Effects of coffee components on the response of GABA(A) receptors expressed in Xenopus oocytes

The effects of both coffee components and coffee extract on the electrical responses of GABA(A) receptors expressed in Xenopus oocytes were studied by injecting cRNAs of the alpha(1) and beta(1) subunits of the bovine receptors. The aqueous extract of coffee dose-dependently inhibited the GABA-elicited responses, whereas the lipophilic extract of coffee by diethyl ether slightly potentiated it at low doses (0.1-0.4 microL/mL) but showed inhibition at high doses (0.5-0.8 microL/mL). Theophylline inhibited the response in a noncompetitive mechanism (K(i) = 0.55 mM), whereas theobromine and trigonelline hydrochloride inhibited it in a competitive manner, K(i) = 3.8 and 13 mM, respectively. Benzothiazole, catechol, 2,4-dimethylstyrene, guaiacol, 1-octen-3-ol, sotolone, and 2,3,5-trimethylphenol potentiated the responses significantly. Potentiation elicited by guaiacol and sotolone was independent of GABA concentrations, whereas that by 1-octen-3-ol was dependent. When 1-octen-3-ol (100 mg/kg) was orally administered to mice prior to intraperitoneal administration of pentobarbital, the sleeping time of mice induced by pentobarbital increased significantly.     

Liquid chromatographic-fluorometric determination of cinnamyl alcohol in perfumes, colognes, and toilet waters

A liquid chromatographic (LC) method is described for the determination of cinnamyl alcohol (3-phenyl-2-propen-1-ol) in fragrance compositions. The fragrance product is partially cleaned up by diluting the fragrance with a 95% ethanol-water mixture and passing it through a short column containing RP-8 packing. An aliquot of the effluent is then analyzed by LC using an RP-18 column interfaced to a spectrophotofluorometer equipped with double monochromators. The fluorescence emission intensity of the eluted cinnamyl alcohol is measured and compared with that of a standard to calculate the amount of cinnamyl alcohol present. Recoveries from fragrance products fortified with cinnamyl alcohol at levels ranging from 0.0020 to 0.060 mg/mL ranged from 85 to 105% with a mean of 94%. The lowest level of determination was 0.0005 mg/mL.     

Antioxidant constituents of almond [Prunus dulcis (Mill.) D.A. Webb] hulls

Almond hulls (Nonpareil variety) were extracted with methanol and analyzed by reversed phase HPLC with diode array detection. The extract contained 5-O-caffeoylquinic acid (chlorogenic acid), 4-O-caffeoylquinic acid (cryptochlorogenic acid), and 3-O-caffeoylquinic acid (neochlorogenic acid) in the ratio 79.5:14.8:5.7. The chlorogenic acid concentration of almond hulls was 42.52 +/- 4.50 mg/100 g of fresh weight (n = 4; moisture content = 11.39%). Extracts were tested for their ability to inhibit the oxidation of methyl linoleate at 40 degrees C. At an equivalent concentration (10 microg/1 g of methyl linoleate) almond hull extracts had higher antioxidant activity than alpha-tocopherol. At higher concentrations (50 microg/1 g of methyl linoleate) almond hull extracts showed increased antioxidant activity that was similar to chlorogenic acid and morin [2-(2,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-1-benzopyran-4-one] standards (at the same concentrations). These data indicate that almond hulls are a potential source of these dietary antioxidants. The sterols (3beta,22E)-stigmasta-5,22-dien-3-ol (stigmasterol) and (3beta)-stigmast-5-en-3-ol (beta-sitosterol) (18.9 mg and 16.0 mg/100 g of almond hull, respectively) were identified by GC-MS of the silylated almond hull extract.     

Sphingomonas sp. strain SB5 degrades carbofuran to a new metabolite by hydrolysis at the furanyl ring

Microorganisms capable of degrading carbofuran were isolated from soils and examined for the degradation of this pesticide at ring structure. An isolate that could degrade carbofuran and carbofuran-7-phenol was selected for further studies. The 16S rRNA analysis results showed that the isolate belongs to the genus of Sphingomonas, close to dioxin and dicamba degraders, and is named Sphingomonas sp. SB5. SB5 did not show any similarity of 16S rRNA to known carbofuran degraders. When time-course degradation of carbofuran by SB5 was examined by solvent extraction combined with liquid chromatographic analysis, almost complete disappearance of carbofuran was observed within 12 h, giving several accumulative metabolites. Bacterial cultures incubated with carbofuran-7-phenol suggested that the accumulated metabolites were derived from carbofuran-7-phenol. The control without SB5 and kanamycin-treated SB5 did not show any metabolite, suggesting a biological involvement in the degradation of carbofuran. GC/MS and LC/MS analyses identified 2-hydroxy-3-(3-methylpropan-2-ol) phenol as one of the accumulated metabolites, suggesting that the strain SB5 could degrade carbofuran-7-phenol by hydrolysis at the furanyl ring. This is the first report to identify 2-hydroxy-3-(3-methylpropan-2-ol) phenol as a new product derived biologically from carbofuran-7-phenol.     

Liquid chromatographic cleanup and determination of low levels of vitamin D3 in sheep plasma

A liquid chromatographic (LC) method is described for the determination of vitamin D3 in sheep plasma. Samples are extracted by one of 2 different methods, depending on the concentration of vitamin D3. The samples are purified by using either a Sep-Pak silica cartridge or a small alumina column, followed by additional cleanup on a Metalsorb LC column. Final analysis was carried out on a 5 micron C18 column using a radial compression separation system with an acetonitrile-methanol solvent system. Vitamin D3 was completely resolved from any interfering compounds in the plasma; total run time was less than 15 min, using a variable wavelength detector set at 264 nm. The method was successfully applied to samples at levels of 1-10 ng added vitamin D3 mL sheep plasma, with recoveries in the range 90-97%.     

Sulfur aroma precursor present in S-glutathione conjugate form: identification of S-3-(hexan-1-ol)-glutathione in must from Vitis vinifera L. cv. Sauvignon blanc

When Sauvignon blanc or Gros Manseng grape must was percolated through an immobilized gamma-glutamyltranspeptidase column, there was a significant increase in the concentration of S-3-(hexan-1-ol)-L-cysteine, the precursor of 3-mercaptohexan-1-ol, a compound that contributes to the varietal aroma of wines made from these grapes. Low- and high-resolution liquid secondary ion mass sepectrometry (LSIMS) analyses established the presence of S-3-(hexan-1-ol)-glutathione in Sauvignon blanc must. The identification of this compound suggests that the S-3-(hexan-1-ol)-L-cysteine in grapes is produced by the catabolism of S-3-(hexan-1-ol)-glutathione. As is the case in other plant or animal organisms, S-glutathione conjugates may be involved in certain detoxification systems in vines.     

3-Sulfanylhexanol precursor biogenesis in grapevine cells: the stimulating effect of Botrytis cinerea

Volatile thiols, compounds that contribute strongly to the varietal aroma, are present in much higher concentrations in sweet wines than in dry wines. This positive effect, due to the presence of Botrytis cinerea on the berries, in fact results from a strong enrichment of cysteine S-conjugate precursors in botrytized berries. In the present study, a convenient model was investigated to reproduce and therefore study this phenomenon. A Vitis vinifera cell culture was used as a simple model, and we focused on S-3-(hexan-1-ol)-l-cysteine (P-3SH), the cysteinylated precursor of 3-sulfanylhexanol. We demonstrated that grapevine cells were able to produce P-3SH and that the presence of B. cinerea considerably increased the precursor level (up to 1000-fold). This positive result was determined to be due to metabolites secreted by the fungus. These molecules were temperature sensitive, unstable over time, and their production was activated in the presence of grapevine cells. Moreover, part of the pathway leading to P-3SH was deciphered: it was directly derived from the cleavage of S-3-(hexan-1-ol)-l-glutathione, which itself was generated after a conjugation of glutathione on (E)-2-hexenal.     

Quantification of cysteine S-conjugate of 3-sulfanylhexan-1-ol in must and wine of petite arvine vine by stable isotope dilution analysis

Making use of a convenient synthetic approach to prepare the deuterated S-3-(hexan-1-ol)-cysteine by a Michael addition reaction, an analytical method was developed to measure the presence of the cysteine S-conjugate, precursor of 3-sulfanylhexan-1-ol (3-mercaptohexan-1-ol), in must and wine from Petite Arvine vine. The method uses a stable isotope dilution assay with a suitable one-step sample preparation and HPLC-MS detection. The method has limits of detection and quantification of 3 and 10 microg/L, respectively. A correlation between the increase of the precursor concentration and the increase of the degree of rot has been established.     

Ethylidene-bridged Flavan-3-ols in red wine and correlation with wine age

Condensed tannins are responsible for astringency and bitterness and participate in the color stability of red wines. During wine making and aging, they undergo chemical changes including, for example, acetaldehyde-induced polymerization. Following this study, the ethylidene-bridged flavan-3-ols were monitored in different vintage wines made from grapes collected in the same vineyard in three wineries in Bordeaux, Pauillac, and Saint Julien. Flavan-3-ol ethylidene bridges were quantified by wine 2,2'-ethylidenediphloroglucinol (EDP) phloroglucinolysis. This method was based upon the analysis of EDP, a product formed after acid-catalyzed cleavage of wine flavan-3-ols in the presence of excess phloroglucinol. The flavan-3-ol ethylidene bridges were then compared to flavan-3-ol contents (phloroglucinolysis), phenolic contents, and color measurements. Low amounts of flavan-3-ol ethylidene bridges (0.8-2.5 mg L(-1)) were quantified in wines. Flavan-3-ol ethylidene bridges represent less than 4% of flavan-3-ol bonds, but the proportion of these linkages relative to native interflavan bonds increased with wine age. This proportion correlated with pigmented polymers.     

Identification of cysteinylated aroma precursors of certain volatile thiols in passion fruit juice

Together with 3-mercaptohexan-1-ol and 3-mercaptohexyl acetate, already known to contribute to the aroma of passion fruit (Passiflora edulis), 3-mercapto-3-methylbutan-1-ol and 3-mercapto-3-methylbutyl acetate have been identified for the first time in this fruit. 3-Mercaptohexan-1-ol and 3-mercapto-3-methylbutan-1-ol may be produced in vitro from nonvolatile extracts of this fruit by the enzymatic action of a cell-free extract of Eubacterium limosum, which has a beta-lyase activity on S-cysteine conjugates (EC 4.4.1.13). This release strongly suggests that these volatile thiols are present in combined form, as S-cysteine conjugates. It was possible to identify the precursor of 3-mercaptohexan-1-ol as S-(3-hexan-1-ol)-L-cysteine, in the form of trimethylsilylated derivatives from the juice of this fruit, using GC/MS analysis. The presence of free and combined forms of these volatile thiols in this fruit has now been demonstrated.     

Occurrence of Odorant Polyfunctional Thiols in Beers Hopped with Different Cultivars. First Evidence of an S-Cysteine Conjugate in Hop ( Humulus lupulus L.)

Forty-one thiols, mainly β-sulfanylalkyl acetates, β-sulfanylalkyl alcohols, and β-sulfanylalkyl carbonyls, were recently evidenced in hop. In a beer hopped with the Tomahawk cultivar, most of them were found at higher levels than expected. The aim of the present work was to investigate the polyfunctional thiols in beers hopped with different varieties. A few thiols proved not to come only from hop (mainly 2-sulfanylethyl acetate, μg/L levels, and 1-sulfanylpentan-3-one and 1-sulfanylpentan-3-ol, ng/L levels, internal standard (IST) equivalents). The thiol profile of Saaz-hopped beer proved similar to that of the reference beer produced without hop. A high level of 3-sulfanyloctan-1-ol emerged as an indicator of the use of Tomahawk hop (140 ng/L, IST equivalents; FD (flavor dilution) = 65536). In both Cascade- and Tomahawk-hopped beers, 3-sulfanylhexan-1-ol and 3-sulfanylheptan-1-ol were smelled at high flavor dilutions, although only for the latter, significant amounts of the unreduced 3-sulfanylheptanal were found in hop. As already claimed for hop authentication, 3-sulfanyl-4-methylpentan-1-ol remains a good marker of Nelson Sauvin-hopped beers (548 ng/L, IST equivalents; FD = 65536), together with 4-sulfanyl-4-methylpentan-2-one (128 ng/L, FD = 4096). As illustrated by the huge production occurring during fermentation, accurate prediction of hop varietal impact requires quantitating thiol adducts in hop. S-3-(1-Hydroxyhexyl)cysteine was evidenced here for the first time in Cascade hop.     

Discrimination of olive oils and fruits into cultivars and maturity stages based on phenolic and volatile compounds

Olive oil and fruit samples from six cultivars sampled at four different maturity stages were discriminated into cultivars and maturity stages. The variables-volatile and phenolic compounds-that significantly (p < 0.01) discriminated cultivars and maturity stage groups were identified. Separation by stepwise linear discriminant analysis revealed that Manzanilla olive cultivar was separated from cultivars Leccino, Barnea, Mission, Corregiola, and Paragon, whereas cultivars Corregiola and Paragon formed a cluster. The volatile compounds hexanol, hexanal, and 1-penten-3-ol were responsible for the discrimination of cultivars. All maturity stages were discriminated, with the separation of early stages attributed to oil phenolic compounds, tyrosol and oleuropein derivatives, whereas the volatile compounds (E)-2-hexenal, hexanol, 1-penten-3-ol, and (Z)-2-penten-3-ol characterized the separation of all maturity stages and in particular the late stages. Hexanol and 1-penten-3-ol characterized the separation of both cultivars and maturity stages.     

Identification of potent odorants formed by autoxidation of arachidonic acid: structure elucidation and synthesis of (E,Z,Z)-2,4,7-tridecatrienal.

The aroma composition of autoxidized arachidonic acid was characterized by aroma extract dilution analysis. The most potent odorant was trans-4,5-epoxy-(E)-2-decenal followed by 1-octen-3-one, (E,Z)-2,4-decadienal, (E,Z,Z)-2,4,7-tridecatrienal, (E,E)-2,4-decadienal, and hexanal. (E,Z,Z)-2,4,7-Tridecatrienal was unequivocally identified by mass spectrometry and nuclear magnetic resonance (NMR) data. The stereochemistry of its extended double-bond system was elucidated on the basis of NMR measurements. The target compound was synthesized in four steps starting with bromination of 2-octyn-1-ol, followed by copper-catalyzed coupling of the bromide with ethylmagnesium bromide and (E)-2-penten-4-yn-1-ol. Partial hydrogenation of the resulting C(13)-compound with triple bonds in the positions C-4 and C-7 gave rise to (E,Z,Z)-2,4,7-tridecatrien-1-ol, which was finally oxidized to the target compound. It exhibits a typical egg-white-like, marine-like odor at low concentrations, and an intense orange-citrus, animal-like odor at higher concentrations. Its odor threshold was estimated by gas chromatography-olfactometry to be 0.07 ng/L air, which is of the same order of magnitude as that reported for 1-octen-3-one and (E,E)-2,4-decadienal.     

Determination of xanthomegnin in grains and animal feeds by liquid chromatography with electrochemical detection

A sensitive, highly selective liquid chromatographic (LC) method is described which uses electrochemical (EC) reduction of the analyte in the determinative step. The method is capable of determining xanthomegnin in mixed animal feeds and grains at levels ranging from 15 to 1200 ng/g. The method can detect as little as 0.5 ng xanthomegnin injected on the LC column. Xanthomegnin is extracted with chloroform and 0.1M phosphoric acid. An aliquot of the crude extract is purified by silica gel column chromatography using a Sep-Pak silica gel cartridge. A novel feature of the method is that xanthomegnin is "backed off" the column by reversing the flow of the eluant through the column. LC is then used to separate xanthomegnin from other interfering substances. Xanthomegnin is detected by EC reduction at -0.16 V. Recoveries of xanthomegnin added to samples at levels ranging from 15 to 1200 ng/g averaged 79% with a coefficient of variation of 7.9%. Results also demonstrate that this LC system can separate the related metabolites viomellein and rubrosulphin from each other and from xanthomegnin and that the same EC detection system can be used to detect these metabolites.     

Variation of major volatile constituents in various green teas from Southeast Asia

A total of 15 green tea samples were prepared from fresh tea leaves obtained from three different countries: two from Laos, seven from Myanmar, and six from Vietnam. The volatile aroma constituents of the 15 samples were analyzed by gas chromatography/mass spectroscopy. Eleven aroma constituents were chosen from over 100 chemicals found in the samples to compare differences among various teas. They were hexanal, 1-penten-3-ol, heptanal, 1-pentenal, (Z)-2-penten-1-ol, (Z)-3-penten-1-ol, linalool oxide (trans-furanoid), linalool oxide (cis-furanoid), linalool, linalyl propanoate, and geraniol. Generally, concentrations of linalool and hexanal seem to play an important role in the quality of green teas. Green teas from Laos and Myanmar contained heterocyclic compounds, such as pyridines and pyrazines, formed by high-temperature processing. The presence of these heterocyclic compounds suggested that the temperature used for tea processing plays an important role in the formation of aroma chemicals in green teas.     

Liquid chromatographic determination of morphine sulfate and some contaminants in injections and bulk drug material

A liquid chromatographic (LC) procedure is described for the assay of morphine sulfate in bulk drug material and injection solutions. The bulk drug and injection samples are prepared by direct dilution with LC mobile solvent. The average bulk drug purity (5 manufacturers) determined by the LC method was 99.9% with a difference of 0.1% from the average purity (anhydrous) found by the official USP XX procedure. The average LC recovery (19 studies) of morphine sulfate added to injection samples was 99.4% with a coefficient of variation (CV) of 1.14%. Morphine sulfate content was determined in triplicate for 53 injection samples (1-15 mg morphine sulfate/mL) formulated by 6 manufacturers, using the proposed LC procedure. Individual sample CV (n = 3) averaged 1.14%. The LC method is simple and specific for morphine sulfate. Major degradation products, preservatives, and some contaminants and related compounds are separated during LC.     

Liquid chromatographic determination of tenuazonic acid and alternariol methyl ether in tomatoes and tomato products

A liquid chromatographic (LC) method for determining tenuazonic acid (TA) and alternariol methyl ether (AME) in tomatoes and tomato products is described. The Alternaria metabolites are extracted from a water slurry of the sample with CHCl3, the mixture is centrifuged, and the extract is fractionated on a silica gel column. Reverse phase LC with an ultraviolet detector (for TA) and a fluorescence detector (for AME) connected in series is used for final separation and determination. The limit of determination for TA and AME is 25 and 3 ng/g, respectively, with average recoveries from catsup of 83 and 68%, respectively. The LC method also detects alternariol, but interfering peaks in some samples prevent accurate quantitation. Chemical ionization mass spectrometry (CIMS) is used to confirm TA. Samples (142) of tomatoes collected from commercial processing lines were analyzed; TA was found in 73 samples (0.4-70 micrograms/g).     

Quantitation of the intense aroma compound 3-mercapto-2-methylpentan-1-ol in raw and processed onions (Allium cepa) of different origins and in other Allium varieties using a stable isotope dilution assay

A stable isotope dilution assay was developed for the quantitation of the potent onion odorant 3-mercapto-2-methylpentan-1-ol (1) using mass chromatography and synthesized [(2)H(2)]-3-mercapto-2-methylpentan-1-ol as the internal standard. Application of the newly developed method on onions from different origins revealed amounts between 8 and 32 microg/kg in raw onions, whereas 34-246 microg was found in sliced, stored (50 min), and then cooked onions. In extracts prepared by simultaneous steam distillation-extraction the highest concentrations of 1 were formed, amounting to >1200 microg/kg. The much higher content of 3-mercapto-2-methylpentan-1-ol in cooked onions suggested its formation from specific, yet unkown, precursors enzymatically formed during cutting of raw onions. 1 was for the first time identified and also quantified in other Allium species such as chives, scallions, and leek, whereas surprisingly garlic and bear's garlic did not contain the aroma compound.     

Comparison of odor-active compounds from six distinctly different rice flavor types

Using a dynamic headspace system with Tenax trap, GC-MS, GC-olfactometry (GC-O), and multivariate analysis, the aroma chemistry of six distinctly different rice flavor types (basmati, jasmine, two Korean japonica cultivars, black rice, and a nonaromatic rice) was analyzed. A total of 36 odorants from cooked samples were characterized by trained assessors. Twenty-five odorants had an intermediate or greater intensity (odor intensity >or= 3) and were considered to be major odor-active compounds. Their odor thresholds in air were determined using GC-O. 2-Acetyl-1-pyrroline (2-AP) had the lowest odor threshold (0.02 ng/L) followed by 11 aldehydes (ranging from 0.09 to 3.1 ng/L), guaiacol (1.5 ng/L), and 1-octen-3-ol (2.7 ng/L). On the basis of odor thresholds and odor activity values (OAVs), the importance of each major odor-active compound was assessed. OAVs for 2-AP, hexanal, ( E)-2-nonenal, octanal, heptanal, and nonanal comprised >97% of the relative proportion of OAVs from each rice flavor type, even though the relative proportion varied among samples. Thirteen odor-active compounds [2-AP, hexanal, ( E)-2-nonenal, octanal, heptanal, nonanal, 1-octen-3-ol, ( E)-2-octenal, ( E, E)-2,4-nonadienal, 2-heptanone, ( E, E)-2,4-decadienal, decanal, and guaiacol] among the six flavor types were the primary compounds explaining the differences in aroma. Multivariate analysis demonstrated that the individual rice flavor types could be separated and characterized using these compounds, which may be of potential use in rice-breeding programs focusing on flavor.     

Behavioral responses of the diamondback moth, Plutella xylostella, to green leaf volatiles of Brassica oleracea subsp. capitata

Green leaf volatiles (GLVs) from Brassica oleracea subsp. capitata L. have been identified as 1-hexanol, (Z)-3-hexen-1-ol, 1-hexen-3-ol, hexanal, (E)-2-hexenal, hexyl acetate, and (Z)-3-hexenyl acetate, by their mass spectra and retention times in comparison with authentic samples. No isothiocyanates were found in the extract. The activity of these chemicals has been determined on mated and unmated males and females of the diamondback moth (DBM) Plutella xylostella in the laboratory (wind tunnel) and in the field. On unmated males, mixtures of (Z)-3-hexenyl acetate, (E)-2-hexenal, and (Z)-3-hexen-1-ol with the pheromone induced attractant/arresting behavior in 80-100% of the males tested, significantly higher than the effect induced by the pheromone alone. On mated males and unmated females the effect of the GLVs alone or in combination with the pheromone was poor, while on mated females these compounds elicited upwind flight and arresting behavior in 40-60% of the females assayed. There was no synergism when these chemicals were mixed with the pheromone. In the field, (Z)-3-hexenyl acetate, the most active GLV in laboratory tests, when mixed with the pheromone in 1:1 ratio, enhanced 6-7-fold the number of females and 20-30% the number of males caught by traps over those baited with the pheromone alone. Our results indicate that the enhancement of the attraction of both males and females of the DBM to traps baited with pheromone blended with the relatively inexpensive and environmentally safe (Z)-3-hexenyl acetate may be important for future control strategies of the pest.