The contribution of phenolic acids to the anti-inflammatory activity of mushrooms: Screening in phenolic extracts, individual parent molecules and synthesized glucuronated and methylated derivatives

In the present study, the ethanolic extracts of fourteen edible mushrooms were investigated for their anti-inflammatory potential in LPS (lipopolysaccharide) activated RAW 264.7 macrophages. Furthermore the extracts were chemically characterized in terms of phenolic acids and related compounds. The identified molecules (p-hydroxybenzoic, p-coumaric and cinnamic acids) and their glucuronated and methylated derivatives obtained by chemical synthesis were also evaluated for the same bioactivity, in order to establish structure-activity relationships and to comprehend the effects of in vivo metabolism reactions in the activity of the compounds. The extracts of Pleurotus ostreatus, Macrolepiota procera, Boletus impolitus and Agaricus bisporus revealed the strongest anti-inflammatory potential (EC50 values 96 ± 1 to 190 ± 6 µg/mL, and also the highest concentration of cinnamic acid (656 to 156 µg/g), which was also the individual compound with the highest anti-inflammatory activity. The derivatives of p-coumaric acid revealed the strongest properties, specially the derivative methylated in the carboxylic group (CoA-M1) that exhibited similar activity to the one showed by dexamethaxone used as anti-inflammatory standard; by contrast, the derivatives of p-hydroxybenzoic revealed the lowest inhibition of NO production. All in all, whereas the conjugation reactions change the chemical structure of phenolic acids and may increase or decrease their activity, the glucuronated and methylated derivatives of the studied compounds are still displaying anti-inflammatory activity.

Delivery as nanoparticles reduces imatinib mesylate-induced cardiotoxicity and improves anticancer activity

Clinical effectiveness of imatinib mesylate in cancer treatment is compromised by its off-target cardiotoxicity. In the present study, we have developed physically stable imatinib mesylate-loaded poly(lactide-co-glycolide) nanoparticles (INPs) that could sustainably release the drug, and studied its efficacy by in vitro anticancer and in vivo cardiotoxicity assays. MTT (methylthiazolyldiphenyl-tetrazolium bromide) assay revealed that INPs are more cytotoxic to MCF-7 breast cancer cells compared to the equivalent concentration of free imatinib mesylate. Wistar rats orally administered with 50 mg/kg INPs for 28 days showed no significant cardiotoxicity or associated changes. Whereas, increased alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase levels, and reduced white blood cell, red blood cell, and hemoglobin content were observed in the animals administered with free drug. While the histological sections from hearts of animals that received INPs did not show any significant cardiotoxic symptoms, loss of normal architecture and increased cytoplasmic vacuolization were observed in the heart sections of animals administered with free imatinib mesylate. Based on these results, we conclude that nano-encapsulation of imatinib mesylate increases its efficacy against cancer cells, with almost no cardiotoxicity.

Physical activity, cardiorespiratory fitness, dietary content, and risk factors that cause a predisposition towards cardiovascular disease

OBJECTIVE: To analyze associations between levels of physical activity, cardiorespiratory fitness, dietary content, and risk factors that cause a predisposition towards cardiovascular disease. METHODS: Sixty-two individuals aged between 20 and 45 years were evaluated. Levels of physical activity were established by estimates of energy demand corresponding to everyday activity; indices for cardiorespiratory fitness were obtained from estimates of maximal oxygen consumption; information about dietary content was obtained from dietary records kept on seven consecutive days. To indicate risk factors that cause a predisposition towards cardiovascular disease, use was made of body mass indexes, waist-hip circumference relationships, levels of arterial pressure and of plasma lipid-lipoprotein concentration. To establish associations between the variables studied, multiple regression analysis was used. RESULTS: Physical activity levels and cardiorespiratory fitness levels were inversely correlated with the amount and distribution of body fat and arterial pressure. Taken together, the two variables were responsible for between 16% and 19% of the variation in arterial pressure. Total and saturated fat ingestion was associated with higher serum lipid levels. Both dietary components were responsible for between 49% and 61% of the variation in LDL-cholesterol. CONCLUSION: High ingestion of food rich in total and saturated fat and decreased levels of physical activity and of cardiorespiratory fitness are associated with an increased risk of cardiovascular disease, which supports previous data.

Effect of Propafenone on the Contractile Activity of Latissimus Dorsi Muscle Isolated in an Organ Chamber: Experimental Study in Rats

OBJECTIVE: To study the effect of propafenone on the contractile function of latissimus dorsi muscle isolated from rats in an organ chamber. METHODS: We studied 20 latissimus dorsi muscles of Wistar rats and divided them into 2 groups: group I (n=10), or control group - we studied the feasibility of muscle contractility; group II (n=10), in which the contralateral muscles were grouped - we analyzed the effect of propafenone on muscle contractility. After building a muscle ring, 8 periods of sequential 2-minute baths were performed, with intervals of preprogrammed electrical stimulation using a pacemaker of 50 stimuli/min. In group II, propafenone, at the concentration of 9.8 µg/mL, was added to the bath in period 2 and withdrawn in period 4. RESULTS: In group I, no significant depression in muscle contraction occurred up to period 5 (p>0.05). In group II, a significant depression occurred in all periods, except between the last 2 periods (p<0.05). Comparing groups I and II only in period 1, which was a standard period for both groups, we found no significant difference (p>0.05). CONCLUSION: Propafenone had a depressing effect on the contractile function of latissimus dorsi muscle isolated from rats and studied in an organ chamber.

Activity of carvacrol against Coagulase-negative Staphylococci biofilm related infections

Dissertação de mestrado em Bioengenharia

Molluscicide activity of the "Avelós" plant (Euphorbia tirucalli, L. ) on Biomphalaria glabrata, the mollusc vector of schistosomiasis

An aqueous solution of the latex of Euphorbia tirucalli collected at sites receiving large amounts of sunlight showed molluscicide action on Biomphalaria glabrata, with LD50 obtained at the concentration of 28,0 ppm and LD90 at the concentration of 85,0 ppm. The toxicity of the product for fish was similar to that of Bayluscide and of copper sulfate used for comparison. However, the wide distribution of the plant, its easy propagation and the simple procedure for extraction of the active substance, which is biodegradable, favor "avelós" as a promising agent in the control of schistosomiasis.

Lactate modulates the activity of primary cortical neurons through a receptor-mediated pathway.

Lactate is increasingly described as an energy substrate of the brain. Beside this still debated metabolic role, lactate may have other effects on brain cells. Here, we describe lactate as a neuromodulator, able to influence the activity of cortical neurons. Neuronal excitability of mouse primary neurons was monitored by calcium imaging. When applied in conjunction with glucose, lactate induced a decrease in the spontaneous calcium spiking frequency of neurons. The effect was reversible and concentration dependent (IC50 ∼4.2 mM). To test whether lactate effects are dependent on energy metabolism, we applied the closely related substrate pyruvate (5 mM) or switched to different glucose concentrations (0.5 or 10 mM). None of these conditions reproduced the effect of lactate. Recently, a Gi protein-coupled receptor for lactate called HCA1 has been introduced. To test if this receptor is implicated in the observed lactate sensitivity, we incubated cells with pertussis toxin (PTX) an inhibitor of Gi-protein. PTX prevented the decrease of neuronal activity by L-lactate. Moreover 3,5-dyhydroxybenzoic acid, a specific agonist of the HCA1 receptor, mimicked the action of lactate. This study indicates that lactate operates a negative feedback on neuronal activity by a receptor-mediated mechanism, independent from its intracellular metabolism.

Cysteine 230 is essential for the structure and activity of the cytotoxic ligand TRAIL.

Unlike other tumor necrosis factor family members, the cytotoxic ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)/Apo-2L contains an unpaired cysteine residue (Cys(230)) in its receptor-binding domain. Here we show that the biological activity of both soluble recombinant TRAIL and cell-associated, full-length TRAIL is critically dependent on the presence of Cys(230). Mutation of Cys(230) to alanine or serine strongly affected its ability to kill target cells. Binding to its receptors was decreased by at least 200-fold, and the stability of its trimeric structure was reduced. In recombinant TRAIL, Cys(230) was found engaged either in interchain disulfide bridge formation, resulting in poorly active TRAIL, or in the chelation of one zinc atom per TRAIL trimer in the active, pro-apoptotic form of TRAIL.

Implication of Nerve Growth Factor in intestinal mucosal mast cell activity and colonic motor alterations in a model of ovalbumin-induced gut dysfunction in rats

We determined NGF involvement in MMCs and colonic motor alterations in an ovalbumin (OVA)-induced gut dysfunction model in rats. Animals received OVA (6 weeks), with/without simultaneous K252a (TrkA antagonist) treatment. MMCs, rat mast cell protease II (RMCPII) levels and colonic contractility in vitro were assessed. OVA increased MMC density and RMCPII concentration. Spontaneous contractility was similar in both groups and inhibited by K252a. Carbachol responses were increased by OVA in a K252a-independent manner. NO-synthase inhibition increased spontaneous activity in OVA-treated animals in a K252a-dependent manner. These observations support an involvement of NGF in the functional changes observed in this model.

Ocular distribution, spectrum of activity, and in vivo viral neutralization of a fully humanized anti-herpes simplex virus IgG Fab fragment following topical application.

Herpes simplex ocular infection is a major cause of corneal blindness. Local antiviral treatments exist but are associated with corneal toxicity, and resistance has become an issue. We evaluated the biodistribution and efficacy of a humanized anti-herpes simplex virus (anti-HSV) IgG FAb fragment (AC-8; 53 kDa) following repeated topical administration. AC-8 was found in the corneal epithelium, anterior stroma, subepithelial stromal cells, and retinal glial cells, with preferential entry through the ocular limbus. AC-8 was active against 13 different strains of HSV-1, with 50% and 90% mean effective concentrations (MEC(50) and MEC(90), respectively) ranging from 0.03 to 0.13 μg/ml, indicating broad-spectrum activity. The in vivo efficacy of AC-8 was evaluated in a mouse model of herpes-induced ocular disease. Treatment with low-dose AC-8 (1 mg/ml) slightly reduced the ocular disease scores. A greater reduction of the disease scores was observed in the 10-mg/ml AC-8-treated group, but not as much as with trifluridine (TFT). AC-8 treatment reduced viral titers but less than trifluridine. AC-8 did not display any toxicity to the cornea or other structures in the eye. In summary, topical instillation of an anti-HSV FAb can be used on both intact and ulcerated corneas. It is well tolerated and does not alter reepithelialization. Further studies to improve the antiviral effect are needed for AC-8 to be considered for therapeutic use.

Increase of a Calcium Independent Transglutaminase Activity in the Erythrocyte during the Infection with Plasmodium falciparum

We have studied the activity of a calcium dependent transglutaminase (EC 2.3.2.13) during the growth of the parasite Plasmodium falciparum inside the infected human erythrocyte. There is only one detectable transglutaminase in the two-cell-system, and its origin is erythrocytic. No activity was detected in preparations of the parasite devoid of erythrocyte cytoplasm. The Michaelis Menten constants (Km) of the enzyme for the substrates N'N'dimethylcaseine and putrescine were undistinguishable whether the cell extracts used in their determination were obtained from normal or from infected red cells. The total activity of transglutaminase in stringently synchronized cultures, measured at 0.5mM Ca2+, decreased with the maturation of the parasite. However, a fraction which became irreversibly activated and independent of calcium concentration was detected. The proportion of this fraction grew with maturation; it represented only 20% of the activity in 20 hr-old-trophozoites while in 48-hr-schizonts it was more than 85% of the total activity. The activation of this fraction of transglutaminase did not depend on an increase in the erythrocyte cytoplasmic calcium, since most of the calcium was shown to be located in the parasite.

Antitumor and Antiviral Activity of Colombian Medicinal Plant Extracts

Extracts of nine species of plants traditionally used in Colombia for the treatment of a variety of diseases were tested in vitro for their potential antitumor (cytotoxicity) and antiherpetic activity. MTT (Tetrazolium blue) and Neutral Red colorimetric assays were used to evaluate the reduction of viability of cell cultures in presence and absence of the extracts. MTT was also used to evaluate the effects of the extracts on the lytic activity of herpes simplex virus type 2 (HSV-2). The 50% cytotoxic concentration (CC50) and the 50% inhibitory concentration of the viral effect (EC50) for each extract were calculated by linear regression analysis. Extracts from Annona muricata, A. cherimolia and Rollinia membranacea, known for their cytotoxicity were used as positive controls. Likewise, acyclovir and heparin were used as positive controls of antiherpetic activity. Methanolic extract from Annona sp. on HEp-2 cells presented a CC50 value at 72 hr of 49.6x103mg/ml. Neither of the other extracts examined showed a significant cytotoxicity. The aqueous extract from Beta vulgaris, the ethanol extract from Callisia grasilis and the methanol extract Annona sp. showed some antiherpetic activity with acceptable therapeutic indexes (the ratio of CC50 to EC50). These species are good candidates for further activity-monitored fractionation to identify active principles.

Antibacterial activity of Ocimum gratissimum L. essential oil

The essential oil (EO) of Ocimum gratissimum inhibited Staphylococcus aureus at a concentration of 0.75 mg/ml. The minimal inhibitory concentrations (MICs) for Shigella flexineri, Salmonella enteritidis, Escherichia coli, Klebsiella sp., and Proteus mirabilis were at concentrations ranging from 3 to 12 mg/ml. The endpoint was not reached for Pseudomonas aeruginosa (>=24 mg/ml). The MICs of the reference drugs used in this study were similar to those presented in other reports. The minimum bactericidal concentration of EO was within a twofold dilution of the MIC for this organism. The compound that showed antibacterial activity in the EO of O. gratissimum was identified as eugenol and structural findings were further supported by gas chromatography/mass spectra retention time data. The structure was supported by spectroscopic methods.

The determinants of YIc's R&D activity

This paper examines the determinants of young innovative companies’ (YICs) R&D activities taking into account the autoregressive nature of innovation. Using a large longitudinal dataset comprising Spanish manufacturing firms over the period 1990-2008, we find that previous R&D experience is a fundamental determinant for mature and young firms, albeit to a smaller extent in the case of the YICs, suggesting that their innovation behaviour is less persistent and more erratic. Moreover, our results suggest that firm and market characteristics play a distinct role in boosting the innovation activity of firms of different age. In particular, while market concentration and the degree of product diversification are found to be important in boosting R&D activities in the sub-sample of mature firms only, YICs’ spending on R&D appears to be more sensitive to demand-pull variables, suggesting the presence of credit constraints. These results have been obtained using a recently proposed dynamic type-2 tobit estimator, which accounts for individual effects and efficiently handles the initial conditions problem.

Chitinolytic activity in viable spores of encephalitozoon species

By employing 4-methylumbelliferyl-beta-D-NN',N"-triacetylchitotriose substrate in a semi quantitative assay, chitinolytic activity in viable spores of Encephalitozoon cuniculi and E. intestinalis was detected and dependence on reaction time, spore concentration, concentration of substrate and temperature were demonstrated. It was possible to block the chitinolytic activity by chitin hydrolysate. By incubation at 80°C for 10 min or at 55°C for 20 min the spores were loosing the chitinolytic activity. Incubation of the spores in trypsin reduced the chitinolytic activity. Cellulase activity could not be detected.