Fundação de Apoio a Pesquisa e à Inovação Tecnológica do Estado de Sergipe - FAPITEC/SE / Eplingiella fruticosa (Lamiaceae) is an important aromatic medicinal herb traditionally used in northeastern Brazil for the treatment of pain, inflammation and convulsion. However, the scientific evidence of these activities has been little explored. Studies using essential oils (EOs) complexed with β-cyclodextrin (βCD) have shown promise for improving the biological effects of EOs in the management of chronic pain. Thus, the goal of the present study was to evaluate the antihyperalgesic effect of the essential oil obtained from E. fruticosa (EplEO) and its inclusion complex with βCD (EplEO-βCD) about chronic musculoskeletal pain in rodents. EplEO was extracted by hydrodistillation and its chemical composition was determined by gas chromatography coupled to mass spectrometry and flame ionization detector (GC-MS/FID). The EplEO-βCD complexes were prepared by two different methods (physical mixing and past complexation) and later characterized in differential scanning calorimetry, thermogravimetry / derivative thermogravimetry, moisture determination and scanning electron microscopy. After approval of the project by CEPA-UFS (Number 65/09) the FM model was induced in adult male albino Swiss mice by two injections of saline solution pH 4.0 (20 μl) in the left gastrocnemius, with a 5-day interval. After induction of hyperalgesia, the mice were treated daily (6th day to day 12) with EplEO (50 mg/kg, p.o.), EplEO-βCD (50 mg/kg, p.o.), vehicle (isotonic saline, p.o.) or Tramadol (4 mg/kg, i.p.) and evaluated for mechanical hyperalgesia. Previously, a time-effect curve was performed to verify the administration of EplEO and EplEO-βCD. In addition, the animals were evaluated for motor coordination and muscle strength using EplEO (50 mg/kg, p.o.), EplEO-βCD (50 mg/kg, p.o.), vehicle (isotonic saline, p.o.) or Diazepam (4 mg/kg, i.p.). After the experiments, the animals were sacrificed and evaluated for expression of Fos-positive cells in the spinal cord by immunofluorescence. EplEO produced a yield of 0.741% (m/m) and GC-MS/FID analysis identified 27 compounds in total, being (E)-cariophylene (14.16%), bicyclogermacrene (12.68%), 1,8-cineole (11.03%), α-pinene (6.79%) and β-pinene (5.10%) the majority. The complexation of EplEO-βCD by past complexation was shown to be more effective by the characterization tests. Treatment with EplEO-βCD produced a long-acting antihyperalgesic effect (p <0.05 vs. vehicle and p <0.05 vs EplEO) which had an effect time of eight hours when compared to EplEO (p <0.01 vs vehicle) with time effect of four hours, without changes in motor coordination or myorelaxant effect. In addition, EplEO and EplEO-βCD produced a significant antihyperalgesic effect (p <0.01 or p <0.001) over 7 consecutive days of treatment. The immunofluorescence assay demonstrated that the group of animals treated with EplEO had a significant (p <0.001) decrease in the number of Fos-positive cells in the spinal cord when compared to the vehicle group. In view of the foregoing, it has been demonstrated that the anti-hyperalgesic effect produced by EplEO has been improved following βCD complexation with a possible relationship in the classic central pain inhibition pathway. / Eplingiella fruticosa (Lamiaceae) é uma importante erva medicinal aromática utilizada tradicionalmente no nordeste do Brasil para o tratamento da dor, inflamação e convulsão. No entanto, as evidências científicas dessas atividades têm sido pouco exploradas. Estudos utilizando óleos essenciais (OEs) complexados com a β-ciclodextrina (βCD) têm se mostrado promissores para melhorar os efeitos biológicos dos OEs no manejo da dor crônica. Assim, o objetivo do presente estudo foi avaliar o efeito anti-hiperalgésico do óleo essencial obtido de E. fruticosa (EplEO) e seu complexo de inclusão com βCD (EplEO-βCD) sobre a dor crônica musculoesquelética em roedores. O EplEO foi extraído por hidrodestilação e sua composição química foi determinada por cromatografia gasosa acoplada a espectrometria de massa e detector por ionização de chama (CG-EM/DIC). Os complexos EplEO-βCD foram preparados através de dois métodos diferentes (mistura física e malaxagem) e posteriormente caracterizados em calorimetria exploratória diferencial, termogravimetria/termogravimetria derivada, determinação da umidade e microscopia eletrônica de varredura. Após a aprovação do projeto pelo CEPA-UFS (Nº 65/09) o modelo FM foi induzido em camundongos Swiss albinos machos adultos através de duas injeções de solução salina pH 4,0 (20 μl) no gastrocnêmio esquerdo, com 5 dias de intervalo. Após a indução da hiperalgesia, os camundongos foram tratados diariamente (6° dia ao 12º dia) com EplEO (50 mg/kg, via oral (v.o.)), EplEO-βCD (50 mg/kg, v.o), veículo (salina isotônica, v.o) ou Tramadol (4 mg/kg, i.p) e avaliados quanto a hiperalgesia mecânica. Também foi realizado uma curva tempo-efeito para verificar a administração de EplEO e EplEO-βCD. Além disso, os animais foram avaliados quanto a coordenação motora e força muscular utilizando EplEO (50 mg/kg, v.o), EplEO-βCD (50 mg/kg, v.o), veículo (salina isotônica, v.o) ou Diazepam (4 mg/kg, i.p). Após os experimentos, os animais foram sacrificados e avaliados quanto a expressão de células Fos positivas na medula espinal por imunofluorescência. O EplEO apresentou rendimento 0,741% (m/m) e a análise por CG-EM/DIC identificou 27 compostos ao total, sendo (E)-cariofileno (14,16%), biciclogermacreno (12,68%), 1,8-cineol (11,03%), α-pineno (6,79%) e β-pineno (5,10%) os majoritários. A complexação do EplEO-βCD por malaxagem mostrou-se mais efetiva pelos testes de caracterização. O tratamento com EplEO-βCD produziu um efeito anti-hiperalgésico de longa duração (p <0,05 vs veículo e p <0,05 vs EplEO) que teve um tempo de efeito de oitos horas quando comparado ao EplEO (p <0,01 vs veículo) com tempo efeito de quatro horas, sem alterações na coordenação motora ou efeito miorrelaxante. Além disso, o EplEO e o EplEO-βCD produziram um efeito anti-hiperalgésico significativo (p <0,01 ou p <0,001) ao longo de 7 dias consecutivos de tratamento. O ensaio de imunofluorescência demonstrou que o grupo dos animais tratados com EplEO obteve uma diminuição significativa (p <0,001) no número de células Fos positivas na medula espinhal quando comparado com o grupo veículo. Diante do exposto, foi demostrado que o efeito anti-hiperalgésico produzido por EplEO foi melhorado após complexação com βCD com possível relação na via clássica de inibição central da dor. / São Cristóvão, SE

Neste trabalho de Mestrado, foram feitos estudos do comportamento fotofísico da fisetina em diversos solventes através de medidas de absorção de luz UV-Visível, fluorescência estática e resolvida no tempo e da interação entre fisetina (3,3\',4\',7-tetrahidroxiflavona) e 7-hidroxiflavona com ciclodextrinas ( beta e gama) (CDs) através de experimentos de absorção de luz UV-Visível, sinal induzido de dicroísmo circular, fluorescência estática e resolvida no tempo, anisotropia de estado estacionário e RMN de 1H, focando a dependência destas medidas em função da temperatura e do pH. Os resultados experimentais foram comparados com estudos mecânico-quânticos baseados no modelo semi-empírico SAM1 (AMPAC), e nos funcionais B3LYP e MPW1PW91, da Teoria do Funcional de Densidade, empregando os conjuntos de funções de base 6-311G* e 3-21G**. Os estudos da fisetina em diferentes solventes próticos e apróticos mostraram que a fluorescência da sonda é fortemente dependente do solvente. Resultados experimentais indicam a formação de complexos de inclusão entre as CDs e os flavonóides acima. A fisetina com ?-CD forma complexo de estequiometria 1:1 nos meios neutro/ácido e básico, cujos valores de constante de complexação são 900 +- 100 +_ 240 +_ 90 (L/mol), respectivamente. Os dados teóricos evidenciaram que a inclusão da fisetina na beta-CD ocorre preferencialmente pelo anel fenila. O complexo com a gama-CD apresenta estequiometria de 1:1 em meio ácido/neutro e de 1:2 em meio básico, com constantes de complexação de 94 +- 30 e 130 +- 10 (L/mol), respectivamente. Os estudos com a 7-hidroxiflavona revelaram que somente ocorre a formação de complexos com a beta-CD de estequiometria 1:1 e não há dependência do pH. As constantes de complexação obtidas nos meios ácido/neutro e básico são similares, 1430 +-- 510 e 1220 +- 165 L/mol, respectivamente. / In this work, the photophysics of fisetin (3,3\',4\',7-tetrahydroxyflavone) in several solvents was studied through UV-vis absorption spectra, steady-state and time-resolved fluorescence measurements. The interaction between fisetin and 7-hydroxyflavone and cyclodextrins (b- e g-) (CDs) was also investigated by UV-vis absorption spectra, induced signal of circular dichroism, steady-state and time-resolved fluorescence, steady-state anisotropy, and 1H NMR, with dependence on pH and temperature. Some experimental data were compared with quantum-mechanics studies based on the SAM1 (AMPAC) semi-empirical model, as well as with the B3LYP and MPW1PW91 functional models from the Density Functional Theory using the 6-311G* and 3-21G* basis sets. The study of the photophysics of fisetin in protic and aprotic solvents showed a complex behavior and a strong dependence on the solvent. The occurrence of many equilibria between the possible structures of fisetin, e.g. the normal, a few deprotonated ones, and the tautomer due to the excited state intramolecular proton transfer can be responsible for the complex analyses of these experimental data. The spectroscopic measurements show that, at pH 4.0 and 6.5, the complex fisetin - b-CD is formed in a Fis:b-CD 1:1 stoichiometry and an equilibrium constant (K) of 900 ± 100 L/mol. In basic medium (pH 11.5), K decreases to 240 ± 90 L/mol. Molecular modeling points out that the inclusion complex is formed preferentially via entry of the fisetin phenyl group into b-CD. On the other hand, the fisetin - g-CD has a stoichiometry of 1:1 in acid/neutral solutions and of 1:2 in basic conditions. The K values are 94 ± 30 e 130 ± 10 (L/mol), respectively. The 7-hydroxyflavone can only form inclusion complexes with b-CD. The stoichiometry is 1:1 and there is no dependence on pH. Both equilibrium constants determined either in acid or basic medium is very similar to each other, 1430 ± 510, and 1220 ± 165 L/mol, respectively. For this reason, we suppose that the inclusion of this compound into b-CD is also through the phenyl ring of the flavonoid.

A preparação de formulações contendo o princípio ativo 17-valerato de betametasona (VB) é amplamente difundida entre as indústrias farmacêuticas, por se tratar de fármaco antiinflamatório de escolha, no tratamento de condições em que a terapia com corticoesteróides é indicada. Muito empregado no tratamento tópico de condições alérgicas e inflamatórias dos olhos, orelhas e nariz, inalação para a profilaxia da asma e também em veterinária. Isto devido ao seu alto poder antiinflamatório, quando comparado a outros corticoesteróides, e sua falta virtual de propriedades mineralocorticóides, causando baixa retenção de sódio e, subsequentemente, de água. Conforme descrita na Farmacopéia Americana USP 32 NF 27, o princípio ativo 17-valerato de betametasona hidrolisa-se em seu isômero 21-valerato de betametasona, seu principal produto de degradação, que possui baixo poder antiinflamatório. Adicionalmente, a norma brasileira em vigência para estudos de estabilidade de medicamentos, RE n°1, de 29 de Julho de 2005, propõe condições estressantes para estudo de estabilidade de longa duração (30°C/75%UR), o que acelera a reação de hidrólise (degradação) do princípio ativo. Conhecidamente, estudos prévios mostram que formulações tópicas contendo o VB (loção, creme, solução e pomada) apresentam uma estabilidade curta. Assim, uma forma de estabilizar o VB é a complexação (inclusão), com compostos de ciclodextrina (CD). O objetivo deste projeto foi estabelecer procedimentos para a obtenção, caracterização físico-química e avaliação de estabilidade térmica do complexo sólido supracitado. Para atender este objetivo técnicas de análise térmica (calorimetria exploratória diferencial e termogravimetria), infravermelho médio com transformada de Fourier, ressonância magnética nuclear e cromatografia líquida de alta eficiência, fizeram-se necessárias. / Preparation of formulations containing the active ingredient betamethasone 17-valerate (VB) is widely defunded within pharmaceutical industry, once it concerns an anti-inflammatory drug and an option, in the treatment of conditions in which corticosteroids therapy is indicated. Often employed in topical treatment of eye, ear and nose allergic and inflammatory conditions, inhalation for asthma prophylaxes, and also in veterinary. This because its high anti-inflammatory activity, when compared to others corticosteroids, and its virtual lack of mineralocorticoids properties, causing a low sodium retention and, subsequently, of water. As described in the United States pharmacopeia USP 32 NF 27, the active ingredient betamethasone 17-valerate hydrolyses into its isomer betamethasone 21- valerate, its main degradation product, that has a low anti-inflammatory activity . Additionally, the Brazilian legislation for drug products stability study, RE n°1, July 29th 2005, introduce long therm stability study stressing conditions (30°C/75%RH), accelerating the reactive hydrolysis (degradation) for the active ingredient. Well known, previous studies show that topical formulations containing VB (lotion, cream, solution and ointment) presents a short stability. Complexation (inclusion) with cyclodextrin (CD) compounds shows a reasonable way to improve the VB stability. The project objective is to establish procedures for the obtainment, physicochemical characterization and solid complex (cited above) thermal stability evaluation. In order to achieve this objective thermal analysis techniques (differential scanning calorimetry and thermogravimetry), Fourier transformation middle infrared, nuclear magnetic resonance and high performance liquid chromatography, were needed.

A furosemida é um fármaco de ação diurética e amplamente utilizado em tratamentos de doenças renais, cardíacas e pulmonares. Sua absorção é problemática e de alta variabilidade inter e intraindividual. Este fármaco tem sido classificado como pertencente às classes II (baixa solubilidade e alta permeabilidade) ou IV (baixa permeabilidade e baixa solubilidade) do Sistema de Classificação de Biofarmacêutica (SCB). Em estudos anteriores da equipe de pesquisa, SPRICIGO e colaboradores (2008) e SILVA (2014) desenvolveram complexos de furosemida com hidroxipropil-&#946;-ciclodextrina que permitiram a otimização da solubilidade deste fármaco. Entretanto, dados sobre a sua permeabilidade intestinal, quando complexado, não foram determinados. Somando-se a isto, a literatura apresenta informações distintas em relação a este parâmetro, o que corrobora a importância de se avaliar a permeabilidade deste fármaco. Diversas técnicas têm sido empregadas para a avaliação da permeabilidade intestinal dos fármacos. No presente trabalho empregou-se o modelo de perfusão in situ de passagem tripla, cuja técnica possibilita avaliar a permeabilidade em três segmentos diferentes em um mesmo animal e ainda, apresenta características interessantes, pois trata-se de um método que proporciona, durante todo o experimento, condições mais próximas daquelas encontradas durante o processo in vivo de absorção de fármacos no intestino tais como: suprimento sanguíneo, inervação intacta, preservação das proteínas transportadoras de membranas e presença da camada de muco. O presente trabalho foi dividido nas seguintes etapas: (i) obtenção da furosemida complexada com hidroxipropil-&#946;-ciclodextrina, (ii) caracterização dos fármacos utilizando técnicas de análises térmicas, (iii) estudo de perfusão in situ de passagem tripla nos três segmentos intestinais (duodeno, jejuno e íleo) de ratos machos Wistar na ausência e na presença de inibidores da glicoproteína P e de enzimas metabolizadoras CYP3A4 com posterior análise estatística do impacto da ciclodextrina e inibidores na permeabilidade da furosemida e; (iv) análise histológica das microvilosidades intestinais após o ensaio de perfusão in situ nos três segmentos intestinais. Os valores encontrados em cada segmento para furosemida complexada foram: 8,58 ± 0,002 x 10-5 cm.s-1; 9,15 ± 0,003 x10-5 cm.s-1 e; 8,06 ± 0,002 x 10-5 cm.s-1, respectivamente para duodeno, jejuno e íleo enquanto que para furosemida pura encontraram-se os seguintes: 3,42 ± 0,08 x 10-5 cm.s-1 para duodeno; 3,87 ± 0,11 x 10-5 cm.s-1 para jejuno e 3,08 ± 0,001 x 10-5 cm.s-1 para íleo. Assim sendo, os valores obtidos para a permeabilidade da furosemida complexada foram significativamente superiores (p < 0,05) aos da furosemida pura, sugerindo que, a ciclodextrina pode ter influência no mecanismo de transporte da furosemida, que é via passiva paracelular. Quanto aos mecanismos envolvidos na permeabilidade da furosemida através dos enterócitos, pode-se sugerir que observou-se pouca influência dos inibidores da glicoproteína P (P-gp) e da enzima CYP3A4, sugerindo que não há uma participação importante destes mecanismos em sua absorção intestinal. / Furosemide, which is a diuretic drug, is widely used in heart, kidney and pulmonary disease treatments. The absorption is problematic with high variability inter and intra individuals. This drug has been classified as belonging to class II (low solubility and high permeability) or IV (low permeability and low solubility) of the Biopharmaceutical Classification System (BCS). In previous studies of the research team, Spricigo and colleagues (2008) and Silva (2014) developed complex of furosemide with hydroxypropyl-&#946;-cyclodextrin which allowed the optimization of the solubility of this drug. However, datas concerning it\'s intestinal permeability, when complexed, have not been determined. Addicted to this, the literature shows many information regarding to this parameter, which confirms the importance of the evaluation of the permeability of this drug. Some techniques have been employed in order to evaluate the intestinal permeability of drugs. In the present work, a triple single-pass intestinal perfusion technique was used for three different segments. This technique enables the evaluation of the permeability of different segments in the same animal and also has interesting features such as: it provides during all the experiment conditions closer to those found in in vivo process of a drug absorption in the gut; blood supply; intact innervations; preservation of membrane transporter proteins and presence of mucus layer. This study was divided into the following steps: (i) obtaining furosemide complexed with hydroxypropyl-&#946;-cyclodextrin, (ii) characterization of drugs using techniques of thermal analysis, (iii) perfusion study in situ triple passage in three segments (duodenum, jejunum and ileum) from male Wistar rats in the absence and presence of inhibitors of P-glycoprotein and metabolizing enzymes CYP3A4 and subsequential statistical analysis of the impact of the cyclodextrin and the inhibitors in the permeability of furosemide and (iv) histological analysis of intestinal microvilli after in situ perfusion assay in three segments. The values found in each segment for complexed furosemide were: 8,58 ± 0,002 x 10-5 cm.s-1; 9,15 ± 0,003 x 10-5 cm.s-1; 8,06 ± 0,002 x 10-5 cm.s-1, respectively for duodenum, jejunum and ileum while for pure furosemide, the values were: 3,42 ± 0,08 x 10-5 cm.s-1 to duodenum; 3,87 ± 0,11 x 10-5 cm.s-1 to jejunum and 3,08 ± 0,001 x 10-5 cm.s-1 to ileum. Thus, the values obtained for the permeability of the complexed furosemide were significantly higher (p < 0,05) than those found for pure furosemide, suggesting that the cyclodextrin might have an influence on the transport mechanism of furosemide, which is passive paracellular route. About the mechanisms involved in the permeability of furosemide through the enterocytes, it can be suggested that there was little effect of P-glycoprotein (P-gp) inhibitors and CYP3A4 enzyme, suggesting that there is an important role of these mechanisms in the furosemide intestinal absorption.

Submitted by isabela.moljf@hotmail.com (isabela.moljf@hotmail.com) on 2017-05-12T13:14:28Z No. of bitstreams: 1 elisagomeslanna.pdf: 3970736 bytes, checksum: 6d637b75ed33e9ab58ac8e2e682d7e88 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-05-12T15:44:54Z (GMT) No. of bitstreams: 1 elisagomeslanna.pdf: 3970736 bytes, checksum: 6d637b75ed33e9ab58ac8e2e682d7e88 (MD5) / Made available in DSpace on 2017-05-12T15:44:54Z (GMT). No. of bitstreams: 1 elisagomeslanna.pdf: 3970736 bytes, checksum: 6d637b75ed33e9ab58ac8e2e682d7e88 (MD5) Previous issue date: 2015-07-17 / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / A Bryophyllum pinnatum(Lam.) Oken (Crassulaceae) é conhecida popularmente como folha-da-fortuna, courona, courona-vermelha, coirama e saião-roxo. Popularmente é usada como agente antimicrobiano (antifúngico e antibacteriano) e para o tratamento de úlceras gástricas, diarreia, vômito, queimaduras e doenças inflamatórias. O objetivo do presente trabalho foi potencializar a bioatividade do seu extrato etanólico através do desenvolvimento de uma composição nanoestruturada, usando -ciclodextrina (CD) como matriz hospedeira, seguida de sua incorporação em formulação semissólida para uso como anti-inflamatório tópico. Inicialmente, o material vegetal seco e pulverizado foi submetido à maceração com etanol seguida de partição, obtendo-se as frações hexânica, diclorometânica, acetato de etila e butanólica. Para o nanoencapsulamento do extrato/frações com βCD, quantidades iguais de extrato/frações e βCD foram pesados e solubilizados em etanol e a solução foi submetida à secagem (40º C) sob agitação. Caracterizações físico-químicas foram realizadas no intuito de mostrar a formação de compostos de inclusão entre a CD e constituintes do extrato e, identificar suas propriedades. Classes de constituintes do extrato/frações foram identificadas através das espectroscopias de UV-Vis e IR. A identificação dos nanoagregados e a avaliação da estabilidade coloidal foram feitas por medidas de espalhamento de luz dinâmico, potencial zeta e condutividade. Análise térmica diferencial (DTA) e termogravimetria (TGA) foram usadas para comprovar o aumento da estabilidade térmica enquanto que ensaios de solubilização foram realizados para mostrar o aumento da solubilidade na presença de CD. Os experimentos de atividade antioxidante do extrato/frações avaliada pelos ensaios com radical DPPH, do poder de redução do Fe3+ e do sistema de cooxidaçãoβ-caroteno/ácido linoleico, permitiram mostrar que a CD foi capaz de melhorar significativamente a atividade antioxidante do extrato etanólico (P < 0,005), fração acetato de etila (P < 0,005) e fração hexânica (P < 0,005), se comparado com o extrato/fração puro. A atividade anti-inflamatória tópica foi avaliada através do modelo de edema de orelha induzida aplicação tópica de óleo de cróton, com posterior análise histológica e ensaio da atividade da enzima mieloperoxidase (MPO). Quando avaliado a atividade anti-inflamatória das formulações semissólidas contendo o composto de inclusão com βCD, percebeu-se uma melhora da atividade quando comparado com as formulações que continham o extrato etanólico na forma livre, mostrando que a complexação dos componentes do extrato com a CD é uma importante estratégia a ser considerada para o desenvolvimento de formulação para uso tópico. / The Bryophyllum pinnatum (Lam.) Oken (Crassulaceae) is popularly known as ―folha-da-fortuna‖, ―courona‖, ―courona-vermelha‖, ―coirama‖ and ―saião-roxo‖. It is popularly used as antimicrobial (antifungal and antibacterial) and for treatment of gastric ulcers, diarrhea, vomiting, burns and inflammatory diseases. The goal of this study was to enhance the bioactivity of the ethanolic extract by developing nanostructured compositions, using β-cyclodextrin (βCD) as host matrix, followed by its incorporation into semisolid formulation for use as anti-inflammatory topic. Initially, the dried and powdered plant material was subjected to maceration with ethanol followed by partition to obtain the hexanic, dichloromethanic, ethyl acetate and butanolic fractions. For the nanoencapsulation of the extract/fractions with βCD, equal amounts of extract/fractions and βCD were weighed and solubilized in ethanol and the solution was subjected to drying (40 °C) under stirring. Physicochemical characterizations were carried out in order to show the formation of inclusion compounds between βCD and constituents of the extract/fraction and, identify their properties. Constituents of the extract/fractions were identified by UV-Vis and IR spectroscopies. The identification of nanoclusters and the evaluation of the colloidal stability was made by dynamic light scattering measurements, zeta potential and conductivity. Differential thermal analysis (DTA) and thermogravimetry (TGA) were used to demonstrate the increase in thermal stability while solubilization tests were performed to show increased solubility in the presence of βCD. The antioxidant activity of the extract/fractions was evaluated by trials with DPPH radical, power reduction of Fe3+ and β-carotene/linoleic acid system, allowed to show that βCD was able to significantly improve the antioxidant activity of ethanol extract (P <0.005), ethyl acetate fraction (P <0.005) and hexane fraction (P <0.005), compared with the extract/fraction pure. The topical anti-inflammatory activity was evaluated through ear edema model induced by croton oil, with subsequent histological analysis and assay of myeloperoxidase activity (MPO). When evaluated the anti-inflammatory activity of semisolid formulations containing the inclusion compound with βCD, it was noticed an improvement in activity when compared to the formulations containing the ethanol extract in the free form, proving that the complexation of the extract components with βCD is an important strategy to consider for the formulation development for topical use.

L'objectif principal de notre étude a été la mise au point des formulations liposomiales encapsulant des monoterpènes (MT) et phénylpropènes (PP). Le manuscrit contient deux axes principaux. Le premier a porté sur l'interaction de l'HP-ß-CD, de MT et PP avec des membranes lipidiques. L'HP-ß-CD interagit avec les têtes polaires et les chaînes acyles des phospholipides et montre un effet fluidifiant sur les liposomes formés de DPPC et de Lipoid S100. De plus, l'HP-ß-CD présent dans le compartiment aqueux des liposomes les protège durant la lyophilisation. Les MT et PP interagissent avec la tête et les chaînes alkyles de DPPC entrainant sa fluidification. D'autre part, les composés les plus hydrophobes de la série, ont été plus actifs contre E. coli. Le deuxième axe a été orienté vers la préparation des liposomes conventionnels et du système mixte CD/liposome (ACL). La technique de double encapsulation (ACL2) a été appliquée. Les préparations sont faites à partir de Phospholipon 90H ou Lipoid S100 par la méthode d'injection éthanolique. Les vésicules ont été caractérisées pour la taille, pdI, le potentiel zêta, la morphologie, le rendement et la cinétique de libération de la molécule. L'anéthol (Ane) est utilisé comme un système modèle. ACL et ACL2 ont permis d'améliorer le rendement d'encapsulation de l'Ane et de ralentir sa libération. Les liposomes composés de Lipoid S100 ont montré une meilleure photostabilité et rendement d'encapsulation. Le Lipoid S100 a été ainsi utilisé pour la préparation à grande échelle. Les vésicules obtenues à petite et grande échelle, ont présenté les caractéristiques proches ce qui témoigne d'une bonne reproductibilité de ces procédés / The main objective of this work was the development of liposomal formulations encapsulating monoterpenes (MT) and phenylpropene (PP). The manuscript contains two main axes: the first axis was oriented to the interaction of HP-ß-CD, MTs and PPs with lipid membranes. The HP-ß-CD interacts with the polar heads and acyl chains of phospholipids and showed a fluidizing effect on liposomes formed with DPPC or Lipoid S100. In addition, the incorporation of HP-ß-CD in the internal aqueous compartment of the liposomes protected them during lyophilization. The studied monoterpenes interact with the choline head group and the alkyl chains of DPPC membrane raising the bilayer’s fluidity. In addition, the most hydrophobic compounds of the series, were more active against E. coli. The second axis was oriented towards the preparation of conventional liposomes and drug-in-cyclodextrin-in-liposomes (ACL). The double loaded technique (double loaded liposomes, ACL2) was applied. Phospholipon 90H or Lipoid S100 were used to prepare liposomes by the ethanol injection method. Vesicles were characterized in terms of size, pdI, zeta potential, morphology, loading rate and drug release kinetics. Anethole (Ane) is used as a model system. Compared to conventional liposomes, ACL and ACL2 improve the loading rate of Ane and reduce Ane release. Liposomes prepared with Lipoid S100 showed the best photostability and loading rate. Lipoid S100 is used to prepare liposomes at large scale by membrane contactor and by pilot scale. The characteristics of the vesicles obtained at laboratory and large scale confirmed the reproducibility of the two methods of preparation

Graphene sheets, one atom thick, two dimensional layers of carbon atoms, have gained enormous importance over the past few years due to their unique attributes - high electronic, thermal conductivities and exceptional mechanical strength. Chemical reduction of graphene oxide (GO) has been considered as a viable route for large scale production of graphene sheets. The reduced graphene oxide (r -GO) sheets although their conductivities are comparatively lower than that of graphene are nevertheless versatile material for applications in thin films and composites. An important consideration in the design of solution processing techniques for the preparation of processable graphene sheets is the dispersibility of GO and r -GO in different solvents, especially aqueous dispersibility. While GO is dispersible in water over a wide range of pH values, r -GO shows poor dispersibility and over a limited range of pH values. Graphene oxide, the oxidized form of graphene, are single atomic layers with lateral dimension that can extend to hundreds of nanometers. The sheets contain a sizable fraction of carbons that are sp3 hybridized and covalently bonded to oxygen in the form of epoxy, carbonyls as well as ionizable hydroxyl and carboxylic functional groups located on the rim of the sheets. The remaining carbons form isolated sp2 graphene like networks. On reduction the oxygen functionalities are removed and the sp2 network partially restored. This thesis focuses on the aqueous dispersibility of GO and r -GO, and describes a strategy to enhance the dispersibility of r -GO by cyclodextrin functionalization. Chapter 1 of the thesis provides a brief review of the synthetic procedures and structure of GO and r -GO while Chapter 2 describes the experimental methods and characterization techniques used in the thesis. The chemistry underlying the aqueous dispersibility of GO and r -GO at different values of pH have been investigated by zeta potential measurements, pH titrations and infrared spectroscopy (Chapter 3). These measurements show that r -GO sheets have ionizable groups with a single pKa value (8.0) while GO sheets have groups that are more acidic (pKa = 4.3), in addition to groups with pKa values of 6.6 and 9.0. Infrared spectroscopy has been used to follow the sequence of ionization events. In both GO and r -GO sheets, it is ionization of the carboxylic groups that is primarily responsible for the build up of charge, but on GO sheets, the presence of phenolic and hydroxyl groups in close proximity to the carboxylic groups lowers the pKa value by stabilizing the carboxylate anion, resulting in superior water dispersibility. Till recently GO was primarily considered only as an easily available precursor for chemical routes to r -GO but it has now been recognized as an interesting material in its own right. Two such attributes that have attracted wide spread attention are the in- trinsic and tunable fluorescence of GO as well as formation of liquid crystalline phases. Aqueous dispersions of GO exhibit strong pH dependent fluorescence in the visible region that originates, in part, from the oxygenated functionalities present. In Chapter 4, the spectral migration on nanosecond timescales of the pH dependent features in the fluores- cence spectra of GO is described. The changes in the steady state fluorescence spectra with pH have been correlated with the sequence of dissociation events that occur in GO dispersions at different values of pH described in Chapter 3, from time resolved emission spectra (TRES) constructed from the wavelength dependent fluorescence decay curves, it is shown that the migration is associated with excited state proton transfer. Both ‘intramolecular’ and ‘intermolecular’ transfers involving the quasimolecular oxygenated aromatic fragments are observed. Aqueous dispersions of GO constitute a distinctive class of 2D-anisotropic colloids with competing interactions - long range electrostatic repulsion, originating from ionized carboxylic groups located on the rim of the sheets and weak dispersive attractive interactions originating from the un-oxidized sp2 graphitic domains. In Chapter 5, it is shown that, colloidal dispersions of GO are intrinsically frustrated, exhibiting a range of arrested or metastable states, encompassing fluid, glass and gels that coexist with liquid crystalline order. These states can be accessed by varying the relative magnitudes of the repulsive and attractive forces by changing the ionic strength of the medium, by addition of salt and/or the concentration of the dispersion. At low salt concentrations, where long range electrostatic repulsions dominates, the formation of a repulsive Wigner glass is observed while at high salt concentrations, when attractive forces dominate, the formation of gels that exhibits a nematic to columnar liquid crystalline transition. These studies highlights how the chemical structure of GO - hydrophilic ionizable groups and hydrophobic graphitic domains coexisting on a single sheet - gives rise to a rich and complex phase diagram. The poor dispersibility of r -GO in aqueous media limits its use in practical applica- tions. To enhance the dispersibility, r -GO sheets have been functionalized by covalently linking -cyclodextrin ( -CD) cavities to the sheets via an amide linkage (Chapter 6). The functionalized -CD: rGO sheets, in contrast to r -GO, are dispersible over a wide range of pH values (2 - 13). Zeta potential measurements indicate that there is more than one factor responsible for the dispersibility. It is shown that planar aromatic molecules adsorbed on the r -GO sheet as well as nonplanar molecules included in the tethered -CD cavities have their fluorescence effectively quenched by the -CD: rGO sheets. The -CD: rGO sheets combine the hydrophobicity associated with r -GO along with the hydrophobicity of the cyclodextrin cavities in a single water dispersible material. Resonance Raman spectroscopy is a powerful analytical tool for detecting and identi- fying analytes, but the associated strong fluorescence background severely limits the use of the technique. In Chapter 7, it is shown that the cyclodextrin functionalized -CD: rGO sheets, described in Chapter 6, provides a versatile platform for resonance Raman detection. Planar aromatic and dye molecules that adsorb on the r -GO graphitic domains and non-planar molecules included within the tethered -CD cavities have their fluorescence effectively quenched. Using the water dispersible -CD: rGO sheets, it is possible to record the resonance Raman spectra of adsorbed and included organic chromophores directly in aqueous media without having to extract or deposit on a substrate. The Raman signal intensities show a linear dependence with the concentration of analyte present in water. This is significant, as it allows for the identification and estimation of organic analytes present in water by resonance Raman spectroscopy.

Capillary electrophoresis (CE) is a modern analytical technique, which is electrokinetic separation generated by high voltage and taken place inside the small capillaries. In this dissertation, several advanced capillary electrophoresis methods are presented using different approaches of CE and UV and mass spectrometry are utilized as the detection methods. Capillary electrochromatography (CEC), as one of the CE modes, is a recent developed technique which is a hybrid of capillary electrophoresis and high performance liquid chromatography (HPLC). Capillary electrochromatography exhibits advantages of both techniques. In Chapter 2, monolithic capillary column are fabricated using in situ photoinitiation polymerization method. The column was then applied for the separation of six antidepressant compounds. Meanwhile, a simple chiral separation method is developed and presented in Chapter 3. Beta cycodextrin was utilized to achieve the goal of chiral separation. Not only twelve cathinone analytes were separated, but also isomers of several analytes were enantiomerically separated. To better understand the molecular information on the analytes, the TOF-MS system was coupled with the CE. A sheath liquid and a partial filling technique (PFT) were employed to reduce the contamination of MS ionization source. Accurate molecular information was obtained. It is necessary to propose, develop, and optimize new techniques that are suitable for trace-level analysis of samples in forensic, pharmaceutical, and environmental applications. Capillary electrophoresis (CE) was selected for this task, as it requires lower amounts of samples, it simplifies sample preparation, and it has the flexibility to perform separations of neutral and charged molecules as well as enantiomers. Overall, the study demonstrates the versatility of capillary electrophoresis methods in forensic, pharmaceutical, and environmental applications.

This thesis consists of two parts. The first part deals with the visible emission of ZnO Nanocrystals and its possible application in Resonance Energy Transfer (RET) studies. The second part of the thesis is on the magnetic properties of the layered transition metal Thiophosphates MPS3 (M = Mn, Fe), their solid solutions and intercalation compounds. Recent advances in semiconductor nanocrystals or quantum dots (QDs) as inorganic fluorophores have pioneered a new direction in the fluorescent based techniques to investigate fundamental processes in lifesciences. Their broad absorption spectra with narrow, Size-tunable emissions with high quantum e±ciency and stability under relative harsh environments have made inorganic QD's the fluorophores of choice in many applications. Among inorganic fluorophores the II-VI semiconductors based on cadmium chalcogenides are the front-runners. The cytotoxicity associated with these QDs is, however, a major drawback and has lead to the search for new nanocrystalline fluorophores that are non-toxic and possess the same favorable fluorescence properties as the Cd based QDs, viz, tunability and narrow spectral profile. ZnO Nano particles are known to exhibit two emission bands; a narrow emission band in UV around 380 nm (3.25 eV) at a wavelength just below the onset of the band gap excitation in the absorption spectra and a broad emission band in the blue-green part of the visible spectrum, with a maximum between 500 and 550 nm (2.5-2.2 eV). The UV Emission originates from the recombination of bound excitons - excited electrons in the Conduction band with holes in the valence band. The visible emission of ZnO nanocrystals is known to involve deep trap states that lie approximately midway between the Conduction and valence bands and surface defects that exist as shallow traps. In principle, visible-light-emitting ZnO nanocrystals would be ideal candidates as replacement for Cd-based fluorescent labels since they are nontoxic, less expensive, and chemically stable in air. Nanoscale ZnO, however, tends to aggregate and/or undergo Ostwald ripening be-Cause of high surface free energy resulting in an increase in crystallite size and consequent Disappearance of the visible emission. Most attempts to stabilize the ZnO nanocrystals by Capping has usually resulted in the quenching of the visible trap emission. The objective of the present study was to stabilize the visible light emission of ZnO nanocrystals, to Understand the origin and mechanism of the visible emission and to explore the possibility Of using the visible emission of ZnO in RET studies. The stabilization of visible light emission in ZnO nanocrystals was achieved by forming ZnO:MgO core-shell nanocrystals. The nanocrystals were synthesized by a sequential preparative procedure that involved formation of a ZnO core followed by an MgO shell. The Nanocrystals were characterized by using XRD, TEM, optical absorption and photoluminescence spectroscopy. These are described in Chapter 2 of the thesis. The ZnO: MgO Core-shell nanostructures exhibit stable emission in the visible for extended periods. Application of the ZnO: MgO nanocrystals either as fluorescent probes or RET studies require that they be dispersible in both polar and non-polar solvents. This as realized by appropriate choice of the capping agents (Chapter 3). ZnO: MgO nanocrystals with hydrophobic surface were obtained by capping the nanocrystals with oleic acid. The oleate capped ZnO: MgO nanocrystals are soluble in a variety of non-polar organic solvents with no change in their emission properties. Water-soluble ZnO: MgO nanocrystals were obtained by capping the ZnO:MgO nanocrystals with carboxymethyl-β-cyclodextrin (CMCD). The hydroxyl groups located at the rim of the cyclodextrin cavity renders the surface hydrophilic. The integrity of the CMCD molecules are preserved on capping and their by hydrophobic cavities available for host-guest chemistry. The visible emission of The ZnO: MgO nanocrystals are unaltered by the nature of the capping agent. The origin and mechanism of the visible emission from ZnO: MgO nanocrystals has been Investigated using time-resolved emission spectroscopy technique (Chapter 4). The time-evolution of the photoluminescence spectra show that there are, in fact, two features in the visible emission whose relative importance and efficiencies vary with time. These features originate from recombination involving trapped electrons and holes, respectively, And with efficiencies that depend on the occupancy of the trap density of states. The application of the visible emission of ZnO: MgO nanocrystals as resonance energy transfer (RET) donors in water and hydrophobic media are demonstrated. In aqueous media, the carboxymethyl β-cyclodextrin (CMCD) capped ZnO: MgO nanocrystals is able to accommodate the organic dye Nile Red by an inclusion in the anchored hydrophobic cyclodextrin cavity forming a 1:1 complex. Nile Red was chosen as the guest molecule because its absorption has appreciable overlap with ZnO: MgO visible emission, a prerequisite for RET to occur. The resonance energy transfer on the band gap excitation of The ZnO core to included Red molecules in the CMCD-ZnO: MgO-Nile Red supramolecular assembly is demonstrated in aqueous media. A similar RET process is shown to occur in the non-polar media in the oleate capped ZnO: MgO nanocrystals when Nile Red is partitioned from the solvent into hydrophobic anchored oleate chains. The wavelength dependent energy transfer in the system has been studied using time-resolved emission spectroscopic technique. The importance of trap states in giving rise to non-Forster distance dependence for the RET is highlighted. The second part of the thesis deals with magnetism in low dimensional layered transition metal thiophophates, MPS3 (M = Mn, Fe). Low dimensional magnetic systems continue to be a fertile ground for discovering new phenomena and properties. Among two-dimensional magnetic systems the insulating transition metal thiophosphates are one of the few known layered systems, in which both magnetic and crystallographic lattices are two dimensional (2D). In the metal chalcogenophosphates, the magnetic MPX3 layers are separated by a van der Waals gap that effectively rules out interlayer exchange and hence these systems are nearly perfect 2D magnetic systems, with the magnetic ions forming a honeycomb arrangement within the layer. Due to the crystallographic two-dimensional nature these materials may be intercalated by variety of molecules or ions leading to change in magnetic properties. The objective of this thesis work is to try and modify the magnetic properties of the transition metal thiophosphates either by forming solid solutions of the type, M1-xMxPS3, (M, M = Mn, Fe) or by intercalating hydrated metal ions within the layers. The structure, Bonding, reactivity and magnetic properties are briefly introduced in Chapter 7. The Scope and nature of the present work in presented towards the end of the chapter. MnPS3 and FePS3 have identical crystal structures and both order antiferromagnetically at low temperatures, TN. The in-plane magnetic structures of the antiferromagnetically ordered the Neel state in the two compounds are, however, different. In MnPS3 the spins Alternate up-down whereas in FePS3 the spins are arranged as ferromagnetic chains with Alternate chains coupled antiferromagnetically. Since the crystal structures are identical, These two compounds can form solid solutions, Mn1-xFexPS3(0≤x≥1) over the entire concentration range. The magnetic properties of the single crystals of the solid solutions was measured by using a SQUID magnetometer. This system is of interest since the contrasting Neel states of the end-members may give rise to new magnetic phenomena at intermediate composition. It is shown that the magnetic behavior falls into three distinct categories. The Mn-rich compositions behave like a dilute MnPS3 lattice, the Fe-rich compositions behave like dilute FePS3 and in the intermediate compositions a spin-glass like phase appears. The phase boundaries for these regime in Mn1-xFexPS3, 0≤x≥1 is shown to be related to the percolation threshold for a honeycomb lattice. MnPS3 is known to undergo a unusual ion-exchange intercalation reaction. Intercalation occurs by the inclusion of hydrated metal ions in the galleries of MnPS3 with charge neutrality maintained by loss of the Mn2+ ions from the layer (Equation). MnPS3 + 2xG+ (aq) → Mn1-xPS3 [G (H2O) y] 2x + xMn2+ (aq) Where G is a neutral guest species. This chemistry has been exploited to intercalate hydrated Mn2+ ions in the interlamellar space to give Mn1-xPS3[Mn(H2O)6]x. the magnetic properties of this 3D analogue of MnPS3 has been investigated in Chapter 9.

Quinones have been the subject of much interest due to their various biological activities, mainly as antitumor and as trypanocidal agents. Quinones are cytotoxic by two main mechanisms: the generation of ROS resulting in oxidative stress and alkylation of cellular nucleophiles, such as DNA and some enzymes such as topoisomerases. Their activity depends on bioreduction, similarly to what happens to nitroaromatic compounds. They also catalyze electron transfer reactions in biological processes and, after reduction generate radical anions (semiquinone radical anion and nitro), which depending on stability, can furnish their free electrons to acceptor molecules. In the present study, compounds with mixed functionalities derived from nor-β-lapachone, including a nitroaniline group were electrochemically studied in protic (acetate buffer) and aprotic (DMF+TBABF4, DMSO+TBAP and Acetonitrile+TBABF4) media, using glassy carbon and mercury as working electrodes. The compounds showed a complex redox behavior and the mechanism was elucidated using electron spin resonance. The electroreduced products of nor-β-lapachone and of the nitroquinones reacted with oxygen, indicative of the generation of reactive oxygen species, reactivity in the order of 2 > nor-β-lapachone > 3 > 1. We investigated their interaction with DNA, which was shown to be positive for nitroquinones and negative for the precursor nor-β-lapachone, in agreement with biological assays which had also shown that the nitroquinones cause DNA damage. The stability of the nitrosemiquinones, their half-life times were measured using mercury electrode, and the reaction rates for the electrochemical process-following-up-isproportionation reaction were measured. From these studies, a lower stability for the meta-substituted nitrophenylaniline (k2 = 5.188 x 103 L mol-1 s-1 and t1/2 = 0.06 s) was evidenced. Upon spectroelectrochemical reduction studies of the nitroquinones, the generation of radicalar intermediates (semiquinone radical anion and nitro radical anion) was observed, with differences between o- and m-derived compounds and the p-substituted one. To increase the solubility of the nitroquinones, in order to allow in vivo studies, the formation of inclusion complexes with β-cyclodextrin were evaluated. Positive results were obtained, leading to a viable formulation alternative for further biological studies with the compounds. / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Quinonas despertam muito interesse devido às suas diversas atividades biológicas, principalmente como agentes antitumoral e tripanossomicida. A citotoxicidade de quinonas decorre de dois mecanismos principais: geração de EROs resultando no estresse oxidativo e alquilação de nucleófilos celulares, como o DNA e algumas enzimas, como as topoisomerases. Sua atividade depende de biorredução, similarmente ao que acontece com compostos nitroaromáticos. Eles também catalisam reações de transferência de elétrons biológicas e, após a redução geram ânions radicais (ânions radicais semiquinona e nitro), que dependendo da estabilidade, podem transferir seus elétrons livres a moléculas aceptoras. Neste trabalho, compostos de funcionalidade mista derivados da nor-β-lapachona, com um grupo nitroanilina, foram estudados eletroquimicamente nos meios prótico (tampão acetato) e aprótico (DMF + TBABF4, DMSO + TBAP e acetonitrila + TBABF4), utilizando carbono vítreo e mercúrio como eletrodos de trabalho. Os compostos apresentaram um comportamento redox complexo e o mecanismo elucidado por espectroeletroquímica. Os produtos da eletro-redução da nor-β-lapachona e nitroquinonas reagiram com oxigênio, indicativo da geração de EROs, na ordem de reatividade 2 > nor-β-lapachona > 3 > 1. Foi investigada a interação com DNA, que se mostrou positiva para as nitroquinonas e negativa para o precursor nor-β-lapachona, concordantes com ensaios biológicos que também evidenciou que as nitroquinonas causam dano ao DNA. A estabilidade do nitrosemiquinona, tempos de meia-vida e as taxas reacionais referentes à reação química de desproporcionamento acoplada ao processo eletroquímico para cada derivado foram determinadas usando eletrodo de mercúrio. A partir desses estudos, foi evidenciada a menor estabilidade para o nitrofenilamina meta-substituída (k2 = 15,188 x 103 L mol-1 s-1 and t1/2 = 0,06 s). Nos estudos por espectroeletroquímica das nitroquinonas, observou-se a geração de intermediários radicalares (ânions radicais semiquinona e nitro), com diferenças entre os nitroderivados o- e m-, assim como para o p- derivado. Com objetivo de aumentar a solubilidade das nitroquinonas, para posteriores estudos in vivo, foi avaliada a formação de complexos de inclusão com β-ciclodextrina. Foram obtidos resultados positivos, refletindo em alternativa viável para formulações posteriores com estes compostos e ensaios biológicos.