Industrial Crops and Products 41 (2013) 172178

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Allium roseum L. volatile compounds prole and antioxydant activity for chemotype discrimination Case study of the wild plant of Sfax (Tunisia)
Sami Zouari a,b , Mouna Ketata a,c , Nourhene Boudhrioua c , Emna Ammar a,
a b c

UR Study & Management of Urban and Coastal Environments, LARSEN National Engineering School in Sfax, B.P. 1173, 3038 Sfax, Tunisia Laboratory of Range Ecology, Institute of Arid Areas, 4119 Medenine, Tunisia UR Mcanique des Fluides Applique et Modlisation, National Engineering School in Sfax, University of Sfax, B.P. 1173, 3038 Sfax, Tunisia

a r t i c l e

i n f o

a b s t r a c t
This study deals with the chemical composition and antioxidant activities of Allium roseum L. a chemotype growing wild in Tunisia, to evidence new bioactive natural compounds in its ower essential oil, and to investigate possible antioxidative methanolic leaves extract properties. This essential oil was extracted by hydrodistillation and analyzed by GC and GC/MS. The main compounds were butylated hydroxytoluene, eugenol, farnesol, dimethyl trisulde, methyl 2-propenyl trisulde, Z-9-octadecenoic acid and tricosane found at respectively 10.0%, 8.4%, 6.9%, 6.1%, 5.3%, 5.2% and 4.8%. Moreover, 1-oxa-4,6-diazacyclooctane5-thione (3.9%) was isolated for the rst time in the Tunisian A. roseum, especially in the country central area (Sfax City). The methanolic leaves extract had antioxidant activities, assessed by two complementary tests: the DPPH radical-scavenging and the reducing power essays, when compared with those of butylated hydroxy anisole and ascorbic acid. These antioxidant activities and the essential oil prole would be a ngerprint for chemotype discrimination. 2012 Elsevier B.V. All rights reserved.

Article history: Received 21 July 2011 Received in revised form 7 April 2012 Accepted 11 April 2012 Keywords: Allium roseum Essential oil Chemical composition Farnesol Antioxidant activity

1. Introduction Traditional medicine offers rich and large unexplored therapeutic resources, useful for the pharmaceutical industry. Through academic research, pharmaceutical sciences may take prot from the available bioresources, and ethnobotanical as well as ethnopharmacological approaches that would be of interest. Plantderived natural products are abundant in nature. Many of them exhibit numerous biological activities and some can be employed as food additives (Gourine et al., 2010). Antioxidants are of great importance in terms of oxidative stress prevention, which may result from several degenerative diseases (Helen et al., 2000). Many fruits and vegetables are potentially useful for risk decrease of several chronic diseases, such as coronary heart disease and some cancers (Block et al., 1992; Lampe, 1999). These protective effects have been particularly attributed to various antioxidant compounds, such as vitamins C and E, -carotene and polyphenolic compounds (Diplock et al., 1998). The Allium genus is one of the major sources of polyphenolic compounds (Shon et al., 2004; Singh et al., 2009), and the antioxydative activity of some Alliums species has been reported and has been mainly attributed

Corresponding author. Tel.: +216 98 41 23 64; fax: +216 74 27 55 95. E-mail address: ammarenis@yahoo.fr (E. Ammar). 0926-6690/$ see front matter 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.indcrop.2012.04.020

to a variety of organo-sulfurous compounds as well as their precursors (Kim et al., 1997; Auger et al., 2004; Arnault and Auger, 2006; Najjaa et al., 2007, 2011). The Allium genus L. (Alliaceae) exhibits a great diversity considering morphological characters, particularly in life forms (bulb or rhizome) and ecological habitats. Moreover, this genus is of a major economical importance as vegetable and herbal crops, and ornamental plant (Ricroch et al., 2005). The genus consists mostly of perennial and bulbous plants widely distributed over holarctic regions from the dry subtropics to the boreal zone (Stearn, 1992). Rosy garlic (Allium roseum L.) is an important medicinal and aromatic plant, specic to the Tunisian ora. This plant is an allogamous perennial spontaneous weed (Jendoubi et al., 2001). Its oblong bulb grows about 3060 cm tall (Quezel and Santa, 1963), and its owers are wide and pink or white colored (Jendoubi et al., 2001). Rosy garlic is mainly found in light and sandy soils, in cultivated elds as well as in fallows and on roadsides. In ancient times, this species was used as a vegetable, spice or herbal remedy (Najjaa et al., 2007). At present, the species is used in traditional pharmacopoeia for its expectorant properties. It is extensively widespread in the South of Tunisia, where it has been empirically used for its numerous therapeutic virtues, especially against rheumatism. However, it is seriously threatened because of the over-collection and the bulbs use (Marcucci and Tornadore, 1997). Furthermore,

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A. roseum is also used as a condiment, substituting onion (Cunod, 1954). Investigations dealing with the chemical composition of A. roseum growing wild in Tunisia are still scarce. Recently, some studies described the essential oil chemical composition of this species collected from some Tunisian regions (Ben Jannet et al., 2007; Najjaa et al., 2007; Zouari et al., 2012). Nevertheless, at the authors knowledge, no available information concerning the antioxidant activities of A. roseum growing wild in Tunisia was published except the recent work of Najjaa et al. (2011) where the antioxidant activities were assessed using two functional analytical methods based on the radicals (ABTS and DPPH) scavenging potential. It was founds that the radical scavenging values depended on both plant material (bulb, leaf and ower) and extraction solvent used (acetone/water, methanol/water, or distilled water). Furthermore, aerial parts (leaves and owers) exhibited relatively higher DPPH and ABTS values when compared to bulb values. Aware of the scarce works on A. roseum, studies on this species did not cover the entire area where this plant is growing wild, especially in the central Tunisia (Sfax City). Therefore, the aim of the present work was to study the chemical composition of central Tunisia A. roseum ower essential oils, in order to evidence a new specic chemotype according to the region location, adapted to climatic and soil conditions. The methanolic extract antioxidant activity of this species was investigated. 2. Materials and methods 2.1. Plant material A. roseum var. odoratissimum was collected from Sfax, a city in the Centre of Tunisia (latitude 34 46 29 N, longitude 10 39 73 E; elevation: 41 m), where the climate is semi arid, characterized by a mean rainfall of 200 mm/year. The plant was collected at the owering stage (March 2008). Botanical identication was carried out at the Laboratory of Ecology in the Faculty of Science (Sfax, Tunisia), according to the Flora of Tunisia (Cunod, 1954). Voucher specimen was deposited at the herbarium of the Faculty of Sciences in Sfax. Flowers were separated from lignied part and were used for hydrodistillation while leaves samples were air-dried (temperature 24 C, humidity 62%) for a week to be used for antioxidant assay. 2.2. Essential oil extraction Hydrodistillation of the fresh owers (100 g) of A. roseum was performed in a Clevenger-type apparatus for 4 h up to the point at which the oil contained in the matrix was exhausted (Najjaa et al., 2007). The collector solvent used was n-hexane (2 ml) as reported by Basar et al. (2001). After evaporation of the solvent under N2 ow, the essential oil was dried over anhydrous sodium sulfate and stored in dark sealed vials at 20 C until chemical analysis. The A. roseum essential oil was then analyzed by GCFID and GCMS. 2.3. Essential oil analysis 2.3.1. Gas chromatography A Hewlett-Packard 5890 series II gas chromatograph equipped with HP-5MS capillary column (30 m 0.25 mm i.d., lm thickness 0.25 m; Hewlett-Packard) and connected to a ame 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 ow of

1.2 ml/min. The analysis was performed on 2 l volume sample. The split ratio was 1/100. Percentages of the constituents were calculated by electronic integration of FID peak areas, without the use of response factor correction. Kovts indices were calculated for separate compounds relative to C9 C26 n-alkanes mixture (Aldrich Library of chemicals standards) (Kovts, 1958). 2.3.2. Gas chromatographymass spectrometry (GC/MS) The isolated volatile compounds were analyzed by GC/MS, using a Hewlett-Packard 5890 series II gas chromatograph. The fused HP5MS 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, the temperature of the detector was set at 280 C. The carrier gas was helium (99.995% purity), with a ow rate of 1.2 ml/min and the analyzed sample volume was 2 l. The mass spectrometer was operated in the electron impact (EI) positive mode (70 eV). The ion source and quadrupole mass analyzer temperature were respectively 230 C and 150 C. The range of mass spectra was 35350 m/z. 2.3.3. Volatile compounds identication The compounds of the essential oil were identied by comparing the mass spectra data with spectra available from the Wiley 275 and NIST 0.5a mass spectra libraries. Further identication conrmations were made referring to retention index data generated from a series of known standards of n-alkanes mixture (C9 C26 ) (Aldrich Library of chemicals standards) (Kovts, 1958) and to those previously reported in the literature (Table 1). 2.4. Antioxidant activity 2.4.1. DPPH radical-scavenging assay The DPPH radical-scavenging activity of the A. roseum leaves methanolic extract was determined as described by Kirby and Schmidt (1997) with some modications (Hajji et al., 2010). Briey, a volume of 500 l of methanolic extract at different concentrations (100500 g/ml) was added to 375 l of 99% methanol and 125 l of a DPPH solution (0.2 mM in methanol) as free radical source. The mixtures were incubated for 60 min in the dark at room temperature. Scavenging capacity was measured spectrophotometrically (T70 UV-visible spectrometer, PG Instruments Ltd., Wibtoft, China) by monitoring the decrease in absorbance at 517 nm. In its radical form, DPPH has an absorption band at 517 nm which disappears upon reduction by an antiradical compound. The lower reaction mixture absorbance, the higher free radical-scavenging activity. The antiradical activity was expressed as IC50 (mg/ml), the concentration required to inhibit 50% of the DPPH, and a lower IC50 value corresponded to a higher methanolic extract antioxidant activity (Patro et al., 2005). The DPPH radical-scavenging activity was calculated as follows: Ac As 100 Ac

Radical-scavenging activity (%) =

where Ac and As are respectively the control and the sample absorbances. The control was conducted in the same manner as the sample, which was replaced by methanol. Butylated hydroxy anisole (BHA) was used as positive control. The test was carried out in triplicate. 2.4.2. Reducing power assay The ability of the A. roseum methanolic leaves extract to reduce iron (III) was determined according to the method of Yildirim et al.

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Table 1 Mean percentage of the fresh owers essential oil components from Allium roseum (Sfax, Tunisia) and RI comparison according to the literature. Peak number Compounda Retention index (RI) Calculated 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 Methyl 2-propenyl disulde Methyl 1-propenyl disulde 1,3-Dithiane Dimethyl trisulde 2,2,4,6,6-Pentamethyl heptane Di-2-propenyl disulde 1-Oxa-4,6-diazacyclooctane-5-thione Methyl 2-propenyl trisulde Camphor cis Methyl propenyl sulde Naphthalene Decanal Dimethyl tetrasulde Linalyl acetate Caprolactam Di-2-propenyl trisulde 2-Methoxy thiophene -Elemene Eugenol Geranyl acetate Tetradecene Tetradecane Methyl eugenol -Caryophylene Isoeugenol Butylated hydroxytoluene Elemicin trans-Nerolidol Hexadecane Methoxyeugenol 8-Heptadecene Heptadecane Farnesol Tetradecanoic acid Benzyl benzoate Octadecane Cyclohexadecane 1-Nonadecene Hexadecanoic acid Eicosane Heneicosane Z-9-Octadecenoic acid Octadecanoic acid Docosane Tricosane Tetracosane Pentacosane Total
a b b

Percentage Literature (reference) 913 (Ansorena et al., 2001) 930 (Pyun and Shin, 2006) 934 (Zouari et al., 2012) 972 (Jarunrattanasri et al., 2007) 997 (Insausti et al., 2005) 1083 (Prez et al., 2007) Not available 1130 (Godevac et al., 2008) 1144 (Tounsi et al., 2011) 1153 (Pyun and Shin, 2006) 1191 (Zeng et al., 2007) 1206 (Bonati et al., 2005) 1215 (Bonati et al., 2005) 1258 (Skaltsa et al., 2001) 1267 (Teyeb et al., 2010) 1296 (Ansorena et al., 2001) Not available 1339 (Skaltsa et al., 2001) 1364 (Ansorena et al., 2001) 1383 (Asuming et al., 2005) 1389 (Mimica-Dukic et al., 2003) 1400 (Kovts, 1958) 1407 (Cole et al., 2007) 1423 (Asuming et al., 2005) 1459 (Zhao et al., 2006) 1518 (Ansorena et al., 2001) 1559 (Cole et al., 2007) 1566 (Zhao et al., 2006) 1600 (Kovts, 1958) 1609 (Ansorena et al., 2001) 1677 (Xian et al., 2006) 1700 (Kovts, 1958) 1722 (Tounsi et al., 2011) 1769 (Conforti et al., 2009) 1770 (Zeng et al., 2007) 1800 (Kovts, 1958) 1875 (Conforti et al., 2009) 1892 (Conforti et al., 2009) 1963 (Zeng et al., 2007) 1995 (Kovts, 1958) 2097 (Kovts, 1958) 2152 (Skaltsa et al., 2001) 2172 (Conforti et al., 2009) 2200 (Kovts, 1958) 2300 (Kovts, 1958) 2400 (Kovts, 1958) 2500 (Kovts, 1958) 3.48 0.84 3.61 6.05 0.37 1.76 3.89 5.25 0.41 0.55 0.28 0.65 0.34 0.41 3.71 0.99 0.74 0.30 8.42 0.62 0.24 0.24 1.55 0.73 1.55 10.03 2.39 2.51 0.58 0.48 0.22 0.26 6.92 0.25 0.25 0.44 0.32 0.83 3.74 0.55 0.54 5.23 1.00 0.56 4.78 0.64 2.85 92.35

918 932 941 973 989 1081 1103 1144 1148 1164 1189 1205 1221 1254 1276 1304 1331 1340 1367 1382 1388 1395 1407 1426 1459 1518 1559 1567 1595 1610 1673 1694 1726 1766 1773 1792 1872 1886 1955 1973 2094 2156 2173 2197 2302 2400 2500

Compounds are listed in order of their elution from the HP 5MS column. Retention index calculated against C9 C26 n-alkanes mixture on HP-5MS column.

(2001). Sample solutions (1 ml) of the extract different concentrations (100500 g/ml) were mixed with 1.25 ml of 0.2 M phosphate buffer (pH = 6.6) and 1.25 ml of potassium ferricyanide solution (10 g/l). The mixtures were incubated at 50 C for 30 min, then 1.25 ml of 10% (w/v) trichloroacetic acid was added. The mixtures were then centrifuged at 3000 g for 10 min. Finally, 1.25 ml of the supernatant solution was mixed with 1.25 ml of distilled water and 0.25 ml of ferric chloride (1 g/l). After 10 min reaction, the resulting solution absorbance was measured at 700 nm. The reaction mixture absorbance increase reected the reducing power intensity increase. The EC50 value (mg/ml) is the methanolic extract concentration at which the absorbance was 0.5 for the reducing power; it was calculated from the graph absorbance at 700 nm against methanolic extract concentration (Tounsi et al., 2009). Ascorbic acid was used as positive control. The presented values were the means of triplicate analyses.

3. Results and discussion 3.1. Essential oil analysis The A. roseum fresh owers subjected to hydrodistillation using a Clevenger apparatus lead to a light yellow-colored essential oil which yielded 0.05% (v/w). The A. roseum essential oil chemical composition was investigated using both GC and GC/MS techniques. The volatile compounds composition was based on the chemical functions (acids, alcohols, aliphatics, aromatics, sulfurs and terpenes). The identied components, their percentages, their calculated RI and their comparison according to the RI-values previously published in the literature are listed in Table 1, considering the order of the compounds elution on the HP-5MS column. A total of 47 compounds were identied, accounting for almost the whole oil constituents (93%). Butylated hydroxytoluene, eugenol, farnesol,

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Fig. 1. S-compounds genesis during hydrodistillation of Allium genus material.

dimethyl trisulde, methyl 2-propenyl trisulde, Z-9-octadecenoic acid and tricosane were the most abundant components with rates varying from 4.8 to 10.0%, constituting 46.7% of the total oil. These were followed by 1-oxa-4,6-diazacyclooctane-5-thione, hexadecanoic acid, caprolactam, 1,3-dithiane and methyl 2-propenyl disulde which were at almost equal rates of about 3.5%. Others components were present at rates inferior to 3%. The A. roseum essential oil chemical classes distribution was reported in Table 2. The identied components may be classied into sulfurous and non-sulfurous compounds. It should be mentioned that, according to previous published data, the S-containing compounds are responsible for the Liliaceae vegetable appropriate odor, avor and also health benets (Block, 1985; Auger et al., 2004; Arnault and Auger, 2006). These characteristics are recognized as the basic studied species value and may explain its important use in traditional medicine. Indeed, in the Allium genus, these S-containing compounds are present as alk(en)yl cysteine sulfoxides, a non volatile precursors, and while distillating the vegetal material, the allinase is activated and catalyzed the reaction generating especially thiosulnates (Jaillais et al., 1999; Arnault and Auger, 2006). These are very unstable compounds, and may give rise to further rearrangements, leading to a wide variety of derived sulfurous compounds (monosuldes, disuldes, trisuldides and tetrasuldes). During hydrodistillation of Allium genus material, the S-compounds geneses are presented according to the reaction (Fig. 1). 3.1.1. Sulfurous compounds Table 2 shows high content of sulfurous compounds (about the third of the total eluted compounds). Over the eleven identied sulfurous compounds, ve were present at relatively high rates, ranged from 3.5 to 6.1%. The most abundant ones among them were the substituted trisuldes, represented by dimethyl trisulde as major compound, followed by methyl 2-propenyl trisulde.

Table 2 Sulfurous compounds and non sulfurous compounds of the essential oil from fresh owers of Allium roseum (Sfax, Tunisia). Compound Sulfurous compounds Cyclic compounds Non cyclic compounds Non sulfurous compounds Phenolic derivatives Hydrocarbons Fatty acids Oxygenated sesquiterpene Cyclic ketones and cyclic amide Esters Hydrocarbons sesquiterpene Aldehydes Aromatic compounds Percentage 27.50 5.47 22.02 64.85 24.42 13.42 10.22 9.43 4.12 1.28 1.03 0.65 0.28

These compounds were also dominant in the A. roseum fresh owers essential oil collected from Bengardane area, in the South-East of Tunisia (Zouari et al., 2012). Considering the cyclic sulfurous compound, the major compound was the 1-oxa-4,6-diazacyclooctane-5-thione, isolated for the rst time in Tunisian A. roseum harvested in the central Tunisia (Sfax city). To our knowledge, no previous report identied this volatile sulfurous compound in A. roseum species. However, Prithiviraj et al. (2004) described the 1-oxa-4,6-diazacyclooctane5-thione isolated from onion bulb inoculated with Fusarium oxysporum, as a specic metabolite and a volatile vegetable compound to detect and discriminate onion basal rots disease. This metabolite would be a useful biosensor in non destructive essay for the early detection system of the disease presence. More recently, the 1-oxa-4,6-diazacyclooctane-5-thione was detected at relatively high rate among the volatile compounds identied in garlic harvested in Xuzhou area of China, extracted by solid phase microextraction and analyzed by GC/MS (Ma et al., 2011). Sulfurous compounds could be separated into two major groups according to their acyclic or cyclic structure. These compounds would be generated from non volatile precursors i.e. alk(en)yl cysteine sulfoxydes as presented in reaction (I) in Fig. 1. Furthermore, the allinase would be the main enzyme involved in these volatile metabolites biosynthesis. The acyclic group included various compounds from mono to polysulde, substituted by different radicals (trisulde, disulde, monosulde and tetrasulde). While in garlic compounds, alliin (Salk(en)yl-l-cysteine sulfoxide) was the major compound, in onion isoalliin with 1-propenyl residue were the main metabolite (Auger et al., 2004; Prithiviraj et al., 2004) and in A. roseum, the polysulde compounds were predominant (Najjaa et al., 2007; Zouari et al., 2012). Furthermore, in the A. roseum essential oil collected from the South-East of Tunisia, the most abundant volatile compounds were diallyl trisuldes followed by diallyl disuldes accounting for 12.3% and 8.9% respectively (Zouari et al., 2012). In the present study, the trisuldes were equally present as dimethylated (6%) and methyl 2-propenylated (5.3%), and nally as di-2-propenylated (1%) at a low rate (Table 1). The abundance of these compounds was also mentioned in Allium cepa L. and Allium stulosum L. essential oils (Prithiviraj et al., 2004; Gyawali et al., 2005; Pyun and Shin, 2006). Monosulde and tetrasulde compounds were detected at minor rates (Table 1), compared to A. roseum essential oil owers harvested in Bengardene. In the latter area, such compounds were mainly represented by methional (Najjaa et al., 2007). It may be suggested that meterological conditions would orchestrate the metabolic pathways. Apart 1-oxa-4,6-diazacyclooctane-5-thione as major cyclic sulfurous compound (3.9%), the 1,3-dithiane was also found at relatively high rate (3,61%), while the 2-methoxy thiophene was detected at low rate (<1%). The rst compound was also found in the A. roseum essential oil collected from the South-East of Tunisia

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Table 3 Mass spectral data of some newly isolated Allium roseum oil compounds. Compound Retention time in min Percentage MW Structure m/z (%)

NH
1-Oxa-4,6-diazacyclooctane-5-thione 10.25 3.89 146

NH

146(100), 101(11), 86(10), 73(39), 61(37), 55(7)

O NH
Caprolactam 13.88 3.71 113 113(95), 85(62), 67(13), 55(100), 42(47)

Elemicin

19.09

2.39

208

CH3O CH3O
OH

208(100), 193(55), 177(13), 133(15), 91(12), 77(15)

OCH3

Farnesol

21.83

6.92

222

136(12), 107(13), 93(25), 81(31), 69(100), 41(39)

and in Allium sativum bulb (Ejaz et al., 2003; Zouari et al., 2012). The second compound was evidenced for the rst time in the essential oil of Allium genus with a relatively low level, and would characterize the essential oil of A. roseum L. var. odoratissimum chemotype harvested in Sfax area. 3.1.2. Non-sulfurous compounds The A. roseum essential oil non-sulfurous compounds contained mainly phenolic derivatives (butylated hydroxytoluene, eugenol and its derivatives), representing the fourth of the total compounds (Table 2). Besides the butylated hydroxytoluene (10.03%), eugenol represented the most dominant phenolic compound in this essential oil, with a rate of 35% of the total phenolic derivatives. Eugenol is used as a avoring agent in cosmetic and in foods and has a strong antioxidant effect which may contribute to aldehydes and ketones reduction (Chevance and Farmer, 1999; Fujisawa et al., 2002). The second non-sulfurous compounds abundant group was hydrocarbons followed by fatty acids group at relatively high rates, considering that the sample was not methylated and hence fatty acids would not be in the suitable conditions to be identied. Oxygenated sesquiterpenes were also found at relatively high rate and included mainly farnesol (74%) of the total sesquiterpene. Farnesol is a very interesting compound promoting epithelial cell defense against Candida albicans, a human pathogenic yeast (Dcanis et al., 2009). This compound was also evidenced to be a quorum sensing molecule produced by the dimorphic fungus C. albicans. Moreover, farnesol acts to suppress C. albicans mycelia development in a quorum-sensing manner and it was suggested as the rst of a novel antifungal compounds class (Hornby et al., 2001; Gregu s et al., 2010). Interestingly, in the present work camphor, an oxygenated

monoterpene, was found at very low rate (0.41%), compared to A. roseum essential oil owers harvested in the Tunisian SouthEast (13.4%) (Najjaa et al., 2007). Based on its concentration, the A. roseum owers would be a safety food when wild grown in Sfax (Tunisia). In Tunisia, the essential oils chemical proles of the A. roseum owers collected from the center (Sfax) and the South-East (Zouari et al., 2012) differed not only in the number of sulfurous compounds but also in their chemotypes (acyclic or cyclic structure) and rates. Indeed, in the Center the essential oil contained much more non sulfurous compounds than those identied in the South-East, explaining the low sulfurous compounds rates found in Sfax and consequently a lower number of sulfurous compounds. These could be attributed to several factors such as climate, soil composition and edaphic factors (Angioni et al., 2006). 3.1.3. Mass spectral characterization of some newly isolated A. roseum oil compounds Mass spectra of some newly isolated A. roseum oil compounds and their relative area percentages are presented in Table 3. The newly sulfurous constituent was the 1-oxa-4,6diazacyclooctane-5-thione with a retention time of 10.25 min and M+ ion at 146 represented the base peak in its mass spectrum. Fragments at m/z 73 and 61, could be attributed to [CH2 NHCS]+ or [CH2 OCH2 CH2 NH]+ and [HOCH2 CH2 NH2 ]+ . Moreover, the newly non sulfurous compound, having a retention time of 13.88 min, was characterized by a mass ion m/z 113 and identied as caprolactam, with the fragment ion ([CH3 CH CHCH2 ]+ ) at m/z 55 as the base peak. Caprolactam MS displayed also peaks at m/z 85 and 42, corresponding to the following fragment ions:

S. Zouari et al. / Industrial Crops and Products 41 (2013) 172178 Table 4 Antiradical (DPPH) and reducing activity of Allium roseum leaves methanolic extract. Compounds A. roseum leaves methanolic extract BHA Ascorbic acid EC50 (g/ml) DPPH 240.4 0.5 14.1 0.2 IC50 (g/ml) reducing power 248.7 0.5 6.7 0.1

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[CH2 CH2 CH2 CH2 CH2 NH]+ and [CH2 CH2 CH2 ]+ . It is noticeable that caprolactam, a hydrophobic organic compound, was recently identied in leachate water samples from landll site, and its MS was in agreement with the A. roseum mass spectral assignments and with their good matching with library spectrum (Uchida et al., 2009). Manfully, another interesting constituent was eluted at 19.09 min and had M+ ion at m/z 208. This compound was identied as elemicin, based on its mass spectral assignments and their good matching with library spectrum. It should be mentioned that elemicin was recently shown to exhibit dose-dependent inhibition of the ADP-induced human platelet serotonin release and would have a health effect (Grice et al., 2010). Finally, another interesting compound was identied at relatively high rate, with a retention time of 21.83 min and a molecular weight of 222. Its MS displayed peaks at m/z 136, 121 and 69, linked to ion arising from loss of hydrogen and methyl of the fragment [(CH3 )2 C CHCH2 CH2 C(CH3 ) CHCH2 ]+ , and [(CH3 )2 C CHCH2 ]+ with the peak at m/z 69 being the base peak. This compound was identied as farnesol, based on its mass spectrum and the spectral match with that of the standard. This compound of health benet would attribute a neutraceutical value to this Allium species, growing in the Centre of Tunisia under semi arid climate. 3.2. Antioxidant activity determination The A. roseum leaves methanolic extract was investigated for possible antioxidative capacities by using two complementary tests: DPPH radical-scavenging and reducing power essays, compared respectively to those of BHA and ascorbic acid. 3.2.1. DPPH radical-scavenging activity DPPH is a stable free radical with a maximum absorbance at 517 nm. When DPPH radicals encounter a proton-donating substrate such as an antioxidant, the radicals would be scavenged and the absorbance is reduced (Shimada et al., 1992). The decrease in absorbance would inform on radical-scavenging activity. This method is widely used to investigate the scavenging activity of some natural compounds (Hajji et al., 2010). The leaves methanolic extract IC50 value was 17 times that of BHA (Table 4). When compared to the synthetic antioxidant BHA, the A. roseum leaves methanolic extract exhibited moderate antioxidant abilities to reduce DPPH radicals. Previous study on the antioxidant activity of ve Allium methanolic extracts species (Allium nevsehirense, Allium sivasicum, Allium dictyoprosum, Allium scrodoprosum subsp. rotundum and Allium atroviolaceum), measured by DPPH, showed an IC50 ranged between 79 and 104 g/ml with an efciency of 3.95 (IC50 extract/IC50 BHT) (Tepe et al., 2005). For A. roseum, the efciency was equal to 17.05 (IC50 extract/IC50 BHA) and antioxidant activity was 4.5 times lower than that of the other Allium species previously studied. For further comparison with other species, Candan et al. (2003) show that the essential oil of Achillea millefolium, rich in eucalyptol and camphor, possesses strong antioxidative activity as evaluated by DPPH method. 3.2.2. Reducing power The reducing power-essay is often used to evaluate the antioxidant ability to donate electrons (Yildirim et al., 2000). The ability of

the A. roseum leaves methanolic extract, and ascorbic acid as a positive control, to reduce Fe3+ to Fe2+ is presented in Table 4. A direct correlation between antioxidative activities and reducing power of some bioactive compounds was exhibited by the conversion of Fe3+ /ferricyanide complex to ferrous form (Shimada et al., 1992; Hajji et al., 2010). The A. roseum leaves methanolic extract EC50 value of reducing power ability was 37 times that of ascorbic acid, exhibiting a low reducing power. 4. Conclusion The results of this work describe the chemical composition of essential oil of A. roseum growing wild in the semi arid region of Tunisia (central Tunisia) which showed the presence of a wide range of compounds, accounting for more than 90% of the whole oil constituents. Butylated hydroxytoluene, eugenol, farnesol, dimethyl trisulde, methyl 2-propenyl trisulde, Z-9-octadecenoic acid and tricosane were the most abundant components, constituting almost half the total oil. Moreover, 1-oxa4,6-diazacyclooctane-5-thione, found at relatively high rate, was isolated for the rst time in the Tunisian A. roseum, wild growing in the Center of the country (Sfax City). The present data reveals the characteristic composition of the essential oil of this species which could be considered as a new chemotype specic to the spices growing in Sfax city, adapted to the climatic and the soil conditions of this region. Since the antioxidant activities of the studied plant, evaluated by two complementary tests: DPPH radical-scavenging and reducing power-essays, have not been reported before, our nding demonstrate that the methanolic leaves extract exhibited moderate antioxidant activities when compared to those of butylated hydroxyl anisole and ascorbic acid. Therefore, it could be also concluded that the study presented herein is the rst report on the essential oil composition and antioxydant activity of the A. roseum L. chemotype, growing wild in the semi arid region of Tunisia (Sfax city). Nevertheless, further investigations with other Allium species from this bioclimatic stage would be interesting to be considered, allowing the conrmation of the essential oil chemotype ngerprint of the species. Acknowledgment The authors are very grateful to Mrs. Hela Chabouni Fourati, an English teacher-trainer in the area of Sfax, for English Language correction. References
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