Desenvolveram-se métodos bioanalíticos para determinação de anfotericina B e fluconazol em apenas 200 L de plasma e líquor (LCR) através da cromatografia líquida de alta eficiência (CLAE UV-VIS). A anfotericina B foi determinada através de CLAE-VIS utilizando p-nitrofenol como padrão interno, após purificação das matrizes biológicas com acetonitrila, seguida da análise em coluna Nova Pak C18 (150 x 3,9mm, 4 micron) e fase móvel constituída por tampão acetato 0,1M pH 5,0 e acetonitrila (50:50,v/v) 0,5mL/min em 385nm; o tempo de corrida foi 15 min. Através da validação o método mostrou-se robusto com 0,2-25,0 µg/mL(linearidade, r2 0,9999), LD 0,1 µg/mL, precisão (5,4% e 6,9%), exatidão expressa através do erro sistemático (3,3% e 2,2%): intra e interdias). Os estudos de estabilidade evidenciaram 1,0% para o erro sistemático e 3% de precisão na bandeja (tempo e condição de análise por 24 h), e os ciclos de congelamento evidenciaram boa estabilidade uma vez que todos os ensaios foram realizados em Laboratório de luz amarela. O fluconazol foi determinado através de CLAE-UV utilizando carbamazepina como padrão interno, após purificação das matrizes biológicas pela extração líquido-líquido com diclorometano em meio alcalino, seguido da análise em coluna Nova Pak C18 (150 x 3,9mm, 4 micron) e fase móvel constituída por água UP e acetonitrila (70:30,v/v) 0,5mL/min em 210nm; o tempo de corrida foi 15 min. O método mostrou-se robusto com 0,2-250 µg/mL(linearidade, r2 0,9998), LD 0,1µg/mL, com boa recuperação absoluta (98%) e relativa (100%), precisão 0,5%/1,3%, exatidão expressa através do erro sistemático (1,2%). Evidenciou-se ótima estabilidade para os extratos em bandeja (tempo e condição de análise por 24 h), na longa duração (20° C, 9 meses) e através dos ciclos de congelamento. Investigaram-se 21 pacientes adultos de ambos os sexos portadores de meningite criptocócica com AIDS após internação emergencial em terapia de alta dose com anfotericina B (1mg/Kg) e fluonazol (400 mg, 12/12 horas) durante 12 semanas. O monitoramento das concentrações de anfotericina B e fluconazol no plasma e no LCR forneceram as razões que permitiram estimar a penetração dos antifúngicos no SNC. Obtiveram-se concentrações de anfotericina B, médias (IC95%): 2,30 (0,02-5,08) µg/mL no plasma e 0,30 (0,19-0,36) µg/mL no LCR. As concentrações do fluconazol, médias (IC95%) foram: 31,7 (20,1-43,3) µg/mL no plasma e 19,4 (11,1-27,7) µg/mL no LCR. Com base nos resultados obtidos conclui-se que a penetração da anfotericina B foi insuficiente (10-27%), enquanto que a do fluconazol mostrou-se adequada com valores médios (IC95%) de 67 (47-87) %. / Analytical methods were developed to determine amphotericin B and fluconazole in only 200 L of plasma and in cerebrospinal fluid (CSF) by liquid chromatography (HPLC UVVIS). Amphotericin B was determined by HPLC - VIS using p-nitrophenol as internal standard, after the purification of biological matrices using acetonitrile, followed by chromatographic analysis in a Nova Pak C18 column (150 x 3.9mm, 4 micron) and mobile phase consisting of acetate buffer 0.1M pH 5.0 plus acetonitrile (50:50,v/v) 0.5mL/min at 385nm; the run time required was 15 min. Bioanalytical method validated showed robustness, 0.2-25,0µg/mL (linearity, r2 0.9999), DL 0.1µg/mL, precision (5.4%/6.0%), accuracy expressed as systematic error (3.3%/2.2%). The stability was investigated, error systematic was 1% for the vials on the rack (time and conditions of drug analysis, 24h). Thawing cycles showed good stability after three freezing-thawing cycles. All procedures were performed under yellow light at room temperature. Fluconazole was determined by HPLC - UV using carbamazepine as internal standard, after the purification of biological matrices using liquid-liquid extraction in alkaline medium, followed by chromatographic analysis in a Nova Pak C18 column (150 x 3.9mm, 4 micron) and mobile phase consisting of purified water plus acetonitrile (70:30,v/v) 0.5mL/min at 210nm; the run time required was 15 min. Bioanalytical method validated showed robustness, 0.2-250 µg/mL(linearity, r2 0.9998), DL 0.1µg/mL. Absolute recovery was 98% and relative recovery was 100%, intra/interday precision were 0,5/-1,3%; accuracy expressed as systematic error were 1.2%/1.2%.and relative recovery was 100%. Good stability for the vials on the rack (time and conditions of drug analysis, 24h) and long term stability (at 20o C for 9 months) were demonstrated. Also thawing cycles showed good stability after three freezing-thawing cycles. Twenty one adult patients of both sex were investigated. Inpatients with meningitis by Cryptococcus neoformans with AIDS were under high dose therapy with amphotericin B 1mg/Kg plus fluonazole 400 mg, every 12h during 12 weeks. Therapeutic monitoring of amphotericin B and fluconazole in plasma and in CSF showed ratios that indicate the penetration of antifungal drugs into CNS. Mean (CI95%) data were for amphotericin B 2.30 (0.02-5.08 ) µg/mL in plasma and 0.30 (0.19-0.36) µg/mL in CSF. Fluconazole showed 31.7 (20.1-43.3) µg/mL in plasma and 19.4 (11.1-27.7) µg/mL in CSF. Based on data obtained we conclude that the penetration of amphotericin B was poor (10-27%) while fluconazole was adequate 67% (47-87%), mean (CI95%).

Desenvolveram-se métodos bioanalíticos para determinação de anfotericina B e fluconazol em apenas 200 L de plasma e líquor (LCR) através da cromatografia líquida de alta eficiência (CLAE UV-VIS). A anfotericina B foi determinada através de CLAE-VIS utilizando p-nitrofenol como padrão interno, após purificação das matrizes biológicas com acetonitrila, seguida da análise em coluna Nova Pak C18 (150 x 3,9mm, 4 micron) e fase móvel constituída por tampão acetato 0,1M pH 5,0 e acetonitrila (50:50,v/v) 0,5mL/min em 385nm; o tempo de corrida foi 15 min. Através da validação o método mostrou-se robusto com 0,2-25,0 µg/mL(linearidade, r2 0,9999), LD 0,1 µg/mL, precisão (5,4% e 6,9%), exatidão expressa através do erro sistemático (3,3% e 2,2%): intra e interdias). Os estudos de estabilidade evidenciaram 1,0% para o erro sistemático e 3% de precisão na bandeja (tempo e condição de análise por 24 h), e os ciclos de congelamento evidenciaram boa estabilidade uma vez que todos os ensaios foram realizados em Laboratório de luz amarela. O fluconazol foi determinado através de CLAE-UV utilizando carbamazepina como padrão interno, após purificação das matrizes biológicas pela extração líquido-líquido com diclorometano em meio alcalino, seguido da análise em coluna Nova Pak C18 (150 x 3,9mm, 4 micron) e fase móvel constituída por água UP e acetonitrila (70:30,v/v) 0,5mL/min em 210nm; o tempo de corrida foi 15 min. O método mostrou-se robusto com 0,2-250 µg/mL(linearidade, r2 0,9998), LD 0,1µg/mL, com boa recuperação absoluta (98%) e relativa (100%), precisão 0,5%/1,3%, exatidão expressa através do erro sistemático (1,2%). Evidenciou-se ótima estabilidade para os extratos em bandeja (tempo e condição de análise por 24 h), na longa duração (20° C, 9 meses) e através dos ciclos de congelamento. Investigaram-se 21 pacientes adultos de ambos os sexos portadores de meningite criptocócica com AIDS após internação emergencial em terapia de alta dose com anfotericina B (1mg/Kg) e fluonazol (400 mg, 12/12 horas) durante 12 semanas. O monitoramento das concentrações de anfotericina B e fluconazol no plasma e no LCR forneceram as razões que permitiram estimar a penetração dos antifúngicos no SNC. Obtiveram-se concentrações de anfotericina B, médias (IC95%): 2,30 (0,02-5,08) µg/mL no plasma e 0,30 (0,19-0,36) µg/mL no LCR. As concentrações do fluconazol, médias (IC95%) foram: 31,7 (20,1-43,3) µg/mL no plasma e 19,4 (11,1-27,7) µg/mL no LCR. Com base nos resultados obtidos conclui-se que a penetração da anfotericina B foi insuficiente (10-27%), enquanto que a do fluconazol mostrou-se adequada com valores médios (IC95%) de 67 (47-87) %. / Analytical methods were developed to determine amphotericin B and fluconazole in only 200 L of plasma and in cerebrospinal fluid (CSF) by liquid chromatography (HPLC UVVIS). Amphotericin B was determined by HPLC - VIS using p-nitrophenol as internal standard, after the purification of biological matrices using acetonitrile, followed by chromatographic analysis in a Nova Pak C18 column (150 x 3.9mm, 4 micron) and mobile phase consisting of acetate buffer 0.1M pH 5.0 plus acetonitrile (50:50,v/v) 0.5mL/min at 385nm; the run time required was 15 min. Bioanalytical method validated showed robustness, 0.2-25,0µg/mL (linearity, r2 0.9999), DL 0.1µg/mL, precision (5.4%/6.0%), accuracy expressed as systematic error (3.3%/2.2%). The stability was investigated, error systematic was 1% for the vials on the rack (time and conditions of drug analysis, 24h). Thawing cycles showed good stability after three freezing-thawing cycles. All procedures were performed under yellow light at room temperature. Fluconazole was determined by HPLC - UV using carbamazepine as internal standard, after the purification of biological matrices using liquid-liquid extraction in alkaline medium, followed by chromatographic analysis in a Nova Pak C18 column (150 x 3.9mm, 4 micron) and mobile phase consisting of purified water plus acetonitrile (70:30,v/v) 0.5mL/min at 210nm; the run time required was 15 min. Bioanalytical method validated showed robustness, 0.2-250 µg/mL(linearity, r2 0.9998), DL 0.1µg/mL. Absolute recovery was 98% and relative recovery was 100%, intra/interday precision were 0,5/-1,3%; accuracy expressed as systematic error were 1.2%/1.2%.and relative recovery was 100%. Good stability for the vials on the rack (time and conditions of drug analysis, 24h) and long term stability (at 20o C for 9 months) were demonstrated. Also thawing cycles showed good stability after three freezing-thawing cycles. Twenty one adult patients of both sex were investigated. Inpatients with meningitis by Cryptococcus neoformans with AIDS were under high dose therapy with amphotericin B 1mg/Kg plus fluonazole 400 mg, every 12h during 12 weeks. Therapeutic monitoring of amphotericin B and fluconazole in plasma and in CSF showed ratios that indicate the penetration of antifungal drugs into CNS. Mean (CI95%) data were for amphotericin B 2.30 (0.02-5.08 ) µg/mL in plasma and 0.30 (0.19-0.36) µg/mL in CSF. Fluconazole showed 31.7 (20.1-43.3) µg/mL in plasma and 19.4 (11.1-27.7) µg/mL in CSF. Based on data obtained we conclude that the penetration of amphotericin B was poor (10-27%) while fluconazole was adequate 67% (47-87%), mean (CI95%).

En raison de sa richesse en ressources génétiques, et des utilisations traditionnelles locales qui en sont faites, la biodiversité végétale issue du bassin amazonien constitue une véritable source de principes actifs à valoriser. L’espèce Euterpe oleracea Mart., vernaculairement appelée baie d’açai, qui connaît un intérêt scientifique important, est le parfait exemple de ressources naturelles bioactives valorisées issues de cette zone géographique. Les études scientifiques lui confèrent de très nombreuses propriétés biologiques, mais, la plus connue et la plus médiatisée est sa capacité antioxydante liée majoritairement à sa composition polyphénolique. En Guyane française, on peut parler de « diversité » au sein de la famille des Palmiers puisque plus de 75 espèces y ont été recensées. Parmi lesquelles, on peut citer une espèce très commune ayant des propriétés surtout alimentaires, et dont la connaissance phytochimique reste, à l’heure actuelle, très limitée : l’Oenocarpus bataua Mart dit patawa. Ce sujet de thèse de doctorat s’articule autour de la mesure de l’activité antioxydante du fruit mûr de ce palmier, et de la détermination des polyphénols responsables de cette dernière. La singularité de ce travail réside dans l’étude des différentes composantes tissulaires du fruit : mésocarpe, épicarpe et mélange épicarpe/mésocarpe (MEM). Dans un premier temps, les conditions les plus favorables d’extraction de biomolécules (notamment de l’épicarpe et du mésocarpe) ont été définies à l’aide du test DPPH. Les tests préliminaires effectués sur les tissus pris séparément, ont conduit à sélectionner un mélange acétone/eau (70/30, v/v) pour révéler, au mieux, la capacité antioxydante de chaque partie du fruit. Une étape de délipidation initiale s’est avérée nécessaire dans le cas de l’étude du mésocarpe. La confirmation de l’activité antioxydante a été réalisée au moyen d’autres tests d’activité chimique (TEAC, FRAP, ORAC), et a été complétée par l’utilisation d’un test d’activité biologique (KRL) en raison de ses mécanismes réactionnels plus complets. Il en ressort que le tissu végétal le plus antioxydant est le mésocarpe qui contient des proanthocyanidines, famille de composés phénoliques connue pour ses nombreuses activités biologiques.Le même travail a été effectué sur les tissus regroupés (fruit global ou MEM). Ainsi, a été retenue l’utilisation du solvant mixte acétone/eau sans étape de délipidation initiale. La capacité antioxydante du fruit étudié a été comparée à celle de l’açai, espèce choisie comme référence. Il s’avère que les extraits d’açai ont une activité antioxydante très supérieure à celle du patawa lorsqu’ils sont testés vis-à-vis de l’ORAC et du KRL. Le tissu mésocarpe a, lui, démontré une capacité antioxydante supérieure à celle de l’açai. Ces résultats sont à associer avec la composition phytochimique propre à chaque fruit. La composition polyphénolique du fruit de patawa, déterminée par UPLC/MSn, supposerait la présence d’anthocyanes, de tanins condensés, de stilbènes et d’acides phénoliques. Ces travaux, qui méritent d’être approfondis, en particulier pour le mésocarpe, ouvrent de nouvelles perspectives d’utilisation du fruit patawa, en particulier l’incorporation de composés phénoliques issus du mésocarpe dans des formulations galéniques ayant attrait aux domaines de la Nutrition, de la Cosmétique et de la Pharmaceutique. / Due to its wealth in genetic resources, and to traditional uses, plant biodiversity issued from the Amazonian Basin is a real source of active process to valorize. The specie Euterpeoleracea Mart., usually called acai berry, which is experiencing a huge scientific interest, is the perfect example of valued natural bioactive resources from the geographic area. Scientific studies give it many biological properties, but the most known is its antioxidant property mainly due to its polyphenolic composition. In French Guiana, we can use the term “diversity” within the palm family with more than 75 species identified. Among them is a common species, Oenocarpus bataua Mart., called “Patawa”, mainly with alimentary properties but for which knowledge of phytochemical properties is until now very poor. The present research deals with determining the antioxidant activity of this palm fruit and with the identification of the polyphenols responsible for it.The uniqueness of this work lays in the study of the different tissue components of this fruit namely the mesocarp, the epicarp and mixing epicarp / mesocarp (MEM). In a first time, the most favorable extraction conditions of biomolecules (particularly of the epicarp and mesocarp) were defined using the DPPH test. The preliminary tests performed on those tissues taken separately, have led to select an acetone / water (70/30, v / v) to reveal, at best, the antioxidant capacity of each part of the fruit. An initial defatting step was necessary in the case of the study of the mesocarp. The confirmation of the antioxidant activity was carried out by other tests of chemical activity (TEAC, FRAP, ORAC), and was supplemented by the use of a bioassay (KRL) due to its more complete reaction mechanisms. Results point out that the most antioxidant tissue is the mesocarp that contains proanthocyanidins, phenolics of a chemical family known for its numerous biological activities.The same work was performed on tissues combined (overall result). The mixed solvent acetone / water, without initial defatting step, has been selected. The antioxidant capacity of fruit was compared to that of the Acai specie chosen as a reference. It turns out that acai extracts have antioxidant activity much greater than that of Patawa when tested vis-à-vis of ORAC and KRL. In contrary, mesocarp tissue has a greater antioxidant capacity than that of Acai. These results can be associated with the phytochemical composition of each fruit. The polyphenolic composition of the fruit of Patawa determined by UPLC / MSn, reflects the presence of anthocyanins, condensed tannins, stilbene and phenolic acids. This work, which deserves to be deepened, especially for the mesocarp tissue, opens new prospects for the use of Patawa fruit, especially the incorporation of phenolic compounds from the mesocarp in pharmaceutical formulations linked to the fields of Nutrition, of Cosmetics and Pharmaceuticals.