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The methodology developed in this study through the acquisition of fingerprint chromatograms by liquid chromatography-diode array detector (LC-DAD), aided by principal component analysis, made it possible to assess, in a rational way, the chemical differences between six accessions of Lippia alba and to verify the influence of the method of extraction, the seasonality and the individuality of each accession on chemical composition of its extracts. Among all extracts analyzed against cancer cell lines, eight of them showed to be more promising against a human leukemia cell line (HL-60), displaying cell growth inhibition percentage ranging between 40.0 and 52.0%. In the inhibitory activity assays against acetylcholinesterase enzyme, all extracts showed weak inhibitory effect, with highlight only for six of them, which displayed inhibition ranging between 27.0 and 32.0%.

Orientador : Prof. Cid Aimbiré M. Santos Co-orientadora : Profª. Vincenza Andrisano Tese (doutorado) - Universidade Federal do Paraná, Setor de Ciências da Saúde, Programa de Pós-Graduação em Ciências Farmacêuticas. Defesa: Curitiba, 17/04/2014 Inclui referências Área de concentração: Insumos, medicamentos e correlatos Resumo: A doença de Alzheimer, uma doença progressiva, multifatorial, degenerativa do cérebro, é a causa mais comum de demência em idosos. Clinicamente, é caracterizada pela perda de memória progressiva e déficit em diferentes domínios cognitivos associados com a perda de marcadores pré-sinápticos do sistema colinérgico nas áreas do cérebro relacionadas com a memória e aprendizagem e com a presença de depósitos de placas amiloides e emaranhados neurofibrilares no cérebro de aflitos indivíduos. Uma abordagem nova para enfrentar a natureza multifatorial da doença pode ser representada pela descoberta de compostos únicos capazes de modular simultaneamente diferentes alvos envolvidos na cascata neurodegenerativa. Portanto, a capacidade inibitória do alcaloide uleína extraídos da casca do caule da planta medicinal brasileira Himatanthus lancifolius Woodson (Müll. Arg.); Apocynaceae contra conhecido alvos validados da doença de Alzheimer, incluindo as enzimas acetilcolinesterase humana, butirilcolinesterase de soro humano, beta-secretase humana e a agregação espontânea do peptídeo beta-amiloide foi realizada resultando em uma atividade inibidora marcante, dentro da gama nanomolar, contra a enzima beta-secretase humana, com um valor de IC50 de 180 (± 22) nM. Além disso, a uleína foi capaz de inibir ambas as colinesterases com valores de IC50 de 279,0 (± 4,5) ?M e 24,0 (± 1,5) ?M, respectivamente. Finalmente, a uleína também foi capaz de inibir significativamente a agregação espontânea do peptídeo beta-amiloide (40 % na proporção 5:1). Um método cromatográfico rápido, reprodutível e executável com detecção por fluorescência para o ensaio de inibição da enzima beta-secretase recombinante humana utilizando um substrato fluorogênico conhecido como substrato IV foi desenvolvido e aplicado para a avaliação de atividade enzima hrBACE1 imobilizada e sua inibição por um composto natural o qual apresentava propriedades auto-fluorescentes. O método provou ser sensível e permitiu a detecção seletiva do produto de reação enzimática e estudos de inibição. Palavras-chaves: Doença de Alzheimer, uleína, aceticholinesterase, butirilcolinesterase, betasecretase, peptídeo beta-amiloide, inibidores de agregação, IMER Abstract: Alzheimer's disease, a progressive, multifactorial, degenerative disorder of the brain, is the most common cause of dementia among the elderly. It is clinically characterized by loss of memory and progressive deficit in different cognitive domains associated with a loss of the presynaptic markers of the cholinergic system in the brain areas related to memory and learning, and with the presence of amyloid deposits and neurofibrillary tangles in the brain of afflicted individuals. An emerging disease modifying approach to face the multifactorial nature of AD may be represented by the discovery of single compounds which may simultaneously modulate different targets involved in the neurodegenerative AD cascade. Therefore the inhibitory capacity of the indole alkaloid uleine extracted from the stem bark of the Brazilian medicinal plant Himatanthus lancifolius (Müll. Arg.) Woodson; Apocynaceae against known Alzheimer's disease validated targets, including human acetylcholinesterase, butyrylcholinesterase from human serum, human beta-secretase and spontaneous ?- amyloid(1-42) aggregation was performed resulting on a remarkable inhibitory activity, within the nanomolar range, against human beta-secretase, with an IC50 value of 180 (±22) nM. In addition, uleine was able to inhibit both cholinesterases showing IC50 values of 279.0 (±4.5) ?M and 24.0 (±1.5) ?M, respectively. Finally, uleine was also capable of significantly inhibit amyloid self-aggregation (40% at the 5x concentration of ?-amyloid(1-42). In addition, High throughput screening techniques are required for the fast hit inhibitors selection in the early discovery process. However, in inhibitors screening campaign, the most frequently used methoxycoumarin based peptide substrate (M-2420) is not widely applicable when aromatic or heterocyclic compounds of natural source show auto-fluorescence interferences. Here, in order to overcome these drawbacks, we propose the use of a highly selective 4-(4-dimethylaminophenylazo)benzoic acid/5-[(2-aminoethyl)amino]naphthalene-1- sulfonic acid based peptide substrate (Substrate IV), whose cleavage product is devoid of spectroscopic interference. The human beta-secretase immobilized enzyme reactor was prepared and characterized in terms of units of immobilized of beta-secretase. Substrate IV cleavage was kinetically characterized on-line in terms of KM and vmax, in a classical Michaelis and Menten study. The online kinetic constants were found consistent with those obtained with the in solution standard method. In order to further validate the use of Substrate IV for inhibition studies, the inhibitory potency of a well-known beta-secretase peptide inhibitor was determined. A further application with uleine as BACE1 inhibitor was carried out to prove the system suitability for natural compounds. Keywords: Alzheimer's disease, uleine, acetycholinesterase, butyrylcholinesterase, ?- secretase, amyloid-? peptide, inhibitors, aggregation, IMER

vorgelegt von: Claudia Seidl Literaturverz. Bl.47 - 50 FH Campus Wien, Masterlehrgang Physiotherapie, Masterthesis, 2010

Orientador : Prof. Dr. Cid Aimbiré de M. Santos Disseratção (mestrado) - Universidade Federal do Paraná, Setor de Ciências da Saúde, Programa de Pós-Graduação em Ciências Farmacêuticas. Defesa: Curitiba, 11/05/2010 Bibliografia: fls. 79-83 Área de concentração: Insumos, medicamentos e correlatos Resumo: A doenca de Alzheimer e uma doenca multifatorial, progressiva e neurodegenerativa caracterizada por alteracoes nas funcoes congitivas de aprendizado e memoria, bem como por alteracoes comportamentais. Os inibidores da enzima acetilcolinesterase constituem o principal tratamento da doenca. Especies vegetais sao excelentes fontes de substancias naturais com potencial anticolinesterasico e os alcaloides, como a galantamina e huperzina A, se destacam como a principal classe de inibidores. O objetivo dessa dissertacao de mestrado foi contribuir com a busca de novas substancias naturais com potencial anticolinesterasico. Os extratos fluidos, fracoes e substancias isoladas foram avaliadas utilizando a associacao dos ensaios em CCD (metodo de Rhee) e microplaca (metodo de Ellman modificado por Rhee) com o reagente de Ellman e o ensaio em CCD com o sal Fast Blue B (metodo de Marston). Dezenove extratos simples de diferentes especies foram avaliados pelos ensaios em CCD e somente duas nao apresentaram atividade inibitoria nas concentracoes testadas. As fracoes DCM, n-butanol e aquosa da especie Rauvolfia sellowii foram as fracoes mais ativas enquanto que para a especie Himatanthus lancifolius foram as racoes DCM e EtOAc. Na fracao rica em alcaloides da especie H. lancifolius foi possivel ainda relacionar a atividade encontrada com a atividade do seu principal alcaloide uleina uma vez que apresentaram o esmo valor de Rf para as manchas de inibicao. A uleina apresentou um valor de IC50 de 0,45 ƒÊM, valor este que se encontra dentro do intervalo de valores de IC50 encontrado na literatura para a galantamina 0,39 a 1,5 ƒÊM). Alem disso, este trabalho apresenta o desenvolvimento e otimizacao de um ensaio para a determinacao da atividade anticolinesterasica utilizando a enzima imobilizada em um suporte de silica epoxidada dentro de uma coluna cromatografica acoplada ao equipamento de CLAE. Abstract: Alzheimer's disease is a multifactorial, progressive neurodegenerative disorder characterized by changes in cognitive functions like learning and memory, as well as behavioural changes. Acetylcholinesterase inhibitors are the disease primary treatment. Plant species are excellent sources of natural substances with potential anticholinesterase activity and alkaloids, such as galantamine and huperzine A, stand out as the major class of inhibitors. The aim of this dissertation was to contribute with the search for new natural substances with potential anticholinesterase activity. The fluid extracts, fractions and isolated compounds were evaluated using a combination of assays in TLC (Rhee's method) and microplate (Ellman's method modified by Rhee) with Ellman's reagent and the assay in TLC with Fast Blue B salt (Marston Ls method). Nineteen simple extracts from different species were evaluated using the TLC assays and only two didn ft show inhibitory activity at the concentrations tested. The DCM, n-butanol and aqueous fractions from Rauvolfia sellowii were the most active fractions while the DCM and EtOAc were the most active for the Himatanthus lancifolius. In the alkaloid rich fraction of the H. lancifolius species was possible to relate the activity found with the activity of its main alkaloid uleine once they showed the same Rf values for the inhibition spots. Uleine presented an IC50 value of 0.45 ƒÊM, within the range of IC50 values found in literature for galantamine (0.39 to 1.5 ƒÊM). Furthermore, this work presents the development and optimization of an assay for the determination of the acetylcholinesterase activity using the enzyme ACHE immobilized on a silica-epoxy support in a chromatography column coupled to the HPLC equipment.