The mechanism of action of Russian propolis ethanol extracts against two antibiotic-resistant biofilm-forming bacteria.

UNLABELLED: The interaction between antibiotic-resistant Staphylococcus aureus and antibiotic-sensitive Escherichia coli biofilm-forming bacteria and Russian propolis ethanol extracts was evaluated. In this study, bacterial cell death occurred when the cell membranes of bacteria interacted specifically with the antibacterial compounds found in propolis. In order to understand the Russian propolis ethanol extract mechanism of action, microscopy and bacterial lysis studies were conducted. Results uncovered from these experiments imply that the mechanism of action of Russian propolis ethanol extracts is structural rather than functional. The results obtained throughout this study demonstrate cell membrane damage, resulting in cell lysis and eventually bacterial death. SIGNIFICANCE AND IMPACT OF THE STUDY: Most strains of bacteria and subsequently biofilms, have evolved and have altered their chemical composition in an attempt to protect themselves from antibiotics. The resistant nature of bacteria stems from the chemical rather than the physical means of inactivation of antibiotics. The results uncovered in this work demonstrate the potential application of Russian propolis ethanol extracts as a very efficient and effective method for bacterial and biofilm inactivation.

Clinical outcomes in reduction mammaplasty: a systematic review and meta-analysis of published studies.

This systematic review and meta-analysis were undertaken to determine whether reduction mammaplasty improves measurable outcomes in women with breast hypertrophy. A systematic review of the literature in 5 languages from 1985 until March 1999 was performed, and data were compared for meta-analysis. Eligible studies were both experimental and observational and involved women with preoperative physical and/or psychosocial signs and symptoms who underwent reduction mammaplasty for breast hypertrophy. Outcomes assessed were postoperative physical signs and symptoms such as shoulder pain, shoulder (bra strap) grooving, and quality-of-life domains, such as physical and psychological functioning, and were expressed primarily as risk differences (RDs). Twenty-nine studies of 4173 patients met all eligibility criteria. Reduction mammaplasty was associated with a statistically significant improvement in physical signs and symptoms involving shoulder pain (RD, 0.71 [95% confidence interval (CI), 0.62-0.80]); shoulder grooving (RD, 0.69 [95% CI, 0.60-0.78]); upper/lower back pain (RD, 0.59 [95% CI, 0.48-0.70]); neck pain (RD, 0.50 [95% CI, 0.37-0.64]); intertrigo (RD, 0.44 [95% CI, 0.34-0.54]); breast pain (RD, 0.36 [95% CI, 0.17-0.55]); headache (RD, 0.28 [95% CI, 0.11-0.46]); and pain/numbness in the hands (RD, 0.11 [95% CI, 0.04-0.18]). The quality-of-life parameter of physical functioning was also statistically significant (RD, 0.58 [95% CI, 0.44-0.71]), while psychological functioning was not significant (RD, 0.46 [95% CI, 0.00-1.00]). The evidence suggests that women undergoing reduction mammaplasty for breast hypertrophy have significant postoperative improvement in preoperative signs and symptoms, quality of life, or both.

Acyloxymethyl acidic drug derivatives: in vitro hydrolytic reactivity.

A series of acyloxymethyl drug derivatives of the NH-acidic drugs, phenytoin and theophylline and of the carboxylic acid drugs, thioctic acid and indomethacin, were prepared in order to determine the effect of varying the nature of the drug on the in vitro rate of hydrolysis catalyzed by porcine liver esterase and human plasma. The acyl portion was comprised of either valeric acid (val) or gamma-linolenic acid (GLA). With the exception of some GLA prodrugs, the derivatives displayed first-order kinetics in both enzyme systems. The NH-acidic drug derivatives were hydrolyzed faster than the carboxylic drug derivatives by porcine liver esterase and human plasma. It was found that the short chain valeric acid derivatives were hydrolyzed faster than the GLA derivatives. The rates of hydrolysis for the relatively smaller prodrugs of theophylline and thioctic acid were greater than the rates of hydrolysis for the bulkier phenytoin and indomethacin prodrugs indicating steric hindrance was important. The lipophilicity index, log K, of the valeric acid drug derivatives was plotted against the logarithm of the hydrolysis rate constant, k, and it was observed that log k decreased with an increase in log K. A comparison of these results with those of previous studies where the alkyl and acyl moieties were varied of acyloxyalkyl theophylline derivatives has provided a rationale, based on lipophilicity, for the structure of a prodrug to be designed based on an in vitro desired rate of hydrolysis.

Comparison of the grignard deacylation TLC and HPLC methods and high resolution 13C-NMR for the sn-2 positional analysis of triacylglycerols containing gamma-linolenic acid.

There is increasing evidence that the fatty acid, gamma-linolenic acid (GLA) is the effective component found in evening primrose oil (EPO) which has been shown to bring about clinical improvement in a number of disease conditions. The two major triacylglycerols (TAGs) in EPO are trilinolein (LLL) and a TAG species containing one GLA and two linoleic (LA) fatty acid chains. This latter TAG, called dilinoleoyl-mono-gamma-linolenin (DLMG or Oenotheral), makes up about 15% by weight of EPO and accounts for over one-half of the total amount of GLA present in EPO. Although DLMG is comprised of three possible isomers, the abbreviation is used to represent the naturally occurring mixture of these isomers. We have isolated DLMG from EPO and also prepared its three possible isomers, sn-GLL, sn-LGL and sn-LLG, and carried out the sn-2 positional analysis using three different approaches, namely, Grignard deacylation TLC and HPLC methods and high resolution 13C-NMR spectroscopy. The results of the sn-2 positional analysis for both the natural and synthetic TAGs containing LA and GLA in this study using the three approaches are all in very good agreement. This indicates that the three positional analysis methods are valid within their acceptable error margin and can be used with confidence in determining the fatty acid composition of the sn-2 position. Given the increased availability of NMR spectrometers this method might prove to be the easiest and most convenient in determining the sn-2 position for oil or TAG samples that contain a small number of different fatty acids providing all the 13C-NMR carbonyl resonances are well resolved.

Effect of salt-loading and spontaneous hypertension on in vitro metabolism of [1-14C]linoleic and [2-14C]dihomo-gamma-linolenic acids.

The present study compared the effect of spontaneous hypertension and salt-loading on in vitro metabolism of 18:2n-6 (linoleic acid) and 20:3n-6 (dihomo-gamma-linolenic acid). Ten weanling spontaneously hypertensive rats (SHR) and 10 normotensive Wistar-Kyoto rats (WKY) maintained on a rodent lab chow were given tap water with (n = 5) or without (n = 5) addition of 1% NaCl for 4 weeks. Thereafter, animals were killed and liver microsomes were prepared. Aliquots of microsomes suspended in the phosphate-sucrose buffer containing MgCl2, ATP, CoA, and NADPH were incubated with 0.3 microCi of [1-14C]-18:2n-6 or [2-14C]-20:3n-6 at 37 degrees C for 15 min. The activity of delta 6- and delta 5-desaturases, and the distribution of radioactivity in different lipid fractions and in phospholipid fatty acids were determined. Results showed that both spontaneous hypertension and salt-loading suppressed the desaturation of radiolabeled 18:2n-6 and of 20:3n-6. Incubation of microsomes with [1-14C]-18:2n-6 resulted in 29% of radioactivity being associated with phospholipid fraction, of which 3% was associated with 18:3n-6. Incubation with radiolabeled 20:3n-6 resulted in 30% of the radioactivity being incorporated into phospholipids, of which 50% was associated with 20:4n-6 (arachidonic acid). Salt-loading suppressed the incorporation of radiolabeled fatty acids into phospholipids, more so in SHR than in WKY. Thus, salt-loading not only suppressed the desaturation of 18:2n-6 and 20:3n-6, but also interfered with the acylation of n-6 fatty acids into the phospholipid fraction.

Effects of repeated gestation and lactation on milk n-6 fatty acid composition in rats fed on a diet rich in 18:2n-6 or 18:3n-6.

The present study examined the effect of repeated gestation and lactation on the levels of long-chain n-6 polyunsaturated fatty acids in rat milk fat, and examined whether such levels might be modulated by supplementing the diet of the lactating dams with either (g/kg) 50 safflower oil (SFO; containing 800 g 18:2n-6/kg), or 50 evening primrose oil (EPO; containing 720 g 18:2n-6 and 90 g 18:3n-6/kg). The milk was collected at three different times (days 1, 8 and 15) in each given lactation period from female Sprague-Dawley rats which were successively bred for four pregnancies and lactations. Results showed that dietary fat and breeding frequency had no significant effects on milk triacylglycerol content, but they modified the pattern of milk fatty acids in both triacylglycerol and phospholipid fractions. After three or four successive breedings rats fed on EPO produced milk containing less saturated but more monounsaturated and polyunsaturated fatty acids compared with those fed on SFO. During the course of lactation the levels of n-6 metabolites, e.g. 18:3n-6, 20:3n-6 and 20:4n-6, in milk fat declined progressively. However, they were consistently higher in the EPO group than in the SFO group. These findings suggest that the levels of long-chain n-6 metabolites in the milk fat may be increased through supplementing the maternal diet with 18:3n-6.

Effect of dietary linoleic acid content on the distribution of triacylglycerol molecular species in rat adipose tissue.

The present study examined the effect of varying dietary linoleate intake (0.01, 0.24, 2.4, 24, 80 or 160 g/kg diet) for 24 weeks on the distribution of triacylglycerol (TG) molecular species in rat epididymal adipose tissue. Adipose TG fractions were purified by thin-layer chromatography and separated into different molecular species by reverse-phase high-performance liquid chromatography. The identification of TG species was based on fatty acid composition, retention time and the theoretical carbon number. When the dietary 18:2n-6 content was equal to or less than 24 g/kg, no significant amounts of n-6 fatty acids (mainly 18:2n-6) were observed in adipose tissue TG despite the fact that the levels of 20:4n-6 in liver phospholipids increased significantly. There were 12 major molecular species in adipose tissue when the dietary 18:2n-6 content was less than 2.4 g/kg. When the dietary 18:2n-6 content reached 24 g/kg, an additional six TG species containing one, two or three molecules of 18:2n-6 were observed. The levels of TG molecules containing two or three 18:2n-6 residues were further increased when the diet contained very large amounts of linoleic acid (160 g/kg). Conversely, those TG species containing only one 18:2n-6 residue became less abundant. It is suggested that the accumulation of these linoleate-rich TG molecular species in adipose tissue, particularly di- and trilinoleoyl containing TG, is the result of an adequate or an excessive intake of linoleic acid.

Effect of maternal dietary fats with variable n-3/n-6 ratios on tissue fatty acid composition in suckling mice.

This report examines the distribution of n-3 and n-6 fatty acids in heart, kidney and liver phosphatidylcholine and phosphatidylethanolamine of suckling mice from dams fed a fat-supplemented diet with variable n-3/n-6 ratios. After conception and throughout the pregnancy and lactation period, dams were fed a fat-free liquid diet supplemented with 20% by energy of oil mixtures (fish oil concentrate, rich in 20:5n-3 and 22:6n-3, and safflower oil concentrate, rich in 18:2n-6). The diets contained similar amounts of combined n-3 and n-6 fatty acids but variable ratios of n-3 to n-6 fatty acids (0, 0.25, 0.5, 1, 2 and 4). In 12-day-old suckling mice, as the n-3/n-6 ratio in the maternal diet increased (up to approx. 0.5), the tissue levels of 20:5n-3, 22:5n-3 and 22:6n-3 increased, whereas those of 18:2n-6 and 20:4n-6 decreased. The responses were similar in both phospholipid subclasses, but varied between different tissues. Generally, the n-3/n-6 ratios were significantly greater in pup tissues than in milk fat, indicating preferential incorporation of n-3 over n-6 fatty acids into phospholipids during growth. However, the incorporation of n-3 fatty acids in pups was significantly suppressed whereas that of n-6 fatty acids was increased when 18:2n-6 was replaced by its delta 6-desaturation product, 18:3n-6 (concentrated from evening primrose oil), as the source of n-6 fatty acid.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of dietary n-3/n-6 ratio on brain development in the mouse: a dose response study with long-chain n-3 fatty acids.

This study examines the effects of the ratio of n-3/n-6 fatty acids (FA) on brain development in mice when long-chain n-3 FA are supplied in the diet. From conception until 12 days after birth, B6D2F1 mice were fed liquid diets, each providing 10% of energy from olive oil, and a further 10% from different combinations of free FA concentrates derived from safflower oil (18:2n-6), and fish oil (20:5n-3 and 22:6n-3). The range of dietary n-3/n-6 ratios was 0, 0.25, 0.5, 1.0, 2.0 and 4.0, with an n-6 content of greater than 1.5% of energy in all diets, and similar levels of total polyunsaturated fatty acids (PUFA). In an additional group of ratio 0.5, 18:2n-6 was partially replaced by its delta 6 desaturation product, 18:3n-6. Biochemical analyses were conducted on 12-day-old pup brains, as well as on samples of maternal milk. No obvious effects on overall pup growth and development were observed, apart from a smaller litter size at ratio 1. Co-variance analysis indicated that increasing the n-3/n-6 ratio was associated with slightly smaller brains, relative to body weight. We found that 18:2n-6 and 20:5n-3 were the predominant n-6 and n-3 FA in the milk; in the brain these were 20:4n-6 and 22:6n-3, respectively. Increasing dietary n-3/n-6 ratios generally resulted in an increase in n-3 FA, with a corresponding decrease in n-6 FA.(ABSTRACT TRUNCATED AT 250 WORDS)

Automated separation and quantitation of lipid fractions by high-performance liquid chromatography and mass detection.

This report describes an improved separation and quantitation of lipid fractions in a total lipid extract by high-performance liquid chromatography using a modified solvent and gradient system delivered by dual pumps and incorporating a mass detector and autosampler. The detector responses for various lipid fractions (cholesteryl esters, triacylglycerols, free cholesterol, and seven major phospholipid classes) were fitted to a quadratic equation, y = ax2 + bx + c, and quantified after detector calibration by a computer. This new system has the advantage of automation and reproducible separation. The present method was applied to rat liver analysis.

Modification of liver fatty acid metabolism in mice by n-3 and n-6 delta 6-desaturase substrates and products.

The effects of dietary supplementation of either alpha-linolenic acid (18:3(n-3)) or stearidonic acid (18:4(n-3)) in combination with either linoleic acid (18:2(n-6)) or gamma-linolenic acid (18:3(n-6)) on liver fatty acid composition in mice were examined. Essential fatty acid deficient male C57BL/6 mice were separated into four groups of seven each and were fed a fat-free semi-purified diet supplemented with 1% (w/w) fatty acid methyl ester mixture (1:1), 18:2(n-6)/18:3(n-3), 18:2(n-6)/18:4(n-3), 18:3(n-6)/18:3(n-3), or 18:3(n-6)/18:4(n-3). After 7 days on the diets, fatty acid compositions in liver phosphatidylcholine and phosphatidylethanolamine fractions were analyzed. In groups fed 18:4(n-3) (18:2(n-6)/18:4(n-3) or 18:3(n-6)/18:4(n-3)) as compared to those fed 18:3(n-3) (18:2(n-6)/18:3(n-3) or 18:3(n-6)/18:3(n-3)), the levels of 20:4(n-3), 20:5(n-3) and 22:5(n-3) were increased, whereas those of 20:3(n-6) and 20:4(n-6) were decreased. When 18:3(n-6) replaced 18:2(n-6) as the source of n-6 acids, the levels of 18:3(n-6), 20:3(n-6), 20:4(n-6) and 22:5(n-6) were increased, whereas those of 20:4(n-3) and 20:5(n-3) were reduced. Replacing 18:3(n-3) by 18:4(n-3) reduced the (n-6)/(n-3) ratio by approx. 30%, whereas replacing 18:2(n-6) by 18:3(n-6) increased the (n-6)/(n-3) ratio by approx. 2-fold. These findings indicated that delta 6-desaturase products were metabolized more readily than their precursors. Both products also competed for the subsequent metabolic enzymes. However, the n-6 fatty acids derived from 18:3(n-6) were incorporated more favourably into liver phospholipids than n-3 fatty acids derived from 18:4(n-3).

The role of n-3 essential fatty acids in brain and behavioral development: a cross-fostering study in the mouse.

A cross-fostering design was used to examine the effects on brain and behavioral development in mice of pre- and/or postnatal dietary supplementation with n-3 fatty acids. Pregnant mice were fed either of two liquid diets, control (con) or experimental (exp). Each diet provided 3% of the calories in the form of n-6 fatty acids; the experimental diet was supplemented with an additional 1.5% from long chain n-3 fatty acids derived from fish oil. There were four treatment groups, with all pups fostered at birth. These groups were (prenatal diet/postnatal diet): Group 1. exp/exp; Group 2, exp/con; Group 3, con/exp; Group 4, con/con; a fifth control group (unfostered) was fed lab chow (LC) throughout the study. Animals from the exp/exp and con/con groups were weaned onto lab chow for later behavioral assessment. Prenatal n-3 supplementation resulted in a small acceleration of behavioral development. The adult animals did not differ on visual discrimination learning nor did they differ in visual acuity. During development the fatty acid composition of the brain membrane phospholipids reflected closely that of the pre- and postnatal dietary conditions. Levels of 22:5n-3 and 22:6n-3 increased in the n-3 supplemented groups, accompanied by a decrease in levels of 22:4n-6 and 22:5n-6; the net effect of these changes was to increase the total levels of C22 fatty acids. While these results support considerable plasticity of the fatty acid composition of the developing brain with respect to the immediate dietary availability of n-3 compounds, they do not support long term effects on learning capacity of n-3 supplementation during the developmental period.