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.

Este trabalho descreve o estudo quÃmico de Croton muscicarpa (Euphorbiaceae), planta nativa do Nordeste do Brasil, popularmente conhecida como âvelame-de-cheiroâ. A espÃcie foi coletada na Chapada Diamantina no estado da Bahia, e sua investigaÃÃo quÃmica foi realizada atravÃs do isolamento e caracterizaÃÃo de metabÃlitos secundÃrios presentes nos extratos etanÃlico dos talos e hexÃnico das raÃzes. Foram isolados dos talos os sesquiterpenos, espatulenol e 6-metÃxi-cipereno, este Ãltimo de descriÃÃo inÃdita na literatura. Das raÃzes foram isolados o triterpeno, Ãcido acetil aleuritÃlico (AAA) e uma polimetoxiflavona, a 5-hidroxi-3,6,7,3â,4â-pentametoxiflavona (artemetina). O isolamento dos constituintes quÃmicos foi realizado por mÃtodos cromatogrÃficos convencionais e a determinaÃÃo estrutural das substÃncias isoladas realizada atravÃs do uso de tÃcnicas espectromÃtricas como: Infravermelho (IV), Espectrometria de Massa (EM) e RessonÃncia MagnÃtica Nuclear de HidrogÃnio (RMN 1H) e Carbono-13 (RMN 13C), incluindo tÃcnicas bidimensionais (COSY, HSQC, HMBC e NOESY) e comparaÃÃo com os dados descritos na literatura. / Este trabalho descreve o estudo quÃmico de Croton muscicarpa (Euphorbiaceae), planta nativa do Nordeste do Brasil, popularmente conhecida como âvelame-de-cheiroâ. A espÃcie foi coletada na Chapada Diamantina no estado da Bahia, e sua investigaÃÃo quÃmica foi realizada atravÃs do isolamento e caracterizaÃÃo de metabÃlitos secundÃrios presentes nos extratos etanÃlico dos talos e hexÃnico das raÃzes. Foram isolados dos talos os sesquiterpenos, espatulenol e 6-metÃxi-cipereno, este Ãltimo de descriÃÃo inÃdita na literatura. Das raÃzes foram isolados o triterpeno, Ãcido acetil aleuritÃlico (AAA) e uma polimetoxiflavona, a 5-hidroxi-3,6,7,3â,4â-pentametoxiflavona (artemetina). O isolamento dos constituintes quÃmicos foi realizado por mÃtodos cromatogrÃficos convencionais e a determinaÃÃo estrutural das substÃncias isoladas realizada atravÃs do uso de tÃcnicas espectromÃtricas como: Infravermelho (IV), Espectrometria de Massa (EM) e RessonÃncia MagnÃtica Nuclear de HidrogÃnio (RMN 1H) e Carbono-13 (RMN 13C), incluindo tÃcnicas bidimensionais (COSY, HSQC, HMBC e NOESY) e comparaÃÃo com os dados descritos na literatura.

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.

B. braunii is a green alga that has attracted attention as a potential renewable fuel source due to its high oil content and the archeological record of its unique contribution to oil and coal shales. Three extant chemotypes of B. braunii have been described, namely race A, race B, and race L, which accumulate alkadienes and alkatrienes, botryococcene and squalene and their methylated derivatives, and lycopadiene, respectively. The methylated triterpenes, particularly botryococcenes, produced by race B can be efficiently converted to high quality combustible fuels and other petrochemicals; however, botryococcene biosynthesis has remained enigmatic. It has been suggested that botryococcene biosynthesis could resemble that of squalene, arising from an initial condensation of two molecules of farnesyl diphosphate (FPP) to form pre-squalene diphosphate (PSPP), which then undergoes a reductive rearrangement to form squalene, or in an alternative reductive rearrangement, botryococcene. Based on the proposed similarities, we predicted that a botryococcene synthase would resemble squalene synthase and hence, isolated squalene synthase-like genes from B. braunii race B. While B. braunii does harbor at least one typical squalene synthase, none of the other three squalene synthase-like (SSL) genes encode for botryococcene biosynthesis directly. SSL-1 catalyzes the biosynthesis of PSPP and SSL-2 the biosynthesis of bisfarnesyl ether and to a lesser extent squalene, while SSL-3 does not appear able to directly utilize FPP as a substrate. However, when SSL-1 is combined with either SSL-2 or SSL-3, in vivo and in vitro, robust squalene or botryococcene biosynthesis was observed, respectively. These findings were unexpected because squalene synthase, an ancient and likely progenitor to the other Botryococcus triterpene synthases, catalyzes a two-step reaction within a single enzyme unit without intermediate release, yet in B. braunii, these activities appear to have separated and evolved inter-dependently for specialized triterpene production. Expression of various configurations of the SSL genes in TN-7 yeast demonstrates that botryococcene can be efficiently produced in a heterologous host. Additionally, three triterpene methyltransferase (TMTs) were isolated which efficiently catalyze the transfer of a methyl group from S-adenosyl methionine (SAM) to either squalene (TMT-1 and TMT-2) or botryococcene (TMT-3) in vivo and in vitro. Co-expression of the various TMT genes with either squalene synthase or botryococcene synthase in TN-7 yeast resulted in the accumulation of C31 and C32 methyl derivatives of squalene or botryococcene, demonstrating their potential for heterologous production. The methylation sites were determined by NMR spectroscopy to be identical to C31 and C32 methyl-derivatives of squalene or botryococcene observed in B. braunii race B. Expression studies of various heterologous squalene synthase genes in S. cerevisiae corroborated an earlier but surprising observation reported in the literature. While the squalene synthase gene of S. cerevisiae was able to complement an erg9 (squalene synthase) knockout in yeast, squalene synthase genes from plants and animals were not. Chemical profiles revealed that squalene accumulated to significant levels in yeast expressing the squalene synthase of plant, animal, or S. cerevisiae. This suggested that it was not the ability of these heterologous synthase enzymes to produce squalene, but their inability to feed squalene into the native sterol biosynthetic pathway that prevented them from restoring normal ergosterol biosynthesis in S. cerevisiae. By examining the ability of chimera squalene synthase enzymes to complement the erg9 mutation, a discrete sequence of amino acids near the C-terminus of the enzyme was identified which is necessary and sufficient for allowing any squalene synthase to restore normal sterol metabolism.

The chemical diversity and biological activities of terpene and terpenoids have served in the development of new flavors, fragrances, medicines and pesticides. While terpenes are made predominantly by plants and microbes in small amounts and as components of complex mixtures, chemical synthesis of terpenes remains technically challenging, costly and inefficient. In this dissertation, methods to create new yeast lines possessing a dispensable mevalonate biosynthetic pathway wherein carbon flux can be diverted to build any chemical class of terpene product are described. The ability of this line to generate diterpenes was next investigated. Using a 5.5 L fed bath fermentation system, about 569 mg/L kaurene and approximately 207 mg/L abietadiene plus 136 mg/L additional isomers were achieved. To engineer more highly modified diterpenes might have greater industrial, agricultural or medicinal applications, kaurenoic acid production reached 514 mg/L with byproduct kaurene and kaurenal at 71.7mg/L and 20.1mg/L, respectively, in fed batch fermentation conditions. Furthermore, ZXM lines for engineer monoterpene and ZXB lines for engineer triterpene were generated by additional specific genomic modification, 84.76 ±13.2 mg/L linalool, 20.54±3.8 mg/L nerolidol and 297.7mg/L squalene were accumulate in ZXM144 line ana ZXB line, respectively, in shake flask conditions.

Terpenes comprise a large diverse class of natural products and many of them attract interest because of their physiological function, therapeutic and industrial values. Triterpene oils including squalene (C30), botrycococcene (C30) and their methylated derivatives (C31-C37) generated by the green algae Botryococcus braunii race B, which have recently received significant attention because of their utility for advanced biofuels. However, the slow growth habit of B. braunii makes it impractical as a robust biofuel production system. In this thesis, we firstly evaluated the potential of generating high levels of triterpene (C30) production in tobacco plants by diverting carbon flux from cytosolic MVA pathway or plastidic MEP pathway by overexpressing avian farnesyl diphosphate synthase along with triterpene synthase targeted to the cytoplasm or the chloroplast of cells. Up to 1,000 µg/g fresh weight of squalene and 544 µg/g fresh weight of botryococcene was achieved in our transgenic plants with this metabolism direct to the chloroplasts, which is about approximately 100-times greater than that accumulating in the plants engineered for cytosolic production. To test if methylated triterpenes can be produced in tobacco, we also engineered triterpene methyltransferases (TMTs) into wild type plants and transgenic tobacco plants selected for high level triterpene accumulation. We observed that up to 91% of the total triterpene content was converted to methylated forms (C31, C32) by targeting the TMTs to the chloroplasts of transgenic plants, whereas only 4-14% of total triterpenes were methylated when TMTs were directed to the cytoplasm. Select transgenic lines were growing in field studies from 2011 to 2014 to evaluate their physiological performance under field conditions. Surprisingly, the field studies suggested that the growth and agronomic performance of the transgenic lines accumulating squalene were not compromised, while those accumulating high levels of botryococcene were only 72%-76% as tall, had about 59%-75% of the leaf area, and about 55%-75% of the biomass as wild type plants. Yet, these transgenic plants had photosynthetic capacity equal to the wild type plants.

CoordenaÃÃo de AperfeÃoamento de Pessoal de NÃvel Superior / The number of cases of morbidity and mortality in patients with chronic wounds has increasing every year. In this sense, wound healing has become one the major dermatological problems. Secondary metabolites from plants have been used in several assays as promising molecules on various diseases attracted importance on treatment of various diseases. However, there are few data about it effect on wound healing. Combretum leprosum Mart. is a native species of the Caatinga and its ethanolic extract is commonly used as a healing agent, as a sedative and treatment of haemorrhages. A of the aim of the study was to evaluate the effect of triterpene 3β, 6β, 16β-trihydroxylup-20(29)-ene (CLF-1) isolated from ethanolic extract from the leaves of Combretum leprosum on skin wounds induced in vitro and in vivo lesions was avaluated. In vitro assay, the CLF-1 (2.5 μg/mL) not showed toxicity to human dermal fibroblasts (HDF). Moreover, the CLF-1 induced fibroblasts migration to the healing of the artificial injury with most effectiveness than control at 24h. In addition, the molecular mechanism of the CLF-1 treatment was studied. The results suggest that migration of fibroblasts occurs by upregulated expression of TGF-β1 and reduced levels of the inflammatory cytokine TNF-α. In the in vivo study, experimental skin lesions were created in the back of mice and treatment with the ethanolic extract from C. leprosum (EECL) and CLF-1 was evaluated for 12 days. The treatment with EECL and CLF-1 induced a faster and more effective epithelialization when compared to control. The histopathological assessment showed that EECL and CLF-1 has similar profiles during the regenerative process. This result suggest that probably the active component in the EECL may be CLF-1. Beyond of the spend about the wound healing chronic diseases as cancer are estimated as one of the main causes of dead and one of the main spending factors to the health systems. The conventional treatments, such as surgery, chemotherapy and radio are effective against primary tumors, hence not have the same effect in the later stages of the disease. In this sense, several biomolecules has attracted interest from the researchers because its antitumor potential. Marine sponjes are a biological reservoir of biomolecules, especially lectins. Lectins are proteins that bind reversibel way to carbohydrate epitops without modified them. Unlike plant lectins, there are few reports describing the mechanism of action of lectins from marine sponges to induce apoptosis in tumor cells. The effect of a lectin isolated from the marine sponge Haliclona caerulea (H3) on human breast cancer MCF-7 cell line was evaluated. H3 caused MCF7 cell reduction of viability in more than 50% (IC50=100 μg/ml) at 6h, 24h and 48h. However, on normal cells, the treatment with H3 induced a reduction in over 50% only at the highest dose tested (500 μg/ml). Furthermore, H3 provoke arrest in the G1 cell cycle phase and induce MCF7 cells apoptosis in 24h and 48h. Lysotracker Red and real-time qPCR assays suggest that effect of H3 may be related to a dynamic balance between apoptosis and autophagic cell death mediated by increase of expression of caspase-9 and LC3. In conclusion, the results suggest that CLF-1 and H3 lectin may be promising biomolecules to treatment of acute and chronic wounds and rare diseases like cancers. / O nÃmero de casos de morbimortalidade em pacientes com feridas crÃnicas vem aumentando a cada ano. Nesse sentido, a cicatrizaÃÃo de feridas tem se tornado um dos principais problemas dermatolÃgicos. MetabÃlitos secundÃrios de plantas tem sido utilizados em vÃrios estudos como molÃculas promissoras no tratamento de vÃrias patologias. Entretanto, sÃo poucos os dados sobre seu efeito na cicatrizaÃÃo de feridas. Combretum leprosum Mart. à uma planta nativa da Caatinga e seu extrato etanÃlico à bastante utilizado na medicina popular para cicatrizaÃÃo de feridas, como sedativo ou tratamento de hemorragias. Um dos objetivos do presente trabalho foi avaliar o efeito do triterpeno 3β, 6β, 16β-trihydroxylup-20 (29)-ene (CLF-1) isolado do extrato etanÃlico de folhas de Combretum leprosum sobre lesÃes induzidas in vitro e in vivo. No ensaio in vitro, o CLF-1 (2,5 μg/mL) nÃo apresentou toxicidade a fibroblastos dermais humanos (HDF). AlÃm disso, o CLF-1 induziu a migraÃÃo de fibroblastos para o fechamento da lesÃo artificial de maneira mais efetiva, comparado ao controle, em 24h. AlÃm disso, o mecanismo molecular do efeito do CLF-1 foi estudado. Os resultados sugerem que a migraÃÃo de fibroblastos pode ocorrer pelo aumento da expressÃo de TGF-β1 e reduÃÃo nos nÃveis da citocina inflamatÃria TNF-α. No estudo in vivo, lesÃes experimentais foram induzidas na regiÃo dorsal de camundongos e o tratamento com o extrato etanÃlico de C. leprosum (EECL) e CLF-1 foi avaliado por um periodo de 12 dias. Os tratamentos com EECL e CLF-1 induziram uma reepitelizaÃÃo mais rÃpida e efetiva em comparaÃÃo ao controle. O estudo histopatolÃgico mostrou que EECL e CLF-1 apresentam perfis similares durante o processo regenerativo. Esses resultados sugerem que, provavelmente o componente ativo no EECL possa ser o CLF-1. AlÃm dos elevados custos relacionados a cicatrizaÃÃo de feridas, patologias crÃnicas como o cÃncer sÃo estimados como uma das principais causas de morte e um dos principais fatores dispendiosos para os sistemas de saÃde. Os tratamentos convencionais como cirurgia, quimioterapia e radioterapia sÃo efetivos contra tumores primÃrios, contudo, nÃo possuem o mesmo efeito nos estÃgios mais avanÃados da doenÃa. Nesse sentido, muitas biomolÃculas tem atraÃdo interesse da comunidade cientÃfica devido ao seu potencial antitumoral. Esponjas marinhas sÃo consideradas reservas biolÃgicas de vÃrias biomolÃculas, sobretudo lectinas. Lectinas sÃo proteÃnas que se ligam a carboidratos de maneira reversÃvel sem alterar sua estrutura. De modo diferente como relaÃÃo a lectinas de plantas, hà poucos relatos na literatura descrevendo o mecanismo de aÃÃo de lectinas de esponjas sobre a induÃÃo de apoptose de cÃlulas tumorais. O efeito antitumoral de uma lectina isolada da esponja marinha Haliclona caerulea (H3) sobre cÃlulas do adenocarcinoma de mama humano MCF7 foi avaliado. H3 induziu a reduÃÃo da viabilidade celular de MCF7 em mais de 50% (IC50=100 μg/mL) em 6h, 24h e 48h. Contudo, sobre cÃlulas normais, o tratamento com H3 induziu a reduÃÃo em mais de 50%, apenas na maior dose testada (500 μg/mL). AlÃm disso, H3 provoca arraste no ciclo celular na fase G1 e induz apoptose das cÃlulas MCF7 em 24 e 48h. Ensaios utilizando Lysotracker Red e PCR em tempo-real sugerem que o efeito de H3 pode estar relacionado a um balanÃo dinÃmico entre apoptose e autofagia, mediados pelo aumento da expressÃo de caspase-9 e LC3. Em conclusÃo, os resultados sugerem que o CLF-1 e H3 podem ser biomolÃculas promissoras para o tratamento de feridas agudas e crÃnicas e enfermidades como cÃncer.

FundaÃÃo de Amparo à Pesquisa do Estado do Amazonas / Os efeitos dos triterpenos pentaciclicos -amirina e -amirina, isolados a partir da resina de Protium heptaphyllum March. (Burseraceae), foram testados preliminarmente em modelos de nocicepÃÃo oral, sendo que -amirina apresentou significantes efeitos antinociceptivos, norteando a pesquisa com este isolado na investigaÃÃo de seus efeitos em modelos de dor orofacial induzida por capsaicina ou formalina e na dor induzida por capsaicina na cÃrnea de camundongos; na dor tÃrmica (testes de imersÃo de cauda em Ãgua quente e placa quente); e na nocicepÃÃo visceral induzida por Ãcido acÃtico 0,6%. Camundongos Swiss machos (n = 8 / grupo) foram prÃ-tratados com β-amirina (10, 30 e 100 mg / kg, v.o.), morfina (5 mg / kg, s.c.) ou controle (Ãgua destilada + 0,05% de Tween 80, v.o.), uma hora antes de capsaicina (20 L, 1,5 g) ou formalina (20L/animal) serem administradas na vibrissa direita. β-amirina tambÃm foi avaliada em teste comportamental relacionado à dor, desta vez por aplicaÃÃo tÃpica de capsaicina na conjuntiva do camundongo (âeye wiping testâ). Neste teste foi medido o tempo, em segundos, que o animal passou âlimpandoâ o olho durante um perÃodo de 10 minutos. O triterpenÃide demonstrou principalmente um efeito antinociceptivo dose-independente em todos os modelos de nocicepÃÃo testados. Na dor orofacial induzida por capsaicina, -amirina (30 e 100 mg/kg) e morfina foram mais eficazes na reduÃÃo da resposta nociceptiva. Nestas doses, as reduÃÃes foram de 81 e 90% para -amirina e 97% para morfina, respectivamente. No modelo de dor orofacial, a nocicepÃÃo produzida pela capsaicina à acompanhada por um aumento na resposta tÃrmica localizada (que foi mensurada por termometria), e reduzida significantemente pelo prÃ-tratamento dos animais com -amirina ou L-NAME, um inibidor da NOS. Em animais diabÃticos, a capsaicina injetada na vibrissa promoveu um menor grau de nocicepÃÃo orofacial comparada com os nÃo-diabÃticos. No teste da formalina, morfina e β-amirina apresentaram antinocicepÃÃo significativa reversÃvel nas duas fases por naloxona. No entanto, β-amirina (30 mg/kg) inibiu a segunda fase com maior eficiÃncia. Os valores de DE50 para β-amirina e morfina foram 16,44 mg/kg (LC 10,0-38,41) e 3 mg/kg (LC 2,5-5,0) na primeira fase e 43,37 mg/kg (LC 30,52-39,30) e 3 mg/kg (LC 2,5-5,0) na segunda fase, respectivamente. A co-administraÃÃo de β-amirina e morfina, em seus respectivos nÃveis de dose de DE50, nÃo apresentou qualquer efeito aditivo ou potencializador antinociceptivo. No entanto, as combinaÃÃes das doses DE25 e DE12,5 apresentaram uma antinocicepÃÃo comparÃvel ao efeito combinado da DE50, sugerindo que atravÃs da utilizaÃÃo de β-amirina, a dose analgÃsica de morfina poderia ser minimizada para evitar a sua alta dose e os efeitos colaterais associados. β-amirina tambÃm foi eficaz em aumentar o limiar de dor tÃrmica no teste da imersÃo da cauda (mais nÃo no teste placa quente) e, na reduÃÃo das contorÃÃes induzidas por Ãcido acÃtico. A antinocicepÃÃo produzida por β-amirina, foi significativamente bloqueada em animais prÃ-tratados com os respectivos antagonistas vermelho de rutÃnio (2 mg/kg, s.c.) e naloxona (1 mg/kg, i.p.), indicando o envolvimento de receptores da capsaicina (TRPV1) e opiÃides em seu mecanismo. No teste da formalina, de forma similar à morfina, β-amirina bloqueou significativamente a inibiÃÃo da ingestÃo alimentar associada a dor. Assim como morfina, β-amirina apresentou aÃÃo inibitÃria sobre o trÃnsito intestinal, efeito esse revertido pelo prÃ-tratamento com antagonista opiÃide nÃo seletivo, naloxona. Estes dados sugerem que β-amirina apresenta um potencial antinociceptivo comparÃvel à analgesia perifÃrica produzida pela morfina, evidencia a exploraÃÃo desta para o desenvolvimento de um analgÃsico nÃo-opiÃide Ãtil na farmacoterapia de patologias do trigÃmeo e visceral. / The effects of pentacyclic triterpene β-amiryn and β-amyrin, isolated from resin of Protium heptaphyllum March. (Burseraceae), were preliminarily showed significant tested in models of nociception oral, and antinociceptives effects, guiding the search with this isolate in the investigation of their effects in models of orofacial pain induced by capsaicin or formalin and against capsaicin-induced corneal pain; thermal pain (tail immersion test in hot water and hot-plate) and in acetic acid 0,6%-induced visceral nociception in mice. Male Swiss mice (n = 8 per group) were pre-treated with β-Amyrin (10, 30, and 100 mg/kg, p.o.), morphine (5 mg/kg, s.c.) or vehicle (distlled water + 0,05% Tween 80), one hour before the capsaicin (20 μl, 1.5 μg) or formalin (20 μl, 1.5%) injection into the right vibrissa. β-Amyrin was also assessed on pain-related behavioral test (Eye-wiping) by topical application of capsaicin (20 μl, 1.5 μg) on to the mouse conjuctiva and the time (sec) that the animal spent in eye wiping was determined during a 10 min period. The triterpenoid demonstrated mostly a dose-unrelated antinociception in all the test models of nociception. Against the orofacial pain induced by capsaicin, β-Amyrin (30 e 100 mg/kg, p.o.) and morphine showed greater potency in reducing the nociceptive response. At the doses employed, the reductions were 81 and 90% to β-Amyrin and 97% for the morphine, respectively. Capsaicin nociception in orofacial test is accompanied by a localized thermal flare (measured by thermometry), which was significantly diminished by pretreatment of animals with β-Amyrin or L-NAME, an NOS inhibitor. In four weeks diabetic mice, capsaicin injected into vibrissa pad demonstrated a lesser degree of orofacial nociception compared to non-diabetics. In formalin test, both morphine and β-Amyrin showed significant naloxone reversible antinociception in both phases. However, β-Amyrin inhibited the second phase response, more prominently, at 30 mg/kg. The caliculated ED50 values for β-Amyrin and morphine were 16,44 mg/kg (CL 10,0 - 38,41) and 3 mg/kg (CL 2,5 - 5,0) in the first phase and 43,37 mg/kg (CL 30,52 - 39,30) and 3 mg/kg (CL 2,5 - 5,0) in the second phase, respectively. Co-administration of β-Amyrin and morphine at their respective ED50 dose levels failed to demonstrate any additive or potentiating effect on anti-nociception. However, at ED25 and ED12.5 dose-combinations exhibited an antinociception that equalled their ED50 combination effect, suggesting that by the use of β-Amyrin, the analgesic dose of morphine could be minimised to avoid its high-dose-associated side-effects. Similar to morphine, β-Amyrin significantly blocked the pain-related suppression of food intake in formalin test. β-Amyrin (30 and 100 mg/kg was also effective in increasing the thermal pain threshold in hot-water tail immersion test (but not in hot-plate test), and in reducing the acetic acid-induced writhes. The antinociception produced by 30 mg/kg β-Amyrin was significantly blocked in animals pre-treated with the respective antagonists capsazepine (5 mg/kg, s.c.), and naloxone (1 mg kg/kg, i.p.), indicating the involvement of capsaicin (TRPV1) and opioid receptors in its mechanism. Like morphine, β-Amyrin showed an inhibitory effect on intestinal transit, an effect reversed by pretreatment with nonseletive opiÃide antagonist, naloxona. These data indicate that β-Amyrin has the antinociceptive potential comparable to peripheral analgesia produced by morphine that could be explored further on its suitability in developing a non-opioid analgesic useful in pharmacotherapy of trigeminal and visceral pathologies.

nÃo hà / The Protium heptaphyllum (almecegueira) exudes an amorphous resin consisting of four binary mixtures of triterpenoids, and the mixture of ,-amyrin its major constituent. In folk medicine, the resin of Protium heptaphyllum is used as anti-inflammatory, gastroprotective, analgesic, expectorant and healing. The mixture of pentacyclic triterpenes ,-amyrin (AB) (63:37) has gastroprotective, antipruritic, antiinflammatory and antioxidant properties. Experimental studies with pentacyclic triterpene compounds belonging to the groups ursan, oleanan and lupan showed inhibition of different enzyme systems intimately related to metabolism/absorption of carbohydrates and lipids. Thus the hypoglycemic and hypolipidemic activity of AB was evaluated in Swiss mice, the models of diabetes induced by streptozotocin (STZ) and alloxan (ALX) and hyperlipidemia induced by triton WR-1339 and hyperlipidemic diet (DH). AB (10, 30 and 100mg/kg) was able to reduce blood glucose in a model of acute treatment in STZ-induced diabetes, we observed two times (3 and 5 hours after administration of AB). In five days after treatment of diabetic animals by ALX, AB (30 and 100 mg/kg) also decreased hyperglycemia, an effect also observed for the use of glibenclamide (10 mg/Kg) in both models. But in normal animals, AB did not affect blood glucose, unlike the positive control glibenclamide (10 mg/kg). Although the model of diabetes for ALX, AB reduced total cholesterol (TC) serum at a dose of 100mg/kg, and triglycerides (TG) at doses of 30 and 100 mg/kg. In this same model, reduced the serum amylase AB (AB 30 and 100 mg/kg) and serum lipase (AB 100 mg/kg), a result also observed in normal animals, the same doses. In the Oral Glucose Tolerance Test (OGTT), AB (30 and 100 mg/kg) was shown to reduce blood glucose levels 60 min after administration of glucose, and AB (10, 30 and 100 mg/kg) in 90 min. In the model of hyperlipidemia by triton, AB (10, 30 and 100 mg/kg) significantly reduced the levels of TC and TG, in times of 24 and 48 h after administration of triton, an effect also observed for fenofibrate (200 mg/kg) used as positive control. The levels of HDL-c elevation experienced with the use of AB at all doses and times observed, as well as fenofibrate. In the model of hyperlipidemia by HD, AB (10, 30 and 100 mg/kg) controlled weight gain of animals receiving HD, as well as reduced TC, TG, LDL-c and VLDL-c, although the latter only in doses 30 and 100 mg/kg. These two doses were also effective in raising HDL-c. All doses of AB administered reduced the atherogenic index. All doses of AB also reduced the hepatic cholesterol in this model. All effects reported were similar to the positive control (fenofibrate 200 mg/kg). AB (10, 30 and 100 mg/kg) was able to significantly improve the antioxidant defenses of the liver, because it elevated the activity of hepatic SOD and CAT when compared to control high-fat diet, and raised the level of NP-SH in dose of 100 mg/kg, as well as reduced lipid peroxidation by decreasing the MDA, at all doses. Together, these results indicate that α,β-amyrin has hypolipidemic and hypoglycemic effect and deserves further evaluation in larger animal models that simulate chronic conditions of diabetes and dyslipidemia, in addition to research on their mechanism of action. / O Protium heptaphyllum (almecegueira) exsuda uma resina amorfa constituÃda de quatro misturas binÃrias de triterpenÃides, sendo a mistura de ,-amirina o constituinte majoritÃrio. Na medicina popular, a resina de Protium heptaphyllum à utilizada como antiinflamatÃria, gastroprotetora, analgÃsica, expectorante e cicatrizante. A mistura de triterpenos pentacÃclicos ,-amirina (AB) (63:37) possui propriedades gastroprotetora, antipruriginosa, antiinflamatÃria e antioxidante. Estudos experimentais com compostos triterpÃnicos pentacÃclicos que pertencem ao grupo ursano, oleanano e lupano mostraram a inibiÃÃo de diferentes sistemas enzimÃticos intimamente relacionados ao metabolismo/absorÃÃo de carboidratos e lipÃdios, deste modo, a atividade hipoglicemiante e hipolipemiante de AB foi avaliada, em camundongos Swiss, nos modelo de diabetes induzida por estreptozotocina (STZ) e por aloxano (ALX) e hiperlipidemia induzida por triton WR-1339 e dieta hiperlipidÃmica (DH). AB (10, 30 e 100mg/Kg) foi capaz de reduzir a glicemia num tratamento agudo no modelo de diabetes induzida por STZ, nos dois tempos observados (3 e 5h apÃs administraÃÃo de AB). Em tratamento apÃs cinco dias de animais diabÃticos por ALX, AB (30 e 100mg/Kg) tambÃm diminuiu a hiperglicemia, efeito este, tambÃm observado para o uso de glibenclamida (10mg/Kg) nos dois modelos. PorÃm em animais normais, AB nÃo alterou a glicose sanguÃnea, ao contrÃrio do controle positivo glibenclamida (10 mg/Kg). Ainda no modelo de diabetes por ALX, AB reduziu o colesterol total (CT) sÃrico na dose de 100mg/Kg, bem como os triglicerÃdeos (TG) nas doses de 30 e 100 mg/Kg. Neste mesmo modelo, AB reduziu a amilase sÃrica (AB 30 e 100 mg/Kg) e a lipase sÃrica (AB 100mg/Kg), resultado este observado tambÃm em animais normais, nas mesmas doses. No Teste Oral de TolerÃncia à Glicose (TOTG), AB (30 e 100 mg/Kg) mostrou reduzir a glicemia 60 min apÃs a administraÃÃo de glicose, bem como AB (10, 30 e 100 mg/Kg) em 90 min. No modelo de hiperlipidemia por triton WR-1339, AB (10, 30 e 100mg/Kg) reduziu de forma significativa os nÃveis de CT e TG, nos tempos de 24h e 48h apÃs a administraÃÃo do triton WR-1339, efeito observado tambÃm para fenofibrato (200mg/Kg) utilizado como controle positivo. Os nÃveis de HDL-c sofreram elevaÃÃo com o uso de AB em todas as doses e tempos observados, assim como o fenofibrato. No modelo de hiperlipidemia por DH, AB (10, 30 e 100mg/Kg) controlou o ganho de peso dos animais que receberam a DH, bem como reduziram CT, TG, LDL-c e VLDL-c, porÃm este Ãltimo somente nas doses de 30 e 100mg/Kg. Estas duas doses tambÃm foram eficazes para elevar o HDL-c. Todas as doses de AB administradas reduziram o Ãndice aterogÃnico. Todas as doses de AB tambÃm reduziram o colesterol hepÃtico neste modelo. Todos os efeitos relatados foram similares ao controle positivo (fenofibrato 200mg/Kg). AB (10, 30 e 100 mg/Kg) foi capaz de melhorar significativamente as defesas antioxidantes do tecido hepÃtico, pois elevou a atividade das enzimas catalase e superÃxido dismutase hepÃticas, quando comparado ao controle dieta hiperlipÃdica, bem como elevou o nÃvel dos grupos sulfidrÃlicos nÃo protÃicos na dose de 100mg/Kg, assim como reduziu a peroxidaÃÃo lipÃdica ao diminuir o malondialdeÃdo, em todas as doses. Em conjunto, esses resultados indicam que α,β-amirina possui efeito hipoglicemiante e hipolipemiante e que merece maior avaliaÃÃo futura em modelos animais que simulem situaÃÃes crÃnicas de diabetes e dislipidemias, alÃm de pesquisa de seu mecanismo de aÃÃo.

Este trabalho consiste em uma introdução teórica dos fundamentos básicos da difração de raios-X por cristais, de uma descrição sucinta do difratômetro automático CAD-4 da Enraf-Nonius, e do método de Patterson e dos métodos diretos usados na determinação de estruturas. Foram resolvidas as estruturas cristalinas e moleculares de um complexo de níquel (II) e de cinco triterpenos. Os cinco triterpenos são produtos naturais extraídos da casca de madeira Austroplenckia populnea (Celastraceae). Monocristais desses produtos foram analisados por difração de raios X usando um difratômetro automático CAD-4 e com radiação de M&#959K&#945 (&#955= 0,71073&#197) monocromatizada por cristal de grafita. Os principais dados cristalográficos estão resumidos na tabela abaixo:   triterpenos T1 T2 T3 T4 T5 Fórmula Química C31H48O4 C30H48O3 C31H48O3 C31H50O3 C30H48O4 Mr 484,73 456,71 468,73 470,74 472,71 Grupo Espacial P21 P1 C2 P212121 P21 a(Å) 6,697(2) 7,271(2) 12,109(2) 6,815(2) 14,695(2) b(Å) 14,714(7) 12,389(8) 7,346(2) 16,127(2) 13,699(2) c(Å) 13,866(3) 15,632(2) 30,570(6) 24,695(4) 6,622(3) α(°) 90 74,95(2) 90 90 90 β(°) 103,53(2) 87,55(2) 99,83(2) 90 104,45(2) γ(°) 90 86,76(4) 90 90 90 Z 2 2 4 4 2 V(Å3) 1328(1) 1357(1) 2679(2) 2714(2) 1288(1) dc(g⋅cm-3) 1,211 1,118 1,162 1,152 1,219 μ(cm-1) 0,726 0,651 0,675 0,668 0,733 F(000) 532 504 1032 1040 520 R 0,0485 0,0996 0,0574 0,0545 0,0420 Nº refl.: I>3σ(I) 1395 1972 751 1438 838 O complexo de níquel, [NiII(DPEH)2](NO3)2 2H2O, foi sintetizado [70] a partir de uma solução alcoólica de 2 moles do ligante DPEH (Diacetilmonooxima - &#946 - Piridil-(2)-Etilimina) para cada mol de [Ni(OH2)6](NO3)2. Um monocristal desse composto foi analisado por difração de raios X, usando um difratômetro automático CAD-4 e radiação de M&#959K&#945 (&#955 = 0,71073&#197) monocromatizada por cristal de grafita. Dados cristalográficos principais: sistema monoclínico Cc, Mr = 629,27, a = 8,502(2), b = 18.702(2), c = 18.653(4)&#197, &#946 = 103,22(2)&#176, V = 2887(2)&#1973, Z = 4, dc = 1,448gcm-3, &#956 = 7,36cm-1, F(000) = 1312, R =0,0594 e 1656 refleções com I &#62 2&#959(I). O átomo de Ni está coordenado por seis átomos de N com uma configuração octaédrica distorcida. / This work consist of a theoretical introduction of the basic principles of x-ray diffraction by crystals, a brief descruption of the automatic diffractometer CAD-4 Enraf-Nonius and the Patterson and direct methods used in structure determination. The crystals structures of one Ni(II) complex and Five triterpenes were solved. The five triterpenes are natural products extracted from the bark of Austroplenckia populnea (Celastraceae). Single crystals of the products were analyzed by X-ray diffraction, using the CAD-4 automatic diffractometer M&#959K&#945 (&#955= 0,71073&#197) monochromatizated by a graphite crystal. The relevant crystallographic data are summarized in the table below:   triterpenos T1 T2 T3 T4 T5 Fórmula Química C31H48O4 C30H48O3 C31H48O3 C31H50O3 C30H48O4 Mr 484,73 456,71 468,73 470,74 472,71 Grupo Espacial P21 P1 C2 P212121 P21 a(Å) 6,697(2) 7,271(2) 12,109(2) 6,815(2) 14,695(2) b(Å) 14,714(7) 12,389(8) 7,346(2) 16,127(2) 13,699(2) c(Å) 13,866(3) 15,632(2) 30,570(6) 24,695(4) 6,622(3) α(°) 90 74,95(2) 90 90 90 β(°) 103,53(2) 87,55(2) 99,83(2) 90 104,45(2) γ(°) 90 86,76(4) 90 90 90 Z 2 2 4 4 2 V(Å3) 1328(1) 1357(1) 2679(2) 2714(2) 1288(1) dc(g⋅cm-3) 1,211 1,118 1,162 1,152 1,219 μ(cm-1) 0,726 0,651 0,675 0,668 0,733 F(000) 532 504 1032 1040 520 R 0,0485 0,0996 0,0574 0,0545 0,0420 Nº refl.: I>3σ(I) 1395 1972 751 1438 838 The Ni complex, [NiII(DPEH)2](NO3)2 2H2O, was synthesized from a 2M alcoholic solution of the ligand DPEH (Diacethylmonoxima - &#946 - Pyridil-(2)-Ethylimina) for each mol of [Ni(OH2)6](NO3)2. A single crystal of this compound was analyzed by X-ray diffraction, using the CAD-4 automatic diffractometer M&#959K&#945 (&#955 = 0,71073&#197) monochromatizated by a graphite crystal. Principal crystallographic data are: monoclinic Cc, Mr = 629,27, a = 8,502(2), b = 18.702(2), c = 18.653(4)&#197, &#946 = 103,22(2)&#176, V = 2887(2)&#1973, Z = 4, dc = 1,448gcm-3, &#956 = 7,36cm-1, F(000) = 1312, R =0,0594 and 1656 reflections with I &#62 2&#959(I). The Ni atom is coordinated to six N atoms in a distorted octahedrical configuration.