes in the form of giant unilamellar vesicles (GUVs) to simulate raft-like structures that have been proposed to govern many important processes in plasma membranes (e.g., intracellular singling and trafficking). Specifically, we use fluorescent microscopy to interrogate how those membrane additives modulate the phase behavior of free-standing GUVs, as well as the miscibility transition temperature (Tm). We find that M2TM increases Tm and causes vesicle budding; polyglutamine aggregates disrupt lipid membranes; and the three polyphenol compounds exert disparate effects on GUV Tm.

Waste-generating industrialisation is intrinsically associated with population and economic proliferation. This places considerable emphasis on South Africa's water shortage due to the integral relationship between population growth rate and infrastructure development. Of the various types of industry-generated effluents, those containing organic pollutants such as phenols are generally difficult to remediate. Much work has been reported in the literature on the use of enzymes for the removal of phenols from these waste-streams but little application of this bioremediation approach has reached practical fruition. This study focuses on integrating and synergistically combining the advantages of enzyme-mediated dephenolisation of synthetic and industrial effluent with that of membrane teclmology. The ability of the enzyme polyphenol oxidase to convert phenol and a number of its derivatives to chemically reactive o-quinones has been reported extensively in the literature. These o-quinones can then physically be removed from solution using various precipitation or adsorption techniques. The enzyme is, however, plagued by a product-induced phenomenon known as suicide inactivation, which renders it inactive and thus limits its application as a bioremediation tool. Integrating membrane technology with the enzyme's catalytic ability by immobilising polyphenol oxidase onto polysulphone and poly(ether sulphone) capillary membranes enabled the physical removal of these inhibitory products from the micro-environment of the immobilised enzyme which therefore increased the phenol conversion capability of the immobilised biocatalyst. Under non-immobilised conditions it was found that when exposed to a mixture of various phenols the substrate preference of the enzyme is a function of the R-group. Under immobilised conditions, however, the substrate preference of the enzyme becomes a function of certain transport constraints imposed by the capillary membrane itself. Furthermore, by integrating a quinone-removal process in the enzyme-immobilised bioreactor configuration, a 21-fold increase in the amount of substrate converted per Unit enzyme was observed when compared to the conversion capacity of the inunobilised enzyme without the product removal step. Comparisons were also made using different membrane bioreactor configurations (orientating the capillaries transverse as opposed to parallel to the module axis) and different immobilisation matrices (poly(ether sulphone) and polysulphone capillary membranes). Conversion efficiencies as high as 77% were maintained for several hours using the combination of transverse-flow modules and novel polysulphone capillary membranes. It was therefore concluded that immobilisation of polyphenol oxidase on capillary membranes does indeed show considerable potential for future development.

The thesis reports the synthesis and characterization of new carbamoylic prodrugs of the natural bioactive polyphenol Curcumin, found in many foodstuff. Curcumin is a promising drug candidate because of its credited beneficial health effects and wide spectrum of action. However, the low bioavailability due to its poor solubility in aqueous media (i.e. physiological fluids) and to its very fast hepatic metabolism, greatly limits the use of Curcumin in the pharmaceutical field. The “prodrug approach” represents a valid solution to overcome these issues. This is based on the conjugation of the active compound with a specific promoiety through a bioreversible linkage. This modification is expected to improve the pharmacokinetic profile of the compound (prevent fast metabolism and enhance bioavailability). The choice of the bioreversible linker is crucial for the pharmacokinetic performance of the prodrug. I chose and used a urethane linkage, which has been recently demonstrated by our group to be the best option to protect and enhance the bioavailability of natural polyphenols in vivo. Curcumin was conjugated with two different types of promoieties: natural amino acids and poly-2-alkyl-oxazolines, a class of hydrophilic synthetic polymers. In particular, leucine, isoleucine and valine were selected as promoieties for the conjugation with Curcumin, since these amino acids show enhanced intestinal absorption due to active transport mechanism. The conjugation of these amino acids with Curcumin could thus improve the absorption of Curcumin following oral administration of the prodrug. Curcumin has two phenolic hydroxyl groups. I functionalized Curcumin on one and both hydroxy groups by conjugation with amino acids through a urethane linkage, obtaining six new prodrugs (mono- and di-substituted, respectively). The stability of the new prodrugs and of Curcumin itself in aqueous media was studied using different experimental approaches: UPLC-UV, HPLC/MS, UV-Vis spectroscopy and 1H-NMR. The study of these processes and systems was severely hindered by the solubility of the new Curcumin prodrugs in aqueous media which turned out to be rather poor, either under acidic or near neutral pH conditions, as are found in the stomach and intestine. The prodrugs proved to be more stable in acidic solutions as expected based on the known reactivity of the N-monosubstituted carbamoylic linkage. In solutions at pH 6.8 (as found in the intestine), some degradation was observed. The stability of the new derivatives against hydrolysis was also studied by 1H-NMR spectroscopy in D2O-DMSO-d6 solutions with different D2O contents. No degradation was observed in these aqueous media over three hours. Preliminary pharmacokinetic experiments were carried out and proved that the di-substituted leucine carbamoyl derivative of Curcumin is capable of improving the bioavailability of the parent phenol in mice model. Finally, the affinity of these prodrugs for plasma proteins was assed, using fluorescence binding assays with human serum albumin (HSA), which is ubiquitous in the blood stream. All of the new derivatives, except for the mono-leucine prodrug, have higher affinity for HSA than Curcumin itself. From these combined data a good distribution in the blood stream and high stability against hepatic enzymes can be expected for the new derivatives developed with this thesis, in particular for the mono-functionalized compounds, which make these compounds potential candidates as Curcumin prodrugs. The last chapter of this thesis is focused on the synthesis of new drug-delivery systems obtained from the self-assembly of amphiphilic polymeric prodrugs of Curcumin. Also in this case a carbamoylic bond was chosen as the linkage between Curcumin and the polymer chain. Poly-2-methyl-2-oxazolines (PMOXAs) were chosen as promoieties for conjugation with Curcumin because of their similarity to the most commonly used poly-ethylene glycols (PEGs), showing the same stealth effects, together with a reduced immune response, and a higher hydrophilicity. In particular, five poly-2-methyl-2-oxazolines of different chain length were conjugated to one hydroxy group of Curcumin. Because of the high hydrophilic character of the PMOXAs, these five new prodrugs can be classified as amphiphiles. Indeed they possess the ability to self-assemble in micelle-like structures when put in aqueous solution, providing good-to-excellent solubility of Curcumin in physiological-like media. The correlation between the chain length and the self-assembly ability of these prodrugs was investigated. All derivatives have a critical micellar concentration (CMC) in the 10-6-10-4 M range. Curcumin-PMOXA30 showed the highest ability to self assemble into micelles at concentrations as low as 10-6 M. On the contrary, conjugates with PMOXA50 and PMOXA100 resulted in a higher CMC value, probably due to the unfavourable hydrophilic/hydrophobic ratio, typical of longer polymeric chain, which prevents the self-assembly process. On the other hand the derivative Curcumin-PMOXA10, showed the tendency to aggregate and precipitate in solution, probably due to a too pronounced hydrophobic character. The size and the stability in water of all micelles were tested using dynamic light scattering (DLS) and transmission electron microscopy (TEM). An inverse correlation between chain length and size was found. In fact, conjugates with PMOXA20 and PMOXA30 form micelles with a diameter around 100-130 nm, whereas analogues with PMOXA50 and PMOXA100 with diameters of about 50-30 nm. For all conjugates, except Curcumin-PMOXA10, a good stability in aqueous environments was observed. Furthermore, I tested the release of Curcumin from each type of micelles by means of UV-Vis analysis. The experiments were carried out in phosphate buffered saline (PBS) at pH 7.4 and at 37°C in order to mimic the blood pH value. The conjugate Curcumin-PMOXA30 showed the best kinetic profile with around 90% of released Curcumin during five hours. The other conjugates showed instead a release of Curcumin around 10-30% during the same time.
La tesi descrive la sintesi e la caratterizzazione di nuovi prodrugs carbamoilici della Curcumina ((1E,6E)-1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione), un importante polifenolo naturale cui vengono accreditate molteplici proprietà benefiche ed un ampio spettro d’azione. L’utilizzo in campo farmaceutico del composto naturale tal quale è però limitato dalla sua bassa biodisponibilità dovuta a scarsa solubilità nei liquidi fisiologici ed a un rapido metabolismo epatico. Difatti, dopo somministrazione orale di Curcumina, solo tracce del composto naturale vengono ritrovate in circolo. L’approccio prodrug, rappresenta quindi un’utile opzione per aggirare questi problemi. Questo approccio è basato sulla modificazione del composto naturale mediante protezione bioreversibile e coniugazione a specifici sostituenti (promoieties) per ottenere le proprietà chimico-fisiche desiderate (solubilità, stabilità ecc). Sulla base di risultati ottenuti precedentemente con altri fenoli naturali, ho scelto ed utilizzato il legame carbamoilico per coniugare la Curcumina a due diversi tipi di promoieties, amino acidi naturali e polimeri sintetici poliossazolinici. Nello specifico, tre amminoacidi, leucina, isoleucina e valina, sono stati selezionati come promoieties più promettenti da coniugare alla Curcumina. Gli amminoacidi sono stati scelti per la loro affinità verso specifici trasportatori presenti a livello intestinale. Questo, dovrebbe favorire l’assorbimento intestinale della Curcumina a seguito della somministrazione orale dei prodrugs. Poiché la Curcumina possiede due gruppi idrossilici, ho scelto di funzionalizzarne rispettivamente uno ed entrambi mediante coniugazione con gli amminoacidi. Sono stati sintetizzati così sei prodrugs della Curcumina classificabili in mono- e di-sostituiti. Successivamente è stata testata la stabilità della Curcumina e dei prodrugs in soluzione acquosa a diversi valori di pH (1 e 6.8) al fine di mimare le condizioni di stomaco e intestino. Sono state effettuate analisi UPLC-UV, HPLC-MS, spettroscopia UV-Vis, e 1H-NMR. Nel condurre questi esperimenti, ho incontrato però numerosi problemi dovuti in particolar modo alla bassa solubilità dei composti in acqua ed alla loro tendenza a precipitare. I prodrugs hanno mostrato una maggiore stabilità in soluzioni acide, confermando le aspettative relative ai carbammati N-mono-sostituiti, la cui reattività è nota. In soluzioni a pH 6.8 (pH intestinale) sono invece state osservate alcune tracce di degradazione. L’idrolisi dei derivati è stata studiata anche tramite spettroscopia 1H-NMR usando soluzioni di D2O/DMSO-d6 a diverse percentuali di D2O. Nessuna traccia di degradazione dei prodrugs è stata osservata in queste condizioni. Gli esperimenti preliminari di farmacocinetica che sono stati condotti su topi modello, hanno inoltre provato che il derivato carbamoilico di-sostituito della Leucina è in grado di aumentare la biodisponibilità della Curcumina dopo somministrazione orale. Infine, ho verificato l’affinità della Curcumina e dei prodrugs nei confronti delle proteine plasmatiche. In particolare, ho effettuato saggi fluorimetrici di binding utilizzando l’albumina umana (HSA), importante per le sue capacità di complessamento e distribuzione di sostanze farmaceutiche nel sangue. Tutti i sei nuovi prodrugs hanno mostrato un’affinità elevata per l’albumina, superiore a quella della Curcumina stessa, ad eccezione del derivato mono-sostituito della leucina. Dall’insieme dei risultati ottenuti dallo studio dei prodrugs descritti in questa tesi è emersa la loro buona distribuzione nel circolo sanguigno e un’alta stabilità nei confronti degli enzimi epatici, evidente soprattutto per i composti mono-sostituiti, incoraggiandone così un potenziale utilizzo in campo terapeutico. L’ultimo capitolo di questa tesi descrive la sintesi di nuovi sistemi di rilascio farmaceutico ottenuti mediante un processo di auto assemblaggio di prodrugs polimerici della Curcumina con caratteristiche anfifiliche. Anche in questo caso è stato scelto il legame carbamoilico per garantire la coniugazione tra i polimeri e la Curcumina. Tra i vari polimeri, la scelta è ricaduta sulle poli-2-metil-2-ossazoline (PMOXAs) la cui somiglianza ai poli-etilenglicoli (PEGs), molto spesso utilizzati in questo campo, sommata ad una minore risposta immunitaria e ad una maggiore idrofilia rende promettente la scelta di questi composti a ruolo di promoieties. In particolare, cinque poli-2-metil-2-ossazoline, differenti tra loro per lunghezza della catena polimerica, sono state coniugate ad un solo gruppo idrossilico della Curcumina. A causa dell’elevata idrofilicità dei polimeri e della alta idrofobicità della Curcumina, questi nuovi cinque prodrugs possono essere classificati come anfifilici. Essi hanno, infatti, mostrato la capacità di auto assemblare in strutture di tipo micellare in soluzioni acquose garantendo così un’elevata solubilità della Curcumina in condizioni simili a quelle fisiologiche. Il lavoro si è concentrato in particolare sulla studio della correlazione tra la lunghezza della catena polimerica e la capacità di auto assemblare. Per tutti i derivati è stata determinata la concentrazione micellare critica (CMC), che si aggira in un intervallo di 10-6-10-4 M. Dai risultati emersi, il coniugato Curcumina-PMOXA30, caratterizzato da una CMC intorno a 10-6 M, si è dimostrato il più incline ad auto assemblare. Al contrario, sono state identificate CMC più alte per i coniugati Curcumina-PMOXA50 e Curcumina-PMOXA100 dovute probabilmente ad un bilancio sfavorevole tra le parti idrofobiche e quelle idrofiliche che, essendo più lunghe, ostacolano il processo di micellizzazione. Dall’altra parte, il derivato Curcumina-PMOXA10 ha mostrato alta tendenza ad aggregare e precipitare a causa della corta catena polimerica che in questo caso non riesce a bilanciare l’idrofobicità della Curcumina. La stabilità e le dimensioni delle micelle in soluzione acquosa sono state stimate utilizzando il light scattering dinamico (DLS) e la microscopia a trasmissione elettronica (TEM). La lunghezza della catena polimerica e la dimensione delle particelle sono risultate essere in un rapporto di inversa proporzionalità. Infatti, i coniugati costituiti da PMOXA20 e PMOXA30 presentano un diametro intorno a 100-130 nm, mentre i coniugati con PMOXA50 e PMOXA100 mostrano diametri inferiori intorno a 50-30 nm. Inoltre, tutti i coniugati, ad eccezione di Curcumina-PMOXA10, hanno dimostrato una buona stabilità in soluzione acquosa. Successivamente, ho testato il rilascio di Curcumina da ogni tipologia di micella mediante analisi UV-Vis in soluzione di buffer salino (PBS) a pH 7.4 ed a 37°C per mimare il pH sanguigno. Il coniugato Curcumina-PMOXA30 è risultato il più promettente mostrando il migliore profilo cinetico e favorendo il rilascio di circa il 90 % della Curcumina che costituisce la micella durante le prime cinque ore. Gli altri coniugati hanno invece mostrato profili diversi rilasciando, nello stesso intervallo di tempo, percentuali di Curcumina intorno al 10-30%.

In this study, the aim was to find an economical plant source for polyphenol oxidase (PPO) production as an alternative to mushroom and possible application areas by characterization and immobilization of the PPOs. For this purpose, tissues of various plants of no commercial value were screened for their PPO activities. Mulberry leaf tissues showed the highest PPO activity against 4-methyl catechol which was comparable to that of mushroom. Average Km and Vmax values of free mulberry leaf PPOs were found as 7 mM and 218 U/ml, respectively. Mulberry leaf PPOs were immobilized in a polypyrole matrix and the Km and Vmax values of immobilized PPOs were calculated as 35 mM and 3 U/ml, respectively. Mulberry leaf PPO was the most active at 45°
C and pH 7. By using electrophoretic analysis, laccase and catechol oxidase type activities of PPOs and in addition, peroxidase activity were detected. Molecular weights of laccase, peroxidase and catechol oxidase were found to be about 62, 64 and 62-64 kDa, with pI values of 8.0-8.5, 4.5 and 10, sequentially.