Dissertations / Theses on the topic 'Huperzine'

Huperzine A is a natural product, originally isolated from the Chinese club moss Huperzia serrata, which is currently undergoing clinical trials in the U.S. and has already been approved in China for the treatment of Alzheimer's disease (AD). This sesquiterpene alkaloid of the Lycopodium family is an eXGellent selective and reversible inhibitor of acetylcholinesterase (AChE), which is an established target in the palliative treatment of AD. Huperzine A has been the target of several syntheses and a number of analogues have also been prepared. However, all of the synthetic approaches to date have been essentially linear and thus not ideal for analogue preparation. This thesis describes the completed development of the first formal convergent synthesis of (±)-huperzine A, starting from commercially available cyclohex-2-enone and aminopicoline. A key step in the synthesis is an intramolecular Heck cyclisation, which gives the core tricycle of the natural product Optimisation of the intramolecular Heck reaction allowed for it to be carried out on a multi-gram scale and in 72% yield. Once completed, th~ converg~nt synthesis could now be used in the preparation ofnovel huperzine A analogues, which would be tested for AChE inhibitory activity and also for broad range CNS activity. Initial analogue work described in this thesis has proved promising, with a nitrogen atom having been successfully incorporated into the tricyclic core at two different positions via Beckmann rearrangements. This thesis also describes the resolution of a key intermedi~te in the newly-developed racemic synthesis, thus allowing for the synthesis of enantiomerically-pure huperzine A or related analogues.

The aim of the research was to develop a flexible, stereocontrolled route to huperzine A and B and other lycopodium alkaloids. One of the main objectives was to investigate whether intramolecular radical cyclisation reactions on to carbon-nitrogen double bonds might provide a new method for the synthesis of key intermediates in the total synthesis. To this end the intramolecular cyclisation reactions of vinyl and aryl radicals on to oxime ethers have been explored and these reactions have been used to prepare a number of useful bicyclic and tricyclic carbocyclic compounds and related heterocyclic compounds in good yields. This represents essentially new synthetic methodology as prior to this work there was only one reported example of the use of an oxime ether as a radical trap. The best reagent for these reactions utilising vinyl and aryl halide precursors was found to be tributyltin hydride. Preliminary attempts to carry out tandem radical cyclisation reactions incorporating oxime ethers have not, so far, been successful on the model systems investigated. This new synthetic strategy has been used to carry out one-carbon ring expansions of five-membered cyclic oxime ethers similar to those reported recently for carbonyl compounds. However, attempts to extend this ring expansion to the two-carbon system resulted only in cyclisation to give a bicyclic compound, rather than ring expansion. Attempts to cleave the N-O bonds of some of the hydroxylamines resulting from these cyclisation reactions proved to be unsuccessful using SmI2, Zn and acetic acid or aluminium amalgam. Attempted cleavage of the N-O alkyl bond of a hydroxylamine group adjacent to an aromatic ring using lithium aluminium hydride resulted in a novel ring expansion reaction, which may occur by an unusual electron transfer process to give radical intermediates.

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The alkaloid cryptolepine (1) and eight synthetic analogues (2-8) were assessed for in vitro activities against Trypanosoma brucei. Four of the analogues were found to be highly potent with IC50 values of less than 3 nM and three of these were assessed against T. brucei brucei infection in rats. The most effective compound was 2,7-dibromocryptolepine (7); a single oral dose of 20 mg/Kg suppressed parasitaemia and increased the mean survival time to 13.6 days compared with 8.4 days for untreated controls. In addition, four huperzine derivatives (9-12) were shown to have in vitro antitrypanosomal activities with IC50 values from 303-377 nM.

Phlegmariurus is the most abundant genus of Lycopodiaceae in Brazil with 40 species, 24 of which are endemic, occurring mainly in the Atlantic Rainforest and Rocky Fields (“Campos Rupestres”) in the Espinhaço range. Some phylogeny works with the group had already been carried out, but not including a representative sample of the Brazilian species. Thus, we performed a molecular phylogeny of Phlegmariurus species endemic in Brazil using three molecular markers of cpDNA. Phlegmariurus showed to be monophyletic, including two clades, one Neotropical and another Paleotropical. All the Brazilian species sampled appeared in the Neotropical clade. The endemic species appeared in several clades alongside Andean species. Epiphytism and the microphylls differentiation were optimized at the obtained clade and they appeared several times in the evolutionary history of the group
Phlegmariurus é o gênero mais abundante de Lycopodiaceae no Brasil com 40 espécies, sendo 24 destas endêmicas, ocorrendo principalmente na Mata Atlântica e Campos Rupestres da Cadeia do Espinhaço. Alguns trabalhos de filogenia com o grupo já haviam sido realizados, contudo sem incluir uma amostragem representativa das espécies brasileiras. Dessa forma, realizamos a filogenia molecular das espécies de Phlegmariurus endêmicas do Brasil utilizando três marcadores moleculares de cpDNA. Phlegmariurus se mostrou monofilético, incluindo dois clados, um Neotropical e outro Paleotropical. Todas as espécies brasileiras amostradas apareceram no clado neotropical. As espécies endêmicas apareceram em vários clados em meio as espécies andinas. O epifitismo e a diferenciação dos microfilos foram otimizados nos clados obtidos e indicaram que essas características surgiram várias vezes na história evolutiva do grupo

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pos-Graduação em Biologia Vegetal, Florianópolis, 2009.
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Lycopodiaceae inclui quatro gêneros, Huperzia distingüe-se dos outros três gêneros por apresentar, entre outras características, caules ramificados isotomicamente e esporofilos semelhantes às folhas vegetativas. Huperzia mandiocana é uma epífita ocorrente nas florestas dos Estados da Bahia, Minas Gerais, São Paulo, Rio de Janeiro, Paraná, Santa Catarina, Rio Grande do Sul, bem como no Paraguai e Norte da Argentina, em altitudes entre 200-1000 m, com as menores altitudes na parte Sul da área de ocorrência junto ao domínio da Mata Atlântica. Na literatura não há registro sobre aspectos morfoanatômicos do esporófito (caule, raiz, esporofilo e esporângio) de H. mandiocana, assim este é o objetivo do presente estudo, incluindo também dados sobre ontogênese. Esporófitos de H. mandiocana foram coletados no Morro do Caçador (Florianópolis, Santa Catarina, Brasil), entre as coordenadas 753991 - 6959613 e 754450 - 6959380 UTM (Lat. 27º 27`30" e Long. 48º25`30"), sendo utilizadas caule, raiz, esporofilos e esporângios em distintas fases de desenvolvimento. O esporófito é ereto e fixa-se ao tronco do forófito por raízes adventícias. O ápice caulinar é formado por túnica e corpo que originam tecidos constituintes dos esporofilos e esporângios. A túnica forma o tecido de revestimento destas duas estruturas, mas também origina tecidos clorofilados dos esporofilos e todos os tecidos esporangiais. O corpo origina o tecido provascular e o meristema fundamental, que formam os tecidos vasculares do caule e esporofilos, o córtex caulinar e o parênquima perivascular do esporofilo respectivamente. As raízes têm origem no periciclo caulinar e seguem, no sentido basípeto, através do córtex paralelamente ao estelo, emergem em tufos na base caulinar. Estas raízes, além da absorção, servem para sustentar o caule ereto, pois são constituídas por abundante tecido esclerenquimático. A espécie é isofila, há apenas um tipo de folha (esporofilo), na base da qual está o esporângio; a folha é microfila tem uma só nervura e não ramificadas. O esporofilo é hipoestomático, com estômatos anomocíticos; a epiderme é uniestratificada, com cutícula espessa. O mesofilo é constituído por clorênquima, com células lobadas, e parênquima aclorofilado. O esporângio origina-se da túnica e quando maduro é reniforme, constituído por epiderme, com paredes espessadas, endotécio, tapete e um único lóculo contendo esporos triletes e de superfície ornamentada.
Lycopodiaceae includes four genera, Huperzia distinguishes itself from the other three genera, among other features, because stems is branched and isotonic sporophyll (with axillary sporangia) similar to vegetative leaves. Huperzia mandiocana is an epiphyte occurring in the forests from Bahia, Minas Gerais, Sao Paulo, Rio de Janeiro, Parana, Santa Catarina, Rio Grande do Sul States, as well as in Paraguay and northern Argentina. It is located at altitudes between 200-1000 m, with the lowest altitudes in the southern area of occurrence along the Atlantic Forest domain. There are no published records on anatomical features of H. mandiocana sporophyte (stem, root, leaf and sporangium), so this is the purpose of this study, including also data on ontogeny. Huperzia mandiocana sporophytes were collected at the "Morro do Caçador" (Florianópolis, Santa Catarina, Brazil), between coordinates 753,991 to 6,959,613 and 754,450 to 6,959,380 UTM (Lat. 27 º 27 '30 "and Long. 48 º 25' 30") and the shoot, root, sporophyll and sporangia in various development stages were analysed. The sporophyte is straight and it is fixed upon the trunk of the host tree by adventitious roots. The shoot apex consists of tunica and corpus, originating tissues of sporophyll and sporangium. The tunica forms the outermost tissue of these two structures, but also creates chlorophyllous tissues of sporophyll and all sporangia tissues. The corpus originates provascular and ground meristem, which form the vascular tissues of stem and sporophyll, the stem cortical parenchyma and perivascular parenchyma. The roots are originated from pericycle in the stem and they followed in the basipetal direction through the cortex parallelly to the stele and they emerge in tufts at the base stem. Besides to absorption, these roots also to sustain the stem erect, they are composed of abundant sclerenchyma. The species is homophily, only one type of leaf (sporophyll) and sporangium is on its basis; sporophyll have only one rib, without branches. The sporophyll is hypostomatic and stomata anomocytic, the epidermis is uniseriate and the cuticle is thick. The mesophyll is consisted of lobed chlorenchyma cells and ground parenchyma. The mature sporangium arises from tunica and kidney-shaped, it´s constituted of epidermis, with thick walls, endotecium, tapetum and only one locule that containing trilete spores and ornamented surface.

Apesar de representarem cerca de 50% de todas as células do encéfalo humano, as células da glia foram negligenciadas pela Doutrina do Neurônio, no início do século XX, por não serem excitáveis e por não se comunicarem como os neurônios. Durante um longo período, os estudos sobre os astrócitos se restringiam a suas funções de suporte do SNC. Atualmente se sabe que os astrócitos expressam receptores para uma variedade de neurotransmissores, como por exemplo, receptores colinérgicos nicotínicos, sugerindo possíveis mecanismos de resposta aos sinais enviados pela atividade neuronal, de modo variável e dinâmico, espontaneamente ou dependente da atividade neuronal e ativado pela alteração dos níveis intracelulares de Ca2+. Entretanto, os mecanismos específicos que explicam como essas células podem ser ativadas e qual sua contribuição para funcionamento do SNC ainda não estão totalmente esclarecidos. A secreção da proteína trófica S100B pelo astrócito pode ser um exemplo de comunicação celular, bem como também marcador biológico para diversas doenças. O nosso grupo já mostrou o envolvimento de diversos sistemas neuronais na modulação da secreção da proteína S100B. Um outro exemplo de comunicação é a liberação de gliotransmissores e a influência aguda astrocítica na transmissão sináptica. O objetivo dessa tese foi investigar se ativação do sistema colinérgico modula a secreção da proteína S100B em culturas primárias de astrócitos. Ainda, investigamos o uso da optogenética para o estudo da gliotransmissão em fatias de hipocampo. As culturas foram tratadas com inibidores da acetilcolinesterase (huperzina-A e tacrina), agonistas (acetiltiocolina, nicotina e carbacol) e antagonistas (mecamilamina e escopolamina) colinérgicos incubados durante 1 e 24 h. Os estudos em optogenética partiram primeiramente da caracterização do sistema cre/lox através da análise da expressão das proteínas de interesse, seletiva para astrócitos, por imunohistoquímica. Os resultados do primeiro estudo mostraram que a huperzina-A aumentou a secreção de S100B em culturas primárias de astrócitos, assim como o tratamento com nicotina; no segundo, discutiu-se as limitações metodológicas quanto a especificidade das proteínas e alteração da fisiologia dos astrócitos. Os resultados obtidos nessa tese ressaltam para a importância do conhecimento da fisiologia do astrócito, contribuindo para o entendimento de um possível mecanismo de ativação colinérgico dos astrócitos em promover secreção de S100B. Também é preciso ressaltar a relevância de novas metodologias que ajudam a descrever melhor o papel dos astrócitos na atividade sináptica.
Although about half of the brain cells are glial cells, the Neuron Doctrine has neglected them, at the beginning of 20th century, because they were not excitable and also, they were unable to communicate as neurons. For a long time, the studies about astrocytes functions were limited to trophic and metabolic support to neurons by which providing for the homeostasis of the nervous system. A variety of studies have been shown the expression of different neurotransmitters receptors in astrocytes, for instance, nicotinic receptors, suggesting the possibility of astrocytic response to neuronal activity. This response is variable and dynamic and mostly can be activated by changes at the intracellular calcium levels. However, the specific mechanisms of astrocytic Ca2+ excitability and how this process could contribute to CNS functioning are still unclear. The S100B secretion could be an example of cellular communication, as well as biomarker for diverse diseases. S100B is calcium binding protein, produced and secreted mainly by astrocytes. Our group has already demonstrated the relevance of various neuronal systems mediating the S100B secretion. Another astrocytic form of communication could be the release of gliotransmitters and the acute influence at synaptic transmission. The aim of this thesis was to investigate whether cholinergic system activation could modulate S100B secretion in primary astrocytes cultures. Moreover, we investigated the optogenetic properties as a tool for gliotransmission studies in hippocampal slices. The astrocytes cultures were treated with acetylcolinesterase inhibitors (huperzine-A and tacrine), agonists (acetylthiocholine, nicotine and carbachol) and antagonists (mecamylamine and scopolamine) for 1 and 24h. The optogenetic studies were conducted, firstly, from the cre/lox characterization of protein expression, selectively for astrocytes, by immunohistochemistry. The first study results showed that huperzine-A increased S100B secretion, as well as nicotine; in the second study, we have discussed the main methodological limitations concerning the protein specificity and astrocytes physiology changes. These results raise the importance of astrocytes functions investigations, especially S100B secretion, contributing for the first time to the understanding of astrocytes excitability likely trough nicotinic system activation. In addition, it is worthy to note that new methodologies are relevant and can help us in a better description of astrocytes role during synaptic activity and brain functioning.

A doença de Alzheimer (DA) resulta de múltiplos eventos patogênicos, que incluem anormalidades no processamento protéico, inflamação e estresse oxidativo, levando à destruição seletiva de populações neuronais, especialmente de neurônios colinérgicos. Tal hipótese colinérgica justifica o uso de inibidores da acetilcolinesterase (AChE) como a estratégia utilizada para tratamento de pacientes com DA. Huperzina A (HupA), um alcalóide Licopodium extraído de uma espécie vegetal (Huperzia serrata) de uso tradicional pela medicina chinesa, parece se apresentar como uma alternativa. A HupA atua como um inibidor potente, reversível e altamente específico da AChE. Sua potência como anticolinesterásico é similar ou superior à da fisostigmina, galantamina, donepezil e tacrina, anticolinesterásicos aprovados para o tratamento da DA Este alcalóide também demonstrou múltiplos efeitos neuroprotetores, incluindo modificação do processamento do peptídeo -amilóide, redução do estresse oxidativo, proteção neuronal contra apoptose e regulação da expressão e secreção de fatores tróficos, tais como fator de crescimento neuronal e em situações de injúria com comprometimento de funções glutamatérgicas. A S100B é uma proteína da família S100 de proteínas ligantes de cálcio, produzida e secretada por astrócitos no SNC. Quando em baixas concentrações no meio extracelular (nanomolar), ela é trófica; já em concentrações maiores (micromolar), é tóxica, podendo levar à apoptose. Sabe-se que a S100B está envolvida na patofisiologia de diversas doenças neurodegenerativas, como por exemplo, na DA, no qual ocorre um aumento dos níveis dessa proteína no líquor em sua fase precoce. Neste trabalho investigamos o efeito da HupA, da tacrina (tetraaminoacridina ou THA) e da acetiltiocolina (ATCh) em astrócitos e em fatias hipocampais, quanto à secreção e conteúdo intracelular de S100B e proteína fibrilar glial ácida (GFAP), durante 1 e 24 h. A HupA (100 μM) aumentou a secreção em cultura de astrócitos em 1 h, mas diferentemente causou uma redução em fatias hipocampais, bem como ATCh (100 μM). Após 24 h de tratamento, foi possível observar em cultura de astrócitos um efeito da tacrina a 100 μM sobre a redução do conteúdo de S100B intracelular, mas não de HupA ou ATCh. O imunocontéudo de GFAP não foi alterado após os tratamentos. Uma análise geral desses resultados nos mostra que a HupA pode regular mecanismos de secreção da S100B. Além disto, a modulação de secreção desta proteína parece ser modulada por receptores colinérgicos.
Alzheimer´s Disease (AD) results in multiple pathogenic events, which include abnormalities of protein process, inflammation and oxidative stress, leading to a neuronal population destruction, especially of cholinergic neurons. This cholinergic hypothesis justifies the use of acetylcholinesterase inhibitors (IAChE) as the strategy for patients with AD. Huperzine A (HupA), a novel Lycopodium alkaloid isolated originally from a traditional Chinese medicine, seems to be an alternative. HupA is a reversible, potent and selective IAChE. Its potency and duration of AChE inhibition rival those of physostigmine, galanthamine, donepezil and tacrine, approved for treatment of DA. HupA also demonstrated multiple neuroprotector effects, including modifying the β-amyloid processing, decreasing oxidative stress, protecting neurons against apoptosis and also expressing and regulating secretion of trophic factors, such as neuronal growth factor (NGF) and also acts reducing glutamatergic excitotoxicity. S100B is a calcium-binding protein, produced and secreted by astrocytes in the central nervous system (CNS) and plays a regulatory role in the cytoskeleton and cell cycle. Moreover, extracellular S100B, a marker of glial activation in several conditions of brain injury, for example in AD, has a trophic or apoptotic effect on neurons, depending on its concentration. Here we investigated the effect of HupA, tacrina (tetraminoacridine or THA) and acetilthiocholine (ATCh), on S100B secretion, S100B and GFAP intracellular contend in cortical astrocytes cultures and hippocampal slices treated for 1 and 24 h. For 1 h, HupA and ATCh (100 μM) increased the secretion in astrocytes for 1 h, but caused a reduction on hippocampal slices (P < 0.05). After 24 h of treatment, tacrine (100 μM) reduced the S100B intracellular contend in astrocytes cultures, but it was not observed with HupA or ATCh. The GFAP immunocontent did not change after these treatments. Generally, our data show that HupA can regulate S100B secretion mechanisms and that this modulation could be activated by cholinergic receptors.

La enfermedad de Alzheimer (AD) es uno de los trastornos neurodegenerativos más importantes que afectan a las personas de edad avanzada, y tan sólo los fármacos anticolinesterásicos han probado tener cierta utilidad en la terapéutica de esta enfermedad. En el presente trabajo de tesis doctoral se han evaluado diversas características farmacológicas de dos nuevos anticolinesterásicos híbridos tacrina-huperzina A, (±)-huprina Y (huprina Y) y (±)-huprina Z (huprina Z). Mediante el método de Ellman se determinó la inhibición de los enzimas acetilcolinesterasa (AChE) bovina y humana y butirilcolinesterasa (BChE) humana. Ambas huprinas resultaron más activas que la tacrina y la huperzina A como inhibidores de la AChE bovina y humana, el mecanismo de inhibición fue de tipo mixto. Además, en los experimentos de reversibilidad de la actividad sobre la AChE bovina, la huprina Y mostró inhibir la enzima con carácter tight-binding. Por otro lado, tanto la huprina Y como la huprina Z actuaron de forma más potente sobre la AChE que sobre la BChE, y de forma más selectiva sobre la AChE humana que sobre la AChE bovina. En los estudios ex vivo realizados en ratón, ambos compuestos mostraron una clara actividad inhibitoria sobre la AChE cerebral, 20 min post-inyección, siendo la huprina Y más potente que la huprina Z [DI50 = 1.09 (0.39-2.98) vs 5.77 (3.29-10.30) mmol/kg]. El tiempo de semivida biológica del efecto fue igual a 1 hora en ambos casos. Se inyectó una única dosis de 10 mmol/kg de diferentes anticolinesterásicos a otro grupo de ratones y se comprobó que la inhibición de la enzima era muy inferior a la mostrada a la misma dosis por las huprinas. Otro aspecto interesante fue observar que las máximas dosis utilizadas de los compuestos (30 mmol/kg huprina Y y 40 mmol/kg huprina Z) sólo producían un ligero aumento de salivación y un leve temblor, mientras que a dosis inferiores de tacrina y huperzina A los efectos indeseables eran muy superiores, llegando en algunos casos a producirse la muerte del animal. A las DI50 calculadas, las huprinas inhibieron la ChE periférica (plasma) con una potencia muy inferior a la mostrada para la AChE central. Por último, se estudió el efecto de estos anticolinesterásicos sobre los receptores muscarínicos. En primer lugar se determinó el desplazamiento de [3H]-pirenzepina y [3H]-QNB, éste último junto a pirenzepina 10 mM, en membranas de hipocampo de rata. Mediante análisis de regresión no lineal se mostró que ambas huprinas tenían una alta afinidad por los receptores muscarínicos M1 (Ki = 2.55 ± 0.89.10-7 huprina Y; 3.89 ± 0.28.10-7 huprina Z), y que esta afinidad era significativamente superior a la que mostraban por los receptores M2 (Ki = 6.91 ± 1.39.10-6 huprina Y; >10-5 huprina Z). A continuación se realizaron estudios de producción de fosfatos de [3H]-inositol inducida por las huprinas con el objeto de discernir si estos compuestos actúan como agonistas o como antagonistas muscarínicos. El carbacol, reconocido agonista muscarínico, mostró una CE50 igual a 8.40 ± 0.42 .10-5 M (efecto revertido en casi su totalidad por los antagonistas atropina y pirenzepina), mientras que el valor para la huprina Y fue de 4.72 ± 1.40 .10-4M y para la huprina Z de 3.80 ± 0.20 .10-4 M. Los aumentos en la producción de fosfatos de [3H]-inositol inducidos por las huprinas fueron revertidos en un 50% por los antagonistas muscarínicos, mientras que el antagonista de los adrenoceptores a1 prazosina revirtió un 30% el efecto, lo cual demuestra que estos fármacos son agonistas muscarínicos M1, pero que además existen otros mecanismos implicados. En resumen, los resultados expuestos en esta tesis doctoral prueban que tanto la huprina Y como la huprina Z son dos potentes anticolinesterásicos, que actúan a nivel del SNC y que presentan un potencial interés para el tratamiento de la EA.
Alzheimer disease (AD) is an important neurodegenerative disorder which affects to elderly people and only acetylcholinesterase inhibitors have shown some efficacy in its treatment. This work evaluates some pharmacological characteristics of two new tacrine-huperzine A hybrids, (±)-huprine Y (huprine Y) and (±)-huprine Z (huprine Z). Bovine and human acetylcholinesterase (AChE) and human butyrylcholinesterase (BChE) inhibition were assayed by Ellman's method. The two huprines were more active than both tacrine and huperzine A as inhibitors of both human and bovine AChE, and they acted as mixed-type AChE inhibitors. Moreover, huprine Y exhibited a tight-binding character seen in the experiments of reversibility of bovine AChE inhibitory activity. In addition, both compounds were more active toward AChE than toward BChE. Also, the selectivity for the human AChE was greater than for the bovine enzyme. In ex vivo studies performed in mice, both drugs showed a clear inhibitory activity of brain AChE, 20 min after i.p. injection, huprine Y being more potent than huprine Z [ID50 = 1.09 (0.39-2.98) vs 5.77 (3.29-10.30) mmol/kg]. The time-course study of the inhibitory effect displayed a t1/2 of 1 hour for the two compounds. A single dose (10 mmol/kg) of different anticholinesterasic drugs was administered to another group of mice and in all cases the effect was lower than the effect displayed by huprines at the same dose. Another interesting point was that the huprines at doses as high as 30 and 40 mmol/kg, respectively, only showed slight increases of salivation and some tremors, while tacrine and huperzine A produced higher undesirable effects, and even the death of some animals. The previous ID50 calculated for the huprines were administered to some mice and the inhibitory activity was determined using plasma. Both drugs inhibited the peripheral enzyme with a lower potency than the central ChE. Lastly, the effect on muscarinic receptors was studied. Displacements of [3H]-pirenzepine and [3H]-QNB, this one in presence of pirenzepine 10 mM were performed in membranes of rat hippocampus. Non-linear regression analysis of the curves showed that both drugs had a higher affinity for the M1 receptor (Ki = 2.55 ± 0.89.10-7 huprine Y; 3.89 ± 0.28.10-7 huprine Z) than for the M2 receptor (Ki = 6.91 ± 1.39.10-6 huprine Y; >10-5 huprine Z). Studies of the huprines production of [3H]-inositol phosphates were done in order to know wheter these drugs act as agonist or antagonist of M1 receptors. Carbachol, a well-known muscarinic agonist, showed a EC50 of 8.40 ± 0.42 .10-5 M (effect reverted in almost 100% by muscarinic antagonist atropine and pirenzepine), while EC50 were 4.72 ± 1.40 .10-4M and 3.80 ± 0.20 .10-4 M for the huprine Y and the huprine Z, respectively. The increases of inositol phosphates production induced by the drugs were antagonized in a 50% in both cases by the muscarinic antagonist. On the other hand, prazosine, an a1-adrenoceptor antagonist, reverted 30% of the effect, which proves that huprines are muscarinic M1 agonists, but there are other mechanisms involved in the production of inositol phosphates. In summary, these results show that huprine Y and huprine Z are two potent anticholinesterasic drugs, which act at CNS and which are of potential interest in the treatment of the AD.

La enfermedad de Alzheimer (EA) es la enfermedad neurodegenerativa más común entre las demencias existentes hoy en día, cursando con alteraciones cognitivas y neuropsiquiátricas. Durante el proceso normal de envejecimiento existe un declive de las funciones sensoriales y motoras, acompañado de una disfunción cognitiva leve. Sin embargo, la demencia comprende un grupo de disfunciones caracterizadas por un deterioro gradual de las habilidades intelectuales, hasta el punto de originar graves problemas sociales. La prevalencia de los casos de demencia en personas de más de 80 años es de aproximadamente un 30% de la población, siendo la EA un 50-70% del total de los casos.
Se han realizado enormes esfuerzos para desarrollar nuevas estrategias terapéuticas para el tratamiento de la EA, estrategias basadas en el uso de fármacos que disminuyan la velocidad de desarrollo de la enfermedad. Los inhibidores de la acetilcolinesterasa (IAChE) que reducen el avance de la enfermedad de forma temporal y por ello retrasan el deterioro cognitivo de los enfermos de EA, siguen siendo, hoy por hoy, los únicos fármacos que están aprobados para el tratamiento de la EA. Sin embargo, el avance en el conocimiento del desarrollo de la enfermedad ha puesto especial atención en el papel de elementos importantes en el deterioro del sistema nervioso central (SNC) como por ejemplo el efecto de la proteína βA en la muerte neuronal o la importancia de los receptores nicotínicos en su papel neuroprotector. Es por ello que en la búsqueda de nuevos tratamientos que incrementen y mantengan la mejoría clínica de los pacientes de EA, se le está dando especial protagonismo a aquellos que combinan más de un mecanismo de acción.
Estudios previos han demostrado que la (±)huprina X o (±) 12-amino-3-cloro-9-etil-6, 7, 10, 11-tetrahidro-7, 11-metenociclooctano[b]-quinolina, presenta una potente acción inhibidora sobre la actividad de la acetilcolinesterasa (AChE). Sin embargo, y como objetivo principal del presente trabajo, se ha pretendido establecer el perfil colinérgico de la (±)huprina X, para observar su interacción con otros elementos del sistema colinérgico de especial interés en la fisiopatología de la EA, como son los receptores muscarínicos, nicotínicos y locus periférico de la AChE (éste último relacionado con el efecto progregante sobre la proteína βA).
El resultado de este estudio nos presentará una molécula activa en distintos niveles del sistema colinérgico y por ello con un potencial terapéutico de gran interés en el tratamiento sintomático de la EA.
Los objetivos específicos planteados fueron los siguientes:
1. El estudio de la interacción de la (±) huprina X con los receptores muscarínicos del tipo M1 y M2:
a) en hipocampo de rata, mediante la técnica de unión de radioligandos, con el fin de determinar la afinidad de la (±)huprina X sobre dichos receptores.
b) en estriado y corteza prefrontal de rata, mediante la técnica de superfusión de sinaptosomas para establecer el tipo de interacción sobre dichos receptores.
2. El estudio de la interacción de la (±)huprina X sobre los receptores nicotínicos de corteza prefrontal de rata mediante la técnica de superfusión de sinaptosomas:
a) determinando la interacción directa con los receptores nicotínicos
b) determinando la potenciación de los receptores nicotínicos
3. El estudio del efecto de la (±) huprina X y de la galantamina (potenciadores de los receptores nicotínicos), sobre la liberación de ACh en cortes de estriado de rata, con el fin de establecer la importancia de la potenciación del receptor nicotínico en un sistema en el cual están presentes otros neurotransmisores y neuromoduladores. Análisis comparativo con la huperzina A (carente del efecto potenciador sobre los receptores nicotínicos).
4. El estudio del efecto de la (±)huprina X sobre la actividad proagregante de la AChE en el proceso de la amiloidogénesis. Dicho estudio, de características preliminares, se lleva a cabo con la proteína priónica PrP106-126 de características amiloidogénicas parecidas a las de la proteína βA.
Alzheimer disease (AD) is one of the most common neurodegenerative diseases among the number of dementias existing today, showing cognitive and neuro-psychiatric alterations in the patient. During the normal process of ageing, it can be observed an alteration in the sensorial functions and also in the movement functions, also with a weak cognitive dysfunction. Nevertheless, dementia is characterized by showing a group of dysfunctions that conclude in a gradual impairment of the intellectual abilities that becomes in a grave social problem for the affected person. People older than 80 years, have a prevalence of dementia around 30%, and 50-70% of it is regarding AD.
High efforts have been done to develop new therapeutic strategies directed to treat EA; these strategies have been based on the use of medicines that are be able to reduce the progression of disease. The acetylcholinesterase inhibitors (IAChE) that have show to able to reduce the progression of the disease but only in a temporal way, are nowadays, the only medicines approved for the treatment of the disease. Nevertheless, the scientific and medical knowledge concerning how the disease progresses, has been a positive effect on research due to it has been developed new targets related with the central nervous system (CNS) impairment, such as the effect of βA protein on the neuronal death or the relevance of nicotinic receptors related its neuroprotective effect. Taking into account this, research on new treatments to maintain and increase the clinic improvement of AD´s patients, is based on the combination of those that combine different approaches.
Previous studies have shown that (±) Huprine X or (±) 12-amino-3-chloro-9-ethyl-6,7,10,11-tetrahydro-7,11-methanocycloocta[b]quinoline hydrochloride, shows a potent inhibitory action on the activity of the acetylcholinesterase (AChE). The main goal of the present study is to establish the cholinergic profile of the (±) Huprine X, observing the interaction with other cholinergic elements, moreover the AChE, which also show special interest in the AD physiopathology, such as muscarinic receptors, nicotinic receptors, or the periferic locus of AChE (related with the pro aggregating effect on βA protein.
The conclusion of this study will show us a molecule, active at different levels of the cholinergic system, and with a therapeutic potential highly interesting in the symptomatic treatment of the AD.
The specific objectives proposed were as follows:
1. The study of the interaction of the (±) Huprine X on the M1 and M2 muscarinic receptors:
a) in rat hippocampus preparation, using radioligand binding, with the objective to determine the affinity degree of the molecule on the receptos.
b) in rat striatum and in rat prefrontal cortex preparations, using a synaptosomal superfusion method to establish the nature of the interaction observed.
2. The study of the interaction of the (±) Huprine X on the nicotinic receptors of rat prefrontal cortex using a synaptosomal superfusion method:
a) it was determined the direct interaction on nicotinic receptors.
b) it was determined the potentiating effect of the (±) Huprine X on nicotinic receptors.
3. The study of the effect of (±) Huprine X and Galantamine (both nicotinic receptor potentiators), on the ACh release in rat striatum slices. The main goal of this study was establish the relevance of the potentiating effect on nicotinic receptors in a more complex system where other neuromodulators and neurotransmitters are presents. Comparative analysis with Huperzine A that lacks the potentiating effect on nicotinic receptors described for (±) Huprine X and Galantamine.
4. Study of the effect of (±) Huprine X on the proaggregating activity of AChE in the amyloidogenesis process. This study, only a preliminary study, was done with prionic protein PrP106-126, with amyloidogenic characteristics similar to βA protein.

The Lycopodiaceae is an ancient and cosmopolitan family of fern allies that include an estimated 35 Huperzia species occurring throughout the South-East Asian and Australian region. Thirteen species naturally occur in Australia and are found mainly in the tropical rainforests of far north Queensland. Over the past decade, there has been renewed interest in Huperzia and their respective Huperzine alkaloid concentrations following the discovery of Huperzine A (HupA) and Huperzine B (HupB) alkaloids in H. serrata (Thunb. ex Murray) Trevis. Both alkaloids are of pharmaceutical interest since they are highly selective and potent reversible inhibitors of acetylcholine esterase. Huperzine alkaloid concentrations of Australasian Huperzia have not been well documented, and no prior studies have been undertaken to investigate the amenability of Australasian Huperzia to alternative propagation techniques such as axenic culture.
This research presents an extensive screen of 16 Australasian Huperzia species to investigate their Huperzine alkaloid concentrations. HupA (0.032 to 1.012 mg g-1 DW) was detected in ten out of the sixteen Huperzia species examined, while HupB (0.008 to 0.339 mg g-1 DW) was detected in eight. From this extensive study, H. elmeri (Herter) Holub was observed as the species with the greatest potential to yield high Huperzine-containing individuals. In addition, the screen established that Australasian Huperzia generally contain higher HupA levels than H. serrata, the main source of commercial HupA, which on average only contains 0.082 mg g-1 DW HupA. The fractionation and spectrometric analysis of alkaloids as part of the screen led to the discovery of three Huperzine alkaloids co-occurring within the same plant: HupA, HupB and Huperzine C, isolated from an individual of an Australian H. carinata (Desv. Ex Poir.) Trevis.
The potential of establishing axenic cultures of Australasian Huperzia was also investigated in this research. Actively growing axenic cultures of H. carinata, H. squarrosa (C.Forster) Trevisan, H. phlegmaria (L.) Rothm. and H. phlegmarioidies (Gaudich.) Rothm., together with callus and cell suspension cultures of H. carinata and H. phlegmaria, were successfully established. The results suggest that culturing in total darkness is essential to allow for optimal callus and cell suspension growth. In addition, this study also investigated the possibilities of germinating various Huperzia spores, by both symbiotic and asymbiotic means. Germination of H. squarrosa spores was achieved by both symbiotic and asymbiotic means, and was only observed in cultures which were kept in the dark, implying that there is a form of photo-inhibition mechanism preventing spores from germinating when they are exposed to light. Beneficial effects of various types of spore treatments prior to sowing, in terms of increased spore germination was also observed.
In conclusion, the results presented suggest that Australasian Huperzia are indeed a potentially valuable resource for Huperzine alkaloids. The investigations into the conditions required for the successful introduction and maintenance of Australasian Huperzia in axenic culture has also further extended our understanding of these plants, and their amenability towards axenic culture conditions as a means of alternative propagation.

碩士
中原大學
化學研究所
78
HuperzineA是由常用於中藥上的植物石松Lycopodium serratum Thunb , 分離出來的 一種生物鹼。 Huperzine A 為一種乙醯膽鹼 的抑制劑 (inhibitor), 在生理實驗上有顯著地抗乙 醯膽鹼 (anticholinesterase) 的活性, 在動物生理實驗上, Huperzine A 對動物 具有增進記憶力及學習能力的作用 。目前在中國大陸對此生物鹼正進行有關因阿爾 滋海默氏病 (Alzheimer'sdisease) 疾病所造成的語言障礙及嚴重的記憶力受損的臨 床實驗以及因重症肌無力症 (myasthenia gravis)所造成肌肉運動後出現程度不等的 無力之臨床實驗。 以1, 4- 環己二酮單伸乙縮酮當起始物, 經內醯胺化得 [A], 氮- 芐基化得 [B], 芳 香化得化合物 [C], 欲引進硝基, 卻得化合物 [D], 而非預期產物 [E]。 使用更簡潔的方法, 一步將 [A]芳香化成化合物 [F], 經氧- 甲基化得 [G], 去內縮 酮得 [H], 烯醇- 乙酸酯化反應得 [I], 欲引進硝基, 卻得 [J], 而非預期產物。 吾等以甲氧羰基取代硝基得 [K]。 吾等自1, 4- 環己二酮單伸乙縮酮, 經還原得 [L], 氧- 芐基化得 [M], 溴化得 [N] , 消去反應得 [O], 去內縮酮得α, β- 不飽和酮 [P]。 自化合物 [M], 經去內縮酮得 [Q], 經烯醇- 乙酸酯化反應得 [R]。 自1, 4- 環己二酮單伸乙縮酮, 經烯醇- 乙酸酯化反應得 [S], 做硝化反應得 [T], 經1, 4- 加成反應- 醛醇縮合反應, 得化合物 [U], 由於 [U]的合成, 將有助於以後 重要中間產物 [Z]的合成。

碩士
大葉大學
生物產業科技學系
99
Base on those research in this following year, Huperzine A what we can find out in Huperzia serrata is kind of potent Acetylcholinesterase blocker; Acetylcholinesterase is the enzyme which decompose the Acetylcholine, and Acetylcholine is the Neurotransmitter of nerve function and memory working. That's why Huperzine A can help treatment the memory loss, especially the Alzheimer's disease and early symptoms of dementia. The purpose of this research is use microwave to assist the extraction method, extraction the Huperzine A inside of Huperzia serrata, and used the HPLC for the qualitative analysis; Also to investigate the best condition of microwave assist and ultrasound assist extraction method though the Orthogonal experimental design (Orthogonal Array) to assist the extraction the Huperzine A inside of Huperzia serrata. In this research, we also looking for compare Antioxidant capacity inside of the active ingredients in the best extraction condition, it can be the helping data to prove in the product development. In the resource show to us the best condition of microwave assist extraction method is ethanol concentration 75%, solid-liquid ratio is 1:30, temperature in 70℃,working time is 5 min, and get the Huperzine A is 0.0062 mg/mL; and the the best condition of ultrasound assist extraction method is ethanol concentration 75%, solid-liquid ratio is 1:30, temperature in 60℃,working time is 90 min, and get the Huperzine A is 0.007 mg/mL. The analysis of active ingredients has also get some content.

PART I. Asymmetric syntheses of both natural (+)- and nonnatural (-)- byssochlamic acid via a [2+2] photoaddition-cycloreversion strategy are described. X-ray crystallographic analysis of the cyclohexylamine salt 99 showed that the structure of the monomethyl ester 100 from esterase hydrolysis of 44 was originally misassigned as 56. The enantiomeric relationship of the two diolides 106 and 70 permitted syntheses of nonnatural byssochlamic acid (-)-3 and natural byssochiamic acid (+)-3 from enantiopure alcohol (+)-64 and from its enantiomer (-)-110, respectively. Through the use of (��)-103 to reach both enantiomers of byssochlamic acid (3) and subsequent epimerization of the npropyl chain, it was proved that the cis configuration of the two alkyl substituents is strongly preferred in the natural product. PART II. An asymmetric approach towards the nootropic agent huperzine A is described. Formation of cyclobutane 122 with the desired stereochemistry was accomplished using intramolecular [2+2] photoaddition of the enantiopure enone 121. Attempts to prepare the methoxypyridine system via an azadiene Diels- Alder reaction were unsuccessful. However, intramolecular Michael addition of 181 produced silyl ether 182 which was converted into the pyridone 187 by treatment with hydrogen fluoride followed by selenoxide elimination. Attempts to effect the key sigmatropic rearrangement of ketone 197 into a direct precursor of huperzine A were unsuccessful.
Graduation date: 2001

Studies of the occurrence of montane and boreomontane species in ravines of the sandstone landscape are scarce and the occurrence of these species are explained by the presence of temperature inversion. The question is, which factors limit the occurrence ofthese species in ravines with temperature inversion. The aim of this diploma thesis is to reveal factors that influence the occurrence of Huperzia selago in inverse ravines of sandstone landscape. This work uses a habitat variables recorded directly for populations of H. selago and variables derived from a digital elevation model. These derived variables are also used for creation of two predictive models of geographic distribution of H. selago in the National Park Bohemian Switzerland. When we summarize the most informative variables of predictive models and habitat conditions significantly different from control sites, we get the typical habitat of H. selago. Such sites will likely be found on the rock at the bottom of the valley. Factors that influence the suitability of habitat are: moisture, vegetation type, slope, and distance to the bottom of the valley.