Influence of condensed tannins from Brazilian semi-arid legumes on ruminal degradability, microbial colonization and ruminal enzymatic activity in Saanen goats

The present study aimed at determining the influence of condensed tannins present in the Brazilian legume species Mimosa hostilis, Mimosa caesalpinifolia and Bauhinia cheilantha on ruminal degradability, microbial colonization and enzymatic activity. Polyethylene glycol (PEG) was used to reduce the astringency and concentration of soluble condensed tannins. Four ruminally-cannulated Saanen goats (60 +/- 8 kg BW) were fed, in two experimental periods, with a hay diet based on the studied legumes treated or non-treated with PEG. Voluntary intake, microbial colonization, DM, CP, NDF, and ruminal degradability of PEG treated and non-treated forage leaves, as well as pH, ammonia and 1,4 P-endoglucanase activity of the rumen content were evaluated. Astringency and soluble tannin concentration of the studied legumes were reduced by approximately 70% and 50%, respectively, with PEG treatment. Average DM intake was higher for the treated diet (16.76 g DM/kg BW/day against 13.06 g DM/kg BW/day). Percentile values for degradation parameters and for potential and effective degradabilities of DM, CP and NDF were also affected by the tannins, but at different intensities. Electron microscopic observations of ruminally-incubated legume leaves showed a more effective microbial colonization of PEG-treated leaves for all legume species. A decrease in pH and an increase in ammonia concentration and in endoglucanase activity in the ruminal content was also observed for PEG-treated diets at all sampling periods. Condensed tannins of the studied legume species have influenced the adhesion conditions, colonization and enzymatic activity of the microbial ecosystem, and consequently the ruminal degradation of the different dietary fractions. For this reason, the reduction in condensed tannin would be of great importance to improve the nutrition of ruminant feeding of these species. (c) 2005 Elsevier B.V. All rights reserved.

Determination of Cd(II) and Cd-metallothioneins in biological extracts using baker's yeast and inductively coupled plasma optical emission spectrometry

The use of Saccharomyces cerevisiae as a sorbent material to separate Cd(II) and Cd-metallothionein complex (Cd-MT) has been explored. Solid-liquid phase extractions were carried out in batch mode and the main parameters of the process (pH, temperature, time of incubation, amount of biomass and analyte) were evaluated. Under optimized conditions, the yeast quantitatively retain (94 +/- 5%) the Cd(II) while 97 +/- 2% of the Cd-MT remain in the supernatant. on base of the findings of this study, a simple method is proposed to determine Cd(II) and Cd-MT in cytosols extracted from mouse kidney and crab hepatopancreas. Inductively coupled plasma optical emission spectrometry was used to quantify the analytes in solid and liquid phase. Determination of Cd in the solid phase was carried out by introducing a slurry of the yeast (0.0625 g/10 mL) directly to the inductively coupled plasma optical emission spectrometer. Mixed standards solutions, which also have been submitted to the extraction procedure, were used to quantify the analytes in the samples. Thus, matrix effects due to nebulization of the slurry were overcame. Limits of detection (3 sigma) for Cd(II) and Cd-MT were 1.5 and 1.2 mu g L-1, respectively. Relative standard deviations of signals were 4.2% for measurements in the slurry of solid phase and 2.1% for measurements in the liquid phase. Recoveries of the analytes in cytosol samples were between 76 and 114%. The concentrations of Cd(II) (2.4 +/- 0.5 mu g L-1) and Cd-MT (3.0 +/- 0.5 mu g L-1) found by using the proposed approach were close to those found by tangential-flow ultrafiltration technique (2.6 +/- 0.7 mu g L-1 for Cd(II) and 3.7 +/- 1.7 mu g L-1 for Cd-MT).

Effect of Mouriri pusa extracts on experimentally induced gastric lesions in rodents: Role of endogenous sulfhydryls compounds and nitric oxide in gastroprotection

Several plants are used in folk medicine to treat gastrointestinal disorders. Mouriri pusa Gardn. (Melastomataceae) is a medicinal plant commonly used in the central region of Brazil against gastric ulcer. Two organic extracts methanolic (MeOH) and dichloromethane (DCM) obtained by sequential extraction from the leaves of Mouriri pusa were evaluated for their ability to protect the gastric mucosa against injuries caused by necrotizing agents (0.3 M HCl/60% EtOH, absolute ethanol, non-steroidal anti-inflammatory drug, stress and pylorus ligature) in mice and rats. The best results were obtained after pretreatment with MeOH extract whereas the DCM extract did not show the same significant antiulcerogenic activity. No acute toxicity was observed in animals treated with 5 g/kg, p.o. of MeOH extract. The mechanism involving the antiulcerogenic action of MeOH extract seemed to be related to NO generation and also suggested the effective participation of endogenous sulfhydryl group in the gastroprotective action. Phytochemical investigation of the MeOH extract of Mouriri pusa yielded tannins, flavonoids and (-)-epicatechin. The presence of these phenolic compounds probably would explain the antiulcerogenic effect of the polar extract of Mouriri pusa leaves. (c) 2006 Elsevier B.V.. All rights reserved.

PHARMACOLOGICAL EVALUATION OF THE INHIBITORY EFFECT OF EXTRACTS FROM ANCHIETIA-SALUTARIS ON THE HISTAMINE-RELEASE INDUCED IN THE RAT AND THE GUINEA-PIG

The tea made with leaves and stems of plant Anchietia salutaris is traditionally used in Brazil to treat allergies. We examined the effects of a crude aqueous extract and of purified fractions of this plant on the histamine release induced in rat and guinea pig tissues. The crude extract (3-10 mu g/ml) inhibits the histamine release induced by compound 48/80 (0.5 mu g/ml) and antigen in rat peritoneal mast cells. The inhibition is significant after 10 s of preincubation and is completed after 3 min. The crude extract dissolved in the perfusion fluid (1-30 mu g/ml) also inhibits the histamine release induced in guinea pig heart by cardiac anaphylaxis and in hearts from pretreated animals (10-100 mg/kg i.p.). In pretreated animals, the effect manifests after 3 h, is maximum after 12 h and disappears after 48 h. The histamine release induced in isolated guinea pig heart by ionophore A23187 is inhibited by similar doses as in antigen-induced histamine release. Extraction with solvents concentrated the active principle (s) in the hexane fractions, as demonstrated by the inhibition of the histamine release induced by antigen in isolated cells from guinea pig heart dispersed with collagenase. In subfractions produced by the fractionation of the hexane fraction, the active principle(s) concentrated in the subfractions obtained by extraction with hexane and ethyl acetate, which shows the low polarity of the compound(s). The same subfractions that inhibit the histamine release induced by antigen in cells from guinea pig heart also inhibit pulmonary cells. Our result show that A. salutaris contains low-polarity compound(s) that inhibit the histamine release induced by three different mechanisms in mast cells from two animal species. These facts suggest that the active principle(s) of A, salutaris could be useful in the treatment of allergies and/or as a tool for the study of mast cell secretions.

Metodologia rápida e eficiente para análise de pesticidas organoclorados em fubá

A rapid and efficient analytical method is presented for the quantitative analysis of 10 organochlorine pesticides in corn meal. The extraction and clean up steps are combined into one step by transferring the sample to a chromatographic column prepacked with alumina and silica gel. The pesticides are eluted with n-hexane-dichloromethane 9:1 (v/v) and the extracts analized by gas-liquid chromatography with electron capture detection, the average recoveries were between 78% and 98% and the detection limits were between 1 and 5 ng/g.

Dissociation of enzymatic and pharmacological properties of piratoxins-I and -III, two myotoxic phospholipases A(2) from Bothrops pirajai snake venom

Piratoxins (PrTX) I and III are phospholipases A(2) (PLA(2)s) or PLA(2) homologue myotoxins isolated from Bothrops pirajai snake venom, which also induce myonecrosis, bactericidal activity against Escherichia coli, disruption of artificial membranes, and edema. PrTX-III is a catalytically active hemolytic and anticoagulant Asp49 PLA(2), while PrTX-I is a Lys49 PLA, homologue, which is catalytically inactive on artificial substrates, but promotes blockade of neuromuscular transmission. Chemical modifications of His, Lys, Tyr, and Trp residues of PrTX-I and PrTX-III were performed, together with cleavage of the N-terminal octapeptide by CNBr and inhibition by heparin and EDTA. The lethality, bactericidal activity, myotoxicity, neuromuscular effect, edema inducing effect, catalytic and anticoagulant activities, and the liposome-disruptive activity of the modified toxins were evaluated. A complex pattern of functional differences between the modified and native toxins was observed. However, in general, chemical modifications that significantly affected the diverse pharmacological effects of the toxins did not influence catalytic or membrane disrupting activities. Analysis of structural changes by circular dichroism spectroscopy demonstrated significant changes in the secondary structure only in the case of N-terminal octapeptide cleavage. These data indicate that PrTX-I and PrTX-III possess regions other than the catalytic site, which determine their toxic and pharmacological activities. (C) 2001 Academic Press.

Anticlastogenic effect of aqueous extracts of Agaricus blazei on CHO-k1 cells, studying different developmental phases of the mushroom

The Agaricus blazei Murill (ABM) mushroom, known as the sun mushroom, is native to Brazil and has become known for its medicinal properties. This study examined the anticlastogenic effect of Agaricus blazei in Chinese hamster ovary cells, CHO-k1, by means of a chromosome aberration test using methyl methanesulphonate (MMS, 10(-4)M) as the DNA damage inducing agent. Two mushroom lines were used, ABM 99/26 and ABM 97/11, and the latter was used in the young (Y) and sporulating (S) developmental phases. The cells were treated for 12 h with MMS alone or combined with aqueous extracts of A. blazei at a final concentration of 0.15%, which were prepared at three different temperatures: (a) hot (60 degreesC), (b) room temperature (25 degreesC) and (c) chilled (4 degreesC). Mushroom extracts showed a marked anticlastogenic effect against DNA damage, as evidenced by a decrease in the number of cells with breaks, regardless of the line used, or the developmental stage or the temperature at which the extract was prepared. Generally, the extracts were more effective in reducing the isochromatid type breaks. The data obtained suggest that extracts of A. blazei mushroom are anticlastogenic under the conditions tested, mainly during the G1 and S stages of the cell cycle, where chromosome breaks of the isochromatid type are produced by the MMS agent. (C) 2003 Elsevier Ltd. All rights reserved.

Cutaneous hypersensitivity induced in rabbits by extracts of the tick Amblyomma cajennense (Acari : Ixodidae)

The cutaneous hypersensitivity test was used to correlate host resistance to ticks and type of reactions elicited by Amblyomma cajennense (Fabricius, 1787) tick extract in rabbits. Rabbits were divided into 3 groups of 2 animals each: naive, pre-infested and control. Cutaneous hypersensitivity was induced by intradermal inoculation of 25 mug extract in 0.03 rut of phosphate buffered saline (PBS) in rabbit ears. Control rabbits were inoculated with PBS only. The ear thickness was measured with a Mitutoyo(R) device before and 10 min, 1, 2,4,18, 24,48,72 and 96 h post-inoculation (PI). Pre-infested rabbits showed an immediate type reaction within the 1st 10 min PI (60 % increase in ear thickness) and a delayed reaction (18 h) (85 % increase), whereas the naive rabbits showed only the immediate reaction within the 1st 4 h (60 % increase). PBS induced only mild reactions. These results point out the crucial role of the cellular immune response of rabbits in the expression of resistance to A. cajennense.

Electrophoretic variants of intracellular catalase of different Candida species

Intracellular and extracellular catalases of different species of Candida were investigated using different culture media. All the Candida strains produced intracellular catalase, whose enzymatic activity was detected by non-denaturating polyacrylamide gradient (4-30%) gel electrophoresis. The cell extracts presented a major 230 kDa catalase band and in some strains variants of catalase with different molecular weights were detected. Candida catalase activity was not affected by heating at 50degreesC and incubation with beta-mercaptoethanol, but treatment with sodium dodecyl sulphate inhibited or reduced enzymatic activity. Extracellular enzyme activity was not detected in any of the culture filtrate extracts tested.

Allergenic extracts from Metarhizium anisopliae: Obtainment and characterization

Metarhizium anisopliae is used as a biopesticide for insects that damage agricultural plantations like sugar cane and forage plants. In a previous study the sensitization to this fungus of asthmatic patients coming from sugar cane areas was showed. The aims of this work were: to compare crude extracts obtained with Tris-HCl and Coca liquid from several growth phases of M. anisopliae concerning the total content of proteins and their electrophoretic analysis profile; to evaluate in vivo allergic sensitization in Balb/c mice and allergic patients from a sugar cane area, and to characterize the allergenic fractions in the sera of patients positive for the prick test by means of Western-blotting. The extract obtained with Coca liquid on the 16(th) day was the one that presented the greatest number of proteic fractions, including all those present in the other extracts. Twelve fractions were verified in this extract with approximate molecular weights from 94 to 14 kDa. The allergenicity of the extract obtained on the 16(th) day was proven by the production of IgE antibodies in Balb/c mice, with titres of 200. Prick tests carried out with the extract of the 16(th) day in 79 atopic individuals (from sugar cane area), 35 atopic individuals (from urban area) and 11 non- atopic individuals showed respective positivity of 29%, 9% and 0%. The allergenic characterization in vitro was performed by means of Western blotting, and the fractions that reacted with the positive individuals' sera were those of approximate molecular weights of 67 kDa (95%); 20 kDa (55%); 94 kDa (36%); 34 and 36 kDa (23%); 43 and 48 kDa (14%): 16 kDa (9%) and 54kDa (5%). It was concluded that the crude allergenic extract, obtained with Coca liquid from the 16(th) day growth of Metarhizium anisopliae, contains allergenic fractions and can be used in diagnostic screening tests.

Dialysable leukocyte extracts modify the course of experimental paracoccidioidomycosis in the Syrian hamster

The effect of dialysable leukocyte extracts (DLE) obtained from hamsters immunized with Paracoccidioides brasiliensis (immune DLE) and from non-immunized hamsters (non-immune DLE) was studied in hamsters inoculated with P. brasiliensis by the intratesticular route. Treatment with immune or non-immune DLE was started during the third week of infection and was repeated at 7, 11, 15 and 19 weeks. A group of untreated infected animals was used as control. Animals were submitted to the delayed hypersensitivity skin test to P. brasiliensis antigen (PbAg) in vivo and assayed in vitro by the macrophage migration inhibition test in the presence of Phytohemagglutinin (PHA) and PbAg and by immunodiffusion for specific antibody. The animals were sacrificed at 4, 8, 12, 16 and 20 weeks. The morphology and extension of the lesions were studied at the inoculation site, and in lymph nodes, lungs, liver, spleen and kidneys. In contrast to the controls, animals treated with both DLEs maintained a positive cell-mediated immune response throughout the experiment and developed less extensive infection with a significantly lower number of fungi in the lesions. The results suggest that immune and non-immune DLE preparations modified the evolution of experimental paracoccidioidomycosis with equal efficiency. This similarity may be explained by the immunoregulatory activities of both extracts.