The Chemical Compositions of the Volatile Oils of Garlic (Allium sativum) and Wild Garlic (Allium vineale)

Garlic, Allium sativum, is broadly used around the world for its numerous culinary and medicinal uses. Wild garlic, Allium vineale, has been used as a substitute for garlic, both in food as well as in herbal medicine. The present study investigated the chemical compositions of A. sativum and A. vineale essential oils. The essential oils from the bulbs of A. sativum, cultivated in Spain, were obtained by three different methods: laboratory hydrodistillation, industrial hydrodistillation, and industrial steam distillation. The essential oils of wild-growing A. vineale from north Alabama were obtained by hydrodistillation. The resulting essential oils were analyzed by gas chromatography-flame ionization detection (GC-FID) and gas chromatography-mass spectrometry (GC-MS). Both A. sativum and A. vineale oils were dominated by allyl polysulfides. There were minor quantitative differences between the A. sativum oils owing to the distillation methods employed, as well as differences from previously reported garlic oils from other geographical locations. Allium vineale oil showed a qualitative similarity to Allium ursinum essential oil. The compositions of garlic and wild garlic are consistent with their use as flavoring agents in foods as well as their uses as herbal medicines. However, quantitative differences are likely to affect the flavor and bioactivity profiles of these Allium species.


Introduction
Garlic (Allium sativum L., Amaryllidaceae) likely originated in Central Asia [1]. The plant has been used as a flavoring agent and a traditional medicine since antiquity, and is now cultivated worldwide [1,2]. Allium vineale L. (wild garlic, crow garlic) is native to Great Britain, most of Europe, North Africa, and the Middle East. The plant has been introduced to North America, Australia, and New Zealand [3].
Allium sativum has been used as a diaphoretic, diuretic, expectorant, and stimulant [4]. Extracts of A. sativum have shown broad-spectrum antibacterial [5] and antifungal [6] activity and the plant has been used to treat tuberculosis, coughs, and colds [7]. Garlic preparations have demonstrated hypotensive activity in moderately hypertensive subjects, and garlic-based phytotherapeutic products are used in France for minor vascular disorders [8]. There is an inverse correlation between regular consumption of garlic and stomach cancer frequency [8], but there seems to be no correlation between garlic consumption and other cancers. Garlic has been used in food preparation not only for its flavor, but also as a digestive aid [4]. Allium vineale has been used as a substitute for A. sativum in cooking; the bulb is used as a flavoring agent and the leaves as an addition to salad [9,10]. Cherokee Native Americans used both A. vineale and A. sativum as carminatives, diuretics, and expectorants [11,12].
Although there have been numerous investigations on the phytochemistry of garlic (A. sativum) [1,13,14], the chemistry of wild garlic (A. vineale) has not been investigated, and because of the history of the uses of Allium species as both condiments and phytopharmaceuticals, we have investigated the essential oil compositions of A. sativum from Spain, obtained by different isolation methods, and A. vineale growing wild in north Alabama, USA.

Allium sativum
Bulbs of Allium sativum were collected from a field in Las Pedroñeras, Spain (39 • 26 59" N, 2 • 40 23" W, 745 m elevation), in December 2015. Garlic bulbs were finely chopped, and were subjected to three different distillation methods: laboratory hydrodistillation using a Clevenger apparatus for 3 h, industrial hydrodistillation for 4 h, and industrial steam distillation for 5 h. Pale yellow essential oils were obtained in 0.2%, 0.22% and 0.18% yields, respectively. The obtained essential oils and hydrosol were separated by decantation; remaining water was removed from the essential oils with sodium chloride. The collected essential oil samples were stored under refrigeration (−4 • C) until analysis.

Allium vineale
Four different samples of Allium vineale were collected from a field in Huntsville, Alabama (34 • 38 46" N, 86 • 33 27" W, 191 m elevation) on 10 April 2017, 8 a.m. Each sample was cleaned of debris, the entire plant (leaves and bulbs) chopped, and hydrodistilled using a Likens-Nickerson apparatus for 4 h with continuous extraction with dichloromethane (CH 2 Cl 2 ). Evaporation of the dichloromethane yielded pale yellow essential oils with an extremely pungent odor (Table 1).

Gas Chromatography-Mass Spectrometry (GC-MS)
GC-MS characterization of A. sativum oils was carried out as previously described using a Shimadzu GCMS-QP2010 Ultra (Shimadzu Scientific Instruments, Columbia, MD, USA) [15,16]. This instrument was operated in the electron impact (EI) mode set at electron energy 70 eV with a scan range of 40-400 amu, a scan rate of 3.0 scans per second, and with GC-MS solution software. A ZB-5 fused silica capillary column (Phenomenex, Torrance, CA, USA), 30 m length × 0.25 mm inner diameter, with a (5% phenyl)-polymethylsiloxane stationary phase and a film thickness of 0.25 µm was used as the GC column. Helium was used as the carrier gas and the pressure was set at 551.6 kPa with a flow rate of 1.37 mL/min on the column head. The temperature of the injector was set at 250 • C and the temperature of the ion source was set at 200 • C. The temperature of the GC oven was programmed to be 50 • C initially and was programmed to increase at a rate of 2 • C/min to a final temperature of 260 • C. The samples were prepared with CH 2 Cl 2 in a 5% w/v solution. Then, 0.1 µL of the solutions were injected into the instrument with a split ratio of 30:1.
The retention indices were determined by reference to a homologous series of n-alkanes. The components of each essential oil sample were identified based on their retention indices and mass spectral fragmentation patterns compared to reference literature [18][19][20][21][22] and our in-house library.

Semi-Quantitative Gas Chromatography
Semi-quantitative GC was performed with an Agilent 6890 GC with Agilent FID (flame ionization detector) (Agilent Technologies), HP-5ms column (30 m length × 0.25 mm inner diameter × 0.25 µm film thickness), He carrier gas, head pressure (144.1 kPa), flow rate (2.0 mL/min); oven temperature program (as above). The percent compositions of the essential oils were determined from raw peak area percentages without standardization.

Conclusions
The essential oils of garlic and wild garlic are shown to be dominated by sulfur-containing compounds, particularly allyl polysulfides. Garlic oils from various geographical locations have shown qualitative similarities, but quantitative differences in the concentrations of organosulfur compounds, and are likely to affect both the medicinal and the organoleptic properties of the garlic. Wild garlic is qualitatively similar in composition to garlic, but there are some key differences: diallyl disulfide and diallyl trisulfide concentrations are higher in garlic than in wild garlic, while allyl 1-propenyl disulfide and dimethyl trisulfide concentrations are higher in wild garlic than in garlic.