Nuolatos plečiantis maisto papildų rinkai ir augant naujai sukuriamų papildų kiekiui būtina sukurti greitas, tikslias ir efektyvias papildų analizės metodikas, norint užtikrinti į rinką tiekiamų farmacinių prepartų kokybę. Vitamino E nustatymui efektyviosios skysčių chromatografijos būdu yra sukurta nemažai įvairių metodikų, tačiau svarbu žinoti, kuri metodika yra efektyviausia būtent vitamino E preparatams. Tyrimo tikslas - atlikti kokybinį ir kiekybinį tokoferolio acetato nustatymą pritaikant efektyviosios skysčių chromatografijos metodą bei surasti efektyviausią tokoferolio acetato ekstrakcijos metodiką vaistinių preparatų analizei. Tyrimo uždaviniai - surinkti ir apibendrinti informaciją apie vitaminą E, jo poveikį organizmui, maisto papildus su tokoferolio acetatu bei jų analizę; optimizuoti metodiką tokoferolio acetato preparatų analizei efektyviosios skysčių chromatografijos būdu ir ją validuoti; atlikti tokoferolio acetato preparatų (maisto papildo ir vaistinio preparato) paruošimą analizei įvairiomis ekstrakcijos metodikomis; išanalizuoti gautus duomenis ir padaryti išvadas apie efektyviausias ekstrakcijos metodikas bandinių paruošimui bei skirtumus tarp vaistinio preparato ir maisto papildo analizės. Metodas – tyrimas atliktas chromatografine sistema su fotodiodų matricos detektoriumi. Mobilioji fazė – metanolis:distiliuotas vanduo (95:5), tekėjimo greitis – 1.2 ml/min, bandinio tūris – 10 µl, trukmė – 14 min, bangos ilgis – 282 nm. Išbandytos preparatų... [toliau žr. visą tekstą] / The food supplements market is constantly growing and new products are entering it everyday. In order to assure the quality of these pharmaceuticals it is important to develop methods of analysis which are fast, precise and effective. There are many methods created for the analysis of vitamin E using high-performance liquid chromatography so it is important to know which of them are the most effective for vitamin E food supplements. The aim – to perform a qualitative and a quantitive assey of tochopherol acetate using high performance liquid chromatography and to find the most effective method of extracting tocopherol acetate from food suplements. The tasks – to collect and to summarize the data on vitamin E, its effects on human body, on the supplements containing tocopherol acetate and their analysis; to develop a high-performance liquid chromatography method of analysis suitable for the pharmaceuticals containing tocopherol acetate ant to validate it; to perform a tocopherol acetate (a food supplement and a drug) samples preparation using different techniques chosen; to analyze the data collected and to make conclusions about the most effective way of preparing samples for the analysis and about the differences between a drug and a food supplement analysis. The method – the study was conducted by high-performance liquid chromatography using a photodiode matrix detector. Mobile phase – methanol:purified water (95:5), flow-rate – 1.2 ml/min, injection volume – 10 µl... [to full text]

Submitted by Biblioteca da Faculdade de Farmácia (bff@ndc.uff.br) on 2017-03-13T18:50:12Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Duarte, Letícia de Souza Guimarães [Dissertação, 2016].pdf: 2499210 bytes, checksum: 961dab593ba2ea593fadba0a224c15cc (MD5) / Made available in DSpace on 2017-03-13T18:50:12Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Duarte, Letícia de Souza Guimarães [Dissertação, 2016].pdf: 2499210 bytes, checksum: 961dab593ba2ea593fadba0a224c15cc (MD5) / A papaína, que corresponde a um complexo de enzimas extraídas do vegetal Carica papaya L., tem sido utilizada pela sua propriedade proteolítica em desbridamento enzimático de feridas. Diversos estudos descrevem o uso da papaína na forma de pó, solução, gel e creme sobre feridas, porém a baixa estabilidade da enzima nesses meios limita sua utilização em larga escala. O objetivo principal do trabalho consiste em desenvolver e avaliar formulações de papaína 10%(p/p) em gel carbômero, contendo antioxidantes como acetato de alfa-tocoferol e metabissulfito de sódio, associados a outros adjuvantes que permitam melhor estabilidade da enzima. As formulações iniciais para o estudo foram planejadas segundo desenho experimental fatorial 23, sendo as variáveis independentes representadas pelas concentrações de cisteína, de polissorbato 80 e de antioxidante (acetato de alfa-tocoferol ou metabissulfito de sódio). As amostras, mantidas sob refrigeração (5°C ± 3) por 7 dias, foram submetidas a ensaios de espectrofluorimetria para avaliação da atividade enzimática. Os resultados iniciais revelaram que as amostras que continham metabissulfito de sódio associado às maiores concentrações de polissorbato 80, apresentaram as melhores condições para manutenção da atividade proteolítica, enquanto que as amostras contendo alfa-tocoferol não foram capazes de manter a atividade. Sendo assim, realizou-se estudo de estabilidade com preparações contendo concentrações fixas de papaína 10% (p/p), polissorbato 80 2,0% (p/p), com variações nas concentrações de cisteína de 0,10, 0,13 e 0,16% (p/p) e metabissulfito de sódio de 0,50, 0,75 e 1,00% (p/p). Durante o período estudado, as amostras apresentaram aspecto homogêneo, sem mudanças na coloração e no odor. Na avaliação de pH não houve variação significativa dos valores (p>0,05). A variação de potencial Zeta foi menor que ǀ10ǀmV, não havendo diferença estatisticamente significativa em função do tempo (p>0,05). A partir dos resultados obtidos, novo desenho fatorial 23 foi traçado considerando como variáveis independentes: o tempo, as concentrações de metabissulfito e de cisteína. A presença de metabissulfito 0,5% ou 1,0% (p/p) associado a cisteína 0,16%(p/p) e polissorbato 80 2,0% (p/p), proporcionaram os menores valores de decaimento na concentração de papaína ativa nas formulações testadas por 28 dias. / Papain, which corresponds to a complex of enzymes from the plant Carica papaya L., has been used for its proteolytic property in enzymatic wound debridement. Several studies describe the use of papain as powder, solution, gel and cream forms over wounds; however, the low stability of the enzyme in the media limits its use in large scale. The main objective of this work is to develop and evaluate formulations with papain 10% (w/w) at carbomer gel, containing antioxidants as alpha-tocopherol acetate and sodium metabisulphite, associated with other adjuvants that enable better stability of the enzymes. The early formulations for the study were planned in a factorial experimental design 23, with the independent variables represented by cysteine, polysorbate 80 and antioxidant (alpha-tocopherol or sodium metabisulphite) concentrations. The samples, kept under refrigeration (5°C ± 3) for 7 days, were submitted to spectrofluorimetry essays for enzyme activity evaluation. Initial findings showed that the samples containing sodium metabisulphite associated with higher concentrations of polysorbate 80, presented the best conditions for proteolytic activity maintenance, whereas the samples containing alpha-tocopherol were not able to maintain activity. Thus, stability study was carried out with preparations with fixed concentrations of papain 10% (w/w), polysorbate 80 2.0% (w/w), with variations in the concentrations of cystein 0.10, 0.13 and 0.16% (w/w) and sodium metabisulphite 0.50, 0.75 and 1.00% (w/w). During the studied period, the samples showed a homogeneous appearance, without color or odor changes. At pH evaluation there was no significant change in values (p> 0.05). Zeta potential have varied less than |10|mV, with no statistically significant difference over time (p> 0.05). From the results, a new factorial design 23 was traced, considering as independent variables: time, metabisulphite and cystein concentrations. The presence of metabisulphite 0.5 % or 1.0 % (w/w) associated with cysteine 0.16 % (w/w), and polysorbate 80 2.0% (w/w), showed the lowest decay values of active papain concentration in the formulations tested for 28 days.

Dans cette thèse, nous avons combiné les approches des cultures single-cell, de cytométrie en flux,des analyses de métabolisme énergétique et de génétique moléculaire afin d’explorer les effets del’Acétate d’α-Tocopherol (α-TOA) sur les cellules stromales mésenchymateuses (MStroC) et leurs souspopulations fonctionnelles (cellules souches et progénitrices mésenchymateuses). L’autre but était de tester une molécule de miR-210 par rapport à son utilisation potentielle comme « hypoxia mimicking molecule ». Après avoir démontré l’hétérogénéité de la population MStroC et conclu que la population de premier passage est appropriée pour des expérimentations ultérieures, nous avons trouvé que l’α-TOA présentait un effet positif sur le maintien de la capacité proliférative élevée des cellules souches mésenchymateuses. Cet effet est accompagné d’une atténuation de l’activité de la chaîne de transport d’électrons (ETC) qui pourrait d’autre part expliquer l’accroissement modéré du niveau des Reactive Oxygen Species mitochondriales (mtROS) que nous avons détectées. L’augmentation du niveau de mtROS pourrait être associée à une dégradation de protéine HIF-1 dans la population MStroC exposée à l’α-TOA. Bien que nous n’ayons pas détecté d’augmentation compensatoire de la glycolyse, les phénomènes observés représentent en partie la réponse cellulaire complexe au faible niveau d’O2. Il a été établi que ce phénomène était relié au maintien de primitivité des cellules souches. Le mécanisme exact reste à clarifier ainsi que son potentiel translationnel. En outre, nous avons apporté la preuve que miR-210 fait partie intégrante de la réponse des MStroC à la faible concentration en O2. Dans cette étude, nous avons montré que l’augmentation de l’expression de miR-210 sur une période courte (jusqu’à 24 heures) et après une période étendue (jusqu’à 72 heures) d’exposition des MStroC à une faible concentration en O2. De plus, nous avons prouvé que ce micro ARN pouvait être régulé par les deux facteurs transcriptionnels HIF-1 et HIF-2, nous laissant penser que ceci faisait partie intégrante de la réponse des MStroC à une faible concentration en O2. Jusqu’à présent, nos données suggèrent que miR-210 est digne d’intérêt en tant que bonne molécule « hypoxia mimicking ». / In this thesis, we combined approaches of single-cell cultures, flow-cytometry, energetic metabolismanalysis and molecular genetics in order to get insight in the effects of α-Tocopherol-Acetate (α-TOA)on Mesenchymal Stromal Cells (MStroC) and their functional subpopulations (mesenchymal stem and progenitor cells). The other aim was to test a miR-210 molecule with respect to its potential use as hypoxia mimicking molecule. After defining MStroC population heterogeneity and concluding that the first passage population is convenient for further experiments, we demonstrated that α-TOA exhibits a positive effect on the maintenance of high proliferative capacity of mesenchymal stem cells. This effect could be associated with an attenuation of electron transport chain (ETC) activity, which, on the other hand could explain moderate increase in the level of mitochondrial Reactive Oxygen Species (mtROS) we detected. The increase in mtROS level could be associated with a decreased HIF-1 alpha protein degradation in MStroC exposed to α-TOA. Although we did not detect a compensatory increase in glycolysis, the observed phenomena depict part of a complex cellular response to the low O2 that is demonstrated to be related with maintenance of stem cell primitiveness. The exact mechanism remains to be elucidated as well as its translational potential. In addition, we provided new evidences that miR-210 is integral part in MStroC response to low O2. In the study, we showed increased in miR-210 expression in a short-term (up to 24 hours) and after extended (up to 72 hours) MStroC exposed to low O2. Moreover, we demonstrated that this micro- RNA could be regulated by both HIF-1 and HIF-2 transcriptional factors, suggesting it as integral part of MStroC response to low O2. So far, our data suggest that miR-210 is worthy to be considered as good hypoxia mimicking molecule.