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.     

Identification of two novel pigment precursors and a reddish-purple pigment involved in the blue-green discoloration of onion and garlic

By using a model reaction system representing blue-green discoloration that occurs when purees of onion (Allium cepa L.) and garlic (Allium sativum L.) are mixed, we isolated two pigment precursors (PPs) and a reddish-purple pigment (PUR-1) and determined their chemical structures. PPs were isolated from a heat-treated solution containing color developer (CD) and either l-valine or l-alanine, and their structures were determined as 2-(3,4-dimethylpyrrolyl)-3-methylbutanoic acid (PP-Val), and 2-(3,4-dimethyl-1H-pyrrolyl) propanoic acid (PP-Ala), respectively. Next, PUR-1 was isolated from a heat-treated solution containing PP-Val and allicin, and its structure was determined as (1E)-1-(1-((1S)-1-carboxy-2-methylpropyl)-3,4-dimethyl-1H-pyrrol-2-yl)-prop-1-enylene-3-(1-((1S)-1-carboxy-2-methylpropyl)-3,4-dimethyl-1H-pyrrol-2-ylidenium). The structure of PUR-1 suggested that PP molecules containing a 3,4-dimethyl pyrrole ring had been cross-linked by an allyl group of allicin to form conjugated pigments. While PUR-1 is a dipyrrole compound exhibiting a reddish-purple color, a color shift toward blue to green can be expected as the cross-linking reaction continues to form, for example, tri- or tetrapyrrole compounds.     

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.     

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.     

Effect of Processing and Cooking Conditions on Onion ( Allium cepa L.) Induced Antiplatelet Activity and Thiosulfinate Content

Allium vegetables serve as sources of antiplatelet agents that may contribute to the prevention of cardiovascular disease. However, onion and garlic, the major Allium species, are usually cooked before consumption. Here, we examined the effect of cooking on onion in vitro antiplatelet activity (IVAA). Two different cooking systems (convection oven and microwaves) and several time-temperature variables were tested on whole bulbs, quarters of bulbs, and completely crushed bulbs, monitoring the degradation of sulfur antiplatelet compounds (e.g., thiosulfinates) by analysis of pyruvate levels. Although heating was, in general, detrimental for onion IVAA, the extent of this effect varied greatly, from unaffected antiplatelet activity (AA) (i.e., similar to raw onion) to a complete lost of activity, depending upon the manner in which onions were prepared prior to heating, the cooking method, and the intensity of the heat treatment. "Whole", "quarters", and "crushed" onions lost their IVAA after 30, 20, and 10 min of oven heating, respectively. The longer retainment of AA in intact bulbs was attributed to a later alliinase inactivation. Proaggregatory effects observed in samples subjected to the most intense oven and microwave heat treatments suggest that extensively cooked onions may stimulate rather than inhibit platelet aggregation. The efficacy of Allium species as antiplatelet agents, as affected by preparation and cooking conditions, is discussed.     

Fate and kinetic modeling of reactivity of alkanesulfenic acids and thiosulfinates in model systems and onion homogenates

The dynamic changes in thiosulfinate profiles were studied in reaction systems containing a crude onion alliinase, S-alk(en)yl-L-cysteine sulfoxide substrates (1) and preformed thiosulfinates (4). Regioisomeric excesses of one of two possible heterologous 4 species (RS(O)SR', where R does not equal R') could be manipulated under conditions where alliinase, 1, and 4 levels were varied. Regioisomeric excesses could be explained by a thiosulfinate (4)/alkanesulfenic acid (2) trapping mechanism, with the greatest control over product profile governed by the rate of 2 generation in the system. The series of reactions existing in this dynamic reaction system was kinetically modeled with reasonable fits to the experimental data. The application of the 4/2 trapping strategy to manipulate thiosulfinate and related organosulfur product profiles in diluted onion homogenates was demonstrated using exogenous MeS(O)SMe (4a), PrS(O)SPr (4c), and AllS(O)SAll (4d) as the preformed thiosulfinate.     

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.     

Increase in the permeability of tonoplast of garlic (Allium sativum) by monocarboxylic acids

Immersion of intact aged garlic (Allium sativum) cloves in a series of 5% weak organic monocarboxylate solutions (pH 2.0) resulted in green color formation. No color was formed upon treatment with other weak organic acids, such as citric and malic acids, and the inorganic hydrochloric acid under the same conditions. To understand the significance of monocarboxylic acids and their differing function from that of other acids, acetic acid was compared with organic acids citric and malic and the inorganic hydrochloric acid. The effects of these acids on the permeability of plasma and intracellular membrane of garlic cells were measured by conductivity, light microscopy, and transmission electron microscopy. Except for hydrochloric acid, treatment of garlic with all three organic acids greatly increased the relative conductivity of their respective pickling solutions, indicating that all tested organic acids increased the permeability of plasma membrane. Moreover, a pickling solution containing acetic acid exhibited 1.5-fold higher relative conductivity (approximately 90%) as compared to those (approximately 60%) of both citric and malic acids, implying that exposure of garlic cloves to acetic acid not only changed the permeability of the plasma membrane but also increased the permeability of intracellular membrane. Exposure of garlic to acetic acid led to the production of precipitate along the tonoplast, but no precipitate was formed by citric and malic acids. This indicates that the structure of the tonoplast was damaged by this treatment. Further support for this conclusion comes from results showing that the concentration of thiosulfinates [which are produced only by catalytic conversion of S-alk(en)yl-l-cysteine sulfoxides in cytosol by alliinase located in the vacuole] in the acetic acid pickling solution is 1.3 mg/mL, but almost no thiosulfinates were detected in the pickling solution of citric and malic acids. Thus, all present results suggest that damage of tonoplast by treatment with monocarboxylates such as acetic acid may be the main reason for the greening of garlic.     

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.     

Assessment of initial stages of Maillard reaction in dehydrated onion and garlic samples

The initial steps of the Maillard reaction in freshly laboratory-freeze-dried and commercial dehydrated onion and garlic samples have been assessed by quantitative determination of 2-furoylmethylamino acids, obtained after acid hydrolysis of the corresponding Amadori compound. In freshly prepared samples, hardly any presence of 2-furoylmethylamino acids was detected, whereas in commercial samples, onion contained much more important levels of 2-furoylmethylamino acids as compared to garlic species. 2-Furoylmethyl-gamma-aminobutyric acid (1), 2-furoylmethyl-lysine (furosine; 2), and 2-furoylmethylarginine (3) were identified in all commercial dehydrated onion samples, with compound 3 being the most abundant. All garlic samples presented slightly higher levels of 2 than 3 with no presence of 1. The observed differences between onion and garlic commercial samples may be due to their very different content of reducing sugars. Moreover, some variations found in 2-furoylmethyl derivatives within both onion and garlic species could be also attributed to different processing and storage conditions during the manufacture of these products. The findings of this study show the first evidence of important levels of Amadori compounds in dehydrated garlic and onion samples, as well as the usefulness of 2-furoylmethyl derivatives as quality indicators for the early detection of the Maillard reaction in onion and garlic products.     

Marker-assisted selection of Ms locus responsible for male fertility restoration in onion (Allium cepa L.)

Genetic Resources and Crop Evolution - The study aimed to identify the male-fertility restoration locus (Ms) among 72 breeding lines of onion (Allium cepa L.) by genotyping, which is crucial in the...     

Determination of S-methyl-, S-propyl-, and S-propenyl-L-cysteine sulfoxides by gas chromatography-mass spectrometry after tert-butyldimethylsilylation

A gas chromatographic-mass spectrometric method for the determination of S-methyl-L-cysteine sulfoxide (1), S-propyl-L-cysteine sulfoxide (2), and S-propenyl-L-cysteine sulfoxide (3), specific marker compounds in the genus Allium, is described. The target amino acids were converted to the tert-butyldimethylsilyl derivatives. The products were silylated on the amino and carboxyl groups and on an additional oxygen atom and were separated on a nonpolar capillary column. That incorporation of three tert-butyldimethylsilyl groups had occurred was verified by mass spectrometry, which gave an m/z 302 fragment as base peak (amino acid side chain eliminated ion) and m/z 436 (1), 464 (2), or 462 (3) as major peaks (tert-butyl function eliminated ion), by electron impact ionization. The detection limits for 1 and 2 under selected ion monitoring at m/z 436 (1) and m/z 464 (2), respectively, were determined to be 0.3 and 1.8 ng per injection. To clean up the analytes from the solvent extract of onion, as a representative food material, onion, the sample solution was subjected to combined solid phase extraction. The eluate from a Sep-Pak C(18) cartridge was applied to a Bond Elut SCX cartridge (H(+) form), followed by washing with 0.1 M hydrochloric acid and elution with 0.5 M ammonia. From a simulated matrix solution containing 5% sucrose, 1 and 2 were extracted quantitatively, and the detection yield was approximately 75%. The contents of 1, 2, and 3 in commercial onion were estimated to be 0.3, 3.1, and 3.0 mg, respectively, per gram of fresh weight.     

辐照对大蒜中SOD活性及硒含量的影响

研究了0.1、1.0kGy的辐照处理对10℃和25℃贮藏大蒜的SOD活性及总硒、无机硒、有机硒含量的影响。结果表明:在两种贮藏温度下,辐照处理都能够延缓大蒜中SOD活性的下降。辐照处理并不能影响大蒜中各类硒含量的变化,但是贮存于25℃大蒜中总硒、无机硒、有机硒含量显著高于10℃贮藏的大蒜。     

Allium species from Central and Southwest Asia are rich sources of marasmin

Marasmin, which is especially known from the two South African species Tulbaghia alliacea and Tulbaghia violacea , but was also described for the garlic mushroom Marasmius alliaceus , is the precursor of the thiosulfinate marasmicin. Marasmicin has attracted considerable attention because of its antifungal and tuberculostatic activities. However, many Allium species of the subgenus Melanocrommyum, especially Allium suworowii , are also very rich in marasmin. A. suworowii revealed concentrations of marasmin up to 1.6%, related to the fresh weight of bulbs, and up to 3.0%, related to air-dried fruiting bodies, of the corresponding γ-glutamylmarsmin was found in M. alliaceus. Both species show much higher amounts of marasmin as Tulbaghia and could be considered as natural sources for the isolation of this compound. Further promising Allium species with considerable amounts of marasmin besides other cysteine sulfoxides are Allium stipitatum and Allium altissimum . (R(S),R(C))-Marasmin is typical for the investigated species of the subgenus Melanocrommyum, whereas γ-glutamyl-(S(S),R(C))-marasmin is the only cysteine sulfoxide for the genus Marasmius known until now. Both cysteine sulfoxides were isolated and described as o-phthaldialdehyde (OPA) derivatives. Furthermore, the cysteine sulfoxides methiin, propiin, S-(2-pyrrolyl)-cysteine sulfoxide, eventually S-(2-pyridyl)-cysteine sulfoxide and S-(2-pyridyl)-L-cysteine N-oxide were found.     

DETERMINING THE ZINC REQUIREMENT OF ONION BY SEED ANALYSIS

Our study analyzed the effect of foliar tissues and seed tissue for determining the micronutrient status of a crop. Zinc (Zn) requirements of onion (Allium cepa L.) leaves and seeds were estimated from yield response curves based on field experiment conducted on a Zn-deficient calcareous soil. Three onion cultivars, i.e., ‘Swat-1’, ‘Phulkara’, and ‘Sariab Red’ were grown by applying 0, 2, 4, 8, and 16 kg Zn ha^?1. Zinc application significantly increased seed yield of all the three cultivars of onion. The order of seed yield response to Zn fertilization was: ‘Swat-1’ < ‘Phulkara’ < ‘Sariab Red’. Fertilizer Zn requirement for near-maximum seed yield was 2 kg Zn ha^?1. Zinc concentration in mature onion seed also appeared to be a good indicator of soil Zn availability status. Critical Zn concentration in seed was 18 mg Zn kg^?1, and in matured leaves was 21 mg kg^?1.     

Mechanism of the greening color formation of "laba" garlic, a traditional homemade chinese food product

While green discoloration during garlic processing is of a major concern, this greening is desirable and required for the traditional homemade Chinese "Laba" garlic. To obtain insights into the mechanism of color formation, simulation of the greening of "Laba" garlic was carried out in the laboratory by soaking aged garlic in 5% (v/v, pH 2.33) acetic acid solution. After 2 days, the garlic cloves turned green. Up to 4 days, pigment(s) diffused from garlic cloves to the pickling solution. The solution exhibits two maximal absorbances at approximately 440 and approximately 590 nm, corresponding to yellow and blue species, respectively, the combination of which creates the green coloration. With increasing time from 4 to 25 days, the concentration of both yellow and blue species increases at nearly the same rate, while after 25 days, the concentration of the yellow species increases faster than that of the blue species. Interestingly, most thiosulfinates ( approximately 85%) in garlic cloves were converted within 4 days, suggesting that thiosulfinate conversion is proportional to the formation of the pigments. Consistent with this conclusion, alliinase and acetic acid were required for the color formation. UV-vis spectral measurements and pH results suggest that the color formation occurs by two kinds of processes: one enzymatic and the other nonenzymatic. Low pH (2.0-3.0) favors nonenzymatic reactions, while high pH (6.0 or above) is conducive to enzymatic reactions. Thus, the ideal pH for the entire process of garlic greening is between 4.0 and 5.0, which is a compromise of the optimal pH of both the enzymatic and nonenzymatic reactions.     

The use of imaging complex based on Mikmed-6 microscope in the record of allium test results for the estimation of soil genotoxicity

We assessed the suitability of a video system based on a Mikmed-6 optical microscope as a tool for objective measurement in the Allium test for estimating soil genotoxicity. Digitized microimages of the apical meristem of common onion (Allium cepa L.) fix the data needed for calculating mitotic and phase indices and create an objective scheme of mitotic errors and chromosome aberrations.     

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.     

Free amino acid and cysteine sulfoxide composition of 11 garlic (Allium sativum L.) cultivars by gas chromatography with flame ionization and mass selective detection

Two garlic subspecies (n = 11), Allium sativum L. var. opioscorodon (hardneck) and Allium sativum L. var. sativum (softneck), were evaluated for their free amino acid composition. The free amino acid content of garlic samples analyzed ranged from 1121.7 to 3106.1 mg/100 g of fresh weight (mean = 2130.7 +/- 681.5 mg/100 g). Hardneck garlic had greater methiin, alliin, and total free amino acids contents compared to softneck garlic. The major free amino acid present in all but one subspecies was glutamine (cv. Mother of Pearl had aspartic acid as the major free amino acid). Cv. Music Pink garlic (a rocambole hardneck variety) contained the most methiin, alliin, and total free amino acids. The solid-phase extraction, alkylchloroformate derivatization, GC-FID, and GC-MS methods used in this study were simple and rapid, allowing 18 free amino acids in garlic to be separated within 10 min.     

Collections of short-day onion germplasm in West Africa: a survey

Results are presented of a 1994 postal survey of holdings of local onion (Allium cepa L.) germplasm by institutions in West Africa. Data obtained from respondents included the number of accessions of onion or shallot held and the conditions in which onion seed was stored. Twenty-four replies were received and eighteen sites where collections were held were identified. The numbers of local A. cepa accessions in local collections varied in number from 38 down to nil, but many local collections were of less than five accessions. The largest number of national onion accessions was held at CERRA, Maradi, in Niger (16), and the largest regional collection was at the Station de Farako-bâ, Burkina Faso (38). Two sites held substantial shallot collections: CRA Bareng in Guinée and IDESSA, Bouaké, Côte d'Ivoire. Conditions under which onion seed was stored varied from low temperature, controlled storage in moisture-proof packs with desiccants, to storage in plastic envelopes under ambient laboratory conditions.