Avaliação do extrato do fungo caripia montagnei e de agonistas de PPAR - α no processo inflamatório

The mushrooms have been object of intense research in view of its potential raising of application in different sectors of the pharmacology and alimentary industry. Among diverse bioactive composites of polyssacharides nature that exist in the fungus the glucans are much searched. These are polymers of glucose and classified as the type of glicosidic linking [α, β]. Peroxisome proliferator-activated receptors (PPARs), ranscription factors belonging to the family of nuclear receptors that bind themselves o specific agonists, have shown their importance in controlling the inflammatory process. The aim of this study was to perform a chemical characterization of extract rom the mushroom Caripia montagnei, assess its antiinflammatory and antibacterial effect and determine if this effect occurs via PPAR. This mushroom is composed of carbohydrates (63.3±4.1%), lipids (21.4l±0.9%) and proteins (2.2± 0.3%). The aqueous solution resulting from the fractionation contained carbohydrates (98.7±3.3%) and protein (1.3±0.25%). Analyses of infrared spectrophotometry and of nuclear magnetic esonance demonstrated that the extract of mushroom C. montagnei is rich in β-glucans. In hioglycolate-induced peritonitis, the C. montagnei glucans (50 mg/kg) educed the inflammatory process in 65.5±5.2% and agonists, pharmacological igands, for PPAR: Wy-14643 (49.3±6.1%), PFOA (48.9±3.8%) and clofibrate in 45.2±3.2%. Sodium diclofenac showed a reduction of 81.65±0.6%. In the plantar edema, the glucans from C. montagnei (50 mg/kg) and L-NAME reduced the edema to a similar degree 91.4±0.3% and 92.8±0,5 %, respectively. In all the groups tested, nitric oxide (NO), an inflammation mediator, showed a significant reduction in the nitrate/nitrite levels when compared to the positive control (P<0.001). The C. montagnei glucans did not show cytotoxicity in the concentrations tested (2.5, 5.0, 10.0, 20.0 and 40.0 µg/100 µL). Antibacterial activity demonstrated that, unlike total extract, there was no inhibition of bacterial growth. The C. montagnei glucans show great potential for antiinflammatory applications. This effect suggests that it is mediated by PPAR activation and by COX and iNOS inhibition

Purificação, caracterização e atividade bioinseticida de um inibidor de tripsina de sementes de Crotalaria pallida

A proteinaceous trypsin inhibitor was purified from Crotalaria pallida seeds by ammonium sulphate fractionation, affinity chromatography on immobilized Trypsin-Sepharose and TCA precipitation. The trypsin inhibitor, named ITC, had Mr of 32.5 kDa by SDS-PAGE and was composed by two subunits with 27.7 and 5.6 kDa linked by disulphide bridges, a typical characteristic of Kunitz-Inhibitor family. ITC was stable until 50°C, and at 100°C its residual activity was of about 60%. Also, ITC was stable at pHs 2 to 12. The inhibition of trypsin by ITC was non-competitive, with a Ki of 8,8 x 10-7M. ITC inhibits weakly other serine proteinases such as chymotrypsin and elastase. The inhibition of papain (44% of inhibition), a cysteine proteinase was an indicative of the bi-functionality of ITC. In vitro assays against digestive proteinases from several Lepdoptera, Diptera and Coleoptera pests were made. ITC inhibited in 100% digestive enzymes of Ceratitis capitata (fruit fly), Spodoptera frugiperda and Alabama argillacea, the last one being a cotton pest. It also inhibited in 74.4% Callosobruchus maculatus (bean weevil) digestive enzymes, a Coleoptera pest. ITC, when added in artificial diet models, affected weakly the development of C. capitata larvae and it had a WD50 of 2.65% to C. maculatus larvae

Purificação e caracterização de uma β-glucuronidase extraída do molusco mesogastropoda ampularidade Pomacea sp

This work studies the involved enzymatic way in the metabolism of glycosaminoglycans sulfateds in the mollusc Pomacea sp. Had been identified endoglycosidases and exoglycosidases in the enzymatic extract of the mollusc Pomacea sp by means of hydrolysis activity in condroitim sulphate of whale cartilage and of the p-Nitrofenil-β-glucuronide, respectively. The enzymatic extracts qere obtained of Pomacea sp. being used of 0.1 sodium acetate buffer, pH 5.0 and later centrifugated the 8,000 x g and the presents proteins in the sobrenadante were submitted to the fractionament with two crescents ammonium sulphate concentrations, the visualized activity biggest in the F2 fraction (50-80%). The β-glucuronidase (F3) was isolated in gel chromatography filtration Biogel 1.5m, the purification degree was ratified in Chromatography Liquid of high efficiency (HPLC). The enzyme was purificated 6.362,5 times with 35,6% yield. The β -glucuronidase isolated in this work showed a molecular mass of 100 kDa, determined for eletroforese in poliacrilamida gel . The determination of the ideal kinetic parameters for the catalysis of the p-nitrofenil- β -glucuronide for β-glucuronidase, showed excellent activity in pH 5,0 and temperature 65ºC for 6 hours and apparent Km of 72 x 10-2 mM. It is necessary for the total degradation of 3mM of p-N-β-glucoronide, the amount of 1,2μg of ss-glucuronidase. The BaCl2 increased the activity of ss-glucuronidase, and the activity was inhibited completely by the composites SDS and NaH2PO4

Atividade β-D-N-Acetilglucosaminidásica de enzimas imobilizadas extraídas da Artemia franciscana e possíveis aplicações biotecnológicas

A β-D-N-acetilglucosaminidase extracted and partially isolated from crustacean Artemia franciscana by ammonium sulfate precipitation and filtration gel chromatography Bio Gel A 1.5m. the enzyme was immobilized on ferromagnetic Dacron yielding a insoluble active derivative with 5.0 units/mg protein and 10.35% of the soluble enzyme activity. β-D-N-acetilglucosaminidase-ferromagnetic Dacron was easily removed from the reaction mixture by a magnetic field, it was reused for ten times without loss in its activity. The ferromagnetic Dacron was better activated at pH 5.0. The particles visualized at scanning electron microscope (SEM) had presented different sizes, varying between 721nm and 100µm. Infra red confirmed immobilization on support, as showed by primary amino peaks at 1640 and 1560 cm-1 . The immobilize enzyme presented Km of 2.32 ± 0.48 mM and optimum temperature of 50°C. Bought presented the same thermal stable of the soluble enzyme and larger enzymatic activity at pH 5.5. β-D-N-acetilglucosaminidase-Dacron ferromagnético showed sensible for some íons as the silver (AgNO3), with loss of activity. The β-D-N acetilglucosaminidase activity for mercury chloride (HgCl2), whom is one of the most toxic substance joined in nature, it was presented activity already diminished at 0,01mM and lost total activity at 4mM, indicating sensitivity for this type of metal. β-D-N-acetilglucosaminidase-ferromagnetic Dacron showed degradative capacity on heparan sulfate, the enzyme still demonstrated degradative capacity on heparan sulphate, suggesting a possible application to produce fractions of this glycosaminoglycan

Efeito inseticida de vicilinas isoladas de sementes de erythrina velutina em condições de semi-campo para moscas das frutas (ceratitis capitata)

Chitin-binding vicilins from legume seeds (Erythrina velutina. Canavalia ensiformes and Phaseolus vulgares) were isolated by ammonium sulfate followed by affinity chromatography on a chitin column. Effect of these vicilins on female adults of Ceratitis capitata was examined by bioassay and in a semi-field assay model. Mechanism of action of the vicilins was determined by in vivo digestibility and chitin affinity. Among the tested vicilins, E. velutina when added to diet caused strong effect on mortality at 10% dose. This insecticidal property was tested in a semi-field assay which showed the same effect observed in laboratory conditions, where doses of 10% and 15% were lethal to female adults of C. capitata. These deleterious effects were not only associated to the binding to chitin structures present in peritrophic membrane, but principally to its low digestibility in the C. capitata digestive tract. This fact was confirmed because chiting binding proteins as WGA and the other tested vicilins were not toxic to female adults of C. capitata due susceptibility of these proteins to digestive enzymes of the insects. By other side EvV was more resistant to digestive enzymes, causing deleterious effects on female adults of C. capitata. These results showed that EvV may be part of the pest management programs or an alternative in plant improvement program in the population control of this fruticulture pest

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

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

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Lectina da esponja marinha Tedania ignis: purificação, caracterização e interação com leishmanias

Lectin obtained from the marine sponge Tedania ignis was purified and characterized by extraction of soluble proteins (crude extract) in 50mM Borax, pH 7.5. The purification procedure was carried out by crude extract precipitation with ammonium sulfate 30% (FI). The precipitated was resuspended in the same buffer and fractionated with acetone 1.0 volume (F1.0). A lectin was purified from this specific fraction by using an affinity chromatography Sepharose 6B. This lectin preferentially agglutinated human erythrocytes from B type previously treated with papain enzyme. The hemagglutinating activity lectin was dependent of divalent Mn2+ cation and was inhibited by the carbohydrates galactose, xylose and fructose. SDS-PAGE analysis indicated a molecular mass of the lectin around 45 kDa. This protein showed stability until 40°C for 1 h. Further, it showed activity between pH 2.5 and 11.5, with an enhanced activity at pH 7.5. Leishmania chagasi promastigotes stained with Coomassie brilliant blue R-250 were agglutinated by F1,0 and in the presence of galactose this interaction was abolished. These results show that this lectin could be implicated in defense procedures and it will can be used as biological tools in studies with this protozoon

Purificação e caracterização de uma ß-N-acetillhexosaminadase extraída do mamífero marinho Sotalia fluviatilis

This report shows 2232 times purification of a βNAcetylhexosaminidase from hepatic extracts from the sea mammal Sotalia fluviatilis homogenate with final recovery of 8,4%. Sequenced steps were utilized for enzyme purification: ammonium sulfate fractionation, Biogel A 1.5 m, chitin, DEAESepharose and hydroxyapatite chromatographies. The protein molecular mass was estimated in 10 kDa using SDSPAGE and confirmed by MALDITOF. It was found to have an optimal pH of 5.0 and a temperature of 60°C. Using pnitrophenylNAcetylβDglycosaminide apparent Km and Vmax values were of 2.72 mM and 0.572 nmol/mg/min, respectively. The enzyme was inhibited by mercury chloride (HgCl2) and sodium dodecil sulfate (SDS)

Purificação e caracterização parcial de duas n-acetil-B-hexosaminidases do equinoderma marinho echinometra lucunter

Two b-N-acetylhexosaminidases (F11 e F15) were purified from Echinometra lucunter gonads extracts. The purified enzymes were obtained using ammonium sulfate fractionation, followed by gel filtration chromatographies (Sephacryl S-200, Sephadex G-75 and Sephacryl S-200). The F11 fraction was purified 192.47 -fold with a 28.5% yield, and F15 fraction 85.41 -fold with a 32.3% yield. The molecular weights of the fractions were 116 kDa for F11 and 42 kDa for F15 using SDS-PAGE. In Sephacryl S-200, F15 was 84 kDa, indicating that it is a dimeric protein. When p-nitrophenyl-β-D-glycosaminide was used as substrate, we determined an apparent Km of 0.257 mM and Vmax of 0.704 for F11 and for F15 the Km was 0.235 mM and Vmax of 0.9 mM of product liberated by hour. Both enzymes have optimum pH and temperature respectively at 5.0 and 45 °C. The enzymes showed inhibition by silver nitrate, while the glucuronic acid was a potent activator. The high inhibition of F15 by N-etylmaleimide indicates that sulphydril groups are involved in the catalysis of synthetic substrate

Estudo comparativo de carragenanas comerciais Kappa, Iota e Lambda no processo inflamatório em ratos :edema intraplantar e pleurisia

The Iota, Kappa and Lambda commercial carrageenans are rarely pure and normally contain varying amounts of the other types of carrageenans. The exact amount of impurity depends on the seaweed source and extraction procedure. Then, different analysis methods have been applied for determination of the main constituents of carrageenans because these three carrageenans are extensively used in food, cosmetic and pharmaceutical industry. The electrophoresis of these compounds proved that the carrageenans are constituted by sulfated polysaccharides. These compounds were characterized by colorimetric methods and was observed that the Lambda carrageenan shown the greater value (33.38%) of sulfate. These polymers were examined by means of 13C NMR spectroscopy and infrared spectra. The polysaccharides consisted mainly of units alternating of sulfated galactoses and anhydrogalactoses. The aim of the study was also to test the inflammatory action of these different polysaccharides. A suitable model of inflammation is acute sterile inflammation of the rat hind limb induced by carrageenan. Paw edema was induced by injecting carrageenans (κ, ι and λ) in saline into the hind paw of a male Wistar rats (175–200 g). The pathway to acute inflammation by carrageenan (kappa, iota and lambda) were expressed as time-edema dependence and measured by paw edema volume. For this purpose, was used an apparatus (pakymeter), which makes it possible to measure the inflammation (swelling of the rat foot) with sufficient accuracy. The results showed that κ-carrageenan (1%) have an edema of 3.7 mm and the paw edema increase was time and dose dependent; the ι-carrageenan (0.2%) caused an edema of 4 mm and the λ-carrageenan (1%) caused an edema of 3.6 mm. Other model was used in this study based in the inflammation of pleura for comparatives studies. Injection of carrageenans into the pleural cavity of rat induced an acute inflammatory response characterized by fluid accumulation in the pleural cavity, a large number of neutrophils and raised NO production. The levels of NO were measured by Griess reactive. The ι-carrageenan caused the greater inflammation, because it has high concentration of nitrite/nitrate (63.478 nmoles/rat), exudato volume (1.52 ml) and PMNs (4902 x 103 cells). Quantitative evaluation of inflammations of rats is a useful and important parameter for the evaluation of the efficacy of anti-inflammatory drugs

Obtenção de nanopartículas de hexaferrita de bário pelo método pechini

In this study barium hexaferrite was (general formulae BaFe12O19) was synthesized by the Pechini method under different conditions of heat treatment. Precursors like barium carbonate and iron nitrate were used. These magnetic ceramic, with magnetoplumbite type structure, are widely used as permanent magnet because of its excellent magnetic properties, such as: high Curie temperature, good magnetic anisotropy, high coercivity and corrosion resistance. The samples were characterized by thermal analysis (DTA and TG), X- ray Diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), Scanning Electron Microscopy (SEM) end Vibrating sample Magnetometer (VSM). The results confirm the expected phase, which was reinforced according to our analysis. A single phase powder at relatively high temperatures with particle sizes around 100 nm was obtained. The characteristic magnetic behavior one of the phases has been noted (probably superparamagnetic material), while another phase was identified as a ferrimagnetic material. The ferrimagnetic phase showed vortex configuration with two central and slightly inclined plateaus. In general, increase of heat treatment temperature and time, directly influenced the technological properties of the samples

Síntese de pigmentos cerâmicos inorgânicos nanométricos e encapsulados com estrutura core-shell pela rota dos precursores poliméricos

The present work has as objective the development of ceramic pigments based in iron oxides and cobalt through the polymeric precursor method, as well as study their characteristics and properties using methods of physical, chemical, morphological and optical characterizations.In this work was used iron nitrate, and cobalt citrate as precursor and nanometer silica as a matrix. The synthesis was based on dissolving the citric acid as complexing agent, addition of metal oxides, such as chromophores ions and polymerization with ethylene glycol. The powder obtained has undergone pre-ignition, breakdown and thermal treatments at different calcination temperatures (700 °C, 800 °C, 900 °C, 1000 °C and 1100 °C). Thermogravimetric analyzes were performed (BT) and Differential Thermal Analysis (DTA), in order to evaluate the term decomposition of samples, beyond characterization by techniques such as BET, which classified as microporous materials samples calcined at 700 ° C, 800 º C and 900 º C and non-porous when annealed at 1000 ° C and 1100 º C, X-ray diffraction (XRD), which identified the formation of two crystalline phases, the Cobalt Ferrite (CoFe2O4) and Cristobalite (SiO2), Scanning Electron Microscopy (SEM) revealed the formation of agglomerates of particles slightly rounded;and Analysis of Colorimetry, temperature of 700 °C, 800 °C and 900 °C showed a brown color and 1000 °C and 1100 °C violet

Síntese e caracterização de TaC e óxido misto de tântalo e cobre nanoestruturados a partir do precursor oxálico de tântalo através de reações gás-sólido e sólido-sólido a baixa temperatura

The research and development of nanostructured materials have been growing significantly in the last years. These materials have properties that were significantly modified as compared to conventional materials due to the extremely small dimensions of the crystallites. The tantalum carbide (TaC) is an extremely hard material that has high hardness, high melting point, high chemical stability, good resistance to chemical attack and thermal shock and excellent resistance to oxidation and corrosion. The Compounds of Tantalum impregnated with copper also have excellent dielectric and magnetic properties. Therefore, this study aimed to obtain TaC and mixed tantalum oxide and nanostructured copper from the precursor of tris (oxalate) hydrate ammonium oxitantalato, through gas-solid reaction and solid-solid respectively at low temperature (1000 ° C) and short reaction time. The materials obtained were characterized by X-ray diffraction (XRD), Rietveld refinement, Scanning Electron Microscopy (SEM), Spectroscopy X-Ray Fluorescence (XRF), infrared spectroscopy (IR), thermogravimetric (TG), thermal analysis (DTA) and BET. Through the XRD analyses and the Reitiveld refinement of the TaC with S = 1.1584, we observed the formation of pure tantalum carbide and cubic structure with average crystallite size on the order of 12.5 nanometers. From the synthesis made of mixed oxide of tantalum and copper were formed two distinct phases: CuTa10O26 and Ta2O5, although the latter has been formed in lesser amounts