Efficient preparation of catechin thio conjugates by one step extraction/depolymerization of pine (Pinus pinaster) bark procyanidins

The skin penetrating antioxidant cysteamine derivative of (-)-epicatechin as well as other thio conjugates were efficiently obtained with high yields from pine (Pinus pinaster) bark by simultaneous one pot extraction and depolymerization using water and cysteamine hydrochloride. The influence of the concentration of bark, acid, and cysteamine, as well as the reaction time on the total conversion, was studied. The total conversion into the epicatechin and catechin conjugates was as high as 47 g/kg pine bark with 1666 g cysteamine/kg bark and 28 g/kg with 166 g cysteamine/kg bark. A fast cleanup step by absorption/desorption on XAD-16 greatly facilitated further purification of the active major component. At a pilot scale, 4beta-(2-aminoethylthio)epicatechin (1) (conversion 263 g, purity 35% by reversed phase high-performance liquid chromatography/weight) was obtained from 17 kg of pine bark after simultaneous extraction/depolymerization followed by cleanup with the polymeric resin in approximately 10 h. The results show that pine (P. pinaster) bark is a suitable source of flavanols for the preparation of active thio derivatives. Conditions are given for the fast and efficient preparation of the conjugates.     

Procyanidin fractions from pine (Pinus pinaster) bark: radical scavenging power in solution, antioxidant activity in emulsion, and antiproliferative effect in melanoma cells

Pine (Pinus pinaster) bark is a rich source of procyanidin oligomers. From a total polyphenolic extract, we have generated fractions of different procyanidin composition. The mixtures, devoid of gallate esters, were active as free radical scavengers against ABTS(*+), DPPH, and HNTTM. Pine bark fractions were tested for antioxidant activity in solution (hydrogen donation and electron transfer) and emulsion (inhibition of lipid peroxidation) and compared with their galloylated counterparts from grape origin. While galloylation clearly influenced the free radical scavenging efficiency in solution, it did not seem to play a determinant role in protection against lipid peroxidation in emulsion. The fractions were very mild inhibitors of cell proliferation. Because gallate esters appear to interfere with crucial cell functions, gallate free pine procyanidins may be the innocuous chemopreventative agents of choice for many applications in food and skin protection.     

Frequency of urinary tract infection among dogs with pruritic disorders receiving long-term glucocorticoid treatment

OBJECTIVE: To determine frequency of urinary tract infection (UTI) among dogs with pruritic disorders that were or were not receiving long-term glucocorticoid treatment. DESIGN: Observational study. ANIMALS: 127 dogs receiving glucocorticoids for > 6 months and 94 dogs not receiving glucocorticoids. PROCEDURE: Bacterial culture of urine samples was performed in dogs receiving long-term glucocorticoid treatment, and information was collected on drug administered, dosage, frequency of administration, duration of glucocorticoid treatment, and clinical signs of UTI. For dogs not receiving glucocorticoids, a single urine sample was submitted for bacterial culture. RESULTS: Multiple (2 to 6) urine samples were submitted for 70 of the 127 (55%) dogs receiving glucocorticoids; thus, 240 urine samples were analyzed. For 23 of the 127 (18.1%) dogs, results of bacterial culture were positive at least once, but none of the dogs had clinical signs of UTI. Pyuria and bacteriuria (present vs absent) were found to correctly predict results of bacterial culture for 89.9% and 95.8% of the samples, respectively. Type of glycocorticoid, dosage, frequency of administration, and duration of treatment were not associated with frequency of UTI. None of the urine samples from dogs not receiving glucocorticoids yielded bacterial growth. The frequency of UTI was significantly higher for dogs treated with glucocorticoids than for dogs that had not received glucocorticoids. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that dogs receiving long-term glucocorticoid treatment have an increased risk of developing a UTI. On this basis, we recommend that urine samples be submitted for bacterial culture at least yearly for such dogs.     

Characterization of antibiotic resistance genes and virulence factors in faecal enterococci of wild animals in Portugal

Antibiotic susceptibility was tested in 140 non-selected enterococci (73 Enterococcus faecalis, 45 E. faecium and 22 of other species) recovered from faecal samples of 77 wild animals in Portugal. Susceptibility testing for 11 antibiotics (vancomycin, teicoplanin, ampicillin, streptomycin, gentamicin, kanamycin, chloramphenicol, tetracycline, erythromycin, quinupristin-dalfopristin and ciprofloxacin) was determined by disk diffusion and agar dilution methods. Forty-four isolates (31.4%) showed susceptibility to all the antibiotics tested (5.5% of E. faecalis; 62.2% of E. faecium; and 78.6% of E. hirae). Neither ampicillin-resistance nor acquired-vancomycin-resistance was detected and 1.4% of the isolates showed high-level-resistance for gentamicin or streptomycin. Tetracycline and erythromycin resistances were shown in 28.6% and 20.1% of the isolates, respectively. Antibiotic resistance genes were studied by polymerase chain reaction (PCR) and sequencing and tet(M) + tet(L), erm(B) or aac(6')-aph(2') genes were detected in most of tetracycline-, erythromycin- or gentamicin-resistant enterococci respectively. Genes encoding virulence factors were studied by PCR and a wide variety of virulence genes were detected in most of E. faecalis isolates but were rarely found in E. faecium and not detected in the other species. The prevalence of genes encoding virulence factors in E. faecalis was as follows: cpd (98.6%), gelE (75.3%), agg (30.1%), fsr (17.8%), ace (9.6%) and esp (4.1%). Low percentages of antibiotic resistance was found in the faecal enterococci of wild animals but a wide variety of virulence genes were detected among E. faecalis isolates although were rare in the other species.     

Towards sustainable control of Lepidopterous pests in olive cultivation

Zusammenfassung Traditionelle, extensiv bearbeitete Olivenhaine, aber auch moderne Intensiv-Plantagen mit künstlicher Bewässerung und hohem Einsatz von Düngern bzw. chemischen Pflanzenschutzmitteln kennzeichnen die derzeitigen verschiedenen Anbauformen der Olive im Mittelmeerraum. Schadlepidopteren wie die Olivenmotte (Prays oleae, Lep.: Yponomeutidae) und die Jasminmotte (Palpita unionalis, Lep.: Pyralidae) werden durch regelmässigen Insektizideinsatz bekämpft. Das von der EU geförderte internationale Forschungsprojekt TRIPHELIO zielte auf die Entwicklung insektizidfreier Alternativmethoden durch (1) die Optimierung der pheromongestützten Überwachung und Verwirrtechnik, (2) der Anwendung von Habitatmanagement-Strategien zur Förderung natürlicher Gegenspieler, und (3) dem Einsatz von Trichogramma-Schlupfwespen. Zusätzlich wurden Module für eine optimale Anwendung biotechnischer und biologischer Methoden bezüglich der Phänologie der Schadinsekten und möglicher Nebenwirkungen von Pestiziden erarbeitet. Die intensive Kooperation zwischen Wissenschaftlern und Praktikern aus mehreren Ländern Europas und Nordafrikas erlaubte den Entwurf möglicher Lösungsansätze für verschiedene Anbaubedingungen und klimatische Regionen des Mittelmeerraumes. Die wichtigsten Ergebnisse und Ausblicke für eine zukünftige praktische Umsetzung werden in dieser Veröffentlichung beschrieben.     

Differential effects of the endocrine-disrupting compounds bisphenol-A and octylphenol on gonadotropin secretion, in prepubertal ewe lambs

This study examined the effects of long-term exposure to the endocrine-disrupting compounds (EDCs) Bisphenol-A (BPA) and Octylphenol (OP) on gonadotrophin secretion in pre-pubertal female sheep. Four-week-old, female lambs were randomly allocated to four groups (n=6), and twice each week treated with i.m. injections of either corn oil (vehicle controls), diethylstilbestrol (DES; 0.175mg/kg), BPA (3.5mg/kg) or OP (3.5mg/kg). After 5 weeks of treatment, animals were ovariectomized (ovx) and ovary weights recorded. Two weeks later, blood samples were collected from lambs every 15min for 6h, for LH pulse analysis. Animals were then euthanased and adrenal and kidney weight recorded. An age-related increase in tonic LH secretion was noted in Control, BPA- and OP-treated lambs, but was absent in DES-treated lambs. Following ovx, LH secretion increased in all except DES-treated lambs; FSH concentrations increased in all groups. BPA and DES significantly suppressed LH pulse frequency (C: 6.7+/-0.3pulses/6h, DES: 1.5+/-0.8pulses/6h, BPA: 2.3+/-0.8pulses/6h) and amplitude (C: 7.1+/-1.0ng/ml, DES: 1.9+/-0.6ng/ml, BPA: 1.6+/-0.4ng/ml). OP had no effect on LH secretion (Frequency: 5.8+/-0.5pulses/6h, amplitude: 8.0+/-2.0ng/ml). Ovary weight was similar among all groups. Results show that chronic in vivo exposure of prepubertal female lambs to BPA, at levels lower than those reported previously, can have significant effects on LH secretion that are comparable to those seen following exposure to the known xenoestrogen, DES. Exposure to an equal dose of the EDC OP, over the equivalent period of time was without effect on gonadotropin secretion in the prepubertal ewe lamb. These results indicate that exposure of prepubertal female lambs to the EDC BPA can induce significant effects on gonadotropin secretion, the potential long-term effects of exposure and the effects of these changes on reproductive performance and efficacy, therefore, merit further study.     

Detection of genes encoding virulence factors and bacteriocins in fecal enterococci of poultry in Portugal

Seventy-six Enterococcus isolates (43 E. faecalis, 30 E. faecium, two E. durans, and one E. hirae) recovered from fecal samples of poultry in a slaughterhouse (one isolate per fecal sample and one fecal sample per lot of animals) were studied for bacteriocin production and for the presence of genes encoding bacteriocins and virulence factors. The presence of genes encoding virulence factors (cpd, geE, fsr, ace, agg, and esp) and bacteriocins (entA, entB, entP, entQ, entAS-48, entL50A/B, cyl, and bac31) were studied by polymerase chain reaction in all enterococci. At least two virulence genes were detected in all 43 E. faecalis isolates, cpd and gelE being the most frequently detected genes (97.7%) followed by ace (62.8%), agg (39.5%), fsr (27.9%), and esp (2.3%). No virulence genes were detected in the other enterococcal species with the exception of one E. faecium and one E. durans isolates that harbored the gelE gene. Antimicrobial activity against eight indicator bacteria (including Listeria monocytogenes) was assayed in the enterococci, and 23 (30.3%) showed inhibitory activity against L. monocytogenes, the other 22 enterococci showing activity against indicator bacteria other than L. monocytogenes. Only the entA, entB, and cyl genes were detected in our study (entA + entB in nine E. faecium isolates and the cyl gene in seven E. faecalis isolates). A wide variety of virulence genes have been detected in fecal E. faecalis isolates from poultry, but not in the other enterococcal species. However, the presence of known bacteriocin structural genes is associated more with the E. faecium species.