Sephadex LH-20 Gel Chromatography Research Articles

Antibacterial Polyketides from the Plant Endophytic Fungus Fusarium sp.

Background: Endophytic fungi have been recognized as new sources of natural products with a variety of biological activities, providing lead compounds for drug discovery and development. Objective: The objective of this study is to isolate and identify the secondary metabolites from the plant endophytic fungus Fusarium sp. HJY2 and evaluate their antibacterial activities. Methods: The compounds were isolated and purified by the methods of silica gel column chromatography, Sephadex LH-20 gel chromatography, and semi-preparative high performance liquid chromatography (HPLC). The structures of the isolated compounds were elucidated by comparing the NMR and MS spectroscopic data with those of pieces of literature. The antibacterial activities were evaluated by the broth microdilution method. Results: Seven polyketides were isolated from the fermented extracts of the fungus Fusarium sp. HJY2 and identified as sydowinol (1), dihydrolateropyrone (2), 13-oxo-9Z,11E-octadecadienoic acid (3), (E)-ferulic acid (4), 4-hydroxyphenylacetic acid (5), methyl 2-(2-hydroxyphenyl)acetate (6) and 4-hydroxy-3-methoxyacetophenone (7). Compound 3 exhibited moderate antibacterial activities against Bacillus subtilis, Mycobacterium smegmatis, Ralstonia solanacearum, and Xanthomonas campestris pv. campestris with MIC values of 40, 40, 80 and 40 μg/mL, respectively. Conclusion: Seven compounds were isolated from the plant endophytic fungus Fusarium sp. HJY2. Compound 1 was isolated from the Fusarium genus for the first time. Compound 3 showed moderate antibacterial activities.

Investigating the efficacy of purified tannin extracts from underutilized temperate forages in reducing enteric methane emissions in vitro

The study investigated how the concentration and composition of purified tannin extracts, at various inclusion rates, affect the ruminal in vitro fermentation parameters. Tannin extracts were isolated from four different forage species: birdsfoot trefoil (Lotus corniculatus), sulla (Hedysarum coronarium), big trefoil (Lotus pedunculatus), and salad burnet (Sanguisorba minor). Plants extracts were purified by Sephadex LH-20 gel chromatography and analyzed by UPLC–ESI–MS/MS. The results showed a large variation among the extracts from different species in terms of tannin composition and structural features. The extracts from salad burnet were dominated by hydrolysable tannins, comprising mainly ellagitannins. The extracts derived from sulla and big trefoil contained predominantly proanthocyanidins (PA), primarily composed of prodelphinidins with high mean degree of polymerisation (mDP). Birdsfoot trefoil extracts comprised procyanidin-rich PAs with low mDP. To determine whether the combined presence of tannins and flavonoid together lead to synergistic or antagonistic effects, the tannin extracts were incubated both with or without rutin at concentrations of 10, 20, and 30 g/kg DM, using a base substrate of perennial ryegrass (Lolium perenne, control). In general, all the tannin extracts decreased methane (CH4) production compared to the control, while no significant effect of rutin was observed on both gas (GP) and CH4 production, neither pure, nor in the simultaneous presence of tannins. The highest CH4 reduction (15%, at 30 g/kg DM) was observed from sulla and big trefoil extracts compared to control, but this was also supplemented with a concomitant reduction in GP (11%) indicating a reduction in feed digestibility. The extracts from birdsfoot trefoil and salad burnet reduced CH4 by up to 12% without significantly reducing GP, indicating the importance of tannin composition on ruminal fermentation.

Secondary Metabolites from the Mangrove-derived Fungus Trichoderma sp.

Background:: Secondary metabolites from the mangrove-derived fungi have great potential to produce natural products with novel structures and significant biological activities. Objective:: The objective of this study was to isolate and identify the secondary metabolites from the mangrove-derived fungus Trichoderma sp. WHUF0342, and evaluate their antibacterial activities. Methods:: The compounds were isolated and purified by silica gel column chromatography, Sephadex LH-20 gel chromatography, and semi-preparative high-performance liquid chromatography (HPLC). Their structures were elucidated by comparing the NMR and MS spectroscopic data with those of literature. The antibacterial activity were evaluated by modified broth microdilution assay. Results:: Eight compounds were isolated from the fermented extracts of the fungus Trichoderma sp. WHUF0342 and identified as chaxine B (1), nafuredin (2), dichlorodiaportin (3), ferulaic acid (4), bis(2-ethylhexyl) benzene-1,2-dicarboxylate (5), methyl 4-hydroxyphenylacetate (6), 4- hydroxyphenyl acetate (7), and 4-hydroxyphenylethyl acetate (8). Chaxine B (1) showed antibacterial activity against the phytopathogenic bacterium Ralstonia solanacearum with a minimum inhibitory concentration (MIC) value of 16 μg/mL. The antibacterial activity against plant pathogen of compound 1 was reported for the first time in this study. Conclusion:: This study not only enriched the secondary metabolites of mangrove-derived fungi but also provided a valuable resource for the prevention of agricultural pathogen infections.

Response surface methodology-optimized extraction of flavonoids from pomelo peels and isolation of naringin with antioxidant activities by Sephadex LH20 gel chromatography

In this study, flavonoids were extracted from pomelo peels and naringin was isolated from the flavonoid extract. The effects of extraction parameters, namely, ethanol concentration, solid-to-liquid ratio, and extraction time, on the yield of flavonoids extracted from pomelo peels were analyzed according to the Box-Behnken design of response surface methodology. The experimental conditions for flavonoid extraction were optimized, and naringin was separated from the extracted flavonoids using Sephadex LH-20 column chromatography. Experimental results showed that the influence of factors on the extraction rate of flavonoids from pomelo peels was in the order of ethanol concentration > solid-to-liquid ratio > extraction time, and the optimal extraction parameters were 85% ethanol concentration, 1:20 solid-to-liquid ratio, and 4-h extraction time for extracting flavonoids from pomelo peels. Under these conditions, the yield of flavonoids was 6.07 ± 0.06 mg/g. After three times of extraction, the flavonoid extraction rate reached 96.55%, and the residual naringin in the pomelo peels was 0.017 mg/g, at which point the bitterness in the pomelo peels disappeared. Two components, namely, PF1 and PF2, were separated from the crude flavonoid of pomelo peels through Sephadex LH20 column chromatography. PF2 was identified as naringin by high-performance liquid chromatography tandem mass spectrometry, with a purity of 95.7 ± 0.23%. Both flavonoids and PF2 exhibited good in vitro radicals scavenging activities on DPPH, ABTS, superoxide anion and hydroxyl.

Purification and characterization of a novel bacteriocin from Lactiplantibacillus plantarum Z057, and its antibacterial and antibiofilm activities against Vibrio parahaemolyticus

Vibrio parahaemolyticus and its biofilm threaten food safety and human health. Bacteriocins are known to inhibit various foodborne bacteria and their biofilms; however, few bacteriocins with antibiofilm activity against V. parahaemolyticus have been reported. Herein, a novel plantaricin Z057 from Lactiplantibacillus plantarum Z057 was extracted with ethyl acetate, purified by Sephadex LH-20 gel chromatography and reversed-phase high-performance liquid chromatography, and identified by nano-liquid chromatography coupled with tandem mass spectrometry. Its molecular mass and amino acid composition were 1272.61 Da and A-G-G-D-G-G-H-G-G-S-I-I-L-Y, respectively. Plantaricin Z057 presented a broad antibacterial spectrum against Gram-positive and Gram-negative pathogens. Moreover, it also exhibited excellent heat tolerance, maintained antibacterial activity at pH 2.0–4.0, and was susceptible to common proteases (pepsin, trypsin, papain, proteinase K). In addition, scanning electron microscopy revealed that plantaricin Z057 destroyed membrane integrity and caused cell deformation and cytoplasmic leakage. The crystal violet staining and XTT reduction assays revealed that plantaricin Z057 markedly decreased biofilm biomass and metabolic activity of V. parahemolyticus. Fluorescence microscopy showed that plantaricin Z057 efficiently inactivated sessile cells, and damaged integrated biofilm structure by reducing the extracellular polymer content. In summary, plantaricin Z057 exhibited significant potential for controlling V. parahaemolyticus and could be used in food production as a biological preservative.

Structural characterization and bioactivity of proanthocyanidins from indigenous cinnamon (Cinnamomum osmophloeum).

Barks and twigs of common species of cinnamon with abundant proanthocyanidins are used as a spice, fold medicine or supplement. Cinnamomum osmophloeum is an endemic species in Taiwan and coumarin was not detected in the oil of the C. osmophloeum twig. The present study aimed to evaluate the relationship between the bioactivities and proanthocyanidins of C. osmophloeum twig extracts (CoTE). The n-butanol soluble fraction from CoTE was divided into 10 subfractions (F1-F10) by Sephadex LH-20 gel chromatography. The antihyperglycemic activities were examined by α-glucosidase, α-amylase and protein tyrosine phosphatase 1B inhibitory assays. Total antioxidant activities were examined by 2,2-diphenyl-1-picrylhydrazyl free radical scavenging and ferrous ion-chelating assays. The results revealed that subfractions F6-F10, with high proanthocyanidin contents, showed excellent antihyperglycemic and antioxidant activities. Subfractions F6-F10 were analyzed further by matrix-assisted laser desorption/ionization-time of flight/mass spectrometry and thiolysis-reversed-phase high-performance liquid chromatography/tandem mass spectrometry methods. The results showed that the mean degrees of polymerization of proanthocyanidins in subfractions F6-F10 ranged from 3.5 to 5.1, with the highest degrees of polymerization of proanthocyanidins reaching 8 in subfractions F8-F10. Two compounds in F6 were identified as cinnamtannin B1 and parameritannin A1. These proanthocyanidins contained at least one A-type and major B-type linkages. These results demonstrate that proanthocyanidins are associated with their antihyperglycemic and antioxidant activities in CoTE. © 2016 Society of Chemical Industry.

Purification and Toxicity Study of a Saponin from Seeds of Albizia odorata , a Fabaceae from Madagascar

In order to continue the research of natural compounds of interest such as pesticides and therapeutic molecules in endemic species of Albizia from Madagascar, potentials of Albizia odorata seed extract were assessed. A toxic saponin (saponoside), named Albodorine, was isolated by extraction with hot ethanol or distilled water followed by purification procedure comprising n-butanol partition, precipitation by aceton-diethyl ether (50/50), Sephadex LH-20 gel chromatography and silica gel chromatography. All these methods were guided by toxicity tests on mice and homogeneity tests by Thin Layer Chromatography (TLC). Albodorine was thermostable, soluble in water and organic solvents and tasted bitter. Its acidic hydrolysis released glucose, arabinose and rhamnose. Tested on different experimental animal models, it was toxic to warm and cold blooded animals. In mouse, when intraperitoneally administered, it caused acute intoxication mainly presented as hyperpnea, ataxia and terminal seizures before the animal died. Its LD50 was about 9 mg/kg of mouse body weight by intraperitoneal route. In different organs, it caused histopathological lesions characterized by vascular congestions and important hemorrhage in liver, lungs and kidneys. In vitro, it reduced the heart rate and force of contraction of isolated rat atria. It had hemolytic activity. Albodorine showed toxicological properties that could be exploited under certain conditions for the control of harmful organisms.

Sulfated steroid – amino acid conjugate from the Irish marine sponge Polymastia boletiformis

Polymastia boletiformis, a brightly coloured orange-yellow fistulose sponge is widespread on the coasts of Britain and Ireland. Previous studies on this genus have yielded a number of interesting polyenes [1] and steroids. [2]. In the course of our on-going investigations within the frame of Beaufort Marine Biosdiscovery Research Programme directed to the biological screening and isolation of bioactive metabolites from Irish marine organisms [3], we have evaluated the antifungal activity of a number of Irish marine sponges against the plant pathogen Cladosporium cucumerinum. The crude extract of P. boletiformis, collected from the west coast of Ireland, showed activity against C. cucumerinum at 1 mg/mL. Antifungal activity-guided fractionation of the aqueous methanol-soluble portion of the crude extract by RP18 Flash CC, Sephadex LH-20 gel chromatography and RP18 HPLC led to the isolation of a new, minor sulfated steroid – amino acid conjugate (1). The structure of the new natural product was elucidated on the basis of extensive spectral data analysis, including 1D and 2D NMR experiments (COSY, NOESY, HSQC, HMBC) and HRMS. The assessment of antifungal activity of this metabolite and the identification of the absolute stereochemistry of the chiral centers on the side chain of the molecule is currently underway.

Isolation of angiotensin I-converting enzyme inhibitor from pepsin hydrolysate of porcine hemoglobin

Angiotensin I-converting enzyme (ACE) inhibitors have been widely used as antihypertensive agents. However, most synthetic ACE inhibitors ineluctably have severe side effects. Researchers have focused on various ACE-inhibitory peptides derived from dietary food. In the present study, we reported peptides produced from porcine blood, an important food in Asian countries. Through enzymatic hydrolysis, we found that peptides from this animal compound have ACE-inhibitory effects. Porcine hemoglobin was hydrolyzed using ordinary proteases, including alcalase, trypsin, neutral, papain, protamex, and pepsin. Results showed that pepsin was the most efficient protease in producing active peptides, and the pepsin hydrolysate of porcine hemoglobin showed the highest activity (IC50 = 1.53 ± 0.03 mg/mL). Combining DA 201-C macroporous resin chromatography, Sephadex LH-20 gel chromatography, and reversed-phase high-performance liquid chromatography, the fraction 2-IV was purified from pepsin hydrolysis of porcine hemoglobin; this compound exhibited the highest ACE-inhibitory activity (IC50 = 0.02 ± 0.01 mg/mL). Through Edman degradation, we also found that the exact amino acid sequence of fraction 2-IV was Gln–Glu–Leu–Pro–Gly. The results indicated that porcine hemoglobin peptides possessed significant ACE-inhibitory effect in vitro, which is an important complement of the previous work.

Antimicrobial activities of stearidonic and gamma-linolenic acids from the green seaweed Enteromorpha linza against several oral pathogenic bacteria.

BackgroundWe found that the edible green seaweed Enteromorpha linza displayed potent antimicrobial activity against Prevotella intermedia and Porphyromonas gingivalis. To elucidate the active component of E. linza, isolation procedures were performed.ResultsThe main active compound was isolated by polarity fractionation, Sephadex LH-20 gel chromatography, and reverse-phase high-performance liquid chromatography (RP-HPLC). The active compounds were eluted at isocratic 95% acetonitrile by RP-HPLC and identified as unsaturated fatty acids, stearidonic acid (SA, C18:4 n-3) and gamma-linolenic acid (GLA, C18:3 n-6) by gas chromatography–mass spectrometry, 1H nuclear magnetic resonance (NMR) spectroscopy, and 13C NMR spectroscopy. The yields of SA and GLA from dried seaweed tissue were 6.33 × 10-3% and 6.47 × 10-3%, respectively. The minimal inhibitory concentration values of SA and GLA were 39.06 μg/mL against P. intermedia and 9.76 μg/mL against P. gingivalis, respectively. SA and GLA were also active against several other oral pathogens, including Aggregatibacter actinomycetemcomitans, Candida albicans, Fusobacterium nucleatum subsp. vincenti, and Streptococcus mutans, at micromolar concentrations.ConclusionsThese data suggest that the E. linza extracts SA and GLA are useful antimicrobial agents for the prevention and/or treatment of periodontitis.Electronic supplementary materialThe online version of this article (doi:10.1186/1999-3110-54-39) contains supplementary material, which is available to authorized users.

Characterization of bioactive plant ellagitannins by chromatographic, spectroscopic and mass spectrometric methods

Ellagitannins are widely distributed plant polyphenols showing potent anti-herbivore activities, but rather complex chemical structures. Here we show how ellagitannins with different structures can be efficiently characterized from plant extracts by utilizing the information obtained from four universal methods used for ellagitannin analysis: Sephadex LH-20 gel chromatography, high-performance liquid chromatography, ultraviolet spectroscopy, and mass spectrometry. We show that by combining the information obtained from the ellagitannins’ chromatographic elution order, specific shapes of the ultraviolet spectra, molecular masses and characteristic mass spectral fragmentations, it is possible to classify these polyphenols into different ellagitannin sub-groups, and even characterize the structures within the sub-groups. These findings aid in the fast screening of complex plant samples for their ellagitannin composition without having to isolate compounds for individual characterization.

Polyphenols from Ginkgo biloba

By Sephadex LH-20 gel chromatography of an extract from Gingko biloba leaves, polymeric proanthocyanidins were eluted after the fractions of flavonol glycosides and biflavone glycosides. A purified proanthocyanidin polymer accounted for 86.6% of the total proanthocyanidins, and for 37.7% of the total antioxidant activity of this leaf extract. For structure elucidation, the polymer was submitted to acidic depolymerization in the presence of phloroglucinol. The structures of the resulting flavan-3-ols and phloroglucinol adducts were determined on the basis of 1D-and reverse 2D-NMR (HSQC, HMBC) spectra of their peracetylated derivatives, MALDI-TOF-MS and CD-spectroscopy. The observations resulting from the degradation with phloroglucinol were confirmed by 13C-NMR spectroscopy of the polymer. The mean molecular weight of the polymeric fraction was estimated to be 9–10 flavan-3-ol-units.

Identification and Quantification of Phenolic Compounds from the Forage Legume Sainfoin (Onobrychis viciifolia)

Phenolic compounds of sainfoin ( Onobrychis viciifolia ) variety Cotswold Common are assumed to contribute to its nutritive value and bioactive properties. A purified acetone/water extract was separated by Sephadex LH-20 gel chromatography. Sixty-three phenolic and other aromatic compounds were identified by means of chemical, chromatographic, and spectroscopic methods. Reverse phase HPLC with diode array and chemical reaction detection was used to investigate the phenolic composition of different plant organs. All plant parts showed specific phenolic profiles. Moreover, there were considerable variations in the phenolic content among individual plants of the same variety. The three most abundant phenolic compounds were found to be arbutin [predominant in petiols, 17.7 mg/g of dry weight (DW)], rutin (predominant in leaves, 19.9 mg/g of DW), and catechin (predominant flavanol in petiols, 3.5 mg/g of DW). The present study reveals that the phenolic profile of sainfoin is even more complex than hitherto assumed.

Isolation and characterization of angiotensin I-converting enzyme inhibitory peptides derived from porcine hemoglobin

Animal blood is potentially an untapped source of drugs and value-added food production. More than 400 million pigs are slaughtered each year but porcine blood is usually discarded in China. This study describes the isolation and characterization of angiotensin I-converting enzyme (ACE) inhibitory peptides derived from porcine hemoglobin. The most active hydrolysate was obtained from the peptic digestion of porcine hemoglobin. After the purification of ACE-inhibitory peptides with Sephadex LH-20 gel chromatography and reversed-phase high-performance liquid chromatography (RP-HPLC) on C 18 column, two active fractions were obtained. They were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) and electrospray ionization tandem mass spectrometry (ESI-MS/MS). They were LGFPTTKTYFPHF and VVYPWT, corresponding to the 34–46 fragment of the α chain and the 34–39 fragment of the β chain of porcine hemoglobin, with IC 50 values of 4.92 and 6.02 μM, respectively. They were the first found from porcine hemoglobin; in particular, LGFPTTKTYFPHF was a novel ACE-inhibitory peptide. In addition, the purified ACE inhibitors both competitively inhibited ACE, and maintained inhibitory activity even after incubation with gastrointestinal proteases. This suggests that these peptides might have a potential antihypertensive effect.

Antioxidant Oligomeric Proanthocyanidins from Sea Buckthorn (Hippophaë rhamnoides) Pomace

After flavonol glycosides, monomeric flavan-3-ols, and dimeric and trimeric proanthocyanidins were fractionated from an extract of sea buckthorn (Hippophae rhamnoides) pomace by Sephadex LH-20 gel chromatography, oligomeric proanthocyanidins were eluted. The oligomeric fraction accounted for 84% of the total proanthocyanidins and 75% of the total antioxidant activity of the sea buckthorn pomace extract. To elucidate the structure of the oligomeric fraction, it was depolymerized by acid catalysis in the presence of phloroglucinol. The structure of the resulting flavan-3-ol monomers and flavan-3-ol-phloroglucinol adducts was determined by electrospray ionization mass spectrometry (ESI-MS) and (1)H and (13)C nuclear magnetic resonance (NMR) spectroscopy. Quantitative high-performance liquid chromatography investigations demonstrated (+)-gallocatechin as the predominating subunit in the oligomeric fraction. This observation was confirmed by ESI-MS, matrix-assisted laser desorption/ionization mass spectrometry, and (13)C NMR spectroscopy. The results showed that the majority of the flavan-3-ol subunits possessed a 2,3-trans configuration. The oligomers consisted mainly of prodelphinidin subunits whereas procyanidins were present in smaller amounts, indicating a very uncommon composition of the sea buckthorn proanthocyanidins. The mean degree of polymerization of the oligomeric proanthocyanidins was between 6 and 9.

Antioxidant gallocatechins, dimeric and trimeric proanthocyanidins from sea buckthorn (Hippopha� rhamnoides) pomace

Residues such as peels and seeds that result from fruit juice production may contain substantial amounts of valuable natural antioxidants. In order to isolate the potential antioxidants gallocatechins and proanthocyanidins, pomace from sea buckthorn (Hippophae rhamnoides) berries was extracted by acetone-water (75:25, v/v) and fractionated by Sephadex LH-20 gel chromatography and semipreparative HPLC. The structures of the monomeric flavan-3-ols isolated, (+)-gallocatechin and (−)-epigallocatechin, were elucidated by electrospray mass spectrometry (ESI-MS) and 1H and 13C NMR spectroscopy. Five dimeric proanthocyanidins were identified by HPLC-ESI-MS(-MS) and by acid-catalyzed cleavage in the presence of phloroglucinol. Moreover, nine trimeric proanthocyanidins were tentatively identified by HPLC-ESI-MS(-MS) in the Sephadex fractions of sea buckthorn pomace. The isolated flavan-3-ols and proanthocyanidins were potent in scavenging Fremy’s salt, a synthetic free radical. They possessed antioxidant capacities that were higher or comparable to that of ascorbic acid or Trolox. On comparing the antioxidant capacities of monomeric flavan-3-ols and dimeric proanthocyanidins, no significant influence from the degree of polymerization (DP) was observed.

Structural investigations of flavonol glycosides from sea buckthorn (Hippophaë rhamnoides) pomace by NMR spectroscopy and HPLC-ESI-MS(n).

Four flavonol glycosides were isolated from an extract of sea buckthorn pomace (Hippophaë rhamnoides) by Sephadex LH-20 gel chromatography and semipreparative HPLC. Their structures were elucidated by hydrolysis studies, ESI-MS(n), UV, and (1)H and (13)C NMR spectroscopy. The occurrence of the major flavonol glycoside kaempferol 3-O-beta-sophoroside-7-O-alpha-rhamnoside in sea buckthorn is described here for the first time. A further 21 flavonol glycosides of Sephadex LH-20 fractions of sea buckthorn pomace were characterized by HPLC-DAD-ESI-MS. The characteristic MS-MS and MS(3) fragmentation pattern of flavonol glycosides previously identified in sea buckthorn juice and of flavonol glycosides identified by NMR spectroscopy gave valuable indications for their identification. The results demonstrate that loss of the sugar moiety from C-7 of the aglycon is more favored than fission of the glycosidic linkage at the C-3 position. Thus, most of the compounds identified were 7-rhamnosides of isorhamnetin, kaempferol, and quercetin, which exhibit different substitution patterns at the C-3 position, mainly glucosides, rutinosides, and sophorosides. In addition, numerous flavonol glycosides were detected lacking a sugar moiety at C-7. Finally, eight flavonol derivatives were identified that are acylated by hydroxybenzoic or hydoxycinnamic acids.

Use of the Zebra Mussel (Dreissena polymorpha) as a Bioindicator for Aromatic Hydrocarbons in Hamilton Harbour

Abstract Three methods for the extraction of polycyclic aromatic hydrocarbons (PAHs) from zebra mussels (Dreissena polymorpha) sampled from Hamilton Harbour were compared. Replicate freeze-dried mussel tissue samples were extracted using acid digestion, tissue homogenization (mechanical extraction) and ultra-sonication. Each extract was submitted to a cleanup procedure (alumina chro-matography and Sephadex LH20 gel chromatography), followed by analysis using gas chromatography-mass spectrometry (GC-MS). The three extraction methods were equally efficient, based on a statistical comparison of mean concentrations of individual PAHs. Mussel extracts, when subjected to bioassays with Salmonella typhimurium strain YG1029 (TA100-like) in the presence of an exogenous metabolic activation system (S9), exhibited significant mutagenic responses; these responses varied with the PAH content of the mussel extracts. Sources of PAHs in mussel extracts were determined by examining the profiles of sulfur-containing polycyclic aromatic compounds (thia-arenes). Comparison of the ratios of certain thia-arenes with ratios in source samples enabled identification of vehicular emissions and coal tar-contaminated sediment as two sources of PAH contamination in Hamilton Harbour.

Identification of polycyclic aromatic hydrocarbons in mutagenic adsorbates to a copper-phthalocyanine derivative recovered from municipal river water

A study was made to identify polycyclic aromatic hydrocarbons (PAHs) in the mutagenic adsorbate to blue cotton recovered from the water of the Katsura River which is a tributary of the Yodo River, a typical municipal river. As blue cotton bears a covalently bound copper-phthalocyanine derivative which can adsorb PAHs over 3 rings, PAHs in the adsorbate were separated into 4 fractions (I–IV) by Sephadex LH-20 gel chromatography. Fractions III and IV showed high direct and indirect frameshift mutagenicity in strains YG1021 and YG1024, the nitroreductase- and O-acetyltransferase-overproducing derivatives of TA98, especially in YG1024 with S9 mix, whereas these fractions showed less mutagenicity in TA98NR or TA98/1,8-DNP 6. These results suggest that mutagenic nitroarenes and aminoarenes are present in both fractions. The retention times of some peaks separated from both fractions using high performance liquid chromatography (HPLC) with a fluorescence detector were identical with those of authentic PAHs. Gas chromatography-mass spectrometry of some HPLC fractions demonstrated that anthraquinone, azulene derivative, quinoline derivative, chrysene and benzo[ b]fluoranthene are probably contained in these fractions.

Isolation, structure, and properties of the beta-carboline formed from 5-hydroxytryptamine by the superoxide anion-generating system.

A yellow substance was isolated by Sephadex LH-20 gel chromatography, silica gel TLC, and reversed-phase HPLC after incubation of 5-hydroxytryptamine (5-HT) with the superoxide anion (O2-)-generating system, i.e., the xanthine-xanthine oxidase system, in the presence of the Fe-EDTA complex and glycine in alkaline medium. The product gave a blue color with Ehrlich's reagent very slowly but no color with xanthydrol and Gibbs' reagent. Its reduced form, however, gave an immediate blue-violet color with all three reagents. No color was developed with ninhydrin, but the reduced form was orange-red. The chemical structure of the yellow substance was identified by 1H-nuclear magnetic resonance and field desorption-mass spectrometry as 4,9-dihydro-3H-pyrido[3,4-b]indol-6-ol (6-hydroxy-3,4-dihydro-beta-carboline, 5-hydroxy-2,3-dihydrotryptoline). The one carbon unit inserted into 5-HT came from glycine, with its 14C-2 being incorporated into C-1 of the yellow substance. The mechanism for the formation of the yellow substance from 5-HT is discussed. This compound inhibited 5-HT uptake into rat brain cortical synaptosomes with an IC50 of 1.5 X 10(-4) M and a Ki value of 1.2 X 10(-5) M.