(EN) Proteinogenic amino acids are key components of living organisms. Thus, the latest metabolomics research has focused on developing fast and sensitive methods for the determination of amino acids. In this context, this thesis contains two studies describing development of high-performance separation techniques for the quantification of amino acids. In the first study, a capillary electrophoresis method was developed for the determination of free amino acids in tobacco plants, particularly focusing on optimizing the extraction of amino acids from solid plant materials. The extraction procedure was optimized using design of experiments (DoE) to obtain the highest possible extraction yield of amino acids. Factors such as volume and concentration of the extraction solvent (hydrochloric acid) were assessed as the most significant. Subsequently, the optimal values of these factors were determined using response surface methodology (RSM). Lastly, proteinogenic amino acids were quantified using capillary electrophoresis with contactless conductivity detection and calibration with internal standard, which improved the precision of the method. The second study aimed at developing a supercritical fluid chromatography method for the determination of free proteinogenic amino acids in human plasma. The most...

Dans le cadre de l’intégration tridimensionnelle (3D) associée à l’utilisation d’un nombre croissant de circuits intégrés (CIs), le besoin en condensateurs de découplage à forte densité de capacité (≥ 1 μF.cm-2), capables d’opérer sur une gamme de fréquences de plus en plus étendue, est crucial afin de limiter les fluctuations de tension d’alimentation au niveau des CIs. Le principal frein au développement de ces condensateurs réside dans l’obtention de couches minces (≤ 100 nm) à partir de matériaux ayant une forte permittivité relative (ε_r > 200 à 1 GHz), des technologies compatibles avec une intégration à grande échelle, et peu coûteux.L’approche proposée dans cette thèse s’appuie d’une part, sur la possibilité de synthétiser des nanoparticules diélectriques à base de Ba0.6Sr0.4TiO3 [BST] (Ø = 16 ± 2 nm, ε_r = 260 à 1 kHz) - obtenues en milieux fluides supercritiques - comme matériau diélectrique et d’autre part, sur la pulvérisation de jets ou spray coating comme technologie de dépôt en couches minces. Dans un premier temps, les nanoparticules de BST ont été synthétisées et fonctionnalisées par l’acide 3-aminopropylphosphonique (APA), dans le but de les disperser dans un solvant et d’obtenir des suspensions colloïdales stables. Les ligands ont aussi pour fonction d’améliorer la tenue mécanique des films (auto-assemblage) sur le substrat de cuivre (Cu). La variation de paramètres liés à la solution à base des nanoparticules (concentration, durée de dispersion mécanique) et à la technique de dépôt (température du substrat, débit), ont permis d’optimiser les conditions pour obtenir des films uniformes à base de nanoparticules fonctionnalisées (BST-APA). Des films de 200 ± 50 nm d’épaisseur ont ainsi pu être obtenus. Après dépôt de plots d’aluminium (Al) sur les films à base de BST-APA et utilisés comme électrode supérieure, les caractéristiques capacité-tension (C-V) et courant-tension (I-V) des condensateurs de structure Al/BST-APA/Cu ont permis d’extraire une densité de capacité élevée (0.71 μF.cm-2) et une densité de courant de fuite (25 μA.cm-2) mesurées à 1 V. Les résultats obtenus au cours de cette thèse montent que la pulvérisation de jets est une alternative aux procédés coûteux reportés (ablation laser, pulvérisation) dans l’état de l’art, pour la fabrication de condensateurs de hautes performances.Mots clés : Condensateurs MIM, couches minces, fluides supercritiques, diélectriques, Ba0.6Sr0.4TiO3, pulvérisation de jets, nanofabrication. / Within the three-dimensional (3D) integration associated with the use of an increasing amount of integrated circuits (ICs), there is strong need of high capacitance density (≥ 1 μF.cm-2) decoupling capacitors, able to operate on large frequency bandwidth, in order to reduce the noise that can compromise the signal integrity in ICs. The main challenge of these capacitors relies on the deposition of thin films (≤ 100 nm) using innovative materials with high relative permittivity (ε_r > 200 à 1 GHz) and «low cost» technologies compatible with large scale integration.On one hand, the proposed approach in this thesis benefits from the possibility of synthetizing – by the supercritical fluid technology – and using Ba0.6Sr0.4TiO3 (BST) nanoparticles (Ø = 16 ± 2 nm, ε_r = 260 at1 kHz) as dielectric material and on the other hand, from the use of spray coating as technique for the deposition of these materials as thin films. First of all, the BST nanoparticles synthesized are functionalized with specific ligands (3-aminopropylphosphonic acid, APA), in order to obtain colloidal suspensions composed by aggregates with size (Ø < 100 nm) showing few fluctuations during two months. The other function of ligands is to improve the adhesion of the deposited films (self-assembling) on the copper (Cu) substrate. Different solvent are studied for the preparation of the solutions : N-méthyl-2-pyrrolidone (NMP), water, methanol and ethanol. The variation of different parameters related to the solution and the deposition technique helped us to define the optimal conditions leading to different thickness of film (200 – 1000 nm) based on pristine (BST) and functionalized nanoparticles (BST-APA). Using ethanol instead of NMP as solvent, enabled us to prevent de formation of a copper oxide layer and organic residues. After deposition of aluminum pads (Al) on BST or BST-APA films and used as top electrode, the capacitance-voltage (C-V) and current-voltage (I-V) characteristics of capacitors with metal-insulator-metal (MIM) structure enabled us to achieve high capacitance density (~ 0.7 μF.cm-2) and low leakage current (~ 25 μA.cm-2) at 1 V.Keywords: MIM capacitors, thin films, supercritical fluids, Ba0.6Sr0.4TiO3, spray coating, nanofabrication in cleanroom.

Raman spectroscopy, laser-induced fluorescence, and sum-frequency generation (SFG) spectroscopy are used to investigate the behavior of compressible mobile phases and stationary phases under a variety of chromatographic conditions. Efforts to understand and optimize separations employing compressible mobile phases have been limited by a lack of understanding of the mobile phase density gradient. Mobile phase compressibility leads to gradients in linear velocity and solute retention and affects separation speed and efficiency, especially in packed columns. This work describes on-column density measurement of CO2, a common carrier fluid for SFC and SGC, in packed capillary columns using Raman microspectroscopy. On-column detection by laser-induced fluorescence is used to observe the effect of the mobile phase density gradient on separation speed and efficiency, and experimental efficiency is compared to a theoretical model. Additionally, SFG spectroscopy allows for probing the structure of model monomeric and polymeric C18 stationary phases under pressure; this provides a basis for correlating selectivity with pressure-induced structural changes in stationary phase materials. Together, this work provides a more complete understanding of the role of column pressure and fluid compressibility on the speed, efficiency, and selectivity of chemical separations.

Listeria monocytogenes continues to challenge the dairy industry in causing post-process contamination of cheeses. To reduce risk of contamination, it is crucial to understand the growth and survival of pathogenic bacteria in cheese products and to develop post-process mitigation strategies. This study evaluated the fate of pathogens in reduced and regular sodium Mozzarella cheese, and the potential of Supercritical Fluid Extraction with CO2 (SFE) to reduce Listeria innocua on Mozzarella and Queso Fresco. The survival of L. monocytogenes, Salmonella, and E.coli O157:H7 (2-3 log CFU/g) in reduced sodium Mozzarella (1.62%), compared to regular sodium Mozzarella cheese (2.15%) at 4ºC and 12ºC for 90 and 30 days, respectively, was evaluated. Salmonella and E. coli O157:H7 populations decreased over incubation time at both temperatures and no difference (pListeria monocytogenes population also decreased during incubation time at 4°C regardless of the sodium concentration in Mozzarella cheese. However, there was a difference in the population of L. monocytogenes for regular and reduced sodium incubated 12°C, and its populations increased 1 log CFU/g in reduced sodium Mozzarella cheese. Additionally, this study determined the bactericidal effect of SFE on the population of L. innocua, a surrogate for L. monocytogenes, in Mozzarella and Queso Fresco cheese (6 log CFU/g) treated with SFE at two pressures and temperatures (120 bar at 40°C and 150 bar at 50°C) for 30 min. SFE treatment at 120 bar, 40°C for 30 min decreased L. innocua by approximately 3.0 and 3.5 log CFU/g in Mozzarella and Queso Fresco cheeses, respectively. SFE at 150 bar and 50°C reduced L. innocua by approximately 3.78 and 5.2 log CFU/g in Mozzarella and Queso Fresco cheeses, respectively. Since SFE had a minimal effect on the physico-chemical characteristics of the cheeses assayed, the results suggest SFE might be used to reduce L. monocytogenes in cheeses without negatively impacting product quality.

<p>U okviru ove doktorske disertacije, proučavana je ekstrakcija bosiljka (<em>Ocimum basilicum L.</em>) primenom postupka hidrodestilacije, Soxhlet ekstrakcije i ugljendioksidom u superkritičnom stanju. Određen je sadržaj etarskog ulja u drogi (0,565%), a primenom GC/MS(FID) metoda određen kvalitativni i kvantitativni sastav detektovanih komponenata. Identifikovane komponente svrstane su u nekoliko grupa (monoterpeni, seskviterpeni i njihovi oksidovani derivati). Rezultati ispitivanja pokazuju da je dominantno jedinjenje etarskog ulja bosiljka linalool (50,09%), i da ono pripada linaloolskom hemotipu (hemotip A).<br />Ispitan je uticaj temperature (40-60&deg;C) i pritiska (100&ndash;300 bar) superkritičnog ugljendioksida na promenu prinosa ekstrakta. Utvrđeno je da se kumulativni prinos ekstrakta povećava sa porastom pritiska ekstrakcije, dok u zavisnosti od temperature, prinos ekstrakcije zavisi od gustine rastvarača i napona pare komponenata u ekstraktu.<br />Primenom sukcesivne (frakcione) superkritične ekstrakcije izvr&scaron;eno je frakcionisanje, a dobijeni ekstrakti upoređivani u pogledu prinosa, kvalitativnog i kvantitativnog sastava.<br />Ispitivanja hemijskog sastava CO₂ ekstrakta (GC/MS i GC/FID) pokazala su da ekstrakti pored komponenata etarskog ulja sadrže i druga lipofilna jedinjenja (voskove, masna ulja, masne kiseline, fitosterole i sl.). Dominantne komponente u svim ispitivanim ekstraktima su linalool, eugenol i &delta;-kadinen, dok su &alpha;-bergamoten, germakren D, &gamma;-kadinen, &beta;-selinen i spatulenol komponente sa manjim saržajem.<br />Na osnovu eksperimentalnih rezultata kinetike superkritične ekstrakcije izvr&scaron;eno je modelovanje ekstrakcionog sistema bosiljak &ndash; superkritični CO₂, primenom tri modela (Kandiah i Spiro, Brunner i Esquivel i sar.). Na osnovu srednjih vrednosti relativnog odstupanja modelovanih i eksperimentalnih vrednosti prinosa ekstrakcije, utvrđeno je da model Kandiah i Spiro pokazuje prihvatljivo slaganje, dok ostali modeli pokazuju lo&scaron;ije slaganje eksperimentalno određenih i modelom izračunatih prinosa ekstrakta.<br />U zavr&scaron;noj fazi rada ispitan je antioksidativni potencijal etarskog ulja i CO₂ ekstrakata, primenom DPPH metode. Najveću antioksidativnu aktivnost pokazao je ekstrakt dobijen na 100 bar, 60&deg;C (IC50 = 18,93 &mu;g/ml), koja je 5 puta veća od antioksidativne aktivnosti etarskog ulja bosiljka.</p> / <p>In this study, the extraction of basil (O. basilicum L.) ussing the hydrodistillation process, Soxhlet extraction and supercritical extraction with carbon dioxide were investigated. The content of essential oil was determined (0,565%), and the qualitative and quantitative composition of detected coumpound was made by GC/MS(FID). The identified coumpounds were classified into five groups (monoterpenes, sesquiterpenes and their oxidative derivates). The results of investigation shows that the dominant compound of basil essential oil is linalool (50,09%), and that it belongs to linalool chemotype (chemotype A).<br />The influence of temperature (40-60&deg;C) and pressure (100&ndash;300 bar) of supercritical carbon dioxide on extraction yields were studied. It was determined that the total yield of extract increases with the increaseing of extraction pressure, while the influence of temperature on the yield depends on the density of the solvent and vapor pressure of components in the extract.<br />Fractional supercritical fluid extraction was performed, and obtained extracts were compared regarding of yield, and the qualitative and quantitative composition of the extract. Chemical analyses of CO2 extract by GC/MS and GC/FID showed that extract contains, besides essential oil componentes, other lipophilic compounds (waxes, fatty oils, fatty acids, phytosterols, etc.). The few compounds were dominant in all analyzed basil extracts: linalool, and to a lesser extent eugenol, &delta;-cadinene, &alpha;-bergamotene, germacrene D, &gamma;-cadinene, &beta;-selinene and spathulenol. Based on the experimental results of the extraction kinetics, a modeling of basil &ndash; supercritical CO2 extraction system was performed, using three models (Kandiah and Spiro, Brunner, and Esquivel et al.). Based on the average values of the relative deviations of modeled and experimental values of extraction yield, it was found that the model Kandiah and Spiro shows acceptable agreement, while other models show lower agreement of the experimentally determined and model calculated extraction yield.<br />In finale phase of work the antioxidant potential of essential oil and CO2 extracts were examined, using DPPH method. The highest antioxidant activity showed CO2 extract obtained at 100 bar, 60&deg;C (IC50 = 18,93 &mu;g/ml), which is 5 time higher then antioxidant activity of basil essential oil.</p>

A soja e seus produtos são ricos em isoflavonóides, estes possuem a concentração aumentada após a fermentação microbiana, devido à liberação de fenólicos, através da conversão das formas glicosídicas em agliconas. O processo fermentativo em estado sólido por fungos filamentosos tem sido amplamente empregado como uma estratégica biotecnológica na busca de agregar valores aos produtos e subprodutos agroindustriais, principalmente elevando os teores de compostos bioativos. No presente estudo, foram avaliados o aumento dessas propriedades, como antioxidante e anti-inflamatória em diferentes substratos utilizando linhagens fúngicas do gênero Rhizopus sp. As 17 linhagens testadas foram capazes de elevar o potencial antioxidante dos grãos de soja. A fermentação de soja e arroz utilizando o R. oligosporus NRRL 2710 resultou em um incremento de 26,5 vezes o potencial antioxidante e 9,33 vezes o teor de compostos fenólicos nos extratos etanólicos, em 72 horas de processo. Além disso, mostraram resposta positiva para a atividade antiinflamatória, apresentando capacidade de inibição máxima de 70,75% e concentração inibitória de 4,2 mg mL. Os produtos fermentados pelo R. oligosporus NRRL 2710 foram submetidos a diferentes processos de extração: Soxhlet com diferentes solventes e supercrítica de CO2 + etanol (scCO2 + EtOH), o que permitiu verificar a relação entre a polaridade e o rendimento de extração. As extrações de Soxhlet foram realizadas usando amostras fermentadas secas e úmidas, os maiores valores de rendimento obtidos foram 42,09% e 32,84%, respectivamente. Este tipo de extração permitiu obter valores de atividade antioxidante na concentração inibitória (IC50) de 0,55 e 0,67 g g-1 e compostos fenólicos de 1,30 e 1,05 mg EAG g-1. Diferentes condições foram testadas no processo de extração com fluido comprimido (scCO2 + EtOH), para estudar a influência da pressão e temperatura no rendimento da extração. Os resultados mostraram que ambas as variáveis influenciam o rendimento do processo. Os extratos deste processo apresentaram um valor de IC50 de 0,17 g g-1, inibindo 94,05% do radical DPPH. Quanto aos compostos fenólicos, estes extratos apresentaram 1,39 mg EAG g-1. / The soybean and its products are rich in isoflavonoids, these have the increased concentration after the microbial fermentation, due to the release of phenolics, through the conversion of the glycosidic forms into aglycones. The solid state fermentative process by filamentous fungi has been widely used as a biotechnological strategy in the search of aggregating values to agroindustrial products and byproducts, mainly raising the levels of bioactive compounds. In the present study, the increase of these antioxidant and anti-inflammatory properties in different substrates using fungal strains of the genus Rhizopus sp. The 17 lines tested were able to raise the antioxidant potential of the soybean grains. The fermentation of soybean and rice using R. oligosporus NRRL 2710 resulted in an increase of 26.5 times the antioxidant potential and 9.33 times the content of phenolic compounds in the ethanolic extracts, in 72 hours of process. In addition, they showed a positive response to anti-inflammatory activity, with a maximum inhibitory capacity of 70.75% and an inhibitory concentration of 4.2 mg mL. The products fermented by R. oligosporus NRRL 2710 were submitted to different extraction processes: Soxhlet with different solvents and supercritical CO2 + ethanol (scCO2 + EtOH), which allowed to verify the relationship between polarity and extraction yield. Soxhlet extractions were performed using dry and wet fermented samples, the highest values of yield obtained were 42.09% and 32.84%, respectively. This type of extraction allowed to obtain values of antioxidant activity at the inhibitory concentration (IC50) of 0.55 and 0.67 g g-1 and phenolic compounds of 1.30 and 1.05 mg GAE g-1. Different conditions were tested in the compressed fluid extraction process (scCO2 + EtOH) to study the influence of pressure and temperature on extraction yield. The results showed that both variables influence the yield of the process. The extracts from this procedure had an IC50 value of 0.17 g g-1, inhibiting 94.05% of the DPPH radical. As for phenolic compounds, these extracts presented 1.39 mg GAE g-1.

<p>The main aim of the thesis was to explore the potential of supercritical fluid (SF) techniques in the field of drug delivery. In particular, the relatively recently developed solution-enhanced dispersion by supercritical fluids (SEDS) technology has been employed in the preparation of particles/powders. </p><p>The manufacturing, stability and bioavailability of a dosage form strongly depend on the physicochemical properties of the formulation particles. For example, dry powder inhalation (DPI) for administering drugs to the respiratory tract require particles in a narrow size range (1-5 μm) to be effective. The identification of polymorphs and control of purity are also important issues since the physicochemical properties and therapeutic effects of the alternative forms of a drug may differ substantially. Solvent-based traditional crystallisation processes provide the product that may require further down-stream processing to obtain particles for advanced drug delivery applications. This can result in unwanted changes in the physicochemical properties of the particles and thus affect the performance of the dosage form. SF processing has addressed many of the challenges in particle formation research. Among several SF technologies developed for particle processing over the last decade, the SEDS process with its specially designed co-axial nozzle with mixing chamber has resulted in improved control over the particle formation process. Carbon dioxide (CO₂) was used as the SF, because it has low critical points and is non-toxic, non-flammable and relatively inexpensive. </p><p>The initial part of the thesis concerns the formation of particles of model drugs such as hydrocortisone, budesonide and flunisolide using SEDS technology and the determination of the influence of processing conditions and solvents on particle characteristics such as size, shape and crystal structure. Particles of model drugs of differing shapes in a size range suitable for inhalation delivery were prepared. In the process, two new polymorphic forms of flunisolide were identified. This was the first report of SEDS technology being shown as a polymorph-screening tool. The remainder of the thesis deals with the development of SEDS technology for precipitating therapeutic proteins such as recombinant human growth hormone (hGH) from aqueous solutions. Powders of hGH were precipitated using SEDS without significant changes in the chemical or physical stability of the protein. The addition of sucrose to hGH in the feed solution promoted precipitation and minimised the detrimental effects of the solvent and/or the process on the physical aggregation of the protein. </p><p>In conclusion, this thesis highlights the applicability of the SEDS process in drug delivery research and advances general understanding of the particle formation phenomenon. The SEDS process may also prove to be a potential alternative technology for the precipitation of stable powders of therapeutic proteins.</p>

The main aim of the thesis was to explore the potential of supercritical fluid (SF) techniques in the field of drug delivery. In particular, the relatively recently developed solution-enhanced dispersion by supercritical fluids (SEDS) technology has been employed in the preparation of particles/powders. The manufacturing, stability and bioavailability of a dosage form strongly depend on the physicochemical properties of the formulation particles. For example, dry powder inhalation (DPI) for administering drugs to the respiratory tract require particles in a narrow size range (1-5 μm) to be effective. The identification of polymorphs and control of purity are also important issues since the physicochemical properties and therapeutic effects of the alternative forms of a drug may differ substantially. Solvent-based traditional crystallisation processes provide the product that may require further down-stream processing to obtain particles for advanced drug delivery applications. This can result in unwanted changes in the physicochemical properties of the particles and thus affect the performance of the dosage form. SF processing has addressed many of the challenges in particle formation research. Among several SF technologies developed for particle processing over the last decade, the SEDS process with its specially designed co-axial nozzle with mixing chamber has resulted in improved control over the particle formation process. Carbon dioxide (CO2) was used as the SF, because it has low critical points and is non-toxic, non-flammable and relatively inexpensive. The initial part of the thesis concerns the formation of particles of model drugs such as hydrocortisone, budesonide and flunisolide using SEDS technology and the determination of the influence of processing conditions and solvents on particle characteristics such as size, shape and crystal structure. Particles of model drugs of differing shapes in a size range suitable for inhalation delivery were prepared. In the process, two new polymorphic forms of flunisolide were identified. This was the first report of SEDS technology being shown as a polymorph-screening tool. The remainder of the thesis deals with the development of SEDS technology for precipitating therapeutic proteins such as recombinant human growth hormone (hGH) from aqueous solutions. Powders of hGH were precipitated using SEDS without significant changes in the chemical or physical stability of the protein. The addition of sucrose to hGH in the feed solution promoted precipitation and minimised the detrimental effects of the solvent and/or the process on the physical aggregation of the protein. In conclusion, this thesis highlights the applicability of the SEDS process in drug delivery research and advances general understanding of the particle formation phenomenon. The SEDS process may also prove to be a potential alternative technology for the precipitation of stable powders of therapeutic proteins.

La délivrance contrôlée de médicaments constitue un enjeu thérapeutique de première importance pour le milieu médical. Elle doit, en effet, permettre de diminuer la toxicité du médicament en limitant sa concentration dans l'organisme et en ciblant son lieu de délivrance. Ce projet de recherche a conduit à la réalisation de nouveaux nano-vecteurs particulaires utilisables pour la délivrance de médicaments par hyperthermie locale. Ces objets sont constitués d'un cœur de polynorbornène et d’une écorce de poly (oxyde d’éthylène) et sont synthétisés par polymérisation par ouverture de cycle de type métathèse (ROMP) en dispersion. L’acide salicylique est encapsulé dans ces particules en tant que molécule modèle. L'originalité de ce sujet pluridisciplinaire réside : dans la nature de la particule, qui permet une libération du principe actif à partir de 35-45°C et dans la technique d'imprégnation des particules par la voie fluide supercritique. / Controlled release systems are attracting increasing interest because of their potential application in biomedical field. Indeed, such systems should enable to lower toxicity of medicines by targeting the therapeutic action and by limiting the concentration of drugs in the organism. In this research project, new nanoparticles have been developed for a thermosensitive control of drugs’ delivery. These particles consist of a polynorbornene core and a polyethylene oxide shell and are developed by dispersion ring opening metathesis polymerization. As a model drug, salicylic acid has been encapsulated in those core-shell materials. This multidisciplinary project is original for two reasons. On the one hand, the nature of the particle which enables to release the drug from 35-45°C. On the other hand, the encapsulation technique that has been chosen is the impregnation under supercritical carbon dioxide.

To contribute to the knowledge of the chemical species P. klotzschianum, P. hispidum and P. arboreum, analyzes of volatile obtained by hydrodistillation-(HD), extracts obtained by supercritical fluid extraction-(SFE) and identification of isolated compounds were performed using techniques such as GC/MS/FID, NMR (1D, 2D) and IV. Sesquiterpenes present in P. klotzschianum, P. hispidum and P. arboreum as the (E)-caryophyllene, bicyclogermacrene and germacrene D observed only in P.hispidum and P. klotzschianum were the main substances responsible for the high values obtained sesquiterpenes identified by HD. For the monoterpenes £]-pinene (27.19¡Ó0.27%) and £-pinene (7.17¡Ó0.50%) identified in the chemical composition of the stems of P. klotzschianum and Ô-3-carene (17.39¡Ó0.15%, 19.13¡Ó0.48%) respectively present in fresh and dry leaves of P. hispidum also deserved highlighted. The highest yield of extract using supercritical carbon dioxide-(SC-CO2) was 1.36% (80 ¢XC/220 bar) P. klotzschianum and 1.92% (80 ¢XC/200 bar) for P. hispidum, already with the addition of modifiers 2.18% for P. klotzschianum and 3.62% P. hispidum both using 5% methanol at 80 ¢XC/220 bar. By analysis of variance and F test the cosolvent was the variable that most influenced the yields of extracts for F = 288.95 (P. klotzschianum) and F = 409.59 (P.hispidum). The major compounds identified by GC/MS/FID of P. hispidum using SC-CO2 and SC-CO2 + co-solvents were germacrene D (21.08%-80 ¢XC/200 bar-isopropanol 1%) and squalene (15.18%-80 ¢X C/200 bar). Four compounds not initially identified by GC/MS/FID were isolated from extracts of SC-CO2 by column-chromatography (CC) followed by preparative layer chromatography-(CCDP): N-[3-(6-methoxy-3´, 4´-methylenedioxyphenyl)-2(Z)-propenoyl]-pyrrolidine (PHC7_1), N-[3-(6´-methoxy-3´, 4´-methylenedioxyphenyl)-2 (E)-propenoyl]-pyrrolidine (PHC7_2), N-[7 (3´, 4´ methylenedioxyphenyl)-2(Z), 4(E)-heptadienoil]-pyrrolidine (PHC6_2) and N-[7 (3´, 4´ methylenedioxyphenyl)-2(E), 4(Z)-heptadienoil]-pyrrolidine (PHC9), among these only PHC7_1 was cited in the literature on P. hispidum, the others were first reported in the species, with an PHC6_2 PHC9 unpublished in the literature. In chemical composition of the extracts of P. klotzschianum, germacrene D (25.00¡Ó0.11%-40 ¢XC/220 bar/isopropanol 3%), bicyclogermacrene (15.41¡Ó0.23%-40 ¢XC/220 bar/isopropanol 3%), 14-oxy-muuroleno-£ (21.23¡Ó0.10%-80 ¢XC/180 bar/ethanol 3%) pipercalosidina (22.06¡Ó0.01%-40 ¢XC/180 bar/methanol 1%) and (E)-caryophyllene (11.66¡Ó0.05%-40 ¢XC/220 bar/isopropanol 3%) are the major constituents present in the extracts obtained with SC-CO2 or addition modifier. Among the samples tested in front of IC50 HepG2 and HL-60 cells, the samples P. hispidum stems oil (94.77¡Ó1.93%-HepG2), P. klotzschianum fruit oil (90.53¡Ó1.10%-HepG2, 76.64¡Ó7.44%-HL-60) and P. klotzschianum oil fresh leaves (93.24¡Ó0.70%-HepG2) had cytotoxic activity. The oil obtained from the dried leaves of P. klotzschianum (122.372¡Ó1.247 £gg mL-1), dried leaves of P. hispidum (141.876¡Ó2.333 £gg mL-1), fresh leaves of P. arboreum (187.901¡Ó2.106 £gg mL-1) and fresh leaves P. klotzschianum (223.051¡Ó1.253 £gg mL-1) showed larvicidal activity against the mosquito Aedes aegypti. / Visando contribuir para o conhecimento quimico das especies P. klotzschianum, P. hispidum e P. arboreum, analises dos volateis obtidos por hidrodestilacao-(HD), extratos obtidos por extracao com fluido supercritico-(EFS) e identificacao de compostos isolados foram realizadas por tecnicas como CG/EM/DIC, RMN (1D, 2D) e IV. Sesquiterpenos presentes na P. klotzschianum, P. hispidum e P. arboreum como o (E)-cariofileno, biciclogermacreno e o germacreno D observados somente na P. klotzschianum e P. hispidum foram as principais substancias responsaveis pelos altos percentuais de sesquiterpenos identificados obtidos por HD. Para os monoterpenos o £]-pineno (27,19¡Ó0,27%) e £-pineno (7,17¡Ó0,50%) identificados na composicao quimica dos caules de P. klotzschianum e Ô-3-careno (17,39¡Ó0,15%-19,13¡Ó0,48%) respectivamente presente nas folhas frescas e folhas secas da P. hispidum mereceram destaque. O maior rendimento de extrato utilizando dioxido de carbono supercritico-(SC-CO2) foi 1,36% (80 ¢XC/220 bar) P. klotzschianum e 1,92% (80 ¢XC/200 bar) para a P. hispidum, ja com adicao de modificadores 2,18% para a P. klotzschianum e 3,62% para a P. hispidum ambas utilizando 5% de metanol a 80 ¢XC/220 bar. Pela analise de variancia e teste F o co-solvente foi a variavel que maior influenciou para os rendimentos dos extratos F=288,95 (P. klotzschianum) e F=409,59 (P.hispidum). Os constituintes majoritarios identificados por CG/EM/DIC para a P. hispidum utilizando SC-CO2 e SC-CO2 + co-solventes foram o germacreno D (21,08%-80 ¢XC/200 bar- isopropanol 1%) e o esqualeno (15,18%-80 ¢XC/200 bar). Quatro substancias inicialmente nao identificadas por CG/EM/DIC foram isoladas dos extratos de SC-CO2 por meio de cromatografia em coluna-(CC) seguida de cromatografia em camada preparativa-(CCDP) N-[3-(6¡¦-metoxi-3¡¦,4¡¦-metilenodioxifenil)-2(Z)-propenoil]-pirrolidina (PHC7_1), N-[3-(6¡¦-metoxi-3¡¦,4¡¦-metilenodioxifenil)-2(E)-propenoil]-pirrolidina (PHC7_2), N-[7(3¡¦,4¡¦ metilenodioxifenil)-2(Z),4(E)-heptadienoil]-pirrolidina (PHC6_2) e N-[7(3¡¦,4¡¦ metilenodioxifenil)-2(E),4(Z)-heptadienoil]-pirrolidina (PHC9), dentre estas apenas a PHC7_1 foi citada na literatura sobre P. hispidum, as demais foram relatadas pela primeira vez na especie, sendo a PHC6_2 e PHC9 ineditas na literatura. Na composicao quimica dos extratos da P. klotzschianum o germacreno D (25,00¡Ó0,11%-40 ¢XC/220 bar/isopropanol 3%), biciclogermacreno (15,41¡Ó0,23%-40 ¢XC/220 bar/isopropanol 3%), 14-oxi-£-muuroleno (21,23¡Ó0,10%-80 ¢XC/180 bar/etanol 3%), pipercalosidina (22,06¡Ó0,01%-40 ¢XC/180 bar/metanol 1%) e (E)-cariofileno (11,66¡Ó0,05%-40¢XC/220 bar/isopropanol 3%) foram os constituintes majoritarios presentes nos extratos obtidos com SC-CO2 ou com adicao de modificadores. Dentre as amostras testadas na IC50 frente as celulas HepG2 e HL60 as amostras P. klotzschianum oleo dos caules (94,77¡Ó1,93%-HepG2), P. klotzschianum oleo dos frutos (90,53¡Ó1,10%-HepG2, 76,64¡Ó7,44%-HL60) e P. klotzschianum oleo das folhas frescas (93,24¡Ó0,70%-HepG2) apresentaram atividade citotoxica. Os oleos obtidos das folhas secas da P. klotzschianum (122,372¡Ó1,247 £gg mL-1), folhas secas da P. hispidum (141,876¡Ó2,333 £gg mL-1), folhas frescas da P. arboreum (187,901¡Ó2,106 £gg mL-1) e folhas frescas da P. klotzschianum (223,051¡Ó1,253 £gg mL-1) apresentaram atividade larvicida contra o mosquito Aedes aegypti.