Orientador: Delia B. Rodriguez-Amaya / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-10T05:46:01Z (GMT). No. of bitstreams: 1 Huber_LisiaSenger_D.pdf: 841058 bytes, checksum: 9612feac8503fceabd312029260ed6cf (MD5) Previous issue date: 2007 / Resumo: Devido a crescente importância atribuída aos flavonóides nos últimos anos, decorrente de suas ações relacionadas à prevenção de doenças degenerativas, e aliada à carência de dados destes compostos em alimentos brasileiros, este estudo teve como objetivo determinar os teores de flavonóis e flavonas em alimentos consumidos no Brasil e avaliar alguns fatores que influenciam seus níveis nestes alimentos. Uma revisão bibliográfica é apresentada no Capítulo 1, na qual são descritos os principais efeitos benéficos à saúde, aspectos analíticos e fatores que influenciam os teores de flavonóides em alimentos. Estes compostos com ação benéfica à saúde, atuam como antioxidantes, inibidores da proliferação celular, antiestrogênicos e mediadores intracelulares, exercendo proteção principalmente contra câncer e doenças cardiovasculares. A determinação desses compostos normalmente é feita utilizando-se cromatografia líquida de alta eficiência com detector de arranjo de diodos. Os níveis de flavonóides em alimentos podem ser influenciados por vários fatores, como estação do ano, preparo e processamento de alimentos. O Capítulo 2 descreve o desenvolvimento e validação da metodologia analítica para determinação de flavonóis e flavonas em hortaliças. Utilizando-se Delineamento Composto Central Rotacional, obtiveram-se as melhores condições para extração e hidrólise dos flavonóides encontrados na natureza na forma glicosídica, a suas respectivas agliconas. Essas condições foram: 1,0M de HCl por 6 horas para espinafre e couve, 1,6M de HCl por 5 horas para rúcula, 1,2M de HCl por 2 horas para alface, 1,7M de HCl por 4,3 horas para salsa e 0,8M de HCl por 2,5 horas para cebola. As condições cromatográficas utilizadas foram coluna Nova-Pak C18 (4ìm, 3,9x150mm), e fase móvel constituída de metanol e água, acidificados com 0,3% de ácido fórmico, em gradiente linear. Utilizando a metodologia analítica validada no Capítulo 2, no Capítulo 3 foram identificados e quantificados os flavonóides de alface lisa (6,73-9,77'g/g de quercetina), alface crespa (7,18-30,8'g/g de quercetina), cebola branca (323-362'g/g de quercetina), cebola roxa (390-423'g/g de quercetina), couve (256-399'g/g de quercetina e 333-339'g/g de kaempferol), espinafre (52,8-62,3'g/g de quercetina e 145-170'g/g de kaempferol), rúcula (137-143'g/g de quercetina e 402-501'g/g de kaempferol) e salsa (1521-1636'g/g de apigenina). Também foi avaliado o efeito sazonal nos teores destes compostos, sendo que estes tenderam a ser maiores no verão que no inverno, embora as diferenças não tenham sido estatisticamente significativas. No Capítulo 4, foram determinados os teores de flavonóides em sucos prontos para o consumo, sucos concentrados e polpas de caju, acerola e pitanga, e em amostras de cebola e salsa desidratadas. Os resultados indicaram perdas de flavonóides durante o processamento destes alimentos. Os teores nas amostras processadas foram nitidamente menores aos obtidos previamente nas amostras in natura. Os derivados de frutas apresentaram teores decrescentes na seguinte ordem: polpas, suco concentrado, suco pronto para consumo. Os teores de quercetina nas amostras de cebola desidratada foram bastante variados, indicando diferenças de variedades utilizadas como matéria-prima ou nas condições de processamento empregadas. Os resultados sugerem que estudos de monitoramento das perdas de flavonóides, da matéria-prima ao produto final, são altamente requeridos. O comportamento de flavonóis em couve, espinafre e rúcula minimamente processados, estocados em atmosfera modificada, em diferentes temperaturas, na presença e ausência de luz, foi avaliado e discutido no Capítulo 5. A qualidade sensorial dessas amostras também foi avaliada, para verificar a vida-de-prateleira. No geral, a vida útil foi negativamente influenciada pelo aumento na temperatura de estocagem na presença de luz. Não ocorreram perdas pronunciadas nos teores destes compostos durante a estocagem das três folhas minimamente processadas, podendo inclusive aumentar em certos períodos do armazenamento / Abstract: Had the increasing importance attributed to the flavonóides in the last years, decurrent of its action related to the prevention of degenerative illnesses, and allied to the lack of data of these composites in Brazilian foods, this study it had as objective to determine texts of flavonóis and flavonas in foods consumed in Brazil and to evaluate some factors that influence its levels in these foods. A bibliographical revision is presented in Chapter 1, in which the main beneficial effect to the health, analytical aspects and factors are described that influence texts of flavonóides in foods. These composites with beneficial action to the health, act as antirust, inhibiting of the proliferation cellular, antiestrogênicos and mediating intracellular, exerting protection mainly against cardiovascular cancer and illnesses. The determination of these composites normally is made using liquid chromatography of high efficiency with detector of arrangement of diodes. The levels of flavonóides in foods can be influenced by some factors, as station of the year, preparation and processing of foods. Chapter 2 describes the development and validation of the analytical methodology for determination of flavonóis and flavonas in hortaliças. Using Rotational Central Composed Delineation, the best conditions for extration and hydrolysis of the flavonóides found in the nature in the glicosídica form, its respective agliconas had been gotten. These conditions had been: 1,0M of HCl for 6 hours for spinach and borecole, 1,6M of HCl for 5 hours for rúcula, 1,2M of HCl for 2 hours for lettuce, 1,7M of HCl for 4,3 hours for parsley and 0,8M of HCl for 2,5 hours for onion. The used chromatographic conditions had been column Nova-Pak C18 (4ìm, 3,9x150mm), and mobile phase consisting of methanol and water, acidified with 0,3% of acid fórmico, in linear gradient. Using the validated analytical methodology in Chapter 2, in Chapter 3 they had been identified and quantified the flavonóides of smooth lettuce (6,73-9,77'g/g of quercetina), lettuce crespa (7,18-30,8'g/g of quercetina), white onion (323-362'g/g of quercetina), purple onion (390-423'g/g of quercetina), borecole (256-399'g/g of quercetina and 333-339'g/g of kaempferol), spinach (52,8-62,3'g/g of quercetina and 145-170'g/g of kaempferol), rúcula (137-143'g/g of quercetina and 402-501'g/g of kaempferol) and parsley (1521-1636'g/g of apigenina). Also the sazonal effect in texts of these composites was evaluated, being that these had tended to be bigger in the summer that in the winter, the differences have even so not been estatisticamente significant. In Chapter 4, the texts of flavonóides in ready juices for the consumption, intent juices and cashew pulps, acerola and pitanga had been determined, and in samples of dehydrated onion and parsley. The results had indicated losses of flavonóides during the processing of these foods. The texts in the processed samples had been nitidamente lesser to gotten previously in the samples in natura. The derivatives of fruits had presented decreasing texts in the following order: pulps, concentrated juice, ready juice for consumption. The texts of quercetina in the samples of dehydrated onion sufficiently had been varied, indicating differences of used varieties as raw material or in the employed conditions of processing. The results suggest that studies of monitoramento of the losses of flavonóides, of the raw material to the end item, highly are required. The behavior of flavonóis in borecole, spinach and rúcula minimamente processings, storaged in modified atmosphere, in different temperatures, na.presença and absence of light, was evaluated and argued in Chapter 5. The sensorial quality of these samples also was evaluated, to verify the life-of-shelf. In the generality, the useful life negative was influenced by the increase in the temperature of stockage na.presença of light. Sharp losses in texts of these composites had not occurred during the stockage of three minimamente processed leves, also being able to increase in certain periods of the storage / Doutorado / Doutor em Ciência de Alimentos

Orientador: Delia B. Rodriguez-Amaya / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-13T18:45:19Z (GMT). No. of bitstreams: 1 Bombonati_AlineYashima_M.pdf: 14727500 bytes, checksum: cb3e5718f8b66916dd2c522a99b17e51 (MD5) Previous issue date: 2009 / Mestrado / Mestre em Ciência de Alimentos

Berry ripening in lowbush blueberry (Vaccinium angustifolium Ait.) is influenced by developmental, physiological and climatic factors resulting in a heterogenous mix of maturities at harvest. This study characterizes the physico-chemical changes which occur during fruit ontogeny and links ripening patterns to micoclimate. Individual clones in five commercial fiels were followed in the 2006 and 2007 growing seasons. Phenolic acids, flavonols, and flavan-3-ols decreased and anthocyanins increased with maturity. Peak maturity consistently occurred at 1200 accumulated growing degree days (GDD). There was a sharp decline in fruit retention at the end of the growing season suggesting a date after which harvested yield declines but no consistent pattern was detected between years or fields.The consistency of GDD accumulation in relation to ripening pattern suggests GDDs can be used as a predictive ripening index. The physico-chemical nature of ripe berries indicates ripe berries could be harvested earlier than is currently the practice.

Abstract Blackcurrants (Ribes nigrum L.) are widely consumed due to their favourable taste and health-promoting effects. The berries and extracts from different parts of the plant show anticarcinogenic, antioxidative and anti-inflammatory properties, and are effective in reducing the risk of cardiovascular disease and in promoting brain health. These health-promoting benefits are due to high concentrations of valuable compounds such as anthocyanins and flavonols in blackcurrants. However, these compounds are sensitive to heat and processing and some are lost when the berries are processed into products such as jams, purees and juices. Industrial processing of juices is a multistep process that typically includes enzyme treatment, pressing, pasteurisation, clarification and usually also thermal concentration. Alternative minimal processing technologies are required to preserve the health-promoting compounds in products by avoiding the use of high temperatures and extensive clarification. Integrated membrane technology, i.e. combined ultrafiltration and reverse osmosis, was used in this thesis for the production of blackcurrant juice concentrate. Pre-treatment methods, such as enzymatic treatment, ultrafiltration, enzymatic treatment combined with ultrafiltration and centrifugation to increase the filtration efficiency in reverse osmosis were evaluated. Processing was modelled to define the resistances, using the resistance-in-series model. The preservation and concentration of anthocyanins and flavonols were analysed. The results indicated that the main resistance in the reverse osmosis process was polarisation resistance, while membrane resistance was lower and fouling resistance was one order of magnitude lower than the other resistances. The filtration efficiency results showed that the highest flux was achieved by ultrafiltered blackcurrant juice, but that the resulting juices were substantially lower in anthocyanins and flavonols, which were retained on the ultrafiltration membrane. Therefore, replacing ultrafiltration with centrifugation as the clarification method for juices is recommended. Value-added processing of blackcurrant was conceptualised by valorisation of the marc left in the berry pressing process for extraction of anthocyanin compounds. Conventional extraction was compared with microwave-assisted extraction (MAE), with the latter optimised using response surface methodology to achieve maximum efficiency in extracting anthocyanins. The optimum parameters found for MAE were: microwave power 700 W, extraction time 10 minutes, pH 2 adjusted with hydrochloric acid and a solid to solvent ratio of 0.05. Conventional extraction showed the best results when carried out at 80 °C for 300 minutes in aqueous solution with pH 2 adjusted by hydrochloric acid. Under these conditions, recovery of anthocyanins was still 10% lower than with MAE for only 10 minutes of extraction time. / Tiivistelmä Mustaherukoita käytetään paljon niiden hyvän maun ja terveyttä edistävien vaikutusten ansiosta. Marjoilla ja marjakasvin eri osien uutteilla on osoitettu olevan antikarsinogeenisia, antioksidatiivisia ja tulehduksia estäviä ominaisuuksia ja ne ovat tehokkaita pienentämään sydän- ja verisuonisairauksia. Ne edistävät myös aivojen terveyttä. Marjojen arvokkailla yhdisteillä kuten antosyanideillä ja flavonoleilla on terveyttä edistäviä vaikutuksia. Mustaherukassa on runsaasti näitä yhdisteitä. Hillojen, soseiden ja mehujen prosessoinnissa menetetään näitä hyödyllisiä yhdisteistä, koska ne ovat herkkiä lämmölle ja prosessoinnin vaikutuksille. Mehujen prosessoinnissa käytetään entsyymikäsittelyjä, puristusta, pastörointia, selkeytystä ja usein myös lämpökonsentrointia. Tuotteiden terveyttä edistävien yhdisteiden säilyttämiseksi tarvitaan uudenlaisia hellävaraisia prosessointitekniikoita ilman korkeita lämpötiloja ja voimakasta selkeyttämistä. Tässä työssä tutkittiin yhdistettyjen kalvotekniikoiden kuten ultrasuodatuksen ja käänteisosmoosin käyttöä mustaherukkatiivistemehun prosessoinnissa. Esikäsittelymenetelmiä, mm. entsyymikäsittelyä, ultrasuodatusta, entsyymikäsittelyn ja ultrasuodatuksen yhdistelmää sekä sentrifugointia, arvioitiin käänteisosmoosin suodatustehokkuuden parantamisessa. Suodatusvastuksen määrittämiseksi prosessi mallinnettiin käyttäen sarja -vastus mallia. Antosyanidien ja flavonolien säilyminen ja konsentroituminen prosesseissa määritettiin. Tulokset osoittivat, että suurin vastus käänteisosmoosissa aiheutui polarisaatiovastuksesta, kun taas kalvon vastus oli pienempi. Mallinnus osoitti myös, että likaantumisen aiheuttama vastus oli yhtä magnitudia alhaisempi kuin muut vastukset. Suodatusteho osoitti, että suurin virtaus saavutettiin ultrasuodatetulla mustaherukkamehulla. Ultrasuodatetussa mehussa oli kuitenkin huomattavasti vähemmän antosyaniineja ja flavonoleja, mikä johtui näiden yhdisteiden tarttumisesta ultrasuodatuskalvoon. Näin ollen, tämän työn tulokset suosittelevat ultrasuodatuksen korvaamista sentrifugoinnilla mehun kirkastusprosessissa. Mustaherukkamehun tuotannossa muodostuu sivutuotteena ns. puristekakkua, joka sisältää runsaasti antosyaaneja. Työssä kehitettiin antosyaanien talteenottoa tästä sivutuotteesta vertaamalla tavanomaista uuttotekniikkaa mikroaaltoavusteiseen uuttoon. Prosessi optimoitiin vastepintamenetelmällä mahdollisimman suuren antosyaanien uuttotehokkuuden saavuttamiseksi. Optimaaliset parametrit saatiin mikroaaltoavusteisessa uutossa teholla 700 W, uuttoajalla 10 minuuttia, kiintoaines-liuotin -suhteella 0,05 pH-arvossa 2, mikä saavutettiin lisäämällä suolahappoa. Tavanomaisessa uutossa parhaat antosyaanisaannot saavutettiin suolahappo-vesiliuoksella pH-arvossa 2 uuttamalla 300 minuuttia lämpötilassa 80 °C. Antosyaanisaanto oli kuitenkin tavanomaisessa uutossa optimiolosuhteissa 10% pienempi kuin mikroaaltoavusteisessa uutossa 10 minuutin uuttoajalla.

Le platane est un arbre de la famille des Platanaceae très commun en ville en particulier en Europe et dans toutes les zones tempérées. Les espèces du genre Platanus et plus particulièrement l’espèce Platanus orientalis, très répandue en Grèce, sont sévèrement attaquées par des agents phytopathogènes provoquant le chancre coloré du platane, l’anthracnose ou l’oïdium. En revanche, Platanus acerifolia, un hybride obtenu entre P. occidentalis and P. orientalis, très commun en France, se montre plus résistant vis-à-vis de ces pathogènes. L’étude par HPLC d’extraits de deux espèces de Platanaceae européennes P. acerifolia et P. orientalis a montré des différences selon le solvant d’extraction. L’extrait dichlorométhanique de P. acerifolia s’est montré plus riche en composés que l’espèce P. orientalis. Par contre, les chromatogrammes obtenus à partir des extraits méthanoliques sont très similaires et dominés par les deux composés majoritaires (tiliroside et platanoside). Des techniques préparatives comme la chromatographie de partage centrifuge (CPC), la moyenne pression (MPLC), la chromatographie sur colonne de Sephadex ou sur résine XAD-7 ont été utilisées afin d’isoler les différents constituants majoritaires et d’identifier les composés qui diffèrent d’une espèce à l’autre. L’élucidation structurale est réalisée grâce à des techniques telles que HR-EIS-MS et RMN 1D & 2D. L’étude de l’extrait dichlorométhanique nous a permis de compléter la connaissance phytochimique de ces deux plantes européennes et a conduit à l’isolement et à la détermination structurale de 38 molécules dont 7 composés nouveaux (1 coumarine, 3 flavonols prenylés, 2 dihydrochalcones, un terpénoïde. Par ailleurs, l’extrait méthanolique a conduit d’une part à l’isolement du tiliroside et du platanoside majoritaires et d’autre part à l’isolement de composés minoritaires, trente-trois autres molécules dont 5 nouveaux composés (1 flavonol glucosylé, 3 coumarines, 1 dihydrochalcone). Enfin, l’évaluation biologique des composés isolés in vitro ou in vivo a été réalisée sur différentes cibles : activités antifongiques, anti-âge, antioestrogéniques. Les activités cytotoxiques sur les cellules de cancer du sein MCF-7 et sur les cellules de cancer de l’endomètre (ISHIKAWA). / Platanus is a small genus of trees belonging to Platanaceae family, very common in Europe and temperate zones. Platanus species, and especially Platanus orientalis (Oriental plane), wide-spread in Greece are known to be severely attacked by phytopathogens such as Ceratocystis fimbriata f. sp. platani for canker stain, Apiognomonia veneta for anthracnose and Microsphaera platini for powdery mildew. However Platanus acerifolia (London plane), a hybrid between the P. occidentalis and P. orientalis, which is very common in France, have been found to be resistant to these pathogenic fungi. The HPLC profile of the dichloromethane extracts of the two species P. orientalis and P. acerifolia revealed a qualititative difference whereas, the methanol extracts were found to be similar with two predominant phenolic constituents (tiliroside and platanoside) present in both species. Further isolation and purification of their secondary metabolites were performed using various chromatographic techniques (CPC, MPLC, CC, XAD-7, Sephadex LH20, prep-TLC) and their identification was performed by HRMS and NMR (1 & 2D) spectroscopy. The studies of dichloromethane extract allowed us to deal with phytochemical knowledge of these two European plants in depth and led to isolation and structural elucidation of 38 compounds including 7 news constituents (1 coumarin, 3 flavonols, 2 dihydrochalcones and 1 terpenoid). Moreover, the methanol extract led to the isolation of their major constituents (tiliroside and platanoside) in one step. Furthermore, 33 minors compounds were isolated including 5 news compounds (one flavonol glycoside 3 coumarins and 1 dihydrochalcone). In addition, the isolated compounds have been subjected to in vitro or in vivo evaluation on different targets: antifungal, anti-ageing, anti-oestrogenic properties. Finally, the cytotoxic activity was studied on breast cancer cells (MCF-7) and endometrial cancer (Ishikawa).

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.:1 Einleitung 1 2 Literaturübersicht 3 2.1 Funktionen von Flavonoiden in Pflanzen 3 2.2 Biosynthese und Struktur von Flavonoiden 4 2.3 Flavonoidwirkungen im menschlichen und tierischen Organismus 7 2.3.1 Antioxidative Eigenschaften 10 2.3.2 Wirkungen auf Enzyme und Transportproteine des Arzneistoffwechsels 12 2.4 Bioverfügbarkeit und Stoffwechsel des Flavonols Quercetin 13 2.4.1 Bioverfügbarkeit von Quercetin 13 2.4.2 Einfluss des Futters 15 2.4.3 Einfluss des Glycosylierungsmusters 16 2.4.4 Intestinale Absorption und Metabolismus 18 2.4.5 Einfluss der intestinalen Mikroflora 21 2.4.6 Bindung an Plasmaproteine 22 2.4.7 Gewebeverteilung 23 2.4.8 Exkretion 24 2.5 Zielsetzung 25 3 Tiere, Material und Methoden 26 3.1 Versuchstiere und Haltungsbedingungen 26 3.2 Verwendete Flavonole 27 3.3 Durchführung des Tierversuchs 28 3.3.1 Allgemeine Durchführung der Versuche 28 3.3.2 Bioverfügbarkeit von Quercetin und Rutin (je 30 mg/kg KM) 29 3.3.3 Absolute Bioverfügbarkeit von Quercetin 30 3.3.4 Relative Bioverfügbarkeit verschiedener Quercetinglycoside 31 3.4 Probenaufarbeitung 31 3.5 HPLC 33 3.5.1 Methodenvalidierung und -kalibrierung 34 3.5.2 Berechnung der pharmakokinetischen Parameter 40 3.5.3 Statistische Auswertung 41 4 Ergebnisse 42 4.1 Bioverfügbarkeit von Quercetin und Rutin (je 30 mg/kg KM) 42 4.1.1 Quercetin-Aglycon 42 4.1.2 Rutin 46 4.2 Absolute Bioverfügbarkeit Quercetin 48 4.3 Relative Bioverfügbarkeit verschiedener Quercetinglycoside 52 4.3.1 Quercetin-Aglycon 53 4.3.2 Rutin 56 4.3.3 Isoquercitrin 57 5 Diskussion 62 5.1 Zielsetzung der Studie 62 5.2 Methodische Aspekte 62 5.2.1 Auswahl der Versuchstiere und Versuchsanordnung 62 5.2.2 Wahl der Testmahlzeit 63 5.2.3 Einfluss der Zeitpunkte für die Probennahme auf die Berechnung der Verfügbarkeit 64 5.2.4 Analysemethode 65 5.3 Plasmametaboliten von Quercetin 66 5.4 Absolute Bioverfügbarkeit von Quercetin 69 5.5 Relative Bioverfügbarkeit verschiedener Quercetinglycoside 70 5.5.1 Isoquercitrin 70 5.5.2 Rutin 71 5.6 Bezug der pharmakokinetischen Daten zu potentiellen In-vivo-Wirkungen 73 5.7 Schlussfolgerungen 75 6 Zusammenfassung 76 7 Summary 78 8 Literaturverzeichnis 80 9 Anhang 102 9.1 HPLC-Chemikaien 102 9.2 Validierung der HPLC-Methode 103 Danksagung 109 / 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.:1 Einleitung 1 2 Literaturübersicht 3 2.1 Funktionen von Flavonoiden in Pflanzen 3 2.2 Biosynthese und Struktur von Flavonoiden 4 2.3 Flavonoidwirkungen im menschlichen und tierischen Organismus 7 2.3.1 Antioxidative Eigenschaften 10 2.3.2 Wirkungen auf Enzyme und Transportproteine des Arzneistoffwechsels 12 2.4 Bioverfügbarkeit und Stoffwechsel des Flavonols Quercetin 13 2.4.1 Bioverfügbarkeit von Quercetin 13 2.4.2 Einfluss des Futters 15 2.4.3 Einfluss des Glycosylierungsmusters 16 2.4.4 Intestinale Absorption und Metabolismus 18 2.4.5 Einfluss der intestinalen Mikroflora 21 2.4.6 Bindung an Plasmaproteine 22 2.4.7 Gewebeverteilung 23 2.4.8 Exkretion 24 2.5 Zielsetzung 25 3 Tiere, Material und Methoden 26 3.1 Versuchstiere und Haltungsbedingungen 26 3.2 Verwendete Flavonole 27 3.3 Durchführung des Tierversuchs 28 3.3.1 Allgemeine Durchführung der Versuche 28 3.3.2 Bioverfügbarkeit von Quercetin und Rutin (je 30 mg/kg KM) 29 3.3.3 Absolute Bioverfügbarkeit von Quercetin 30 3.3.4 Relative Bioverfügbarkeit verschiedener Quercetinglycoside 31 3.4 Probenaufarbeitung 31 3.5 HPLC 33 3.5.1 Methodenvalidierung und -kalibrierung 34 3.5.2 Berechnung der pharmakokinetischen Parameter 40 3.5.3 Statistische Auswertung 41 4 Ergebnisse 42 4.1 Bioverfügbarkeit von Quercetin und Rutin (je 30 mg/kg KM) 42 4.1.1 Quercetin-Aglycon 42 4.1.2 Rutin 46 4.2 Absolute Bioverfügbarkeit Quercetin 48 4.3 Relative Bioverfügbarkeit verschiedener Quercetinglycoside 52 4.3.1 Quercetin-Aglycon 53 4.3.2 Rutin 56 4.3.3 Isoquercitrin 57 5 Diskussion 62 5.1 Zielsetzung der Studie 62 5.2 Methodische Aspekte 62 5.2.1 Auswahl der Versuchstiere und Versuchsanordnung 62 5.2.2 Wahl der Testmahlzeit 63 5.2.3 Einfluss der Zeitpunkte für die Probennahme auf die Berechnung der Verfügbarkeit 64 5.2.4 Analysemethode 65 5.3 Plasmametaboliten von Quercetin 66 5.4 Absolute Bioverfügbarkeit von Quercetin 69 5.5 Relative Bioverfügbarkeit verschiedener Quercetinglycoside 70 5.5.1 Isoquercitrin 70 5.5.2 Rutin 71 5.6 Bezug der pharmakokinetischen Daten zu potentiellen In-vivo-Wirkungen 73 5.7 Schlussfolgerungen 75 6 Zusammenfassung 76 7 Summary 78 8 Literaturverzeichnis 80 9 Anhang 102 9.1 HPLC-Chemikaien 102 9.2 Validierung der HPLC-Methode 103 Danksagung 109

This work describes the chemical and biological studies carried out using two species of plants, which belongs to the Polygonaceae family: Coccoloba mollis Casaretto (1844) and Triplaris americana Linnaeus. The study aimed to monitoring the antioxidant (DPPH, total phenolic content, reduction power by Fe3+-Fe2+ and total oxidation by FTC method), anticholinesterasic and larvicidal (4th larval instars of the Aedes aegypti) activities of extracts and isolated compounds from C. mollis and T. americana. Phytochemical investigation of some extracts with promising results in at least one of the activities mentioned above of C. mollis lead to the isolation of two phytosteroids (sitosterol and sitostenone), a diterpene (transphytol), a triterpene (simiarenol) and a benzenoid (vanilic acid); while from T. americana were isolated two benzenoids (gallic acid and protocatechuic acid methyl ester), a phenetylamide (moupinamide), a phenylpropanoid derivative (vanicoside D), two phytosteroids (sitosterol and sitostenone), two flavonols (quercetin and 3-O-α-arabinofuranosylquercetin) and two triterpenes (friedelin and friedelinol). In general, these results have been contributed to extend the chemiotaxonomic profile of the genera Coccoloba and Triplaris, and as a consequence it increased the knowledge of the Polygonaceae. These compounds were identified on the basis of spectroscopic analyses (IR, MS and NMR, including DEPT, APT, COSY, HSQC and HMBC). Regarding the to antioxidant assays, fractions from fruits, leaves, stem and roots of T. americana showed comparable results to those presented by the standards (α-tocopherol, BHT and ascorbic acid). From active fractions extracts of the fruits were isolated four compounds that were also active (gallic acid, vanicoside D, quercetin and 3-O-α- arabinofuranosylquercetin). The qualitative assays used to demonstrate the inhibition of the enzyme acetylcholinesterase by fractions from partition and filtration on silica gel of C. mollis (bark, leaves and stem) and of T. americana (fruits, leaves, stem and roots) showed positive results as well. Chromatographic guided-fractionation of some these fractions resulted in the isolation of seven compounds five of them (sitosterol, vanilic acid, gallic acid, protocatechuic acid methyl ester and vanicoside D) showed positive results. In the assays against 4th larval instars of the Aedes aegypti, the fractions promising more were derived from hexane extracts of stem, leaves and bark of C. mollis and from stem and roots, as well as EtOAc sub-fraction from the CHCl3 extract of fruits of T. americana. Conclusion, these results presented data that can be used in further studies. / Conselho Nacional de Desenvolvimento Científico e Tecnológico / O presente trabalho descreve o estudo químico, monitorado pelas atividades antioxidantes (DPPH, quantificação de fenóis totais, poder de redução Fe3+-Fe2+ e oxidação total pelo método FTC), anticolinesterásica e larvicida (larvas do 4o ínstar do Aedes aegypti), de extratos e substâncias isoladas das espécies Coccoloba mollis Casaretto (1844) e Triplaris americana Linnaeus (Polygonaceae). A investigação fitoquímica de alguns dos extratos com resultados promissores em pelo menos uma das atividades acima mencionadas de C. mollis conduziu ao isolamento de dois fitoesteróides (sitosterol e sitostenona), um diterpeno (transfitol), um triterpeno (simiarenol) e de um benzenóide (ácido vanílico); enquanto que da espécie T. americana foram isolados dois benzenóides (ácido gálico e éster metílico do ácido protocatecóico), uma fenetilamida (moupinamida), um derivado fenilpropanóide (vanicosídeo D), dois fitoesteróides (sitosterol e sitostenona), dois flavonóis (quercetina e 3-O-α-arabinofuranosilquercetina) e dois triterpenos (fridelina e fridelinol). De um modo geral, este resultado contribuiu para ampliar o perfil químiotaxonômico dos gêneros Coccoloba e Triplaris e, conseqüentemente da química de Polygonaceae. Estas substâncias foram identificadas com base na análise de dados espectroscópicos (IV, EM e RMN, incluindo DEPT, APT, COSY, HSQC e HMBC). Em relação aos ensaios antioxidantes, as frações oriundas dos frutos, folhas, caule e raízes de T. americana apresentaram resultados comparáveis aos dos padrões utilizados (α-tocoferol, BHT e ácido ascórbico). Das frações ativas dos frutos foram isoladas quatro substâncias que também foram ativas (ácido gálico, vanicosídeo D, quercetina e 3-O-α-arabinofuranosilquercetina). No que diz respeito aos ensaios qualitativos frente à enzima acetilcolinesterase, frações oriundas de partição e de filtração em gel de sílica de C. mollis (cascas, folhas e caule) e de T. americana (frutos, folhas, caule e raízes) forneceram resultados positivos. O fracionamento cromatográfico monitorado de alguns dessas frações resultou no isolamento de sete substâncias, das quais cinco (sitosterol, ácido vanílico, ácido gálico, éster metílico do ácido protocatecóico e vanicosídeo D) forneceram resultados positivos. Nos ensaios frente larvas do 4º ínstar do Aedes aegypti, as frações mais promissoras foram em hexano do caule, folhas e cascas de C. mollis e em hexano de caule e raízes e a subfração em AcOEt, oriunda da filtração da fração em CHCl3 dos frutos de T. americana. De um modo geral, esses resultados forneceram dados que poderão servir de base para estudos posteriores.