Production of secondary metabolites in plant tissue cultures The goal of this study is to determine the influence of sodium molybdate and sodium tungstate as elicitors on production of scopoletin in cell suspension culture of Angelica archangelica L. The culture was grown in a liquid culture medium Murashige and Skoog on a roller apparatus in the dark and light. The content of scopoletin was in cells and in the culture medium determined by high performance liquid chromatography with fluorometric detection. The results show that sodium molybdate served as an elicitor to the production of scopoletin positively, application of sodium tungstate did not increase scopoletin production in any case. The highest production of scopoletin after application of sodium molybdate versus the control cells was reached in the suspension culture of Angelica archangelica L. cultured in the dark at a concentration of 25,50 mg/l. Scopoletin content increased by 166,7 %. After application of sodium tungstate the production always decreased, in the medium of a suspension culture of Angelica archangelica L. cultivated in the light at a concentration of 66.00 mg/l, the production decrease was by up 62,5 %.

D.Phil. / The objective of the research described in the first part of this thesis was to develop a general method utilising palladium catalysed reactions for the synthesis of the anti-cancer compound, lavendamycin and analogs thereof. Therefore, the development of a general route to synthetic euivalents of the lavendamycin AB quinoline system, 2-hydroxyquinolines, with potential for coupling to the CDE or CD moiety, was addressed. The first protocol for the synthesis of 2-hydroxyquinolines invloved to the use of appropriately substituted o-nitrophenyltriflates (readily prepared from phenols) in a Heck reaction under neutral conditions followed by a one-pot reduction and cyclisation step. The synthetic potential of such an approach was demonstrated by the preparation of a suitable lavendamycin AB synthon from commecially avalaible guiacol. A second general strategy towards the synthesis of the AB synthon utilising a performed ring system commecially available 8-hydroxyquinoline has been successfully developed. This approach requiring the introduction of a suitable leaving group in the 2-position involved the following sequence of reactions: protection of 8-hydroxyl group N-oxidation, and a rearrangement step. This methodology yielded five different key intermediates all possessing suitable functionality in the 2 position which would allow further cross-coupling to an appropriate CDE ring equivalent.

A comprehensive method for the analysis of 11 target pharmaceuticals representing multiple therapeutic classes was developed for biological tissues (fish) and water. Water samples were extracted using solid phase extraction (SPE), while fish tissue homogenates were extracted using accelerated solvent extraction (ASE) followed by mixed-mode cation exchange SPE cleanup and analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Among the 11 target pharmaceuticals analyzed, trimethoprim, caffeine, sulfamethoxazole, diphenhydramine, diltiazem, carbamazepine, erythromycin and fluoxetine were consistently detected in reclaimed water. On the other hand, caffeine, diphenhydramine and carbamazepine were consistently detected in fish and surface water samples. In order to understand the uptake and depuration of pharmaceuticals as well as bioconcentration factors (BCFs) under the worst-case conditions, mosquito fish were exposed to reclaimed water under static-renewal for 7 days, followed by a 14-day depuration phase in clean water. Characterization of the exposure media revealed the presence of 26 pharmaceuticals while 5 pharmaceuticals including caffeine, diphenhydramine, diltiazem, carbamazepine, and ibuprofen were present in the organisms as early as 5 h from the start of the exposure. Liquid chromatography ultra-high resolution Orbitrap mass spectrometry was explored as a tool to identify and quantify phase II pharmaceutical metabolites in reclaimed water. The resulting data confirmed the presence of acetyl-sulfamethoxazole and sulfamethoxazole glucuronide in reclaimed water. To my knowledge, this is the first known report of sulfamethoxazole glucuronide surviving intact through wastewater treatment plants and occurring in environmental water samples. Finally, five bioaccumulative pharmaceuticals including caffeine, carbamazepine, diltiazem, diphenhydramine and ibuprofen detected in reclaimed water were investigated regarding the acute and chronic risks to aquatic organisms. The results indicated a low potential risk of carbamazepine even under the worst case exposure scenario. Given the dilution factors that affect environmental releases, the risk of exposure to carbamazepine will be even more reduced.

Physalis angulata L. (Solanaceae), popularmente conhecida como camapu ou balãozinho, é uma espécie que vem ganhando importância econômica em decorrência de suas atividades biológicas, como citotóxica, antibacteriana e anti-inflamatória. Dentre os diversos metabólitos secundários conhecidos para esta espécie, destacam-se os flavonoides e alcaloides. É amplamente difundido que o metabolismo secundário das plantas pode ser alterado por fatores bióticos e abióticos. Nesse sentido, esse trabalho objetivou destacar a influência do Potato virus Y, estirpe O (PVYO), isolado de camapu, no metabolismo secundário, avaliando as quantidades e os perfis de flavonoides e alcaloides também em plantas de camapu infectadas. A infecção viral foi induzida em plantas obtidas por sementes, os metabólitos foram extraídos a partir de folhas, foram analisados em CLAE e CG-EM e comparados com aqueles provenientes de plantas sadias. Não foram observadas diferenças qualitativas nos perfis tanto dos flavonoides quanto dos alcaloides. Por outro lado, houve diminuição no teor de flavonoides totais nas plantas infectadas na infecção sistêmica. Além disso, houve aumento significativo do derivado de atropina nas plantas infectadas em comparação com os demais tratamentos. Dessa forma, sugerimos que a infecção induzida pelo PVYO em plantas de camapu teve maior impacto na via das substâncias nitrogenadas em relação às fenólicas / Physalis angulata L. (Solanaceae), popularly known as \"camapu\" or \"balãozinho\", has become economically important due to its biological activities, such as cytotoxic, antibacterial and anti-inflammatory. Among the secondary metabolites, flavonoids and alkaloids have already been described for this species. Secondary metabolism can be influenced by both biotic and environmental agents. The main goal of this work was to analyze the influence of Potato virus Y (PVYO) on the secondary metabolism of infected \"camapu\" plants, evaluating total amount and profiles of flavonoids and alkaloids. Viral infection was induced in plants obtained from seeds. The compounds were extracted from leaves, analyzed in HPLC and GC/MS and compared with those from healthy plants. There were no qualitative differences between healthy and infected plants concerning to alkaloid and flavonoid profiles. On the other hand, it was noticed a lower flavonoid concentration in the systemic infection of infected plants. An increase of an atropine derivative was observed in infected plants. In conclusion, according to the results we propose that the PVYO induced infection in plants of \"camapu\" had stronger impact on the nitrogenous compounds than on phenolics

Plantas parasitas são aquelas que retiram os recursos necessários para a sua sobrevivência de outras plantas (hospedeiras) por meio de uma estrutura denominada haustório, que permite que a parasita se conecte ao sistema vascular da hospedeira. As plantas podem responder ao ataque parasita ativando alguns mecanismos de defesa, dentre eles, a produção de substâncias fenólicas. Os viscos constituem um grupo de plantas parasitas aéreas pertencentes a Santalales que podem ser divididos em viscos holoparasitas ou hemiparasitas. Alguns gêneros de viscos são de importância econômica, dentre eles, Phoradendron, um gênero de visco hemiparasita. Esse trabalho teve como objetivos determinar os conteúdos de taninos, flavonoides, fenilpropanoides, carboidratos solúveis, amido, lipídeos, a razão carbono/nitrogênio e avaliar como a relação parasítica afeta o metabolismo das espécies envolvidas. Para tanto, coletou−se no município de Campanha (MG) folhas e ramos de seis indivíduos não parasitados de Tapirira guianensis (FT e RT) e, a partir de nove indivíduos parasitados de T. guianensis, foram coletados ramos e folhas de Phoradendron crassifolium (FP e RP), ramos não infestados da hospedeira e suas folhas (RNI e FRNI) e ramos infestados de T. guianensis e suas folhas (RI e FRI), sendo que ramos infestados foram divididos em região proximal, galha e distal (RPRI, RGRI e RDRI). Como principais resultados estão o aumento no teor de proantocianidinas e de carboidratos solúveis em FRNI e FRI em relação a FT. Ramos infestados sofreram redução nos conteúdos de carboidratos solúveis, amido, lipídeos, nitrogênio e substâncias fenólicas, exceto na região da galha (RGRI) em que aumentos de amido e flavonoides foram verificados. Observou-se ainda que RNI parece ter sofrido um acúmulo de substâncias fenólicas. O conjunto dos resultados obtidos mostra que P. crassifolium afeta o metabolismo primário e o secundário de T. guianensis, com influência mais pronunciada nos ramos do que nas folhas da hospedeira. Pode−se sugerir que a parasita obtêm nitrogênio e carboidratos solúveis de sua hospedeira e que pode ocorrer uma possível realocação de carbono para crescimento, desenvolvimento, manutenção e, ainda, defesa de ramos ainda não infestados. Além disso, sugere−se que o acúmulo de fenólicos em ramos não infestados (RNI) possa ser um mecanismo de defesa usado pela hospedeira com o objetivo de evitar, ou mesmo diminuir, novas infestações em ramos ainda não parasitados. / Parasitic plants are those that withdraw necessary resources for its survival from other plants (hosts) through a structure so-called haustorium which enables the parasite to connect to the host\'s vascular system. Plants can respond to the parasite attack activating some defense mechanisms, including the production of phenolic compounds. Mistletoes are a group of stem parasitic plants belonging to Santalales that can be grouped into holoparasite or hemiparasite mistletoes. Some mistletoes genera are of economic importance among them Phoradendron, a hemiparasite mistletoe. This study aimed to determine the contents of tannins, flavonoids, phenylpropanoids, soluble carbohydrates, starch, lipids, the carbon⁄nitrogen ratio and evaluate how the parasitic relationship affects the metabolism of the involved species. Were collected in Camapanha (MG) leaves and branches from six non−parasitized individuals of Tapirira guianensis, host plant (LT and BT). From nine parasitized individuals of T. guianensis were collected branches and leaves of Phoradendon crassifolium, the parasitic plant (LP and BP), non-infested host branches and their leaves (NIHB and NIHB-L) and infested branches and their leaves (IHB and IHB-L). Infested branches of T. guianensis were divided into proximal, gall, and distal regions (IHB-P, IHB-G and IHB-D). Main results were an increase in the proanthocyanidin and soluble carbohydrates content in NIHB−L and IHB−L in relation to LT. There was a reduction of soluble carbohydrate, starch, lipids, nitrogen, and phenolic compounds in infested branches, except in the gall region (IHB-G), which showed an increase of flavonoids and starch contents. It was also observed that NIHB appears to accumulate phenolic compounds. Results showed that P. crassifolium affects the primary and secondary metabolism of T. guianensis, and branches were affected more intense than the leaves. It can be suggested that the parasite withdraws nitrogen and soluble carbohydrates from its host and this could result in possible carbon relocation for growth, development, maintenance, and also defense of branches not yet infested. In addition, it could be suggested the accumulation of phenolic compounds in non−infested branches (NIHB) as a defense mechanism used by the host in order to prevent or even reduce new infestations of an individual already parasitized.

O gênero Croton abriga aproximadamente 1300 espécies, muitas delas reconhecidas pela sua utilização terapêutica, que se deve a presença de grande diversidade de metabólitos secundários. Tais propriedades foram ponto de partida para investigações sobre as propriedades fitoquímicas do gênero. C. vulnerarius e C. echinocarpus são espécies endêmicas do Brasil pertencentes à seção Cyclostigma e até então pouco estudadas. Neste projeto foram extraídos, identificados e isolados componentes químicos destas espécies e analisadas as suas propriedades bioativas (antioxidante, antimicrobiana e anti-HIV). Foi verificado que, diferentemente de outras espécies da mesma seção, C. echinocarpus e C. vulnerarius não apresentam diterpenos em sua composição. No entanto, C. echinocarpus possui entre seus principais componentes um alcaloide, identificado por espectrometria de massas como coridina. Em relação às substâncias fenólicas, observamos que as espécies possuem perfis semelhantes, com alguns flavonoides em comum, sendo que o principal componente de C. echinocarpus é a apigenina vitexina e de C. vulnerarius , o derivado de campferol tilirosídeo. Em relação às atividades biológicas, foi verificado que ambas as espécies possuem potencial antioxidante e antimicrobiano contra espécies de bactérias Gram negativas, especialmente nos extratos mais ricos em compostos fenólicos. Quanto à ação anti-HIV, foi observado que a fração contendo o alcaloide coridina isolado apresentou um grande potencial de atividade anti transcriptase reversa, atingindo valores acima de 90% de atividade, mesmo para a menor concentração testada / Croton comprises approximately 1,300 species, many of them known for their therapeutic uses, which is due to the presence of a large diversity of secondary metabolites. Those properties were the starting point for research on the phytochemical characteristics of the genus. C. vulnerarius e C. echinocarpus are Brazilian endemic species belonging to section Cyclostigma. This project aimed the extraction, identification and isolation of chemical constituents from both species and the analyze of the bioactive properties of such constituents (antioxidant, antimicrobial and anti-HIV). It was found that, unlike other species of the same section, C. echinocarpus and C. vulnerarius does not present diterpenes in their composition. However, C. echinocarpus showed alkaloids among its main constituents, one of them identified by mass spectrometry as coridine. Regarding the phenolic substances, we observed that the two species have similar profiles, with some flavonoids in common. C. echinocarpus presents the flavone C-glycosylated vitexin as its main flavonoid constituent, while C. vulnerarius has the acylated derivative of kaempferol, tiliroside. Regarding the biological activities, both species showed antioxidant potential; antimicrobial effects against Gram-negative bacteria, especially the extracts richer in vitexin; and anti-HIV activity, where the fraction containing the alkaloid coridine showed great potential, reaching values above 90% of the anti-HIV activity, even when the lowest concentration was tested (100 mg.mL-1)

A endocrinologia reprodutiva do muriqui do sul B. arachnoides foi avaliada em quatro fêmeas adultas pela dosagem de metabólitos fecais de estrógenos e em quatro machos adultos e um macho subadulto pela dosagem de metabólitos de testosterona e glicocorticóides durante um período de onze meses em duas diferentes condições restritivas, ilha de 600m2, Curitiba (PPC) e viveiro (15,40x5,85x4,70m) Rio de Janeiro (CPRJ). As fêmeas apresentaram grande variação individual nas concentrações de estrógenos e progestinas fecais ao longo do período de estudo, três não apresentaram atividade ovariana no período de outubro-dezembro e apenas uma apresentou atividade durante todos os períodos amostrados. Mesmo com níveis baixos de esteróides as fêmeas apresentaram comportamento perceptivo e cópulas. Os machos do PPC apresentaram níveis significativamente mais altos de glicocorticóides e níveis significativamente mais baixos de testosterona do que os machos do CPRJ (p<0,05) apresentaram. O macho subadulto apresentou níveis significativamente mais baixos para metabólitos de testosterona. Nos machos as cópulas ocorreram próximo às elevações das concentrações de testosterona. Em três situações grandes elevações dos níveis de glicocorticóides fecais puderam ser relacionadas a situações estressantes. Nosso estudo comprovou a eficácia do método empregado para monitoramento reprodutivo bem como para avaliar situações estressantes. / The reproductive endocrinology of the southern muriqui (Brachyteles. Arachnoids) was evaluated through the dosage of fecal metabolites steroids for 04 adult females and fecal metabolites of testosterone and glucocorticoids for 04 adult males and 01 subadult male. The study was conducted over an eleven month period at two environmental conditions: a) an island of 600 m2, with natural vegetation, at Curitiba Zoo (PPC) and b) a large cage of 15,40X5,85X4,70m at Rio de Janeiro Primatological Centre (CPRJ). It was observed that females had large strogen interindividual variation and fecal progestins: three females did not show ovarian activity over a partial period of the study, while ovarian activity was observed for all sample periods for one female. Despite the detection of low levels of steroids, proceptive behaviour and copulations were observed. The PPC males showed significant levels of glicorticoids and testosteorne when compared to the CPRJ males (p<0.05). The subadult male exhibited the lowest level of testosterone metabolites and those differences were significant when compared to the adult males (p<0.05). For all males, copulations occurred when testosterone levels were highest and peaks of glucorticoids were linked to stressfull situations. This study have shown that the method used was effective for reproductive monitoring as well as for evaluating stressfull situations.

The endogenous arachidonate-based lipids that activate cannabinoid receptors have been well characterized in mammals. In plants only 12-18 carbon fatty acid ethanolamides have been identifi ed so far and have been shown to modulate a number of physiological processes including seed and seedling development. However, since moss plants contain arachidonic acid, we hypothesized the occurrence of arachidonate-based metabolites in their tissues. Using selective lipidomics approach, we identifi ed the presence of anandamide or arachidonylethanolamide (a 20C polyunsaturated fatty acid ethanolamide) and its precursors, in Physcomitrella patens that were previously not reported in plants. Comprehensive lipid profi les for protonema and gametophyte tissues of moss also revealed the occurrence of other saturated and unsaturated fatty acid ethanolamides and a distinct phospholipid and galactolipid composition. Further studies showed that anandamide, like abscisic acid, inhibits the growth of gametophytes more severely than saturated fatty acid ethanolamides. Our current studies are focused on understanding the physiological and developmental role of polyunsaturated fatty acid ethanolamides in nonseed plants. In conclusion, discovery of anandamide in moss provided us with an exciting possibility to identify fatty acid ethanolamide metabolic pathway in early land plants and elucidate receptor-mediated endocannabinoid signaling responses in plants that is akin to mammals.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacognosy Student: Jiří Olšar Supervisor: doc. PharmDr. Tomáš Siatka, CSc. Title of diploma thesis: Podophyllotoxin in eastern red cedar (Juniperus virginiana) This thesis describes the optimisation of the extraction process for quantity determination of podophyllotoxin in the leaves of Juniperus virginiana L. This determination was conducted through three different methods: the extraction in ultrasound bath, the extraction in the orbital shaker and the maceration in cold environment of 4 řC. Extractions were affected by various factors which were monitored, for instance time of extraction, temperature, and extraction solvents. The most efficient method appears to be the extraction in ultrasound bath with the use of methanol or a combination of phosphate buffer with ethyl-acetate.

Objectives The gut microbiota and its metabolites – namely short chain volatile fatty acids (SCVFAs) – interact with the digestive, immune, and nervous systems. States of microbiome dysbiosis are highly associated with obesity and GI symptoms, and profiles of SCVFAs, which serve functions as fuel sources and signaling molecules, mimic this dysbiotic state. This study aimed to further our understanding of associations between bacterial diversity and GI symptoms, BMI, immune markers, and SCVFAs and to identify bacteria differentially abundant with changes to the previously mentioned variables. Methods Data (measures of GI distress, BMI, immunoglobulins, fecal proximate analysis, SCVFAs, and 16s RNA sequences) was extracted from a study containing non-celiac gluten-sensitive and control participants. QIIME2 was used to process 16s RNA data, analyze quantitative, qualitative, phylogenetic quantitative, and phylogenetic qualitative measures of alpha and beta diversity and to perform an analysis of composition of microbes (ANCOM) for differential abundances data. Results Many significant differences were seen, namely in multiple measures of alpha diversity for IgG4 (P < 0.018), propionate (P < 0.014), caproate (P < 0.003), heartburn (P < 0.004), urgent need to defecate (P < 0.027), and feelings of incomplete evacuation (P < 0.024). Statistical significance was seen in multiple measures of beta diversity between normal and overweight (P < 0.01), normal and obese (P < 0.005), and overweight and obese BMI (P < 0.016), IgG4 (P < 0.033), propionate (P < 0.001), increased gas (P < 0.024), and urgent need for defecation (P < 0.026). The ANCOM identified multiple species of bacteria differentially abundant with changes to variables. Conclusions Findings suggest differences in both alpha and beta diversity with various GI symptoms, SCVFAs, and BMI. This research supports plans to apply analysis to larger sample sizes to train machine learning classifiers to identify important features of microbial profiles associated with certain SCVFA and markers of health. Funding Sources ETSU (CCRHS, Deans's Research Enhancement Award and CPH, Health Sciences Funding, Honors College Summer Research Fellowship) and Shield Nutraceuticals, LLC.