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Nešplja (Mespilus germanica L.) ali mušmula spada v družino rožnic (Rosaceae) in izvira iz jugozahodne Azije. Poznamo več sort nešpelj, in sicer 'Domača' ali 'Navadna' nešplja, 'Kraljevska' ali 'Royal', 'Holandska' in 'Nottingham' nešplja. Plodovi so značilne temno rjave barve z veliko muho. Tehnološko zreli plodovi niso užitni, zato jih po obiranju shranimo v temnem in hladnem prostoru, da se umedijo ali pa jih pustimo na drevesu, da so izpostavljeni slani. Plodovi nešplje so bogat vir primarnih in sekundarnih metabolitov, ki imajo koristne in zdravilne učinke na zdravje ljudi. V magistrski nalogi smo preučevali vpliv različnih načinov shranjevanja na vsebnost izbranih primarnih in sekundarnih metabolitov v plodovih nešplje. Ugotovili smo, da plodovi nešplje vsebujejo največ fruktoze (59,2 g/kg sveže mase (FW)) in glukoze (54,4 g/kg FW), med organskimi kislinami pa jabolčne (8,4 g/kg FW) in kinske kisline (8,8 g/kg FW). Izmed fenolov predstavljajo največje vsebnosti predstavljajo fenolne kisline (532,85 mg/kg FW) in flavanoli (375,21 mg/kg FW). Ugotovili smo, da se s shranjevanjem nešpelj zmanjšuje vsebnost vseh primarnih in sekundarnih metabolitov. Umedene nešplje, ki so skladiščene v kleti, imajo značilno najmanjše vsebnosti vseh analiziranih metabolitov. Ugotavljamo, da je zaradi precej velike vsebnosti metabolitov v plodovih nabranih po slani, to najprimernejši način shranjevanja za nešpljo. Takrat so plodovi užitno zreli in hkrati vsebujejo velike vsebnosti fenolnih snovi. Medlar (Mespilus germanica L.) belongs to Rosaceae family and is indigenous to southwest Asia. There are several cultivars of common medlar plant: ‘Domača’ or ‘Navadna’, 'Royal', 'Hollandia' and 'Nottingham'. The fruits are typically dark brown with a big calyx. Technologically ripe fruits are not edible, therefore they need to be stored in a dark and cold place after harvesting in order to undergo bletting. Alternatively, they can be left on a tree to soften and become bletted by frost. The medlar fruits are rich in primary and secondary metabolites, both of which have beneficial and healing health effects. In this master thesis the influence of different ways of storage procedures on the content of selected primary and secondary metabolites in medlar fruits were studied. The major determined sugars in fruits were fructose (59.2 g/kg fresh weight (FW)) and glucose (54.4 g/kg FW), whereas malic acid (8.4 g/kg FW) and quinic acid (8.8 g/kg FW) were major acids in medlar. Among the phenolics phenolic acids (532.85 mg/kg FW) and flavanols (375.21 mg/kg FW) were major groups. It was determined that storage reduces the content of all primary and secondary metabolites. Bletted medlars which were stored in a cellar had the lowest content of all metabolites. It was established that the most appropriate method of storage is harvesting fruit after natural hoarfrost. The fruits are at that time edibly ripe and at the same time possess high contents of metabolites.

ilustraciones, fotografías a color, gráficas, tablas En el presente documento se presentan los resultados del estudio fitoquímico y de actividad biológica del extracto etanólico de raíz de Zanthoxylum caribaeum (Rutaceae) y la obtención de metabolitos secundarios con posible actividad neuroprotectora aplicable en el tratamiento de la enfermedad de Alzheimer. Para el estudio fitoquímico biodirigido se usaron métodos de extracción y purificación clásicos como la extracción liquido – liquido (usando solventes de polaridad creciente: hexano, acetato de etilo, butanol y agua), cromatografía en columna y seguimiento de pureza por cromatografía en capa delgada, mientras que los métodos de caracterización e identificación de metabolitos se aplicaron técnicas instrumentales como cromatografía liquida de alta eficiencia, espectrometría de masas y métodos espectroscópicos como la resonancia magnética nuclear de hidrogeno y carbono, en una y dos dimensiones. El estudio de actividad bilógica se planteó en dos etapas; la primera por bioatugrafía directa de seguimiento al proceso de extracción y purificación del extracto crudo, facciones y compuestos puros usando DPPH y -Caroteno para evaluar el potencial antioxidante y Acetilcolinesterasa para evaluar el potencial inhibitorio de la encima; de esta manera se pretendió orientar la purificación hacia la obtención de metabolitos secundarios con capacidad inhibitoria de la encima y/o actividad antioxidante o captadores de radicales libres ya que estas dos propiedades han sido asociadas a la pérdida de las capacidades cognitivas de las personas que padecen el síndrome de Alzheimer. Adicionalmente se incluyó el reactivo de Dragendorff, con el fin de establecer que fracciones contenían metabolitos tipo alcaloide, ya que se ha evidenciado que estas moléculas presentan actividad neuroprotectora. En la segunda etapa del estudio biológico permitió evaluó de forma cuantitativa la capacidad antioxidante y anticolinérgica de las fracciones principales procedentes de la partición con solventes y de los metabolitos aislados aplicando métodos colorimétricos en microdilución usando Fisostigmina como control positivo para la actividad acticolinergica y Quercetina para la actividad antioxidante; en los casos en los que fue posible, se calculó la IC50. Es así como la purificación bioguiada condujo al aislamiento y caracterización de los siguientes compuestos: 6,8-dimetoxi-2-H-1-quinolin-2-ona, 3-((1-metilciclopentil) amino) quinolin-2(1H)-ona, 104-ozaxol[3,2-a]quinolina y ácido antranilico y la evaluación de su posible actividad neuroprotectora, basada en los resultados de actividad antioxidante y anticolinérgica, demostró que los compuestos de manera individual presentan actividades antioxidantes medias a bajas y que la actividad anticolinérgica se pierde a medida que se avanza en la putificación, siendo más activas las fracciones que los compuestos puros. (Texto tomado de la fuente). The present document presents the results of the phytochemical and biological activity study of the ethanolic extract of the root of Zanthoxylum caribaeum (Rutaceae) and the obtaining of secondary metabolites with possible neuroprotective activity applicable in the treatment of Alzheimer's disease. For the bio-directed phytochemical study, classic extraction and purification methods were used such as liquid-liquid extraction (using solvents of increasing polarity: n-hexane, ethyl acetate, n-butanol and water), column chromatography and purity monitoring by thin layer chromatography. While the methods of characterization and identification of metabolites were applied instrumental techniques such as high efficiency liquid chromatography, mass spectrometry and spectroscopic methods such as nuclear magnetic resonance of hydrogen and carbon, in one and two dimensions. The study of biological activity was planned in two stages; the first by direct bioatugraphy of follow-up to the extraction and purification process of the crude extract, factions and pure compounds using DPPH and -Carotene to evaluate the antioxidant potential and Acetylcholinesterase to evaluate the inhibitory potential of the enzyme; In this way, it was intended to orient purification towards obtaining secondary metabolites with inhibitory capacity of the enzyme and / or antioxidant activity or free radical scavengers since these two properties have been associated with the loss of cognitive abilities of people who suffer from Alzheimer's syndrome. Additionally, the Dragendorff reagent was included, in order to establish which fractions contained alkaloid-like metabolites, since it has been shown that these molecules have neuroprotective activity. In the second stage of the biological study, it was possible to quantitatively evaluate the antioxidant and anticholinergic capacity of the main fractions from the partition with solvents and the isolated metabolites applying colorimetric methods in microdilution using Physostigmine as a positive control for acticholinergic activity and Quercetin for the antioxidant activity; In the cases where it was possible, the IC50 was calculated. Thus, the bioguided purification led to the isolation and characterization of the following compounds: 6,8-dimethoxy-2-H-1-quinolin-2-one, 3-((1-methylciclopentil) amino) quinolin-2(1H)-one, 104-ozaxol [3,2-a] quinoline and Anthranilic acid and the evaluation of its possible neuroprotective activity, based on the results of antioxidant and anticholinergic activity, showed that the compounds individually present medium to low antioxidant activities and that the anticholinergic activity is lost as the putification progresses, the fractions being more active than the pure compounds. Ministerio de Ciencias, Tecnología e Innovación Maestría Magíster en Ciencias - Química Productos Naturales

Za poliploidne rastline je značilno, da imajo zaradi pomnoženih kromosomov zmožnost utišanja ali izražanja novih genov, ki boljše vplivajo na rast, razvoj in druge lastnosti rastline. Na primeru konoplje (Cannabis sativa L.) in hmelja (Humulus lupulus L.), katera se gojita večinsko za uporabo sekundarnih metabolitov, ima indukcija poliploidnosti velik pomen. Konoplja se po svetu prideluje v medicinske namene za pridobivanje kanabinoidov CBD-ja in THC-ja. Slednji ima psihoaktivne učinke in zaradi tega večina držav na svetu konopljo uvršča med ilegalne droge. Z indukcijo poliploidnosti lahko mi povečamo ali zmanjšamo vsebnost določenih kanabinoidov ter tako omogočimo bolj ekonomično pridelavo. Pri hmelju je podobno in sicer so glavni pridelek ženska socvetja, s prisotnostjo alfa-kislin, katera se uporabljajo v pivovarski industriji. Z indukcijo poliploidnosti, specifično triploidnosti, lahko pridelujemo partenokarpa socvetja, katera ni potrebno predčasno tretirani z raznimi substancami, z namenom odstranitve semen, ki bi znižala ekonomsko vrednost in okus pridelka. Namen diplomskega dela je predstavitev do sedaj uporabljenih metod indukcije poliploidnosti pri teh dveh vrstah. Polyploid plants are characterized by the ability to use or express new genes, which have a beneficial influence on the growth, development and other characteristics of the plant, due to the multiplied chromosomes. In the case of cannabis (Cannabis sativa L.) and hops (Humulus lupulus L.), which are grown mostly for the use of secondary metabolites, the induction of polyploidy is of great importance. Cannabis is grown worldwide for medical purposes to obtain cannabinoids CBD and THC. The Latter has psychoactive effects and as a result most countries in the world classify cannabis as an illegal drug. By inducing polyploidy, we can increase or decrease the content of particular cannabinoids to enable more economical farming. In the case of hops, it is similar, the main yield are female inflorescences, which contain alpha-acids, that are used in the brewing industry. By inducing polyploidy, especially triploidy, we can grow parthenocarpic inflorescences, which do not need to be prematurely treated with various substances, to remove seeds, which would reduce the economic value and taste of the crop. The aim of the thesis is to present techniques that have been used to induce poliploidy in cannabis and hop.

Hmelj je trajna ovijalka, primarno gojena zaradi uporabe v pivovarstvu. Za pivovarstvo so uporabni storžki hmelja, ki s svojimi bioaktivnimi snovmi pivo konzervirajo in mu dajejo prepoznavno aromo in okus. Storžke uporabljajo tudi v proizvodnji farmacevtskih pripravkov. Poleg storžkov so uporabni tudi mladi poganjki, ki se uporabljajo kot zelenjava, preizkušajo pa tudi potencialno uporabnost listov in plodov. Storžki in poganjki ter tudi listi in plodovi vsebujejo snovi, ki imajo bioaktivno delovanje. Bioaktivne snovi, ki so lahko primarni ali sekundarni metaboliti, hmelju dajejo sedativne, estrogene, protimikrobne, antioksidativne, antikancerogene in druge lastnosti. Najbolj so raziskane bioaktivne snovi v storžkih, med njimi zlasti eterična olja, hmeljeve smole in hmeljevi fenoli. Med fenoli sta najbolj zanimiva ksantohumol in 8-prenilnaringenin, ki velja za enega najmočnejših fitoestrogenov. Zaradi enostavne uporabe in brez primerov klinično obravnavanih alergijskih reakcij in stranskih učinkov ima hmelj velik potencial v farmaciji. Hops is a perrenial climbing plant, primarily cultivated for its use in brewing. Hop cones are used in brewing, whose bioactive compounds conserve beer and give it its distinctive aroma and taste. Hop cones are also used in manufacturing pharmaceuticals. In addition to cones, young shoots are used as vegetables, potential uses for leaves and seeds are also being investigated. Cones, shoots, as well as leaves and seeds contain bioactive compounds. Bioactive substances, which are primary or secondary metabolites, give the hops sedative, estrogenic, antimicrobial, antioxidant, anticancer and other properties. Bioactive compounds found in hop cones are the most studied, especially essential oils, hop resins and hop phenolics. Among the phenolics, the most interesting ones are xanthohumol and 8-prenylnaringenin, the latter is considered to be one of the most potent phytoestrogens. Because of its simple use and unregistered allergic reactions or side effects, hops have great potential in pharmaceutical use.

Izvedli smo lončni poskus na dveh vrstah čilija C. annuum L. 'Chili-AS Rot' in C. chinense Jacq. 'Naga Morich'. Rastline smo namakali pod dvema različnima namakalnima režimoma, in sicer optimalno namakanje do poljske kapacitete in deficitno namakanje do 50 % poljske kapacitete (nadzorovan sušni stres). Preučevali smo, kako zmanjšana količina vode oziroma sušni stres vplivata na količino in kakovost pridelka (primarne in sekundarne metabolite v plodovih čilijev). Deficitno namakanje je imelo vpliv na količino, saj so imele rastline pri vseh terminih vzorčenja manjše pridelke. Pri vrsti C. chinense 'Naga Morich' je deficitno namakanje vplivalo na samo cvetenje, saj so proti koncu cvetovi začeli odpadati. Vsebnosti sladkorjev so bile manjše pri rastlinah v sušnem stresu, tako pri vrsti C. annuum 'Chili-AS Rot' kot pri vrsti C. chinense 'Naga Morich'. Deficitno namakanje je vplivalo na vsebnost organskih kislin. Pri sorti C. annuum 'Chili-AS Rot' so bile vsebnosti citronske, kinske, fumarne, šikimske in oksalne kisline večje pri optimalno namakanih rastlinah ter vsebnosti jabolčne kisline so bile identične pri obeh tipih namakanja. Sorta C. chinense 'Naga Morich' je vsebovala več citronske kisline pri optimalnem namakanju. Vsebnosti ostalih organskih kislin so bile večje pri rastlinah v sušnih razmerah. Sorta C. annuum 'Chili-AS Rot' je vsebovala več askorbinske kisline in skupnih fenolnih spojin ter manj kapsaicina in dihidrokapsaicina pri optimalnemu namakanju pri poljski kapaciteti. Sorta C. chinense 'Naga Morich' je vsebovala več askorbinske kisline, skupnih fenolnih spojin, kapsaicina in dihidrokapsaicina pri optimalnemu namakanju. A pot experiment was conducted on two species of chilies C. annuum L. 'Chili-AS Rot' and C. chinense Jacq. 'Naga Morich'. Plants were irrigated at two different irrigation regimes, optimal irrigation or field capacity and deficit irrigation or 50 % field capacity, which can be treated as drought stress. The effects of drought stress on quantity and quality of the yield (primary and secondary metabolites), were studied. Deficit irrigation affected yield quantity, as both chili species had lower yield at deficit irrigation. Species C. chinense 'Naga Morich' under deficit irrigation abscised the flowers, thus no fruits were harvested at the third picking. Concentrations of sugars were lower at both plant species under deficit irrigation. Concentrations of organic acids in fruits of C. annuum 'Chili-AS Rot', such as citric acid, tartaric acid, oxalic acid, fumaric acid, quinic acid were higher at optimal irrigation. Malic acid concentrations were similar at both irrigation treatments. In the fruits of C. chinense Jacq. 'Naga Morich', the concentration of citric acid was higher with optimal irrigation. Concentrations of other organic acids were higher at deficit irrigation. C. annuum 'Chili-AS Rot' had higher concentration of ascorbic acid and total phenolic content at optimal irrigation and higher capsaicin and dihydrocapsaicin concentrations in stressed plants. C. chinense 'Naga Morich' had higher concentrations of ascorbic acid, total phenolic content, capsaicin and dihydrocapsaicin at optimal irrigation regime.

Na območju gorenjske regije smo v treh poskusih, v obdobju 2011–2015, analizirali morfološke parametre korenov in vsebnost metabolitov v korenčku (Daucus carota L.), pridelanem po smernicah ekološke in integrirane pridelave. Vsebnost primarnih in sekundarnih metabolitov smo analizirali s pomočjo sistema visokotlačne tekočinske kromatografije v kombinaciji z masno spektrometrijo (HPLS-MS), glede na kultivar ('Rodelika', 'Fanal' in 'Rolanka'), vrsto uporabljenega gnojila (organsko in mineralno), povečano količina dušika in kalija v gnojilu ter leto pridelave. Dodana hranila so v obeh pridelovalnih sistemih značilno povečala (od 47–262 %) pridelek korenčka vseh preizkušenih kultivarjev, glede na kontrolno obravnavanje (negnojene parcele). V ekološko pridelanem korenčku je bilo značilno manj jabolčne (43 %), fumarne (19 %) in šikimske kisline (23 %) ter več piruvične (80 %) in askorbinske kisline (12 %) v primerjavi z integrirano pridelanim korenčkom. Vsebnost fenolnih kislin in karotenoidov je bila sortno značilna. Koreni kultivarja 'Fanal' so vsebovali 30 % manj karotenoidov kot koreni kultivarjev 'Rodelika' in 'Rolanka'. V korenih kultivarja 'Rolanka' je bilo 21 % in 48 % več fenolnih kislin v 'Rodelika' in 'Fanal'. Povečana količina dodanega dušika in kalija je povečala vsebnost saharoze (10 %) in zmanjšala vsebnost derivatov ferulne kisline (105 %) glede na manj gnojen korenček. V korenih negnojenega korenčka je bilo 37 % manj saharoze, 64 % manj jabolčne kisline in 10 % več askorbinske kisline ter 74 % več derivatov ferulne kisline glede na povečano gnojenje z N in K. Zrelost korena je imela značilen vpliv na vse analizirane metabolite, leto pridelave pa le na vsebnost glukoze (65 % manj), jabolčne kisline (36 % več), askorbinske kisline (26 % manj) in fenolnih kislin, ki jih je bilo v letu 2012 160 % več glede na leto 2011). V tretjem tj. lončnem poskusu smo pridelali korenček za analizo izotopske sestave dušika, ki bi služil kot marker za ločevanje ekološko in integrirano pridelanega korenčka. Uporaba organskega ali mineralnega gnojila je značilno vplivala na izotopsko sestavo dušika v korenčku (organsko gnojen korenček je imel 47–107 % več težjega izotopa 15N kot korenček, gnojen z mineralnimi gnojili), vendar pa metoda izotopske sestave δ15N ni dovolj natančna, da bi jo uporabili kot edini kriterij za preverjanje pristnosti ekološke pridelave. In the period 2011–2015 we conducted three experiments in the Gorenjska region in which we analysed morphological parameters of the taproots and the content of metabolites in carrots (Daucus carota L.) grown according to the organic and integrated growth guidelines. The content of primary and secondary metabolites was analysed with high performance liquid chromatography and mass spectrometry (HPLS-MS). We considered cultivar ('Rodelika', 'Fanal' and 'Rolanka'), type of fertiliser (organic and mineral), the increased amount of nitrogen and potassium and the year of growth. In both growing systems the added nutrients significantly increased the yield of all cultivars (from 47 % to 262 %) as compared to the control plots (not fertilised). Organically grown carrots contained significantly less malic acid (43 %), fumaric acid (19 %), and shikimic acid (23 %). On the other hand, they contained more pyruvic acid (80 %) and ascorbic acid (12 %) compared to the carrots from the integrated growth system. The content of phenolic acids and carotenoids dependend on the cultivar. 'Fanal' contained 30 % less carotenoids than 'Rodelika' and 'Rolanka'. 'Rolanka' taproots contained 21 % and 48 % more phenolic acids than 'Rodelika' and 'Fanal' respectively. The increased amount of added nitrogen and potassium influenced saccharose content which was increased by 10 %, and reduced the amount of ferulic acid derivatives by 105 % as compared to the less heavily fertilised carrots. The taproots of unfertilised carrots contained less saccharose (37 %), less malic acid (64 %), more ascorbic acid (10 %) and more ferulic acids derivatives (74 %) with regard to the increased fertilisation with nitrogen and potassium. Taproot maturity significantly influenced all the metabolites we had analysed while the year of growth only influenced glucose content (65 % less), malic acid (36 % more), ascorbic acid (26 % less) and phenolic acids (160% more in 2012 as compared to 2011). In the third experiment, which was a pot experiment, we grew carrots for the analysis of the isotopic composition of nitrogen which would serve as a marker in determining growing system (organic or integrated). Using organic or mineral fertiliser, respectively, significantly influenced the isotopic composition of nitrogen in the carrots (organically grown carrots contained from 47 % to 107 % more of the heavier isotope 15N than the ones fertilised with mineral fertilisers, however, the method of isotopic composition δ15N is not accurate enough to be used as a sole criterion in determining the authenticity of organic growth.

Merremia aegyptia (Convolvulaceae) is not affected by the predominant aridity in most of guajiro territory (La Guajira, Colombia). However, this species has been underestimated and considered weed since its medicinal properties and other possible uses are unknown. Thus, the present investigation was tried to establish the influence of seasons and the phenology on the production of polyphenols in M. aegyptia and to estimate the antioxidant potential of its extracts. Foliar tissue samples were taken for one year and subjected to methanol extraction, total polyphenol concentration was established by Folin-Ciacolteau method and correlated with monthly precipitation and temperature. In addition the percentage of inhibition of its leaf extracts on the DPPH radical was estimated. Results suggest variation in monthly concentration of total polyphenols, obtaining the highest concentration in January 2018 (6,03%). Furthermore, the extracts have a higher antioxidant activity (85,15%) than ascorbic acid (38,65%). It was concluded that the availability of water in soil affects the production of polyphenols in M. aegyptia and that this species represents a plant resource capable of being cultivated and exploited under the environmental conditions of La Guajira., Merremia aegyptia (Convolvulaceae) no se ve afectada por la aridez que presenta la mayor parte del territorio guajiro (La Guajira, Colombia). Sin embargo, ha sido subestimada y considerada arvense al desconocerse sus propiedades medicinales y otros posibles usos. Así, con la presente investigación se pretendió establecer la influencia de los periodos climáticos y la fenología sobre la producción de polifenoles en dicha especie y estimar el potencial antioxidante de sus extractos. Se tomaron muestras de tejido foliar durante un año y se sometieron a extracción con metanol, la concentración de polifenoles totales fue establecida mediante el método de Folin-Ciacolteau y se correlacionó con la precipitación y temperatura mensual. Además, se estimó el porcentaje de inhibición de los extractos sobre el radical DPPH. Los resultados obtenidos indicaron variación en la concentración mensual de polifenoles totales, siendo enero de 2018 el mes donde se registró la mayor concentración (6,03%). Adicionalmente, los extractos presentaron mayor actividad antioxidante (85,15%) que el ácido ascórbico (38,65%). Se concluyó que la disponibilidad de agua en el suelo afecta la producción de polifenoles en M. aegyptia y que esta especie representa un recurso vegetal susceptible de ser cultivado y explotado bajo las condiciones ambientales de La Guajira., Merremia aegyptia (Convolvulaceae) não é afetada pela aridez da maior parte do território guajiro (La Guajira, Colômbia). Porém tem sido subestimada e considerada como erva daninha pelo que nao são conhecidas suas propriedades medicinais e outros usos possíveis. Nesse sentido, a presente investigação procurou-se estabelecer a influência dos períodos climáticos e a fenologia na produção de polifenóis nessa espécie e estimar o potencial antioxidante de seus extratos. Amostras do tecido foliar foram colhidas por um ano e submetidas à extração de metanol e, a concentração de polifenóis totais foi estabelecida pelo método de Folin-Ciacolteau e correlacionada com precipitação e temperatura mensais, além da porcentagem de inibição da extratos sobre o radical DPPH. Os resultados obtidos indicaram variação na concentração mensal de polifenóis totais, sendo janeiro de 2018 o mês em que a maior concentração foi registrada (6,03%); além disso, os extratos apresentaram maior atividade antioxidante (85,15%) que o ácido ascórbico (38,65%). Concluiu-se que a disponibilidade de água no solo afeta a produção de polifenóis em M. aegyptia e que essa espécie representa um recurso vegetal capaz de ser cultivado e explorado nas condições ambientais do estado La Guajira.

No presente trabalho foi realizado o estudo químico de Eriotheca pubescens com a finalidade de isolar e identificar estruturalmente metabólitos secundários presentes no caule e nas folhas desta espécie. Além disso, foi realizada avaliação do potencial antioxidante através do método de captura dos radicais livres com DPPH e avaliação do potencial de inibição frente às catepsinas K, L e V através de ensaio in vitro utilizando o substrato fluorogênico Z-FR-MCA. O estudo químico do extrato etanólico do caule levou ao isolamento e identificação dos triterpenoides α-amirina e β-amirina; dois diterpenoides, ácido 7-oxo-labda-8,13-dien-15-oico e o ácido Δ13,14-ent-labd-8β-ol-15-oico; quatro derivados do ácido benzoico, o ácido p-hidroxibenzoico, o ácido 3,4- diidroxibenzoico, o ácido 4-hidroxi-3,5-dimetoxibenzoico, o ácido 3-metoxi-4- hidroxibenzoico e o flavonoide quercetina. Do extrato etanólico das folhas foram isolados e identificados dois triterpenoides, sendo estes a lupenona e o lupeol. No ensaio de atividade antioxidante o ácido 3,4-diidroxibenzoico (EC50 de 0,34 ± 0,0083 mM) e a quercetina (EC50 de 0,11 ± 0,0057 mM) apresentam elevado potencial antioxidante. Nos ensaios frente às catepsinas K, L e V, os extratos e a maior parte das frações apresentaram atividade inibitória superior a 50% e das substâncias isoladas e avaliadas o ácido p-hidroxibenzoico mostrou-se um moderado inibidor (IC50 de 135,81 ± 17,07 μM) e a quercetina um potente e seletivo inibidor (IC50 de 2,2 ± 0,2 μM), ambos para catepsina V. Estes resultados são importantes para contribuir com o conhecimento químico e biológico desta espécie, que até o momento não possui relatos de estudos químicos e biológicos. In the present work the chemical study of Eriotheca pubescens lead to the isolation and structural identification of secondary metabolites present in the stems and leaves of this species. Furthermore, it was evaluated of antioxidant potential by the capture method of free radicals using DPPH and the study also was lead to search cathepsins K, L, and V inhibitors through in vitro test using the fluorogenic substrate Z-FR-MCA. The chemical study of the ethanolic extract from the stem led to the isolation and identification of triterpenoids α-amyrin and β-amyrin; two diterpenoids, 7-oxo-labda-8,13-dien-15-oic acid and ent-Δ13,14- labd-8β-ol-15-oic acid; four derivatives of benzoic acid, p-hydroxybenzoic acid, 3,4-dihydroxybenzoic acid, 4-hydroxy-3,5-dimethoxybenzoic acid, 3-methoxy-4- hydroxybenzoic acid and flavonoid quercetin. From the ethanolic extract of the leaves, it has been isolated and identified two triterpenoids, lupenona and lupeol. The compounds 3,4-dihydroxybenzoic acid (EC50 0,34 ± 0,0083 mM) and quercetina (EC50 de 0,11 ± 0,0057 mM) showed high antioxidant potential using DPPH assays. The extracts and the major fractions showed above 50% inhibitory activity against cathepsins K, L and V. The p-hydroxybenzoic acid was shown to be a moderate inhibitor (IC50 135,81 ± 17,07 μM) and quercetin a potent and selective inhibitor (IC50 2,2 ± 0,2 μM), both against cathepsin V. These results are important to contribute the chemical and biological knowledge of this species, which so far has no reports of chemical and biological studies. Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES

Pilocarpus species have been exploited as the only source of the imidazole alkaloid pilocarpine (used in glaucoma treatment), since its isolation up to the present. Almost all Pilocarpusspecies are native from Brazil. Because of the medicinal importance of pilocarpine, several of them are in the path of extinction. Other secondary metabolites, such as coumarins, flavonoids and terpenes, were described for Pilocarpusspecies. In this review the secondary metabolites, other than pilocarpine, isolated from Pilocarpusspecies and their biological activities were compiled. Although the variety of structures and the importance of the biological activities described in literature for Pilocarpusspecies this is an unexploited field of research in Natural Products and Pharmacology.Espécies de Pilocarpus têm sido exploradas como única fonte do alcalóide imidazólico pilocarpina (utilizado no tratamento do glaucoma) desde o isolamento dessa substância até os dias atuais. A maioria das espécies de Pilocarpus conhecida é nativa do Brasil e, devido à importância medicinal que a pilocarpina possui e ao desmatamento, várias se encontram em risco de extinção. Outros metabólitos secundários entre os quais cumarinas, flavonóides e terpenos foram descritos em espécies desse gênero. Nesta revisão foram relacionados os metabólitos secundários isolados em diversas espécies de Pilocarpus bem como suas atividades biológicas. Apesar da variedade de estruturas e as importantes atividades biológicas já descritas na literatura para as outras classes de metabólitos secundários, ainda há um vasto campo de estudo para as espécies de Pilocarpus.

Nieuwe biosyntheseroutes leveren 50 secundaire metabolieten op In zijn proefschrift beschrijft Hazrat Ali drie nieuwe biosynthetische routes van secundaire metabolieten, die meer dan vijftig secunaire metalbolieten opleverden met belangrijke biologische eigenschappen. De ontdekking van levensreddende medicatie en antibiotica die door de natuur geproduceerd worden heeft de levensverwachting van de mens de laatste eeuw enorm doen verlengen. Echter, door het ongecontroleerd toepassen van voornamelijk antibiotica zijn er steeds meer incidenten waarbij resistentiemechanismen de behandeling te niet doen. Hierdoor is er een urgente behoefte ontstaan aan nieuwe actieve verbindingen,die een alternatief werkingsmechanisme bieden voor de eliminatie van pathogenen micro-organismen. In dit opzicht kunnen de isolatie en structuurophelderingen van natuurlijke producten helpen bij het zoeken naar nieuwe actieve verbindingen met antibioticawerking. Bio-informatische technieken zoals de identificatie van potentiële secundaire metabolietgenclusters, heterologe genexpressie en functionele analyse van synthetasegenen ondersteunt middels vergelijkende metabolietprofileringsmethodieken, kunnen bijdragen aan de isolatie van nieuwe natuurlijke producten. Dit soort metabolieten worden voornamelijk gesynthetiseerd door middel van grote multifunctionele eiwitcomplexen genaamd polyketide synthases (PKS) en niet-ribosomale peptidesynthetases (NRPS), welke simpele bouwstenen als aminozuren en organische zuren assembleren tot complexe metabolieten.

Bacteriën, schimmels en planten produceren allerlei chemische stofjes, waarvan velen medicinaal worden toegepast als bijvoorbeeld antibiotica, chemotherapeutica, cholesterolverlagende middelen of immunosuppressiva. Er is een groeiende vraag naar meer van zulke stofjes, met name omdat bacteriën op grote schaal resistent worden tegen de antibiotica die momenteel gebruikt worden. De mate waarin nieuwe moleculen ontdekt worden is in de afgelopen tijd echter juist afgenomen. Om deze trend te keren, hebben wij een nieuwe strategie ontworpen om sneller meer moleculen te ontdekken die potentie hebben om tot medicijnen ontwikkeld te worden: door slimme computerprogramma’s te schrijven hebben we het mogelijk gemaakt om de enorme hoeveelheid erfelijke informatie van allerlei verschillende levensvormen, die recent beschikbaar is gekomen door nieuwe technologieën, efficiënt door te spitten. Dit maakt het mogelijk om te voorspellen wat voor moleculen deze levensvormen kunnen maken, enkel en alleen op basis van de informatie in hun DNA. Zo hoefden wij ons niet meer te beperken tot het onderzoeken van relatief willekeurig gekozen specifieke bacteriën of schimmels, maar konden wij duizenden verschillende levensvormen tegelijk verkennen, en vonden wij daarin talloze systemen die moleculen maken met farmaceutische potentie. Om deze systemen vervolgens ook in grote getale snel te kunnen uittesten, bedachten wij een nieuwe benadering: door ze op de computer te herontwerpen, kunnen de systemen ingebracht worden in bacteriën die speciaal ontworpen zijn om in het lab efficiënt zulke moleculen in elkaar te zetten. De nieuwe strategieën hebben de potentie om een radicale verandering te bewerkstelligen in de manier waarop medicijnen ontwikkeld worden uit door de natuur gemaakte stofjes.

Bacteriën, schimmels en planten produceren allerlei chemische stofjes, waarvan velen medicinaal worden toegepast als bijvoorbeeld antibiotica, chemotherapeutica, cholesterolverlagende middelen of immunosuppressiva. Er is een groeiende vraag naar meer van zulke stofjes, met name omdat bacteriën op grote schaal resistent worden tegen de antibiotica die momenteel gebruikt worden. De mate waarin nieuwe moleculen ontdekt worden is in de afgelopen tijd echter juist afgenomen. Om deze trend te keren, hebben wij een nieuwe strategie ontworpen om sneller meer moleculen te ontdekken die potentie hebben om tot medicijnen ontwikkeld te worden: door slimme computerprogramma’s te schrijven hebben we het mogelijk gemaakt om de enorme hoeveelheid erfelijke informatie van allerlei verschillende levensvormen, die recent beschikbaar is gekomen door nieuwe technologieën, efficiënt door te spitten. Dit maakt het mogelijk om te voorspellen wat voor moleculen deze levensvormen kunnen maken, enkel en alleen op basis van de informatie in hun DNA. Zo hoefden wij ons niet meer te beperken tot het onderzoeken van relatief willekeurig gekozen specifieke bacteriën of schimmels, maar konden wij duizenden verschillende levensvormen tegelijk verkennen, en vonden wij daarin talloze systemen die moleculen maken met farmaceutische potentie. Om deze systemen vervolgens ook in grote getale snel te kunnen uittesten, bedachten wij een nieuwe benadering: door ze op de computer te herontwerpen, kunnen de systemen ingebracht worden in bacteriën die speciaal ontworpen zijn om in het lab efficiënt zulke moleculen in elkaar te zetten. De nieuwe strategieën hebben de potentie om een radicale verandering te bewerkstelligen in de manier waarop medicijnen ontwikkeld worden uit door de natuur gemaakte stofjes.

Nieuwe biosyntheseroutes leveren 50 secundaire metabolieten op In zijn proefschrift beschrijft Hazrat Ali drie nieuwe biosynthetische routes van secundaire metabolieten, die meer dan vijftig secunaire metalbolieten opleverden met belangrijke biologische eigenschappen. De ontdekking van levensreddende medicatie en antibiotica die door de natuur geproduceerd worden heeft de levensverwachting van de mens de laatste eeuw enorm doen verlengen. Echter, door het ongecontroleerd toepassen van voornamelijk antibiotica zijn er steeds meer incidenten waarbij resistentiemechanismen de behandeling te niet doen. Hierdoor is er een urgente behoefte ontstaan aan nieuwe actieve verbindingen,die een alternatief werkingsmechanisme bieden voor de eliminatie van pathogenen micro-organismen. In dit opzicht kunnen de isolatie en structuurophelderingen van natuurlijke producten helpen bij het zoeken naar nieuwe actieve verbindingen met antibioticawerking. Bio-informatische technieken zoals de identificatie van potentiële secundaire metabolietgenclusters, heterologe genexpressie en functionele analyse van synthetasegenen ondersteunt middels vergelijkende metabolietprofileringsmethodieken, kunnen bijdragen aan de isolatie van nieuwe natuurlijke producten. Dit soort metabolieten worden voornamelijk gesynthetiseerd door middel van grote multifunctionele eiwitcomplexen genaamd polyketide synthases (PKS) en niet-ribosomale peptidesynthetases (NRPS), welke simpele bouwstenen als aminozuren en organische zuren assembleren tot complexe metabolieten., In his thesis Hazrat Ali reported three new secondary metabolite biosynthetic pathways with more than fifty secondary metabolites with important biological properties. These metabolites may be utilized for human benefits as they have antimicrobial, antibiotic, anti-cancer and neurotoxin properties The remarkable feature of all three biosynthetic pathways is the large number of intermediates and/or products that is the result of a distinct unspecificity of the NRPS and/or modifying enzymes involved. Through the use of highly sensitive detection techniques based on mass spectrometry, novel compounds with important biological activity could be detected that had not been seen before.Overall, this demonstrates that P. chrysogenum has the capability to synthesize multiple metabolites at a time. We also observed that by the deletion of the biosynthetic roquefortine/meleagrine pathway resulted in increase in production of chrysogen and related metabolites and vice versa even though the expression of the genes involved remained unaltered. This phenomenon is likely due to a re-direction of the metabolic (nitrogen) flux yielding increased levels of particular metabolites. This implies that for an optimal P. chyrsogenum strain, it is desirable to inactivate all unnecessary secondary metabolite genes. Remarkably, this is a phenomenon that to some extent already happened during the classical strain improvement that resulted in the silencing or mutational inactivation of various secondary metabolite genes.

Bacteriën, schimmels en planten produceren allerlei chemische stofjes, waarvan velen medicinaal worden toegepast als bijvoorbeeld antibiotica, chemotherapeutica, cholesterolverlagende middelen of immunosuppressiva. Er is een groeiende vraag naar meer van zulke stofjes, met name omdat bacteriën op grote schaal resistent worden tegen de antibiotica die momenteel gebruikt worden. De mate waarin nieuwe moleculen ontdekt worden is in de afgelopen tijd echter juist afgenomen. Om deze trend te keren, hebben wij een nieuwe strategie ontworpen om sneller meer moleculen te ontdekken die potentie hebben om tot medicijnen ontwikkeld te worden: door slimme computerprogramma’s te schrijven hebben we het mogelijk gemaakt om de enorme hoeveelheid erfelijke informatie van allerlei verschillende levensvormen, die recent beschikbaar is gekomen door nieuwe technologieën, efficiënt door te spitten. Dit maakt het mogelijk om te voorspellen wat voor moleculen deze levensvormen kunnen maken, enkel en alleen op basis van de informatie in hun DNA. Zo hoefden wij ons niet meer te beperken tot het onderzoeken van relatief willekeurig gekozen specifieke bacteriën of schimmels, maar konden wij duizenden verschillende levensvormen tegelijk verkennen, en vonden wij daarin talloze systemen die moleculen maken met farmaceutische potentie. Om deze systemen vervolgens ook in grote getale snel te kunnen uittesten, bedachten wij een nieuwe benadering: door ze op de computer te herontwerpen, kunnen de systemen ingebracht worden in bacteriën die speciaal ontworpen zijn om in het lab efficiënt zulke moleculen in elkaar te zetten. De nieuwe strategieën hebben de potentie om een radicale verandering te bewerkstelligen in de manier waarop medicijnen ontwikkeld worden uit door de natuur gemaakte stofjes.

Nieuwe biosyntheseroutes leveren 50 secundaire metabolieten op In zijn proefschrift beschrijft Hazrat Ali drie nieuwe biosynthetische routes van secundaire metabolieten, die meer dan vijftig secunaire metalbolieten opleverden met belangrijke biologische eigenschappen. De ontdekking van levensreddende medicatie en antibiotica die door de natuur geproduceerd worden heeft de levensverwachting van de mens de laatste eeuw enorm doen verlengen. Echter, door het ongecontroleerd toepassen van voornamelijk antibiotica zijn er steeds meer incidenten waarbij resistentiemechanismen de behandeling te niet doen. Hierdoor is er een urgente behoefte ontstaan aan nieuwe actieve verbindingen,die een alternatief werkingsmechanisme bieden voor de eliminatie van pathogenen micro-organismen. In dit opzicht kunnen de isolatie en structuurophelderingen van natuurlijke producten helpen bij het zoeken naar nieuwe actieve verbindingen met antibioticawerking. Bio-informatische technieken zoals de identificatie van potentiële secundaire metabolietgenclusters, heterologe genexpressie en functionele analyse van synthetasegenen ondersteunt middels vergelijkende metabolietprofileringsmethodieken, kunnen bijdragen aan de isolatie van nieuwe natuurlijke producten. Dit soort metabolieten worden voornamelijk gesynthetiseerd door middel van grote multifunctionele eiwitcomplexen genaamd polyketide synthases (PKS) en niet-ribosomale peptidesynthetases (NRPS), welke simpele bouwstenen als aminozuren en organische zuren assembleren tot complexe metabolieten.

Karakteristiek voor eukaryote cellen is dat ze afzondelijke door een of meerdere membranen afgesloren compartimenten bevatten, orgallellen genoemd. Organellen zijn zeer dynamische structuren met een specifieke architectuur en een grote verscheidenheid aan cellulaire functies waarvan sommige uniek zijn voor een bepaaJde ontwikkelingsfase van het organisme waarin ze voorkomen. Gebaseerd op hun atkomst en de manier waaop ze worden gevormd kunnen organellen in het algemeen in twee groepen worden onderverdeeld: auto nome (zoals mitochondria, chloroplasten, celkernen en ER) en niet-autonome organellen (zoals Golgi, vacuo len en cellulaire transport blaasjes). Peroxisomen zijn organellen omvflt door een enkele membraan en kunnen een grote verscheidenheid aan functies vervullen. Naast hun geconserveerde functie in de afbraak van vetzuren en ontgifting van waterstotperoxide, zijn peroxisomen ook betrokken bij totosynthese, synthese van secundaire metabolieten, methanol metabolisme, purine degradatie etc. De betrokkenheid van peroxisomen in de vorming van de~ecundaire metaboliet penicilline is een van de ondemerpen van dit proefschrift (voor uitgebreide informatie, zie hoordstuk I en 2). Op dit moment is de wijze hoe peroxisomen \\orden gevormd sterk onderhevig aan debat. Er zijn in principe twee stromingen. Een hiervan claimt dat peroxisomen zich kLlnnen gedragen als autonome organellen die groeien, delen en overerven bij celdeling, zodat volgende generaties cellen steeds peroxisomen ebvatten. Echtre, recente publicaties geven aan dat peroxisomen L1it het ER ontstaan (de 110\'0 synthese genoemd) en dus atllankelijk zijn van het niet-autonome cel endomembraan systeem.

Karakteristiek voor eukaryote cellen is dat ze afzondelijke door een of meerdere membranen afgesloren compartimenten bevatten, orgallellen genoemd. Organellen zijn zeer dynamische structuren met een specifieke architectuur en een grote verscheidenheid aan cellulaire functies waarvan sommige uniek zijn voor een bepaaJde ontwikkelingsfase van het organisme waarin ze voorkomen. Gebaseerd op hun atkomst en de manier waaop ze worden gevormd kunnen organellen in het algemeen in twee groepen worden onderverdeeld: auto nome (zoals mitochondria, chloroplasten, celkernen en ER) en niet-autonome organellen (zoals Golgi, vacuo len en cellulaire transport blaasjes). Peroxisomen zijn organellen omvflt door een enkele membraan en kunnen een grote verscheidenheid aan functies vervullen. Naast hun geconserveerde functie in de afbraak van vetzuren en ontgifting van waterstotperoxide, zijn peroxisomen ook betrokken bij totosynthese, synthese van secundaire metabolieten, methanol metabolisme, purine degradatie etc. De betrokkenheid van peroxisomen in de vorming van de~ecundaire metaboliet penicilline is een van de ondemerpen van dit proefschrift (voor uitgebreide informatie, zie hoordstuk I en 2). Op dit moment is de wijze hoe peroxisomen \\orden gevormd sterk onderhevig aan debat. Er zijn in principe twee stromingen. Een hiervan claimt dat peroxisomen zich kLlnnen gedragen als autonome organellen die groeien, delen en overerven bij celdeling, zodat volgende generaties cellen steeds peroxisomen ebvatten. Echtre, recente publicaties geven aan dat peroxisomen L1it het ER ontstaan (de 110\'0 synthese genoemd) en dus atllankelijk zijn van het niet-autonome cel endomembraan systeem.

Karakteristiek voor eukaryote cellen is dat ze afzondelijke door een of meerdere membranen afgesloren compartimenten bevatten, orgallellen genoemd. Organellen zijn zeer dynamische structuren met een specifieke architectuur en een grote verscheidenheid aan cellulaire functies waarvan sommige uniek zijn voor een bepaaJde ontwikkelingsfase van het organisme waarin ze voorkomen. Gebaseerd op hun atkomst en de manier waaop ze worden gevormd kunnen organellen in het algemeen in twee groepen worden onderverdeeld: auto nome (zoals mitochondria, chloroplasten, celkernen en ER) en niet-autonome organellen (zoals Golgi, vacuo len en cellulaire transport blaasjes). Peroxisomen zijn organellen omvflt door een enkele membraan en kunnen een grote verscheidenheid aan functies vervullen. Naast hun geconserveerde functie in de afbraak van vetzuren en ontgifting van waterstotperoxide, zijn peroxisomen ook betrokken bij totosynthese, synthese van secundaire metabolieten, methanol metabolisme, purine degradatie etc. De betrokkenheid van peroxisomen in de vorming van de~ecundaire metaboliet penicilline is een van de ondemerpen van dit proefschrift (voor uitgebreide informatie, zie hoordstuk I en 2). Op dit moment is de wijze hoe peroxisomen \\orden gevormd sterk onderhevig aan debat. Er zijn in principe twee stromingen. Een hiervan claimt dat peroxisomen zich kLlnnen gedragen als autonome organellen die groeien, delen en overerven bij celdeling, zodat volgende generaties cellen steeds peroxisomen ebvatten. Echtre, recente publicaties geven aan dat peroxisomen L1it het ER ontstaan (de 110\'0 synthese genoemd) en dus atllankelijk zijn van het niet-autonome cel endomembraan systeem.

This work describes the isolation of endophytic fungi from plant species Hancornia speciosa and led to the isolation of 14 pure strains. Of which 4 were selected for the chemical study. The chemical study allowed identied 12 substances isolated from fungi Phomopsis sp., Guignardia sp., Aspergillus niger e Aspergillus sp., grown in different culture medium. The crude extracts of fungus Phomopsis sp., grown in PDB medium led to isolation of five compounds, 5- methylmelein, nectriapyrone, succnic acid, 5-hydroxylmethylmelein and a diketopiperazine. Already when the same fungus was grown in ME medium, led to isolation of two compounds, tyrosol and tryptofol. The crude extract of Guignardia sp grown in ME medium led to isolation of compound salicylic acid, which has a plant for ecological importance.The crude extract of Aspergillus niger, grown in PDB medium led to isolation of compounds pyrophen and nigragillin. Already when the same fungus was grown in ME medium, led to isolation of compound itaconic acid. And of crude extract of Aspergillus sp., grown in ME medium was identification of their major compound kojic acid, which was quantified by HPLC in the crude extract (0.7 mg/g of crude extract). O crude extract was tested for its antioxidant activity with DPPH, NO. and H2O2, and exhibited significantly potent inhibition of NO (IC50 de 150 μg/mL) production, showed taht this fungi it owns big biotchenology potential. The structures of these substances were established by spectroscopic methods, including the application of bidimensional NMR techniques, mass spectrum and comparison with published data. Este trabalho descreve o isolamento de fungos endofíticos da espécie vegetal Hancornia speciosa, o qual conduziu a 14 linhagens puras, dos quais quatro foram selecionados para o estudo químico. O estudo químico possibilitou foi identicar 12 substâncias, isoladas dos fungos Phomopsis sp., Guignardia sp., Aspergillus níger e Aspergillus sp., cultivados em diferentes meios de culturas. O extrato bruto do fungo Phomopsis sp cultivado no meio liquido PBD levou ao isolamento de cinco compostos, 5- metilmeleína, nectapirona, ácido succínico, 5-hidroxilmetilmeleína e uma dicetopiperazina. Já o mesmo fungo quando cultivado no meio líquido-ME, levou ao isolamento de dois compostos, tirosol e triptofol. O estudo do extrato bruto de Guignardia sp., cultivado no meio liquido ME conduziu ao isolamento do composto ácido salicílico, o qual tem um importancia ecológica para planta. O extrato bruto do fungo Aspergillus niger cultivado no meio liquido PBD levou ao isolamento dos compostos pirofen e nigragillin. Já o mesmo fungo quando cultivado em extrato de malte, levou ao isolamento do ácido itacônico. E do extrato bruto do fungo Aspergillus sp., cultivado no meio liquido PBD foi identificada a substância majoritária ácido kójico, a qual foi quantificada por CLAE no extrato bruto (0,7g/ g de extrato bruto). O extrato desse fungo foi testado quanto a sua atividade antioxidante com DPPH, NO. , e H2O2, e mostrou uma forte atividade de inibição da produção de NO (IC50 de 150 μg/mL), indicando que este fungo possui grande potencial biotecnológico. As estruturas das substâncias foram determinadas por métodos espectrométricos, incluindo RMN bi-dimensional, espectros de massas e comparação com a literatura.

Universidade Federal de Sao Carlos Studies of marine natural products in 20 years of research have revealed a wide variety of molecules with complex structures and hitherto unknown, and potent biological activities. The aims of this study were to isolate microbial strains associated with Didemnum granulatum, to fermentate such strains for the production of crude extracts and the screening of these extracts for bioactive secondary metabolites. For the isolation of the strains, six different culture media were prepared with artificial sea water: malt extract 2% and 3%, carrot/potato, corn, oat and GPY. The strains were cultivated for 30 days in 250mL of the same culture media. The crude extracts were obtained by partition with ethyl acetate. The extracts were sent for the cytotoxic activity bioassay and analyzed by thin layer chromatography (TLC) on silica gel plate (with absorption in UV-vis). Among the 26 extracts, samples DG (M3) 6'C and DG (M3) 5'C showed potent cytotoxic activity, samples DG (B) 13, DG (M3) 1 and DG (G) 2B a moderate activity. The analysis by TLC indicated the presence of bioactive secondary metabolites in the extracts. The strains were identified as bioactive three fungal: DG (M 3) 6'C (Penicillium sp.) DG (M3) 5'C (Cladosporium sp.) DG (M3) 1 (Aspergillus sp.) And two bacterial: DG (B) 13 (Aurantimonas sp.) DG (G) 2B (Nocardiopsis sp.). These were grown on a larger scale of broth (500 mL) and extracted after 7, 14, 21 and 30 days of incubation. We carried out a cleaning process of these extracts by solid phase extraction (SPE) column of silica gel derivatized with C18 (35 mL of eluent: 100% H2O, MeOH/H2O 1:1 and MeOH 100%). The fractions generated were analyzed by HPLC-UV-MS trying to find characteristic profiles of secondary metabolites. From the DG sample (M3) 6'C (A + B), was isolated and identified the compound 13- deoxy-fomenona previously obtained by Tirilly et al. in 1983. Although this compound is not unheard of, no literature reports of studies involving bioassays cytotoxic against tumor cells and even this compound has been isolated from other species of microorganisms other than the fungus Dicyma pulvinata. Os estudos sobre produtos naturais marinhos, durante os ultimos 20 anos de pesquisa, revelaram uma grande variedade de moleculas com estruturas complexas e ate entao desconhecidas, alem de potentes atividades biologicas. Assim, os objetivos deste trabalho foram isolar linhagens de micro-organismos associadas a ascidia Didemnum granulatum, sua posterior fermentacao para a producao de extratos brutos e a analise destes extratos na busca por metabolitos secundarios bioativos. Para o isolamento das linhagens foram utilizados os seguintes meios de cultura preparados com agua do mar artificial: extrato de malte 2% e 3%, cenoura/batata, fuba, aveia e GPY. A fermentacao das linhagens foi feita em 250 mL dos mesmos meios de cultura sem agar, durante 30 dias. Os extratos brutos foram obtidos por particao com acetato de etila. Estes extratos foram enviados para bioensaio de atividade citotoxica contra celulas tumorais e analisados por cromatografia em camada delgada (CCD) em placa de silica gel (com diferentes reveladores). Dentre os 26 extratos, as amostras DG(M3) 6 C e DG(M3) 5 C apresentaram potente atividade citotoxica, as amostras DG(B)13, DG(M3)1 e DG(G)2B uma atividade moderada. As analises por CCD indicaram a presenca de metabolitos secundarios nos extratos bioativos. As linhagens bioativas foram identificadas como sendo tres fungicas: DG(M3)6 C (Penicilium sp.); DG(M3)5 C (Cladosporium sp.); DG(M3)1 (Aspergillus sp.) e duas bacterianas: DG(B)13 (Aurantimonas sp.); DG(G)2B (Nocardiopsis sp.). Estas foram cultivadas em escala maior de meio liquido (500 mL) e extraidas apos 7, 14, 21 e 30 dias de incubacao. Realizou-se um processo de limpeza destes extratos por extracao em fase solida (SPE) em coluna de silica-gel derivatizada com C18 (35 mL dos eluentes: H2O 100%, MeOH/H2O 1:1 e MeOH 100%). As fracoes geradas foram analisadas por CLAE-UV-EM buscando-se encontrar perfis caracteristicos de metabolitos secundarios. A partir da amostra DG(M3) 6 C (A+B), foi isolado e identificado o composto 13-desoxi-fomenona, previamente obtido por Tirilly et al. em 1983. Apesar deste composto nao ser inedito, nao ha relatos na literatura de estudos envolvendo bioensaios citotoxicos contra celulas tumorais e nem mesmo deste composto ter sido isolado de outras especies de microrganismos que nao seja do fungo Dicyma pulvinata.

The monitoring of biomass production showed positive reaction on amount of proteins in media. The best media for biomass production were mSeed and Seed at temperatures of 20 and 25⁰C. Slow growth of strain shows very high amount of secondary metabolites. During investigation of secondary metabolites almost all detected compounds were completely new. From known compounds, Bassianolide, a compound known to be produced by Beauveria family, first time was detected to be produced by Beauveria brongniartii. Also for two other completely new compounds, bb_1 and bb_2, structural proposal was made. All compounds that have been produced in higher amount are non-polar. For future research, to identify other compounds it is necessary to perform a MS3 experiment for determination of fragments that it is impossible to identify only by MS/MS experiment. Farther more, for detection of exact position of all atoms in compound, NMR technology is needed. After isolation of certain compounds, biological tests are needed to prove entomolophatogenic abilities. Except testing it’s affiances on Melolontha melolontha, it is necessary to test Beauveriana brongniartii's effects on other insects and its effect on balance in nature.

Pilocarpus species have been exploited as the only source of the imidazole alkaloid pilocarpine (used in glaucoma treatment), since its isolation up to the present. Almost all Pilocarpusspecies are native from Brazil. Because of the medicinal importance of pilocarpine, several of them are in the path of extinction. Other secondary metabolites, such as coumarins, flavonoids and terpenes, were described for Pilocarpusspecies. In this review the secondary metabolites, other than pilocarpine, isolated from Pilocarpusspecies and their biological activities were compiled. Although the variety of structures and the importance of the biological activities described in literature for Pilocarpusspecies this is an unexploited field of research in Natural Products and Pharmacology. Espécies de Pilocarpus têm sido exploradas como única fonte do alcalóide imidazólico pilocarpina (utilizado no tratamento do glaucoma) desde o isolamento dessa substância até os dias atuais. A maioria das espécies de Pilocarpus conhecida é nativa do Brasil e, devido à importância medicinal que a pilocarpina possui e ao desmatamento, várias se encontram em risco de extinção. Outros metabólitos secundários entre os quais cumarinas, flavonóides e terpenos foram descritos em espécies desse gênero. Nesta revisão foram relacionados os metabólitos secundários isolados em diversas espécies de Pilocarpus bem como suas atividades biológicas. Apesar da variedade de estruturas e as importantes atividades biológicas já descritas na literatura para as outras classes de metabólitos secundários, ainda há um vasto campo de estudo para as espécies de Pilocarpus.