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

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Avaliação da ação bioinseticida de SBTI e vicilina de Erythrina velutina em enzimas digestivas e membrana peritrófica de larvas de Plodia interpunctella (Lepidoptera: Pyralidae)

Plodia interpunctella (Indian meal moth) is a cosmopolitan pest that attacks not only a wide range of stored grain as well other food products. Due to its economic importance several researches have focused in a method with ability to control this pest with few or no damage to the environment. The study of digestive enzymes inhibitors, lectins and chitin-binding proteins, has often been proposed as an alternative to reduce insect damage. In this study we report the major classes of digestive enzymes during larval growth in P. Interpunctella, being those proteinases actives at pH 9.5 and optimum temperature of 50 oC to both larvae of the 3rd instar and pre-pupal stage of development. In vitro and zymogram assays presented the effects of several inhibitors, such as SBTI, TLCK and PMSF to intestinal homogenate of 3rd instar larvae of 62%, 92% and 87% of inhibition and In pre-pupal stage of 87%, 62 % and 55% of inhibition, respectively. Zymograms showed inhibition of two low molecular masses protein bands by TLCK and that in presence of SBTI were retarded. These results are indicative of predominance of digestive serine proteinases in gut homogenate from Plodia interpunctella larvae. This serine proteinase was then used as a target to evaluate the effect of SBTI on larvae in in vivo assay. Effect of SBTI on mortality and larval mass was not observed at until 4% of concentration (w/w) in diets. Chitin, another target to insecticidal proteins, was observed by chemical method. Moreover, optic microscopy confirmed the presence of a peritrophic membrane. Established this target, in vivo effect of EvV, a chitin binding vicilin, evaluated during the larval development of P. interpunctella and was obtained a LD50 of 0,23% and WD50 of 0,27% to this protein. Mechanism of action was proposed through of the in vivo digestibility of EvV methodology. During the passage through the larval digestive tract was observed that EvV was susceptible to digestive enzymes and a reactive fragment, visualized by Western blotting, produced by digestion was recovered after dissociation of the peritrophic membrane. The bound of EvV to peritrophic membrane was confirmed by immunohystochemical assays that showed strong immunofluorescent signal of EvV-FITC binding and peritrophic membrane. These results are a indicative that vicilins could be utilized as potential insecticide to Plodia interpunctella and a control methods using EvV as bioinsecticide should be studied to reduce lost caused by storage insect pests

Diversity of new root nodule bacteria from Erythrina velutina Willd., a native legume from the Caatinga dry forest (Northeastern Brazil).

This study aims to evaluate the phenotypical characteristics of bacterial isolates from mulungu (Erythrina velutina Willd.) nodules and determinate their Box-PCR fingerprinting. All bacteria were evaluated by the following phenotypic characteristics: growth rate, pH change, colony color and mucus production. The bacterial isolates able to re-nodulate the original host were also evaluated regarding its tolerance to increased salinity and different incubation temperatures, ability to growth using different carbon sources, intrinsic antibiotic resistance and ?in vitro? auxin biosynthesis. The molecular fingerprints were set up using the Box-PCR technique and the isolates were clustered by their profiles. Among the 22 bacterial isolates obtained, eight were able to re-nodulate the original host. Among the nodule inducing isolates, some were tolerant to 1% of NaCl and 39° C and all of them metabolized the maltose, fructose, glucose, sucrose and arabinose, were resistant to rifampicin and produced auxin. The bacteria showed low genetic similarity among them and reference strains, which indicates the great genetic variability of the isolates. The results of this work are the first reports about the bacterial isolates able to nodulate this species. A more deep study of these bacteria may reveal the existence of isolates tolerant to environmental stresses and suitable as a future mulungu inoculant.

Reserve carbohydrates and lipids from the seeds of four tropical tree species with different sensitivity to desiccation

Considering the importance of water content for the conservation and storage of seeds, and the involvement of soluble carbohydrates and lipids for embryo development, a comparative study was carried out among the seeds of Inga vera (ingá), Eugenia uniflora (pitanga), both classified as recalcitrant, and Caesalpinia echinata (brazilwood) and Erythrina speciosa (mulungu), considered as orthodox seeds. Low concentrations of cyclitols (0.3-0.5%), raffinose family oligosaccharides (ca. 0.05%) and unsaturated fatty acids (0-19%) were found in the seeds of ingá and pitanga, while larger amounts of cyclitols (2-3%) and raffinose (4.6-13%) were found in brazilwood and mulungu, respectively. These results, in addition to higher proportions of unsaturated fatty acids (53-71%) in orthodox seeds, suggested that sugars and lipids played important role in water movement, protecting the embryo cell membranes against injuries during dehydration.

Molecular characterization of a miraculin-like gene differentially expressed during coffee development and coffee leaf miner infestation

The characterization of a coffee gene encoding a protein similar to miraculin-like proteins, which are members of the plant Kunitz serine trypsin inhibitor (STI) family of proteinase inhibitors (PIs), is described. PIs are important proteins in plant defence against insects and in the regulation of proteolysis during plant development. This gene has high identity with the Richadella dulcifica taste-modifying protein miraculin and with the tomato protein LeMir; and was named as CoMir (Coffea miraculin). Structural protein modelling indicated that CoMir had structural similarities with the Kunitz STI proteins, but suggested specific folding structures. CoMir was up-regulated after coffee leaf miner (Leucoptera coffella) oviposition in resistant plants of a progeny derived from crosses between C. racemosa (resistant) and C. arabica (susceptible). Interestingly, this gene was down-regulated during coffee leaf miner herbivory in susceptible plants. CoMir expression was up-regulated after abscisic acid application and wounding stress and was prominent during the early stages of flower and fruit development. In situ hybridization revealed that CoMir transcripts accumulated in the anther tissues that display programmed cell death (tapetum, endothecium and stomium) and in the metaxylem vessels of the petals, stigma and leaves. In addition, the recombinant protein CoMir shows inhibitory activity against trypsin. According to the present results CoMir may act in proteolytic regulation during coffee development and in the defence against L. coffeella. The similarity of CoMir with other Kunitz STI proteins and the role of CoMir in plant development and plant stress are discussed.

Dormancy as exaptation to protect mimetic seeds against deterioration before dispersal

Mimetic seeds simulate the appearance of fleshy fruits and arilled seeds without producing nutritive tissues as a reward for seed dispersers. In this strategy of seed dispersal, seeds may remain attached to the mother plant for long periods after maturity, increasing their availability to naive seed dispersers. The hypothesis that seed coat impermeability in many tropical Fabaceae with mimetic seeds serves as an exaptation to protect the seeds from deterioration and rotting while awaiting dispersal was investigated. Seed coat impermeability was evaluated in five mimetic-seeded species of tropical Fabaceae in south-eastern Brazil (Abarema langsdorffii, Abrus precatorius, Adenanthera pavonina, Erythrina velutina and Ormosia arborea) and in Erythrina speciosa, a `basal` species in its genus, which has monochromatic brown seeds and no mimetic displays. Seed hardness was evaluated as a defence against accelerated ageing (humid chamber at 41 degrees C for 144 h). Seed development and physiological potential of O. arborea was evaluated and the effect of holding mature seeds in pods on the mother plant in the field for a period of 1 year under humid tropical conditions was compared with seeds stored under controlled conditions (15 degrees C and 40 % relative air humidity). All five mimetic-seeded species, and E. speciosa, showed strong coat impermeability, which protected the seeds against deterioration in accelerated ageing. Most O. arborea seeds only became dormant 2 months after pod dehiscence. Germination of seeds after 1 year on the plant in a humid tropical climate was 56 %, compared with 80 % for seeds stored in controlled conditions (15 degrees C, 45 % relative humidity). Seedling shoot length after 1 year did not differ between seed sources. Dormancy acts in mimetic-seeded species as an exaptation to reduce seed deterioration, allowing an increase in their effective dispersal period and mitigating the losses incurred by low removal rates by naive avian frugivores.

Pollen and stigma morphology of some Phaseoleae species (Leguminosae) with different pollinators

Pollen transport to a receptive stigma can be facilitated through different pollinators, which submits the pollen to different selection pressures. This study aimed to associate pollen and stigma morphology with zoophily in species of the tribe Phaseoleae. Species of the genera Erythrina, Macroptilium and Mucuna with different pollinators were chosen. Pollen grains and stigmas were examined under light microscopy (anatomy), scanning electronic microscopy (surface analyses) and transmission electronic microscopy (ultrastructure). The three genera differ in terms of pollen wall ornamentation, pollen size, pollen aperture, thickness of the pollen wall, amount of pollenkitt, pollen hydration status and dominant reserves within the pollen grain, while species within each genus are very similar in most studied characteristics. Most of these features lack relationships to pollinator type, especially in Erythrina and Mucuna. Pollen reserves are discussed on a broad scale, according to the occurrence of protein in the pollen of invertebrate- or vertebrate-pollinated species. Some pollen characteristics are more associated to semi-dry stigma requirements. This apical, compact, cuticularised and secretory stigma occurs in all species investigated. We conclude that data on pollen and stigma structure should be included together with those on floral morphology and pollinator behaviour for the establishment of functional pollination classes.

Desempenho de doze espécies arbóreas nativas e introduzidas com potencial de uso múltiplo no estado do Acre, Brasil

O objetivo deste estudo foi avaliar o desempenho de espécies arbóreas com potencial de uso múltiplo, nas condições ambientais da Amazônia ocidental brasileira, visando recomendá-las como componentes de sistemas agroflorestais e para recuperação de áreas degradadas. O experimento foi estabelecido na Estação Experimental da Embrapa Acre, com 12 espécies nativas e introduzidas, no espaçamento de 1,50 m entre linhas, por 1,50 m entre plantas. O delineamento experimental foi de blocos casualizados, com quatro repetições. Cada espécie foi representada por cinco plantas em cada repetição. As avaliações, realizadas até quatro anos e meio após o plantio mostraram que: a) todas as espécies apresentaram índices de sobrevivência superiores a 80%, com exceção de Cedrela odorata (75%), Aspidosperma vargasii (65%) e Spondias lutea (65%); b) o incremento médio anual em altura total foi maior em Acacia mangium (2,91 m) e Acacia pollyphylla (2,66 m), seguida por Leucaena leucocephala, Calycophyllum spruceanum e Gliricidia sepium com médias de 2,38, 1,56 e 1,40 m, respectivamente; c) o incremento médio anual em diâmetro basal também foi maior em A. mangium (4,98 cm) e A. pollyphylla (3,92 cm), seguidas de Erythrina fusca (3,06 cm), Spondias lutea (2,37 cm), Erythrina poeppigiana (2,17 cm) e E. berteroana (2,12 cm); d) o incremento anual em diâmetro da copa foi maior em A. pollyphylla (2,50 m), G. sepium (1,03 m), Torresea acreana (0,98 m) e Erythrina fusca (0,87 m). Estes resultados sugerem a boa adaptação das espécies introduzidas que apresentaram desenvolvimento superior à maioria das nativas, principalmente em termos de capacidade de estabelecimento e crescimento inicial.

Catálogo sistemático dos pólens das plantas arbóreas do brasil meridional: V - Leguminosae: Papilionatae

In this paper of the catalogue of south brazilian arboreal pollen grains, the autor deals with the Papilionatae. The Mimosoideae and Caesalpinioideae are yet in preparation, so that a discussion of the three subfamilies (or families) is not possible. In relation with the systematical subdivision of the Papilionatae, we found a large correspondence with the morphology of the present pollen grains. The group of Phaseoleae contains the genera Mucuna, Erythrina and Dioclea; the grains of the studied species are very different one from another; the first of the genera possesses very volumous grains, with three colpori and a reticulated superficies; the second has three-porated pollen grains with a large reticulated superficies, and the third, Dioclea, is yet different; it possesses oblated grains, each three-colporated, with a thick sexine and a psilated superficies. So, we can say, that Phaseoleae is a erypalynous group. Dalbergieae, with the genera: Andira, Dalbergia, Lonchocarpus, Machaerium, Platymiscium and Pterocarpus (and Dahlstedtia, the only exception), has very uniform pollen grains, and may be considered stenopalynous. It is not possible to include the genus Dahlstedtia into this group. A little exception is represented by Pterocarpus violaceus, because of the reticulated sexine of its grains, while the others, also three-colporated, possess a tectate-reticulated sexine. The genera Myrocarpus and Ormosia, from Sophoreae, are very more similar to the Dalbergieae as to any other genus of the Phaseoleae.

Screening of some plants used in the Brazilian folk medicine for the treatment of infectious diseases

Extracts of 13 Brazilian medicinal plants were screened for their antimicrobial activity against bacteria and yeasts. Of these, 10 plant extracts showed varied levels of antibacterial activity. Piper regnellii presented a good activity against Staphylococus aureus and Bacillus subtilis, a moderate activity on Pseudomonas aeruginosa, and a weak activity against Escherichia coli. Punica granatum showed good activity on S. aureus and was inactive against the other standard strains. Eugenia uniflora presented moderate activity on both S. aureus and E. coli. Psidium guajava,Tanacetum vulgare, Arctium lappa, Mikania glomerata, Sambucus canadensis, Plantago major and Erythrina speciosa presented some degree of antibacterial activity. Spilanthes acmella, Lippia alba, and Achillea millefolium were considered inactive. Five of the plant extracts presented compounds with Rf values similar to the antibacterial compounds visible on bioautogram. Of these, three plants belong to the Asteraceae family. This may mean that the same compounds are responsible for the antibacterial activity in these plants. Anticandidal activity was detected in nine plant extracts (P. guajava, E. uniflora, P. granatum, A. lappa, T. vulgare, M. glomerata, L. alba, P. regnellii, and P. major). The results might explain the ethnobotanical use of the studied species for the treatment of various infectious diseases.

Efeitos do fracionamento da adubação mineral, da adubação orgânica e da calagem na produtividade e incidência de vassoura-de-bruxa em cacaueiros da Amazônia

A resposta do cacaueiro à aplicação de micronutrientes, matéria orgânica, calcário e fracionamento da adubação foi determinada em dois experimentos, instalados em solos das unidades Terra Roxa estruturada eutrófica (TR) e Latossolo Amarelo (LA), no Estado do Pará, no período de 1988 a 1993. As lavouras de cacau do híbrido Sca 6 x Be 10 foram implantadas pelo sistema de derrubada total e queima da floresta primária, utilizando-se sombreamento provisório de bananeira (Musa spp.) e permanente de Erythrina poeppigiana ou Gmelina arborea. Os tratamentos foram distribuídos em blocos casualizados, com três repetições, sendo a parcela constituída de 20 plantas úteis. Os resultados de produção de cacau demonstraram que não houve interação significativa entre tratamentos e solos ou ano de condução do experimento. O fracionamento da adubação NPK em três aplicações/ano e a adubação NPK + Zn foram os melhores tratamentos, provocando incrementos (P < 0,05) na produtividade do cacaueiro da ordem de 27,5% e 10,9%, respectivamente. A menor incidência (P < 0,05) de frutos atacados pela enfermidade vassoura-de-bruxa, causada pelo fungo Crinipellis perniciosa, foi observada nos tratamentos em que se aplicaram uma mistura de micronutrientes (B, Cu, Zn, Fe e Mo) ou esterco de gado na dosagem de 5 t ha-1; as porcentagens de frutos doentes dos demais tratamentos não diferiram da adubação NPK (testemunha). Para o aumento da produtividade, a estratégia de adubação mais eficiente foi o fracionamento da adubação NPK (60 kg ha-1 de N, P(2)0(5) e K(2)0) em três aplicações ao ano. O efeito de micronutrientes e do esterco de gado na infecção da vassoura-de-bruxa merece ser investigado com mais atenção.

Cultivo orgânico de cultivares de café a pleno sol e sombreado

O objetivo deste trabalho foi avaliar a influência de dois sistemas de cultivo orgânico sobre crescimento, fenologia, nutrição e produtividade de cultivares de cafeeiro, e sobre as características químicas do solo. Seis cultivares de café foram avaliadas em monocultura a pleno sol e em cultivo sombreado com banana e Erythrina verna, em delineamento de blocos ao acaso, com quatro repetições. O sombreamento reduziu a taxa de crescimento das cultivares nos primeiros 15 meses de cultivo. Depois de três anos reduziu, também, o diâmetro, o número de ramos produtivos e de nós dos cafeeiros; aumentou a área foliar e o tamanho dos grãos; reduziu o teor de K do solo, e aumentou os teores de N e de Mg nas folhas do café. Na primeira etapa da colheita (março/2004), a produtividade média das cultivares foi menor no sistema sombreado, o que evidencia que o sombreamento retarda a maturação dos grãos. Na segunda etapa (junho/2004), houve diferença entre cultivares, porém não entre os sistemas de cultivo. A produtividade média total (soma das duas etapas) foi igual nos dois sistemas de cultivo. As cultivares Tupi, Icatu e Obatã são as mais promissoras para o cultivo sombreado.

Enraizamento de corticeira-da-serra em função do tipo de estaca e variações sazonais

Erythrina falcata Benth. tem sido usada na recuperação de ecossistemas degradados, como planta ornamental e em sistemas agroflorestais. Entretanto, a produção de mudas por sementes é difícil. O objetivo deste trabalho foi avaliar a propagação vegetativa de E. falcata utilizando estacas caulinares provenientes de árvores adultas (estacas herbáceas, semilenhosas e de rebrota) e mudas, coletadas em quatro épocas do ano e o efeito do ácido indolbutírico (AIB) no enraizamento de estacas dessa espécie. Depois de preparadas, as estacas foram imersas em ácido indolbutírico (AIB, 0, 1,5 e 3 g L-1). O material se desenvolveu em tubetes de plástico (55 mL), em casa de vegetação climatizada, a uma temperatura de 25 a 30ºC e umidade relativa do ar acima de 80%, em substrato de vermiculita granulometria média. Maior porcentagem de enraizamento (73%), comprimento das quatro maiores raízes (46 mm) e número de raízes (6,2) foram obtidos em estacas de mudas coletadas no verão. Não houve enraizamento em estacas semilenhosas, provenientes de árvores adultas. A imersão das estacas em AIB não exerceu qualquer influência no enraizamento. As estacas de mudas coletadas no verão são as de melhor propagação vegetativa em virtude de melhor porcentagem de enraizamento e sobrevivência.