Ces travaux de thèse décrivent l’hémisynthèse de dérivés de l’acide grandiflorénique (AG). Ce produit naturel a dans un premier temps été converti en ent-kaur-9(11),16-dien-19-ol, composé qui a ensuite été estérifié dans les conditions de Steglich par une série d’acides benzoïques diversement substitués. Aux douze nouveaux composés ainsi obtenus a été ajoutée la synthèse du 9(11),16-dien-19-(2- méthyl-5-nitro-1H-imidazol-1-yl)éthyl-ent-kaurène. Ces dérivés de l’AG ont été soigneusement purifiés, puis complètement caractérisés (IR, SM, RMN 1D et 2D) en vue de leur évaluation biologique. L’activité antibactérienne a été testée sur différentes souches bactériennes comme Staphylococcus aureus, Enterococcus faecalis, Klebsiella pneumoniae, Pseudomonas aeruginosa et Escherichia coli. Les propriétés fongicides ont été évaluées sur Candida albicans et C. krusei. L’activité anti-inflammatoire in vivo a été étudiée sur des souris BIO:NMRI en utilisant le test de l’oedème de l’oreille induit par le xylol. Les dérivés d’acide grandiflorénique n’ont malheureusement montré aucune activité antibactérienne, ni antifongique, mais leur activité anti-inflammatoire s’est montrée prometteuse dans le cas de l’inflammation aiguë, le 9(11),16-dien-18-[2-(4-isobutylphényl)propyl]-ent-kaurène se révélant être le meilleur composé. / This thesis work describes the hemisynthesis of grandiflorenic acid (GA) derivatives. GA natural product was first converted into ent-kaur-9(11),16 dien-19-ol, which was next esterified under Steglich conditions with a series of diversely substituted benzoic acids. To the twelve new compounds thus obtained, was added the preparation of the 9(11),16-dien-19-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl-entkaurene. All these new GA derivatives were carefully purified, and then fully characterized (IR, MS, 1D and 2D NMR) in the aim of their biological evaluation. The antibacterial activity was tested against several bacteria strains, such as Staphylococcus aureus, Enterococcus faecalis, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Escherichia coli. The fungicide properties were evaluated against Candida albicans and C. krusei. The in vivo anti-inflammatory activity was studied on BIO:NMRI mice using the xylol-induced ear-edema test. Unfortunately, no antibacterial nor antifungal activity was exhibited by the grandiflorenic acid derivatives, but their anti-inflammatory activity revealed promising against acute inflammation, with the 9(11),16-dien-18-[2-(4-isobutylphenyl)propyl]- ent-kaurene being the best compound.

A família Euphorbiaceae destaca-se pela produção de compostos de importância industrial e alimentícia. Diversos representantes são de interesse devido à produção de óleos voláteis. São exemplos, espécies do gênero Croton que apresentam propriedades medicinais tais como anti-inflamatória, antimicrobiana e cicatrizante. Algumas espécies de Croton nativas do Rio Grande do Sul foram investigadas visando à análise dos óleos voláteis obtidos por hidrodestilação das partes aéreas das plantas. Foram coletadas três espécies em diversas localidades do Estado, sendo elas Croton pallidulus, C. isabelli e C. ericoides. A composição química dos óleos foi determinada por cromatografia à gás/ espectrômetria de massas. Sesquiterpenos e Monoterpenos foram os principais compostos identificados nos óleos de C. pallidulus, C. isabelli e C. ericoides. Adicionalmente, os óleos voláteis obtidos de cada espécie foram testados quanto a atividade frente a Acanthamoeba polyphaga. Tanto o óleo volátil de C. pallidulus quanto o de C. ericoides apresentaram atividade amebicida, porém este último foi mais ativo: na concentração de 0,5 mg/mL, foi capaz de inviabilizar 87% dos trofozoitos, enquanto que o óleo de C. pallidulus, na mesma concentração, inviabilizou apenas 29% dos trofozoítos. O óleo volátil de C. isabelli foi o que apresentou menor atividade, pois, numa concentração de 10 mg/mL, inviabilizou apenas 4% dos trofozoitos. Os óleos voláteis das três espécies mostraram efeito citotóxico através do método do MTT em células VERO. O óleo de C. ericoides, de maior atividade amebicida, foi também o que apresentou maior citotoxicidade. Além disso, os óleos voláteis de C. pallidulus e C. ericoides foram analisados quanto à atividade antibacteriana contra Staphylococcus aureus, Escherichia coli e Pseudomonas aeruginosa usando o método de bioautografia. Verificou-se que os óleos de C. pallidulus e C. ericoides inibiram o crescimento de E. coli e S. aureus. Com relação a P. aeruginosa não houve inibição do crescimento da bactéria pelas duas espécies de Croton. / The family Euphorbiaceae has been highlighted by the production of compounds of industrial interest and importance for the food. Several representatives species are of interest due to production of volatile oils. Examples are species of Croton that have medicinal properties, such as anti-inflammatory, antifungal, antimicrobial and healing. Some species of the genus Croton, widely distributed in Rio Grande do Sul, were investigated in order to analyze the chemical composition of volatile oils obtained by hydrodistillation from the aerial parts of the plants. Three species were collected in different localities of Rio Grande do Sul, Croton pallidulus, C. isabelli and C. ericoides. The chemical composition of the oils was analyzed using gas chromatography coupled to mass spectrometry. Sesquiterpenes and Monoterpenes were the major compounds identified in the oils of C. pallidulus, C. isabelli and C. ericoides. Additionally, the volatile oils obtained from each species were tested for amebicidal activity against Acanthamoeba polyphaga. Both the volatile oil of C. pallidulus and that of C. ericoides showed activity against A. polyphaga, but the latter was more active: the concentration of 0.5 mg/mL, was able to kill 87% of trophozoites, while the oil of C. pallidulus, at the same concentration, killed only 29% of trophozoites. The volatile oil of C. isabelli was the one that showed less activity; in a concentration of 10 mg/mL, the oil killed only 4% of trophozoites. The volatile oils of the three species showed a cytotoxic effect in VERO cells determined by the MTT method. The oil of C. ericoides, the most active against the Acanthamoeba, presented also the highest cytotoxicity. .In addition, the volatile oils of C. pallidulus and C. ericoides were assayed for antibacterial activity against Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa using the bioautography method. It was found that the oils of C. pallidulus and C. ericoides inhibited the growth of E. coli and S. aureus. With respect to P. aeruginosa no inhibition of bacterial growth by both species of Croton was observed.

Através de Testes de Diluição em Sistema de Tubos Múltiplos determinou-se in vitro atividade antibacteriana em inflorescências de Achyrocline satureioides (Lam.) DC. – Asteraceae (“macela”, “marcela”), expressa como Intensidade de Atividade de Inibição Bacteriana (IINIB / bacteriostasia) e Intensidade de Atividade de Inativação Bacteriana (IINAB / bactericidia), a partir de formas de extração etanólica (hidroalcoolaturas) e hídrica (decoctos), sobre inóculos padronizados de Enterococcus faecalis (ATCC 19433), Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 11229) e Salmonella Enteritidis (ATCC 11076). Enterococcus faecalis apresentou a maior sensibilidade, seguido por Staphylococcus aureus, enquanto Salmonella Enteritidis e Escherichia coli apresentaram-se mais resistentes. Dentre as formas de extração, a hidroalcoolatura apresentou capacidade de inibição e/ou inativação intensa e seletiva frente aos quatro inóculos bacterianos. Os decoctos mostraram-se completamente ineficazes frente às bactérias Gram-negativas, enquanto que as Gram-positivas apresentaram somente bacteriostasia/inibição. / Dilutions Tests in Multiple Tubes System were used to establish the antibacterial activity “in vitro” of inflorescences of Achyroclines satureioides (Lam.) DC. – Asteraceae – (“macela”, “marcela”), from ethanolic extracts (hydroalcoholics) and hydrics (decoction) on pattern baterial suspension such as Enterococcus faecalis (ATCC 19433), Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 11229) and Salmonella Enteritidis (ATCC 11076) expressed as Intensity Activity of Bacterial Inhibition (IINIB) and Intensity Activity of Bacterial Inativation (IINAB). The mentioned extract showed to be more sensibility with Enterococcus faecalis, followed by Staphylococcus aureus, however Salmonella Enteritidis and Escherichia coli showed to be more resistent. Behind the extraction forms, the hydroalcoholic extract presented intense and selective inhibition and / or inactivation capacity in relation of the four agents. However the hydroalcoholic extract presented better antibacterial activity intensity (inhibition / inativation) in relation of the four bacterial suspensions. Decoctions showed to be completely without capacity in front of the Gram-negatives bacterias, on the other hand the Gram-positive the bacteriostasy / inhibition were observed.

Pacientes ortodônticos apresentam grande prevalência de lesões de mancha branca devido ao maior acúmulo de biofilme em torno dos brackets. O objetivo deste estudo foi desenvolver um adesivo ortodôntico que apresentasse atividade antibacteriana e que estimulasse a deposição mineral para evitar o aparecimento e/ou a evolução destas lesões. Adesivos experimentais foram formulados contendo 75% de BisGMA e 25% de TEGDMA por peso; sistema fotoiniciador a base de canforoquinona e 5% de sílica coloidal. Os compostos 1,3,5-triacriloilhexahidro-1,3,5-triazina (TAT) e vidro fosfato invertido contendo pentóxido de nióbio (PIG-Nb) foram adicionados ao adesivo base, como agente antibacteriano e remineralizante, respectivamente. Foram analisados grupos contendo somente TAT nas concentrações de 10, 15 e 20% por peso; grupos contendo somente PIG-Nb nas concentrações de 1, 2,5 e 5% por peso e um grupo contendo 20% TAT e 5% PIG-Nb, por peso. Utilizou-se um grupo sem a adição de TAT e PIG-Nb como Grupo Controle, dentre os experimentais. O adesivo ortodôntico comercial Transbond XT foi utilizado para comparação. Avaliou-se a atividade antibacteriana, capacidade de deposição mineral, variação de pH, grau de conversão, amolecimento em solvente e resistência de união dos adesivos ortodônticos. Os adesivos desenvolvidos apresentaram atividade antibacteriana e capacidade de estimular deposição mineral sem prejudicar as propriedades dos adesivos. Os resultados obtidos a partir dos ensaios realizados neste trabalho permitem concluir que se trata de material com propriedades favoráveis e potencialidade de, após ajustes necessários, tornar-se viável para uso na prática odontológica. / White spot lesions are a concern for orthodontic patients due to biofilm accumulation around brackets. The aim of this study was to develop an antibacterial and remineralizing orthodontic adhesive to overcome these lesions. Experimental orthodontic adhesives were formulated containing 75wt% BisGMA and 25wt% TEGDMA; camphorquinone-based photoinitiator system and 5wt%fummed silica. The compounds 1,3,5-tryacryloylhexahydro-1,3,5-triazine (TAT) and phosphate invert glass containing niobium pentoxide (PIG-Nb) were added to the adhesive as antibacterial and remineralizing agents, respectively. Experimental groups obtained containing only TAT at 10, 15 and 20wt% concentration; groups containing only PIG-Nb at 1, 2.5 and 5wt%, and one group containing 20wt% TAT and 5wt% PIG-Nb. A groups without TAT and PIG-Nb was used as Control and Transbond XT was used as a commercial control. Antibacterial activity, mineral deposition, changes in pH, degree of conversion, softening in solvent and shear bond strength of orthodontic adhesives was assessed. The experimental adhesives presented antibacterial activity and were capable to induce mineral deposition with no negative effect on adhesive's properties. The results of this study allow the conclusion that this material has favorable properties and potential, after necessary adjustments become viable for use in dental practice.

Focus of this study was on the development of one of the faster, simpler, cost effective and environmentally friendly sample pre-treatment techniques which employs a supported liquid membrane, in this case a Hollow-fiber supported liquid membrane (HF-SLM) for determination of seven (7) quinolone antibiotics (enrofloxacin, ciprofloxacin, danofloxacin, difloxacin, norfloxacin, nalidixic acid and sarafloxacin) in bovine kidney samples followed by LC-MS/MS analysis. The key parameters of the method were optimized and the method was validated following the 2002/657 EC guidelines. The optimum HF-SLM conditions were therefore; NaH2PO4 as a donor phase at pH 7, 0.1% formic acid at pH 3 as acceptor phase. Triethylamine was the optimized liquid membrane and the stirring time was optimized at 1 hour. Separation of the 7 quinolones including 3 internal standards (enrofloxacin-d5, norfloxacin-d5 and difloxacin-d3) was carried out on a Phenomenex Kinetex 2.6 μm XB-C18, 100 mm x 4.6 mm, 100Å column. Validation parameters such as Correlation coefficients (r2) ranging from 0.9714-0.9975 were obtained, while limit of detection (LOD) ranged between 3-39 ug kg-1 and limit of quantification (LOQ) ranged between 10-130 ug kg-1. The obtained limits at which it can be concluded with an error probability of α = 95% that a sample is non-compliant (CCα) ranged from 28 – 422 ug kg-1 while CCβ; the smallest content of the substance that may be detected, identified or quantified in a sample with an error probability of β = 95%, ranged from 29 – 454 ug kg-1. The method was found to be reproducible with CVs ≤ 23 %. The tested samples from Botswana local abattoirs showed no presence of quinolone antibiotics when the method was applied to real bovine kidney samples. Hollow-fiber supported liquid membrane can therefore be used for extraction of biological samples since it is a “greener technique” which uses less solvent which are less harmful to the environment when disposed as compared to dispersive Solid Phase Extraction (dSPE). / Chemistry / M. Sc. (Chemistry)

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No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) tese_6676_Dissertao_Christine Facco_2013.pdf: 1315436 bytes, checksum: 39ff1bc59d15b9b7a17e711efee2949b (MD5) / Essa dissertação teve o apoio financeiro do Edital Universal 14/2011 CNPq (nº processo: 483036/2011-0) e do Edital PROEX nº 04/2011. / Na tentativa de obter novas substâncias com atividade antibacteriana, o objetivo desse trabalho foi avaliar o potencial antibacteriano de quatro peptídeos sintetizados, dos quais dois possuem sequência derivada da fragmentação in silico do G-CSF humano, enquanto os outros dois foram planejados teoricamente, verificando, assim, seu interesse como novos agentes terapêuticos na saúde humana. A avaliação foi realizada em duas etapas: análise in silico, que consistiu em predições de propriedades e parâmetros associados à ação antibacteriana, por meio de ferramentas computacionais; e o experimento in vitro para a determinação da concentração inibitória mínima (MIC) dos peptídeos contra bactérias Gram positivas e negativas. A maioria das predições foi favorável para os quatro peptídeos, pois os resultados determinados para hidrofobicidade, anfipaticidade, tamanho, estrutura secundária, carga líquida, potencial de ligação em membrana, meia-vida e índice de Boman encontram-se dentro de valores desejados para o potencial antibacteriano. Na análise in silico, apenas a predição algorítmica da atividade antimicrobiana gerou resultados desfavoráveis para os peptídeos com sequências derivadas do G-CSF (peptídeos 1 e 2), porém essa mesma predição foi positiva para os outros dois. O ensaio in vitro demonstrou que até a concentração mais alta utilizada dos quatro peptídeos (500 μg/mL) foi insuficiente para a determinação de sua concentração inibitória mínima, porém, observou-se considerável diminuição no crescimento de E. coli (58,7%) pelo peptídeo 4 e de E. fecalis (86,1%) e E. coli (54,9%) pelo peptídeo 3, em comparação com o controle de viabilidade. Esses valores indicam a presença de ação antibacteriana por parte dos peptídeos planejados teoricamente (peptídeos 3 e 4), corroborando com as predições computacionais. Dessa forma, é possível concluir que a análise in silico foi de suma importância para a seleção dos peptídeos a serem sintetizados, os quais apresentaram nos ensaios in vitro resultados em concordância com a predição computacional de atividade antimicrobiana. / In attempt to obtain new substances with antibacterial activity, the aim of this study was to evaluate the antibacterial potential of four synthesized peptides, where two of them have sequence derived from human G-CSF in silico fragmentation, while the other two were theoretically planned, allowing the verification of their interest as new therapeutic agents at human health. The evaluation was performed in two stages: in silico analysis, consisting of predictions of properties and parameters associated with antibacterial effect, through computational tools; and the in vitro experiment for determination of the minimum inhibitory concentration (MIC) of the peptides against Gram positive and negative bacteria. Most predictions was favorable for all four peptides, showed by determined results of hydrophobicity, amphipathicity, size, secondary structure, net charge, membrane binding potential, half-life and Boman Index, considered as desirable values for antibacterial potential. In the in silico analysis, only algorithmic prediction of antimicrobial activity revealed unfavorable results for peptides with sequences derived from G-CSF (peptides 1 and 2), nonetheless, the predictions were positive for the other two. The in vitro assay showed that up to the highest concentration used of the four peptides (500 μg/mL) was insufficient for determination of minimum inhibitory concentration, however it was possible to observe significant growing decrease of E. coli (58.7%) by peptide 4 and E. fecalis (86.1%) and E. coli (54.9%) by peptide 3, when compared with the viability control. These values indicate the presence of antibacterial activity in the theoretically planned peptides (peptides 3 and 4), confirming the computational predictions. Thus, it is possible to conclude that the in silico analysis was very important for the selection of the peptides to be synthesized, which showed results of in vitro assays in agreement with the computational prediction of antimicrobial activity.

TiO2 nanotubes grown on titanium alloy are known to increase the biocompatibility of the alloy when used in dental/orthopaedic implants. Furthermore, their nanotubular structures can act as antibacterial agent carrier and as a scaffold for tissue engineering with the aim of adding antibacterial properties to the implant. This study aims at fabricating an antibacterial and biocompatible nanocomposite coating on Ti-6Al-4V involving nano-ZnO and nano-Ag. Initially, TiO2 nanotubes were self-assembled on the polished surface of medical grade Ti-6Al-4V alloy discs using anodisation. First silver nanoparticles were chemically reduced from silver ammonia using delta-δ-gluconolactone for different duration on the nanotubes to form TiO2-Ag composite coating. Nano HA was added to the latter coating with the aim of reducing toxicity from silver, hence forming TiO2-Ag-HA coating. Secondly, nano-ZnO was thermo-chemically grown on the TiO2 nanotubes using zinc nitrate and hexamethylenetetramine. They were then annealed at 350-550 oC hence forming TiO2-ZnO. HA was grown on the latter coating by a biomimetic method whereby the coated discs were placed in a concentrated simulated body fluid at 37 oC forming TiO2-ZnO-HA. The stability of the 4 coatings, TiO2-Ag, TiO2-Ag-HA, TiO2-ZnO and TiO2-ZnO-HA were assessed using the dialysis method (n=3 each) and then exposed to S.aureus for 24 hours in BHI broth. Their antibacterial properties were assessed using different assays and microscopic imaging with respect to different controls (n=6 each for assays and n=3 for imaging). Their biocompatibility properties were assessed in the presence of primary human osteoblast cells in DMEM media with the help of biochemical assays, molecular gene expression and microscopic imaging (n=3). Both silver and zinc coated nanotubes showed significant level of antibacterial properties with silver coating being more bactericidal than the coating containing zinc. Nonetheless, the zinc oxide coatings were more biocompatible than the silver coating. Nano silver and zinc oxide containing composite coatings were successfully synthesised and tested in the presence of bacteria and human cells. The final conclusion was that nano-silver was still toxic and nano-ZnO coatings were more biocompatible.

Através de Testes de Diluição em Sistema de Tubos Múltiplos determinou-se a Intensidade de Atividade de Inibição Bacteriana (IINIB/bacteriostasia) e a Intensidade de Atividade de Inativação Bacteriana (IINAB/bactericidia) de soluções contendo extratos hidroetanólicos e hídricos (decocto e infuso) de três acessos de Bixa orellana L. (urucum) a saber: Arroio do Meio/RS, Eldorado do Sul/RS e Maringá/PR, sobre inóculos padronizados de Salmonella Enteritidis (ATCC 11076), Escherichia coli (ATCC 11229), Staphylococcus aureus (ATCC 25923), Enterococcus faecalis (ATCC 19433) e Listeria monocytogenes (ATCC 19114). Determinou-se, paralelamente, o teor de bixina presente nas sementes. Os extratos hídricos apresentaram baixa atividade de inibição e/ou inativação sobre os inóculos bacterianos, enquanto que a forma de extração hidroetanólica apresentou atividade antibacteriana seletiva e significativamente mais intensa (inibição/inativação) entre as cinco bactérias testadas. Independente da forma de extração, as bactérias Enterococcus faecalis e Listeria monocytogenes foram as mais sensíveis à atividade antibacteriana, enquanto que Escherichia coli apresentou a menor sensibilidade. Houve diferença significativa entre os teores de bixina dos três acessos, e, consequentemente, a atividade antibacteriana determinada mostrou-se diretamente proporcional a estes teores. / Through of Dilution Tests in Multiple Tubes System it was determined the intensity of bacterial inhibition activity (IINIB/bacteriostasy) and the intensity of bacterial inactivation activity (IINAB/bactericidie) from solutions containing hidroetanolic and hydric (decoction and infusion) extracts of tree accesses from Bixa orellana L. (annatto) at know: Arroio do Meio/RS, Eldorado do Sul/RS and Maringá/PR, on standardized inocula of Salmonella Enteritidis (ATCC 11076), Escherichia coli (ATCC 11229), Staphylococcus aureus (ATCC 25923), Enterococcus faecalis (ATCC 19433) and Listeria monocytogenes (ATCC 19114). It was determined, in parallel, the content of bixin present in the three different accesses of the seeds. The forms of hydric extraction showed low inhibition and/or inactivation activity of the bacterial inocula, and the hidroetanolic extract form showed selective antibacterial activity and significantly pronounced inhibition/inactivation against the five bacteria tested. Independent of the extraction forms, Enterococcus faecalis and Listeria monocytogenes were the more sensitive agents to the antibacterial activity. Escherichia coli had the lowest sensitivity to all forms of extraction. The bixin contents were significantly different between the accesses and, consequently, the antibacterial activity was directly proportional to this contents.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Ocimum gratissimum L. (Lamiaceae), known as alfavaca, is widely distributed in tropical and warm-temperature regions. It is used as a food flavoring and for medicinal purposes against infectious diseases. Its biological properties are attributed to the presence of thymol or eugenol-rich essential oils. This study describes qualitative and quantitative variations in essential oils from stems, inflorescences and leaves of O. gratissimum L. grown in southern Brazil, in two harvest periods. The evaluation of eugenol content, antibacterial activity and toxicity of the different ethanolic extracts from dried aerial parts of O. gratissimum were studied. Afterwards, the essential oil from fresh inflorescences had its antibacterial activity, toxicity and chemical composition determined. The essential oils were obtained by hydrodistillation and analyzed by GC/MS. The extracts were obtained by maceration, soxhlet, ultrasonic cleaning bath, ultrasonic horn and microwave-assisted extractions. Eugenol content was determined by GC-FID using the pure compound isolated from aerial parts of the plant as reference. Antibacterial activity was assayed by microdilution method as established by CLSI/NCCLS. Toxicity was verified by brine shrimp (Artemia salina L.) lethality test. The major compounds of all essential oils were eugenol (52.6 - 89.2 %) and γ-muurolene (6.2 35.3 %). The harvest season influenced only the yields of inflorescence and stem essential oils. The low seasonal variation of eugenol in the essential oil of the leaves indicated that these are the best source to obtain this compound. Inflorescences essential oil was bacteriostatic (MIC between 0.5-2 mg/mL) and bactericidal (MBC between 1-4 mg/mL) against all strains tested. Significant differences (P<0.05) among the extractive methods used to the obtention of ethanolic extracts were observed in all analyses. Higher antimicrobial activity was verified to extracts compared to the essential oil tested against S. aureus ATCC 25923 and clinical isolate of B. cereus, MRSA (MIC of 0.66 mg/mL), ESBL and Shigella flexneri (MIC of 1.32 mg/mL). Bactericidal activity of the extracts was verified in Grampositive bacteria. Eugenol content did not explain all differences obtained among the antimicrobial activities of the tested extracts. The results suggest synergistic action among phyto-constituents. Maceration was considered the best extraction process considering the biological activities of all extracts. The antimicrobial avaliation results confirmed the brine shrimp lethality test as predictive method for this activity. / Ocimum gratissimum L. (Lamiaceae), conhecida popularmente como alfavaca, é uma planta amplamente distribuída em regiões de clima tropical e temperado. É utilizada como aromatizante de alimentos e medicinalmente no combate a doenças infecciosas. Suas propriedades biológicas têm sido atribuídas à presença de óleo essencial rico em timol ou eugenol. Este trabalho descreve as variações qualitativas e quantitativas nos óleos essenciais obtidos a partir dos talos, inflorescências e folhas desta espécie cultivada no Sul do Brasil em relação ao período de coleta. Também descreve a avaliação da atividade antibacteriana, toxicidade e teor de eugenol nos extratos etanólicos brutos das partes aéreas secas de O. gratissimum obtidos por diferentes metodologias. O óleo essencial obtido a partir das inflorescências frescas deste vegetal teve sua composição química, atividade antibacteriana e toxicidade estudadas. Os óleos essenciais foram obtidos por hidrodestilação e analisados por CG/EM. Os extratos etanólicos foram obtidos através dos métodos de maceração, soxhlet, banho de ultrassom, sonda de ultrassom e micro-ondas. O conteúdo de eugenol nos extratos foi determinado por CG-FID utilizando eugenol obtido das partes aéreas de O. gratissimum como padrão. A avaliação da atividade antibacteriana foi realizada pelo método de microdiluição em caldo (CLSI/NCCLS). O teste de letalidade frente à Artemia salina L. foi utilizado para análise da toxicidade. Os constituintes majoritários de todos os óleos foram eugenol (52,6 - 89,2 %) e γ-muuroleno (6,2 35,3 %). Apenas os óleos essenciais das inflorescências e talos tiveram seus rendimentos influenciados pela estação de coleta. A pequena variação no teor de eugenol presente no óleo essencial das folhas indicou ser esta a melhor parte do vegetal para a obtenção deste composto. O óleo essencial das inflorescências foi bacteriostático (CIM entre 0,5-2 mg/mL) e bactericida (CBM entre 1-4 mg/mL) frente a todas as cepas bacterianas testadas. Os extratos etanólicos demonstraram diferenças significativas (P< 0.05) entre si em relação a rendimento, teor de eugenol, atividade antibacteriana e toxicidade. Maior atividade antimicrobiana foi verificada para os extratos comparado ao óleo essencial testado frente à S. aureus ATCC 25923 e aos isolados de B. cereus, MRSA (CIM de 0,66 mg/mL), ESBL e Shigella flexneri (CIM de 1,32 mg/mL). Ação bactericida dos extratos foi obtida principalmente frente a bactérias Gram-positivas. Os teores de eugenol detectados nos extratos de O. gratissimum não puderam ser correlacionados com a atividade bacteriostática, o que sugere efeito sinérgico entre fito-constituintes. A maceração foi considerada como a melhor metodologia para a obtenção de extratos etanólicos das partes aéreas desta espécie. Os resultados de atividade antibacteriana confirmaram o teste de toxicidade frente à Artemia salina L. como um método preditivo deste tipo de atividade.

Made available in DSpace on 2014-06-11T19:31:33Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-05-30Bitstream added on 2014-06-13T21:02:44Z : No. of bitstreams: 1 pappen_fg_dr_arafo.pdf: 4993789 bytes, checksum: ca121f0b3da3d2618c6caa7970c772de (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Universidade Estadual Paulista (UNESP) / O objetivo deste estudo foi avaliar a atividade antibacteriana das soluções Tetraclean®, MTAD® e modificações desta formulação sobre uma cepa de Enterococcus faecalis pelo teste de contato direto e frente ao biofilme. Na metodologia de contato direto, uma suspensão de E. faecalis foi exposta às soluções irrigadoras, por períodos de 30 s, 1, 3 e 10 min, e um agente neutralizante foi utilizado. As colônias viáveis foram quantificadas após diluições seriadas e cultura em placas de ágar. Para determinação do número inicial de UFC, água esterilizada foi usada como controle. Para o crescimento do biofilme, amostras de biofilme dentário foram suspensas em BHI (Brain Heart Infusion), e incubadas por 14 dias em anaerobiose, tendo como substrato, discos de hidroxiapatita. Após o período de incubação, os biofilmes foram expostos às soluções irrigadoras por períodos de 30 s, 1 e 3 min. Os biofilmes corados com o Live/Dead Baclight stain (Molecular Probes, Europe BV), que diferencia células viáveis (verde) e células não-viáveis (vermelho) foram observados na microscopia confocal. As imagens do biofilme obtidas pelo software EZ-C1 for Nikon foram transferidas para análise quantitativa da proporção de células viáveis sobre o total de células do biofilme para o software Imaris 5.0. No teste de contato direto, o Tetraclean® eliminou completamente o E. faecalis em 30 s, enquanto o MTAD® e o MTAD® + CTR 0,01% eliminaram após 10 min, e o MTAD® + CTR 0,1% resultou em culturas negativas após 3 min de contato com a suspensão bacteriana. Quando removido o Tween 80 do MTAD®, e acrescida a CTR 0,01%, obteve-se culturas negativas após 1 minuto. Os resultados obtidos pelo método de exposição ao biofilme confirmaram os achados do teste de contato direto. / The aim of this study was to evaluate the antibacterial effect of MTAD® and Tetraclean®. The tests were performed on Enterococcus faecalis using the direct exposure test and an in vitro biofilm model. Alternative formulations to MTAD® were also included in the experiments: MTAD® + 0.01% cetrimide (CTR); MTAD® + 0.1% CTR; MTAC (the detergent Tween 80 was substituted for cetrimide) with 0.01% CTR and MTAC (0.1% CTR). In the direct exposure test, a bacterial suspension was mixed with the irrigation solutions. After 30 seconds, 1 minute, 3 minutes and 10 minutes, an inactivator agent was used. The number or viable colonies were calculated after serial 10-fold dilutions and culture in agar plates. To calculate the initial number of colonies, sterilized water was used as a control. Dental biofilm samples were suspended in BHI broth and incubated in anaerobic conditions in hidroxiapatite discs. After incubation for 14 days, the biofilms were exposed to the irrigation solutions for 30 seconds, 1 minute and 3 minutes. Live/Dead Baclight stain (Molecular Probes, Europe BV) was used to diferentiate viable cells (Green) and non viable cells (Red). The samples were observed in the Confocal Laser Microscope. The biofilm images in 3D obtained in the EZ-C1 for Nikon software were transfered to quantitative analysis in the Imaris 5.0 software. The software calculated the ratio of green cells. In the direct exposure test, Tetraclean® and MTAC (0.1% CTR) erradicated E. faecalis in 30 s. MTAD® and MTAD® + 0.01% CTR eliminated E. faecalis after 10 min. MTAD® + 0.1% CTR resulted in negative culture after 3 min. Negative cultures were obtained after 1 min when MTAC (0.01% CTR) was used. The findings from the biofilm exposure method confirmed the results from direct exposure test. The Kruskal-Wallis statistical analysis showed a significant difference in time exposure to irrigant (P<0.001).