Submitted by isabela.moljf@hotmail.com (isabela.moljf@hotmail.com) on 2017-08-24T11:08:43Z No. of bitstreams: 1 dalyaramendonçadematos.pdf: 2975621 bytes, checksum: 30567d62bd6e7554f436a9bbad1397de (MD5) / Rejected by Adriana Oliveira (adriana.oliveira@ufjf.edu.br), reason: on 2017-08-24T11:38:38Z (GMT) / Submitted by isabela.moljf@hotmail.com (isabela.moljf@hotmail.com) on 2017-08-24T11:54:12Z No. of bitstreams: 0 / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-08-24T13:01:20Z (GMT) No. of bitstreams: 0 / Made available in DSpace on 2017-08-24T13:01:20Z (GMT). No. of bitstreams: 0 Previous issue date: 2016-09-23 / O ácido caurenoico é um diterpeno caurânico encontrado em diversas espécies vegetais tais como Mikania glomerata (guaco), Copaifera langsdorffi (copaíba) e Smallanthus sonchifolia (yacon). Essa substância apresenta ação tripanocida, larvicida, antimicrobiana, analgésica, anti-inflamatória, relaxante da musculatura lisa, hipotensora, diurética, hipoglicêmica e citotóxica. Por tratar-se de uma molécula promissora para o desenvolvimento de novos fármacos, o objetivo de nosso estudo é determinar o perfil farmacocinético e a biodisponibilidade oral do ácido caurenoico em ratos. Foram utilizados Ratos Wistar (n=6), aos quais foram administrados 50 ou 100 mg/kg de ácido caurenóico por via IV ou oral. Os animais foram canulados pela veia jugular, permitindo a administração intravenosa do ácido caurenoico e coletas seriadas de sangue do tempo zero até 10 horas. A extração do ácido caurenoico do plasma foi realizada após acidificação com ácido acético 1% (v/v), seguida de precipitação de proteínas com acetonitrila. A quantificação do analito foi realizada utilizandose cromatografia líquida de alta eficiência com detecção ultravioleta (CLAE-UV), empregando-se as seguintes condições analíticas: coluna C18 (150 x 4.6 mm, 5 μm) mantida a 40°C, eluição isocrática com fase móvel composta por acetonitrila:água acidificada com ácido ortofosfórico 0,1% (70:30 v/v), fluxo de 1 mL/min, detecção em 200 nm e volume de injeção de 80 μL. A metodologia proposta foi validada e mostrou-se precisa, exata, robusta, confiável e linear entre 0,75 e 100 μg/mL. A partir do decaimento plasmático dos animais que receberam 50 mg/kg do ácido caurenoico por via intravenosa, foram determinados os seguintes parâmetros farmacocinéticos: Cmax = 22,2 ± 1,6 mg/L, Vd = 14,5 ± 1,5 L/kg, CL = 17,7 ± 1,5 mL/min/kg, ASC = 2859 ± 278 mg/L.h e T1/2 = 9,5 ± 0.6 h. Os resultados obtidos apontaram que o ácido caurenoico, administrado por via intravenosa, apresentou um comportamento cinético linear e bicompartimental na dose testada. Não foram encontrados níveis quantificáveis da substância nas amostras provenientes de ratos tratados com 50 ou 100 mg/kg de ácido caurenoico, por via oral, impossibilitando a definição de sua biodisponibilidade oral e sugerindo uma baixa absorção por esta via. Este é o primeiro estudo farmacocinético desta molécula e, esta avaliação, mesmo que pré-clínica, pode contribuir para o processo de desenvolvimento de novos medicamentos a partir desse diterpeno. / Kaurenoic acid is a kaurane-type diterpene found in several plant species such as Mikania glomerata (guaco), Copaifera langsdorffi (copaíba) and Smallanthus sonchifolia (yacon). Previous studies described several biological activities for this substance such as antitrypanosomal, antimicrobial, analgesic, anti-inflammatory, smooth muscle relaxant, hypotensive, diuretic, hypoglycemic and cytotoxic. As this molecule represents a lead compound for the development of new drugs, the aim of our study is to determine the pharmacokinetics profile and oral bioavailability of kaurenoic acid in rats. Wistar rats (n = 6) received 50 or 100 mg/kg of kaurenoic acid by intravenous or oral routes. The insertion of a cannula into the right external jugular vein of Wistar rats allowed intravenous administration of kaurenoic acid and collection of blood samples within predetermined time intervals. Extraction procedures from plasma consisted of acidification with 1% acetic acid (v/v), followed by precipitation of proteins with acetonitrile. The supernatant was submitted to highperformance liquid chromatography with UV detection (HPLC-UV) for quantification of kaurenoic acid. The established analytical conditions were: C18 column (150 x 4.6 mm, 5 μm) maintained at 40 ° C, isocratic elution with a mobile phase consisting of acetonitrile: acidified water with 0.1% orthophosphoric acid (70:30 v/v), a flow of 1 mL/min, UV detection at 200 nm and injection volume of 80 μL. The proposed methodology proved to be precise, accurate, robust and reliable. The linearity range is between 0.75 and 100 μg/mL. Plasma decay of animals receiving an intravenously dose of 50 mg/kg allowed the determination of the following pharmacokinetic parameters: Cmax = 22.2 ± 1.6 mg/L; Vd = 14.5 ± 1.5 L/kg; CL = 17.7 ± 1.5 mL/min/kg; AUC = 2859 ± 278 mg/L.h and T1/2 = 9.5 ± 0.6 h. Kaurenoic acid administered intravenously showed a linear and two-compartment kinetic behavior at the tested dose. As no measurable levels of substance were found in the samples from mice treated orally with kaurenoic acid, the determination of oral bioavailability was not possible, suggesting poor absorption through this route. This is the first pharmacokinetic study of this molecule and this preclinical assessment can contribute to the process of development of new drugs with this diterpene.

Through the ICBG (International Cooperative Biodiversity Group) program and a continuing search for anticancer compounds, plant extracts were obtianed from Suriname and Madagascar and screened for cytotoxic activity in the A2780 human ovarian cancer cell line. Fractionation of a leaf and flower extract of Casearia nigrescens led to the isolation of six new clerodane diterpenes. Four were new natural products and the other two were previously unreported hydrolysis products. Their structures were determined using mass spectrometry and 1-D and 2-D NMR. All six compounds were cytotoxic in the A2780 human ovarian cancer cell line. Fractionation of a leaf extract of Vernonia pachyclada led to the isolation of four new sesquiterpene lactones. Their structures were determined using mass spectrometry, 1-D and 2-D NMR, and (in one case) single crystal X-ray diffraction. All four compounds were cytotoxic in the A2780 human ovarian cancer cell line. Fractionation of an extract of Casimirella ampla led to the isolation of three new diterpenes and two known diterpenes. Their structures were determined using mass spectrometry and 1-D and 2-D NMR. All five compounds were cytotoxic in the A2780 human ovarian cancer cell line. Fractionation of root and stem extracts of Mendoncia cowanii led to the isolation of two new naphthaquinones, and two known naphthaquinones. Their structures were determined using mass spectrometry and 1-D and 2-D NMR. All four compounds were cytotoxic in the A2780 human ovarian cancer cell line and three compounds exhibited weak inhibition of Akt kinase. The fractionation of five additional extracts resulted in the isolation of twelve known compounds. Their structures were determined using mass spectrometry, 1-D and 2-D NMR, and comparison to literature data. All twelve compounds were cytotoxic in the A2780 human ovarian cancer cell line. / Ph. D.

As a part of a continuing search for bioactive compounds with the International Cooperative Biodiversity Group (ICBG), and in collaboration with the Natural Products Discovery Institute of the Institute for Hepatitis and Virus Research (IHVR), twelve plant extracts were investigated for their antiproliferative activity against the A2780 cell line, three plant extracts were investigated for their antimalarial activity against Plasmodium falciparum, and three plant extracts were investigated for their anti-inflammatory activity (PPAR-y inhibition). Bioassay-guided fractionation of extracts led to the identification of four new antiproliferative compounds (2.1-2.3, 3.1), five new anti-inflammatory compounds (6.4a, 6.5a-b, 6.6a, 6.6c), and twenty-eight known compounds from eight of the extracts. In addition, mallotojaponin C, an antimalarial natural product, and derivatives were synthesized and investigated for their antimalarial activity. / Ph. D.

As part of an International Cooperative Biodiversity Group (ICBG) program and a continuing search for antiproliferative natural products from the Madagascar rainforest, and a collaborative research project established between Virginia Tech and the Institute for Hepatitis and Virus Research (IHVR) focusing on searching for bioactive natural products from tropical forests in South Africa, 20 extracts were selected for investigation based on their antiproliferative activities against A2780 human ovarian cancer cell line or antimalarial activities against the Dd2 strain of Plasmodium falciparum. Bioassay-guided fractionation of seven of the extracts yielded twenty new compounds and twenty-four known compounds, and their structures were elucidated by using a combination of 1D (1H and 13C) and 2D NMR spectroscopy including COSY, HASQC, HMQC, HMBC, and NOESY sequences, mass spectrometry, UV, IR, ECD, optical rotation, and chemical conversions. In addition, ten known compounds were isolated from another five of the extracts, while studies on the remaining extracts were suspended due to loss of activity, unworkable small amounts of material, or low structural interest. The plants and their metabolites are discussed in the following order: five new antimalarial 5,6-dihydro-𝛼-pyrones and six bicyclic tetrahydro-𝛼-pyrone derivatives from Cryptocarya rigidifolia (Lauraceae); two new and five known antiproliferative lignans from Cleistanthus boivinianus (Phyllanthaceae); two new and two known antiproliferative sesquiterpenes lactones from Piptocoma antillana (Asteraceae); one new antiproliferative 1,4-naphthoquinone, one known antiproliferative isoflovonoid, and five known antiproliferative stilbenoids from Stuhlmannia moavi (Leguminosae); four known antiproliferative bisbenzylisoquinoline alkaloids from Anisocycla grandidieri (Menispermaceae); one new and two known antiproliferative butanolides, and two new antiproliferative secobutanolides from Ocotea macrocarpa (Lauraceae); one new antiproliferative and five known antiproliferative diterpenoids from Malleastrum rakotozafyi (Meliceae); and 10 known compounds from Monoporus sp. (Myrsinaceae), Premna corymbosa (Verbenaceae), Premna perplexanes (Verbenaceae), Epallage longipes (Asteraceae), and Cinnamosma fragrans (Canellaceae). / Ph. D.

As a part of the continuing search for bioactive compounds with the Madagascar International Cooperative Biodiversity Group (ICBG), and in collaboration with the Natural Products Discovery Institute of the Institute for Hepatitis and Virus Research (IHVR), thirteen plant extracts were investigated for antiplasmodial activity, thirteen plant extracts were investigated for antiproliferative activity, and one extract was investigated for inhibitors of the shikimate pathway in Plasmodium falciparum. Bioassay-guided fractionation of the extracts led to the identification of nineteen compounds with both antiplasmodial and antiproliferative activity, and thirteen compounds with only antiproliferative activity. Thirteen of these compounds (2.1 – 2.9, 3.3, 3.4, 4.5, and 5.1) were previously unknown. In addition total synthesis was used to confirm the structure of one new compound (4.5) and two other new natural-product like compounds (4.6 and 4.7) were also synthesized and investigated for antiplasmodial activity. / Ph. D. / Plants have a long history of producing compounds (Natural Products) that have been used as medicines. This dissertation focuses on the isolation of potential anticancer and antimalarial natural products from plants and their structure determination. The isolation of compounds was aided by the use of cell-based bioassays to determine the inhibition of cell growth. Growth inhibition of human ovarian cancer cells (the A2780 cell line) was used to test for potential anticancer activity, and growth inhibition of the malaria-causing parasite Plasmodium falciparum was used to test for potential antimalarial activity. Twenty-seven plant extracts from two different plant libraries were found to have biological activity in one of these bioassays, and bioassay-guided isolation performed on nine of these extracts led to the isolation of thirteen new compounds and fourteen known compounds. The isolation, structure determination, and biological evaluation of all isolated compounds are discussed in this work.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / O triterpeno lupeol e o tanino hidrolisÃvel dilactona do Ãcido valonÃico (DAV), componentes majoritÃrios de Cenostigma macrophyllum Tul. (Leguminoseae), foram avaliados no modelo experimental de lesÃo gÃstrica induzida por etanol e em modelos comportamentais. O lupeol (3, 10 e 30mg/kg, v.o.) e a DAV (3, 10 e 30mg/kg, i.p.) atenuaram significativamente (p<0,05) as lesÃes gÃstricas induzidas por etanol. No estudo mecanÃstico, o lupeol (30mg/kg) e a DAV (10mg/kg) mostraram aÃÃo antioxidante, previnindo a depleÃÃo de grupos sulfidrilas nÃo protÃicos e a participaÃÃo do Ãxido nÃtrico, de prostaglandinas, de canais de potÃssio ATP-dependentes e canais de cÃlcio. Foi observada ainda a participaÃÃo de receptores alfa-adrenÃrgicos, mas nÃo de receptores opiÃides, no mecanismo gastroprotetor das substÃncias. Tanto o lupeol (30 mg/kg) quanto a DAV (10mg/kg) reduziram a acidez gÃstrica total sem alterar o volume secretÃrio gÃstrico no modelo de ligadura do piloro. Na avaliaÃÃo sobre a motilidade intestinal normal, o tratamento com lupeol (3, 10 e 30mg/kg) nÃo alterou o percentual de trÃnsito em relaÃÃo ao controle, contudo a DAV (3, 10 e 30mg/kg) reduziu de forma significativa (p<0,05) o percentual de trÃnsito, por um mecanismo que nÃo envolve receptores opiÃides ou adrenÃrgicos. DAV (10 mg/kg) tambÃm foi capaz de inibir significativamente (p<0,05) o trÃnsito estimulado por Ãleo de rÃcino. Nos modelos comportamentais, o lupeol (3, 10 e 30mg/kg, v.o.) nÃo produziu alteraÃÃo na atividade locomotora dos animais no teste da movimentaÃÃo espontÃnea, entretanto o tratamento com DAV (3, 10 e 30mg/kg, i.p.) produziu uma diminuiÃÃo significativa (p<0,05) da atividade locomotora no mesmo teste, sem alterar a coordenaÃÃo motora dos animais no teste do rota rod. O tratamento com DAV (3,10 e 30mg/kg) reduziu a latÃncia e aumentou a duraÃÃo do sono induzido por pentobarbital sÃdico, assim como aumentou o tempo de imobilidade no teste da suspensÃo da cauda. A administraÃÃo de DAV nas doses de 3, 10 e 30mg/kg induziu catalepsia nos animais e a dose de 10mg/kg foi capaz de inibir a hiperlocomoÃÃo induzida por anfetamina (5 mg/kg). Estes dados sugerem que tanto o lupeol quanto a DAV possuem um potencial efeito gastroprotetor possivelmente relacionado a um mecanismo antioxidante, ao aumento de grupos NP-SH, com participaÃÃo do NO, das PGs, dos canais de K+ATP e do cÃlcio. A DAV demonstrou alteraÃÃes comportamentais que sugerem um efeito depressor do Sistema Nervoso Central com possÃvel envolvimento da dopamina. / The Lupeol triterpene and valoneic acid dilactone (VAD), two major chemical components isolated from Cenostigma macrophyllum Tul. (Leguminoseae) were evaluated in the experimental model gastric lesion induced by ethanol and in animal models of behavioral. Both lupeol (3, 10 and 30 mg/kg, p.o.), and VAD (3, 10 and 30 mg/kg, i.p.) afforded significant gastroprotection (p<0,05) against absolute ethanol-induced gastric lesions. In the mechanistic studies, lupeol (30mg/kg) and VAD (10mg/kg) demonstrated an antioxidant action by preventing the ethanol-evoked depletion of non-protein sulfhydryls (NP-SHs), the involvement of nitric oxide (NO), prostaglandins (PGs), and the ATP-dependent potassium and calcium channels. Also observed were the participation of &#61537;2-adrenoceptors but not the opioid receptors in the gastroprotective effect of these substances. Lupeol (30 mg/kg) as well as DAV (10mg/kg) effectively reduced the total acidity (p<0,05) in the stomach without altering the gastric secretory volume in pylorus-ligatet rat. While normal intestinal transit was unalttered by lupeol (3, 10 e 30 mg/kg). VAD (3, 10 e 30 mg/kg) significantly (p<0,05) reduced by a mechanism that do not involve either opioid or adrenergic receptors. In addition, VAD (10 mg/kg) was also able to inhibit significantly (p<0, 05) the intestinal transit promoted by castor oil in mice. In open field test, lupeol (3, 10 e 30mg/kg) failed to demonstrate no significant change in locomotor activity of animals. However VAD (3, 10 e 30mg/kg) showed significant diminution (p<0,05) of locomotor activity in this test, but did not affect the motor coordination in rota-rod test. Mice treated with VAD (3,10 and 30mg/kg) demonstrated reduced latency and increase duration of sleeping time induced by pentobarbital sodium and showed enhanced immobility time in tail-suspension test. VAD at the doses 3, 10 and 30 mg/kg induced catalepsy in animals and at 10 mg/kg, it could effectively counteract the hypermotility induced by amphetamine (5mg/kg). These results suggest that both lupeol and VAD can affored gastroprotection against ethanol induced gastric injuri primarily through anantioxidant mechanism by restoring glutathione (NS-PH). The study futher indicate the possible involviment of NO, PGs, KATP channel activationaswell as membrane calcium. Besides the gastroprotection, DAV evidenced depressant effects in behavioral tests, possibly involving monoaminergic or adenosinergic systems which need to be clarifield in a future study.

A presente tese relata o estudo químico de duas espécies vegetais pertencentes à família Asteraceae: Mikania sp. nov; e Wunderlichia crulsiana. Da espécie Mikania sp. nov. foram estudados os extratos diclorometânicos das folhas e dos galhos, tendo sido isolados e identificados sete ácidos diterpênicos; dos óleos voláteis das folhas e dos galhos foram identificadas vinte substâncias entre monoterpenos e sesquiterpenos. Da espécie Wunderlichia crulsiana foram estudados os extratos diclorometânicos das flores e dos galhos. Das flores foram isolados e identificados palmitatos e acetatos de triterpenoílas. Dos galhos foram isolados e identificados acetatos de triterpenoílas e triterpenonas e identificados por CG triterpenóis acetilados e palmitatos de triterpenoílas hidrolisados e posteriormente acetilados. Deste extrato foram também isolados e identificados três lactonas sesquiterpênicas e um sesquiterpeno. A identificação das substâncias foi realizada através de RMN de 1H, de 13C (BBD e DEPT 135º), CG/EM e co-injeção de padrões. Os extratos brutos de ambas as plantas apresentaram discreta atividade, quando submetidos a um ensaio antifúngico. As frações, contendo os triterpenóis, acetatos e palmitatos de triterpenoflas e as lactonas sesquiterpênicas, foram testadas quanto à atividade anti-infíamatória mostrando-se bastante ativas. Tanto os extratos brutos quanto as fraqões supracitadas não se mostraram ativos em um ensaio antitumoral. Estes resultados indicam a seletividade da atividade dos extratos e frações, possivelmente não contendo substâncias citotóxicas. / This work describes the chemical study of two plants which belong to the family Asteraceae: Mikania sp. nov. and Wunderlichia crulsiana. The dichlorometane extracts from leaves and stems of Mikania sp. nov. we studied and seven diterpenic acids were isolated and identified. The volatile oil from leaves and stems of this plant were also studied and twenty substances among monoterpenes and sesquiterpenes were identified. From Wunderlichia crulsiana we analysed the dichlorometane extracts from flowers and stems. From flowers we isolated and identified triterpenes esterified with palmitic acid and acetic acid. From stems we isolated and identified triterpenes esterified with acetic acid and 3-oxo-triterpenes. By GC, we identified hydroxylated triterpenes that were acetylated, and triterpenes esterified with palmitic acid that were hydrolysed and acetylated. From stems we also isolated and identified three sesquiterpene lactones and a sesquiterpene. The compounds were identified by PMR, CMR (BBD and DEIT 135º), GC/MS and co-injection with authentic samples The extracts of both plants showed low activity when submitted to bioassay with Cladosporium sphaerospermum. The fractions which contain the hydroxylated triterpenes, triterpenes esterified with acetic and palmitic acids and sesquiterpene lactones were submitted to anti-inflammatory bioassay and showed 42%, 29%, 29% and 47% of activity, respectively. The above mentioned extracts and the fractions did not show significant activity on the Saccharomyces cerevisae bioassay, so there were not citotoxic substances in them.

Made available in DSpace on 2015-05-14T12:59:35Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 1945153 bytes, checksum: 2c1abc7e6111c3fc75af32ff4400e496 (MD5) Previous issue date: 2012-02-17 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / From Xylopia langsdorfiana A. St-Hil. & Tul. stem bark was isolated the diterpene of trachylobane class, ent-7α-hydroxytrachyloban-18-oic acid (trachylobane-318) that in previous studies showed to be able to relax guinea-pig trachea pre-contracted by carbachol (CCh). Thus, we aimed to investigate the action mechanism underlying in this trachylobane-318 relaxant activity. Trachea rings were suspended in organ baths, containing Kreb s solution, at 37 ºC and aired with carbogenic mixture. Isometric contractions were registered using a digital acquisition system. In order to evaluate a direct effect of the diterpene in Ca2+-calmodulin complex was used chlorpromazine (CPZ) (10-6 M), a calmodulin inhibitor, and we observed that trachylobane-318 relaxation effect (pD2 = 4.38 ± 0.07, n = 5) was not significantly altered in presence of this inhibitor (pD2 = 4.25 ± 0.07, n = 5). Then, was performed a protocol using different potassium (K+) extracellular concentrations which indicated that trachylobane-318 would be acting as a possible potassium channels activator since its relaxation was more potent when guinea-pig trachea was pre-contracted by KCl 18 mM (pD2 = 4.90 ± 0.25, n = 5) than by KCl 60 mM (pD2 = 3.88 ± 0.01, n = 5). To confirm the potassium channels participation was used a non-selective potassium channels blocker, tetraethylammonium (TEA+) 10 mM, that was pre-incubated before CCh addition, that resulted in an attenuation of diterpene relaxation (pD2 = 4.01 ± 0.06, n = 5). To determinate which potassium channels subtypes would be involved in the trachylobane-318 action, the diterpene relaxation curve was assessed in the presence of several potassium channels selective blockers. The fact of the trachylobane-318 relaxation curve was shifted to the right in a significant manner in the presence of 4-AP, a selective blocker of voltage activated K+ channels (Kv) (pD2 = 4.00 ± 0.06, n = 5); glibenclamide, a selective blocker of ATP-sensitive K+ channel (KATP) (pD2 = 3.91 ± 0.003, n = 5); apamin, a selective blocker of small conductance calcium-activated K+ channels (SKCa) (3.45 ± 0.14, n = 5) and big conductance calcium-activated K+ channels (BKCa) (3,80 ± 0,05, n = 5) is suggestive that the diterpene is modulating positively these channels to exert its relaxant effect. On the other hand, the inwardly rectifying K+ channels (Kir) was discarded since the relaxation curve was not altered (pD2 = 4.15 ± 0.10, n = 5) in the presence of BaCl2, selective blocker of these channels. Cyclic nucleotides participation was discarded since the relaxation curve obtained with aminophylline on guinea-pig contracted by CCh (pD2 = 4.27 ± 0.09, n = 5), a phosphodiesterases (PDEs) non-selective inhibitor, was not altered in trachylobane-318 presence (pD2 = 4.46 ± 0.08, n = 5). Thus, trachylobane-318 relaxant effect seems to involve the positive modulation of potassium channels subtypes Kv, KATP, SKCa and BKCa on guinea pig trachea. / A partir das cascas do caule de Xylopia langsdorfiana A. St-Hil. & Tul. foi isolado o diterpeno da classe dos traquilobanos, ent-7α-hidroxitraquiloban-18-oico (traquilobano-318) que em estudos anteriores mostrou-se capaz de relaxar a traqueia de cobaia pré-contraída com carbacol (CCh). Assim, o objetivo deste trabalho foi investigar o mecanismo de ação envolvido nesta atividade relaxante do traquilobano-318. Os anéis de traqueia foram suspensos em cubas para órgão isolado, contendo solução de Krebs, a 37º C e aerados com carbogênio. As contrações isométricas foram registradas com o auxílio de um sistema de aquisição digital. Para avaliar uma possível ação direta do diterpeno sobre o complexo Ca2+-calmodulina foi utilizado a clorpromazina (CPZ) (10-6 M), um inibidor da calmodulina, e observou-se que o efeito relaxante do traquilobano-318 (pD2 = 4,38 ± 0,07, n = 5) não foi significantemente alterado na presença deste inibidor (pD2 = 4,25 ± 0,07, n = 5). Em seguida foi realizado um protocolo usando diferentes concentrações extracelulares de potássio (K+) que indicaram que o traquilobano-318 estaria agindo como um possível ativador dos canais de K+, uma vez que seu efeito relaxante mostrou-se mais potente quando a traqueia era pré-contraída por 18 mM de KCl (pD2 = 4,90 ± 0,25, n = 5) do que quando pré-contraída por 60 mM de KCl (pD2 = 3,88 ± 0,01, n = 5). Para confirmar a participação dos canais de K+ utilizou-se um bloqueador não seletivo destes canais, tetraetilamônio (TEA+) (10 mM) que foi pré-incubado antes da adição de CCh, o que resultou na atenuação do relaxamento promovido pelo diterpeno (pD2 = 4,01 ± 0,06, n = 5). Com o objetivo de se determinar quais subtipos de canais de potássio estariam envolvidos na ação relaxante do traquilobano-318, a curva de relaxamento do diterpeno foi avaliada na presença de vários bloqueadores seletivos destes canais. O fato da curva de relaxamento do traquilobano-318 (pD2 = 4,38 ± 0,07) ter sido desviada para direita, de maneira significante, na presença de 4-AP, bloqueador dos canais de K+ sensíveis a voltagem (Kv) (pD2 = 3,91 ± 0,003); glibenclamida, bloqueador dos canais de K+ sensíveis ao ATP (KATP) (pD2 = 4,00 ± 0,06); apamina, bloqueador dos canais de K+ ativados pelo cálcio de pequena condutância (SKCa) (pD2 = 3,45 ± 0,14) e na presença de iberiotoxina, bloqueador dos canais de K+ ativados pelo cálcio de grande condutância (BKCa) (pD2 = 3,80 ± 0,05) sugere que o diterpeno está modulando positivamente estes canais para exercer seu efeito relaxante. Por outro lado a participação dos canais de potássio retificadores de entrada (Kir) foi descartada pois a curva de relaxamento do traquilobano-318 não foi alterada (pD2 = 4,15 ± 0,10, n = 5) na presença do BaCl2, bloqueador seletivos destes canais. A participação dos nucleotídios cíclicos foi descartada, uma vez que a curva de relaxamento em traqueia pré-contraída por CCh obtida com aminofilina (pD2 = 4,27 ± 0,09, n = 5), um inibidor não seletivo das fosfodiesterases (PDEs), não foi alterada na presença do traquilobano-318 (pD2 = 4,46 ± 0,08, n = 5). Assim, o efeito relaxante do traquilobano-318 parece envolver a modulação positiva dos subtipos de canais de potássio KATP, Kv, SKCa e BKCa em traqueia isolada de cobaia.

Submitted by Cristhiane Guerra (cristhiane.guerra@gmail.com) on 2017-02-03T16:05:21Z No. of bitstreams: 1 arquivototal.pdf: 6455028 bytes, checksum: ad59284d305f31e21541e47aaeb18f12 (MD5) / Made available in DSpace on 2017-02-03T16:05:21Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 6455028 bytes, checksum: ad59284d305f31e21541e47aaeb18f12 (MD5) Previous issue date: 2015-02-23 / Phytochemical studies with Xylopia langsdorffiana (Annonaceae), have described isolation of trachylobane, atisane, kaurane, and labdane diterpenes, from the leaves, fruits, stems and roots of the species. Maytenus distichophylla (Celastraceae), popularly known as espinheira santa – (sacred thorn bush), is used in folk medicine to treat stomach ulcers, and data from the literature report the isolation of triterpenoids friedelane, and pyrimidine sesquiterpene alkaloids. Computer aided drug design brings optimizing techniques that facilitate the discovery of new drugs, offering features that allow the connection of compounds to known proteins in three dimensions, and the resulting models are able to predict reliable experimental values for biologically active molecules, making the search for biologically active compounds more efficient. Our work associated experimental tests and computational investigations based on plants selected from the Paraiban flora: X. langsdorffiana, M. distichophylla, and other Maytenus species. We report the experimental tests performed with roots and stems of Maytenus distichophylla that enabled extraction and structural determination of flavonoids, steroids, and friedelane and lupane triterpenoids. Parallel studies were developed using computational docking, PLS, and hydrophobicity, with 15 triterpenoids isolated from Maytenus species (including M. distichophylla), and 17 diterpenes isolated from X. langsdorffiana. Further, we determined chemical compositions (by GC-MS and NMR), and modulation activity against multi-resistant bacteria. Essential oils of three X. langsdorffiana specimens were analyzed by Principal Component Analysis (PCA). Three compounds were isolated from stems of M. distichophylla by chromatographic techniques: 3-oxo-12α-hydroxyfriedelane, 30- hydroxyfriedelan-3one and 29-hydroxyfriedelan-3-one. Another four compounds were isolated from the roots: β-sitosterol, 11α-hydroxyglochidone, rigidenol and 4’-Omethylepigallocatechin, this being the first report in the species of the latter three. In the docking study, the diterpenes ent-atisan-7α-acetoxy-16α-ol, labdorfianic C acid e labdorfianic B acid had the lowest formation energy values when respectively complexed with cruzaine, CYP2C9, and COX. The triterpenoids with the lowest formation energy values when complexed with these enzymes were, 3β-hydroxy-9,12-en-ursane (cruzaine), 3,4-secofriedelan- 3-oic acid (CYP2C9) and 3-oxo-12α-hydroxyfriedelane (COX). In predicting antitumor activity, as calculated from the constructed PLS model, the diterpene labdorfianic C acid, and triterpene 3β-hydroxyfriedelane, showed the best predicted values. The majority compounds identified in the essential oils were: ent-atisan-16α-ol, germacrene D, limonene and β-pinene. The monoterpenes, β-pinene and limonene, were identified in all analyzed essential oils in different percentages. Through analysis of the essential oil of the X. langsdorffiana stem (specimen A), NMR (¹H and ¹³C) were able to identify ent-atisan-16α-ol, the first time reported for this species. Some essential oils from specimen A when modulated with oxacillin against S. aureus, decreased concentration of the antibiotic by 99%. Principal component analysis (PCA) performed with the essential oil of three X. langsdorffiana specimens allowed identifying a similarity between the chemical species A and B, and a difference in C compared to A and/or B. This result may be related to the age or size of the specimens, since they share the same geographic coordinates. The models obtained in the CADD studies of were satisfactory and generated predictive results. / Estudos fitoquímico com Xylopia langsdorffiana (Annonaceae), descrevem o isolamento de diterpenos do tipo traquilobano, atisano, caurano e labdano, das folhas, frutos, caule e raízes da espécie. Maytenus distichophylla (Celastraceae), conhecida popularmente como espinheira santa, é utilizada na medicina popular para o tratamento de ulceras estomacais, e dados da literatura relatam o isolamento de triterpenos do tipo friedelano e alcaloides sesquiterpênicos pirimidínicos. O planejamento de fármacos auxiliado por computador, vem otimizando técnicas que facilitam na descoberta de novos fármacos, oferecendo recursos que permitem o acoplamento de compostos à proteínas tridimensionalmente conhecidas e originando modelos capazes de predizer valores experimentais confiáveis para moléculas biologicamente ativas, tornando a busca por compostos biologicamente ativos mais eficiente. Este trabalho associou ensaios experimentais e investigações computacionais baseadas nas plantas extraídas da flora Paraibana: X. langsdorffiana, M. distichophylla, e outras espécies do gênero Maytenus. Reportaremos os testes experimentais realizados com raízes e caule de Maytenus distichophylla que possibilitaram a extração e determinação estrutural de flavonoide, esteroide e, triterpenos do tipo friedelano e lupano. Paralelamente realizamos estudos computacionais de docking, PLS e hidrofobicidade, com 15 triterpenos isolados de espécies do gênero Maytenus (incluindo M. distichophylla) e, 17 diterpenos isolados de X. langsdorffiana. Além disso, determinamos a composição química (por CG-EM e RMN), e atividade moduladora frente a bactérias multirresistentes. Os óleos essenciais das 3 espécimes de X. langsdorffiana foram analisadas pela Análise de Componente Principal (PCA). Três compostos foram isolados do caule de M. distichophylla por técnicas cromatográficas: 3-oxo-12α-hidroxifriedelano, 30-hidroxifriedelan- 3ona e 29-hidroxifriedelan-3-ona. Outros quatro compostos foram isolados das raízes: β- sitosterol, 11α-hidroxigloquidona, rigidenol e 4’-O-metilepigalocatequina, sendo os três últimos relatados pela primeira vez na espécie. No estudo de docking, os diterpenos ent-atisan- 7α-acetoxi-16α-ol, ácido labdorfiânico C e ácido labdorfiânico B apresentaram os menores valores de energia de formação quando complexados com a cruzaína, CYP2C9 e COX, respectivamente. Os triterpenos que apresentaram os menores valores de energia de formação complexados com essas enzimas foram, 3β-hidroxi-9,12-en-ursano (cruzaína), ácido 3,4-secofriedelan- 3-óico (CYP2C9) e 3-oxo-12α-hidroxifriedelano (COX). Na predição da atividade antitumoral, calculada a partir do modelo PLS construído, o diterpeno ácido labdorfiânico C, e o triterpeno 3β-hidroxifriedelano, apresentaram os melhores valores preditos. Os compostos majoritários identificados nos óleos essenciais foram: ent-atisan-16α-ol, germacreno D, limoneno e β-pineno. Os monoterpenos, β-pineno e limoneno, foram identificados em todos os óleos essenciais analisados, com diferentes porcentagens. Através da análise do óleo essencial do caule (espécime A) de X. Langsdorffiana, por RMN (¹H e ¹³C) foi possível identificar o entatisan- 16α-ol, relatado pela primeira vez na espécie. Alguns óleos essenciais do espécime A, quando modulados com oxacilina frente a S. aureus, diminuíram a concentração do antibiótico em 99%. A análise de componente principal (PCA) realizada com o óleo essencial de três espécimes de X. langsdorffiana, permitiu identificar uma similaridade química entre os espécimes A e B, e uma diferença em C, quando comparados com A e/ou B. Este resultado pode estar relacionado a idade ou porte dos espécimes, já que os mesmos se encontram na mesma coordenada geográfica. Os modelos obtidos nos estudos de CADD foram satisfatórios e geraram resultados preditivos.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / O diterpeno, lactona do Ãcido hawtriwaico (LAHT) isolado dos capÃtulos florais de Egletes viscosa Less. (Asteraceae) foi avaliado nos modelos de edema de orelha e nocicepÃÃo induzida por capsaicina em camundongos. A LAHT (12,5; 25 e 50 mg/kg, v.o.) atenuou significativamente a resposta ao edema de orelha induzido pela aplicaÃÃo tÃpica de capsaicina (250 Âg), de maneira dose dependente (45,7; 86,9 e 100 % respectivamente). A resposta ao edema de orelha induzido pela capsaicina foi tambÃm significativamente inibida em 74,8 % pelo vermelho de rutÃnio (VR; 3mg/kg, s.c.), um antagonista nÃo competitivo do receptor da capsaicina (TRPV1). A LAHT (50 mg/kg, v.o.) nÃo modificou a resposta ao edema de pata induzido por composto 48/80 (10 Âg), histamina (10 Âg) ou serotonina (10 Âg), demonstrando que a LAHT nÃo bloqueia a desgranulaÃÃo de cÃlulas mastocitÃrias ou os receptores de histamina e serotonina. No entanto, o edema de pata induzido por substÃncia P (SP) foi significativamente suprimido pela LAHT (25 e 50 mg/kg, v.o.), em 29,4 e 53,3 % respectivamente, bem como o aumento da permeabilidade capilar induzido pela injeÃÃo intraperitoneal de Ãcido acÃtico. No modelo de nocicepÃÃo, a LAHT (12,5; 25 e 50 mg/kg, v.o.) suprimiu de forma significativa o comportamento nociceptivo de lamber a pata induzido pela injeÃÃo intraplantar de 1,6 Âg de capsaicina (27,1; 32,3 e 52 % respectivamente). A resposta à nocicepÃÃo induzida pela capsaicina foi significativamente inibida pelo VR (3 mg/kg, s.c.) em 64,9 %. O efeito antinociceptivo da LAHT (50 mg/kg, v.o.) nÃo foi afetado pelo prÃ-tratamento por naloxona, mas foi significativamente antagonizado pela teofilina e glibenclamida, respectivos bloqueadores de adenosina e canais de KATP dependentes. A LAHT (50 mg/kg, v.o.) nÃo alterou o tempo de sono, nÃo prejudicou a atividade locomotora e nÃo alterou a coordenaÃÃo motora dos camundongos como evidenciado nos testes do tempo de sono induzido por pentobarbital, do campo aberto e rota-rod respectivamente. Esses dados sugerem que a LAHT inibe a inflamaÃÃo neurogÃnica aguda, possivelmente pela inibiÃÃo da liberaÃÃo de SP ou bloqueio de seus receptores, e a nocicepÃÃo possivelmente pelo envolvimento de adenosina endÃgena e canais de KATP dependentes / The diterpene, 12-acetoxy-hawtriwaic acid lactone (AHAL) isolated from the flower buds of Egletes viscosa Less. (Asteraceae), a popular medicinal plant largely encountered in Cearà State was evaluated in mice for its anti-inflammatory and antinociceptive potential, using capsaicin-induced ear edema and hindpaw nociception as experimental models. AHAL (12.5, 25 and 50 mg/kg, p.o.) significantly attenuated the ear edema response to topically applied capsaicin (250Âg), in a dose-related manner (45.7, 86.9 and 100 % respectively). This response to capsaicin was also greatly inhibited by ruthenium red (3 mg/kg, s.c.), a non-competitive capsaicin receptor (TRPV1) antagonist by 74.8%. The anti-edema effect of AHAL (50 mg/kg, p.o.) seems unrelated either to inhibition of mast cell degranulation or to antagonism at histamine and serotonin receptor since AHAL did not modify the paw edema response-induced by intraplantar injections of compound 48/80 (10 Âg), histamine (10 Âg) or serotonin (10 Âg). However the substance P (SP) induced hindpaw edema was significantily inhibited by AHAL (25 and 50 mg/kg, p.o.) by 29.4 and 53.3 % respectively, as well as the increase in capillary permeability induced by intra-peritoneal acetic acid. In the hindpaw nociceptive model, AHAL suppressed the nocifensive paw-licking behavior induced by intraplantar injection of capsaicin (1.6 Âg). This response to capsaicin was also greatly inhibited by ruthenium red (3 mg/kg, s.c.) by 64.9 %. The antinociceptive effect of AHAL (50 mg/kg, p.o.) was unaffected by naloxone pre-treatment but was significantly antagonized by theophylline and glibenclamide, the respective blockers of adenosine receptor and KATP-sensitive channels. AHAL (50 mg/kg, p.o.) did not impair the ambulation or motor coordination of mice in open-field and rota-rod tests. These data allow us to conclude that AHAL possesses anti-inflammatory and antinociceptive properties. It possibly inhibits acute neurogenic inflammation by the inhibition of SP release or its receptor blockade and the nociception through mechanisms that involve the endogenous adenosine and opening of ATP-sensitive potassium channels