Made available in DSpace on 2014-06-11T19:22:59Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-06-08Bitstream added on 2014-06-13T20:29:34Z : No. of bitstreams: 1 silva_avr_me_rcla.pdf: 1706887 bytes, checksum: eda68ea29b93397b581e427121535611 (MD5) / Neste trabalho estudou-se a estrutura, função e mecanismo de ação do peptídeo antibacteriano Protonectarina-MP (isolado de veneno da vespa social Protonectarina sylveirae) tendo seu resíduo C-terminal nas formas ácida (-OH) e amidada (-NH2). Os peptídeos foram sintetizados, utilizando-se a estratégia Fmoc, purificados por cromatografia líquida de alta performance. O monitoramento do material sintético foi feito por espectrometria de massas ESI-MS e por seqüenciamento através de Química Degradativa de Edman. A estrutura secundária foi investigada pelo uso de espectroscopia de dicroísmo circular e modelagem molecular. Atividade lítica (extravasamento) e interação do resíduo de triptofano em vesículas foram investigadas pelo uso de espectrômetro de fluorescência. Foram realizados ensaios sobre as interações desses peptídeos em meio de vesículas zwitteriônicas e aniônica, formando complexos proteolipossomos que foram submetidos à troca isotópica H/D monitorada por espectrometria de massas ESI-MS e MS/MS. Além disso, foram realizados ensaios biológicos de atividade hemolítica, de desgranulação de mastócito, de liberação da enzima citoplasmática Lactato Desidrogenase e de atividade antimicrobianas. Os dados de CD revelam uma tendência dos peptídeos se estruturarem em hélice-α em ambiente hidrofóbico e em ambiente de membranas. Porém, o mesmo não pode ser observado em meio aquoso. Os modelos obtidos para ambos os peptídeos por modelagem molecular mostram uma estruturação em hélice-α anfipática. Nos ensaios de atividade lítica em vesículas, os peptídeos apresentaram um processo com cooperatividade positiva, com curvas de dose-resposta que mostram uma dependência sigmoidal com a concentração do peptídeo. Os resultados da fluorescência do triptofanos mostram um deslocamento da emissão para a região de onda do azul para o peptídeo... / In the present work was studied the structure, function and mechanism of action of the antibacterial peptide Protonectarina-MP (isolated from venom of social wasp Protonectarina sylveirae) with its carboxyamidation (-NH2) and carboxyl-free (-OH) Cterminal forms. The peptides were manually synthesized on-solid phase by using Fmoc strategy and purified under HPLC. The homogeneity of the synthetic material was analyzed by ESI mass spectrometry and Edman Degradation Chemistry. The secondary structure was investigated through circular dichroism (CD) spectroscopy and molecular modeling. Lytic activity and peptides interaction with the membranes was also investigated through tryptophan emission, by fluorescence spectrometry. The interaction of peptides with zwitterionic and anionic vesicles was investigated through the combination of H/D exchange and ESI-mass spectrometry. Some biological activities, like: mast cell degranulation, release of cytoplasmic enzyme lactate dehydrogenase, hemolysis and antibiosis were investigated for both peptides. The CD spectra revealed that the peptides in hydrophobic environments or in presence of biological membranes have the tendency to form helix conformations; however, organized structures were not observed in aqueous or buffer solutions. The models obtained by molecular modeling show that both peptides form an amphipathic α-helix. The peptides presented a positive cooperative process in the lytic activity of vesicles, with dose-response curves presenting a sigmoidal dependence with the peptide concentration. The results of the fluorescence of tryptophans showed a shift of the emission wavelength to the blue region of the peptide Protonectarina-MP (-NH2), which was not observed for its analogue presenting the C-terminal residue in free acid form. This is indicating a greater interaction of the amidated peptide in membranes, when compared to the peptide... (Complete abstract click electronic access below)

Made available in DSpace on 2014-12-17T14:03:32Z (GMT). No. of bitstreams: 1 CinthiaBST.pdf: 1239764 bytes, checksum: b0f986e10d6574ec55e837255b634b41 (MD5) Previous issue date: 2010-02-11 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / The exopolysaccharides are extracellular compounds produced by some species of fungi and bacteria. It is suggested that these molecules, even when in the form of complex polysaccharide-peptide, are the main bioactive molecules of many fungus. Some of the biological activities displayed by these compounds can be accentuated and others may arise when you add chemically polar or nonpolar groups to polysaccharides. The fruiting body of Pleurotus sajor-caju produces a heteropolysaccharide with antineoplastic and antimicrobial activity, but other biological activities of this polymer have not been evaluated. In this work the exopolysaccharide of Pleurotus sajor-caju was sulfated chemically and structurally characterized. We also evaluated the antiproliferative, antioxidant and anticoagulant activities from native exopolysaccharide (PN) and its sulfated derivated (PS). Polyacrylamide gel electrophoresis, infrared spectroscopy and nuclear magnetic resonance (??C) proved successful in sulfation of PN to obtain PS. Analysis by gas chromatography-mass spectroscopy showed that PN and PS are composed of mannose, galactose, 3-O-methyl-galactose and glucose in proportion percentage of 44,9:16,3:19,8:19 and 49, 7:14,4:17,7:18,2, respectively. The percentage of sulfate found in PS was 22.5%. Antioxidants assays revealed that the sulfation procedure affects differently the activities of exopolysaccharides, while the total antioxidant capacity, the scavenging activity of superoxide radical and ferric chelating were not affected by sulfation, on the other hand the chemical modification of PN enhanced the scavenging activity of hydroxyl radical and reducing power. PS also showed anticoagulant activity in a dose-dependent manner and clotting time was 3.0 times higher than the baseline value in APTT at 2 mg/mL. The exopolysaccharide not presented antiproliferative activity against HeLa tumor cells, but PS affects the cellular proliferation in a time-dependent manner. After 72 h, the inhibition rate of PS (2.0 mg/mL) on HeLa cells was about 60%. The results showed that PN sulfation increase some of their activities. / Os exopolissacar?deos s?o compostos extracelulares produzidos por algumas esp?cies de fungos e bact?rias. ? sugerido que estas mol?culas, inclusive quando na forma de complexo polissacar?deo-pept?deo, s?o as principais mol?culas bioativas de v?rios fungos. Muitas das atividades biol?gicas apresentadas por esses compostos podem ser acentuadas e outras podem surgir quando se adiciona quimicamente aos polissacar?deos grupamentos polares ou apolares. O corpo de frutifica??o de Pleurotus sajor-caju produz um heteropolissacar?deo com atividade antioneopl?sica e antimicrobiana, contudo outras atividades biol?gicas desse pol?mero ainda n?o foram avaliadas. Neste trabalho o exopolissacar?deo de Pleurotus sajor-caju foi sulfatado quimicamente e caracterizado estruturalmente. Tamb?m foram avaliadas as atividades antiproliferativa, antioxidante e anticoagulante do exopolissacar?deo nativo (PN) e de seu derivado sulfatado (PS). Eletroforese em gel de agarose, espectroscopia de infravermelho e resson?ncia magn?tica nuclear (??C) comprovaram o sucesso na sulfata??o de PN para a obten??o de PS. An?lise por cromatografia gasosa acoplada a espectroscopia de massa mostrou que PN e PS s?o constitu?dos de manose, galactose, 3-O-metil-galactose e glicose na propor??o percentual de 44,9:16,3:19,8:19 e 49,7:14,4:17,7:18,2, respectivamente. O percentual de sulfato encontrado em PS foi de 22,5%. Testes antioxidantes revelaram que o processo de sulfata??o influencia de forma diferente nas atividades do exopolissacar?deo. Enquanto a capacidade antioxidante total, a capacidade de seq?estro de radical super?xido e a quela??o f?rrica n?o foram influenciadas pela sulfata??o, essa potencializou a atividade seq?estradora de radicais hidroxila e o poder redutor do exopolissacar?deo. Ap?s o processo de sulfata??o o exopolissacar?deo passou a apresentar atividade anticoagulante de forma dose-dependente, triplicando o tempo de coagula??o em rela??o ao controle numa concentra??o de aproximadamente 2 mg/mL. O exopolissacar?deo n?o apresentou atividade antiproliferativa frente ?s c?lulas tumorais HeLa, por?m ap?s sulfata??o ele passou a apresentar essa atividade de forma tempo- ependente, chegando a inibir em 60% a taxa de prolifera??o das c?lulas com 2 mg/mL, ap?s 72 h de exposi??o. Os resultados aqui obtidos mostraram que a sulfata??o do exopolissacar?deo potencializou algumas de suas atividades.

As estruturas e propriedades biológicas das amidas piplartina e a piperina, isoladas respectivamente de Piper tuberculatum e P. nigrum, inspiraram a síntese de 89 derivados e 7 esters estruturalmente relacionadas. As preparações envolveram metodologias tradicionais e os compostos purificados tiveram suas estruturas caracterizadas por análises espectroscópicas e espectrométricas. Os estudos de fragmentação por IE e IES indicaram a clivagem preferencial da ligação N-CO no caso das cinamamidas, dienamidas e cinamimidas. Estudos computacionais envolvendo afinidade protônica e energias de ligação confirmaram a fragmentação preferencial da ligação amídica para as amidas. A citotoxicidade de 89 substâncias foi avaliada contra três células leucêmicas (K562, Nalm6 e Raji) e a partir dos valores de IC50 foram realizados estudos de relação estrutura-atividade (SAR). As linhagens K562 e a Nalm6 foram a mais resistente e vulnerável, respectivamente, e as amidas piplartina (1a), N-Ciclohexil-N-(ciclohexilcarbamoil)-3-(3,4,5-trimetoxifenil)propanamida (1n), e (E)-N,N-dibutil-3-(3,4-dimetoxifenil)acrilamida (13h) foram as mais ativas com IC50 de 0,34 µM; 0,84 µM e 1,88 µM contra K562 e (E)-N-ciclohexil-N-(ciclohexilcarbamoil)-3-(3,4-dimetoxifenil)acrylamida (13i) com IC50 de 0,98 µM contra Nalm6. A avaliação de atividade leishmanicida de 18 substâncias não se mostrou promissora. As abordagens qualitativas e quantitativas foram feitas baseadas nos descritores moleculares gerados pelo programa VolSurf+. A partir de métodos quimiométricos tais com PLS, algoritmo genético, árvores de decisão foi possível gerar modelos para correlacionar às propriedades moleculares com a atividade biológica. As propriedades de absorção, distribuição, metabolismo e excreção e os equilíbrios entres as regiões hidrofílicas e hidrofóbicas foram importantes para atividade citotóxica. O estudo de ancoragem molecular mostrou que as amidas (E)-N,N-dibutil-3-(3,4,5-trimetoxifenil)acrilamida (1l), 1n, (E)-3-(4-clorofenil)-N-ciclohexil-N-(ciclohexilcarbamoil)acrilamida (5a), 13h e 13i podem atuar como inibidores das histonas desacetilases particularmente HDAC4 e HDAC8. / The structures and biological properties of the amides piplartine and piperine isolated from Piper tuberculatum and P. nigrum respectively, inspired the synthesis of derivatives 89 and 7 esters structurally related. Their preparations were achieved using classical procedures and the purified amides were submitted to spectroscopic and spectrometric characterization. The study of fragmentation process by EI and ESI suggested the preferential cleavage of the N-CO bond of cinnamamides, dienamides and cinnamimides. The cytotoxicity of 89 compounds was evaluated against three leukemic cells (K562, Nalm6 and Raji) and based on IC50 values the structure-activity relationship (SAR) was performed. While the K562 and Nalm6 cells were the more resistant and more sensitive, respectively, the amides piplartine (1a), N-cyclohexyl-N-(ciclohexylcarbamoyl)-3-(3,4,5-trimethoxyphenyl)propanamide (1n) and (E)-N,N-dibutyl-3-(3,4-dimethoxyphenyl)acrylamide (13h) were in general the most active with IC50 of 0.34 µM, 0.84 µM and 1.88 µM against K562 and (E)-N-cyclohexyl-N-(ciclohexylcarbamoyl)-3-(3,4-dimethoxyphenyl)acrylamide (13i) with IC50 of 0.98 µM against Nalm6. The evaluation of leishmanicidal activity of 18 substances was also performed but was not promising. Qualitative and quantitative approaches were made based on molecular descriptors generated by VolSurf+ program. The chemometric methods such as PLS, genetic algorithm, decision trees generated models to correlate molecular properties with the biological activity. The absorption, distribution, metabolism and excretion properties and a balance between hydrophilic and hydrophobic moieties of the amides were important for an optimized activity. The molecular docking revealed that amides such as (E)-N,N-dibutyl-3-(3,4,5-trimethoxyphenyl)acrylamide (1l), 1n, (E)-3-(4-chlorophenyl)-N-cyclohexil-N-(cyclohexylcarbamoyl)acrylamide (5a), 13h and 13i have potential to act as possible inhibitors of histone deacetylase proteins particularly HDAC4 and HDAC8.

La région sahariennne est une des zones les plus défavorables à la croissance et développement des espèces animales. Cependant, peu d’espèces possèdent à la fois des mécanismes morphologiques et écophysiologiques, assurant leur survie dans les sols arides et sur les dunes de sable.En effet, on peut estimer que le métabolisme secondaire biosynthétise des quantités considérables de composés bioactifs, destinés à assurer le développement et la continuité de ces espèces, notamment leur survie dans ces conditions sahariennes précaires.Les propriétés pharmacologiques des extraits de plantes sahariennes peuvent apporter des bénéfices dans la guérison de certaines maladies microbiennes ou prolifératives ou également contribuer au développement de certaines activités antioxydantes.L'étude des propriétés toxiques vise donc à enrichir la connaissance du potentiel bioactif des plantes sahariennes.Ces aspects de la puissance du métabolisme furent étudiés chez certaines espèces végétales strictement sahariennes. Il s’agit de : Anthyllis henoniana Coss., Centropodiaforskalii (Vahl) Cope, Cornulaca monacantha Delile, Ephedra alata var. alenda (Stapf.)Trabut, Euphorbia guyoniana Boiss & Reut., Henophyton deserti Coss. & Durieu,Hélianthemum confertum Dunal, Moltkiopsis ciliata (Forssk.) I.M.Johnst. et Spartidiumsaharae (Coss. & Durieu) Pomel. Les principaux résultats obtenus montrent que ces espèces possèdent des propriétés intéressantes, susceptibles d’être utiles pour le traitement de certaines maladies humaines.En revanche, parfois à certaines concentrations, des extraits de ces espèces peuvent présenter des effets toxiques sur les organismes.En dépit des conditions extrêmes, le Sahara représente la zone de développement d'une certaine diversité biologique, et principalement des espèces de plantes précieuses dont une meilleure connaissance scientifique de leur propriétés phytochimiques se révèle indispensable. / The Saharan desert is one of the most unfavorable areas, to the plant life. However, a small number of plants possesses both morphological and ecophysiological mechanisms ensuring their survival in the arid soil and on the sand dunes.It can be estimated that the secondary metabolism biosynthesizes considerable amounts of bioactive compounds, meant to ensure the development and the continuity of these species.The pharmacological properties of saharan plant extracts may bring benefits in the healing of certain microbial or proliferative diseases or contributes to the supply of antioxidants activities.The study of toxic properties is meant to enrich the knowledge of Saharan plants’bioactive potential.The biological activities of Anthyllis henoniana Coss., Centropodia forskalii (Vahl)Cope, Cornulaca monacantha Delile, Ephedra alata var. alenda (Stapf.) Trabut,Euphorbia guyoniana Boiss & Reut., Henophyton deserti Coss. & Durieu, Helianthemum confertum Dunal, Moltkiopsis ciliata (Forssk.) I.M.Johnst. and Spartidium saharae (Coss.& Durieu) Pomel have been studied. It can be seen that these species possess interesting properties, capitalized in the treatment of some human diseases. But, on the other hand in certain concentrations, extracts from these species may exhibit toxic effects onorganisms.Despite the extreme conditions, the Saharan desert represents the area of development for some valuable plant species, whose scientific knowledge is necessary.

La résistance aux antibiotiques ou la difficulté de plus en plus croissante à traiter les maladies actuelles avec les composés disponibles sur le marché ont contraint les chercheurs à trouver de nouvelles sources de molécules actives. Les lichens produisent divers composés biologiquement actifs en raison de la grande diversité de leur écosystème. Ils représentent ainsi une source prometteuse de composés bioactifs. Le profilage chimique de Nephroma laevigatum a été effectué. Une analyse LC-MS/MS avec des approches en réseaux moléculaires ont permis d’appréhender la diversité chimique de ce lichen et quatre composés différents ont été isolés et identifiés par RMN puis testés pour leur activité antiproliférative. Cependant, les ressources en lichens sont limitées, ce qui restreint leur utilisation. De plus, le thalle lichénique constitue une niche écologique de choix pour d’autres microorganismes, ce qui en fait une source potentielle de nouvelles molécules d’intérêts. La culture de champignons endolichéniques a été entreprise. Ainsi, 46 souches ont été isolées et identifiées par DNA barcoding (amorces ITS4 et ITS5). Les souches identifiées appartiennent au genre Nemania, Daldinia, Peziza et Coniochaeta. Une investigation biologique a été réalisée sur six souches sélectionnées appartenant à deux espèces (Nemania aenea var. aureolatum et N. serpens). Ainsi, deux souches se sont démarquées par leurs activités antiprolifératives et anti-biofilms. Des études chimiques et biologiques plus approfondies de ces dernières (Gir_20 N. aenea var. aureolatum et Cor_08 N. serpens) ont été par la suite effectuées et huit composés différents ont été isolés et identifiés par RMN 1D et 2D. L’étude de l’effet des extraits sur les lignées cancéreuses humaines HT- 29, HCT116, PC-3 et DU145 a permis de mettre en évidence des changements morphologiques au niveau cellulaire. L’analyse de l’expression de marqueurs protéiques pro- et anti-apoptotiques ainsi que la fragmentation de l’ADN mettent en évidence l’induction de l’apoptose. Le profilage chimique par LC-MS/MS de ces souches a ensuite été réalisé et comparé par des approches en réseaux moléculaires permettant ainsi de visualiser la diversité chimique entre les deux espèces de champignons endolichéniques. / Antibiotics resistance or increase of difficulty to treat for current diseases with commercially available compounds has obligated researchers to find new sources of active molecules. Lichens produce various biologically active compounds due to the great diversity of their ecosystem. Thus, they represent a promising source of bioactive compounds. Chemical profiling of Nephroma laevigatum was performed. LC-MS/MS analysis with molecular network approach allowed understanding chemical diversity of this lichen and four different compounds were isolated and identified by NMR and tested for their antiproliferative activity. However, lichen resources are limited, which limits their use. In addition, lichen thalli are an ecological niche for other microorganisms and a wide reservoir for access to bioactive molecules. Cultivation of endolichenic fungi was undertaken. Thus, 46 strains were isolated and identified by DNA barcoding (primers ITS4 and ITS5). The isolated fungi belong to genus Nemania, Daldinia, Peziza and Coniochaeta. Biological investigation was carried out on six selected strains belonging to two species (Nemania aenea var. aureolatum and N. serpens). So, two strains distinguished by their antiproliferative and anti-biofilm activities. Further chemical and biological studies of these strains (Gir_20 N. aenea var. aureolatum and Cor_08 N. serpens) were subsequently performed and eight different compounds were isolated and identified by 1D and 2D NMR. Study of effect of the extracts on the human cancer lines HT-29, HCT116, PC-3 and DU145 made it possible to highlight morphological changes at the cellular level. Analyses of the expression of pro- and anti-apoptotic protein markers as well as DNA fragmentation demonstrate the induction of apoptosis. LC-MS/MS chemical profiling of these strains was performed and compared with molecular network approach, to visualize chemical diversity between the two species of endolichenic fungi.

La présente étude s’attache à étudier l’effet d’une source de carbone organique sélectionnée sur la croissance et la production de métabolites de la microalgue rouge Porphyridium marinum. Cette dernière s’est montrée incapable de se multiplier en hétérotrophie, en absence totale de lumière. Cependant, en condition mixotrophique la production de biomasse, lipides et de phycobiliprotéines par P. marinum a été améliorée en comparaison avec la condition autotrophique. Les teneurs en exopolysaccharides ont été presque similaires dans les deux conditions. Dans le but de valoriser ces métabolites, l’effet antioxydant, antibactérien, antibiofilm et anticancéreux ont été testés. L’exopolysaccharide de P. marinum ainsi que ses dérivés de faible poids moléculaire (EPS-2P et EPS-5P) ont tous présenté des activités antibactériennes et antibiofilm à différentes concentrations. Cependant l’EPS-2P et l’EPS-5P ont été jugés plus efficaces pour l’activité anticancéreuse contre les cellules de cancer de sein. D’autre part, la production du pigment majoritaire des microalgues rouges, la B-phycoerythrine (B-PE), a été optimisée moyennant des plans d’expériences adaptés. Une teneur de 40 mg/g MS a été obtenue en faisant varier la concentration en NaNO3, K2HPO4 et solution métallique ainsi que l’intensité lumineuse. Après purification, la molécule optimisée a montré une activité antioxydante en termes de piégeage des radicaux libres DPPH, chélation et réduction des ions de fer et inhibition de la décoloration du β-carotène. Nos données suggèrent alors que les métabolites produits par la microalgue rouge P. marinum peuvent être potentiellement utilisé dans plusieurs applications à savoir cosmétiques et pharmaceutiques. / The present study investigates the effect of a selected organic carbon source on the growth and production of metabolites of the red microalga Porphyridium marinum. The latter has been unable to multiply in heterotrophy, in total absence of light. However, in mixotrophic condition the productions of biomass, lipids and phycobiliproteins by P. marinum have been improved in comparison with the autotrophic condition. The contents of exopolysaccharide were almost similar under both conditions. In order to valorize these metabolites, the antioxidant, antibacterial, antibiofilm and anticancer effect were tested. The exopolysaccharide of P. marinum as well as its low molecular weight derivatives (EPS-2P and EPS-5P) have all exhibited antibacterial and antibiofilm activities at different concentrations. However, EPS-2P and EPS-5P were found to be more effective for anti-cancer activity against breast cancer cells. On the other hand, the production of the majority pigment of red microalgae, B-phycoerythrin (B-PE), has been optimized by means of adapted experimental plans. A content of 40 mg/g DW was obtained by varying the concentration of NaNO3, K2HPO4 and metal solution as well as the light intensity. After purification, the optimized molecule showed antioxidant activity in terms of free radical scavenging DPPH, chelation and reduction of iron ions and β-carotene bleachinginhibition. Our data suggest that the metabolites produced by the red microalga P. marinum may be potentially used in several applications namely cosmetic and pharmaceutical.

La composition en huiles essentielles et en lipides de réserve des graines et fruits saisonniers de quatre espèces végétales d’origine congolaise (Aframomum stipulatum (Gagnep) K. Schum, Aframomum giganteum (Oliv. and Hand) K. Schum, Spondias mombin L. et Nephelium lappaceum L.), a été déterminée par l’utilisation du couplage chromatographie en phase gazeuse/spectrométrie de masse. Les résultats obtenus ont permis de comprendre la bioaccumulation des huiles essentielles et des lipides de réserve dans les organes des différentes espèces. Dans le cas des espèces du genre Aframomum, les graines et les coques de fruits sont sources à la fois des huiles essentielles et des lipides de réserve. Les huiles essentielles des graines des deux espèces du genre Aframomun sont riches en composés monoterpéniques hydrocarbonés et oxygénés (β-pinène, 1,8-cinéole, phellandrène) avec des taux non négligeables de sesquiterpènes (α-sélinène et β-caryophyllène). Les huiles essentielles des coques des deux mêmes espèces se révèlent plus riches en monoterpénes oxygénés que celles des graines avec des composés très caractéristiques (β-pinène, 1,8-cineole, linalool pour A. giganteum et β-pinène, terpinéol, 1,8-cinéole pour A. stipulatum). On note dans les deux espèces, en dépit des procédés unitaires différents utilisés, la présence de lipides de réserve avec un profil acides gras du type palmitique-linoléique. Seuls les lipides de réserve ont été caractérisés dans Spondias mombin L. et Nephelium lappaceum L.. Les différentes parties du fruit de Spondias mombin sont dominées, comme dans le cas des Aframomum, par la présence des acides palmitique-linoléique avec une présence marquée de l’hentriacontane dans l’huile de peaux de fruits. L’huile d’amande de Nephelium lappaceum se distingue par sa richesse en acide arachidique C20:0 et acide 11- eicosénoïque C20:1. Les quatre espèces présentent un apport en phytostérols et en tocophérols. Des essais prometteurs de formulation de phytoproduits ont été réalisés avec des mélanges de tensioactifs, des a-monoglycérides et des huiles essentielles de graines de A. giganteum. Les activités antimicrobienne, anticancéreuse, antipaludique et anti-radicalaire ont été évaluées sur les extraits d’huiles essentielles et sur les lipides de réserve ainsi que sur les analogues synthétiques de lipides pris seuls ou en combinaison. Les résultats obtenus sont prometteurs / The composition of essential oils and fat of seasonal fruits and seeds of four plant species from the Congo (Aframomum stipulatum (Gagnep) K. Schum, Aframomum giganteum (Oliv. and Hand) K. Schum, Spondias mombin L. and Nephelium lappaceum L.), was determined by the combined use of mass spectrometry and gas chromatography. The results were used for understanding the bio-accumulation of essential oils and the storage of lipids in the organs of different species. In the case of the genus Aframomum, seeds and fruit shells are both sources of essential oils and fat. Seeds essential oils of the two species of Aframomun were rich in hydrocarbon and oxygenated monoterpene compounds such as β-pinene, 1,8-cineole, phellandrene, with significant levels of sesquiterpenes (α-selinene and β-caryophyllene). The shells essential oils of the same two species appear to be more rich in oxygenated monoterpenes than seeds, with a large structural features (β-pinene, 1,8-cineole, linalool for A. giganteum and b-pinene, terpineol, 1.8 -cineole for A. stipulatum). It is notified in both species that despite the different unit processes used, the presence of lipid with a fatty acid profile of the type palmitic-linoleic. Only lipids have been characterized from Spondias mombin L. and Nephelium lappaceum L.. The different parts of the fruit of Spondias mombin are dominated, like Aframomum, by the presence of palmitic-linoleic with a large presence of hentriacontane in the oil fruit skins. The kernel oil Nephelium lappaceum is distinguished by its wealth of arachidic acid C20:0 and 11-eicosénoïc acid C20:1. The four species contained in phytosterols and tocopherols. Testing promising formulation of herbal products were made with mixtures of surfactants, α-monoglycerides and essential oils from seeds of A. giganteum. Antimicrobial, anticancer, antimalarial and anti-radical activities were evaluated on the essential oils on the lipid as well as on the synthetic analogs of lipid alone or in combination. The results are promising

Le manque de thérapies innovantes en chimiothérapie humaine incite la communauté scientifique à s'intéresser à de nouvelles sources de composés bioactifs. Nous pouvons citer les métabolites secondaires de plantes, auxquels appartiennent les acides hydroxy pentacycliques triterpénoiques (AHPTs) et plus particulièrement les Acides Ursolique (AU), Oléanolique (AO) et Bétulinique (AB). Ces molécules font l'objet de nombreuses études qui tendent à démontrer leurs propriétés : anti-infectieuses, anticancéreuses, antiprolifératives, anti-inflammatoires, hépatoprotectrices. Le principal obstacle à leur utilisation à des fins thérapeutiques, reste l'insolubilité de ces AHPTs dans l'eau. L'objectif de ce travail a donc été d'augmenter leur hydrosolubilité. Dans un premier temps, en accord avec les recommandations et/ou normes existantes, nous avons démontré que le spectre d'activité antibactérienne de l'AU et de l'AO se limitait aux bactéries à Gram positif. Aucun AHPT n'a montré d'activité antifongique. Seul l'AB a montré une activité intéressante sur le Cytomégalovirus humain (hCMV) ; aucune activité antivirale n'ayant été retrouvée sur le Poliovirus. Enfin, l'AB, mais encore plus l'AU ont montré une activité anticancéreuse à l'encontre de cellules modèles de leucémie myéloïde chronique (LMC). Dans un deuxième temps, nous avons procédé à la fabrication et à l'étude de complexes entre les AHPTs et des cyclodextrines. Nous avons retenu la gamma-cyclodextrine (gamma-CD), qui présentait l'avantage de complexer les 3 AHPTs avec une constante de formation « moyenne » à « élevée ». Ces complexes AHPTs :gamma-CD ont été caractérisés en utilisant diverses techniques : chromatographiques, thermiques et spectrométriques. Nous avons conclu à l'obtention de complexes d'inclusion qui ont permis d'augmenter la solubilité des AHPTs. Dans une dernière partie, nous avons évalué les activités biologiques des complexes AHPTs : gamma-CD. Les résultats montrent que les complexes AU : gamma-CD et AO : gamma-CD restent actifs à l'encontre des bactéries à Gram positif (mais avec une efficacité plus faible) ; tandis que le complexe AB : gamma-CD se révèle être actif sur certaines bactéries. Le complexe AB : gamma-CD, et de façon surprenante le complexe AU : gamma-CD présentent une activité antivirale à l'encontre du hCMV. Enfin, la diminution de la cytotoxicité liée à la complexation des AHPTs accroit l'intérêt des molécules d'AU et d'AB sur les cellules de LMC / The lack of innovative therapies in human chemotherapy incites the scientific community to be interested in new sources of bioactive compounds. We can quote the secondary metabolites of plants, to which belong hydroxy pentacyclic triterpene acids (HPTAs) and more particularly Ursolic (UA), Oleanolic (OA) and Betulinic Acids(BA). These molecules are the subjetc of numerous studies which tend to demonstrate their properties: anti-infective, anticancer, antiproliferatives, anti-inflammatory and hepatoprotectrive. The main obstacle to their use in therapeutic purposes stays the insolubility of these AHPTs in the water. Thus the objective of this work was to increase their hydrosolubility. At first, in agreement with the recommendations and/or the existing standards, we demonstrated that the antibacterial spectra of UA and OA are limited to Gram-positive bacteria. No HPTA showed antifungal activity. Only the BA showed an interesting antiviral activity on human Cytomegalovirus (hCMV); no antiviral activity was on Poliovirus. Finally, the BA, but even more UA showed an anticancer activity against cellular model of chronic myeloid leukemia (CML). Secondly, we proceeded to the manufacturing and to the study of complexes between HPTAs and cyclodextrines. We retained gamma-cyclodextrine (gamma-CD), which presented the advantage to complex the 3 AHPTs with a constant of "average" to "raised". These HPTAs:gamma-CD complexes were characterized by using diverse techniques: chromatographic, thermal and spectrometric. We concluded in the obtaining of inclusion complexes which allowed increasing the solubility of the HPTAs. In a last part, we estimated the biological activities of the HPTAs:gamma-CD complexes. The results show that the UA:gamma-CD and OA:gamma-CD complexes remain active against Gram-positive bacteria (but with a weaker efficiency); whereas the BA:gamma-CD complex shows to be active on certain bacteria. The BA:gamma-CD complex, and in a surprising way the UA:gamma-CD complex presents an antiviral activity against the hCMV. Finally, the decrease of the cytotoxicity linked to the complexation of the HPTAs believes the interest of UA and of BA on CML cells

Cinque varietà di sorgo (Sorghum bicolor (L.) Moench) provenienti da Nigeria, Senegal, Burkina Faso e due dall’Italia, sono state caratterizzate per il loro profilo nutrizionale, analizzando la modulazione di due marcatori cellulari (contenuto in proteine totali e attività della GPx), il contenuto di fattori chelanti (tannini e fitati), l’attività fitasica e il contenuto, la bioaccessibilità e la biodisponibilità di ferro e zinco e analizzando l’impatto di due importanti processi alimentari, quali fermentazione e cottura. I risultati mostrano che sia la varietà sia il processo influenzano la maggior parte dei parametri. Tannini e fitati, elementi in traccia, contenuto proteico totale e il processo (soprattutto la fermentazione) sono modulati dalla varietà. La fermentazione riduce il contenuto di fattori chelanti e aumenta l’attività fitasica, incrementando, così, la biodisponibilità degli elementi in traccia; mentre la cottura non ha un impatto significativo. D’altra parte, la fermentazione ha un impatto negativo su entrambi i marcatori cellulari, mentre la cottura solo sul contenuto proteico totale. Le varietà africane, soprattutto quella dal Senegal, hanno mostrato il migliore profilo nutrizionale, mentre, globalmente, le italiane il peggiore. I risultati indicano che la selezione di varietà e di adeguati processi alimentari può avere un impatto significativo sul valore nutrizionale del sorgo. / Five sorghum cultivars (Sorghum bicolor (L.) Moench) from Nigeria, Senegal, Burkina Faso and Italy (two), were characterized for their nutritional values by analysing the modulation of cellular functional markers (total protein content and GPx activity), chelating factors (iron-binding phenolic groups and phytates) content and phytase activity, as well as iron and zinc content, bioaccessibility and bioavailability, also considering the impact of food processing methods as fermentation and cooking. The results revealed that both variety and food processing affect most of the tested parameters. Chelating factors, trace elements, cellular total protein content, as well as the effect of processing (mainly fermentation) were modulated by variety. Fermentation decreased chelating factors content, as well as increased phytase activity, leading to enhancement of iron and zinc estimated bioavailability, whereas cooking had no substantial impact. On the other hand, fermentation process had a negative impact on both cellular functional markers while cooking only on the cellular total protein content. The African varieties, particularly the Senegal one, showed the most promising nutritional profile, whereas the two Italian varieties overall showed a poor one. The results indicate that selection of traditional varieties and processing methods can have a significant impact on parameters relevant to sorghum nutritional value.

O trevo-vermelho (Trifolium pratense L.), uma das leguminosas forrageiras mais utilizadas na agricultura mundial, contém as isoflavonas formononetina e biochanina A, e em menores concentrações daidzeína e genisteína. Estes compostos têm ganhado muito interesse devido aos relatos de seus benefícios à saúde humana. Recentemente, no ano de 2009, esta espécie foi incluida na lista da Relação Nacional de Plantas Medicinais de Interesse ao SUS (RENISUS) que contém plantas medicinais com potencial de gerar produtos para serem produzidos e distribuídos pelo SUS. Mas é importante destacar que não existem estudos de quantificação de isoflavonas em plantas de trevo-vermelho cultivados no Brasil. Os objetivos deste trabalho foram validar método de análise de amostras de trevo-vermelho por Cromatografia Líquida de Alta Eficiência (CLAE); quantificar quatro isoflavonas em 78 acessos de trevo-vermelho; verificar as modificações sazonais na concentração de três acessos de trevo-vermelho; aumentar os níveis de isoflavonas através de melhoramento genético; e determinar a atividade anti-inflamatória in vivo e in vitro e de atividade de inibição da acetilcolinesterase de um extrato seco de trevo-vermelho. Para analisar as plantas por CLAE, estas foram hidrolisadas, separadas por diclorometano, e ressuspendidas em metanol. O método de CLAE usado foi validado. As isoflavonas (daidzeína, genisteína, formononetina e biochanina A) foram quantificadas em 78 acessos de trevo-vermelho, os conteúdos das agliconas (expressos em μg/g de planta seca, n=3) destes compostos variaram entre 0,00 a 137,91 para daidzeína; 14,70 a 516,51 para genisteína; 452,97 a 28548,65 para formononetina; e 1967,64 a 20145,27 para biochanina A, e a concentração total de isoflavonas variou entre 9,81 e 36,36 mg/g. O estudo sazonal mostrou que a concentração total das isoflavonas não difere entre as estações, mas verifica-se que na primavera (quando o trevo-vermelho está no estágio reprodutivo) se observam concentrações inferiores, e no inverno (quando a planta está no estágio vegetativo) são encontradas concentrações mais elevadas. No estudo de melhoramento genético foi possível observar um aumento na concentração total de isoflavonas. A atividade anti-inflamatória in vitro foi medida através da mobilização de leucócitos, pelo ensaio de quimiotaxia na câmara de Boyden. A atividade anti-inflamatória in vivo foi avaliada pelo teste de edema de pata de rato induzido por carragenina. Os resultados do teste anti-inflamatório in vitro mostram que houve significante inibição da migração dos leucócitos nas concentrações de 100,0 (94,73% de inibição), 50,0 (95,39% de inibição), 25,0 (94,73% de inibição), 10,0 (84,68% de inibição) e 5,0 (78,75% de inibição) μg/mL de extrato seco de trevo-vermelho. O teste anti-inflamatório in vivo demonstrou significante atividade nas doses testadas, 100 e 50 mg/kg de extrato seco de trevo-vermelho. O percentual médio de inibição do edema foi 63,37%. O teste da inibição da acetilcolinesterase não demonstrou atividade. Os resultados deste estudo sugerem que se pode produzir plantas com concentrações mais elevadas de isoflavonas para a produção de fitomedicamentos com maior qualidade, e o extrato de trevo-vermelho pode ser adequado para o tratamento de doenças inflamatórias. / Red clover (Trifolium pratense L.) is one of the most utilized forage legume in the world agriculture and contains the isoflavones formononetin and biochanin A, and in smaller concentration daidzein and genistein. These compounds have gained a high interest due to their human health benefits. Recently this species was included in the list of Relação Nacional de Plantas Medicinais de Interesse ao SUS (RENISUS) which contains medicinal plants that have potential to generate products to be produced and distributed by SUS. But it is important to highlight that does not exist any study of isoflavones quantification in red clover plants cultivated in Brazil. The aims of this work were to validate a method to analize red clover samples by High Performance Liquid Cromatography (HPLC); to quantify four isoflavones compounds in 78 red clover accessions; to verify seasonal modifications in the isoflavone concentration of three red clover accessions; to increase isoflavones levels through plant breeding; and to determine in vivo and in vitro anti-inflammatory activity, and inhibitory activity of acetylcholinesterase of a red clover dry extract. To analyze the plants by HPLC, they were hydrolyzed, separated by dichloromethane, and resuspended with methanol. The HPLC method used was validated. Isoflavones (daidzein, genistein, formononetin and biochanin A) were quantified in 78 red clover accessions, the aglycone contents (expressed in μg/g of dry material, n=3) of these compounds varied from 0.00 to 137.91 to daidzein; 14.70 to 516.51 to genistein; 452.97 to 28548.65 to formononetin; and 1967.64 to 20145.27 to biochanin A, and the total isoflavone concentration ranged between 9.81 and 36.36 mg/g. The seasonal study showed that the total concentration of isoflavones is not statistically different at different seasons, but is possible to verify that at spring (when red clover is at reproductive stage) we could observe the lowest concentration, and at winter (when red clover is at vegetative stage) we found the highest concentration. In the breeding study we could observe an increase in the total concentration of isoflavones. The in vitro anti-inflammatory activity was measured towards by leucocytes mobilization, by chemotaxis assay in Boyden´s chamber. The in vivo anti-inflammatory activity was tested by a carrageenan-induced rat paw edema test. The results of anti-inflammatory in vitro test showed that there was a significant inhibition of leukocyte migration at concentrations of 100.0 (94.73% of inhibition), 50.0 (95.39% of inhibition), 25.0 (94.73% of inhibition), 10.0 (84.68% of inhibition) and 5.0 (78.75% of inhibition) μg/mL of red clover dry extract. The in vivo anti-inflammatory test demonstrated significant activity in the tested doses, 100 and 50 mg/kg of red clover dry extract. The average of edema inhibition percentage was 63.37%. The inhibition of acetylcholinesterase test did not show activity. The results of this study suggest that we can select or produce plants with higher concentrations of isoflavones to produce phytomedicines with higher quality, and the red clover extract might be suitable for the treatment of inflammatory diseases.