The seed coat of the common bean (Phaseolus vulgaris L.) can be considered an important source of flavonoids. Flavonoids are well known for their beneficial effects on health long before they were isolated as effective compounds. Dry beans are typically processed and the seed coats may be removed and discarded prior to consumption. Therefore, a better understanding of the antioxidant and anti-inflammatory activity of coloured dry bean seed coats would be beneficial in determining their potential use as an ingredient in the functional food and nutraceutical industry. Flavonol glycosides were identified from acetone extracts of seed coats of black beans, pinto beans, and dark and light red kidney beans, representing nine varieties grown in Manitoba. Based on HPLC-MS/MS, black beans contained the 3-O-glycosides of kaempferol, quercetin, and myricetin. Pinto beans contained kaempferol 3-O-glycosides, while light and dark red kidney beans contained quercetin 3-O-glycosides. In addition, we reported the presence of a flavonol triglycoside for the first time in dry bean seed coats. Concentrations of kaempferol-3-O-glucoside were the greatest varying from 0.44 to 7.08 to mg/g of dried seed coat weight, followed by quercetin-3-O-glucoside varying from 0.91 to 3.84 mg/g of dried seed coat weight, and then rutin varying from 0.13 to 0.22 mg/g of dried seed coat weight. The DPPH method demonstrated seed coat crude extracts of Eclipse, a black bean, and Windbreaker, a pinto bean, to have the highest antioxidant activities among the samples. Eclipse, with the maximum concentration of total phenolic compounds, exhibited an antioxidant activity of 57,816 μmol trolox equivalent/100g of dried seed coat weight and Windbreaker, with the maximum concentration of total flavonoid compounds, exhibited an antioxidant activity of 57,451 μmol trolox equivalent/100g of dried seed coat weight. Cellular measures of anti-inflammatory activity of seed coat crude extracts in LPS-induced murine macrophage RAW 264.7 cells showed both anti- and pro-inflammatory effects. Extracts of Windbreaker and Eclipse decreased TNF-α levels, suggesting anti-inflammatory properties, while other varieties showed increased levels of TNF-α production, or pro-inflammatory activity. The results indicate seed coats of Windbreaker and Eclipse may have the potential to be used as a functional food ingredient and possibly prevent disease and enhance human health.

The seed coat of the common bean (Phaseolus vulgaris L.) can be considered an important source of flavonoids. Flavonoids are well known for their beneficial effects on health long before they were isolated as effective compounds. Dry beans are typically processed and the seed coats may be removed and discarded prior to consumption. Therefore, a better understanding of the antioxidant and anti-inflammatory activity of coloured dry bean seed coats would be beneficial in determining their potential use as an ingredient in the functional food and nutraceutical industry. Flavonol glycosides were identified from acetone extracts of seed coats of black beans, pinto beans, and dark and light red kidney beans, representing nine varieties grown in Manitoba. Based on HPLC-MS/MS, black beans contained the 3-O-glycosides of kaempferol, quercetin, and myricetin. Pinto beans contained kaempferol 3-O-glycosides, while light and dark red kidney beans contained quercetin 3-O-glycosides. In addition, we reported the presence of a flavonol triglycoside for the first time in dry bean seed coats. Concentrations of kaempferol-3-O-glucoside were the greatest varying from 0.44 to 7.08 to mg/g of dried seed coat weight, followed by quercetin-3-O-glucoside varying from 0.91 to 3.84 mg/g of dried seed coat weight, and then rutin varying from 0.13 to 0.22 mg/g of dried seed coat weight. The DPPH method demonstrated seed coat crude extracts of Eclipse, a black bean, and Windbreaker, a pinto bean, to have the highest antioxidant activities among the samples. Eclipse, with the maximum concentration of total phenolic compounds, exhibited an antioxidant activity of 57,816 μmol trolox equivalent/100g of dried seed coat weight and Windbreaker, with the maximum concentration of total flavonoid compounds, exhibited an antioxidant activity of 57,451 μmol trolox equivalent/100g of dried seed coat weight. Cellular measures of anti-inflammatory activity of seed coat crude extracts in LPS-induced murine macrophage RAW 264.7 cells showed both anti- and pro-inflammatory effects. Extracts of Windbreaker and Eclipse decreased TNF-α levels, suggesting anti-inflammatory properties, while other varieties showed increased levels of TNF-α production, or pro-inflammatory activity. The results indicate seed coats of Windbreaker and Eclipse may have the potential to be used as a functional food ingredient and possibly prevent disease and enhance human health.

Obesity is considered an underlying risk factor for metabolic disease including cardiovascular disease (CVD) and diabetes. The fractions containing flavonols from apple peel were evaluated for their antioxidant, anti-inflammatory, and hypolipidemic properties using in vitro and in vivo experimental model systems. The fractionated polyphenolics from apple peels showed a strong antioxidant property protecting against heat-induced oxidation of polyunsaturated fatty acids present in fish oil. Apple flavonols (AF), eicosapentaenoic acid (EPA) and the isoquercitrin-EPA ester (QE) significantly reduced serum triacylglycerols and elevated the high density lipoprotein (HDL)-cholesterol compared to the high fat control group. C-reactive protein and interleukin-6 were also reduced compared to the high fat control group and inflammation induced by lipopolysaccharides. Serum adiponectin and interferon-? concentrations were significantly altered by QE treatment. Overall, AF and QE exhibited anti-inflammatory and hypolipidemic effects under in vivo conditions. These beneficial physiological properties and mode of action of AF and QE need to be further investigated.

Carbon monoxide (CO) is an extremely useful molecule with applications in industrial manufacturing, synthetic procedures as a C1 building block, and as a potential pharmaceutical to produce anti-inflammatory effects and vasodilation. However, the toxicity associated with CO has prevented its full utilization. In order to safely handle CO, compounds and molecules have been developed that act as storage materials for the gas. Ideal storage platforms only release CO upon stimulation via a trigger. Light activation is the most desirable trigger as it can be regulated in terms of the intensity and the wavelength of light used. The majority of light-induced CO-storage platforms that have been reported to date consist of metal carbonyl compounds where CO is bound directly to a metal center. However, disadvantages inherent to this motif, such as potential toxicity associated with the metal and lack of characterization of CO release remnant(s), has pushed the research community to search for alternative CO storage structures. The research presented in this dissertation outlines our approach toward the development of safe-to-handle, light-induced CO release platforms. We use a flavonol structure similar to those found in fruits and vegetables, such as quercetin, as a light-induced CO release unit. Through changes in the structure of the flavonol and its surrounding environment in chemical compounds, we have found ways to strategically control the light-induced CO release reactivity of the flavonol. Chemical compounds developed in this project are of interest for studying the effects of CO in biological systems and applications in synthetic processes.

Abstract Flavonoids are a class of secondary metabolites in plants that are involved in many important functions. Various flavonoid compounds have also been reported to be beneficial for human health. Bilberry (Vaccinium myrtillus L.) is the characteristic field layer species in boreal forests and the fruits of bilberry are rich in anthocyanin pigments, a subclass of flavonoids. In the present work, flavonoid biosynthesis was examined in different tissues of bilberry. The focus was on the developing fruits of wild type and natural color mutants of bilberry, and on effect of solar radiation on flavonoid biosynthesis in bilberry leaves. For the isolation of RNA for gene expression analysis, a method was optimized for different tissues of bilberry. The cDNA fragments of five genes from the flavonoid pathway, coding phenylalanine ammonia-lyase, chalcone synthase, flavanone 3-hydroxylase, dihydroflavanol 4-reductase and anthocyanidin synthase, were isolated from bilberry using polymerase chain reaction technique, sequenced, and labelled with dioxigenin-dUTP label. These homologous, bilberry-specific probes were used for determining the expression of the flavonoid pathway genes in bilberry fruits, flowers and leaves with a modified non-radioactive method developed in the course of the study. The anthocyanins, catechins, proanthocyanidins, flavonols and hydroxycinnamic acids in fruits, leaves and different fractions of bilberry were identified and quantified with high-performance liquid chromatography combined with a diode array detector and mass spectrometer. The results demonstrate a correlation between anthocyanin accumulation and expression of the flavonoid pathway genes during the ripening of berries. A correlation between flavonol and anthocyanin biosynthesis was detected in bilberry and also in previous literature collected from flavonol and anthocyanin analyses from other fruits. Accordingly, models for the connection between flavonol and anthocyanin synthesis in fruit species were suggested. Activation of the expression of flavonoid pathway genes and accumulation of flavonoids and hydroxycinnamic acids was detected in leaves growing under direct solar radiation, compared to the shadow leaves of the same plants. Based on the results, it is suggested that cyanidin of anthocyanins and flavonol quercetin play a predominant role in the defence against high solar radiation in Vaccinium leaves. The results give new information about the biosynthesis of flavonoids in bilberry at the gene level, in addition to the information of the composition and content of flavonoids during fruit development and in different parts of the bilberry plant. Also, new information was obtained of the roles of flavonoids in protecting plants from excess solar radiation.

Microwave hydrodiffusion and gravity (MHG) technique is an attempt towards development of green extraction, as this environment friendly technique has completely eliminated the use of organic solvents. After describing the effectiveness of microwave radiations in extraction field in the first part of this manuscript, we have optimized this noval extraction method to get antioxidants rich extract. Along with studying the temperature distributions in different parts of plant material under the effect of microwave irradiations, we have analyzed the influence of microwaves in enhancing the antioxidant activity of extracts by using different tests. We have got the promising results concerning about the antioxidant rich extracts of different onion varieties and sea buckthorn in generalization step against the conventional solvent extracts. The application of vacuum system in this extraction system helped in restraining the limitations like dry extract yield and flavonol contents. Incomparison to traditional and recent extraction systems, the MHG extracts doesn’t require any filtration and purification steps as it works in absence of any solvent and water and are highly recommended for direct application in industrial products / L'hydrodiffusion générée par micro-ondes est une nouvelle technique d'extraction mise au point au sein de l'Université d'Avignon et des Pays de Vaucluse. Ce procédé est une combinaison entre une technique traditionnelle et une technologie innovante. En effet, le chauffage par micro-ondes a permis d'initier et de générer le transfert de matière et de chaleur de l"intérieur des matrices végétales (oignons) vers l'extérieur et de réduire de façon considérable les temps d'extraction des antioxydants sans aucune intervention de solvant. A titre de comparaison, les polyphénols de différentes variétés d'oignons ont été extraits par l'hydrodiffusion générée par micro-ondes et par la technique conventionnelle, l'extraction par solvant. Les rendements obtenus par micro-ondes sont presque identiques à ceux obtenus à l'aide d’un solvant alors que les temps d'extraction sont réduits. La capacité antioxydante des extraits micro-ondes est supérieure à celle obtenue par technique conventionelle. Ce qui présage des potentialités d'application dans le domaine agroalimentaire en particulier pour la valorisation des co-produits. Une étude cinétique de l'extraction, ainsi qu'une observation au microscope optique(cytologie) des matrices traitées soumises aux micro-ondes et au solvant ont mis en évidence la spécificité de l'extraction sans solvant assistée par micro-ondes au niveau des mécanismes de libération et d'extraction des molécules antioxydantes au sein du végétal. L'effet des micro-ondes a pour conséquence une libération plus rapide des principes actifs contenue dans la plante grâce à l'ouverture quasi instantanée des glandes et l'explosion des cellules. L'explication de la différence de composition chimique entre les procédés d'extraction par solvant et par micro-ondes pourrait être basée principalement sur des phénomènes de solubilité, de polarisation diélectrique ainsi qu'un transfert de matière et de chaleur inversé

Carbon monoxide (CO) is now well established as one of the signaling molecules in higher organisms, including humans. Due to its physiological roles, CO is now accepted as a potential therapeutic agent. The use of CO gas has been studied in multiple clinical trials. Vasodilation, anti-inflammatory, anti-apoptotic, anti-proliferative and cytoprotective effects are just a few of the pharmacological actions attributed to CO gas in various models of diseases. Use of inhaled CO gas as a therapeutic has many limitations which necessitate the development of a new approach for CO delivery. In order to handle CO safely, compounds that release CO (CO-releasing molecules, CORMs) have been developed. CORMs that release CO only when triggered, and with the ability to target certain tissue sites, are of particular interest. Our lab is developing molecules that release CO only when illuminated with visible light (photoCORMs). These photoCORMs are based on a motif found in naturally-occurring flavonols, which are chemical compounds found in wide variety of foods including fruits, vegetables, tea and dark chocolate. The research presented in this dissertation outlines the results of studies on extended flavonols as CO release agents. The specific studies described herein focus on understanding visible light-induced CO-releasing flavonols in terms of their: a) structure/reactivity relationships, especially in biological environments; b) interactions with metal ions and proteins; c) reaction pathway of CO release; and d) their properties when combined with a CO-sensing motif.

Cancer is one of the leading causes of death in countries throughout the world. Increase in the production of Reactive Oxygen Species (ROS) has been implicated in many human disease processes, including aging and carcinogenesis. Detoxification of ROS in the cell is provided by both enzymatic and non-enzymatic systems, which constitute the antioxidant defence systems and is crucial to the survival of organisms. The cancer inhibitory propierties of antioxidant compounds such as polyphenols have been well established in experimental and epidemiological studies, showing the intake of these antioxidants within our diet can carry out an effective protective action toward the oxidative stress created in the body by imbalance between ROS and its endogenous defence mechanism. However, the molecular mechanisms responsibles for these effects are not so well known and more studies are needed to provide clear evidence of their protective effects.The aim of this study is to determine and compare the posible antitumoral properties of several polyphenolic fractions, obtained from the extraction and fractionation of wine by-products consisting of grape skins, seeds and stems. These polyphenolic fractions have high antiradical potential and are mainly composed by flavanol monomers with or without gallate groups, glycosylated flavonols and mostly procyanidin oligomers. The effect of these fractions is analysed on cancer cells at cellular and metabolic levels. Moreover, as solar radiation in the UV range is the major source of adverse reactions in the skin and is one of the most efficient environmental carcinogen known, the possible capacity of these fractions to protect against cellular damage induced by ultraviolet radiation is evaluated and compared. The results obtained in this study let us to confirm the polyphenolic fractions studied are very specific antiproliferative agents with very low cytotoxicity to non-proliferative normal cells, such as peripherial blood lymphocites (PBLs). Moreover, treatment with these fractions results in intracellular metabolic changes, restricting the ability of tumoral cells to proliferate and inhibiting glycolysis, being higher for the fraction rich in ECG containing oligomeric flavanols.Like for the study of antitumoral effect at cellular and metabolic levels, the results obtained in the analysis of the protective capacity of these polyphenolic fractions against UV-induced damage, confirm them as potential natural chemopreventive agents. Briefly, the results obtained in this study let us to conclude the polyphenolic fractions rich in procyanidin oligomers and gallate esters are the most efficient as antitumoral agents, active at both cellular and metabolic levels with low cytotoxicity. Additionally, polymerization and percentge of galloylation are also important in the efficacy of the polyphenolic fractions as protectors against damage induced by ultraviolet radiation, suggesting they may be useful for the prevention and treatment of a variety of solar UV light-induced human skin disorders.

Flavonols are hydroxyl-substituted flavonoids and naturally occur as secondary metabolites in plants. Several studies have discovered extensive medicinal properties of flavonols. The present work reports on structural and functional investigation of the B ring substituted flavonols based on spectroscopic and electrochemical techniques. The purpose of this study is to determine the influence of the B ring substitutions on the hydrogen bonding interactions, the electronic effects in ruthenium complexes and the Al3+ chelation of B ring substituted flavonols. The electronic effects of the B rings were changed by introducing methyl, methoxy and nitro groups at position 4ʹ on the B ring. The 3ʹ-methyl substitution was performed in order to increase the electronic density of the B ring via inductive effects. The 2ʹ-methyl and 2ʹ, 6ʹ-dimethyl substitutions increased the steric effects around the inter-ring bond between the B and the C rings, and the B ring was highly deconjugated from the AC rings. The intramolecular hydrogen bonding distances at 3-hydroxy-4-carbonyl units of the B ring substituted flavonols were elongated while the dihedral angles between the B and AC increased. Strong intermolecular hydrogen bonding interactions were also observed in the crystal structures of 4ʹ-methylflavonol, 4ʹ-methoxyflavonol, 4ʹ-nitroflavonol and 2ʹ,6ʹ-dimethylflavonol. Furthermore, several crystal packing patterns were observed, and it is postulated that dihedral angles and intramolecular hydrogen bonding distances are both affected by the intermolecular hydrogen bonding interactions and the crystal packing forces. In addition, the ruthenium complexes of B ring substituted flavonols were synthesized and characterized by spectroscopic and electrochemical techniques. B ring substitution effects were minimal in IR spectroscopy and X-ray crystallography. The levels of the conjugation of the rutheniumlavonolate complexes were demonstrated by electronic absorption spectra recorded in methanol at room temperature. The most positive oxidation potential was obtained with the electron withdrawing nitro group substitution, and the electron donating substitutions resulted in more negative oxidation potentials. The spectroscopic investigation of the complex formation of Al(III) with flavonols and 3-hydroxychromone is described. The stoichiometric composition and stability constants are also given. The comparison of the results obtained from Al(III) chelation shows significant effects of the B ring substitutions.

6 Zusammenfassung Marianne Reinboth Bioverfügbarkeit des Flavonols Quercetin beim Hund Veterinär-Physiologisches Institut der Veterinärmedizinischen Fakultät der Universität Leipzig Eingereicht im Juni 2010 79 Seiten, 20 Abbildungen, 6 Tabellen, 211 Literaturangaben, 1 Anhang Schlüsselwörter: Quercetin, Bioverfügbarkeit, Hund, absolute Bioverfügbarkeit, Isoquercitrin, Rutin, Flavonole Für das pflanzliche Flavonol Quercetin werden vielfältige gesundheitsfördernde Wirkungen postuliert, so auch bei Hunden. Über die Bioverfügbarkeit des Flavonols bei dieser Spezies liegen bislang jedoch keinerlei Daten vor. Daher hatte diese Arbeit das Ziel, Bioverfügbarkeit und pharmakokinetische Parameter von Quercetin und wichtigen Quercetinglycosiden bei Hunden nach deren Verabreichung mit einer Testmahlzeit in einer praxisrelevanten Dosierung von 10 mg/kg Körpermasse zu untersuchen. Dazu erhielten 9 adulte Beagles beiderlei Geschlechts das zuckerfreie \"Aglycon\" Quercetin bzw. seine Glycoside Isoquercitrin (Quercetin-3-O-Glucosid) und Rutin (Quercetin-3-O-Glucorhamnosid) in jeweils äquimolarer Dosierung in einer Testmahlzeit verabreicht. Anschließend wurden Blutproben über einen Zeitraum von bis zu 72 Stunden entnommen und mittels HPLC die Konzentrations-Zeitverläufe der Metaboliten im Blutplasma, die Bioverfügbarkeit sowie weitere pharmakokinetische Parameter bestimmt. Weiterhin wurde die absolute Bioverfügbarkeit von Quercetin aus dem Vergleich einer oralen mit einer intravenösen Applikation bestimmt. Der weitaus größte Teil der Plasmametaboliten von Quercetin sowie seiner beiden Glycoside bestand aus glucuronidierten bzw. sulfatierten Quercetinkonjugaten. Nicht konjugiertes Quercetin-Aglycon kam nur in einem Anteil von etwa 20 % vor. Neben Quercetin machten seine Metaboliten Isorhamnetin und Kämpferol weniger als 10 % aller im Plasma zirkulierenden Flavonole aus. Die absolute Bioverfügbarkeit von Quercetin betrug nur etwa 4 %. Die relative Bioverfügbarkeit aus dem 3-O-Glucosid Isoquercitrin war mehr als doppelt so hoch wie aus dem Aglycon, die maximalen Plasmaspiegel lagen aber auch hier unter 1 µmol/l. Sowohl nach Aufnahme von Quercetin als auch nach Isoquercitrin kam es zu einer relativ schnellen Absorption aus dem Dünndarm mit einem ersten Plasmapeak ungefähr eine Stunde nach der Ingestion. Vier Stunden nach Aufnahme der beiden Flavonole trat ein zweiter Plasmapeak auf, der in der Regel höher als der erste ausfiel. Dies deutet auf einen enterohepatischen Kreislauf der über die Galle ausgeschiedenen Metaboliten hin. Nach Aufnahme von Rutin kam es zu einer verzögerten Absorption, da eine Deglycosylierung durch bakterielle Glycosidasen im Dickdarm Voraussetzung für die Absorption des Flavonols ist. Maximale Plasmakonzentrationen wurden im Mittel erst 11 Stunden nach Ingestion dieses Glycosids erreicht. Die maximalen Plasmakonzentra-tionen nach Rutin waren geringer als nach Quercetin oder Isoquercitrin, jedoch war die mittlere Verweildauer der Plasmametaboliten mit 18 Stunden auch wesentlich länger. Im Unterschied zu anderen Spezies war die relative Bioverfügbarkeit von Rutin gegenüber Quercetin nicht verringert. Obwohl Rutin eine relativ gute Quercetinquelle für Hunde zu sein scheint, muss bei der Einschätzung möglicher In-vivo-Wirkungen die relativ geringe Bioverfügbarkeit sowie die intensive Metabolisierung seines Aglycons Quercetin berücksichtigt werden. / 7 Summary Marianne Reinboth Bioavailability of the Flavonol Quercetin in Dogs Institute of Physiology of the Faculty of Veterinary Medicine, University of Leipzig Submitted in June 2010 79 pages, 20 figures, 6 tables, 211 references, 1 appendix Keywords: quercetin, bioavailability, dog, absolute bioavailability, isoquercitrin, rutin, flavonols The plant flavonol quercetin is supposed to exert multiple health-related effects in dogs. To date no information on its bioavailability in this particular species is avai-lable. This study intended to investigate bioavailability and pharmacokinetics of quercetin and certain quercetin glycosides in dogs after ingestion of a test meal sup-plemented with a quercetin dose equivalent to 10 mg/kg body weight. Nine adult beagle dogs of both sexes received the aglycon quercetin (sugarfree) or its glycosides isoquercitrin (quercetin-3-O-glucoside) and rutin (quercetin-3-O-glucorhamnoside) in equimolar amounts together with a test meal. Blood samples were taken over a period of up to 72 hours; bioavailability and pharmacokinetics were calculated from the HPLC-derived plasmaconcentration-time-curves. Absolute bioavailability was calculated by comparing an oral to an intravenous administration of quercetin. The majority of analysed plasma metabolites were glucuronidated and sulfated con-jugates of quercetin. Non-conjugated quercetin aglycon comprised only 20 %. Be-sides quercetin, its metabolites isorhamnetin and kaempferol made up less than 10 % of all circulating metabolites. The absolute bioavailability of quercetin was only 4 %. The relative bioavailability of quercetin from isoquercitrin was more than twice as high than from the aglycon, but even there maximal plasma concentrations were generally less than 1 μmol/l. Absorption from the small intestine was rather fast with a first plasma peak after 1 hour after ingestion of quercetin or isoquercitrin. A second, generally higher plasma peak occurred 4 hours after ingestion. This suggests an in-tensive enterohepatic recycling of biliary secreted metabolites. Absorption was significantly delayed after ingestion of rutin due to the necessity of bacterial deglycosilation in the large intestine. Plasma concentrations peaked only after 11 hours. Plasma concentrations after rutin were lower than after quercetin or isoquercitrin, but mean residence time of plasma metabolites was as long as 18 hours after rutin ingestion. Consequently, a once daily feeding of dogs with rutin might lead to relatively constant plasma metabolite concentrations. In contrast to other species, bioavailability from rutin was not smaller than that from quercetin. Although rutin seems to be a relative good quercetin source for dogs, estimations about potential in-vivo-effects of quercetin have to take into consideration its low bioavailabilty and intensive metabolism.