Luehea divaricata, known as açoita-cavalo , is a Brazilian native species and has few reported in the literature. Phytochemical investigation of the methanolic crude extract and its fractions (acid ethereal, basic ethereal, basic ethyl acetate, neutral dichloromethane, neutral ethyl acetate and neutral hexane) from stem bark of L. divaricata (Malvaceae) resulted in the isolation of seven compounds: bis (2-ethyl-heptyl) ftalate (26), one triterpene friedelin (28), mixture of the terpenes α- (27) and β-amyrin (1), two steroids β-sitosterol (29) and glycosyl β-sisotsterol (14), and two flavonoids known as catechin (22) and epicatechin (8), which are diasteroisomers, respectively. The compound friedelin (28), isolated from ethereal acid fraction, was identified for first time in genus Luehea. The structures of the isolated metabolites were elucidated by 1H and 13C NMR in one- and two-dimensional analysis, by X-ray diffraction, melting point, beyond comparison with standard samples where possible and data available from literature. The extract, fractions and isolated compounds were tested for their antimicrobial activity and antioxidant capacity. Crude methanolic extract, fractions and isolated compounds from acid-base fractionation showed effective antibacterial (Gram-positive and Gram-negative) and antifungal activities for the strains tested. Basic and neutral ethyl acetate fractions showed good antioxidant potential when tested against their capacity. Thus, the study of phytochemical and pharmacological activities of the identified compounds from stem bark of L. divaricata expands knowledge alluding to chemotaxonomy and justifies its use in ethnopharmacology because, to date, only studies with other aerial parts were performed. / Luehea divaricata, conhecida como açoita-cavalo, é uma espécie nativa brasileira e com poucos relatos na literatura. A investigação fitoquímica do extrato bruto metanólico e de suas frações (etérea ácida, etérea básica, acetato de etila básica, diclorometano neutra, acetato de etila neutra e hexânica neutra) das cascas do caule de L. divaricata (Malvaceae) resultou no isolamento de sete compostos: Ftalato de bis (2-etil-heptila) (26), triterpeno friedelina (28), mistura triterpênica de α- (27) e β-amirina (1), dois esteroides β-sitosterol (29) e β-sisotsterol glicosilado (14), além de dois flavonoides conhecidos como Catequina (22) e epicatequina (8), estes sendo diasteroisômeros, respectivamente. O composto friedelina (28) isolado da fração etérea acida, foi identificado pela primeira vez no gênero Luehea. As estruturas dos metabólitos isolados foram elucidadas através de RMN 1H e 13C, uni e bidimensionais, por análise de difração de raio-X, ponto de fusão além de comparação com amostra padrão quando existente e dados disponíveis na literatura. O extrato, as frações e os compostos isolados foram testados quanto à sua atividade antimicrobiana e a sua capacidade antioxidante. Extrato bruto metanólico, suas frações e compostos isolados obtidos no fracionamento ácido-base apresentaram atividade antibacteriana (Gram-positivas e Gram-negativas) e antifúngica efetivas, para as cepas testadas. As frações acetato de etila básico e acetato de etila neutro quando testadas frente a sua capacidade antioxidante apresentaram um bom potencial. Dessa forma, o estudo fitoquímico e das atividades farmacológicas dos compostos identificados da casca do caule de L. divaricata amplia os conhecimentos alusivos à quimiotaxonomia, bem como justifica seu uso na etnofarmacologia pois, até o momento, apenas estudos com outras partes aéreas foram realizados.

L’objectif de ce projet de thèse était de caractériser le métabolisme de terpénoïdes (ou isoprénoïdes) chez les plantes supérieures. L’essentiel du travail a consisté à caractériser des triterpènes synthases (TTPS) d’Arabidopsis thaliana, un modèle végétal, ainsi que celles de l’épurge (Euphorbia lathyris), une euphorbe pour laquelle des applications agronomiques sont envisagées. Au cours de ma thèse, j’ai aussi contribué à l’étude du métabolisme et des fonctions des précurseurs de triterpènes et de stérols, ainsi qu’à leurs fonctions biologiques.Les triterpènes synthases, ou 2,3-oxydosqualène cyclases (OSCs), convertissent le substrat 2,3-oxydosqualene (SqO) en une multitude d'alcools triterpéniques, et ainsi amorcent la biosynthèse de dérivés triterpénoïdes (triterpènes oxydés, conjugués, etc ..). Arabidopsis thaliana contient 13 OSCs produisant divers squelettes triterpéniques, de type stéroïdien ou non-stéroïdien. Les produits de cyclisation du SqO ont été élucidés structuralement (GC-MS, RMN) après expression hétérologue des enzymes en levure erg7. Cette levure est déficiente en lanostérol synthase (ERG7), ce qui permet d'accumuler le SqO, substrat des cyclases. Lorsque le mutant est transformé avec un ADNc codant une triterpène synthase, il est capable de convertir le SqO en un ou plusieurs triterpènes. Cependant, la caractérisation des 13 OSCs d'Arabidopsis réalisée de façon hétérologue en levure n’a pas été établie inplanta. De façon surprenante, certains des composés produits dans les levures erg7 transformées n'ont jamais été détectés chez Arabidopsis. C'est pourquoi il a été nécessaire de reconsidérer les fonctions biochimiques exactes de ces enzymes dans un contexte végétal. / The subject of this PhD thesis is part of a research project entitled "Production of energy-rich triterpenoids in Euphorbia lathyris, a potential crop for third generation biofuels," whose acronym is EULAFUEL. This project is funded by a multipartner program ANR-KBBE and has been extended until December 2013. The aim of this PhD project is to get new insights into the aspects related with the biosynthesis and accumulation of latex triterpenoids. In addition, for comparison, a major objective of the thesis is to characterize functionally the enzymes involved in the synthesis of triterpenes in the model plant Arabidopsis thaliana. Triterpene synthases, also named oxidosqualene cyclases (OSCs), convert 2,3-oxidosqualene (OS) into a multitude of triterpene alcohols and there by initiate triterpene biosynthesis. Arabidopsis thaliana for instance has 13 OSCs producing diverse skeletons of steroidal or non-steroidal triterpenes. Cyclization products of a given enzyme have been characterized biochemically using a yeast heterologous expression system. However, for the majority of Arabidopsis triterpene synthases, inplanta studies are lacking. In fact, most of the compounds produced in yeast expressing such enzymes have never been detected in wild-type Arabidopsis. This is a reason why we should reconsider the exact biochemical function of triterpene synthases in the plant context. Then, in a comparative approach in E. lathyris, we project to study the specific triterpene accumulation in the laticifers, a specialized cell type where high amounts of lanosterol, an unusual OS cyclization product for plants, accumulate.

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.

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 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.

Oleanolic and ursolic acid (OA and UA) are triterpenic acids with diverse biological activities that are of interest to the pharmaceutical industry. To investigate the scope for producing these compound using cell suspension cultures of Salvia species, calli from S. officinalis, S. virgata and S. fruticosa were induced using several plant growth regulator (PGR) combinations. Eleven lines were selected for suspension induction from a pool of calli. Six suspension cultures were established successfully and cultivated in the Respiration Activity MOnitoring System® (RAMOS®) to obtain online data on their growth kinetics and to establish appropriate sampling schedules for the determination of their OA and UA production. Based on their observed growth behaviour, OA and UA contents, and aggregation properties, one suspension culture from each studied Salvia species was selected for further optimisation. The μmax values for these suspension cultures ranged from 0.20 to 0.37°d-1, their OA and UA contents were greater than 1.3 and 1.2 mg g-1, respectively, and they afforded maximum volumetric yields of 21.0 mg l-1 for OA and 32.8 mg l-1 for UA. These results will be useful in the development of a refined Salvia suspension-based process for OA and UA production.

Squalene synthase (SS) is an essential enzyme in eukaryotic systems responsible for an important branch point in isoprenoid metabolism that leads to sterol formation. The mechanistic complexity of SS has made it a difficult enzyme to study. The green alga Botryococcus braunii race B possesses several squalene synthase-like (SSL) enzymes that afford a unique opportunity to study the complex mechanism of triterpene biosynthesis. SSL-1 catalyzes presqualene diphosphate (PSPP) formation, which can either be converted to squalene by SSL-2 or botryococcene by SSL-3. A rationally designed mutant study of B. braunii squalene synthase (BbSS) and SSL-3 was conducted to understand structure-function relations among these enzymes. These studies revealed two amino acid positions in SSL-3 (N171, G207) that appeared to control 1’-3 versus 1’-1 linkages. The reciprocal mutations in the corresponding positions of BbSS did not convert this enzyme into a botryococcene synthase. Next, a genetic selection was developed to evolve SSL enzymes towards a fully functional SS. Previous studies have shown that Saccharomyces cerevisiae squalene synthase (ScSS) can be knocked out and although lethal, growth can be restored by providing an exogenous source of ergosterol. Additional studies have shown that successful complementation of the ScSS knockout with a non-fungal SS is possible but requires a fungal SS carboxy- terminus region. Given these observations, proof-of-principle experiments were conducted to demonstrate that SSL-SSL fusion enzymes could complement the ScSS knockout followed by construction of a mutant SSL-SSL fusion enzyme library that was screened in the ScSS knockout yeast line. From this library, mutant SSL-SSL fusion enzymes were identified that were able to complement, which demonstrated the feasibility of this approach as a genetic selection for mutant SSL enzymes. Squalene and botryococcene have valuable industrial applications in vaccine adjuvant formations, cosmetic products, and renewable energy feedstock material. Limitations in natural sources of these molecules have made heterologous production of them an important research target. Algae represent a desirable group of organisms that could be engineered to produce these metabolites because they are photosynthetic and capable of using non-arable farmland. The feasibility, approach, and progress for engineering green algae to produce squalene and botryococcene are discussed.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Este trabalho descreve a investigaÃÃo fitoquÃmica da espÃcie vegetal Caesalpinia ferrea (Leguminosae), conhecida como âjucÃâ e usada popularmente no nordeste do Brasil como antirreumÃtico, antimicrobiano e entre outros usos. Os extratos de C. ferrea atravÃs da cromatografia em coluna tendo como fase estacionÃria gel de sÃlica e Sephadex LH-20 levaram ao isolamento de quatro substancias do tipo polifenÃis {Ãcido gÃlico (Ãcido-3,4,5-tri-hidrÃxi-benzÃico), Ãcido elÃgico (4,4â,5,5â,6,6â-hexahidroxidifÃnico-2,6,2â,6â-dilactona), amentoflavona (8-[5-(5,7-dihidroxi-4-oxo-cromeno-2-il)-2-hidroxi-fenil]-5,7-dihidroxi-2-(4-hidroxifenil) cromeno-4-one) e resveratrol (trans-3,5,4â-trihidroxiestilbeno)}; uma mistura constituÃda de um esterÃide {β-sitosterol (24-etilcolest-5-en-3β-ol) e de dois Ãcidos graxos [Ãcido palmÃtico (n-hexadecanÃico) e esteÃrico (n-octadecanÃico)]}; uma outra mistura constituÃda dos triterpenos [Lupeol (Lup-20(29)-en-3β-ol) e β-amirina (olean-12-en-3β-ol), do esterÃide 24-metilenocicloartanol (3β-9,19-Ciclolanostan-3-ol-24-metileno) e do Ãlcool lignocÃrico (n-tetracosonol). A caracterizaÃÃo dos compostos envolveu o uso das tÃcnicas de RMN 1H e 13C (1D e 2D), IV, CG-EM-IE e CLAE-EM-IES. Os extratos brutos das vagens e algumas de suas fraÃÃes foram submetidos a testes de atividades antiacetilcolinesterase e antioxidante com resultados promissores. O bi-flavonÃide, a fitoalexina e os constituintes das misturas mencionadas sÃo relatados pela primeira vez na literatura em C. ferrea. / This work describe the phytochemical investigation of pods and stems of specie Caesalpinia ferrea (Leguminosae) known as âjucÃâ. Popularly, this plant is used as antirheumatic, antimicrobial and among other uses. The hexane and ethanol extracts to pods and stems of C. ferrea were submitted to column chromatography having silica gel and Sephadex LH-20 as stationary phase that led to the isolation of four compounds of the type polyphenol: {gallic acid (3,4,5-trihydroxy-benzoic acid), ellagic acid (4,4', 5,5', 6,6'-hexahydroxydifÃnico-2,6,2',6'-dilactone), amentoflavone (8-[5-(5,7-dihydroxy-4-oxo-chromene-2-yl)-2-hydroxyphenyl]-5,7-dihydroxy-2-(4-hydroxyphenyl)) chromen-4-one and resveratrol (trans-3,5,4â trihydroxystilbene)}; a mixture consisting of a steroid {-sitosterol(24-etilcolest-5-en-3-ol) and two fatty acids [palmitic acid (nhexadecanoic) and stearic acid (n-octadecanoic acid)]}; another mixture consisting of the triterpenes [Lupeol (Lup-20(29)-en-3-ol) and -amyrin (olean-12-en-3-ol), steroid 24-metilenocicloartanol (3-9,19 Ciclolanostan-3-ol-24-methylene) and lignoceric alcohol (n-tetracosonol). The characterization of the chemical components involved the use 1H and 13C NMR (1D and 2D), IR, GC-MS-EI e HPLC-MS-ESI. The extracts of pods and some of its fractions were submitted in test of the antioxidant activities and antiacetylcholinesterase with promising results. The bi-flavonoid, phytoalexin and components of mixtures mentioned are first reported in the literature to C. ferrea.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Os triterpenos pentacÃclicos sÃo compostos naturais com atividade antiinflamatÃria e citoprotetora e sÃo relativamente atÃxicos. A pancreatite aguda, uma inflamaÃÃo aguda do pÃncreas, pode levar à sÃndrome da resposta inflamatÃria sistÃmica (SRIS) e à sÃndrome da disfunÃÃo mÃltipla de ÃrgÃos (MODS), condiÃÃes que podem levar o paciente ao Ãbito. Neste trabalho, a mistura de triterpenos pentacÃclicos alfa,β-amirina, isolada do Protium hepthaphyllum, foi investigada quanto aos seus efeitos nos modelos de pancreatite aguda induzida por L-arginina em ratos e por ceruleÃna em camundongos. No modelo de pancreatite aguda induzida por L-arginina, ratos Wistar machos foram tratados com a mistura de alfa,β-amirina (10, 30 e 100 mg/kg, v.o.) ou com o veÃculo (2% de Tween 80 em Ãgua destilada, 10ml/kg) 48, 24 e 1,5h antes da administraÃÃo de L-arginina (2 x 2,5 g/kg; 1 h de intervalo) ou com metilprednisolona (30 mg/kg, i.m.) 30 min antes da administraÃÃo de L-arginina. Na pancreatite induzida por ceruleÃna, camundongos Swiss machos foram tratados com a mistura de alfa,β-amirina (10, 30 e 100 mg/kg, v.o.) ou com o veÃculo (2% de Tween 80 em Ãgua destilada, 10ml/kg) 48, 24 e 1,5h antes da administraÃÃo de ceruleÃna (5 x 50 μg/kg; 1 h de intervalo) ou com talidomida (200 mg/kg, v.o.) 1h antes da administraÃÃo de ceruleÃna. Animais tratados apenas com salina (0,9%, NaCl) foram incluÃdos nos dois modelos. Foram analisados o edema pancreÃtico, nÃveis sÃricos de amilase, lipase e citocinas (TNF alfa, IL-6), mieloperoxidase, substÃncias reativas ao Ãcido tiobarbitÃrico (TBARS), histologia e imunohistoquÃmica (TNF-alfa, iNOS e nitrotirosina) pancreÃtica. L-arginina e ceruleÃna aumentaram significativamente o edema pancreÃtico e os nÃveis sÃricos de amilase, lipase, TNF alfa, IL-6. A avaliaÃÃo histopatolÃgica do pÃncreas revelou a presenÃa de edema, infiltraÃÃo neutrofÃlica, hemorragia, vacuolizaÃÃo e necrose acinar. Foi observado um aumento acentuado na expressÃo de TNF alfa, iNOS e nitrotirosina na avaliaÃÃo por imunohistoquÃmica. O prÃ-tratamento com alfa e beta-amirina (10, 30 e 100 mg/kg, v.o.), metilprednisolona (30 mg/kg, i.m.) ou talidomida (200 mg/kg, v.o.) atenuaram significativamente a severidade da pancreatite aguda induzida tanto por L-arginina, quanto por ceruleÃna, evidenciado pela reduÃÃo do edema pancreÃtico, amilase, lipase e citocinas sÃricas, mieloperoxidase e TBARS pancreÃtico. AlÃm disso, o tratamento com alfa,β-amirina e com as drogas de referÃncia suprimiram as alteraÃÃes histopatolÃgicas e a expressÃo de citocinas e nitrotirosina pancreÃticas. Em conjunto, esses resultados indicam que alfa,β-amirina melhora a severidade da pancreatite aguda induzida por L-arginina ou ceruleÃna por agir como antiinflamatÃrio e antioxidante. / Triterpenes are natural compounds with anti-inflammatory and cytoprotective effects relatively non-toxic. Acute pancreatitis, an inflammation of the pancreas, may lead to systemic inflammatory response syndrome (SIRS) and multiple organ dysfunction syndrome (MODS), conditions that can lead the patient to death. In this study, a mixture of triterpenes isolated from Protium hepthaphyllum was investigated for their effects in models of acute pancreatitis. In the model of acute pancreatitis induced by L-arginine, male Wistar rats were treated with a mixture of ,β-amyrin (10, 30 and 100 mg/kg, p.o.) or with vehicle (2% Tween 80 in distilled water, 10 ml/kg) 48, 24 and 1.5 h before the administration of L-arginine (2 x 2.5 g/kg, 1 h apart) or with metilprednisolone (30 mg/kg, i.m.) 30 min before the administration of L-arginine. In cerulein-induced pancreatitis, male Swiss mice were treated with a mixture of ,β-amyrin (10, 30 and 100 mg/kg, p.o.) or with vehicle (2% Tween 80 in distilled water, 10 ml/kg) 48, 24 and 1.5 h before administration of cerulein (5 x 50 mg/kg, 1 h apart) or with thalidomide (200 mg/kg, p.o.) 1h before administration of cerulein. Animals treated with saline (0.9% NaCl) were included in both models. We analyzed the pancreatic edema, serum amylase, lipase and cytokines (TNF-, IL-6), myeloperoxidase, reactive substances to thiobarbituric acid (TBARS), histology and immunohistochemistry (TNF-, iNOS and nitrotyrosine) pancreatic. L-arginine and cerulein significantly increased pancreatic edema and serum levels of amylase, lipase, TNF- and IL-6. Histopathologic evaluation of pancreas revealed the presence of edema, neutrophil infiltration, hemorrhage, acinar vacuolization and necrosis. We observed a marked increase in the expression of TNF-, iNOS and nitrotyrosine in the evaluation by immunohistochemistry. The pre-treatment with alpha and beta-amyrin (10, 30 and 100 mg/kg, p.o.), metilprednisolone (30 mg/kg, i.m.) or thalidomide (200 mg/kg, p.o.) significantly attenuated the severity of acute pancreatitis induced by either L-arginine, and by cerulein, as evidenced by the reduction of pancreatic edema, amylase, lipase and serum cytokines, myeloperoxidase and pancreatic TBARS. Furthermore, treatment with ,β-amyrin and the reference drugs suppressed the histopathological changes and expression of cytokines and nitrotyrosine pancreatic. Together, these results indicate that the mixture of ,-amyrin reduces the severity of acute pancreatitis induced by L-arginine or cerulein acting as anti-inflammatory and antioxidant agent.

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.