Garlicnin A from the fraction regulating macrophage activation of Allium sativum

Garlicnin A (1), a new stable, sulfur-containing compound isolated from a fraction of the acetone extracts of Allium sativum L. garlic bulbs, showed the potential to suppress tumor cell proliferation by inhibiting the polarization of M2 alternatively activated macrophages, and its structure was characterized as 3,4-dimethyl-5-(4,5-dithia-1E,7-octadiene)-tetrahydrothiophene-2-sulfoxide-S-oxide, on the basis of the results of spectroscopic analysis results.     

Uptake and speciation of selenium in garlic cultivated in soil amended with symbiotic fungi (mycorrhiza) and selenate

The scope of the work was to investigate the influence of selenate fertilisation and the addition of symbiotic fungi (mycorrhiza) to soil on selenium and selenium species concentrations in garlic. The selenium species were extracted from garlic cultivated in experimental plots by proteolytic enzymes, which ensured liberation of selenium species contained in peptides or proteins. Separate extractions using an aqueous solution of enzyme-deactivating hydroxylamine hydrochloride counteracted the possible degradation of labile selenium species by enzymes (such as alliinase) that occur naturally in garlic. The selenium content in garlic, which was analysed by ICP–MS, showed that addition of mycorrhiza to the natural soil increased the selenium uptake by garlic tenfold to 15 μg g⁻¹ (dry mass). Fertilisation with selenate and addition of mycorrhiza strongly increased the selenium content in garlic to around one part per thousand. The parallel analysis of the sample extracts by cation exchange and reversed-phase HPLC with ICP–MS detection showed that γ-glutamyl-Se-methyl-selenocysteine amounted to 2/3, whereas methylselenocysteine, selenomethionine and selenate each amounted to a few percent of the total chromatographed selenium in all garlic samples. Se-allyl-selenocysteine and Se-propyl-selenocysteine, which are selenium analogues of biologically active sulfur-containing amino acids known to occur in garlic, were searched for but not detected in any of the extracts. The amendment of soil by mycorrhiza and/or by selenate increased the content of selenium but not the distribution of detected selenium species in garlic. Finally, the use of two-dimensional HPLC (size exclusion followed by reversed-phase) allowed the structural characterisation of γ-glutamyl-Se-methyl-selenocysteine and γ-glutamyl-Se-methyl-selenomethionine in isolated chromatographic fractions by quadrupole time-of-flight mass spectrometry.     

Bioactive S-alk(en)yl cysteine sulfoxide metabolites in the genus Allium: the chemistry of potential therapeutic agents

S-Alk(en)yl cysteine sulfoxides are odourless, non-protein sulfur amino acids typically found in members of the family Alliaceae and are the precursors to the lachrymatory and flavour compounds found in the agronomically important genus Allium. Traditionally, Allium species, particularly the onion (Allium cepa) and garlic (A. sativum), have been used for centuries in European, Asian and American folk medicines for the treatment of numerous human pathologies, however it is only recently that any significant progress has been made in determining their mechanisms of action. Indeed, our understanding of the role of Allium species in human health undoubtedly comes from the combination of several academic disciplines including botany, biochemistry and nutrition. During tissue damage, S-alk(en)yl cysteine sulfoxides are converted to their respective thiosulfinates or propanethial-S-oxide by the action of the enzyme alliinase (EC 4.4.1.4). Depending on the Allium species, and under differing conditions, thiosulfinates can decompose to form additional sulfur constituents including diallyl, methyl allyl, and diethyl mono-, di-, tri-, tetra-, penta-, and hexasulfides, the vinyldithiins and (E)- and (Z)-ajoene. Recent reports have shown onion and garlic extracts, along with several principal sulfur constituents, can induce phase II detoxification enzymes like glutathione-S-transferases (EC 2.5.1.18) and quinone reductase (QR) NAD(P)H: (quinine acceptor) oxidoreductase (EC 1.6.99.2) in mammalian tissues, as well as also influencing cell cycle arrest and apoptosis in numerous in vitro cancer cell models. Moreover, studies are also beginning to highlight a role of Allium-derived sulfur compounds in cardiovascular protection. In this review, we discuss the chemical diversity of S-alk(en)yl cysteine sulfoxide metabolites in the context of their biochemical and pharmacological mechanisms.     

Determination of neonicotinoid pesticides residues in agricultural samples by solid-phase extraction combined with liquid chromatography-tandem mass spectrometry

This work reports a new sensitive multi-residue liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for detection, confirmation and quantification of six neonicotinoid pesticides (dinotefuran, thiamethoxam, clothiandin, imidacloprid, acetamiprid and thiacloprid) in agricultural samples (chestnut, shallot, ginger and tea). Activated carbon and HLB solid-phase extraction cartridges were used for cleaning up the extracts. Analysis is performed by LC-MS/MS operated in the multiple reaction monitoring (MRM) mode, acquiring two specific precursor-product ion transitions per target compound. Quantification was carried by the internal standard method with D(4)-labeled imidacloprid. The method showed excellent linearity (R(2)≥0.9991) and precision (relative standard deviation, RSD≤8.6%) for all compounds. Limits of quantification (LOQs) were 0.01 mg kg(-1) for chestnut, shallot, ginger sample and 0.02 mg kg(-1) for tea sample. The average recoveries, measured at three concentrations levels (0.01 mg kg(-1), 0.02 mg kg(-1) and 0.1 mg kg(-1) for chestnut, shallot, ginger sample, 0.02 mg kg(-1), 0.04 mg kg(-1) and 0.2 mg kg(-1) for tea sample), were in the range 82.1-108.5%. The method was satisfactorily validated for the analysis of 150 agricultural samples (chestnut, shallot, ginger and tea). Imidacloprid and acetamiprid were detected at concentration levels ranging from 0.05 to 3.6 mg kg(-1).     

Garlicnins B(1), C(1), and D, from the fraction regulating macrophage activation of Allium sativum

Several novel sulfides from acetone extracts of bulbs of garlic (Allium sativum L.), were identified and investigated. These were named garlicnins B(1) (1), C(1) (2), and D (3), and they were found to have the ability to control macrophage activation. Garlicnins B(1) (1) and C(1) (2) possess a new skeleton of cyclic sulfoxide and their structures of garlicnins B(1) (1) and C(1) (2) were characterized as 3,4-dimethyltetrahydrothiophene-S-oxide derivatives carrying the substitutions of a propenyl and a sulfenic acid, and an allyldithiine and a 1-propene-sulfenic acid (a), respectively. The mechanism of the proposed production of these compounds is discussed. Garlicnin D (3), dithiine-type, was estimated to be derived by addition of (a)+allyl thiosulfenic acid (b) derived from allicin. The identification of these novel sufoxides from onion and garlic accumulates a great deal of new chemistry to the Allium sulfide field, and future pharmacological investigations aid the development of natural, healthy foods and anti-cancer agents that could potentially prevent or combat disease.     

Speciation of Selenium in Enriched Garlic Sprouts by High-Performance Liquid Chromatography Coupled with Inductively Coupled Plasma-Mass Spectrometry

Selenium species in enriched garlic (Allium Sativum L) sprouts were determined by high-performance liquid chromatography coupled with inductively coupled plasma-mass spectrometry (HPLC-ICP-MS). The garlic was grown in a 10 mg/L selenite nutrient solution, and the total selenium accumulated by the sprouts was 250 µg/g. Three mobile phase systems were investigated and a buffer of 20 mmol/L ammonium acetate/5% methanol was chosen for subsequent analysis. Comparative experiments were performed to determine the selenium species in the garlic sprouts with four extraction solutions: 0.1 mol/L HCl, 0.1 mol/L NaOH, 20 mmol/L ammonium acetate/5% methanol and protease XIV. The most suitable results were obtained using 0.1 mol/L HCl as the extracting solution. Validation was performed; all selenium compounds had recoveries of 102.5% to total selenium, with good linearity and precision. The major compound accumulated in the garlic sprouts was methylselenocysteine, which accounted for 65.01% of the selenium.     

Microdetermination of steroid and triterpene saponin glycosides in various plant materials I. Allium species

Summary From plant material (Allium sativum, Allium cepa, Allium porrum), extracts characterised by a high haemolytic activity were obtained. In the raw saponin preparation the percentage of saponins in garlic, onion and leek was determined, applying the densitometric method on thin layer chromatograms, previously proposed⁵. Purified crystalline or oil-like saponin preparations were hydrolysed or complexed with cholesterol. The isolated sapogenins were identified by mass spectrometry, IR, NMR and UV analysis. The following sapogenins were found: sitosterol, gitogenin, oleanolic acid and amyrin.

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Mikrobestimmung von Steroid- und Triterpen-Saponinen in verschiedenem Pflanzenmaterial

Summary Aus Allium sativum, Allium cepa und Allium porrum wurden Extrakte erhalten, die stark hämolytisch wirken. Die rohen Saponin-Präparate wurden auf Dünnschicht-Platten mit unserer densitometrischen Methode untersucht und ihr Saponingehalt bestimmt. Die gereinigten, kristallisierten oder öligen Saponinpräparate wurden einer Hydrolyse und einer Komplexierung mit Cholesterin unterzogen. Die isolierten Sapogenine wurden massenspektrometrisch, mit IR, NMR und UV geprüft. Folgende Sapogenine wurden gefunden: Sitosterol, Gitogenin, Oleanolsäure und Amyrin.

     

Quantitative determination of S-alk(en)ylcysteine-S-oxides by micellar electrokinetic capillary chromatography

A novel method for determination of S-alk(en)ylcysteine-S-oxides by capillary electrophoresis has been developed and validated. The method is based on extraction of these sulfur amino acids by methanol, their derivatization by fluorenylmethyl chloroformate and subsequent separation by micellar electrokinetic capillary chromatography. Main advantages of the new method are simplicity, sensitivity, high specificity and very low running costs, making it suitable for routine analysis of a large number of samples. Employing this method, the content of S-alk(en)ylcysteine-S-oxides was determined in 12 commonly consumed alliaceous and cruciferous vegetables (e.g. garlic, onion, leek, chive, cabbage, radish, cauliflower and broccoli). The total content of these amino acids in the Allium species evaluated varied between 0.59 and 12.3mg g(-1) fresh weight. Whereas alliin was found only in garlic, isoalliin was the major S-alk(en)ylcysteine-S-oxide in onion, leek, chive and shallot. On the other hand, the cruciferous species analyzed contained only methiin in the range of 0.06-2.45mg g(-1) fresh weight.     

Solid-phase microextraction-gas chromatographic-mass spectrometric analysis of garlic oil obtained by hydrodistillation

Garlic (Allium sativum L.) is highly consumed worldwide. This crop is mainly known for its flavor and odor, although the many medicinal properties that are attributed to it, including anticarcinogenic, antiatherosclerotic, and antithrombotic potential, among several others, have called the attention of scientists since very early times. It is known that sulfur-containing volatiles are the principal compounds responsible for such properties. The aims of this work were to develop a solventless extraction method for sulfur-containing volatiles from garlic, as well as their chemical characterization. Since garlic volatiles are rather thermolabile, low-pressure hydrodistillation was chosen as the extracting method. The analysis of all compounds was performed on an HP-FFAP chromatographic column mounted in a GC-MS system. For volatile transfer and injection method, solid-phase microextraction was selected, with the use of eight different fibers. The most abundant volatile compound was diallyl disulfide, followed by diallyl trisulfide. Among the 47 totally identified compounds, 18 were linear sulfur-containing volatile compounds, 6 were of non-sulfur nature, and the other 23 were cyclic compounds. However, linear sulfur volatiles accounted for 94% of the total amount.     

Speciation of selenium in selenium-enriched shiitake mushroom, Lentinula edodes

The major selenium compound in an aqueous extract of the most popular mushroom in Eastern Asian countries, shiitake (Lentinula edodes), fortified with selenium (Se) was identified by means of hyphenated techniques, i.e. HPLC-inductively coupled argon plasma mass spectrometry and HPLC-electrospray ionization mass spectrometry (HPLC–ICP MS and HPLC–ESI MS). Sixty-eight per cent of the total Se in the selenized shiitake was extracted with water, and 49.8% of the Se in the water extract was eluted in the high molecular mass fraction (>40,000 kDa) before incubation at 37 °C. After incubation, 40.6% of the Se in the water extract was eluted in a lower molecular mass fraction and the Se eluted in the high molecular mass fraction had decreased to 14.0%, suggesting that the major selenium compound in the water extract was initially in a form bound to macromolecule(s) and was then enzymatically liberated from the macromolecule(s). The retention time of the liberated selenium compound in HPLC–ICP MS matched that of selenomethionine (SeMet), and the masses of molecular and fragment ions detected by HPLC–ESI MS also suggested that the selenium compound was SeMet. The selenized shiitake accumulated Se as SeMet, and SeMet might be bound to the water extractable high molecular mass protein(s).Electronic Supplementary Material Supplementary material is available for this article at     

Development of an analytical method for polycyclic aromatic hydrocarbons and their derivatives

Garlic and onion, are well known for their medical value, especially in against cancer and anticardiovacular diseases. "Alliins" (S-alk(en)yl-L-cysteine sulphoxides) are sources of major active compounds in Allium plants. Se incorporation into garlic significantly increases activities of garlic in cancer prevention and inhibition. Selenomethionine, selenocysteine and Se-methylselenocysteine have been identified in garlic and onion. Previously we identified gamma-glutamyl-Se-methyl-L-selenocysteine, in extracts of garlic cultivated in Se-rich soil [Med. Res. Rev. 16 (1) (1996) 111], suggesting the possible existence of Se-alk(en)yl-L-cysteine selenoxides (Se-"alliins") in garlic. Several comparative experiments were carried out to demonstrate the existence of Se-"alliins" in Se-enriched garlic and onion. We found that there was one similar time-dependent Se signal in HPLC-inductively coupled plasma MS chromatograms of cold-water extracts of freeze-dried garlic powder and fresh garlic. This signal was lost when the extracts of garlic powder and fresh garlic were stored for 1 day at >4 degrees C, but remained in fresh onion extract at the same storage conditions. These phenomena and possible mechanisms are discussed. An additional experiment showed that Allium species cultivated in Se-rich soil might contain two different Se-"alliins".     

Single‐step modified QuEChERS for determination of chlorothalonil in shallot (Allium ascalonicum) using GC‐μECD and confirmation via mass spectrometry

A single extraction method was developed for chlorothalonil in shallot using gas chromatography with an electron capture detector (GC‐μECD). Samples were extracted with single‐step modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) method using ethyl acetate as an extraction solvent. Significant matrix effects were observed, and the calibration curve was constructed from the matrix. The linearity of the analytical response across the studied range of concentrations (0.01–1.00 mg/L) was excellent, obtaining a correlation coefficient (r²) of 0.996. >0.996. Recovery studies were carried out on spiked shallot blank samples, at two concentration levels (0.4 and 2.0 mg/kg) with three replicates performed at each level. Mean recoveries of 97.2–104.9% with RSDs of 1.3–2.7% were obtained. The method is demonstrated to be suitable for the determination of chlorothalonil in shallot. The dissipation rates of chlorothalonil were described using first‐order kinetics, and its half‐life was 2.8 days. Based on the dissipation pattern of the pesticide residues, the pre‐harvest residue limit (PHRL) was also calculated. Residues were confirmed via mass spectrometry. Copyright © 2012 John Wiley & Sons, Ltd.     

Alliin Lyase (Alliinase) from Garlic (Allium sativum)

The garlic plant (Allium sativum) alliinase (EC 4.4.1.4), which catalyzes the synthesis of allicin, was purified to homogeneity from bulbs using various steps, including hydrophobic chromatography. Molecular and biochemical studies showed that the enzyme is a dimer of two subunits of MW 51.5 kDa each. ItsK _m using synthetic S-allylcysteine sulfoxide (+isomer) as substrate was 1.1 mM, its pH optimum 6.5, and its isoelectric point 6.35. The enzyme is a glycoprotein containing 6% carbohydrate. N-terminal sequences of the intact polypeptide chain as well as of a number of peptides obtained after cyanogen bromide cleavage were obtained. Cloning of the cDNAs encoding alliinase was performed by a two-step strategy. In the first, a cDNA fragment (pAli-1-450 bp) was obtained by PCR using a mixed oligonucleotide primer synthesized according to a 6-amino acid segment near theN- terminal of the intact polypeptide. The second step involved screening of garlic λgt11 and λZAPII cDNA libraries withpAli-1, which yielded two clones; one was nearly full length and the second was full length. These clones exhibited some degree of DNA sequence divergence, especially in their 3′ noncoding regions, suggesting that they were encoded by separate genes. The nearly full length cDNA was fused in frame to a DNA encoding a signal peptide from a wheat gliadin, and expressed inXenopus oocytes. This yielded a 50 kDa protein that interacted with the antibodies against natural bulb alliinase. Northern and Western blot analyses showed that the bulb alliinase was highly expressed in bulbs, whereas a lower expression level was found in leaves, and no expression was detected in roots. Strikingly, the roots exhibited an abundant alliinase activity, suggesting that this tissue expressed a distinct alliinase isozyme with very low homology to the bulb enzyme.     

气相色谱-负化学离子源质谱法测定含硫蔬菜中残留的14种农药

建立了含硫蔬菜(大葱、大蒜、蒜薹及韭菜等)中14种农药残留的气相色谱-负化学离子源质谱(GC-NCI/MS)检测方法。样品先采用微波加热处理除去大部分的含硫干扰物,然后用乙腈均质提取,提取液用凝胶渗透色谱(GPC)和N-丙基乙二胺(PSA)固相萃取小柱净化后用GC-NCI/MS在选择离子监测模式下测定。在50 μg/kg加标水平下回收率为49.2%～113.1%,相对标准偏差为1.42%～8.70%,检出限(以3倍信噪比计)为0.5～10.0 μg/kg。方法的选择性好,抗干扰能力强,能消除复杂基质带来的干扰,适合于含硫蔬菜中农药残留的确证分析。     

HPLC Fingerprint Characteristics of Active Materials of Garlic and Other Allium Species

The bioactive molecules of garlic are classified according to their enzymatic activities as either alliinase activators or alliinase inactivators. The alliinase activation reaction system is mainly composed of sulfur-containing compounds, whereas the alliinase inactivation system is mainly composed of amino acid-based materials. The purpose of this study was to develop an effective way to digitally express features of complex active compounds of garlic as a basis for quality control. HPLC was used to develop the fingerprints of plants from different Allium species and different geographical regions according to whether the compounds were sulfur containing or based on amino acids. Using the Shannon equation, I values of sulfur-containing compounds from garlic ranged from 3.55 to 3.94, whereas I values of sulfur-containing compounds from other Allium plants, onions, leek, and Welsh onion, ranged from 3.38 to 3.53. The I values of amino acid-based compounds from garlic ranged from 3.67 to 3.91, whereas I values of these compounds from other Allium plants ranged from 3.88 to 3.99. This method effectively distinguished garlic from different species of Allium plants. This method also provided a way to digitally monitor the presence of complex active compounds of garlic and may allow evaluation of quality. This method may also provide a theoretical basis for quality control of bioactive compounds from other medicinal plants.     

Preconcentration and determination of tellurium in garlic samples by hydride generation atomic absorption spectrometry

A procedure for the determination of traces of total tellurium (Te) in garlic (Allium sativa) is described that combines hydride generation atomic absorption spectrometry with preconcentration of the analyte by coprecipitation. The samples, each spiked with lanthanum nitrate (20 mg/L), are introduced into an Amberlite XAD-4 resin and mixed with ammonium buffer (pH 9.1). Te is preconcentrated by coprecipitation with the generated lanthanum hydroxide precipitate. The precipitate is quantitatively collected in the resin, eluted with hydrochloric acid, and then transferred into the atomizer device. Considering a sample consumption of 25 mL, an enrichment factor of 10 was obtained. The detection limit (3sigma) was 0.03 microg/L, and the precision (relative standard deviation) was 3.5% (n = 10) at the 10 microg/L level. The calibration graph using the preconcentration system for Te was linear with a correlation coefficient of 0.9993. Satisfactory results were obtained for the analysis of Te in garlic samples.     

Identification of non-peptide species in selenized yeast by MALDI mass spectrometry using post-source decay and orthogonal Q-TOF detection

The potential of tandem mass spectrometry following matrix-assisted laser desorption ionization (MALDI) was studied for speciation of selenium. Non-peptide selenium-containing compounds were isolated from a selenized yeast aqueous extract by size-exclusion chromatography. Post-source decay (PSD) was compared with orthogonal quadrupole collision cell dissociation for the purpose of obtaining fragmentation and structural information. In the PSD mode, the use of ion gate covering the whole isotopic cluster of the parent compound allowed the immediate recognition of fragments containing Se and those in which this element was absent. The tandem mass spectra obtained by orthogonal MALDI Q-TOF were equally informative in terms of the number of fragments but suffered from a poorer sensitivity. The mass accuracy was ca. 20 times better in the oMALDI configuration than in the PSD mode. An unknown selenium compound with an m/z 388 was detected with a mass accuracy of 3 ppm according to the proposed empiric formula.     

用GC/MS分析几种葱属植物挥发油中的有机硫化物

采用蒸汽蒸馏的方法,分别提取了大葱(Allium fistulosum L)、薤白(Allium macrostemonBunge)和大蒜(Allium sativum L)中的挥发油,用GC/MS方法鉴别了它们中的主要硫化合物,并进行了比较。     

高效液相色谱-等离子体质谱联用方法研究富硒大米中硒的形态

根据Osborne溶解性蛋白质分类法,用去离子水、2%NaCl溶液、70%乙醇、0.5%KOH4种溶液分别提取出大米中相应的清蛋白、球蛋白、醇溶蛋白及谷蛋白。利用电感耦合等离子质谱(ICP-MS)测量各类蛋白提取液中的硒含量;用色谱与质谱联用法对大米蛋白提取液中含硒蛋白组分及含硒氨基酸组成进行初步分析。结果表明:大米中的硒主要与蛋白质结合,在4类蛋白提取液中硒含量由高到低的分布顺序为:谷蛋白、醇溶蛋白、清蛋白和球蛋白。清蛋白提取液中,分子量为12.6 kDa蛋白是主要的含硒蛋白组分;大米中约30%的硒以硒代半胱氨酸形式存在。     

Development and validation of a thin-layer chromatography-densitometric method for the quantitation of alliin from garlic (Allium sativum) and its formulations

A selective, precise, and accurate high-performance thin-layer chromatographic (HPTLC) method has been proposed for the analysis of garlic and its formulations for their alliin content. The method involves densitometric evaluation of alliin after resolving it by HPTLC on silica gel plates with n-butanol-acetic acid-water (6 + 2 + 2, v/v) as the mobile phase. For densitometric evaluation, peak areas were recorded at 540 nm after derivatizing the resolved bands with ninhydrin reagent. The relation between the concentration of alliin and corresponding peak areas was found to be linear within the range of 250 to 1500 ng/spot. The method was validated for precision (interday and intraday), repeatability, and accuracy. Mean recovery was 98.36%. The method was applied for the quantitation of alliin in bulbs of Allium sativum Linn. (garlic) and its formulations. The proposed TLC method was found to be precise, specific, sensitive, and accurate and can be used for routine quality control of garlic and its formulations.