Comparative study between the effects of hyaluronic acid and acid galactan purified from eggs of the mollusk Pomacea sp in wound healing

To compare the effect of hyaluronic acid (HA) and of AG on the healing of intestine wounds. Methods: The semi-purified extract of the eggs of the mollusc was obtained by fractionation with ammonium sulfate and purification for ion-exchange chromatography. The obtained galactans were eluted in water (neutral galactan) and in 0.1 and 0.2M NaCl (acidic galactans). The in vivo study was performed with 45 “Wistar” rats, separated in three groups (n=15). Solutions containing HA 1%, GA 1% or saline solution 0,9%, was placed topically on the sutures of wounds in the small intestine of the rats. After 05, 10 and 21 days the animals were sacrificed and biopsy of the healing tissue was done. Results: The hystologic grading was more significant for HA and AG groups when compared to the group C. AG stimulated the appearance of macrophages, giant cells and increase in the concentration of collagen in the area of the wound when compared to HA. Conclusion: The topical use of GA in intestinal wounds promoted the anticipation of events that are important in the wound healing

Purificação, caracterização e efeitos imunomodulatório e antiproliferativo de uma lectina do fungo Clavaria cristata (Holmsk.) Pers

A 140,0 kDa lectin was purified and characterized from the mushroom Clavaria cristata. The purification procedures from the crude extract of the mushroom comprised gel filtration chromatography on Sephacryl s200 and ion exchange on Resource Q column. The purified lectin agglutinated all types of human erythrocytes with preference for trypsinized type O erythrocytes. The haemagglutinating activity is dependent of Ca 2+ ions and was strongly inhibited by the glycoprotein bovine submaxillary mucin (BSM) up to the concentration of 0, 125 mg/mL. The C. cristata lectin (CcL) was stable in the pH range of 2,5-11,5 and termostable up to 80 °C. CcL molecular mass determined by gel filtration on a Superose 6 10 300 column was approximately 140,3 kDa. SDS polyacrilamide gel electrophoresis revealed a single band with a molecular mass of approximately 14,5 kDa, when the lectin was heated at 100 ⁰C in the presence or absence of β-mercaptoethanol. CcL induced activation of murine peritoneal macrophages in vitro resulting in the release of nitric oxide (NO), reaching the maximum production at 24 h. In experimental paw oedema model in mice, CcL showed proinflammatory activity being able to induce oedema formation. Cell viability of HepG2, MDA 435 e 3T3 cell lines was examined after 72 h of incubation with CcL in different concentrations (0,5-50 μg/mL). CcL inhibited HepG2 cells growth with an IC50 value of 50 μg/mL. In the present work, the observed immunomodulatory and antiproliferative effects indicate CcL as a possible immunomodulator compound, interfering in the macrophages immune response, taking possible anti-parasitic, anti-tumoral effects or diagnostic and/or therapeutic

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

A infecção por Leishmania infantum chagasi altera o metabolismo lipídico do hospedeiro

American visceral leishmaniasis (AVL), caused by Leishmania infantum chagasi (L.i.chagasi), stands as a public health problem in Brazil, with human and canine cases related in all states..Lipid metabolism can be modified in several status of infection. For example, experimental studies show that the cholesterol is necessary to internalization and replication of L.i.chagasi in macrophages through caveolar domains. Patients with AVL present low levels of cholesterol and a visible triglycerides increase. This work aimed to evaluate the lipid metabolism in several post-infection status by L.i.chagasi, including individuals with symptomatic infection (AVL), and asymptomatic. The levels of cholesterol, triglycerides, HDL and reactive C protein, were measured. Individuals with AVL were compared with individuals with assymptomatic infection and presented low levels of total cholesterol (128 ± 6.180 mg/dL vs. 158 ±5.733 mg/dL, p=0.0001), HDL (29 ± 1.746 mg/dL vs. 37 ± 1.647 mg/dL, p=0.0001), increased levels of triglycerides (149.5 mg/dL ± 12.72 vs. 78.00 ± 10.43 mg/dL, p=0.0095) and higher levels of reactive C protein (1.750± 0.4939 mg/dL vs. 0.40 ± 0.1707 mg/dL; p=0.0001). The expression of genes related to lipid metabolism, such as LXR-a, LXR-b, PPAR-a, PPAR-d, PPAR-g and APOE was evaluated by real time PCR. A reduction in the expression of those genes was found in the group of AVL patients corroborating the serum levels of the metabolites earlier quantified. Our findings suggest a modulation of metabolism of lipids, in the chronic phase of AVL, this could facilitate the survival of leishmania, due to the known reduction on the ability of macrophages in presenting antigens efficiently to the T cells due to the reduction in the cholesterol available, it results in a subversion of the host immunity.

Avaliação do potencial anti-inflamatório de composto tipo heparina (cCTH) extraído do caranguejo Goniopsis cruentata em modelo experimental de peritonite

Heparin, a sulfated polysaccharide, was the first compound used as an anticoagulant and antithrombotic agent. Due to their structural characteristics, also has great potential anti-inflammatory, though such use is limited in inflammation because of their marked effects on coagulation. The occurrence of heparin-like compounds that exhibit anticoagulant activity decreased in aquatic invertebrates, such as crab Goniopsis cruentata, sparked interest for the study of such compounds as anti-inflammatory drugs. Therefore, the objective of this study was to evaluate the potential modulator of heparin-like compound extracted from Goniopsis cruentata in inflammatory events, coagulation, and to evaluate some aspects of its structure. The heparin-type compound had a high degree of N-sulphation in its structure, being able to reduce leukocyte migration into the peritoneal cavity at lower doses compared to heparin and diclofenac sodium (anti-inflammatory commercial). Furthermore, it was also able to inhibit the production of nitric oxide and tumor necrosis factor alpha by activated macrophages, inhibited the activation of the enzyme neutrophil elastase in low concentrations and showed a lower anticoagulant effect in high doses as compared to porcine mucosal heparin. Because of these observations, the compound extracted from crab Goniopsis cruentata can be used as a structural model for future anti-inflammatory agents

Estudo clínico-laboratorial e histopatológico de cães naturalmente infectados por Leishmania chagasi com diferentes graus de manifestação física

Canine Visceral Leishmania (CVL) is an important zoonotic disease that has a world wide distribution and has a large impact on public health on the American Continent, especially in Brazil, where the nature of endemic diseases in humans affects a large part of the nation. The influence of the prevalence of CVL in the increased rate of human cases in endemic areas and in the unleashing of epidemic outbreaks shows the need for a more profound understanding, that would generate significant advances in the current measures used to control the reservoirs of sickness that are practiced by the Programa Nacional de Vigilância e Controle da Leishmaniose Visceral. The present work describes and compares the clinical-laboratorial and histopathological findings of twenty-three dogs that were naturally infected by Leishmania chagasi, from endemic areas in metropolitan Natal, Rio Grande do Norte, Brazil. These animals, that were selected and given physical and serological exams (IFI and ELISA rK-39), were classified according to the degree of clinical severity and had blood samples drawn (whole blood and serum) for a complete hemogram and a coagulogram to be done as well as biochemical tests for kidney and liver function. The confirmation of infection by L. chagasi was done after the euthanasia of the animals, through the direct demonstration of the parasite in the impression of the spleen and liver crowned with GIEMSA and through a cultivation by means of NNN/Schneider. According to the clinical evaluation, the animals were classified as asymptomatic (7), oligosymptomatic (7) and polysymptomatic (9). Among the animals that were chosen to be autopsied, there were 2 asymptomatic, 3 oligosymptomatic and 3 polysymptomatic, for the purpose of studying their histopathology, having collected fragments of the spleen, liver, kidneys and skin and were fixed in 10% tamponed formol. The comparison between the average parameters of the clinical-laboratory tested animals in the groups was done through the Student t test (a<0.05). The main clinical signals observed were lymphadenomegaly, alopecy, dermatitis, exfoliation, cutaneous ulcers, onicogriphosis and emaciation. The main clinical-laboratorial alterations established, mainly in the polysymptomatic group, were anemia, hyperproteinemia, hyperglobulinemia, alterations in the albumin/globulin ratio and increased ALT activity. Renal alterations were not verified (urea and creatinine levels were normal). Thrombocytopenia was observed in three clinical groups. However, the other indicators of coagulation function (TAP and TTPA) did not have abnormal variations. There were inflammatory infiltrations and leishmania amastigotes in the skin of polysymptomatic dogs, however, they were not found in the skin of asymptomatic animals. Hypertrophy and hyperplasia of the phagocyte mononuclear system, leishmania amastigote parasites were found in the macrophages, extramedullary hematopoiesis and degenerative alterations were detected in the spleen and liver of 8 of the animals submitted to histopathological exams. In accord with these results, it was demonstrated that the expected alterations in the hematological and biochemical parameters in function of their viscerotropic nature of CVL are mainly observed in the more advanced stages of the disease. The absence of inflammatory infiltration and parasite load in the skin suggest that infected animals without symptoms may have an importance irrelevant to the infectiousness of the vector

Avaliação da atividade antimalárica de extratos obtidos de algas marinhas no litoral do Rio Grande do Norte

Malaria is a major parasitic disease worldwide, accounting for about 500 million cases and causing 2 million to 3 million deaths annually. Four species are responsible for transmitting this disease to humans: Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium ovale. The parasite resistance to antimalarial drugs and the usual limitations of the vector control implications are contributing to the spread of the disease. The most of significant advances in the search for new antimalarial drugs is based on natural components, the main ones being currently used antimalarial drugs derived from plants. Research on natural products of marine origin (particularly algae) show that some species possess antiplasmodial activity. Knowing that the coast of Rio Grande do Norte is home to several species of algae, the present study was to evaluate, for the first time, the antimalarial activity of ethanolic extracts of seaweed Spatoglossum schroederi, Gracilaria birdiae and Udotea flabellum against Plasmodium falciparum 3D7 strain tests and in vitro using the murine model (Plasmodium berghei) for evaluation in vivo. These species were ground, macerated with ethanol for 24 hours and the extracts concentrated in rotaevaporador (45 ° C ± 5 ° C). For in vitro tests, the extracts were diluted and tested at concentrations between 100 and 1.56 μg/ml (seven concentrations in triplicate), in order to obtain IC50 of each extract. The cytotoxicity tests with macrophages and BGM were performed using the MTT colorimetric assay. BGM macrophages and cells were distributed in 96 wells per plate (1x 105 to macrophages and 1x104 cells per well for BGM) and incubated for 24h at 37 ° C. The ethanol extracts were diluted and tested at concentrations of 100 to 1,56 μg/ml (seven concentrations in triplicate). After periods of 24 hours of incubation with the extracts, 100 μg of MTT was added to each well, and 3 hours elapsed, the supernatant was removed and added 200 μl of DMSO in each well. The absorbance of each well was obtained by reading on a spectrophotometer at 570 nm filter. To evaluate the acute toxicity in vivo, Swiss mice received a single dose (oral) 2000 mg/kg/animal of each extract tested. The parameters of acute toxicity were observed for 8 days. For in vivo tests, Swiss mice were inoculated with 1x105 erythrocytes infected with P. berghei. The treatment was given first to fourth day after infection with 0.2 ml of the extracts in doses of 1000 and 500 mg//g animal. The negative control group received 0.2 ml of 2% Tween-20, whereas the positive control group received sub-dose of chloroquine (5 mg/kg/animal). The assessment of antimalarial activity was done by suppressing suppressing the parasitemia at 5 and 7 days after infection. The growth inhibition of parasites was determined relative to negative control (% inhibition = parasitaemia in control - parasitemia in sample / parasitemia control x 100), the mortality of animals was monitored daily for 30 days The results showed that algae Spatoglossum schroederi and Udotea flabellum showed antimalarial activity in vitro, with reduced parasitemia of 70.54% and 54, respectively. The extracts of the three algae tested showed moderate to high cytotoxicity. Algae S. schroederi and U. flabellum were active against P. berghei only at doses of 500 mg / kg with reduction ranging from 54.58 to 52.65% for the fifth day and from 32.24 to 47.34% for the seventh day, respectively. No toxicity was observed in vivo at the dose tested, over the 8 days of observation. Although preliminary data, the bioactive components in those possible seaweed may be promising for the development of new anti-malarial drugs

Avaliação das atividades anti-toxoplásmica, antioxidante e antiinflamatória dos monoterpenos timol (lippia sidoides) e estragol (croton zenhtneri)

Toxoplasmosis, a benign disease in normal healthy individuals, can have serious effects in pregnant women and immunocompromised patients. It is a parasitic disease caused by Toxoplasma gondii (Tg), an obligatory intracellular protozoan. The prophylactic and therapeutic arsenal against this parasite is very restricted. Thus, there is an ongoing search for novel drugs and therapeutic strategies. A promising alternative is a rational approach using medicinal plants. This study aimed to standardize methodologies for assessing the toxicological, antiproliferative, antioxidant, antiinflammatory and anti-Toxoplasma effects of Estragole and Thymol compounds isolated from species of plants (Lippia sidoides and Croton zenhtneri) commonly used in the Cariri region of Ceara State, Brazil. First we evaluated in vivo toxicity and conducted a pathological analysis of mice livers. In vivo antiinflammatory activity was assessed using air pouch and paw edema methods. Cytotoxicity assays were performed and antiproliferative, antioxidant and nitric oxide production analyzed. Anti-Toxoplasma activity was evaluated in a congenital experimental model with varying stages of maternal infection using the ME-49 strain and a non- congenital model by using ME-49 and RH strains. The results suggest low to moderate toxicity for both compounds. Thymol was more toxic in vivo and in vitro, having greater pathological repercussion than Estragole. The compounds were inactive for antiproliferative activity. Thymol showed better antioxidant activity, while Estragole stimulated nitric oxide production in macrophages. Both showed significant antiinflammatory activity. In non-congenital Tg infection, both compounds were active only against the ME49 strain. In congenital infection, Estragole (oral route) improved the newborn weight of infected mothers compared with untreated controls. Subcutaneous administration of the two compounds increased the weight of offspring born to infected mothers compared with untreated controls. We concluded that Estragole and Thymol exhibit important biological and anti-Toxoplasma activities. Further studies are needed to elucidate the mechanism of action of these compounds and other possible activities not investigated in the present study

Analise estrutural do galactano acidico dos ovos de pomacea lineata e avaliacao de suas atividades proliferativa e antiinflamatoria

The acidic galactan (AG) was obtained by extraction and proteolysis by acetone precipitation of the eggs of the mollusc Pomacea lineata. Its structure was elucidated by a combination of chemical analysis, the intrinsic viscosity and NMR spectroscopy 1D and 2D. Biological aspects of AG were evaluated by in vivo testing of healing and peritonitis induced (anti-inflammatory activity) and in vitro assays of cytotoxicity (MTT). This polymer showed a simple structure without the presence of sulfate and uronic acids in its structure. Its intrinsic viscosity and relative were evaluated at 0.44 ± 0.05 and 1.744± 0.07 dl.g-1. Spectroscopy showed that the AG has a constitution composed predominantly of β-D-galactosis, and β-D-glucosamine-NAcetil that comes in a smaller proportion in chain. The character of this acidic polysaccharide is given by the presence of pyruvate in the molecule, forming a cyclic acetal of six states, located in β-D-galactosis. The involvement of AG in the healing process was evaluated and the histological analysis revealed that there was so early in the process of healing, a great stimulation of macrophages with granuloma formation. Suggesting that AG may have promoted the advance of biological events required for tissue healing. In the trial of the GA-induced peritonitis showed dose dependent, demonstrating the anti-inflammatory effect at concentrations above 20 mg/kg, and confirming its inflammatory character and the concentration of 1mg/kg. In vitro tests used in the GA concentration of 1000 μg/mL showed proliferative activity by stimulating the growth of 3T3 cells, corroborating the findings in vivo and demonstrating the absence of cytotoxic activity

Atividade antiinflamatória de uma heterofucana da alga marrom Padina gymnospora

Fucans, sulfated polysaccharides extracted from brown algae and some echinoderms, have been extensively studied for its diverse biological activities and because of its interference with molecular mechanisms of cell to cell recognition, including leukocyte trafficking from blood vessels into sites of inflammation mediated by selectin, a family of adhesion molecules. In the present study, we examined structural features of a heterofucan extracted from brown algae Padina gymnospora and its effect on the leukocyte migration to the peritoneum. The sulfated polysaccharides were extracted from the brown seaweed by proteolysis with the proteolytic enzyme maxatase. The presence of protein and uronic acid contamination was detected in the crude polysaccharide extract. Fractionation of the crude extract with growing concentrations of acetone produced five fractions with different concentrations of fucose, xylose, uronic acid, galactose, glucose and sulfate. The fraction precipitated with 1.5 volumes of acetone was characterized by infrared and nuclear magnetic resonance, through which can be observed the presence of sulfate groups in the C4 of -L-fucose. The anti-inflammatory action of this composite was assessed by a sodium thioglycollate-induced peritonitis assay and through nitric oxide production by the peritoneal macrophages using Griess reagent. Fraction F1.5 was efficient in reducing leukocyte influx into the peritoneal cavity when 10 mg/kg and 25mg/kg were used, resulting in a decrease of 56 and 39%, respectively. A decrease of nitric oxide production occurred when high concentrations of fucana were used. The cytotoxicity of the composite was also assessed using the reduction of 3-(4,5 dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT). Fraction F1.5 had no cytotoxicity when 500 μg/mL of the fraction was used. This study suggests the use of fraction F1.5 (heterofucan) as an anti-inflammatory