A doença periodontal é uma das doenças inflamatórias crônicas mais comuns que acometem a população. A grande destruição tecidual observada durante o seu desenvolvimento tem sido atribuída ao processo inflamatório exacerbado e ao desequilíbrio favorável à geração de espécies reativas de oxigênio em relação àcapacidade de defesa dos antioxidantes. A epigalocatequina-3-galato (EGCG) obtida da Camellia sinensis é uma substância que apresenta potencial antioxidante e antiinflamatório e, mais recentemente, testes in vitro têm mostrado que também possui atividade anti-osteoclastogênica, sendo apontada como uma possível droga para uso terapêutico nas patologias ósseas com excessiva atividade osteoclástica e destruição óssea. O objetivo do trabalho foi verificar morfométricamente em imagens obtidas pela tomografia microcomputadorizada (micro-CT) e cortes histológicos se a administração diária de EGCG reduz o processo inflamatório e a perda óssea alveolar na doença periodontal induzida por ligadura em ratos. O primeiro molar inferior direito de 60 ratos foi amarrado com fio de seda 3.0 e divididos em grupo sem tratamento (GST), grupo tratado com EGCG (GTEGCG) que recebeu diariamente por gavagem 100mg/Kg de EGCG e grupo Sham (GTsalina) que recebeu apenas solução salina. Nos períodos de 0, 7, 14 e 21 dias (n=5 animais/período/grupo) imagens digitais foram obtidas no microtomógrafo sendo submetidos à análise do nível ósseo periodontal (PBL) e da densidade óssea (BV/TV) inter-radicular. Nos cortes longitudinais do M1 corados pela HE foi avaliado o PBL e morfometricamente o percentual e volume de processo inflamatório e tecido ósseo, além do número osteoclastos/cm2. Os dados foram submetidos à ANOVA a dois critérios e ao teste de Tukey (p<0,05). O PBL determinado nas imagens microtomográficas e histológicas mostraram que a perda óssea aumenta em todos os grupos durante a fase aguda da doença (0 a 14 dias) e estabiliza na fase crônica (14 dias-21 dias). Em geral, o PBL foi menor no GTEGCG (média de 0,839 mm) comparado aos GST e GTsalina (média de 0,953 ). Quanto à densidade óssea o BV/TV foi maior no GTEGCG (68%) comparados aos GST (62,06%). O percentual do processo inflamatório e o número de osteoclastos foram menores no GTEGCG, com pico aos 14 dias (3,4% de processo inflamatório e 32 osteoclastos/cm2), comparados aos GST e GTsalina cujo o pico foi aos 7 dias (média de 8,6% de processo inflamatório e 68 osteoclastos/cm2). Concluímos que, no modelo de periodontite induzida por ligadura, o tratamento com EGCG diminui o processo inflamatório e a osteoclastogênese e consequentemente a perda óssea e a severidade da doença. / Periodontal disease is currently one of the most common chronic inflammatory diseases affecting the population. The large tissue destruction observed during its development, has been attributed to exacerbated inflammatory process and unbalance response between production of reactive oxygen species and antioxidant defense capacity. Recently, the substance epigallocatechin-3-gallate (EGCG) obtained from Camellia sinensis have been associated to antioxidant and antiinflammatory actions. In vitro studies have shown that EGCG has also antiosteoclastogenic activity suggesting to be a potencial drug for use in therapeutic treatment of bone diseases with excessive osteoclast formation and bone destruction. The aim of this study was to verify morphometrically in micro-ct and histological images whether daily administration of EGCG inhibits/decreases alveolar bone loss in periodontal disease induced in rats by ligature. The lower right first molar of 60 rats was tied with surgical suture thread 3.0. The animals were divided into untreated group (GST), EGCG treated group (GTEGCG) which received 100mg/kg of EGCG by gavage daily and Sham group (GT saline) which received saline solution only. In periods of 0, 7, 14 and 21 days (n=5 animals/period/group) digital images were obtained in microtomography (SkyScan1176) and subjected to analysis of PBL in the mesial, distal, buccal and lingual root and BV/TV bone volume percentage. In the sagittal slides PBL volumetric points and inflammatory process as well as the number of osteoclasts/cm2 was analyzed. Data were submitted to twoway ANOVA and Tukey test (p <0.05). PBL determined in microtomographic and histological images showed that bone loss increased and stabilized, respectively, in the all groups acute phase (days 0 to 14) and chronic phase (14 days, 21 days) of the disease. In general, the PBL was lower in GTEGCG (average 0,839 mm) compared to GST and GTsaline (average 0,953). Regarding bone density BV/TV in GTEGCG was higher (68%) compared to GST (62.06%). The percentage of inflammation and the number of osteoclasts was more mild in GTEGCG, reaching peak at 14 days (3.4% inflammatory process and 32 osteoclasts / cm2) compared to GST and GTsaline whose peak was at 7 days (average 8.6% inflammatory process and 68 osteoclasts / cm2). It was concluded that in the current model of periodontal disease induced by ligature, EGCG treatment decreases inflammatory process, osteoclastogenesis activity, bone loss, and consequently the severity of the disease.

A hallmark of type 2 diabetes (T2D) is β-cell dysfunction and the eventual loss of functional β-cell mass. Therefore, mechanisms that improve or preserve β-cell function could be used to improve the quality of life of individuals with T2D. Studies have shown that monomeric, oligomeric and polymeric cocoa flavanols have different effects on obesity, insulin resistance and glucose tolerance. We hypothesized that these cocoa flavanols may have beneficial effects on β-cell function. INS-1 832/13 derived β-cells and primary rat islets cultured with a monomeric catechin-rich cocoa flavanol fraction demonstrated enhanced glucose-stimulated insulin secretion, while cells cultured with total cocoa extract, oligomeric, or polymeric procyanidin-rich fractions demonstrated no improvement. The increased glucose-stimulated insulin secretion in the presence of the monomeric catechin-rich fraction corresponded with enhanced mitochondrial respiration, suggesting improvements in β-cell fuel utilization. Mitochondrial complex III, IV and V components were upregulated after culture with the monomer-rich fraction, corresponding with increased cellular ATP production. The monomer-rich fraction improved cellular redox state and increased glutathione concentration, which corresponds with Nrf2 nuclear localization and expression of Nrf2 target genes, including NRF-1 and GABPA, essential genes for increasing mitochondrial function. We propose a model by which monomeric cocoa catechins improve the cellular redox state, resulting in Nrf2 nuclear migration and upregulation of genes critical for mitochondrial respiration, and, ultimately, enhanced glucose-stimulated insulin secretion and β-cell function. These results suggest a mechanism by which monomeric cocoa catechins exert their effects as an effective complementary strategy to benefit T2D patients.

A phytochemical investigation on methanolic extract of roots of yellow rose led to the isolation of a catechin gallate. The crude extract first underwent fractionation using petroleum ether, chloroform, ethylacetate, butanol with water as solvents in their respective order. The emerging solvent fractions were subjected to further separation using lipophilic sephadex (LH-20), and silica gel column chromatography to isolate pure compounds. Analytical thin layer chromatography (TLC) was used to confirm the presence of a catechin in butanol fraction. Purified catechin compound was subjected to 2,2-diphenyl-1-picrylhydrazyl (DPPH) experiment to determine its Radical Scavenging Capacity, which was found quite promising. Chemical structure of purified compound was established by using Nuclear Magnetic Resonance (NMR) and Mass Spectrometry (MS) experiments.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Photoaged skin caused by chronic ultraviolet radiation (UVR) is characterised clinically with hyperpigmentation, coarse skin texture and deep wrinkles; the worst outcome is skin cancer. Histological investigation of the alteration within major extracellular matrices (ECM; elastic fibres, fibrillar collagens) is essential study to understand the cellular effect on skin structure from UVR. This thesis used an acute dose of radiation to examine in humans in vivo the effect of UVR on ECM components before assessing whether a dietary intervention could protect skin from UVR damage. Green tea catechins (GTCs) have anti-oxidant properties and may be an interesting option as a systemic photoprotection agent. Hence this thesis assesses: 1) the effect of acute irradiation of skin on dermal ECM damage to see whether it mimics the changes observed in photoageing and; 2) whether dietary supplementation with GTC will provide dermal ECM protection. UV-induced change in elastic fibre network. Initially, the effect of two different UV light sources on elastic fibre protein (elastic fibres, fibrillin-rich microfibrils and fibulin-2 and -5 microfibrils) remodelling was performed. The effect of ultraviolet B vs full-spectrum solar simulated radiation (SSR) were investigated in a small sample of healthy Caucasian volunteers (n = 6 per group). At 24 hour after 3× MED irradiation, Weigert's resorcin–fuchsin stained elastic fibres showed a significant reduction regardless of irradiation protocol (UVB, P<0.01; SSR P<0.05). Specific components were identified by immunohistochemistry; a significant reduction in fibrillin-rich microfibrils (FRM) was observed in UVB-irradiated skin (P<0.05), whilst fibulin-5-positive microfibrils were only affected by SSR (P<0.05). The data revealed, therefore, differential effects on UV wavelength on ECM remodelling. SSR, the more physiologically relevant light source was used in subsequent studies Supplement effect in SSR-induced damage in elastic fibre. Fifty healthy volunteers were recruited to this randomised control trial to investigate whether GTC can protect skin from photodamage. Volunteers were randomized to GTC (1080 mg plus 100 mg vitamin C; n=25) or placebo (maltodextrin; n = 25) daily for 12-weeks with compliance assessed biochemically in urine samples. Of the n = 50 recruited, 44 volunteers completed the study. In baseline, UVR challenge resulted in a significant remodeling of the cutaneous elastic fiber system (P<0.001), particularly fibulin-2 and fibulin-5-positive microfibrils at 24-hr after 3×MED irradiation. In post-supplementation, fibulin-5 positive microfibrils were protected from UVR remodeling (% staining, mean ± SE; no UV, 18.1±0.89; UVR, 17.1±0.61; P=0.30) whilst no protection was seen in the placebo group (no UVR, 19.41±0.79; UVR, 17.69±0.61; P<0.05). Supplement effect in SSR-induced damage in collagenous matrix. In the identical experiment, collagenous matrices including synthesis of procollagen I was also examined as fibrillar collagens are the major ECM components providing strength within dermis. The fibrillar collagen and newly synthesised procollagen I were stained by Picrosirius red and immunohistochemistry respectively. At baseline, acute irradiation significantly reduced papillary dermal fibrillar collagens (P<0.001) and induced deposition of newly synthesised pro-collagen I (P=0.02). In post-supplementation, GTC enhanced the deposition of thin collagen fibres in the dermis. Whilst placebo showed no effect on the altered organisation of fibrillar collagens or deposition of pro-collagen I following the irradiation challenge, GTC protected the organisation of fibrillar collagens in the papillary dermis (P=0.97).This novel in vivo human study may be used to recapitulate elastic fibre and collagen changes associated with photoageing and may be useful for dissecting out the mechanisms underlying extracellular matrix damage in response to chronic sunlight exposure. Furthermore, in a randomized control trial, dietary GTC protected fibulin-5 microfibrils and collagen fibres in the papillary dermis from UV-mediated degradation. The mechanism by which this protection occurs requires further study.

Submitted by Bruna de Oliveira null (bruna_dol@hotmail.com) on 2017-08-28T18:37:05Z No. of bitstreams: 1 Dissertação FINAL.pdf: 2838275 bytes, checksum: 88512cc2a992ddfbe636d76b31127af8 (MD5) / Approved for entry into archive by Luiz Galeffi (luizgaleffi@gmail.com) on 2017-08-29T18:02:32Z (GMT) No. of bitstreams: 1 reis_bo_me_araca.pdf: 2838275 bytes, checksum: 88512cc2a992ddfbe636d76b31127af8 (MD5) / Made available in DSpace on 2017-08-29T18:02:32Z (GMT). No. of bitstreams: 1 reis_bo_me_araca.pdf: 2838275 bytes, checksum: 88512cc2a992ddfbe636d76b31127af8 (MD5) Previous issue date: 2017-08-15 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Objetivo: Avaliar a influência de inibidores de proteases no potencial de degradação do colágeno das dentina sadia, esclerótica e afetada por cárie. Materiais e métodos: Trinta e nove molares humanos foram utilizados, treze para cada condição dentinária. Três fatias foram obtidas de cada dente, cada uma imersa em diferentes soluções: 1) saliva artificial; 2) clorexidina 2%; 3) EGCG 0,5%. Após incubação nas soluções por 1h, amostras foram sujeitas à degradação enzimática pela colagenase derivada da Clostridium histolyticum. Propriedades mecânicas de nanodureza (HIT) e módulo de elasticidade (Er) dos três diferentes tipos de dentina foram mensuradas antes e após a degradação, bem como a resistência à tração do colágeno. Resultados do teste de resistência à tração e nanoindentação foram submetidos à ANOVA dois e três fatores para medidas repetidas, e pós-teste de Tukey (α=0,05). Resultados: Maiores valores de resistência à tração foram encontrados para dentina sadia, nos grupos controle (40,30 ± 21,38 MPa) e EGCG 0,5% (30,05 ± 19,67 MPa). Antes da degradação, maiores valores de HIT (0,237 ± 0,062 GPa) e Er (5,58 ± 1,75 GPa) foram encontrados para o grupo EGCG 0,5%, na dentina afetada por cárie. Após a degradação, grupo clorexidina 2% apresentou maiores valores de HIT e Er para dentinas sadia (0,134 ± 0,020 GPa e 3,57 ± 0,40 GPa) e esclerótica (0,201 ± 0,048 GPa e 4,30 ± 0,56 GPa). Conclusões: O uso da clorexidina 2%, principalmente em dentina esclerótica, mostrou-se mais efetivo em promover aumento na resistência à tração e nas propriedades mecânicas, antes e após a degradação. A EGCG 0,5% apresentou melhor efeito sobre as propriedades mecânicas na dentina afetada por cárie, especialmente antes da degradação enzimática. Relevância Clínica: O efetivo conhecimento do mecanismo de ação de inibidores de proteases em diferentes tipos de dentina poderia contribuir para melhoria da resistência do substrato e para maior longevidade dos processos de união sobre este tecido. / Objetive: To evaluate the influence of proteases inhibitors on the collagen degradation from sound, sclerotic and caries-affected dentin. Materials and Methods: Thirty-nine human molars were used, thirteen for each dentin condition. Three slices were obtained from each tooth, each one immersed in different solutions: 1) artificial saliva; 2) 2% chlorhexidine; 3) 0.5% EGCG. After incubation in the solutions for 1h, samples were subjected to enzymatic degradation by collagenase derived from Clostridium histolyticum. Mechanical properties of nanohardness (HIT) and elastic modulus (Er) of the three types of dentin were measured before and after degradation, as well as the microtensile strength. Results of the microtensile strength and nanoindentation tests were submitted to ANOVA two and three factors for repeated measurements, and Tukey post-test (α = 0.05). Results: Higher values ​​of tensile strength were found for sound dentin in control (40.30 ± 21.38 MPa) and 0.5% EGCG (30.05 ± 19.67 MPa) groups. Before degradation, higher values ​​of HIT (0.237 ± 0.062 GPa) and Er (5.58 ± 1.75 GPa) were found for the 0.5% EGCG group in caries-affected dentin. After degradation, 2% chlorhexidine group had higher values ​​of HIT and Er for sound (0.134 ± 0.020 GPa and 3.57 ± 0.40 GPa) and sclerotic (0.201 ± 0.048 GPa and 4.30 ± 0.56 GPa) dentin. Conclusions: The use of 2% chlorhexidine, mainly in sclerotic dentin, was shown to be more effective in promoting increase in the microtensile strength and mechanical properties, before and after the degradation. The 0.5% EGCG showed a better effect on mechanical properties in caries-affected dentin, before the enzymatic degradation. Clinical Relevance: The effective knowledge of the mechanism of action of protease inhibitors in different types of dentin could contribute to the improvement of the resistance of the substrate and to the longer longevity of the bonding processes on this tissue. / FAPESP: 2015/10566-7

A doença periodontal é uma das doenças inflamatórias crônicas mais comuns que acometem a população. A grande destruição tecidual observada durante o seu desenvolvimento tem sido atribuída ao processo inflamatório exacerbado e ao desequilíbrio favorável à geração de espécies reativas de oxigênio em relação àcapacidade de defesa dos antioxidantes. A epigalocatequina-3-galato (EGCG) obtida da Camellia sinensis é uma substância que apresenta potencial antioxidante e antiinflamatório e, mais recentemente, testes in vitro têm mostrado que também possui atividade anti-osteoclastogênica, sendo apontada como uma possível droga para uso terapêutico nas patologias ósseas com excessiva atividade osteoclástica e destruição óssea. O objetivo do trabalho foi verificar morfométricamente em imagens obtidas pela tomografia microcomputadorizada (micro-CT) e cortes histológicos se a administração diária de EGCG reduz o processo inflamatório e a perda óssea alveolar na doença periodontal induzida por ligadura em ratos. O primeiro molar inferior direito de 60 ratos foi amarrado com fio de seda 3.0 e divididos em grupo sem tratamento (GST), grupo tratado com EGCG (GTEGCG) que recebeu diariamente por gavagem 100mg/Kg de EGCG e grupo Sham (GTsalina) que recebeu apenas solução salina. Nos períodos de 0, 7, 14 e 21 dias (n=5 animais/período/grupo) imagens digitais foram obtidas no microtomógrafo sendo submetidos à análise do nível ósseo periodontal (PBL) e da densidade óssea (BV/TV) inter-radicular. Nos cortes longitudinais do M1 corados pela HE foi avaliado o PBL e morfometricamente o percentual e volume de processo inflamatório e tecido ósseo, além do número osteoclastos/cm2. Os dados foram submetidos à ANOVA a dois critérios e ao teste de Tukey (p<0,05). O PBL determinado nas imagens microtomográficas e histológicas mostraram que a perda óssea aumenta em todos os grupos durante a fase aguda da doença (0 a 14 dias) e estabiliza na fase crônica (14 dias-21 dias). Em geral, o PBL foi menor no GTEGCG (média de 0,839 mm) comparado aos GST e GTsalina (média de 0,953 ). Quanto à densidade óssea o BV/TV foi maior no GTEGCG (68%) comparados aos GST (62,06%). O percentual do processo inflamatório e o número de osteoclastos foram menores no GTEGCG, com pico aos 14 dias (3,4% de processo inflamatório e 32 osteoclastos/cm2), comparados aos GST e GTsalina cujo o pico foi aos 7 dias (média de 8,6% de processo inflamatório e 68 osteoclastos/cm2). Concluímos que, no modelo de periodontite induzida por ligadura, o tratamento com EGCG diminui o processo inflamatório e a osteoclastogênese e consequentemente a perda óssea e a severidade da doença. / Periodontal disease is currently one of the most common chronic inflammatory diseases affecting the population. The large tissue destruction observed during its development, has been attributed to exacerbated inflammatory process and unbalance response between production of reactive oxygen species and antioxidant defense capacity. Recently, the substance epigallocatechin-3-gallate (EGCG) obtained from Camellia sinensis have been associated to antioxidant and antiinflammatory actions. In vitro studies have shown that EGCG has also antiosteoclastogenic activity suggesting to be a potencial drug for use in therapeutic treatment of bone diseases with excessive osteoclast formation and bone destruction. The aim of this study was to verify morphometrically in micro-ct and histological images whether daily administration of EGCG inhibits/decreases alveolar bone loss in periodontal disease induced in rats by ligature. The lower right first molar of 60 rats was tied with surgical suture thread 3.0. The animals were divided into untreated group (GST), EGCG treated group (GTEGCG) which received 100mg/kg of EGCG by gavage daily and Sham group (GT saline) which received saline solution only. In periods of 0, 7, 14 and 21 days (n=5 animals/period/group) digital images were obtained in microtomography (SkyScan1176) and subjected to analysis of PBL in the mesial, distal, buccal and lingual root and BV/TV bone volume percentage. In the sagittal slides PBL volumetric points and inflammatory process as well as the number of osteoclasts/cm2 was analyzed. Data were submitted to twoway ANOVA and Tukey test (p <0.05). PBL determined in microtomographic and histological images showed that bone loss increased and stabilized, respectively, in the all groups acute phase (days 0 to 14) and chronic phase (14 days, 21 days) of the disease. In general, the PBL was lower in GTEGCG (average 0,839 mm) compared to GST and GTsaline (average 0,953). Regarding bone density BV/TV in GTEGCG was higher (68%) compared to GST (62.06%). The percentage of inflammation and the number of osteoclasts was more mild in GTEGCG, reaching peak at 14 days (3.4% inflammatory process and 32 osteoclasts / cm2) compared to GST and GTsaline whose peak was at 7 days (average 8.6% inflammatory process and 68 osteoclasts / cm2). It was concluded that in the current model of periodontal disease induced by ligature, EGCG treatment decreases inflammatory process, osteoclastogenesis activity, bone loss, and consequently the severity of the disease.

No description available.

In both Type 1 diabetes (T1D) and Type 2 diabetes (T2D), inadequate beta-cell mass and beta-cell dysfunction lead to impaired insulin secretion, and ultimately worsen glycemic control. Green tea has drawn wide attention due to its possible health-promoting properties, including enhancement of beta-cell function. We assessed the acute and relative long-term effects of epigallocatechin gallate (EGCG) on insulin secretion and synthesis from clonal beta-cells (INS1E cells), rat islets, and human islets, using 0.1, 1, or 5 µM. We determined if EGCG decreased blood glucose in healthy rats acutely, using 50 or 150 mg/kg body weight (BW), and after 12 days of supplementation in drinking water, using 0.1% and 0.5%. In the in vitro studies, EGCG significantly potentiated glucose-stimulated insulin secretion (GSIS) in rat islets (at 0.1, 1, and 5 µM) and human islets (at 1 µM), and elevated insulin content within INS1E cells (at 0.1, 1, and 5 µm) and human islets (at 1 µM), (P<0.05). Nutritional supplementation of EGCG (0.5% in drinking water) for 12 days in healthy rats significantly increased insulin synthesis, compared to that of controls, from 0.2 ± 0.02 to 1.4 ± 0.2 ng/mg protein, without alteration of insulin secretion in isolated islets (P<0.05). These findings demonstrate that EGCG may play a role in the regulation of pancreatic beta-cell function, thereby contributing to an anti-diabetic effect of this agent. / Master of Science