Thesis (M. Phil.)--University of Hong Kong, 1982.

published_or_final_version / Botany / Master / Master of Philosophy

CoordenaÃÃo de AperfeÃoamento de Pessoal de NÃvel Superior / Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Periconia hispidula is an endophytic fungus isolated from dried leaves from semi-arid dunes of Bahia. The fungus was subjected to cultivation by varying nutritional factors in four different culture mediuns: MPD (malt, peptone and dextrose), BD (potato dextrose), BDL (potato, dextrose and yeast) and MntPL (mannitol, peptone and yeast), and analyzing the extracts on different incubation days (7, 14, 21, 28 days). A survey of primary cytotoxic activity was carried out from the extracts against tumor cell line of colon cancer (HCT-116). The MPD extracts - 28 days BDL - 28 days and BD - 21 days showed a promising cytotoxic activity and were preliminarily selected for the chemical study. The chromatographic fractionation of MPD-28 days extract resulted in the isolation of 11 secondary metabolites characterized as {4-chromanone, 6-hydroxy-(R) -methyl- (PS-1)}, {4-chromanone, 6-hydroxy-(S) -methyl- (PS-2)}, {E, 1-(2,5 dihydroxyphenyl) but-2-en-1-one (PS-3)}, {1-(2, 5 dihydroxyphenyl)-butan-1-one (PS-4)}, {Z-methyl-3-(3-hydroxyphenyl) propenoate (PS-5)}, modiolide A (PS-7), fusanolide B (PS-8), stagonolide E (PS-9), {(3R,4R)-3,4-dihydro, 3,4,8- trihdroxy, naphthalen-1(2H)-one (PS-11)}, {(4S) isosclerona (PS-12)}, furtermore PS-10 without structural characterization yet. The BDL-BD-28 days and 21 days extracts presented a chromatographic profile very similar to MPD-28 days extract, thus, the chromatographic fractionations of both extracts were targeted for isolation of substances absent in MPD-28 days. The 3,4 dihydoxy-benzoic acid was isolated just from the BD-extract 21 days, while the fractionation of the extract BDL 28days gave (PS-13 without structural characterization yet) as different. Among the isolated metabolites, {4-chromanone, 6-hydroxy-(S) -methyl- (PS-2)}, fusanolido B and PS10 (probably) showed were new compounds. The isolated compounds were shown to be inactive in cytotoxicity assays against strains of HCT-116 and MC-27 (breast adenocarcinoma). However, the 1- (2,5-dihydroxyphenyl) but-2-en-1-one showed strong inhibition with a MIC of 62.5 Âg/mL to be subjected to antimicrobial test against strains of fungi Candida Krusei (ATCC 142432TM) and Candida albicans (ATCC 10231TM) and CIM of 125 Âg/mL against Candida parapsilosis (ATCC 22019TM), while {4-chromanone, 6-hydroxy-(S) -methyl-}, Z-3-(3-hydroxyphenyl) propenoate methyl, modiolide A, estagonolide E, fusanolido B and 3R,4R-dihydro-3,4,8-trihydroxy-1-(2H) -naftalelone showed moderate activity with MIC of 500 Âg / mL.Usual chromatographic techniques including liquid-liquid partitioning, flash chromatography and high pressure liquid chromatography (HPLC) were used for the isolation of secondary metabolites, while the structural characterization was possible through the use of spectrometric techniques using infrared (IR), mass spectrometry (MS), and uni and bidimensional techniques of nuclear magnetic resonance (NMR), and comparison with literature data. / Periconia hipidula à um fungo endofÃtico isolado de folhas secas provenientes de dunas do semiÃrido do estado da Bahia. O fungo foi submetido ao cultivo atravÃs da variaÃÃo de fatores nutricionais em quatro meios diferentes: MPD (malte, peptona e dextrose), BD (batata, dextrose), BDL (batata, dextrose e levedura) e MntPL (manitol, peptona e levedura), e anÃlise dos extratos em diferentes dias de incubaÃÃo (7, 14, 21, 28 dias). A prospecÃÃo da atividade citotÃxica preliminar foi realizada a partir dos extratos obtidos frente à linhagem de cÃlulas tumorais de cÃncer de cÃlon (HCT-116). Os extratos MPD â 28 dias, BDL - 28 dias e BD - 21 dias apresentaram uma atividade citotÃxica promissora e foram preliminarmente selecionados para o estudo quÃmico. O fracionamento cromatogrÃfico do extrato MPD-28 dias, resultou no isolamento de 11 metabÃlitos secundÃrios caracterizados como {(R), 6-hidroxi-2-metil, 4-cromanona (PS-1)}, { (S), 6-hidroxi-2-metil, 4-cromanona (PS-2)}, {E, 1-(2,5 diidroxi-fenil) but-2-en-1-ona (PS-3)}, {1-(2, 5 diidroxi-fenil)-butan-1-ona (PS-4)}, Z-3-(3 hidroxifenil) propenoato de metila (PS-5), modiolido A (PS-7), fusanolido B (PS-8), estagonolido E (PS-9), {(3R,4R)-3,4diidro,3,4,8 triidroxi,naftalen-1-(2H)-ona (PS-11)}, (4S)-isosclerona (PS-12), alÃm de PS-10 que se encontram em fase de caracterizaÃÃo estrutural. Os extratos BDL-28 dias e BD-21 dias apresentaram um perfil cromatogrÃfico bastante semelhante ao extrato MPD-28 dias, desta forma, os fracionamentos cromatogrÃficos de ambos os extratos foram direcionados para o isolamento de substÃncias ausentes em MPD-28 dias. O Ãcido 3,4 diidroxi-benzÃico (PS-6) foi isolado apenas do extrato BD-21 dias, enquanto que o fracionamento do extrato BDL 28d forneceu PS-13 (em fase de caracterizaÃÃo estrutural) como diferente. Dentre os metabÃlitos isolados, os compostos (2S)-6-hidroxi-2-metil-4-cromanona, fusanolido B apresentaram carÃter inÃdito na literatura. Os compostos isolados mostraram-se inativos em ensaios de atividade citotÃxica frente a cepas de HCT-116 e MC-27 (adenocarcinoma de mama). No entanto, o composto 1-(2,5-diidroxifenil)-but-2-en-1-ona apresentou elevada inibiÃÃo com CIM de 62,5 Âg/mL ao ser submetido à ensaio antimicrobiano frente a cepas de fungos Candida Krusei (ATCC 142432TM) e Candida albicans (ATCC 10231TM) e 125 Âg/mL frente à Candida parapsilosis (ATCC 22019TM), enquanto que o (2R)-6-hidroxi-2-metil-4-cromanona, Z-3-(3-hidroxifenil)-propenoato de metila, o modiolido A, o estagonolido E, o fusanolido B e a 3,4-diidro- 3,4,8-triidroxi-1(2H)-naftalelona apresentaram moderada atividade com CIM de 500 Âg/mL. TÃcnicas cromatogrÃficas usuais, incluindo partiÃÃo lÃquido-lÃquido, coluna de sÃlica flash e cromatografia de alta eficiÃncia (CLAE) foram utilizadas para o isolamento dos metabÃlitos secundÃrios, enquanto que a caracterizaÃÃo estrutural foi possÃvel atravÃs do uso de tÃcnicas espectromÃtricas utilizando infravermelho (IV), espectrometria de massa (EM) e ressonÃncia magnÃtica nuclear (RMN) com experimentos uni e bidimensionais, alÃm de comparaÃÃo com dados da literatura.

NASCIMENTO, Hélio Oliveira do. Estudo do potencial químico e citotóxico dos metabólitos secundários do fungo endofítico Periconia hispidula. 2016. 154 f. Dissertação (Mestrado em Química)-Universidade Federal do Ceará, Fortaleza, 2016. / Submitted by Jairo Viana (jairo@ufc.br) on 2017-08-01T20:21:47Z No. of bitstreams: 1 2016_dis_honascimento.pdf: 8663458 bytes, checksum: 7310ad2651bf300ddf8cd835199d769a (MD5) / Approved for entry into archive by Jairo Viana (jairo@ufc.br) on 2017-08-01T20:22:06Z (GMT) No. of bitstreams: 1 2016_dis_honascimento.pdf: 8663458 bytes, checksum: 7310ad2651bf300ddf8cd835199d769a (MD5) / Made available in DSpace on 2017-08-01T20:22:06Z (GMT). No. of bitstreams: 1 2016_dis_honascimento.pdf: 8663458 bytes, checksum: 7310ad2651bf300ddf8cd835199d769a (MD5) Previous issue date: 2016 / Periconia hipidula é um fungo endofítico isolado de folhas secas provenientes de dunas do semiárido do estado da Bahia. O fungo foi submetido ao cultivo através da variação de fatores nutricionais em quatro meios diferentes: MPD (malte, peptona e dextrose), BD (batata, dextrose), BDL (batata, dextrose e levedura) e MntPL (manitol, peptona e levedura), e análise dos extratos em diferentes dias de incubação (7, 14, 21, 28 dias). A prospecção da atividade citotóxica preliminar foi realizada a partir dos extratos obtidos frente à linhagem de células tumorais de câncer de cólon (HCT-116). Os extratos MPD – 28 dias, BDL - 28 dias e BD - 21 dias apresentaram uma atividade citotóxica promissora e foram preliminarmente selecionados para o estudo químico. O fracionamento cromatográfico do extrato MPD-28 dias, resultou no isolamento de 11 metabólitos secundários caracterizados como {(R), 6-hidroxi-2-metil, 4-cromanona (PS-1)}, { (S), 6-hidroxi-2-metil, 4-cromanona (PS-2)}, {E, 1-(2,5 diidroxi-fenil) but-2-en-1-ona (PS-3)}, {1-(2, 5 diidroxi-fenil)-butan-1-ona (PS-4)}, Z-3-(3 hidroxifenil) propenoato de metila (PS-5), modiolido A (PS-7), fusanolido B (PS-8), estagonolido E (PS-9), {(3R,4R)-3,4diidro,3,4,8 triidroxi,naftalen-1-(2H)-ona (PS-11)}, (4S)-isosclerona (PS-12), além de PS-10 que se encontram em fase de caracterização estrutural. Os extratos BDL-28 dias e BD-21 dias apresentaram um perfil cromatográfico bastante semelhante ao extrato MPD-28 dias, desta forma, os fracionamentos cromatográficos de ambos os extratos foram direcionados para o isolamento de substâncias ausentes em MPD-28 dias. O ácido 3,4 diidroxi-benzóico (PS-6) foi isolado apenas do extrato BD-21 dias, enquanto que o fracionamento do extrato BDL 28d forneceu PS-13 (em fase de caracterização estrutural) como diferente. Dentre os metabólitos isolados, os compostos (2S)-6-hidroxi-2-metil-4-cromanona, fusanolido B apresentaram caráter inédito na literatura. Os compostos isolados mostraram-se inativos em ensaios de atividade citotóxica frente a cepas de HCT-116 e MC-27 (adenocarcinoma de mama). No entanto, o composto 1-(2,5-diidroxifenil)-but-2-en-1-ona apresentou elevada inibição com CIM de 62,5 µg/mL ao ser submetido à ensaio antimicrobiano frente a cepas de fungos Candida Krusei (ATCC® 142432TM) e Candida albicans (ATCC® 10231TM) e 125 µg/mL frente à Candida parapsilosis (ATCC® 22019TM), enquanto que o (2R)-6-hidroxi-2-metil-4-cromanona, Z-3-(3-hidroxifenil)-propenoato de metila, o modiolido A, o estagonolido E, o fusanolido B e a 3,4-diidro- 3,4,8-triidroxi-1(2H)-naftalelona apresentaram moderada atividade com CIM de 500 µg/mL. Técnicas cromatográficas usuais, incluindo partição líquido-líquido, coluna de sílica flash e cromatografia de alta eficiência (CLAE) foram utilizadas para o isolamento dos metabólitos secundários, enquanto que a caracterização estrutural foi possível através do uso de técnicas espectrométricas utilizando infravermelho (IV), espectrometria de massa (EM) e ressonância magnética nuclear (RMN) com experimentos uni e bidimensionais, além de comparação com dados da literatura. / Periconia hispidula is an endophytic fungus isolated from dried leaves from semi-arid dunes of Bahia. The fungus was subjected to cultivation by varying nutritional factors in four different culture mediuns: MPD (malt, peptone and dextrose), BD (potato dextrose), BDL (potato, dextrose and yeast) and MntPL (mannitol, peptone and yeast), and analyzing the extracts on different incubation days (7, 14, 21, 28 days). A survey of primary cytotoxic activity was carried out from the extracts against tumor cell line of colon cancer (HCT-116). The MPD extracts - 28 days BDL - 28 days and BD - 21 days showed a promising cytotoxic activity and were preliminarily selected for the chemical study. The chromatographic fractionation of MPD-28 days extract resulted in the isolation of 11 secondary metabolites characterized as {4-chromanone, 6-hydroxy-(R) -methyl- (PS-1)}, {4-chromanone, 6-hydroxy-(S) -methyl- (PS-2)}, {E, 1-(2,5 dihydroxyphenyl) but-2-en-1-one (PS-3)}, {1-(2, 5 dihydroxyphenyl)-butan-1-one (PS-4)}, {Z-methyl-3-(3-hydroxyphenyl) propenoate (PS-5)}, modiolide A (PS-7), fusanolide B (PS-8), stagonolide E (PS-9), {(3R,4R)-3,4-dihydro, 3,4,8- trihdroxy, naphthalen-1(2H)-one (PS-11)}, {(4S) isosclerona (PS-12)}, furtermore PS-10 without structural characterization yet. The BDL-BD-28 days and 21 days extracts presented a chromatographic profile very similar to MPD-28 days extract, thus, the chromatographic fractionations of both extracts were targeted for isolation of substances absent in MPD-28 days. The 3,4 dihydoxy-benzoic acid was isolated just from the BD-extract 21 days, while the fractionation of the extract BDL 28days gave (PS-13 without structural characterization yet) as different. Among the isolated metabolites, {4-chromanone, 6-hydroxy-(S) -methyl- (PS-2)}, fusanolido B and PS10 (probably) showed were new compounds. The isolated compounds were shown to be inactive in cytotoxicity assays against strains of HCT-116 and MC-27 (breast adenocarcinoma). However, the 1- (2,5-dihydroxyphenyl) but-2-en-1-one showed strong inhibition with a MIC of 62.5 µg/mL to be subjected to antimicrobial test against strains of fungi Candida Krusei (ATCC® 142432TM) and Candida albicans (ATCC® 10231TM) and CIM of 125 µg/mL against Candida parapsilosis (ATCC® 22019TM), while {4-chromanone, 6-hydroxy-(S) -methyl-}, Z-3-(3-hydroxyphenyl) propenoate methyl, modiolide A, estagonolide E, fusanolido B and 3R,4R-dihydro-3,4,8-trihydroxy-1-(2H) -naftalelone showed moderate activity with MIC of 500 µg / mL.Usual chromatographic techniques including liquid-liquid partitioning, flash chromatography and high pressure liquid chromatography (HPLC) were used for the isolation of secondary metabolites, while the structural characterization was possible through the use of spectrometric techniques using infrared (IR), mass spectrometry (MS), and uni and bidimensional techniques of nuclear magnetic resonance (NMR), and comparison with literature data.

Doctor of Philosophy / Department of Biology / Ari M. Jumpponen / Dark septate endophytes (DSE) are darkly pigmented microfungal ascomycetes commonly observed in the healthy plant roots. Studying the functional roles of DSE is challenging as fundamental information about their identity, nutritional requirements, host range or host preference are lacking. Objective 1: root colonizing fungi were isolated from Konza plants roots and DSE fungi were identified by testing Koch’s postulates using leek plants. Periconia macrospinosa and Microdochium sp., were identified as DSE as they produced microsclerotia and chlamydospores in the root cortex. Select DSE were tested for their enzymatic capabilities and ability to utilize nitrogen sources: fungi tested positive for amylase, cellulase, polyphenol oxidases and gelatinase. Periconia isolates utilized organic and inorganic nitrogen suggesting facultative biotrophic and saprotrophic habits. Objective 2: a Microdochium isolate and three Periconia isolates were screened on 16 plant species (six native grasses and forbs, four crops) in a resynthesis system to test host range. DSE colonized all plant species, albeit to varying degrees. Host biomass and nutritional levels to DSE colonization varied within and among host species confirming the broad host range. Based on % responsiveness to DSE colonization, a metric similar to ‘mycorrhizal dependency’, grasses responded positively, while forbs and crops responded negatively. To test this observed ‘host preference’ under natural conditions, Konza roots from seven grass and nine forb species were surveyed for DSE colonization. Grasses hosted 50% greater DSE than forbs, supporting the broad host range and host preference of DSE fungi. Objective 3: three conspecific Arabidopsis ecotypes, Col-0, Cvi-0 and Kin-1 were inoculated with 25 P. macrospinosa isolates in resynthesis system. The three ecotypes responded differently to inoculation: Col-0 and Cvi-0 responded negatively, while Kin-1 response was neutral. Despite the negative or neutral response, each ecotype responded positively to one or two isolates. The outcomes were along the mutualism-parasitism continuum precluding an unambiguous assignment to any particular life-style. This study shows that the outcomes along this continuum are dictated by host and fungal genotypes. However, the more important question about their function remains. Additional studies with Arabidopsis microarrays are likely to provide unique insights into the potential roles of DSE.