Thesis (Ph. D.)--Chemistry and Biochemistry, Georgia Institute of Technology, 2009. / Committee Chair: Kubanek, Julia; Committee Member: Fernandez, Facundo M.; Committee Member: Harvey, Stephen C.; Committee Member: Hay, Mark E.; Committee Member: Hud, Nicholas V. Part of the SMARTech Electronic Thesis and Dissertation Collection.

Caesalpinia férrea Martius, popularmente conhecida como pau-ferro e jucá, é utilizada na medicina tradicional para o tratamento de problemas hepáticos, respiratórios e, em especial, para distúrbios gastrintestinais e como cicatrizante. Deste modo, os objetivos do presente trabalho visaram avaliar os extratos brutos liofilizados de folha (EBLF) e caule (EBLC) quanto a caracterização botânica, o estudo químico e farmacológico, direcionando principalmente, às ações antiúlcera, antioxidante e cicatrizante, e toxicidade destes órgãos vegetais de C. ferrea. A triagem fitoquímica foi realizada com a droga vegetal constituída de folha (DF) e de caule (DC), bem como com os seus EBLF e EBLC. Os métodos empregados foram preconizados por Farnsworth (1966) e Matos (1988) onde foram pesquisados os seguintes compostos: flavonóides, glicósidos cardiotônicos, saponinas, antraderivados, alcalóides, cumarinas, taninos e óleo essencial. Além disso, foi realizada a quantificação de taninos e flavonóides segundo a metodologia proposta na Farmacopéia Européia (2001) e na Farmacopéia Brasileira (2003), respectivamente. Para a avaliação da Toxicidade de C. ferrea foram realizadas a Toxicidade Aguda de ambos os órgãos vegetais, a DL50 do EBLF e a Toxicidade subcrônica do EBLF e EBLC, todos os modelos seguiram a metodologia de Brito (1994). Para análise da atividade antiulcerogênica da espécie em estudo foi realizado o teste da indução de lesão gástrica aguda por etanol/HCI e lesão subcrônica por ácido acético. Grupos Tratados receberam EBLF ou EBLC ou frações ou extratos enriquecidos em flavonóides, Grupo Controle água ou tween 80 e Grupo de Referência Misoprostol ou Cimetidina. Três parâmetros foram avaliados neste modelo: Área Total de Lesão (ATL) , Área Relativa de Lesão (ARL) e índice de Lesão Ulcerativa (ILU). A atividade antioxidante \"in vitro\" foi medida através da inibição da autoxidação de homogenato de cérebro de Ratos (Stocks et aI., 1974). Os extratos foram solubilizados em etanol 70% e as diluições (0.05-0.003mg/mL) foram efetuadas em etanol 35%. O etanol 35% foi utilizado como controle. E na avaliação da Atividade Cicatrizante, os animais (ratos) sofreram uma incisão na região dorsal com auxílio de punch. Os Grupos Tratados receberam diariamente 1 mL de EBLF ou EBLC, solubilizados a 15% em água, e o Grupo Controle água destilada na mesma proporção por um período de 14 dias. Na triagem fitoquímica foram detectados para ambos os extratos, flavonóides, taninos, além de antraderivados e cumarinas nas Folhas. A porcentagem encontrada de Taninos na DF foi de 7.13% e no EBLF de 23.95% e na DC foi de 2.26% e no EBLC de 11.77%. Já a quantificação de flavonóides foi de 0.0095% na DF, 0.026% no EBLF, 0.00014% na DC e de 0.0017% no EBLC. No teste de toxicidade aguda, somente os animais que receberam EBLF apresentaram alterações comportamentais a partir dos primeiros tempos de observação e morte de 3 animais machos e 2 fêmeas (n=5/sexo). Dessa forma, a DL50 encontrada para este extrato vegetal foi de 5471.64 mg/Kg para as fêmeas e de 3112.94 mg/Kg para os machos. Na Toxicidade subcrônica, apenas os animais fêmeas que receberam EBLC (800 mg/Kg) apresentaram uma diferença significativa, em relação ao grupo controle, quanto ao peso do rim, porém não foi encontrada nenhuma alteração histológica neste órgão. EBLF e EBLC apresentaram significativa atividade antiulcerogênica no modelo de lesão gástrica aguda dentro dos parâmetros avaliados. O EBLC reduziu em 37% a ARL. Já o EBLF foi tão ativo como o Misoprostol reduzindo em 95%, 81% e 63% a ATL, a ARL e o ILU, respectivamente contra 92%, 70% e 59% do fármaco de referência. Porém, as frações e os extratos enriquecidos em flavonóides obtidos de ambos os extratos brutos liofilizados não apresentaram atividade antiulcerogênica em nenhum dos 3 parâmetros. Esses mesmos resultados foram obtidos no modelo de lesão gástrica subcrônica para ambos os extratos vegetais. Os EBLF e EBLC de C. ferrea promoveram uma atividade antioxidante de 94% e 84%, respectivamente, na concentração de 0.8196 µg/mL, e um Q 1/2de 0.2331 (Folha) e 0.5061 (Caule) µg/mL. Na avaliação da atividade cicatrizante de ambos os extratos vegetais, não foi encontrada diferença significativa entre os Grupos Tratados e Controle. No laudo histológico não se observou nenhum sinal de cicatrização tecidual. Apesar de C. ferrea ser utilizada pela população como cicatrizante, não foi possível confirmar tal atividade nas folhas e nos caules desta espécie. / Caesalpinia ferrea Martius , populary, known as iron-wood or juca, is utilized in traditional medicine in the treatment of both hepatic and respiratory problems and, in special, for gastrointestinals disturbances and eventual healing. The objective of the present work is to evaluate the leiophyllized brute extracts of the leaf (lBEl) and the stem (lBES), the botanic caracterization, the chemical and pharmacological studies, focuzing principally, the antiulcer and healing action, and also the toxicity of these vegetable organs of C. ferrea. The phytochemical was ma de with the drug of the leaf (DL) and of the stem (DS)- LaEL and lBES. The method was precognized by Famsworth (1966) e Matos (1988) where doing research of: flavonoids, cardiotonic glicosids, saponins, antraderivates, alkaloids, coumarins, tannins and essential oi!. Beyond that, the quantification of tannins and flavonoids according to the metodology proposed in European Pharmacopeia (2001) and Brazilian Pharmacopeia (2003) were also undertaken, respectively. For the evaluation of the C. ferrea toxicity, the acute toxicity of both vegetable organs, the DL50 of LBEL and the subcronical toxicity of LBEL and LBES, were analyzed following the metodology of Brito (1994).The test of induction of acute gastric lesion for ethanol/HCI was used for antiulcerogenic activity analysis of the species studied. Treated Groups received LBEL or LBES or fractions or extracts enrich in flavonoids, Controls Groups water or tween 80 and misoprostol Reference Group. Three parameters were evaluated considering: Total Area of Lesion (TAL), Relative Area of Lesion (RAL) and Rate of Ulcerative Lesion (RUL). And for evaluation of subcronic gastric lesion by acetic acid 30%, the Treated Group received LBEL or LBES, Control Group water and cimetidine Reference Group. The ulcerative lesions were evaluated only in 2 parameters: RAL and RUL. An antioxidant activity \"in vitro\" was measured through inibition of antioxidation of homogenate of rat brain (Stocks et aI., 1974). The extracts were solubilized in ethanol 70% and dilutions (0.05-0.003mg/mL) were performed in ethanol 35%. The ethanol 35% was utilized like control. And for evaluation of healing activity, the animals (rats) suffered na incision in the dorsal region with a punch aid. The Treated Groups received daily 1mL of LBEL or LBES, solubilized by 15% in water, and Control Group distilled in the same proportion during a 14 day period. In phytochemical were detected for both extacts, flavonoids, tannins., beyond antradderivate and coumarins in leaves. The percentages of tannins found were 7.13% in DL, 23.95% in LBEL, 2.26% in OS and 11.77% in LBES. The quantification of flavonoids was 0.0095% in DL, 0.026% in LBEL, 0.00014% in DS and 0.0017% in LBES. During the acute toxicity test, it was observed a behaviour alteration among animais that received LBEL up tp the first time of observation and death pof 3 males and 2 females. The DL50 found to this vegetable extract was 5471.64 mg/Kg for the females and 3112.94 mg/Kg for the males. In subronic toxicity, only the females receiving LBES (800mg/Kg) presented a significant difference, according to the Control Group, as much as the weight of a kidney. However, no histologic alteration in this organ was found. LBEL and LBES presented antiulcerogenic significant activity in acute gstric lesions, based on the parameters evaluated. The LBES was reduced to 37% in RAL. The LBEL was so active as the misoprostol, being reducid to 95%,81% and 63% TAL, RAL and the RUL, respectively against 92%, 70% and 59% of pharmaco of reference. Nevertheless, nor the fractions nor the flavonoids enriched extracts obtained from both leiophyllized brute extracts showed antiulcerogenic activity at the 3 studied parameters. Up to the present time, in model subcronic gastric lesion, none of both vegetable extracts in question has showed active similar to TAL, RAL and RUL in relation to the Control Group. The LBEL and LBES of C. ferrea promoved an antioxidant activity of 93,56% and 84,38%, respectively, in concentration of de 0.8196 µg/mL, and a Q1/2 of 0.2331 (leaf) and 0.5061 (Stem) µg/mL. Conceming the healing activity evaluation of both vegetable extracts, no significant differences between Treats and Control Groups were found. Also in histologic award, no sign of tecidual cicatrization was observed. In spite of the fact that C. ferrea has been utilized by the population as cicatrizant, no clear evidence of its leaves and stems healing activity has been confirmed.

This study aimed to evaluate the antimicrobial efficacy of medicinal hemp plant extracts to determine the antibacterial effects of indigenous Sansevieria species and exotic Cannabis sativa phytotherapy varieties. This study also assessed whether aqueous o

Diversas plantas com atuação no trato gastrointestinal - ricas em taninos e antraquinonas - ainda deixam muitas dúvidas quanto a sua caracterização estrutural, o seu mecanismo de ação e toxicidade. O presente trabalho objetivou o estudo químico e biológico de duas plantas utilizadas para doenças relacionadas ao sistema gastrointestinal: barbatimão (Stryphnodendron rotundifolium), rico em procianidinas e taninos (utilizada para combater gastrite); e cáscara sagrada (Rhamnus purshiana), rica em antraquinonas (utilizada para constipação). Inicialmente foi realizado um estudo químico com as plantas envolvendo isolamento e caracterização de componentes do extrato por RMN (1H, 13C, DEPT e NOESY), espectrometria de massas (MS), infravermelho e dicroísmo circular vibracional (VCD). Essas substâncias foram utilizadas para a padronização dos extratos utilizados em estudo biológico. O estudo biológico envolveu testes in vitro para avaliar a toxicidade de diferentes componentes do extrato de cáscara sagrada e teste in vivo com barbatimão, visando, através do tratamento de animais e remoção do estômago para estudo em microscopia confocal, entender o mecanismo de proteção gástrica. Com o desenvolvimento do trabalho foi possível desenvolver novas metodologias para a identificação de antraquinonas utilizando espectrometria de massas e provar a veracidade de regras empíricas utilizadas para a determinação da configuração absoluta de moléculas dessa classe. Os testes in vitro com antraquinonas isoladas e padrões comerciais indicam a possibilidade de aprimoramento dos perfis químicos utilizados para a produção de medicamentos a base de extratos de cáscara sagrada, visando à redução de toxicidade. Quanto ao barbatimão, o estudo químico possibilitou a aplicação da técnica de VCD para a diferenciação de procianidinas diasteroisomericas. Os estudos in vivo confirmaram a eficácia do uso de taninos para combater gastrite através da formação de um revestimento protetor. / Several plants with action in the gastrointestinal tract - rich in tannins and anthraquinones - cause many doubts concerning their chemical characterization, mechanism of action and toxicity. The present work aimed to develop a chemical and biological study of two plants used for gastrointestinal system related diseases: barbatimão (Stryphnodendron rotundifolium), rich in procyanidins and tannins (used to treat gastritis); and cascara sagrada (Rhamnus purshiana), rich in anthraquinones (used for constipation). Initially, a chemical study with plants was performed comprising isolation and characterization of components of the extract through NMR (1H, 13C, DEPT and NOESY), mass spectrometry (MS), and infrared and vibrational circular dichroism (VCD). These substances were used for extract standardization used in biological studies. The in vitro tests were performed in order to evaluate the toxicity of different components of the cascara extract. The in vivo tests with barbatimão aimed to better understand the mechanism of gastric protection, by treating animals with the extract, removing their stomachs, and analyzing them with fluorescence microscopy. Through this work we were able to develop new methods for identifying anthraquinones by mass spectrometry and to prove empirical NMR rules used to determine the absolute configuration of molecules of this class. The tests with isolated anthraquinones indicated the possibility of improving chemical profile to further reduce cascara sagrada toxicity. The barbatimão chemical study allowed the application of VCD technique for the differentiation of diasteroisomeric procyanidins, and confirmed the effectiveness of using tannins for gastritis treatment through the formation of a protective coating.

Cordia ecalyculata Vell. (Boraginaceae), espécie medicinal vulgarmente conhecida como porangaba e chá-de-bugre, é utilizada popularmente como emagrecedor, diurético, para curar tosses catarrais e reumatismos além de ser usada no tratamento de úlceras externas em forma de banhos. No Brasil existe grande confusão com relação aos nomes populares das espécies medicinais, fato que motivou a realização do presente trabalho. A espécie estudada é confundida com outras espécies vegetais, particularmente com Casearia sylvestris Swartz (Flacourtiaceae), também conhecida pelos nomes de guaçatonga e erva-de-bugre. O estudo farmacobotânico de C. ecalyculata revelou estruturas papilosas na epiderme e células esclereificadas no parênquima cortical, que não haviam sido descritas anteriormente. Foram listadas as principais diferenças morfoanatômicas entre as folhas de C. sylvestris e C. ecalyculata. auxiliares na diagnose da droga vegetal comercializada. As amostras adquiridas no comércio como porangaba não apresentaram características de C. ecalyculata, mas sim, de C. sylvestris, como: duas a três camadas de parênquima paliçádico; cavidades secretoras; drusas e cristais prismáticos em abundância; estômatos paracíticos; células epidérmicas poligonais; ausência de litocistos e areia cristalina. Fotomicrografias ilustram o trabalho. A triagem fitoquímica do extrato hidroetanólico liofilizado (EHEL) de C. ecalyculata apresentou resultado positivo para taninos, flavonóides e saponinas. A análise cromatográfica permitiu verificar a presença de substância coincidente com a alantoína no extrato de C. ecalyculata, além de possibilitar o desenvolvimento do perfil cromatográfico, auxiliar na diferenciação de C. sylvestris. Os ensaios de quantificação de alantoína no EHEL foram realizados por meio de dois métodos espectrofotométricos, cujos valores encontrados foram 0,68% e 0,70% de alantoína. O EHEL não apresentou róxicidade no ensaio de toxicidade aguda, na dose de 5 g/kg de massa corpórea do animal, por via oral. O experimento de atividade antiúlcera do EHEL de C. ecalyculata não apresentou diferenças significativas entre os animais controle e tratados. / Cordia ecalyculata Vell. (Boraginaceae) is a medicinal species most known as porangaba and chá-de-bugre. It is commonly used as diuretic, weight controller, for phleem cough healing, rheumatism healing and also in baths for externai ulcers treatment. The present work aims at clarifying the popular names of medicinal species often misnamed in Brazil. For example, the species cited above has always been mistaken as Casearia sylvestris Swartz. (Flacourtiaceae), also known as guaçatonga and erva-de-bugre. The pharmacological-botanical study of C. ecalyculata revealed papillary structures on the epidermis and sclerenchymatous cells on the cortical parenchyma which had not yet been described in the literature. The main morphological-anatomical differences between the C. sylvestris and the C. ecalyculata leaves were listed. These differences have been of great help concerning the diagnosis of commercialized vegetal drug. The commercialized samples do not show the features of the C. ecalyculata, but the features of the C. sylvestris, such as: two or three palisade parenchyma layers; secretive cavities; plenty of druses and prismatic crystals; paracytic stomata; polygonal epidermical cells; absence of lythocysts and crystal-sand, as it can be seen in the illustrative photomicrographies. The phytochemical screening of the lyophilized hydroethanolical extract (EHEL) of the C. ecalyculata showed a positive outcome for tannins, flavonoids and saponins. The chromatographic analysis not only allowed the evaluation of the coincident substance with the allantoin in the C. ecalyculata extract, but also enable the development of its chromatographic aspect, what eventually helped to differenciate it from C. sylvestris. The quantification assays of allantoin in the EHEL extract of the C. ecalyculata were performed through two spectrophotometric methods and the values found were 0,68% and 0,70% of allantoin. The EHEL extract displayed no toxicity in the acute toxicity trial in the 5 g/kg oral dosage per animal body weight. The anti-ulcer assay of this extract showed no meaningful differences among the subject and the treated animais.

Actinomycins are a class of compounds consisting of phenoxazinone-like core attached to two peptidolactone rings, denoted as α and β. A unique component of a few families—actinomycins G, Y, and Z—is a chlorinated β-ring threonine residue. Families G and Y also contained an actinomycin that possess a β-ring heterocycle (actinomycins G5 and Y5, respectively); prior to this work, no β-ring heterocycle-containing actinomycins were reported for the Z family. Unlike other actinomycin derivatives, Y5’s cytotoxicity was abolished while still maintaining some antibacterial potency. We constructed a model compound to probe the physical properties of the actinomycin core to test conditions under which heterocycle formation would occur. We also analyzed the gene clusters of these actinomycin producers for gene candidates to from this structural motif. We found the the actinomycin core aniline to have pKa values of 2.976 and 8.429 and a significant shift in UV absorption between 300-310nm when the group becomes charged. We also found cyclization conditions and no obvious gene candidates to form the β-ring heterocycle based on our gene cluster analysis. We hypothesize that the familial diversity of the actinomycin G, Y and Z familes is due to the reactivity of the phenoxazinone-like core.

<p>The process of drug discovery from natural products starts with the selection of study object. In this project recent knowledge and methods are incorporated to investigate the process of such selection for pharmacognostic investigations. As the model and object of study mistletoes and their content of the small cytotoxic peptides thionins are chosen.</p><p>The thionins are compared in silico to other proposed plant innate defense peptides. Utilizing analysis of amino acid sequences and secondary structures, the thionins are shown to be one of eight distinct groups of cystein-rich plant polypeptides analysed. Common features of thionins are exploited in an investigation of isolation methods, where a simple acidic extraction is equally efficient to isolate thionins as the laborious methods hitherto used. </p><p>An effort to study the relationships of the order Santalales was done. To infer phylogenetic relationships from DNA sequences, we increased the taxon sampling for utilized genes and regions such as <i>rbcL</i>, <i>atpB</i> and ribosomal 18S and 26S rDNA sequences within the Santalales. Analysing these together with published sequences for other tricolpate taxa a position for Santalales as sister to caryophyllids and basal to asterids is implied. This indication is supported by chemical characters such as the presence of cyclopeptide alkaloids of a kind only known from Gentianales.</p><p>To validate the chemosystematic implications from thionin distribution extracts of mistletoes collected in Panama, Taiwan and Madagascar, and the relative <i>Osyris alba</i> (Santalaceae) collected in Spain, were screened with the established fluorescence microculture cytotoxicity assay using the thionin-sensitive human lymphoma cell-line U937GTB. Bioassay guided isolation concludes that the cytotoxic compounds in Loranthaceae may however constitute another group of peptides.</p><p>In conclusion this work shows that the incorporation of informatic techniques may aid prediction and decision making when planning pharmacognostic research.</p>

The process of drug discovery from natural products starts with the selection of study object. In this project recent knowledge and methods are incorporated to investigate the process of such selection for pharmacognostic investigations. As the model and object of study mistletoes and their content of the small cytotoxic peptides thionins are chosen. The thionins are compared in silico to other proposed plant innate defense peptides. Utilizing analysis of amino acid sequences and secondary structures, the thionins are shown to be one of eight distinct groups of cystein-rich plant polypeptides analysed. Common features of thionins are exploited in an investigation of isolation methods, where a simple acidic extraction is equally efficient to isolate thionins as the laborious methods hitherto used. An effort to study the relationships of the order Santalales was done. To infer phylogenetic relationships from DNA sequences, we increased the taxon sampling for utilized genes and regions such as rbcL, atpB and ribosomal 18S and 26S rDNA sequences within the Santalales. Analysing these together with published sequences for other tricolpate taxa a position for Santalales as sister to caryophyllids and basal to asterids is implied. This indication is supported by chemical characters such as the presence of cyclopeptide alkaloids of a kind only known from Gentianales. To validate the chemosystematic implications from thionin distribution extracts of mistletoes collected in Panama, Taiwan and Madagascar, and the relative Osyris alba (Santalaceae) collected in Spain, were screened with the established fluorescence microculture cytotoxicity assay using the thionin-sensitive human lymphoma cell-line U937GTB. Bioassay guided isolation concludes that the cytotoxic compounds in Loranthaceae may however constitute another group of peptides. In conclusion this work shows that the incorporation of informatic techniques may aid prediction and decision making when planning pharmacognostic research.

This thesis is an account of investigation on the natural products deriving from various marine algae and has resulted in the discovery of eleven novel bioactive metabolites. Isolation and characterization of these new molecules were carried out using different chromatographic techniques and by analyses of different spectroscopic data, respectively. Using bioassay guided fractionation (brine shrimp toxicity assay), I isolated and identified five new, biologically active compounds [2β,3α-epitaondiol, flabellinol, flabellinone, stypoaldehyde and stypohydroperoxide], together with five known compounds [2-geranylgeranyl-6-methyl-1, 4-benzoquinone, (-) epistypodiol, (-) stypoldione, fucoxanthin and iditol] from the marine brown alga Stypopodium flabelliforme, collected from Papua New Guinea. All of the new compounds were found to have cytotoxic activity (EC₅₀ ranges from 0.8-10 μg/ml) in human lung cancer (NCI-H460). 2β,3α-epitaondiol and flabellinol exhibited strong sodium channel blocking activity (EC₅₀=0.3 and 0.9 μg/ml, respectively). As a result of efforts to identify bioactive agents from marine algae, I have isolated and identified one new halogenated monoterpene [(-)-(5E,7Z)-3,4,8- trichloro-7-dichloromethyl-3-methyl-1,5,7-octatriene] in addition to another three known halogenated monoterpene compounds from the red alga Plocamium cartilagineum collected from the eastern coast of South Africa. [(-)-(5E,7Z)-3,4,8- trichloro-7-dichloromethyl-3-methyl-1,5,7-octatriene] was found to be active as a cytotoxic agent in human lung cancer (NCI-H460) and mouse neuro-2a cell lines (EC₅₀ 4 μg/ml). As part of continued search for bioactive secondary metabolites from marine sources using a bioassay guided fractionation approach (anti-trypanosome activity), I examined the organic extract of a Papua New Guinean collection of the green alga Udotea orientalis growing on a coral wall and collected in September 1998. Successive HPLC separations resulted in the isolation of three new compounds; (+) curcuepoxide A, (+) curcuepoxide B and (+)-l0α-hydroxycurcudiol. In addition I isolated four known compounds; (+)-10β- hydroxycurcudiol, (+) curcuphenol, (+) curcudiol and (+) curcudiol-10-one. A bioassay guided investigation approach (anti-Sirt2) of a Lyngbya majuscula collection from Key West Florida, led to the discovery of two novel bioactive natural products [(+)-malyngamide X and one cyclic depsipeptide, (+)-floridamide]. The new cyclic depsipeptide, (+)-floridamide contains four amino acids units beside the unique unit, 2,2-dimethyl-3-hydroxyoctanoic acid (Dhoaa). / Graduation date: 2004

Natural products and small molecules play a major role in drug development. However, using natural products as a source of medicine comes with many challenges, such as lack of natural abundance and difficulty in isolation. Consequently, synthetic organic chemistry is a solution in order to access these compounds in usable quantities. However, synthetic chemisty comes with its own challenges such as efficiency, chemoselectivity, stereoselectivity and enantioselectivity. Therefore, synthetic tools that addresses these challenges are required solve these limitations. This thesis discusses new methodologies using strained carbocycles (cyclopropanes and cyclopropenes) as the reactive subunit for the construction of different carbocyclic and heterocyclic compounds. The homo-Nazarov cyclization of alkenyl and heteroaryl cyclopropyl ketones was used in order to construct cyclohexenones, cyclohexenols, heteroaryl ring-fused cyclohexenones, dihydrofurans, furans and furanones in a mild and efficient manner. Benzofused heteroaromatic compounds were achieved via the Lewis acid-catalyzed cycloisomerization of cyclopropene-3,3-dicarbonyls and furan-3-carboxylates. These heteroaromatic compounds can be applied to medicinal chemistry and material science.