Das Metabolische Syndrom stellt eine Kombination verschiedener metabolischer Anomalien in einem Individuum dar. Starkes Übergewicht gilt als maßgebende Größe in der Genese des Syndroms, welches mit einem enormen Risiko für kardiovaskuläre Erkrankungen einhergeht. Um die stark steigende Prävalenz des Metabolischen Syndroms einzudämmen, sind dringend Konzepte für die Behandlung, vor allem jedoch für die Prävention von Übergewicht erforderlich. Einen wichtigen Beitrag leisten diesbezüglich Ballaststoffe in der Ernährung. Sie tragen auf unterschiedlichen Wegen zur Gewichtskontrolle bei und beeinflussen zudem verschiedene mit dem Metabolischen Syndrom assoziierte Blutparameter. Ebenso werden protektive Effekte von Polyphenolen, welche zur Gruppe der sekundären Pflanzenstoffe zählen, beschrieben. Diese wirken u. a. auf den Glukose- sowie den Insulinhaushalt und greifen darüber hinaus in die Regulation der Fettverbrennung sowie des Energieverbrauches ein. Die Kombination beider Substanzgruppen verspricht bedeutendes gesundheitsförderndes Potential; dieses wurde gegenwärtig jedoch kaum untersucht. Carobballaststoff ist ein polyphenolreicher und vorwiegend unlöslicher Extrakt der Frucht des Johannisbrotbaumes (Ceratonia siliqua L). Bislang publizierte Studien zur physiologischen Wirksamkeit dieses Ballaststoffpräparates weisen sowohl beim Tier als auch beim Menschen bemerkenswerte hypocholesterinämische Eigenschaften nach. Inwiefern sich der Verzehr des Carobballaststoffes ebenso auf die Entwicklung von Übergewicht sowie anderen Messgrößen des Metabolischen Syndroms auswirkt, ist allerdings nicht bekannt. Die Zielstellung der Promotionsarbeit bestand darin, die postprandialen Wirkungen des Carobballaststoffverzehrs mit Hilfe einer Humanstudie aufzuzeigen. In die randomisierten, einfach verblindeten Untersuchungen im cross-over-Design wurden 20 gesunde Erwachsene im Alter zwischen 22 und 62 Jahren eingeschlossen. Unter Verwendung variierender Begleitmahlzeiten wurden die postprandialen Effekte verschiedener Mengen des Carobballaststoffes untersucht. Hierbei standen die Veränderungen der Plasmakonzentrationen von Glukose, Triglyceriden (TG), totalem und acyliertem Ghrelin sowie der Serumkonzentrationen von Insulin und nicht-veresterten Fettsäuren (NEFA) im Mittelpunkt der Betrachtungen. Der Verzehr des Carobballaststoffes in Kombination mit 200 ml Wasser und 50 g Glukose erhöhte die postprandialen Glukose- und Insulinkonzentrationen gegenüber der Glukoselösung ohne Ballaststoffzusatz. In Kombination mit 400 ml einer Flüssigmahlzeit verzehrt, senkte Carobballaststoff die postprandialen TG-, NEFA- und Ghrelin- (acyliert) Antworten. Die Untersuchung des respiratorischen Quotienten nach Zusatz von Carobballaststoff zur Flüssigmahlzeit mittels indirekter Respirationskalorimetrie bekräftigte die bereits bekannten Effekte auf den Lipidmetabolismus und wies zudem eine Steigerung der Fettverwertung unter Verminderung der Glukoseoxidation nach. Wurde Carobballaststoff schließlich in Lebensmittel eingebracht, sanken nach dem Verzehr dieser Lebensmittel erneut die postprandialen Konzentrationen an TG und NEFA. Gleichzeitig erhöhten sich die Glukose-, Insulin- sowie Ghrelin- (acyliert) Antworten. Carobballaststoff löst in Abhängigkeit von der jeweils verzehrten Begleitmatrix unterschiedliche Effekte aus. Das Präparat weist beachtliche Wirkungen auf die Blutlipide sowie den Energieverbrauch auf, hat indes ungünstige Wirkungen auf die Blutglukose, sofern er in Kombination mit einer veränderten Nährstoffmatrix aufgenommen wird. Carobballaststoff besitzt starkes gesundheitsförderndes Potential; jedoch sind weitere Studien notwendig, um seine Wirkungen sowie deren Voraussetzungen besser zu verstehen. Ferner sollten Untersuchungen über einen längeren Zeitraum vorgenommen werden, um die langfristige Relevanz der gewonnenen Ergebnisse darzulegen. Danach stellt die Anreicherung spezieller Lebensmittel mit Carobballaststoff einen geeigneten Weg dar, um von den viel versprechenden protektiven Wirkungen des Präparates zu profitieren. / The metabolic syndrome is a clustering of metabolic abnormalities and cardiovascular risk factors that occur in one individual. Since many individuals suffering of the metabolic syndrome are overweight, dietary treatment should primarily focus on weight reduction and control to handle and, much more importantly, to prevent the genesis of the syndrome. Dietary fiber could play a role in the management of the metabolic syndrome through its ability to control body weight as well as several parameters associated to the syndrome. Beside this, dietary polyphenols have been shown to influence glucose and insulin metabolism. Furthermore, studies in mice and men showed that polyphenols might increase fat oxidation and energy expenditure. The combination of both, dietary fiber and polyphenols, may exert beneficial health effects, which are not known up to now. A polyphenol-rich insoluble dietary fiber preparation from carob pulp (Ceratonia siliqua L; carob fiber) showed significant hypocholesterolemic activity in different animal trails and two human studies indicating that carob fiber may have potent health effects. However, short term effects on parameters associated with the control of the metabolic syndrome are not known. Therefore, a study was conducted to investigate the postprandial effects of carob fiber in healthy humans using different background food matrices. The study was designed as a randomized single-blind cross-over study in 20 subjects, aged 22 to 62 years. During several examinations plasma glucose, triglycerides (TG), total and acylated ghrelin, cholecystokinin (CCK) and peptide YY (PYY) as well as serum insulin and non-esterified fatty acids (NEFA) were repeatedly assessed before and after ingestion of carob fiber in combination with different test meals. The study results showed that carob fiber, consumed within a glucose load, increased plasma glucose and serum insulin compared to control. Plasma PYY increased after consumption of carob-enriched glucose solution. In a second part of the study, postprandial changes were assessed before and after ingestion of an isocaloric standardized liquid meal with or without carob fiber. Carob fiber intake lowered acylated ghrelin, triglycerides, and NEFA compared to control meal. Postprandial energy expenditure was increased and RQ was reduced after liquid meal with carob fiber compared to control meal. Finally, effects of the consumption of carob fiber-enriched foods were investigated at the subsequent day. Enrichment of foods increased plasma glucose and acylated ghrelin responses. Plasma triglycerides and serum NEFA responses were lower after bread ingestion compared to control. These results indicate that the effects of polyphenol-rich carob fiber on different blood parameters depend on the meal composition. Carob fiber showed beneficial effects on blood lipids as well as energy homeostasis, but also a deteriorated glycemic control when administered within a different food matrix. Further studies are needed to clarify observed effects and the mechanisms behind. Additionally, present study shows only short-term effects of carob fiber consumption, which might be less pronounced after long-term consumption due to adaptation mechanisms. Thus, future studies should elucidate the relevance of observed effects in long-term.

Universiẗat, Diss., 2007--Kassel.

Universiẗat, Diss., 2001--Gießen.

München, Techn. Univ., Diss., 2004.

Nahrungsinhaltsstoffe sind im Organismus an Steuerungsprozessen und Stoffwechselvorgängen beteiligt, wobei die Mechanismen ihrer Wirkung noch nicht völlig aufgeklärt sind. Wie Vitamin E zeigen auch sekundäre Pflanzeninhaltsstoffe in Zellsystemen sowie <I>in vivo</I> eine Reihe biologischer Wirkungen, deren Erklärung jedoch häufig auf ihre antioxidative Eigenschaft reduziert wird. Ziel der Dissertation war es, den Einfluss von Vitamin E und anderen Pflanzeninhaltsstoffen (in Form von Pflanzenextrakten oder isolierten sekundären Pflanzeninhaltsstoffen, z.B. Polyphenole), die bisher alle hauptsächlich als Antioxidanz klassifiziert wurden, auf die transkriptionelle Regulation von Phase I- und Phase II-Enzymen zu untersuchen. Dazu wurde die Aktivierung des PXR (pregnane X receptor) und des Nrf2 (NF-E2-related factor-2) als zentrale Transkriptionsfaktoren der Phase I- bzw. Phase II-Enzyme getestet. <br><br> Der Einfluss von verschiedenen Vitamin E-Formen und antioxidativen Pflanzeninhaltsstoffen in Form von Reinsubstanzen (Curcumin, EGCG, Medox, Quercetin, Resveratrol und Sulforaphan) oder Pflanzenextrakten (aus Blaubeeren, Gewürznelken, Himbeeren, Nelkenpfeffer, Thymian oder Walnüssen) auf die Aktivierung von PXR und Nrf2 sowie des Promotors eines jeweiligen Zielgens (CYP3A4 bzw. GI-GPx) wurde <I>in vitro</I> mit Reportergenplasmiden untersucht. Es zeigte sich, dass sowohl Vitamin E-Formen als auch verschiedene sekundäre Pflanzeninhaltsstoffe PXR und/oder Nrf2 sowie die Promotoren der jeweiligen Zielgene CYP3A4 bzw. GI-GPx aktivieren. In einem Tierexperiment konnte diese genregulatorische Wirkung von Vitamin E auf die <I>in vivo</I>-Situation übertragen werden. In Lebern von Mäusen, deren Futter unterschiedliche Mengen von Vitamin E enthielt (Mangel-, Normal- und Überflussdiät), wurde eine direkte Korrelation zwischen der alpha-Tocopherol-Konzentration und der Cyp3a11 mRNA-Expression nachgewiesen (Cyp3a11 ist das murine Homolog zum humanen CYP3A4). Entgegen der <I>in vitro</I>-Situation hatte gamma-Tocotrienol <I>in vivo</I> einen nur kaum nachweisbaren Effekt auf die Expression der Cyp3a11 mRNA, induzierte aber die Expression der alpha-TTP mRNA. Es konnte gezeigt werden, dass Vitamin E und sekundäre Pflanzeninhaltsstoffe Phase I- und Phase II-Enzyme transkriptionell regulieren können. <br><br> Die Wirkungen des Vitamin E können sich allerdings nur entfalten, wenn die Vitamin E-Formen ausreichend vom Körper aufgenommen werden. Gegenstand der Dissertation waren daher auch Untersuchungen zur Bioverfügbarkeit (zelluläre Akkumulation und Metabolismus) verschiedener Vitamin E-Formen. Es konnte gezeigt werden, dass Unterschiede in der chemischen Struktur der Vitamin E-Formen deren zelluläre Akkumulation und Metabolisierung beeinflussen. <br><br> Unter Berücksichtigung der Ergebnisse der Dissertation lassen sich protektive Wirkungen von antioxidativen Nahrungsinhaltsstoffen auch unabhängig von ihren antioxidativen Eigenschaften über die Induktion zelleigener Schutzsysteme, einschließlich der Phase I- und Phase II-Enzyme, erklären. Die Induktion der zelleigenen Abwehr lässt sich auch als adaptive Antwort (sog. "adaptive response") des Organismus gegenüber zellschädigenden Ereignissen betrachten. / In the organism food compounds are involved in regulatory and metabolic processes although the mechanisms of their effects have not been completely elucidated yet. Like vitamin E, secondary plant compounds have diverse biological effects, both in cell systems as well as <I>in vivo</I>. However, the explanation thereof is often reduced to their antioxidative capacity. The aim of this thesis was to investigate the influence of vitamin E and other plant compounds (in form of plant extracts or isolated secondary plant compounds, e.g. polyphenols), which were up to now classified primarily as antioxidants, on the transcription of phase I- and phase II-enzymes. For this, the activation of central transcription factors of the phase I- or phase II enzymes, PXR (pregnane X receptor) and Nrf2 (NF-E2-related factor-2), was tested. <br><br> The influence of different vitamin E forms and antioxidative plant compounds in form of pure substances (curcumin, EGCG, Medox, quercetin, resveratrol, and sulforaphane) or plant extracts (from blueberries, clove, raspberries, allspice, thyme, or walnuts) on the activation of PXR and Nrf2 as well as on the promoter of a respective target gene (CYP3A4 or GI-GPx) was investigated <I>in vitro</I> by reporter gene assays. It appeared that vitamin E forms as well as different secondary plant compounds activate PXR and/or Nrf2 as well as the promoter of the respective target genes CYP3A4 and GI-GPx. The effects of vitamin E were confirmed <I>in vivo</I> by an animal experiment. In livers of mice whose diet contained different amounts of vitamin E (deficient, adequate and supra-nutritional), a direct correlation between alpha-tocopherol content and Cyp3a11 mRNA expression was shown (Cyp3a11 is the murine homolog to the human CYP3A4). In contrast to the <I>in vitro</I> observations, gamma-tocotrienol <I>in vivo</I> only had a small effect on the expression of Cyp3a11 mRNA. However, it induced the expression of alpha-TTP on mRNA level. It could be shown that vitamin E and secondary plant compounds can influence the transcriptional regulation of phase I- and/or phase II-enzymes. <br><br> However, these effects of vitamin E can only be seen if the vitamin E forms are taken up by the body sufficiently. Therefore, another aim of the thesis was to investigate the bioavailability of different vitamin E forms (i.e., cellular accumulation and metabolism). It could be shown that differences in the chemical structure of vitamin E forms influence their cellular accumulation and metabolism. <br><br> Regarding the results of this thesis, protective effects of antioxidative food compounds can be explained independent of their antioxidative properties by the induction of cellular protective systems, including phase I- and phase II-enzymes. The induction of cellular defence mechanism can also be considered as an adaptive response of the organism towards cell-damaging events.

Objectives: In the present in situ/ex vivo study the impact of tannic acid on the erosion-protective properties of the enamel pellicle was tested. Additionally, the antiadherent and antibacterial effects of tannic acid were evaluated. Methods: The pellicle was formed in situ on bovine enamel samples fixed on individual splints worn by 6 subjects. Following 1 min of pellicle formation the volunteers rinsed for 10 min with tannic acid. After further oral exposure for 19 min, 109 min, and 8 h overnight, respectively, slabs were incubated in HCl ex vivo (pH 2.0, 2.3, 3.0) over 120 s. Subsequently, kinetics of calcium and phosphate release were measured photometrically. Samples after a 1-min fluoride mouth rinse as well as enamel samples with and without a 30-min in situ pellicle served as controls. Antiadherent effects were evaluated after a 1-min rinse with tannic acid and oral exposure of the slabs overnight. DAPI (4 ′ ,6-diamidino2-phenylindole) combined with concanavalin A staining and live/dead staining was used for fluorescence microscopic visualization and quantification of adherent bacteria and glucans. Modification of the pellicle’s ultrastructure by tannic acid was evaluated by transmission electron microscopy (TEM). Results: Tannic acid significantly improved the erosion-protective properties of the pellicle in a pH-dependent manner. Bacterial adherence and glucan formation on enamel were significantly reduced after rinses with tannic acid as investigated by fluorescence microscopy. TEM imaging indicated that rinsing with tannic acid yielded a sustainable modification of the pellicle; it was distinctly more electron dense. Conclusion: Tannic acid offers an effective and sustainable approach for the prevention of caries and erosion.

Durch die Freisetzung allelopathisch aktiver Substanzen können Makrophyten das Wachstum von Phytoplankton beeinflussen und damit den Klarwasserzustand von Flachseen stabilisieren. Umweltfaktoren beeinflussen allelopathische Effekte und wurden untersucht, um die Bedeutung allelopathischer Effekte im Ökosystem einzuschätzen. Der Gehalt allelopathisch aktiver polyphenolischer Substanzen zeigte Schwankungen bis zu einer Größenordung im Apikalmeristem von Myriophyllum verticillatum L innerhalb von vier Jahren 2004-2007, die nur teilweise durch den Nährstoffgehalt in der Pflanze erklärt wurden. Der Gehalt an Polyphenolen in der Trockenmasse der Pflanze korrelierte mit der Wachstumshemmung von Anabaena variabilis im Biotest und zeigte Maxima im Mai bis Juli wenn Konkurrenz mit dem Phytoplankton um Licht beim Wachstum zur Wasseroberfläche besteht. Mittels HPLC und MS gelang die Identifizierung von Hexahydroxydiphenoyl di- und -tri-galloylglucose Isomeren in den hauptaktiven Fraktionen des Pflanzenextraktes. Abiotische und biotische Einflüsse der Umwelt auf die Allelochemikalie z.B. photolytische und mikrobielle Umwandlungsprozesse führten bei der Modellsubstanz Tanninsäure (TA), die in Struktur und Funktion den gefundenen Allelochemikalien ähnelt, zur Bildung von refraktären hochmolekularen Verbindungen mit anhaltender allelopathischer Wirkung, die mittels LC-OCD den Huminstoffe zugeordnet wurden. Temperatur als weiterer Umweltfaktor beeinflusste artspezifisch die Reaktion der untersuchten Algen auf TA. Dabei waren die Art und das Ausmaß der Reaktion von der Anwesenheit von Bakterien abhängig. Bakterien der Gattung Pseudomonas wurden isoliert, die in der Lage waren, TA abzubauen und deren allelopathische Effekte zu mindern. Es konnten gezeigt werden, dassl Umwelteinflüsse auf die Allelochemikalie, mutualistische Phytoplankton-Bakterien Interaktionen und die Zusammensetzung der Bakteriengemeinschaft allelopathische Effekte qualitativ als auch quantitativ beeinflussten. / Dissolved organic compounds released by macrophytes can have allelopathic effects on phytoplankton and thereby contribute to stabilize the clear water state of shallow lakes. Identifying factors influencing allelopathy enables evaluating allelopathic effects in the ecosystem.One factor is the temporal dynamic of allelopathically active substances and was investigated as total phenolic compounds(TPC). TPC ranged by an order of magnitude in apicals of Myriophyllum verticillatum L. during four years (2004-2007). Nutrient content partly explained TPC dynamic. The highest amounts of TPC in plant tissue corresponded to maximal growth inhibition of Anabaena variabilis in biotest from May to July when macrophytes compete with algae for light to grow to the water surface. Isomers of Hexahydroxydiphenoyl -di- and -trigalloylglucose identified by HPLC and LC-MS were found in the most allelopathically active fractions in the biotest. By the use of analytical and molecularbiological methods photolytic transformation and degradation by bacteria, changes in mutualistic interaction of bacteria and phytoplankton and shifts in bacterial community composition were identified as factors influencing the allelochemical and the phytoplankton response to TA quantitatively and qualitatively. Photolytic and microbial transformation formed long lasting allelopathically active degradation products of an allelopathic test substance, tannic acid (TA). Temperature was shown to influence the phytoplankton response to TA species specifically varying with presence or absence of bacteria. Bacteria community composition mediated phytoplankton response and specific bacteria as Pseudomonas sp.were able to degrade allelochemicals as TA and thereby lowered the allelopathic effect. Thus, allelopathic effects can be influenced by abiotic and biotic factors acting on the allelochemical, the target organisms and on mutualistic interaction between target organisms altering the outcome of allelopathic effects.

The Maillard reaction is a network of different non-enzymatic reactions between carbonyl groups of reducing sugars and amino groups from amino acids, peptides, or proteins, which progresses in three major stages and originates a very heterogeneous mixture of reaction products. It is also known as non-enzymatic browning, due to the brown macromolecular pigments formed in the final stage of the reaction. The chemistry underlying the Maillard reaction is complex. It encloses not only one reaction pathway, but a whole network of various transformations. As virtually all foods contain both proteins and carbohydrates, Maillard reaction products are present in the daily diet in considerable amounts. The endogenous formation of Maillard reaction products, especially related to ageing and diabetes, aroused intense discussions about the health consequences of the “glycation”, the term that describes the in vivo reaction corresponding to the Maillard reaction in foods. Melanoidins are the final brown products of the Maillard reaction. They are responsible for the color formed during the heat processing of foods like coffee, bread, malt, and beef. Melanoidins are high molecular weight polydisperse polymers containing nitrogen. Their structure is largely unknown. Coffee melanoidins, which are object of the present study, contain thermally transformed polysaccharides, proteins, and phenolic compounds. Since the mechanisms involved on the formation of these macromolecules, and the chemical transformations which take place during the heat treatment are not completely elucidated, key structural features were analyzed. Especially the incorporation of chlorogenic acids in the melanoidin skeleton was object of attention of the present work. Another major aim of this work was to investigate the influence of the Maillard reaction on the inhibitory potential of food components against zinc metalloproteases. The studied enzymes were three human matrix metalloproteases (MMP-1, -2 and -9), which are able to degrade matrix proteins and participate in many physiological processes, including tissue turnover and repair, but also constitute important targets in malignant and degenerative diseases. A microbial collagenase from Chlostridium histolyticum was chosen due to its subtract similarity to MMPs. Furthermore, Angiotensin Converting Enzyme (ACE), which plays a central role in cardiovascular pathologies such as hypertension and cardiac hypertrophy, was investigated. As a prototypical Maillard reaction product, coffee melanoidin was adopted. Due to the roast dependent inhibitory activity of the coffee melanoidin fractions against matrix metalloproteases, the functionalization caused by the non-enzymatic browning was closer investigated. Na-carboxyalkylated derivatives of a sequence of relevant peptides were synthesized, in a variation of the process-induced formation of Nε-carboxymethyllysine, a major advanced glycation end-product (AGE). The inhibitory activity against zinc metalloproteases of the sequence of selected peptides and their Na-carboxymethyl- (CM-) and Na-carboxyethyl- (CE-) derivates was investigated.

Background: A variety of microbial communities exist throughout the human and animal body. Genetics, environmental factors and long-term dietary habit contribute to shaping the composition of the gut microbiota. For this reason the study of the gut microbiota of a mammal exhibiting an extraordinary life span is of great importance. The naked mole-rat (Heterocephalus glaber) is a eusocial mammal known for its longevity and cancer resistance. Methods: Here we analyzed its gut microbiota by cultivating the bacteria under aerobic and anaerobic conditions and identifying their species by mass spectrometry. Results: Altogether, 29 species of microbes were identified, predominantly belonging to Firmicutes, and Bacteroidetes. The most frequent species were Bacillus megaterium (45.2 %), followed by Bacteroides thetaiotaomicron (19.4 %), Bacteroides ovatus, Staphylococcus sciuri and Paenibacillus spp., each with a frequency of 16.1 %. Conclusion: Overall, the gut of the naked mole-rat is colonized by diverse, but low numbers of cultivable microbes compared with humans and mice. The primary food plants of the rodents are rich in polyphenols and related compounds, possessing anti-microbial, anti-inflammatory, anti-oxidative as well as anti-cancer activity which may contribute to their exceptionally healthy life.

The Maillard reaction is a network of different non-enzymatic reactions between carbonyl groups of reducing sugars and amino groups from amino acids, peptides, or proteins, which progresses in three major stages and originates a very heterogeneous mixture of reaction products. It is also known as non-enzymatic browning, due to the brown macromolecular pigments formed in the final stage of the reaction. The chemistry underlying the Maillard reaction is complex. It encloses not only one reaction pathway, but a whole network of various transformations. As virtually all foods contain both proteins and carbohydrates, Maillard reaction products are present in the daily diet in considerable amounts. The endogenous formation of Maillard reaction products, especially related to ageing and diabetes, aroused intense discussions about the health consequences of the “glycation”, the term that describes the in vivo reaction corresponding to the Maillard reaction in foods. Melanoidins are the final brown products of the Maillard reaction. They are responsible for the color formed during the heat processing of foods like coffee, bread, malt, and beef. Melanoidins are high molecular weight polydisperse polymers containing nitrogen. Their structure is largely unknown. Coffee melanoidins, which are object of the present study, contain thermally transformed polysaccharides, proteins, and phenolic compounds. Since the mechanisms involved on the formation of these macromolecules, and the chemical transformations which take place during the heat treatment are not completely elucidated, key structural features were analyzed. Especially the incorporation of chlorogenic acids in the melanoidin skeleton was object of attention of the present work. Another major aim of this work was to investigate the influence of the Maillard reaction on the inhibitory potential of food components against zinc metalloproteases. The studied enzymes were three human matrix metalloproteases (MMP-1, -2 and -9), which are able to degrade matrix proteins and participate in many physiological processes, including tissue turnover and repair, but also constitute important targets in malignant and degenerative diseases. A microbial collagenase from Chlostridium histolyticum was chosen due to its subtract similarity to MMPs. Furthermore, Angiotensin Converting Enzyme (ACE), which plays a central role in cardiovascular pathologies such as hypertension and cardiac hypertrophy, was investigated. As a prototypical Maillard reaction product, coffee melanoidin was adopted. Due to the roast dependent inhibitory activity of the coffee melanoidin fractions against matrix metalloproteases, the functionalization caused by the non-enzymatic browning was closer investigated. Na-carboxyalkylated derivatives of a sequence of relevant peptides were synthesized, in a variation of the process-induced formation of Nε-carboxymethyllysine, a major advanced glycation end-product (AGE). The inhibitory activity against zinc metalloproteases of the sequence of selected peptides and their Na-carboxymethyl- (CM-) and Na-carboxyethyl- (CE-) derivates was investigated.:LIST OF CONTENTS I LIST OF TABLES IV LIST OF FIGURES V LIST OF ABBREVIATIONS VII 1 INTRODUCTION 1 2 BACKGROUND 3 2.1 Maillard reaction in food 3 2.1.1 Melanoidins 8 2.2 Coffee 11 2.2.1 General aspects 11 2.2.1.1 Coffee production 12 2.2.1.2 General chemical composition 14 2.2.1.3 Coffee and health 20 2.2.2 Coffee melanoidins 24 2.2.2.1 Chemistry of coffee melanoidins 24 2.2.2.2 Properties of coffee melanoidins 29 2.3 Zinc metallopeptidases 32 2.3.1 Matrix metalloproteinases (MMPs) 33 2.3.1.1 Functions of MMPs 35 2.3.1.2 Structure of MMPs 37 2.3.1.3 Inhibition of MMPs 39 2.3.2 Clostridium histolyticum collagenase (ChC) 43 2.3.2.1 Functions of ChC 43 2.3.2.2 Structure of ChC 43 2.3.2.3 Inhibition of ChC 44 2.3.3 Agiotensin converting enzyme (ACE) 45 2.3.3.1 Functions of ACE 45 2.3.3.2 Structure of ACE 46 2.3.3.3 Inhibition of ACE 48 3 EXPERIMENTAL SECTION 50 3.1 Chemicals, materials and equipment 50 3.1.1 Chemicals 50 3.1.2 Material 52 3.1.3 Equipment 52 3.1.4 Solutions 54 3.2 Synthesis of Nα-carboxyalkylated peptides 55 3.2.1 Nα-carboxyalkylation of GP, LL, IA, GA, GL, AP, IP and IPP by reductive alkylation 55 3.2.2 Nα-carboxyalkylation of IW using sodium cyanoborohydride 56 3.3 Purification 57 3.3.1 Ion Exchange Chromatographic purification 57 3.3.1.1 Spotting test 58 3.3.2 HPLC purification of CM-IW 58 3.3.3 Overview of the synthesis and elution conditions 59 3.4 Characterization of carboxyalkylated peptides 61 3.4.1 Mass spectrometry 61 3.4.2 Elemental Analysis 61 3.4.3 Analytical characteristics of carboxyalkylated peptides 62 3.5 Preparation of coffee fractions 65 3.5.1 Roasting conditions 65 3.5.2 Fractionation of coffee samples: Isolation of coffee melanoidins 67 3.6 Structural studies 69 3.6.1 Estimation of the molecular weight 69 3.6.2 C/N ratio 70 3.6.3 Amino acid analysis 70 3.6.3.1 Acid hydrolysis 70 3.6.3.2 General amino acid analysis 70 3.6.3.3 Lysinoalanine 71 3.6.3.4 Pentosidine 72 3.6.4 Total phenols 74 3.6.5 Raman spectroscopy 74 3.7 Study on inhibition of zinc metalloproteases 75 3.7.1 Inhibition of ACE 75 3.7.1.1 General enzymatic assay 75 3.7.1.2 Quantification 78 3.7.2 Inhibition of MMP-1, -2 and -9 79 3.7.2.1 General enzymatic assay 80 3.7.2.2 Effect of zinc addition on the inhibition of MMP-1 by melanoidins 81 3.7.3 Inhibition of ChC 82 3.7.3.1 General enzymatic assay 82 3.7.3.2 Quantification 84 3.7.4 Calculation of IC50 84 4 RESULTS AND DISCUSSION 86 4.1 Coffee melanoidins 86 4.1.1 Isolation of coffee fractions 86 4.2 Inhibition of zinc-dependent peptidases by coffee fractions 89 4.2.1 Inhibition of MMPs 89 4.2.2 Inhibition of other zinc metalloproteases 98 4.2.3 General considerations 99 4.3 Structural studies on coffee melanoidins 101 4.3.1 Gel permeation chromatography 102 4.3.2 Elemental analysis: C/N ratio 113 4.3.3 Amino acid analysis 116 4.3.4 Total phenolics 120 4.3.5 Correlation between total phenols content and C/N ratio in coffee melanoidins 123 4.3.6 Raman spectroscopy 124 4.4 Derivatization of peptides 129 4.4.1 Nα-carboxyalkylation of peptides by reductive alkylation 130 4.5 Preliminary investigations on the inhibitory potential of Nα-carboxyalkyl derivatives of peptides against metalloproteases 133 4.5.1 Inhibition against ACE 134 4.5.2 Inhibition against other zinc metalloproteases 138 5 SUMMARY 141 6 REFERENCES 145 LIST OF PUBLICATIONS AND CONFERENCE CONTRIBUTIONS 168 AKNOWLEDGMENTS 169 ERKLÄRUNG 170