Parâmetros biológicos de Herpsilochmus pectoralis Sclater 1857 (Aves: Thamnophilidae) em fragmentos de mata atlântica do Nordeste do Brasil

The study of birds represents an important tool for the understanding of the processes involved in behavioral and morphological patterns. The species we have studied belongs to Thamnophilidae family, the third largest family restricted to the Neotropic ecozone. They are popularly known as antbirds and comprise 209 species. A large portion of the species has cryptic behavior, making the acoustic communication an important tool for maintaining contact among birds. Herpsilochmus pectoralis Sclater 1857 has evident sexual dimorphism, measured between 10 and 12 cm height and it is found in forest fragments in the Northeast and it is also categorized as vulnerable to extinction process. This study was conducted in three sandbank fragments on the east coast of the state of Rio Grande do Norte, Brazil. With the help of tape recordings between 2006 and 2012 it was possible to describe and characterize the sing of H. pectoralis. The sing from male birds has more and longer length than the female sing (16% of dimorphism). No differences were found in the dominant frequency between the sexes. We describe four types of calls from this species repertoire. Through capturing with ornithological nets between 2009 and 2012 it was possible to describe and compare the morphology of H. pectoralis. The species have shown lower corporal mass in the dry season. The young birds showed morphometric similarities in comparison to adults. The species has no accentuated dimorphism in their morphometric characteristics. The young ones with flying capabilities have morphometric characteristics of adults, even though they do not show a characteristic adult plumage. The moult pattern in the species is a characteristic of tropical birds, but it shows short reproductive period, typical of temperate species. Even being in the tropical region, the species suffers with seasonal rainfall, which influences their reproductive phenology and moult (remex and rectrix) cycle. Thus, this dissertation provides information on the biology of H. pectoralis to support the understanding of the relationship of this species to the environment and also to know the variations of morphology and vocal aspects, in order to understand patterns and general characteristics of Thamnophilidae.

Efeito do ácido ascórbico sobre a espermatogênese de camundongos com distrofia muscular de Duchenne

The objective was to evaluate spermatogenesis alterations caused by DMD and the effect of the treatment using ascorbic acid in preventing those injuries. Twenty four mice were used, 12 from the C57BL/10 lineage (non-dystrophic) and 12 from the C57BL/10Mdx (dystrophic). The sample was divided in six groups containing 4 animals each, as: C30 = 30 days control; D30 = Dystrophic with 30 days; C60 = 60 days control; D60 = Distrophic with 60 days; CS60 = 60 days control supplemented with ascorbic acid and DS60 = Dystrophic with 60 days supplemented with ascorbic acid. The ascorbic acid supplementation was given in the water, 0,005 mg/day. After euthanasia, the testicles (right and left) were collected, weighted and cross sectioned. The material was fixed in the Karnovsky solution for 24 hours, included in resin for histological studies (morphological and morphometric analyzes) submitted to ultrastructural analysis and immunohistochemistry for caspase-3. There was a significant increase in the tunica propria percentage in D30 compared to C30 and D60. The ultrastructural analysis showed mitochondrial apoptosis evidence of Sertoli cells that can reduce sperm efficiency in CS60 and DS60. A higher volume density of apoptotic cells postivas to Caspase-3 in C30 and D30 versus DS60 compared to CS60. There was severe hypertrophy of the Leydig cells between D30 and D60. However, with supplementation was observed reversal of this change in DS60. The ultrastructure of Leydig cells to early presence of lipid vesicles was observed in the group pre-pubertal dystrophic (D30). Thus, the DMD affect the organization of the seminiferous tubules and intertubule, however, the ascorbic acid supplementation used for the treatment of DMD has been just enough to reduce the hypertrophy of the Leydig cells.

Extração, caracterização e prospecção de atividades farmacológicas de polissacarídeos sulfatados da macroalga verde Caulerpa prolifera

This study aimed to extract, characterize and conduct a prospective analysis of pharmacological activities of sulfated polysaccharides from green seaweed Caulerpa prolifera. Seven fractions (CP-0.3/CP-0.5/CP-0.7/CP-0.9/CP-1.1/CP-1.5/CP-2.0) were obtained from C. prolifera by alkaline proteolysis followed by sequential precipitation in acetone. The physicochemical analyzes indicated that C. prolifera synthesizes a homogalactan (CP-0.9) and different populations of sulfated heteropolysaccharides. In the analysis of anticoagulant activity, all fractions except CP-0.3, influenced the intrinsic coagulation pathway. All fractions showed antioxidant activity in six different assays being more pronounced in hydrogen peroxide scavenging assay, especially CP-0.3, CP-0.7 and CP-0.9 (which obtained 61% of hydrogen peroxide scavenging), in ferric chelation assay (especially CP-0.9 with 56% chelation) and cupric chelation assay (especially CP-2.0 with 78% chelation). With respect to immunomodulatory activity, the presence of CP-0.3, CP-0.7 and CP-0.9 showed an immunogenic potential, increasing the production of nitric oxide (NO) by 48, 142 and 163 times, respectively. Conversely, the NO synthesis fell 73% after the activation of macrophages by LPS, incubated concurrently with CP-2.0. The anti-adipogenic activity of the fractions was also evaluated and CP-1.5 was able to reduce the differentiation of pre-adipocytes (3T3-L1) into adipocytes by 60%, without affecting the cell viability. The fractions CP-0.3, CP-0.5 and CP-0.9 reduced the viability of the HeLa cells (human cervical adenocarcinoma) by 55% and CP-1.5 reduced the viability of the 786-0 cells (human renal adenocarcinoma) by 75%. Leishmanicidal activity and microbicide effect against Carbapenem-resistant Klebsiella pneumoniae (KPC) have not been identified. However, the viability of Staphylococcus epidermidis was reduced by 23.8% in the presence of CP -1.5. All fractions were able to change the formation of calcium oxalate crystals. CP-0.3, CP-0.5 and CP-1.1 only promoted the formation of COD type crystals with a very small size (1 μm). Confocal microscopy and zeta potential data of crystals formed in the presence of the samples showed that the polysaccharides present in the fractions must interact with calcium ions present throughout the crystal lattice, affecting the growth and morphology of crystals The results described herein indicate that the fractions rich in polysaccharides obtained from the green seaweed C. prolifera present a multi therapeutic potential, and subsequent purification steps, as well as research on the mechanisms of action by which these polymers act should be investigated.

Um dermatam sulfato antitrombótico do camarão Litopenaeus vanammei inibe a inflamação e angiogênese

Inflammation is combined of a vascular and a cellular reaction, resulting in different cells and tissue responses, both the intravascular and extravascular environment. As the inflammatory process occurs, coagulation proteases, in particular thrombin (FIIa), are able to initiate various cellular responses in vascular biology and therefore is often observed activation of other biological systems, leading to complications during an event inflammatory, such as thrombosis and angiogenesis. Thus, antagonists molecules of these events are interesting models for the development of novel anti-inflammatory drugs. Thereby, it is worth stressing the glycosaminoglycans (GAGs), which are able to interact with several proteins involved in important biological processes, including inflammation and coagulation. Therefore, this study aimed to evaluate the anti-inflammatory, antithrombotic and anti-angiogenic potentials, as well anticoagulant of a dermatan sulfate-like GAG (DS) extracted from the Litopenaeus vannamei cephalotorax. The compound was obtained after proteolysis and purification by ion-exchange chromatography. After total digestion by DS-like compounds digesting lyases (chondroitinase ABC), the DS-like nature was revealed, and then called DSL. The shrimp compound showed reduced anticoagulant effect by the aPTT assay, but high anti-IIa activity, directly and through heparin cofactor II. On inflammation, the compound had a significant inhibitory effect with the reduction of proinflammatory cytokines. Potential Inhibitory were reported in the antithrombotic and anti-angiogenic assay, the latter being dose dependent. As for anti-hemostatic activity, the polysaccharides did not induced significant bleeding effect. Thus, the results shown by the shrimp DS-like compound indicate this glycosaminoglycan as a biotechnology target with prospects for the development of new multipotent drugs.

Biologia reprodutiva de Mimus gilvus (Aves: Mimidae) em área de restinga no nordeste do Brasil

The thrush beach, Mimus gilvus (Aves: Mimidae) is a passerine widely distributed in Central and South America. In Brazil occurs mainly in the areas of the resting and vegetation near the beach. In southeastern Brazil this species has disappeared, mainly due to urbanization. Many attributes of their reproductive biology are unknown, especially in relation to reproductive success. During the years 2010-2011, 2011-2012 and 2014-2015 were made visits to the area of restinga forest in Centro de Lançamento da Barreira do Inferno (CLBI), located between the cities of Natal and Parnamirim-RN (5 ° 54'S 35 ° 10'W ) where they were made systematic searches to trying describe reproductive biology of the characteristics of M. gilvus, estimate their reproductive success using the Mayfield method, and identify the main factors that influence their reproductive success in environment the resting. For this, forty fifth active nests monitored were used. Only during the breeding season of 2011-2012 and 2014-2015 were made systematic visits to the study area. The reproductive period ranged August to March. Clutch size ranged from two, three and six eggs (n = 22). Broods of two eggs were more common, with an average of eggs laid per nest of 2 ± 0,51 (n = 20 nests). The incubation period was approximately 13 ± 1,9 days (n = 11 nests). The period of stay of the nestlings was approximately 11 ± 1,6 days (n = 9 nests). With approximately 11 days old the nestlings were able to leave the nest. The apparent success was 37,8% and the success estimated by Mayfield method was 26,6%. Predation was the main cause of loss of nests in the study area. The daily survival rates (TDS) were obtained from 0,9593 incubation and 0,9313 for nestling period respectively. Survival estimates for each period was 0,5827 for incubation and 0,4571 for nestling period. The cumulative average rainfall for each month influenced negatively the hatching rates of M. gilvus nests. In addition, the number hatch eggs among the most rainy season (rainy season) and the period of lowest rainfall (dry season) were different. The number of lost nests of M. gilvus was lower in scrubs than cactus, which may account for the largest number of nests of this species found in scrubs. Survival rates in nestling period were lower compared with the incubation period. The fact that the survival rates nests of M. gilvus be lower in the nestling period compared to the incubation period may result from increased activity of adults during this phase of the nest, which in turn would increase predation rates. M. gilvus seems to avoid the rainy season during their reproduction, concentrated most of their nests in periods of low rainfall.

Potencial terapêutico de um heparinóide isolado do invertebrado marinho Litopenaeus vannamei

The occurrence of bioactive compounds in marine organisms comes awaking the interest of the pharmaceutical industry. Heparin, a sulfated polysaccharide which presence was already identified in several marine invertebrates, is very attractive due its remarkable functional versatility. Besides to intervene in blood coagulation, this molecule has a great anti-inflammatory potential. However, its strong anticoagulant activity difficult the clinical exploitation of its anti-inflammatory properties. Thus, the aims of this work were to evaluate the effect of a heparin-like compound (heparinoid), isolated from the cephalotorax of the Litopenaeus vannamei shrimp, on the inflammatory response, hemostasia and synthesis of antithrombotic heparan sulfate by endothelial cells, besides studying some aspects concerning its structure. The purified heparinoid was structurally characterized following an analytical boarding, involving electrophoresis and chromatography. The structural analysis have shown that this compound possess a high content of glucuronic acid residues and disulfated disaccharide units. In contrast to mammalian heparin, the heparinoid was incapable to stimulate the synthesis of heparan sulfate by endothelial cells in the tested concentrations, beyond to show reduced anticoagulant activity and hemorrhagic effect. In a model of acute inflammation, the compound isolated from the shrimp reduced more than 50% of the cellular infiltration. Besides reduce the activity of MMP-9 and proMMP-2 of the peritoneal lavage of inflamed animals, the heparinoid also reduced the activity of MMP-9 secreted by activated human leukocytes. These results demonstrate the potential of heparinoid from L. vannamei to intervene in the inflammatory response. For possessing reduced anticoagulant activity and hemorrhagic effect, this compound can serve as a structural model to direct the development of more specific therapeutical agents to the treatment of inflammatory diseases

Avaliação do efeito citotóxico da lectina da esponja marinha Cliona varians contra células de leucemia mielóide crônica

In this study, a BCR-ABL expressing human chronic myelogenous leukaemia cell line (K562) was used to investigate the antitumoral potential of a novel lectin (CvL) purified from the marine sponge Cliona varians. CvL inhibited the growth of K562 cells with an IC50 value of 70 g/ml, but was ineffective to normal human peripheral blood lymphocytes in the same range of concentrations tested (180 g/ml). Cell death occurred after 72 h of exposure to the lectin and with sign of apoptosis as analysed by DAPI staining. Investigation of the possible effectors of this process showed that cell death occurred in the presence of Bcl-2 and Bax expression, and involved a caspase-independent pathway. Confocal fluorescence microscopy indicated a major role for the lysosomal protease cathepsin B in mediating cell death. Accordingly, pre-incubation of K562 cells with the cathepsin inhibitor L-trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64) abolished the cytotoxic effect of CvL. Furthermore, we found upregulation of tumor necrosis factor receptor 1 (TNFR1) and down-modulation of p65 subunit of nuclear factor kappa B (NFB) expression in CvL-treated cells. These effects were accompanied by increased levels of p21 and downmodulation of pRb, suggesting that CvL is capable of cell cycle arrest. Collectively, these findings suggest that cathepsin B acts as death mediator in CvL-induced cytotoxicity possibly in a still uncharacterized connection with the membrane death receptor pathway

Atividades biológicas de polissacarídeos sulfatados extraídos da alga vermelha Gracilaria caudata

In recent years, sulfated polysaccharides (SP) from marine algae have emerged as an important class of natural biopolymers with potential pharmacology applications. Among these, SP isolated from the cell walls of red algae have been study due to their anticoagulant,antithrombotic and anti-inflammatory activities. In the present study, three sulfated polysaccharides fractions denominated F1.5v, F2.0v and F3.0v were obtained from seaweed G. caudate by proteolysis followed to acetone fractionation. Gel electrophoresis using 0.05 M 1,3-diaminopropane-acetate buffer, pH 9,0, stained with 0.1% toluidine blue, showed the presence of SP in all fractions. The chemical analysis demonstrated that all the fractions are composed mainly of galactose. These compounds were evaluated in anticoagulant, antioxidant and antiproliferative activities. In anticoagulant activity evaluated through aPTT and PT tests, no one fractions presented anticoagulant activity at tested concentrations (0.1 mg/mL; 1.0 mg/mL; 2.0 mg/mL).The antioxidant activities of the three fractions were evaluated by the following in vitro systems: Total antioxidant capacity, superoxide and hydroxyl radical scavenging, ferrous chelating activity and reducing power. The fractions were found to have different levels of antioxidant activity in the systems tested. F1.5v shows the highest activity, especially in the ferrous chelating system, with 70% of ferrous inhibiting at 1.0 mg.mL-1. Finally, all the fractions showed dose-dependent antiproliferative activity against HeLa cells. The fractions F1.5v and F2.0v presented the highest antiproliferative activity at 2.0 mg/mL with 42.7% and 37.0% of inhibition, respectively. Ours results suggests that the sulfated polysaccharides from seaweed G. caudata are promising compounds in antioxidant and/or antitumor therapy

Determinação de motivos de ligação à quitina em vicilinas de Canavalia ensiformis e Vigna unguiculata através de métodos in silico e relação com suas toxicidades para o bruquídeo Callosobruchus maculatus (Coleoptera:Bruchidae)

Chitin is an important structural component of the cellular wall of fungi and exoskeleton of many invertebrate plagues, such as insects and nematodes. In digestory systems of insects it forms a named matrix of peritrophic membrane. One of the most studied interaction models protein-carbohydrate is the model that involves chitin-binding proteins. Among the involved characterized domains already in this interaction if they detach the hevein domain (HD), from of Hevea brasiliensis (Rubber tree), the R&R consensus domain (R&R), found in cuticular proteins of insects, and the motif called in this study as conglicinin motif (CD), found in the cristallography structure of the β-conglicinin bounded with GlcNac. These three chitin-binding domains had been used to determine which of them could be involved in silico in the interaction of Canavalia ensiformis and Vigna unguiculata vicilins with chitin, as well as associate these results with the WD50 of these vicilins for Callosobruchus maculatus larvae. The technique of comparative modeling was used for construction of the model 3D of the vicilin of V. unguiculata, that was not found in the data bases. Using the ClustalW program it was gotten localization of these domains in the vicilins primary structure. The domains R&R and CD had been found with bigger homology in the vicilins primary sequences and had been target of interaction studies. Through program GRAMM models of interaction ( dockings ) of the vicilins with GlcNac had been gotten. The results had shown that, through analysis in silico, HD is not part of the vicilins structures, proving the result gotten with the alignment of the primary sequences; the R&R domain, although not to have structural similarity in the vicilins, probably it has a participation in the activity of interaction of these with GlcNac; whereas the CD domain participates directly in the interaction of the vicilins with GlcNac. These results in silico show that the amino acid number, the types and the amount of binding made for the CD motif with GlcNac seem to be directly associates to the deleterious power that these vicilins show for C. maculatus larvae. This can give an initial step in the briefing of as the vicilins interact with alive chitin in and exert its toxic power for insects that possess peritrophic membrane

Análise toxicológica in vitro e in vivo de uma fucana antitrombótica da alga marrom Spatoglossum schdöederi

Fucan is a term used to denominate a family of sulfated polysaccharides rich in L-fucose. They are extracted mainly from the extracellular matrix of brown algae and echinoderms. The brown alga Spatoglossum schröederi (Dictyotaceae) has three heterofucans named A, B and C. Our research group have been extracted non anticoagulant heterofucan from S. schröederi which possess antithrombotic activity in vivo. However, their toxicity in vitro and in vivo has not yet been determined. For the results in toxicity in vitro, we observed that the fucan A at 20, 500 and 1000 μg/plate showed no mutagenic activity in Kado test (Microsuspension), when the bacterial strains TA97a, TA98, TA100 and TA102, with and without S9 were used. The comet assay showed that fucan A (from 20 to 1000 μg/mL) did not cause any genotoxic effect on CHO cells. There was no damage to the DNA of these cells, as evidenced by the tail length and tail moment, which were similar to that found for the negative control. The fucan A from S. schröederi was administered at 20 μg/g of rat (dose which it showed high antithrombotic activity) during two months. After that, the animals were killed and examined. The data showed that fucan A did not cause any change in biochemistry and hematological parameters, as well as, in the morphology and size of the rat s organs analyzed. In conclusion, this study indicates that fucan is a compound with potential pharmacological that has no toxicity

Avaliação do potencial anti-inflamatório, anticoagulante e hemorrágico de heparinóides presentes em "Artemia franciscana"

Heparan sulfate (HS) and Heparin (Hep) glycosaminoglycans (GAGs) are heterogeneous and highly charged polysaccharides. HS is structurally related to Hep but is much less substituted with sulfo groups than heparin and has a more varied structure (or sequence). Because of structural similiarities between these two polymers, they have been described together as heparinoids . Both chains bind a variety of proteins and mediate various physiologically important processes including, blood coagulation, cell adhesion and growth factor regulation. Heparinoids with structural characteristics similar to these described from HS and/or Hep from mammalian tissues have been isolated from different species of invertebrates, although only a few heparinoids from unusual sources have been characterized. The present study describes the presence of unusual heparinoids population from Artemia franciscana, isolated after proteolysis and fractionation by ion exchange resin and named, F-3.0M. The study model in vivo were hemostasis (rat tail scarification) and inflamatoty activity. The tests in vitro were used for coagulations assays (PT and APTT). The analyse of the heparinoids eluted with 3,0M NaCl showed electrophoretic migration in different buffer systems a single band with a behaviour intermediate between those of mammalian HEP and HS. The main products obtained from Artemia heparinoids after enzymatic degradation with heparitinases I and II from F. heparinum were N-sulphated disaccharides (∆U-GlcNS,6S/ ∆U,2S-GlcNS and ∆U-GlcNS) and N-acetylated disaccharides (∆U, GlcNAc). This heparinoid had a lower hemorrhagic effect (400μg/ml) when compared to unfractiionated heparins(25μg/ml).The results also suggest a negligible APTT activity of this heparinoid (62.2s). No action was observed on PT indicating that F-3.0M haven t action on the extrinsic pathway. The results showed that the fraction F- 3.0M have inhibitory effect on migration of leukocytes, 64.5% in the concentration of 10 μg/ml (P<0.001). The search for new heparin and/or heparan sulphates analogs devoid of anticoagulant activity is an atractive alternative and may open up a wide variety of new therapeutic applications

Avaliação das propriedades farmacológicas de polissacarídeos do fungo Scleroderma nitidum

Several pharmacological properties have been attributed to isolated compounds from mushroom. Recently, have these compounds, especially the polysaccharides derived from mushrooms, modulate the immune system, and its antitumor, antiviral, antibiotic and antiinflammatory activities. This study assesses the possible pharmacological properties of the polysaccharides from Scleroderma nitidum mushroom. The centesimal composition of the tissue showed that this fungus is composed mainly of fibers (35.61%), ash (33.69%) and carbohydrates (25.31%). The chemical analysis of the polysaccharide fraction showed high levels of carbohydrates (94.71%) and low content of protein (5.29%). These polysaccharides are composed of glucose, galactose, mannose and fucose in the following molar ratios 0.156, 0.044, 0.025, 0.066 and the infrared analysis showed a possible polysaccharide-protein complex. The polysaccharides from Scleroderma nitidum showed antioxidant potential with concentration-dependent antioxidant activity compared to ascorbic acid. The analysis scavenging of superoxide radical and inhibition of lipid peroxidation showed that the polysaccharides from S. nitidum have an IC50 of 12.70 mg/ml and EC50 10.4 μg/ml, respectively. The antioxidant activity was confirmed by the presence of reducing potential of these polysaccharides. The effect of these polymers on the inflammatory process was tested using the carrageenan or histamine-induced paw edema model and the sodium thioglycolate or zymosan-induced model. The polysaccharides were effective in reducing edema (73% at 50 mg/kg) and cell infiltrate (37% at 10 mg/kg) in both inflammation models tested. Nitric oxide, a mediator in the inflammatory process, showed a reduction of around 26% at 10 mg/kg of body weight. Analysis of pro- and anti-inflammatory cytokines showed that in the groups treated with polysaccharides from S. nitidum there was an increase in cytokines such as IL-1ra, IL-10, and MIP-1β concomitant with the decrease in INF-γ (75%) and IL-2 (22%). We observed the influence of polysaccharides on the modulation of the expression of nuclear factor κB. Thus, polysaccharides from S. nitidum reduced the expression of NF-κB by up to 64%. The results obtained suggest that NF-κB modulation is one of the possible mechanisms that explain the anti-inflammatory effect of polysaccharides from the fungus S. nitidum.

Avaliação do potencial antioxidante e antiinflamatório de galactomanana do fungo Tyllopiillus ballllouiii.

Polymers of mushroom cellular wall are recognized for presenting a lot of biological activities such as anti-inflammatory, antioxidant and anti-tumoral action. Polysaccharides from mushrooms of different molecular mass obtained mushrooms can activate leucocytes, stimulate fagocitic, citotoxic and antimicrobial activity including oxygen reactive species production. In this study were investigated chemical characteristics, in vitro antioxidant activity and anti-inflammatory action in an acute inflammation model of the polysaccharides extracted from Tylopilus ballouii. Results showed that were mainly extracted polysaccharides and that it primarily consisted of mannose and galactose with variable amounts of xylose and fucose. Infrared analysis showed a possible interation between this polysaccharides and proteins. In addition, molecular mass was about 140KDa. Antioxidant activity was tested by superoxide and hydroxyl radical scavenging assay, total antioxidant activity and lipid peroxidation assay. For superoxide and hydroxyl radical generation inhibition, polysaccharides have an IC50 of 2.36 and 0.36 mg/mL, respectively. Lipid peroxidation assay results showed that polysaccharides from Tylopilus ballouii present an IC50 of 3.42 mg/mL. Futhermore, anti-inflammatory assay showed that polysaccharides cause an paw edema decreasing in 32.8, 42 and 56% in 30, 50 and 70 mg/Kg dose, respectively. Thus, these results can indicate a possible use for these polysaccharides from Tylopilus ballouii as an anti-inflammatory and antioxidant.

Atividades biológicas de xilana de sabugo de milho

The corn cob is an agricultural by-product still little used, this in part due to the low knowledge of the biotechnological potential of their molecules. Xylan from corn cobs (XSM) is a polysaccharide present in greater quantity in the structure of plant and its biotechnology potential is little known. This study aimed to the extraction, chemical characterization and evaluation of biological activities of xylan from corn cobs. To this end, corncobs were cleaned, cut, dried and crushed, resulting in flour. This was subjected to a methodology that combines the use of alkaline conditions with waves of ultrasound. After methanol precipitation, centrifugation and drying was obtained a yield of 40% (g/g flour). Chemical analysis indicated a high percentage of polysaccharides in the sample (60%) and low contamination by protein (0.4%) and phenolic compounds (> 0.01%). Analysis of monosaccharide composition indicated the presence of xylose:glucose:arabinose:galactose:mannose:glucuronic acid in a molar ratio 50:20:15:10:2.5:2.5. The presence of xylan in the sample was confirmed by nuclear magnetic resonance (¹H and ¹³C) and infrared spectroscopy (IR). Tests were conducted to evaluate the antioxidant potential of XSM. This showed a total antioxidant capacity of 48.45 EAA/g sample. However, did not show scavenging activity of superoxide and hydroxyl radical and also reducing power. But, showing a high capacity chelating iron ions with 70% with about 2 mg/mL. The ability to XSM to influence cell proliferation in culture was also evaluated. This polymer did not influence the proliferation of normal fibroblast cells (3T3), however, decreased the rate of proliferation of tumor cells (HeLa) in a dose-dependent, reaching an inhibition of about 50% with a concentration around 2 mg/mL. Analyzing proteins related to cell death, by immunoblotting, XSM increases the amount of Bax, Bcl-2 decrease, increase cytochrome c and AIF, and reduce pro-caspase-3, indicating the induction of cell death induced apoptosis dependent and independent of caspase. XSM did not show anticoagulant activity in the PT test. However, the test of activated partial thromboplastin time (aPTT), XSM increased clotting time at about 5 times with 600 μg of sample compared with the negative control. The presence of sulfate on the XSM was discarded by agarose gel electrophoresis and IR. After carboxyl-reduction of XSM the anticoagulant activity decreased dramatically. The data of this study demonstrate that XSM has potential as antioxidant, antiproliferative and anticoagulant compound. Future studies to characterize these activities of XSM will help to increase knowledge about this molecule extracted from corn and allow their use in functional foods, pharmaceuticals and chemical industries.

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