FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico / Moringa oleifera Lam. à uma planta nativa do nordeste da Ãndia muito utilizada na medicina popular devido Ãs suas vÃrias propriedades farmacolÃgicas. Estudos etnofarmacolÃgicos tÃm demonstrado atividade hipoglicemiante de compostos oriundos de partes dessa planta, principalmente de suas folhas, em modelos de animais diabÃticos e, tambÃm, em humanos, embasando sua utilizaÃÃo na medicina popular para o tratamento do diabetes. A aÃÃo hipoglicemiante de compostos de folhas de M. oleifera tem sido creditada a componentes oriundos do metabolismo secundÃrio vegetal, sendo escassos trabalhos abordando a participaÃÃo de proteÃnas nessa aÃÃo farmacolÃgica. O objetivo deste trabalho foi obter uma fraÃÃo proteica a partir de folhas de M. oleifera e verificar seu efeito hipoglicemiante em modelos de animais diabÃticos. Para isso, foi realizada extraÃÃo de proteÃnas a partir de folhas de M. oleifera, seguida de precipitaÃÃo com sulfato de amÃnio (0-90%). ApÃs diÃlise exaustiva, o material foi liofilizado para obtenÃÃo da fraÃÃo proteica denominada Mo-FPF. Como modelo de animal experimental, foram utilizados camundongos com diabetes quimicamente induzido por aloxano. Inicialmente, doses de 100, 300 e 500 mg/Kg de peso corpÃreo de Mo-FPF foram administradas intraperitonealmente, tendo sido a dose de 500 mg/Kg de peso corpÃreo a mais efetiva na reduÃÃo glicÃmica apÃs 1, 3 e 5 horas da administraÃÃo. Tal efeito hipoglicemiante nÃo foi verificado pela rota intragÃstrica. AlÃm disso, esse efeito sofreu reduÃÃo quando Mo-FPF foi previamente fervida e administrada por via intraperitoneal. Mo-FPF, administrada diariamente pela rota intraperitoneal, na dose de 500 mg/Kg, durante 20 dias, resultou em reduÃÃo glicÃmica, alÃm de ter exercido efeito hipocolesterolemiante e antioxidante. O perfil eletroforÃtico de Mo-FPF mostrou uma diversidade de bandas proteicas, que foram suscetÃveis à aÃÃo da pepsina e tripsina em ensaio de digestibilidade in vitro. AtravÃs de imunoensaio por Dot Blot, foi verificada reaÃÃo cruzada entre o anticorpo anti-insulina humana e Mo-FPF, sugerindo a existÃncia de epÃtopos antigÃnicos do tipo insulina em proteÃnas de folhas de M. oleifera. Assim, o conjunto de dados obtidos mostra que proteÃnas oriundas de folhas de M. oleifera contribuem para o efeito hipoglicemiante demonstrado neste trabalho. / Moringa oleifera is a plant native to northeastern India widely used in Indian folk medicine due to its various pharmacological properties. Ethnopharmacological studies have demonstrated that compounds derived from parts of this plant, especially leaves, have hypoglycemic activity in diabetic animal models and in humans, allowing its use in folk medicine. The hypoglycemic action of M. oleifera leaves has been attributed to compounds from plant secondary metabolism, however studies showing the involvement of proteins as antidiabetic substances are scarce. The aim of this study was to obtain a protein fraction of M. oleifera leaves and evaluate its hypoglycemic effects on diabetic animal models. For this, the leaf proteins were extracted followed by ammonium sulfate precipitation (0-90%). After exhaustive dialysis, this material was lyophilized to obtain the protein fraction named Mo-PFL. As experimental animal model, alloxan-induced diabetic mice were used. Initially Mo-PFL was intraperitoneally administered at doses of 100, 300 and 500 mg/Kg body weight. The dose of 500 mg/Kg body weight was more effective in reducing blood glucose after 1, 3 and 5 hours of Mo-PFL administration. This hypoglycemic effect was not observed by the intragastric route. This effect was also reduced when boiled Mo-PFL was intraperitoneally administered. Daily intraperitoneal administration of Mo-PFL at a dose of 500 mg/Kg body weight for 20 days caused a significant reduction in blood glucose level and also exerted antioxidant and hypocholesterolemic effects. Electrophoretic pattern of Mo-PFL showed a variety of protein bands, which were susceptible to in vitro pepsin and trypsin digestion. A Dot Blot immunoassay showed cross reactivity between human anti-insulin antibody and Mo-PFL, suggesting the presence of insulin-like epitopes in M. oleifera leaf protein. Overall, these data show that some proteins derived from M. oleifera leaves may contribute to the hypoglycemic effect observed in the present work.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Mo-CBP3 à uma proteÃna ligante à quitina, purificada de sementes de Moringa oleifera, com amplo espectro de aÃÃo contra fungos fitopatogÃnicos. No presente trabalho, novas propriedades estruturais da Mo-CBP3 sÃo descritas, revelando correlaÃÃo entre sua estabilidade estrutural e atividade antifÃngica. Em adiÃÃo, para melhor compreensÃo dos mecanismos pelos quais essa proteÃna exerce aÃÃo antifÃngica, sua habilidade de induzir a produÃÃo endÃgena de espÃcies reativas de oxigÃnio e de causar alteraÃÃes morfolÃgicas e ultraestruturais foi analisada, usando Fusarium solani como modelo. F. solani à uma espÃcie de fÃcil manuseio e desenvolvimento rÃpido, ideal para ensaios in vitro, e de relevÃncia, por se tratar de um fungo que ataca culturas economicamente importantes. Com foco na utilizaÃÃo segura da Mo-CBP3 como agente quÃmico contra fungos, seus efeitos citotÃxicos sobre cÃlulas eucariÃticas tambÃm foram investigados. Mo-CBP3 à uma proteÃna ligante à quitina de 18,0 kDa, de acordo com PAGE-SDS. Todavia, anÃlise por espectrometria de massas revelou que essa proteÃna consiste de mÃltiplas isoformas com massas moleculares variando entre 12,2 e 12,3 kDa. Mo-CBP3 à composta por duas cadeias polipeptÃdicas de 5,0 e 9,0 kDa, denominadas de cadeia A e cadeia B, respectivamente. A cadeia B contÃm a sequÃncia NH2-terminal representada por CPAIQRCCQQLRNIQPPCRCCQ, enquanto que a cadeia A tem o resÃduo NH2-terminal bloqueado. cDNA codificador da cadeia B foi obtido com iniciadores sintetizados a partir de sua sequÃncia NH2-terminal. AnÃlises in silico das sequÃncias de nucleotÃdeos e de aminoÃcidos deduzida confirmaram a presenÃa de isoformas e massa molecular da Mo-CBP3 e identificaram sÃtios potenciais de O-glicosilaÃÃo e fosforilaÃÃo. AlÃm disso, similaridades entre Mo-CBP3 e outras proteÃnas de M. oleifera, bem como com albuminas 2S, foram detectadas. A estrutura secundaria da Mo-CBP3 à composta por 30,3% α-hÃlices, 16,3% folhas β, 22,3% voltas e 30,4% estruturas ao acaso. Na espectroscopia de fluorescÃncia, excitaÃÃes de uma soluÃÃo da Mo-CBP3 a 280 nm e 295 nm produziram emissÃo mÃxima a 303 e 309 nm, respectivamente. A estrutura da Mo-CBP3 à altamente estÃvel, se apresentando indiferente Ãs mudanÃas de temperatura e pH. Mo-CBP3 (0,05-0,1 mg/mL) se mostrou capaz de inibir a germinaÃÃo de conÃdios de vÃrios fungos fitopatogÃnicos, incluindo F. solani, F. oxysporum, Colletotrichum musae e C. gloeosporioides. Similarmente, Mo-CBP3 (0,05 mg/mL) foi capaz de inibir o crescimento micelial de F. solani e apresentou tanto efeito fungistÃtico como fungicida, dependendo da concentraÃÃo usada. LigaÃÃo da Mo-CBP3 à superfÃcie de cÃlulas fÃngicas ocorre, pelo menos em parte, via interaÃÃo eletrostÃtica, jà que NaCl 0,15 M aboliu seu efeito inibitÃrio. Mo-CBP3 induziu a produÃÃo de espÃcies reativas de oxigÃnio e causou perda de assimetria e deformaÃÃes em cÃlulas de F. solani. DesorganizaÃÃo do sistema de endomembranas, condensaÃÃo do citosol e aumento de vacuolizaÃÃo tambÃm foram observados. Mo-CBP3 nÃo mostrou atividade hemolÃtica e nem foi capaz de alterar a viabilidade das cÃlulas MCF-7 e Caco-2, sugerindo que essa proteÃna nÃo à tÃxica para cÃlulas humanas. Com base na alta estabilidade e no amplo espectro de aÃÃo contra fungos fitopatogÃnicos em baixas concentraÃÃes e, tambÃm, na ausÃncia de citotoxicidade para cÃlulas humanas testadas, Mo-CBP3 tem grande potencial para desenvolvimento de novas drogas antifÃngicas ou na produÃÃo de plantas transgÃnicas mais resistentes a fungos. / Mo-CBP3 is a chitin-binding protein purified from Moringa oleifera seeds that displays broad inhibitory activity against phytopathogenic fungi. In this work, we report new structural features of Mo-CBP3 that reveal a correlation between its structural stability and antifungal activity. In addition, to gain better insights into the mechanisms by which this protein acts as an antifungal agent, its ability to induce the endogenous production of reactive oxygen species and to trigger morphologic and ultrastructural alterations were analysed using Fusarium solani as a model. F. solani is an easy-to-handle and fast-developing species, making it ideal for in vitro assays, and it holds relevance as a phytopathogenic fungus that attacks economically important crop plants. To fully explore the biosafety of Mo-CBP3 as a chemical agent against fungi, its cytotoxic effects on eukaryotic cells were also investigated. Mo-CBP3 is a chitin-binding protein of 18.0 kDa, according to SDS-PAGE. However, by mass spectrometry analysis, it was observed that this protein consists of multiple isoforms with molecular masses ranging between 12.2 and 12.3 kDa. Mo-CBP3 is composed by two polypeptide chains of 5.0 and 9.0 kDa, named A and B chain, respectively. The B chain contains the following NH2-terminal sequence CPAIQRCCQQLRNIQPPCRCCQ while the A chain has a blocked NH2-terminal residue. cDNA encoding the B chain was obtained with primers of its NH2-terminal sequence. In silico analyses of nucleotide and deduced amino acid sequences confirmed the presence of isoforms and molecular mass of Mo-CBP3 and identified potential sites of O-glicosylation and phosphorilation. Moreover, similarities between Mo-CBP3 and other M. oleifera proteins as well as 2S albumins were detected. The secondary structure of Mo-CBP3 showed 30.3% α-helices, 16.3% β-sheets, 22.3% turns and 30.4% unordered forms. In the fluorescence spectroscopy, excitation of Mo-CBP3 solution at 280 nm and 295 nm gave emission maxima at 303 and 309 nm, respectively. The Mo-CBP3 structure is highly stable and retains its antifungal activity regardless of temperature and pH. Mo-CBP3 (0.05-0.1 mg/mL) was able to inhibit the conidia germination of several phytopathogenic fungi, including F. solani, F. oxysporum, Colletotrichum musae and C. gloeosporioides. Similarly, Mo-CBP3 was inhibitory to the mycelial mass development of F. solani at 0.05 mg/mL and has both fungistatic and fungicidal effects, depending on the concentration used. Binding of Mo-CBP3 to the fungal cell surface is achieved, at least in part, via electrostatic interactions, as 150 mM NaCl abolished its inhibitory effect. Mo-CBP3 induced the production of reactive oxygen species and caused in F. solani cells a marked loss of asymmetry, deformations and deep wrinkles in comparison to control cells. Disorganisation of the endomembrane system and condensation and shrinkage of cytosol with increased vacuolation and the loss of normal structure and content were also observed. Mo-CBP3 did not show haemolytic activity and it was not capable to alter de viability of both MCF-7 and Caco-2 cells, suggesting that this protein is not toxic for human cells. Based on its high stability and broad-spectrum efficacy against important phytopathogenic fungi at low inhibitory concentrations and absence of cytotoxicity to human cells, Mo-CBP3 has great potential in the development of new antifungal drugs or in transgenic crops with enhanced resistance to fungi.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Mo-CBP4 à uma proteÃna ligante à quitina, com atividade anti-inflamatÃria e antinociceptiva, isolada de sementes de Moringa oleifera Lamarck, uma Ãrvore conhecida pelo seu valor medicinal. Este trabalho teve como objetivo a caracterizaÃÃo estrutural, farmacolÃgica e toxicolÃgica de Mo-CBP4, com foco na sua utilizaÃÃo como agente terapÃutico. Mo-CBP4 à um heterodÃmero de 11,8 kDa, composto por cadeias de 3,9 kDa e 8,4 kDa. O sequenciamento de Mo-CBP4 por degradaÃÃo de Edman e espectrometria de massas mostrou a presenÃa de muitos resÃduos de aminoÃcidos bÃsicos e alta similaridade com proteÃnas isoladas de sementes de M. oleifera. AnÃlises espectroscÃpicas utilizando dicroÃsmo circular demonstraram que Mo-CBP4 à composta por α-hÃlices (36%), folhas-β (15%), voltas (19%) e estruturas ao acaso (30%), com alta estabilidade estrutural frente a variaÃÃes de temperatura e pH. Mo-CBP4 (0,1, 1,0 e 10 mg/kg), por via endovenosa, mostrou atividade anti-inflamatÃria no modelo de peritonite induzida por carragenina em ratos, com inibiÃÃo (79%) da migraÃÃo de neutrÃfilos jà na menor dose. Atividade anti-inflamatÃria de Mo-CBP4 (40 mg/kg) tambÃm ocorreu por via oral em camundongos, usando o mesmo modelo citado, com significativa inibiÃÃo (48%) da migraÃÃo de neutrÃfilos. Tal atividade permaneceu mesmo apÃs aquecimento da proteÃna a 100 ÂC por 1 hora, porÃm, a prÃ-incubaÃÃo com N-acetil-D-glucosamina 0,1 M foi capaz de reverter sua aÃÃo. Os nÃveis sÃricos de IL-1β e IL-10, apÃs induÃÃo de peritonite, sofreram alteraÃÃo com o prÃ-tratamento da proteÃna. Adicionalmente, Mo-CBP4 (10 mg/kg) mostrou atividade antinociceptiva, verificada atravÃs do teste da formalina (2,5%), quando administrada via intraperitoneal, inibindo apenas a dor perifÃrica. No modelo de hipernocicepÃÃo mecÃnica induzida por carragenina, animais tratados via oral com a proteÃna (20, 40 e 80 mg/kg), 1 hora antes do agente inflamatÃrio, mostraram-se mais resistentes à sensibilizaÃÃo apÃs 1, 3 e 5 horas, apresentando inibiÃÃo mÃxima na dose de 40 mg/kg, estando essa atividade relacionada com a reduÃÃo da migraÃÃo de neutrÃfilos. A hipernocicepÃÃo direta causada por PGE2 ou epinefrina nÃo sofreu alteraÃÃo com o prÃ-tratamento de Mo-CBP4. A proteÃna tambÃm nÃo causou comprometimento na atividade locomotora dos animais, quando submetidos ao teste do campo aberto. A administraÃÃo oral de Mo-CBP4 em dose Ãnica (2000 mg/kg) ou em doses repetidas (10, 40 e 100 mg/kg) nÃo resultou em efeitos tÃxicos ou adversos. Similarmente, avaliaÃÃo da citotoxicidade e genotoxicidade de Mo-CBP4, utilizando cÃlulas tumorais ou normais, mostrou ausÃncia de toxicidade. Os resultados obtidos sugerem que devido Ãs propriedades anti-inflamatÃria e antinociceptiva, alta estabilidade e aparente ausÃncia de toxicidade, Mo-CBP4 possui grande potencial como um futuro biofÃrmaco. / Mo-CBP4 is a chitin-binding protein that is associated with the antinociceptive and anti-inflammatory effects of Moringa oleifera seeds, a tree known for its medicinal value. The aim of this study was the structural, pharmacological and toxicological characterization of Mo-CBP4, focusing on its use as a therapeutic agent. Mo-CBP4 is a 11.8 kDa heterodimer, composed by 3.9 kDa and 8.4 kDa chains. Mo-CBP4 sequencing by Edman degradation and mass spectrometry showed the presence of many basic amino acid residues and high similarity with other proteins from M. oleifera seeds. Circular dichroism spectroscopy showed that the Mo-CBP4 contains 36% α-helix, 15% β-sheet, 19% turn and 30% unordered and high structural stability to temperature and pH variations. Mo-CBP4 (0.1, 1.0 and 10 mg/kg) by intravenous administration showed anti-inflammatory activity on the model of carrageenan-induced peritonitis in rats, with inhibition (79%) of neutrophil migration already at the lowest dose. Mo-CBP4 (40 mg/kg), when administered by oral route on mice, using the same mentioned model, also presented anti-inflammatory activity, causing a significant inhibition (48%) of neutrophil migration. Mo-CBP4 retained this activity even after heating at 100 ÂC or pre-incubation with 0.1 M N-acetyl-D-glucosamine, both for 1 hour. Serum levels of IL-1β and IL-10 after peritonitis induction changed in Mo-CBP4-pretreated mice. In addition, Mo-CBP4 (10 mg/kg), administered intraperitoneally, also showed antinociceptive activity by using the formalin (2.5%) test in mice model, inhibiting just the peripheral pain. In the carrageenan-induced mechanical hypernociception model, animals pretreated with oral Mo-CBP4 (20, 40 and 80 mg/kg), 1 hour prior to inflammatory agent use, proved to be more resistant to sensitization after 1, 3 and 5 hours, with maximal inhibition at a dose of 40 mg/kg, being this activity correlated with the reduction of neutrophil migration. Direct hypernociception caused by epinephrine or PGE2 was not affected with Mo-CBP4 pretreatment. Mo-CBP4 did not impair the locomotor activity of animals in the open field test. In general, no toxic signs or adverse effects were seen after oral administration of Mo-CBP4 at a single dose (2000 mg/kg) or after repeated doses (10, 40 and 100 mg/kg). Similarly, cytotoxicity and genotoxicity tests with Mo-CBP4, using tumour or normal cells, showed no toxicity. The results suggest that due to anti-inflammatory and antinociceptive properties, high stability and apparent lack of toxicity, Mo-CBP4 has great potential as a future biopharmaceutical drug.

Moringa oleifera Lam (Moringa) is a kind of easy adaptation to the Brazilian semiarid region, but its cultivation is rudimentary. Missing information about the physical and physiological quality of seeds, seedlings assessment under conditions of water stress and the development of plantations in different spacings. Thus, the present study aimed to characterize the internal morphology and identification of fungi from the seeds, the physiological assessment of seedlings subjected to water restriction and evaluation of the initial growth of moringa under gradient spacing. The morphology of the seeds was observed using the X-ray test with an intensity of 22 kV for 14.8 s in Laboratório de Análise de Sementes of the Universidade Federal de Laras (UFLA) using 200 seeds of two different genotypes. The identification and quantification of fungi were made by the blotter test the Laboratório de Patologia de Sementes of the UFLA using 200 seeds of two different genotypes. The seedlings from seeds of a genotype were evaluated under water restriction at 40, 60, 80 and 100% of field capacity for 21 days to ecophysiological parameters and biometric. The gradient spacing planting was installed in the Experimental Campus of the Universidade Federal de Sergipe using the systematic design, with 10 different densities, and the plants were grown from seeds of four genotypes. Seeds in relation to their internal morphology were divided into three classes: (a) full and well formed, (b) with space and malformed and (c) stained or damage. The health test were observed following fungi: Aspergillus niger, Aspergillus flavus, Alternaria sp., Fusarium sp., Penicillium sp. and Phomopsis sp. Moringa seedlings are more sensitive to water deficit restriction under 40% of field capacity with respect to gas exchange parameters. Plants under gradient spacing showed better development unit of area equal to or greater than 9.5 m2 plant-1. We conclude that planting moringa can be deployed with a density less than or equal to 1,000 plants per hectare and irrigation between 100% and 60% of field capacity. / Moringa oleifera Lam. (moringa) é uma espécie de fácil adaptação ao semiárido brasileiro, contudo o seu cultivo é rudimentar. Faltam informações sobre a qualidade física e fisiológica das sementes, avaliação de mudas sob condições de restrição hídrica e o desenvolvimento de plantios em diferentes espaçamentos. Assim, o presente trabalho teve por objetivo a caracterização da morfologia interna e identificação de fungos das sementes, a avaliação fisiológica de mudas submetidas à restrição hídrica e a avaliação do desenvolvimento inicial de plantas de moringa sob gradiente de espaçamento. A morfologia interna das sementes foi observada empregando o teste de raios X com intensidade de 22 kV por 14,8 s no Laboratório de Análise de Sementes da Universidade Federal de Lavras (UFLA), utilizando 200 sementes de dois diferentes genótipos. A identificação e quantificação de fungos foram realizadas por meio do blotter test no Laboratório de Patologia de Sementes da UFLA, utilizando 200 sementes de dois diferentes genótipos. As mudas, provenientes de sementes de um genótipo, foram avaliadas sob restrição hídrica a 40, 60, 80 e 100% da capacidade de campo durante 21 dias para os parâmetros ecofisiológicos e biométricos. O gradiente de espaçamento em plantio foi instalado no Campus Experimental da Universidade Federal de Sergipe empregando o delineamento sistemático, com 10 diferentes densidades, e as plantas foram oriundas de sementes de quatro genótipos. As sementes com relação à sua morfologia interna foram divididas em três classes: (a) cheias e bem formadas, (b) com espaço e mal formadas e (c) manchadas ou com dano. No teste de sanidade foram observados os seguintes fungos: Aspergillus niger, Aspergillus flavus, Alternaria sp., Fusarium sp., Penicillium sp. e Phomopsis sp. As mudas de moringa são mais sensíveis à restrição déficit hídrica sob 40% da capacidade de campo com relação aos parâmetros de trocas gasosas. As plantas sob gradiente de espaçamento apresentaram melhor desenvolvimento em unidade de área igual ou superior à 9,5 m2 planta-1. Conclui-se que plantios de moringa podem ser implantados com densidade igual ou inferior a 1.000 plantas por hectares e com irrigação entre 100% e 60% da capacidade de campo.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Moringa oleifera Lam. à uma Ãrvore conhecida pelo seu valor medicinal, uma vez que extratos de todas as partes desta planta mostraram propriedades farmacolÃgicas reconhecidas pelo uso popular e corroboradas pela comunidade cientÃfica. Dentre as atividades farmacolÃgicas, destacam-se Ãquelas relacionadas a processos inflamatÃrios. Este trabalho teve como objetivo o isolamento e caracterizaÃÃo de uma proteÃna ligante à quitina de sementes de Moringa oleifera e avaliaÃÃo de suas atividades antinociceptiva e anti-inflamatÃria. As proteÃnas foram extraÃdas da farinha delipidada de sementes e o extrato total foi fracionado em albuminas e globulinas. A fraÃÃo albumÃnica foi submetida à cromatografia de afinidade em matriz de quitina. A fraÃÃo ligante à quitina eluÃda com N-acetil-D-glucosamina 0,1 M foi submetida à cromatografia de troca iÃnica acoplada a um sistema de FPLC, rendendo cinco picos protÃicos. O material eluÃdo com tampÃo acetato de sÃdio 0,5 M, pH 5,2, contendo NaCl 0,6 M, denominado Mo-CBP4 (Mo â Moringa oleifera; âCBP â âchitin binding proteinâ), teve um rendimento final de 0,54% e foi escolhido para dar continuidade ao trabalho. Mo-CBP4 apresentou-se como duas bandas protÃicas de massas moleculares aparentes de 27,5 e 16,5 kDa, em condiÃÃes nÃo-redutoras, e de 9,8 kDa em condiÃÃes redutoras. Eletroforese bidimensional desta proteÃna revelou a presenÃa de dois spots (18,7 e 13,4 kDa), com mesmo ponto isoelÃtrico (pI de 10,55). Trata-se de uma glicoproteÃna com 2,85% de carboidratos, nÃo apresentando atividade hemaglutinante. Mo-CBP4 mostrou-se tÃo eficiente quanto o sulfato de alumÃnio e potÃssio na capacidade de coagular material em suspensÃo na Ãgua. A proteÃna em questÃo foi resistente à proteÃlise no teste de digestibilidade in vitro utilizando pepsina, tripsina e quimotripsina. Para avaliar os efeitos antinociceptivo e anti-inflamatÃrio de Mo-CBP4, foi utilizado o modelo de contorÃÃes abdominais induzidas pela injeÃÃo de Ãcido acÃtico 0,8% (via i.p.) em camundongos. A proteÃna, nas doses de 1,0, 3,5 e 10 mg/kg, foi capaz de prevenir as contorÃÃes de maneira dose dependente, chegando a 98,9% de inibiÃÃo na dose de 10 mg/kg. Mo-CBP4 tambÃm apresentou atividade antinociceptiva por via oral (10 mg/kg), atingindo uma inibiÃÃo de 52,9% quando administrada 60 minutos antes da injeÃÃo de Ãcido acÃtico. Mo-CBP4 inibiu o aumento da permeabilidade vascular (89,1%) e a migraÃÃo leucocitÃria (60,9%) na dose de 10 mg/kg via i.p. A propriedade antinociceptiva parece ser independente do sÃtio de interaÃÃo ao carboidrato N-acetil-D-glucosamina, enquanto que a atividade anti-inflamatÃria mostrou um decrÃscimo quando a regiÃo de ligaÃÃo ao carboidrato foi bloqueada. Os resultados apresentados suportam cientificamente o uso popular da M. oleifera e mostram que uma proteÃna ligante à quitina, Mo-CBP4, està associada aos efeitos antinociceptivo e anti-inflamatÃrio das sementes / Moringa oleifera Lam. is a perennial multipurpose tree with a strong scientific evidence of its curative power and used in folk medicine to cure several inflammatory processes. This work aimed to isolate and characterize a chitin-binding protein from Moringa oleifera seeds, and evaluate its antinociceptive and anti-inflammatory effects. Chitin-binding proteins were obtained after application of albumin fraction from crude extract of M. oleifera seeds into a chitin column and the adsorbed fraction was applied in a Resource-S matrix attached to FPLC system. The fraction eluted with 50 mM sodium acetate buffer, pH 5.2, containing 0.6 M NaCl, named Mo-CBP4 was used for the experiment. Mo-CBP4 showed a single band on SDS-PAGE (molecular mass 9.8 kDa) in presence of reducing agent, however in the absence of 2-mercaptoethanol two bands corresponding to 27.5 and 16.5 kDa were observed. Bidimensional electrophoresis of this protein revealed the presence of two spots (18.7 and 13.4 kDa), with the same isoeletric point value corresponding to 10.55. Mo-CBP4 is a glycoprotein containing 2.85% neutral sugar, which failed to agglutinate untreated or trypsin-treated erythrocytes from rabbit or human origin. This protein showed coagulant activity, similar to aluminum and potassium sulfate, the coagulant most widely used in water treatment. Mo-CBP4 was subjected to in vitro digestion with pepsin, trypsin, or chymotrypsin and appeared to be markedly resistant to digestion. Acetic acid-induced abdominal constrictions, increase in vascular permeability and leukocyte migration tests were used for the antinociceptive and anti-inflammatory activities assessment. Mo-CBP4 (1.0, 3.5 and 10 mg/kg) into mice potently and significantly reduced the occurrence of abdominal writhing in a dose dependent manner by 18.9, 44.6% and 98.9%, respectively. In addition, the oral administration of the protein (10 mg/kg) resulted in 18% and 52.9% reductions in abdominal writhing when given 30 and 60 min prior to acetic acid administration, respectively. Mo-CBP4 also caused a significant and dose-dependent inhibition of peritoneal capillary permeability induced by acid acetic and significantly inhibited leukocyte accumulation in the peritoneal cavity. The effect antinociceptive appeared to be independent on the carbohydrate recognition site. However the anti-inflammatory activity was partially reversed when Mo-CBP4 was pre-incubated with specific carbohydrate ligand, showing that this effect is dependent on the carbohydrate recognition site

Orientador: Edésio Luiz Simionatto. / Coorientador: Martinho Rau. / Dissertação (Mestrado em Química) - Programa de Pós-Graduação em Química, Centro de Ciências Exatas e Naturais, Universidade Regional de Blumenau, Blumenau.

Thesis (MSC. Agriculture (Animal Production)) --University of Limpopo, 2014 / Two experiments were conducted to determine the effect of moringa seed meal supplementation on productivity and carcass characteristics of Ross 308 broiler chickens. The first experiment determined the effect of moringa seed meal supplementation on productivity of Ross 308 broiler chickens aged one to 21 days. Two hundred and fifty unsexed day-old Ross 308 broiler chicks were randomly allocated to five dietary treatments, replicated five times, and each replication having 10 chickens. A completely randomized design was used. The chickens were fed on a grower diet supplemented with 0 (M0), 5 (M5), 10 (M10), 15 (M15) and 20 (M20) g of moringa seed meal/bird/day. Moringa seed meal supplementation had no effect (P>0.05) on feed intake, metabolisable energy intake, nitrogen retention, feed conversion ratio and live weight of unsexed Ross 308 broiler chickens. Moringa seed meal supplementation improved (P<0.05) growth rates of unsexed Ross 308 broiler chickens aged one to 21 days. A moringa seed meal supplementation level of 13.3 g/kg DM feed optimized growth rate of Ross 308 broiler chickens aged one to 21 days. The second experiment determined the effect of moringa seed meal supplementation on productivity and carcass characteristics of female Ross 308 broiler chickens aged 22 to 42 days. The chickens weighing 558 ± 10 g/bird were randomly allocated to five treatments with five replications having 10 birds. The chickens, aged 21 days, were allocated to the treatments in a completely randomized design. The chickens were fed on a grower diet supplemented with 0 (FM0), 5 (FM5), 10 (FM10), 15 (FM15) and 20 (FM20) g of moringa seed meal per kg DM. Moringa seed meal supplementation had no effect (P>0.05) on feed intake, growth rate, feed conversion ratio, live weight, metabolisable energy intake, carcass weight, breast meat weight, abdominal fat pad weight, liver weight, heart weight, thigh weight, meat flavour, juiciness and tenderness of female Ross 308 broiler chickens. However, moringa seed meal supplementation improved (P<0.05) nitrogen retention and gizzard weights of female Ross 308 broiler chickens. vi It was concluded that moringa seed meal supplementation improved growth rate of unsexed Ross 308 broiler chickens aged one to 21 days. Similarly, moringa seed meal supplementation increased nitrogen retention and gizzard weights of female Ross 308 broiler chickens aged 22 to 42 days.

Objetivo: Evaluar el efecto antibacteriano del extracto metanólico de Moringa oleifera (Moringa) combinado con Hidróxido de calcio sobre cepas de Enterococcus faecalis (ATCC29212). Materiales y métodos: Se preparó extractos metanólicos de tallos, hojas y tallos más hojas de Moringa oleifera, los cuales fueron combinados con Hidróxido de calcio en igual proporción. Posteriormente se procedió a evaluar el efecto antibacteriano de los extractos en presencia y ausencia del Hidróxido de calcio contra la cepa bacteriana de Enterococcus faecalis utilizando el método de Kirby-Bauer y el recuento de Unidades Formadoras de Colonias (UFC). Resultados: Se observó que al comparar el Hidróxido de calcio con los extractos metanólicos de Moringa oleifera en presencia y ausencia del Hidróxido de calcio se demuestra que, el Hidróxido de calcio por si solo tiene alto efecto antibacteriano a diferencia de las diversas combinaciones. Conclusiones: La importancia de este estudio fue evaluar si el extracto metanólico de Moringa oleifera repotencia el efecto antibacteriano del Hidróxido de calcio. Los resultados muestran que los diferentes extractos metanólicos de Moringa oleifera no tienen la capacidad de repotenciar al Hidróxido de calcio. / Objective: Evaluate the antibacterial effect of the methanolic extract of Moringa oleifera (Moringa) combined with calcium hydroxide on strains of Enterococcus faecalis (ATCC29212). Materials and methods: Methanolic extracts of stems, leaves and stems plus Moringa oleifera leaves were prepared, which were combined with calcium hydroxide in equal proportion. Subsequently, the antibacterial effect of the extracts in the presence and absence of calcium hydroxide against the bacterial strain of Enterococcus faecalis was evaluated using the Kirby-Bauer method and the count of Colony Forming Units (CFU). Results: It was observed that when comparing calcium hydroxide with the methanolic extracts of Moringa oleifera in the presence and absence of calcium hydroxide, it is shown that calcium hydroxide alone has a high antibacterial effect unlike the various combinations. Conclusions: The importance of this study was to evaluate if the methanolic extract of Moringa oleifera enhances the antibacterial effect of calcium hydroxide. The results show that different methanolic extracts of Moringa oleifera do not have the capacity to repower the calcium hydroxide. / Tesis

Solar water disinfection (SODIS) is the process of treating microbiologically contaminated water in clear plastic bottles through exposure to sunlight. One of the major limiting factors of this treatment is source water quality. This work investigates the impact of organic matter and turbidity on SODIS efficiency. Organic matter was found to decrease bacterial inactivation to a much greater extent than the presence of inorganic particles. The ability of moringa oleifera seed emulsion to clarify source waters was investigated as a coagulation pretreatment. This coagulant is most effective in highly turbid, high humic content waters, and achieves up to 1-log bacterial removal. The combined moringa oleifera coagulation-SODIS treatment sequence was tested in highly coloured natural source water and was found to reduce the sunlight exposure time required by up to 2 hours. Treated water should be consumed immediately following the individual or combined treatments due to the potential for bacterial regrowth.

Solar water disinfection (SODIS) is the process of treating microbiologically contaminated water in clear plastic bottles through exposure to sunlight. One of the major limiting factors of this treatment is source water quality. This work investigates the impact of organic matter and turbidity on SODIS efficiency. Organic matter was found to decrease bacterial inactivation to a much greater extent than the presence of inorganic particles. The ability of moringa oleifera seed emulsion to clarify source waters was investigated as a coagulation pretreatment. This coagulant is most effective in highly turbid, high humic content waters, and achieves up to 1-log bacterial removal. The combined moringa oleifera coagulation-SODIS treatment sequence was tested in highly coloured natural source water and was found to reduce the sunlight exposure time required by up to 2 hours. Treated water should be consumed immediately following the individual or combined treatments due to the potential for bacterial regrowth.