Background: Platinum group metal (PGM) mining and refining is a large constituent of the mining sector of South Africa and contributes significantly to the gross domestic product. The PGMs include the rare metals platinum (Pt), palladium (Pd), rhodium (Rh), ruthenium (Ru), iridium (Ir) and osmium (Os). During the refining process workers are potentially exposed to various chemical forms of the PGMs via the respiratory and dermal exposure routes. Historically, emphasis has been on respiratory exposure while the extent of skin exposure is still unknown. Among the different forms of PGMs, the salts are potential sensitisers, with platinum being a known respiratory sensitiser. Workers occupationally exposed to platinum and rhodium have reported respiratory as well as skin symptoms. However, it is unknown if these metals in the salt form are permeable through human skin, and whether dermal exposure could contribute to sensitisation. Evidence regarding differences between African and Caucasian skin anatomy and structure, as well as permeation through skin is contradictory, and no information is available on metal permeation through African skin. The in vitro diffusion method has been utilised successfully in occupational toxicology to demonstrate that metals such as chromium, cobalt and nickel, to name a few, permeate through human skin. The permeability of platinum and rhodium has not been investigated previously. Aims and objectives: The research aim was to obtain insight into the permeability of platinum and rhodium through intact human skin and to provide information needed to determine the potential health risk following dermal exposure to these metals. The specific objectives included: (i) to critically review the in vitro diffusion method that is used to determine the permeability of metals through human skin, (ii) to investigate the permeation of potassium tetrachloroplatinate (K2PtCl4) and rhodium chloride (RhCl3) as representative PGM salts through intact human skin over a 24-hour period, (iii) to evaluate the difference in permeability of platinum and rhodium through intact human skin, (iv) to evaluate the difference in permeability of platinum through intact African and Caucasian human skin. Methods: Abdominal skin obtained after cosmetic procedures was obtained from five female Caucasian and three female African donors between the ages of 28 and 52 with ethical approval from the North-West University. Full thickness skin tissue was mounted in a vertical Franz diffusion cell. Skin integrity was tested by measuring the electrical resistance across the skin before and after conclusion of the experiments, using a Tinsley LCR Data bridge Model 6401. The donor solution of 32.46 mg K2PtCl4 in 50 ml of synthetic sweat (pH 6.5), and 43.15 mg RhCl3 in 50 ml of synthetic sweat (pH 6.5) was prepared. The donor solution was applied to the stratum corneum side of the skin and physiological receptor solution (pH 7.35) was added to the receptor compartment. The concentration of the metals in the receptor solution was determined by high resolution inductively coupled plasma-mass spectrometry after extraction at various intervals during the 24 hours of the study. After completion of the study, the skin was rinsed four times to remove any platinum or rhodium remaining on the skin surface. The skin was digested using hydrogen peroxide, nitric acid and hydrochloric acid during different steps to determine the mass of the metals remaining in the skin by inductively coupled plasma-optical emission spectrometry. Results: The comparison of published in vitro skin permeation studies involving metals is impeded by the variations in the experimental design and dissimilarity in the reporting of results. Differences in experimental design included, most noticeably, the use of various donor and receptor solutions, different temperatures wherein the receptor compartment was placed, differences in skin thickness and variations in exposed skin surface areas. The metals considered in the review, namely chromium, cobalt, gold, lead, mercury, nickel, platinum, rhodium and silver, permeate through intact human skin under physiological conditions. Large variations in the permeability results were observed, with the notable differences in methodology as the probable reason. Results obtained from the in vitro experiments indicate that platinum and rhodium permeated through intact Caucasian skin with flux values of 0.12 and 0.05 ng/cm2/h, respectively. The cumulative mass of platinum (2.57 ng/cm2) that permeated after 24 hours of exposure was statistically significantly (p = 0.016) higher than rhodium permeation (1.11 ng/cm2). The mass of platinum (1 459.47 ng/cm2) retained in the skin after 24 hours of exposure was statistically significantly (p < 0.001) higher than rhodium retention (757.04 ng/cm2). The comparison of permeability between two different racial groups indicates that platinum permeated through the skin of both racial groups with the flux through African skin found as 1.93 ng/cm2/h and 0.27 ng/cm2/h through Caucasian skin. The cumulative mass of platinum permeated after 24 hours of exposure was statistically significantly (p = 0.044) higher through African skin (37.52 ng/cm2) than Caucasian skin (5.05 ng/cm2). The retention of platinum in African skin (3 064.13 ng/cm2) was more than twice the mass retained in Caucasian skin (1 486.32 ng/cm2). Conclusions: The in vitro diffusion method is an applicable method to determine skin permeability of metals. However, the experimental design and format of data reporting should be standardised to enable comparison of results from different studies. Platinum and rhodium permeated through intact human skin, with platinum permeation significantly higher. African skin was significantly more permeable by platinum than Caucasian skin. Both platinum and rhodium were retained inside the skin after 24 hours of exposure, possibly forming a reservoir which could contribute to continued permeation through the skin even after removal thereof from the skin. Platinum and rhodium permeated through full thickness skin and thereby could possibly contribute to local skin symptoms such as dermatitis and urticaria found in occupationally exposed workers. By permeating through the upper layers of the skin, these metals could potentially reach the viable epidermis and contribute to sensitisation. / PhD (Occupational Hygiene), North-West University, Potchefstroom Campus, 2015

Skin is the largest immune organ and an ideal site to administer vaccines. However, by nature, skin is not permeable to antigens, which are macromolecules. The major hurdle in skin permeation is the outermost stratum corneum layer. Microneedles have proven feasible to create micron-sized channels in the epidermis of the skin, through which protein and plasmid DNA antigens can penetrate into the viable skin epidermis and dermis. However, the immune responses induced by microneedle-mediated transcutaneous immunization with protein or plasmid DNA alone are generally weak, and a vaccine adjuvant is often required to induce strong immune responses. Data from numerous previous studies have shown that nanoparticles as a vaccine carrier can significantly enhance the immunogenicity of antigens, but the feasibility of utilizing nanoparticles as a vaccine carrier to enhance the immune responses induced by microneedle-mediated transcutaneous immunization has rarely been studied. In this dissertation, using protein antigen (OVA) chemically conjugated onto the surface of solid-lipid nanoparticles and plasmid DNA (pCMV-beta, pVax/opt-BoNT/C-Hc50, and pCI-neo-sOVA) physically coated on the surface of cationic polymeric nanoparticles, we showed that the immune responses induced by microneedle-mediated transcutaneous immunization with protein antigens or plasmid DNA vaccines are significantly enhanced by delivering the proteins and plasmid DNA with nanoparticles. Importantly, microneedle-mediated transcutaneous immunization with proteins or plasmid DNA induces not only systemic immune responses, but also mucosal immune responses. In addition, it is generally believed that microneedles are safe. However, it remained unclear whether the micropores created by microneedles on the skin will also facilitate the permeation of microbes such as bacteria into the skin. In this dissertation, we also designed an unique ex vivo model to evaluate the permeation of live bacteria through mouse skin pretreated with microneedles. The results demonstrated that the risk of potential bacterial infection associated with microneedle treatment is not greater than that associated with a hypodermic needle injection. / text

No presente trabalho foi realizado o isolamento da 3-O-metilquercetina, a partir de produto seco do extrato de inflorescências de Achyrocline satureioides, e sua caracterização. Em etapa farmacotécnica, foi realizado o estudo da associação deste flavonóide com b-ciclodextrina, bem como testes preliminares de permeação cutânea das associações, incorporadas ou não em gel de hidroxipropilmetilcelulose. As técnicas espectroscópicas, infravermelho e ressonância magnética de hidrogênio, confirmaram a estrutura do flavonóide isolado. Para o doseamento da 3-Ometilquercetina, realizou-se a validação de metodologia analítica por cromatografia líquida de alta eficiência, obtendo-se linearidade, na faixa de concentração de 0,05 a 1,5 μg/mL, precisão e exatidão adequadas. A análise da associação da 3-Ometilquercetina com b-ciclodextrina por infravermelho, ressonância magnética de hidrogênio e a análise pelo método empírico de Mecânica Molecular (MM2) do software Chem3D Ultra (Versão 9.0, CambridgeSoft) indicam possível inclusão do anel B da 3-O-metilquercetina na cavidade da b-ciclodextrina, sendo a inserção do flavonóide pela borda das hidroxilas secundárias mais favorável do que pela borda das hidroxilas primárias. A b-ciclodextrina e o gel de hidroxipropilmetilcelulose promoveram a permeação do flavonóide através da pele. A realização de ensaios in vivo para a seleção da melhor formulação constitui-se na principal perspectiva de continuidade de investigação científica do tema. / 3-O-methylquercetin (3-OMQ) was isolated from spray dried powder of Achyrocline satureioides and characterized by IR and 1H NMR. The study of association of this flavonoid with b-cyclodextrin (bCD) was performed, as well as preliminary skin permeation tests of these associations, incorporated or not in hydroxypropyl methylcellulose (HPMC) hydrogel. A LC method for 3-OMQ assay was validated in the concentration range from 0.05 to 1.5 μg/mL, with suitable precision and accuracy. The complexation of 3-OMQ with bCD was analyzed by IR, 1H NMR and Molecular Mechanics (Chem3D Ultra 9.0, CambridgeSoft) and the results indicated the possible insertion of B ring of the flavonoid into the bCD cavity, being the insertion through the secondary OH rim more favorable than through the primary OH rim. bCD and HPMC promoted the permeation of the flavonoid through the skin. In vivo assay is required to select the appropriate formulation.

Diversos estudos têm demonstrado a atividade antioxidante de extratos de Achyrocline satureioides. Essa atividade tem sido atribuída à presença de compostos flavonoídicos, em especial, da quercetina. Neste contexto, o objetivo da presente dissertação foi desenvolver nanoemulsões de uso tópico contendo o extrato deste vegetal, bem com o flavonóide quercetina isolado. Numa primeira etapa, nanoemulsões constituídas de octildodecanol, lecitina de gema de ovo, solução extrativa de Achyrocline satureioides (NEE) ou quercetina (NEQ) e água foram preparadas por emulsificação espontânea. O procedimento conduziu à obtenção de nanoemulsões monodispersas (IP<0,2) com um diâmetro médio de gotícula de cerca de 200 e 300 nm e potencial zeta de cerca de -27 e -42 mV, respectivamente, para NEQ e NEE. Esses resultados sugerem a localização de componentes do extrato de Achyrocline satureioides na interface das nanoestruturas, como indicado pelas imagens obtidas por microscopia eletrônica de transmissão. Numa segunda etapa, foi revalidado um método isocrático para a quantificação da quercetina por CLAE, mostrando-se este linear, preciso, exato e específico. A quantidade de quercetina associada com as nanoemulsões foi de 100%, para a concentração de 100 μg/mL de quercetina. Após, foi realizado o estudo de retenção cutânea da quercetina a partir das formulações utilizando pele de orelha suína, em células de difusão de Franz. Os resultados indicam um maior acúmulo de quercetina na pele a partir da NEE (~16 μg/g) em comparação a NEQ (~6 μg/g), após 8 horas de permeação, indicando a influência de componentes do extrato sobre esta propriedade. Finalmente, determinou-se a atividade antioxidante da quercetina através de medidas de substâncias reativas ao ácido tiobarbitúrico (TBA-RS). Encontrou-se uma menor lipoperoxidação nos testes realizados com o extrato de Achyrocline satureioides (56%) em relação aos testes utilizando quercetina isolada (91%), sem diferença na lipoperoxidação entre a NEQ (27 %) e a NEE (22 %). A proteção oferecida pelas formulações foi determinada nas peles submetidas ao estudo de permeação, sendo detectada uma diminuição da lipoperoxidação no tecido que recebeu tratamento com NEE, contudo, essa atividade não foi detectada para NEQ. Em conclusão, os resultados obtidos nesse estudo demonstram o efeito dos constituintes das formulações sobre propriedades físico-químicas das nanoemulsões, bem como na sua retenção na pele e na atividade antioxidante. / Several studies have demonstrated the antioxidant activity of Achyrocline satureioides extracts. This activity has been attributed to the presence of flavonoids, mainly quercetin. In this context, the aim of the present work was to develop topical nanoemulsions containing extract of this plant, as well as isolated quercetin. In a first step, nanoemulsions composed by octyldodecanol, egg lecithin, Achyrocline satureioides extract (NEE) or quercetin (NEQ) and water were prepared by spontaneous emulsification procedure. This process led to monodisperse nanoemulsions presenting mean droplet size of approximately 200 and 300 nm, and zeta potencial of –27 and –42 mV for NEQ and NEE, respectively. These results suggest that Achyrocline satureioides extract compounds are located in the O/W interface of nanoemulsions, as attested by the transmition electron microscopy. In a second step, an isocratic method for quercetin quantification was revalidated. This showed to be linear, accurate, precise and specific. The amout of quercetin associated with nanoemulsions was close to 100%, for a 100 μg/mL of quercetin. After that, a study of quercetin skin retention from the formulations using pig´s skin ear was performed, in Franz diffusion cell. The results revealed a higher accumulation of quercetin in skin from NEE (~16 μg/g) compared to NEQ (~6 μg/g), after 8h of permeation, indicating the influence of the extract compounds on this property. Finally, the antioxidant activity of quercetin was measured by tiobarbituric acid reactive species (TBA-RS). It shows a minor lipoperoxidation for Achyrocline satureioides extract (56%) than isolated quercetin (91%). No difference for NEQ (27 %) and NEE (22 %) lipoperoxidation was found. The protection against oxidation by the formulations was measured in the skin and it was found a reduction in tissue treated with NEE. This reduction was not observed for NEQ. In conclusion, this study shows the effect of formulation over the physico-chemical properties of nanoemulsion’s formulation, as well as on the skin retention and its antioxidant activity.

No presente trabalho foi realizado o isolamento da 3-O-metilquercetina, a partir de produto seco do extrato de inflorescências de Achyrocline satureioides, e sua caracterização. Em etapa farmacotécnica, foi realizado o estudo da associação deste flavonóide com b-ciclodextrina, bem como testes preliminares de permeação cutânea das associações, incorporadas ou não em gel de hidroxipropilmetilcelulose. As técnicas espectroscópicas, infravermelho e ressonância magnética de hidrogênio, confirmaram a estrutura do flavonóide isolado. Para o doseamento da 3-Ometilquercetina, realizou-se a validação de metodologia analítica por cromatografia líquida de alta eficiência, obtendo-se linearidade, na faixa de concentração de 0,05 a 1,5 μg/mL, precisão e exatidão adequadas. A análise da associação da 3-Ometilquercetina com b-ciclodextrina por infravermelho, ressonância magnética de hidrogênio e a análise pelo método empírico de Mecânica Molecular (MM2) do software Chem3D Ultra (Versão 9.0, CambridgeSoft) indicam possível inclusão do anel B da 3-O-metilquercetina na cavidade da b-ciclodextrina, sendo a inserção do flavonóide pela borda das hidroxilas secundárias mais favorável do que pela borda das hidroxilas primárias. A b-ciclodextrina e o gel de hidroxipropilmetilcelulose promoveram a permeação do flavonóide através da pele. A realização de ensaios in vivo para a seleção da melhor formulação constitui-se na principal perspectiva de continuidade de investigação científica do tema. / 3-O-methylquercetin (3-OMQ) was isolated from spray dried powder of Achyrocline satureioides and characterized by IR and 1H NMR. The study of association of this flavonoid with b-cyclodextrin (bCD) was performed, as well as preliminary skin permeation tests of these associations, incorporated or not in hydroxypropyl methylcellulose (HPMC) hydrogel. A LC method for 3-OMQ assay was validated in the concentration range from 0.05 to 1.5 μg/mL, with suitable precision and accuracy. The complexation of 3-OMQ with bCD was analyzed by IR, 1H NMR and Molecular Mechanics (Chem3D Ultra 9.0, CambridgeSoft) and the results indicated the possible insertion of B ring of the flavonoid into the bCD cavity, being the insertion through the secondary OH rim more favorable than through the primary OH rim. bCD and HPMC promoted the permeation of the flavonoid through the skin. In vivo assay is required to select the appropriate formulation.

L’acné est la dermatose la plus courante dans le monde. Cette pathologie, à causes multiples, peut impliquer des traitements longs dont l’efficacité reste à améliorer. Au niveau topique, le ciblage de la surface cutanée et en particulier des foyers infectieux (impliquant notamment la bactérie P. acnes) est un axe de recherche visant un meilleur traitement de la pathologie. Ces travaux de thèse s’inscrivent dans cet objectif, et portent sur l’utilisation de particules submicroniques minéral-organiques à base d’apatites bio-inspirées pour le traitement de l’acné en vue de la vectorisation d’un antibiotique via une nouvelle formulation galénique. Dans un premier temps, les recherches se sont focalisées sur l’élaboration et la caractérisation physico-chimique de particules apatitiques préparées en présence d’un agent dispersant. Parmi les conditions testées, un protocole de référence permettant d’obtenir une suspension colloïdale de particules d’apatite de diamètre hydrodynamique moyen (DLS) de 180 nm, stabilisées à l’aide d’un polyéthylène glycol phosphonaté, a été retenu. L’analyse des particules par DRX et IRTF a mis en évidence le caractère nanocristallin biomimétique de la phase apatitique. Dans un second temps, l’adsorption d’une molécule modèle phosphatée puis d’un antibiotique – le phosphate de clindamycine (ClindP) – a été quantifiée et analysée à l’aide de différents modèles d’adsorption. Par ailleurs, la possibilité d’une incorporation d’ions biologiquement actifs (ex : antibactériens, antiinflammatoires) tels que Cu2+ et/ou Zn2+ dans l’apatite colloïdale a été établie. Dans un troisième temps, des évaluations biologiques ainsi que divers essais de suivi des particules ont été entrepris. L’interaction avec des éléments du sang – globules rouges et protéines plasmatiques – a été explorée (dans l’éventualité d’une application sur peau lésée), mettant en évidence l’excellente hémocompatibilité de ces particules colloïdales. Différentes techniques de suivi des particules ont ensuite été abordées sur membranes synthétiques et sur explants d’oreilles de porcs, telles que l’utilisation de cellules de Franz en modes statique et dynamique, ou encore la microscopie confocale Raman. Les résultats obtenus indiquent que cette dernière technique est adaptée à l’étude de la localisation cutanée de ces particules colloïdales, et montrent une accumulation de celles-ci au niveau de l’épiderme et des follicules pileux. Enfin, une étude préliminaire d’élaboration et de caractérisation d’une forme galénique (bigel) a été abordée / Acne is the most frequent dermatosis in the world. This multifaceted pathology may necessitate long-term treatments which can be improved. For a topical application, the idea of targeting the skin surface and in particular infected pilosebaceous units (involving bacteria such as P. acnes) is one approach for a better treatment of this pathology. This thesis work follows this objective, and deals with the use of submicron mineral-organic particles based on bio-inspired apatite for the treatment of acne, in view of drug delivery via a new galenic formulation. In a first stage, research was focused on the synthesis and physicochemical characterization of apatitic particles prepared in the presence of a dispersing agent. Among the tested conditions, a reference protocol was retained, allowing the obtainment of a colloidal suspension of apatite particles with a mean hydrodynamic diameter (DLS) of 180 nm, stabilized with phosphonated polyethyleneglycol. XRD and FTIR evidenced the biomimetic nanocrystalline nature of the apatitic phase. In a second stage, the adsorption of a model phosphate molecule and then of an antibiotic – clindamycin phosphate (ClindP) – was quantified and analyzed with regard to various adsorption models. Moreover, the possibility to incorporate biologically-active ions (e.g. antibacterial, antiinflammatory) such as Cu2+ and/or Zn2+ in colloidal apatite was established. In a third part, biological evaluations as well as particle follow-up experiments were performed: the interaction with blood components – red blood cells and plasma proteins – was explored (in the eventuality of application on damaged skin), evidencing the excellent hemocompatibility of these colloidal particles. Various particle follow-up techniques were then considered, involving synthetic membranes and porcine ear skin, such as the use of Franz cells in static and dynamic modes or else Raman confocal microscopy. Results indicate that the latter technique is suitable for the study of the localization of these colloidal particles within the skin, and point out their accumulation on the epidermis and hair follicles. Finally, a preliminary study was carried out on the setup and characterization of a galenic form (bigel).

Made available in DSpace on 2014-06-11T19:25:27Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-02-21Bitstream added on 2014-06-13T20:33:03Z : No. of bitstreams: 1 martini_pc_me_arafcf.pdf: 595100 bytes, checksum: bcb255af52349311710acfecbb49c0e8 (MD5) / Universidade Estadual Paulista (UNESP) / O aumento da expectativa de vida tem provocado grande demanda por produtos que auxiliem na prevenção do envelhecimento da pele. O ácido hialurônico (AH) está sendo muito utilizado em formulações antienvelhecimento por se acreditar que ele possa atenuar os efeitos que o tempo produz na pele. Porém, para o desenvolvimento de um cosmético, é necessário avaliar o risco potencial dos ingredientes que compõem a formulação, que devem estar em concentração que apresente margem de segurança adequada, sendo importante a realização de teste de absorção, estudo do potencial de risco irritativo e testes de mutagenicidade. O objetivo deste estudo foi desenvolver um gel de ácido hialurônico com alta massa molecular e incorporar ácido hialurônico de massas moleculares menores, realizar o controle microbiológico dos géis, avaliar a toxicidade dérmica aguda e a citotoxicidade, determinar o comportamento reológico das formulações, analisar seu perfil de liberação, permeação e retenção cutânea in vitro. Foi preparado como gel base, a mistura de água e AH de alta massa molecular e, posteriormente, neste gel formado foram incorporados os AHs de menores massas moleculares. O controle de qualidade microbiológico foi realizado de acordo com a Farmacopeia Brasileira (2010), a avaliação da toxicidade dérmica aguda foi realizada utilizando-se 25 ratos wistar, que foram divididos em 5 grupos com 5 animais em que cada grupo recebeu a aplicação de um dos géis para posterior analise dos resultados. Para a avaliação da citotoxicidade utilizou-se o método colorimétrico 3-(4,5-dimetiltiazol-2-il)2,5-difenil brometo de tetrazoilium (MTT) descrito por MOSMANN, utilizando-se linhagens celulares de HepG2 (Human Epidermoide Cancer Cells) e HaCaT. As amostras tiveram seu comportamento reológico avaliado em reômetro HAAKE. O estudo de liberação, permeação e retenção cutânea... / Increased life expectancy has caused a great demand for products that help in the prevention of skin aging. Hyaluronic acid is being used in anti-aging formulations because it is expected that it can decrease the effects of time in the skin. However, for the development of a cosmetic it is necessary to evaluate the potential risk of the ingredients in the formulation that should be with adequate concentration to provide a margin of safety, being important to carry out absorption test, studies of the potential risk of irritating the skin and mutagenicity tests. The aim of this study was to develop a gel of hyaluronic acid with high molecular weight and incorporate hyaluronic acid with small molecular weights in it, analyze the microbiological control of the gels, evaluate the acute dermal toxicity and cytotoxicity, determine rheological characteristics, analyze the release profile, skin permeation and retention in vitro. It was prepared like gel base, the mixture of water and AH of high molecular weight, and after was incorporate AH of smaller molecular weight. The quality control microbiological was accomplished in agreement with Brazilian Pharmacopoeia (2010), the evaluate the acute dermal toxicity was made with 25 rat wistar, that were divided in 5 groups with 5 animals in each group received the application from one of the gels for subsequent analyzes of the results. For the evaluation of the cytotoxicity was used the method colorimeter 3-(4,5-dimetiltiazol-2-il)2,5-difenil tetrazoilium bromide (MTT) described by MOSMANN (1983), being used cellular lineages of HepG2 (Human Epidermoide Câncer Cells) and HaCaT. The samples had its behavior rheological characteristics. The analyze the release profile, skin permeation and retention were accomplished using cells of Franz modified. The microbiological was in agreement... (Complete abstract click electronic access below)

No presente trabalho foi realizado o isolamento da 3-O-metilquercetina, a partir de produto seco do extrato de inflorescências de Achyrocline satureioides, e sua caracterização. Em etapa farmacotécnica, foi realizado o estudo da associação deste flavonóide com b-ciclodextrina, bem como testes preliminares de permeação cutânea das associações, incorporadas ou não em gel de hidroxipropilmetilcelulose. As técnicas espectroscópicas, infravermelho e ressonância magnética de hidrogênio, confirmaram a estrutura do flavonóide isolado. Para o doseamento da 3-Ometilquercetina, realizou-se a validação de metodologia analítica por cromatografia líquida de alta eficiência, obtendo-se linearidade, na faixa de concentração de 0,05 a 1,5 μg/mL, precisão e exatidão adequadas. A análise da associação da 3-Ometilquercetina com b-ciclodextrina por infravermelho, ressonância magnética de hidrogênio e a análise pelo método empírico de Mecânica Molecular (MM2) do software Chem3D Ultra (Versão 9.0, CambridgeSoft) indicam possível inclusão do anel B da 3-O-metilquercetina na cavidade da b-ciclodextrina, sendo a inserção do flavonóide pela borda das hidroxilas secundárias mais favorável do que pela borda das hidroxilas primárias. A b-ciclodextrina e o gel de hidroxipropilmetilcelulose promoveram a permeação do flavonóide através da pele. A realização de ensaios in vivo para a seleção da melhor formulação constitui-se na principal perspectiva de continuidade de investigação científica do tema. / 3-O-methylquercetin (3-OMQ) was isolated from spray dried powder of Achyrocline satureioides and characterized by IR and 1H NMR. The study of association of this flavonoid with b-cyclodextrin (bCD) was performed, as well as preliminary skin permeation tests of these associations, incorporated or not in hydroxypropyl methylcellulose (HPMC) hydrogel. A LC method for 3-OMQ assay was validated in the concentration range from 0.05 to 1.5 μg/mL, with suitable precision and accuracy. The complexation of 3-OMQ with bCD was analyzed by IR, 1H NMR and Molecular Mechanics (Chem3D Ultra 9.0, CambridgeSoft) and the results indicated the possible insertion of B ring of the flavonoid into the bCD cavity, being the insertion through the secondary OH rim more favorable than through the primary OH rim. bCD and HPMC promoted the permeation of the flavonoid through the skin. In vivo assay is required to select the appropriate formulation.

Diversos estudos têm demonstrado a atividade antioxidante de extratos de Achyrocline satureioides. Essa atividade tem sido atribuída à presença de compostos flavonoídicos, em especial, da quercetina. Neste contexto, o objetivo da presente dissertação foi desenvolver nanoemulsões de uso tópico contendo o extrato deste vegetal, bem com o flavonóide quercetina isolado. Numa primeira etapa, nanoemulsões constituídas de octildodecanol, lecitina de gema de ovo, solução extrativa de Achyrocline satureioides (NEE) ou quercetina (NEQ) e água foram preparadas por emulsificação espontânea. O procedimento conduziu à obtenção de nanoemulsões monodispersas (IP<0,2) com um diâmetro médio de gotícula de cerca de 200 e 300 nm e potencial zeta de cerca de -27 e -42 mV, respectivamente, para NEQ e NEE. Esses resultados sugerem a localização de componentes do extrato de Achyrocline satureioides na interface das nanoestruturas, como indicado pelas imagens obtidas por microscopia eletrônica de transmissão. Numa segunda etapa, foi revalidado um método isocrático para a quantificação da quercetina por CLAE, mostrando-se este linear, preciso, exato e específico. A quantidade de quercetina associada com as nanoemulsões foi de 100%, para a concentração de 100 μg/mL de quercetina. Após, foi realizado o estudo de retenção cutânea da quercetina a partir das formulações utilizando pele de orelha suína, em células de difusão de Franz. Os resultados indicam um maior acúmulo de quercetina na pele a partir da NEE (~16 μg/g) em comparação a NEQ (~6 μg/g), após 8 horas de permeação, indicando a influência de componentes do extrato sobre esta propriedade. Finalmente, determinou-se a atividade antioxidante da quercetina através de medidas de substâncias reativas ao ácido tiobarbitúrico (TBA-RS). Encontrou-se uma menor lipoperoxidação nos testes realizados com o extrato de Achyrocline satureioides (56%) em relação aos testes utilizando quercetina isolada (91%), sem diferença na lipoperoxidação entre a NEQ (27 %) e a NEE (22 %). A proteção oferecida pelas formulações foi determinada nas peles submetidas ao estudo de permeação, sendo detectada uma diminuição da lipoperoxidação no tecido que recebeu tratamento com NEE, contudo, essa atividade não foi detectada para NEQ. Em conclusão, os resultados obtidos nesse estudo demonstram o efeito dos constituintes das formulações sobre propriedades físico-químicas das nanoemulsões, bem como na sua retenção na pele e na atividade antioxidante. / Several studies have demonstrated the antioxidant activity of Achyrocline satureioides extracts. This activity has been attributed to the presence of flavonoids, mainly quercetin. In this context, the aim of the present work was to develop topical nanoemulsions containing extract of this plant, as well as isolated quercetin. In a first step, nanoemulsions composed by octyldodecanol, egg lecithin, Achyrocline satureioides extract (NEE) or quercetin (NEQ) and water were prepared by spontaneous emulsification procedure. This process led to monodisperse nanoemulsions presenting mean droplet size of approximately 200 and 300 nm, and zeta potencial of –27 and –42 mV for NEQ and NEE, respectively. These results suggest that Achyrocline satureioides extract compounds are located in the O/W interface of nanoemulsions, as attested by the transmition electron microscopy. In a second step, an isocratic method for quercetin quantification was revalidated. This showed to be linear, accurate, precise and specific. The amout of quercetin associated with nanoemulsions was close to 100%, for a 100 μg/mL of quercetin. After that, a study of quercetin skin retention from the formulations using pig´s skin ear was performed, in Franz diffusion cell. The results revealed a higher accumulation of quercetin in skin from NEE (~16 μg/g) compared to NEQ (~6 μg/g), after 8h of permeation, indicating the influence of the extract compounds on this property. Finally, the antioxidant activity of quercetin was measured by tiobarbituric acid reactive species (TBA-RS). It shows a minor lipoperoxidation for Achyrocline satureioides extract (56%) than isolated quercetin (91%). No difference for NEQ (27 %) and NEE (22 %) lipoperoxidation was found. The protection against oxidation by the formulations was measured in the skin and it was found a reduction in tissue treated with NEE. This reduction was not observed for NEQ. In conclusion, this study shows the effect of formulation over the physico-chemical properties of nanoemulsion’s formulation, as well as on the skin retention and its antioxidant activity.

Made available in DSpace on 2014-06-11T19:24:11Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-02-18Bitstream added on 2014-06-13T19:51:30Z : No. of bitstreams: 1 zambon_aplb_me_arafcf.pdf: 681314 bytes, checksum: 5de628ca0606efd6f2252b3fc61e5c74 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Universidade Estadual Paulista (UNESP) / Os raios ultravioletas podem desencadear queimadura solar, fotoenvelhecimento e câncer de pele e, por este motivo, foram desenvolvidos os filtros solares. Atualmente considera-se que uma formulação fotoprotetora ideal e de maior fotoestabilidade deve conter em sua formulação associação de filtros solares com ampla capacidade de absorção da radiação UVB e UVA. Dentre os filtros solares utilizados neste trabalho estão o p-metoxicinamato de octila (OMC), um dos mais utilizados globalmente na proteção frente à radiação UVB, a Benzofenona-3, absorvedor de radiação UVB e UVA II, o Ácido Sulfônico Fenilbenzimidazol, um filtro solar UV-B hidrossolúvel e o Bemotrizinol, com amplo espectro. O sítio de ação desejável de um filtro solar é restrito à superfície da pele e sua função é perdida quando este permeia a pele e atinge a circulação sistêmica. Estudos in vivo e in vitro têm demonstrado a permeação e absorção sistêmica de filtros solares através da pele. Este trabalho teve como objetivo avaliar a interferência da associação dos filtros solares benzofenona 3, ácido sulfônico fenilbenzimzidazol e BEMT nos estudos de estabilidade, liberação, permeação e retenção cutânea in vitro do OMC. A metodologia de identificação e quantificação do OMC foi validada, levando-se em consideração a análise dos limites de confiança. Foram realizados estudos de estabilidade preliminar e acelerada das formulações e do OMC em virtude da fotoinstabilidade do mesmo. Os estudos de liberação, permeação e retenção cutânea foram realizados utilizando-se o equipamento de célula de difusão vertical de Franz modificada e as membranas utilizadas foram acetato de celulose para o estudo de liberação e orelha de porco para permeação e retenção cutânea. Os resultados obtidos permitiram concluir que as formulações estudadas apresentaram comportamento... / Ultraviolet rays can cause sunburn, photoaging and skin cancer and, because of this, sunscreens were developed. Currently it is considered that an ideal sunscreen formulation and greater photostability must contain in its formulation association of solar filters with high capacity to absorb UVB and UVA. Among the solar filters used in this work are p-octyl methoxycinnamate (OMC), one of the most globally used in the protection against UVB radiation, Benzophenone-3, UVB and UVA II absorber Fenilbenzimidazol Sulfonic Acid, a UV-B hidrosoluble solar filter and Bemotrizinol with broad spectrum. The desirable site of action of a solar filter is limited to the skin surface and its function is lost when it permeates the skin and reaches the systemic circulation. In vivo and in vitro studies have demonstrated the permeation and systemic absorption of solar filters across the skin. This study objective was to evaluate the interference of association of the solar filters benzophenone-3, fenilbenzimzidazol sulfonic acid and BEMT on the stability, release, in vitro skin permeation and retention of the OMC. The methodology for OMC’s identification and quantification was validated, taking into account the confidence limits analysis. Preliminary and accelerated stability studies of the formulations and the OMC, because of it’s photoinstability. The release studies, skin permeation and retention were performed using the equipment Franz’s vertical diffusion cell modified, and the membranes used were cellulose acetate for the release study and pig ear skin for permeation and retention. The results showed that the formulations studied showed similar behavior stability and OMC’s release, regardless of the association of the solar filters... (Complete abstract click electronic access below)