Precursors and formation of pyrithione and other pyridyl-containing sulfur compounds in drumstick onion, Allium stipitatum

Two novel, structurally unusual cysteine derivatives were isolated from the bulbs of Allium stipitatum (Allium subg. Melanocrommyum) and shown to be S-(2-pyridyl)cysteine N-oxide and S-(2-pyridyl)glutathione N-oxide. The former compound is the first example of a naturally occurring alliinase substrate that contains an N-oxide functionality instead of the S-oxide group. In addition, S-methylcysteine S-oxide (methiin) and S-(methylthiomethyl)cysteine 4-oxide (marasmin) were found in the bulbs. Presented data suggest that the previously reported identification of S-(2-pyridyl)cysteine S-oxide was most likely erroneous. The alliinase-mediated formation of pyridyl-containing compounds following disruption of A. stipitatum bulbs was studied by a combination of HPLC-MS, HPLC-PDA, DART-MS, and NMR techniques. It was found that no pyridyl-containing thiosulfinates are present in homogenized bulbs in detectable quantities. Instead, various pyridine N-oxide derivatives are formed, including N-hydroxypyridine-2(1H)-thione (pyrithione), 2-(methyldithio)pyridine N-oxide, 2-[(methylthio)methyldithio]pyridine N-oxide, di(2-pyridyl) disulfide N-oxide, and di(2-pyridyl) disulfide N,N'-dioxide. This represents the first report of pyrithione formation as a natural product.     

Isolation and identification of a red pigment from Allium subgenus Melanocrommyum

Three new sulfur-containing compounds were identified in Allium L. species belonging to the subgenus Melanocrommyum as the first examples of sulfur-containing pyrrole derivatives in nature. Some of these species are traditionally used in Southwest and Central Asia as vegetables and herbal drugs. A hypothetical biogenetic scheme is proposed in which L-(+)- S-(3-pyrrolyl)cysteine sulfoxide is enzymically degraded. The resulting 2-lactyl-3'-pyrrolyl sulfoxide is condensed readily to the red pigment 3,3'-dithio-2,2'-dipyrrole. All compounds are chemically unstable, rendering the analysis extremely difficult. Correlation NMR in combination with diffusion NMR allowed the identification of these low molecular weight compounds. For the first time, the compounds involved in the coloring process of Allium plant material have been identified from native plant material.     

Allium discoloration: the precursor and formation of the red pigment in giant onion (Allium giganteum Regel) and some other subgenus Melanocrommyum species

The precursor of the orange-red pigment formed upon wounding the bulbs of Allium giganteum (Allium subg. Melanocrommyum) was isolated and shown to be S-(2-pyrrolyl)cysteine S-oxide. In addition, two other pyrrolylsulfinyl derivatives were found in an extract from the bulbs, namely, 3-(2-pyrrolylsulfinyl)lactic acid and S-(3-pyrrolyl)cysteine S-oxide. Contrary to a previous report, the latter compound was shown not to serve as the precursor of the pigment, being in fact only an artifact formed during isolation. The formation of pyrrolyl-containing compounds following disruption of A. giganteum bulbs was studied by a combination of LC-MS, LC-NMR and DART-MS. It was found that S-(2-pyrrolyl)cysteine S-oxide is cleaved by a C-S lyase (alliinase) to yield 2-pyrrolesulfenic acid. Two molecules of the latter compound give rise to highly reactive S-(2-pyrrolyl) 2-pyrrolethiosulfinate which in turn converts into red 2,2'-epidithio-3,3'-dipyrrole (dipyrrolo[2,3-d:2',3'-e]-1,2-dithiin). Several other pyrrolyl-containing compounds were detected in A. giganteum for the first time, including S-methyl 2-pyrrolethiosulfinate, S-(2-pyrrolyl) methanethiosulfinate, di(2-pyrrolyl) disulfide, and S-(2-pyrrolyl) 2-pyrrolethiosulfonate. It can be concluded that the formation of the orange-red pigment in Allium subg. Melanocrommyum species, despite sharing several analogous features, is of a different nature than the pink discoloration of onion (A. cepa).     

Characterization of some Allium hybrids by aroma precursors, aroma profiles, and alliinase activity

Various Allium hybrids, obtained by the crossbreeding of Allium cepa (onion) as the mother plant and six taxonomically distant wild species obtained by embryo rescue, were investigated with special respect to their individual profiles of cysteine sulfoxides as well as enzymically and nonenzymically formed aroma substances. Alliinase (EC 4.4.1.4) catalyzes the conversion of odorless (+)-S-alk(en)yl-L-cysteine sulfoxides into volatile thiosulfinates. These thiosulfinates were converted to a variety of sulfides by steam distillation. SPME-gas chromatography (GC) and high-performance liquid chromatography (HPLC) used for the analysis of aroma components and their precursors permitted a high sample throughput, so that numerous gene bank accessions and Allium breeding materials were analyzed within a comparatively short time. Cysteine sulfoxides as well as alliinase activity were found in all investigated samples at different levels, but (+)-S-methyl-L-cysteine sulfoxide (methiin) was the most abundant sulfoxide present. (+)-S-(trans-1-Propenyl)-L-cysteine sulfoxide (isoalliin) is typical for onion and was found in all investigated hybrids. The pattern of the other cysteine sulfoxides depended strongly on the parent plants used. The profile of aroma components corresponded with the related pattern of aroma precursors (cysteine sulfoxides). Successful hybridization was proven by randomly amplified polymorphic DNA analysis. Together with these established marker techniques, HPLC and SPME-GC analysis provide support to breeding projects designed to improve the health and aroma properties of Allium hybrids.     

Distribution of S-Alk(en)ylcysteine sulfoxides in some Allium species. Identification Of a new flavor precursor: S-ethylcysteine sulfoxide (Ethiin)

The content of S-alk(en)ylcysteine sulfoxides, important nonvolatile flavor precursors, was determined in 15 different Allium species by means of gas chromatography. The method employed is based on derivatization of S-alk(en)ylcysteine sulfoxides with ethyl chloroformate followed by their reduction with sodium iodide. The total content of S-alk(en)ylcysteine sulfoxides varied considerably in the wide range between 0.02 and 1.3% fresh weight. Not only the total content but also relative proportions of individual derivatives varied to a great extent. A novel S-alkylcysteine derivative, S-ethylcysteine sulfoxide (ethiin), not previously reported to occur in Allium species, was found in most of the samples examined in trace amounts. None of the other S-alk(en)ylcysteine sulfoxides, for example, isopropyl, (Z)-1-propenyl, butyl, or pentyl, were detected in any of the samples analyzed, limiting possible levels of each of these components to 相似文献   

Cysteine sulfoxides and alliinase activity of some Allium species

The flavor precursors of 17 species belonging to the Alliaceae family were analyzed by HPLC, and results were evaluated with respect to the classification of species into their genus, subgenus, and section. Identification and quantification of these precursors were carried out by synthetic and natural reference materials. In addition, nine of these species were investigated in terms of their alliinase activity. Alliinase (EC 4.4.1.4) catalyzes the conversion of odorless (+)-S-alk(en)yl-L-cysteine sulfoxides into volatile thiosulfinates. Cysteine sulfoxides as well as alliinase activity were found in all investigated samples, and (+)-S-methyl-L-cysteine sulfoxide was most abundant. (+)-S-Propyl-L-cysteine sulfoxide was detected in only a few, not closely related, species. Analysis of the crude protein extract of nine species gave evidence that alliinase activities of samples were similar in terms of pH and temperature optimum, K(M) value, and substrate specificity. For all investigated protein extracts, the highest specific alliinase activity was found for (+)-S-(2-propenyl)-L-cysteine sulfoxide (alliin). The substrate specificity of these enzymes was not related to relative abundance of the cysteine sulfoxides. However, SDS-PAGE yielded some significant differences among species in terms of their total protein compositions. Species belonging to different subgenera exhibited a specific protein pattern with molecular masses between 13 and 35 kDa.     

Quantitative determination of allicin in garlic: supercritical fluid extraction and standard addition of alliin

A quantitative method is described for the determination of allicin (2-propene-1-sulfinothioic acid S-2-propenyl ester) in garlic, using standard additions of alliin (l-(+)-S-allylcysteine sulfoxide) in conjunction with supercritical fluid extraction (SFE) and high performance liquid chromatography analysis with UV-vis absorbance detection. Optimum CO(2)-SFE conditions provided 96% recovery for allicin with precision of 3% (RSD) for repeat samples. The incorporation of an internal standard (allyl phenyl sulfone) in the SFE step resulted in a modest improvement in recovery (99%) and precision (2% RSD). Standard additions of alliin were converted to allicin in situ by endogenous alliinase (l-(+)-S-alk(en)ylcysteine sulfoxide lyase, EC 4.4.1.4). Complete conversion of the spiked alliin to allicin was achieved by making additions after homogenization-induced conversion of the naturally occurring cysteine sulfoxides to thiosulfinates had taken place, thus eliminating the likelihood of competing reactions. Concentration values for allicin determined in samples of fresh garlic (Allium sativum L. and Allium ampeloprasum) and commercially available garlic powders (Allium sativum L.) by standard addition of alliin were found in all cases to be in statistical agreement (95% confidence interval) with values determined using a secondary allicin standard (concentration determined using published extinction coefficients). This method provides a convenient alternative for assessing the amount of allicin present in fresh and powdered garlic, as alliin is a far more stable and commercially prevalent compound than allicin and is thus more amenable for use as a standard for routine analysis.     

Chemical characterization of Allium ursinum L. depending on harvesting time

Sulfur-containing compounds of ramson (Allium ursinum L.) are responsible for its traditional use in terms of culinary and medicinal purposes. Leaves and bulbs were investigated for their contents of cysteine sulfoxides (volatile precursors) as well as volatile compounds released from minced plant material. Plants were analyzed during the whole vegetation period, focused on the months from March to June. Additionally, within the dormancy period bulbs were analyzed again and alliinase activity was determined. The pattern of volatile compounds was analyzed both by SPME/GC-MS and by SDE/GC-MS. Compared to each other, SDE exhibited a wider spectrum of detectable volatile compounds. The quality and quantity of volatiles significantly depended on the time of harvest. The highest amounts of volatile precursors can be gained in March and April, shortly before flowering time (up to 0.4% of total cysteine sulfoxides). The main cysteine sulfoxides were alliin and isoalliin. It has been found that alliinase of A. ursinum exhibited properties similar to those of alliinase of garlic (Allium sativum L.), but differing in terms of substrate specificity.     

Chemical characterization of interspecific hybrids between Allium cepa L. and Allium kermesinum Rchb

Interspecific hybridization between wild and cultivated species of the genus Allium has been performed to generate plant material possessing biochemical properties of both parental plants. These cross-breeding experiments should lead to Allium plants with higher amounts of valuable constituents. The chemical characterization of interspecific hybrids between A. cepa and A. kermesinum is described on the basis of their sulfur-containing constituents and secondary metabolites. In addition, the hybrid character has been proven by random amplified polymorphic DNA (RAPD) analysis of the progenies obtained from the crosses. It has been shown that the distribution of the cysteine sulfoxides as well as the volatile secondary metabolites in the hybrids is not uniform. The profiles are mainly determined by the paternal wild species A. kermesinum. It has been ascertained that the gas chromatography profiles of the hybrids show increasing amounts of unsymmetrical substituted oligosulfides, which are known to be physiologically active substances. On the basis of statistical calculations, three different types of hybrids can be separated. The chemical analysis of cysteine sulfoxides and volatile sulfur-containing substances is shown to be a useful tool for breeding purposes as it allows an effective selection with regard to optimal distribution and amount of valuable constituents.     

Influence of Nitrogen and Sulfur Fertilization on the Alliin Content of Onions and Garlic

Abstract

Onion (Allium cepa L.) and garlic (Allium sativum L.) were among the earliest cultivated crops and have been popular in folk medicine for centuries. Alliins (cysteine sulfoxides) are the characteristic sulfur (S) containing secondary metabolites of Allium species like onions, shallot, garlic, leek and chives and they cause taste and sharpness and are criteria for the pharmaceutical quality. The influence of the S nutritional status on the content of secondary S containing metabolites was shown for different crops such as oilseed rape, mustard, nasturtium, and allium species. It was the aim of this study to investigate the influence of the S and nitrogen (N) supply on the alliin content of onion and garlic and to evaluate the significance for crop quality. In a greenhouse experiment three levels of N and S were applied in factorial combinations of 0, 50, and 250 mg pot^?1 S and 250, 500, and 1000 mg pot^?1 N. 8 plants were grown in a Mitscherlich pot containing 8 kg sand. Leaves and bulbs were sampled twice during the growth period in order to follow up translocation processes. The first sampling was carried out when leaves were developed, but bulb growth had not yet started and the second one during main bulb growth. An increasing S supply was related to an increasing alliin content in leaves and bulbs of both crops, whereas nitrogen fertilization had only a minor influence. The alliin content in bulbs could be doubled by S fertilization. A translocation of alliin from leaves to bulbs was found so that time of harvest has a strong influence on the alliin content. At the beginning of plant development high alliin contents were found in leaves, while with bulb development they were translocated into this plant organ. The results show that the potential health benefits of Allium species could be distinctly improved by S fertilization.     

Allium discoloration: precursors involved in onion pinking and garlic greening

Precursors involved in the formation of pink and green-blue pigments generated during onion and garlic processing, respectively, have been studied. It has been confirmed that the formations of both pigments are of very similar natures, with (E)-S-(1-propenyl)cysteine sulfoxide (isoalliin) serving as the primary precursor. Upon disruption of the tissue, isoalliin and other S-alk(en)ylcysteine sulfoxides are enzymatically cleaved, yielding 1-propenyl-containing thiosulfinates [CH3CH=CHS(O)SR; R = methyl, allyl, propyl, 1-propenyl] among others. The latter compounds have been shown to subsequently react with amino acids to produce the pigments. Whereas the propyl, 1-propenyl, and methyl derivatives form pink, pink-red, and magenta compounds, those containing the allyl group give rise to blue products after reacting with glycine at pH 5.0. The role of other thiosulfinates [RS(O)SR'] (R, R' = methyl, allyl, propyl) and (Z)-thiopropanal S-oxide (the onion lachrymatory principle) in the formation of the pigments is also discussed.     

Antioxidative activity of sulfur-containing compounds in Allium species for human low-density lipoprotein (LDL) oxidation in vitro

Sulfur-containing compounds contributing to health promotion in Allium species are produced via enzymic and thermochemical reactions. Sulfur-containing amino acids and volatile organosulfur compounds were prepared for an antioxidative assay. The inhibitory activity of S-alk(en)yl-l-cysteines and their sulfoxides, volatile alk(en)yl disulfides and trisulfides, and vinyldithiins in Allium species against lipid hydroperoxide (LOOH) formation in human low-density lipoprotein (LDL) was examined. It was elucidated that the alk(en)yl substituents (methyl, propyl, and allyl) and the number of sulfur atoms in the compounds were important for the antioxidative activity. 3,4-Dihydro-3-vinyl-1,2-dithiin, which is produced by a thermochemical reaction of allyl 2-propenethiosulfinate, exhibited the highest antioxidative activity of human LDL among sulfur-containing compounds.     

Effect of genetic characteristics and environmental factors on organosulfur compounds in garlic ( Allium sativum L.) grown in Andalusia, Spain

The content of organosulfur compounds was determined in selected garlic cultivars grown at four locations in Andalusia, Spain. The organosulfur compounds studied were three γ-glutamyl peptides, namely, γ-l-glutamyl-S-(2-propenyl)-l-cysteine (GSAC), γ-l-glutamyl-S-(trans-1-propenyl)-l-cysteine (GSPC), and γ-l-glutamyl-S-methyl-l-cysteine (GSMC), and four cysteine sulfoxides (alliin, isoalliin, methiin, and cycloalliin). There was a significant effect of the location, cultivar, and garlic ecotype on individual organosulfur compound contents. Purple-type cultivars showed on average the highest contents of GSMC, GSAC, alliin, and methiin but the lowest isoalliin content. The impact of genotype was relatively high for GSAC, whereas this factor hardly contributed to the total variability in alliin and isoalliin content. Planting date had a significant effect on the content of alliin and isoalliin. Discriminant analysis evidenced the ability of organosulfur compounds to distinguish among garlic bulbs from different locations or ecotypes with 81 or 86% accuracy, respectively.     

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.     

Synthesis of the individual diastereomers of the cysteine conjugate of 3-mercaptohexanol (3-MH)

The individual diastereoisomers of the cysteine conjugate of 3-mercaptohexanol (4) were synthesized with high isomeric purity (>98%). On treatment with Apotryptophanase enzyme, the 3R diastereoisomer of 4 gave an 82% yield of the R enantiomer of 1, with no trace of the 3S enantiomer present. Conversely, the 3S diastereoisomer of 4 gave the 3S enantiomer of 1 (43%) accompanied by a trace of the 3R form (S/R = 98.5:1.5), reflecting the diastereomeric purity of the cysteine conjugate. The same stereochemical outcome was observed when the individual diastereoisomers of 4 were added to fermentations with the Saccharomyces cerevisiae AWRI 1655 yeast strain, which gave 1 in 1% yield. A d(10)-analogue of 1 was synthesized and used as an internal standard to determine, by gas chromatography-mass spectrometry (GC-MS), the amounts of 1 formed in these transformations.     

Mercury Extraction from Contaminated Soils by l-Cysteine: Species Dependency and Transformation Processes

A new approach in soil remediation washing techniques is the use of l-cysteine based on the formation of organic complexes. In this study, the applicability of l-cysteine for the mobilisation of different mercury species from contaminated soils was evaluated. Soils were treated with l-cysteine solutions with S?CHg molar ratios of 1, 2, 10, 20, 100 and 200. In 24?h batch experiments, leachates with water could mobilise 1% of Hg. The addition of l-cysteine led to an increase of Hg mobilisation of 42% for soils with inorganically bound Hg. In column experiments, the maximum Hg removal rate was 75%. For soils with organically bound Hg or HgS, only 1?C5% of Hg was mobilised. Thus, the extraction of Hg from soils with l-cysteine is highly dependent on the Hg-binding form. Hg speciation analyses of leachates indicate that Hg?Cl-cysteine complexes are labile complexes which can be easily transformed. Soil samples speciation analysis revealed that reduction to elemental mercury takes place at low S?CHg ratios (1 to 10), assumingly by microbial activity. At higher S?CHg ratios of 10 and 100, precipitation of stable Hg?CS complexes could be observed. These species transformation processes are limitations for considering l-cysteine leaching as a remediation strategy.     

Nonvolatile S-alk(en)ylthio-L-cysteine derivatives in fresh onion (Allium cepa L. cultivar)

The L-cysteine derivatives (R)-2-amino-3-(methyldisulfanyl)propanoic acid (S-methylthio-L-cysteine), (R)-2-amino-3-(propyldisulfanyl)propanoic acid (S-propylthio-L-cysteine), (R)-2-amino-3-(1-propenyldisulfanyl)propanoic acid (S-(1-propenylthio)-L-cysteine), and (R)-2-amino-3-(2-propenyldisulfanyl)propanoic acid (S-allylthio-L-cysteine) were prepared from 3-[(methoxycarbonyl)dithio]-L-alanine, obtained from the reaction of L-cysteine with methoxycarbonylsulfenyl chloride. The occurrence of these S-(+)-alk(en)ylthio-L-cysteine derivatives in onion (Allium cepa L.) was proven by using UPLC-MS-ESI(+) in SRM mode. Their concentrations in fresh onion were estimated to be 0.19 mg/kg S-methylthio-L-cysteine, 0.01 mg/kg S-propylthio-L-cysteine, and 0.56 mg/kg (S-(1-propenyllthio)-L-cysteine, concentrations that are about 3000 times lower than that of isoalliin (S-(1-propenyl-S-oxo-L-cysteine). These compounds were treated with Fusobacterium nucleatum, a microorganism responsible for the formation of mouth malodor. These L-cysteine disulfides were demonstrated to predominantly produce tri- and tetrasulfides. Isoalliin is almost entirely consumed by the plant enzyme alliin lyase (EC 4.4.1.4 S-alk(en)yl-S-oxo-L-cysteine lyase) in a few seconds, but it is not transformed by F. nucleatum. This example of flavor modulation shows that the plant produces different precursors, leading to the formation of the same types of volatile sulfur compounds. Whereas the plant enzyme efficiently transforms S-alk(en)yl-S-oxo-L-cysteine, mouth bacteria are responsible for the transformation of S-alk(en)ylthio-L-cysteine.     

Model studies on precursor system generating blue pigment in onion and garlic

Reactions involved in blue-green discoloration in a mixture of onion (Allium cepa L.) and garlic (Allium sativum L.) were investigated. Vivid-blue color was successfully reproduced by using a defined model reaction system comprising only trans-(+)-S-(1-propenyl)-L-cysteine sulfoxide (1-PeCSO) from onion, S-allyl-L-cysteine sulfoxide (2-PeCSO) from garlic, purified alliinase (EC 4.4.1.4), and glycine (or some other amino acids). Four reaction steps identified and factors affecting the blue color formation were in good agreement with those suggested by earlier investigators. When crude onion alliinase was used in place of garlic alliinase, less pigment was formed. This result was explained by a difference in the amount of thiosulfinates, colorless intermediates termed color developers, yielded from 1-PeCSO by these enzymes.     

In vitro biogeneration of pure thiosulfinates and propanethial-S-oxide

A model reaction system was developed for generating pure thiosulfinates and propanethial-S-oxide (PTSO) using an isolated alliinase (EC 4.4.1.4) and isolated or synthetic alk(en)yl-L-cysteine sulfoxides (ACSO). Reaction yields ranged from 30 to 60% after 3 h at 21-23 degrees C, and organosulfur reaction products were extracted into CHCl3 to yield product preparations of controlled composition. A pure thiosulfinate or PTSO was derived from a single ACSO, and a preparation containing a mixture of four thiosulfinate species was derived from reaction mixtures employing binary ACSO substrate systems. Identities of homologous thiosulfinates and PTSO were confirmed by 1H NMR. This approach has the potential to be used as a preparative tool for yielding pure thiosulfinates and PTSO to facilitate the study of chemical and biological properties of this group of compounds or as a means to study the dynamics of organosulfur chemistry in preparations from Allium spp.     

Structure and bioactivity of thiosulfinates resulting from suppression of lachrymatory factor synthase in onion

In normal onion (Allium cepa), trans-S-1-propenyl-L-cysteine sulfoxide is transformed via 1-propenesulfenic acid into propanethial S-oxide, a lachrymatory factor, through successive reactions catalyzed by alliinase and lachrymatory factor synthase (LFS). A recent report showed that suppression of the LFS activity caused a dramatic increase in thiosulfinates previously reported as "zwiebelane isomers". After purification by recycle high-performance liquid chromatography and subsequent analyses, we established the planar structure of the putative "zwiebelane isomers" as S-3,4-dimethyl-5-hydroxythiolane-2-yl 1-propenethiosulfinate, in which two of the three molecules of 1-propenesulfenic acid involved in the formation gave the thiolane backbone, and the third molecule gave the thiosulfinate structure. Of at least three stereoisomers observed, one in the (2'R,3'R,4'R,5'R)-configuration was collected as an isolated fraction, and the other isomers were collected as a combined fraction because spontaneous tautomerization prevented further purification. Both fractions showed inhibitory activities against cyclooxygenase-1 and α-glucosidase in vitro.