Thesis (Ph.D.) - University of St Andrews, April 2010.

Made available in DSpace on 2014-06-11T19:32:09Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-08-26Bitstream added on 2014-06-13T18:43:05Z : No. of bitstreams: 1 silva_hr_dr_arafcf.pdf: 2348065 bytes, checksum: c63f75a77b4cb78f5b87a6170be20a8f (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Universidade Estadual Paulista (UNESP) / Nas últimas décadas, tem ocorrido um aumento expressivo na incidência de infecções fúngicas. Por outro lado, o tratamento das micoses nem sempre é efetivo, pois os fármacos antifúngicos disponíveis apresentam espectro de atividade e perfil farmacocinético inadequados, em especial no tratamento das lesões cutâneas. Portanto, o objetivo do presente trabalho foi desenvolver e caracterizar sistemas nanoestruturados estabilizados com álcool cetílico etoxilado 20 OE e propoxilado 5 OP (PROC) contendo fluconazol (FLU), empregando ácido oléico (AO) e óleo de copaíba como fases oleosas. A caracterização química, seguida de ensaios biológicos, dos óleos de copaíba de diferentes fontes, foi determinante para a escolha do óleo a ser usado no desenvolvimento das formulações. A construção de diagramas de fases com ambos os óleos mostrou que diferentes sistemas como microemulsões (ME) e cristais líquidos (CL) podem ser formados. A caracterização físico-química (MLP, SAXS e Reologia) mostrou que na região de 40% de PROC, o aumento da quantidade de água favorece a formação de sistemas mais estruturados como CL de fase lamelar e hexagonal quando se utiliza AO e óleo de copaíba... / In the last decades, there has been an expressive increase in incidence of fungal infections. On the other hand, the treatment of mycoses is not always effective, because available antifungal drugs show inappropriate activity spectrum and pharmacokinetic profile. The aim of this work was to develop and characterize nanostructured systems stabilized by propoxyl (5OP) ethoxyl (20 OE) cethyl alcohol (PROC) containing fluconazole, using oleic acid and copaiba oil from different origins as oily phases. Chemical and biological characterization of copaiba oils from different origins was decisive for the choice of oil to be used in the development of the formulations. The construction of phase diagrams with studied copaiba oils showed that different systems such as microemulsions (ME) and liquid crystals (CL) can be formed. The characterization by polarized light microscopy, rheological behavior and SAXS confirmed the results obtained in phase diagrams, showing that in the region of 40% of PROC, the increase in the quantity of water favors the formation of more structured systems as CL of lamellar and hexagonal phase... (Complete abstract click electronic access below)

Resumo: Nas últimas décadas, tem ocorrido um aumento expressivo na incidência de infecções fúngicas. Por outro lado, o tratamento das micoses nem sempre é efetivo, pois os fármacos antifúngicos disponíveis apresentam espectro de atividade e perfil farmacocinético inadequados, em especial no tratamento das lesões cutâneas. Portanto, o objetivo do presente trabalho foi desenvolver e caracterizar sistemas nanoestruturados estabilizados com álcool cetílico etoxilado 20 OE e propoxilado 5 OP (PROC) contendo fluconazol (FLU), empregando ácido oléico (AO) e óleo de copaíba como fases oleosas. A caracterização química, seguida de ensaios biológicos, dos óleos de copaíba de diferentes fontes, foi determinante para a escolha do óleo a ser usado no desenvolvimento das formulações. A construção de diagramas de fases com ambos os óleos mostrou que diferentes sistemas como microemulsões (ME) e cristais líquidos (CL) podem ser formados. A caracterização físico-química (MLP, SAXS e Reologia) mostrou que na região de 40% de PROC, o aumento da quantidade de água favorece a formação de sistemas mais estruturados como CL de fase lamelar e hexagonal quando se utiliza AO e óleo de copaíba... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: In the last decades, there has been an expressive increase in incidence of fungal infections. On the other hand, the treatment of mycoses is not always effective, because available antifungal drugs show inappropriate activity spectrum and pharmacokinetic profile. The aim of this work was to develop and characterize nanostructured systems stabilized by propoxyl (5OP) ethoxyl (20 OE) cethyl alcohol (PROC) containing fluconazole, using oleic acid and copaiba oil from different origins as oily phases. Chemical and biological characterization of copaiba oils from different origins was decisive for the choice of oil to be used in the development of the formulations. The construction of phase diagrams with studied copaiba oils showed that different systems such as microemulsions (ME) and liquid crystals (CL) can be formed. The characterization by polarized light microscopy, rheological behavior and SAXS confirmed the results obtained in phase diagrams, showing that in the region of 40% of PROC, the increase in the quantity of water favors the formation of more structured systems as CL of lamellar and hexagonal phase... (Complete abstract click electronic access below) / Orientador: Maria Palmira Daflon Gremião / Coorientador: Georgino Honorato de Oliveira / Banca: Nereide Stela Santos Magalhães / Banca: Marlus Chorilli / Banca: Silvia Stanisçuaski Guterres / Banca: Renata Fonseca Vianna Lopez / Doutor

Dissertação(mestrado) - Universidade Federal do Rio Grande, Programa de Pós-Graduação em Engenharia e Ciência de Alimentos, Escola de Química e Alimentos, 2008. / Submitted by Caroline Silva (krol_bilhar@hotmail.com) on 2012-09-13T19:06:33Z No. of bitstreams: 1 avaliao da atividade antifngica e antimicotoxinas de extratos de farelo de arroz cebola e microalga chlorella.pdf: 988335 bytes, checksum: 8413be9e7b4208c1612064781396a048 (MD5) / Approved for entry into archive by Bruna Vieira(bruninha_vieira@ibest.com.br) on 2012-09-24T22:59:35Z (GMT) No. of bitstreams: 1 avaliao da atividade antifngica e antimicotoxinas de extratos de farelo de arroz cebola e microalga chlorella.pdf: 988335 bytes, checksum: 8413be9e7b4208c1612064781396a048 (MD5) / Made available in DSpace on 2012-09-24T22:59:35Z (GMT). No. of bitstreams: 1 avaliao da atividade antifngica e antimicotoxinas de extratos de farelo de arroz cebola e microalga chlorella.pdf: 988335 bytes, checksum: 8413be9e7b4208c1612064781396a048 (MD5) Previous issue date: 2008 / A contaminação fúngica acarreta alterações na qualidade nutricional e no valor econômico dos produtos alimentícios podendo causar danos patológicos em plantas, animais e humanos. A identificação da atividade antifúngica e antimicotoxinas, em extratos de diferentes fontes que exibem propriedades de inibir naturalmente o crescimento de fungos e subseqüente produção de micotoxinas (metabólitos secundários produzidos por fungos toxigênicos), abre a perspectiva de empregar de forma mais eficiente os tecidos vegetais empregando-os como conservadores naturais. Entre os compostos com propriedades inibidoras de crescimento fúngico e produção de micotoxinas, naturalmente presentes em alimentos, destacam-se os compostos fenólicos, que por sua estrutura química dificultam a atividade de enzimas metabólicas de microrganismos. As matérias-primas escolhidas foram: o farelo de arroz, a cebola e a microalga Chlorella phyrenoidosa. Aos três tecidos são atribuídas propriedades funcionais, sendo que os dois primeiros são abundantes na região sul e comercializados com baixo valor agregado. A chlorella é empregada em dietas especiais como fonte de compostos bioativos especialmente aminoácidos essenciais e compostos antioxidantes. Este trabalho teve como objetivo determinar o teor de compostos fenólicos, a atividade antifúngica sobre o desenvolvimento dos fungos dos gêneros Rhyzopus sp., Aspergillus oryzae, Aspergillus flavus, e Fusarium graminearum e a atividade antimicotoxinas sobre o fungo Aspergillus flavus, em extratos de farelo de arroz, cebola e Chlorella. Os compostos fenólicos da cebola foram extraídos em três sistemas solventes: aquoso, metanólico e acetato de etila. Os compostos fenólicos do farelo de arroz e da chlorella foram extraídos com metanol, sendo quantificados colorimetricamente com reagente de Folin-Ciocalteau. O conteúdo de fenóis totais nos vegetais variou de 68 μgfenóis.g-1 em extrato aceto-etílico de cebola a 3012 μgfenóis.g-1 em extrato aquoso de cebola. Os extratos foram triados sobre os fungos Rhyzopus sp e Aspergillus oryzae que foram os modelos para estimar a inibição de crescimento fúngico. Esporos de Aspergillus flavus foram utilizados para estudar o efeito inibidor da produção de aflatoxina B1 e B2. Os extratos testados apresentaram algum grau de inibição do desenvolvimento fúngico, sendo a chlorella a que apresentou maior inibição em relação aos outros extratos, em todos os fungos testados, chegando a 31% de inibição/μg fenol total. Após o 7°, 14° e 21° dia de incubação foram realizadas extrações de micotoxinas do meio de crescimento pelo método adaptado de TANAKA et al., (2000). O extrato fenólico de Chlorella inibiu totalmente a produção de micotoxinas em relação ao controle. Estes resultados mostram que a ação antifúngica está naturalmente presente em alguns tecidos vegetais e que encontrar a forma de extraí-los e aplicá-los como conservadores de alimentos é muito promissor para agregar valor aos alimentos, principalmente aqueles de baixo valor comercial. / The fungal contamination cause alterations in nutritional quality and economic value of food products and can cause pathological damages in plants, animals and humans. The identification of the antifungal activity and antimycotoxin in extracts of sources different that show properties of naturally inhibiting the fungal growth and subsequent mycotoxins production (metabolites secondary produced by toxic fungal), open the perspective of employing in the more efficient form the vegetable tissue employing them like natural conservatives. Between compounds with properties inhibiting of fungal growth and mycotoxins production, naturally present in foods, stand out the phenolics, what for your chemical structure make difficult the activity of metabolic enzymes of microrganisms. The chosen raw materials were: the rice bran, the onion and the microalga Chlorella phyrenoidosa. To three tissues functional properties are attributed being that two first ones are abundant in the south region and marketed with low collected value. The Chlorella is employed in special diets like source of bioactives compounds and antioxidant compounds. This objective study was determine the tenor of phenolics compounds, the antifungal activity on the development of the fungal Rhyzopus sp, Aspergillus oryzae, Aspergillus flavus and Fusarium graminearum and antimycotoxin activity on the fungal Aspergillus flavus, in extracts of bran of rice, onion and Chlorella. The phenolic compounds of the onion were extracted in three solvent systens: aqueous, methanolic and ethila acetate. The phenolic compounds of the bran of rice and of the Chlorella were extracted with methanol, and quantified with reagent of Folin-Ciocalteau. The total phenolics levels in vegetables ranged between 68 μgphenolicg-1 in aceto-ethylic extract of anion and 3012 μgphenolic g-1 in aqueous extract of onion. The extracts was used in antifungal tests against strains of Rhyzopus sp and Aspergillus oryzae that were the models to appreciate the inhibition of fungal growth. Spores of Aspergillus flavus were used to study the inhibiting effect of the aflatoxins B1 and B2 production. The tested extracts presented some degree of inhibition of the fungal development, being the Chlorella Who presented tested bigger inhibition regasding other extracts, in all the fungal, is reaching 31% of inhibition/μgphenolic . After 7°, 14 and 21° Day of incubation were carried out extractions of mycotoxins from growth médium were determined by TANAKA et al., method (2000). The phenolic extract of chlorella inhibited totally the mycotoxins production regarding the control. These results show that the antifungal activity is present naturally in some vegetable tissues and that to find the formo of extracting to apply as conservatives of foods is very promising to values collect to the foods, principally those of low commercial value.

Orientadores: Altair Antoninha Del Bel Cury, Pedro Luiz Rosalen / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-05T09:42:20Z (GMT). No. of bitstreams: 1 Silva_WanderJoseda_M.pdf: 3982460 bytes, checksum: cc52755a091670127495a82d48ac39c9 (MD5) Previous issue date: 2005 / Resumo: Na candidose, ou estomatite induzida por prótese, a Candida albicans atua como o patógeno mais importante e mais virulento. A terapêutica indicada no tratamento são os agentes antifúngicos (AA) como Fluconazol (FLU) e Nistatina (NYS) e mais recentemente tem sido indicado soluções a base de própolis. Entretanto, a literatura é escassa sobre os efeitos destes agentes nos materiais utilizados para confecção de bases de próteses. O objetivo deste estudo foi verificar a influência de NYS, FLU, e gel orobase (GO) de própolis sobre a superfície de resinas acrílicas durante período de 14 dias. Água destilada e GO manipulado sem princípio ativo foram utilizadas como grupos controle. Espécimes (3,0 x 2,5 x 0,5 cm) foram fabricados a partir de moldes de cera, com resina acrílica polimerizada em banho de água (Jet Clássico, grupos 1 a 5) ou com energia de microondas (OndaCryl, grupos 6 a 10), ambas processadas de acordo com as instruções dos fabricantes. Após a confecção, os espécimes receberam acabamento e polimento e a rugosidade superficial (RS), ângulo de contato (AC), energia livre de superfície (ELS) e dureza de superfície (DS) foram mensuradas. Os tratamentos de superfície foram feitos com os AA e grupos controles e os espécimes foram escovados com dentifrício e escova macia 3 vezes ao dia durante os 14 dias. Os meios de imersão foram substituídos diariamente. Novas mensurações para as variáveis foram executadas em 3, 7, 10 e 14 dias de exposição. Os compostos dos AA que poderiam ser incorporados à resina acrílica foram mensurados por cromatografia líquida. Os resultados mostraram que a RS aumentou para ambas as resinas acrílicas, mas não diferiram entre si (p>0,05); os grupos G2 e G7, contendo propolis mostraram valores maiores e estatisticamente diferente dos demais agentes nos tempos de T7, T10 e T14 (p<0,05). Também com o passar do tempo os grupos G1, G3, G4 e G8 mostraram valores de rugosidade aumentada e significantemente diferente dos demais tempos (p<0,05, teste de Tukey). Nas análises de cromatografia não foi detectada a presença de FLU ou NYS, assim como não foi detectada diferença entre as resinas acrílicas (p>0,05, teste de Tukey) quanto a compostos do GO. A microscopia eletrônica de varredura mostrou as alterações sobre a superfície dos espécimes após o tratamento. Dentro dos limites deste estudo, é possível concluir que os agentes antifúngicos podem interferir com propriedades da superfície de resina acrílica que são associadas a adesão de Candida sp / Abstract: The high prevalence of candidosis in denture wearers, and its association to Candida spp. as principal pathogen is well established. Besides, there are many studies about candidosis etiology, predisposing factors and its treatment using antifungal agents such as Fluconazole (FLU) and Nystatin (NYS). However, little work has been performed to explore the effects of these antifungal agents on acrylic resin surface. The aim of this study was to verify the influence of NYS, FLU, and propolis orobase gel over surface acrylic resins. Distillate water and orobase gel without any active component were used as control groups. Specimens (3.0 x 2.5 x 0.5 cm) were fabricated from wax moulds using heat cured (HC = Clássico; groups 1 to 5) or microwave cured (MW = Onda Cry; groups 6 to 10) acrylics resin, both processed according to manufacturers¿ instructions. After polymerized the specimens were polished and had their surface roughness (SR), contact angle (CA), surface free energy (SFE) and hardness (HD) measured. The surface treatments were done with antifungal agents and controls. The specimens had their surface toothbrushed 3 times a day during the 14 days of exposure to antifungal agents. The antifungal agents¿ solutions and gels were replaced daily. Measurements to all variables were performed in days 3, 7, 10 and 14. Components from antifungal agents that could be incorporated to acrylic resin measured with liquid chromatography. The results showed that SR increased for both acrylic resins and the treatment with propolis showed higher values and statistically different from the others (p<0.05). No statistical difference (Tukey test, p>0.05) was found regarding CA and SFE. No presence of FLU or NYS were detected in chromatography and no difference (p<0.05) between acrylic resins were detect to orobase gels detection. Scanning electronic microscopy evaluation showed surface alterations after treatment. Within the limits of this study, it is possible to conclude that antifungal agents are able to interfere with the surface properties of acrylic resins that are associated to Candida spp. adhesion / Mestrado / Protese Dental / Mestre em Clínica Odontológica

Orientador: Francisco Benedito Teixeira Pessine / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Quimica / Made available in DSpace on 2018-08-14T22:28:32Z (GMT). No. of bitstreams: 1 Brescansin_EdeilzaGomes_D.pdf: 1646931 bytes, checksum: 9e5e045b99495a199511f36621650953 (MD5) Previous issue date: 2006 / Resumo: No presente trabalho, buscamos desenvolver formulação lipossomal sistêmica de nistatina (NYS) com o propósito de aplicações futuras na terapêutica de micoses sistêmicas, que tanto afligem indivíduos imunodeprimidos. Na primeira parte do projeto caracterizamos o fármaco através de análises térmicas (TGA e DSC), análises espectroscópicas (IV, UV e fluorescência), teste de solubilidade, avaliação da umidade segundo o método de Karl-Fischer e coeficiente de partição octanol/meio aquoso. Na segunda parte do trabalho procuramos desenvolver e otimizar formulações lipossomais, onde o fármaco apresentasse maior estabilidade química. Foram preparadas formulações lipossomais pelo método do filme lipídico que foram analisadas por HPLC. As preparações passaram por processo de diminuição de tamanho e separação entre fármaco livre e encapsulado. A terceira e última fase do projeto consistiu da caracterização de duas preparações, através de análises do raio hidrodinâmico, determinação da carga de superfície das partículas (potencial zeta), teor de fosfolipídios totais (teor de fosfato), eficiência e avaliação da encapsulação por análises de HPLC, anisotropia e supressão de fluorescência. Ainda na fase de caracterização investigamos a atividade biológica in vitro, destas formulações, pelo teste de susceptibilidade. As preparações foram desafiadas contra leveduras e dermatófitos. Pela seleção e otimização das formulações desenvolvidas concluímos que a melhor formulação foi a que apresenta uma composição de 70 % de Epikuron 200 SH; 20 % de Colesterol; 10 % de Diestearoilfosfatidilglicerol, 1,3 % de 2,6 - bis (1,1-dimetil) - 4 - metil fenol (BHT) e 7 % de NYS. Duas preparações foram caracterizadas L1 (hidratada a 55°C) e L2 (hidratada a 60°C). Os resultados para anisotropia e supressão de fluorescência evidenciam a encapsulação do fármaco nas preparações estudadas, tanto na bicamada lipídica quanto na superfície dos lipossomas. Verificamos também que L2 apresentou menor polidisperdidade de seu raio hidrodinâmico, maior carga de superfície e maior eficiência de encapsulação, evidenciando ser L2 a preparação, físico-quimicamente, mais estável. Todavia, a preparação L1 mostrou, após sonicação, atividade biológica in vitro maior que L2. Estes resultados podem refletir a possível degradação do fármaco, quando aquecido a 60°C. / Abstract: This study was designed to develop liposomal formulations containing Nystatin (NYS), looking forward its future applications in systemic mycosis therapy, which often affect immunodepressed individuals. In the first part of the project, the drug was characterized by means of thermal analyses (TGA and DSC), IR, UV/V and fluorescence spectroscopies, solubility tests, humidity contents evaluation (Karl-Fischer method) and octanol-water partition coefficient. In the second part of the study, liposomal formulations were developed and optimized, in which the drug presented improved chemical stability. Liposomal formulations were prepared by the dry lipid film hydration method and were analyzed by HPLC. The vesicles were submitted to the processes of size reduction and separation of non encapsulated drug. The last part of the project consisted in the characterization of the two formulations by means of analysis of the is hydrodynamic radius; determination of the particles superficial charge (zeta-potential); total phospholipids content (phosphate); degree of NYS with encapsulation by HPLC; degree of anisotropy and fluorescence quenching of NYS. Still during the characterization phase, the in vitro biological activity of the formulations was investigated by means of susceptibility tests. The formulations were challenged against some yeasts and filamentous fungi. Conclusions pointed that the best formulation was the one which presented in its compositions 70% Epikuron 200 SH, 20% Cholesterol, 10 % Distearoylphosphatidylglycerol, 1.3 % 2,6 - bis (1,1-dimethylethyl) - 4 - methylphenol (BHT) and 7 % NYS. Two formulations were characterized: L1 (hydrates at 55°C) and L2 (hydrated at 60°C). Results regarding the degree of anisotropy and fluorescence quenching evidenced by NYS encapsulation at the liposomal lipid bilayers. Moreover, it was observed that L2 presented lower polydispersity in size distribution, higher superficial load and higher encapsulation effectiveness, i.e. L2 presented higher physico-chemical stability. Nevertheless the L1 preparation showed, after sonication, higher biological activity in vitro than L2, which might reflect possible thermal degradation of NYS when heated to 60°C. / Doutorado / Físico-Química / Doutor em Ciências

Phytochemical analysis of both HEA1 and the crude plant extract showed the presence of phenolics, tannins and saponins. Saponins were the predominant secondary metabolites and were mostly abundant in the plant extract and to a lesser extent in the active compound. Steroidal saponins, tannins and phenolics were also detected in the plant extract, but only the phenolics were detected in the active compound. The results of the phytochemical analysis showed that those compounds that were not present in the active compound could be removed from the crude extract during the TLC purification process. Investigation on the mechanism of action of the crude plant extract on the sterol production by A. flavus showed that the plant extract affected ergosterol biosynthesis by causing an accumulation of oxidosqualene in the ergosterol biosynthetic pathway resulting in a decline in ergosterol production. An oscillatory response in lanosterol production was observed in the presence of the plant extract, which may be an adaptation mechanism of A. flavus to unfavourable conditions and compensation for the loss of enzyme activity which may have occurred as a result of the accumulation of oxidosqualene. The antifungal activity of the plant extract on ergosterol production by A. flavus may also be due to saponins which target the cell membrane and ergosterol production in fungi. The effect of the plant extract on the fungal cell wall of A. flavus also showed that the plant extract caused a decline in β-(1, 3) glucan production by inhibiting β-glucan synthase. The plant extract also affected the chitin synthesis pathway of A. flavus, by causing a decline in chitin production, which was due to the inhibition of chitin synthase. Investigation of chitinase production using 4MU substrates showed that the plant extract caused an accumulation of chitobioses, by activating chitobiosidases and endochitinases. A decline in N-acetylglucosaminidase activity in the presence of the plant extract was observed and this prevented the formation of N-acetylglucosamine. The accumulation of chitobiosidase and endochitinase may be as a result of autolysis that may be triggered by A. flavus as a survival mechanism in the presence of the plant extract and as a compensatory mechanism for the loss of β-glucans and chitin. The antifungal effect of the plant extract on various components of the cell wall of A. flavus, makes T. violacea aqueous plant extract an ideal chemotherapeutic agent against both human and plant pathogens of Aspergillus. The broad spectrum of antifungal activity of T. violacea against A. flavus also eliminates any chances of the fungus developing resistance towards it and would make it a candidate for use as a potential antifungal agent. Further identification and possible chemical synthesis is needed to shed light on the safety and efficacy of the active compound for further development as a chemotherapeutic agent.

Chemical and biological aspects of thiarubrine, a highly antifungal dithiacyclohexadiene polyine, were investigated. A tissue culture system for the production of thiarubrines was developed by culturing hairy roots of Chaenactis douglasii induced by Agrobacterium rhizogenes strain TR7. One culture line accumulated two times the levels of thiarubrines of nontransformed control root cultures, while maintaining rapid growth. The combination of fast growth and high thiarubrine accumulation could not be duplicated in controls by adding exogenous NAA to the culture medium. Hairy root cultures also produced less thiarubrine B relative to thiarubrine A compared to controls. Thiarubrine synthesis appears to be closely correlated with degree of tissue differentiation; it is suggested that it may be more practical to improve the growth rate of thiarubrine-producing root cultures by transformation rather than seek to induce synthesis in fast-growing suspension cultures. The biosynthetic relation between thiarubrines and the always co-occurring thiophenes was investigated by performing ³⁵S tracer experiments with C. douglasii hairy root cultures. It is possible that the thiophenes are not actively synthesized by the roots but rather are products of thiarubrine decomposition resulting from the extraction procedures and other manipulations of the cultures. The in vitro conversion of thiarubrine to thiophene can be induced by light, heat and other agents. No turnover of thiarubrines could be detected in the cultures in late logarithmic or stationary phases of the growth cycle. I Thiarubrines show strong light-independent antibacterial and antifungal activity. The mechanism of action of thiarubrine against E. coli and S. cerevisiae was investigated using comparative disk bioassays. A very similiar polyine from Rudbeckia hirta was as active as thiarubrine in the dark, indicating the central role of the disulfide ring in toxicity of the compounds. Visible light enhanced this activity suggesting that decomposition of the disulfide ring is important for its antibiotic effects. The photodegradation product, a thiophene, is phototoxic, probably via both type I and type II photosensitization mechanisms. The root culture extracts of Rudbeckia hirta yielded a new isomer of a known dithiacyclohexadiene polyine. MS and NMR analyses confirmed the cis configuration of this isomer. / Science, Faculty of / Botany, Department of / Graduate

The aim of this study was to investigate plant species to develop a product with the potential of protecting plants or plant products against plant fungal pathogens. Hexane, dichloromethane, acetone, and methanol leaf extracts of six plant species (Bucida buceras, Breonadia salicina, Harpephyllum caffrum, Olinia ventosa, Vangueria infausta and Xylotheca kraussiana) were evaluated for antifungal activity against seven plant fungal pathogens (Aspergillus niger, A. parasiticus, Colletotrichum gloeosporioides, Penicillium janthinellum, P. expansum, Trichoderma harzianum and Fusarium oxysporum). These plant species were selected from more than 400 plant species evaluated in the Phytomedicine Programme that had good activity against two animal fungal pathogens. All the leaf extracts were active against at least one or more of the phytopathogenic fungi in a serial microdilution assay. Of the six plant species, B. buceras had the best antifungal activity against four of the fungi, with MIC values as low as 0.02 mg/ml and 0.08 mg/ml against Penicillium expansum, P. janthinellum, Trichoderma harzianum and Fusarium oxysporum. The number of active compounds in the plant extracts was determined using bioautography with the above-mentioned plant pathogens. No active compounds were observed in some plant extracts against the fungal plant pathogens indicating possible synergism between metabolites responsible for the antifungal activity of the extract. B. salicina and O. ventosa were the most promising plant species, with at least three antifungal compounds. The antioxidant activities of plant extracts were determined using the qualitative method by spraying TLC chromatograms developed in three eluent systems BEA, CEF and EMW with 1, l-diphenyl -2 picrylhydrazyl (DPPH). The plant extracts of five of these species did not have a strong antioxidant activity. The methanol extract of X. kraussiana was the most active radical scavenger in the DPPH assay amongst the six medicinal plants screened. Based on good activity against Aspergillus niger and A. parasiticus, leaf extracts of the six plant species were also tested for antifungal activity against A. fumigatus, a very important animal fungal pathogen. The acetone extracts of B. buceras, B. salicina, V. infausta and X. kraussina had good antifungal activity against the animal pathogens, with MIC values ranging between 0.02 and 0.08 mg/ml. This indicates that crude extracts of these species may be more valuable in combating Aspergillus infections in animals than in humans. Based on the results discussed above, B. salicina was selected for in-depth study. Serial exhaustive extraction was used to extract plant material with solvents of increasing polarities namely, hexane, chloroform, acetone and MeOH. Amongst the four extractants, MeOH extracted the largest quantity of plant material 12.3% (61.5g), followed by acetone 5.6% (27.8 g), hexane 2.6% (12.8 g) and chloroform 2.1% (10.3 g). The chloroform fraction was selected for further work because it had the best antifungal activity against A. niger, C. gloeosporioides, P. janthinellum and T. harzianum and the bioautography assay showed the presence of several antifungal compounds in the chloroform fraction. Column chromatography was used in a bio-assay guided fractionation and led to isolation of four compounds. The antimicrobial activity was determined against seven plant pathogenic fungi and three bacteria, including the Gram-positive Staphylococcus aureus (ATCC 29213) and the Gram-negative Escherichia coli (ATCC 25922) and Pseudomonas aureus (ATCC 27853). The isolated compounds had good antifungal activity against A. parasiticus with an MIC of 10 μg/ml, while in other cases it ranged from 20 to 250 μg/ml. Amongst the four compounds tested, only three had a clear band, indicating that the growth of the pathogenic fungi was inhibited in the bioautography assay. Nuclear magnetic resonance spectroscopy (NMR) and mass spectroscopy (MS) were used for identification of isolated compounds. Only one compound was identified as the triterpenoid ursolic acid. Ursolic acid has been isolated from several plant species and has antifungal activity against Candida albicans (Shai et al. 2008). This is the first report on the isolation of antifungal compounds from leaves of Breonadia salicina. The other compounds isolated appeared to be mixtures of fatty acids based on mass spectroscopy and the structures were not elucidated. The cytotoxicity of acetone extracts and the four isolated compounds were determined against Vero cells using a tetrazolium-based colorimetric (MTT) assay. The acetone extract was selected based on good in vitro antifungal activity and was used in an in vivo fruit experiment. The acetone extract was less toxic toward the Vero cells with an LC50 of 82 μg/ml than ursolic acid and compound 4 which had LC50 values of 25 and 36 μg/ml respectively. Compounds 2 and 3 had low toxicity against the cells with LC50 values greater than 200 μg/ml. The potential use of the extract or isolated compound(s) against three plant fungal pathogens Penicillium expansum and P. janthinellum as well as P. digitatum (isolated from infected oranges) were tested after treating the oranges with the extract and ursolic acid. The model used gave good reproducible results. The concentration that inhibited growth correlated reasonably well with MIC values determined by serial microplate dilution. There were substantial differences in the susceptibility of the different isolates tested. The activity of ursolic acid was in the same order as that of the crude acetone leaf extract of B. salicina. The LC50 of the extract varied from 1 to 1.8 mg/ml. Penicillium digitatum was more resistant to amphotericin B in comparison to other Penicillium species. It has been reported that the fungus was resistant to the three fungicides: sodium ï-phenylphenate (ï-phenylphenol), imazalil, and thiabendazole used commercially in the fruit industry to reduce postharvest decay (Holmes and Eckert 1999). The toxicity of the extract to Vero cells was in the order of 10 times lower than the LC50 of the extracts to the fungal pathogens. Although much work still has to be done, there is good potential that a commercial product can be developed from an acetone leaf extract of B.salicina leaves, especially if the activity of this extract can be improved by removing inactive compounds. The results confirm the traditional use of B. salicina and demonstrate the potential value of developing biopesticides from plants. / Thesis (PhD)--University of Pretoria, 2009. / Paraclinical Sciences / unrestricted

Please read the abstract in the dissertation / Dissertation (MSc)--University of Pretoria, 2004. / Plant Science / unrestricted