Marine bacteria associated with marine macroorganisms: the potential antimicrobial resources

In order to explore marine microorganisms with medical potential, marine bacteria were isolated from seawater, sediment, marine invertebrates and seaweeds collected from different coastal areas of the China Sea. The antimicrobial activities of these bacteria were investigated. Ethyl acetate extracts of marine bacterial fermentation were screened for antimicrobial activities using the method of agar diffusion. The results showed that 42 strains of the isolates have antimicrobial activity. The proportion of active bacteria associated with marine invertebrates (20%) and seaweeds (11%) is higher than that isolated from seawater (7%) and sediment (5%). The active marine bacteria were assigned to the genera Alteromonas, Pseudomonas, Bacillus and Flavobacterium. The TLC autobiographic overlay assay implied that the antimicrobial metabolites produced by four strains with wide antimicrobial spectrum were different. Due to a competitive role for space and nutrient, the marine bacteria associated with marine macroorganisms (invertebrates and seaweeds) could produce more antibiotic substances. These marine bacteria were expected to be potential resources of natural antibiotic products.

A preliminary study on the mechanism of harmful algal bloom mitigation by use of sophorolipid treatment

in order to investigate a new method of mitigating the deleterious effect of harmful algal blooms (HABs), the inhibition of the glycolipid biosurfactant sophorolipid on three common harmful algae Alexandrium tamarense, Heterosigma akashiwo and Cochlodinium polykrikoides was studied. The optimum preparation condition for sophorolipid, the inhibition capability of sophorolipid and the interaction mechanism of sophorolipid on the three algal species were investigated. Results showed that sophorolipid prepared by extraction with ethyl acetate exhibited the most prominent inhibition effect and that storage time of one year had little influence on the inhibition effect of sophorolipid. The optimum concentration of 10-20 mg/l sophorolipid inhibited the motility of about 90% of the tested harmful algal cells without recovery. Investigation of the algicidal process revealed that sophorolipid induced ecdysis of A. tamarense, quick lysis of H. akashiwo and swelling of C. polykrikoides in a relatively short time. Investigation of the nucleotides showed that more than 15% of the nucleotides were released from the cytoplasm under the effect of 10-20 mg/l sophorolipid, indicating the irreversible damage on the cellular membrane, which resulted in the disintegration of the harmful algal cells. (C) 2004 Elsevier B.V. All rights reserved.

Bromophenols from the red alga Rhodomela confervoides

Six new bromophenols, 3-bromo-4,5-bis(2,3-dibromo-4,5-dihydroxybenzyl)pyrocatechol (1), 2,2',3-tribromo-3',4,4',5-tetrahydroxy-6'-hydroxymethyldiphenylmethane (2), 2,2',3-tribromo-3',4,4',5-tetrahydroxy-6'-ethyloxymethyldiphenylmethane (3),(+/-)-2-methyl-3-(2,3-dibromo-4,5-dihydroxyphenyl)propylaldehyde (4), (+/-)-2-methyl-3-(2,3-dibromo-4,5-dihydroxyphenyl)propylaldehyde dimethyl acetal (5), and 3-bromo-4,5-dihydroxybenzoic acid methyl ester (6), together with eight known bromophenols, 3-bromo-4,5-dihydroxybenzaldehyde (7), 2,3-dibromo-4,5-dihydroxybenzyl alcohol (lanosol, 8), 2,3-dibromo-4,5-dihydroxybenzyl methyl ether (9), 2,3-dibromo-4,5-dihydroxybenzyl ethyl ether (10), 2,3-dibromo-4,5-dihydroxybenzylaldehyde (11), bis(2,3-dibromo-4,5-dihydroxybenzyl) ether (12), 3-bromo-4-(2,3-dibromo-4,5-dihydroxybenzyl)-5-methoxymethylpyrocatechol (13), and 2,2',3,3'-tetrabromo-4,4',5,5'-tetrahydroxydiphenyl methane (14), were isolated from the red alga Rhodomela confervoides. Their structures were elucidated by chemical and spectroscopic methods including IR, HRFABMS, and 1D and 2D NMR techniques.

An efficient combination of supercritical fluid extraction and high-speed counter-current chromatography to extract and purify homoisoflavonoids from Ophiopogon japonicus (Thunb.) Ker-Gawler

Supercritical fluid extraction (SFE) was used to extract homoisoflavonoids from Ophiopogon japonicus (Thunb.) Ker-Gawler. The optimization of parameters was carried out using an orthogonal test L-9 (3)(4) including pressure, temperature, dynamic extraction time and the amount of modifier. The process was then scaled up by 100 times with a preparative SFE system under the optimized conditions of 25 MPa, 55 degrees C, 4.0 h and 25% methanol as a modifier. Then crude extracts were separated and purified by high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of n-hexane/ethyl acetate/methanol/ACN/water (1.8:1.0:1.0:1.2:1.0 v/v). There three homoisoflavonoidal compounds including methylophiopogonanone A 6-aldehydo-isoophiopogonone A, and 6-formyl-isoophiopogonanone A, were successfully isolated and purified in one step. The collected fractions were analyzed by HPLC. In each operation, 140 mg crude extracts was separated and yielded 15.3 mg of methylophiopogonanone A (96.9% purity), 4.1 mg of 6-aldehydo-isoophiopogonone A (98.3% purity) and 13.5 mg of 6-formyl-isoophiopogonanone A (97.3% purity) respectively. The chemical structure of the three homoisoflavonoids are identified by means of ESI-MS and NMR analysis.

A NOVEL BROMOPHENOL FROM MARINE RED ALGA Symphyocladia latiuscula

2,3,6-Tribromo-4,5-dihydroxybenzyl ethyl ether (1), a new bromophenol, was isolated from the ethanol extract of marine red alga Symphyocladia latiuscula, with a known compound, 2,3,6-tribromo-4,5-dihydroxybenzyl methyl ether ( 2). Their structures were elucidated by spectroscopic analysis, including high-resolution mass spectroscopy, and 1 and 2-dimensional NMR techniques. Compounds 1 and 2 showed inhibitory activity against Staphyloccocus aureus with IC50 102 and 50 mu g/mL, respectively.

One step isolation and purification of liquiritigenin and isoliquiritigenin from Glycyrrhiza uralensis Risch using high-speed counter-current chromatography

High-speed counter-current chromatography (HSCCC) technique in semi-preparative scale has been successfully applied to the separation of bioactive flavonoid compounds, liquiritigenin and isoliquiritigenin in one step from the crude extract of Glycyrrhiza uralensis Risch. The HSCCC was performed using a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-acetonitrile-water (2:2:1:0.6:2, v/v). Yields of liquiritigenin (98.9% purity) and isoliquiritigenin (98.3% purity) obtained were 0.52% and 0.32%. Chemical structures of the purified liquiritigenin and isoliquiritigenin were identified by electrospray ionization-MS (ESI-MS) and NMR analysis. (c) 2005 Published by Elsevier B.V.

Studies on synthesis, structure and biological activities of novel triazole compounds containing thioamide

Four novel triazole compounds containing thioamide group were designed and synthesized by using triazole, phenyl isothiocyanate and aryl ethyl ketone as raw material. Their structures were conformed by elemental analysis, H-1 NMR, IR and MS spectra. The crystal structure of 1-[1-anilinothiocarbonyl-1-(4-fluorobenzoyl)methyl]-1,2,4-trizole has been determined by X-ray diffraction analysis. The preliminary bioassays have shown that the title compounds exhibit certain antifungal activity.

Electrochemical, quantum chemical and SEM investigation of the inhibiting effect and mechanism of ciprofloxacin, norfloxacin and ofloxacin on the corrosion for mild steel in hydrochloric acid

The inhibiting effect and mechanism of 1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-piperazinyl)-3-quinoline carboxylicacid(ciprofloxacin), 1-ethyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-piperazinyl)-3-quinoline carboxylic acid (norfloxacin) and (-)-(S)-9-fluoro-2,3-dihydro-3-methyl-10-(4-methyl-1-piperazinyl)-7-oxo-7 H-pyrido(1,2,3-de)-1,4-benzoxazine-6 carboxylic acid (ofloxacin) on the corrosion of mild steel in 1 mol/L HCl have been studied using electrochemical method, quantum chemical method and SEM at 303 K. The potentiodynamic results showed that these compounds suppressed both cathodic and anodic processes of mild steel corrosion in 1 mol/L HCl. The impedance spectroscopy showed that R-p values increased, and C-dl values decreased with the rising of the working concentration. Quantum chemical calculation showed that there was a positive correlation between some inhibitors structure properties and the inhibitory efficiency. The inhibitors function through adsorption followed Langmuir isotherm, and chemisorption made more contribution to the adsorption of the inhibitors on the steel surface compared with physical adsorption. SEM analysis suggested that the metal had been protected from aggressive corrosion because of the addition of the inhibitors.

The inhibition of mild steel corrosion in 1 M hydrochloric acid solutions by triazole derivative

The title compound 1-(4,5-dihydro-3-phenylpyridine-1-yl)-2-(1H-1,2,4-triazole-1-yl)ethyl ketone (DTE) was synthesized and its inhibiting action on the corrosion of mild steel in 1 M hydrochloric acid solutions was investigated by means of weight loss, potentiodynamic polarization, electrochemical impedance spectroscopy (EIS) and scanning electronic microscope (SEM). Results obtained revealed that DIE performed excellently as a corrosion inhibitor for mild steel in 1 M hydrochloric acid media and its efficiency attains more than 90.9% at 1.0 x 10(-3) M at 298 K. Polarization curves indicated that the inhibitor behave mainly as mixed-type inhibitor. EIS showed that the charge transfer controls the corrosion process in the uninhibited and inhibited solutions. Adsorption of the inhibitor on the mild steel surface followed Langmuir adsorption isotherm. And the values of the free energy of adsorption Delta G(ads) indicated that the adsorption of DTE molecule was a spontaneous process and was typical of chemisorption. (c) 2008 Elsevier B.V. All rights reserved.

Simultaneous determination of monoamine and amino acid neurotransmitters in rat endbrain tissues by pre-column derivatization with high-performance liquid chromatographic fluorescence detection and mass spectrometric identification

A sensitive and efficient method for simultaneous determination of glutamic acid (Glu), gamma-amino-butyric acid (GABA), dopamine (DA), 5-hydroxytryptamine (5-HT) and 5-hydroxyindole acetic acid (5-HIAA) in rat endbrains was developed by high-performance liquid chromatography (HPLC) with fluorescence detection and on-line mass spectrometric identification following derivatization with 1,2-benzo-3,4-dihydrocarbazole-9-ethyl chloroformate (BCEOC). Different parameters which influenced derivatization and separation were optimized. The complete separation of five neurotransmitter (NT) derivatives was performed on a reversed-phase Hypersil BDS-C-18 column with a gradient elution. The rapid structure identification of five neurotransmitter derivatives was carried out by on-line mass spectrometry with electrospray ionization (ESI) source in positive ion mode, and the BCEOC-labeled derivatives were characterized by easy-to-interpret mass spectra. Stability of derivatives, repeatability, precision and accuracy were evaluated and the results were excellent for efficient HPLC analysis. The quantitative linear range of five neurotransmitters were 2.441-2 x 10(4) nM, and limits of detection were in the range of 0.398-1.258 nM (S/N = 3:1). The changes of their concentrations in endbrains of three rat groups were also studied using this HPLC fluorescence detection method. The results indicated that exhausting exercise could obviously influence the concentrations of neurotransmitters in rat endbrains. The established method exhibited excellent validity, high sensitivity and convenience, and provided a new technique for simultaneous analysis of monoamine and amino acid neurotransmitters in rat brain. (C) 2008 Elsevier B.V. All rights reserved.

Analysis of primary aromatic amines using precolumn derivatization by HPLC fluorescence detection and online MS identification

2-(2-Phenyl-1H-phenanthro-[9,10-d]imidazole-1-yl)-acetic acid (PPIA) and 2-(9-acridone)-acetic acid (AAA), two novel precolumn fluorescent derivatization reagents, have been developed and compared for analysis of primary aromatic amines by high performance liquid chromatographic fluorescence detection coupled with online mass spectrometric identification. PPIA and AAA react rapidly and smoothly with the aromatic amines on the basis of a condensation reaction using 1-ethyl-3-(3dimethylaminopropyl)-carbodiimide (EDC) as dehydrating catalyst to form stable derivatives with emission wavelengths at 380 and 440 nm, respectively. Taking six primary aromatic amines (aniline, 2-methylaniline, 2-methoxyaniline, 4-methylaniline, 4-chloroaniline, and 4-bromoaniline) as testing compounds, derivatization conditions such as coupling reagent, basic catalyst, reaction temperature and time, reaction solvent, and fluorescent labeling reagent concentration have also been investigated. With the better PPIA method, chromatographic separation of derivatized aromatic amines exhibited a good baseline resolution on an RP column. At the same time, by online mass spectrometric identification with atmospheric pressure chemical ionization (APCI) source in positive ion mode, the PPIA-labeled derivatives were characterized by easy-to-interpret mass spectra due to the prominent protonated molecular ion m/z [M + H](+) and specific fragment ions (MS/MS) m/z 335 and 295. The linear range is 24.41 fmol-200.0 pmol with correlation coefficients in the range of 0.9996-0.9999, and detection limits of PPIA-labeled aromatic amines are 0.12-0.21 nmol/L (S/N = 3). Method repeatability, precision, and recovery were evaluated and the results were excellent for the efficient HPLC analysis. The most important argument, however, was the high sensitivity and ease-of-handling of the PPIA method. Preliminary experiments with wastewater samples collected from the waterspout of a paper mill and its nearby soil where pollution with aromatic amines may be expected show that the method is highly validated with little interference in the chromatogram.

LC determination of amino acids in rat plasma with fluorescence detection: Application to exercise physiology

An LC method for the determination of 20 amino acids (AAs), using 1,2-Benzo-3,4-dihydrocarbazole-9-ethyl chloroformate (BCEOC) as fluorescent labeling reagent, has been validated and applied for the analysis of AAs in rat plasma at three different states concerning exercise physiology. Identification of AA derivatives was carried out by LC-MS with electrospray ion (ESI), and the MS-MS cleavage mode of the representative tyrosine (Tyr) derivative was analyzed. Gradient elution on a Hypersil BDS C-18 column gave good separation of the derivatives. Excellent linear responses were observed and good compositional data could be obtained from as little as 50-200 mu L of plasma samples. The contents of 20 AAs in rat plasma of three groups (24 rats, group A: quiet state, group B: at exercising exhaust, group C: 12 h after exercising exhaust) exhibited evident difference corresponding to the physiological states. Facile BCEOC derivatization coupled with LC-FLD-ESI-MS analysis allowed the development of a highly sensitive method for the quantitative analysis of trace level of AAs from plasma or other biochemical samples.

Extraction of Nitraria tangutorum seed oil by supercritical carbon dioxide and determination of free fatty acids by HPLC/APC/IMS with fluorescence detection

The seed oil from Nitraria tangutorum samples was obtained by supercritical carbon dioxide extraction methods. The extraction parameters for this methodology, including pressure, temperature, particle size and extraction time, were optimized. The free fatty acids in the seed oil were separated with a pre-column derivation method and 1,2-benzo-3,4-dihydrocarbazole-9-ethyl-p-toluenesulfonate (BDETS) as a labeling regent, followed by high-performance liquid chromatography (HPLC) with fluorescence detection. The target compounds were identified by mass spectrometry with atmospheric pressure chemical ionization (APCI in positive-ion mode). HPLC analysis shows that the main compositions of the seed oil samples were free fatty acids (FFAs) in high to low concentrations as follows: linoleic acid, oleic acid, hexadecanoic acid and octadecanoic acid. The assay detection limits (at signal-to-noise of 3:1) were 3.378-6.572 nmol/L. Excellent linear responses were observed, with correlation coefficients greater than 0.999. The facile BDETS derivatization coupled with mass spectrometry detection allowed the development of a highly sensitive method for analyzing free fatty acids in seed oil by supercritical CO2 extraction. The established method is highly efficient for seed oil extraction and extremely sensitive for fatty acid profile determination. (C) 2007 Elsevier B.V. All rights reserved.

Determination of free fatty acids in bryophyte plants and soil by HPLC with fluorescence detection and identification by online MS

A method for the determination of long and short chain free fatty acids (FFAs), using 1-[2-(ptoluenesulfonate)-ethyll-2-phenylimidazole-[4,5-f-9,10-phenanthrene (TSPP) as labeling reagent, has been developed. Identification of FFA derivatives was carried out by HPLC-MS with atmospheric pressure chemical ionization (APCI) in positive ion mode. Gradient elution on an Agilent Eclipse XDB-C-8 column gave good separation of the derivatives. Excellent linear responses were observed and good compositional data could be obtained from as little as 200 mg of bryophyte plants and soil samples. Facile TSPP derivatization coupled with HPLC-APCI-MS analysis allowed the development of a highly sensitive method for the quantitative analysis of trace level of FFAs from biological and natural environmental samples.

Determination of aliphatic amines from soil and wastewater of a paper mill by pre-column derivatization using HPLC and tandem mass spectrometry (HPLC-MS/MS)

A pre-column derivatization method for the sensitive determination of aliphatic amines using the labeling reagent 1,2-benzo-3,4-dihydrocarbazole-9-ethyl chloroformate (BCEOC) followed by HPLC with fluorescence detection and APCI/NIS identification in positive-ion mode has been developed. The chromophore of 2-(9-carbazole)-ethyl chloroformate (CEOC) reagent was replaced by the 1,2-benzo-3,4-dihydrocarbazole functional group, which resulted in a sensitive fluorescence derivatizing reagent, BCEOC, that could easily and quickly label amines. Derivatives were stable enough to be efficiently analyzed by HPLC and showed an intense protonated molecular ion corresponding m/z [M + H](+) with APCI/MS in positive-ion mode. The collision induced dissociation of the protonated molecular ion formed characteristic fragment ions at m/z 264.1, m/z 246.0 and m/z 218.1, corresponding to the cleavages of CH2CH2O-CO, CH2CH2-OCO, and N-CH2CH2O bonds. Studies on derivatization conditions demonstrated that excellent derivatization yields close to 100% were observed with a 3 to 4-fold molar reagent excess in acetonitrile solvent, in the presence of borate buffer (pH 9.0) at 40 degrees C for 10 min. In addition, the detection responses for BCEOC derivatives were compared with those obtained with CEOC and FMOC as labeling reagents. The ratios I-BCEOC/I-CEOC and I-BCEOC/I-FMOC were, respectively, 1.40-2.76 and 1.36-2.92 for fluorescence responses (here, I was the relative fluorescence intensity). Separation of the amine derivatives had been optimized on an Eclipse XDB-C-8 column. Detection limits calculated from an 0.10 pmol injection, at a signal-to-noise ratio of 3, were 18.65-38.82 fmol (injection volume 10 mu L for fluorescence detection. The relative standard deviations for intraday determination (n = 6) of standard amine derivatives (50 pmol) were 0.0063-0.037% for retention times and 3.36-6.93% for peak areas. The mean intra-and inter-assay precision for all amines were <5.4% and 5.8%, respectively. The recoveries of amines ranged from 96 to 113%. Excellent linear responses were observed with correlation coefficients of >0.9994. The established method provided a simple and highly sensitive technique for the quantitative analysis of trace amounts of aliphatic amines from biological and natural environmental samples.