Дисертації з теми "Proanthocyanidols"

Els ritmes circadians permeten als organismes anticipar-se als canvis mediambientals i adaptar el metabolisme al patró d’alimentació i la disponibilitat d’aliments. De fet, les alteracions del ritme circadià provoquen trastorns del metabolisme com la síndrome metabòlica. Els ritmes circadians es mantenen pel rellotge central que es troba a l’ hipotàlem, tot i que els teixits perifèrics també presenten oscil•ladors circadians . Des d’un punt de vista molecular, el sistema circadià està constituït per gens rellotge que interacciones entre si, formant un cicle de regulació. La senyal més important per sincronizar aquest cicle amb l’exterior, és la llum. No obstant, hi ha altres senyals externes, com els cicle d’alimentació-dejuni i alguns components dels aliments, que també poden actuar sincronitzant el rellotge molecular. Les proantocianidines, que són una subclasse de flavonoides, indueixen una amplia gama d’efectes beneficiosos per la salut, millorant totes les patologies de la síndrome metabòlica. Per lo tant, l’objectiu principal d’aquesta tesi va ser avaluar la capacitat de les proantocianidines per modular el rellotge molecular a nivell central i perifèric, sota condicions estàndard o alterades. Els resultats demostren que les proantocianidines modulen els ritmes de expressió dels gens rellotge al llarg de les 24 hores, tant a nivell central con perifèric, tot i que els seus efectes depenen de si el tractament amb proantocianidines ha set durant la fase de llum o la fase de foscor. A més, els efectes sobre el sistema estan associats a canvis de la fluctuació ciracadiana d’alguns metabòlits importants en plasma, o dels nivells de NAD en el fetge. En conjunt, aquests resultats suggereixen que les proantocianidines podrien promoure els seus efectes beneficiosos sobre el metabolisme a través de la seva interacció amb el sistema circadià.
Circadian rhythms allow organisms to anticipate environmental changes and to adapt the metabolism to feeding regime and food availability. In fact, alterations of circadian rhythm induce metabolic disturbances, such as metabolic syndrome. Circadian rhythms are maintained by a central clock in the hypothalamus, but circadian clocks are also present in peripheral tissues. At molecular level, the clock system is composed by feedback loops of core-clock and clock-controlled genes. The most important synchronizer of the clock system is light, but other external cues, such as fasting-feeding time or food components, also act as synchronizers. Proanthocyanidins, a flavonoid sub-class, are reported to have a vast range of beneficial effects improving all the components of the metabolic syndrome. Therefore, the main objective of this thesis was to evaluate the capacity of proanthocyanidins to modulate the central and peripheral molecular clocks under standard or disrupted conditions. Results show that proanthocyanidins modulate the 24-h rhythm expression of clock-core and clock-controlled gens in the central and peripheral clocks. However, the time of proanthocyanidin administration, in the light or dark phase, determine the precise effect on the molecular clock. The modulation of the clock system is associated with variations of the circadian fluctuation of some important metabolites in plasma or NAD levels in liver. Overall these results suggest that proanthocyanidins could mediate their beneficial metabolic effects through their interaction with the clock machinery.
Los ritmos circadianos permiten a los organismos anticiparse a los cambios medioambientales y adaptar el metabolismo al patrón de alimentación y la disponibilidad de alimentos. De hecho, alteraciones del ritmo circadiano provocan trastornos del metabolismo como el síndrome metabólico. Los ritmos circadianos se mantienen por la acción del reloj central ubicado en el hipotálamo, aunque la mayoría de tejidos periféricos también disponen de reloj molecular. Desde un punto de vista molecular, el sistema circadiano está compuesto por genes “reloj” que interaccionan entre sí, formando un bucle de regulación. La señal externa más importante que sincroniza el sistema circadiano con el exterior es la luz, pero los ciclos de alimentación y ayunas, así como algunos componentes de los alimentos también actúan como señales externas sincronizadoras del reloj molecular. Las proantocianidinas, que son una subclase de los flavanoides, ejercen una amplia gama de efectos beneficiosos sobre la salud, mejorando todas las patologías del síndrome metabólico. Por lo tanto, el objetivo de la presente tesis fue evaluar la capacidad de las proantocianidinas para modular el reloj molecular a nivel central y periférico, bajo situaciones estándar o alteradas. Los resultados muestran que las proantocianidinas modulan el ritmo de expresión de los genes reloj a lo largo de las 24 horas, tanto a nivel central como periférico, aunque sus efectos dependen de si el tratamiento con proantocianidinas se ha realizado durante la fase de luz o la fase de oscuridad. Además, los efectos sobre el sistema circadiano están asociados a modificaciones en la fluctuación circadiana de algunos metabolitos importantes en plasma, o de los niveles de NAD en el hígado. En conjunto, estos resultados sugieren que las proantocianidinas podrían mediar sus efectos beneficiosos sobre el metabolismo a través de su interacción con el reloj molecular.

Nosocomial, or hospital acquired, infections, are ubiquitous within the modern clinical setting leading to over $5 billion annually of related healthcare costs in North America. All indwelling devices are highly susceptible to bacterial colonization where physico-chemical interactions between bacteria and biomaterial surfaces have been implicated as determinant factors in the fate of the initial adhesion processes. It has been proposed that by exploiting interference strategies within this critical step of infection the ability to create 'non-infective' biomaterials may be developed.
This thesis demonstrates the effectivity of North American cranberry (Vaccinium macrocarpon) derived proanthocyanidins in preventing the adhesion of pathogenic bacteria to biomaterial surfaces. Specifically, using a model of catheter associated urinary tract infection, significant reductions in initial adhesion of uropathogenic Escherichia coli and Enterococcus faecalis to PVC and PTFE were observed. With the application of colloidal theory, a mechanism of steric interference was determined as responsible for these effects.
The evidence presented implicates PAC as a molecule of interest for the development of novel biomaterials with increased resistance to bacteria colonization.

Anthocyanins are one of the richest pigments, which belong to flavonoid compounds in plant kingdom. They have many biological and ecological functions. Over the past many years, numerous efforts have been made to determine the biosynthetic pathway of anthocyanins and also to identify several regulatory proteins mainly in flowers and fruits of model plants and crop plants. However, many questions concerning the metabolism of anthocyanins in foliage remains unsolved. One example is âHow can developmental processes impact on accumulation patterns of anthocyanins in leavesâ. In this study, we choose several cultivars from one of the most popular ornamental plants Acer palmatum Thunb. to understand the mechanism of developmental changes of pigmentation in leaf. Several other maple species were also analyzed. We propose that the metabolism of anthocyanins play an essential role in such changes. We use an integrated approach of phytochemistry and metabolic profiling to determine the biosynthesis and metabolism of anthocyanins and their impacts on foliage color. Proanthocyanidin analysis was carried out as well to determine their relationship to both anthocyanin production and foliar coloration. We have found that even for green leaves with no/trace amount of detectable anthocyanins, the biosynthetic pathway of anthocyanidin/proanthocyanidin is still activated. Our results indicate that metabolic channeling directing the anthocyanin pathway to the proanthocyanidin biosynthesis plays a very important role in pigmentation pattern change along developmental processes.

While cranberry consumption has been linked with the prevention of bacterial infections in the urinary tract for many years, our understanding of the bioavailability and mechanisms by which cranberry prevents bacterial infection is limited. Despite frequent use in clinical trials, it is hypothesized that the bioavailability of cranberry derived materials in the urinary tract may be limited due to their interaction with human serum proteins such as albumin, α-1-acid glycoprotein (AAG), and fibrinogen. Upon reaching the urinary tract, cranberry derived materials may interfere with bacterial pathogenesis via interaction with the lipopolysaccharides (LPS) on the bacterial cell surface and the secreted biosurfactant, rhamnolipid. Quartz crystal microbalance with dissipation monitoring (QCM-D) is a sensitive mass sensor that is used in this study to directly investigate the interactions between cranberry derived materials and human serum proteins (albumin, AAG, and fibrinogen) or bacterial components (LPS and rhamnolipid). The binding of cranberry proanthocyanidins (CPAC) to all three serum proteins, the rhamnolipids, and LPS from the uropathogen Escherichia coli O111:B4 can be described by Langmuir-type isotherms allowing the determination of the apparent adsorption affinity constant between the CPAC and each biomolecule. CPAC interacts most strongly with fibrinogen with a binding constant of 2.2×108 M-1. CPAC exhibits weaker interactions with albumin and AAG, with binding constants of 2.4×106 M-1 and 1.5×106 M-1, respectively. These binding interactions will limit the bioavailability of the CPAC at the site of action thus highlighting the need for an improved understanding of the bioavailability and pharmacokinetics of cranberry consumption before further clinical trials. Furthermore, CPAC interacts with LPS from Pseudomonas aeruginosa 10 in a fundamentally different manner than it interacts with E. coli O111:B4 LPS or P. aeruginosa rhamnolipids, supporting the theory that there are multiple mechanisms via which cranberry prevents bacterial infections and that cranberry may be more effective at preventing certain bacterial infections.
Tandis que la consommation de canneberge a été liée à la prévention d'infections bactériennes dans les voies urinaires pendant de nombreuses années, notre compréhension de la biodisponibilité et les mécanismes par lequel canneberge empêche les infections bactériennes est limitée. Malgré l'utilisation fréquente dans les essais cliniques, la biodisponibilité de matières dérivées de canneberges dans les voies urinaires peut être limitée en raison de leur interaction avec les protéines de sérum humain tels que l'albumine, α-1 glycoprotéine acide (AAG) et du fibrinogène. Dès qu'elles arrivent à la voie urinaire, les matières dérivées de canneberges peuvent interférer avec la pathogenèse bactérienne via l'interaction avec les lipopolysaccharides (LPS) sur la surface bactérienne et les biosurfactants sécrétées, rhamnolipide. Microbalance à quartz avec la surveillance de la dissipation (QCM-D) est un instrument capable de détecter de mass avec une haute sensibilité qui est utilisé dans cette étude d'enquêter directement sur les interactions entre les matières dérivées de canneberges et protéines de sérum humain ou des composants bactériens (LPS et rhamnolipide). Les liaisons entre les proanthocyanidines de canneberge (CPAC) et les trois protéines sériques, les rhamnolipides, et le LPS de Escherichia coli O111:B4 uropathogènique peut être décrite par les isothermes de type Langmuir permettant la détermination de la constante d'affinité apparente d'adsorption entre la CPAC et chaque biomolécule. CPAC interagit fortement avec le fibrinogène avec une constante de fixation de 2.2x108 M-1. CPAC a des interactions plus faibles avec l'albumine et AAG, avec les constantes de fixation de 2.4x106 M-1 et 1.5x106 M-1, respectivement. Ces interactions de liaison limiteront la biodisponibilité de la CPAC au site d'action, mettant ainsi en évidence la nécessité d'une meilleure compréhension de la biodisponibilité et la pharmacocinétique de la consommation de canneberge avant d'autres essais cliniques. De plus, CPAC interagit avec le LPS de Pseudomonas aeruginosa 10 de manière fondamentalement différente qu'il interagit avec le LPS de E. coli O111:B4 ou rhamnolipides de P. aeruginosa, soutenant la théorie selon laquelle il y a plusieurs mécanismes par laquelle canneberge empêche les infections bactériennes et la canneberge peut-être plus efficace pour prévenir certaines infections bactériennes.

Les catequines i els seus polímers, les procianidines, són flavonoids presents en hortalisses
i fruits amb efectes beneficiosos sobre la salut. Actuen com a antioxidants segrestant
espècies reactives d'oxigen (ROS) i quelant els metalls ferro i coure. També es comporten
com a molècules senyalitzadores, modulant múltiples vies de senyalització i metabòliques i
l'expressió gènica, incloent-hi la d'enzims antioxidants. Resultats previs del Grup de Recerca
en Nutrigenòmica mostren que una dosi oral aguda d'un extracte de procianidines de llavor
de raïm (GSPE) reprimeix l'expressió de les metal·lotioneïnes (MT), proteïnes lligadores de
zinc, a fetge de rates, i tanmateix incrementa l'expressió del receptor nuclear orfe small
heterodimer partner.(SHP/Nr0b2) (Del Bas et al., 2005). Igualment, es va demonstrar que les
procianidines actuen com a coactivadors transcripcionals del receptor nuclear d'àcids biliars
Farnesoid X Receptor (FXR), el qual es responsable de la sobre-expressió de SHP causada
per GSPE a cèl·lules hepàtiques, i de l'efecte hipotrigliceridèmic de les prociandines effect
(Del Bas et al., 2008; Del Bas et al., 2009).
Els objectius d'aquesta Tesi van ser determinar si les catequines i procianidines
interaccionen amb el zinc, avaluar el seu efecte sobre l'homeòstasi del zinc en cèl·lules
hepàtiques -incloent l'efecte sobre l'expressió de genes MT, utilitzats aquí com a
biomarcadors de l'activitat de les procianidines a cèl·lules hepàtiques-, i disseccionar els
mecanismes pels quals les procianidines afecten l'homeòstasi del zinc, en particular
confirmar si els gens MT són dianes de SHP i FXR.
Els resultats obtinguts mostren que GSPE, així com diverses catequines i procianidines
pures, incloent-hi el flavonoid del te verd (-)-epigallocatechin-3-gallate (EGCG), lliguen
cations de zinc en solució amb una afinitat més gran que el quelant específic de zinc
Zinquin. En cèl·lules d'hepatocarcinoma humanes HepG2, GSPE inhibeix l'acumulació
intracel·lular de zinc i contraresta els efectes tòxics de dosis elevades de zinc sobre la
viabilitat cel·lular. GSPE reprimeix l'expressió de gens de MTs i d'exportadors de zinc mentre
que estimula l'expressió d'importadors de zinc. L'expressió dels importadors de zinc de la
xarxa Trans-Golgi és estimulada per GSPE. A més a més, GSPE bloqueja la inducció de
l'expressió de MTs per la citoquina proinflamatoria IL-6, pel generador de ROS tBOOH, per
l'agonista de receptors de glucocorticoids dexametasona, i pels metalls coure i zinc.
EGCG reprodueix els efectes de GSPE sobre l'homeòstasi del zinc en HepG2, reprimint
l'expressió de MTs i d'exportadors de zinc, estimulant l'expressió d'importadors de zinc, i
UNIVERSITAT ROVIRA I VIRGILI
EFFECTS OF DIETARY CATECHINS AND PROANTHOCYANIDINS ON ZINC HOMEOSTASIS IN HEPATIC CELLS
Isabel Maria Quesada
ISBN:978-84-694-1258-9/DL:T-322-2011
inhibint l'acumulació de zinc intracel·lular i la toxicitat de dosis elevades de zinc. La
procinidina dimèrica B1 i la trimèrica C1 es comporten tenen efectes contraris als de GSPE i
EGCG pel que fa a l'expressió de MT i l'acumulació de zinc total en cèl·lules HepG2.
Pel que fa al zinc làbil citoplasmàtic, la minúscula fracció del total del zinc cel·lular que
modula múltiples vies metabòliques i senyalitzadores, tant GSPE com EGCG i C1 eleven en
gran manera els nivells de zinc làbil detectable per Zinquin a cèl·lules HepG2.
Experiments amb ratolins KO per SHP o per FXR han demonstrat que GSPE reprimeix
l'expressió postprandrial de gens MT a fetge per una via que no depen de SHP però que és
depenent de FXR. A més, l'àcid biliar CDCA, un lligand fisiològic i activador de FXR,
reprimeix l'expressió de gens MT a cèl·lules HepG2. Per tant, els gens MT són diana de FXR
i, conseqüentment, FXR apareix com un receptor nuclear que modula l'homeòstasi del zinc.
Per explicar aquests resultats, proposem que catequines i procianidines poden actuar tant
com a segrestadors de zinc -evitant la seva entrada a la cèl·lula a través dels transportadors
de zinc de membrana plasmàtica-, com d'ionòfors de zinc -cotransportant cations zinc a
través de la bicapa lipídica i incrementant així els nivells de zinc làbil citoplasmàtic. La
repressió de gens MT induïda per l'activació de FXR per GSPE podria també contribuir a
l'increment de zinc làbil, en impedir que els cations zinc siguin segrestats per apo-tioneïna
sintetitzada de novo.
Donat el paper del zinc làbil com a modulador de múltiples víes de senyalització i
metabòlics, formulem la hipòtesi que la quelació extracel·lular de cations de zinc i l'elevació
de zinc làbil citoplasmàtic són mecanismes subjacents a l'activitat biològica de catequines i
procianidines i, per tant, que les vies metabòliques i de senyalització afectades pel zinc làbil,
ho seràn també per aquests flavonoids.
Effects of dietary catechins and proanthocyanidins on zinc homeostasis in
hepatic cells.
Catechins and their polymers procyanidins are health-promoting flavonoids found in edible
vegetables and fruits. They act as antioxidants by scavenging reactive oxygen species and
by chelating the redox-active metals iron and copper. They also behave as signaling
molecules, modulating multiple cell signaling and metabolic pathways and gene expression,
including that of antioxidant enzymes. Previous results of the Nutrigenomics Reseach Group
showed that an oral acute dose of a grape-seed procyanidin extract (GSPE) represses the
expression of the zinc-binding protein metallothionein (MT) genes in rat liver, and enhances
the expression of the orfan nuclear receptor small heterodimer partner (SHP/Nr0b2) (Del Bas
et al., 2005). In addition, it was shown that procyanidins act as transcriptional coactivators of
the nuclear bile acid receptor Farnesoid X Receptor (FXR), which in turns upregulates SHP
expression, thereby exerting an hypotrygliceridemic effect (Del Bas et al., 2008; Del Bas et
al., 2009).
The objectives of this Ph.D. Thesis were to determine whether catechins and procyanidins
interact with the redox-inactive metal zinc, to evaluate their effect on zinc homeostasis in
hepatic cells -including the expression of MT genes, used here as a biomarkers of
procyanidin activity in hepatic cells-, and to disect the mechanisms by which procyanidins
affect cellular zinc homeostasis, in particular to asses whether MT genes are targets of SHP
and FXR.
Our results show that GSPE, as well as individual catechins and procyanidins tested,
including the green tea flavonoid (-)-epigallocatechin-3-gallate (EGCG), bind zinc cations in
solution with higher affinity than the zinc-specific chelator Zinquin. In human
hepatocarcinoma HepG2 cells, GSPE inhibits intracellular zinc accumulation and counteracts
the toxic effects of excess zinc on cell viability. At the mRNA expression level, GSPE
downregulates MTs and zinc-efflux transporters while upregulating zinc-influx transporters.
Zinc importers of the Trans-Golgi network are upregulated by GSPE. In addition, GSPE
blocks the induction of MTs expression by the proinflammatory cytokine IL-6, the ROS
generator tBOOH, the glucocorticoid receptor agonist dexamethasone, and the metals
copper and zinc.
EGCG reproduces the major effects of GSPE on zinc homeostasis in HepG2, downreguling
the expression of MTs and zinc-efflux transporters, while upregulating the expression of zincinflux
transporters, concomitantly inhibiting intracellular zinc accumulation and the toxicity of high zinc doses. Procyanidin dimer B1 and trimer C1 behave opposite to GSPE and EGCG
with regard to MT expression and intracellular zinc accumulation in HepG2 cells.
Concerning cytoplasmic labile zinc, the tiny fraction of total cellular zinc that modulates
signaling and metabolic pathways, we found that GSPE, EGCG and trimeric procyanidin C1
greatly elevate Zinquin-detectable labile zinc in HepG2 cells.
Experiments with SHP-null and FXR-null mice demonstrate that GSPE downregulates
postprandial expression of MT genes in the liver, in a SHP-independent but FXR-dependent
manner. In addition, chenodeoxycholic acid, a physiological ligand and activator of FXR,
represses the expression of MT genes in HepG2 cells. Thus, MT genes are targets of FXR
and, consequently, FXR is revealed as a modulator of zinc homeostasis.
To explain these results, we postulate that catechins and procyanidis may act both as
sequestrants of zinc -thereby impeding the entrance of zinc cations to the cell through
plasma membrane zinc transporters-, and as zinc ionophores -thereby cotransporting zinc
cations through the lipid bilayer and increasing the levels of cytoplasmic labile zinc.
Repression of MT expression by procyanidin-activated FXR might also contribute to the
increment of the labile pool of zinc, by hindering the sequestration of zinc-cations by de novo
synthesized apo-thionein.
Given the role of labile zinc as modulator of multiple intracellular signaling and metabolic
pathways, we forward the hypothesis that extracellular complexation of zinc cations and
subsequent elevation of cytoplasmic labile zinc may be relevant mechanisms underlying the
health-promoting activity of catechins and procyanidins and, therefore, that the signaling and
metabolic pathways modulated by labile zinc will be aslo a target of these flavonoids.

El tracte intestinal és un lloc d'interacció amb microorganismes i factors ambientals potencialment nocius. En aquest sentit, un alt consum de components majoritaris de la dieta occidental com la fructosa i els greixos saturats s'ha associat amb la disfunció intestinal (disrupció de la funció de barrera i inflamació) i l'entrada d'endotoxines bacterianes amb efecte proinflamatori en la circulació. Addicionalment, una alta concentració d'endotoxina en plasma (endotoxemia metabòlica) es vincula amb la síndrome metabòlica. Així, l'intestí està emergint com un blanc terapèutic per a la prevenció i tractament de malalties. Les proantocianidines (PACs) són compostos fenòlics naturals amb potent efecte antiinflamatori en la mucosa intestinal d'acord amb evidència preclínica. Per tant, l'administració de PACs és prometedora com a estratègia terapèutica complementària, però la seva eficàcia s'ha de confirmar en humans. El principal objectiu de la present tesi doctoral va ser avaluar l'impacte d'un extracte de PACs de la pinyol de raïm (GSPE) en models preclínics de disfunció intestinal i explorar la seva efectivitat en l'humà. Trobem que una dieta estil occidental (dieta de cafeteria) indueix disfunció intestinal en rates i que l'alteració de permeabilitat al còlon contribueix en gran mesura a la endotoxemia metabòlica. Aquests efectes van ser atribuïts parcialment a altes concentracions luminals de fructosa i van poder ser revertits amb dosis farmacològiques de GSPE in vivo. Finalment, contrastem aquests resultats amb evidència derivada d'un model humà ex vivo de disfunció colònica. En aquest model vam poder replicar la reducció de permeabilitat i la millora de l'estat inflamatori reportats in vivo. En conclusió, l'administració de GSPE pot millorar la disfunció intestinal i la endotoxemia metabòlica associada. Les dosis efectives en humans són probablement farmacològiques i hauran de ser establertes en estudis clínics posteriors.
El tracto intestinal es un sitio de interacción con microorganismos y factores ambientales potencialmente dañinos. En este sentido, un alto consumo de componentes mayoritarios de la dieta occidental como la fructosa y las grasas saturadas se ha asociado con la disfunción intestinal (disrupción de la función de barrera e inflamación) y la entrada de endotoxinas bacterianas con efecto proinflamatorio en la circulación. Adicionalmente, una alta concentración de endotoxina en plasma (endotoxemia metabólica) se vincula con el síndrome metabólico. Así, el intestino está emergiendo como un blanco terapéutico para la prevención y tratamiento de enfermedades. Las proantocianidinas (PACs) son compuestos fenólicos naturales con potente efecto antiinflamatorio en la mucosa intestinal, de acuerdo con evidencia preclínica. Por consiguiente, la administración de PACs es prometedora como estrategia terapéutica complementaria, pero su eficacia debe ser confirmada en humanos. El principal objetivo de la presente tesis doctoral fue evaluar el impacto de un extracto de PACs de la pepita de uva (GSPE) en modelos preclínicos de disfunción intestinal y explorar su efectividad en el humano. Encontramos que una dieta estilo occidental (dieta de cafetería) induce disfunción intestinal en ratas y que la alteración de permeabilidad en el colon contribuye en gran medida a la endotoxemia metabólica. Estos efectos fueron atribuidos parcialmente a altas concentraciones luminales de fructosa y pudieron ser revertidos con dosis farmacológicas de GSPE in vivo. Por último, contrastamos estos resultados con evidencia derivada de un modelo humano ex vivo de disfunción colónica. En este modelo pudimos replicar la reducción de permeabilidad y el mejoramiento del estado inflamatorio reportados in vivo. En conclusión, la administración de GSPE puede mejorar la disfunción intestinal y la endotoxemia metabólica asociada. Las dosis efectivas en humanos son probablemente farmacológicas y tendrán que ser establecidas en estudios clínicos posteriores.
The intestinal tract is a site of interaction with microorganisms and potentially detrimental environmental factors. The high intake of fructose and saturated fats typical of the Western diet has been associated with intestinal dysfunction (disruption of barrier function and inflammation) and an increased influx of proinflammatory bacterial endotoxins into the systemic circulation. In turn, high concentrations of plasma endotoxins (metabolic endotoxemia) are a precursor to the onset of metabolic syndrome. In view of the above, the intestine is emerging as a target for disease prevention and therapy. Proanthocyanins (PACs) are naturally occurring phenolic compounds with remarkable anti-inflammatory properties in the intestinal mucosa, according to preclinical studies. Thus, PAC administration is a promising adjunctive therapeutic strategy for the treatment of intestinal dysfunction, but its efficacy in humans is yet to be confirmed. The main objective of this doctoral thesis was to evaluate the impact of a grape-seed PAC extract (GSPE) on a rat and cell culture-based model of intestinal dysfunction and to investigate its effectiveness in humans. We found that a long-term Western-style diet (cafeteria diet) induces intestinal dysfunction in rats, and that alterations in the permeability of the colon largely contribute to metabolic endotoxemia. These effects are partially driven by high luminal concentrations of fructose and could be effectively reversed in vivo by pharmacological doses of GSPE. Lastly, we compared these findings with evidence derived from an ex vivo human model of chemically-induced colonic dysfunction in which we were able to replicate the reduction of intestinal permeability and the amelioration of inflammatory status by means of GSPE found in vivo. In conclusion, the administration of GSPE results in the overall improvement of intestinal dysfunction and associated metabolic endotoxemia. Effective doses in humans are probably pharmacological and will have to be determined in clinical trials.

Sainfoin proanthocyanidins (PAs) are complex mixtures of homo- and hetero- polymers consisting of B-type procyanidins and prodelphinidins. Direct analysis by thiolytic degradation revealed a wide range of PA contents and compositions within the HealthyHay germplasm collection (46 accessions). PA contents varied from 0.57% to 2.80% (g PAl100g freeze-dried samples). PAs polymer size ranged from 12 to 84 flavan-3-ol units in terms of their mean degree of polymerisation. Prodelphinidin/procyanidin ratios ranged from 53/47 to 95/5 and trans/cis ratios varied from 12/88 to 34/66. Purified PAs fractions from 4 selected accessions showed a positive correlation between polymer size and prodelphinidin content within each accession (R2 from 0.69 to 0.92). Careful selection of MALDI-TOF MS matrices and analytical conditions made it possible to detect PAs up to 12 subunits and also ion signals that could be assigned to A-type and rarely reported glycosylated A-type PAs. For detecting and confirming the polymer size of underivatised higher molecular weight PAs a new HPLC-GPC technique consisting in a single calibration curve for galloyl glucoses, ellagitannins and PAs was developed. Peak-average molecular weights of sainfoin PA fractions were overestimated by 42.0% at 2436 Dalton and underestimated by 13.9% at 8318 Dalton. Number-average molecular weights were overestimated by 30.0% and underestimated by 25.8%, respectively. Cluster analysis of the HealthyHay germplasm collection revealed that accessions clustered into two main clusters, Western Europe and Eastern Europe/Asia, and that accessions from Armenia, Canada and USA clustered into another group. This seems to be in agreement with the strong links between geographic origin and accession performance found in the HealthyHay sainfoin germplasm. This research made significant contributions to the fields of PA analysis and germplasm screening in terms of novel analytical techniques for determining the average molecular weight distribution, content, subunit composition and linkages, purification and fractionation of proanthocyanidins in sainfoin.

Peanut skin, a low-value agriculture waste product, has drawn lots of research interest in recent years, due to its high content of A-type proanthocyanidins. A-type proanthocyanidins have been believed to contribute to cranberries' anti-UTI (urinary tract infection) effect. In this study, we compared the A-type proanthocyanidins in cranberry and peanut skin crude extracts using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Many similarities were found in the proanthocyanidin composition of cranberries and peanut skins. New oligomeric and polymeric proanthocyanidins in peanut skins, including heteroproanthocyanidins and proanthocyanidins with sugar moieties or galloyl esters, were tentatively identified. Solid phase extraction (SPE) and HPLC fractionation largely improved MALDI-TOF's ability to detect proanthocyanidins with high degrees of polymerization (DP). By analyzing the identified compounds in each fraction, we were also able to find some interesting elution pattern of the proanthocyanidins on the SPE cartridges and on the HPLC column. For example, the elution order on both the SPE cartridges and the diol phase column generally followed the DP. A-type proanthocyanidins tended to elute earlier than the B-type. Prodelphinidins retained much longer than other proanthocyanidins with the same DP. These findings may help researcher to identify future research directions and develop new separation methods to facilitate the identification of bioactive components in proanthocyanidin-rich plant extracts.
Ph. D.

Mestrado em Viticultura e Enologia - Instituto Superior de Agronomia / Faculdade de Ciências. Universidade do Porto
This study aims to evaluate the influence of altitude on the phénolic compounds and the precursors of aroma glycosides in Touriga Nacional and Touriga Franca. It was gathered from several samples along the maturation of two vineyard with different altitudes, Quinta Cova da Barca. In Douro Superior, is at an altitude of 125m and 700m at Quinta do Cardo, in Beira Interior (Sub-region Castelo Rodrigo). The results show that there is a tendency to higher values of anthocyanins, color intensity and % prodelfinidyn of condensed tannins in the vineyards at a higher altitude. In the case of proanthocyanidins and the % esterification with acid gálhico this tendency is reversed, since this is higher than in the vineyards that have a lower altitude, which is indeed visible in both variety. As for the degree of polymerization, there are differences when comparing both variety. Touriga Nacional in this case is higher than in Quinta do Cardo (high altitude), the same is not true of Touriga Franca, since it is at a low altitude that the degree is higher. Finally, analyzing the glycosylated precursors, there are still differences that are not statistically significant. Highest in the vineyard, the precursors of aroma tend to take a higher value, except when matured.

Mestrado Vinifera EuroMaster - Instituto Superior de Agronomia
Different methods have been established in order to perform accurately the quantification of the condensed tannins in various plant products and beverages. The method of reaction in acid medium has been widely used for the quantification of condensed tannins. This method is based on the reaction of vanillin with the phenolic rings of condensed tannins and more specifically with the fusel aromatic rings of their flavan-3-ol units. In a previous study (Sun et al., 1998), several parameters that can affect the accuracy of the determination of condensed tannins have been examined by this method, and among them the influence of phenolic compounds other than tannins, in particular non-flavonoids such as phenolic acids (cinnamic acid, p-hydroxybenzoic acid, caffeic acid, gallic acid, p-coumaric acid, syringic acid), but also flavonols (quercetin dihydrate, kaempferol, myricetin, rutin) and the anthocyanin malvidin-3-glucoside; that may interfere the reaction of proanthocyanidins with vanillin assay. According to this analytical procedure proposed by Sun et al. (1998), other phenolic compounds of oenological interest not tested so far were analyzed to assess their possible interference with the reaction of proanthocyanidins in its quantification by the vanillin assay. In details, the phenolic compounds that have been studied were flavonols and flavones, stilbenes, various volatile phenols, and other phenols from wood such as ellagitannins, coumarins, aldehydes and still other compounds such as tyrosol and 2-phenylethanol. The chemical compounds examined at different concentrations did not produce any reaction with the vanillin. Therefore, the modified vanillin assay can be interpreted as a method for quantification of condensed tannins in grape and wine samples without any important analytical interference from other compounds not condensed tannins

La disfunció intestinal es basa en un estat proinflamatori en l'intestí i en una funció de barrera defectuosa, característiques comunes de diverses malalties cròniques intestinals, però també associades amb patologies metabòliques relacionades amb la dieta, com és l’obesitat. En aquest sentit, les proantocianidines són compostos bioactius naturals de la família dels flavonoides que han mostrat posseir propietats antiinflamatòries i que podrien tenir efectes significatius en l’entorn intestinal, influint en la fisiologia i la bioquímica de l'intestí. En aquest marc, la present tesi va ser dissenyada per aclarir el possible paper de les proantocianidines en la modulació de la resposta inflamatòria intestinal i la funció de barrera en diferents models animals complementaris de disfunció intestinal. Per assolir aquest objectiu global, primer vam examinar l'impacte d'una dieta obesogènica en l'estat de salut intestinal al llarg del temps i posteriorment vam analitzar l'efecte protector d'un extracte de proantocianidines de la llavor de raïm, comparant l'eficàcia de les diferents dosis i dels temps d'administració a l’hora de protegir de la disfunció intestinal. En segon lloc, vam analitzar el paper del mateix extracte de proantocianidines en un model animal d’inflamació intestinal aguda i d’alteració de la permeabilitat intestinal induïda per injecció amb lipopolisacàrids. En resum, la present tesi va revelar que l'obesitat induïda per la dieta i l'exposició aguda a lipopolisacàrids desencadenen un grau similar d’inflamació intestinal i deteriorament de l'estat funcional de la barrera, i més important, que l'administració oral de les proantocianidines millora la inflamació intestinal i la funció de barrera.
La disfunción intestinal se basa en un estado proinflamatorio en el intestino y en una función de barrera defectuosa, características comunes de diversas enfermedades crónicas intestinales, pero también asociadas con patologías metabólicas relacionadas con la dieta, como es la obesidad. En este sentido, las proantocianidinas son compuestos bioactivos naturales de la familia de los flavonoides que han mostrado poseer propiedades antiinflamatorias y que podrían tener efectos significativos en el entorno intestinal, influyendo así en la fisiología y la bioquímica del intestino. En este marco, la presente tesis fue diseñada para aclarar el posible papel de las proantocianidinas en la modulación de la respuesta inflamatoria intestinal y la función de barrera en diferentes modelos animales complementarios de disfunción intestinal. Para alcanzar este objetivo global, primero examinamos el impacto de una dieta obesogénica en el estado de salud intestinal a lo largo del tiempo y posteriormente analizamos el efecto protector de un extracto de proantocianidinas de la semilla de la uva, comparando la eficacia de las diferentes dosis y los tiempos de administración a la hora de proteger de la disfunción intestinal. En segundo lugar, analizamos el papel del mismo extracto de proantocianidinas en un modelo animal de inflamación intestinal aguda y de alteración de la permeabilidad intestinal inducida por inyección con lipopolisacáridos. En resumen, la presente tesis reveló que la obesidad inducida por la dieta y la exposición aguda a lipopolisacáridos desencadenan un grado similar de inflamación intestinal y deterioro del estado funcional de la barrera, y más importante, que la administración oral de las proantocianidinas mejora la inflamación intestinal y la función de barrera.
Intestinal dysfunction is based on a pro-inflammatory state in the intestine and on a defective barrier function, both considered common features of intestinal chronic diseases. However, intestinal dysfunction has also been associated with obesity and other metabolic diet-related pathologies. In this regard, proanthocyanidins are natural bioactive compounds from the flavonoid family with anti-inflammatory properties that might have significant effects on the intestinal environment, the physiology and the biochemistry of the intestine. In this framework, the present thesis was designed to elucidate the role of proanthocyanidins in the modulation of the intestinal inflammatory response and barrier function in complementary animal models of intestinal dysfunction To accomplish this global objective, firstly we examined the impact of an obesogenic diet on intestinal health status over time to then analyse the protective effect of a grape seed proanthocyanidin extract and compare the effectiveness of different doses and times of administration to protect against intestinal dysfunction. Secondly, the role of the same extract of proanthocyanidins was analysed in an animal model of acute intestinal inflammation and impaired intestinal permeability induced by lipopolysaccharides injection. To sum up, the present thesis revealed that diet induced obesity or acute lipopolysaccharide exposition trigger similar degree of intestinal inflammation and impaired barrier function state, and more importantly, that the oral administration of proanthocyanidins improves intestinal inflammation and barrier function.

Proanthocyanidins (PA) in some forage legumes have been linked to contradictory effects in animal health and nutrition. Ruminants fed with PA-containing plants do not suffer from bloat and can also reduce gastrointestinal parasite infections, improve protein use efficiency and reduce greenhouse gas emissions. Plants have a wide range of PA contents and compositions. Therefore, screening tools are required to determine the optimal contents and types to exploit PA bioactivities on farms. This research initially focused on the identification and isolation of PAs with contrasting characteristics from various plant species. These samples were then used to develop novel methods such as UPLC-ESI-MS/MS and 13C HSQC NMR for extractable PAs and 13C CPMAS NMR for PAs within plants, and to probe structure-activity relationships. These PAs were also subjected to complementary analytical methods, which demonstrated that depolymerisation techniques can provide quantitative information on PA contents and compositions and mass spectrometric techniques on molecular distributions. These analyses revealed an enormous range of molecular profiles. This diversity, however, led to good but not excellent correlations between the degradation methods. It also affected mass spectrometric and liquid-state NMR responses. In particular, there were some discrepancies between thiolysis-HPLC and UPLC-ESI-MS/MS results of sainfoin PA extracts. For solid-state NMR, the PAs from model plants proved too homogeneous for the analysis of the highly complex PAs in sainfoin plants. Nevertheless, this method could rank accessions on the basis of PA composition and discriminated between plant organs via signature spectra. Therefore, final decisions on which of these methods to use will depend on the research objectives and sample numbers. Finally, anti-parasitic assays discovered that the in vitro exsheathement inhibition of the abomasal parasite, Haemonchus contortus was dependent on the average molecular size of purified PA mixtures. In addition, collaborative studies showed that prodelphinidins or PA size also affected some anti-parasitic and ruminal fermentation results.

Consumers view natural antioxidants as a safe means to reduce spoilage in foods. In addition, these compounds have been reported to be responsible for human health benefits. Identification of these compounds in peanut skins may enhance consumer interest, improve sales, and increase the value of peanuts. This study evaluated analytical methods which have not been previously incorporated for the analysis of peanut skins. Toyopearl size-exclusion chromatography (SEC) was used for separating phenolic size-classes in raw methanolic extract from skins of Gregory peanuts. This allowed for an enhanced analysis of phenolic content and antioxidant activity based on compound classes, and provided a viable preparatory separation technique for further identification. Toyopearl SEC of raw methanolic peanut skin extract produced nine fractions based on molecular size. Analysis of total phenolics in these fractions indicated Gregory peanut skins contain high concentrations of phenolic compounds. Further studies revealed the fractions contained compounds which exhibited antioxidant activities that were significantly higher than that of butylated hydroxyanisole (BHA), a common synthetic antioxidant used in the food industry. This indicates peanut skin extracts are a viable antioxidant source, and that synthetic antioxidants can be replaced with those naturally-derived from peanut by-products. Structures contained in each fraction were identified using high performance liquid chromatography (HPLC) coupled with electrospray ionization (ESI) ion trap mass spectrometry (MSn). Prior to this study, approximately 20 compounds have been identified in peanut skins. The combination of Toyopearl SEC with ESI-HPLC-MSn allowed for the identification of 314 phenolic-based compounds, most of which are newly discovered compounds in peanut skins. Many compounds identified are known to have powerful antioxidant effects, and also have been reported to exhibit numerous beneficial chemical and biological activities, including the treatment of various human health-related conditions. It is evident that peanut skins may be a potential untapped source for the extraction of natural food antioxidants, nutracueticals, and even pharmaceuticals. Because peanut skins are largely a wasted resource to peanut processors, the novel polyphenols identified in this research could have a significant financial impact on the peanut industry.
Ph. D.

Thesis (Ph. D.)--Georgia State University, 2008.
Title from title page (Digital Archive@GSU, viewed June 24, 2010) Sidney Crow Jr., committee chair; Kuk-Jeong Chen, Robert Simmons, George Pierce, committee members. Includes bibliographical references (p. 66-68).

Ce travail de thèse est une contribution à l’étude phytochimique par Chromatographie de Partage Centrifuge (CPC), de trois plantes utilisées en médecine traditionnelle au Soudan : Aristolochia bracteolata (plante entière), Ziziphus spina-christi (feuilles) et Hydnora abyssinica (rhizomes). Ce travail a permis de mettre au point trois méthodologies de purification par CPC, applicables au fractionnement des acides aristolochiques, des flavonoïdes ou des proanthocyanidols (PAC). Dans ce contexte, la première partie de ce manuscrit est consacrée aux notions générales portant sur la CPC. La deuxième partie porte sur l’étude d’Aristolochia bracteolata. Cette plante est utilisée en médecine traditionnelle, malgré la présence d'acides aristolochiques qui confèrent une néphrotoxicité élevée. Ce travail a permis de mettre au point une méthode innovante pour l’isolement et la purification, avec un très haut niveau de pureté, des acides aristolochiques I, II et IIIa à partir d’un extrait brut, en une étape par CPC en mode d’échange d’ions forts (SIX-CPC). L’acide aristolochique IIIa n’avait jamais été décrit dans cette plante auparavant. Ces résultats ont fait l’objet d’une publication en 2015 dans Separation and Purification Technology. Dans la troisième partie de cette thèse, la CPC a été appliquée à l’isolement de flavonosides présents dans Z. spina-christi. Nous appuyant sur l’expérience du laboratoire dans l’extraction par CPC des flavonosides du Ginkgo biloba, nous proposons une méthodologie de purification utilisant les systèmes de solvant biphasiques EtOAc/n-BuOH/MeOH/H2O et EtOAc/n-BuOH/H2O à différents ratios en fonction de la polarité des flavonosides. Dans la dernière partie, l’étude phytochimique de Hydnora abyssinica a mis en évidence la présence de PACs, polymères de hauts poids moléculaires de flavanols. La méthodologie de fractionnement CPC, précédée d’un pré-fractionnement sur résine LH-20, a permis l’isolement pour la première fois dans cette plante de la katsumadine et du rhodioloside
This work was a contribution to the phytochemical study of three Sudanese medicinal plants: Aristolochia bracteolata (Whole plant), Ziziphus spina-christi (Leaves) and Hydnora abyssinica (Rhizomes). The specificity of this research program was to emphasize the application of Centrifugal Partition Chromatography (CPC) for the fractionation of these plants. Three specific CPC methodologies were developed for the purification of either aristolochic acids, flavonoids or proanthocyanidins (PACs). In this context, the first part of this manuscript was devoted to the presentation of the CPC methodology. The second part focused on the fractionation of crude extract of Aristolochia bracteolata. This plant is used in traditional medicine, in spite of the presence of aristolochic acids that confer a high nephrotoxicity. In this work was developed an innovating procedure for the isolation and purification in high purity of aristolochic acids I, II and IIIa, in one step from crude extract, using Strong Ions eXchange CPC (SIX-CPC). These results were published in 2015 in Separation and Purification Technology. In the third part, the flavonosides present in Z. spina-christi were isolated using CPC, either in normal or reverse elution mode, using two phases solvent systems EtOAc/n-BuOH/MeOH/H2O or EtOAc/n-BuOH/H2O with different ratios. In the last part, the phytochemical study of Hydnora abyssinica led to the fractionation of PACs, polymers of high molecular weight of flavanols. The CPC fractionation methodology, preceded by LH-20 resin pre-fractionation, allowed the isolation of katsumadine and rhodioloside

Doutoramento em Biologia - Instituto Superior de Agronomia / UL
The periderm is the frontier tissue that protects plants with secondary growth from external stresses. It is mostly composed of empty-lumened suberized cork cells, resulting from the activity of the phellogen or cork cambium. Cork oak produces an exceptionally thick periderm used for important industrial applications. Given the periderm importance in plant survival and cork industrial quality, the goal of this research project was to contribute for disclosing the cork ontogenic program by studying the periderm development from the first-formed cell layer until the raw cork used in the industry and to identify epigenetic factors that might be associated with cork quality
N/A

Las proantocianidinas ejercen efectos beneficiosos sobre algunos desordenes metabólicos que se consideran factores de riesgo de las enfermedades cardiovasculares. La desregulación del metabolismo lipoproteico juega un papel muy importante en los estados lipídicos alterados. Por lo tanto, los objetivos de esta Tesis fueron: estudiar la contribución del hígado y el intestino en la respuesta hipolipidémica de las proantocianidinas y evaluar los efectos de las proantocianidinas en modelos dislipidémicos nutricionales. Para realizar los experimentos se utilizaron tres modelos experimentales: ratas, ratones y células Caco2. Los resultados obtenidos fueron: que en un test de tolerancia lipídica tanto los quilomicrones como las VLDL contribuyen al efecto hipotrigliceridémico de las proantocianidinas pero su influencia depende del tiempo. Además, las proantocianidinas reprimen la secreción de TG por el hígado in vivo y por el intestino in vitro. ACSL se manifiesta como un gen diana de ellas en las células intestinales. Y finalmente, las proantocianidinas corrigen la dislipidemia pero no contrarrestan la ganancia de peso inducida por la dieta aunque estas tienen un efecto bimodal sobre la retención de energía in vivo.
Proanthocyanidins have been shown to exert advantageous actions on several metabolic disorders that are risk factors of cardiovascular diseases. Lipoprotein metabolism plays an important role in altered lipid states. Therefore, the aims of this Thesis were: To assess the contribution of the liver and the intestine in the hypolipidemic response triggered by proanthocyanidins and to evaluate the short-term effect of an oral intake of proanthocyanidins in dyslipidemic nutritional models. For these purposes, three experimental models have been used: Rats, mice and human Caco2 cells. The obtained results are: In a fat tolerance test, both chylomicrons and VLDL contribute to the hypotriglyceridemic action of proanthocyanidins but their influence depends on time. Moreover, proanthocyanidins repress TG secretion by the liver in vivo and by the intestine in vitro. Furthermore, ACSL manifests as their target gene in intestinal cells. Finally, proanthocyanidins correct dyslipidemia but not the weight gain induced by diet though these have a bimodal effect on energy retention in vivo.

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.

Mestrado em Viticultura e Enologia - Instituto Superior de Agronomia
Several analytical methods have been developed for the quantification of condensed tannins, however the time required and complexity of these methods difficult to implement in routine analysis. In this work the objective was to evaluate the application of Fourier transform mid-infrared spectroscopy for the quantification of total condensed tannins, as well as the determination of their polymeric fractions (FIII), oligomeric (FII) and monomeric flavan-3-ols (FI) in 55 red wines. Calibration models were developed using two methodologies as reference: fractionation by reverse phase method and quantification by reaction with vanillin and the method of precipitation with methyl cellulose. The results show that the coefficients of determination obtained by the external validation of the models were less than 0.80, except for the prediction model of polymeric tannins at concentrations greater than 400 mg / L, but this model was not considered robust, because the high standard error of calibration obtained in cross validation (SEC = 512,4 mg / L) and low number of samples in the external validation (n = 5), which leads to the conclusion that in this study the models are not robust enough to estimation of concentrations of total condensed tannins, and fractions FI, FII and FIII.

La mise au point de méthodes d'extraction et de purification des composés phénoliques d'un vin rouge de Bordeaux a permis, pour la première fois, l'isolement de cinq familles de dérivés dont deux dont nouvelles. Leur caractérisation structurale, par spectroscopie et par résonance magnétique nuclaire en particulier, conduit à l'identification des castavinols, d'un nouveau dérivé glycosylé bensofuranique, ainsi que de deux aurones hydratées (la maesopsine 3-0-glucoside et l'alphitonine 3-0-glucoside), deux acides cinnamiques glycosylés (le coumaroyl-6-glucopyranose et un hétéroside de ce dérivé) et quatre lignanes (le méso-sécoisolaricirésinol, le ent-isolaricirésinol, la (+_)-isolaricirésinol et le 5-méthoxy-isolaricirésinol). La catéchine, l'épicatéchine et les dimères procyanidoliques B1 et B2 ont également été isolés à partir du vin rouge et de ce fait caractérisés avec certitude. L'étude par chromatographie liquide haute performance sur trois millésimes (1988 à 1990) basée sur la recherche de cinq molécules nouvellement isolées, montre que ces substances sont des constituants permanents de la composition phénolique
We performed the extracting and purification processes of phenolic compounds in Bordeaux red wines. This method allows us to isolate for the first time compounds from five different families, among which two are new. Their structural elucidation, by spectroscopic means and particularly nuclear magnetic resonance, leads to the identification of four compounds the castavinol series, a new glycosyl, a new glycosyl bensofuranic compound, and two hydrated aurones (maesopsine 3-0-glucoside and alphitonine : 3-0-glucoside), two glycosylated cinnamic acids (coumaroyl-6-glucopyranose and an heteroside derivative) and four lignans (mesosecoisolariciresinol, ent-isolariciresinol, (+_)-isolariciresinol and 5-methoxy-isolariciresinol). Catechin, epicatechin and the procyanidins B1 and B2 have been also isolated from red wine and thus characterized with no ambiguity. Aiming to detect the presence of five compounds newly isolated shows that these substances are constant constituents of phenolic composition of Bordeaux red wines

Orientador: Alba Regina Monteiro Souza Brito
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
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Resumo: As doenças inflamatórias intestinais (DII) compreendem doença de Crohn e reto-colite ulcerativa inespecífica, ambas caracterizadas por processo inflamatório crônico e descontrolado no intestino mediado por células do sistema imune. DII tem sido objeto de extensas pesquisas nos últimos anos; indefinição sobre etiologia e ausência de tratamento eficaz os justificam. Nesse cenário, produtos naturais surgem como alternativas terapêuticas, uma vez que moléculas com atividade farmacológica pronunciada, especialmente compostos fenólicos, têm merecido destaque. Neste trabalho foram avaliadas três frações orgânicas (aquosa - Aq, butanólica - BuOH, e acetato de EtOAc) obtidas a partir das cascas de Rhizophora mangle em modelos experimentais de colite induzida por dextrana sal sódico (DSS) em camundongos e ácido 2,4,6-tri-nitro-benzeno-sulfônico (TNBS) em ratos. BuOH (0.5 e 1.5 mg.Kg-1) e EtOAc (1.5 mg.Kg-1) foram efetivas no modelo de colite por DSS, enquanto BuOH 0.5 mg.K-1 e EtOAc 1.5 mg.Kg-1 mostraram-se eficazes na colite aguda por TNBS. No modelo sub-crônico de colite os tratamentos BuOH e EtOAc reduziram novamente os danos causados pelo TNBS, embora BuOH tenha se mostrado mais eficiente. A avaliação de mediadores envolvidos nesses modelos experimentais mostrou que tanto BuOH quanto EtOAc exercem ação sobre a atividade da glutationa peroxidase (GSH-Px) e mieloperoxidase (MPO) bem como sobre os níveis das citocinas pró-inflamatórias fator de necrose tumoral-? (TNF-?), interferon-? (IFN-?), interleucinas 1?, 6 e 12 (IL-1?, IL-6 e IL-12), além de reduzirem a expressão de ciclo-oxigenase-2 (COX-2) no modelo de DSS. No modelo sub-crônico de colite induzida por TNBS, às duas frações atuaram sobre a atividade da GSH-Px e catalase (CAT) e sobre os níveis de glutationa (GSH), TNF-? e IL-12; EtOH também reduziu os níveis de IL-6 e levou ao aumento de citocina anti-inflamatória interleucina-10 (IL-10), BuOH por sua vez mostrou-se eficaz em reduzir o índice de peroxidação lipídica (TBARS) e atividade da MPO, atuando também sobre a expressão de ciclo-oxigenase-1 (COX-1). A análise fitoquímica revelou presença de procianidinas oligoméricas nas duas frações ativas, BuOH e EtOAc, sugerindo que estas substâncias sejam responsáveis pelos efeitos produzidos pelos tratamentos experimentais. As frações BuOH e EtOAc apresentaram efeitos importantes sobre os componentes imune, inflamatório e oxidante em modelos experimentais de colite induzida por TNBS e DSS em doses consideradas baixas para uma mistura de substâncias. As procianidinas de R. mangle surgem então como alternativa terapêutica interessante sobre as DII
Abstract: Inflammatory bowel diseases (IBD) comprises Crohn's disease and ulcerative colitis, both are characterized by an immune-mediated chronic inflammatory process in the gut. IBD have been studied over the past few years; undefined etiology and the absence of effective treatments justify these studies. In this scenario, natural products arises as therapeutic alternative, once various studies in animal models have shown efficient treatments. Nowadays, molecules with pronounced pharmacological activities, especially phenolic compounds, have been highlighted. In this work three organic fractions (aqueous - Aq, butanolic - BuOH and ethyl acetate - EtOAc) obtained from Rhizophora mangle barks were evaluated in experimental models of dextran sodium sulphate (DSS)-induced colitis in mice and 2,4,6-tri-nitro-benzene sulphonic acid (TNBS)-induced colitis in rats. BuOH (0.5 and 1.5 mg.Kg-1) and EtOAc (1.5 mg.Kg-1) were effective in the DSS-induced model, while BuOH (0.5 mg.Kg-1) and EtOAc (1.5 mg.Kg-1) were also effective in acute TNBS-induced model of colitis, which lead to the evaluation of these last ones in sub-chronic TNBS-induced models of colitis. In this model, both treatments reduced the injury provoked by the instillation of TNBS, although BuOH has shown more efficiency. The evaluation of the mediators involved in these experimental models showed that either BuOH as EtOAc act on the glutathione peroxidase (GSH-Px) and myeloperoxidase activities as well as on pro-inflammatory cytokines tumor necrosis factor- (TNF-?), interferon- (IFN-?) and interleukins -1?, 6 and 12 (IL-1, IL-6 and IL-12) levels, and down-regulation of ciclo-oxygenase-2 (COX-2) expression in the DSS-induced model of colitis. Whereas in the sub-chronic TNBS-induced model of colitis, both fractions showed effects on the GSH-Px and catalase (CAT) activities, and glutathione (GSH), TNF-? and IL-12 levels; EtOAc also reduced IL-6 levels and lead to the enhancement of the levels of an anti-inflammatory cytokine, interlukin-10 (IL-10), on the other hand BuOH showed to be efficient in reducing lipid peroxidation index (TBARS) and MPO activity, acting on the upregulation of cyclo-oxygenase-1 (COX-1). Phytochemistry analysis reveals the presence of oligomeric procyanidins in both fractions, BuOH and EtOAc, suggesting that these substances are responsible for the effects produced by the experimental treatments. BuOH and EtOAc fractions presented important effects on the immune, inflammatory and oxidant components in the experimental models of TNBS and DSS-induced colitis using doses, considered, lower for a mixture of substances. The procyanidins of R. mangle became an interesting alternative therapeutic for IBD
Doutorado
Farmacologia
Doutor em Farmacologia

A cultura popular atribui ao gênero Passiflora várias propriedades medicinais e funcionais. Frutas frescas e secas, cascas, infusões e suco da polpa de maracujás silvestres são consumidos e comercializados para controlar ansiedade, insônia, tremores, diabetes e obesidade, entre outras indicações. Dessa forma, os objetivos deste trabalho foram caracterizar e comparar as folhas e frutos (pericarpo, polpa e sementes) das espécies P. edulis Sims variedades BRS Sol do Cerrado, Ouro Vermelho e Gigante Amarelo, P. alata Curtis, P. setacea DC e P. tenuifila Killip em relação ao seu potencial funcional, avaliando-se conteúdos de compostos fenólicos totais, proantocianidinas, vitamina C, alcaloides, tipos e teores de flavonoides, controle potencial de glicemia pós-prandial (inibição de α-amilase e α-glicosidase in vitro) e capacidade antioxidante in vitro. O principal flavonoide encontrado tanto em folhas como em frutos foi a homoorientina e as maiores concentrações foram encontradas em folhas, seguido de pericarpo. Os maiores teores de alcaloides foram detectados nas polpas dos frutos de P. edulis (0,1-0,2 mg/100 g b.s.). As maiores concentrações de compostos fenólicos foram detectadas nas folhas de P. edulis e P. alata, porém as sementes de P. tenuifila e principalmente P. setacea apresentaram os teores mais elevados em comparação com as outras frações. Em relação às proantocianidinas, este é o primeiro estudo que mostra a presenças destes compostos em Passiflora spp, em concentrações significativamente maiores nas sementes. Além disso, o estilbeno piceatanol (3,4,3\',5\'-tetrahidroxi-trans-estilbeno) também foi encontrado nas sementes de P. edulis variedades BRS Ouro Vermelho (2 mg/100 g b.s.) e Gigante Amarelo (3 mg/100 g b.s.) e P. alata (10 mg/100 g b.s.). Os compostos fenólicos encontrados nas sementes de todas as espécies de Passiflora analisadas se mostraram potentes inibidores das enzimas α-amilase e α-glicosidase. Este trabalho mostrou que os frutos e as folhas de Passiflora spp podem ser considerados boas fontes de compostos bioativos, especialmente com atividade antioxidante, apresentando, portanto, potenciais efeitos benéficos sobre a saúde humana.
Folk culture assigns to the genus Passiflora various medicinal and functional properties. Fresh and dried fruits , barks , teas and wild passion fruit pulp juice are consumed and sold to control anxiety, insomnia , tremor , diabetes and obesity , among other indications .Thus, the aim of this study were to characterize the leaves and fruit (pericarp , pulp and seeds) of the species P. edulis Sims varieties BRS Sol do Cerrado, Ouro Vermelho e Gigante Amarelo, P. alata Curtis, P. setacea DC e P. tenuifila Killip in relation to their functional potential , evaluating content of total phenolic compounds , proanthocyanidins , vitamin C , alkaloids , types and levels of flavonoids, potential control of postprandial glycemia (inhibition of α-amylase and α-glucosidase in vitro) and in vitro antioxidant capacity. The main flavonoid found in both leaves and fruits was homoorientina and the highest concentrations were found in leaves, followed by pericarp . The highest levels of alkaloids were detected in the pulp of the fruits of P. edulis ( 0.1-0.2 mg/100 g b.s. ) . The highest concentrations of phenolic compounds were identified in the leaves of P. edulis and P. alata, but the seeds of P. tenuifila and especially P. setacea showed higher levels compared with the other fractions . With respect to proanthocyanidin, this is the first study to show the presence of these compounds in Passiflora spp at concentrations significantly increased in seeds. Furthermore, piceatannol stilbene ( 3,4,3\',5\'- tetrahydroxy -trans- stilbene ) was also found in the seeds of P. edulis varieties BRS Ouro Vermelho (2 mg/100 g d.w.) and Gigante Amarelo (3 mg/100 g d.w.) and P. alata (10 mg/100 g d.w.). Phenolic compounds found in the seeds of all species analyzed are good inhibitors of α-amylase and α-glicosidase. This work showed that the fruit and leaves of Passiflora spp. can be considered good sources of bioactive compounds, especially with antioxidant activity, thus presenting potential beneficial effects on human health.

Doutoramento em Engenharia Alimentar - Instituto Superior de Agronomia
Mannoproteins interact with several wine compounds and they seem to enhance the product sensory characteristics. This way, it was essential to understand more deeply the behaviour and the interactions established by mannoproteins through the industrial wine production process. The mannoproteins release in a white wine was studied, evaluating two different bâtonnage methods by comparing mannoproteins profile and content. The wines submitted to wood barrel bâtonnage presented a higher polysaccharide concentration and a better body mouth-feel. Subsequent to this work, the impact of fining and tartaric stabilization processes on the released mannoproteins was studied. Bentonite, gelatine and static cold tartaric stabilization removed high molecular weight mannoproteins and diatomaceous earth filtration removed mannoproteins with no selectivity. The interaction between mannoproteins and red wine phenolic compounds was also evaluated, by adding commercial mannoprotein enriched products and by adding winery produced yeast lees. Mannoproteins did not show an interaction with colour compounds but they seemed to delay the polymerization reaction of proanthocyanidins with a polymerization degree between eight and fourteen

En esta Tesis doctoral se han estudiado las proantocianidinas de pieles y semillas de uvas de las variedades Monastrell, Cabernet Sauvignon y Syrah, así como la transferencia de las mismas al vino y el efecto de algunas técnicas enológicas en la modulación de su extracción. Los resultados obtenidos muestran que la concentración de estos compuestos es máxima al inicio de la maduración, disminuyendo conforme se acerca el momento de la vendimia. La cantidad de proantocianidinas en los vinos depende tanto de la variedad como de la técnica enológica aplicada. Tiempos de maceración largos y técnicas como la maceración prefermentativa en frío o el uso de enzimas de maceración promueven una mayor extracción de proantocianidinas, aunque las de las uvas de la variedad Syrah son mucho más fácilmente extraíbles que las de Monastrell, en igualdad de condiciones.
In this thesis, skin and seed proanthocyanidins of Monastrell, Cabernet Sauvignon and Syrah grape varieties have been studied, as well as their transfer from grapes to wine and the effect of some oenological techniques in modulating their extraction. The results show that the concentration of these compounds was maximum at the beginning of ripening, decreasing up to moment of harvest. The concentration of proanthocyanidins in wines depends both on the variety and the applied oenological technique. Long maceration times and techniques such as cold soak or the use of maceration enzymes promote a large extraction of proanthocyanidins, although, under similar conditions, the proanthocyanidins of Syrah grapes are more easily transferred to must/wine than those of Monastrell grapes.

A Lesão Renal Aguda (LRA) é a complicação mais grave da rabdomiólise. Nessa síndrome, a liberação do pigmento heme desencadeia uma lesão que se caracteriza por vasoconstrição glomerular e toxicidade celular direta com componente oxidativo. A lesão oxidativa desencadeada é uma das linhas fisiopatológicas mais intrigantes. A renoproteção com antioxidantes tem demonstrado efeito satisfatório. As proantocianidinas são antioxidantes naturais encontrados no extrato da semente da uva. O objetivo deste estudo foi avaliar o efeito antioxidante da Vitis vinifera (extrato da semente de uva) sobre a função renal de ratos submetidos à lesão por rabdomiólise. Foram utilizados ratos Wistar, adultos machos, pesando entre 250-300 gramas. A LRA foi induzida pela administração de glicerol 50% i.m (intramuscular). Os animais foram distribuídos em 4 grupos: grupo Salina (6ml/Kg de NaCl 0,9% via intraperitoneal (i.p) 1 vez ao dia), grupo Glicerol (6ml/Kg de glicerol i.m, cada região femoral recebeu 3ml/Kg de glicerol, 1 vez ao dia), grupo Vitis vinifera (3mg/Kg v.o por 5 dias) e grupo Glicerol+Vitis vinifera que recebeu Vitis vinifera por 5 dias antes do glicerol. Foram avaliados o marcador de lesão muscular (CK), a função renal (FR), a função tubular (FENa e FEK), o perfil oxidativo (peróxidos urinários-FOX-2 e MDA-TBARS) , a histologia e morfometria renal. O grupo Glicerol tratado com Vitis vinifera apresentou melhora da FR e tubular, redução dos níveis da peroxidação lipídica e melhora da histologia renal. Os resultados deste estudo confirmaram a proteção antioxidante, com repercussão histológica, da Vitis vinifera na LRA induzida por glicerol.
The Acute Kidney Injury (AKI) is the worst complication of rhabdomyolysis. In this syndrome, the delivery of heme pigment induces an injury that distinguishes itself by glomerular vasoconstriction and direct cellular toxicity with oxidative component. The oxidative injury is an intriguing one of the pathophysiological mechanism. The renoprotection with antioxidants has demonstrated satisfactory effect. The proanthocyanidins are natural antioxidants found in grape seed extract. The aim of this study was to evaluate the antioxidant effect of Vitis vinifera (grape seed extract) on the renal function of rats submitted to the injury by rhabdomyolysis. Wistar rats, male, adults, weight ranging from 250-300 g were used. The AKI was induced intramuscular (i.m.) administration of glycerol 50%. The animals were distributed in 4 groups: Saline group (6ml/Kg of NaCl 0,9% intraperitoneal (i.p) once a day), Glycerol group (6ml/Kg of glycerol i.m, each femoral region received 3ml/Kg of glycerol, once a day), Vitis vinifera group (3mg/Kg v.o by 5 days) and Glycerol+Vitis vinifera group that has received Vitis vinifera by 5 days before glycerol. Marker of muscular injury (CK), renal function (RF), the tubular function (FENa and FEK), the oxidative profile (urinary peroxides -FOX-2 and MDA-TBARS), the histological and kidney morphometric were evaluated. The Glycerol group treated with Vitis vinifera has shown improvements in RF and tubular, reduction of levels of lipid peroxidation and amelioration of kidney histology. The results of this study have confirmed the antioxidant protection, with histological repercussion of Vitis vinifera in AKI induced by glycerol.

Orientador: Fernanda Gonçalves Basso
Resumo: A mucosite é um dos principais efeitos adversos do tratamento oncológico e apresenta influência significativa na morbidade e qualidade de vida dos pacientes submetidos a este tipo de tratamento. A etiopatogenia desta condição vem sendo relacionada não só a toxicidade direta dos quimioterápicos e radiação ionizante, mas também a uma ativação exacerbada da resposta inflamatória dos tecidos. Recentemente, o desenvolvimento e a severidade da mucosite oral também tem sido relacionada a expressão exacerbada de metaloproteinases (MMPs), que resulta na desorganização dos tecidos epitelial e conjuntivo e consequentemente, em atraso do processo de reparo. O manejo da mucosite oral tem sido baseado na utilização de agentes analgésicos tópicos, antibióticos e antifúngicos, bem como na aplicação da laserterapia de baixa intensidade (LBI). A LBI apresenta resultados positivos sobre as lesões de mucosite oral, porém, esta modalidade terapêutica requer a utilização de um equipamento de custo moderado e deve ser feita sob supervisão. Desta forma, novas estratégias preventivas e terapêuticas, que possam atuar na modulação da resposta inflamatória e na expressão de MMPs para diminuir a incidência e a severidade das lesões de mucosite oral e também acelerar o processo de reparo têm sido propostas. Estudos recentes demonstraram a efetividade de compostos naturais, como as antocianinas, na redução da incidência e severidade destas lesões. O objetivo desta pesquisa in vitro foi avaliar comparativam... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Mucositis is the major adverse effect of oncological treatment and presents huge impact on oncological patients' morbidity and quality of life. Etiopathogenesis of oral mucositis has been not only related to a direct toxicity of chemotherapy drugs and radiant therapy, but also to an extended inflammatory reaction. Recently, development and severity of oral mucositis has also been associated to increased expression of metalloproteinases (MMPs), that results in disorganization of epithelial and connective tissues, leading to a delayed tissue healing. Treatment of oral mucositis is based on the application of topic analgesic and antimicrobial compounds, as also low-level lasertherapy (LLLT). LLLT shows positive outcomes on oral mucositis, however, this therapy requires the use of a moderate cost equipament and professional supervision. Therefore, new preventive and therapeutic strategies that can act on the modulation of inflammatory response as well as on MMPs expression could decrease the incidence and severity of oral mucositis, therefore enhancing oral mucosa healing. Recent studies demonstrated the effectiveness of natural compounds, such as antocyanins on the decreased incidence and severity of these lesions. Consequently, the aim of the present in vitro study were to assess the effects of proanthocyanidins (PAs, grape seed extract), naringenin (NA, proanthocyanidins-derived synthetic compound), also denominated flavanones, and LLLT as modulators for synthesis of interleukin-6 and MMPs from gingival fibroblasts. Cells were isolated from the papillary gingival tissue of a young (24 years) and healthy individual by enzymatic digestion, then cultured and submitted to different treatments. The influence of two chemotherapeutic agents (5-fluorouracil - 5-FU and methotrexate - MTX) and inflammatory cytokines (TNF-α, IL-6 and -8) on the induction of MMP-2 and -9 synthesis and gene expression were ... (Complete abstract electronic access below)
Mestre

Parmi les métabolites secondaires impliqués dans la qualité du raisin et du vin, les tanins condensés ou proanthocyanidines (PAs) jouent un rôle majeur, en particulier dans l'astringence et la stabilité de la couleur du vin. Ces molécules sont également impliquées dans la défense des plantes contre des stress biotiques et abiotiques. En outre, les effets bénéfiques des PAs pour la santé humaine sont bien documentés. Les PAs de la vigne ont la particularité d’être estérifiées avec de l’acide gallique. Une réaction d’acylation appelée galloylation est responsable de cette modification. Les études montrent que la galloylation influence les propriétés œnologiques et pharmacologiques des PAs. Dans la baie de raisin, les PAs sont synthétisés dans les premiers stades de développement, principalement dans les pellicules et les pépins. Un nombre relativement faible d'étapes enzymatiques sont nécessaires pour la biosynthèse de la structure de base de ces métabolites et les gènes correspondants sont aujourd'hui largement connus chez les plantes modèles, y compris chez la vigne. Cependant, les mécanismes moléculaires impliqués dans les étapes finales, y compris la galloylation, ne sont encore que partiellement connus. Des résultats antérieurs obtenus après la recherche de QTL influençant la composition du raisin, et en particulier le taux de galloylation des PAs, et des études transcriptomiques après surexpression de facteurs de transcription régulant la biosynthèse de la voie des PAs, ont permis l'identification de gènes potentiellement impliqués dans ces étapes. Des gènes de shikimate déshydrogénase (SDH) ont été identifiés. Ces gènes interviendraient en amont, pour la biosynthèse de l'acide gallique. Trois glucosyltransférases ainsi identifiées et déjà caractérisées au laboratoire sont impliquées dans la biosynthèse de l'ester de glucose de l'acide gallique (β-glucogalline), qui servirait d'intermédiaire pour la galloylation des PAs. Ces méthodes de criblage ont également permis d’identifier 2 acyltransférases de type sérine carboxypeptidase, nommées glucose acyltransférases (GATs) qui seraient capables de catalyser la dernière étape de galloylation: le transfert de l'acide gallique depuis la β-glucogalline sur les PAs. Le premier objectif de cette thèse a été de déterminer la fonction des SDHs codées par les gènes de vigne. Certaines SDHs recombinantes produites de façon hétérologue chez E.coli ont la capacité à produire de l'acide gallique in vitro. Leur niveau d’expression au cours du développement et dans différents tissus de la baie a également été établi. Les résultats obtenus in vitro sont étayés par le profil métabolique (acide gallique, β-glucogalline et PAs) de hairy-roots de vigne transformées avec un gène de SDH. Le second objectif de cette thèse a été de valider la fonction des GATs par expression transitoire dans des feuilles de tabac et des tests enzymatiques in vitro. La transformation transitoire de feuilles de vigne avec les GATs a permis de moduler la concentration d’esters phénoliques et nomment des flavan-3-ols galloylés in planta. L’étude de ces gènes a été étendue aux plantes vasculaires par des analyses phylogénétiques et a permis d’identifier des motifs peptidiques potentiellement impliqués dans les mécanismes étudiés et reflétant la sub-fonctionnalisation de certains gènes. Ce travail a fourni des informations sur les bases génétiques et les mécanismes moléculaires impliqués dans la biosynthèse de l'acide gallique et son transfert en deux étapes sur les flavan-3-ols (galloylation). De nouvelles hypothèses sur l'intervention de différents transporteurs et la nature des molécules transportées pourront être formulées
Among the secondary metabolites involved in grape berry and wine quality, condensed tannins or proanthocyanidins (PAs) play a major role, especially in astringency and color stability of wine. These molecules are also involved in plant defence against biotic and abiotic stresses. Furthermore, the beneficial effects of PAs to human health are well documented. In grapevine, PAs have the distinctive feature of being esterified with gallic acid. An acylation reaction called galloylation is responsible for this modification. Studies show that the galloylation influences oenological and pharmacological properties of PAs. In the grape berry, PAs are synthesized in the early stages of development, mainly in skin and seeds. A relatively small number of enzymatic steps are required for the biosynthesis of the basic structure of these metabolites and the corresponding genes are now widely known in model plants, including in grapevine. However, the molecular mechanisms involved in the final steps, including galloylation, are only partially known. Earlier results obtained after the search of QTL influencing the composition of the grape berry, especially the galloylation ratio of PAs, and transcriptomic studies after overexpression of transcription factors that regulate PAs biosynthesis pathway, have allowed the identification of genes potentially involved in these steps. Shikimate dehydrogenase (SDH) genes were identified. These genes would intervene upstream, for the biosynthesis of gallic acid. Three identified glucosyltransferases, already characterized in the laboratory, are involved in the biosynthesis of glucose ester of gallic acid (β-glucogalline), which could serve as an intermediary for PAs galloylation. These screening methods have also helped to identify 2 serine carboxypeptidase-like acyltransferases, called glucose acyltransferases (GATs) which are capable of catalyzing the last step of galloylation: the transfer of gallic acid from β-glucogalline to PAs. The first objective of this thesis was to determine the function of the SDHs encoded by grapevine genes. Recombinant SDHs, produced heterologously in E. coli, have the capacity to generate gallic acid in vitro. Their level of expression during development and in different tissues of the berry was also established. In vitro results are supported by the metabolic profile (gallic acid, β-glucogallin and PAs) of grapevine hairy -roots transformed with a SDH gene. The second objective of this thesis was to validate the function of the GATs by transient expression in tobacco leaves and in vitro enzyme assays. The transient transformation of grapevine leaves with GATs allowed to modulate the concentration of phenolic esters and notably galloylated flavan-3-ols in planta. The study of these genes was extended to vascular plants by phylogenetic analyses which allowed to identify peptide motifs potentially involved in the studied mechanisms and reflecting the sub-functionalization of certain genes. This work has provided informations on the genetic basis and molecular mechanisms involved in the biosynthesis of gallic acid and its two-step transfer on flavan-3-ols (galloylation). New hypotheses on the intervention of different carriers and nature of transported molecules can be proposed

Donats els problemes de salut associats al sobrepès, en aquesta tesi hem investigat el possible us d'un extracte de proantocianidines de pinyol de raïm (GSPE) com agent saciant, fent servir rates com a model d'experimentació. Hem observat que sota una pauta d'administració adequada, el GSPE disminueix la ingesta tant de manera aguda com de manera continuada, durant períodes de 8 dies consecutius. Aquestes propietats saciants, sumades a un efecte lipolític, resulten en un descens significatiu del pes corporal. A l'investigar les vies de senyalització implicades, hem observat que l'administració de GSPE modifica la producció i secreció de diverses hormones gastrointestinals que afecten l'apetit, entre les que destaquen el GLP-1, d'efectes saciants, i la ghrelina, inductora de l'apetit. En estudis amb antagonistes hem observat que de manera aguda l'administració de GSPE augmenta la concentració plasmàtica de GLP-1, i que l'efecte saciant del GSPE i d'un dels seus compostos, l'àcid gàlic, són directament mediats pel receptor de GLP-1. En estudis de 8 dies consecutius hem observat que els efectes saciants de l'àcid gàlic no es mantenen al llarg del temps, reforçant la importància d'altres compostos de l'extracte per a mantenir un efecte continuat. En aquests estudis subcrònics, l'administració de GSPE comporta un gran descens en la síntesi de ghrelina, un fet que hem observat estretament relacionat amb l'increment de senyalització de GLP-1 a l'hipotàlem, la inducció de la sacietat i l'efecte lipolític del GSPE. Esperem aquests estudis permetin iniciar estudis per a l'aplicació del GSPE en humans.
Dados los problemas de salud asociados al sobrepeso, en esta tesis hemos investigado el posible uso de un extracto de proantocianidinas de pepita de uva (GSPE) como agente saciante, utilizando ratas como modelo de experimentación. Hemos observado que bajo una pauta de administración adecuada, el GSPE disminuye la ingesta tanto de manera aguda como de forma continuada, durante períodos de 8 días consecutivos. Estas propiedades saciantes, sumadas a un efecto lipolítico, resultan en un descenso significativo del peso corporal. Al investigar las vías de señalización implicadas, hemos observado que la administración de GSPE modifica la producción y secreción de varias hormonas gastrointestinales que afectan el apetito, entre las que destacan el GLP-1, de efectos saciantes, y la ghrelina , inductora del apetito. En estudios con antagonistas hemos observado que de manera aguda la administración de GSPE aumenta la concentración plasmática de GLP-1, y que el efecto saciante del GSPE y de uno de sus compuestos, el ácido gálico, son directamente mediados por el receptor de GLP-1. En estudios de 8 días consecutivos hemos observado que los efectos saciantes del ácido gálico no se mantienen a lo largo del tiempo, reforzando la importancia de otros compuestos del extracto para mantener un efecto continuado. En estos estudios subcrónicos, la administración de GSPE conlleva un gran descenso en la síntesis de ghrelina, un hecho que hemos observado estrechamente relacionado con el incremento de señalización de GLP-1 en el hipotálamo, la inducción de la saciedad y el efecto lipolítico del GSPE. Esperamos estos estudios permitan iniciar estudios para la aplicación del GSPE en humanos.
Given the health problems associated with overweight, in this thesis we have investigated the possible use of a grape seed proanthocyanidin extract (GSPE) as a satiating agent, using rats as an experimental model. We have observed that under an adequate administration pattern, GSPE decreases intake both acutely and continuously along periods of 8 consecutive days. These satiating properties, added to a lipolytic effect, result in a significant decrease in body weight. In investigating the signaling pathways involved, we have observed that the administration of GSPE modifies the production and secretion of several gastrointestinal hormones that affect appetite, including GLP-1, with satiating effects, and the appetite-inducing hormone ghrelin. In studies with antagonists we have observed that the administration of GSPE increases the plasma concentration of GLP-1 and that the satiating effect of GSPE and one of its compounds, gallic acid, is directly mediated by the GLP-1 receptor. In studies of 8 consecutive days we have observed that the satiating effects of gallic acid are not maintained over time, reinforcing the importance of other compounds in the extract to maintain a continued effect. In these subchronic studies, GSPE administration leads to a large decrease in ghrelin synthesis, a fact that we have observed closely related to the increase in GLP-1 signaling in the hypothalamus, the satiety induction and the lipolytic effect of GSPE . We hope these studies will allow translational studies for the application of GSPE in humans.

O estresse oxidativo causado pela hiperglicemia desempenha um importante papel no desenvolvimento e progressão do diabetes mellitus (DM) e suas complicações, como a cicatrização deficiente de feridas. Nós avaliamos os efeitos da administração tópica de nanopartículas lipídicas sólidas (NLS) contendo extrato de semente de uva sobre a cicatrização de feridas cutâneas de camundongos controles e diabéticos. Os resultados obtidos na caracterização do nosso modelo experimental mostraram um atraso no fechamento da ferida dos animais diabéticos, acompanhado de uma resposta inflamatória atrasada, mas persistente, bem como um grau significativo de peroxidação lipídica na derme dos animais diabéticos. O tratamento tópico com gel, com a NLS branca e com a NLS contendo extrato de semente de uva foram capazes de recuperar o atraso na cicatrização da ferida diabética e atenuar a peroxidação lipídica, de forma mais acentuada com a aplicação da NLS branca e com a NLS associada ao extrato de semente de uva, comprovando a suas propriedades antioxidantes.
Oxidative stress caused by hyperglycemia plays an important role in the development and progression of diabetes mellitus (DM) and its complications, such as impaired wound healing. We evaluated the effects of topical administration of solid lipid nanoparticles (SLN) containing grape seed extract on skin wound healing in control and diabetic mice. The results showed a retarded wound closure in diabetic animals, accompanied by a delayed but persistent inflammatory reaction, as well as a significant degree of lipid peroxidation in the dermis. Topical treatment with gel (vehicle), white NLS and NLS associated with grape seed extract were able to efficiently accelerate wound closure and attenuate lipid peroxidation, especially with the application of white NLS and NLS containing Vitaflavan®, proving its antioxidant properties.

Thesis (MSc)--University of Stellenbosch, 2011.
ENGLISH ABSTRACT: High molar mass tannin extracts are complex mixtures which are distributed in both molar mass and chemical composition. Condensed tannins from quebracho and mimosa and hydrolysable tannins of tara, chestnut wood and turkey gall were studied. Application of a single analytical technique is not sufficient to elucidate the complete structures present in the extracts. 13C Nuclear Magnetic Resonance (NMR) spectroscopy and Matrix Assisted Laser Desorption/Ionisation Time-of-Flight (MALDI-TOF) mass spectrometry were applied in order to determine the chemical composition and molar mass, respectively. A new mass spectrometric method that can uniquely determine the oligomer microstructure was developed using Collision Induced Dissociation (CID) experiments. Bulk analysis only showed the average composition of the extracts, in order to obtain specific information on the molar mass and chemical composition distributions. Hydrophilic Interaction Liquid Chromatography (HILIC) was used for analysis of the condensed tannins and for the hydrolysable tannins Normal Phase Liquid Chromatography (NP-LC) was utilised. The HILIC separation was up-scaled and the fractions were collected and analysed by MALDI-TOF, and this coupling revealed that separation occurs by molar and chemical composition. For separation of the molecules only by size, Size Exclusion Chromatography (SEC) analyses were carried out; this allowed for relative comparison of the tannin molecules. In conclusion, for characterisation of high molar mass tannins a multi-dimensional approach was necessary since the various distributions present in these extracts are superimposed.
AFRIKAANSE OPSOMMING: Hoë molekulêre massa tannienekstrakte is komplekse mengsels, in terme van beide molekulêre massa en chemiese samestelling. Gekondenseerde tanniene vanaf quebracho en mimosa, en hidroliseerbare tanniene vanaf tara, kastaaiinghout en Turksegal is bestudeer. Die gebruik van ‘n enkele analitiese tegniek is nie voldoende om die volledige struktuur van komponente teenwoordig in die ekstrakte te analiseer nie. 13C KMR-spektroskopie en MALDI-TOF-massaspektroskopie is gebruik om die chemiese samestelling en molekulêre massa, onderskeidelik, te bepaal. ‘n Nuwe metode is ontwikkel vir die bepaling van die oligomeer-mikrostruktuur deur gebruik te maak van botsings-geïnduseerde dissosiasie eksperimente. Grootmaat analise het net die gemiddelde samestelling van die ekstrak bepaal. Hidrofiliese-interaksie-vloeistofchromatografie (HILIC) is gebruik vir die analise van gekondenseerde tanniene en gewone fase-vloeistofchromatografie is gebruik vir die hidroliseerbare tanniene. Die HILIC-skeiding is op groter skaal uitgevoer en die fraksies is versamel en gebruik vir MALDI-TOF analise. Hierdie koppeling het getoon dat skeiding plaasvind op grond van molekulêre massa en chemiese samestelling. Grootte-uitsluitingschromatografie is gebruik vir die skeiding van molekules alleenlik op grootte. Hierdeur kon ‘n relatiewe vergelyking van die tannienmolekules gemaak word. Vir die karakterisering van hoë molekulêre massa tanniene is ‘n multi-dimensionele benadering nodig aangesien die verskeie verspreidings teenwoording in hierdie ekstrakte supergeponeerd is.

Recent studies have demonstrated that peanut and grape processing by-products may be richer sources of bioactive compounds as compared to their original raw material and feedstock; however, before their application as a source of nutraceuticals or in the prevention of lipid oxidation in food systems, certain technological challenges have to be addressed. This study discusses recent advances in the application of plant food processing by-products as sources of phenolic compounds with special emphasis on the profiling and screening of phenolics using high-performance liquid chromatography-mass spectrometry, their potential health benefits, and microbiologial safety. The major findings are summarized in chapters 2, 3, and 4. The first chapter deals with phenolics from grape by-products. In general, insoluble-bound phenolics were more effective in inhibiting copper-induced human LDL-cholesterol oxidation in vitro than free and esterified phenolics. Phenolic extracts from all fractions inhibited peroxyl radical-induced DNA strand breakage. The third chapter brings about the effects of gamma-irradiation on the microbial growth, phenolic composition, and antioxidant properties of peanut skin. Gamma-irradiation at 5.0 kGy decreased the microbiological count of the product. Total phenolic and proanthocyanidin contents, ABTS radical cation, DPPH radical, hydrogen peroxide, and hydroxyl radical scavenging capacities as well as the reducing power of the sample were increased upon gamma-irradiation in both the free and insoluble-bound phenolic fractions. The bioactivity of the free phenolics against in vitro human LDL-cholesterol oxidation and copper induced DNA strand breakage was improved upon gamma-irradiation. Phenolic compounds were positively or tentatively identified and their distribution was in the decreasing order of free > esterified > insoluble-bound forms. Procyanidin dimer A was increased in all phenolic fractions, whereas procyanidin dimer B decreased. Gamma-irradiation induced changes may be explained by molecular conversion, depolymerization, and cross-linking. In the fourth chapter, the ability of selected enzymes in improving the extraction of insoluble-bound phenolics from the starting material (experiment I) or the residues containing insoluble-bound phenolics (experiment II) were evaluated. Pronase and Viscozyme improved the extraction of insoluble-bound phenolics. Viscozyme released higher amounts of gallic acid, catechin, and prodelphinidin dimer A compared to Pronase treatment. Furthermore, p-coumaric and caffeic acids, as well as procyanidin dimer B, were extracted with Viscozyme but not with Pronase treatment. Solubility plays an important role in the bioavailability of phenolic compounds, hence this study may assist in better exploitation of phenolics from winemaking by-products as functional food ingredients or supplements.
Estudos recentes têm demonstrado que subprodutos da indústria processadora de amendoim e uva podem ser mais ricos em compostos bioativos em comparação às suas matérias-primas. No entanto, alguns desafios tecnológicos precisam ser enfrentados antes da sua aplicação como fonte de compostos nutracêuticos ou na prevenção da oxidação lipídica em sistemas alimentares. Este estudo discute os recentes avanços na aplicação de subprodutos da indústria processadora de amendoim e uva como fontes de compostos fenólicos. Especial ênfase foi dada a sua caracterização por cromatografia líquida acoplada à espectrometria de massas, aos potenciais benefícios à saúde e à segurança microbiológica. As principais conclusões estão apresentadas nos capítulos 2, 3 e 4. O primeiro capítulo trata de compostos bioativos de subprodutos da indústria de suco de uva e da produção vinícola. A fração da qual foram extraídos os compostos fenólicos ligados à parede celular foi predominante. Em geral, esta fração também foi a mais eficaz na inibição da oxidação do LDL - colesterol in vitro quando comparada à fração que continha os fenólicos livres e os esterificados. Os compostos fenólicos de todas as frações inibiram o dano oxidativo ao DNA induzido por radicais peroxila. O terceiro capítulo fala sobre os efeitos da irradiação gama sobre a carga microbiana, a composição fenólica e as propriedades antioxidantes da película de amendoim. A irradiação gama (5,0 kGy) diminuiu a contagem microbiana do produto. Os compostos fenólicos totais, o teor de proantocianidinas e a capacidade dos extratos em neutralizar radicais como o ABTS, DPPH e espécies reativas de oxigênio como o peróxido de hidrogênio e radicais hidroxila, assim como o poder redutor da amostra, aumentaram devido à irradiação gama em ambas as frações (contendo fenólicos livres e ligados à parede celular). A bioatividade dos compostos fenólicos livres contra a oxidação do LDL-colesterol in vitro e contra os danos oxidativos ao DNA aumentou com a irradiação gama. Os compostos fenólicos foram positivamente ou tentativamente identificados, distribuindo-se entre: fenólicos livres > esterificados > ligados. Houve aumento na concentração de dímeros de procianidina A em todas as frações, enquanto a concentração de dímeros de procianidina B diminuiu. Essas alterações podem ser explicadas pela conversão molecular, despolimerização e formação de ligações cruzadas. No quarto e último capítulo, enzimas selecionadas foram aplicadas à matéria-prima inicial (experimento I) ou nos resíduos contendo apenas compostos fenólicos insolúveis (experimento II). Pronase e Viscozyme aumentaram a extração de compostos fenólicos insolúveis (ligados à parede celular). Viscozyme liberou maiores quantidades de ácido gálico, catequina e dímero de prodelfinidina A em comparação ao tratamento com Pronase. Além disso, os ácidos p-cumárico e ácido caféico, bem como o dímero de procianidina B, foram extraídos com Viscozyme, mas não com Pronase. A solubilidade desempenha um papel importante na biodisponibilidade de compostos fenólicos. Desta forma, o terceiro estudo oferece uma alternativa para a exploração de compostos fenólicos de subprodutos da indústria vinícola como ingredientes alimentares com propriedades funcionais ou suplementos alimentares.

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O presente trabalho descreve o estudo fitoquímico de Dioclea violacea Mart. além da avaliação das atividades antioxidante e imunomoduladora das substâncias isoladas. Dioclea violacea é uma trepadeira pertencente à família Leguminosae (Fabaceae), subfamília Faboideae (Papilionoideae), tribo Phaseoleae, que ocorre no litoral desde a Guiana até o estado de São Paulo. O caule de um espécime foi coletado no município de Umburanas, Bahia, em área de vegetação de caatinga e relevo de tabuleiro. Após secagem e moagem o material foi submetido à maceração com metanol. O extrato metanólico obtido foi particionado fornecendo os extratos hexânico, clorofórmico e acetato de etila. Os extratos foram submetidos a purificação separadamente, sendo seus constituintes isolados e purificados através de técnicas de CC e CCDP em gel de sílica 60, 60H, 60PF, Poliamida 6 e 11 e permeação em gel de Sephadex LH20. Deste modo, as substâncias isoladas tiveram suas estruturas elucidadas através da análise de dados de RMN de 1H, 13C incluindo experimentos DEPT, ?nOe diff?, além de técnicas bidimensionais como HETCOR, HOMO/COSY, HMQC, HMBC, NOESY, auxiliadas por EMIE, FAB, UV, IV, [a]D25 e CD. Do extrato hexânico foram isolados e identificados a-tocoferol, estigmast-4-en-3-ona, b-sitosterol, estigmasterol, lupeol e b-amirina. Do extrato clorofórmico foram isolados ácido oleanólico, a nova flavanona 7,4?-diidroxi-6-metoxiflavanona, além das 7-hidroxi-6-metoxiflavanona, 5,7-diidroxiflavanona, 5,7-diidroxi-8-metoxiflavanona, 7-hidroxiflavanona, 4?,7-diidroxiflavanona, 7,3?,4?-triidroxiflavanona já anteriormente isoladas de outras fontes. Além disso, foram também isolados deste extrato o 7-hidroxi-6-metoxiflavanonol, 2?,4? diidroxichalcona, 2?,4,4?-triidroxi-3-metoxichalcona, 2?,3,4,4?-tetraidroxichalcona, além da lactona lasiodiplodina e de um novo biflavonóide a,b?-epoxi,-2,2?,4?-triidroxichalcona-(b®4?)-7,4?-diidroxi-6-metoxiflavanona. Enquanto que, do extrato acetato de etila foram isolados a epicatequina, a proantocianidina do tipo A denominada 3?,4?,7-triidroxiflavana-(2b®7,4b®8)-3-prenil-fustina, e outras duas proantocianidinas do tipo A2; epicatequina-(2b®7,4b®8)-epicatequina e epigalocatequina-(2b®7,4b®8)-epicatequina. As substâncias isoladas foram submetidas a testes de atividade antioxidante pelos métodos de inibição da autooxidação do b-caroteno, e seqüestro do radical livre DPPH, sendo que a epicatequina e as proantocianidinas epicatequina-(2b®7,4b®8)-epicatequina e epigalocatequina-(2b®7,4b®8)-epicatequina foram as mais ativas. Algumas das substâncias foram submetidas a testes de atividade imunomoduladora pelos métodos de inibição da proliferação de linfócitos e inibição da síntese de NO, onde a 7 hidroxiflavanona demonstrou maior atividade. A substância mais ativa 7-hidroxiflavanona foi sintetizada a partir da reação de esterificação do ácido cinâmico com o resorcinol em presença de ácido polifosfórico, seguida de rearranjo de Fries e ciclização, porém com baixo rendimento (1,6%). Os produtos principais da reação foram a 7-hidroxi-3,4-diidro-4-fenilcumarina (68,7%) e a 5-hidroxi-3,4-diidro-4-fenilcumarina (11,2%).
Salvador

Le contrôle combinatoire de l’ expression des gènes est une caractéristique importante du profil spatio-temporel de l'accumulation des flavonoïdes chez les plantes. Des résultats précédents avaient démontré chez Arabidopsis thaliana, que la régulation de l’accumulation des anthocyanes et des proanthocyanidines repose sur l'activité de facteurs de régulation de la transcription de type R2R3-MYB et bHLH qui forment des complexes ternaires ("MBW") avec la protéine TTG1 (WDR). L'objectif de la thèse était de caractériser les complexes MBW impliqués et leurs cibles, pour avoir une compréhension globale des mécanismes transcriptionnels qui contrôlent la biosynthèse des flavonoïdes.En utilisant différentes approches génétiques et moléculaires, nous avons montré que seuls les gènes « tardifs » (c’est à dire DFR, LDOX, BAN, TT19, TT12 et AHA10) sont des cibles directes des complexes MBW. Bien que le complexe de régulation impliquant les protéines TT2-TT8-TTG1 ait un rôle majeur dans la régulation de ces gènes structuraux dans la graine d’Arabidopsis, trois autres complexes contenant MYB5, GL3 ou EGL3 sont également impliqués de façon tissu-spécifique. Comme l’expression du gène TT8 joue un rôle clef dans ces régulations, une dissection fonctionnelle de son promoteur a été entreprise. Elle a montré la nature modulaire de ce promoteur avec deux domaines impliqués dans l’expression tissu-spécifique et un troisième dans la force du promoteur. Les résultats obtenus suggèrent également l’existence d’autres régulateurs qui restent à caractériser. Enfin, nous avons développé une nouvelle technique de caractérisation des interactions entre les facteurs de transcription et les promoteurs, basée sur l’expression transitoire dans des protoplastes de mousse (i.e. Physcomitrella patens). Nous avons ainsi pu identifier les éléments cis des promoteurs impliqués dans la régulation de l’expression de TT8 et de chacun des promoteurs cibles des complexes MBW.L’ensemble de ces travaux permet de fournir un modèle plus complet du réseau de régulations transcriptionnelles qui contrôle la biosynthèse des proanthocyanidines et des anthocyanes, ainsi que des outils et de nouvelles pistes pour poursuivre ces études chez Arabidopsis et d’autres plantes
The combinatorial control of gene expression is a key feature of the spatio-temporal pattern of flavonoid accumulation in plants. Previous results have shown that the regulation of anthocyanins and proanthocyanidins (PAs or tannins) pigmentation relies on the transcriptional activity of R2R3-MYB and bHLH proteins that form “MBW” ternary complexes with TTG1 (WD-Repeats), in Arabidopsis thaliana. The purpose of the thesis was to figure out the nature and spatio-temporal activity of these MBW complexes and to identify their direct targets, which were essential steps toward a comprehensive understanding of the transcriptional mechanisms that control flavonoid biosynthesis. Using different molecular and genetic approaches this thesis has demonstrated that only late biosynthetic genes (namely DFR, LDOX, BAN, TT19, TT12 and AHA10) are direct targets of the MBW complexes. Interestingly, although the TT2-TT8-TTG1 complex was shown to play the major role in regulating the expression of these structural genes in developing seeds, three additional MBW complexes that contain MYB5, GL3 or EGL3 are also involved, in a tissue-specific manner. Because the expression of TT8 is largely involved in these regulations, a functional dissection of its promoter was carried out. Two modules drive the tissue-specific activity of the TT8 promoter in PA- and anthocyanin-accumulating cells, and a third module is responsible for the strength of the promoter. Interestingly, this regulation involves at least six different MBW complexes. Our results also suggest that some putative new regulators remain to be discovered. Last, use of a newly developed fast and sensitive transient expression system that relies on protoplasts of the moss Physcomitrella patens has allowed the identification of both, specific cis-regulatory elements through which TT8 expression is regulated and the minimal promoter for each of the genes that are targeted by the MBW complexes.Altogether, by answering fundamental questions and by demonstrating or invalidating previously made hypotheses, we have provided a new and comprehensive view of the regulatory mechanisms controlling PA and anthocyanin biosynthesis in Arabidopsis, as well as new clues and tools for further investigation of this pathway in Arabidopsis and other plant species

En els últims anys ha augmentat la presència de l’obesitat i de malalties associades a aquesta. En vista d’aquest fet, la cerca de teràpies preventives així com tractaments per aquestes malalties esdevé de gran interès per la salut publica. Les hormones intestinals secretades per les cèl•lules enteroendocrines juguen un paper clau en la regulació de la ingesta i la homeòstasis de la glucosa. En aquest marc, la recerca d’aquesta tesi doctoral s’ha centrat en l’acció dels compostos naturals bioactius sobre el sistema enteroendocrí. En estudis previs, el nostre grup de recerca va observar que un extracte de proantocianidines del pinyol del raïm (GSPE) augmentava els nivells plasmàtics de GLP-1 en rates. En aquesta tesi doctoral, s’ha esbrinat que aquest augment podria ser en part explicat per l’acció directa del GSPE sobre les cèl•lules enteroendocrines. D’altra banda, s’ha demostrat que l’acció directa del GSPE també modula la secreció de les principals hormones intestinals, induint un augment de la secreció de GIP i PYY, així com una reducció de la secreció de CCK. Els resultats obtinguts en el cultiu d’organoids han demostrat que el GSPE incrementa els principals marcadors de les cèl•lules L i modula els factors de transcripció involucrats en la diferenciació d’aquestes. Conseqüentment, aquests resultats senyalen que promoure la diferenciació de les cèl•lules L és un mecanisme d’acció del GSPE en tractament prolongats. D’altra banda, la recerca realitzada en tractaments subcrònics ha revelat que el GSPE modifica la composició de la biota intestinal i que el perfil microbià correlaciona amb els paràmetres metabòlics de l’hoste, del qual destaca la correlació amb els nivells plasmàtics incrementats de la hormona GLP-1 activa. Altrament, s’ha trobat una nova font de compostos naturals bioactius, l’hidrolitzat de pota de pollastre, la qual s’ha demostrat que actua com un agent antihiperglicèmic en rates que presenten una homeòstasis de la glucosa alterada, degut a la capacitat d’inhibir l’activitat de DPP-IV i d’augmentar la secreció endògena de GLP-1. En conclusió, els resultats obtinguts en aquesta tesi doctoral mostren que els compostos naturals bioactius actuen en el sistema enteroendocrí, per tant aquests podrien ser un agents terapèutics adequats per al tractament de la obesitat i la diabetes.
En los últimos años ha aumentado la presencia de la obesidad y de enfermedades asociadas a ésta. Ante este hecho, la búsqueda de remedios preventivos, así como de tratamientos para estas enfermedades, se vuelve de gran interés para la salud pública. Las hormonas intestinales secretadas por las células enteroendocrinas juegan un papel en la regulación de la ingesta y la homeostasis de la glucosa. En este contexto, la investigación de esta tesis doctoral se ha centrado en la acción de los compuestos naturales bioactivos sobre el sistema enteroendocrino. En estudios previos, nuestro grupo de investigación observó que un extracto de proantocianidinas de hueso de uva (GSPE) aumentaba los niveles plasmáticos de GLP-1 en ratas. En esta tesis doctoral se ha descubierto que este aumento podría ser explicado en parte por la acción directa del GSPE sobre las células enteroendocrinas. Por otro lado, se ha demostrado que la acción directa del GSPE también modula la secreción de GIP y PYY, así como una reducción de la secreción de CCK. Los resultados obtenidos en el cultivo de organoides han demostrado que el GSPE aumenta los principales marcadores de las células L y modula los factores de transcripción involucrados en la diferenciación de éstas. Consecuentemente, estos resultados señalan que promover la diferenciación de las células L es un mecanismo de acción del GSPE en tratamientos prolongados. Por otro lado, la investigación realizada en tratamientos subcrónicos ha revelado que el GSPE modifica la composición de la microbiota intestinal y que el perfil microbiano correlaciona con los parámetros metabólicos del huésped, del cual destaca la correlación con los niveles plasmáticos aumentados de la hormona GLP-1 activa. Además, se ha encontrado una nueva fuente de compuestos naturales bioactivos, el hidrolizado de pata de pollo, el cual se ha demostrado que actúa como un agente antihiperglicémico en ratas que presentan una homeóstasis de la glucosa alterada, debido a la capacidad de inhibir la actividad de DPP-IV y de aumentar la secreción endógena de GLP-1. En conclusión, los resultados obtenidos en esta tesis doctoral muestran que los compuestos naturales bioactivos actúan en el sistema endocrino, con lo cual éstos podrían ser unos agentes terapéuticos adecuados para el tratamiento de la obesidad y diabetes.
In the last decade, there has been an increasing prevalence of obesity and metabolic-associated diseases. In view of this fact, finding preventive therapies, as well as treatments for these diseases is of great interest for public health. Gut hormones secreted from enteroendocrine cells (EECs) play a key role in the regulation of food intake and glucose homeostasis. In this context, the research of this thesis has focused on the role of natural bioactive compounds on the enteroendocrine system. Our research group reported in previous studies that grape seed proanthocyanidin extract (GSPE) increased GLP-1 plasma levels in rats. In this thesis, we elucidated that such increase might be in part explained by the direct action of GSPE on enteroendocrine cells. Moreover, we demonstrate that GSPE also modulates the secretion of the main gut hormones by directly acting on EECs, inducing an increase of GIP and PYY release, while reducing CCK release. The results obtained in this thesis using organoids culture demonstrated that GSPE up-regulate the main markers of L-cell and modulate transcription factors involved in L-cell differentiation, and thereby point out that the promotion of L-cell differentiation is a mechanism by which GSPE act in prolonged treatments. Moreover, our findings in mid-term treatments revealed that gut microbiota composition is modulated by GSPE and such microbial composition profile correlates with host metabolic parameters, and remarkably with increased active GLP-1 plasma levels. Furthermore, we found a new source of natural bioactive compounds, chicken leg hydrolyzate, and demonstrated that it acts as antihyperglycemic agent in disrupted-glucose homeostasis animals due to the capacity of inhibiting DPP-IV activity and enhancing endogenous GLP-1 release . In conclusion, the findings obtained in this thesis show that natural bioactive compounds act through different mechanisms on the enteroendocrine system, and thereby could be good therapeutic agents to treat obesity and glucose homeostasis disruption.

Ugljenične nanocevi (UNC) imaju sve veću primenu u elektronici, kompjuterskoj i optičkoj industriji, kao i u biomedicini. Dok proizvodnja jednoslojnih UNC nanocevi beleži sve veći rast poslednjih godina, rizik koji nosi izlaganje ovom nanomaterijalu ostaje nerazjašnjen. Oskudni i često kontradiktorni podaci o toksičnim efektima jednoslojnih UNC ukazuju na potrebu za daljim ispitivanjima. U našim istraživanjima ispitivane su promene u ćelijskom odgovoru kao i morfološke promene nakon delovanja jednoslojnih ugljeničnih nanocevi na ćelijskoj liniji humanih fetalnih fibroblasta pluća MRC-5 i ćelijskoj liniji humanog adenokarcinoma pluća A549. U ovoj studiji korišćene su jednoslojne ugljenične nanocevi koje su sadržale rezidualne nečistoće poput gvožđa. Citotoksičnost jednoslojnih UNC (engl. single-walled carbon nanotubes – SWCNT) je ispitivana kolorimetrijskim MTT testom. Tokom 24 i 48h niske koncentracije jednoslojnih ugljeničnih nanocevi (<250 μg/mL) pokazale su nisku toksičnost na proliferaciju i vijabilnost u obe ispitivane ćelijske linije. Ipak, pri visokim koncentracijama UNC (250-750 μg/mL) antiproliferativni efekat je bio blizu IC50 vrednostima. Na osnovu rezultata dobijenih MTT testom može se zaključiti da su maligne A549 ćelije osetljivije na delovanje jednoslojnih UNC u odnosu na normalne MRC-5 ćelije. Kombinacija ugljeničnih nanocevi sa prirodnim polifenolima (resveratrolom i proantocijanidolima) nije značajno uticala na citotoksičnost u MRC-5 ćelijama, za razliku od A549 ćelija gde je tretman kombinacijama umanjio toksičnost ugljeničnih nanocevi. Transmisionom elektronskom mikroskopijom ispitivan je efekat jednoslojnih ugljeničnih nanocevi na ćelijsku morfologiju i preživljavanje. Intracelularni agregati ugljeničnih nanocevi primećeni su u obe ćelijske linije, čime je potvrđeno da ugljenične nanocevi ulaze u ćelije. Imajući u vidu da nanomaterijali poput ugljeničnih nanocevi indukuju oksidativni stres i njime posredovanu apoptozu, na protočnom citometru je određivano prisustvo ćelija u apoptozi i nekrozi. Tretman ćelija sa jednoslojnim ugljeničnim nanocevima nije doveo do značajnog porasta broja apoptotskih ili nekrotičnih ćelija, što ide u prilog niskoj toksičnosti ovog nanomaterijala, odnosno ukazuje na alternativne mehanizme toksičnosti. Međutim kombinacija jednoslojnih ugljeničnih nanocevi sa antioksidantima, resveratrolom i proantocijanidolima indukuje veći procenat apoptoze i nekroze u odnosu na tretman samo sa nanocevima. Promene u ekspresiji gena praćene su lančanom reakcijom polimeraza (PCR). Komparativna analiza rezultata genske ekspresije MRC-5 i A549ćelija nakon tretmana sa jednoslojnim ugljeničnim nanocevima pojedinačno i u kombinaciji sa antioksidantima ukazala je na kompleksnost i raznolikost biološkog odgovora ispitivanih ćelija. U našem istraživanju ispitivana je i promena aktivnosti enzima antioksidativne zaštite i količine glutationa u ćeliji. Primena jednoslojnih ugljeničnih nanocevi u MRC-5 ćelijama dovodi po smanjenja specifične aktivnosti enzima SOD i GR, povećava specifičnu aktivnost GPx i ne utiče na promenu specifične aktivnosti GST i količine glutationa u ćeliji Primena jednoslojnih ugljeničnih nanocevi u A549 ćelijama dovodi po smanjenja specifične aktivnosti enzima SOD, ne utiče na promenu specifične aktivnost enzima GR, GST i GPx, i dovodi do povećanja količine glutationa u ćeliji. Ćelijska vijabilnost, morfološke promene, redoks homeostaza i ekspresija ispitivanih gena bile su promenjene nakon tretmana sa jednoslojnim ugljeničnim nanocevima. Iako su dobijeni rezultati značajni za procenu toksičnosti ugljeničnih nanocevi, neophodna su dalja istraživanja koja treba da doprinesu boljem razumevanju toksičnih efekata ugljeničnih nanocevi.
Carbon nanotubes are being actively introduced in electronics, computer science, and optics as well as for various biomedical applications. While production of single-walled carbon nanotubes (SWCNT) has escalated in recent years, the knowledge on risk associated with exposure remains unclear. Contradictory data on the toxic effects of single-walled carbon nanotubes highlights the urgent need for further studies. In this study we investigated the alterations in cellular response along with morphological changes induced by single-walled carbon nanotubes in human lung fibroblast cell line MRC-5 and adenocarcinoma human alveolar basal epithelial cells A549. In this study we used SWCNT containing large amounts of residual metallic impurities such is iron, and the iron concentration increased in dose dependent manner in cells exposed to SWCNT. Cytotoxicity was evaluated by MTT assay and SWCNT showed little cytotoxic effect on the proliferation and viability of two cell lines tested at the concentrations used (<250 μg/mL) within 24 and 48h. However exposing both cell lines to high concentrations (250-750 μg/mL) resulted in near IC50 values. Based on MTT test SWCNT were more cytotoxic to A549 cell line. Cytotoxicity of SWCNT in combination with natural polyphenols (resveratrol and proanthocyanidins) did not noticeably affect the cytotoxicity of SWCNT to MRC-5 cells. However introduction of polyphenols did reduce the cytotoxicity of SWCNT to A549 cells. Transmission electron microscopy was used to complement cytotoxicity assays and to investigate the pathological effect of internalized SWCNT on cell morphology and survival. Intracellular bundles of CNTs, possibly aggregated/agglomerated were observed in both cell lines, confirming internalization after 24h exposure. Since nanoparticles like carbon nanotubes are toxic mainly because they cause oxidative stress, often associated with an increased apoptosis we checked for apoptotic and necrotic cells using flow cytometry. Incubation with SWCNT did not result in pronounced apoptosis or necrosis supporting its low toxicity and possibly alternative mechanism of cell damage. However incubation with SWCNT in combination with resveratrol and proanthocyanidins induced higher levels of both apoptosis and necrosis than SWCNT alone. Changes in gene expression following exposure to SWCNT were evaluated by polymerase chain reaction PCR array which indicated complex and diverse change in expression of genes involved in apoptosis, cell proliferation and oxidative stress. Finally we investigated the modulation of the antioxidant enzyme system and the changes in the cytosolic levels of GSH. SWCNT reduced the specific activity of SOD and GR enzymes, increased GPx activity. No changes in intracellular levels of GSH were observed in MRC-5 cell line. Same treatment in A549 cell reduced the specific activity of SOD, had no effect on GR, GST and GPx activity, but increased intracellular levels of GSH. Cell viability, morphologic changes, redox homeostasis and gene expression were affected by the presence of SWCNT. Although our findings are useful in predicting human response against SWCNT exposure, further study is needed for better understanding of the effects of SWCNT.

碩士
中華醫事科技大學
醫學檢驗生物技術系碩士班
100
Adipose tissues are energy store and metabolic tissues. Obesity is a result of an imbalance between energy intake and expenditure. Some medical conditions often accompany obesity, especially diabetes, cardiovascular disorders, and certain cancers. Therefore, study of methods for preventing obesity is necessary for improving human health. It has been shown that pine bark extract represents lipolysis effect, which may be mediated by the major components oligomeric proanthocyanidins (OPC). Natural products containing OPC, kinds of polyphenols, are popular healthy food to serve as a scavenger of free radical and for preventing inflammation, cancers, and cardiovascular diseases. It has been shown that OPC may decrease adipose accumulation through promoting lipolysis and inhibiting lipogenesis. However, its mechanism is still unclear. To address this issue, we used insulin to stimulate pre-adipocyte 3T3-L1 cells and co-treated OPC to study the effects of OPC in lipid accumulation. We also used cell counting for measuring cell viability, Oil red O staining for detecting lipid and addition of 3H-glucose for investigating glucose uptake. We found that treatment of OPC significantly decreased cell viability at day 1 to 3. To study whether this effect through apoptosis, we used DAPI to stain nuclei, and found that OPC-induced cell death was not through apoptosis. Then, we found that addition of insulin could induce differentiation of 3T3-L1 cells. We confirmed this effect by Oil red O staining. To quantify lipid accumulation, we extracted Oil red O by isopropanol, measured the absorbance under OD 510 nm and found that OPC increased lipid accumulation. We found that OPC promoted lipid accumulation in 11 day-insulin treated cells but inhibited this process in 14 day-insulin treated cells. We added 3H-glucose to access glucose uptake and found that OPC did not involve in undifferentiated cells but inhibited glucose uptake in 14 day-insulin treated cells. Our findings in this study did not confirm the results from previous reports totally. Therefore, the effects of OPC in lipid accumulation are remained to be further clarified.

碩士
朝陽科技大學
應用化學系碩士班
92
Proanthocyanidins is a natural antioxidant from grape seeds, which have great potential in medical applications: antibacterial, antineoplastics and cure of heart disease. In this study, NaCl solutions were used for extracting proanthocyanidins from grapeseed meal. Before the extraction of proanthocyanidins, grapeseed was defatted by supercritical CO2 fluid extraction at 40 oC and 230 bar for two hours . To remore the containing 12% oil from grapeseed. Then, defatted grapeseed meal was extracted by NaCl solution and the optimal condition of extraction is 0.2M NaCl solution for fifteen hours. Defatted grapeseed meal contained a small quantity of dimeric procyanidins, which was 33 mg/100g at dry basis. The extracted proanthocyainidins contained 62% monomer, 21% dimmer, 1% trimer, 1% dimmer gallate, and 15% others. Finally, antioxidation testing experiments of proanthocyanidins were performed as following: inhibiting lipid peroxidation , reducing power, and DPPH scaveng ing activity. In the inhibiting lipid peroxidation tests, inhibition of PA extract and dimmer PA were up to 99% at a concentration of 1 mg/mL. In the reducing power tests, dimmer PA (1.25) was better than PA extract (1.01) at a concentration of 1 mg/mL. In the analysis of DPPH scavenging activity tests, dimmer PA (96%) was better than PA extract (90%) at a concentration about 1 mg/mL.

Tese de doutoramento em Ciência Alimentar
As características sensoriais dos vinhos bem como a sua estabilidade são factores de grande importância para a sua competitividade e consequentemente para a sua comercialização nos mercados internacionais e nacionais. Uma das operações tecnológicas mais utilizada na elaboração dos vinhos é a colagem proteica, pois influência as características sensoriais (amargor e adstringência) bem como a estabilidade do vinho. A eficiência e a actuação das colas proteicas dependem por um lado da composição em proantocianidinas existentes nos vinhos que tem por base a casta e o processo de vinificação utilizado, por outro das características físico-químicas das diversas colas proteicas. Um dos objectivos do presente trabalho foi procurar conhecer os perfis tânicos de algumas variedades de castas Vitis vinifera bem como dos vinhos monovarietais delas resultantes. Pretendeu-se também caracterizar as principiais colas proteicas comercializadas no mercado português bem como avaliar a sua eficácia em relação às diferentes fracções de proantocianidinas dos vinhos tintos e brancos. Por fim, foram adicionadas as colas proteicas anteriormente caracterizadas a soluções modelo semelhantes ao vinho, em que cada uma das soluções era constituída por proantocianidinas com diferentes graus médios de polimerização com o intuito de aprofundar o conhecimento sobre a influência da estrutura das proantocianidinas, da concentração de proantocianidinas, do pH e da temperatura no processo de colagem. Com este estudo pretende-se disponibilizar informação de suporte à escolha do tipo de cola a usar em função do tipo de produto final a obter, e consequentemente optimizar a operação de colagem. Os resultados mostraram que a quantidade e as características estruturais das proantocianidinas presentes nas grainhas e nas películas são diferentes entre as castas estudadas. Os vinhos monovarietais obtidos a partir dessas castas apresentavam proantocianidinas com um grau médio de polimerização que oscila entre 2,1 e 9,6. Nos vinhos monovarietais obtidos em dois anos consecutivos, verificou-se haver uma variação da concentração em proantocianidinas, no entanto o grau médio de polimerização das proantocianidinas manteve-se inalterado para cada casta. Os vinhos monovarietais analisados após seis meses mostraram uma redução de 39-59 % na quantidade de proantocianidinas e também se constatou uma modificação quanto à distribuição das diferentes proantocianidinas com distintos graus médios de polimerização. Parece ter ocorrido em simultâneo uma polimerização das proantocianidinas com um grau médio de polimerização mais baixo e uma perda das proantocianidinas com um grau médio de polimerização mais elevado. As colas proteicas comerciais caracterizadas mostraram diferentes características físico-químicas, quanto à distribuição da sua massa molecular, ponto isoeléctrico e quanto à densidade de carga de superfície. Essas variações verificam-se não só entre colas de diferentes tipos, como seria de esperar, mas também entre cada tipo de cola. Assim, o caseinato de potássio, a caseína, a albumina de ovo e a ictiocola sólida (obtida da bexiga natatória de peixes) são caracterizados por bandas individualizadas, respectivamente nos 30,0 kDa, na vizinhança dos 43,0 kDa e por várias bandas bem definidas superior a 94,0 kDa, entre 94,0 e 43,0 kDa e nos 20,1 kDa, enquanto que o perfil electroforético das gelatinas e ictiocolas líquidas, de uma gelatina sólida e da ictiocola sólida (obtida da hidrólise da pele dos peixes) são caracterizados por uma polidispersão na distribuição das suas massas moleculares. Em duas outras gelatinas sólidas não foram detectadas bandas entre as massas moleculares de 14,4 a 94,0 kDa. A densidade de carga de superfície também apresenta valores diferentes entre as colas estudadas. Assim, a albumina de ovo, a ictiocola e a gelatina (em soluções a 1%) mostraram densidade de carga de superfície superior quando estas se apresentavam sobre a forma sólida. A adição de colas proteicas com diferentes características físico-químicas a vinhos tintos e brancos mostrou que estas actuam diferenciadamente sobre as fracções de proantocianidinas de diferentes graus médios de polimerização. Foi também mostrado que o decréscimo depende da cola proteica mas também do grau médio de polimerização da fracção de proantocianidina. As duas ictiocolas estudadas decresceram as fracções de grau médio de polimerização 1,5 e de 3,4 do vinho tinto, no entanto a ictiocola obtida da bexiga natatória do peixe reduziu o dobro essas fracções do que a ictiocola caracterizada por uma polidispersão inferior a 20,1 kDa. Os resultados sugerem que a acção das colas depende do tipo de moléculas de proantocianidina com que interage e não tanto se a operação se efectua em vinho tinto ou branco. Assim, a adição de ictiocolas, não induziu uma diminuição notória nas proantocianidinas com um grau médio de polimerização de 3,8 no vinho branco bem como na fracção de proantocianidinas com um grau médio de polimerização de 3,4 no vinho tinto. Por outro lado, foram a caseína e a gelatina caracterizada por uma baixa massa molecular as que mais reduziram a fracção de grau médio de polimerização 1,5 em ambos os vinhos. Após aplicação de colas proteicas as características estruturais das proantocianidinas que permanecem no vinho colado são diferentes das do vinho inicial. Constatou-se um decréscimo do grau médio de polimerização das proantocianidinas induzido pela albumina de ovo na fracção mais polimerizada de 26 % no vinho branco e de 24 % no vinho tinto, e nos ensaios com as outras proteínas foi registrado um decréscimo de 6 a 14 % no vinho tinto e de 3 a 24 % no vinho branco. A intensidade corante bem como as moléculas relacionadas com a cor foram menos influenciadas pela colagem proteica comparativamente às proantocianidinas. Pelo método do CIELab verificou-se que em todos os vinhos tintos colados, a luminosidade (L*) aumentou acentuadamente o que parece estar associado a uma redução dos vermelhos (a*), proporcionado pela redução dos pigmentos. Estes dados estão em concordâncias com os resultados obtidos para as antocianinas monoméricas bem como para os pigmentos totais e poliméricos. No que diz respeito à limpidez, foi constatado, que quanto maior for a densidade de carga de superfície da proteína maior é a capacidade de clarificação do vinho. Foi estabelecida uma correlação linear entre a densidade de carga de superfície total e o decréscimo da turvação. Nos estudos efectuados em soluções modelo, mostrou-se que as ictiocolas e as gelatinas apresentam uma correlação (r=0.52 e r=- 0.49, respectivamente; P< 0,05) estatística significativa entre a percentagem de decréscimo das proantocianidinas e o grau médio de polimerização das fracções de proantocianidinas presentes nas soluções. Foi ainda mostrado que o decréscimo de proantocianidinas era sempre superior à temperatura de 10 ºC do que à temperatura de 20 ºC. Para uma concentração de proantocianidinas superior, verificou-se um maior decréscimo para as fracções de proantocianidinas de grau médio de polimerização mais elevado. Não se verificou influencia do pH, quando se aplicou a ictiocola obtida da bexiga natatória de peixe na fracção de proantocianidinas com um grau médio de polimerização superior e quando se aplicou a ictiocola obtida da hidrólise da pele de peixe na fracção de proantocianidinas com um grau médio de polimerização inferior.
Wine sensory characteristics and stability are of great importance for the wine competitivity and consequently for their commercialization on the national and international market. One of the most required technological process in winemaking is protein fining which influenced wine sensory (bitterness and astringency) and stability. The effect of protein fining depends on the wine proanthocyanidin composition, which is influenced by the grape variety and the wine production process employed as well as on the physic-chemical characteristics of the protein fining agents. The aim of this work was to know the tannic profile of grapes from Vitis vinifera varieties and from their monovarietal wines as well as to characterise commercial protein fining agents. The characterised proteins were added to white and red wine in order to better understand their action on the proanthocyanidin fractions and on the sensory characteristics. The characterised proteins were also added to wine-like model solutions containing each one proanthocyanidin fractions with an identified mean degree of polymerisation to enhance the information of the influence of environmental factors (pH and temperature), proanthocyanidin structural characteristics and concentration on the fining process. With these work we want to improve the knowledge of the protein fining agents and consequently allow the optimisation of the fining process. The results showed that the quantity and the structural characteristics of the proanthocyanidins of grape seeds and skins differed between the V. vinifera L. cv grape varieties studied. On the monovarietal wines obtained from these grape varieties, the mean degree of polymerisation ranged from 2.1 to 9.6. In monovarietal wines obtained from two different vintages was observed that the concentration altered but the mean degree of polymerisation remained unchanged. The monovarietal wines analysed after 6 month showed a reduction of 39-59 % on the amount of proanthocyanidins and the distribution of the diverse proanthocyanidin fractions is different. The different proteins characterised showed distinct physic-chemical characteristics such as molecular weight distribution, isolectric point and surface charge densities. These differences are not only confirmed among the different proteins as it would be accepted, but also in fining agents obtained from the same type of protein The addition of proteins with different physic-chemical characteristics to red and white wines showed that they decrease differently the proanthocyanidin fractions with diverse mean degree of polymerisation. The decrease depends on the fining agent but also on the mean degree of polymerisation of the proanthocyanidin fraction. Therefore, the two isinglasses assayed decreased the proanthocyanidin fractions with mean degree of polymerisation 1.5 and 3.4 from red wine, however isinglass obtained from fish swim bladder decreases these fractions more than the twice as effectively as isinglass obtained from fish skin. The results suggested that the proteins acted in function of the mean degree of polymerisation of the proanthocyanidins independently they come from red or white wine. Any of the isinglass diminished the proanthocyanidins with a mean degree of polymerisation of 3.8 in white wine as well as with 3.4 in red wine. After employ of proteins the structural characteristics of the proanthocyanidin remained in the fined wine were different from that presented on the initial wine. Regarding the mean degree of polymerisation of fined wines, the egg albumin induced a decrease on the mean degree of polymerisation of 24 % in red wine and 26 % in white wine for the more polymerised tannin fraction; although within all assays were observed a decrease ranged from 6 to 14 % in red wine and from 3 to 24 % in white wine. Colour intensity and molecules related to wine colour were shown to be less influenced by proteins than proanthocyanidins. A linear correlation was found between total surface charge density and decrease of turbidity. In wine-like model solution was shown that isinglasses and gelatines presented a statistical significant correlation between the decrease of the percentage of proanthocyanidins and the mean degree of polymerisation of the proanthocyanidin fractions presented in the solution. The proanthocyanidin decrease was always higher at 10 ºC than at 20 ºC. At a higher proanthocyanidin concentration, a greater decrease was shown for the proanthocyanidin fractions with higher mean degree of polymerisation. The pH did not influenced the decrease of proanthocyanidin fractions with higher mean degree of polymerisation after fining with swim bladder isinglass, and the proanthocyanidin fractions with lower mean degree of polymerisation after fining with isinglass obtained from fish skin.

Phytochemicals found in grains complement those found in fruits and vegetables. These phytochemicals, though minor compounds, contribute to the antioxidant properties which are related to the health benefits associated with the consumption of whole grain. In this thesis project, nine barley genotypes of diverse origin namely CI2230 from Nepal, CI1248 from Israel, 3 Peruvian genotypes; Peru 3, Peru 16 and Peru 35, Hokuto Hadaka from Japan, EX116; a cross between Moroccan and Canadian genotype, EX83; a cross between two Canadian genotypes and EX127; a cross between Canadian and German genotypes were studied. The genotypes were categorized based on appearance into purple, black and yellow grains. Phenolic acids and flavonoids were identified and quantified in these diverse genotypes using HPLC-ESI-MS analysis. The main classes of dietary flavonoids studied in the barleys were anthocyanins and flavan-3-ols. Phenolic acids were identified and quantified (p-coumaric, ferulic, sinapic, caffeic, vanillic). Three ferulic acid dehydrodimers (8-0-4’ DFA, 8-5’ benzofuran form and 5-5’ DFA) were also identified. The most abundant dimeric flavan-3-ols were procyanidins B3 and prodelphinidin B3. The monomeric unit, (+)-catechin, was the most abundant while catechin glucoside (m/z 451) was also identified. Among the Peruvian genotypes, Peru 16 and Peru 35 exhibited relatively high levels of total PA content. Total phenolic content and antioxidant activities of methanolic, acetone and alkali hydrolyzed extracts of the nine barley genotypes was determined by the Folin- Ciocalteau assay, DPPH radical scavenging assay and oxygen radical absorbance capacity (ORAC assay).The acetone extract exhibited the highest antioxidant capacity using all the methods of analysis. Furthermore, dark colored grains were found to exhibit higher contents of phenolic compounds. The phenolic acids, PAs and anthocyanins identified and quantified had significant contribution to the overall antioxidant capacity of the barley whole grain. Four hull-less genotypes namely CI2230, EX127, CI1248 and Peru 35 were further partially sprouted to establish the effects of sprouting on phenolic acid composition. Partial sprouting was observed to significantly increase the soluble conjugated phenolic acids. The barley genotypes studied were found to contain different quantities of phytochemicals and had high proanthocyanidin content thereby rendering them as alternative sources of antioxidants. Barley sprouts present a possible novel food ingredient with improved properties such as phenolic acid composition and other benefits such as easier incorporation into food products under development.

Thesis (M.S.)--University of Wisconsin--Madison, 1995.
Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 94-105).