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Highly infectious pathogenic strains of bacteria are becoming increasingly resistant to the current clinical antibiotics which have created a dire need for the development of novel antibiotics. O-Acetylpeptidoglycan esterase 1 (Ape1) is a periplasmic esterase present in several peptidoglycan (PG) O-acetylating pathogenic species of Gram-positive and all Gram-negative bacteria that perform this modification to this essential cell wall polymer. Inhibition of this growth-limiting enzyme may prove the principle that Ape1 has the potential to be the target for the development of a novel class of antibiotics. Ape1 plays a crucial role in bacterial growth by regulating PG turnover through catalytic removal of the C-6 acetyl group from O-acetylPG. This activity is required for the continued metabolism of PG because the major autolytic enzymes involved, the lytic transglycosylases, require a free C-6 hydroxyl group to produce their reaction product, 1,6-anhydromuramic acid. Several of the compounds that have been identified to effectively inhibit Ape1, were re-evaluated by determining their kinetic parameters. Work presented in this thesis explored the inhibitory potential of these compounds, belonging to the anthraquinone (alizarin, quinizarin, quinalizarin, emodin, sennoside A) or tannin (ellagic acid) families of compounds, both in vitro and in vivo, among species of bacteria that are known to O-acetylate their PG. Of the inhibitory compounds tested, ellagic acid was found to be most effective in vitro, with an IC50 value of 0.91 µM ± 0.06, Ki 1.18 ± 0.04 and in vivo it was shown to reduce bacterial growth.

Breast cancer is the most frequently diagnosed cancer among women around the world. The hormone 17-estradiol (E2) is strongly implicated as a causative agent in this cancer. Since estrogen acts as a complete carcinogen, agents that interfere with the carcinogenic actions of E2 are required. Most agents effective against experimental mammary carcinogenesis have been employed as pure compounds disregarding the synergy that exists between several phytonutrients in a whole food. In these studies we have taken a unified approach, by employing a pure phytonutrient ellagic acid and whole foods that contain the phytonutrient at various levels berries, in the prevention of E2- induced mammary cancer in ACI rats. We have also used a tiered approach by screening several phytochemicals in vitro and implementing these results in both short- and long-term studies. Initially, several phytochemicals were tested as pure compounds against oxidative DNA damage induced by 4-hydroxy estradiol and CuCl2. Ellagic acid, was the most effective agent (andgt;98% reduction). In a short-term in vivo study, both dietary blueberry and strawberry (5% w/w), were ineffective in reducing the baseline oxidative DNA damage in the livers of CD-1 mice. However, red raspberry (5% w/w) was highly effective (50% reduction) and ellagic acid (400 ppm) was moderately effective (25% reduction). Further both diets up-regulated hepatic DNA repair genes in a similar fashion. In a long-term estradiol-induced mammary carcinogenicity study in ACI rats, dietary berries (2.5% w/w) and ellagic acid (400 ppm) reduced both tumor volume and tumors per animal to different extents (50-75%). One mechanism by which these dietary interventions inhibit mammary tumorigenesis may be via modulation of E2 metabolism, especially at the early stages of carcinogenesis. At 6 weeks after E2 treatment both berries and ellagic acid or berries alone significantly offset E2- induced changes in CYP1B1 and CYP1A1 expressions respectively. In addition, no toxicity or adverse effects are observed when rodents were fed either berries (1 - 5%) or ellagic acid (400 ppm). These data taken collectively support the possibility of using natural foods such as berries as an adjuvant to current pharmacological therapies in the prevention and treatment of breast cancer.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第22494号 / 農博第2398号 / 新制||農||1076(附属図書館) / 学位論文||R2||N5274(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 小川 順, 教授 栗原 達夫, 教授 加納 健司 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Eleven subjects completed a clinical trial to determine the safety/tolerability of freeze-dried black raspberries (BRB) and to measure, in plasma and urine, specific anthocyanins-cyanidin-3-glucoside, cyanidin-3-sambubioside, cyanidin-3-rutinoside, and cyanidin-3- xylosylrutinoside, as well as ellagic acid. Subjects were fed 45 g of freeze-dried BRB daily for 7 days. Blood samples were collected predose on days 1 and 7 and at 10 time points postdose. Urine was collected for 12 hours predose on days 1 and 7 and at three 4-hour intervals postdose. Maximum concentrations of anthocyanins and ellagic acid in plasma occurred at 1 to 2 hours, and maximum quantities in urine appeared from 0 to 4 hours. Overall, less than 1% of these compounds were absorbed and excreted in urine. None of the pharmacokinetic parameters changed significantly between days 1 and 7. In conclusion, 45 g of freeze-dried BRB daily are well tolerated and result in quantifiable anthocyanins and ellagic acid in plasma and urine.

Submitted by Marlene Santos (marlene.bc.ufg@gmail.com) on 2016-06-01T20:07:51Z No. of bitstreams: 2 Dissertação - Liliane de Sousa Silva - 2015.pdf: 4894695 bytes, checksum: ef4469d862b59150ebb61ed28e1293ff (MD5) license_rdf: 19874 bytes, checksum: 38cb62ef53e6f513db2fb7e337df6485 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-06-02T15:32:33Z (GMT) No. of bitstreams: 2 Dissertação - Liliane de Sousa Silva - 2015.pdf: 4894695 bytes, checksum: ef4469d862b59150ebb61ed28e1293ff (MD5) license_rdf: 19874 bytes, checksum: 38cb62ef53e6f513db2fb7e337df6485 (MD5) / Made available in DSpace on 2016-06-02T15:32:33Z (GMT). No. of bitstreams: 2 Dissertação - Liliane de Sousa Silva - 2015.pdf: 4894695 bytes, checksum: ef4469d862b59150ebb61ed28e1293ff (MD5) license_rdf: 19874 bytes, checksum: 38cb62ef53e6f513db2fb7e337df6485 (MD5) Previous issue date: 2015-09-30 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / The gallic acid (GA) and its derived ellagic acid (EA) are widely distributed in nature tannins, present in tea, red wine, fruit, drinks and various medicinal plants. AG and AE are known to have anti inflammatory properties, antimutagenic, anticancerígenica and antioxidant activity. To produce functionalized derivatives of such compounds by biotransformation with filamentous fungi is an efficient and sustainable alternative, due to their ability to catalyze further reactions regio and stereo-selective. The aim of this study was to apply strains of Cerrado in the biotransformation of AG and AE. For this Thin Layer Chromatography (TLC) and High Performance Liquid Chromatography (HPLC) to monitor the production of derivatives developed analytical methodologies. Five strains of the Brazilian Cerrado were selected, these are Beauveria sp. IP06, IP08, IP94, IP98 and IP153. Incubation was performed in two different culture media: Glucose and "corn steep" for 96 hours at 27 C and 200 rpm. At the end of incubation the extraction and purification of the derivatives was performed. All strains were effective for biotranformação compounds. The culture medium influenced quali-quantitatively the production of derivatives, "corn steep" provided greater variety of substrates for both derivatives. The obtained derivatives were characterized by MS, ¹H NMR, HSQC, HMBC, UV and CCD. Derivatives of fatty acids are: benzoic acid, 3-O-methyl-β-glucopyranosyl galatoester (Labiocon 465); benzoic acid, 4 - [[6-O- (3,4-dihydroxy-5-methoxybenzoic acid)-α-D-glucopyranoside]oxy]-3-hydroxy-5metoxi (Labiocon 484); gallic acid 3-O-β-glucopiranoside (510 Labiocon) and 3,4-dihydroxy-5-methoxy-benzoic acid 2-hydroxy-5-methoxycarbonyl-phenyl ester (Labiocon 530). The AE is derived from 3,8-Dimethoxy-dibenzo [b, d] pyran-6-one ellagic acid (511 Labiocon) and 4-O, 3'-O-methyl -β-D-glucose (Labiocon 534) . / O ácido gálico (AG) e o seu derivado o ácido elágico (AE), são taninos amplamente distribuído na natureza, presente no chá, vinho tinto, frutas, bebidas e várias plantas medicinais. AG e AE são conhecidos por apresentarem propriedades antiinflamatórias, antimutagênica, anticancerígenica e atividade antioxidante. Produzir derivados funcionalizados desses compostos por meio da biotransformação com fungos filamentosos é uma alternativa eficiente e sustentável, devido à sua capacidade para catalisar novas reações regio e estereo-seletivas. O objetivo deste trabalho foi aplicar cepas do Cerrado na biotransformação do AG e AE. Para isso foram desenvolvidas metodologias analíticas por Cromatografia em Camada Delgada (CCD) e Cromatografia Líquida de Alta Eficiência (CLAE), a fim de monitorar a produção dos derivados. Foram selecionadas cinco cepas isoladas do Cerrado brasileiro, sendo estas Beauveria sp. IP06, IP08, IP94, IP98 e IP153. A incubação foi realizada em dois meios de cultura diferentes: glicose e “Corn Steep” durante 96 horas a 27º C e 200 rpm. Ao término da incubação foi realizada a extração e purificação dos derivados. Todas as cepas mostraram-se eficazes para a biotranformação dos compostos. O meio de cultura influenciou quali-quantitativamente na produção dos derivados, o “Corn Steep” proporcionou maior variedade de derivados para ambos substratos. Os derivados obtidos foram caracterizados por EM, RMN ¹H, HSQC, HMBC, UV e CCD. Os derivados do AG são: ácido benzoico, 3-O-metil β-glucopiranosil-galatoester (Labiocon 465); ácido benzóico,4-[[6-O-(3,4-diidroxi-5-metoxibenzoico)-α-D-glucopiranosideo]oxi]-3-hidroxi-5metoxi (Labiocon 484); ácido gálico 3-O-β-glucopiranoside (Labiocon 510) e ácido 3,4-Diidroxi-5-metoxi-benzoico 2-hidroxi-5-metoxicarbonil-fenil ester (Labiocon 530). O derivado do AE é 3,8-Dimetoxi-dibenzo[b,d]piran-6-one (Labiocon 511) e o ácido elágico 4-O,3’-O-metil -β-D-glicose (Labiocon 534).

A diet rich in polyphenols has been proposed to have beneficial health effects and to reduce risk of disease. Ellagic acid, a polyphenol common in red berries and pomegranates, has potential anti-tumorigenic effects that make it interesting to further study in different cancer cell systems. Neuroblastoma is a childhood cancer that arises during development of the peripheral nervous system. Neuroblastoma, being an embryonal tumor, show loss of function of genes controlling differentiation and apoptosis. Neuroblastoma is a heterogenic tumor disease, and highly malignant neuroblastomas are difficult to treat despite different treatment modalities, identifying a need for new and combinatory treatments. A common model for human neuroblastoma is the SH-SY5Y cell line resembling immature neuroblasts that can be differentiated in vitro with several agents including the phorbol ester 12-O-tetradecanoylphorbol-13-acetate and the vitamin A-derivative all-trans retinoic acid. Here, the effect of ellagic acid on proliferation, cell detachment and apoptosis in non-differentiated and in vitro-differentiated SH-SY5Y cells were studied with the aim of identifying cellular target mechanisms and a possible therapeutic potential for ellagic acid. In non-differentiated cells, ellagic acid reduced cell number, inhibited cell cycle activity, and induced cell detachment and apoptosis. Apoptosis was partly mediated by the intrinsic pathway. 12-O-tetradecanoylphorbol-13-acetate and all-trans retinoic acid both induced morphological differentiation, while only the latter induced G0/G1-arrest. Single-cell analysis revealed that 12-O-tetradecanoylphorbol-13-acetate-treated cells continued cycling during neuritogenesis while these two read-outs were mutually exclusive in all-trans retinoic acid-treated cells. 12-O-tetradecanoylphorbol-13-acetate- and especially all-trans retinoic acid-differentiated cells showed lower sensitivity to ellagic acid-dependent cell detachment and apoptosis. / <p>Artikel 4 ("Altered sensitivity...") ingick som manuskript i avhandlingen, nu publicerad.</p>

Os benefícios à saúde oferecidos pelos leites fermentados probióticos podem ser ampliados pela adição de polpa de frutas vermelhas uma vez que estas apresentam reconhecidas propriedades funcionais. Ácido elágico e elagitaninos presentes nas frutas apresentam função fisiológica e metabólica na alimentação. Porém, a estabilidade destes compostos bioativos em produtos probióticos durante armazenamento refrigerado ainda não foi estudada. Sendo assim, este projeto tem por finalidade investigar o comportamento do ácido elágico em bebidas lácteas simbióticas suplementadas com L-triptofano e polpa de frutas vermelhas durante armazenamento refrigerado. O estudo foi realizado através de um delineamento experimental do tipo simplex centróide para modelagem de misturas. Foram preparadas 16 bebidas lácteas divididas em duas diferentes matrizes de ensaios segundo: (i) enriquecimento ou não da bebida láctea com triptofano e (ii) composição em frutas vermelhas do preparado de frutas. As amostras de bebida láctea foram avaliadas quanto as suas características físico-químicas (pH, reologia e atributos de cor), microbiológicas, microestrutura e quanto ao teor de ácido elágico. As bebidas lácteas apresentaram reduzidas pós-acidificação sem variações significativas para as bebidas contendo triptofano. As mesmas bebidas apresentaram melhora significativa na viabilidade da bactéria probiótica Lactobacillus acidophilus ao longo do período estudado. Os parâmetros de cor se mostraram estáveis nas bebidas lácteas durante o armazenamento refrigerado. O comportamento reológico das bebidas lácteas foi semelhante e adição de polpa de fruta proporcionou um efeito benéfico na consistência e viscosidade dos produtos. Foi possível estimar o conteúdo ótimo de ácido elágico nas bebidas lácteas simbióticas pela metodologia de superfície de resposta. As bebidas lácteas adicionadas de polpa de morango apresentaram maiores teores de ácido elágico, principalmente nas bebidas contendo triptofano. As fotos micrografias obtidas através da microscopia eletrônica de varredura permitiram avaliar a interação dos componentes da matriz das bebidas lácteas simbióticas de forma satisfatória. / The health benefits provided by probiotic fermented milk can be enhanced by the addition of native fruit pulp as they have recognized functional properties. Ellagic acid and ellagitannins present in fruis show physiological and metabolic function. Identification of these bioactive compounds in the formulation of symbiotic fermented beverages supplemented with L-tryptophan and red fruit pulp is innovative. Therefore, this project aimed to investigate the behaviour of ellagic acid in symbiotic lactic beverages supplemented with L-tryptophan and red pulp fruits during cold storage. Through an experimental simplex centroid design for mixtures modelling 16 beverages were prepared divided into four different matrices according to : (i) enrichment or not with L-tryptophan and (ii) composition of the prepared fruit pulp. The responses to be evaluated will be defined between the physical-chemical (pH, rheology and colour attributes), microbiological, microstructure and the amounts of ellagic acid. Lactic beverages have shown low post-acidification without significant variations for beverages containing tryptophan. The improvement in the viability of the probiotic bacteria Lactobacillus acidophilus throughout the study period was achieved. The colour parameters were stable during cold storage. The rheological behaviour was similar and the addition of red fruit pulps provided a beneficial effect on the consistency and viscosity of the products. It was possible to estimate the optimum amount of ellagic acid throughout response surface methodology. Lactic beverages added with strawberry pulp have had higher amounts of ellagic acid, particularly when L-tryptophan was supplemented. The photomicrographs obtained by scanning electron microscopy allowed adequate assessing the interaction of the matrix components of symbiotic lactic beverages.

Os benefícios à saúde oferecidos pelos leites fermentados probióticos podem ser ampliados pela adição de polpa de frutas vermelhas uma vez que estas apresentam reconhecidas propriedades funcionais. Ácido elágico e elagitaninos presentes nas frutas apresentam função fisiológica e metabólica na alimentação. Porém, a estabilidade destes compostos bioativos em produtos probióticos durante armazenamento refrigerado ainda não foi estudada. Sendo assim, este projeto tem por finalidade investigar o comportamento do ácido elágico em bebidas lácteas simbióticas suplementadas com L-triptofano e polpa de frutas vermelhas durante armazenamento refrigerado. O estudo foi realizado através de um delineamento experimental do tipo simplex centróide para modelagem de misturas. Foram preparadas 16 bebidas lácteas divididas em duas diferentes matrizes de ensaios segundo: (i) enriquecimento ou não da bebida láctea com triptofano e (ii) composição em frutas vermelhas do preparado de frutas. As amostras de bebida láctea foram avaliadas quanto as suas características físico-químicas (pH, reologia e atributos de cor), microbiológicas, microestrutura e quanto ao teor de ácido elágico. As bebidas lácteas apresentaram reduzidas pós-acidificação sem variações significativas para as bebidas contendo triptofano. As mesmas bebidas apresentaram melhora significativa na viabilidade da bactéria probiótica Lactobacillus acidophilus ao longo do período estudado. Os parâmetros de cor se mostraram estáveis nas bebidas lácteas durante o armazenamento refrigerado. O comportamento reológico das bebidas lácteas foi semelhante e adição de polpa de fruta proporcionou um efeito benéfico na consistência e viscosidade dos produtos. Foi possível estimar o conteúdo ótimo de ácido elágico nas bebidas lácteas simbióticas pela metodologia de superfície de resposta. As bebidas lácteas adicionadas de polpa de morango apresentaram maiores teores de ácido elágico, principalmente nas bebidas contendo triptofano. As fotos micrografias obtidas através da microscopia eletrônica de varredura permitiram avaliar a interação dos componentes da matriz das bebidas lácteas simbióticas de forma satisfatória. / The health benefits provided by probiotic fermented milk can be enhanced by the addition of native fruit pulp as they have recognized functional properties. Ellagic acid and ellagitannins present in fruis show physiological and metabolic function. Identification of these bioactive compounds in the formulation of symbiotic fermented beverages supplemented with L-tryptophan and red fruit pulp is innovative. Therefore, this project aimed to investigate the behaviour of ellagic acid in symbiotic lactic beverages supplemented with L-tryptophan and red pulp fruits during cold storage. Through an experimental simplex centroid design for mixtures modelling 16 beverages were prepared divided into four different matrices according to : (i) enrichment or not with L-tryptophan and (ii) composition of the prepared fruit pulp. The responses to be evaluated will be defined between the physical-chemical (pH, rheology and colour attributes), microbiological, microstructure and the amounts of ellagic acid. Lactic beverages have shown low post-acidification without significant variations for beverages containing tryptophan. The improvement in the viability of the probiotic bacteria Lactobacillus acidophilus throughout the study period was achieved. The colour parameters were stable during cold storage. The rheological behaviour was similar and the addition of red fruit pulps provided a beneficial effect on the consistency and viscosity of the products. It was possible to estimate the optimum amount of ellagic acid throughout response surface methodology. Lactic beverages added with strawberry pulp have had higher amounts of ellagic acid, particularly when L-tryptophan was supplemented. The photomicrographs obtained by scanning electron microscopy allowed adequate assessing the interaction of the matrix components of symbiotic lactic beverages.

No description available.