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Avaliação do efeito osteogênico por diferentes fitoestrógenos em cultura de osteoblastos derivados de células tronco mesenquimais / Evaluation of the osteogenic effect of different phytoestrogens in osteoblasts culture derived from mesenchymal stem cellsFaria, Amanda Natalina de 15 March 2013 (has links)
A menopausa é provocada pela falência da produção de hormônios ovarianos e tem como consequências alterações desfavoráveis no metabolismo e perda de massa óssea. O declínio da produção de estrógeno é considerado um grande fator de risco para o desenvolvimento da osteoporose em mulheres e como tratamento faz-se o uso da Terapia de Reposição Hormonal. No entanto, esta terapia tem trazido riscos á saúde de alguns grupos de mulheres. Como alternativa ao tratamento tradicional, tem-se os fitoestrógenos, e com eles as isoflavonas, encontradas principalmente na soja, Trifolium pratense e Cimicifuga racemosa. Este estudo teve como objetivo comparar a capacidade de estimular a osteogênese in vitro, a partir de cultura de osteoblastos derivados de células tronco mesenquimais, em duas preparações de fitoestrógenos: O extrato de soja biotransformado pelo fungo Aspergillus awamori (ESBF), e o Menoflavon® 40mg (Melbrosin International) composto pela isoflavona Trifolium pratense. Para este objetivo foram realizadas: a) Avaliação do crescimento e proliferação celular b) Viabilidade e crescimento das culturas de osteoblastos. c) Dosagem de proteína total das culturas de osteoblastos. d) Determinação da atividade específica da enzima fosfatase alcalina. e) Formação da matriz mineralizada. O Menoflavon® foi testado nas concentrações de 28,75 nM de daidzeína (D) + 7,5 nM de genisteína (G) (0,5 ?g/mL de Menoflavon®); 57,5 nM de D + 15 nM de G (1 ?g/mL de Menoflavon®); e 230 nM de D + 60 nM de G (4 ?g/mL de Menoflavon®); controle padrão de 57,5 nM de D + 15 nM de G comercial e controle de dimetilsulfóxido (DMSO). O ESBF foi testado nas concentrações de 1,181 nM de D + 0,922 nM de G (0,5 ?g/mL de ESBF); 2,361 nM de D + 1,845 nM de G (1 ?g/mL de ESBF); 9,445 nM de D + 7,379 nM de G (4 ?g/mL de ESBF); controle padrão de 2,361 nM de D + 1,845 nM de G comercial e controle de DMSO. Com a metodologia do MTT (3[4,5-dimetiltiazol-2-il]-2,5-brometo difenil tetrazolium) e da Resazurina comprovamos que não houve morte celular nas concentrações testadas com as duas formulações. A dosagem de proteínas totais manteve-se constante com as duas formulações e a formação de matriz mineralizada também manteve-se constante em relação ao controle para ambos. A atividade específica da fosfatase alcalina teve um decréscimo significativo ao 14º dia com todas as concentrações testadas e ao 21º dia com algumas concentrações de Menoflavon e com o ESBF decresceu em alguns dias, no entanto manteve-se estável no restante do teste. O ESBF mostrou ser melhor que o Menoflavon®, já que obtivemos resultados semelhantes e sua concentração é 24 vezes menor. No entanto, o estudo realizado mostrou que tanto o ESBF quanto o Menoflavon® não são capazes de estimular a osteogênese in vitro, a partir de cultura de osteoblastos derivados de células tronco mesenquimais. / Menopause is caused by failure in the production of ovarian hormones and its consequences are unfavorable changes in metabolism and bone loss. The decline in estrogen production is considered a major risk factor for the development of osteoporosis in women, and the Hormone Replacement Therapy is used as treatment. However, this therapy has brought some risks to the health of some groups of women. As an alternative to the traditional treatment, phytoestrogens as isoflavones, found mainly in soy, Trifolium pratense, Cimicifuga racemosa and rye can be used. This study aimed to compare the ability of two preparations of phytoestrogens to stimulate osteogenesis in vitro (from cultures of osteoblasts derived from mesenchymal stem cells): soy extract biotransformed by the fungus Aspergillus awamori (ESBF), and Menoflavon® 40mg (Melbrosin International) composed by the isoflavone Trifolium pratense. With this objective, were used: a) Evaluation of cell growth and proliferation; b) Viability of the cultures of osteoblasts; c) Determination of total protein from the cultures of osteoblasts; d) Determination of the specific activity of the enzyme alkaline phosphatase; e) Formation of mineralized matrix. Menoflavon® was tested at the concentrations of 28.75 nM of daidzein (D) + 7.5 nM of genistein (G) (Menoflavon® 0.5 ?g/mL); 57.5 nM D + 15 nM G (Menoflavon® 1 ?g/mL); and 230 nM D + 60 nM G (Menoflavon® 4 ?g/mL); standard control of commercial 57.5 nM D + 15 nM G and DMSO control. ESBF was tested at the concentrations of 1.181 nM D + 0.922 nM G (ESBF 0.5 ?g/mL); 2.361 nM D + 1.845 nM G (ESBF 1 ?g/mL); 9.445 nM D + 7.379 nM G (ESBF 4 ?g/mL); standard control of commercial 2.361 nM D + 1.845 nM G and dimetilsulfoxide (DMSO) control. With the MTT and Resazurin methods we verified that there was no cell death for all concentrations tested with the two formulations. The amount of total protein remained constant with the two formulations, and the formation of mineralized matrix also were the same as the control. The specific activity of alkaline phosphatase decreased significantly on day 14 for all concentrations tested, and at day 21 for some concentrations of Menoflavon®, with the ESBF decreased some days, however remained constant in the other tests. Therefore, ESBF proved better than Menoflavon®, since we obtained similar results for both, but the concentration of ESBF is 24 times smaller than the concentration of Menoflavon®. However, both the ESBF and the Menoflavon® were not capable of stimulating osteogenesis in vitro from cultures of osteoblasts derived from mesenchymal stem cells.
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Avaliação do efeito osteogênico por diferentes fitoestrógenos em cultura de osteoblastos derivados de células tronco mesenquimais / Evaluation of the osteogenic effect of different phytoestrogens in osteoblasts culture derived from mesenchymal stem cellsAmanda Natalina de Faria 15 March 2013 (has links)
A menopausa é provocada pela falência da produção de hormônios ovarianos e tem como consequências alterações desfavoráveis no metabolismo e perda de massa óssea. O declínio da produção de estrógeno é considerado um grande fator de risco para o desenvolvimento da osteoporose em mulheres e como tratamento faz-se o uso da Terapia de Reposição Hormonal. No entanto, esta terapia tem trazido riscos á saúde de alguns grupos de mulheres. Como alternativa ao tratamento tradicional, tem-se os fitoestrógenos, e com eles as isoflavonas, encontradas principalmente na soja, Trifolium pratense e Cimicifuga racemosa. Este estudo teve como objetivo comparar a capacidade de estimular a osteogênese in vitro, a partir de cultura de osteoblastos derivados de células tronco mesenquimais, em duas preparações de fitoestrógenos: O extrato de soja biotransformado pelo fungo Aspergillus awamori (ESBF), e o Menoflavon® 40mg (Melbrosin International) composto pela isoflavona Trifolium pratense. Para este objetivo foram realizadas: a) Avaliação do crescimento e proliferação celular b) Viabilidade e crescimento das culturas de osteoblastos. c) Dosagem de proteína total das culturas de osteoblastos. d) Determinação da atividade específica da enzima fosfatase alcalina. e) Formação da matriz mineralizada. O Menoflavon® foi testado nas concentrações de 28,75 nM de daidzeína (D) + 7,5 nM de genisteína (G) (0,5 ?g/mL de Menoflavon®); 57,5 nM de D + 15 nM de G (1 ?g/mL de Menoflavon®); e 230 nM de D + 60 nM de G (4 ?g/mL de Menoflavon®); controle padrão de 57,5 nM de D + 15 nM de G comercial e controle de dimetilsulfóxido (DMSO). O ESBF foi testado nas concentrações de 1,181 nM de D + 0,922 nM de G (0,5 ?g/mL de ESBF); 2,361 nM de D + 1,845 nM de G (1 ?g/mL de ESBF); 9,445 nM de D + 7,379 nM de G (4 ?g/mL de ESBF); controle padrão de 2,361 nM de D + 1,845 nM de G comercial e controle de DMSO. Com a metodologia do MTT (3[4,5-dimetiltiazol-2-il]-2,5-brometo difenil tetrazolium) e da Resazurina comprovamos que não houve morte celular nas concentrações testadas com as duas formulações. A dosagem de proteínas totais manteve-se constante com as duas formulações e a formação de matriz mineralizada também manteve-se constante em relação ao controle para ambos. A atividade específica da fosfatase alcalina teve um decréscimo significativo ao 14º dia com todas as concentrações testadas e ao 21º dia com algumas concentrações de Menoflavon e com o ESBF decresceu em alguns dias, no entanto manteve-se estável no restante do teste. O ESBF mostrou ser melhor que o Menoflavon®, já que obtivemos resultados semelhantes e sua concentração é 24 vezes menor. No entanto, o estudo realizado mostrou que tanto o ESBF quanto o Menoflavon® não são capazes de estimular a osteogênese in vitro, a partir de cultura de osteoblastos derivados de células tronco mesenquimais. / Menopause is caused by failure in the production of ovarian hormones and its consequences are unfavorable changes in metabolism and bone loss. The decline in estrogen production is considered a major risk factor for the development of osteoporosis in women, and the Hormone Replacement Therapy is used as treatment. However, this therapy has brought some risks to the health of some groups of women. As an alternative to the traditional treatment, phytoestrogens as isoflavones, found mainly in soy, Trifolium pratense, Cimicifuga racemosa and rye can be used. This study aimed to compare the ability of two preparations of phytoestrogens to stimulate osteogenesis in vitro (from cultures of osteoblasts derived from mesenchymal stem cells): soy extract biotransformed by the fungus Aspergillus awamori (ESBF), and Menoflavon® 40mg (Melbrosin International) composed by the isoflavone Trifolium pratense. With this objective, were used: a) Evaluation of cell growth and proliferation; b) Viability of the cultures of osteoblasts; c) Determination of total protein from the cultures of osteoblasts; d) Determination of the specific activity of the enzyme alkaline phosphatase; e) Formation of mineralized matrix. Menoflavon® was tested at the concentrations of 28.75 nM of daidzein (D) + 7.5 nM of genistein (G) (Menoflavon® 0.5 ?g/mL); 57.5 nM D + 15 nM G (Menoflavon® 1 ?g/mL); and 230 nM D + 60 nM G (Menoflavon® 4 ?g/mL); standard control of commercial 57.5 nM D + 15 nM G and DMSO control. ESBF was tested at the concentrations of 1.181 nM D + 0.922 nM G (ESBF 0.5 ?g/mL); 2.361 nM D + 1.845 nM G (ESBF 1 ?g/mL); 9.445 nM D + 7.379 nM G (ESBF 4 ?g/mL); standard control of commercial 2.361 nM D + 1.845 nM G and dimetilsulfoxide (DMSO) control. With the MTT and Resazurin methods we verified that there was no cell death for all concentrations tested with the two formulations. The amount of total protein remained constant with the two formulations, and the formation of mineralized matrix also were the same as the control. The specific activity of alkaline phosphatase decreased significantly on day 14 for all concentrations tested, and at day 21 for some concentrations of Menoflavon®, with the ESBF decreased some days, however remained constant in the other tests. Therefore, ESBF proved better than Menoflavon®, since we obtained similar results for both, but the concentration of ESBF is 24 times smaller than the concentration of Menoflavon®. However, both the ESBF and the Menoflavon® were not capable of stimulating osteogenesis in vitro from cultures of osteoblasts derived from mesenchymal stem cells.
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Évaluation de l’effet antidiabétique de plantes médicinales de la forêt boréale et identification des principes actifs de deux espèces prometteusesEl Hamaoui El Nachar, Abir 03 1900 (has links)
Le diabète de type 2 est une maladie chronique dont l’incidence est en augmentation
continuelle. Le risque de développer le diabète de type 2 chez les populations autochtones du
Canada est de trois à cinq fois plus élevé que le reste de la population canadienne. La forêt
boréale comporte plusieurs plantes médicinales ayant un potentiel pour le traitement ou la
prévention du diabète. Certaines de ces plantes font partie de la médecine traditionnelle et
alternative Crie. Des enquêtes ethnobotaniques ont amené notre équipe de recherche à
identifier 17 extraits de plantes médicinales utilisées par les Cris d’Eeyou Istchee (Baie James,
Québec) pour traiter les symptômes du diabète. Parmi ces extraits, certains ont montré des
activités anti-diabétiques au niveau des cellules musculaires, des adipocytes et dans des études
in vivo réalisées chez des animaux. Le but de cette thèse est d’élucider l’effet de ces 17 plantes
sur l’homéostasie hépatique de glucose, d’identifier l’espèce la plus prometteuse et isoler ces
constituants actifs. De même, le bleuet nain du genre Vaccinium angustifolium fait partie de la
forêt boréale canadienne et est connu pour ses activités anti-diabétiques. Une
biotransformation du jus de bleuet lui confère une activité antioxydante accrue et un profil
biologique différent. Le deuxième but de cette thèse est d’élucider les mécanismes d’action
par lesquels le jus de bleuet biotransformé (BJ) exerce son effet anti-diabétique et d’identifier
ses principes actifs.
Les résultats ont montré que trois extraits de plantes Cris se sont démarqués par leur effet sur
l’homéostasie hépatique de glucose. Picea glauca exerce son effet en diminuant la production
de glucose alors que Larix laricina agit en augmentant le stockage de glucose. Abies balsamea
a montré le profil le plus prometteur, elle agit simultanément en diminuant l’activité de la
Glucose-6-phosphatase (G6Pase) via la stimulation des voies insulino-dépendante et -
indépendante et en augmentant l’activité de la Glycogène synthétase (GS) suite à la
phosphorylation de la Glycogène synthase kinase-3. Le fractionnement de l’extrait d’Abies
balsamea guidé par les deux bioessais a mené à l’isolation de trois composés actifs; l’acide
abiétique (AA), l’acide déhydroabiétique (DAA) et le squalène (SQ). Les principes actifs ont
montré le même mécanisme d’action que l’extrait brut en diminuant l’activité de la G6Pase et
augmentant celle de la GS ainsi qu’en activant les voies de signalisation impliquées. Le DAA
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s’est démarqué par son effet le plus puissant et très comparable à celui de l’extrait d’Abies
balsamea dans toutes les expériences.
De son côté le BJ a montré un effet sur la diminution de la production hépatique de glucose,
l’augmentation de son stockage ainsi que l’augmentation de son transport dans le muscle. Son
fractionnement guidé par les bioessais a permis d’isoler sept fractions dont trois étaient les
plus actives. L’identification des constituants de ces fractions actives a mené à isoler quatres
composés phénoliques; l’acide chlorogénique, l’acide gallique, l’acide protocatéchique et le
catéchol. Le catéchol s’est démarqué avec ses effets les plus puissants en diminuant l’activité
de la G6Pase, augmentant celle de la GS et en stimulant le transport de glucose dans le
muscle.
Les résultats de cette thèse indiquent que la diminution de la production hépatique de glucose
peut s’ajouter au profil anti-diabétique de certaines plantes médicinales Cries et surtout à celui
d’A.balsamea dont les composés actifs peuvent aider dans le développement de nouvelles
molécules anti-diabétiques. De plus, les résultats de cette thèse ont montré que l’activité antidiabétique
du BJ implique le contrôle de l’homéostasie de glucose au niveau du foie et du
muscle. L’identification du catéchol comme principe actif avec potentiel anti-diabétique
prometteur pourra servir pour des fins thérapeutiques ultérieures. / Type 2 diabetes is a chronic disease for which incidence is continuously increasing.
The risk of developing type 2 diabetes among Aboriginal people in Canada is three to five
times higher than the rest of the Canadian population. The boreal forest has several medicinal
plants with potential for the treatment or prevention of diabetes. Some of these plants are part
of the Cree traditional and alternative medicine. Ethnobotanical surveys led our research team
to identify 17 medicinal plant extracts used by the Crees of Eeyou Istchee (James Bay,
Quebec) to treat symptoms of diabetes. Some extracts showed anti-diabetic activities in
muscle cells, adipocytes and in vivo studies in animals. The aim of this thesis is to elucidate
the effect of these 17 plants on hepatic glucose homeostasis, to identify the most promising
species and isolate its active constituents. Similarly, Canadian lowbush blueberry, Vaccinium
angustifolium.Ait, is part of the Canadian boreal forest and is known for its anti-diabetic
activities. Biotransformation of blueberry juice gives it an increased antioxidant activity and a
different biological profile. The second aim of this thesis is to elucidate the mechanisms of
action by which biotransformed blueberry juice (BJ) exerts its anti-diabetic effect and identify
its active constituents.
The results showed that three Cree plants stood out with their effect on hepatic glucose
homeostasis. Picea glauca exerts its effect by reducing glucose production whereas Larix
laricina works by increasing its the storage. Abies balsamea showed the most promising
profile, simultaneously and powerfully reducing glucose-6-phosphatase (G6Pase) involving
both insulin-dependent and -independent pathways and stimulating Glycogen synthase (GS)
via phosphorylation of Glycogen synthase kinase-3 (GSK-3). Bioassay-guided fractionation of
Abies balsamea led to the isolation of three active compounds; Abietic acid (AA),
dehydroabietic acid (DAA) and squalene (SQ). The active constituents have shown the same
mechanism of action as the crude extract by decreasing the activity of G6Pase, increasing that
of the GS and activating signaling pathways. DAA stood out for its most powerful effect close
to that of the crude extract in all experiments.
Our results showed that anti-diabetic activity of BJ involves decrease in hepatic glucose
production, increase of storage and enhancement of glucose uptake in muscle. Its bioassayiv
guided fractionation led to isolate seven fractions, three of which were the most active.
Identification of components in the active fractions resulted in four isolated phenolic
compounds; chlorogenic acid, gallic acid, protocatechuic acid and catechol. Catechol stood
out with its most powerful effects by decreasing the activity of G6Pase, increasing the GS and
stimulating glucose transport in muscle.
Our results thus confirm that the reduction of hepatic glucose production likely contributes to
the therapeutic potential of several anti-diabetic Cree traditional plant and especially that of
Abies balsamea whose active compounds may help in the development of new anti-diabetic
molecules. In addition, the results of this thesis showed that the anti-diabetic activity of BJ
involves control of glucose homeostasis in the liver and muscle. Identification of catechol as
an active compound with anti-diabetic promising potential can be used for future therapeutic
purposes.
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