L’attrait des compagnies pharmaceutiques pour des structures cycliques possédant des propriétés biologiques intéressantes par les compagnies pharmaceutiques a orienté les projets décrits dans ce mémoire. La synthèse rapide, efficace, verte et économique de ces structures suscite de plus en plus d’attention dans la littérature en raison des cibles biologiques visées qui deviennent de plus en plus complexes. Ce mémoire se divise en deux projets ciblant la synthèse de deux structures aromatiques importantes dans le monde de la chimie médicinale. Dans un premier temps, l’amélioration de la synthèse de dérivés phénoliques a été réalisée. L’apport de la chimie en flux continu dans le développement de voies synthétiques plus vertes et efficaces sera tout d’abord discuté. Ensuite, une revue des antécédents concernant l’hydroxylation d’halogénure d’aryle sera effectuée. Finalement, le développement d’une nouvelle approche rapide de synthèse des phénols utilisant la chimie en flux continu sera présenté, suivi d’un survol de ses avantages et ses limitations. Dans un deuxième temps, le développement d’une nouvelle méthodologie pour la formation de 3-aminoindazoles a été réalisé. Tout d’abord, un résumé de la littérature sur la synthèse de différents indazoles sera présenté. Ensuite, une présentation de deux méthodes efficaces d’activation de liens sera effectuée, soit l’activation d’amides par l’anhydride triflique et l’activation de liens C–H catalysée par des métaux de transition. Finalement, le développement d’une nouvelle méthodologie pour la synthèse de 3-aminoindazole utilisant ces deux approches sera discuté. / The continuous attraction towards accessing cyclic structures that possess interesting biological properties by pharmaceutical companies has guided the projects described in this M.Sc. thesis. Due to the increasing complexity of drug targets, methodologies encompassing efficient, rapid, economical and environmentally friendly syntheses are highly sought in the organic chemistry literature. The present work consists of two projects targeting the synthesis of two important aromatic structures in the field of medicinal chemistry. The first part of the thesis will present an improved synthesis of phenol derivatives. The recent chemical contributions in the continuous development of greener and efficient synthetic routes will be discussed, followed by a quick review of the literature on the hydroxylation of aryl halides. Then, the development of a new approach for rapid synthesis of phenol derivatives using continuous flow chemistry will be presented, including an overview of its benefits and limitations. The second part of the thesis will put forward the development of a novel methodology for the formation of 3-aminoindazoles. A summary of the literature on the synthesis of various indazoles will be presented, followed by an overview of two effective bond activation methods: the amide activation using triflic anhydride and transition metal catalyzed C–H activation. Finally, the evolution of a new method for the synthesis of 3-aminoindazole using the previously mentioned two approaches will be discussed.

Auf Basis moderner unabhängiger Analysenmethoden wurden selektive und spezifische Analysenverfahren für die Analytik von Phenolen, unter besonderer Berücksichtigung der Alkylphenole entwickelt, optimiert und teilweise validiert. Es wurden Verfahren für die Hochleistungs-Flüssigchromatographie - Massenspektrometrie (HPLC-MS), Hochleistungs-Flüssigchromatographie - Kernmagnetische Resonanzspektrometrie (HPLC-NMR), Gaschromatographie - Massenspektrometrie (GC-MS) und Kapillarelektrophorese (CE) entwickelt. Die HPLC-MS wurde mit den Atmosphärendrucktechniken "Electrospray Ionization" (HPLC-ESI-MS) und "Atmospheric Pressure Chemical Ionization" (HPLC-APCI-MS) eingesetzt. Die Gaschromatographie wurde in Kopplung zu einem hochauflösenden Massenspektrometer (GC-HRMS) und in Kopplung zu einem niederauflösenden Quadrupol-Spektrometer (GC-LRMS) verwendet. Mit der Kapillarelektrophorese wurden Verfahren in den Modi "Kapillar-Zonen-Elektrophorese (CZE) und "Micellare Electrokinetische Chromatographie (MEKC) mit verschiedenen internen Anreicherungstechniken ("Stacking") entwickelt. Für die Validierung des MEKC-Verfahrens mit interner Anreicherung ("Direktes-MEKC-Stacking") wurde ein Ringversuch organisiert und durchgeführt. Anhand von representativen Modellgemischen wurden die Verfahren geprüft und miteinander verglichen. Die Modellgemische sind reproduzierbar herstellbar; ihre Stabilität und Homogenität wurde abgesichert, so daß sie als synthetische Referenzmaterialien zur laborinternen Qualitätssicherung, zur Methodenbewertung und zum Laboratoriumsvergleich eingesetzt werden können. Anhand von charakteristischen Kenngrößen wie Bestimmungsgrenzen, Selektivität, Zeitaufwand, Kosten und Meßunsicherheiten wurden die Verfahren auf ihre Eignung als Standard- bzw. Referenzverfahren bewertet. Die Verfahren der HPLC-NMR, HPLC-ESI-MS, HPLC-APCI-MS und GC-HRMS können mit unterschiedlichem Einsatzbereich als Referenzverfahren für die Analytik von Phenolen eingesetzt werden. Die GC-HRMS ist dabei das leistungsstärkste Referenzverfahren. Die Verfahren der GC-LRMS und CE ("Direktes-MEKC-Stacking") sind als Standardverfahren geeignet und besitzen alle Voraussetzungen um auch als Normverfahren für die Analytik von Phenolen eingeführt zu werden. / Different selective and specific analysis procedures were developed for the determination of phenol derivatives, in particular alkylphenols, in environmental relevant matrices using High-Performance Liquid Chromatography - Mass Spectrometry (HPLC-MS), High-Performance Liquid Chromatography- Nuclear-Magnetic Resonance Spectrometry (HPLC-NMR), Gas Chromatography- Mass Spectrometry (GC-MS) and Capillary Electrophoresis (CE). HPLC-MS was used with atmospheric pressure ionization techniques, Electrospray Ionization (HPLC-ESI-MS) and Atmospheric Pressure Chemical Ionization (HPLC-APCI-MS). Analysis procedures for GC-MS were developed for a high resolution mass spectrometer (GC-HRMS) and a low resolution benchtop quadrupole mass spectrometer (GC-LRMS). CE was used in the Capillary Zone Electrophoresis (CZE) and Micellar Electrokinetic Chromatography (MEKC) mode with different internal stacking techniques. The MEKC procedure with internal stacking (called "Direct-MEKC-Stacking") was validated by an interlaboratory comparison test. These procedures were tested and optimized using representative model mixtures and the most efficient then validated. The model mixtures were produced reproducably and their stability and homogeneity were demonstrated for the application as synthetic reference materials, e.g. for internal laboratory control, quality assurance or method development. The different analysis procedures were categorized as standard- or reference procedures on the basis of characteristic properties, e.g. detection limits, selectivity, analysis time, cost and uncertainty. HPLC-NMR, HPLC-ESI-MS, HPLC-APCI-MS, GC-HRMS can be used as reference procedures for different fields of use. Best performance can be reached with GC-HRMS. GC-LRMS and "Direct-MEKC-Stacking" can be used as standard procedures for the analysis of phenol derivatives and they fulfill all conditions for their implementation in standardization.

Oxidation reactions are of prime importance at an industrial level and correspond to a huge market. Oxidation reactions are widely practiced in industry and are thoroughly studied in academic and industrial laboratories. Achievements in oxidation process resulted in the development of many new selective oxidation processes. Environmental protection also relies mainly on oxidation reactions. Remarkable results obtained in this field contributed to promote the social image of chemistry which gradually changes from being the enemy of nature to becoming its friend and savior. This study dealt with two aspects regarding oxidation process. The first aspect represented an experimental study for the partial oxidation of benzene to phenol using Pd membrane in the gaseous phase. The second part was a theoretical study for some of the advanced oxidation process (AOPs) which are applied for contaminant destructions in polluted waters. Niwa and coworkers reported a one step catalytic process to convert benzene to phenol using Pd membrane. According to their work, this technique will produce a higher yield than current cumene and nitrous oxide based industrial routes to phenol. A similar system to produce phenol from benzene in one step was studied in this work. Results at low conversion of benzene to phenol were obtained with a different selectivity from the reported work. High conversion to phenol was not obtained using the same arrangement as the reported one. High conversion to phenol was obtained using a scheme different from the one reported by Niwa et al1. It was found that producing phenol from benzene is not related to Pd-membrane since phenol was produced by passing all reactants over a Pd catalyst. Within the studied experimental conditions, formation of phenol was related to Pd catalyst since Pt catalyst was not capable of activating benzene to produce phenol. Other evidence was the result of a blank experiment, where no catalyst was used. From this experiment no phenol was produced. A kinetic model for the advanced oxidation process using ultraviolet light and hydrogen peroxide (UV/H2O2) in a completely mixed batch reactor has been tested for the destruction of humic acid in aqueous solutions. Known elementary chemical reactions with the corresponding rate constants were taken from the literature and used in this model. Photochemical reaction parameters of hydrogen peroxide and humic acid were also taken from the literature. Humic acid was assumed to be mainly destroyed by direct photolysis and radicals. The rate constant for the HA- reaction was optimized from range of values in the literature. Other fitted parameters were the rate constant of direct photolysis of hydrogen peroxide and humic acid. A series of reactions were proposed for formation of organic byproducts of humic acid destruction by direct photolysis and radicals. The corresponding rate constants were optimized based on the best fit within the range of available published data. This model doesn't assume the net formation of free radicals species is zero. The model was verified by predicting the degradation of HA and H2O2 for experimental data taken from the literature. The kinetic model predicted the effect of initial HA and H2O2 concentration on the process performance regarding the residual fraction of hydrogen peroxide and nonpurgeable dissolved organic carbon (NPDOC). The kinetic model was used to study the effect of the presence of carbonate/bicarbonate on the rate of degradation of NPDOC using hydrogen peroxide and UV (H2O2/UV) oxidation. Experimental data taken from literature were used to test the kinetic model in the presence of carbonate/bicarbonate at different concentrations. The kinetic model was able to describe the trend of the experimental data. The kinetic model simulations, along with the experimental data for the conditions in this work, showed a retardation effect on the rate of degradation of NPDOC due to the presence of bicarbonate and carbonate. This effect was attributed to the scavenging of the hydroxyl radicals by carbonate and bicarbonate. A kinetic model for the degradation of methyl tert-butyl ether (MTBE) in a batch reactor applying Fenton's reagent (FeII/ H2O2) and Fenton-like reagent (Feo/ H2O2) in aqueous solutions was proposed. All of the rate and equilibrium constants for hydrogen peroxide chemistry in aqueous solutions were taken from the literature. Rate and equilibrium constants for ferric and ferrous ions reactions in this model were taken from the reported values in the literature, except for the rate constant for the reaction of ferric ions with hydrogen peroxide where it was fitted within the range that was reported in the literature. Rate constant for iron dissolution was also a fitted parameter. The mechanism of MTBE degradation by the hydroxyl radicals was proposed based on literature studies. The kinetic model was tested on available experimental data from the literature which involved the use of Fenton's reagent and Fenton-like reagent for MTBE degradation. The degradation of MTBE in Fenton's reagent work was characterized to proceed by two stages, a fast one which involved the reaction of ferrous ions with hydrogen peroxide (FeII/H2O2 stage) and another, relatively, slower stage which involved the reaction of ferric ions with hydrogen peroxide (FeIII/H2O2 stage). The experimental data of MTBE degradation in the FeII/H2O2 stage were not sufficient to validate the model, however the model predictions of MTBE degradation in the FeIII/H2O2 stage was good. Also, the model was able to predict the byproducts formation from MTBE degradation and their degradation especially methyl acetate, and tert-butyl alcohol. The effect of each proposed reaction on MTBE degradation and the byproducts formation and degradation was elucidated based on a sensitivity analysis. The kinetic model predicted the degradation of MTBE for Fenton-like reagent for the tested experimental data. Matlab (R13) was used to solve the set of ordinary nonlinear stiff differential equations that described rate of species concentrations in each advanced oxidation kinetic model. Niwa, S. et al., Science 295 (2002) 105

Made available in DSpace on 2017-07-21T18:53:09Z (GMT). No. of bitstreams: 1 Mary Dias Queji.pdf: 909281 bytes, checksum: 0364cd27c13b09019850a0944168a898 (MD5) Previous issue date: 2008-04-05 / The agro industry of the apple generates, during the processing, the pomace which is considered the main by-product. Studies show that of the total amount of fruit that is processed to obtain the apple juice, 20 to 40 correspond to this by-product, which, is usually destined as a complement in animal feed or delivered onto the soil as organic fertilizer. The chemical composition, in moist base, is constituted by moisture (80), fibers (5) and soluble solids (14) represented, mainly, by fructose, glucose and sucrose, as well as, by organic acids, represented, in the most part by malic acid. The apple pomace also presents phenolic compounds that are target nutrients of great sensory and nutritional importance. Therefore, the quantification of its constituents represents an important source of data in the characterization of apple pomace for biotechnological purposes, seeking to attribute an appropriate use for this by-product. The conventional methodologies used for the quantification of sugars, organic acids and total phenols, although being part of the routine analyses in the quality control laboratories, are onerous, time consuming and generate residues. The aim of this study was to develop a fast, versatile analytical technique, of low cost and no pollutant, using the diffuse reflectance infrared spectroscopy (DRIFTS) allied to methods of multivariate calibration (PLSR). For the construction of the multivariate models, the averages of the concentrations of the simple sugars, malic acid and total phenols were used, obtained by the conventional methodologies, as well as the data in the medium infrared spectroscopy (MID) and near (NIR), obtained in duplicate, and of the 52 spectra obtained for the samples of apple pomace, 47 made part of the set calibration and 5 of the set validation. The regression models for the prediction of the concentration of fructose, sucrose, total phenols and malic acid obtained better results in MID, with averages of relative standard errors of 3.9 (with 5 Latents Variable), 6.6 (with 5 Latents Variable), 6.4 (with 4 Latents Variable) and 5.9 (with 5 Latents Variable), respectively. Already the best capacity prediction for glucose concentration was obtained by NIR, in which the average of relative standard error was 7.4 (with 6 Latents Variable). The obtained results demonstrate the good capacity of prediction of the multivariate models based in infrared spectroscopy and characteristic advantages of the association DRIFTPLSR. / A agroindústria da maçã gera, durante o processamento, o bagaço que é considerado o principal subproduto deste setor. Levantamentos mostram que da quantidade total de fruta que é processada para a obtenção do suco de maçã, 20 a 40 correspondem a este subproduto, que, normalmente, é destinado como complemento na alimentação animal ou dispensado no solo como adubo orgânico. Sua composição química, em base úmida, é constituída por umidade (80), fibras (5) e sólidos solúveis (14), estes últimos representados, principalmente, por frutose, glucose e sacarose, bem como, ácidos orgânicos, representados, majoritariamente pelo ácido málico. O bagaço da maçã também apresenta compostos fenólicos, que são fito-nutrientes de grande importância sensorial e nutricional. Portanto, a quantificação desses constituintes representa uma importante fonte de dados na caracterização do bagaço de maçã para finalidades biotecnológicas, visando atribuir um fim mais nobre a este subproduto. As metodologias convencionais empregadas para a quantificação de açúcares, ácidos orgânicos e fenóis totais, embora façam parte das análises de rotina nos laboratórios de controle de qualidade, são onerosas, demoradas e geram resíduos. O objetivo deste trabalho foi desenvolver uma técnica analítica rápida, versátil, de baixo custo e não poluente. Para tal, utilizou-se a espectroscopia no infravermelho por refletância difusa (DRIFTS) aliada a métodos de calibração multivariada (Regressão de Mínimos Quadrados Parciais - PLSR). Para a construção dos modelos multivariados, foram utilizadas as médias das concentrações dos açúcares simples, ácido málico e fenóis totais, obtidas pelas metodologias convencionais, bem como, os dados de espectroscopia no infravermelho médio (MID) e próximo (NIR). Os espectros foram obtidos em duplicata, sendo que dos 52 espectros das amostras de bagaço de maçã, 47 fizeram parte do conjunto de calibração e 5 do conjunto de validação externa. Os modelos de regressão para a previsão da concentração de frutose, sacarose, fenóis totais e ácido málico obtiveram melhores resultados no MID, com médias de erro padrão relativo de 3.9 (com 5 Variáveis Latentes), 6.6 (com 5 Variáveis Latentes), 6.4 (com 4 Variáveis Latentes) e 5.9 (com 5 Variáveis Latentes), respectivamente. Já a melhor capacidade de previsão para a concentração de glucose foi obtida pelo NIR, na qual a média de erro padrão relativo foi de 7.4 (com 6 Variáveis Latentes). Os resultados obtidos demonstram a boa capacidade de previsão dos modelos multivariados fundamentados em espectroscopia no infravermelho e vantagens características da associação DRIFT-PLSR.

<p>Bosiljak (Ocimum basilicum L.) pripada familiji Lamiaceae i jedna je od najrasprostranjenijih i najče&scaron;će gajenih biljaka &scaron;irom sveta. Njegova upotreba je &scaron;iroko rasprostranjena zbog bogatog sadržaja i skladnog odnosa vitamina, minerala kao i različitih fenolnih jedinjenja kao glavnih nosilaca antioksidativne aktivnosti. Poslednjih godina velika pažnja je usmerena ka fenolnim fitokomponentama kao potencijalnim promoterima zdravlja. Ciljevi ovog rada bili su ispitivanje kvalitativnih i kvantitativnih karakteristika, kao i biohemijskih aktivnosti ekstrakata bosiljka dobijenih različitim rastvaračima, kao i ispitivanje uticaja farmaceutsko-tehnolo&scaron;ke formulacije u obliku mikrovezikula ekstrakta bosiljka na glikemijski, lipidni i oksido-redukcioni status kod oglednih životinja, u odnosu na vodeni ekstrakt. In vitro ispitivanja su uključivala analizu 100 ekstrakata dobijenih različitim rastvaračima i stepenom usitnjenosti. Ukupni fenolni i flavonoidni sadržaj, kao i antioksidativna aktivnost određena je spektrofotometrijskom metodom. Takođe, kvantifikovane i kvalifikovane su fenolne komponente (fenolne kiseline i flavonoidi) primenom visokoefikasne tečne hromatografije (HPLC). In vivo ispitivanje je rađeno na 84 albino laboratorijska pacova soja Wistar. Per os su primenjivani vodeni ekstrakt bosiljka, rastvor natrijumove soli monoketoholne kiseline, njihove kombinacije, kao i ekstrakt u obliku farmaceutsko-tehnolo&scaron;ke formulacije, mikrovezikula. Posle sedmodnevnog tretmana, merene su koncentracije glukoze u krvi, a nakon žrtvovanja, u serumu su određeni parametri lipidnog statusa i antioksidativnog stresa. Ex vivo analizama procenjivani su efekti ekstrakta bosiljka na enzimske i neenzimske parametre antioksidativnog odbrambenog sistema i oksidativne modifikacije lipida. Praćen je i uticaj tretmana na akutno o&scaron;tećenje jetre usled primene paracetamola. Svi analizirani ekstrakti bosiljka su pokazali prisustvo velikog broja fenolnih jedinjenja iz klase fenolnih kiselina i flavonoida. Detektovane i kvantifikovane aktivne komponente bile su fenolne kiseline: hlorogenska, p-hidroksibenzoeva, kafena, vanilinska, ferulna, rozmarinska i cimetna, kao i apigenin, kvercetin, naringenin i rutin kao predstavnici flavonoidnih jedinjenja. Primena ekstrakta bosiljka i natrijumove soli monoketoholne kiseline, same ili u kombinaciji, a takođe i u farmaceutsko-tehnolo&scaron;koj formulaciji, pokazale su smanjenja o&scaron;tećenja tokom oksidativnog stresa, značajno antidijabetesno delovanje, povoljan uticaj na lipidni status i protektivni efekat na funkcije jetre i bubrega. Na osnovu dobijenih rezultata može se zaključiti da ekstrakt bosiljka sadrži značajnu količinu fenolnih jedinjenja odgovornih za antioksidativnu aktivnost. Vodeni ekstrakt bosiljka, sam ili u kombinaciji sa monoketoholnom kiselinom, i u obliku farmaceutsko-tehnolo&scaron;ke formulacije snižava nivo glukoze u krvi, ubrzava obnovu &beta; ćelija, ima povoljan efekat na lipidni status i ne dovodi do toksičnih promena na jetri i bubrezima kod eksperimentalnih životinja.</p> / <p>Basil (Ocimum basilicum L.) belongs to the family Lamiaceae and represents one of the most widespread and most commonly cultivated plant worldwide. Its use is widespread due to its rich content and harmonious relationship of vitamins, minerals and various phenolic compounds which are responsible for the antioxidant activity. In recent years, great attention is directed to phenolic phytocomponents as potential promoters of health. The objectives of this study were to test the qualitative and quantitative characteristics, as well as the biochemical activity of basil extracts obtained by various solvents and investigate the impact of pharmaceutical technological formulation of basil extract on glycemic, lipid and oxidation-reduction status of the aqueous extract in the experimental animals. In vitro studies have included 100 extracts obtained by various solvents and degrees of fragmentation. Total phenolic and flavonoid content, as well as antioxidant activity was determined by spectrophotometric method. Also, phenolic compounds (phenolic acids and flavonoids) were qualified and quantified using high performance liquid chromatography (HPLC). In vivo testing was performed on 84 laboratory albino Wistar rats. The aqueous extract of basil, a solution of sodium salt of monoketocholic acid, their combinations, as well as an extract in the form of the pharmaceutical technological formulation were administered per os. After seven-day-treatment, the concentrations of glucose were measured in the blood and after sacrificing of animals, the lipid and antioxidative parameters were determined in serum. Ex vivo analysis assessed the effect of the extract of basil on the parameters of enzymatic and non-enzymatic antioxidant defense system and the oxidative modification of lipids. The effect of paracetamol on acute liver injury was also monitored. All analyzed basil extracts showed the presence of the large number of phenolic compounds from the class of phenolic acids and flavonoids. Chlorogenic, p-hydroxybenzoic, caffeic, vanillic, ferulic, rosmarinic, and cinnamic acid, as well as apigenin, quercetin, naringenin and rutin were detected and quantified. The application of basil extracts and the sodium salt of monoketocholic acid, either alone or in combination, and also in pharmaceutical technological formulation of microvesicles has shown a reduction in damage during oxidative stress, then a significant antidiabetic activity, favorable effect on the lipid profile and protective effect on the liver and kidney function. Based on these results it can be concluded that basil extract contains significant amounts of phenolic compounds responsible for antioxidant activity. The aqueous extract of basil, alone or in combination with monoketocholic acid, and in the form of the pharmaceutical technological formulations lowers glucose levels in the blood, accelerates the restoration of &beta; cells, has a favorable effect on the lipid status and does not lead to toxic changes in the liver and kidneys in experimental animals.</p>

L'oxydation de l'acide férulique et de son ester (le férulate d'éthyle) par la laccase de Myceliophtora thermophila a été étudiée en milieu aqueux et dans des conditions expérimentales douces (30 °C et pH 7,5) afin de synthétiser de nouvelles molécules naturelles grâce à un procédé vert. L'oxydation enzymatique a permis l'obtention d'intermédiaires colorés pour l'acide férulique et incolores pour le férulate d'éthyle. En outre, le férulate d'éthyle a été plus rapidement totalement oxydé que l'acide férulique. De plus, cette procédure a abouti à des produits majoritairement dimériques avec MM = 443 g/mol et MM = 386 g/mol pour le férulate d'éthyle et l'acide férulique, respectivement. Les nouvelles molécules synthétisées ont présenté des propriétés antioxydantes importantes avec de faibles propriétés antibactériennes et cytotoxiques. En présence du chitosane insoluble dans le milieu réactionnel, la laccase a été protégée de l'inhibition liée aux produits d'oxydation et le degré de polymérisation de ces produits a été contrôlé. De plus, les produits d'oxydation ont réagi avec les groupements NH2 libres permettant la formation de liaisons covalentes de type base de Schiff (C=N) au niveau du C2 sur le chitosane. La majorité des produits d'oxydation greffés sur le chitosane était de forme dimérique. Cette procédure a permis d'obtenir du chitosane coloré avec l'acide férulique et incolore avec le férulate d'éthyle tout en présentant de nouvelles propriétés dues au greffage de composés phénoliques. Ces chitosanes dérivés ont présenté des propriétés fonctionnelles intéressantes telles que antioxydantes, physico-chimiques (stabilisation thermique) et biologiques (adhésion cellulaire) ainsi que la conservation des propriétés antibactériennes du chitosane natif / Oxidation of ferulic acid and its ester (ethyl ferulate) by Myceliophtora thermophila laccase has been studied in aqueous medium under mild experimental conditions (30°C and pH 7.5) as a green process to synthesize natural neo-molecules. Enzymatic oxidation led to colored and colorless intermediaries for ferulic acid and ethyl ferulate, respectively. Additionally, ethyl ferulate was oxidized faster than ferulic acid. This procedure has led to dimeric major products with MM = 443 g/mol and MM = 386 g/mol for ethyl ferulate and ferulic acid, respectively. New synthesized molecules demonstrated important antioxidants properties with weak antibacterial and cytotoxic properties. With insoluble chitosan particles in the reaction medium, laccase was protected from inhibition due to oxidation products and the polymerization degree of these products was checked. In addition, the oxidation products reacted with the free NH2 groups forming covalent bonds of Schiff base type (C=N) at C-2 region. The majority of the oxidation products grafted onto chitosan was of dimeric form. This procedure led to colored and colorless chitosans by ferulic acid and ethyl ferulate, respectively, with new properties due to grafting of phenolic compounds. These chitosan derivatives presented interesting functional properties such as antioxidant, physico-chemical (thermal stability) and biological (cell adhesion) as well as the preservation of antibacterial properties of native chitosan

<p>Rod Salix pripada porodici Salicaceae i obuhvata oko 450 vrsta &scaron;irom sveta, od kojih u Republici Srbiji raste oko 18. Kora vrbe ispoljava antiinflamatorno, antireumatsko, analgetičko i antipiretičko delovanje sinergističkim efektom njenih glavnih aktivnih supstanci &ndash; glikozida salicina, fenolnih i flavonoidnih jedinjenja. Ciljevi ovog rada bili su ispitivanje uticaja klasične i savremenih metoda ekstrakcije na hemijski sastav i biolo&scaron;ke aktivnosti ekstrakata različitih vrsta vrba, utvrđivanje razlika u sadržaju aktivnih komponenti i biolo&scaron;kom potencijalu između ekstrakata kore i ekstrakata lista iste vrste vrbe, kao i utvrđivanje razlika u sadržaju sekundarnih metabolita i antioksidantnoj aktivnosti kod ekstrakata različitih vrsta vrba. Ispitivanja su uključivala analizu 92 ekstrakta kore i lista osam predstavnika roda Salix, dobijenih različitim metodama (maceracija, ultrazvučna i mikrotalasna ekstrakcija) i pri različitim uslovima ekstrakcije (rastvarač, vreme, veličina čestica). Sadržaj ukupnih fenola i flavonoida, kao i antioksidativna aktivnost određeni su spektrofotometrijski. Hemijska karakterizacija ekstrakata vr&scaron;ena je primenom visokoefikasne tečne hromatografije (HPLC). Metodom in silico molekularnog dokinga ispitan je inhibitorni potencijal sastojaka ekstrakata prema enzimima ciklooksigenaze (COX-1 i COX-2) i acetilholinesterazi (AChE). Savremenim metodama ekstrakcije izolovan je veći broj pojedinačnih komponenata u najvećoj koncentraciji i dobijeni su ekstrakti sa jačim potencijalom neutralizacije hidroksilnih radikala. Klasična metoda maceracije 70% etanolom (v/v) bila je pogodnija za dobijanje ekstrakata sa jačim antioksidativnim potencijalom prema DPPH radikalu kod gotovo svih ispitivanih Salix vrsta. Kod većine Salix vrsta jači antioksidantni potencijal prema DPPH radikalu ispoljili su ekstrakti kore. Kod polovine ispitivanih vrsta ekstrakti lista su inhibisali hidroksilne radikale u većoj meri. Veći sadržaj pojedinačnih jedinjenja u ekstraktima kore ili lista zavisio je od same vrste vrbe. Najjaču antioksidantu aktivnost ispoljila je vrsta S. alba, dok je u pogledu hemijskog sastava najraznovrsnija vrsta bila S. fragilis. In silico analizom metodom molekularnog dokinga utvrđen je jak inhibitorni potencijal flavonoidnih jedinjenja kvercetina, naringenina i epikatehina, kao i hlorogenske kiseline među fenolnim kiselinama prema enzimima COX-1 i COX-2. Epikatehin, salicin i hlorogenska kiselina ispoljili su značajno inhibitorno delovanje na enzim AChE u doking studijama. Dobijeni rezultati ukazuju da pored kore i list vrbe ima veliki potencijal primene kao izvor značajnih fitokomponenata, kao i na mogućnost eksploatacije vrsta vrba koje nisu u komercijalnoj upotrebi kao lekovitih sirovina za izolovanje antioksidanasa i farmakolo&scaron;ki aktivnih supstanci.</p> / <p>The genus Salix belongs to the family Salicaceae and comprises about 450 species worldwide, out of which 18 grow in the Republic of Serbia. Willow bark exhibits anti-inflammatory, anti-rheumatic, analgesic and antipyretic effects through synergistic action of its main ingredients -glycosidesalicin, phenolic and flavonoid compounds. Aims of this paper were to analyse the impact of classical and modern extraction methods on chemical composition and biological activities of extracts of different willow species, determine the differences in the amount of active compounds and biological potential between bark and leaf extracts within the same willow species, as well as between extracts of various species. The analysis included 92 bark and leaf extracts of eight species from the genus Salix, obtained by different extraction methods (maceration, ulrasound and microwave assisted extraction) and conditions (solvent, time, particle size). Total phenolics and flavonoids content, as well as antioxidant activity were determined spectrophotometrically. Chemical characterization was carried out by high performance liquid chromatography (HPLC). Cyclooxygenase (COX-1 and COX-2) and acetylcholine esterase (AChE) inhibitory potential of compounds isolated from the extracts was examined by in silico method of molecular docking. Greater number of individual components in highest concentration, as well as exracts with stronger hydroxyl radical scavenging potential were obtained by modern extraction methods. Classical method of maceration with 70% ethanol (v/v) was more suitable for obtaining extracts with higher DPPH radical scavenging activity in the vast majority of tested species. In half of the analysed species leaf extracts inhibited hydroxyl radicals more than bark. Higher amount of individual compounds in bark or leaf extracts depended on the species itself. S. alba exhibited the strongest antioxidant activity, whereas S. fragilis had the most diverse chemical composition. Strong COX-1 and COX-2 inhibitory potential of flavonoids quercetin, naringenin and epicatechin, as well as chlorogenic acid among phenolic acids was determined by in silico molecular docking analysis. Molecular docking studies also demonstrated the inhibitory activity of epicatechin, salicin and chlorogenic acid toward AChE. The obtained results indicate that not only bark, but willow leaf as well could be used as source of significant phytochemicals and also the possibility of exploitation of willow species that are not commercially used as medicinal raw material for isolation of antioxidants and pharmacologycally active substances.</p>

This research explored fate of organic contaminants in aquatic plant systems through (i) experimental development of relationships to describe sorption, uptake and enzymatic processing of contaminants by plants and inhibition of aquatic plants by contaminants and (ii) incorporation of experimental relationships into a conceptual model which describes contaminant fate in aquatic plant systems. This study focused on interactions of aquatic plants L. minor and M. aquaticum with halogenated phenols. 2,4,5-trichlorophenol (2,4,5-TCP) and 2,4-dichlorophenol (2,4-DCP) are precursors for the highly toxic and heavily applied herbicides 2,4,5-T and 2,4-D and were examined in detail. Chlorophenols are generally resistant to microbial degradation, a property which may limit microbial remediation options as effective alternatives for clean up of contaminated sites. Relationships for fundamental interactions between plants and contaminants that dictate uptake, enzymatic processing and sequestration of contaminants by aquatic plants were established. An assay which quantified production of oxygen by plants was developed to quantify plant metabolic activity and inhibition. Uptake of chlorinated phenols depended on plant activity and aqueous phase concentration of contaminant in the protonated form. Therefore, plant activity, contaminant pKa and media pH were established as critical parameters controlling rate of contaminant uptake. A conceptual model was developed which incorporated plant activity and inhibition into a mathematical description of uptake of organic contaminants by aquatic plants. The conceptual model was parameterized using experimental data delineating effect of plant activity, inhibition and speciation on contaminant uptake and the model was verified using independently gathered data. Experimentation with radio-labeled chlorinated phenols established that contaminants were sequestered internal to plants by plant enzymatic processing. 19F NMR was established as a technique to quantify transformation and conjugation products internal to plants and contaminant assimilation by plants and demonstrated that multiple metabolites containing the parent compound were present and quantifiable internal to plants. Finally, fate of plant-sequestered contaminants in an anaerobic bioassay was examined using Desulfitobacterium sp. strain Viet1. The results of this study address the role of aquatic plants in sequestration of contaminants in surface waters that indicate the potential and limitations of use of aquatic plants in natural and engineered treatment systems.

Wine grape pomace (WGP), the byproduct from winemaking, is a good source of polyphenols and dietary fibers, and may be utilized as antioxidant dietary fibers (ADF) for food applications. The objectives of this thesis research were to first determine the phenolic compounds, antioxidant and antimicrobial activities in red WGP under different drying processes for long-term storage, and to further evaluate the feasibility of using WGP as a functional food ingredient in yogurt and salad dressing for enhancing the nutritional value and improving storability of the products. Two types of WGP samples, pomace containing seeds and skins (P) and pomace with skins only (S) from Pinot Noir (PN) and Merlot (M) were studied. Samples were subjected to four different drying conditions: 40 °C conventional and vacuum oven, 25 °C ambient air and freeze dry. Total phenolic content (TPC, by Folin-Ciocalteu assay), anthocyanins (ACY, by pH differential method) and flavanols content (TFC, by vanillin assay) of the samples along with their antioxidant activity (DPPH radical scavenge method, RSA) and antibacterial activity (minimum inhibition concentration, MIC) were determined during 16 weeks of storage under vacuum condition at 15±2 °C. Meanwhile, dietary fiber profile was evaluated by using gravimetric-enzyme method. Results showed that dietary fiber contents of PN-P, PN-S, M-P and M-S were 57-63% d.m. with the majority of insoluble fraction. Freeze dried WGP retained the highest bioactive compounds with TPC 21.19-67.74 mg GAE/g d.m., ACY of 0.35-0.76 mg Mal-3-glu/g d.m., TFC of 30.16-106.61 mg CE/g d.m. and RSA of 22.01-37.46 mg AAE/g d.m., followed with ambient air dried samples. Overall, TPC, TFC and RSA were higher in PN than in M, and higher in pomace than in skins, while reverse results were observed in ACY. All samples lost significant amount of bioactive compounds during storage, in which ambient air and freeze dried samples had TPC reduction of 32-56% and 35-58%, respectively at the end of 16 weeks of storage. RSA in PN-P and M-P remained more than 50 mg TE/g d.m., meaning WGP still met the criteria of ADF definition after 16 weeks of storage. WGP extracts showed higher antibacterial efficiency against L. innocua than that of E. coli with MIC of 2, 7, 3 and 8% against L. innocua, and 3, 6, 4 and 9% against E. coli for PN-P, PN-S, M-P and M-S samples, respectively. This study demonstrated that Pinot Noir and Merlot pomace are good sources of ADF even after 16 weeks of storage at 15 °C and vacuum condition. Due to the highest antioxidant activity (RSA 37.46 mg AAE/g) and dietary fiber content (61%), PN-P was selected as ADF to be fortified in yogurt and salad dressing. Three types of WGP: whole powder (WP), liquid extract (LE) and freeze dried extract (FDE) with different concentrations were incorporated into yogurt (Y), Italian (I) and Thousand Island (T) salad dressings. TPC, RSA and dietary fiber content, major quality attributes including pH and peroxide value (PV) during the shelf life and consumer acceptance of fortified products were evaluated. The highest ADF were obtained in 3% WP-Y, 1% WP-I and 2% WP-T samples with the dietary fiber contents of 1.98%, 2.12% and 1.83% and RSA of 935.78, 585.60 and 706.67 mg AAE/kg, respectively. WP fortified products had more dietary fiber content than that of LE and FDE fortified ones because of the insoluble fractions. The pH dropped from 4.52 to 4.32 for 3% WP-Y during three weeks of storage at 4 °C, but remained stable in WGP-I and WGP-T samples after four weeks of storage at 4 °C. Adding WGP resulted in 35-65% reduction of PV in all samples compared to the control. In WGP-Y, the viscosity increased, but syneresis and lactic acid percentage were stable during storage. The 1%WP-Y, 0.5%WP-I and 1%WP-T samples were mostly liked by consumers. Study demonstrated that WGP can be used as a functional food ingredient for enhancing nutraceutical content and extending shelf-life of the food products. This study provided important information about the economically feasible drying methods for retaining the bioactive compounds in WGP during processing and storage and also suggested that WGP can be utilized as antioxidant dietary fiber to be fortified in consumer products to promote nutritional benefit and extend product shelf-life. / Graduation date: 2013

The black rhinoceros remains one of the world's extremely endangered species despite a variety of policies to protect it. The black rhinoceros population at the Great Fish River Reserve (GFRR) in the Eastern Cape in South Africa has increased steadily since their re-introduction in 1986. This megaherbivore is a browser, with a diet obtained largely from the short and medium succulent thicket of the GFRR. Knowledge of the preferential diet of the black rhinoceros on the reserve is an important factor for the effective management of the land and the herbivores that compete for its resources. The dietary preferences of the black rhinoceros at the reserve have been established using backtracking methods. In this study the rbcL gene was used to establish an rbcL gene database of the plants from the GFRR and determine the botanical composition of the black rhinoceros dung from the GFRR. Due to the limited number of rbcL gene plant sequences from the GFRR deposited in the GenBank database, 18 plant species from the GFRR were sequenced. Sequence analyses between the partial rbcL gene sequences generated were able to distinguish between plants down to species level. Plant species from the family Euphorbiaceae and Fabaceae showed sequence variation at intra-specific level compared to those of Tiliaceae which were more conserved. The generated rbcL gene sequences from seasonal dung samples were compared to the rbcL gene sequenced from 18 plant species obtained from the GFRR and those from the GenBank database. A wide range of plant species were identified from the dung samples. There were no major differences in botanical composition between the dung samples, except that Grewia spp. were found to dominate in almost all seasons. The results obtained on the free radical scavenging activity of the extracts against 2,2-Diphenyl-l-picrylhydrazyl (DPPH) increased in the order of methanol > ethyl acetate > chloroform. The DPPH free radical scavenging activity of the methanol plant extracts increased in the order Brachylaena elliptica > Plumbago auriculata > Grewia robusta > Azima tetracantha. Methanol extracts on the TLC plate sprayed with Fe³⁺-2,4,6-Tri-2-pyridyl-s-triazine (TPTZ) showed that the compounds present in the extracts react differently to ferric ion, with most compounds unable to reduce ferric ion. Furthermore the methanol extracts were able to exhibit reduction potentials vs. Ag/AgCl at low concentrations. The compounds in the extracts were shown to be phenolic acids and flavonoid glycosides.