Las procianidinas son compuestos bioactivos presentes en frutas y vegetales. Aunque se conocen los efectos beneficiosos de estos compuestos en la homeostasis de la glucosa, su acción en la funcionalidad de la célula β no es clara. La presente tesis doctoral se ha centrado en describir los efectos de las procianidinas en la síntesis y secreción de insulina. Nuestros resultados muestran la capacidad de las procianidinas de modificar la funcionalidad de la célula β aumentando la relación insulina plasmática/mRNA, aunque la efectividad del tratamiento depende de la situación fisiológica. En situaciones no patológicas, las procianidinas afectan la insulinemia modificando la síntesis, secreción y/o degradación de la insulina. En situaciones de resistencia a la insulina, el tratamiento crónico con procianidinas disminuye la síntesis y secreción de insulina gracias a su acción limitando el acúmulo de lípidos. En cambio, en un modelo más dañado (obesidad genética), las procianidinas ejercen efectos similares pero no son capaces de mejorar la hipersinulinemia. En conclusión, las procianidinas, en las dosis ensayadas, pueden utilizarse únicamente como compuestos bioactivos limitando la disfuncionalidad de la célula β en sus estados iniciales. / Les procianidines són compostos bioactius presents en fruites i vegetals. Tot i que es coneixen els efectes beneficiosos d’aquests compostos en l’homeòstasi de la glucosa, la seva acció en la funcionalitat de la cèl•lulaβ no és clara. La present tesi doctoral s’ha centrat en descriureels efectes de les procianidines en la síntesi i secreció d’insulina. Els nostres resultats mostren la capacitat de les procianidines de modificar la funcionalitat de la cèl•lula β augmentant la relació insulina plasmàtica/mRNA, tot i que l’efectivitat del tractamentdepèn de la situaciófisiològica. En situacions no patològiques, les procianidines afecten la insulinèmia modificant la síntesi, secreciói/o degradació d’insulina. En situacions de resistència a la insulina, el tractamentcrònicamb procianidines disminueix la síntesi i secreció d’insulina gràcies a la seva acció limitant l’acumulació de lípids. En canvi, en un model més danyat (obesitat genètica), les procianidines exerceixen efectes similars però no son capaces de millorar la hiperinsulinèmia. En conclusió, les procianidines, en les dosis assajades, podenutilitzar-seúnicament coma compostos bioactiuslimitant la disfuncionalitat de la cèl•lula β en els seus estats inicials. / Procyanidins are bioactive compounds found in fruits and vegetables widely consumed. It has been reported that procyanidins show some beneficial effects on glucose homeostasis, although their effects on β-cell functionality remain unresolved. This doctoral thesis is focus on describing the effects of procyanidins on insulin synthesis and secretion. Our results showed that procyanidins modify β-cell functionality through increasing the plasma insulin/mRNA ratio, although the effectiveness of the treatment depends on the physiological situation. Under non-pathological situation, procyanidins affected insulinaemia by modifying insulin synthesis, secretion and/or degradation activity. Under insulin-resistance situation, chronic procyanidins administration decreased insulin synthesis and secretion, thanks to its lipid-lowering effect. Otherwise in a more damaged model, Zucker fatty rat, procyanidins treatment is not able to reduce insulin plasma levels although they repress insulin expression. In conclusion, procyanidins could be used as bioactive compound to limit β-cell dysfunctions under high-palatable diets, but at the assayed doses, it is not enough to counteract a strong metabolic disruption.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20565号 / 医博第4250号 / 新制||医||1022(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 川口 義弥, 教授 小泉 昭夫, 教授 岩井 一宏 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Afin d’étudier l’impact de la surmaturation des poires sur les interactions parois-procyanidins, les parois et les procyanidines ont été isolées et caractérisées à partir d’une variété de poire à poiré à deux stade de maturité : mûres et surmatures. Leurs associations ont été quantifiées par la méthode des isothermes de Langmuir et par calorimétrie à titration isotherme.Les poires à poiré sont riches en procyanidines très polymérisées dont les unités constitutives sont principalement constituées de (-)-épicatéchine. Les parois du parenchyme et des cellules parenchymateuses sont principalement constituées de pectines hautement methylées et ramifiées alors que les parois des cellules pierreuses et de l’épiderme sont plutôt lignifiées et riches en hémicelluloses.La surmaturation n’induit pas de modification de la composition et ni de la distribution de la masse molaire des procyanidines. Par contre, la distribution des aggrégats de procyanidines dans les vacuoles change, de dispersée (poires mûres) à adjacente au tonoplaste (poire surmatures). Entre le stade mûr et surmature, les parois de poire perdent des chaînes latérales pectiques, principalement les chaînes d’arabinanes et de galactanes des parois du parenchyme et des cellules parenchymateuses.L'affinité des procyanidines vis-à-vis des parois diminue dans l’ordre suivant : cellules parenchymateuses> parenchyme>cellules pierreuses>épiderme d’après les isothermes de Langmuir. La quantité de procyanidines fixées sur les parois augmente avec la surmaturation surtout pour les parois de cellules pierreuses (et le parenchyme total). La calorimétrie à titration isotherme montre que la surmaturation induit également une modification du mécanisme de l’association parois-procyanidines en modifiant la balance hydrophile hydrophobe pour les cellules parenchymateuses.L'augmentation de la capacité d’adsorption des parois vis-à-vis des procyanidines à la surmaturation a été confirmée par des pressages de jus. Les jus de poires mûres sont plus riches en procyanidines que le jus extraits à partir poires surmatures alors que la concentration en procyanidines dans les fruits n’a pas changé. / In order to study overripening impact on cell wall–procyanidin interactions, perry pear cell walls and procyanidins were isolated and characterized at ripe and overripe stage. Their associations were quantified using Langmuir isotherms and isothermal titration calorimetry.Perry pears were rich in procyanidins with high degree of polymerization and their constitutive units were mainly (-)-epicatechin. Cell walls isolated from the whole flesh and parenchyma cells were characterized by extremely methylated pectin and highly branched rhamnogalacturonans whereas cell walls from stone cells and skin were more lignified and rich in hemicelluloses.Overripening did not modify procyanidin structure and molar mass distribution, but the distribution of the procyanidin-containg aggregates in the vacuoles was modified, from dispersed in ripe pears to close to the tonoplast in overripe pears. Between the ripe and overripe stage, pear cell walls lost pectic side chains, arabinans and galactan, mostly from cell walls isolated from parenchyma cells and the whole flesh.The affinity between procyanidins and cell walls decreased as follows: parenchyma cells > flesh > stone cells > skin as reported by Langmuir isotherms. The amount of bound procyanidins increased at the overripe stage notably for the cell walls from stone cells and whole flesh. Isothermal titration calorimetry indicated that overripening induced the modification of cell wall binding mechanism especially for parenchyma cells where interactions were driven by hydrophobic interactions.The increase of procyanidin binding capacity at the overripe stage was confirmed by juice pressing where overripe pear juices were poorer in procyanidins than ripe pear juices whereas fruit procyanidin contents did not change with overripening.

Cocoa beans (Theobroma cacao) are a highly concentrated source of dietary flavanols- bioactive compounds associated with the health protective properties of cocoa. Cocoa beans undergo processing steps, such as fermentation, roasting, winnowing, grinding, pressing, etc., to produce a final product with specific desirable sensory attributes. It is well established that these processing steps, specifically fermentation and roasting, result in dramatic degradation of cocoa's native flavanols, but it is possible that these processing steps may generate compounds with novel activities, potentially preserving or enhancing bioactivity. Raw unfermented cocoa beans were processed by way of a partial factorial approach to produce cocoa powders from the same batch of raw beans using various combinations of fermentation [unfermented, cool fermented (maximum 46°C), hot fermented (maximum 60°C))] and roasting [unroasted, cool roasted (120°C), hot roasted (170°C)]. To simulate cocoa fermentation in a highly controlled environment, a pilot-scale fermentation model system was employed to eliminate many external unknowns and ensure that the differences between our cocoa powders were due to our various treatments, rather than unknown factors occurring during fermentation and roasting. Low and high molecular weight fractions (8-10 kDa cutoff) were produced from cocoa powder extracts (CPE) of each treatment to quantify Maillard reaction products (MRP). A HILIC-UPLC MS/MS method was developed to more efficiently and sensitively quantify cocoa flavanols with high degrees of polymerization (DP) produced during processing. Overall, cocoa processing significantly (p<0.05) decreased the total phenolic and total flavanol concentrations of CPEs. Hot roasting had the greatest impact on native flavanol degradation yet produced CPEs with the highest mean degree of polymerization (mDP). All CPEs dose-dependently inhibited α-glucosidase enzyme activity, with cool fermented/cool roasted cocoa powder exhibiting the best inhibition (IC50 of 62.2 µg/mL). Increasing flavanol mDP was correlated with decreasing IC50 values, suggesting that the complex flavanols produced during processing enhance cocoa's bioactivity (or their production is associated with other products that enhance bioactivity). Alternatively, high molecular weight CPE fractions were correlated with increasing IC50 values, suggesting that MRPs interfere with enzyme inhibition or are associated with other products (polyphenols, macronutrients, etc.) that interfere with enzyme inhibition. Overall, the data presented within this work indicate that the components of processed cocoa powders are promising inhibitors of α-glucosidase, despite a significant reduction in native flavanol composition induced by processing, and moreover that fermentation and roasting conditions can positively influence the bioactivity of cocoa despite losses of native flavanols. / Master of Science in Life Sciences / According to the Centers for Disease Control and Prevention, obesity-related chronic conditions such as cardiovascular disease and type 2 diabetes mellitus (T2D) are the leading cause of preventable and/or premature death, with 51% of the American population predicted to be obese by 2030. Cocoa (Theobroma cacao) is a highly concentrated source of polyphenols, and these compounds have been shown to interact with and inhibit digestive enzymes responsible for carbohydrate breakdown. By inhibiting the activity of these digestive enzymes, it is possible to slow down carbohydrate absorption after a meal and ultimately reduce large spikes in blood glucose levels, being a promising strategy in the prevention and maintenance of T2D. Cocoa beans undergo processing steps to produce a final product, such as cocoa powder, and it is known that these processing steps reduce the levels of beneficial polyphenols. Yet, how this processing-induced degradation effects the health protective activities of cocoa is still widely unknown and is the focus of this work. Through highly controlled cocoa bean processing, cocoa powders of different processing conditions were produced and used to assess how various processing parameters impacted digestive enzyme activity. Overall, processing steps did reduce levels of native polyphenols. However, these losses did not demonstrate a reduction in enzyme inhibition and certain processing conditions actually enhanced digestive enzyme inhibition. This research shows promise for the potential use of processed cocoa powder as an effective strategy in the prevention and maintenance of T2D and further work must be done to understand the mechanisms behind this relationship.

Fermentation and roasting of cocoa (Theobroma cacao) decrease levels of polyphenolic flavanol compounds. However, it is largely unknown how these changes in polyphenol levels caused by processing affect cocoa's anti-diabetic and anti-obesity bioactivities, such as inhibition of certain enzymes in the body. Polyphenol profiles, protein-binding abilities, presence of compounds termed oxidative polymers, and abilities to inhibit α-glucosidase, pancreatic α-amylase, lipase, and dipeptidyl peptidase-IV (DPP4) in vitro were compared between unfermented bean (UB), fermented bean (FB), unfermented liquor (UL), and fermented liquor (FL) cocoa extracts. Overall, there were significant decreases (p<0.05) in total polyphenols, flavanols, and anthocyanins between the two sets of unfermented and fermented cocoa extracts (CEs). All CEs effectively inhibited α-glucosidase (lowest IC50 = 90.0 ug/mL for UL) and moderately inhibited α-amylase (lowest IC50=183 ug/mL for FL), lipase (lowest IC25=65.5 ug/mL for FB), and DPP4 (lowest IC25=1585 ug/mL for FB) in dose-dependent manners. Fermentation and roasting of the samples affected inhibition of each enzyme differently (both processes enhanced α-amylase inhibition). Improved α-glucosidase and α-amylase inhibitions were correlated with presence of different classifications of oxidative polymers, suggesting that these compounds could be contributing to the bioactivities observed. Some α-glucosidase inhibition might be due to non-specific protein-binding. Improved DPP4 inhibition was strongly correlated to increased CE degree of polymerization. In conclusion, potential enzyme inhibition activities of cocoa were not necessarily negatively affected by the large polyphenol losses that occur during fermentation and roasting. Additionally, it is possible that more complex compounds could be present in cocoa that contribute to its potential anti-diabetic and anti-obesity bioactivities. / Master of Science in Life Sciences

Prediabetes is a condition affecting 35% of US adults and about 50% of US adults age 65+. Foods rich in polyphenols, including flavanols and other flavonoids, have been studied for their putative beneficial effects on many different health conditions including type 2 diabetes mellitus and prediabetes. Studies have shown that some flavanols increase glucagon-like peptide 1 (GLP- 1) levels. GLP-1 is a feeding hormone that increases insulin secretion after carbohydrate consumption and increased GLP-1 levels may be responsible for some of the beneficial effects on glycemic control after flavanol consumption. The present study explored the effects of grape powder consumption on metrics of glycemic health in normoglycemic and prediabetic C57BL/6J mice; additionally, the mechanism of action of grape powder was investigated. Grape powder significantly reduced (p<0.01) blood glucose levels following oral glucose gavage after GLP-1 receptor antagonism by exendin-3 (9-39) compared to sugar-matched control; indicating that it was able to attenuate the hyperglycemic effects of GLP-1 receptor antagonism. Grape powder was employed in acute (1.6 g grape powder/kg bodyweight) and long-term high fat diet (grape powder incorporated into treatment diets at 5% w/w) feeding studies in normoglycemic and prediabetic (diet-induced obesity) mice; grape powder did not improve glycemic control in these studies versus sugar-matched control. The mechanisms by which grape powder ameliorates the deleterious effects of GLP-1 receptor antagonism warrants further study. / Master of Science in Life Sciences

Flavanols are polyphenols that are known to have many beneficial effects on the body. Cocoa is a major source of these flavanols. However, research on the potential anti-obesity and anti-diabetic health benefits of cocoa flavanols is lacking in the literature. Furthermore, the effect that the size of these flavanols has on the extent of its beneficial properties is also unclear. The objective of this study was to evaluate the dietary effects of cocoa flavanols on the onset of obesity, insulin resistance and impaired glucose tolerance and to determine the impact that the size of these compounds has on the magnitude of this effect. Cocoa extract was fractionated into a monomer-, an oligomer-, and a polymer-rich fraction. Six groups (n=9) of C57BL/6J mice were fed either a control low-fat diet, a control high-fat diet, or a high-fat diet supplemented with 25 mg/kg*BW of cocoa extract or one of the three cocoa fractions. After 12 weeks on these diets, the oligomer-rich fraction proved to be most effective in preventing weight gain, fat mass accumulation, elevated fasting blood glucose and impaired glucose tolerance in diet-induced obese mice. This is the first long-term feeding study to examine the relative activities of cocoa constituents on diet-induced obesity and insulin resistance. / Master of Science in Life Sciences

No description available.

Chronic conditions such as obesity, inflammatory bowel disease (IBD), and colitis are associated with gastrointestinal (GI) inflammation and compromised GI barrier integrity. Cocoa may be a potential dietary strategy to mitigate gut-related conditions and been shown to elicit anti-inflammatory, antioxidant, and prebiotic effects. Alkali treatment of cocoa was once thought to reduce its bioactivity, but new evidence suggests it may enhance cocoa's health properties, through the formation of new, potentially bioactive high molecular weight compounds. Inulin, a fructose-containing plant polymer, exerts prebiotic effects and has also been investigated in the mitigation of IBD. This study aims to 1) investigate effects of alkali processing on gut health related bioactivity and phytochemical composition of cocoa and 2) evaluate potential additive benefits of combining cocoa and inulin. Polyphenolic and flavanol compounds in natural cocoa, alkalized cocoa, and inulin powders were characterized using Folin-Ciocalteu (total polyphenols) and 4-dimethylaminocinnamaldehyde (total flavanols) assays, thiolysis , and HILIC UPLC-MS/MS. Treatments of cocoa and inulin were made in 1:2 cocoa:inulin and 1:4 cocoa:inulin mixtures for both natural and alkalized cocoas. Cocoa mixtures, in addition to both cocoa powders and inulin alone, were subjected to an in-vitro digestion to generate material for an in-vitro fecal fermentation. Samples collected from the fermentation at 0, 6, 12, and 24 hours were analyzed via HPLC-MS for microbial metabolites, applied to HT-29 colon cancer cells to assess anti-inflammatory activity, and applied to a florescence assay measuring PLA2 inhibitory activity. The alkalized cocoa powder was found to have a significantly lower concentration of total polyphenols and total flavanols, as well as a lower mDP, suggesting that alkalization may affect larger procyanidins more than smaller flavanol compounds. Inulin enhanced the inhibition of the PLA2 enzyme and enhanced the IL-8 anti-inflammatory properties of cocoa, although the trends were weak. Overall, we did not see any clear, significant effects of alkalization or the addition of inulin to cocoa's colonic metabolite formation or its gut bioactivity in vitro. However, we have demonstrated that colonic fermentation of cocoa may have a negative effect on its bioactivity in vitro. Future research should further explore flavanol DP and bioactivity, fiber's interaction with polyphenols, colonic metabolism of cocoa, and cocoa's gut health effects in vivo. / Master of Science in Life Sciences / Gut conditions like obesity-associated inflammation and inflammatory bowel disease are highly prevalent, debilitating, and currently have no cure. Cocoa has been investigated as a possible dietary strategy for the mitigation and prevention of chronic inflammatory gut conditions due to its anti-inflammatory and enzyme inhibiting properties. Most attribute these effects of cocoa to its abundance of compounds called polyphenols. It is widely thought that the ability of cocoa to promote health is lost when cocoa beans are processed, because of the loss of polyphenols. Alkalization, or "Dutching", is an optional step in cocoa processing that some manufacturers perform to enhance flavor and color formation. Dutching cocoa can promote the polymerization of many smaller, flavanol, protein, and other compounds into larger, indigestible compounds. These indigestible compounds will not be absorbed in the small intestine and may be broken down in the large intestine by colonic bacteria, forming new metabolites. We obtained cocoa powders, one natural (not alkalized) and one alkalized and compared them in terms of content of polyphenols, bioactivities, and anti-inflammatory abilities. Additionally, we added a known prebiotic, inulin, to our cocoa formulations to determine if there are additive benefits of cocoa and inulin together. Ultimately, we found that alkalized cocoa contained lower concentrations of all polyphenolic compounds, even the larger compounds. Inulin enhanced the inhibition of digestive enzymes and the anti-inflammatory properties of cocoa, though not significantly. Inulin also reduced the pH (i.e. increased the acidity) of a simulated gut environment, which may be beneficial. Alkalization did not significantly affect cocoa's enzyme inhibitory activity or anti-inflammatory activity. Overall, the addition of inulin to cocoa does not seem to be effective in increasing cocoa's ability to treat and prevent gut diseases, but more information is needed.

L’objectif de cette thèse est d’étudier l’influence des procyanidines, du sucre, de l’acide, de l’éthanol, des arômes et du CO2 sur le goût sucré, l’acidité, l’amertume et l’astringence du cidre. Des plans d’expériences ont été réalisés en solutions modèles et avec des cidres commerciaux afin d’étudier les effets directs des composés chimiques mais également les interactions entre eux sur la perception de ces quatre attributs sensoriels.Les résultats confirment le rôle déterminant des procyanidines,du sucre, de l’acide, de l’éthanol sur les quatre caractéristiques organoleptiques étudiées. Il faut souligner l’influence de la concentration et du degré de polymérisation des procyanidines qui modifient les perceptions sucrées et acides. Ces travaux montrent également une sur expression de l’amertume pour les procyanidines tétramères et pentamères mais seulement pour des concentrations de 750 mg/L, et une interaction entre la concentration et le degré de polymérisation pour l’astringence.Excepté pour l’amertume, la présence de gaz carbonique modifie la perception sensorielle des échantillons testés :l’astringence augmente, le goût sucré diminue pour les solutions les plus sucrées et finalement, il semble que les dégustateurs aient plus de mal percevoir les différences d’acidité entre les échantillons. Enfin, les arômes peuvent modifier la perception du goût sucré de certains cidres par un effet de congruence. En effet, la présence de notes aromatiques « fruitées » est accompagnée d’une surévaluation du goût sucré et des notes « terreux-foin »conduisent à une diminution du goût sucré perçu. Cependant,ce phénomène de congruence semble être concentration dépendante. / The aim of this thesis is to study the influence of procyanidins, sugar, acid, ethanol, CO2 and aroma on the perception of sweetness, sourness, bitterness and astringency of cider. Experimental designs were carried out with model solutions or commercial ciders to study the direct effects and interactions of the chemical compounds on these four organoleptic characteristics.The results confirm the key role of procyanidins, sugar,acid, ethanol on the four organoleptic characteristics. We notably underline the influence of procyanidins concentration and polymerization degree on sweetness and sourness when literature only focused on bitterness and astringency. Our works also indicate an overexpression of bitterness for tetrameric and pentameric procyanidins but only for concentration of 750 mg/L, and an interaction between concentration and polymerization degree for astringency.Except for bitterness, adding CO2 modify sensorycharacteristics of the samples: astringency increases,sweetness decreases for the sweetest solutions and it seems that tasters have more difficulty perceive acidity differences between samples. Finally, the aromatic characteristics can modify sweetness perception of some ciders due to acongruency effect. Indeed, “fruity” notes generate an over evaluation of sweetness when “hay-earthy”aromas lead to a decrease of this taste. Nevertheless,this phenomenon appears to be concentration dependent.