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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
101

Bioprospecção de biomoléculas isoladas de fungos endofíticos de Combretum leprosum do bioma Caatinga / Bioprospection of biomolecule isolated from endophytic fungal of Combretum leprosum from Caatinga biome

Suikinai Nobre Santos 03 September 2012 (has links)
Os micro-organismos que habitam o interior das plantas (endofíticos ou endófitos) tornaram-se foco de interesse por estarem envolvidos na produção de compostos químicos como enzimas, alcalóides, antibióticos, anticancerígenas e diferentes metabólitos. Os ecossistemas de regiões tropicais tem sido alvo de busca de compostos naturais por causa da riqueza de espécies e nichos ecológicos presentes nestas comunidades. O objetivo deste trabalho o isolamento, identificação e a bioprospecção de fungos endofíticos obtidos de Combretum leprosum e a detecção nos extratos de planta e micro-organismos da presença do composto combretastatin (CA4). Folhas, galhos, frutos e raízes de C. leprosum foram coletados de cinco estados dentro da zona de semiárido brasileiro: Bahia, Piauí, Ceará, Paraíba, Rio Grande do Norte. Partes das amostras foram triturados e submetidos à maceração primeiramente em diclorometano, seguidos de tetrahidrofurano e acetona de acordo com Pettit et al.(1987) para possível extração da CA4. Além disso, para avaliação in vitro da atividade citotóxica e antimicrobiana foram realizadas extrações em acetato de etila, clorofórmio e metanol. Foram detectados a possível presença da CA4 em todos os órgãos das plantas extraídos com tetrahidrofurano e as maiores concentrações foram observadas nas folhas. A atividade antitumoral dos extratos vegetais apresentaram as maiores inibições contra carcinoma (ovário IC50 10µg/mL-1, rim IC50 8,7µg/mL-1 e mama IC50 14,1µg/mL-1) e glioma.IC50 13,5µg/mL-1. A outra parte das amostras (folhas, caules e raízes) foram desinfetadas, fragmentadas e colocadas em meios de cultivo (Martin, BDA, Agar água) por 60 dias, 28°C. Foram isolados 405 fungos endofíticos e 159 apresentaram atividade contra fitopatogênicos, 72% para Rhizoctonia solani e 28% para Pythium aphanidermatum. As vinte e três linhagens que apresentaram as melhores atividades antifitopatogênicas foram submetidas a crescimento em Czapec em cultura estacionaria, por 30 dias, a 28°C, os respectivos metabólitos foram obtidos em múltiplo (3.0 e 11.0) e avaliados a atividade antimicrobiana contra bactérias patogênicas e fungos. Quatro linhagens foram selecionadas, identificadas pelo sequenciamento da região 18S, CFE177 como Fusarium oxysporum, CFE03 como Hypocrea koningii, linhagem CFE108 como Aspesgillus oryzae e CFE391 como Fusarium solani e avaliadas in vitro pelos testes biológicos: atividade antitumoral, antioxidante e antimicobactéria. Os compostos produzidos por A. oryzae CFE108 apresentaram potencial para bioprospecção, e de acordo com as atividade citotóxicas as maiores ações foram contra as linhagens linfoma histiocística (J744), mieloma murino (B16F10) e baixa citotóxidade para carcinoma de bexiga (ECV304) e leucemia eritroblástica humana (k562) na concentração de 1mg/mL-1. Foram isolados dois compostos: SS-XL-32-01 identificado como bis-(2-etilhexil) ftalato (DEHP) e SS-XL-20-1 identificado como fenol, 2.2 metilenobis[6-(1,1-dimetiletil)-4- etil], ambos com atividade anticâncer para células HeLa com percentual de ate 98% e 71%, de morte, respectivamente. Alem disso, a modificação através da reação de metilação do composto SS-XL-32-1 resultou na quebra do anel aromático, formação de 4 subprodutos e perda da atividade, sendo um indicativo do sitio ativo da molécula responsável pela atividade observada. Portanto, fungos endofíticos de 18 plantas do semiárido brasileiro podem ser considerados fonte de bioprospecção para novas moléculas bioativas com atividade antitumoral. / The micro-organisms that reside in the aerial tissues and roots of plants (endophytic or endophyte) became the focus of interest for being involved in the chemical production such as enzymes, alkaloids, antibiotics, anticancer and different metabolites. The ecosystems of tropical region have been targeted search of natural compounds because of the richness of species and ecological niches present in these communities. The aim of this work was the isolation, identification and bioprospection for endophytic fungi from Combretum leprosum and detection in extracts of the plant and micro-organisms for the presence of the combretastatin (CA4). Leaves, stems, fruits and roots of C. leprosum were collected from five states within the semi-arid zone of Brazil: Bahia, Piaui, Ceara, Paraiba, Rio Grande do Norte. Part of the samples were crushed and subjected to maceration in dichloromethane, followed by tetrahydrofuran and acetone according to Pettit et al. (1987) for extracting the possible CA4. Moreover, for in vitro evaluation of the cytotoxic and antimicrobial activity extractions were carried out in ethyl acetate, chloroform and methanol. Were detected the possible presence of CA4 all plant organs extracted with tetrahydrofuran and the highest concentrations were observed on the leaves. The antitumor activity of plant extracts showed the highest inhibition against carcinoma (ovary IC50 10µg/mL-1, kidney IC50 8.7 µg/mL-1 and breast IC50 14.1 µg/mL-1) glioma IC50 and 13.5 mg-/mL-1. The other part of the samples (leaves, stems and roots) were disinfected, fragmented and placed in culture media (Martin, PDA, water agar) for 60 days, 28°C. 405 Endophytic fungi were isolated and 159 showed activity against phytopathogenic, 72% for Rhizoctonia solani and 28% for Pythium aphanidermatum. Twenty-three strains that showed good activities antiphytopathogenic, were grow on medium Czapec in static culture, for 30 days at 28°C, the respective metabolites were obtained in multiples pH (3.0 and 11.0) and evaluated the antimicrobial activity against pathogenic bacteria and fungi. Four strains were selected, identified by sequencing the 18S region, CFE177 as Fusarium oxysporum, CFE03 as Hypocrea koningii, strain CFE108 as Aspesgillus oryzae and CFE391 Fusarium solani, and evaluated by in vitro biological tests: antitumor, antioxidant and antimicobactérium activity. The compounds produced by A. oryzae CFE108 had biological potential and in accordance with the cytotoxic activity, showed the highest activities against lymphoma lines (J744), murine myeloma (B16F10) and low cytotoxicity for carcinoma of the bladder (ECV304) and leukemia erythroblastic human (K562) in 1mg/mL-1 concentration. Two compounds were isolated: SS-XL-32- 01 identified as bis-(2-ethylhexyl)phthalate (DEHP), and SS-XL-20-1 as phenol 2.2methylenobis [6-(1,1-dimethylethyl) - 4-ethyl], both with anticancer activity for HeLa cells with a percentage of up to 98% and 71%, of death, respectively. In addition, modified by methylation reaction of the compound SS-XL-32-1 resulted in the breaking of the aromatic ring and result in formation of four product and loss of activity being indicative of the active site of the molecule can be the aromatic ring. Therefore, endophytic fungi in semiarid Brazil plant can be considered a source of bioprospection for new bioactive molecules with anticancer activity.
102

Études phytochimique, cytotoxique et antibactérienne de champignons endophytes issus de plantes médicinales du Soudan / Phytochemical study, cytotoxic and antibacterial potentialities of endophytic fungi from medicinal plants from Sudan

Khiralla, Afra 16 September 2015 (has links)
Pour la première fois, l’étude de la flore fongique endophytique de cinq plantes médicinales soudanaises : Calotropis procera (Ait.), Catharanthus roseus (L.), Euphorbia prostrata (Ait.), Trigonella foenum-graecum (L.), and Vernonia amygdalina (Del.) a été réalisée. Un total de 23 souches de champignons endophytes ont été isolées à partir des plantes après la stérilisation de surface puis les différentes analyses biologiques ont été effectuées. Les extraits bruts d’acétate d’éthyle de 21 endophytes ainsi que de leurs plantes hôtes ont été évalués pour leur teneur en phénols totaux et leur activité antioxydante en utilisant respectivement la méthode colorimértrique Folin-Ciocalteu et le piégeage des radicaux libres par la méthode 1,1,-diphényl-2-picrylhydrazil (DPPH) in vitro. Une évaluation générale de la cytotoxicité de 16 endophytes sélectionnés ainsi que de leurs plantes hôtes a été réalisée selon le test MTT sur trois types de cellules cancéreuses : carcinome du sein humain (MCF7), adénocarcinome du côlon (HT29 et HCT116). Ces extraits ont été aussi testés, selon la méthode de dilution en bouillon, sur deux souches bactériennes représentatives, Escherichia coli et la souche résistante à la méthicilline de Staphylococcus aureus. La teneur en phénols totaux (89,9 ±7,1 mg Equivalent d’Acide Gallique EAG/g) ainsi que l’activité antioxydante (IC50: 18±0,1 µg/mL) les plus élevées ont été observées pour l’endophyte, Aspergillus terreus 2 isolé à partir des graines de T. foenum-graecum. Byssochlamys spectabilis a montré l’activité cytotoxique la plus importante (1,51 ± 0,2 µg/mL), suivi par Cladosporium cladosporioides 2 (10,5 ± 1,5 µg/mL), puis par Alternaria sp. (13,5 ± 1,8 µg/mL). Seules six souches ont montré une activité contre S. aureus avec des valeurs de MIC qui se situent entre 0,125 et 2 mg/mL dont: Alternaria alternata (0,125 mg/mL), Alternaria sp. (0,250 mg/mL), Byssochlamys spectabilis (0,5 mg/mL). 10 composés purs (0,3 à 40 mg) ont été isolés à partir des extraits bruts d’acétate d’éthyle de Curvularia papendorfii. Le nouveau composé pur (AFB) 3,7,11,15-Tetrahydroxy-18-hydroxymethyl-14,16,20,22,24-pentamethyl-hexacosa-4E,8E,12E,16,18-pentaenoic acid (acide Khartomique) a montré une activité antibactérienne modérée contre S. aureus avec une CIM de 62,5 µg/mL et une faible activité cytotoxique sur les cellules MCF7 avec une IC50 > 100 µM. Le composé pur AF1 a montré une activité cytotoxique modérée sur les cellules HT29 avec une IC50 de 29,78 µM et une très faible activité antibactérienne contre S. aureus. Ces deux composés ne présentent pas d’activité antioxydante. / This study investigated, for the first time, the endophytic fungi flora of five Sudanese medicinal plants: Calotropis procera (Ait.), Catharanthus roseus (L.), Euphorbia prostrata (Ait.), Trigonella foenum-graecum (L.) and Vernonia amygdalina (Del.). A total of 23 endophytic fungal strains were isolated from the plants after surface disinfection and different biological tests were performed. Total phenolic content (TPC) and total antioxidant activity of ethyl acetate crude extracts of 21 endophytes and their host plants were estimated using respectively the Folin-Ciocalteu colorimetric method and 1,1,-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging in vitro method. General evaluation of the cytotoxicity of 16 selected endophytes and their host plants was performed by the MTT assay using cancer cells type: Human breast carcinoma (MCF7) and Colon adenocarcinoma (HT29 and HCT116). Preliminary antibacterial screening was done for the 16 endophytes. These extracts were also tested against two representative bacterial strains, Escherichia coli and methicillin-resistant Staphylococcus aureus, by broth dilution tests. The endophyte, Aspergillus terreus 1 from T. foenum-graecum seeds had the highest TPC in term of Gallic Acid Equivalent (89.9 ± 7.1 mg GAE/g) and antioxidant activity (IC50: 18±0.1µg/mL). Byssochlamys spectabilis showed strong cytotoxicity (1.51 ± 0.2 µg/mL) followed by Cladosporium cladosporioides 2 (10.5 ± 1.5 µg/mL), then Alternaria sp. (13.5 ± 1.8 µg/mL). Only six strains showed activity against methicillin-resistant S. aureus with MIC values ranging between 0.125-2 mg/mL, Alternaria alternata (0.125 mg/mL) Alternaria sp. (0.250 mg/mL) and Byssochlamys spectabilis values (0.5 mg/mL). Ten pure compounds (0.3 to 40 mg) were isolated from ethyl acetate crude extract of Curvularia papendorfii .The new pure compound (AFB) 3,7,11,15-Tetrahydroxy-18-hydroxymethyl-14,16,20,22,24-pentamethyl-hexacosa-4E,8E,12E,16,18-pentaenoic acid (Khartoumic acid) revealed moderate antibacterial activity against S. aureus with MIC value 62.5 µg/mL and weak cytotoxicity with a IC50 > 100 µM against MCF7 cells. The pure compound AF1 showed moderate cytotoxic activity with IC50 value of 29.78 µM against HT29 and weak antibacterial activity with MIC 250 µg/mL against S. aureus. Both compounds displayed no antioxidant activity.
103

Isolation And Identification of Tropane Alkaloid Producing Endophytic Fungi from Datura Metel L., And Studies on Colletotrichum Boninense Recombinant Putrescine N-mehtyltransferase

Naik, Tanushree January 2016 (has links) (PDF)
Datura metel is a herbaceous plant found in almost all tropical parts of the world. It belongs to the family Solanaceae whose members, viz. Duboisia, Atropa, Hyoscyamus and Datura plants are known to produce tropane alkaloids- hyoscyamine and scopolamine which are most noted for their therapeutic use as anti-cholinergic agents. Since these alkaloids are produced in very low amounts in plants, alternative sources and methods of production for these alkaloids have been crucial in meeting the demands for these drugs. Endophytic fungi inhabiting a plant may have the potential to produce the same compounds as the host plants. The aim of the present study was to search for tropane alkaloid producing endophytic fungal isolates from Datura metel. Eighteen endophytic fungi were isolated from various tissues of Datura metel and screened for the presence of three tropane alkaloid biosynthetic genes- putrescine N-methyltransferase (PMT), tropinone reductase I (TRI) and hyoscyamine 6β-hydroxylase (H6H) using PCR-based screening approach. Six endophytic fungal isolates were found to possess the PMT, TR1 and H6H genes. The fungi were identified using molecular taxonomy as Col letotrichum boninense, Phomopsis sp., Fusarium solani, Col letotrichum incarnatum, Col letotrichum siamense and Col letotrichum gloeosporioides and the identity was confirmed using colony and spore morphology. The production of tropane alkaloids hyoscyamine and scopolamine by the fungi has been ascertained using various techniques like TLC, HPLC and ESI-MS/MS by comparison with the authentic reference standards. The amount of tropane alkaloids produced by all six fungi in liquid cultures was quantified using HPLC analysis. Among the six tropane alkaloid-producing fungi Col letotrichum incarnatum gave the highest yields of hyoscyamine and scopolamine which were 3.906 mg/L and 4.13 mg/L, respectively. With an aim to characterize the tropane alkaloid biosynthetic genes in these fungi, the PMT gene was isolated from five of the endophytic fungi- Col letotrichum boni-nense, Fusarium solani, Col letotrichum incarnatum, Col letotrichum siamense and Col-letotrichum gloeosporioides for the first time and the sequence analysis showed high ho-mology (98%) to the Datura metel PMT cDNA. The gene was found to be devoid of introns in the fungi. Further phylogenetic analysis of the full length PMT sequence from the fungi strongly supports the hypothesis of horizontal gene transfer between the host plant and endophytic fungi. For further in detail characterization of fungal PMT, the Col letotrichum boninense PMT gene was taken as a representative. CbPMT gene was cloned in pRSET A expres-sion vector and heterologously expressed in E. coli and biochemically characterized. For optimal yield of soluble protein upon heterologous expression different conditions such as IPTG concentration, temperature and time post induction were optimized. Optimal yield was obtained by inducing the culture by 0.25 mM IPTG once it had reached and O.D. of 0.6 and incubating at 37◦ C for 3 h. The recombinant CbPMT enzyme expressed as histidine tagged fusion protein was purified using Ni-NTA affinity chromatography. Gel elution studies were carried out to determine molecular weight of the protein and it was found that the protein exists as a homodimer in solution with some amount also present as a monomer. Catalytic activity of the purified recombinant enzyme was studied for its dependence on both substrates putrescine as well as S-adenosylmethionine (SAM). The Km and Vmax values for putrescine were found to be 464 µM and 18.55 nkat/mg, respectively, while those for S-adenosylmethionine were found to be 628 µM and 18.63 nkat/mg, respectively. Optimum temperature for activity was found to be 37◦ C and optimum pH range was found to be 8-9. Fluorescence spectroscopy was used to study the binding affinity of both the sub-strates to the enzyme. Fluorescence quenching data for each substrate was analysed by using a nonlinear regression curve fit and Kd values were found to be 0.309 mM for pu-trescine and 0.118 mM for SAM, respectively. Circular dichroism spectrum of the enzyme indicated a pattern typical for alpha helix in the secondary structure. Binding of either substrate led to increase in ellipticity of the protein. Fluorescence quenching studies with collisional quenchers- acrylamide, potassium iodide, and cesium chloride indicated that the native protein is folded in a conformation that allows tryptophan residues to be acces-sible for quenching. The fraction of tryptophan residues (fa ) accessible for quenching by acrylamide (1.06) was found to be higher than that for potassium iodide (0.54) while that cesium ions was the least (0.38). The neutral quencher acrylamide could access all the tryptophans meaning that none of tryptophans are completely buried inside hydrophobic cores. the differential accessibility to the charged quenchers, however, indicates that more of the tryptophans are surrounded by positively charged amino acids. The unfolding of the protein was studied with the aid of chaotropic agents guanidine-HCl and urea and thermodynamic parameters were determined. The denaturant m-values were found to be 2.313 kcal/mol/M for Gdn-HCl and 2.345 kcal/mol/M for urea respectively. The free energy of unfolding was estimated to be 2.635 kcal/mol for Gdn-HCl and 4.630 kcal/mol for urea. Since no reports are available about the thermodynamics of folding and unfolding of PMT from any plant source, this study contributes towards the understanding of protein stability. Although a lot of reports are available on the biochemical characterization of PMT from different plant sources, the crystal structure of PMT is not yet available. In the current work, homology based modelling studies on CbPMT were carried out to get some idea about the protein tertiary structure. Homology based modelling studies showed that a significant amount of protein is present as α-helices which are present on the surface while the β-sheets are present in the interior of the protein. Each monomer of the protein is capable of binding both the substrates and hence the dimerization property of the enzyme could be a purely structural one leading to more stability and solubility of the protein. In conclusion, this study has shown for the first time that endophytic fungi have significant potential to be used for tropane alkaloid production and six such fungal strains have been identified. Although the production of tropane alkaloids by endophytic fungi is not very high, it can be scaled up by over-expressing the biosynthetic gene putrescine N-methyltransferase in the highest producer- Col letotrichum incarnatum to further increase the yield. These endophytic fungi have significant potential to be applied in fermentation technology to meet the demands for these drugs economically.
104

Isolamento e identificação de fungos endofíticos de Abarema cochliocarpos (Gomes) Barneby & Grimes e Dalbergia ecastophyllum (L.) Taub., suas relações antagônicas e atividade antioxidante de seus extratos / Isolation and identification of Abarema cochliocarpos (Gomes) Barneby & Grimes and Dalbergia ecastophyllum (L.) Taub. their antagonism and antioxidant activity of their extracts

Silva, Ricardo Manoel dos Santos 11 May 2011 (has links)
The search for new substances of pharmaceutical interest in the area or even in the most diverse agricultural industry uses, has led researchers to seek alternatives before not properly explored. One such alternative would be to isolate the microorganisms that inhabit plant asymptomatically to produce new substances, studies have shown that these microorganisms, called endophytes, through the coevolution of host plants, has given them many benefits such as protection and resistance pest attack. In parallel, it is seen that many compounds produced by host plants, can also be produced by these microorganisms, and thus its industrial production could be facilitated. This study aimed to isolate and identify fungal endophytes from two species A. cochliocarpos and D. ecsatophyllum popularly known as barbatimão and rabo – de- bugio, respectively, and to examine its antagonism between fungi isolated and the antioxidant activity are also the focus of this work. Endophytic fungi were isolated from six. Of these, five were identified as two of the genus Phomopsis fungi, a fungus of the genus Guignardia and one identified as being of order Pleosporales, the fungus was identified as the fifth genus Nigrospora. One of the fungus could not be identified by the technique used and was called DeF2. The fungi grew better when kept in dark on PDA growth medium and BSA in the laboratory. As antagonists, we observed that the isolates coming from the host D. ecstophyllum were the most efficient and that one gender may have different behaviors depending on the host that originated. From experiments with mycelia of these fungi were obtained organic extrados of different polarities, and the analysis of the antioxidant activity of these extracts showed that the fungus Phomopsis sp. isolated from D. ecastophyllum presented extracts with antioxidant activity than those of other fungi analyzed. / Fundação de Amparo a Pesquisa do Estado de Alagoas / A procura de novas substâncias de interesse na área farmacêutica ou mesmo agroindústria nos mais variados usos, tem levado os pesquisadores a buscar alternativas antes não devidamente exploradas. Uma dessas alternativas seria o isolamento de microrganismos que habitam vegetais de forma assintomática para a produção novas substâncias, estudos tem demonstrado que estes microrganismos, chamados de endofíticos, através da coevolução em suas plantas hospedeiras, tem conferido a elas diversos benefícios como proteção e resistência ao ataque de pragas. Paralelamente, é visto que muitos compostos produzidos pelas plantas hospedeiras, também podem ser produzidos por esses microrganismos, e com isso a sua produção industrial poderia ser facilitada. Este trabalho teve como objetivo isolar e identificar fungos endofíticos de duas espécies vegetais a A. cochliocarpos e a D. ecastophyllum conhecidas popularmente como barbatimão e rabo-de-bugio, respectivamente, bem como analisar suas inter-relações entre os fungos isolados e a atividade antioxidante também são objetivos deste trabalho. Foram isolados seis fungos endofíticos. Destes, cinco foram identificados como sendo dois fungos do gênero Phomopsis, um fungo do gênero Guignardia e um identificado como sendo da ordem Pleosporales, o quinto fungo identificado foi do gênero Nigrospora. Um dos fungos não pôde ser identificado pela técnica utilizada e foi chamado de DeF2. Os fungos tiveram crescimento superior quando mantidos em ambiente escuro e em meio de cultura BDA e BSA em laboratório. Como antagonistas, foi possível observar que os isolados provindos do hospedeiro D. ecstophyllum foram os mais eficientes e que um mesmo gênero pode apresentar comportamentos diferenciados dependendo do hospedeiro que foi originado. A partir de experimentos realizados com micélios destes fungos foi possível obter extrados orgânicos de diferentes polaridades e, a análise da atividade antioxidante destes extratos, mostrou que o fungo Phomopsis sp. isolado de D. ecastophyllum apresentou extratos com atividade antioxidante superior aos de outros fungos analisados.
105

Produtos naturais de fungos endofíticos associados a espécies de Asteraceae e ensaio antibiótico no modelo de infecção em \"Caenorhabditis elegans\" / Natural products from endophytic fungi found in association with Asteraceae species and antibiotic assay in the \"Caenorhabditis elegans\" infection model

Guimarães, Denise Oliveira 05 February 2010 (has links)
O estudo de fontes naturais pouco exploradas e que exibem interações ecológicas específicas no seu habitat tem sido enfatizado como estratégico para a descoberta de novas substâncias bioativas. Microrganismos endofíticos são fontes de produtos naturais relativamente pouco estudadas. Estes microrganismos vivem no interior de um vegetal sem causar danos aparentes ao hospedeiro. A interação ecológica específica entre os microrganismos endofíticos e sua planta hospedeira pode estimular a biossíntese de novos produtos naturais bioativos. Os fungos endofíticos \"Glomerella cingulata\" e \"Guignardia mangiferae\" foram isolados da folhas de \"Viguiera arenaria\". O fungo \"G. mangiferae\" foi cultivado em diferentes condições: meios líquidos Czapek, extrato de malte e caldo batata-dextrose e meio sólido de arroz. Extratos e frações obtidas com o cultivo de \"G. mangiferae\" foram avaliados em ensaios antimicrobianos, citotóxicos e enzimáticos. Foram realizados procedimentos cromatográficos com os extratos brutos, obtendo-se 13 substâncias puras, sendo cinco delas inéditas na literatura. Entre as novas substâncias, quatro são derivados tricicloalternarenos. O extrato bruto etanólico oriundo do cultivo em extrato de malte foi o mais promissor nos ensaios biológicos realizados. A partir desse extrato foi isolado a chaetoglobosina D, um composto já descrito na literatura como citotóxico em células cancerígenas. Os demais extratos, frações e substâncias ensaiadas apresentaram apenas atividade fraca ou moderada. O fungo \"G. cingulata\" foi investigado sobre a produção do metabólito secundário tirosol, previamente isolado de várias linhagens de fungos endofíticos. Devido à ampla ocorrência em fungos endofíticos, o tirosol pode estar correlacionado com uma função sinalizadora para a produção de outros metabólitos secundários. Para a determinação da quantidade de tirosol produzido por \"G. cingulata\", um método analítico quantitativo foi desenvolvido obedecendo-se padrões descritos pelo Guidance of Industry. Foi realizada a análise dos fluídos das culturas de \"G. cingulata\" em diferentes condições de cultivo em meio líquido Czapek. Observou-se que a melhor condição para produção do tirosol ocorre em pH 5,0, 120 rpm, 30 ºC, seis dias de incubação. O cultivo de \"G. cingulata\" com e sem adição de tirosol no meio líquido Czapek não permitiu resultados conclusivos sobre a possível função sinalizadora do tirosol na produção de metabólitos secundários em fungos endofíticos. Durante o período de estágio sanduíche no Departament of Molecular Biology do Massachusetts General Hospital (Harvard Medical School), sob a supervisão do Prof. Dr. Frederick M. Ausubel, foram avaliadas cerca de 170 amostras de origem natural do Brasil, sendo a maioria delas oriundas de fungos endofíticos, frente ao nematóide \"Caenorhabditis elegans\" infectado com linhagem patogênica de \"Enterococcus faecalis\". Cerca de 29% das substâncias puras e 2% dos extratos ensaiados apresentaram boa capacidade de recuperação do nematóide infectado. Os policetídeos chaetoviridinas foram os compostos mais ativos neste modelo de ensaio antibacteriano \"in vivo\". / The search for small bioactive molecules from under-explored natural sources that possess specific ecological interaction in their respective habitat has been emphasized as strategic for the discovery of bioactive compounds. Endophytic microorganisms are still relatively under-explored sources of natural products. These microorganisms live inside the host plant tissues without causing symptoms of disease to the host. The specific ecological interaction between the endophytes and their host plants may trigger the biosynthesis of new small bioactive molecules. The endophytic fungi \"Glomerella cingulata\" and \"Guignardia mangiferae\" were isolated from \"Viguiera arenaria\" leaves. G. mangiferae was cultured using different conditions: Czapek, malt extract and potato-dextrose broth liquid media and rice solid medium. Crude extracts and fractions obtained after \"G. mangiferae\" cultivation were evaluated in antimicrobial, cytotoxic and enzymatic assays. Chromatographic procedures were carried out with crude extracts and led to the isolation of 13 compounds, five of them new in the literature. From the novel compounds four are tricycloalternarene derivatives. The crude ethanolic extract obtained from malt extract cultivation was the most promising extract in the biological assays. Chaetoglobosin D, a cytotoxic compound against cancer cell lines previously described in the literature, was isolated from this bioactive extract. The other extracts, fractions and compounds assayed showed weak or moderate activity in the performed assays. The production of tyrosol by \"G. cingulata\" was investigated, since several endophytic fungal strains from Asteraceae species had already produced this compound. Tyrosol may act as a signaling molecule for the production of other metabolites in endophytic fungi. In order to quantify the production of tyrosol by \"G. cingulata\", an analytical method was validated under Guidance of Industry recommendations. Fluid culture analyses were performed after the cultivation of \"G. cingulata\" under different conditions using Czapek liquid medium. The best condition for tyrosol production occurred at pH 5.0, 120 rpm, 30 ºC, six days of incubation. Cultivation of \"G. cingulata\" with and without tyrosol addition in the Czapek liquid medium did not allow the conclusion that tyrosol act as a signaling molecule for metabolite production by endophytic fungi. During sandwich training at Department of Molecular Biology of Massachusetts General Hospital (Harvard Medical School), under Dr. Frederick M. Ausubel supervision, 170 samples from Brazil, the most part obtained from endophytic fungi, were assayed using the nematode \"Caenorhabditis elegans\" infected with the pathogen \"Enterococcus faecalis\". About 29% of pure compounds and 2% of extracts assayed were able to promote the survival of the infected nematode. The polyketides chaetoviridins were found to be the most active compounds in this \"in vivo\" antibacterial assay.
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Prospecção química e biológica das algas vermelhas Asparagopsis taxiformis e Pyropia spiralis e seus fungos endofíticos / Chemical and biological prospection of red algae Asparagopsis taxiformis and Pyropia spiralis and their endophytic fungi

Medina, Rebeca Previate [UNESP] 22 June 2016 (has links)
Submitted by REBECA PREVIATE MEDINA null (rebecapm_2@hotmail.com) on 2016-07-12T01:22:08Z No. of bitstreams: 1 tese completa final.pdf: 17929997 bytes, checksum: 2ca69cdbb8845123f617feff1388a3eb (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-07-12T20:30:01Z (GMT) No. of bitstreams: 1 medina_rp_dr_araiq_par.pdf: 1819041 bytes, checksum: 6a9b96f8294269d0ff2fc5f05f6dd74b (MD5) / Made available in DSpace on 2016-07-12T20:30:01Z (GMT). No. of bitstreams: 1 medina_rp_dr_araiq_par.pdf: 1819041 bytes, checksum: 6a9b96f8294269d0ff2fc5f05f6dd74b (MD5) Previous issue date: 2016-06-22 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Os Produtos Naturais desempenham um papel dominante na descoberta e desenvolvimento de novos fármacos para o tratamento de diversas doenças. Com isso, a busca por novas fontes de substâncias bioativas tem se intensificado, com destaque para estudos realizados com organismos e micro-organismos marinhos. As algas vermelhas marinhas (Rhodophyta) são as principais produtoras de metabólitos secundários biologicamente ativos dentre as algas e produzem principalmente substâncias halogenadas. Dentre os micro-organismos, podemos destacar os fungos endofíticos de algas marinhas, pois apresentam alta diversidade de espécies e diversidade estrutural dos metabólitos secundários, os quais têm mostrado potencial antitumoral, antimicrobiano e antifúngico. Sendo assim, este trabalho teve como objetivo explorar o potencial das algas vermelhas Asparagopsis taxiformis (estágio Falkenbergia) e Pyropia spiralis, bem como de seus fungos endofíticos, como fontes de substâncias bioativas. O estudo de A. taxiformis e de P. spiralis levou à identificação de dezesseis substâncias por CG-EM, como os ácidos eicosapentaenóico e araquidônico, além de nucleosídeos, como a uridina e a timidina, obtidos de A. taxiformis, e a uridina e a inosina, obtidas de P. spiralis. Os extratos brutos e frações das algas estudadas foram avaliados frente ao fungo fitopatogênico Cladosporium sphaerospermum, frente à enzima acetilcolinesterase e frente a linhagens tumorais de carcinoma de ovário (OVCAR-8), adenocarcinoma de cólon humano (HCT-116) e glioblastoma - humano (SF-295), sendo que os mesmos não apresentaram atividades antifúngica e anticolinesterásica significativas e apenas as frações hexânicas e butanólicas de ambas as algas apresentaram atividade citotóxica moderada frente à linhagem SF-295. Em relação ao estudo dos micro-organismos associados às algas, foi possível isolar sete linhagens de fungos endofíticos da alga A. taxiformis e três linhagens de P. spiralis, que foram cultivadas em meio de batata e dextrose (MBD) preparado com água do mar ou água ultra pura. Após o período de incubação, foram obtidos os extratos brutos em acetato de etila, que foram avaliados quanto ao perfil químico por CLAE e RMN, e perfil biológico por meio dos bioensaios in vitro. Após a prospecção inicial, Nemania bipapillata (AT-05), Sarocladium strictum (PS-01), Annulohypoxylon stygium (AT-03) e Hypoxylon sp. (AT-06) foram selecionados para investigação. O estudo de N. bipapillata, cultivado em água do mar e MBD, levou ao isolamento da dicetopiperazina ciclo(Pro-Tir), tirosol e das isocumarinas, (3R)-5-hidroximetil-meleína e (3R)-5-carbóxi-meleína. Ao cultivar essa linhagem fúngica em água ultra pura e MDB, foram isolados dois norsesquiterpenos e três sesquiterpenos do tipo botriano inéditos na literatura e um conhecido, 4β-acetoxi-9β,10β,15α-triidroxiprobotridial. A análise do extrato bruto de S. strictum, cultivado em água ultra pura e MDB, levou à identificação em mistura de duas dicetopiperazinas e ao isolamento da isoflavona daidzeína e de seis ftalatos naturais, dos quais dois ainda não foram descritos na literatura e nem sintetizados. A. stygium e Hypoxylon sp. foram cultivados em água do mar e MDB, sendo que A. stygium forneceu o pirogalol, que apresentou atividade contra Escherichia coli e Staphylococcus aureus resistente a meticilina, além do tirosol e (˗)-(3R,4R)-3,4,5-triidróxi-tetralona, enquanto Hypoxylon sp. forneceu (3R)-scytalona, (3R,4R)-4-hidróxi-scytalona e daidzeína. Portanto, esses resultados ressaltam a importância da investigação dos organismos marinhos, bem como de seus micro-organismos associados, e contribuem para o conhecimento da quimiodiversidade de organismos de origem marinha da costa brasileira, ainda sub-explorada do ponto de vista químico. / Natural Products have a major role in the discovery and development of new drugs used for diseases treatments. Thus, the search for new sources of bioactive compounds has been intensified and studies have increasingly focused on marine organisms and microorganisms. Marine red algae (Rhodophyta) are the major producers of biologically active secondary metabolites among algae, and biosynthesize mainly halogenated compounds. Among microorganisms, we highlight endophytic fungi from marine algae, as they present marked species diversity as well as structural diversity of secondary metabolites. Such metabolites have shown interesting potential as antitumor, antimicrobial and antifungal agents. Therefore, this project aimed to explore the potential of red algae Asparagopsis taxiformis (Falkenbergia stage) and Pyropia spiralis and their endophytic fungi, as novel sources of bioactive compounds. The study of A. taxiformis and P. spiralis led to identification of sixteen compounds by GC-MS, as eicosapentaenoic and arachidonic acids, in addition to nucleosides uridine and thymidine, obtained from A. taxiformis, and uridine and inosine, obtained from P. spiralis. The crude extracts and fractions from the studied algae were evaluated against phytopathogenic fungus Cladosporium sphaerospermum, against acetylcholinesterase enzyme and against tumor cell lines of ovarian carcinoma (OVCAR-8), human colon adenocarcinoma (HCT-116) and human glioblastoma (SF-295). No significant antifungal or acetylcholinesterase activities were observed, whereas the hexane and butanol fractions from both algae showed moderate cytotoxicity against SF-295 cell line. As for the study on the microorganisms associated to algae, seven endophytic fungi strains were isolated from A. taxiformis and three strains from P. spiralis, which were cultivated in potato dextrose broth (PDB) prepared with either sea water or ultra pure water. After incubation, the ethyl acetate crude extracts were obtained and evaluated for their chemical and biological profiles, by HPLC, NMR and in vitro bioassays, respectively. After the preliminary prospection, Nemania bipapillata (AT-05), Sarocladium strictum (PS-01), Annulohypoxylon stygium (AT-03) and Hypoxylon sp. (AT-06) were selected for further investigation. The study of N. bipapillata, cultivated in sea water and PDB, led to isolation of diketopiperazine cyclo(Pro-Tyr), tyrosol and isocoumarins, (3R)-5-hydroxymethylmellein and (3R)-5-carboxymellein. Cultivation of this fungal strain in ultra pure water and PDB afforded two botryane-type norsesquiterpenes and three sesquiterpenes, unprecedent compounds in the literature and one known, 4β-acetoxy-9β,10β,15α-trihydroxyprobotrydial. Analysis of the crude extract from S. strictum, cultivated in ultra pure water and PDB, afforded a mixture of two diketopiperazines, in addition to isoflavone daidzein and six natural phthalates, including two novel ones which had been neither described in the literature nor synthesized. A. stygium and Hypoxylon sp. were cultivated in sea water and PDB, and A. stygium afforded pyrogallol, which was active against Escherichia coli and methicilin-resistant Staphylococcus aureus (MRSA), in addition to tyrosol and (˗)-(3R,4R)-3,4,5-trihydroxy-1-tetralone. Hypoxylon sp. afforded (3R)-scytalone, (3R,4R)-4-hydroxy-scytalone and daidzein. Such results highlight the importance of research on marine organisms and their associated microorganisms, and contribute to the knowledge of chemodiversity of marine organisms from the still chemically underexplored Brazilian coast.
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Fungos micorrízicos arbusculares e endofíticos do tipo Dark Septate em plantas nativas de Cerrado / Mycorrhizal and Dark Septate endophytic fungi in native Cerrado plants.

Detmann, Kelly da Silva Coutinho 03 August 2007 (has links)
Made available in DSpace on 2015-03-26T13:36:32Z (GMT). No. of bitstreams: 1 texto completo.pdf: 623531 bytes, checksum: 3e10a939648d01ae404b0a09438fda4c (MD5) Previous issue date: 2007-08-03 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The high acidity, the intense intemperism, and high aluminum content are limiting factors to plant development in Brazilian Cerrado Upon abiotic stress conditions the association with mycorrhizal fungi can assist the host plant in absorbing nutrients and water from soil. The high availability of these elements to the host plant improved by arbusculares mycorrhizal fungi (AMF) is determinant in plant survival and structuring of plant ecosystems. The presence of dark septate entophytic fungi in plant roots is another type of symbiotic association related to abiotic conditions such as low humid and day length. The reduced number of publications about these associationsin cerrado species is mainly a function of edaphic conditions and root structural characteristics of native species which difficult the in loco studies of these characters. In this work, it was aimed to study the association between AMF and cerrado native species. It was evaluated and adapted methods for fungal structures observation in symbiosis with Annonaceae, Anacardiaceae, Leguminosae, Melastomataceae, Myrsinaceae, Myrtaceae and Rubiaceae families, which were collected during dry season on senso strictu Cerrado in Floresta Nacional (FLONA) in the municipal district of Paraopeba, Minas Gerais State. Root samples were submitted to three protocols for fungal structures observationg. All evaluated species were colonized by AMF and dark septate fungi, except Xylopia aromatica, which not presented dark septate strutctures. The best diaphanization procedure was obtained when the root samples were autoclaved at 121ºC by 20 minutes in a 2% KOH solution and transferred to a 2% KOH solution by 24 hours at room temperature. This procedure was repeated twice and, after that, the samples were submerged in a 2% KOH solution for 2 hours. The arbuscules were observed with more detail after been included in resin, sectioned and stained with toluidine blue. The non-specific characteristic of AMF observed in sensu stricto Cerrado species was confirmed and suggested for dark septate associations. It indicates that these associations were important as adaptive strategies to cerrado conditions. Roupala montana Aubl. is a species from Proteaceae family which presents a large distribution on different phytophysiognomies of cerrado. Recent studies have been showed that several mycorrhizal fungi structures occurred in these species said to be typically non- mycorrhizal. The low pH and nutrients deficiency in cerrado soils propitiate good conditions for development and establishment of mycorrhizal association in R. montana, as observed in other Proteaceae species. Therefore, in this work it was also aimed to confirm the presence of AMF in R. montana specimens growing at natural cerrado conditions, and correlated the mycorrhizal frequency with nutritional status and edaphic conditions. Three individuals of R. montana were collected in FLONA on cerrado senso strictu, dense cerrado senso strictu, and cerradão during dry and rain seasons. The mycorrhizal occurrence was positively correlated with P, Al, and Mg contents in the soil, and P, K, and Ca in the leaves, and negatively correlated with Ca, Mn, and N contents in the soil. The cerrado soils are acid and have high Al contents which can inhibit the transport of Ca and K to the plants. In this way, the presence of mycorrhizal fungi in R. montana could be a adaptive mechanism for plant surviving under Brazilian cerrado conditions. / A elevada acidez, o intenso intemperismo e os altos teores de alumínio nos solos do Cerrado são fatores limitantes do desenvolvimento vegetal. Em condições de estresses abióticos a associação com fungos micorrízicos auxilia a planta hospedeira na absorção de nutrientes e água do solo. A maior disponibilidades desses elementos para a planta hospedeira pelos fungos micorrízicos arbusculares (FMAs) é determinante na sobrevivência das plantas e na estruturação de formações vegetais. A presença de fungo endofítico do tipo dark septate (DSEF) nas raízes das plantas é outro tipo de associação simbiótica relacionada às condições abióticas como baixa umidade e comprimento do dia. O reduzido número de trabalhos encontrados na literatura dessas associações em espécies de cerrado se deve em parte, pelas condições edáficas e características estruturais radiculares das espécies nativas que dificultam a realização de estudos in loco. Neste trabalho teve-se por objetivo estudar a associação de FMAs em espécies de plantas nativas do cerrado. Para isso, foram testados e adaptados métodos para observação das estruturas fúngicas em simbiose em espécies das famílias Annonaceae, Anacardiaceae, Leguminosae, Melastomataceae, Myrsinaceae, Myrtaceae e Rubiaceae coletadas durante a estação seca em cerrado senso strictu na Floresta Nacional (FLONA) do município de Paraopeba, Minas Gerais. Amostras radiculares foram submetidas a três tipos de protocolos para observação de estruturas fúngicas. Todas as espécies investigadas encontravam-se colonizadas por FMAs e DSEF, exceto DSEF em Xylopia aromática. O melhor método de diafanização foi observado quando as raízes foram autoclavadas a 121 °C em KOH 2 %, por 20 min e, subseqüentemente transferidas para solução nova de KOH 2 % por 24 horas à temperatura ambiente. Este procedimento foi repetido por duas vezes e, em seguida, essas amostras foram imersas em H2O2 2 % por 2 horas. Os arbúsculos foram observados com maiores detalhes após as raízes serem incluídas em resina, seccionadas e coradas com azul de toluidina. O caráter generalista dos FMAs observado nas espécies vegetais do cerrado sensu stricto foi confirmado e sugerido para DSEFs, indicando a importância destas simbioses como estratégia adaptativa às condições de cerrado. Roupala montana Aubl. é uma espécie da família Proteaceae com ampla distribuição nas diferentes fitofisionomias do cerrado. Estudos recentes têm relatado estruturas típicas de fungos micorrízicos em condições de estresses abióticos em espécies dessa família tipicamente não micorrizável. A acidez e deficiência dos solos de cerrado provem apropriadas condições para o desenvolvimento e formação de associações micorrízicas em R. montana, como observado em outros espécies da família Proteaceae. Assim, este trabalho também teve por objetivo confirmar a presença de fungos micorrízicos arbusculares em R. montana crescidas naturalmente em cerrado, correlacionando a freqüência de micorrização com o estado nutricional e condições edáficas. Em três repetições, indivíduos de R. montana foram coletados na FLONA em cerrado senso strictu, cerrado senso strictu denso e cerradão nas estações seca e chuvosa. A micorrização em R. montana foi correlacionada positivamente com o teor de P, Al, e Mg, no solo e de P, K e Ca nas folhas, e negativamente com Ca, Mn e N no solo. Os solos de Cerrado são ácidos com altas concentrações de Al, os quais podem inibir o transporte de Ca e K para as plantas, sugerindo que a presença de fungos micorrízicos em R. montana é um mecanismo adaptativo de sobrevivência as condições do cerrado brasileiro.
108

Fungos promotores do crescimento vegetal e da fitorremediação de metais pesados em combinação com biochar / Fungi promoting plant growth and phytoremediation of heavy metals in combination with biochar

Farias, Christyan Paiva 27 February 2018 (has links)
A busca por medidas sustentáveis para melhorar e suprir a demanda por alimentos que o crescimento populacional exige, aliado a escassez de terras produtivas, além do acúmulo excessivo de metais no ambiente devido a atividades como mineração e aplicação de fertilizantes no solo, foram as duas problemáticas que motivaram o presente estudo. O uso de fungos promotores do crescimento vegetal: Beauveria bassiana, Metarhizium anisopliae, Pochonia chlamydosporia, Purpureocillium lilacinum e Trichoderma asperella, pode tanto vir a influenciar o aumento da produtividade como potencializar a fitorremediação de metais pesados no solo. Esses fatos são explicados pela capacidade do consórcio fúngico de sintetizar e induzir as plantas a produzir fitohormônios direcionados para o melhor desenvolvimento das raízes do hospedeiro. O maior desenvolvimento de raízes pode estimular a fitoextração de metais do solo e conferir tolerância das plantas à contaminação. Na intenção de avaliar a influência dos fungos no desenvolvimento das plantas, foram realizados tratamentos das sementes de soja e milho e aplicação na parte aérea da cultura da cana-de-açúcar. Já para analisar a influência desses organismos no processo de fitorremediação de Cu, Zn e Mn em colunas de solos cultivados com Jacaranda mimosifolia D. Don, os fungos foram inoculados no solo e na parte aérea das plantas. Todos os tratamentos tiveram 6 repetições e suas médias foram submetidas à análise de variância e teste de média ao nível de 5% de probabilidade. O consórcio fúngico condicionou o crescimento de raízes de soja e milho, aumentou a colonização micorrízica arbuscular em soja e cana-de-açúcar. No jacarandá, os fungos melhoraram o desenvolvimento de raízes e parte aérea, maior acúmulo de Mn e Zn na planta, melhor translocação de Mn e Zn das raízes para a parte aérea, além de diminuir o estresse fisiológico. Além disso, a aplicação conjunta de fungos e biochar nas mudas proporciona proteção à sua atividade fotossintética e reduz o risco ambiental de contaminação por metais por diminuir o total de Cu, Mn e Zn lixiviado. O biochar foi adquirido por meio da pirólise lenta do resíduo do beneficiamento da madeira de eucalipto e aplicado superficialmente no solo. Com isso, esse estudo mostrou que os fungos utilizados apresentam potencial para colaborar com o aumento da produtividade de culturas comerciais e colaborar com a melhoria na remediação de solos contaminados com íons metálicos. / The search for sustainable measures to improve and meet the demand for food that population growth requires, combined with the scarcity of productive land, in addition to the excessive accumulation of metals in the environment due to activities such as mining and application of soil fertilizers, were the two problems that motivated the present study. The use of fungi plant growth promoters: Beauveria bassiana, Metarhizium anisopliae, Pochonia chlamydosporia, Purpureocillium lilacinum and Trichoderma asperella can both influence on increasing productivity as the phytoremediation of heavy metals in the soil. These facts are explained by the capacity of the fungal Consortium to synthesize and induce plants to produce phytohormone targeted for the better development of the roots of the host. The further development of roots can stimulate phytoextraction of metals from the soil and give plants tolerance to contamination. In order to evaluate the influence of fungi on the development of plants, were done seed treatments of soybeans and corn and application in aerial part of the culture of sugar cane. To analyze the influence of these organisms in the process of phytoremediation of Cu, Zn and Mn in soil columns cultivated with Jacaranda mimosifolia D. Don, fungi were inoculated in soil and aerial part of plants. All treatments had 6 reptitions and their averages were subjected to analysis of variance and average test at 5% probability. The fungal consortium has conditioned the growth of roots of soybeans and corn, mycorrhizal colonization increased in soybeans and sugar cane. In jacarandá, fungi have improved the development of roots and shoots, greater accumulation of Mn and Zn in plant, best of Mn and Zn translocation from roots to shoots, in addition to decrease physiological stress. In addition, the joint application of fungus and biochar in seedlings provides protection to photosynthetic activity and reduces the risk of environmental contamination by metals by decreasing the amount of Cu, Mn and Zn leached. The biochar was acquired through the slow pyrolysis of the eucalyptus wood processing residue and applied superficially to the soil. With that, this study showed that fungi used present potential to contribute to the increased productivity of commercial crops and contribute to the improvement in the remediation of soils contaminated with metal ions. / Dissertação (Mestrado)
109

Produtos naturais de fungos endofíticos associados a espécies de Asteraceae e ensaio antibiótico no modelo de infecção em \"Caenorhabditis elegans\" / Natural products from endophytic fungi found in association with Asteraceae species and antibiotic assay in the \"Caenorhabditis elegans\" infection model

Denise Oliveira Guimarães 05 February 2010 (has links)
O estudo de fontes naturais pouco exploradas e que exibem interações ecológicas específicas no seu habitat tem sido enfatizado como estratégico para a descoberta de novas substâncias bioativas. Microrganismos endofíticos são fontes de produtos naturais relativamente pouco estudadas. Estes microrganismos vivem no interior de um vegetal sem causar danos aparentes ao hospedeiro. A interação ecológica específica entre os microrganismos endofíticos e sua planta hospedeira pode estimular a biossíntese de novos produtos naturais bioativos. Os fungos endofíticos \"Glomerella cingulata\" e \"Guignardia mangiferae\" foram isolados da folhas de \"Viguiera arenaria\". O fungo \"G. mangiferae\" foi cultivado em diferentes condições: meios líquidos Czapek, extrato de malte e caldo batata-dextrose e meio sólido de arroz. Extratos e frações obtidas com o cultivo de \"G. mangiferae\" foram avaliados em ensaios antimicrobianos, citotóxicos e enzimáticos. Foram realizados procedimentos cromatográficos com os extratos brutos, obtendo-se 13 substâncias puras, sendo cinco delas inéditas na literatura. Entre as novas substâncias, quatro são derivados tricicloalternarenos. O extrato bruto etanólico oriundo do cultivo em extrato de malte foi o mais promissor nos ensaios biológicos realizados. A partir desse extrato foi isolado a chaetoglobosina D, um composto já descrito na literatura como citotóxico em células cancerígenas. Os demais extratos, frações e substâncias ensaiadas apresentaram apenas atividade fraca ou moderada. O fungo \"G. cingulata\" foi investigado sobre a produção do metabólito secundário tirosol, previamente isolado de várias linhagens de fungos endofíticos. Devido à ampla ocorrência em fungos endofíticos, o tirosol pode estar correlacionado com uma função sinalizadora para a produção de outros metabólitos secundários. Para a determinação da quantidade de tirosol produzido por \"G. cingulata\", um método analítico quantitativo foi desenvolvido obedecendo-se padrões descritos pelo Guidance of Industry. Foi realizada a análise dos fluídos das culturas de \"G. cingulata\" em diferentes condições de cultivo em meio líquido Czapek. Observou-se que a melhor condição para produção do tirosol ocorre em pH 5,0, 120 rpm, 30 ºC, seis dias de incubação. O cultivo de \"G. cingulata\" com e sem adição de tirosol no meio líquido Czapek não permitiu resultados conclusivos sobre a possível função sinalizadora do tirosol na produção de metabólitos secundários em fungos endofíticos. Durante o período de estágio sanduíche no Departament of Molecular Biology do Massachusetts General Hospital (Harvard Medical School), sob a supervisão do Prof. Dr. Frederick M. Ausubel, foram avaliadas cerca de 170 amostras de origem natural do Brasil, sendo a maioria delas oriundas de fungos endofíticos, frente ao nematóide \"Caenorhabditis elegans\" infectado com linhagem patogênica de \"Enterococcus faecalis\". Cerca de 29% das substâncias puras e 2% dos extratos ensaiados apresentaram boa capacidade de recuperação do nematóide infectado. Os policetídeos chaetoviridinas foram os compostos mais ativos neste modelo de ensaio antibacteriano \"in vivo\". / The search for small bioactive molecules from under-explored natural sources that possess specific ecological interaction in their respective habitat has been emphasized as strategic for the discovery of bioactive compounds. Endophytic microorganisms are still relatively under-explored sources of natural products. These microorganisms live inside the host plant tissues without causing symptoms of disease to the host. The specific ecological interaction between the endophytes and their host plants may trigger the biosynthesis of new small bioactive molecules. The endophytic fungi \"Glomerella cingulata\" and \"Guignardia mangiferae\" were isolated from \"Viguiera arenaria\" leaves. G. mangiferae was cultured using different conditions: Czapek, malt extract and potato-dextrose broth liquid media and rice solid medium. Crude extracts and fractions obtained after \"G. mangiferae\" cultivation were evaluated in antimicrobial, cytotoxic and enzymatic assays. Chromatographic procedures were carried out with crude extracts and led to the isolation of 13 compounds, five of them new in the literature. From the novel compounds four are tricycloalternarene derivatives. The crude ethanolic extract obtained from malt extract cultivation was the most promising extract in the biological assays. Chaetoglobosin D, a cytotoxic compound against cancer cell lines previously described in the literature, was isolated from this bioactive extract. The other extracts, fractions and compounds assayed showed weak or moderate activity in the performed assays. The production of tyrosol by \"G. cingulata\" was investigated, since several endophytic fungal strains from Asteraceae species had already produced this compound. Tyrosol may act as a signaling molecule for the production of other metabolites in endophytic fungi. In order to quantify the production of tyrosol by \"G. cingulata\", an analytical method was validated under Guidance of Industry recommendations. Fluid culture analyses were performed after the cultivation of \"G. cingulata\" under different conditions using Czapek liquid medium. The best condition for tyrosol production occurred at pH 5.0, 120 rpm, 30 ºC, six days of incubation. Cultivation of \"G. cingulata\" with and without tyrosol addition in the Czapek liquid medium did not allow the conclusion that tyrosol act as a signaling molecule for metabolite production by endophytic fungi. During sandwich training at Department of Molecular Biology of Massachusetts General Hospital (Harvard Medical School), under Dr. Frederick M. Ausubel supervision, 170 samples from Brazil, the most part obtained from endophytic fungi, were assayed using the nematode \"Caenorhabditis elegans\" infected with the pathogen \"Enterococcus faecalis\". About 29% of pure compounds and 2% of extracts assayed were able to promote the survival of the infected nematode. The polyketides chaetoviridins were found to be the most active compounds in this \"in vivo\" antibacterial assay.
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Structure Elucidation and Biological Evaluation of a Novel Steroidal Saponin, Cholestanol Glucoside Isolated from Saraca Asoca Enodophytic Fuungus, Lasiodiplodia Theobromae

Valayil, Jinu Mathew January 2015 (has links) (PDF)
Although the molecular mechanisms underlying the onset and progression of cancer has been unraveled to a great extend, cancer continues to remain a leading cause of death around the world. Clinical efficacy of the existing anticancer drugs are largely compromised by the inherent and acquired resistance of cancer cell types and the severe side effects evoked by chemotherapeutic agents. Hence, the search for novel anticancer drugs with minimum side effects remains an active area of cancer research. Although molecular targeted drugs are preferred over the conventional cytotoxic chemotherapy, the screening of natural compounds with cytotoxic potentialities continues as they can serve as lead structures for the development of tumor selective anticancer drugs. Plants and microorganisms have been the prominent sources of therapeutic agents. Microorganisms being readily renewable, inexhaustible sources of diverse bioactive secondary metabolites are preferred over plants as sources of bioactive compounds. Endophytes are microorganisms that reside within the living tissue of host plant and they enhance the survival value of the host plant by mediating various stress tolerance mechanisms. Endophytic fungi have gained attention as potential sources of bioactive secondary metabolites following the discovery of a taxol producing endophytic fungus Taxomyces adrenae, from Taxus brevifolia. Moreover, endophytes occupy a unique biological niche in which they maintain a balanced interaction with the host organism and other co-inhabiting microorganisms. All these factors contribute to the chemical diversity of the metabolites they produce. Plants restricted to extreme or unique habitats or those with ethnobotanical value are likely to lodge endophytes that possess a unique hoard of secondarymetabolites. Saraca asoca is a traditionalmedicinal plant with its occurrence restricted to countries such as India, Sri Lanka, Burma and Malaysia. The purpose of the present study is to explore the endophytic fungal population associated with S. asoca in search of novel anticancer lead structures. S. asoca was found to house a diverse endophytic fungal population belonging to 37 different species. Identification of the fungal isolates was based on ITS (internal transcribed spacer region) sequence analysis as well as colony and spore characteristics. The organic extracts of all fungal species were assessed for their in vitro cytotoxicities in three human cancer cell lines, HeLa, HepG2 and PC3 byMTT assay. 18 species exhibited remarkable cytotoxic activities, among which Pestalotiopsis sp. 6 exhibited themost significant cytotoxicity. The strain with second highest activity was Lasiodiplodia theobromae. In order to identify the active principle present in the organic extracts of Pestalotiopsis sp. 6 and L. theobromae, the organic extracts were chromatographed on TLC plates and individual compounds were recovered by scraping off from the TLC plates and extracting with methanol. The cytotoxicity assay of the TLC purified compounds suggested the cytotoxic activity of Pestalotiopsis sp.6 to be a synergetic effect of two or more compounds whereas the cytotoxicity of L. theobromae organic extract was largely due to a single compound. Hence the active principle present in L. theobromae organic extract was purified by bioassay - guided column chromatography. Repeated chromatography of the crude extract using three silica gel columns resulted in the isolation of anticancer compound. Based on the analysis of ESI-MS, IR, NMR and UV spectral data, the isolated compound was identified as a novel steroidal saponin, cholestan-3-O-¯-Dglucopyranoside (cholestanol glucoside - CG). The in vitro cytotoxic effects of CG towards seven human cancer cell lines, HeLa, HepG2, PC3, U251,MCF 7, OVCAR3 and A549 were examined. Among the cell lines screened, HeLa cells weremost vulnerable to CG treatment, with an IC50 value of 3.2 ¹M. Hence themode of cell death induction in HeLa cells by CG was further investigated. Analysis of cell cycle progression by propidium iodide (PI) staining revealed that CG arrests the cells in S phase of cell cycle prior to the induction of cell death. The morphological and biochemical features of apoptosis were investigated by nuclear staining, DNA fragmentation assay and Annexin V-FITC/ PI dual staining. All these results suggested that CG effectively induced apoptosis in HeLa cells in a concentration dependent manner. It was also found that CG treatment induced remarkable ROS generation and mitochondrial membrane potential loss. The pretreatment of cells with an antioxidant, N-acetyl cysteine (NAC), blocked CG induced ROS generation, mitochondrialmembrane depolarization and apoptotic cell death. Hence it could be concluded that CG kills the cancer cells by augmenting their basal oxidative stress and hence is less likely to be toxic to normal cells. Moreover, a high Bax to Bcl-2 ratio, high levels of Apaf-1 and p53, activation of procaspase-3 and procaspase-9 and cleavage of PARP were observed in CG treated HeLa cells. Taken together, our results suggested that CG induced apoptosis in HeLa cells via ROS mediated mitochondria dependent pathway. Biosynthesis of secondarymetabolites by filamentous fungi is influenced by the availability of nutrient factors. Therefore, it is essential to optimize the culturemedium components to ensure a maximum and consistent yield of desired metabolite by the fungal isolate. We designed a chemically defined production medium for CG production by L. theobromae. Carbon source, nitrogen source and microelements in the production medium were further optimized in stationary flask cultures to improve the mycelial growth and yield of CG by L. theobromae. The conventional one-factor at a time (OFAT)method was employed for the optimization of carbon and nitrogen sourceswhose contribution effects towards the final yield are large. Response surface methodology (RSM) was employed for the optimization of microelements. Optimization of culturemedium enhanced the yield of CG from 10mg L¡1 to 50mg L¡1. Various secondarymetabolites are produced by organisms in response to different stress conditions. This knowledge has been exploited in plant cell culture systems to increase the yield of particular secondary metabolites by artificial implementation of stress conditions. We investigated the effect of oxidative, osmotic and heat shock stresses on the production of CG by L. theobromae. Heat shock and osmotic stresses in liquid cultures were found to enhance the yield of CG by 1.2-fold, relative to the controls. Oxidative stress by both menadione and H2O2 enhanced the yield by 1.8-fold compared to the controls. Thus oxidative stress proved to be an efficient enhancer of CG production by L. theobromae. These findings ensure a large scale, cost-effective production of CG.

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