Anti-inflammatory and antinociceptive effects of Baccharis dracunculifolia DC (Asteraceae) in different experimental models

Aim of the study: The aerial parts of Baccharis dracunculifolia D.C., popularly known as ""alecrim do campo"" are used in folk medicine as anti-inflammatory. The aim of the present study was to evaluate the anti-inflammatory and antinociceptive activities of the crude hydroalcoholic extract obtained from leaves of Baccharis dracunculifolia (BdE), which have not been reported. Matetials and methods: BdE was analyzed by HPLC and in vivo evaluated (doses ranging from 50 to 400 mg/kg, p.o.) by using the acetic acid-induced abdominal constrictions, paw oedema induced by carrageenan or histamine, overt nociception models using capsaicin, glutamate or phorbol myristate acetate (PMA), formalin-induced nociception and mechanical hypernociception induced by carrageenan or complete Freund adjuvant (CFA). As positive controls it was used paracetamol in both acetic acid and formalin tests; dipyrone in capsaicin, glutamate and PMA-induced nociception; indomethacin in CFA and carrageenan-induced hypernociception models. In addition, the in vitro effects of BdE on COX-2 activity and on the activation of NF-kappa B were also evaluated. Results: BdE (50-400 mg/kg, p.o.) significantly diminished the abdominal constrictions induced by acetic acid, glutamate and CFA. Furthermore, BdE also inhibited the nociceptive responses in both phases of formalin-induced nociception. BdE, administered orally, also produced a long-lasting anti-hypernociceptive effect in the acute model of inflammatory pain induced by carrageenan. It was also observed the inhibition of COX-2 activity by BdE. Conclusion: In summary, the data reported in this work confirmed the traditional anti-inflammatory indications of Baccharis dracunculifolia leaves and provided biological evidences that Baccharis dracunculifolia, like Brazilian green propolis, possess antinociceptive and anti-inflammatory activities. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Environmental Exposure to Methylmercury is Associated with a Decrease in Nitric Oxide Production

Some studies have recently suggested that mercury (Hg)-exposed populations face increased risks of cardiovascular diseases, and experimental data indicate that such risks might be due to reductions in nitric oxide bioavailability. However, no previous study has examined whether Hg exposure affects plasma nitrite concentrations in humans as an indication of nitric oxide production. Here, we investigated whether there is an association between circulating nitrite and Hg concentrations in whole blood, plasma and hair from an exposed methylmercury (MeHg) population. Hair and blood samples were collected from 238 persons exposed to MeHg from fish consumption. Hg concentrations in plasma (PHg), whole blood (BHg) and hair Hg (HHg) were determined by inductively coupled plasma-mass spectrometry. Mean BHg content was 49.8 +/- 35.2 mu g/l, mean PHg was 7.8 +/- 6.9 mu g/l and HHg 14.6 +/- 10.6 mu g/g. Mean plasma nitrite concentration was 253.2 +/- 105.5 nM. No association was found between plasma nitrite concentration and BHg or HHg concentrations in a univariate model. However, multiple regression models adjusted for gender, age and fish consumption showed a significant association between plasma nitrite and plasma Hg concentration (beta = -0.1, p < 0.001). Our findings constitute preliminary clinical evidence that exposure to MeHg may cause inhibitory effects on the production of endothelial nitric oxide.

Low level and sub-chronic exposure to methylmercury induces hypertension in rats: nitric oxide depletion and oxidative damage as possible mechanisms

Increased risk of hypertension after methylmercury (MeHg) exposure has been suggested. However, the underlying mechanisms are not well explored. In this paper, we have analyzed whether sub-chronic exposure to MeHg increases systolic blood pressure even at very low levels. In addition, we analyzed if the methylmercury-induced hypertension is associated with a decreased plasmatic nitric oxide levels and with a dysregulation of the activities of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT), as well as the levels of MDA and glutathione. For this study, Wistar rats were treated with methylmercury chloride (100 mu g/kg per day) or vehicle. Total treatment time was 100 days. Malondialdehyde (MDA) and circulating NOx levels and superoxide dismutase (SOD) and catalase (CAT) activities were determined in plasma, whereas glutathione levels were determined in erythrocytes. Our results show that long-term treatment at a low level of MeHg affected systolic blood pressure, increasing and reducing the levels of plasmatic MDA and NOx, respectively. However, the activity of SOD did not decrease in the MeHg exposed group when compared to the control. We found a negative correlation between plasmatic nitrite/nitrate (NOx) levels and systolic blood pressure (r = -0.67; P = 0.001), and a positive correlation between MDA and systolic blood pressure (r = 0.61; P = 0.03), thus suggesting increased inhibition of NO formation with the increase of hypertension. In conclusion, long-term exposure to a low dose of MeHg increases the systolic pressure and is associated, at least in part, with increased production of ROS as judged by increased production of malondialdehyde and depressed NO availability.

Synthesis, antichagasic in vitro evaluation, cytotoxicity assays, molecular modeling and SAR/QSAR studies of a 2-phenyl-3-(1-phenyl-1H-pyrazol-4-yl)-acrylic acid benzylidene-carbohydrazide series

Chagas disease (American trypanosomiasis) is one of the most important parasitic diseases with serious social and economic impacts mainly on Latin America. This work reports the synthesis, in vitro trypanocidal evaluation, cytotoxicity assays, and molecular modeling and SAR/QSAR studies of a new series of N-phenylpyrazole benzylidene-carbohydrazides. The results pointed 6k (X = H, Y = p-NO(2), pIC(50) = 4.55 M) and 6l (X = F, Y = p-CN, pIC(50) = 4.27 M) as the most potent derivatives compared to crystal violet (pIC(50) = 3.77 M). The halogen-benzylidene-carbohydrazide presented the lowest potency whereas 6l showed the most promising pro. le with low toxicity (0% of cell death). The best equation from the 4D-QSAR analysis (Model 1) was able to explain 85% of the activity variability. The QSAR graphical representation revealed that bulky X-substituents decreased the potency whereas hydrophobic and hydrogen bond acceptor Y-substituents increased it. (C) 2008 Elsevier Ltd. All rights reserved.

Evidence of caspase-mediated apoptosis induced by L-amino acid oxidase isolated from Bothrops atrox snake venom

The aim of this work was to investigate the involvement of caspases in apoptosis induced by L-amino acid oxidase isolated from Bothrops atrox snake venom. The isolation of LAAO involved three chromatographic steps: molecular exclusion on a G-75 column; ion exchange column by HPLC and affinity chromatography on a Lentil Lectin column. SDS-PAGE was used to confirm the expected high purity level of BatroxLAA0. It is a glycoprotein with 12% sugar and an acidic character, as confirmed by its amino acid composition, rich in ""Asp and Glu"" residues. It displays high specificity toward hydrophobic L-amino acids. The N-terminal amino acid sequence and internal peptide sequences showed close structural homology to other snake venom LAAOs. This enzyme induces in vitro platelet aggregation, which may be due to H(2)O(2) production by LAAOs, since the addition of catalase completely inhibited the aggregation effect. It also showed cytotoxicity towards several cancer cell lines: HL60, Jurkat, B16F10 and PC12. The cytotoxicity activity was abolished by catalase. A fluorescence microscopy evaluation revealed a significant increase in the apoptotic index of these cells after BatroxLAAO treatment. This observation was confirmed by phosphatidyl serine exposure and activation of caspases. BatroxLAAO is a protein with various biological functions that can be involved in envenomation. Further investigations of its function will contribute to toxicology advances. Published by Elsevier Inc.

Antiviral and antiparasite properties of an L-amino acid oxidase from the Snake Bothrops jararaca: Cloning and identification of a complete cDNA sequence (Retracted article. See vol. 80, pg. 288, 2010)

L-Amino acid oxidases (LAAOs, EC 1.4.3.2) are flavoenzymes that catalyze the stereospecific oxidative deamination of an L-amino acid substrate to the corresponding a-ketoacid with hydrogen peroxide and ammonia production. The present work describes the first report on the antiviral (Dengue virus) and antiprotozoal (trypanocidal and leishmanicide) activities of a Bothrops jararaca L-amino acid oxidase (BjarLAAO-I) and identify its cDNA sequence. Antiparasite effects were inhibited by catalase, suggesting that they are mediated by H(2)O(2) production. Cells infected with DENV-3 virus previously treated with BjarLAAO-I, showed a decrease in viral titer (13-83-fold) when compared with cells infected with untreated viruses. Untreated and treated promastigotes (T. cruzi and L. amazonensis) were observed by transmission electron microscopy with different degrees of damage. Its complete cDNA sequence, with 1452 bp, encoded an open reading frame of 484 amino acid residues with a theoretical molecular weight and pl of 54,771.8 and 5.7, respectively. The cDNA-deduced amino acid sequence of BjarLAAO shows high identity to LAAOs from other snake venoms. Further investigations will be focused on the related molecular and functional correlation of these enzymes. Such a study should provide valuable information for the therapeutic development of new generations of microbicidal drugs. (C) 2008 Elsevier Inc. All rights reserved.

Anti-inflammatory effects of Lafoensia pacari and ellagic acid in a murine model of asthma

We have shown that the ethanolic extract of Lafoensia pacari inhibits eosinophilic inflammation induced by Toxocara canis infection, and that ellagic acid is the secondary metabolite responsible for the anti-eosinophilic activity seen in a model of beta-glucan peritonitis. In the present study, we investigated the preventive and curative effects of L. pacari extract and ellagic acid on allergic lung inflammation using a murine model of ovalbumin-induced asthma. In bronchoalveolar lavage fluid, preventive (22-day) treatment with L. pacari (200 mg/kg) and ellagic acid (10 mg/kg) inhibited neutrophil counts (by 75% and 57%) and eosinophil counts (by 78% and 68%). L. pacari reduced IL-4 and IL-13 levels (by 67% and 73%), whereas ellagic acid reduced IL-4, IL-5 and IL-13 (by 67%, 88% and 85%). To investigate curative anti-inflammatory effects, we treated mice daily with ellagic acid (0.1, 1, or 10 mg/kg), also treating selected mice with L. pacari (200 mg/kg) from day 18 to day 22. The highest ellagic acid dose reduced neutrophil and eosinophil numbers (by 59% and 82%), inhibited IL-4, IL-5, and IL-13 (by 62%,61%, and 49%). Neither L. pacari nor ellagic acid suppressed ovalbumin-induced airway hyperresponsiveness or cysteinyl leukotriene synthesis in lung homogenates. In mice treated with ellagic acid (10 mg/kg) or L. pacari (200 mg/kg) at 10 min after the second ovalbumin challenge, eosinophil numbers were 53% and 69% lower, respectively. Cytokine levels were unaffected by this treatment. L. pacari and ellagic acid are effective eosinophilic inflammation suppressors, suggesting a potential for treating allergies. (c) 2007 Elsevier B.V All rights reserved.

Cell cycle arrest evidence, parasiticidal and bactericidal properties induced by L-amino acid oxidase from Bothrops atrox snake venom

The present article describes an L-amino acid oxidase from Bothrops atrox snake venom as with antiprotozoal activities in Trypanosoma cruzi and in different species of Leishmania (Leishmania braziliensis, Leishmania donovani and Leishmania major). Leishmanicidal effects were inhibited by catalase, suggesting that they are mediated by H(2)O(2) production. Leishmania spp. cause a spectrum of diseases, ranging from self-healing ulcers to disseminated and often fatal infections, depending on the species involved and the host`s immune response. BatroxLAAO also displays bactericidal activity against both Gram-positive and Gram-negative bacteria. The apoptosis induced by BatroxLAAO on HL-60 cell lines and PBMC cells was determined by morphological cell evaluation using a mix of fluorescent dyes. As revealed by flow cytometry analysis, suppression of cell proliferation with BatroxLAAO was accompanied by the significant accumulation of cells in the G0/G1 phase boundary in HL-60 cells. BatroxLAAO at 25 mu g/mL and 50 mu g/mL blocked G0-G1 transition, resulting in G0/G1 phase cell cycle arrest, thereby delaying the progression of cells through S and G2/M phase in HL-60 cells. This was shown by an accentuated decrease in the proportion of cells in S phase, and the almost absence of G2/M phase cell population. BatroxLAAO is an interesting enzyme that provides a better understanding of the ophidian envenomation mechanism, and has biotechnological potential as a model for therapeutic agents. (C) 2011 Elsevier Masson SAS. All rights reserved.

Isolation and Characterization of a Natriuretic Peptide from Crotalus oreganus abyssus (Grand Canyon Rattlesnake) and its Effects on Systemic Blood Pressure and Nitrite Levels

Studies on the therapeutic potential of venom peptides have significantly advanced the development of new peptide drugs. A good example is captopril, a synthetic peptide drug, which acts as an anti-hypertensive and potentiating bradykinin, inhibiting the angiotensin-converting enzyme, whose precursor was isolated from the venom of Bothrops jararacussu. The natriuretic peptide (NPs) family comprises three members, ANP (atrial natriuretic peptide), BNP (B-type natriuretic peptide) and CNP (C-type natriuretic peptide), and has an important role in blood pressure regulation and electrolyte homeostasis. In this study, we describe, for the first time, the isolation and characterization of a novel natriuretic-like peptide (Coa_NP), isolated from Crotalus Oreganus abyssus venom. The peptide has 32 amino acids and its complete sequence is SKRLSNGCFGLKLDRIGAMSGLGCWRLINESK. The Coa_NP has an average molecular mass of 3510.98 Da and its amino acid sequence presents the loop region that is characteristic of natriuretic peptides (17 amino acids, NP domain consensus; CFGXXXDRIXXXSGLGC). Coa_NP is a natriuretic peptide of the ANP/BNP-like family, since the carboxy terminal region of CNP has its own NP domain. The functional experiments showed that Coa_NP produced biological effects similar to those of the other natriuretic peptides: (1) a dose-dependent decrease in mean arterial pressure; (2) significant increases in plasma nitrite levels, and (3) vasorelaxation in thoracic aortic rings that were pre-contracted with phenylephrine. The structural and biological aspects confirm Coa_NP as a natriuretic peptide isolated from snake venom, thus expanding the diversification of venom components.

Snake Venom L-Amino Acid Oxidases: Some Consideration About their Functional Characterization

Snake Venom L-amino acid oxidases (LAAOs E.C. 1.4.3.2) are flavoenzymes broadly found in various snake venom compositions. LAAOs have become an attractive subject for molecular biology, biochemistry, physiology and medicine due to their actions on various cells and biological effects on platelets, apoptosis, hemorrhage and others. In this review we try to summarize some of these reports, with special emphasis on apoptosis, anti-protozoa, bactericidal and anti-viral activities.

BLOCKING CENTRAL LEUKOTRIENES SYNTHESIS AFFECTS VASOPRESSIN RELEASE DURING SEPSIS

Recent studies revealed that vasopressinergic neurons have a high content of cys-leukotriene C(4) (LTC(4)) synthase, a critical enzyme in cys-leukotriene synthesis that may play a role in regulating vasopressin secretion. This study investigates the role of this enzyme in arginine vasopressin (AVP) release during experimentally induced sepsis. Male Wistar rats received an i.c.v. injection of 3-[1-(p-chlorobenzyl)-5-(isopropyl)-3-tert-butylthioindol-2-yl]-2, 2-dimethylpropanoic acid (MK-886) (1.0 mu g/kg), a leukotrienes (LTs) synthesis inhibitor, or vehicle, 1 h before cecal ligation and puncture (CLP) or sham operation. In one group of animals the survival rate was monitored for 3 days. In another group, the animals were decapitated at 0, 4, 6, 18 and 24 h after CLP or sham operation, and blood was collected for hematocrit, serum sodium and nitrate, plasma osmolality, protein and AVP determination. A third group was used for blood pressure measurements. The neurohypophysis was removed for quantification of AVP content, and the hypothalamus was dissected for LTC4 synthase analysis by Western blot. Mortality after CLP was reduced by the central administration of MK-886. The increase in plasma AVP levels and hypothalamus LTC4 synthase content in the initial phase of sepsis was blocked, whereas the decrease in neurohypophyseal AVP content was partially reversed. Also the blood pressure drop was abolished in this phase. The increase of serum nitric oxide and hematocrit was reduced, and the decrease in plasma protein and osmolality was not affected by the LTs blocker. In the final phase of sepsis, the plasma AVID level and the hypothalamic LTC4 synthase content were at basal levels. The central administration of MK-886 increased the hypothalamic LTC4 synthase content but did not alter the plasma and neurohypophysis AVID levels observed, or the blood pressure during this phase. These results suggest that the central LTs are involved in the vasopressin release observed during sepsis. (C) 2009 IBRO. Published by Elsevier Ltd. All rights reserved.

Phagocytosis, Oxidative Burst, and Produced Reactive Species are Affected by Iron Deficiency Anemia and Anemia of Chronic Diseases in Elderly

Iron and oxidative stress have a regulatory interplay. During the oxidative burst, phagocytic cells produce free radicals such as hypochlorous acid (HOCl). Nevertheless, scarce studies evaluated the effect of either iron deficiency anemia (IDA) or anemia of chronic disease (ACD) on phagocyte function in the elderly. The aim of the present study was to determine the oxidative burst, phagocytosis, and nitric oxide ((aEuro cent)NO) and HOCl, reactive species produced by monocytes and neutrophils in elderly with ACD or IDA. Soluble transferrin receptor, serum ferritin, and soluble transferrin receptor/log ferritin (TfR-F) index determined the iron status. The study was constituted of 39 patients aged over 60 (28 women and 11 men) recruited from the Brazilian Public Health System. Oxidative burst fluorescence intensity per neutrophil in IDA group and HOCl generation in both ACD and IDA groups were found to be lower (p < 0.05). The percentages of neutrophils and monocytes expressing phagocytosis in ACD group were found to be higher (p < 0.05). There was an overproduction of (aEuro cent)NO from monocytes, whereas the fundamental generation of HOCl appeared to be lower. Phagocytosis, oxidative burst, and (aEuro cent)NO and HOCl production are involved in iron metabolism regulation in elderly patients with ACD and IDA.

Structural and functional properties of Bp-LAAO, a new L-amino acid oxidase isolated from Bothrops pauloensis snake venom

An L-amino acid oxidase (Bp-LAAO) from Bothrops pauloensis snake venom was highly purified using sequential chromatography steps on CM-Sepharose, Phenyl-Sepharose CL4B, Benzamidine Sepharose and C18 reverse-phase HPLC. Purified Bp-LAAO showed to be a homodimeric acidic glycoprotein with molecular weight around 65 kDa under reducing conditions in SDS-PAGE. The best substrates for Bp-LAAO were L-Met, L-Leu, L-Phe and L-Ile and the enzyme showed a strong reduction of its catalytic activity upon L-Met and L-Phe substrates at extreme temperatures. Bp-LAAO showed leishmanicidal, antitumoral and bactericidal activities dose dependently. Bp-LAAO induced platelet aggregation in platelet-rich plasma and this activity was inhibited by catalase. Bp-LAAC-cDNA of 1548 bp codified a mature protein with 516 amino acid residues corresponding to a theoretical isoelectric point and molecular weight of 6.3 and 58 kDa, respectively. Additionally, structural and phylogenetic studies identified residues under positive selection and their probable location in Elp-LAAO and other snake venom LAAOs (svLAAOs). Structural and functional investigations of these enzymes can contribute to the advancement of toxinology and to the elaboration of novel therapeutic agents. (C) 2009 Elsevier Masson SAS. All rights reserved.

Suramin inhibits macrophage activation by human group IIA phospholipase A(2), but does not affect bactericidal activity of the enzyme

Suramin is a polysulphonated napthylurea antiprotozoal and anthelminitic drug, which also presents inhibitory activity against a broad range of enzymes. Here we evaluate the effect of suramin on the hydrolytic and biological activities of secreted human group IIA phospholipase A(2) (hsPLA(2)GIIA). The hsPLA(2)GIIA was expressed in E. coli, and refolded from inclusion bodies. The hydrolytic activity of the recombinant enzyme was measured using mixed dioleoylphosphatidylcholine/dioleoylphosphatidylglycerol (DOPC/DOPG) liposomes. The activation of macrophage cell line RAW 264.7 by hsPLA(2) GIIA was monitored by NO release, and bactericidal activity against Micrococcus luteus was evaluated by colony counting and by flow cytometry using the fluorescent probe Sytox Green. The hydrolytic activity of the hsPLA(2) GIIA was inhibited by a concentration of 100 nM suramin and the activation of macrophages by hsPLA(2) GIIA was abolished at protein/suramin molar ratios where the hydrolytic activity of the enzyme was inhibited. In contrast, both the bactericidal activity of hsPLA(2) GIIA against Micrococcus luteus and permeabilization of the bacterial inner membrane were unaffected by suramin concentrations up to 50 mu M. These results demonstrate that suramin selectively inhibits the activity of the hsPLA(2) GIIA against macrophages, whilst leaving the anti-bacterial function unchanged.

Amphotericin B in poly(lactic-co-glycolic acid) (PLGA) and dimercaptosuccinic acid (DMSA) nanoparticles against paracoccidioidomycosis

The present study reports on the preparation and testing of a desoxycholate amphotericin B (D-AMB) sustained delivery system based on poly(lactic-co-glycolic acid) (PLGA) and dimercaptosuccinic acid (DMSA) polymeric blends (Nano-D-AMB) aimed at reducing the number of AMB administrations required to treat mycosis. BALB/c mice were infected with the yeast Paracoccidioides brasiliensis intravenously to mimic the chronic form of paracoccidioidomycosis. At 30 days post-infection, the animals were treated with Nano-D-AMB [6 mg/kg of encapsulated D-AMB, intraperitoneally (ip), interval of 72 h] or D-AMB (2 mg/kg, ip, interval of 24 h). Drug efficacy was investigated by the fungal burden recovery from tissues. Toxicity was assessed by renal and hepatic biochemical parameters, physical appearance of the animals and haematological investigation. The control groups used were non-infected and the infected mice mock treated with PBS. Nano-D-AMB presented results comparable to free D-AMB, with a marked antifungal efficacy. The Nano-D-AMB-treated group presented lower loss of body weight and absence of stress sign (piloerection and hypotrichosis) observed after D-AMB treatment. No renal [blood urea nitrogen (BUN), creatinine] or hepatic (pyruvic and oxalacetic glutamic transaminases) biochemical abnormalities were found. The micronucleus assay showed no significant differences in both the micronucleus frequency and percentage of polychromatic erythrocytes for Nano-D-AMB, indicating the absence of genotoxicity and cytotoxic effects. The D-AMB-coated PLGA-DMSA nanoparticle showed antifungal efficacy, fewer undesirable effects and a favourable extended dosing interval. Nano-D-AMB comprises an AMB formulation able to lessen the number of drug administrations. Further studies would elucidate whether Nano-D-AMB would be useful to treat systemic fungal infections such as paracoccidioidomycosis, candidiasis, aspergillosis and cryptococcosis.