ix, 179 leaves xxiv : ill. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology, 1970

xxiii, 171 leaves : ill. + appendix / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Animal Physiology, 1973

Thesis (Ph.D.) -- University of Adelaide, Dept. of Microbiology, 1970.

Thesis (Ph.D.)--Flinders University, School of Biological Sciences. / Typescript bound. Includes bibliographical references: (leaves 135-157) Also available online.

The fleshy hips of the briar rose, Rosa rugosa were cultured on a modified medium developed by Gamborg (1963) to produce callus and suspension cultures. These cells were very light, almost white. Reduced ascorbic acid, determined by the 2,6-dichlorophenolindophenol titration method, was found in the suspension cultures. This finding prompted an investigation into culture techniques for obtaining an optimum concentration of ascorbic acid. During the growth of the cells, the pH rose and ascorbic acid concentration increased after the sixth day. Age of culture was also an important factor as cultures older than three or four weeks contained virtually no ascorbic acid. Possible precursors of ascorbic acid in plants were added to cultures and their effect on the ascorbic acid level was determined over a period of twenty-four hours. D-glucose, D-(-)-levulose, D-galactose and D-glucurono-ɣ-lactone caused no increase. L-gulono-ɣ-lactone brought about a slight increase and a comparatively large increase was obtained with L-galactono-ɣ-lactone. Confirmation for the latter was obtained using the 2,4-dinitrophenylhydrazine method to rule out the possibility or microbial contamination and to be sure that another metabolite was not responsible for the increased reducing capacity of the system. Omission of myoinositol from the vitamin solution of the medium decreased total cell yield but had no effect on the level of ascorbic acid. Decreasing the sucrose content of the medium by one-half caused a sharp decrease in ascorbic acid concentration, indicating that an adequate supply of sugar is a prerequisite for optimum production of ascorbic acid. Growing liquid suspensions under controlled illumination resulted in increased ascorbic acid values with each successive transfer; however, total cell yields decreased each time. In addition, there was no evidence of chlorophyll production in the cultures. This exploratory work shows that ascorbic acid is present in the tissue culture of Rosa rugosa. Therefore, tissue culture may be useful for elucidating the complete pathway for ascorbic acid production which is as yet still unclear. This information coupled with the effects of altering the physical environment of the cells may direct cultures of Rosa rugosa to produce ascorbic acid in quantities that would make its recovery industrially attractive. / Land and Food Systems, Faculty of / Graduate

O acabamento superficial ou rugosidade das fibras é um fator importantíssimo no estudo da interface fibra/matriz, no que tange à integridade da estrutura, principalmente em estudos de mecanismos de falhas em compósitos, um assunto de suma importância para a indústria aeronáutica, onde esses materiais são amplamente utilizados. O presente trabalho teve como objetivo o estudo da resistência da interface bem como o mecanismo de absorção de energia durante o processo de pull-out em compósitos, incorporando-se a rugosidade superficial das fibras. Para tanto, foi realizada uma profunda análise da superfície das mesmas por meio de um levantamento da topografia das fibras de carbono T300 (com size e sem size) através de Microscopia de Força Atômica, para a determinação da rugosidade superficial, e conseqüentemente, da área e perímetro real das fibras. Novas metodologias de análise de imagens de Microscopia de Força Atômica foram desenvolvidas, para este objetivo, por meio de ajustes mais aderentes à superfície cilíndrica das fibras. Também foi realizada uma análise fractal da superfície das fibras bem como a sugestão de um modelo de ajuste por uma função matemática. Foi desenvolvido um modelo matemático para análise do processo de pull-out utilizando o método de slab. A inclusão de fatores de correção da rugosidade (convencional, ajuste de função e fractal) juntamente com condições de contorno adequadas nas equações diferenciais obtidas no modelo, possibilitou prever-se, de forma razoavelmente boa, valores de tensões e deformações, possibilitando o cálculo da energia necessária para o pull-out e a comparação desses parâmetros com os resultados experimentais dos ensaios de pull-out. Outros parâmetros relevantes também serão apresentados.

Submitted by (edna.saturno@ufrpe.br) on 2016-06-28T16:31:50Z No. of bitstreams: 1 Manoel Messias da Silva Costa.pdf: 985826 bytes, checksum: 6352d38d9d787ac4df432b7949059fd1 (MD5) / Made available in DSpace on 2016-06-28T16:31:50Z (GMT). No. of bitstreams: 1 Manoel Messias da Silva Costa.pdf: 985826 bytes, checksum: 6352d38d9d787ac4df432b7949059fd1 (MD5) Previous issue date: 2008-02-19 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Marine macroalgae are of fundamental importance in coastal ecosystems once they are primary producers and responsible for energy transference to several trophic levels. Besides, their stems support several organisms, both animals and plants which contribute to productivity increase in the coastal zones of the whole oceans. The Rhodophyta specimens, Galaxaura rugosa (J. Ellis & Solander) J.V. Lamouroux, were collected in June, 2006 and June, 2007 at three localities from Fernando de Noronha Archipelago (Atalaia, Porto and Cagarras) aiming to identify and quantify the epiphyte diatoms flora inhabiting the algae’ thallus. A total of 52 taxa were identified being distributed in the classes Coscinodiscophyceae (19%), Fragilariophyceae (21%) and Bacillariophyceae (60%), with 81% dominance of individuals with pennate symmetry. The following species characterized the floristic diatoms structure being considered very frequent once they are present in more than 70% samples analyzed: Diploneis bombus Ehrenberg, Nitzschia sp., Amphora sp., Biddulphia biddulphiana (J.E. Smith) Boyer, Grammatophora marina (Lyngbye) Kützing, Mastogloia binotata (Grunow) Cleve, Tryblionella coarctata (Grunow) Mann, Naviculalonga Grunow, Trachyneis aspera (Ehrenberg) Cleve, Psammodiscus nitidus (Gregory) Round in Mann, Rhabdonema adriaticum Kützing and Cocconeis scutellum Ehrenberg. Total diatoms density presented no significant variations among the several parts of the algae as well as among the three sampling sites presenting the minimum value of 5.000 cell.g-1.algae fresh weight in the apical part of specimens sampled in the Atalaia beach, and the maximum of 60.000 cell.g-1.algae fresh weight in the basal part of specimens from Porto beach. Results confirm that the Rhodophyta Galaxaura rugosa is a good hostess for epiphyte diatoms fixation having been observed the occurrence of these organisms in the whole stem of specimens collected with the higher densities in the basal parts, confirming the importance of these microalgae as primary producers at the diverse beaches of Fernando de Noronha. / As macroalgas marinhas são de primordial importância nos ecossistemas costeiros, pois são consideradas como produtores primários e responsáveis pela transferência de energia para diversos níveis tróficos. Além disso, seus talos suportam vários organismos, tanto animais como vegetais, os quais contribuem para o aumento da produtividade de zonas costeiras de todos os oceanos. Exemplares da rodofíta Galaxaura rugosa (J. Ellis & Solander) J.V. Lamouroux, foram coletados nos meses de junho de 2006 e junho de 2007, em três localidades do Arquipélago de Fernando de Noronha (Praias de Atalaia, Porto e Cagarras), com o objetivo de identificar e quantificar a flora das diatomáceas epífita que habita o talo da alga. Foram identificados 52 táxons distribuídos nas classes Coscinodiscophyceae (19%), Fragilariophyceae (21%) e Bacillariophyceae (60%), com dominância de indivíduos de simetria penada, correspondente a 81%. As seguintes espécies caracterizaram a estrutura florística das diatomáceas, por estarem presentes em mais de 70% das amostras analisadas e, assim, consideradas muitofreqüentes: Diploneis bombus Ehrenberg, Nitzschia sp., Amphora sp., Biddulphia biddulphiana (J.E. Smith) Boyer, Grammatophora marina (Lyngbye)Kutzing, Mastogloia binotata (Grunow) Cleve, Tryblionella coarctata (Grunow) Mann, Navicula longa Grunow, Trachyneis aspera (Ehrenberg) Cleve, Psammodiscus nitidus (Gregory) Round in Mann, Rhabdonema adriaticum Kützing e Cocconeis scutellum Ehrenberg. A densidade total das diatomáceas não apresentou variações significativas entre as diversas partes da alga como também entre os três locais de coleta, com o valor mínimo de 5.000 cel.g-1.peso fresco da alga, encontrado na parte apical de exemplares coletados na praia de Atalaia, e o máximo de 60.000 cel.g-1.peso fresco da alga, na parte basal de exemplares da Praia de Porto. Os resultados confirmaram que a rodofíta Galaxaura rugosa, mostrou-se um bom hospedeiro para fixação das diatomáceas epífitas, tendo sido observado a ocorrência desses organismos em todo o talo dos exemplares coletados, com maiores densidades nas partes basais, confirmando a importância dessas microalgas como produtores primários nas diversas praias de Fernando de Noronha.

L’étude phytochimique des extraits MeOH/H2O (7:3) des fruits de P. rugosa, P. icosandra et C. ruizteranianum, ainsi que de l’écorce de G. giganteum a conduit à l’isolement et à la caractérisation structurale de vingt-trois composés (1-23). Quatre saponines triterpéniques (1-4) dérivées de l’acide serjanique ont été isolées à partir de P. rugosa; ces composés ont précédemment été rapportés pour ce genre, mais ils sont décrits pour cette espèce pour la première fois. L’étude de P. icosandra a permis d’obtenir huit composés parmi lesquels figurent deux acides triterpéniques [l’acide spégulagénique (6) et l’acide épiacétylaleuritolique (5)], deux lignanes (7) et (8) (8, rapporté pour la première fois pour le genre et la famille), un mélange de glycosides stéroïdiens (9 et 9a), deux saponines triterpéniques (10 et 11) dérivées de l’acide serjanique et un composé aromatique (12) appartenant au groupe des peltogynoïdes. L’analyse des fruits de C. ruizteranianum a conduit à l’isolement d’un mélange de triterpènes [incluant les acides ursolique et oléanolique (13)] et sept saponines stéroïdiennes (14-20) de types spirostane (14-15) et furostane (16-20). Tous les composés ont été décrits antérieurement, mais c’est la première fois qu’il sont signalés pour l’espèce et pour le genre Cestrum. Enfin, l’écorce de G. giganteum a fourni trois saponines triterpéniques (21-23) dérivées de l’acide zanhique. L’acide 3-O-(2-O-acétyl--D-glucopyranosyl)-28-O--D-glucopyranosyl-serjanique (10), le 9,10-méthylènedioxy-5-méthoxy-peltogynane (12), l’acide 28-O-{4-O-acétyl--D-fucopyranosyl-[(12)--L-rhamnopyranosyl]-(13)--D-xylopyrano-syl}-3-O--D-gluco-ronopyranosyl-zanhique (21), l’acide 28-O-{4-O-acétyl--D-fucopyranosyl-[(12)--D-glucoronopyranosyl]-(13)--D-xylopyranosyl}-3-O--L-rhamnopyranosyl-zanhique (22) et l’acide 28-O-{4-O-acétyl--D-fuco-pyranosyl-[(12)--D-glucoronopyranosyl]-(13)--D-xylopyranosyl-(12)--D-glucopyrano-syl}-3-O--L-rhamnopyranosyl-zanhique (23) sont décrits pour la première fois comme nouveaux produits naturels. / Phytochemical analysis of the MeOH/H2O (7:3) of the fruits of Phytolacca rugosa, P. icosandra and Cestrum ruizteranianum, as well as the barks of Ganophyllum giganteum, leaded to the isolation and structural characterization of twenty three (1-23) compounds. From P. rugosa, four (1-4) known triterpenoidal saponins belonging to the serjanic acid series were obtained, all saponins are reported for the first time for P. rugosa. P. icosandra was studied and eight compounds were isolated, two triterpenic acids [spergulagenic acid (5) and epiacetylaleurytolic acid (6)], two lignanes (7) and (8) [8, reported for the first time in Phytolaccaceae], a mixture of steroidal glycosides (9) and (9a), two triterpenoidal saponins (10 - 11) and a peltoginoid-type compound (12). Analysis of C. ruizteranianum leaded to the isolation of a triterpene mixture [oleanolic and ursolic acids (13)] and seven known (14-20) spyrostane (14, 15) and furostane-type (16-20) saponins. All saponins are reported for the firs time in Cestrum genus. Finally, from the bark of G. giganteum, three saponins (21-23) belonging to the zanhic acid series were obtained. Compounds determined as: 3-O-(-D-2-acetoxy-glucopyranosyl) 28-O--D-glucopyranosyl-serjánic acid (10) ; 9,10-metylendioxy, 5-metoxy-peltoginane (12); 28-O-{-D-4-O-acetylfucopyranosyl [(12)--L-rhamno-pyranosyl] (13)--D-xylo-pyranosyl}-3-O--D-glucoronopyranosyl-zanhic acid (21); 28-O-{-D-4-O-acetyl-fucopyranosyl [(12)--D-glucoronopyranosyl] (13)--D-xylopyranosyl}-3-O--L-rhamnopyranosyl-zanhic acid (22) and 28-O-{-D-4-O-acetylglucopyranosyl [(12)--D-glucoronopyranosyl] (13)--D-xylopy-ranosyl (12)--D-glucopyrano-syl}-3-O--L-rhamnopyranosyl-zanhic acid (23), are described in this study as new natural products.

Les lipases, protéines ubiquitaires, sont les enzymes les plus étudiées et les plus utilisées dans l’industrie. Elles catalysent un très grand nombre de réactions, d’hydrolyse et de synthèse, conduisant à une grande diversité de molécules, acides, esters, amides…. Les domaines d’applications sont nombreux : les bio-énergies, les arômes, bio-lubrifiants, bio-plastifiants, émulsifiants, produits phytosanitaires et détergents, cosmétiques, synthons pour la chimie fine, produits pharmaceutiques… Aujourd’hui, grâce aux outils génétiques, il est possible de modifier leur activité, spécificité et thermostabilité pour les adapter idéalement aux contraintes industrielles. Dans ce travail de doctorat, nous nous sommes intéressés à quatre lipases d’intérêt industriel. Les 3 premières appartiennent à la famille des lipases de Candida rugosa (Lip1, Lip3 et Lip4). Bien que très homologues, leurs spécificités sont très différentes. Elles se distinguent de toutes les autres lipases par un site actif composé d’un long tunnel avec la triade catalytique à l’entrée de celui-ci. Cela en fait une enzyme particulièrement intéressante pour la conversion et la purification d’acides gras à longue chaîne. La quatrième est une nouvelle lipase identifiée chez la levure oléagineuse, Yarrowia lipolytica. Elle est très active sur les acides gras à longue chaîne, active à pH acide et présentant une grande énantiosélectivité sur des molécules d’intérêt pharmaceutique, les esters d’acide 2- halogéno-aryl acide acétique. Dans un premier temps, un nouveau système d’expression, une souche spécifique de Yarrowia lipolytica, a été étudié pour l’expression de variants construits par mutagenèse dirigée. Cette souche JMY1212 permet une intégration ciblée dans le génome de Y. lipoytica. Nous avons démontré qu’il s’agissait du premier système d’expression permettant de comparer statistiquement l’activité de variants directement à partir du surnageant de culture. Trois des lipases de Candida rugosa ont été clonées avec succès dans cette souche et leurs activités et spécificités vis-à-vis de la longueur de chaines des acides gras ont été étudiées. Lip1 et Lip3 présentent une spécificité pour les acides gras à longueur de chaine moyenne (C8-C10) alors que Lip4 préfère les C18:1. De, plus, pour la première fois, la purification, à partir d’un mélange d’esters éthyliques issu d’huile de poissons, d’acides gras poly-insaturés (PUFAs); acides cis-5, 8, 11, 14, 17-eicosapentaenoic (EPA) et cis-4, 7, 10, 13, 16, 19-docosahexaenoic (DHA), molécules bonnes pour la santé, a été réalisée avec les trois lipases séparées de C. rugosa. Quelle que soit l’enzyme, le rendement de récupération du DHA est supérieur à 93 % (97, 100 et 93 % pour Lip1, Lip3 et Lip4 respectivement. Une pureté maximale en DHA de ~60 % a été obtenue avec Lip3 et Lip4, à partir d’un mélange initial d’esters éthyliques contenant 25% de DHA. Une différence remarquable entre ces trois enzymes est que Lip4 est capable de mieux hydrolyser l’ester d’EPA (60% contre 14 et 16% pour Lip1 et Lip3). Lip4 est même capable d’hydrolyser le DHA (7% contre 3 et 0 % pour Lip1 et Lip3). La deuxième partie de ce travail a été consacrée à l’amélioration de l’énantiosélectivité des deux enzymes étudiées vis-à-vis de synthons d’intérêt dans l’industrie pharmaceutique, les esters de 2-bromo aryl acide acétique. La construction raisonnée d’un double variant de la lipase Lip2 de Y. lipolytica, D97AV232F, a permis d’obtenir une enzyme totalement énantiosélective (E >200). Celle-ci reconnaît l’énantiomère R alors que la lipase sauvage avait une faible préférence pour l’énantiomère S (E=5). Par ailleurs, cette exceptionnelle augmentation de l’énantiosélectivité s’accompagne d’une amélioration de l’activité de l’enzyme qui est ainsi multipliée par 4,5. Sur ce même mélange d’énantiomères, les 3 lipases de C. rugosa se sont avérées remarquables. Malgré leur grande homologie, leur spécificité est différente. Lip1 et Lip3 sont totalement S spécifiques (E>200), alors que Lip4 est R spécifique (E=15). Le docking moléculaire des énantiomères S et R dans le site actif des lipases Lip1 et Lip4 a permis de mieux comprendre ces différences de spécificité et de proposer des cibles de mutagenèse dirigée. L’encombrement et la nature de l’acide aminé présent en position 296 sont cruciaux pour la discrimination de l’enzyme / Lipases, ubiquitous proteins, are the most studied enzymes and the most used in industry. They catalyse a great number of reactions, hydrolysis and synthesis, leading to a great diversity of molecules, acids, esters, amides. There are numerous fields of applications: bio-energies, flavours, bio-lubricants, bio-plasticizers, emulsifiers, detergents, cosmetics, synthons for fine chemistry, and pharmaceutical products. Nowadays, thanks to genetic tools, it is possible to modify their activity, specificity and thermostability to ideally adapt enzymes for the industrial constraints. In this work, we were interested in four lipases of industrial interest. The third ones belong to the lipase family of Candida rugosa (Lip1, Lip3 and Lip4). Although they present high homology, their specificities are very different. They are distinct from the other lipases by the active site composed of a long tunnel with the catalytic triad at the entry of the tunnel. It leads to enzymes particularly interesting for the conversion and the purification of long chain fatty-acids. The fourth one is a new lipase identified from oleaginous yeast, Yarrowia lipolytica. It is one of the most active lipase on long chain fatty-acids; it is very active and stable at acid pH and presents a high enantioselectivity on molecules of pharmaceutical interest, the esters of 2- halogeno-aryl acetic acid. In this work, we first tested a new expression system, a specific strain of Y. lipolytica, for expression of variants obtained by site-directed mutagenesis. This strain JMY1212 enables integration to be targeted to a special locus of the Y. lipoytica genome. We demonstrated that it is the first expression system in which it is possible to compare statistically variant activities directly from the supernatant of the culture. Secondly, three lipases of C. rugosa were cloned successfully in this strain and their activities and specificities with respect to fatty acid chain lengths were studied. Lip1 and Lip3 have specificity for the fattyacids of medium chain (C8-C10) whereas Lip4 prefers C18: 1. Moreover, for the first time, purification, from a mixture of ethyl esters issued from fish oil, polyunsaturated fatty acids (PUFAs); cis-5, 8, 11, 14, 17- eicosapentaenoic acid (EPA) and cis-4, 7, 10, 13, 16, 19-docosahexaenoic acid (DHA), molecules with health benefits, was realised with the three C. rugosa lipases, separately. Whatever the enzyme the recovery of DHA is superior to 90 % (97, 100 and 93 % for Lip1, Lip3 and Lip4 respectively. The maximal DHA purity ~60 % was obtained with Lip3 and Lip4, with an initial ethyl ester mixture containing 25% DHA. A remarkable difference between these enzymes lies in the fact that Lip4 is able to better hydrolyse the EPA esters (60% against 13% and 16% respectively for Lip1 and Lip3). Lip4 is also able to hydrolyse DHA (7% against 3 and 0 % for Lip1 and Lip3 respectively). The third part of this work was devoted to the improvement of the enantioselectivity of the two enzymes studied with respect to the resolution of a racemic mixture of pharmaceutical industry, the R, S esters of 2-bromo aryl acetic acid. The rational construction of a double variant of Lip2 lipase from Y. lipolytica, D97A V232F was realized to obtain a total enantioselective enzyme (E > 200). This variant recognizes the enantiomer R whereas wild-type lipase had a weak preference for the enantiomer S (E=5). In addition, this exceptional increase in the enantioselectivity is accompanied by a 4.5 fold improvement of the activity. With the same mixture of enantiomers, the 3 lipases of C. rugosa proved to be remarkable from the point of view of enantioselectivity. In spite of their high homology, their specificity is different. Lip1 and Lip3 are completely specific for the S enantiomer, whereas Lip4 is R specific (E=15). The molecular docking of the S and R enantiomers in the active site of Lip1 and Lip4 lipases enables the observed differences in specificity to be better understood and targets for site-directed mutagenesis to be proposed. We demonstrated that the nature of the amino acid present in position 296 is crucial for the discrimination of these enzymes

Plants of the genus Kielmeyera, family Clusiaceae are used by the population of the Northeast of the Brazil in traditional medicine to treat various diseases, such as pain and inflammation diseases. A recent study demonstrated antitumor activity for K. rugosa, however, other pharmacological activities have never been studied, for example antihyperalgesic and anti inflammatory activities. Thus, the aim of this study was to evaluate the antihyperalgesic and anti - inflammatory effects of the methanol extract from the stems of K. rugosa (MEKR) in rodents. Swiss mice (25-35 g ) were divided into groups and subjected to treatment with MEKR (., 100, 200 and 400 mg/kg, oral administration, p.o.) , vehicle (0.9% saline solution + 0.2% Tween 80) or standard drug (i.p.). The hypernociception was evaluated at times 0.5 , 1, 2 , and 3 hours after administration (i.pl.) of carrageenan (CG; 300 mg/paw), tumor necrosis factor-α (TNF-α, 100 pg/paw), Prostaglandin E2 (PGE2; 100ƞg/pata) or dopamine (DA, 30 μg/paw) using the digital analgesymeter (von Frey). In the evaluation of anti-inflammatory activity two protocols were used, the first induced by GC (300 mg/well) at 4 hours after pleurisy which the full and differential counts were made of leukocytes as well as the dosage levels of TNF-α and IL-1β pleural lavage. In another protocol conducted to investigate the anti-inflammatory activity corresponded to paw edema induced by GC (1 %/40μL), the paw volume was measured with the aid of plethysmometer at 0-6h after CG. The cytotoxicity of MEKR was evaluated by the MTT colorimetric method. To determine the possible involvement of areas of the Central Nervous System (CNS), the animals were treated and ninety minutes, were anesthetized, perfused, the brains extracted and cut into the cryostat. The brain sections were subjected to immunofluorescence protocol for Fos protein. The motor coordination of the animal was assessed by the Rota Rod test (7 rpm, 180 s). The experimental protocols were approved by the ethics committee of the UFS (CEPA/UFS: 102/11) The results were expressed as mean ± standard error of the mean. Differences between groups were analyzed using the ANOVA one way followed by Tukey s test. The acute pretreatment with MEKR significantly inhibited hyperalgesia induced by nociceptive agents, CG, TNF-α, PGE2 and DA (p < 0.001). The MEKR inhibited the leukocyte recruitment into the pleural cavity (p < 0.01). This was due to the inhibition of leukocyte migration inhibition of neutrophils. The levels of cytokines, TNF-α (p < 0.01) and IL-1β (p < 0.001) were also reduced when the animals were treated with MEKR. MEKR decreased edema formation induced by CG (p < 0.001). However, MEKR showed no cytotoxic effect or change in motor coordination animals. In the Fos protein immunofluorescence test, MEKR showed that the olfactory bulb (p < 0.01), piriform cortex (p < 0.01) and periaqueductal gray (p < 0.001) for the active significantly of the CNS. Therefore, we conclude that MEKR has antihyperalgesic activity probably by activating the central nervous system areas associated with pain modulation and by reduction the production of proinflammatory cytokines, without traces of cytotoxicity. / Diversas plantas do gênero Kielmeyera, família Clusiaceae são utilizadas pela população do Nordeste brasileiro na medicina tradicional para o tratamento de diversas doenças, incluindo distúrbios dolorosos e inflamatórios. Um estudo recente demonstrou atividade antitumoral para a K. rugosa, no entanto, outras atividades farmacológicas nunca foram estudadas para esta espécie vegetal, por exemplo seu possível perfil analgésico e anti-inflamatório. Dessa forma, o objetivo do trabalho foi avaliar o efeito antihiperalgésico e anti-inflamatório do extrato metanólico obtido do caule de K. rugosa (EMKR) em roedores. Foram utilizados camundongos Swiss machos (25-35 g), divididos em grupos e submetidos ao tratamento agudo com EMKR (100, 200 e 400 mg/kg; por via oral, v.o.), veículo (solução salina 0,9% + tween 80 0,2%; v.o.) ou droga padrão (i.p.). A hipernocicepção foi avaliada nos tempos 0,5, 1, 2 e 3 h após a administração (i.pl.) de carragenina (CG; 300 μg/pata), Fator de necrose tumoral-α (TNF-α; 100 pg/pata), Prostaglandina E2 (PGE2; 100ƞg/pata) ou Dopamina (DA; 30 μg/pata), e foram avaliados utilizando-se o analgesímetro digital (Von Frey). Na avaliação da atividade anti-inflamatória foram utilizados dois protocolos, o primeiro de pleurisia induzida por CG (300 μg/cavidade), no qual após 4 horas foram realizadas as contagens total e diferencial de leucócitos, bem como as dosagens dos níveis de TNF-α e IL-1β do lavado pleural. Em outro protocolo realizado para investigar à atividade anti-inflamatória correspondeu ao edema de pata induzida por CG (1%/40μL); o volume da pata foi medido com auxílio do pletismomêtro nos tempos 0-6h após a CG. A citotoxicidade do MEKR foi avaliada através do método colorimétrico do MTT. Para determinar o possível envolvimento de áreas do Sistema Nervoso Central (SNC), os animais foram tratados e, noventa minutos após, foram anestesiados, perfundidos, os cérebros extraídos e cortados em criostato. As secções cerebrais foram submetidas ao protocolo de imunofluorescência para proteína Fos. A coordenação motora do animal foi avaliada através do teste do Rota Rod (7 rpm, 180 s). Os protocolos experimentais foram aprovados pelo comitê de ética da UFS (CEPA/UFS: 102/11) Os resultados foram expressos como média ± erro padrão da média. As diferenças entre os grupos foram analisadas por meio do teste de variância ANOVA, uma via, seguido pelo teste de Tukey. O pré-tratamento com EMKR inibiu significativamente a hiperalgesia induzida pelos agentes álgicos, CG, TNF-α, PGE2 e DA (p < 0,001). O EMKR também foi capaz de inibir o recrutamento leucocitário para a cavidade pleural (p < 0,01). Esta inibição leucocitária ocorreu devido à inibição na migração dos neutrófilos. Os níveis das citocinas, TNF-α (p < 0,01) e IL-1β (p < 0,001) também foram reduzidos quando os animais foram tratados com EMKR. Quanto ao edema, o EMKR diminuiu a formação induzida pela CG (p < 0,001). No entanto, o MEKR não apresentou efeito citotóxico ou alteração da coordenação motora dos animais. No teste de imunofluorescência para proteína Fos, o tratamento com MEKR ativou significativamente o bulbo olfatório (p < 0,01), córtex piriforme (p < 0,01) e a substância cinzenta periaquedutal (p < 0,001), áreas do SNC. Os resultados sugerem que o MEKR possui atividade antihiperalgésica e anti-inflamatória provavelmente por ativação de áreas do sistema nervoso central relacionadas com a modulação da dor e por reduzir a produção de citocinas pró-inflamatórias, sem traços de citotoxicidade.