GC and Mass-Spectrometric Comparison of Organo Sulfur Compounds in Two Varieties of Iranian Garlic

Abstract

Garlic antibiotic action is well known; it is reputed to offer protection against coronary thrombosis and atherosclerosis exhibiting antioxidant and anticancer effects. ^1,2 In this work ¹ H NMR and gas chromatography in conjunction with mass spectrometry have been used for the identification and determination of these compounds in two varieties of Iranian garlic [Allium sativum var. sativum (I) and Allium sativum var. holmense (II)]. The organosulfur compounds in (I) exhibiting a concentration higher than 1% are diallyl sulfide (1.3%), diallyl disulfide (8%), methyl allyl disulfide (19%), methyl allyl trisulfide (3.2%), diallyl trisulfide (5.5%), diallyl tetrasulfide (2%), 2,3-dimethyl thiophen (1.8%), 5-methyl-1,2,3-thiadiazol (5%), and 1,2-ditiolan-3-carboxilic acid (1.5%). The amount of organosulfur compounds in (II) are diallyl disulfide (2%), methyl allyl trisulfide (6.3%), diallyl tetrasulfide (5%), and cyclopentanthiol (2.5%).     

选择离子流动管质谱法测定大蒜酶解液顶空中17种有机硫化合物

采用选择离子流动管质谱(SIFT-MS)法,顶空取样后,以H3O+,NO+或O2+为初始离子,定量分析了17种大蒜酶解液挥发性有机硫化合物。结果表明:有机硫化合物主要为二烯丙基硫醚、甲基烯丙基硫醚、硫代亚磺酸酯和硫醇,其中二烯丙基二硫醚浓度为137 g/m3,二烯丙基三硫醚浓度为13.4 g/m3,甲基烯丙基二硫醚浓度为8.98 g/m3,它们分别占17种有机硫化合物总含量的77.3%,7.58%和5.08%(相对含量);本研究采用顶空取样方法测出大蒜素的浓度为5.63 g/m3,相对含量为3.20%。     

冷柱头程序升温进样-气相色谱/质谱法研究新鲜大蒜中的有机硫化合物

采用冷溶剂提取新鲜大蒜中的有机硫化合物,结合冷柱头程序升温进样,对大蒜原始组分进行了气相色谱/质谱分析 。该法实现了从提取到色谱分离的“冷过程”,因而可以准确地鉴定大蒜提取液在热分解前的原始组分。分析结果表明, 在大蒜提取液中含有3-乙烯基-1,2-二硫杂-5-环己烯及其异构体2-乙烯基-1,3-二硫杂-4-环己烯两种主要组分以及少量 的S-甲基甲烷硫代亚磺酸酯、二烯丙基二硫醚、二烯丙基三硫醚。对大蒜的冷溶剂提取液和水蒸气蒸馏提取的大蒜油中 的有机硫化合物进行了比较,对一些主要有机硫化合物的形成进行了初步探讨。     

Comparative study of extraction techniques for determination of garlic flavor components by gas chromatography–mass spectrometry

Several sampling techniques based on steam distillation (SD), simultaneous distillation and solvent extraction (SDE), solid-phase trapping solvent extraction (SPTE), and headspace solid-phase microextraction (HS-SPME) have been compared for the determination of Korean garlic flavor components by gas chromatography–mass spectrometry (GC–MS). Diallyl disulfide (57.88%), allyl sulfide (23.59%), and diallyl trisulfide (11.40%) were found to be the predominant flavor components of garlic samples extracted by SDE whereas these components were at levels of 89.77%, 2.43%, and 3.89% when the same sample was extracted by SD, 97.77%, 0.17%, and 0.10% by SPTE, and 97.85%, 0.01%, and 0.01% by HS-SPME using the 50/30-m divinyl benzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) fiber. Thermal degradation of components such as allyl methyl sulfide, dimethyl disulfide, and thiirane were observed for SDE and SD but not for SPTE or HS-SPME. HS-SPME had several advantages compared with SD, SDE, and SPTE—rapid solvent-free extraction, no apparent thermal degradation, less laborious manipulation, and less sample requirement. Five different fiber coatings were evaluated to select a suitable fiber for HS-SPME of garlic flavor components. DVB/CAR/PDMS was most efficient among the five types of fiber investigated.     

GC-MS法测定大蒜中的挥发性物质

大蒜(AlliumsatiuvmL.)是多年生草本百合科植物大蒜的鳞茎,按皮的颜色不同可分为紫皮蒜和白皮蒜两种。大蒜原产于意大利的西西里岛,约在两千多年前传入我国。大蒜不仅是极佳的调味食品,而且还有很好的药用功能。近年来,科学家研究证明,大蒜是一种健康食品,有助人体防病保健、延年益寿,是药食兼优的佳品,对人体具有多种药用价值。大蒜具有一种强烈的蒜臭气味,这种特殊的气味由许多复杂的硫化物组成。这类有机硫化物可促进消化,健胃整肠,强化抗菌作用,消肿止痛,改善机体机能的功能,大蒜中还含有乙烷硫代磺酸乙酯和二烯丙基三硫化物等,能阻断强烈致癌物质亚硝胺类在胃部的形成和积累,可预防胃癌、食道癌、肝癌、鼻咽癌的发生。本试验采用"同时蒸馏-萃取方法"(simultaneousdistillationandextraction,SDE)提取白皮和紫皮大蒜的挥发性成分,经GC-MS分析,确定了化合物的化学组成和相对百分含量。     

二烯丙基硫醚化合物的合成

二烯丙基一硫醚（DS）,二烯丙基二硫醚（DDS）,二烯丙基三硫醚（DTS）和二烯丙基四硫醚（DTTS）等是大蒜提取液中的主要有效成分,具有十分广泛独特的生理药理活性。作者以甲酸、丙三醇为超始原料合成了这些化合物,烯丙基硫醚化合物总含量达83%,其中DDS,DTS含量分别达30%。产物均GC-MS鉴定确证。合成方法简单,原料易得,反应步骤少,实用性较强。     

Diallyl Trisulfide,the Antifungal Component of Garlic Essential Oil and the Bioactivity of Its Nanoemulsions Formed by Spontaneous Emulsification

The aim of this study was to evaluate the chemical compounds of garlic essential oil (EO), and determine the antifungal efficacy of garlic EO and its major components, diallyl trisulfide and its nanoemulsions against wood-rotting fungi, Trametes hirsuta and Laetiporus sulphureus. GC-MS analysis revealed that the major constituents of garlic EO were diallyl trisulfide (39.79%), diallyl disulfide (32.91%), and diallyl sulfide (7.02%). In antifungal activity, the IC₅₀ value of garlic EO against T. hirsuta and L. sulphureus were 137.3 and 44.6 μg/mL, respectively. Results from the antifungal tests demonstrated that the three major constituents were shown to have good antifungal activity, in which, diallyl trisulfide was the most effective against T. hirsuta and L. sulphureus, with the IC₅₀ values of 56.1 and 31.6 μg/mL, respectively. The diallyl trisulfide nanoemulsions showed high antifungal efficacy against the examined wood-rotting fungi, and as the amount of diallyl trisulfide in the lipid phase increases, the antifungal efficacy of the nanoemulsions increases. These results showed that the nanoemulsions and normal emulsion of diallyl trisulfide have potential to develop into a natural wood preservative.     

Detection of volatile organic sulfur compounds in water by headspace gas chromatography and membrane inlet mass spectrometry

Two gas chromatographic methods, GC-FID (flame ionization detection) and GC-ELCD (electrolytic conductivity detector) are compared in tlie analysis of volatile organic sulfur compounds (VOSCs) in water samples with a membrane inlet mass spectrometry (MIMS) technique. Carbon disulfide, ethanethiol, dimethyl sulfide, ethyl-methyl sulfide, thiophene, and dimethyl disulfide were used as test compounds. Linear dynamic ranges were found to be two decades with the GC-ELCD method and four decades with the GC-FID and MIMS methods. Detection limits were at low (μg/1 levels with the two gas chromatographic methods and clearly below μg/1 level with the MIMS method. Analysis of one sample takes 40 min with the gas chromatographic methods and five minutes with the MIMS method. The selectivity was good, especially with the GC-ELCD and the MIMS method. In addition, quantitative results obtained with spiked water samples by the three methods are compared.     

Characterization of Key Odorants in Scallion Pancake and Investigation on Their Changes during Storage

To characterize key odorants in scallion pancake (SP), volatiles were extracted by solvent extraction-solvent assisted flavor evaporation. A total of 51 odor-active compounds were identified by gas chromatography-olfactometry (GC-O) and chromatography–mass spectrometry (GC-MS). (Z/E)-3,6-Diethyl-1,2,4,5-tetrathiane was detected for the first time in scallion food. Application of aroma extract dilution analysis to extracts showed maltol, methyl propyl disulfide, dipropyl disulfide and 2-pentylfuran had the highest flavor dilution (FD) factor of 4096. Twenty-three odorants with FD factors ≥ 8 were quantitated, and their odor active values (OAVs) were calculated. Ten compounds with OAVs ≥ 1 were determined as the key odorants; a recombinate model prepared from the key odorants, including (E,E)-2,4-decadienal, dimethyl trisulfide, methyl propyl disulfide, hexanal, dipropyl trisulfide, maltol, acetoin, 2-methylnaphthalene, 2-pentylfuran and 2(5H)-furanone, successfully simulated the overall aroma profile of SP. The changes in odorants during storage were investigated further. With increasing concentrations and OAVs during storage, hexanal became an off-flavor compound.     

Effects of allyl sulfur compounds and garlic extract on the expression of Bcl-2, Bax, and p53 in non small cell lung cancer cell lines

Allyl sulfur compounds play a major role in the chemoprevention against carcinogenesis. The present study compared the antiproliferative effects of diallyl sulfide (DAS), diallyl disulfide (DADS) and garlic extract on p53-wild type H460 and p53-null type H1299 non small cell lung cancer cells (NSCLC). The DAS and DADS treatment of both H460 and H1299 cells resulted in the highest numbers of cells in apoptotic state as measured by acridine orange staining, however, garlic extract treatment did not induce any significant apoptotic cells by MTT assay. DADS was found to be more effective in inducing apoptosis on NSCLC. The level of p53 protein in H460 cell was increased following DADS treatment. DAS and garlic extract treatment of H460 cells induced a rise in the level of Bax and a fall of Bcl-2 level. These results demonstrate that DAS, DADS and garlic extract are effective in reduction of anti-proliferative gene in NSCLC and suggest that modulation of apoptosis-associated cellular proteins by DAS, DADS and garlic extract may be the mechanism for apoptosis which merit further investigation as potential chemoprevention agents.     

Determination of Stability Constants of Cadmium(II) Complexes with Diallyl Disulfide,Dimethyl Disulfide and Diallyl Sulfide Using Differential Pulse Voltammetry

The study of the binding of heavy metal ions by organosulfur compounds of garlic is especially important for the understanding of many biological systems. Cadmium is a non-essential heavy metal known to be one of the most toxic environmental pollutant.Interactions between cadmium and diallyl disulfide (DADS), dimethyl disulfide (DMDS) and diallyl sulfide (DAS) were investigated by differential pulse voltammtery. Determination of the stability constants of cadmium(II) with DADS, DMDS and DAS complexes was based on the DeFord–Hume methodology that calculated from the dependence of the shift of cadmium( II) peak potential upon addition of the ligands DADS, DMDA and DAS separately. The results of this study provide evidence of the formation of 1: 1 and 1: 2 complexes between cadmium(II) and DADS, DMDS and DAS with stability constants (β) in the range of ca. 10^5.8–10^6.2 and 10^9.6–10^10.6 respectively.     

New procedure for the control of the treatment of industrial effluents to remove volatile organosulfur compounds

We present a new procedure for the determination of volatile organosulfur compounds in samples of industrial effluents using dispersive liquid–liquid microextraction and gas chromatography with flame photometric detection. Initially, the extraction parameters were optimized. These included: type and volume of extraction solvent, volume of disperser solvent, salting out effect, pH, time and speed of centrifugation as well as extraction time. The procedure was validated for 30 compounds. The developed procedure has low detection limits of 0.0071–0.49 μg/L and a good precision (relative standard deviation values of 1.2–5.0 and 0.6–4.1% at concentrations of 1 and 10 μg/L, respectively). The procedure was used to determine the content of volatile organosulfur compounds in samples of effluents from the production of bitumens before and after chemical treatment, in which six compounds were identified, including 2‐mercaptoethanol, thiophenol, thioanisole, dipropyl disulfide, 1‐decanethiol, and phenyl isothiocyanate at concentrations ranging from 0.47 to 8.89 μg/L. Problems in the determination of organosulfur compounds related to considerable changes in composition of the effluents, increase in concentration of individual compounds and appearance of secondary pollutants during effluent treatment processes are also discussed.     

Determination of flavone C-glucosides in antioxidant of bamboo leaves (AOB) fortified foods by reversed-phase high-performance liquid chromatography with ultraviolet diode array detection

A sensitive solid-phase microextraction and gas chromatography-pulsed flame photometric detection technique was developed to quantify volatile sulfur compounds in wine. Eleven sulfur compounds, including hydrogen sulfide, methanethiol, ethanethiol, dimethyl sulfide, diethyl sulfide, methyl thioacetate, dimethyl disulfide, ethyl thioacetate, diethyl disulfide, dimethyl trisulfide and methionol, can be quantified simultaneously by employing three internal standards. Calibration curves were established in a synthetic wine, and linear correlation coefficients (R2) were greater than 0.99 for all target compounds. The quantification limits for most volatile sulfur compounds were 0.5 ppb or lower, except for methionol which had a detection limit of 60 ppb. The recovery was studied in synthetic wine as well as Pinot noir, Cabernet Sauvignon, Pinot Grigio, and Chardonnay wines. Although the sulfur compounds behaved differently depending on the wine matrix, recoveries of greater than 80% were achieved for all sulfur compounds. This technique was applied to analyze volatile sulfur compounds in several commercial wine samples; methionol concentrations were found at the ppm level, while the concentrations for hydrogen sulfide, methanethiol, and methyl thioacetate were at ppb levels. Only trace amounts of disulfides and trisulfides were detected, and ethanethiol was not detected.     

Volatile sulfur compounds in Cheddar cheese determined by headspace solid-phase microextraction and gas chromatograph-pulsed flame photometric detection

The aim of this study was to develop a methodology for the analysis of volatile sulfur compounds (VSCs) in Cheddar cheese. Solid-phase microextraction (SPME) was employed to extract VSCs from the cheese matrix using a CAR-PDMS fiber. This extraction method was combined with gas chromatography-pulsed flame photometric detection (GC-PFPD) to achieve high sensitivity for sulfur compounds. The impact of extraction parameters, including time, temperature and sample size, was evaluated to determine the best conditions to analyze sulfur compounds in Cheddar cheese. Hydrogen sulfide, methanethiol, and dimethyl sulfide were found to constitute the majority of the overall sulfur profile while dimethyl disulfide and dimethyl trisulfide were present in lesser amounts. Artifact formation of volatile sulfur compounds was found to be minimal. Two commercial cheese samples were analyzed and differences in sulfur content were observed. Overall, SPME-GC-PFPD was found to be a highly sensitive technique for the analysis of sulfur compounds in Cheddar cheese.     

Trace determination of volatile sulfur compounds by solid-phase microextraction and GC-MS

A method was developed for the simultaneous determination of the following nine volatile sulfur compounds in gas samples: carbon disulfide, carbonyl sulfide, ethyl sulfide, ethyl methyl sulfide, hydrogen sulfide, isopropanethiol, methanethiol, methyl disulfide and methyl sulfide. The target compounds were preconcentrated by solid-phase microextraction (SPME) and determined by gas chromatography combined with mass spectrometry. Experimental design was employed to optimize the extraction time and temperature and concurrent detection of the nine compounds was achieved by using an SPME fiber coated with Carboxen-polydimethylsiloxane (75 microns). Detection limits ranged from 1 ppt (v/v) for carbon disulfide to 350 ppt (v/v) for hydrogen sulfide and calibration functions were linear up to 20 ppb (v/v) for all the compounds investigated.     

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.     

Evaluation of desulfurization procedures for the elimination of sulfur interferences in the organotin analysis of sediments

Different clean-up and desulfurization procedures were compared in order to check their efficiency in eliminating elemental sulfur and organosulfur compounds from sediment extracts. Adsorption column chromatography cannot remove elemental sulfur or organosulfur compounds. Treatment with activated copper powder only removes elemental sulfur, but organosulfur compounds remain in the extract, and phenyltins are partially lost (up to 50%). Ligand exchange chromatography with AgNO₃-coated silica gel as adsorbent effectively removes elemental sulfur and interfering organosulfur compounds from the sediment extract allowing the quantitation of butyltins with recoveries >80%. Since the phenyltin compounds do not survive the desulfurization step, they should be measured in the untreated extract.     

A quantified ginseng (Panax ginseng C.A. Meyer) extract influences lipid acquisition and increases adiponectin expression in 3T3-L1 cells

A Panax ginseng extract (PGE) with a quantified amount of ginsenosides was utilized to investigate its potential to inhibit proliferation, influence lipid acquisition and adiponectin expression in 3T3-L1 cells. Seven fingerprint ginsenosides were quantified using high performance liquid chromatography and their respective molecular weights were further confirmed via LC-ESI-MS analysis from four different extraction methods. Extraction using methanol under reflux produced significantly higher amounts of ginsenosides. The methanol extract consisted of Rg1 (47.40 ± 4.28 mg/g, dry weight of extract), Re (61.62 ± 5.10 mg/g), Rf (6.14 ± 0.28 mg/g), Rb1 (21.73 ± 1.29 mg/g), Rc (78.79 ± 4.15 mg/g), Rb2 (56.80 ± 3.79 mg/g), Rd (5.90 ± 0.41 mg/g). MTT analysis showed that PGE had a concentration-dependent cytotoxic effect on 3T3-L1 preadipocyte and the LC(50) value was calculated to be 18.2 ± 5 μg/mL. Cell cycle analysis showed minimal changes in all four phases. Differentiating adipocytes treated with ginseng extract had a visible decrease in lipid droplets formation measured by Oil red O staining. Consequently, triglycerides levels in media significantly (P < 0.05) decreased by 39.5% and 46.1% when treated at concentrations of 1 μg/mL and 10 μg/mL compared to untreated control cells. Western blot analysis showed that the adiponectin protein expression was significantly (P < 0.05) increased at 10 μg/mL, but not at 1 μg/mL. A quantified PGE reduced the growth of 3T3-L1 cells, down-regulated lipid accumulation and up-regulated adiponectin expression in the 3T3-L1 adipocyte cell model.     

超临界CO_2萃取-分子蒸馏对大蒜化学成分的提取与分离

采用超临界CO2流体萃取技术和分子蒸馏对大蒜化学成分进行萃取与分离，用气相色谱－质谱联用技术测定其化学成分；从超临界CO2萃取物中鉴定出16种成分，经分子蒸馏后，得到4种主要成分：二烯丙基二硫、3－乙烯基－1，2－二硫代环己－5－烯、2－乙烯基－1，3－二硫代环己－5－烯及二烯丙基三硫。     

Simultaneous separation of triterpenoid saponins and flavonoid glycosides from the roots of Glycyrrhiza uralensis Fisch by pH‐zone‐refining counter‐current chromatography

Glycosides including triterpenoid saponins and flavonoid glycosides are the main constituents of Glycyrrhiza uralensis Fisch (licorice) and exhibit prominent pharmacological activities. However, conventional methods for the separation of glycosides always cause irreversible adsorption and unavoidable loss of sample due to their high hydrophilicities. The present paper describes a convenient method for the simultaneous separation of triterpenoid saponins and flavonoid glycosides from licorice by pH‐zone‐refining counter‐current chromatography. Ethyl acetate/n‐butanol/water (2:3:5, v/v) with 10 mM TFA in the upper organic stationary phase and 10 mM ammonia in the lower aqueous mobile phase was used as the biphasic solvent system. Three triterpenoid saponins and two flavonoid glycosides including licorice‐saponin A3 (63.3 mg), glycyrrhizic acid (342.2 mg), 3‐O‐[β‐d ‐glucuronopyranosyl‐(1 → 2)‐β‐d ‐galactopyranosyl]glycyrrhetic acid (56.0 mg), liquiritin apioside (232.6 mg), and liquiritin (386.5 mg) were successfully obtained from licorice ethanol extract (2 g) in one step. This method subtly takes advantage of the common acidic properties of triterpenoid saponins and flavonoid glycosides, and obviously is much more efficient and convenient than the previous methods. It is also the first time that the separation of acidic triterpenoid saponins by using pH‐zone‐refining counter‐current chromatography has been reported.