Purificação, caracterização e avaliação da atividade antiproliferativa de um inibidor de quimotripsina tipo kunitz de semente de Eryhrina velutina

A chymotrypsin inhibitor was purified from Erythrina velutina seeds by ammonium sulphate fractionation, affinities chromatographies on Trypsin-Sepharose, Quimotrypsin-Sepharose and reversed phase C-18 FPLC/AKTA system. The inhibitor, named EvCI, shown molecular mass of 17 kDa, as determined by SDSPAGE. 2D-PAGE showed four isoinhibitors with pI values of 4,42, 4,63, 4,83 and 5,06, with molecular mass of 17 kDa each. The aminoacid sequence of EvCI was determined by MALDI-TOF-MS and showed a high similarity with other Kunitz-type inhibitor of Erythrina variegata. EvCI competitively inhibited chymotrypsin, with Ki of 4 x10-8 M, but did not inhibited trypsin, pancreatic elastase, bromelain and papain. The inhibitory activity of EvCI was stable over wide pH and temperature ranges. In the presence of DTT 100 mM for 120 min, EvCI lost 50 % of activity. Cytotoxicity was studied in HeLa, MDA, HepG2, K562 and PC3 cells after 72-h incubation period. EvCl inhibited HeLa cells growth with an IC50 value of 50 μg/ml. Subsequent studies in HeLa cells analysis of cell death by annexin V/PI double-staining and cell cycle, using flow cytometry. The results provide evidence for a cytostatic activity of EvCl and support further studies on potential application of this inhibitors as an antiproliferative agent in combined therapy against cervical cancer

Avaliação das atividades anti-inflamatória, anticoagulante e antiproliferativa do inibidor de quimotripsina das sementes de erythrina velutina (EvCI)

Studies indicate that several components were isolated from medicinal plants, which have antibacterial, antifungal, antitumor and anti-inflammatory properties. Sepsis is characterized by a systemic inflammation which leads to the production of inflammatory mediators exacerbated by excessive activation of inflammatory cells and disseminated intravascular coagulation (DIC), in which the human neutrophil elastase plays an important role in its pathogenesis. Several epidemiological studies suggest that components of plants, especially legumes, can play a beneficial role in reducing the incidence of different cancers. A chymotrypsin inhibitor of Kunitz (Varela, 2010) was purified from seeds of Erythrina velutina (Mulungu) by fractionation with ammonium sulfate, affinity chromatography on Trypsin-Sepharose, Chymotrypsin-Sepharose and ion exchange chromatography on Resource Q 1 ml (GE Healthcare) in system FPLC / AKTA. The inhibitor, called EvCI, had a molecular mass of 17 kDa determined by SDS-PAGE. The purified protein was able to inhibit human neutrophil elastase (HNE), with an IC50 of 3.12 nM. The EvCI was able to inhibit both pathways of HNE release stimulated by PAF and fMLP (75.6% and 65% respectively). The inhibitor also inhibited leukocyte migration in septic mice about 87% and prolonged the time of coagulation and inhibition factor Xa. EvCI showed neither hemolytic activity nor cytotoxicity. EvCI showed a selective antiproliferative effect to HepG2 cell lines with IC50 of 0.5 micrograms per milliliter. These results suggest EvCI as a molecule antagonist of PAF / fMLP and a potential use in fighting inflammation related disorders, disseminated intravascular coagulation (DIC) and cancer

Clonagem e expressão do gene que codifica o inibidor de quimotripsina de Erythrina velutina WILLD. - caracterização e avaliação de seu potencial farmacológico

Proteinases are enzymes distributed widely founded in several organisms and perform many different functions, from maintaining homeostasis to the worsening of some diseases such as cancer, autoimmune diseases and infections. The proteins responsible of controlling the action of these enzymes are the inhibitors, that are classified based on their target proteases and are founded since simple organisms, such as bacteria, to higher organisms, such as larger plants and mammals. Plant proteinase inhibitors act by reducing or inactivating the activity of target proteases, thus, these proteins have been studied as potential tools in the treatment of diseases related to protease activities. In this context, an inhibitor of chymotrypsin from Erythrina velutina, called EvCI was previously purified and it was observed that this protein plays in vitro anticoagulant activity and anti-inflammatory activity in in vivo model. Aiming to reduce the environmental impact caused by the purification EvCI in high amounts and to facilitate the process of obtaining this protein, the recombinant chymotrypsin inhibitor from Eryhrina velutina was produced after cloning and expression in Escherichia coli. The bacteria were grown in LB medium and after induction of the expression this material was subjected to procedures for cell lysis and the product was applied on Nickel-affinity column. The proteins adsorbed were digested by thrombin and applied on Chymotrypsin-Sepharose affinity column, obtaining the purified inhibitor, named recEvCI. After electrophoresis, the recombinant inhibitor showed an approximately molecular mass of 17 kDa, and reduced the chymotrypsin and elastase activities in vitro. The recombinant inhibitor was sequenced and was found similar amino acids residues when compared to other inhibitors deposited in the database, with some modifications. recEvCI showed high stability under pH variations and reducing conditions, maintaining its activity around 80%. This protein increased the blood coagulation time in vitro by acting on the intrinsic pathway and did not show cytotoxicity against strains of mouse 3T3 fibroblasts and RAW 264.7 macrophages. recEvCI showed microbicide activity related to release of nitric oxide and consequently the activation of macrophages, futhermore having proinflammatory effects assessed by increased release of TNF-α. These results indicate that recEvCI can be biotechnologically used as a new tool in the control of coagulation-related diseases as well as can be an activating agent of the immune system in immunosuppressed individuals

Um novo inibidor de tripsina multifuncional de sementes de Erythrina velutina: caracterizacao e propriedades farmacologicas

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior

Efeito sacietogênico de um novo inibidor de tripsina da paçoca do amendoim com aumento plasmático de colecistocinina (cck)

Obesity is increasing, reaching epidemic levels in many regions of the world. Studies have shown that consumption of peanuts influences on weight control and this influence may be due to the action of trypsin inhibitors sacietogênica that condition increased plasma colescistocinina (CCK). Moreover, the peanut has other health benefits, and these assignments are guaranteed to increase their production and consumption of several of its products, including the paçoca peanut. The aim of this study was to identify the presence of a trypsin inhibitor in paçoca peanut and evaluate its effect on food intake, weight gain and histomorphological changes in swiss mice (n = 8) and Wistar rats (n = 6). Experimental diets were prepared based on the AIN-93G and supplemented with tack or peanut trypsin inhibitor partially purified paçoca peanut (AHTI). After each treatment, the animals were anesthetized and euthanized, their bloods were collected by cardiac puncture for the determination of CCK and other biochemical parameters (glucose, triglycerides, total cholesterol, high density lipoprotein, low density lipoprotein, glutamic-pyruvic transaminase, glutamic oxaloacetic transaminase and albumin) and their pancreas removed for histologic and morphometric analysis. The supplementation with paçoca peanut and the AHTI showed a decrease of body weight gain and food intake in both mice and rats, due to the satiety, since the animals showed no evidence of impairment of nutritional status conditioned by consumption the AHTI. There were also observed biochemical or morphological important when compared with controls. However, AHTI led to increased secretion of CCK, a peptide sacietogênico. Thus, these results indicate that AHTI present in paçoca peanut, is able to enhance the secretion of plasma CCK and thereby reduce the weight gain associated with lower food intake of experimenta animals

Ação Sacietogênica de um inibidor de tripsina da semente de tamarindo (Tamarindus indica L.)

The seeds are excellent sources of proteinase inhibitors and have been highlighted owing to various applications. Among these applications are those in effect on food intake and weight gain that stand out because of the increasing number of obese individuals. This study evaluated the effects of trypsin inhibitor present in the seed of tamarind (Tamarindus indica L.) reduction in weight gain, biochemical and morphological alterations in Wistar rats. For this, we partially purified a trypsin inhibitor tamarind seed. This inhibitor, ITT2 at a concentration of 25 mg / kg body weight, over a period of 14 days was able to reduce food intake in rats (n = 6) by approximately 47%, causing a reduction in weight gain approximately 70% when compared with the control group. With the evaluation of the in vivo digestibility was demonstrated that the animals lost weight due to satiety, presented by the reduction of food intake, since there were significant differences between true digestibility for the control group (90.7%) and the group treated with inhibitor (89.88%). Additionally, we checked the deeds of ITT2 on biochemical parameters (glucose, triglycerides, total cholesterol, high-density lipoprotein, low-density lipoprotein, glutamic-pyruvic transaminase, glutamic oxaloacetic transaminase, gamma glutamyl transferase albumin, globulin, total protein and C-reactive protein) and these, when assessed in the study groups showed no statistically significant variations. We also evaluate the histology of some organs, liver, stomach, intestine, and pancreas, and showed no changes. And to evaluate the effect of trypsin inhibitor on food intake due to the satiety is regulated by cholecystokinin (CCK) were measured plasma levels, and it was observed that the levels of CCK in animals receiving ITT2 were significantly higher ( 20 + 1.22) than in animals receiving only solution with casein (10.14 + 2.9) or water (5.92 + 1.15). Thus, the results indicate that the effect caused ITT2 satiety, reducing food intake, which in turn caused a reduction in weight gain in animals without causing morphological and biochemical changes, this effect caused by the elevation of plasma levels CCK

Estudo de revestimentos inorgânicos com inibidor de corrosão aplicado na superfície do concreto

The corrosive phenomenon on reinforced concrete structures is one of the most founded pathologies on the coastal area. With the objective to prevent the process development, or even, retard its beginning, it was studied the application of inorganic covering over concrete surfaces, after its cure, as well as, evaluate the efficiency of the covering applied on the concrete in reducing its porosity of concrete preventing the entrance of aggressive agents to preserve the integrity of the existing armor inside it, comparing the result obtained with the body-of-proof reference, that didn´t receive covering protection. On the concrete production it was used Portland Cement CP II 32, coarse aggregate, fine aggregate and water from the local distributive. Two types of covering were used, one resin based of silicon and solvent and other white cement based, selected sands and acrylic resin. The concrete mixture adopted was 1:1,5:2,5 (cement, fine aggregate, coarse aggregate) and 0.50 water/cement ratio. With the concrete on fresh state was made the experiment test to determinate the workability. On the hardened state was made the concrete resistance experiment, absorption of water and electrochemical experiments, through polarization curves. Also was held optical microscopy and Scanning Electron Microscopy experiments to analyze the layer of the covering applied to the concrete surface and the interface between the concrete and the layer. The obtained results shows that the covering applied to the concrete surface didn´t affect the resistance towards compression. On the absorption of water occurred a diminution of the percentage absorbed, improving the concrete development by making it more impermeable towards the entrance of aggressive agents. The electrochemical experiment results confirmed the water absorption results; the body-of-proof covered presented larger protection towards the development of corrosives process and retarded the evolution of the corrosive phenomenon

Avaliação do efeito de particulados sólidos na eficiência de um inibidor de corrosão recomendado para meios salinos com CO2

The main problem on the exploration activity on petroleum industry is the formation water resulted on the fields producing. The aggravating of this problem is correlated with the advancing technologies used on the petroleum extractions and on its secondary approach objecting the reobtainment of this oil. Among the main contaminants of the water formation are corrosives gases such as: O2, CO2 and H2S, some solids in suspension and dissolved salts. Concerning to those gases the CO2 is the one that produce significant damage for carbon steel on corrosion process of the petroleum and gas industries. Corrosion inhibitors for carbon steel in formation water is one of the most used agents in control of those damages. In this context, the poor investigations of carbon steel corrosion proceeding from solids in suspension is an opened field for studies. On this work the inhibitor effect of the commercial CORRTREAT 703 was evaluated on some specific solids in suspension at saline medium containing 10.000 ppm of de-aerated chloride using CO2 until non oxygen atmosphere been present. For that, quartz, calcium carbonate, magnetite and iron sulphide were subjected to this investigation as the selected solids. The effect of this inhibitor on corrosion process correlated with those specific solids, was measured using electrochemical (resistance of linear polarization and galvanic pair) and gravimetrical techniques. During all the experimental work important parameters were monitored such as: pH, dissolved oxygen, temperature, instantaneous corrosion rate and galvanic current. According to the obtained results it was proved that the suspension solids calcium carbonate and iron sulphide decrease the corrosion process in higher pH medium. Meanwhile the quartz and magnetite been hardness increase corrosion by broking of the passive layer for erosion. In the other hand, the tested inhibitor in concentration of 50 ppm, showed to be effective (91%) in this corrosion process

Avaliação da espécie vegetal Croton cajucara Benth como inibidor de biocorrosão em aço carbono Aisi 1020

Actually in the oil industry biotechnological approaches represent a challenge. In that, attention to metal structures affected by electrochemical corrosive processes, as well as by the interference of microorganisms (biocorrosion) which affect the kinetics of the environment / metal interface. Regarding to economical and environmental impacts reduction let to the use of natural products as an alternative to toxic synthetic inhibitors. This study aims the employment of green chemistry by evaluating the stem bark extracts (EHC, hydroalcoholic extract) and leaves (ECF, chloroform extract) of plant species Croton cajucara Benth as a corrosion inhibitor. In addition the effectiveness of corrosion inhibition of bioactive trans-clerodane dehydrocrotonin (DCTN) isolated from the stem bark of this Croton was also evaluated. For this purpose, carbon steel AISI 1020 was immersed in saline media (3,5 % NaCl) in the presence and absence of a microorganism recovered from a pipeline oil sample. Corrosion inhibition efficiency and its mechanisms were investigated by linear sweep voltammetry and electrochemical impedance. Culture-dependent and molecular biology techniques were used to characterize and identify bacterial species present in oil samples. The tested natural products EHC, ECF and DCTN (DMSO as solvent) in abiotic environment presented respectively, corrosion inhibition efficiencies of 57.6% (500 ppm), 86.1% (500 ppm) and 54.5% (62.5 ppm). Adsorption phenomena showed that EHC best fit Frumkin isotherm and ECF to Temkin isotherm. EHC extract (250 ppm) dissolved in a polar microemulsion system (MES-EHC) showed significant maximum inhibition efficiency (93.8%) fitting Langmuir isotherm. In the presence of the isolated Pseudomonas sp, EHC and ECF were able to form eco-compatible organic films with anti-corrosive properties

Germinação de sementes e otimização de técnicas de micropropagação de umbuzeiro (spondias tuberosa, arr.) anacardiaceae

The brazilian-plum (Spondias tuberosa, His) is a tropical fruit tree that has been consolidated in the market for agribusiness processing, due to its characteristic flavor of fruit. Accordingly, studies to optimize the propagation of plants are necessary for production of seedlings with agronomic and quality assurance measures. This study aimed at determining the efficient techniques for uniform seed germination, as brazilian-plum seed present mechanical dormancy, and establish optimal culture media for multiplication of shoots from the in vitro micropropagation. Firstly, in a greenhouse at the Universidade Federal do Rio Grande do Norte, was evaluated the influence of different methods of breaking dormancy in the emergence of seedlings of brazilian-plum and speed of germination (IVG) of seeds. After 60 days of cultivation, it was found that splay in the distal portion of the seed was the best treatment, with rates of 85.33% in germinability and 3.415 of IVG, compared with the treatment of seed-soaking in water for 12h + humus and the control group. Subsequently, new sources of seedling explants were obtained in studies of tissue culture. Laboratory of Plant Biotechnology that the university, was used stem apex, nodal segments and internodes in search of decontamination with various concentrations of calcium hypochlorite [Ca(OCl)2] and micropropagation, inoculating them in half WPM (1980) with various concentrations of 6-benzylaminopurine (BAP). We used 10 sample units with three replications for different concentrations of [Ca(OCl)2], BAP and explants type. After thirty days, which was observed for the control of contamination, during the establishment in vitro, concentrations of [Ca(OCl)2] between 0.5% and 2.0% were effective in combating exogenous contamination of the apex. In nodal segments and internodes, concentrations of [Ca(OCl)2] between 1.0% and 2.0% and 1.5% and 2.0% were respectively, sufficient to reduce the percentage of losses in these infestations explants. For micropropagation, the culture medium supplemented with 0.1 mg.L-1 BAP promotes better development of multiple shoots per explants from nodal segment. However, success does not get to shoot training in internodal segment

Germinação e armazenamento de sementes de COCA (Erythroxylum ligustrinum DC. - Erythroxylaceae)

O objetivo deste trabalho foi avaliar o efeito da temperatura sobre a germinação e o armazenamento de sementes de Erythroxylum ligustrinum DC. As sementes foram colocadas germinar caixas de plásticas sobre duas folhas de papel, umedecidas com Nistatina 0, 2%, mantidas em germinadores nas temperaturas constantes de 5, 10, 15, 20, 25, 30, 35, 40 e 45ºC e com fotoperíodo de 12 horas. Para o armazenamento, as sementes foram colocadas em sacos de papel, mantidos à temperatura ambiente e em geladeira. em seguida, mensalmente, foi avaliada a germinação das sementes. A temperatura mínima, a faixa de temperatura ideal e a temperatura máxima para germinação de sementes de coca estão entre 10 e 15ºC, 25 e 30ºC e 40 e 45ºC, respectivamente. O armazenamento de sementes coca por longo período é inviável em embalagens de papel na geladeira (10ºC ± 2) e em temperatura ambiente (27ºC ± 5).

Atividade alelopática de extratos aquosos de plantas medicinais na germinação de Lactuca sativa L. e Bidens pilosa L.

O potencial alelopático de quatro diferentes extratos de plantas medicinais (Eclipta alba, Gomphrena globosa, Tabernaemontana catharinensis e Tithonia diversifolia) foi avaliado na germinação de Bidens pilosa e Lactuca sativa. Dentre os resultados obtidos no teste de germinação, notou-se um maior poder inibitório do extrato de G. globosa sobre sementes e plântulas de Bidens pilosa e Lactuca sativa independente da concentração utilizada e aumento da atividade inibitória de extratos T. catharinensis e de T. diversifolia com aumento da concentração destes. Quanto ao Índice de Velocidade de Germinação, os dados demonstraram redução do índice devido a elevada mortalidade das plântulas submetidas aos extratos de G. globosa e de T. catharinensis e a germinação tardia das sementes submetidas ao extrato de T. diversifolia. Quanto ao vigor das sementes, as menores porcentagens de sobrevivência ocorreram extratos à 2,5% para B. pilosa e 10% para L. sativa.

Influência de extratos aquosos de sorgo sobre a germinação e o desenvolvimento de plântulas de soja

Estudos conduzidos em laboratório ou em condições de campo demonstram o potencial alelopático da espécie Sorghum bicolor L., seja inibindo a germinação e o desenvolvimento de plantas daninhas ou até mesmo de plantas cultivadas em sucessão. Com o objetivo de avaliar os efeitos potencialmente alelopáticos de extratos aquosos das folhas, caules e raízes de cinco híbridos de sorgo (SARA, DKB860, DKB 599, XBG00478 e XBG06020), sobre a germinação e o desenvolvimento de plântulas de soja, cv. MG/BR 46 (Conquista), foram conduzidos dois bioensaios no período de março a junho de 2002. Foram estabelecidas como testemunhas: água destilada (com pH 8,0); água destilada com pH ajustado para 4,5; água destilada com pH ajustado para 6,0 e solução de polietilenoglicol (PEG) equivalente ao potencial osmótico de 0,1 MPa. Dentre as características avaliadas, houve efeito significativo apenas no comprimento de radícula das plântulas de soja, sendo que aquelas tratadas com os extratos de sorgo apresentaram menor radícula, diferindo significativamente das plântulas testemunhas. Observou-se que, com os extratos de raízes dos híbridos SARA, DKB860, XBG00478 e XBG06020 ocorreram os menores comprimentos de radículas de soja, diferindo do DKB 599. O extrato de folhas do XBG00478 resultou em menor média, não diferindo dos extratos de DKB860, DKB599 e XBG06020. No entanto, para o extrato de caule, o SARA resultou em menor radícula, diferindo apenas do XBG00478. Verificou-se, também, que, para os extratos de DKB860 e XBG00478, os extratos de folhas proporcionaram menor média, diferindo dos extratos de raiz e caule, enquanto para DKB599, XBG06020 e SARA, não houve diferença significativa entre os extratos de folha e caule.

Germinação de sementes de Sida rhombifolia e Brachiaria decumbens influenciada por vinhaça, flegmaça e óleo de fúsel

Este trabalho objetivou avaliar, em condições de laboratório, a germinação de sementes de Sida rhombifolia e Brachiaria decumbens submetidas à aplicação de vinhaça, flegmaça e óleo de fúsel. Esses subprodutos, nas concentrações de 12,5; 25,0; 50,0; e 100,0% (v/v), e as testemunhas (água com pH e osmolalidade corrigidos, em função da caracterização realizada nos subprodutos e em suas diluições) foram aplicados diretamente em 100 sementes acondicionadas em caixas de plástico, utilizando papel como substrato. O delineamento experimental foi o inteiramente casualizado, e as médias obtidas foram submetidas ao teste de Tukey. As sementes de Sida rhombifolia submetidas ao óleo de fúsel não germinaram e apresentaram redução na viabilidade, principalmente nas maiores concentrações de aplicação. As sementes de Brachiaria decumbens submetidas à maior concentração de flegmaça apresentaram tendência de redução da viabilidade e do índice de velocidade de germinação (IVG). Na presença do óleo de fúsel as sementes de Brachiaria decumbens não germinaram e apresentaram-se totalmente inviáveis.

Germinação de sementes de melão-de-são-caetano sob variação de água, luz e temperatura

The purpose of this study was to evaluate germination of Momordica charantia L. seeds under different conditions of: water availability (0.0, -0.2, -0.4, -0.6, -0.8 and -1.0 MPa), temperature (10, 15, 20, 25, 30 and 35 degrees C) and quality of incident light (filter dark, red, green, yellow, red off, blue, orange and without filter). For each variable evaluated, experiments were carried out in a completely randomized design with four replicates kept under controlled conditions (germination chamber). There were higher percentages of germination under conditions of mild water restriction (-0.2 MPa) and temperatures around 25 degrees C, regardless of the quality of light. Temperatures below 20 degrees C and above 30 degrees C reduced germination, although lower temperatures had strongly effects on germination. Water availability lower than -0.2 MPa strongly reduced germination, which was inhibited in the treatments of -0.8 and 1.0 MPa.