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Capillary electrophoretic separation of vitamins in sodium dodecyl sulfate containing buffers with lower aliphatic alcohols and n-hexane as organic modifiers.

The effect of lower organic alcohols as co-surfactants (methanol, ethanol, n-propanol, isopropanol, propanediol, n-butanol and isoamylalcohol) and n-hexane as an organic modifier in 12.5 mol/l phosphate buffer with varying SDS concentration was investigated using a set of vitamins and p-hydroxybenzoic acid as the test mixture. It was demonstrated that optimum separations can be achieved particularly at high concentrations of the surfactant; the selectivity can be changed by adding a co-surfactant; while propanol and isopropanol show the same properties as co-surfactants, the most efficient alcohols were isoamylalcohol and propanediol. n-Butanol was capable of selective separation of p-hydroxybenzoic acid in the test mixture. Addition of ethanol appears most effective at higher concentrations (while all the other alcohols are effective already at 5% concentration, the best results with ethanol were obtained when it constituted 20% of the background electrolyte). 5% Concentration of methanol resulted in poor separation of the test mixture, however if 300 microl/10 ml of hexane were added to 20 mmol/l SDS containing phosphate buffer, the resulting separation was practically the same as with 50 mmol/l SDS.

Alcohols↗

Biomonitoring of environmental polycyclic aromatic hydrocarbon exposure by simultaneous measurement of urinary phenanthrene, pyrene and benzo[a]pyrene hydroxides.

A high-performance liquid chromatographic method with fluorescence detection was developed which enables the simultaneous determination of the urinary polycyclic aromatic hydrocarbon metabolites 3-hydroxyphenanthrene, 1-hydroxypyrene and 3-hydroxybenzo[a]pyrene. The method has small solvent consumption because of the use of a microbore RP C18 column and a relatively short run time. Low detection limits of 0.02 nmol/l for 3-hydroxypyrene to 0.19 nmol/l for 3-hydroxybenzo[a]pyrene were attained. In contrast, the detection limits of alpha-naphthol and 9,10-dihydroxy-9,10-dihydrophenanthrene were not adequate for the determination of environmental exposure. The developed method was successfully used for the analysis of urine samples from children.

Air Pollutants↗

The influence of type and quantity of model drug on the extrusion/spheronization of mixtures with microcrystalline cellulose. I. Extrusion parameters.

Five model drugs, (methyl, propyl and butyl paraben, 4-hydroxybenzoic acid and propyl gallate), similar in their chemical structure were mixed with microcrystalline cellulose and water in different proportions and forced through a ram extruder. The overall water movement was measured by the difference between the initial water in the formulation and the water content in the plug remaining after extrusion was completed. The differences in theoretical and practical volume occupancy of the materials inside the barrel were calculated to look for trapped air inside the barrel. The steady-state extrusion force for each formulation was recorded. All five materials demonstrated differences in behaviour during extrusion. The relationship between each of the three properties measured and both the drug-load and initial water content was examined, to establish the potential relationship that existed between the differences due to the drug models. The five drug models were divided into two sub-groups, when examining the way that they underwent extrusion. Methyl, propyl and butyl paraben formed one group while 4-hydroxybenzoic acid and propyl gallate formed the other group. Within the former group the relationship between steady-state extrusion force and the percentage of drug and water present tended to be lower than those in the latter group. For the former group these relationships were non-linear.

Antioxidants↗

Comparison of quantitative high performance thin layer chromatography and the high performance liquid chromatography of parabens.

A method is described for the densitometric determination of the p-hydroxybenzoic esters and p-hydroxybenzoic acid in mixtures or in drugs. This method is compared with the one used in high performance liquid chromatography (HPLC). The calibration curves were linear in interval 0.250-3.60 mumol ml-1 per 200 nl per spot. The limit of detection and the relative standard deviation (RSD) are higher than in HPLC (RSD is 6% in HPTLC. 3% in HPLC; limit of detection about 40 pmol in HPTLC and 25 pmol in HPLC) but HPTLC quantitative determination of parabens in drugs is faster.

Chromatography, High Pressure Liquid↗

Biodegradation of hydroxylated and methoxylated benzoic, phenylacetic and phenylpropenoic acids present in olive mill wastewaters by two bacterial strains.

Two aerobic bacterial strains, a chlorophenol-degrading bacterium characterized in this work as a Ralstonia sp. LD35 on the basis of the sequence of the gene encoding for 16S ribosomal RNA, and Pseudomonas putida DSM 1868, capable of metabolizing 4-methoxybenzoic acid, were tested for their capacity to degrade monocyclic aromatic acids responsible for the toxicity of olive mill wastewaters (OMWs). Both strains possess interesting and complementary degradation capabilities in resting cell conditions: Ralstonia sp. LD35 was found to metabolize 4-hydroxybenzoic, 4-hydroxyphenylacetic, 3,4-dihydroxycinnamic and cinnamic acid, whereas DSM 1868 was capable of metabolizing 4-hydroxy-3-methoxybenzoic, 3,4-dimethoxybenzoic and 4-hydroxy-3,5-dimethoxybenzoic acid, as well as 4-hydroxybenzoic and 4-hydroxyphenylacetic acid. The kinetic parameters describing the growth of the two strains on the same compounds were determined in growing-cell batch conditions, and showed that both strains presented high affinity and high specific growth rates towards all assayed substrates. In addition, the two strains were capable of growing on and extensively biodegrading a mixture of monocyclic aromatic acids commonly found at high concentrations in OMWs, and of growing on a 20% dilution of a natural OMW. All these features make the two strains attractive candidates for the development of a biotechnological process for the biodegradation of aromatic compounds found in OMWs.

Betaproteobacteria↗

Isolation and characterization of the three polypeptide components of 4-chlorobenzoate dehalogenase from Pseudomonas sp. strain CBS-3.

The three genes encoding the 4-chlorobenzene dehalogenase polypeptides were excised from a Pseudomonas sp. CBS-3 DNA fragment and separately cloned and expressed in Escherichia coli. The three enzymes were purified from the respective subclones by using an ammonium sulfate precipitation step followed by one or two column chromatographic steps. The 4-chlorobenzoate:coenzyme A ligase was found to be a homodimer (57-kDa subunit size), to require Mg2+ (Co2+ and Mn2+ are also activators) for activity, and to turn over MgATP (Km = 100 microM), coenzyme A (Km = 80 microM), and 4-chlorobenzoate (Km = 9 microM) at a rate of 30 s-1 at pH 7.5 and 25 degrees C. Benzoate, 4-bromobenzoate, 4-iodobenzoate, and 4-methylbenzoate were shown to be alternate substrates while 4-hydroxybenzoate, 4-aminobenzoate, 2-aminobenzoate, 2,3-dihydroxybenzoate, 4-coumarate, palmate, laurate, caproate, butyrate, and phenylacetate were not substrate active. The 4-chlorobenzoate-coenzyme A dehalogenase was found to be a homotetramer (30 kDa subunit size) to have a Km = 15 microM and kcat = 0.3 s-1 at pH 7.5 and 25 degrees C and to be catalytically inactive toward hydration of crotonyl-CoA, alpha-methylcrotonyl-CoA, and beta-methylcrotonyl-CoA. The 4-hydroxybenzoate-coenzyme A thioesterase was shown to be a homotetramer (16 kDa subunit size), to have a Km = 5 microM and kcat = 7 s-1 at pH 7.5 and 25 degrees C, and to also catalyze the hydrolyses of benzoyl-coenzyme A and 4-chlorobenzoate-coenzyme A. Acetyl-coenzyme A, hexanoyl-coenzyme A, and palmitoyl-coenzyme A were not hydrolyzed by the thioesterase.

Bacterial Proteins↗

Quantification of the oxidizing capacity of nanoparticulate zero-valent iron.

Addition of nanoparticulate zero-valent iron (nZVI) to oxygen-containing water results in oxidation of organic compounds. To assess the potential application of nZVI for oxidative transformation of organic contaminants, the conversion of benzoic acid (BA) to p-hydroxybenzoic acid (p-HBA) was used as a probe reaction. When nZVI was added to BA-containing water, an initial pulse of p-HBA was detected during the first 30 min, followed by the slow generation of additional p-HBA over periods of at least 24 h. The yield of p-HBA increased with increasing BA concentration, presumably due to the increasing 'ability of BA to compete with alternate oxidant sinks, such as ferrous iron. At pH 3, maximum yields of p-HBA during the initial phase of the reaction of up to 25% were observed. The initial rate of nZVI-mediated oxidation of BA exhibited a marked reduction at pH values above 3. Despite the decrease in oxidant production rate, p-HBA was observed during the initial reaction phase at pH values up to 8. Competition experiments with probe compounds expected to exhibit different affinities for the nZVI surface (phenol, aniline, o-hydroxybenzoic acid, and synthetic humic acids) indicated relative rates of reaction that were similar to those observed in competition experiments in which hydroxyl radicals were generated in solution. Examination of the oxidizing capacity of a range of Fe0 particles reveals a capacity in all cases to induce oxidative transformation of benzoic acid, but the high surface areas that can be achieved with nanosized particles renders such particles particularly effective oxidants.

Hydrogen-Ion Concentration↗

Phenolic composition of strawberry genotypes at different maturation stages.

The effects of maturation (green, pink, and ripe) on phenolic composition of strawberry cultivars Camarosa, Dorit, Chandler, and Osmanli and their hybrids were investigated using a high-pressure liquid chromatography (HPLC) method. p-Hydroxybenzoic acid, p-coumaric acid, ellagic acid, cyanidin-3-glucoside, pelargonidin-3-glucoside, kaempferol, quercetin, and myricetin were individually quantified for each stage. The highest amounts of anthocyanins were obtained from ripe fruits whereas ellagic acid was found as the main phenolic in the green fruits. Phenolic concentrations were found statistically different in green and ripe fruits. One hybrid was found to have higher phenolic contents than the other genotypes. The p-hydroxybenzoic and p-coumaric acid levels changed during maturation, but no differences in contents of flavonoids in green and ripe fruit were detected.

Acids, Carbocyclic↗

Metabolism of gallic acid and catechin by Lactobacillus hilgardii from wine.

The ability of Lactobacillus hilgardii 5w to metabolize gallic acid and catechin was evaluated. It was grown in a complex medium containing gallic acid or catechin. The metabolites were analyzed by high-performance liquid chromatography and identified by comparing the retention times and spectral data with the standards of a database. In gallic acid-grown cultures, gallic acid, pyrogallol, catechol, protocatechuic acid, p-hydroxybenzoic acid, p-hydroxybenzaldehyde, and p-hydroxybenzyl alcohol were detected. In catechin-grown cultures, catechin, gallic acid, pyrogallol, catechol, p-hydroxybenzoic acid, acetovanillone, and homovanillic acid were detected. This work presents evidence of gallic acid and catechin degradation by L. hilgardii from wine.

Adsorption↗

New bitter-masking compounds: hydroxylated benzoic acid amides of aromatic amines as structural analogues of homoeriodictyol.

Starting from the known bitter-masking flavanones eriodictyol and homoeriodictyol from herba santa some structurally related hydroxybenzoic acid amides of benzylamines were synthesized and evaluated as masking agents toward bitterness of caffeine by sensory methods. The closest structural relatives of homoeriodictyol, the hydroxybenzoic acid vanillylamides 5-9, were the most active and were able to reduce the bitterness of a 500 mg L(-1) caffeine solution by about 30% at a concentration of 100 mg L(-1). 2,4-Dihydroxybenzoic acid vanillylamide 7 showed a clear dose-dependent activity as inhibitor of the bitter taste of caffein between 5 and 500 mg L(-1). Additionally, it was possible to reduce the bitterness of quinine and salicine but not of the bitter peptide N-l-leucyl-l-tryptophan. Combinations of homoeriodictyol and amide 7 showed no synergistic or antagonistic changes in activity. The results for model compound 7 suggested that the hitherto unknown masking mechanism is probably the same for flavanones and the new amides. In the future, the new amides may be alternatives for the expensive flavanones to create flavor solutions to mask bitterness of pharmaceuticals or foodstuffs.

Amides↗

Effects of skin metabolism on percutaneous penetration of lipophilic drugs.

Effects of skin metabolism on percutaneous penetration of drugs with high lipophilicity were studied in vitro using rat skin pretreated with and without an esterase inhibitor, diisopropyl phosphorofluoridate [also known as diisopropyl fluorophosphate (DFP)]. Without DFP, about 96% of the total penetrated amount appeared as metabolized p-hydroxybenzoic acid in the receptor fluid after application of butylparaben, whereas about 30% penetrated as intact form after application of propylparaben. On the other hand, metabolized p-hydroxybenzoic acid was not defected in the receptor fluid under pretreatment with DFP. DFP significantly decreased (p < 0.05) the total amount that penetrated after application of butylparaben, but it did not significantly affect that of propylparaben. The results indicate that skin metabolism directly affects total amount that penetrated in the case of highly lipophilic drugs, and it was found that the higher metabolic rate to hydrophilic drugs is, the greater the amount that penetrated the skin would be. Thus, when optimal prodrugs are designed for the purpose of enhancing percutaneous penetration, we propose that the bioconversion rate to parent drugs as well as the lipophilicity of prodrugs becomes an important consideration.

Animals↗

Microbial transformations of p-coumaric acid by Bacillus megaterium and Curvularia lunata.

p-Coumaric acid (1) is an abundant phenolic natural product that exhibits both chemoprotectant and antioxidant properties. Microbial transformation screening studies showed that 1 was converted to 4-vinylphenol (4), 4-hydroxybenzoic acid (2), caffeic acid (5), protocatechuic acid (6), and other unidentified metabolites over 144 h. 4-Vinylphenol (4) and its dimer, (2R,2S)-4-(2,3-dihydro-5-hyroxy-2-benzofuranyl) phenol (11), were produced by Bacillus megaterium, and 5-[(E)-2-carboxyethenyl]-2,3-dihydro-2-(4-hydroxyphenyl)-3-benzofurancarboxylic acid (15) and 4-hydroxybenzoic acid (2) were produced by Curvularia lunata. On the basis of deuterium-labeling experiments, B. megaterium catalyzes the nonoxidative enzymatic tautomerization of 1 to a vinylogous beta-keto acid intermediate that decarboxylates to 4. The presence of peroxidase and laccase activities in C. lunata extracts suggests that these enzymes may be involved in one-electron, p-coumaric acid dimerization in this organism.

Bacillus megaterium↗

Agastinol and agastenol, novel lignans from Agastache rugosa and their evaluation in an apoptosis inhibition assay.

Investigation of the whole plant of Agastache rugosa resulted in the isolation of two new lignan compounds. Their structures were elucidated as (8S,7'R,8'S)-4-hydroxybenzoic acid 4-(4-hydroxy-3-methoxybenzyl)-2-(4-hydroxy-3-methoxyphenyl)tetrahydrofuran-3-ylmethyl ester (agastinol, 1) and (7'R,8'S)-4-hydroxybenzoic acid 4-(hydroxy-3-methoxybenzylidene)-2-(4-hydroxy-3-methoxyphenyl)-tetrahydrofuran-3-ylmethyl ester (agastenol, 2). Agastinol and agastenol inhibited etoposide-induced apoptosis in U937 cells with IC50 values of 15.2 and 11.4 microg/mL, respectively.

Apoptosis↗

Metabolic fate of orally administered phyllodulcin in rats.

Naturally occurring phyllodulcin (1) was orally administered to rats to investigate its metabolic fate. Urinary metabolites were analyzed by three-dimensional HPLC. Phyllodulcin-3'-O-sulfate (2), phyllodulcin-3'-O-beta-glucuronide (3), 2-[2-(3,4-dihydroxyphenyl)ethyl]-6-hydroxybenzoic acid (4), and one novel bibenzyl derivative, 2-[2-(3-hydroxy-4-methoxyphenyl)ethyl]-6-hydroxybenzoic acid (5), together with thunberginol G (6) and hydrangenol (7) were isolated from the phyllodulcin-treated urine. 1 was extensively metabolized to 4-6 by a rat fecal suspension after incubation for 24 h. Urinary excretion of 4-6 in rats administered phyllodulcin orally was substantially reduced when the rats were treated with antibiotics to suppress their intestinal flora. On the other hand, the incubation of 1 with rat liver S-9 mix showed the presence of 7 together with 4 and 5.

Administration, Oral↗

Structure--activity relationships of the estrogenic sesquiterpene ester ferutinin. Modification of the terpenoid core.

Esterification of p-hydroxybenzoic acid, a very weak estrogenic compound, with the daucane alcohol jaeschkeanadiol (1b) leads to a spectacular magnification of the estrogenic activity. To identify the structural elements responsible for this effect, the terpenoid core of jaeschkeanadiol p-hydroxybenzoate (ferutinin, 1a) was modified, capitalizing on the presence of two functionalities, the monoacylated, hydrogen-bonded 1,3-diol system and the double bond. The hydrogen bonding, while possibly useful, was not critical for activity, while hydrogenation and cyclopropanation of the double bond were tolerated. Conversely, oxidative modifications of the double bond that placed a hydroxyl on the alpha-face of the molecule proved detrimental. Taken together, these observations identified the substitution at C-8/C-9 as critical for activity.

Benzoates↗

Geranyl Phenyl Ethers from the New Zealand Liverwort Trichocolea hatcheri

Methyl 4-(geranyloxy)-3-hydroxybenzoate (6), previously unreported, has been identified from the New Zealand liverwort Trichocolea hatcheri. Four new related 3-hydroxybenzoates 7-10 were also found, with a carbonyl at C-5 of the geranyl group and various double bond arrangements. Two known geranyl phenyl ethers 1 and 3, found in other Trichocolea species, were also identified.

Journal Article↗

Rat liver metabolism of benzo[b]naphtho[2,1-d]thiophene.

Thioarenes, sulfur-containing polycyclic aromatic hydrocarbons, have been detected in a number of environmental sources. The metabolism of one thioarene, benzo[b]naphtho[2,1,d]thiophene ([2,1]BNT), by F344 rat liver 9000g supernatant (S-9) was studied. [2,1]BNT which is structurally analogous and has similar carcinogenic potency to chrysene, was metabolized to six ethyl acetate-extractable metabolites when incubated with S-9 from Aroclor 1254-treated F344 rats. Each metabolite was collected from reverse-phase HPLC, and their identities were determined by analysis of MS and NMR data. In order of elution from HPLC they are as follows: (1) trans-1,2-dihydroxy-1,2-dihydrobenzo[b]naphtho[2,1-d]thiophene, (2) benzo[b]naphtho[2,1-d]-thiophene sulfone, (3) benzo[b]naphtho[2,1-d]thiophene sulfoxide, (4) trans-3,4-dihydroxy-3,4-dihydrobenzo[b]naphtho[2,1-d]thiophene, (5) 8- or 9-hydroxybenzo[b]naphtho[2,1-d]thiophene, and (6) 7-hydroxybenzo[b]naphtho[2,1-d]thiophene. In addition, the identities of metabolites 1, 2, 3, and 4 were confirmed by comparison to standards. The syntheses of the sulfone and sulfoxide of [2,1]BNT are reported here. The syntheses of the dihydrodiols were reported previously. Metabolite 5, a hydroxy[2,1]BNT, was the major metabolite formed by liver S-9 from untreated F344 rats. Microsomal preparations from these rats also produced significant amounts of the dihydrodiols, 1 and 4, and the sulfoxide, 3. Microsomes prepared from Wistar rats produced dihydrodiols and the sulfone and sulfoxide of [2,1]BNT. Therefore, [2,1]BNT is metabolized by both ring oxidation and sulfur oxidation in these two strains of rats.

Animals↗

Phenolic compounds in a Sahelian sorghum (Sorghum bicolor) genotype (CE145-66) and associated soils.

CE145-66 is an improved early-maturing grain sorghum genotype, increasingly grown by farmers in the Sahelian part of Senegal. This genotype is known to have negative effects on the following groundnut crop, because of the release of allelopathic phenolic compounds into the soil. We have assessed the synthesis of phenolics in sorghum vegetative parts and the variations in synthesis between years and sites. Total phenols and phenolic acids in the aerial parts and roots of flowering sorghum plants from 52 farmers' fields at two sites (Sagnanème and Médina) in Senegal in 1996 and 1997 were measured. Thirty-eight soil samples, collected after the sorghum harvest, from sorghum rows and interrows also were analyzed for their phenolic content. Total phenols reached 1.1-1.5% of root dry weight and 1.1-2.2% of aerial parts dry weight, with little variation between sites, and large variability between years, presumably due to climatic conditions. Eight phenolic acids and three associated aldehydes were identified by HPLC, with p-hydroxybenzoic, p-coumaric, and ferulic acids the most abundant. Their totals reached 2.9-3.2 mg/g in 1996 and 2.6-2.8 mg/g in 1997 for the aerial part; and 3.3-3.6 mg/g in 1996 and 2.8-3.3 mg/g in 1997 for roots. In soils under sorghum rows, the mean water-soluble total phenols increased from 4.6 in 1997 to 6.7 micrograms/g in 1998 in Sagnanème, and from 3.8 in 1997 to 5 micrograms/g in 1998 in Médina. The concentrations of total phenols and phenolic acids were higher in rows than in interrows. All the phenolic monomers identified in vegetative parts were recovered in associated soil samples, with vanillic and p-hydroxybenzoic acids the most abundant. Finally, variability in plant phenolic content seemed more due to climatic than to cropping or soil factors, as differences between years appear more important than differences between or within sites.

Flavonoids↗