A New Essential Oil Chemotype of Allium Roseum Analyzed by an Apolar Column

Abstract

This study deals with the chemical characterization of a medicinal and an aromatic plant of the Tunisian flora: Allium roseum var. odoratissimum, and aimed to identify new bioactive natural compounds in its flower essential oil. These compounds were extracted by hydrodistillation and were analyzed by GC and GC/MS, using an apolar column. The most important compounds characterized were organo-sulphurous (46%), including methyl 2-propenyl trisulfide, di-2-propenyl trisulfide, di-1-propenyl disulfide, di-2-propenyl disulfide, dimethyl trisulfide, methyl 2-propenyl disulfide, and di-1-propenyl trisulfide, found as 10.75, 9.07, 5.81, 4.98, 3.90, 3.30, and 2.53%, respectively. Moreover, heneicosane and pentacosane were identified for the first time at relatively high rates (8.18 and 4.49%, respectively) in the Allium roseum essential oil. This essential oil composition exhibited newly identified sulphurous compounds at relatively high rates (46.53%) when compared with what was found while using polar column.

Keywords:

• Allium roseum
• Essential oil
• Apolar column
• GCMS
• Bioactive compounds
• Sulphurous compounds

INTRODUCTION

For years, many plants have been used as remedies for human illness because of their issued natural substances having therapeutic values,[1] as secondary metabolites that have witnessed pharmacological interest.[2–4 Among such famous plants, Allium is the largest and the most important representative genus of the Liliaceae family, which includes 780 species and is widely distributed in the Northern hemisphere.[5] Besides the well known garlic (Allium sativum L.) and onion (Allium cepa L.), several other species are widely growing for culinary use, such as leek (Allium porrum L.), scallion (Allium fistulosum L.) and shallot (Allium ascalonicum Hort.). Within the garlic species, the most common ones are wild garlic (Allium ursinum L.), elephant garlic (Allium ampeloprasum L.), chive (Allium schoenoprasum L.), and Chinese chive (Allium tuberosum L.).[6]

For many centuries, several species of Liliaceae family have been used as vegetables and spices as well as medicinal properties, curing various diseases with their anticarcinogenic, antithrombotic, antiatherosclerotic, hypolipidemic, antihypertensive, and antimicrobial potential.[7–10 The Allium genus has an important economical value because of its variety of uses: in food or dietary supplement, in health issues, and in ornament (its flowers).[5] Furthermore, the first citation of these plants is found in the Codex Ebers (1550 BC), an Egyptian medical papyrus reporting several therapeutic formulas, based on garlic and onion. In fact, they were used as remedies for a variety of diseases, such as heart problems, headache, bites, worms, several epidemic diseases, antipyretic, and tumours.[6] At present, epidemiological, clinical, and laboratory data have proved that garlic contains many biologically and pharmacologically important beneficial compounds to human health, especially in the prevention and treatment of cancer.[11] Moreover, antioxidative activity of some of Allium's species has been reported and has mainly been attributed to a variety of organo-sulphurous compounds and their precursors.[12] Indeed, the majority of Allium species, especially garlic, contain a variety of secondary sulphur compounds,[13] which are responsible for the distinctive smell and taste, and at the origin of the major bioactive compounds, attributing to Allium's plants as well as their well known medicinal properties.[6,7,14,15]

The Allium roseum, a very polymorphous species, is represented in North Africa by 12 different taxa, including equal varieties, subvarieties, and forms. In Tunisia, only three varieties are available: var. grandiflorum, var. perrotii, and var. odoratissimum.[16] Considered as an endemic taxon in North Africa, the odoratissimum variety is a perennial spontaneous weed. Its oblong bulb grows to about 30–60 cm in height, its flowers are wide, rosy or white colored, and characterized by eyelet-smell.[17] The plant, found in grassy and bushy places, can adapt to poor and sandy soil; for a long time, Allium roseum var. odoratissimum has been used as a vegetable, spice, or herbal remedy for headache and rheumatism treatments.[16] Indeed, according to the World Health Organization, 80% of the world population uses traditional medicine as an alternative to prevent the secondary effects of medicines.[18]

At present, the main orientation to food rich in secondary metabolites, known as nutraceuticals, is a general trend because of its beneficial effects on health with their preventive and therapeutic roles ensuring human well-being. In this perspective, the analysis of these secondary metabolites from the edible Allium genera was conducted especially on garlic and onion evidencing the presence of organo-sulphurous compounds in these food plants.[6]

Nevertheless, apart from our previous work[19] and that of Ben Jannet et al.,[20] scarce information is available concerning the chemical composition of Allium roseum var. odoratissimum flowers essential oil. In our former work, essential oil extracted from the flower gathered in Bengardane and analyzed by means of a polar column, evidenced limited number of eluted compounds. In the latter study, the essential oil composition of Allium roseum (flower and stem) gathered in the region of Monastir (center of Tunisia) and analyzed by means of an apolar column allowed the identification of other compounds. In the present study, we investigated the flower essential oil composition gathered from Bengardane, analyzed by an apolar column, and compared the identified compounds with what was previously reported in the bibliography.

MATERIALS AND METHODS

Plant Material

Allium roseum var. odoratissimum was gathered from Bengardane area, in the South-East of Tunisia at the flowering stage (February 2006) and identified according to the “Flora of Tunisia.”[16] Specimens collected were deposited at the herbarium of the Institute of Arid Areas in Medenine (Tunisia). The fresh flowers were separated from the lignified part and were used for hydrodistillation.

Essential Oil Extraction

The fresh flowers (100 g) were submitted to hydrodistillation for 4 h in a Clevenger-type apparatus.[21] The recovered essential oil was dried through anhydrous sodium sulphate. Allium roseum essential oil yield based on a wet weight basis was 0.05%.

Essential Oil Analysis

Gas chromatography

A Hewlett-Packard 5890 series II gas chromatograph equipped with HP-5MS capillary column (Agilent Technologies, USA) (30 m × 0.25 mm i.d., film thickness 0.25 μm; Hewlett-Packard, Genova, Switzerland) and connected to a flame ionization detector (FID) was used. The column temperature was programmed at 50°C for 1 min, then 7°C/min to 250°C for 5 min. The injection port temperature was 240°C and that of the detector 250 °C. The carrier gas was helium (99.995% purity) at a constant flow of 1.2 ml/min. The analysis was performed on 2 μl volume sample. Percentages of the constituents were calculated by electronic integration of FID peak areas, without the use of response factor correction. Kovàts indices were calculated for separate compounds relative to C₅-C₃₀ n-alkanes mixture (Aldrich Library of chemicals standards).[22] All the essays were performed three times.

Gas chromatography—Mass spectrometry (GC/MS)

The isolated volatile compounds were analyzed by GC-MS, using a Hewlett-Packard 5890 series II gas chromatograph. The fused HP-5MS capillary column (the same as that used in the GC analysis) was coupled to a HP 5972A masse-selective detector (Hewlett-Packard, Palo Alto, CA, USA). The oven temperature was programmed from 50°C (1 min) to 250°C (5 min) at 7°C/min. The temperature of the injector port was held at 250°C, split ratio, 1/100, the temperature of the detector was set at 280°C. The carrier gas was helium (99.995% purity), with a flow rate of 1.2 ml/min and the analyzed sample volume was 2 μl. The mass spectrometer (MS) conditions were as follows: ionization voltage, 70 eV; ion source temperature, 150°C; electron ionization mass spectra were acquired in the mass range 35–350 Da.

Volatile Compounds Identification

The compounds of the essential oil were identified by comparing the mass spectra data with spectra available from the Wiley 275 and NIST 0.5a mass spectra libraries. Further identification confirmations were made referring to retention index data generated from a series of known standards of n-alkanes mixture (C₅-C₃₀) (Aldrich Library of chemicals standards)[22] and to those previously reported in the bibliography.[23–39

RESULTS AND DISCUSSION

The A. roseum var. odoratissimum, fresh flowers essential oil extracted through hydrodistillation had a light yellow color and a pungent odor at room temperature. The essential oil global chromatographic analysis by GC and GC/MS showed 44 compounds representing 79.29% of the whole oil constituents. Among these, some compounds were at relatively low rates (Table 1). The studied Allium roseum species identified compounds can be classified into sulphurous and non-sulphurous compounds (Table 2).

Table 1 Composition in percentage of the fresh flowers essential oil from Allium roseum (Bengardane, Tunisia) and RI comparison according to the literature (LRI)

Peak number	Compound	RI*	Percentage	LRI
1	Methional	901	0.13	906 [23]
2	2,5-Dimethyl thiophene	905	0.23	905 [24]
3	Methyl 2-propenyl disulfide	923	3.30	913 [25]
4	Methyl 1-propenyl disulfide	930	0.64	930 [24]
5	1,3-Dithiane	934	1.65	Not available
6	Dimethyl trisulfide	955	3.9	956 [26]
7	1,3,5-Trimethyl benzene	970	0.11	975 [27]
8	Decane	1000	0.18	1000 [28]
9	p-Cymene	1025	0.22	1023 [29]
10	Limonene	1029	0.29	1028 [29]
11	1,8-Cineole	1031	0.05	1030 [29]
12	γ-Terpinene	1059	0.14	1057 [30]
13	Di-2-propenyl disulfide	1071	4.98	1076 [25]
14	Undecane	1100	0.42	1100 [29]
15	1-Propenyl 2-propenyl disulfide	1104	2.42	1093 [31]
16	Di-1-propenyl disulfide	1107	5.81	Not available
17	Methyl 2-propenyl trisulfide	1130	10.75	1130 [31]
18	Camphor	1147	1.35	1144 [29]
19	2,5-Dichloro thiophene	1165	2.05	Not available
20	Decanal	1180	1.16	1188 [32]
21	2-Vinyl-4H-1,3-dithin	1195	0.33	1206 [25]
22	1,4-Dimethyl tetrasulfide	1203	1.86	1215 [30]
23	Carvacrol	1302	0.83	1300 [29]
24	Di-2-propenyl trisulfide	1312	9.07	1312 [24]
25	Di-1-propenyl trisulfide	1313	2.53	Not available
26	Eugenol	1358	2.32	1358 [28]
27	5-Methyl-1,2,3,4-tetrathia cyclohexane	1383	0.55	Not available
28	Methyl eugenol	1404	0.24	1407 [33]
29	Methyl cis-3-chloropropenoate	1412	0.14	Not available
Peak number	Compound ^a	RI ^b	Percentage	LRI ^c
30	β-Caryophylene	1420	0.68	1423 [34]
31	Isoeugenol	1445	0.51	1436 [35]
32	Diallyl tetrasulfide	1555	2.14	1555 [24]
33	D-Nerolidol	1563	1.17	1561 [36]
34	(E,E)-Farnesol	1722	0.27	1722 [37]
35	Benzyl benzoate	1762	0.16	1764 [37]
36	Pentadecanoic acid	1873	0.14	1870 [38]
37	Nonadecane	1900	0.39	1900 [30]
38	Dibutyl phthalate	1968	0.26	1965 [39]
39	Hexadecanoic acid	1972	2.18	1975 [25]
40	Eicosane	2000	0.34	2000 [30]
41	Heneicosane	2100	8.18	2100 [30]
42	Octadecanoic acid	2178	0.18	2172 [38]
43	Tetracosane	2400	0.55	2400 [30]
44	Pentacosane	2500	4.49	2500 [30]
^aCompounds are listed in order of their elution from the HP 5MS column.
^bRetention Index: Calculated against C5-C30 n-alkanes mixture on HP-5MS column.
^cReferences: [23] Jarunrattanasri et al., 2007; [24] Pyun and Shin, 2006 [25] Ansorena et al., 2001; [26] Bonaïti et al., 2005; [27] Tzakou et al., 2004; [28] Salido et al., 2004; [29] Hudaib et al., 2002; [30] Harzallah-Skhiri et al., 2005; [31] Godevac et al., 2008; [32] Mahmood et al., 2004; [33] Cole et al., 2007; [34] Asuming et al., 2005; [35] Zeng et al., 2007; [36] Sarouglou et al., 2006; [37] Skaltsa et al., 2001; [38] Conforti et al., 2009; [39] Zhao et al., 2005.

Table 2 Sulphurous compounds and non sulphurous compounds of the essential oil from fresh flowers of Allium roseum (Bengardane, Tunisia)

Compounds	Percentages
Sulphurous compounds	46.53
Cyclic compounds	04.81
Non cyclic compounds	41.72
Non sulphurous compounds
Hydrocarbons	15.66
Phenolic derivatives	4.06
Aldehydes	1.16
Cyclic ketones and cyclic ethers	1.40
Aromatic compounds	6.14
Fatty acids	2.50
Alcohols	1.44
Esters and diacids	0.40

Sulphurous Compounds

Table 2 shows that almost half of the eluted compounds included sulphur. Within this group, 10 compounds were identified at relatively high rates, ranging from 2.05 to 10.75%. Among these sulphurous organic compounds, the most abundant ones were the substituted propenyl trisulfides, the major was the methyl 2-propenyl trisulfide followed by the di-2-propenyl trisulfide and these were isolated for the first time in the Tunisian Allium roseum. According to the peak areas of the eluted sulphurous compounds, the trisulfides (26.2%) were the most widely distributed in Allium roseum flowers essential oil, followed by the disulfides (17.15%), and finally the tetrasulphide (4%) (Table 1). While considering the sulfurous compounds eluted by polar column, the methional—a monosulfide compound—was identified at a high concentration (17%).[19] Nevertheless, this compound was minor with a rate of 0.1% in the present study. Most of these compounds were reported as therapeutically active, mainly as anticancerous and cholesterol-lowering agents.[7,8] This organic sulphurous compounds' content is twice as high as that of the previously published data for the same essential oil where their concentration was 23.6%.[19] This doubled rate of sulphurous compounds noticed in the same species could be attributed to the analytical technique used.[24] This group can be further classified into two major groups according to their acyclic or cyclic structure.

Acyclic sulphurous compounds

The acyclic group consisted of various compounds from mono to polysulfide substituted by different radicals. It included both saturated and unsaturated compounds, but only the total relative percentage of unsaturated compounds was considered (39.5%). The volatile components in the Allium species are mainly represented by thiosulfinates, which are very unstable constituents, giving rise to further rearrangements, leading to a wide variety of derived organo-sulphurous compounds. Their structure, biogenesis and variability were well studied.[6]

In garlic essential oil, the most abundant volatile compounds were diallyl disulfide followed by diallyl trisulfide,[8] while in Allium roseum species, the trisulfide compounds (26.25%) were higher than the disulfide compounds (17.15%) (Table 1). The abundance of these compounds was also mentioned in Allium cepa L. and Allium fistulosum L. essential oils,[24,40] where six acyclic sulphurous compounds were mainly identified (methyl 2-propenyl trisulfide, di-2-propenyl trisulfide, 1-propenyl 2-propenyl disulfide, di-1-propenyl disulfide, di-(2-propenyl) disulfide, di-1-propenyl disutfide, and dimethyl trisulfide).

The essential oil composition of the studied plant originating from the south of Tunisia, by means of an apolar column has not been reported so far and the results of the composition of this unique and endemic species are evidenced for the first time. It should be mentioned that, according to many other authors,[14,15,41] the S-containing compounds are responsible for the appropriate smell and taste and also for the health benefits of Liliaceae family plants. These characteristics indicated the important pharmacological value of the studied species and may justify its diverse uses in traditional medicine.

It would be noteworthy to point out that the composition of volatile oils differs greatly among species, and even in the same species, possibly due to analytical technique, chemotype, and several meteorological factors, such as culture climate, harvesting conditions, and other factors that may affect biological activities.[24] The thiosulfonates are very unstable and would further decompose to produce a series of degraded compounds including diallyl disulphides that are partly responsible for: (i) anti-cancer effect, (ii) cardiovascular diseases protection,[7,41,42] (iii) food-bone pathogenic bacteria antimicrobial activity.[43] All of the allyl derivatives were demonstrated to interact with membrane lipids modifying its fluidity.[44]

Cyclic sulphurous compounds

The major cyclic sulphur components were found to be the 2,5-dichlorothiophene and the 1,3-dithiane, evidenced for the first time in the essential oil of Allium genus with a relatively low level and would qualitatively characterize the essential oil of Allium roseum L. var. odoratissimum. Other cyclic compounds were found at minor rates and included the 5-methyl-1,2,3,4-tetrathia cyclohexane, the 2-vinyl-4H-1,3-dithin and the 2,5-dimethyl thiophene. The first compound was also found in the essential oil of Allium sativum with a relatively low rate.[45] The 2-vinyl-4H-1,3-dithin, found in trace amounts, would be generated from allicin degradation during thermal gas chromatographic analysis,[46] as follows:

However, Abu-Lafi et al. reported that the 2-vinyl-4H-1,3-dithin was the major organo-sulphurous compound in the fresh Allium sativum that undergoes decomposition during various extracting procedures (steam distillation, hydrodistillation, different solvents extraction), generating many other degradation-derivatives sulphurous compounds.[47] Low temperature extraction of fresh garlic followed by cryogenic GC-MS and HPLC analysis have been used to support this hypothesis.[46] Thus, the effect of heat seems to be the crucial factor enhancing such degradative reactions. Indeed, it was noted that in garlic, the 2-vinyl-4H-1,3-dithin rate decreased as the microwave heating proceeded.[48] More recently, Sowbhagya et al. evidenced such compounds in garlic volatile oil.[49]

Non-Sulphurous Compounds

The Allium roseum essential oil non-sulphurous compounds contained essentially hydrocarbons (saturated aliphatic compounds, particularly heneicosane and pentacosane), presenting 15.66% of the total compounds (Table 2). Besides, the aromatic compounds particularly the eugenol and its derivatives (3.07%), were the second abundant group of this oil, and were responsible for the species eyelet-smell. Moreover, the studied volatile oil included fatty acids (2.50%) at relatively low rates. Indeed, the sample was not methylated and hence fatty compounds would not be in the suitable conditions for being identified.

The range of non-sulphurous compounds rates eluted by an apolar column was completely different from that found while using a polar column.[19] Indeed, eugenol was a major compound (12.7%) and its methylated derivative was at a higher concentration compared to the present results where eugenol and methyl eugenol concentrations were reduced by fivefold and cis-isoeugenol was newly isolated. The same trend was noticed for the benzyl benzoate with its relatively low concentration, but detected at a higher rate while using a polar column (0.7%). Comparatively, camphor at 13.4% rate determined by the latter column was reduced by tenfold in the present work. However, carvacrol rate had slightly increased while eluted with an apolar column. The difference noticed in rates is related to the column characteristics. Furthermore, it was demonstrated that the Allium sativum L. treatment has many effects on the volatile compounds contents.[45,47–53 As a result, according to the used column, new compounds having a health interest may be found and not evidenced, especially the new chemotypes 1-propenyl 2-propenyl disulfide and di-1-propenyl disulfide.

Mass Spectral Characterization of the Newly Isolated Allium roseum Oil Sulphurous Compounds

Mass spectral of some newly isolated sulphurous compounds from Allium roseum oil and their relative area percentages are presented in Table 3. The major constituent was methyl 2-propenyl trisulfide. It had M⁺ ion at 152 in its mass spectrum. Fragments at m/z 111, 79, 73 corresponded to [SSSCH₃]⁺, [SSCH₃]⁺, and [SCH₂CH=CH₂]⁺. Furthermore, fragment at m/z 87 was the base peak. Moreover, a closely related compound to this major constituent, having a retention time of 5.52 min, was characterized by a mass ion m/z 120 and identified as methyl 2-propenyl disulfide. It is noticeable that this compound was previously isolated in distilled oil from Welsh onions (Allium fustulosum L. variety Maichuon) and scallions (Allium fustulosum L. variety Caespitosum).[54]

Table 3 Mass spectral data of some new sulphurous compounds isolated from Allium roseum essential oil

Compound (retention time in min)	Percentage	MW	Structure	m/z (%)
2,5-Dimethyl thiophene (5.38)	0.2	112		112 (73), 111 (100), 97 (45), 77 (10), 71 (4), 69 (6)
Methyl 2-propenyl disulfide (5.52)	3.3	120		120 (100), 80 (6), 79 (10), 73 (8), 71 (4), 64 (6)
1,3-Dithiane (5.78)	1.6	120		120 (100), 105 (4), 87 (4), 80 (12), 75 (20), 73 (12), 72 (24)
Dimethyl trisulfide (6.24)	3.9	126		126(100), 111(15), 80(10), 79(30), 78(4), 64(11)
Di-2-propenyl disulfide (7.64)	4.9	146		146 (77), 113 (62), 105 (55), 85 (11), 81 (100), 79 (38), 73 (40), 64 (15)
1-Propenyl 2-propenyl disulfide (7.84)	2.4	146		146 (100), 105 (32), 81 (53), 73 (57), 71 (22), 61 (17)
Di-1-propenyl disulfide (7.91)	5.8	146		146 (100), 105 (45), 81 (64), 73 (75), 71 (28), 61 (28)
Methyl 2-propenyl trisulfide (8.53)	10.7	152		152 (12), 111 (16), 88 (12), 87 (100), 79 (14), 73 (78), 64 (12)
2-Vinyl-4H-1,3-dithin (9.60)	0.3	144		144 (74), 111 (60), 103 (17), 97 (21), 85 (15), 79 (13), 72 (100), 71 (85), 69 (13)
1,4-Dimethyl tetrasulfide (9.71)	1.9	158		158 (100), 111 (8), 94 (12), 80 (12), 79 (50), 64 (16)
Di-2-propenyl trisulfide (10.77)	9.1	178		178 (11), 113 (100), 104 (9), 103 (9), 73 (89), 72 (11), 71 (11), 64 (9)
Diallyl tetrasulfide (13.95)	2.1	210		210 (21), 146 (100), 136 (34), 105 (55), 93 (68), 81 (55), 73 (81), 69 (89), 64 (89)

The second major compound with a retention time of 10.77 min, exhibited an M^+. ion at m/z 178. The peak at m/z 114 and the base peak at m/z 113 corresponded to [CH₂=CHCH₂SCH₂CH=CH₂]⁺ and to ion from hydrogen loss of the latter. The other fragments at m/z 73, 72, and 71 were linked to [SCH₂CH=CH₂]⁺ and to ions resulting from hydrogen loss. The compound was identified as di-2-propenyl trisulfide based on these mass spectral assignments and their good matching with library spectrum. Apart from these compounds, other important constituents were isomeric di-1-propenyl disulfide, 1-propenyl 2-propenyl disulfide, and di-2-propenyl disulfide with a specific molecular weight of 146. Their MS displayed the same peaks at m/z 105 and 73 were linked to the fragment ions [SSCH₂CH=CH₂]⁺ and [SCH₂CH=CH₂]⁺. Finally, another interesting compound was eluted at 6.24 min and had M^+. ion at m/z 126. Its MS displayed peaks at m/z 111, 79, and 64, corresponding to the following fragment ions: [SSSCH₃]⁺, [SSCH₃]⁺, and [SS]⁺. This compound was identified as dimethyl trisulfide, based on its mass spectrum compared to the library spectrum data.

Bioactive Molecules of the Inter Alia Genus Allium

In many parts of the world, the common garlic Allium sativum L. is used widely as food flavoring and is well known in folk medicine.[6–8] However, the pink garlic (Allium roseum L.), less spread throughout the world and found as an endemic plant in some countries,[16] has not yet been studied regarding its bioactive molecules, and hence very little is known about its secondary metabolites activities.[16,19,55] These compounds, characteristic of each species in their flavors and odors, are generated by chemical transformations of a series of volatile sulfur compounds. It is well known that plants produce a very large number of small molecular weight compounds or secondary metabolites, and 10 to 15% of each plant genome is devoted to secondary metabolism. The Allium genomes are particularly large (2n = 16, with 15901 Mbp). Among the genes identified were an alliinase and a glutathione-S-transferase. Both enzymes are involved in volatile sulfur compounds synthesis by cleavage of S-alkyl(en)yl cysteine sulphoxide flavors precursors, generating the volatile and reactive sulphur-containing chemicals.[6,9,41] The alliinase enzyme transforms alliins into very unstable thiosulphinates including allicin, the most labile, and degraded into diallyl disulfide, 2-vinyl-(4H)-1,3-dithiin and 3-vinyl-(4H)-1,3-dithiin.[46] Many studies reported the garlic protective effect against cancers and cardiovascular diseases by reducing serum cholesterol concentrations, blood pressure, and by inhibiting platelet aggregation.[6–9 41–46]

Actually, S-compounds increase the reactivity of enzymes involved in the detoxification of carcinogens.[41] Furthermore, the antibacterial activity of these compounds may serve to inhibit the bacterial conversion of nitrate into nitrite, thereby reducing the amount of nitrite available for reaction with amines to form carcinogenic nitrosamines. The chemopreventive effect of Allium vegetables against stomachal and esophageal cancers may be related to their antibacterial properties. Indeed, inhibition of bacterial growth in the gastric cavity may result in less conversion of nitrate to nitrite in the stomach, a probable decrease of endogenous formation of carcinogenic N-nitroso compounds, and a reduction in Helicobacter pilori infection specifically,[7] responsible for ulcer and gastric cancer.[6] The antibacterial effect of Allium roseum essential oil was recently demonstrated, and the anticancer action should be confirmed by more specific studies. Diallyl disulfide, diallyl sulfide, and allicin were the active compounds in garlic, and were partly responsible for its anti-cancer effect and its protection against cardiovascular diseases.[7,41]

Allyl sulfides and alkylsulfides have been shown to increase phase II enzymes, such as glutathione S-transferase and UDP-glucuronosyl transferase, involved in mechanisms of cancer prevention and/or modulate cytochromes P450 activities responsible for carcinogen activation. The contribution of each compound (sulphur, flavanols, and seleno) was hardly quantifiable but the global effect is certainly due to the presence of the compounds myriad.[41] Recent characterization of the pharmacokinetics and metabolism of organosulfur compounds in garlic has revealed that in Allium, other compounds than allicin were biologically active in the body, and would be behind the beneficial activities including those against human pathogenic fungi.[24]

The comparative study of the chemical composition between the essential oil obtained from hydrodistillation of Allium roseum flowers[19] and volatiles compounds of Allium sativum cloves extracted by various procedures[24,45,49] showed similar organo-sulfur compounds, but major quantitative differences. Moreover, the essential oil of Allium roseum contained much more non sulfurous compounds than those identified in Allium sativum, explaining the relatively very high sulphurous compounds rates found in the latter species.

CONCLUSION

The qualitative and the quantitative analysis of essential oil showed the presence of forty-four compounds in the whole essential oil of Allium roseum var. odoratissimum. The most important characterized compounds were organic-sulphurous including methyl 2-propenyl trisulfide, di-(2-propenyl) trisulfide, 1-propenyl 2-propenyl disulfide, di1-propenyl disulfide, di-(2-propenyl) disulfide, dimethyl trisulfide and methyl 2-propenyl disulfide. The most abundant were the substituted propenyl trisulfides, the major was the di-2-propenyl trisulfide followed by the methyl 2-propenyl trisulfide. The number of identified sulphurous compounds was much higher than that of the non sulphurous compounds. Most of them were acyclic unsaturated compounds and common to essential oil of Allium sativum, Allium cepa, and Allium fistulosum. The major cyclic sulphur components evidenced for the first time in the essential oil of Allium genus with a relatively low level, were found to be the 2,5-dichlorothiophene and the 1,3-dithiane. Since the biological activities of essential oil compounds of the studied plant, using an apolar column, have not been reported before, the present data reveals for the first time the characteristic composition of the essential oil of this endemic variety. Further research on experimental animals is needed to support causality between such properties and the cancer-preventive activity. Identification and studies of more other enzymes and genes in Allium involved in flavour precursor biosynthesis should be more developed, and elusive sources of the Allium alky(en)yl groups characteristics remain unknown and their identification would be both a challenge and a goal.

ACKNOWLEDGMENTS

The authors would like to thank Mrs. Hela Chabouni Fourati, an English teacher-trainer in the area of Sfax, for her help with the English language.