Received date: July 29, 2013; Accepted date: August 21, 2013; Published date: August 24, 2013
Citation: Watson CJ, de Souza D, Silva C, Tull D, Garland SM, et al. (2013) Attempt to Detect Garlic Allyl Sulphides from Saliva after Consumption of Garlic Tablets Using GC-MS. J Microb Biochem Technol 5:081-083. doi:10.4172/1948-5948.1000104
Copyright: © 2013 Watson CJ, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Allicin is an organosulfur compound of Allium sativum (garlic) with anti-fungal and antimicrobial properties. However, to date, neither allicin nor its metabolites has been detected in human organs or bodily fluids following oral consumption of garlic. Bioavailability has only been demonstrated using breath studies. Enteric-coated formulations are designed to pass through the acidic stomach environment; hence detection of garlic compounds in saliva following consumption of enteric-coated garlic tablets would provide evidence of bioavailability in a body secretion. An attempt was made to identify metabolites dially disulphide and allyl methyl sulphide using Gas Chromatography Mass Spectrometry (GC-MS). We completed a single participant mass spectrometry study using 13 time points over 24 hours following consumption of 20 enteric-coated garlic tablets. There was no detection of allicin derived sulfides at any time point. In summary, it is possible that these highly volatile compounds may be more readily detectable using a solid phase micro-extraction GC-MS, or headspace analysis methodology, although the less volatile allyl methyl sulfone and allyl methyl sulfoxide may be the main metabolites present.
Allium sativum; GC-MS; Allicin bioavailability; Garlic; Diallyl disulphide; Allyl methyl sulphide
DADS: Diallyl disulphide; DAS: Diallyl Sulphide; DATS: Diallyl Trisulphide; DMTS: Dimethyl Trisulphide AM: Allyl Mercaptan; AMDS: Allyl mMethyl Disulphide; AMS: Allyl Methyl Sulphide; AMSO: Allyl Methyl Sulfoxide; AMSO2: Allyl Methyl Sulfone; AMTS: Allyl Methyl Trisulphide; GC-MS: Gas Chromatography Mass Spectrometry
Garlic produces volatile sulfur compounds, to which many antifungal and antimicrobial properties have been attributed. The authors were interested in the main volatile sulphur compound, allicin, and its potential as a systemic anti-fungal agent. However, the presence of allicin or its metabolites in human fluids has not been reported [1,2].
During the digestive process, allicin quickly metabolizes into other sulphur compounds [3-5]. Thus far, bioavailability has only been measurable by breath studies, which have detected the presence of metabolites AM, AMS and DADS after oral consumption [5,6]. Only traces of allicin were found in blood after being incubated for five minutes . In another in vivo study, DADS was spiked into rat’s blood and identified using GC-MS .
Previously in animals, organosulphur compounds from garlic have been detected using GCMS [9,10]. In rats, DADS, AM, AMSO and AMSO2 were found in stomach, liver, plasma and urine after consumption of 200 mg/kg of body weight. Time-dependent tissue concentrations of DADS were highest within 24 hours and not detectable by 72 hours. In plasma, DADS appeared transiently for 20 minutes then was undetectable. Only trace amounts of AMS were detectable in urine on Day 2, while AMSO and AMSO2 were excreted for a longer time periods, up to 48 hours . Breath AMS has been used to measure bioavailability of garlic thiosulfinates .
In this study, an attempt was made to extract and detect allicin transformation products, AMS and DADS, from saliva following ingestion of enteric-coated garlic tablets.
Ethics approval was obtained from University of Melbourne Human Research Ethics Committee (ID 1033568.1).
Garlic allyl sulphide standards were purchased from MP Biomedicals Australia for validation and refinement of methods. 25 mg of each of the following standards was purchased: allyl disulphide (purity 91.2%; molecular weight 146.27 g/mol), allyl trisulphide (molecular weight 178.3 g/mol) and allyl methyl sulphide (molecular weight 88.17 g/mol). Standards were diluted in the concentration range of 0.50-50 μg/mL in hexane (w/v) for use as points for a linear standard curve, and to determine limits of detection. Additional testing was performed using steam-distilled garlic oil, diluted in hexane to 0.0005% v/v, as a source of a large variety of allyl sulphides.
Using an Agilent 7890a Gas Chromatograph coupled to a 5975C mass selective detector, cryogenic Gas Chromatography (GC) conditions were refined using standards and essential oils. Samples were separated using a 30 m Varian Factor Four VF-5 ms column with a 10m ezi-guard column attached. Column dimensions were 250 μm inner diameters with 0.25 μm film thickness. Oven ramp parameters used were -20°C held for 2 minutes, 10°C/min to 200°C, 35°C/min to 300°C held for 1 minute. Total run time was 27.85 min. Scan range was 30-300 amu, scan rate 15.26 scans/sec. Ionisation voltage was 70eV.
A number of common ions were selected for the allicin transformation products, these were: m/z 41, 45, 73, 88, 111 and 144.
Allyl disulfide and diallyl trisulfide were tested using a concentration range of 0.50-50 μg/Ml (w/v) in hexane. Samples were run on the cryogenic SIM method  and areas quantified for the expected peak.
Garlicin™ tablets, each containing 350 mg garlic powder and yielding 3200 μg of allicin (label claim), with batch number 591678 (expiry date 10/2011), were purchased from an online distributor. This brand has been reported to give high allicin bioavailability . As the time for AMS to reach maximum breath concentrations (tmax) varies from individual to individual , estimates were made for the sole participant of this study. Consumption of three or more Garlicin™ tablets commonly causes temporary abdominal discomfort upon disintegration. By recording the time delay between tablet consumption and abdominal discomfort or garlic breath/taste experienced in five tests, a mean tablet disintegration time of 5.5 hours was calculated. Following consumption of 20 Garlicin™ tablets at 0800, saliva samples were collected every hour for 12 hours, and a further sample at 0800h the following day. The samples were collected in 0.5 ml Eppindorf tubes and placed immediately in the freezer and stored at -80°C until processing. Samples were extracted into hexane at a ratio of 3:1 hexane: saliva to precipitate salivary proteins.
A chromatogram of steam-distilled garlic oil, containing a variety of allyl sulphides, is shown in Figure 1a. An example standard curve for allyl disulfide is shown in Figure 2. No allyl sulphides matching known standards were detected in the saliva at any of the time points (Figure 1b).
The method used had a sensitivity of 0.013 umol for AMS or 0.010 umol DADS mL-1 saliva. The detection of AMS or DADS would have been possible only if at least 10-50% of the consumed allicin had been metabolized to these compounds. Equal availability in saliva as in the total body water or blood would also have been necessary for detection of these compounds. Organosulphur compounds from garlic have never been detected in human organs or bodily fluids following oral consumption. Detection of garlic compounds in saliva following ingestion of enteric-coated tablets may, with methodological refinement, provide an alternative to breathe studies to prove bioavailability by enabling direct detection of organosulphur compounds in body fluids following consumption. In this small study, standards for allyl sulphides from Allium sativum were detected by GC-MS when injected alone and when spiked into saliva (not shown), which enabled full spectral identification of these compounds. However, the sensitivity of the method outlined was not sufficient for detection of physiological levels of allicin metabolites AMS and DADS in saliva after consumption of large amounts of garlic tablets. The presence of electrolytes in saliva may have affected the solubility of AMS. Measuring peroxidases in saliva  for kinetics of oxidation of AMS to AMSO and AMSO2 may have clarified breakdown products present. It is possible that these highly volatile compounds may be more readily detectable using a solid phase micro-extraction GC-MS, or headspace analysis methodology [14-16]. Nevertheless, it appears clear that AMS and DADS are not significantly found in saliva after consuming large amounts of garlic, although the presence of allicin metabolites AMSO and AMSO2 needs yet to be evaluated.
Ashley Dowell, Manager, Analytical Division, Organisational Unit, Southern Cross Plant Science Lismore Australia is gratefully acknowledged for his advice with GC-MS techniques. Professional Development Scholarship: ASHHNA Australasian Sexual and HIV Nurses Association / CSIRO was used to purchase the garlic allyl sulphide standards. Metabolomics Australia, Bio21 is gratefully acknowledged for undertaking the GC-MS Cathy Watson was supported by a student stipend from the University of Melbourne.