Este estudo visou à utilização da tecnologia que emprega CO2 em estado supercrítico (SFE) para estudar a bioatividade dos diterpenos presentes no óleo de café verde, cafestol (C) e caveol (K), contudo propondo também a otimização de extração com líquido pressurizado (PLE) em batelada, utilizando para isto um Delineamento Composto Central Rotacional (DCCR2). O referido tema tem caráter inovador e inédito, já que a tecnologia de extração com líquido pressurizado, até então empregada para extração de analitos, passa a ser estudada em processos de extração de óleos vegetais tais como, óleo de pequi, óleo de café verde, extrato de sementes de pitanga no LTAPPN. Esta técnica utiliza solventes orgânicos e emprega elevada temperatura de extração, o que aumenta a capacidade de solubilização do solvente, e o emprego de altas pressões que acelera a difusão nos poros da matriz já que a viscosidade do solvente é diminuída. Este comportamento ocasiona maior penetração do solvente na matriz, aumentando sua capacidade de extração. A extração supercrítica (SFE) é uma tecnologia limpa, pois não emprega solventes orgânicos sendo promissora na obtenção de extratos enriquecidos com compostos bioativos que possam desempenhar alguma atividade. O estudo da atividade aguda dos diterpenos presentes no óleo de grãos de café verdes obtidos via SFE e PLE demonstrou que o óleo extraído com CO2 supercrítico, na dose de 2.000 mg/Kg no estudo de toxicidade aguda e nas doses de 25, 50 e 75 mg/Kg no estudo de toxicidade de doses repetidas, não apresentou letalidade aos animais, porém parâmetros bioquímicos, hematológicos e histológicos, apresentaram alterações. Todavia para aplicações do óleo de café verde em produtos desenvolvidos pelas indústrias farmacêuticas, alimentícias e/ou cosméticas, mais estudos de avaliação dos efeitos do óleo de café verde in vivo são necessários. Igualmente, o estudo de inovação tecnológica para obtenção de óleo de café verde visa obter extratos enriquecidos em diterpenos, evitando a degradação e tornando-os mais estáveis. Os resultados obtidos indicam que o óleo dos grãos de café verde extraídos via SFE e PLE (em batelada) possuem altas concentrações dos compostos ativos cafestol e caveol, sendo a condição de melhor rendimento a condição 4 (70º C e 8 min. ) a de maior rendimento de óleo 9,78%. / This study aimed to evaluate the bioactivity of the diterpenes in green coffee oil, cafestol (C), and kahweol (K) using supercritical fluid extraction (SFE- CO2), and to optimize the extraction with pressurized liquid extraction (PLE) in batch by using a rotatable central composite design (RCCD2). This issue has innovative and novel character, since the pressurized liquid extraction technology available until now for analyte extraction has been studied in vegetable oil extraction processes such as Pequi oil, green coffee oil, and Pitanga seeds extracts, at LTAPPN. This technique uses organic solvents and high extraction temperature, which increases the solvent solubilization capacity. The use of high pressure accelerates the diffusion rates into the pores of the matrix, due to the lower solvent viscosity. This behavior provides greater penetration of the solvent into the matrix, increasing solvent extraction capacity. The supercritical fluid extraction (SFE) is a clean technology as it does not employ organic solvents, besides being a promising alternative to obtain extracts enriched with bioactive compounds. The results of the acute activity of diterpenes in green coffee oil extracted by SFE and PLE showed that although no lethality was observed in the animals using the oil extracted by SFE at a dose of 2,000 mg / kg in an acute toxicity study, and 25, 50, and 75 mg / kg in a repeated dose toxicity study, changes were observed in biochemical, hematological, and histological parameters. However, more in vivo studies about the effects of green coffee oil are required for pharmaceutical, food, and cosmetic applications. Similarly, the technological innovation to obtain the green coffee oil aims at obtaining extracts enriched in diterpenes, preventing degradation and increasing stability. The results indicate that green coffee oil extracted by SFE and PLE (in batch) have high concentrations of the active compounds cafestol and kahweol, and the best extraction condition was the trial 4 (70 º C, and 8 min), and the highest oil yield was 9.78%.

Enquanto as amêndoas de babaçu são conhecidas por fornecer óleo rico em zinco, fósforo e potássio, seu mesocarpo possui propriedades anti-inflamatórias ainda pouco exploradas. Este estudo visou determinar a composição físico-química e o potencial de toxicidade in vivo do óleo de amêndoas de babaçu e da farinha de seu mesocarpo (ambos extraídos com CO2 supercrítico) bem como avaliar a atividade anti-inflamatória do mesocarpo (extraído com etanol pressurizado). Os extratos foram obtidos via técnicas inovadoras de extração que evitam a degradação de compostos termossensíveis e os resultados aqui alcançados apontam para a composição de extratos obtidos via tecnologia verde e sua caracterização. A extração com fluido supercrítico (SFE) é uma tecnologia limpa (pois não emprega solventes orgânicos) e promissora para obter extratos enriquecidos em compostos com atividade biológica como, por exemplo, fitosterois. Por sua vez, a extração com líquido pressurizado (PLE) é considerada rápida, eficiente e seletiva, sendo este trabalho o primeiro a isolar os flavonoides do extrato etanólico do mesocarpo de babaçu extraído via PLE. Os resultados indicaram que os maiores rendimentos de extração do óleo foram de 55,34 g/100g usando álcool isopropílico como solvente pressurizado e de 53,12 g/100g usando etanol. Tais rendimentos representam 92,4 e 88,7% do total de óleo nas sementes, respectivamente. Rico em ácidos láurico, mirístico, oleico e palmítico, o perfil de ácidos graxos foi o mesmo para os dois solventes nas condições operacionais utilizadas. Com uso de álcool isopropílico, o extrato apresentou a maior concentração de β-sitosterol mediante 3 min de contato com o solvente (St) e 66% de volume de solvente (≅ 7,48 mL, SV). Com uso de álcool etílico, a maior concentração de β-sitosterol ocorreu para 3 min de tempo de contato (St) e 94% (≅ 10,25 mL) de volume de solvente (SV). Em função da composição de ácidos graxos, os principais TAGs presentes no óleo foram COL, LOL, LOM, LOP e MOP. Na SFE, os maiores rendimentos do óleo de babaçu variaram de 51,94 a 57,71 g/100g (para 25 MPa) e de 53,65 a 59,93 g/100g (para 35 MPa), para temperaturas de 40, 50, 60, 70 e 80 °C. A maior concentração de β-sitosterol (27,43 mg/100g de óleo) foi obtida na extração a 70 °C e 25 MPa. Os extratos do mesocarpo de babaçu também foram obtidos via PLE com etanol pressurizado. O maior rendimento (2,95%) e a maior concentração de fenólicos totais (1.257,25 mgGAE/100g) de pó dos extratos do mesocarpo de babaçu foram obtidos a 86 °C e 3 min (St). A maior atividade antimicrobiana na inibição de Staphylococcus aureus ocorreu extrações com menores tempos de contato (St) e elevadas temperaturas (na faixa supra), condições que também propiciaram os melhores rendimentos de extração e atividade antioxidante. O estudo de toxicidade foi feito para o óleo de amêndoas de babaçu obtido via SFE. Na dose aguda de 2.000 mg/kg e no estudo subagudo nas doses de 1, 3 e 5 mg/kg de óleo (doses repetidas), os resultados indicaram que este óleo não apresenta letalidade aos animais. Para avaliar a atividade anti-inflamatória foi utilizado o extrato do mesocarpo de babaçu obtido com etanol pressurizado a 86 °C e 3 min (St). Tal atividade foi confirmada para a dose de 30 mg/kg, administrada em ratos com processo inflamatório induzido com carragenina a 3%. Embora tenham sido constatadas a baixa toxicidade do óleo e a atividade anti-inflamatória do extrato do mesocarpo, mais estudos de avaliação toxicológica são necessários para aplicações do óleo de amêndoas e do extrato de mesocarpo de babaçu em produtos farmacêuticos, alimentícios e/ou cosméticos. / While babassu almonds are known for providing oil rich in zinc, phosphorus and potassium, its mesocarp has anti-inflammatory properties yet underexplored. The present study aimed at determining physical-chemical composition and in vivo toxicity potential of babassu almonds oil and its mesocarp flour (both extracted with supercritical CO2) as well as anti-inflammatory activity of babassu mesocarp (extracted with pressurized ethanol). Extracts were obtained using innovative extraction techniques that avoid the degradation of thermosensitive compounds and results here obtained point to the composition of extracts obtained via green technology and their characterization. Supercritical fluid extraction (SFE) is a promising clean technology (as organic solvents are not employed) to obtain extracts enriched in compounds with biological activity (e.g. phytosterols). In its turn, pressurized fluid extraction (PFE) is considered fast, efficient and selective, this work being the first to isolate flavonoids from ethanolic extract of babassu mesocarp extracted via PLE. Results indicated that highest extract yields of babassu oil were 55.34 g/100 g with isopropyl alcohol as pressurized solvent and 53.12 g/100 g with ethanol as solvent. Aforesaid extracts respectively represent 92.4% and 88.7% of total oil in seeds. Rich in lauric, myristic, oleic and palmitic acids, the fatty acid profile was the same for the two solvents and applied operating conditions. Using isopropyl alcohol, extract had the highest β-sitosterol concentration for 3 min contact time with solvent (St) and 66% of volume of solvent, ≅ 22.44 mL (SV). Using ethyl alcohol as solvent, highest β-sitosterol concentration occurred for St = 3 min and SV = 94% ≅ 31.96 mL. According fatty acids composition, main TAGs present in oil were COL, LOL, LOM, LOP and MOP. In SFE, highest yields of babassu oil ranged from 51.94 to 57.71 g/100 g (at 25 MPa) and from 53.65 to 59.93 g/100 g (at 35 MPa) at temperatures of 40, 50, 60, 70 and 80 °C. The highest concentration of β-sitosterol (namely, 27.43 mg/100g-oil) was obtained for extraction at 70 °C and 25 MPa. Babassu mesocarp extracts were also obtained via PLE with pressurized ethanol. The highest yield (2.95%) and the highest total phenolic concentration (1,257.25 mgGAE/100g) of babassu mesocarp extract were obtained at 86 °C and 3 min (St). The highest antimicrobial activity in Staphylococcus aureus inhibition occurred for lower contact time (St) values and higher temperatures (in aforesaid values), which were conditions also providing the best extraction yields and antioxidant activity. Toxicity study was carried out for babassu almond oil obtained via SFE. At the acute dose of 2,000 mg/kg and in subacute dose study at 1, 3 and 5 mg/kg of oil (repeated doses), results indicated that this oil did not present lethality in animals. Anti-inflammatory activity was evaluated by using babassu mesocarp extracted with pressurized ethanol at 86 °C and 3 min (St). Such activity was confirmed at 30 mg/kg dose administered in rats with induced inflammatory process with carrageenan at 3%. While low toxicity of oil and anti-inflammatory activity of mesocarp extract were both observed, further evaluation studies are required for applications of almond oil and babassu mesocarp extracts in pharmaceutical, food and/or cosmetic products.

Polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs), two groups of struc-turally related chlorinated aromatic hydrocarbons, generally referred to as “dioxins” are of great concern due to their extreme toxicity and presence in all compartments of the environment. Since they occur at very low levels, their analysis is complex and challenging, and there is a need for cost-efficient, reliable and rapid analytical alternatives to the expensive methods in-volving use of gas chromatography high-resolution mass spectrometry (GC-HRMS). It is im-portant to routinely monitor food and feed items to detect contaminations at an early stage. For the regulation of dioxins and dioxin-like PCBs in food and feed according to current legis-lation, large numbers of samples have to be analysed. Furthermore, soils at many industrial sites are also contaminated with dioxins and need remediation. In order to optimize the cost-efficiency of reclamation activities it is important to acquire information about the levels and distribution of dioxins in the contaminated areas. The aim of the studies underlying this thesis was to investigate the potential of comprehen-sive two-dimensional gas chromatography with a micro-electron capture detector (GC × GC-µECD) as a cost-effective method for analysing dioxins and dioxin-like PCBs in food, feed, fly ash and contaminated soils. Quantification studies of dioxins and dioxin-like PCBs were con-ducted and results were compared with GC-HRMS reference data. Generally, there was good agreement between both the congener-specific results and data expressed as total toxic equiva-lents (TEQs). The developed GC × GC-µECD method meets the European Community (EC) requirements for screening methods for control of dioxins and dioxin-like PCBs in food and feed. The presented results also indicate that GC × GC-µECD has potential to be used as a routine method for the congener-specific analysis of 2,3,7,8-PCDD/Fs and dioxin-like PCBs in matrices such as food and feed, fly ash and soil. However, to fully exploit the potential of the GC × GC-µECD technique, it should be combined with a fast and cost-efficient sample preparation procedure. Therefore, a number of certified reference materials (CRMs) were extracted using a new shape-selective pressurized liquid extraction technique with integrated carbon fractionation (PLE-C), and the purified extracts were analysed for PCDD/Fs using GC × GC-µECD. The results compared well with the certified values of a fly ash and a sandy soil CRM, but they were much too high for a com-plex clay soil CRM. It was concluded that this combination of techniques was very promising for screening ash and highly permeable soils. Further assessments and method revisions are still required before GC × GC-µECD can be used on a routine basis, and available software packages need to be refined in order to accelerate the data-handling procedures, which currently restrict the sample throughput.

Enquanto as amêndoas de babaçu são conhecidas por fornecer óleo rico em zinco, fósforo e potássio, seu mesocarpo possui propriedades anti-inflamatórias ainda pouco exploradas. Este estudo visou determinar a composição físico-química e o potencial de toxicidade in vivo do óleo de amêndoas de babaçu e da farinha de seu mesocarpo (ambos extraídos com CO2 supercrítico) bem como avaliar a atividade anti-inflamatória do mesocarpo (extraído com etanol pressurizado). Os extratos foram obtidos via técnicas inovadoras de extração que evitam a degradação de compostos termossensíveis e os resultados aqui alcançados apontam para a composição de extratos obtidos via tecnologia verde e sua caracterização. A extração com fluido supercrítico (SFE) é uma tecnologia limpa (pois não emprega solventes orgânicos) e promissora para obter extratos enriquecidos em compostos com atividade biológica como, por exemplo, fitosterois. Por sua vez, a extração com líquido pressurizado (PLE) é considerada rápida, eficiente e seletiva, sendo este trabalho o primeiro a isolar os flavonoides do extrato etanólico do mesocarpo de babaçu extraído via PLE. Os resultados indicaram que os maiores rendimentos de extração do óleo foram de 55,34 g/100g usando álcool isopropílico como solvente pressurizado e de 53,12 g/100g usando etanol. Tais rendimentos representam 92,4 e 88,7% do total de óleo nas sementes, respectivamente. Rico em ácidos láurico, mirístico, oleico e palmítico, o perfil de ácidos graxos foi o mesmo para os dois solventes nas condições operacionais utilizadas. Com uso de álcool isopropílico, o extrato apresentou a maior concentração de β-sitosterol mediante 3 min de contato com o solvente (St) e 66% de volume de solvente (≅ 7,48 mL, SV). Com uso de álcool etílico, a maior concentração de β-sitosterol ocorreu para 3 min de tempo de contato (St) e 94% (≅ 10,25 mL) de volume de solvente (SV). Em função da composição de ácidos graxos, os principais TAGs presentes no óleo foram COL, LOL, LOM, LOP e MOP. Na SFE, os maiores rendimentos do óleo de babaçu variaram de 51,94 a 57,71 g/100g (para 25 MPa) e de 53,65 a 59,93 g/100g (para 35 MPa), para temperaturas de 40, 50, 60, 70 e 80 °C. A maior concentração de β-sitosterol (27,43 mg/100g de óleo) foi obtida na extração a 70 °C e 25 MPa. Os extratos do mesocarpo de babaçu também foram obtidos via PLE com etanol pressurizado. O maior rendimento (2,95%) e a maior concentração de fenólicos totais (1.257,25 mgGAE/100g) de pó dos extratos do mesocarpo de babaçu foram obtidos a 86 °C e 3 min (St). A maior atividade antimicrobiana na inibição de Staphylococcus aureus ocorreu extrações com menores tempos de contato (St) e elevadas temperaturas (na faixa supra), condições que também propiciaram os melhores rendimentos de extração e atividade antioxidante. O estudo de toxicidade foi feito para o óleo de amêndoas de babaçu obtido via SFE. Na dose aguda de 2.000 mg/kg e no estudo subagudo nas doses de 1, 3 e 5 mg/kg de óleo (doses repetidas), os resultados indicaram que este óleo não apresenta letalidade aos animais. Para avaliar a atividade anti-inflamatória foi utilizado o extrato do mesocarpo de babaçu obtido com etanol pressurizado a 86 °C e 3 min (St). Tal atividade foi confirmada para a dose de 30 mg/kg, administrada em ratos com processo inflamatório induzido com carragenina a 3%. Embora tenham sido constatadas a baixa toxicidade do óleo e a atividade anti-inflamatória do extrato do mesocarpo, mais estudos de avaliação toxicológica são necessários para aplicações do óleo de amêndoas e do extrato de mesocarpo de babaçu em produtos farmacêuticos, alimentícios e/ou cosméticos. / While babassu almonds are known for providing oil rich in zinc, phosphorus and potassium, its mesocarp has anti-inflammatory properties yet underexplored. The present study aimed at determining physical-chemical composition and in vivo toxicity potential of babassu almonds oil and its mesocarp flour (both extracted with supercritical CO2) as well as anti-inflammatory activity of babassu mesocarp (extracted with pressurized ethanol). Extracts were obtained using innovative extraction techniques that avoid the degradation of thermosensitive compounds and results here obtained point to the composition of extracts obtained via green technology and their characterization. Supercritical fluid extraction (SFE) is a promising clean technology (as organic solvents are not employed) to obtain extracts enriched in compounds with biological activity (e.g. phytosterols). In its turn, pressurized fluid extraction (PFE) is considered fast, efficient and selective, this work being the first to isolate flavonoids from ethanolic extract of babassu mesocarp extracted via PLE. Results indicated that highest extract yields of babassu oil were 55.34 g/100 g with isopropyl alcohol as pressurized solvent and 53.12 g/100 g with ethanol as solvent. Aforesaid extracts respectively represent 92.4% and 88.7% of total oil in seeds. Rich in lauric, myristic, oleic and palmitic acids, the fatty acid profile was the same for the two solvents and applied operating conditions. Using isopropyl alcohol, extract had the highest β-sitosterol concentration for 3 min contact time with solvent (St) and 66% of volume of solvent, ≅ 22.44 mL (SV). Using ethyl alcohol as solvent, highest β-sitosterol concentration occurred for St = 3 min and SV = 94% ≅ 31.96 mL. According fatty acids composition, main TAGs present in oil were COL, LOL, LOM, LOP and MOP. In SFE, highest yields of babassu oil ranged from 51.94 to 57.71 g/100 g (at 25 MPa) and from 53.65 to 59.93 g/100 g (at 35 MPa) at temperatures of 40, 50, 60, 70 and 80 °C. The highest concentration of β-sitosterol (namely, 27.43 mg/100g-oil) was obtained for extraction at 70 °C and 25 MPa. Babassu mesocarp extracts were also obtained via PLE with pressurized ethanol. The highest yield (2.95%) and the highest total phenolic concentration (1,257.25 mgGAE/100g) of babassu mesocarp extract were obtained at 86 °C and 3 min (St). The highest antimicrobial activity in Staphylococcus aureus inhibition occurred for lower contact time (St) values and higher temperatures (in aforesaid values), which were conditions also providing the best extraction yields and antioxidant activity. Toxicity study was carried out for babassu almond oil obtained via SFE. At the acute dose of 2,000 mg/kg and in subacute dose study at 1, 3 and 5 mg/kg of oil (repeated doses), results indicated that this oil did not present lethality in animals. Anti-inflammatory activity was evaluated by using babassu mesocarp extracted with pressurized ethanol at 86 °C and 3 min (St). Such activity was confirmed at 30 mg/kg dose administered in rats with induced inflammatory process with carrageenan at 3%. While low toxicity of oil and anti-inflammatory activity of mesocarp extract were both observed, further evaluation studies are required for applications of almond oil and babassu mesocarp extracts in pharmaceutical, food and/or cosmetic products.

Large numbers of samples often need to be prepared and analysed in surveys of organic pollutants in the environment, but while the methods commonly used in such surveys can provide abundant detail they are generally costly, time-consuming and require large amounts of resources, so there is a need for simpler techniques. The work underlying this thesis assessed the potential utility of more convenient sample preparation and bioanalytical techniques for rapidly screening various environmental matrices that could be useful complements to higher resolution methods. Initially, the utility of a simplified extraction technique followed by an enzyme-linked immunosorbent assay (ELISA) for detecting polycyclic aromatic hydrocarbons (PAHs) in authentic (i.e. unspiked) contaminated soils was explored. The results showed that there are relationships between the structure and cross-reactivity among compounds that often co-occur with target PAHs. However, their potential contribution to deviations between estimates of total PAH contents of soils obtained using ELISA and gas chromatography-mass spectrometry (GC-MS) based reference methods were limited. Instead, the cross-reactivity of target PAHs and the failure to extract all of the PAHs prior to the ELISA determinations were the main reasons for these deviations. Polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) were detected in food and feed matrices, as well as in authentic contaminated soils using different bioanalytical techniques – ELISA and two cell-based bioassays: CAFLUX and CALUX (chemically activated fluorescent/luciferase gene expression) assays. In addition, enhanced sample preparation techniques based on accelerated solvent extraction (ASE) were developed. ASE with integrated carbon fractionation (ASE-C) in combination with CAFLUX produced estimates of PCDD and PCDF contents in fish oil and fish meal that agreed well with results obtained using reference methods. Furthermore, results from ELISA and GC-high resolution MS analyses of extracts of PCDD- and PCDF-contaminated soil samples obtained using an adjusted ASE-C technique were strongly correlated. Finally, the thesis reports the first experiments in which the results of CAFLUX, CALUX, and ELISA determinations of PCDDs and PCDFs in extracts of authentic contaminated soil samples were evaluated and compared to those obtained using a reference method. All of the bioanalytical techniques were found to be sufficiently sensitive, selective, and accurate for use in screening in compliance with soil quality assessment criteria. Overall, the improved sample preparation and bioanalytical techniques examined proved to be useful potential complements to conventional methods, enhancing the analytical framework for PAHs, PCDDs, and PCDFs. However, further validation has to be undertaken before they are applied on a large-scale.

Submitted by Marilene Donadel (marilene.donadel@unioeste.br) on 2018-04-27T00:49:59Z No. of bitstreams: 1 Katia_A_Santos_2018.pdf: 1940481 bytes, checksum: dd10357b3e5c25bdf7e64bb4148af513 (MD5) / Made available in DSpace on 2018-04-27T00:49:59Z (GMT). No. of bitstreams: 1 Katia_A_Santos_2018.pdf: 1940481 bytes, checksum: dd10357b3e5c25bdf7e64bb4148af513 (MD5) Previous issue date: 2018-03-02 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Candeia (Eremanthus erythropappus) is a native species of the Brazilian Atlantic forest from which an essential oil with high concentration of sesquiterpene α-bisabolol is extracted. α-Bisabolol is an active principle of important application in the cosmetic and pharmaceutical industries due to its anti-inflammatory, antispasmodic, sedative, antiallergic, anti-irritant, cicatrizant and vermifugal properties. Steam distillation is the most common method used to obtain this oil, with requires long periods of time extraction besides the degradation of thermosensitive compounds. Within this context, the aim of this study was to evaluate the quality of the candeia wood oils obtained by non-conventional methods of extraction (supercritical technology by using carbon dioxide and cosolvents, pressurized liquid and ultrasound-assisted extraction), in terms of oil yield, bisabolol content and antioxidant activity. In addition, the oil re-extraction from the industrial residue was also evaluated. The extractions were carried out with CO2 at temperatures of 40, 55 and 70 oC and pressures of 160, 200 and 240 bar, with a solvent mass flow rate of 1.96 x 10−3 kg min−1 and 120 min of total extraction. The highest extraction yield obtained was 1.42 wt% for the candeia wood and 0.41 wt% for the residue, both at 70 oC and 240 bar, and this condition was selected to perform the extractions using cosolvents. Ethanol and ethyl acetate were added to supercritical CO2 at concentrations of 1, 3 and 5 % (v/v), obtaining up to 2.35 wt% of yield. The Soxhlet (360 min) and pressurized liquid (40, 55 and 70 oC; 100 bar and 20 min) extractions showed the affinity of the candeia compounds for polar solvents and the positive effect of the temperature on the yield, which varied from 0.53 to 7.23 wt%. A Box-Benhken design was employed to evaluate the effect of the variables temperature (40, 50 and 60 oC), n-hexane volume to wood mass (10, 15 and 20 mL g-1) and nominal power (150, 300 and 450 W) on the yield of ultrasound-assisted extractions, obtaining in 7 minutes of extraction, up to 83% of the yield obtained in the conventional technique in Soxhlet (1.57 wt%), with a solvent volume 2.5 times smaller. The major compounds identified in the candeia oil were the sesquiterpenes α-bisabolol, eremanthin and costunolide, and the α-bisabolol content in the oil is favored by the lowest CO2 density, with 74.45 % being obtained for the candeia wood and 50.62 % for the residue, in both cases in oil extracted at 70 oC and 160 bar. The addition of ethanol and ethyl acetate cosolvents to CO2 increased the α-bisabolol yield by 41 %. Also, the cosolvents increased the amount of total phenolic content in the oil, and consequently, its antioxidant capacity. The oil fractionation by column chromatography was efficient for α-bisabolol isolation. However, unlike the candeia wood oil, this compound was not effective in inhibiting the Staphylococcus aureus growth. In relation to the supercritical extractions, the Sovová mathematical model presented a good adjustment to the experimental data for all the conditions used. / A candeia (Eremanthus erythropappus) é uma árvore nativa da Mata Atlântica da qual se extrai um óleo essencial com elevada concentração do sesquiterpeno α-bisabolol, princípio ativo de grande aplicação nas indústrias de cosméticos e farmacêutica devido às suas propriedades anti-inflamatória, antiespasmódica, sedativa, antialérgica, anti-irritante, cicatrizante e vermífuga. A obtenção deste óleo em escala industrial é realizada pelo processo de destilação por arraste a vapor, com elevado tempo de extração, além da degradação de compostos termossensíveis. Neste contexto, o presente trabalho tem como objetivo avaliar a qualidade dos óleos da madeira de candeia, obtidos por métodos não convencionais de extração (tecnologia supercrítica com dióxido de carbono e cossolventes, líquidos pressurizados e solvente assistido por ultrassom), em termos de rendimento, teor de α-bisabolol e atividade antioxidante. Ainda, avaliar a re-extração do óleo do resíduo industrial. As extrações foram realizadas com CO2 nas temperaturas de 40, 55 e 70 oC e pressões de 160, 200 e 240 bar, com vazão mássica de solvente de 1,96 x 10-3 kg min-1 e tempo total de 120 minutos. O maior rendimento obtido para o óleo da candeia nas extrações supercríticas foi de 1,42 % e de 0,41 % para o resíduo, ambos em 70 oC e 240 bar, condição selecionada para os experimentos com cossolventes. Os solventes etanol e acetato de etila foram utilizados nas concentrações de 1, 3 e 5 % (v/v) junto ao CO2, proporcionando rendimentos de até 2,35 %. As extrações Soxhlet (360 min) e com líquidos pressurizados (40, 55 e 70 oC; 100 bar e 20 min) evidenciaram a afinidade dos compostos por solventes polares e o efeito positivo da temperatura sobre o rendimento, de 0,53 a 7,23 %. Um planejamento Box-Behnken foi empregado para avaliar efeitos da temperatura (40, 50 e 60 oC), razão volume de n-hexano/massa de madeira (10, 15 e 20 mL g-1) e potência ultrassônica nominal do equipamento (150, 300 e 450 W) sobre rendimento em óleo, obtendo-se, em 7 minutos de extração, até 83 % do rendimento obtido na técnica convencional em Soxhlet (1,57 %), com volume de solvente 2,5 vezes menor. Os compostos majoritários identificados no óleo da candeia foram os sesquiterpenos α-bisabolol, eremantina e costunolida, e o conteúdo do α-bisabolol no óleo foi favorecido pela menor densidade do CO2 supercrítico, sendo de até 74,5 % para a madeira de candeia e 50,6 % para o resíduo, ambos extraídos na condição de 70 oC e 160 bar. A adição dos cossolventes etanol e acetato de etila ao CO2 elevou o rendimento de α-bisabolol em até 41 %. Também aumentaram a quantidade de fenólicos totais no óleo e, consequentemente, sua capacidade antioxidante. O fracionamento do óleo por cromatografia em coluna foi eficiente para o isolamento do α-bisabolol. No entanto, diferentemente do óleo da madeira de candeia, este composto não foi efetivo na inibição do crescimento de Staphylococcus aureus. Em relação às cinéticas das extrações supercríticas, o modelo matemático de Sovová se ajustou aos dados experimentais em todas as condições utilizadas.

Polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs), two groups of structurally related chlorinated aromatic hydrocarbons, are of high concern due to their global distribution and extreme toxicity. Since they occur at very low levels, their analysis is complex, challenging and hence there is a need for efficient, reliable and rapid alternative analytical methods. Developing such methods was the aim of the project this thesis is based upon. During the first years of the project the focus was on the first parts of the analytical chain (extraction and clean-up). A selective pressurized liquid extraction (SPLE) procedure was developed, involving in-cell clean-up to remove bulk co-extracted matrix components from sample extracts. It was further streamlined by employing a modular pressurized liquid extraction (M-PLE) system, which simultaneously extracts, cleans up and isolates planar PCDD/Fs in a single step. Both methods were validated using a wide range of soil, sediment and sludge reference materials. Using dichloromethane/n-heptane (DCM/Hp; 1/1, v/v) as a solvent, results statistically equivalent to or higher than the reference values were obtained, while an alternative, less harmful non-chlorinated solvent mixture - diethyl ether/n-heptane (DEE/Hp; 1/2, v/v) – yielded data equivalent to those values. Later, the focus of the work shifted to the final instrumental analysis. Six gas chromatography (GC) phases were evaluated with respect to their chromatographic separation of not just the 17 most toxic congeners (2,3,7,8-substituted PCDD/Fs), but all 136 tetra- to octaCDD/Fs. Three novel ionic liquid columns performed much better than previously tested commercially available columns. Supelco SLB-IL61 offered the best overall performance, successfully resolving 106 out of the 136 compounds, and 16 out of the 17 2,3,7,8-substituted PCDD/Fs. Another ionic liquid (SLB-IL111) column provided complementary separation. Together, the two columns separated 128 congeners. The work also included characterization of 22 GC columns’ selectivity and solute-stationary phase interactions. The selectivities were mapped using Principal Component Analysis (PCA) of all 136 PCDD/F’s retention times on the columns, while the interactions were probed by analyzing both the retention times and the substances’ physicochemical properties.

Soil that is heavily contaminated with polycyclic aromatic hydrocarbons (PAHs) is often found at the sites of former gasworks and wood-impregnation plants. Since PAHs are toxic these sites represent a hazard to human health and the environment, and therefore they need to be treated, preferably by a method that destroys the contaminants, and thus eliminates the problem permanently. However, during biological and chemical degradation of PAHs other toxic compounds may be formed. If these transformation products are sufficiently persistent they could potentially accumulate during remedial processes. In the work underlying this thesis the degradation and transformation of PAHs were studied in three remedial processes: viz. a pilot-scale bioslurry reactor, microcosms with wood-rotting fungi and lab-scale treatments with Fenton's reagent. A group of transformation products referred to as oxygenated-PAHs (oxy-PAHs) was found to be particularly important, as these compounds are toxic and were shown to be relatively persistent in the environment. The oxy- PAHs were, for instance, found at significant concentrations in the gasworks soil used in most of the studies. This soil was highly weathered and had therefore been depleted of the more readily degradable compounds. In addition, experiments in which earthworms were exposed to the gasworks soil showed that the oxy-PAHs were more easily taken up in living organisms than PAHs. To facilitate the studies, new extraction and fractionation methods were developed. For instance, pressurized liquid extraction (PLE) was investigated for its reliability and efficiency to extract PAHs and oxy-PAHs from soil. Furthermore, a selective PLE-method was developed that can simultaneously extract and separate the PAHs and oxy-PAHs into two different fractions. This was accomplished by adding a chromatographic material (silica or Florisil) to the extraction cell. Under certain conditions all three remedial processes resulted in increasing amounts of oxy- PAHs in the soil. For example, 1-acenaphthenone and 4-oxapyrene-5-one accumulated in the bioslurry reactor. Similarly, in the soil inoculated with a white-rot fungus 9-fluorenone, benzo[a]anthracene-7,12-dione, 4-hydroxy-9-fluorenone and 4-oxapyrene-5-one accumulated. Finally, in an ethanol-Fenton treatment the concentration of some PAH-quinones increased in the soil. The results show that it might be necessary to monitor oxy-PAHs as well as PAHs during the remediation of PAH-contaminated sites. Otherwise, the soil may be considered detoxified too early in the process. In the long term it would be desirable to include analyses with sufficient marker compounds to follow the possible production and elimination of the oxy-PAHs. However, until such compounds can be identified it is suggested that contaminated soil should be screened for oxy-PAHs in general. The selective PLE-method presented in this thesis could be a useful tool for this.