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1	Análise da atividade antimicrobiana de extratos e frações purificadas da planta arrabidaea chica verl Mota, Milena Rodrigues Soares 25 March 2011 (has links) Submitted by Alisson Mota (alisson.davidbeckam@gmail.com) on 2015-07-13T19:08:16Z No. of bitstreams: 1 Tese - Milena Rodrigues Soares Mota.pdf: 10830184 bytes, checksum: b6f2977c2b749cd675523693c33de0ff (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2015-07-15T17:58:31Z (GMT) No. of bitstreams: 1 Tese - Milena Rodrigues Soares Mota.pdf: 10830184 bytes, checksum: b6f2977c2b749cd675523693c33de0ff (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2015-07-15T18:07:20Z (GMT) No. of bitstreams: 1 Tese - Milena Rodrigues Soares Mota.pdf: 10830184 bytes, checksum: b6f2977c2b749cd675523693c33de0ff (MD5) / Made available in DSpace on 2015-07-15T18:07:20Z (GMT). No. of bitstreams: 1 Tese - Milena Rodrigues Soares Mota.pdf: 10830184 bytes, checksum: b6f2977c2b749cd675523693c33de0ff (MD5) Previous issue date: 2011-03-25 / Não Informada / This study describes the therapeutic potential of extracts and standardized fractions of Arrabidaea chica leaves. A. chica is a Bignoniaceae popularly known as “crajiru”. The genus Arrabidaea occurs in Tropical America, from the Southern Mexico to the Southern Brazil. The red color of its dried leaves is attributed to two flavonoidal pigments: carajurina (main pigment) and carajurona. Chemical studies described the isolation of saponins and flavonoids from the plant leaves; purified 3- desoxyanthocyanidins were reported as anti-inflammatory. The infusion or decoction of the plant leaves is used in the folk medicine to treat anemia, inflammation and in skin wound-healing. In this work, the antimicrobial activity of the standardized extracts and fractions of A. chica cultivated at Embrapa Amazônia Ocidental, Manaus, were evaluated against fungal and bacterial microorganisms grown either from local domestic dogs and cats, or from human samples supplied by the Microorganism Collection of FIOCRUZ, in Manaus, Brazil. The plant dried leaves were extracted with increasing polarity solvents and progressively purified in preparative thin layer chromatography (TLC) and silica-gel column; the semi-purified extracts were standardized in TLC. The agar diffusion method; bioautography; minimum inhibitory concentrations tested against Staphylococcus epidermidis (CBAM 293), Staphylococcus aureus (CBAM 324), Pseudomonas aeruginosa (CBAM 232), Escherichia coli (CBAM 002), Trichophyton mentagrophytes (CFAM 1288), Microsporum canis (CFAM 1289), Malassezia pachydermatis (CFAM 1290) e Candida albicans (CFAM 1285). The standardized fractions were effective against all these microorganisms, but more intensively against Microsporum canis and Staphylococcus epidermidis. The results might favour the use of the standardized sub-fractions of A. chica as topic phytotherapic agent to treat canine external otitis. So far oleanolic and ursolic acids were identified as the main compounds in the active semi-purified fraction but other compounds of the leaves extract were not discarded. Later studies will consider the veterinarian use of the standardized extract, of the active pure entities and the convenience of the natural active antibiotic mix. / Este estudo analisou o potencial terapêutico de extratos e frações purificadas da planta amazônica Arrabidaea chica visando seu uso tópico como medicamento e eficácia comprovada em doenças cutâneas. A. chica Verl., é uma Bignoniaceae conhecida popularmente como crajiru. O gênero Arrabidaea ocorre na América tropical, do sul do México ao sul do Brasil. A cor avermelhada da folha seca e sua propriedade tintorial são devidas a dois pigmentos flavonoídicos: a carajurina, que é o pigmento principal e a carajurona. Dela foram isolados saponinas e flavonóides; As 3-desoxiantocianidinas, descritas na planta parecem possuir atividade antiinflamatória. A medicina popular utiliza o decocto ou a infusão das folhas para tratar anemia, inflamações e na cicatrização da pele. Neste trabalho, a atividade antimicrobiana de extratos e frações padronizadas da A. chica cultivada na Embrapa Amazônia Ocidental, em Manaus/AM, foi avaliada contra fungos e bactérias de amostras clínicas coletadas de animais domésticos e contra amostras humanas depositadas na coleção de Microrganimos da FIOCRUZ, Manaus/AM. Para isso, as folhas secas da planta foram extraídas com solventes de polaridade crescente, as frações foram progressivamente purificadas em cromatografia de placa ou coluna de sílica-gel, os extratos semi-purificados foram padronizados em cromatografia líquida de alta eficiência acoplada à espectrometria de massas. Os testes de difusão em ágar, bioautografia e concentração inibitória mínima foram usados para avaliar a atividade antimicrobiana das subfrações padronizadas frente aos microrganismos Staphylococcus epidermidis (CBAM 293), Staphylococcus aureus (CBAM 324), Pseudomonas aeruginosa (CBAM 232), Escherichia coli (CBAM 002), Trichophyton mentagrophytes (CFAM 1288), Microsporum canis (CFAM 1289), Malassezia pachydermatis (CFAM 1290) e Candida albicans (CFAM 1285). As frações padronizadas foram ativas contra todos esses microrganismos, com melhores resultados contra M. pachydermatis e S. epidermidis. Os resultados foram favoráveis à utilização das subfrações padronizadas na formulação de um produto fitoterápico para uso tópico em otite canina. Nas frações ativas foram identificados os ácidos oleanólico e ursólico. Estudos posteriores deverão avaliar a possibilidade de uso humano das frações purificadas ou dos compostos identificados. Crajiru Difusão em ágar Bioautografia Concentração inibitória mínima Malassezia pachydermatis Staphylococcus epidermidis Agar diffusion method Bioautography Minimum inhibitory concentrations Malassezia pachydermatis Staphylococcus epidermidis CIÊNCIAS BIOLÓGICAS
2	Development of Sensitive In Vitro Assays to Assess the Ocular Toxicity Potential of Chemicals and Ophthalmic Products McCanna, David January 2009 (has links) The utilization of in vitro tests with a tiered testing strategy for detection of mild ocular irritants can reduce the use of animals for testing, provide mechanistic data on toxic effects, and reduce the uncertainty associated with dose selection for clinical trials. The first section of this thesis describes how in vitro methods can be used to improve the prediction of the toxicity of chemicals and ophthalmic products. The proper utilization of in vitro methods can accurately predict toxic threshold levels and reduce animal use in product development. Sections two, three and four describe the development of new sensitive in vitro methods for predicting ocular toxicity. Maintaining the barrier function of the cornea is critical for the prevention of the penetration of infections microorganisms and irritating chemicals into the eye. Chapter 2 describes the development of a method for assessing the effects of chemicals on tight junctions using a human corneal epithelial and canine kidney epithelial cell line. In Chapter 3 a method that uses a primary organ culture for assessing single instillation and multiple instillation toxic effects is described. The ScanTox system was shown to be an ideal system to monitor the toxic effects over time as multiple readings can be taken of treated bovine lenses using the nondestructive method of assessing for the lens optical quality. Confirmations of toxic effects were made with the utilization of the viability dye alamarBlue. Chapter 4 describes the development of sensitive in vitro assays for detecting ocular toxicity by measuring the effects of chemicals on the mitochondrial integrity of bovine cornea, bovine lens epithelium and corneal epithelial cells, using fluorescent dyes. The goal of this research was to develop an in vitro test battery that can be used to accurately predict the ocular toxicity of new chemicals and ophthalmic formulations. By comparing the toxicity seen in vivo animals and humans with the toxicity response in these new in vitro methods, it was demonstrated that these in vitro methods can be utilized in a tiered testing strategy in the development of new chemicals and ophthalmic formulations. in vitro Alternative Methods alamarBlue rhodamine tight junctions bovine lens viability toxicity mitochondria mitochondria integrity benzalkonium chloride sodium dodecyl sulphate sodium lauryl sulfate scanning electron microscopy Draize Draize maximal average scores zonula occludens cosmetic directive PETA humane society animal league ICCVAM ECVAM JaCVAM BCOP bovine cornea ocular irritants ocular irritation ocular toxicity human corneal epithelial cells vitro confocal confocal microscopy metabolic activity optical quality reactive oxygen species contact lens contact lens care solutions barrier function microbial keratitis mulitipurpose solutions tight junction cell damage claudin ZO-1 occludin MDCK Madin Madin-Darby canine kidney cells cornea human cornea human corneal epithelium epithelial cell line human corneal epithelial cell line sodium fluorescein sodium fluorescein permeability fluorescein permeability ocular surface cell monolayer Araki-Sasaki cell physiology risk assessment safety assessment ScanTox scanning laser lens epithelium corneal cells in vitro model ophthalmic formulations multiple instillation back vertex animal testing rabbit testing alternatives to animal testing cultured bovine lens toxicity of chemicals irritation irritants laser scanner organ culture delayed toxicity toxins hydrogen peroxide cornea toxicity cornea toxicity models prediction of human toxicity no observable adverse effect level lowest observable adverse effect level NOAEL LOAEL toxicity thresholds safety factors cornea uncertainty factors preservatives disinfectants ophthalmic products preclinical preclinical testing epithelial barrier drug penetration clinical confocal microscopy animal rights rabbit cornea human cornea human clinical effects toxic animal rights activist sensitive measures toxic effect toxicity threshold agar overlay agar diffusion agar overlay method agar diffusion method cytochrome cytochrome c apoptosis necrotic apoptotic necrosis caspase rhodamine 123 resazuran resorufin cell death cell viability metabolic dye microsomal microsomal enzymes cytotoxicity cytotoxic cytotoxic effect MTT XTT WST-1 plasma membrane mitochondrial mitochondrial morphology ocular toxicity potential ocular toxicity human corneal epithelial cell line confocal analysis corneal epithelium cell fluorescence alamarBlue assay rhodamine dye animal welfare toxic injury degraded mitochondria epithelial monolayer disinfectants membrane integrity eye toxicity eye viability dye toxicity in humans human toxicity effects on the mitochondria mitochondrial toxicity in vitro battery in vitro test battery ophthalmic eye drop direct contact product safety cytotoxicity potential molecular molecular biology refine reduce replace sensitivity and relevance sensitivity relevance rabbit ocular irritation test product development cytotoxicity models cytotoxicity alternative methods replacements for animal testing three r's beagle tiered testing tiered testing strategy replacements animal testing mechanistic toxicity cornea mitochondria dose response threshold Vision Science and Biology
3	Development of Sensitive In Vitro Assays to Assess the Ocular Toxicity Potential of Chemicals and Ophthalmic Products McCanna, David January 2009 (has links) The utilization of in vitro tests with a tiered testing strategy for detection of mild ocular irritants can reduce the use of animals for testing, provide mechanistic data on toxic effects, and reduce the uncertainty associated with dose selection for clinical trials. The first section of this thesis describes how in vitro methods can be used to improve the prediction of the toxicity of chemicals and ophthalmic products. The proper utilization of in vitro methods can accurately predict toxic threshold levels and reduce animal use in product development. Sections two, three and four describe the development of new sensitive in vitro methods for predicting ocular toxicity. Maintaining the barrier function of the cornea is critical for the prevention of the penetration of infections microorganisms and irritating chemicals into the eye. Chapter 2 describes the development of a method for assessing the effects of chemicals on tight junctions using a human corneal epithelial and canine kidney epithelial cell line. In Chapter 3 a method that uses a primary organ culture for assessing single instillation and multiple instillation toxic effects is described. The ScanTox system was shown to be an ideal system to monitor the toxic effects over time as multiple readings can be taken of treated bovine lenses using the nondestructive method of assessing for the lens optical quality. Confirmations of toxic effects were made with the utilization of the viability dye alamarBlue. Chapter 4 describes the development of sensitive in vitro assays for detecting ocular toxicity by measuring the effects of chemicals on the mitochondrial integrity of bovine cornea, bovine lens epithelium and corneal epithelial cells, using fluorescent dyes. The goal of this research was to develop an in vitro test battery that can be used to accurately predict the ocular toxicity of new chemicals and ophthalmic formulations. By comparing the toxicity seen in vivo animals and humans with the toxicity response in these new in vitro methods, it was demonstrated that these in vitro methods can be utilized in a tiered testing strategy in the development of new chemicals and ophthalmic formulations. in vitro Alternative Methods alamarBlue rhodamine tight junctions bovine lens viability toxicity mitochondria mitochondria integrity benzalkonium chloride sodium dodecyl sulphate sodium lauryl sulfate scanning electron microscopy Draize Draize maximal average scores zonula occludens cosmetic directive PETA humane society animal league ICCVAM ECVAM JaCVAM BCOP bovine cornea ocular irritants ocular irritation ocular toxicity human corneal epithelial cells vitro confocal confocal microscopy metabolic activity optical quality reactive oxygen species contact lens contact lens care solutions barrier function microbial keratitis mulitipurpose solutions tight junction cell damage claudin ZO-1 occludin MDCK Madin Madin-Darby canine kidney cells cornea human cornea human corneal epithelium epithelial cell line human corneal epithelial cell line sodium fluorescein sodium fluorescein permeability fluorescein permeability ocular surface cell monolayer Araki-Sasaki cell physiology risk assessment safety assessment ScanTox scanning laser lens epithelium corneal cells in vitro model ophthalmic formulations multiple instillation back vertex animal testing rabbit testing alternatives to animal testing cultured bovine lens toxicity of chemicals irritation irritants laser scanner organ culture delayed toxicity toxins hydrogen peroxide cornea toxicity cornea toxicity models prediction of human toxicity no observable adverse effect level lowest observable adverse effect level NOAEL LOAEL toxicity thresholds safety factors cornea uncertainty factors preservatives disinfectants ophthalmic products preclinical preclinical testing epithelial barrier drug penetration clinical confocal microscopy animal rights rabbit cornea human cornea human clinical effects toxic animal rights activist sensitive measures toxic effect toxicity threshold agar overlay agar diffusion agar overlay method agar diffusion method cytochrome cytochrome c apoptosis necrotic apoptotic necrosis caspase rhodamine 123 resazuran resorufin cell death cell viability metabolic dye microsomal microsomal enzymes cytotoxicity cytotoxic cytotoxic effect MTT XTT WST-1 plasma membrane mitochondrial mitochondrial morphology ocular toxicity potential ocular toxicity human corneal epithelial cell line confocal analysis corneal epithelium cell fluorescence alamarBlue assay rhodamine dye animal welfare toxic injury degraded mitochondria epithelial monolayer disinfectants membrane integrity eye toxicity eye viability dye toxicity in humans human toxicity effects on the mitochondria mitochondrial toxicity in vitro battery in vitro test battery ophthalmic eye drop direct contact product safety cytotoxicity potential molecular molecular biology refine reduce replace sensitivity and relevance sensitivity relevance rabbit ocular irritation test product development cytotoxicity models cytotoxicity alternative methods replacements for animal testing three r's beagle tiered testing tiered testing strategy replacements animal testing mechanistic toxicity cornea mitochondria dose response threshold Vision Science and Biology

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