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Les substances naturelles issues de la biomasse des végétaux ont des intérêts multiples mis à profit dans la biotechnologie tant dans l’industrie alimentaire, cosmétique que pharmaceutique. Parmi ces composés on retrouve une grande partie des métabolites secondaires qui se sont surtout illustrés en thérapie. On a longtemps employé des remèdes traditionnels à base de plantes sans savoir à quoi étaient dues leurs actions, les études des métabolites secondaires font l’objet de nombreuses recherches basées sur les cultures in vitro et in vivo de tissus végétaux. C’est le cas notamment des composés phénoliques, polyphénols, flavonoïdes, anthocyanines et tanins, qui font l’objet de notre étude, composés largement utilisés en thérapeutique comme vasculoprotecteurs, anti-inflammatoires, inhibiteurs enzymatiques, antioxydant et anti radicalaires. Nos travaux ont portés sur les extraits issus des 6 plantes (Daniella oliveri, Desmodium adscendens, Ficus capensis, Securidaca longependuculata, Stevia rebaudiana, Vitex doniana) utilisées traditionnellement pour traiter plusieurs maladies dans diverses parties du monde (Amérique, en Afrique). La méthodologie mise au point pour l’analyse de ces extraits a été appliquée pour le dosage des exsudats racinaires de Miscanthus x géant). La combinaison de certaines méthodes d’analyses chimiques, spectrophotométries (UV, RMN, SM), chromatographies (CCM, CC, HPLC, GC-MS) et biologiques nous ont permis de faire une évaluation quantitative, qualitative des composés phénoliques extraits des 7 plantes d’origines diverses utilisés par la médecine traditionnelle. Les analyses complémentaires ont permis de mettre en évidence les capacités antioxydantes et anti-radicalaires de ces extraits. Tandis que les tests biologiques ont été utilisés pour l’évaluation de certaines propriétés telles que les propriétés antimicrobiennes et anti-inflammatoires. Les résultats de ces travaux nous ont permis d’affirmer que l’ensemble des extraits de plantes étudiés présente des très bonnes propriétés antioxydantes qui pourrait nous permettre de les recommander dans la biotechnologie. / Natural substances from biomass plant have taken advantage of multiple interests in the biotechnology industries both in food, cosmetic and pharmaceutical. Between these compounds are found much of secondary metabolites which are mainly illustrated in therapeutics. Traditional medicines has used for long time plant materials for healing without knowing what had caused their actions, then studies of secondary metabolites are the subject of numerous studies based on in vitro and in vivo cultures of plant tissues. These include phenolic compounds, polyphenols, flavonoids, anthocyanins, tannins ... which are the subject of our study, compounds widely used in therapeutics as vasculoprotective, anti-inflammatory, enzyme inhibitors, antioxidant and anti free radical. Our work has brought the excerpts from six plants (Daniella oliveri, Desmodium adscendens, Ficus capensis, Securidaca longependuculata, Stevia rebaudiana, and Vitex doniana) traditionally used to treat many diseases in various parts of the world (America, Africa, Asia). The methodology developed for the analysis of these extracts has been applied for the determination of root exudates of Miscanthus x giganteus. The combination of some methods of chemical analysis, spectrophotometry (UV, NMR, MS), chromatography (TLC, CC, HPLC, GC-MS) and biology allowed us to evaluate quantitative and qualitative phenolic extracts from these plants materials. Further analysis helped to highlight the antioxidant capacity and anti-radical of these extracts. While biology tests were used for the evaluation of certain properties such as antimicrobial and anti inflammatory. The results of this work have allowed us to conclude that all plant extracts studied has very good antioxidant properties that may allow us to recommend them in biotechnology.

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Nos últimos anos, a pesquisa ambiental tem se defrontado com a questão dos chamados disruptores endócrinos (EDCs). A estes compostos, tais como: produtos farmacêuticos, hormônios naturais e sintéticos, pesticidas, substâncias tensoativas, polímeros de baixa massa molecular e diversos outros contaminantes orgânicos presentes em efluentes municipais e industriais, atribui-se à capacidade de alterar o funcionamento do sistema endócrino. Estrogênios e progestogênios, naturais ou sintéticos, são excretados pela urina de mamíferos, e uma pequena porção nas fezes, e via efluentes de estações de tratamento de esgoto (ETE) entram em vias aquáticas, podendo causar alterações em organismos aquáticos, tais como feminização ou hermafroditismo. Neste contexto, no presente trabalho foi descrito uma metodologia analítica para a extração em fase sólida empregando cartucho C18, dos hormônios naturais, estrona (E1) e 17?-estradiol (E2), e dos hormônios sintéticos, levonorgestrel e 17?-etinilestradiol (EE2) (presentes em anticoncepcionais orais), a partir de uma matriz de esgoto sintético. Foram utilizados dois sistemas cromatográficos neste estudo, ambos de mesmo modelo (SLC-10A, Shimadzu), os quais consistiram em um injetor manual (seringa), com um volume de injeção ajustado para 20µL, e duas bombas modelo LC-10ADVP (Shimadzu). Foram utilizados dois tipos de detectores, um sistema DAD modelo SPD-M10AVP (Shimadzu) e um espectrofluorímetro modelo RF-551 versão 2.4 (Shimadzu). A separação foi feita em coluna C18 (250 X 4,6 mm, 5 µm) com um fluxo de 1 mL min-1. A condição ideal para separação foi o modo isocrático: 48/52 ACN:H2O. O comprimento de onda selecionado para quantificação no sistema de detecção DAD foi: 240nm para o levonorgestrel e 280nm para E1, E2 e EE2. No detector espectrofluorímetro, os comprimentos de onda selecionados para a excitação e emissão dos analitos E1, E2 e EE2 foram: 280nm e 306nm, respectivamente. Para se efetuar o estudo de recuperação, uma matriz simulando esgotos sanitários foi utilizada com o intuito de se ter uma amostra controle (testemunha) livre dos analitos de interesse, devido à dificuldade em se obter uma amostra de esgoto “real" livre destes hormônios. As amostras de esgoto sintético foram fortificadas em três níveis de concentração. Tomou-se 5 replicatas de 100,0mL de amostra testemunha (esgoto sintético) para cada nível de fortificação, estas amostras foram dopadas com os hormônios estudados. Após adaptações de metodologias descritas na literatura, a extração foi feita segundo o procedimento: condicionamento do cartucho (500mg/6mL) com 7mL de acetonitrila, 5mL de metanol e 5mL de água em uma razão de fluxo de 3mL min-1; percolagem de 250,0mL de amostras de esgoto bruto e efluentes tratados pela ETE-Araraquara com fluxo de 1mL min-1; secagem do cartucho por 1 hora a vácuo; eluição dos analitos com 6mL de acetonitrila com fluxo de 1mL min-1; secagem do extrato eluído em corrente de nitrogênio; reconstituição da amostra em 0,5mL de metanol. As amostras de esgoto sintético foram dopadas com os padrões estudados, para análise realizada no sistema DAD, a 0,250?g L-1 para levonorgestrel e 2,50?g L-1 para E1, E2 e EE2 (nível 1); 0,375 ?g L-1 para levonorgestrel e 3,75 ?g L-1 para E1, E2 e EE2 (nível 2); 0,500?g L-1 para levonorgestrel e 5,00?g L-1 para E1, E2 e EE2 (nível 3). Para análise realizada no sistema fluorescente, as amostras de esgoto sintético foram dopadas com os padrões estudados a 0,750?g L-1 para E1; 0,150?g L-1 para E2 e 0,250?g L-1 para EE2 (nível 1); 1,00 ?g L-1 para E1; 0,150?g L-1 para E2 e 0,200?g L-1 para EE2 (nível 2); 1,25?g L-1 para E1; 0,250?g L-1 para E2 e 0,300 ?g L-1 para EE2 (nível 3). Valores de recuperação entre 83-123% com coeficientes de variação menores do que 13,5% foram obtidos para todos os hormônios analisados pelos dois sistemas de detecção (DAD e Fluorescente). Esses dados demonstram a eficiência do método quanto à exatidão e precisão para os níveis de fortificação estudados. O método proposto foi utilizado para avaliar a presenças dos hormônios em afluentes (esgoto bruto) e efluentes da ETE-Araraquara. As amostras coletadas na ETE foram analisadas em triplicata. Foi identificado e quantificado o hormônio natural E2 (31ng L-1) em amostras obtidas antes do tratamento de esgoto. Não foram detectadas concentrações dos analitos em amostras obtidas após o tratamento. / In recent years environmental research has been faced with the issue of the endocrine disrupting chemicals (EDCs). Such compounds, such as: pharmaceutical products, natural and synthetic hormones, pesticides, tensive active substances, low mass molar polymers and many other organic contaminants that appear in municipal and industrial effluents, have the capacity of altering the manner in which the endocrine system works. Natural or synthetic estrogens and progestogens are excreted through the urine of mammals, and a small portion through faeces, and via effluents from sewage treatment plants (STP) flow into aquatic ducts, with the possibility of causing alterations in the aquatic organisms, such as feminization or hermaphroditism. Within this context, the present work describes an analytic methodology for solid phase extraction (SPE) using C18 cartridge of the natural hormones, estrone (E1) and 17?-estradiol (E2), and the synthetic hormones levonorgestrel and 17?-ethinylestradiol (EE2) (found in oral contraceptives) from a synthetic waste matrix. Two chromatographic systems were used in this study, both from the same model (SLC-10A, Shimadzu), a DAD system model SPD-M10AVP (Shimadzu) and a spectrofluorimeter model RF-551 type 2.4 (Shimadzu). Separation was performed in column C18 (250 X 4,6 mm, 5 ?m) with a flux of 1 mL min-1. The ideal separation condition was the isocratic mode: 48/52 ACN:H2O. To carry out the recuperation study, a matrix simulating sewers was used with the objective of having a control sample (witness) free of the samples under scrutiny of the difficulty in obtaining a “real" sewage sample free of these hormones. The samples of synthetic sewage were boosted in three levels of concentration. Recuperation values between 83-123% with variation coefficients lower than 13,5% were obtained for all studied hormones by both systems of detections (DAD and Fluorescent). These data demonstrate the efficiency of the method concerning the accuracy and precision for the fortification levels that were studied. The proposed method was used to assess the presence of hormones in inffluents (raw sewage) and effluents of Araraquara-STP. The natural hormone E2 (31ng L-1) was identified and quantified in samples obtained prior to sewage treatment. No concentrations of analytes in the samples were obtained after the treatment.

Superoxidanionen (O2˙‾) sind eine von mehreren sogenannten reaktiven Sauerstoffspezies, die im menschlichen Körper intra-, aber auch extrazellulär vorkommen. Verschiedene Enzyme, z.B. in der mitochondrialen Atmungskette, die NADPH-Oxidase oder endotheliale NO-Synthasen bilden O2˙‾. Da es sich um eine sehr reaktive Substanz handelt, die mit der DNA sowie mit Proteinen und Lipiden interagiert und diese schädigen kann, spielt sie bei kardiovaskulären Erkrankungen wie etwa der chronischen Herzinsuffizienz, Hypertonie oder Arteriosklerose eine große Rolle, ist aber auch an vielen anderen Erkrankungen wie z.B. dem Diabetes mellitus pathophysiologisch beteiligt. Dies macht verständlich, dass es für die Forschung von entscheidender Bedeutung ist, Methoden zu entwickeln, die zur Erkennung und Quantifizierung von O2˙‾ geeignet sind. Bereits heute gibt es verschiedene Methoden, O2˙‾ nachzuweisen. Jede dieser Methoden hat jedoch ihre ganz spezifischen Vor- aber auch Nachteile. Wir haben eine neue, einfache, sehr schnelle und sensitive HPLC-Methode mit einem internen Standard entwickelt, mit der die O2˙‾-Produktion in Endothelzellen und aortalem Gewebe gut zu messen ist. Sie beruht auf der Tatsache, dass Dihydroethidium (DHE) mit O2.- zu 2-Hydroxyethidium (2-OH-E+) reagiert. Nach Trennung mittels HPLC wurde die Menge an entstandenem 2-OH-E+ durch einen elektrochemischen Detektor gemessen. Die Proben wurden durch isokrate Elution aufgetrennt, was bisher bei der Detektion von 2-OH-E+, DHE und O2˙‾ mit vielen Nachteilen verbunden war. Durch eine spezielle mobile Phase, die ein Ionen-Paar-Reagens enthielt, konnte diese Form der Elution nun auch zur Erkennung von O2˙‾ angewandt werden. DHE und seine Reaktionsprodukte konnten nicht nur eindeutig aufgetrennt werden, sondern die Auftrennung erfolgte auch sehr schnell in nur etwa 15min, was gegenüber älteren Methoden einen eindeutigen Zeitvorteil bringt. Anstatt zwei benötigten wir darüber hinaus nur eine Pumpe, was ebenfalls ein Vorteil der isokraten Elution ist. Wir erreichten auch über längere Messreihen stabile Bedingungen, da für die isokrate Elution die mobile Phase nicht verändert werden muss. Des Weiteren haben wir 3,4-Dihydroxyzimtsäure als internen Standard eingeführt, der sich hinsichtlich seiner Retentionszeit als sehr geeignet erwies und mit einem elektrochemischen Detektor klar und eindeutig nachweisbar war. Dies bietet große Vorteile gegenüber Methoden ohne internen Standard. Veränderungen der Konzentrationen von DHE, 2-OH-E+ und Ethidium aufgrund von Verdampfen des Lösungsmittels Methanol können ebenso erkannt werden wie Ungenauigkeiten während der Präparation sowie Schwankungen im HPLC-System, wie sie etwa bei langen Messreihen durch Auswaschungs-Effekte oder Verunreinigungen auftreten können. Da sich die Konzentration des internen Standards 3,4-Dihydroxyzimtsäure stets mitverändert, können die Messwerte normalisiert werden und somit die Verfälschungen aufgehoben werden. Dem zu Folge sind Messungen mit einem internen Standard gegenüber solchen ohne internen Standard deutlich valider. Sowohl die Stimulation von humanen aortalen Endothelzellen (HAEC) mit Glukose bzw. Tumornekrosefaktor α, als auch die Infusion von Angiotensin II bei männlichen Mäusen mit anschließender Untersuchung der Aorta führt bekanntermaßen zu einem Anstieg von O2˙‾. Dieser Effekt konnte nun auch mit unserer neu etablierten HPLC-Methode nachgewiesen werden. Ebenfalls war ein Anstieg des aortalen O2˙‾-Spiegels bei Ratten nach induziertem Myokardinfarkt bereits in mehreren früheren Arbeiten beschrieben worden. Dieser lag auch bei Messung mit unserer neu etablierten HPLC-Methode eindeutig vor. Die Signale waren hierbei für die untersuchten Substanzen 2-OH-E+, DHE sowie für den internen Standard 3,4-Dihydroxyzimtsäure eindeutig und gut voneinander getrennt. Zusammenfassend konnte somit gezeigt werden, dass sich anhand mehrerer etablierter in vitro und in vivo Modelle erhöhter Sauerstoffradikal-Produktion der Anstieg von O2˙‾ auch mit unserer neuen Variante der HPLC mit isokrater Elution, internem Standard und Messung mittels elektrochemischem Detektor nachweisen ließ. Es handelt sich um eine zuverlässige und sensitive Methode, die zusätzliche Vorteile für die Messung von O2˙‾ mit sich bringt. / A new method of measuring superoxid anion in cardiovasvascular tissue.

Die Natur eröffnet mit der strukturellen Vielfalt ihrer Sekundärmetaboliten einen nahezu unerschöpflichen Pool in der Leit- und Wirkstoffsuche nach pharmazeutisch wirksamen Substanzen. Insbesondere die Alkaloide zeichnen sich durch ihre biologischen Wirksamkeiten aus. Eine noch junge, sehr vielversprechende Substanzklasse stellen die sogenannten Naphthylisochinolin-Alkaloide dar, die bislang ausschließlich in den beiden Pflanzenfamilien der Ancistrocladaceae und Dioncophyllaceae gefunden wurden. Im Rahmen dieser Arbeit wurden Extrakte von Ancistrocladus congolensis (A.c.), Triphyophyllum peltatum (T.p.) und Dioncophyllum thollonii (D.t.) untersucht. Hierbei gelang die Isolation und Strukturaufklärung des bereits bekannten Korupensamin A (A.c.) sowie von sechs bislang unbekannten Alkaloiden: Ancistrocongolin A-D (A.c.), Habropetalin A (T.p.) und Dioncophyllin E (D.t.). Zu dem letztgenannten wurde ein synthetischer Zugang evaluiert. Alle neu isolierten Naturstoffe wurden einer biologischen Aktivitätstestung zugeführt. Im analytischen Bereich der Arbeit gelang die vollständige Strukturzuordnung des bereits seit mehreren Jahren bekannten Tetralons Isoshinanolon, was somit nun ein einfache Analytik für die Bestimmung der absoluten Konfiguration an die Hand gibt. Des Weiteren wurde die HPLC-CD-Kopplung als schnelle und praktikable chirale on-line-Analytik an mehreren Beispielen (Phyllin, TaClo, Murrastifolin F, Cyclorocaglamid, Thalidomid) sowohl im phytochemischen als auch synthetischen Bereich eingeführt und etabliert. / Nature with its wide structural diversity of its secondary metabolites offers an almost inexhaustible source of new structures in the search for pharmaceutical active substances. Especially the alkaloids show remarkable biological activity. A young and very promising substance class are the so-called naphthylisoquinoline alkaloids, which are so far exclusively found in the two plant families of the Ancistrocladaceae and Dioncophyllaceae. In this thesis the extracts of Ancistrocladus congolensis (A.c.), Triphyophyllum peltatum (T.p.) and Dioncophyllum thollonii (D.t.) were examined. Hereby the isolation and structural elucidation of the already known korupensamine A (A.c.) and of six so far unknown alkaloids succeeded: Ancistrocongoline A-D (A.c.), habropetaline A (T.p.) and dioncophylline E (D.t.). The synthetic access to the latter was evaluated. All isolated natural products were subjected to biological testing. Regarding the analytical field, the entire identification of the absolute configuration of the tetralone isoshinanolone was achieved, which gives a simple chiral analytical method at hand to determine the absolute configuration. Furthermore, the HPLC-CD coupling was successfully introduced to phytochemical as well as chemical analysis. This was demonstrated with several examples (phylline, TaClo, murrastifoline F, cycloracaglamide, thalidomide), establishing this technique as a fast and practicable chiral on-line analytic device.

In this work, the radiolysis mechanisms in solids as well as in liquid and frozen aqueous solutions have been studied. Liquid chromatography tandem mass spectrometry has evolved so that mass spectral information can now be used to determine the most probable structures of radiolysis products, even those present in traces amounts. Theoretical routes explaining the formation of radiolysis products can be deduced from their structures. The development of strategies to limit the degradation of the active pharmaceutical ingredient during irradiation of the drug requires a better knowledge of the radiolysis mechanisms responsible for the drug degradation. Metoclopramide, an antiemetic drug, has been selected as a model, due to the variety of its chemical bonds. In the solid state, radiation-induced degradation of the drug was very low (<0.1%) and only four radiolysis products were detected in traces. The “major” radiolysis product was formed after the loss of the chlorine by dissociative electron capture. For metoclopramide liquid aqueous solutions, the loss of the drug was important (~30% loss at 25 kGy) and several radiolysis products were detected. The majority of the degradation products were generated following the attacks of hydroxyl radicals and aqueous electrons. The loss of metoclopramide could be lowered up to acceptable levels (<10% loss) provided that radioprotective additives were added and the irradiation dose was limited to 15 kGy, which could be sufficient to reach the required SAL. The selected excipients were mannitol (which reacts mainly with the hydroxyl radical), nicotinamide and pyridoxine that react with both the aqueous electron and the hydroxyl radical. The irradiation of frozen aqueous solutions allowed to minimize the loss of active substance even for a 25 kGy dose. This approach seems to be the most promising method for terminal sterilization of aqueous solutions by ionizing radiations. The major radiolysis product was formed after the attack of the electron. Some of the radiolysis products detected were attributed to the attack of the hydroxyl radical, demonstrating the feasibility of a reaction between the hydroxyl radical from ice radiolysis and the solute. A comparison was performed with irradiated frozen solutions of metoprolol, which has been studied in liquid aqueous solutions (C. Slegers’ thesis). Degradation of metoprolol when irradiated in frozen solutions was negligible.

The emerging scientific evidences on the role of food components in prevention of several chronic diseases are the momentum for shifting from a traditional focus on production to enhancement of nutritional quality. To further understand the role of these phytochemicals this dissertation describes the development of rapid analytical and isolation methods, and the effect of production systems and processing techniques on the levels of phytochemicals in citrus fruits. In the first study, a simultaneous high performance liquid chromatography (HPLC) method for the rapid analysis of amines and organic acids was developed. The simultaneous extraction and analysis of samples provides an economical method for analyzing a large number of samples. In the second study, rapid separation method of potent health beneficial phytochemicals such as polymethoxyflavones from citrus peels using flash chromatography was developed. Using the developed method, five polymethoxyflavones were separated and isolated with high purity in gram level quantity. In the third study, the levels of phytochemicals in organically and conventionally grown lemons and their storage at market simulated conditions were determined. Results suggest that organically produced citrus fruits have higher content of organic acids and flavonoids than conventionally produced. The fourth and fifth study determined the influence of household processing (blending, juicing, hand squeezing techniques) and emerging processing (high pressure processing [HPP], thermal processing) on the phytochemicals content of ‘Rio Red’ grapefruits. Fruits processed by blending had significantly higher levels of flavonoids, furocoumarins and limonin compared to juicing and hand squeezing, while HPP enabled in extending the shelf life of the processed juice without any adverse effects. Therefore, consuming grapefruit juice processed by blending may provide higher levels of health beneficial phytochemicals. The sixth study describes a rapid flash chromatography method for isolation of PMFs and furocoumarins from citrus industrial by products such as peel oil. In the seventh study the developed method was applied to isolate 10 different phytochemicals from an unexplored citrus species, Miaray mandarin (Citrus miaray TAN.). Among them, the 5,7,8,3',4' pentamethoxyflavone was isolated for the first time from the genus Citrus.