Dissertationen zum Thema „Natural bioactive metabolite“

The past several decades have seen a rise in the numbers of fungal infections due to an increase in the population of immune-compromised individuals. Some of the most common types of invasive fungal infections are caused by Candida, Aspergillus, and Cryptococcus species. The current treatments for systemic fungal infections remain unsatisfactory because of resistance and toxicity problems. Because of this, there is a growing need for classes of antifungal agents effective against these pathogens. Fungi have proven to be an excellent source of novel secondary metabolites possessing a range of bioactivities, including antifungal effects. Most of the research described in this thesis stems from an ecology-based approach for selecting types of fungi for investigation in search of novel bioactive metabolites. Members of three ecological niche groups are discussed: mycoparasitic/fungicolous, endophytic, and coprophilous fungi. Mycoparasitic or fungicolous fungi are those that colonize other fungi and often cause some level of damage to the host. If damage occurs, it may be caused in part by antifungal natural products. Chemical investigations of nine isolates of fungicolous or mycoparasitic fungi (eight of which displayed antifungal activity) described in this thesis afforded five new compounds and 12 known compounds. Three of these known compounds have been reported to display antifungal properties, however, several others have not been tested in bioactivity assays. Due to this and unexpected loss of access to several of the antifungal assays explored in the initial prioritization process, the source of the original extract activity was not identified. Endophytic fungi are those that inhabit plant tissue and may or may not cause damage to the host. In some cases, colonization of a plant by fungi may be beneficial to the plant by providing protection against unwanted insect or microbial pests, which may be the result of bioactive compounds being produced by the colonizing fungus. Chemical investigation of 10 isolates of endophytic fungi described herein (five of which produced antifungal extracts) afforded 21 known compounds and four new metabolites. Of these, three extracts contained known antifungal compounds as major components, indicating that they were responsible for the original observed extract activity. In other cases, loss of access to the antifungal assays employed in the initial selection resulted in the source of the extract activity remaining undetermined. Coprophilous fungi, those that colonize the dung of herbivores, inhabit a nutrient-rich environment populated by many other organisms including other fungi, bacteria, insects, and protists. The production of bioactive compounds by coprophilous fungi might help provide them with a competitive advantage in this environment. Studies of 14 coprophilous fungal isolates (many of which displayed antifungal activity) are described in this thesis, leading to identification of twenty-five known compounds and nine new compounds. The majority of extracts yielded at least one metabolite known to have antifungal activity or one or more new compounds displaying these properties. In addition to the fungal isolates described in this thesis, six new stilbene-type phytoalexins were isolated from peanut seeds (Arachis hypogaea). These compounds are produced by peanut seeds that had been challenged by a mycotoxin-producing fungal invader and may serve to some degree as chemical defenses. A variety of separation techniques were utilized to isolate compounds from a range of classes, including terpenoids, polyketides, and peptides. Structures were established mainly based on analysis of various 1D and 2D NMR data aided by mass spectrometry. Relative stereochemical configurations were determined, where feasible, based on coupling constants and NOE experiments, whereas absolute configurations were determined using Mosher’s or chemical degradation methods. Several new secondary metabolites with distinctive chemical features were isolated, as well as many known compounds, some of which displayed bioactivity in medically or agriculturally relevant assays. The work described here illustrates that these targeted groups of fungi are capable of producing a structurally diverse array of bioactive natural products and supports their selection as targeted sources of potentially useful compounds.

The work undertaken explored the potential for discovery of new bioactive metabolites from soil fungi. The research developed selective mycological isolation techniques and maximised metabolite production from active selected fungi by application of the OSMAC approach and concept of hormesis. Novel active compounds were discovered and many other known compounds characterised.

Immobilised metal affinity chromatography (IMAC) is a privileged separation technique that relies on the fundamentals of coordination chemistry to retain compounds with metal binding properties. While originally established to isolate human serum proteins, histones, and metalloenzymes, and then recombinant proteins with surface-exposed histidine or cysteine residues, its applications have expanded considerably to include the purification of phosphorylated peptides and low molecular weight compounds. The aim of this thesis was to further develop IMAC to first isolate phosphorus-containing compounds and second evaluate its capacity as a value-added step in current natural product drug discovery workflows. A bespoke IMAC resin was synthesised and characterised containing immobilised desferrioxamine B, which is a natural product with extraordinary metal binding affinity towards Zr(IV). The Zr(IV)-immobilised resin was selective in coordinating phosphorus-containing compounds. The resin was used to selectively retain the antiviral drug tenofovir and the herbicide glyphosate at concentrations of approximately 2 and 1 mg g-1 resin under both aqueous and organic conditions, respectively. As compared to existing glyphosate adsorption technologies, the resin demonstrated improved desorption and recycling capacity with at least 93% of the adsorbed glyphosate recovered after three re-uses over seven days. Secondly, a standard IMAC system was re-cast as surrogate of a coordinatively-unsaturated metalloenzyme active site to selectively bind compounds with metal binding motifs. This could be used to select compounds as inhibitors of disease-relevant metalloproteins. A mixture of 16 metalloenzyme inhibitors and an antibiotic were adsorbed to IMAC resin beds charged with Co(II), Cu(II), Fe(III), Ga(III), Ni(II), Yb(III), or Zn(II). Of the 17 compounds, 11 were reversibly retained in quantitative amounts by at least one of the IMAC resin beds with each system offering a distinct selection profile. Remarkably, the Zn(II)-IMAC platform was the only system, which reversibly bound the thiol-containing Zn(II)-ACE1 inhibitors captopril and omapatrilat while the Fe(III)-IMAC system was the only system which reversibly bound the Fe(II)/(III)-5-LO inhibitor licofelone. Furthermore, the Fe(III)-mimicking Ga(III)-IMAC system was the only system that reversibly bound the Fe(II)/(III)-5-LO inhibitor 4-aminosalicylic acid. Finally, IMAC was integrated into a modern natural product drug discovery workflow to examine its capacity as an upstream screening technique for metalloenzyme inhibitor drug leads. Cultures of native and chemically perturbed Salinispora tropica were processed on Cu(II)-, Ni(II)-, and Zn(II)-IMAC systems and analysed using metabolomics and genome mining approaches to identify natural products with metal binding affinity. From an initial collection of 23,000 LC-MS signals, 783 analytes were identified as metal-selective S. tropica metabolites. Metabolomics and genome mining analysis subsequently revealed 18 natural products previously undetected in S. tropica, 9 of which were unique to the perturbed cultures. This project extended the applications of IMAC for the discovery, isolation, and purification of bioactive molecules with metal binding groups. The results reported demonstrate the potential of IMAC as a platform to support environmental remediation, address manufacturing process purification challenges, and streamline natural product drug discovery.

Fungi have been an important source for producing a wide range of secondary metabolites of widely differing chemical structures, as well as biological activities. Many of their metabolites now play a major role in pharmaceutical and agricultural industries. A number of fungi were isolated from soil and leaf litter collected from Arthur’s Pass, West Coast and Kaituna Valley using a particle filtration technique. Fungi were selected based on their unusual morphology or observed cytotoxicity and antimicrobial activity for large scale culture and extraction. A pale yellow compound was isolated from cytotoxic extracts from the culture of Aspergillus versicolor. This compound was identified as sterigmatocystin and the identity confirmed by UV profile and mass spectrometry. Five compounds were isolated from extracts prepared from two different species of Penicillium of which three were active against P388 cells (mycophenolic acid, cycloaspeptide A and mevastatin), one was active against dermatophytes (griseofulvin) and one was not active (3,4,6,8-tetrahydroxy-3-methyl-3,4-dihydroisocoumarin). Two compounds were isolated from extracts prepared from two different species of Phoma. A dark red compound was found to be novel and showed activity against P388 cells and Bacillus subtilis. A second compound also showing cytotoxicity was identified as the known compound phomenone. A further new compound was isolated from extracts of an identified dematiaceous fungus. This alkyl glucoside, however, was not bioactive.

Despite the critical role algae serve as primary producers, increases or accumulation of certain algae may result in Harmful Algal Blooms (HABs). Algal toxins from these blooms contribute significantly to incidences of food borne illness, and evidence suggests HABs are expanding in frequency and distribution. Mitigation of these HABs without knowledge of the ecological purpose and biochemical regulation of their toxins is highly unlikely. The production, function, and potential of secondary metabolites produced by the dinoflagellates Karenia brevis and Pyrodinium bahamense, were investigated. Brevetoxins were demonstrated by two different methods to localize within the cytosol of Karenia brevis. Differential and density-dependent centrifugation followed by Enzyme Linked Immunosorbant Assays (ELISAs) indicated that brevetoxin was not contained by any cellular organelles. Light microscopy of brevetoxin immunolabeled preserved cells visually confirmed these results, showing stain to be distributed throughout the cytosol and notably absent from the nucleus. These results have implications for brevetoxin synthesis and function. The complex cyclization process of brevetoxin therefore likely occurs in the cytosol after export of a polyketide precursor from the chloroplast. Functionally, this cellular location suggests use of brevetoxin in cytosolic functions such as signaling and chelation. Culture experiments of Pyrodinium. bahamense var. bahamense were undertaken to determine the effects of nutrients and environmental conditions on growth requirements and toxin production. HPLC analysis was employed to separate and quantify the saxitoxins. As eutrophication is a concern where this species is most problematic, in the Indian River Lagoon area of Florida, utilization of urea and ammonium were explored and compared to nitrate. While all nitrogen conditions yielded similar growth curves in P. bahamense, the cultures using urea contained a substantially lesser amount of the potent STX congener. This difference implies the urease enzyme utilized by P. bahamense is inefficient and urea based fertilizers are unlikely to create blooms with greater toxicity. Cyst production in P. bahamense was found to depend on nutrient limitation. Cultures utilizing ammonium displayed a smaller proportion of cysts, presumably attributable to the bioavailablility of ammonium. The total toxin content of P. bahamense was found to vary inversely with growth rate, although mole percents of the saxitoxins were largely unchanged over a suite of environmental parameters including temperature, salinity, and pH. Possible reasons for the reported increase in HABs include global warming, dumping of ballast water, and nutrient influx. These studies outline controls on toxin synthesis and production and conditions needed for growth and will aid in predicting environmental and human health effects pending these global changes. Extracts of K. brevis and P. bahamense cultures were assayed against various pathogenic agents. Growth of K. pneumoniae was inhibited by extracts of both K. brevis and P. bahamense. An extract of K. brevis additionally inhibited MRSA, while a P. bahamense extract additionally inhibited both S. aureus and MRSA as well as the most common protozoan vector of malaria, P. falciparum. The activity of a dinoflagellate against an Apicomplexan (P. falciparum) found in this study is especially interesting as the phyla are closely phylogenetically related. Differences in activity of extracts against P. falciparum between a clonal culture on P. bahamense from the Indian River Lagoon and a 2011 bloom sampled from Tampa Bay were observed. Drugs are losing their effectiveness against these infectious agents, making pursuit of new drugs an important field. These results suggest that HAB dinoflagellates hold promise in drug discovery similar to other phytoplankton.

As espécies patogênicas do gênero Colletotrichum apresentam importância mundial, pois causam danos a várias culturas de interesse agronômico. Diversas medidas de controle são empregadas, mas estas nem sempre são eficazes devido à ocorrência de linhagens resistentes. Desta forma, se faz necessário a busca por novos compostos que possam ser utilizados no manejo integrado desta doença. Os produtos naturais isolados de micro-organismos podem ser uma alternativa para o desenvolvimento de novos defensivos agrícolas. Dentre os micro-organismos, as actinobactérias são conhecidas pela produção de inúmeros compostos antimicrobianos. Neste contexto, o presente estudo teve como objetivo o isolamento e a identificação de compostos antifúngicos produzidos por actinobactérias da rizosfera de guaranazeiros. Para isto, a seleção de actinobactérias foi baseada em dois ensaios. No primeiro, as 65 actinobactérias foram avaliadas em ensaio de cultivo pareado frente ao fitopatógeno Colletotrichum gloeosporioides. Destas, os isolados mais promissores foram AM1 (43,78 % de inibição do crescimento micelial), AM3 (43,98 %), AM18 (37,86 %), AM25 (43,17 %), AM30 (47,12 %), AM61 (40,12 %) e AM68 (47,94 %). No segundo ensaio, estes isolados foram cultivados em meio BD e, após o cultivo, o meio metabólico foi submetido a três métodos de extração: (a) partição líquido-líquido com n-butanol; (b) partição líquido-líquido com acetato de etila e (c) coluna sílica gel C18. As frações obtidas a partir das três metodologias foram avaliadas pelo método de difusão em disco de papel contra C. gloeosporioides. Neste ensaio de difusão em disco foram selecionadas as linhagens AM1(n-butanol), AM3 (acetato de etila) e AM25 (C18) para o estudo de bioprospecção. Estas foram identificadas por técnicas moleculares como pertencentes ao gênero Streptomyces. A partir do extrato bruto da Streptomyces sp. AM1 foi isolado um composto análogo do ácido proclavamínico, o qual apresentou atividade mínima inibitória (MIC) de 1,25 mg mL-1 contra o fitopatógeno C. gloeosporioides. Da linhagem Streptomyces sp. AM3 foi isolado o composto streptimidona que apresentou MIC de 1,25 mg mL-1. Já no estudo de Streptomyces sp. AM25 um composto não identificado apresentou MIC de 2,50 mg mL-1. Estes três compostos apresentaram atividade superior aos fungicidas Captan SC® (Captana) e Dithane NT® (Mancozeb), e inferior ao Score® (Difenoconazol). A atividade antifúngica destes compostos ao C. gloeosporioides está sendo relatada pela primeira vez.
The pathogenic species of the genus Colletotrichum present importance worldwide because they cause damage to numerous crops of agronomic interest. Several control methods are employed, but they are not always effective due to the occurrence of resistant strains. Thus, it is necessary searching for new compounds that can be used in the integrated management of this disease. Natural products isolated from microorganisms can be an alternative for the development of new agricultural pesticides. Among microorganisms, actinobacteria are known to produce numerous antimicrobial compounds. In this context, the present study aimed to isolate and identify antifungal compounds produced by actinobacteria from guarana rhizosphere. For this, the selection of actinobacteria was based on two tests. In the first one, the 65 actinobacteria were evaluated in paired cultivation test against the plant pathogen Colletotrichum gloeosporioides. Among them, the most promising isolates were AM1 (43.78% inhibition), AM3 (43.98%), AM18 (37.86%), AM25 (43.17%), AM30 (47.12%), AM61 (40.12%) and AM68 (47.94%). In the second assay, these isolates were cultured in BD medium and, after culturing, the metabolic medium was subjected to three extraction methods: (a) liquid-liquid partition with n-butanol; (B) liquid-liquid partition with ethyl acetate and (c) silica gel column C18. The fractions obtained from the three methodologies were evaluated by paper disc diffusion method against C. gloeosporioides. In this disk diffusion assay, the strains AM1 (n-butanol), AM3 (ethyl acetate) and AM25 (C18) were selected for the bioprospecting study. These were identified by molecular techniques as belonging to the genus Streptomyces. From the crude extract of Streptomyces sp. AM1 the analogous compound proclavaminic acid was isolated, which presented minimal inhibitory activity (MIC) of 1.25 mg mL -1 against the plant pathogen C. gloeosporioides. From Streptomyces sp. AM3, the compound streptimidone was isolated, which presented MIC of 1.25 mg mL-1. In the study of Streptomyces sp. AM25 an unidentified compound had MIC of 2.50 mg mL-1. These three compounds presented superior activity to the fungicides Captan SC® (Captan) and Dithane NT® (Mancozeb), and inferior to the Score® (Difenoconazole). The antifungal activity of these compounds to C. gloeosporioides is being reported here for the first time.

A piplartina é um alcaloide natural conhecido por apresentar diversas atividades biológicas, onde se destaca a ação anticancerígena. Esse produto natural apresentou atividade seletiva frente a vários tipos de células cancerígenas, sendo assim considerado promissor para o desenvolvimento de fármacos. O conhecimento do metabolismo de produtos naturais bioativos é uma importante e necessária etapa para avaliar a eficácia e segurança dessas substâncias. Os micro-organismos são amplamente utilizados em estudos de metabolismo, uma vez que catalisam reações quimio-, régio-, e estereoespecíficas, que muitas vezes são semelhantes às catalisadas pelos seres humanos. Nesse contexto, esse trabalho teve o objetivo de estudar o metabolismo microbiano da piplartina pelos fungos endofíticos Papulaspora immersa SS13 e Penicillium crustosum VR4, de solo Mucor rouxii NRRL 1894, e de coleção comercial Cunninghamella echinulata ATCC 8688a e Beauveria bassiana ATCC 7159. Os experimentos de biotransformação foram monitorados por UPLC-DAD-MS e UPLC-DAD-MS/MS. Todos os fungos utilizados biotransformaram a piplartina, sendo que 14 substâncias majoritárias foram identificadas como produtos de biotransformação nos experimentos em pequena escala. A piplartina e seus derivados apresentaram fragmentações características em IES-EM/EM que foram explicadas utilizando cálculos computacionais. O estudo dessas fragmentações permitiu a identificação e proposição das alterações estruturais que ocorreram nos metabólitos formados. Os fungos P. crustosum VR4 e B. bassiana ATCC 7159 foram selecionados para realizar os experimentos de biotransformação em escala ampliada, pois foram capazes de formar a maior diversidade de derivados da piplartina. Cinco substâncias foram isoladas e identificadas por RMN de 1H, RMN de 13C, HMQC, HMBC, COSY e HRESIMS. Essas substâncias não tinham sido obtidas por biotransformação microbiana anteriormente, sendo que uma ainda não foi descrita na literatura. Foram identificados principalmente produtos formados a partir de reações semelhantes às do metabolismo humano de fase I, como reduções, hidroxilações e hidrólises. Dessa forma, podemos concluir que as culturas microbianas são uma ferramenta útil para estudos preliminares de metabolismo, e para obter padrões de metabólitos que podem ser formados pelo metabolismo humano.
Piplartine is a natural alkaloid recognized by its biological properties, especially the anticancer activity. This natural product showed selective activity against several cancer cells lines, thus being considered a promising hit for drug development. Studies of bioactive natural products metabolism are an important and necessary step for the evaluation of their efficacy and safety. Microorganisms have been widely employed in metabolism studies, since they may catalyze chemo-, regio- and stereospecific reactions that are similar to human metabolism. This work aimed to study the microbial metabolism of piplartine by different fungal strains: the endophytes Penicillium crustosum VR4 and Papulaspora immersa SS13, the soil strain Mucor rouxii NRRL 1894, and the commercial collection strains Cunninghamella echinulata ATCC 8688a and Beauveria bassiana ATCC 7159. Biotransformation experiments were monitored by UPLC-DAD-MS and UPLC-DADMS/ MS. All the screened fungi were able to biotransform piplartine, and 14 compounds were identified as major biotransformation products in the small scale experiments. Piplartine and its derivatives showed characteristics fragmentations on ESI-MS/MS, which were explained using computer calculations. These fragmentation studies allowed the identification and structural proposition of piplartine metabolites. The fungi P. crustosum VR4 and B. bassiana ATCC 7159 were selected to perform the large scale biotransformation experiments, since they were capable to produce a large diversity of piplartine derivatives. Five compounds were isolated and identified by 1H NMR, 13C NMR, HMQC, HMBC, COSY and HRESIMS data. The isolated products had never been previously identified by microbial biotransformation, and one of them was found to be novel in the literature. All the identified and isolated compounds have been produced by reactions similar to those that occur in phase I of human metabolism, such as reduction, hydroxylation and hydrolysis reactions. Thus, we can conclude that the microbial cultures are useful tools for preliminary metabolism studies, and to obtain chemical standards similar to those produced by human metabolism

Abstract Liquid chromatography (LC) combined with mass spectrometry (MS) is one of the most widely used techniques in modern analytical laboratories. Remarkable developments during the two previous decades in both techniques has made LC-MS the method of choice in various environmental, pharmaceutical and biochemical laboratories due to selectivity, sensitivity and versatility. The main focuses in this study were to develop new LC-MS methods to identify and quantify phenolic secondary metabolites in bilberry, lingonberry and hybrid bilberry, to study the biosynthesis of the main secondary metabolites (hypericin and hyperforin and their derivatives) in St John’s wort (SJW) both in vitro and in vivo (in plant), to identify in vitro metabolites of hyperforin in human liver microsomes and to identify the cytochrome P450 (CYP) enzymes responsible for their formation. Both high-performance liquid chromatography (HPLC) and ultra high-performance liquid chromatography (U-HPLC) in combination with time-of-flight (TOF) and triple quadrupole (QqQ) mass spectrometry were used in this study. Identification of 52 phenolic compounds from the leaves of bilberry, lingonberry and hybrid bilberry was accomplished. In total, seven of the identified compounds were reported for the first time in Vaccinium plants and several other compounds were reported for the first time in the studied plants. Incorporation of valine and isoleucine into acyl side chains of phloroglucinols (hyperforin and adhyperforin) via biosynthesis in shoot cultures of SJW was confirmed by using isotopically labeled amino acids and HPLC-MS/MS. Also, 29 biosynthetic in vitro products for HpPKS2 enzyme originating from SJW were identified based on accurate mass data. The metabolism of hyperforin was studied in human liver microsomes (HLM) for the first time. 57 metabolites for hyperforin were identified in the incubations with HLMs, using a substrate concentration of 1 μM. The phase I metabolism of hyperforin was suggested to rely mainly on CYP3A4 and on CYP2C family
Tiivistelmä Nestekromatografia (LC) yhdistettynä massaspektrometriaan (MS) on yksi eniten käytetyistä analyysimenetelmistä nykyaikaisissa analytiikkalaboratorioissa. Viimeisten parin vuosikymmenen aikana LC-MS -laitteet ovat kehittyneet merkittävästi, ja nykyään LC-MS onkin paras menetelmä moniin ympäristö-, lääkeaine- ja biokemiallisiin laboratorioihin sen selektiivisyyden, herkkyyden ja monipuolisuuden vuoksi. Tässä väitöskirjassa kehitettiin uusia LC-MS –menetelmiä mustikan, puolukan ja mustikkapuolukan fenolisten sekundäärimetaboliittien tunnistamiseksi ja kvantitoimiseksi, mäkikuisman pääasiallisten sekundäärimetaboliittien (hyperisiini, hyperforiini ja niiden johdannaiset) tutkimiseksi in vitro- ja kasvinäytteistä sekä hyperforiinin aineenvaihduntatuotteiden tunnistamiseksi ja niitä muodostavien sytokromi P450 (CYP) entsyymien tunnistamiseksi ihmisen maksamikrosomeissa in vitro -menetelmin. Tässä työssä käytettiin sekä korkean erotuskyvyn nestekromatografia (HPLC) että ultra-korkean erotuskyvyn nestekromatografia (U-HPLC) yhdistettynä lentoaikamassaspektrometriin (TOF-MS) ja kolmoiskvadrupolimassaspektrometriin (QqQ-MS). Mustikan, puolukan ja mustikkapuolukan lehdistä tunnistettiin yhteensä 52 fenolista yhdistettä. Seitsemää näistä tunnistetuista yhdisteistä ei oltu aiemmin löydetty Vaccinium -suvun kasveista ja useat muut yhdisteistä löydettiin ensimmäistä kertaa nyt tutkituista kasveista. Valiinin ja isoleusiinin liittyminen floroglusinolien (hyperforiini ja adhyperforiini) asyylisivuketjuihin biosynteesin välityksellä varmistettiin isotoppileimattujen aminohappojen ja HPLC-MS/MS –mittausten avulla. Tässä työssä tunnistettiin myös 29 mäkikuismasta peräisin olevan HpPKS2 -entsyymin in vitro biosynteesituotetta tarkan massan mittausten avulla. Hyperforiinin metaboliaa tutkittiin ensimmäistä kertaa ihmisen maksamikrosomeissa (HLM). Hyperforiinille tunnistettiin yhteensä 57 aineenvaihduntatuotetta ihmisen maksamikrosomi-inkubaatioissa, kun hyperforiinin alkukonsentraatio oli 1 μM. Tämän tutkimuksen tulosten perusteella hyperforiinin faasi I metabolia tapahtuu pääasiassa CYP3A4:n ja CYP2C-perheen välityksellä

Malgré l’intérêt et la diversité des propriétés pharmacologiques des 2,5-dicétopipérazines (DKP), les voies de biosynthèse de ces molécules d’origine microbienne sont très peu connues. L’objectif de mes travaux de thèse a été i) de documenter de nouvelles voies de biosynthèse de DKP qui se caractérisent par la présence d’une synthase de cyclodipeptides (CDPS) travaillant souvent de concert avec une ou plusieurs enzymes de modification des cyclodipeptides et ii) d’explorer la diversité chimique codée par ces voies. Dans un premier temps, je me suis intéressée aux CDPS. Après la sélection par bioinformatique de candidats dans les bases de données génomiques, j’ai pu identifier 51 nouvelles CDPS actives et montrer que ces enzymes peuvent incorporer 17 des 20 acides aminés naturels. Par ailleurs, ce travail a permis de mieux caractériser la famille des CDPS, de définir l’existence de plusieurs sous-familles aux signatures fonctionnelles spécifiques et d’établir les premiers éléments d’un code de spécificité pour la synthèse de cyclodipeptides. Dans un second temps, je me suis attachée à caractériser les enzymes de modification associées aux nouvelles CDPS et, en particulier, les dioxygénases dépendant du Fe(II) et du 2-oxoglutarate (OG) qui sont très représentées dans ces voies. J’ai ainsi pu détecter une activité in vivo pour 11 OG et poursuivre la caractérisation in vitro pour l’une de ces OG, ce qui a permis de caractériser les DKP qu’elle synthétise et d’ainsi montrer la complexité des modifications chimiques introduites. L’ensemble de ces travaux a donc permis d’identifier et de caractériser de nouvelles voies de biosynthèse qui donnent accès à une diversité accrue de DKP
Despite the interest and diversity of the pharmacological properties of 2,5-diketopiperazines (DKPs), the biosynthetic pathways of these microbial molecules are poorly documented. The aim of my doctoral work was i) to identify new DKP biosynthetic pathways that are characterized by the presence of a cyclodipeptide synthase (CDPS) often associated with one or more cyclodipeptide-tailoring enzymes and ii) to explore the chemical diversity encoded by these pathways. First of all, my study focused on CDPSs. After the bioinformatics-based selection of candidates, 51 novel CDPS were characterized, revealing the incorporation of 17 of the 20 proteinogenic amino acids. Moreover, this work has allowed a better characterization of the CDPS family, by showing the existence of several subfamilies with specific functional signatures and laying the foundations of a specificity conferring code for the synthesis of cyclodipeptides. Second, I characterized the tailoring enzymes associated with the newly identified CDPSs and, in particular, the Fe(II) and oxoglutarate dependent dioxygenases (OGs) that are highly represented in these pathways. I detected the in vivo activity for 11 OGs and characterized the in vitro activity for one of them, showing the complexity of the chemical modifications introduced into the cyclodipeptide. This work has led to identify and characterize novel biosynthetic pathways that provide access to a greater diversity of DKPs

A biodiversidade brasileira é conhecida devido a sua riqueza de espécies, sendo considerada uma fonte promissora de produtos naturais. Dentre as plantas vasculares, a família Solanaceae A. Juss. (Solanaceae) é considerada uma das maiores, apresentando distribuição em todas as regiões tropicais e temperadas do mundo. A família Solanaceae apresenta alta diversidade de espécies de importância econômica como fonte de alimentos, propriedades medicinais e ornamentais. Plantas desta família são fontes de metabólitos secundários de diversas classes químicas com as mais diversas aplicações. Fungos fitopatogênicos são responsáveis por causar diversas doenças e consideráveis perdas na agricultura. O controle das doenças é realizado através de métodos químicos, físicos e biológicos, porém o uso excessivo e ininterrupto de produtos químicos pode resultar na seleção de micro-organismos resistentes. Além disto, muitos fungicidas apresentam toxicidade alta e a sua utilização indiscriminada pode causar efeitos indesejáveis sobre outros organismos no ambiente. O objetivo deste estudo é explorar o potencial biológico e químico de metabólitos secundários produzidos por plantas da família Solanaceae com potencial fungitóxico a fitopatógenos. Foram selecionadas 15 espécies de plantas da família Solanaceae que tiveram os extratos de suas folhas avaliados em ensaios biológicos in vitro sobre o crescimento micelial de 6 fitopatógenos de importância na agricultura. Dentre estas, três foram selecionadas para uma investigação mais aprofundada, as espécies Solanum americanum, Acnistus arborescens e Physalis peruviana. No estudo da planta S. americanum, foram identificados compostos bioativos pertencentes à classe dos glicoalcalóides, que inibiram o crescimento micelial do fitopatógeno Moniliophthora perniciosa. No estudo de A. arborescens foi identificado a presença de um composto ativo pertencente à classe dos vitanolidos, provavelmente o 7β-acetoxivitanolido D, e sua ação antifúngica está sendo relatada pela primeira vez. No estudo da planta P. peruviana a fração semipura bioativa indicou a presença de um composto pertencente à classe dos vitanolidos. Esses resultados evidenciaram que os compostos presentes nas plantas, apresentam bioatividade que inibem o crescimento micelial dos fungos fitopatogênicos Moniliophthora perniciosa e Phytophthora cinnamomi, podendo ser uma nova opção coadjuvante no controle de fitopatógenos.
The Brazilian biodiversity is known due to its richness of species, and is considered a promising source of natural products. Among the vascular plants, the family Solanaceae A. Juss. (Solanaceae) is considered one of the largest, with distribution in all tropical and temperate regions of the world. The Solanaceae family has a high diversity of species of economic importance as a source of food, medicinal and ornamental properties. Plants of this family are sources of secondary metabolites from different chemical classes with many different applications. Plant fungi are responsible for causing various diseases and considerable losses in agriculture. The disease control is accomplished through chemical, physical and biological methods, but the excessive and continuous use of chemical products, may result in selection of resistant micro-organisms, in addition, many fungicides have a high toxicity and its indiscriminate use can cause undesirable effects on other organisms in the environment. The objective of this study is to explore the biological and chemical potential of secondary metabolites produced by plants of the Solanaceae family with potential fungitoxic the pathogens. We selected 15 species of Solanaceae plants that had the extracts of its leaves evaluated in vitro biological assays on the mycelial growth of 6 plant pathogens of importance in agriculture. Among these, three were selected for further investigation, the species Solanum americanum, Physalis peruviana and Acnistus arborescens. In the study of plant S. americanum, it was identified bioactive compounds belonging to the class of glycoalkaloids that inhibited the mycelial growth of the pathogen M. perniciosa. In the study of A. arborescens it was identified the presence of the active compound belonging to the class of withanolides, probably the 7β-acetoxywithanolide D, and its antifungal activity is being reported for the first time. In the study of plant P. peruviana semipure bioactive fraction indicated the presence of the compound belonging to the class of withanolides. These results showed that the compounds present in plants, have bioactivity that inhibit the mycelial growth of pathogenic fungi M. perniciosa and P. cinnamomi and may be a new option in the adjuvant control pathogens.

Le retour à la naturalité a favorisé le développement des compléments alimentaires à base de ressources végétales qui apparaissent comme un réservoir quasi-infini de nutriments et de substances naturelles bioactives. Ceci fait de l’extraction solide/liquide une étape incontournable au sein des industries intéressées par ce type de molécules. Avec les préoccupations environnementale set sociétales, il est devenu nécessaire d’inventer et développer de nouveaux procédés qui répondent aux six principes de l’éco-extraction. Cette démarche a totalement inspiré cette thèse qui a pour principal objectif le développement d’une technique d’éco-extraction assistée par ultrasons en substitution de la technique conventionnelle. Ce travail a permis de montrer qu’il était possible d’intensifier l’extraction de composés d’intérêt en utilisant les ultrasons avec une meilleure sélectivité et de meilleurs rendements d’extraction. Une attention particulière a été accordée à la compréhension des mécanismes d’action des ultrasons via une étude macroscopique et microscopique approfondie des structures végétales. Cette investigation a prouvé que les ultrasons agissent différemment en fonction des structures végétales et de leurs propriétés morphologiques et chimiques qui leur confèrent un degré de résistance plus ou moins important face à l’action des ultrasons. Partant de ces résultats, l’étude macroscopique et microscopique a été définie comme un outil de décision pour une extraction ciblée. Cette variabilité a été aussi constatée à l’échelle industrielle prouvant davantage l’importance de l’analyse microscopique. Enfin, le procédé d’extraction par ultrasons a été adopté à l’échelle industrielle pour ses performances d’extraction et pour son empreinte environnementale significativement réduite par rapport au procédé CV. Ce travail a également conduit à des travaux complémentaires sur l’étude du potentiel de solubilisation des produits naturels en vue d’une utilisation pour l’extraction de composés végétaux difficiles à solubiliser dans l’eau. Des résultats prometteurs ont été obtenus en termes de pouvoirs de solubilisation et d’extraction notamment à partir de la matière végétale broyée. Les résultats de cette dernière partie soulèvent cependant des questions qui pourraient faire l’objet de futures recherches et de perspectives pour ce travail qui sont principalement liés à l’étude des problèmes liés au changement du solvant et au prétraitement de la matière première et de la faisabilité industrielle de ce nouveau procédé
With recent trends in the increasing interest to environmental, economic and safety considerations,extraction techniques have largely focused on finding solutions with sustainable and green values toimplement in food processing, cosmetic and pharmaceutical industries. In this context, new “green”extraction techniques were developed such as Ultrasound-Assisted Extraction (UAE). The mainobjective of this thesis is industrial implementation of this new process in substitution to theconventional (CV) process. It has been shown in this work that the extraction of compounds ofinterest from rosemary and other plant matrices could be intensified using ultrasound, and thatdifferent performance gain could be achieved according to the plant matrix structural properties.Indeed, macroscopic and microscopic investigation of untreated and treated raw materials provedthat US act through different mechanisms and its resulting impacts can be extremely limited by plantstructural morphological and chemical properties, especially those of the specialized structures.Significant variability in performance gain was also observed at the industrial scale. Overall, USappears as a promising technique with a significant performance gain in terms of extraction yield andselectivity. Moreover, this process presents low environmental footprint compared to the CV one.Finally, it has been shown that natural products, such as honey and fruit juices, can be used toimprove solubilization and extraction of molecules that are poorly soluble in water. Encouragingresults were obtained in terms of solubilization and extraction abilities, especially from ground rawmaterials. However, these results raise questions related to the feasibility of industrialimplementation of this new process

La résistance aux antimicrobiens est l’un des défis majeurs du XX1eme siècle. Pseudomonas aeruginosa est inscrit sur la liste des organismes pathogènes qui deviennent résistants aux antibiotiques conventionnels. De nouvelles stratégies visant à atténuer la virulence sans perturber la croissance et la viabilité bactériennes, également connues sous le nom de stratégie anti-virulence, sont développées. Les plantes sont connues pour produire de nombreux métabolites secondaires. Des extraits de fruits de Pistachia Lentiscus originaires d'Algérie et de 40 extraits de plantes originaires du Nord-Chili ont été criblés pour leur capacité à atténuer la production de la pyocyanine, un facteur de virulence majeur de P. aeruginosa, dans le but d’évaluer leur potentiel effet antivirulence. Les extraits sélectionnés (Pistacia lentiscus, Azorella atacamensis, Baccharis grisebachii, Haplopappus rigidus et Parastrephia terestiucula), ont été fractionnés et l’ensemble de ces extraits et fractions a montré une atténuation de la production d’autres facteurs de virulence (élastase, rhamnolipides), qui a pu être attribuée, au moins partiellement à une diminution de la communication bactérienne via le mécanisme du quorum sensing. Ces extraits et fractions altèrent également la fluidité membranaire de P. aeruginosa. Cet effet anti-virulence a été validé dans un modèle d'infection cellulaire, et sur le nématode Caenorhabditis elegans. Dans toutes ces conditions, la croissance de P. aeruginosa n'a pas été affectée. Un profilage chimique des extraits et fractions de P. lentiscus et d'A atacamensis a révélé la présence d'acide gingkolique et de diterpenoides de type azorellane/mulinane comme potentiels composés bioactifs. De futures études visent à identifier les composés bioactifs sur P. aeruginosa H103, ainsi que sur un panel de souches cliniques, et à évaluer un potentiel effet potentialisateur de l'activité des antibiotiques. Ces travaux visent in fine à proposer ces composés d’origine végétale comme adjuvants dans le traitement des infections à P. aeruginosa
Antimicrobial resistance has become a great challenge in therapeutic medicine so much so that the World health organization forecasts the possibility of a post-antibiotic era where minor injuries may lead to mortality. Pseudomonas aeruginosa is among the list of organisms that are highly resistant to conventional antibiotics, partly due to its broad genome, which facilitates the elaboration of virulence determinants and rapid adaptation to various environments, in addition to its inherent resistance mechanisms. In view of this, alternative measures of controlling microbial virulence activities using novel approaches that do not disturb its growth and viability, also known as anti-virulence strategy, are gaining wider attention. Since plants are repositories of several metabolites with chemical defense system against environmental pathogens, through ethnobotanical led studies, the effect of Pistacia lentiscus fruit extracts originating from Algeria and forty plant extracts originating from North-Chile were biologically and chemically evaluated with the aim of deciphering their anti-virulence effects against P. aeruginosa. Furthermore, this study tried to gain more insight into the bioactive compounds and possible mechanism of action. From the results obtained, selected plant extracts attenuated P. aeruginosa mainly pyocyanin activity and /or elastase and rhamnolipids virulence production which appears to be associated with the inhibition of quorum sensing activities and the alteration in membrane activities. The anti-virulence effect of the selected extracts (P. lentiscus, Azorella atacamensis, Baccharis grisebachii, Haplopappus rigidus and Parastrephia terestiucula) were also validated in biological models of infections where they mediated the toxicity of P. aeruginosa towards A549 human monolayer cells and/or Caenorhabditis elegans nematode. Interestingly, growth of the pathogen was not affected. Further chemical profiling of P. Lentiscus, and A atacamensis extracts revealed the presence of gingkolic acid and azorellane/mulinane diterpenoids as the putative bioactive compounds. Future studies intend to explore these extracts and their derived compounds on the potentiation of antibiotic activity in a panel of clinical strains. In general, this study sets the pace for the possible use of these plant extracts as adjuvants in treatment of P. aeruginosa infections

Dissertação de mestrado em Biologia Molecular, Biotecnologia e Bioempreendedorismo em Plantas
The global quality and characteristics of grape berries, and ultimately of wine, are influenced by their polyphenolic composition. Therefore, potential strategies to improve berry quality by targeting secondary metabolism pathways of phenolic compound synthesis are useful, particularly in an ongoing context of climate change. These pathways are modulated by several molecular mechanisms, including regulation of gene transcription by specific transcription factors and post-transcriptional regulation by microRNAs. Recently, it was discovered that the primary (non-mature) miRNAs transcripts (pri-miRNAs) could encode for small regulatory peptides (micropeptides – miPEPs). In a positive loop, these miRNA-encoded peptides enhance the transcription and accumulation of their corresponding pri-miRNAs and, consequently, of their mature miRNAs, subsequently leading to an accentuated negative regulation of miRNA-regulated target genes. The objective of this work was to explore this recent discovery and to experiment the exogenous application of a micropeptide (miPEP396a) that putatively promotes the inhibition of the transcription factor VvMYB5b, an activator of the expression of several genes involved in the flavonoid pathway. Designated in this study as miPEP-MYB5b, this micropeptide may serve as a fine-tuning tool for modulation of secondary metabolic pathways in grape berry cells and, consequently, improve their global quality-traits. MiPEP-MYB5b was identified in silico and exogenously added to a Gamay Freaux grape berry cells in two different concentrations (0.1 μM and 0.5 μM). Its effect in the concentration of secondary metabolites such as anthocyanins, total flavonoids, total phenolics and stilbenes as well as in the transcription of key genes involved in biosynthetic routes that produce secondary metabolites with bioactive properties and important for grape berry quality, particularly in flavonoid- and stilbene-synthesizing pathways was analyzed. Both concentrations of miPEP-MYB5b resulted in downregulation of key genes involved in the flavonoid pathway, such as VvLAR1, VvLAR2, and VvCHI, while 0.5 μM resulted in downregulation of flavonoid-related genes VvANR, VvFLS1, VvCHS1. A parallel stimulation of the expression of the stilbene-synthesizing gene VvSTS1 was also observed in miPEP-treated cells. This upregulation of the stilbene pathway was probably due to a miPEP-MYB5b-mediated inhibition of MYB5b and, thus, of the flavonoid pathway, that competes directly with the stilbene pathway for substrate. Concordantly with the inhibition of the flavonoid pathway and stimulation of the stilbene pathway, a higher stilbene content and lower concentration of flavonoids (including anthocyanins) were quantified in grape berry cells. Thus, miPEP-MYB5b exogenous application may be a promising strategy to modulate secondary metabolic pathways in order to produce and accumulate higher quantity of stilbenes in grape berry cells in a near future, by exploring mechanisms of microRNA-mediated gene regulation in plants.
A qualidade e características globais dos bagos de uva e do vinho são influenciadas pela composição em polifenólicos. Por isso, estratégias que melhorem a qualidade dos bagos tendo como alvo as vias do metabolismo secundário que sintetizem compostos fenólicos são úteis, particularmente, no contexto atual das alterações climáticas. Estas vias são moduladas por vários mecanismos moleculares, incluindo a regulação da transcrição de genes através de fatores de transcrição específicos e regulação pós-transcricional através de microRNAs. Recentemente, descobriu-se que os transcritos primários (não-maduros) dos miRNAs codificam pequenos péptidos reguladores (micropéptidos- miPEPs). Num “loop” positivo, estes micropéptidos aumentam a transcrição e a acumulação do pri-miRNA e miRNA correspondentes e, posteriormente, levam a uma regulação negativa dos genes alvo de uma forma mais acentuada. O objetivo deste trabalho consistiu em explorar esta recente descoberta e experimentar a aplicação exógena de um micropéptido (miPEP396a) que, putativamente, promove a inibição do fator de transcrição VvMYB5b, um ativador da expressão de vários genes envolvidos na via dos flavonoides. Designado por miPEP-MYB5b neste estudo, este micropéptido poderá permitir a manipulação das vias do metabolismo secundário nas células dos bagos e, consequentemente, melhorar a sua qualidade global. O miPEP-MYB5b foi identificado in silico e depois adicionado a uma cultura celular de bagos da variedade Gamay Freaux, em duas concentrações diferentes (0,1 μM e 0,5 μM), sendo analisado o seu efeito na concentração de metabolitos secundários como antocianinas, flavonoides totais, fenólicos totais e stilbenos, bem como na transcrição de genes-chaves envolvidos nas vias metabólicas que produzem estes compostos secundários com propriedades bioativas e de grande importância para a qualidade dos bagos, particularmente nas vidas de síntese de flavonoides e stilbenos. Ambas as concentrações de micropéptido levaram à regulação negativa dos genes VvLAR1, VvLAR2 e VvCHI, enquanto a 0,5 μM provocaram uma regulação negativa de genes relacionados com a via dos flavonoides, como VvANR, VvFLS1 e VvCHS1. Foi também observada uma estimulação da expressão do gene VvSTS1, responsável pela síntese de stilbenos, em células tratadas com o micropéptido. Esta regulação positiva da via dos stilbenos ocorreu, provavelmente, devido a uma inibição do MYB5b mediada pelo micropéptido que, em consequência, inibiu a via dos flavonoides, que compete por substrato diretamente com a via dos stilbenos. Para além da inibição da via dos flavonoides e estimulação da via dos stilbenos, detetaram-se maiores quantidades de stilbenos e menor concentração de flavonoides (incluindo antocianinas) em células de bagos de uva. Desta forma, a aplicação exógena do miPEP-MYB5b poderá ser uma estratégia promissora para modular as vias do metabolismo secundário para produzir e acumular mais stilbenos em células dos bagos de uva, explorando os mecanismos de regulação genética mediados por miRNAs em plantas.
The work was supported by National Funds by FCT - Portuguese Foundation for Science and Technology, under the strategic programmes UID/AGR/04033/2019 and UID/BIA/04050/2019. The work was also supported by FCT and European Funds (FEDER/POCI/COMPETE2020) through the research project “MitiVineDrought - Combining "omics" with molecular, biochemical and physiological analyses as an integrated effort to validate novel and easy-to-implement drought mitigation strategies in grapevine while reducing water use” with the ref. PTDC/BIA-FBT/30341/2017 and ref. POCI-01-0145-FEDER-030341, respectively; through the research project “BerryPlastid - Biosynthesis of secondary compounds in the grape berry: unlocking the role of the plastid” with the ref. POCI-01-0145-FEDER-028165 and ref. PTDC/BIA-FBT/28165/2017, respectively; and also through the FCT-funded research project “GrapeInfectomics” (PTDC/ASP-HOR/28485/2017). This work was also supported by the project “INTERACT - VitalityWine - ref. NORTE-01-0145-FEDER-000017 – (through FEDER/COMPETE and NORTE2020/CCDR-N). Artur Conde was supported with a post-doctoral fellow of the mentioned INTERACT/VitalityWine project with the ref. BPD/UTAD/INTERACT/VW/218/2016, and also supported by a post-doctoral researcher contract/position within the project “MitiVineDrought” (PTDC/BIA-FBT/30341/2017 and POCI-01-0145-FEDER-030341). This work also benefited from the networking activities within the European Union-funded COST Action CA17111 – “INTEGRAPE - Data Integration to maximize the power of omics for grapevine improvement”.

Marine organisms are a source of a diverse range of secondary metabolites. This thesis describes the isolation and structure elucidation of novel alkaloid and terpenoid metabolites from marine invertebrates which were collected from the Mackay - Capricorn Section of the Great Barrier Reef Marine Park (Keppel Bay and the Capricorn Bunker Group).

B-Carboline, its N,N symmetrical dimer and a series of novel asymmetrical dimers of B-carboline, were isolated from a didemnid ascidian (genus Didemnum). The asymmetrical dimers, however, were isolated in such low yield from the ascidian that their structures could not be conclusively determined. Derivatization of B-carboline allowed the preparation of these compounds in sufficient quantities to allow the elucidation of their structures by NMR spectroscopy. Two other new asymmetrical dimers of B-carboline, not observed in the ascidian, were also prepared and their structures elucidated.

Tetronic acids are commonly isolated from sponges of the genus Ircinia. A novel sesterterpene tetronic acid was isolated from the sponge Ircinia (= Psammocinia) wistarii. This novel compound (a sulfate ester) was highly unstable; rapid decomposition of the sulfate ester resulted in the formation of the known compounds ircinianin and wistarin. The isolation and structure elucidation of the novel sulfate ester is described in Chapter 3 of this thesis.

Alcyonolide 5, a novel diterpene triacetate, was isolated from two soft corals of the order Alcyonacea. This compound, is one of a series isolated from corals of the genera Alcyonium and Efflatounaria.

The bastadins are a series of (typically macrocyclic) tyrosine derivatives, commonly isolated from the sponge lanthella basta. During these investigations a new bastadin was isolated from lanthella quadrangulata.

The norcembrenolides, a series of norditerpenes, are commonly isolated from the soft corals of the genus Sinularia. The soft coral Sinularia numerosa was found to contain two of these compounds. One was the known compound norcembrenolide (the first representative of this class), the second was a stereoisomer of norcembrenolide. The chemical literature reports several compounds of this type, however, there appears to be a degree of confusion surrounding the structures of these stereoisomers. This thesis resolves the confusion surrounding the stereochemistry of these isomers, and assigns a structure to the minor metabolite of S. numerosa.

Approximately 200 marine invertebrates were collected during the course of these investigations. Many known compounds were isolated from the marine invertebrates that were collected from the Mackay - Capricorn Section of the Great Barrier Reef Marine Park. This afforded an opportunity to survey the natural products which may be found in this region, and highlights some of the difficulties associated with finding novel compounds within this region.

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.

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.