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Industrial Crops and Products 41 (2013) 172– 178

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Industrial Crops and Products

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llium roseum L. volatile compounds profile and antioxydant activity forhemotype discrimination – Case study of the wild plant of Sfax (Tunisia)

ami Zouaria,b, Mouna Ketataa,c, Nourhene Boudhriouac, Emna Ammara,∗

UR Study & Management of Urban and Coastal Environments, LARSEN – National Engineering School in Sfax, B.P. 1173, 3038 Sfax, TunisiaLaboratory of Range Ecology, Institute of Arid Areas, 4119 Medenine, TunisiaUR Mécanique des Fluides Appliquée et Modélisation, National Engineering School in Sfax, University of Sfax, B.P. 1173, 3038 Sfax, Tunisia

r t i c l e i n f o

rticle history:eceived 21 July 2011eceived in revised form 7 April 2012ccepted 11 April 2012

eywords:

a b s t r a c t

This study deals with the chemical composition and antioxidant activities of Allium roseum L. a chemotypegrowing wild in Tunisia, to evidence new bioactive natural compounds in its flower essential oil, and toinvestigate possible antioxidative methanolic leaves extract properties. This essential oil was extracted byhydrodistillation and analyzed by GC and GC/MS. The main compounds were butylated hydroxytoluene,eugenol, farnesol, dimethyl trisulfide, methyl 2-propenyl trisulfide, Z-9-octadecenoic acid and tricosane

llium roseumssential oilhemical compositionarnesolntioxidant activity

found at respectively 10.0%, 8.4%, 6.9%, 6.1%, 5.3%, 5.2% and 4.8%. Moreover, 1-oxa-4,6-diazacyclooctane-5-thione (3.9%) was isolated for the first time in the Tunisian A. roseum, especially in the country centralarea (Sfax City). The methanolic leaves extract had antioxidant activities, assessed by two complementarytests: the DPPH radical-scavenging and the reducing power essays, when compared with those of buty-lated hydroxy anisole and ascorbic acid. These antioxidant activities and the essential oil profile wouldbe a fingerprint for chemotype discrimination.

© 2012 Elsevier B.V. All rights reserved.

. Introduction

Traditional medicine offers rich and large unexplored thera-eutic resources, useful for the pharmaceutical industry. Throughcademic research, pharmaceutical sciences may take profitrom the available bioresources, and ethnobotanical as well asthnopharmacological approaches that would be of interest. Plant-erived natural products are abundant in nature. Many of themxhibit numerous biological activities and some can be employeds food additives (Gourine et al., 2010).

Antioxidants are of great importance in terms of oxidative stressrevention, which may result from several degenerative diseasesHelen et al., 2000). Many fruits and vegetables are potentially use-ul for risk decrease of several chronic diseases, such as coronaryeart disease and some cancers (Block et al., 1992; Lampe, 1999).hese protective effects have been particularly attributed to variousntioxidant compounds, such as vitamins C and E, �-carotene andolyphenolic compounds (Diplock et al., 1998). The Allium genus is

ne of the major sources of polyphenolic compounds (Shon et al.,004; Singh et al., 2009), and the antioxydative activity of somellium’s 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).

926-6690/$ – see front matter © 2012 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.indcrop.2012.04.020

to a variety of organo-sulfurous compounds as well as their precur-sors (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 con-sidering morphological characters, particularly in life forms (bulbor rhizome) and ecological habitats. Moreover, this genus is ofa major economical importance as vegetable and herbal crops,and ornamental plant (Ricroch et al., 2005). The genus consistsmostly of perennial and bulbous plants widely distributed overholarctic regions from the dry subtropics to the boreal zone(Stearn, 1992).

Rosy garlic (Allium roseum L.) is an important medicinal and aro-matic plant, specific to the Tunisian flora. This plant is an allo-gamous perennial spontaneous weed (Jendoubi et al., 2001). Itsoblong bulb grows about 30–60 cm tall (Quezel and Santa, 1963),and its flowers are wide and pink or white colored (Jendoubi et al.,2001). Rosy garlic is mainly found in light and sandy soils, in culti-vated fields as well as in fallows and on roadsides. In ancient times,this species was used as a vegetable, spice or herbal remedy (Najjaaet al., 2007). At present, the species is used in traditional pharma-copoeia for its expectorant properties. It is extensively widespread

in the South of Tunisia, where it has been empirically used forits numerous therapeutic virtues, especially against rheumatism.However, it is seriously threatened because of the over-collectionand the bulbs use (Marcucci and Tornadore, 1997). Furthermore,

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S. Zouari et al. / Industrial Cro

. roseum is also used as a condiment, substituting onion (Cuénod,954).

Investigations dealing with the chemical composition of A.oseum growing wild in Tunisia are still scarce. Recently, sometudies described the essential oil chemical composition of thispecies collected from some Tunisian regions (Ben Jannet et al.,007; Najjaa et al., 2007; Zouari et al., 2012).

Nevertheless, at the author’s knowledge, no available informa-ion concerning the antioxidant activities of A. roseum growingild in Tunisia was published except the recent work of Najjaa

t al. (2011) where the antioxidant activities were assessed usingwo functional analytical methods based on the radicals (ABTS andPPH) scavenging potential. It was founds that the radical scaveng-

ng values depended on both plant material (bulb, leaf and flower)nd extraction solvent used (acetone/water, methanol/water, oristilled water). Furthermore, aerial parts (leaves and flowers)xhibited relatively higher DPPH and ABTS values when comparedo bulb values.

Aware of the scarce works on A. roseum, studies on this speciesid not cover the entire area where this plant is growing wild,specially in the central Tunisia (Sfax City). Therefore, the aim ofhe present work was to study the chemical composition of cen-ral Tunisia A. roseum flower essential oils, in order to evidence aew specific chemotype according to the region location, adaptedo climatic and soil conditions. The methanolic extract antioxidantctivity of this species was investigated.

. Materials and methods

.1. Plant material

A. roseum var. odoratissimum was collected from Sfax, city in the Centre of Tunisia (latitude 34◦46′29′′N,ongitude 10◦39′73′′E; elevation: 41 m), where the cli-

ate is semi arid, characterized by a mean rainfall of00 mm/year. The plant was collected at the flower-

ng stage (March 2008). Botanical identification was carriedut at the Laboratory of Ecology in the Faculty of Science (Sfax,unisia), according to the “Flora of Tunisia” (Cuénod, 1954).oucher specimen was deposited at the herbarium of the Facultyf Sciences in Sfax. Flowers were separated from lignified partnd were used for hydrodistillation while leaves samples wereir-dried (temperature 24 ◦C, humidity 62%) for a week to be usedor antioxidant assay.

.2. Essential oil extraction

Hydrodistillation of the fresh flowers (100 g) of A. roseum waserformed in a Clevenger-type apparatus for 4 h up to the point athich the oil contained in the matrix was exhausted (Najjaa et al.,

007). The collector solvent used was n-hexane (2 ml) as reportedy Basar et al. (2001). After evaporation of the solvent under N2ow, the essential oil was dried over anhydrous sodium sulfate andtored in dark sealed vials at −20 ◦C until chemical analysis. The A.oseum essential oil was then analyzed by GC–FID and GC–MS.

.3. Essential oil analysis

.3.1. Gas chromatographyA Hewlett-Packard 5890 series II gas chromatograph equipped

ith HP-5MS capillary column (30 m × 0.25 mm i.d., film thickness.25 �m; Hewlett-Packard) and connected to a flame ionization

etector (FID) was used. The column temperature was programmedt 50 ◦C for 1 min, then 7 ◦C/min to 250 ◦C for 5 min. The injec-ion port temperature was 240 ◦C and that of the detector 250 ◦C.he carrier gas was helium (99.995% purity) at a constant flow of

Products 41 (2013) 172– 178 173

1.2 ml/min. The analysis was performed on 2 �l volume sample.The split ratio was 1/100. Percentages of the constituents were cal-culated by electronic integration of FID peak areas, without the useof response factor correction. Kovàts indices were calculated forseparate compounds relative to C9–C26 n-alkanes mixture (AldrichLibrary of chemicals standards) (Kovàts, 1958).

2.3.2. 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) wascoupled to a HP 5972A masse-selective detector (Hewlett-Packard,Palo Alto, CA, USA). The oven temperature was programmed from50 ◦C (1 min) to 250 ◦C (5 min) at 7 ◦C/min. The temperature of theinjector port was held at 250 ◦C, the temperature of the detectorwas set at 280 ◦C. The carrier gas was helium (99.995% purity), witha flow 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 ana-lyzer temperature were respectively 230 ◦C and 150 ◦C. The rangeof mass spectra was 35–350 m/z.

2.3.3. Volatile compounds identificationThe compounds of the essential oil were identified by compar-

ing the mass spectra data with spectra available from the Wiley 275and NIST 0.5a mass spectra libraries. Further identification confir-mations were made referring to retention index data generatedfrom a series of known standards of n-alkanes mixture (C9–C26)(Aldrich Library of chemicals standards) (Kovàts, 1958) and to thosepreviously reported in the literature (Table 1).

2.4. Antioxidant activity

2.4.1. DPPH radical-scavenging assayThe DPPH radical-scavenging activity of the A. roseum leaves

methanolic extract was determined as described by Kirby andSchmidt (1997) with some modifications (Hajji et al., 2010). Briefly,a volume of 500 �l of methanolic extract at different concentrations(100–500 �g/ml) was added to 375 �l of 99% methanol and 125 �lof a DPPH solution (0.2 mM in methanol) as free radical source. Themixtures were incubated for 60 min in the dark at room tempera-ture. 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 radicalform, DPPH has an absorption band at 517 nm which disappearsupon reduction by an antiradical compound. The lower reactionmixture absorbance, the higher free radical-scavenging activity.The antiradical activity was expressed as IC50 (mg/ml), the concen-tration required to inhibit 50% of the DPPH, and a lower IC50 valuecorresponded to a higher methanolic extract antioxidant activity(Patro et al., 2005).

The DPPH radical-scavenging activity was calculated as follows:

Radical-scavenging activity (%) =[

Ac − AsAc

]× 100

where Ac and As are respectively the control and the sampleabsorbance’s. The control was conducted in the same manner asthe sample, which was replaced by methanol. Butylated hydroxyanisole (BHA) was used as positive control. The test was carried outin triplicate.

2.4.2. Reducing power assayThe ability of the A. roseum methanolic leaves extract to reduce

iron (III) was determined according to the method of Yildirim et al.

174 S. Zouari et al. / Industrial Crops and Products 41 (2013) 172– 178

Table 1Mean percentage of the fresh flowers essential oil components from Allium roseum (Sfax, Tunisia) and RI comparison according to the literature.

Peak number Compounda Retention index (RI) Percentage

Calculatedb Literature (reference)

1 Methyl 2-propenyl disulfide 918 913 (Ansorena et al., 2001) 3.482 Methyl 1-propenyl disulfide 932 930 (Pyun and Shin, 2006) 0.843 1,3-Dithiane 941 934 (Zouari et al., 2012) 3.614 Dimethyl trisulfide 973 972 (Jarunrattanasri et al., 2007) 6.055 2,2,4,6,6-Pentamethyl heptane 989 997 (Insausti et al., 2005) 0.376 Di-2-propenyl disulfide 1081 1083 (Pérez et al., 2007) 1.767 1-Oxa-4,6-diazacyclooctane-5-thione 1103 Not available 3.898 Methyl 2-propenyl trisulfide 1144 1130 (Go –devac et al., 2008) 5.259 Camphor 1148 1144 (Tounsi et al., 2011) 0.41

10 cis Methyl propenyl sulfide 1164 1153 (Pyun and Shin, 2006) 0.5511 Naphthalene 1189 1191 (Zeng et al., 2007) 0.2812 Decanal 1205 1206 (Bonaïti et al., 2005) 0.6513 Dimethyl tetrasulfide 1221 1215 (Bonaïti et al., 2005) 0.3414 Linalyl acetate 1254 1258 (Skaltsa et al., 2001) 0.4115 Caprolactam 1276 1267 (Teyeb et al., 2010) 3.7116 Di-2-propenyl trisulfide 1304 1296 (Ansorena et al., 2001) 0.9917 2-Methoxy thiophene 1331 Not available 0.7418 �-Elemene 1340 1339 (Skaltsa et al., 2001) 0.3019 Eugenol 1367 1364 (Ansorena et al., 2001) 8.4220 Geranyl acetate 1382 1383 (Asuming et al., 2005) 0.6221 Tetradecene 1388 1389 (Mimica-Dukic et al., 2003) 0.2422 Tetradecane 1395 1400 (Kovàts, 1958) 0.2423 Methyl eugenol 1407 1407 (Cole et al., 2007) 1.5524 �-Caryophylene 1426 1423 (Asuming et al., 2005) 0.7325 Isoeugenol 1459 1459 (Zhao et al., 2006) 1.5526 Butylated hydroxytoluene 1518 1518 (Ansorena et al., 2001) 10.0327 Elemicin 1559 1559 (Cole et al., 2007) 2.3928 trans-Nerolidol 1567 1566 (Zhao et al., 2006) 2.5129 Hexadecane 1595 1600 (Kovàts, 1958) 0.5830 Methoxyeugenol 1610 1609 (Ansorena et al., 2001) 0.4831 8-Heptadecene 1673 1677 (Xian et al., 2006) 0.2232 Heptadecane 1694 1700 (Kovàts, 1958) 0.2633 Farnesol 1726 1722 (Tounsi et al., 2011) 6.9234 Tetradecanoic acid 1766 1769 (Conforti et al., 2009) 0.2535 Benzyl benzoate 1773 1770 (Zeng et al., 2007) 0.2536 Octadecane 1792 1800 (Kovàts, 1958) 0.4437 Cyclohexadecane 1872 1875 (Conforti et al., 2009) 0.3238 1-Nonadecene 1886 1892 (Conforti et al., 2009) 0.8339 Hexadecanoic acid 1955 1963 (Zeng et al., 2007) 3.7440 Eicosane 1973 1995 (Kovàts, 1958) 0.5541 Heneicosane 2094 2097 (Kovàts, 1958) 0.5442 Z-9-Octadecenoic acid 2156 2152 (Skaltsa et al., 2001) 5.2343 Octadecanoic acid 2173 2172 (Conforti et al., 2009) 1.0044 Docosane 2197 2200 (Kovàts, 1958) 0.5645 Tricosane 2302 2300 (Kovàts, 1958) 4.7846 Tetracosane 2400 2400 (Kovàts, 1958) 0.6447 Pentacosane 2500 2500 (Kovàts, 1958) 2.85

Total 92.35

a Compounds are listed in order of their elution from the HP 5MS column.n.

(tb(1ws0stitimam

b Retention index calculated against C9–C26 n-alkanes mixture on HP-5MS colum

2001). Sample solutions (1 ml) of the extract different concentra-ions (100–500 �g/ml) were mixed with 1.25 ml of 0.2 M phosphateuffer (pH = 6.6) and 1.25 ml of potassium ferricyanide solution10 g/l). The mixtures were incubated at 50 ◦C for 30 min, then.25 ml of 10% (w/v) trichloroacetic acid was added. The mixturesere then centrifuged at 3000 × g for 10 min. Finally, 1.25 ml of the

upernatant solution was mixed with 1.25 ml of distilled water and.25 ml of ferric chloride (1 g/l). After 10 min reaction, the resultingolution absorbance was measured at 700 nm. The reaction mix-ure absorbance increase reflected the reducing power intensityncrease. The EC50 value (mg/ml) is the methanolic extract concen-ration at which the absorbance was 0.5 for the reducing power;

t was calculated from the graph absorbance at 700 nm against

ethanolic extract concentration (Tounsi et al., 2009). Ascorbiccid was used as positive control. The presented values were theeans of triplicate analyses.

3. Results and discussion

3.1. Essential oil analysis

The A. roseum fresh flowers subjected to hydrodistillation usinga Clevenger apparatus lead to a light yellow-colored essential oilwhich yielded 0.05% (v/w). The A. roseum essential oil chemicalcomposition was investigated using both GC and GC/MS tech-niques. The volatile compounds composition was based on thechemical functions (acids, alcohols, aliphatics, aromatics, sulfursand terpenes). The identified components, their percentages, theircalculated RI and their comparison according to the RI-values pre-

viously published in the literature are listed in Table 1, consideringthe order of the compounds elution on the HP-5MS column. A totalof 47 compounds were identified, accounting for almost the wholeoil constituents (93%). Butylated hydroxytoluene, eugenol, farnesol,

S. Zouari et al. / Industrial Crops and Products 41 (2013) 172– 178 175

hydro

darTadceTniraAsdcvacTrpDg

3

tsrwfi

TSfl

Fig. 1. S-compounds genesis during

imethyl trisulfide, methyl 2-propenyl trisulfide, Z-9-octadecenoiccid and tricosane were the most abundant components withates varying from 4.8 to 10.0%, constituting 46.7% of the total oil.hese were followed by 1-oxa-4,6-diazacyclooctane-5-thione, hex-decanoic acid, caprolactam, 1,3-dithiane and methyl 2-propenylisulfide which were at almost equal rates of about 3.5%. Othersomponents were present at rates inferior to 3%. The A. roseumssential oil chemical classes distribution was reported in Table 2.he identified components may be classified into sulfurous andon-sulfurous compounds. It should be mentioned that, accord-

ng to previous published data, the S-containing compounds areesponsible for the Liliaceae vegetable appropriate odor, flavor andlso health benefits (Block, 1985; Auger et al., 2004; Arnault anduger, 2006). These characteristics are recognized as the basictudied species value and may explain its important use in tra-itional medicine. Indeed, in the Allium genus, these S-containingompounds are present as alk(en)yl cysteine sulfoxides, a nonolatile precursors, and while distillating the vegetal material, thellinase is activated and catalyzed the reaction generating espe-ially thiosulfinates (Jaillais et al., 1999; Arnault and Auger, 2006).hese are very unstable compounds, and may give rise to furtherearrangements, leading to a wide variety of derived sulfurous com-ounds (monosulfides, disulfides, trisuldides and tetrasulfides).uring hydrodistillation of Allium genus material, the S-compoundseneses are presented according to the reaction (Fig. 1).

.1.1. Sulfurous compoundsTable 2 shows high content of sulfurous compounds (about the

hird of the total eluted compounds). Over the eleven identifiedulfurous compounds, five were present at relatively high rates,

anged from 3.5 to 6.1%. The most abundant ones among themere the substituted trisulfides, represented by dimethyl trisul-de as major compound, followed by methyl 2-propenyl trisulfide.

able 2ulfurous compounds and non sulfurous compounds of the essential oil from freshowers of Allium roseum (Sfax, Tunisia).

Compound Percentage

Sulfurous compounds 27.50Cyclic compounds 5.47Non cyclic compounds 22.02

Non sulfurous compounds 64.85Phenolic derivatives 24.42Hydrocarbons 13.42Fatty acids 10.22Oxygenated sesquiterpene 9.43Cyclic ketones and cyclic amide 4.12Esters 1.28Hydrocarbons sesquiterpene 1.03Aldehydes 0.65Aromatic compounds 0.28

distillation of Allium genus material.

These compounds were also dominant in the A. roseum fresh flow-ers essential oil collected from Bengardane area, in the South-Eastof Tunisia (Zouari et al., 2012).

Considering the cyclic sulfurous compound, the major com-pound was the 1-oxa-4,6-diazacyclooctane-5-thione, isolated forthe first time in Tunisian A. roseum harvested in the centralTunisia (Sfax city). To our knowledge, no previous report identifiedthis volatile sulfurous compound in A. roseum species. However,Prithiviraj et al. (2004) described the 1-oxa-4,6-diazacyclooctane-5-thione isolated from onion bulb inoculated with Fusariumoxysporum, as a specific metabolite and a volatile vegetable com-pound to detect and discriminate onion basal rots disease. Thismetabolite would be a useful biosensor in non destructive essayfor the early detection system of the disease presence. Morerecently, the 1-oxa-4,6-diazacyclooctane-5-thione was detected atrelatively high rate among the volatile compounds identified ingarlic harvested in Xuzhou area of China, extracted by solid phasemicroextraction and analyzed by GC/MS (Ma et al., 2011).

Sulfurous compounds could be separated into two major groupsaccording to their acyclic or cyclic structure. These compoundswould be generated from non volatile precursors i.e. alk(en)yl cys-teine sulfoxydes as presented in reaction (I) in Fig. 1. Furthermore,the allinase would be the main enzyme involved in these volatilemetabolites biosynthesis.

The acyclic group included various compounds from mono topolysulfide, substituted by different radicals (trisulfide, disulfide,monosulfide and tetrasulfide). While in garlic compounds, alliin (S-alk(en)yl-l-cysteine sulfoxide) was the major compound, in onionisoalliin with 1-propenyl residue were the main metabolite (Augeret al., 2004; Prithiviraj et al., 2004) and in A. roseum, the polysulfidecompounds were predominant (Najjaa et al., 2007; Zouari et al.,2012). Furthermore, in the A. roseum essential oil collected fromthe South-East of Tunisia, the most abundant volatile compoundswere diallyl trisulfides followed by diallyl disulfides accounting for12.3% and 8.9% respectively (Zouari et al., 2012). In the presentstudy, the trisulfides were equally present as dimethylated (6%)and methyl 2-propenylated (5.3%), and finally as di-2-propenylated(1%) at a low rate (Table 1). The abundance of these compounds wasalso mentioned in Allium cepa L. and Allium fistulosum L. essentialoils (Prithiviraj et al., 2004; Gyawali et al., 2005; Pyun and Shin,2006). Monosulfide and tetrasulfide compounds were detected atminor rates (Table 1), compared to A. roseum essential oil flow-ers harvested in Bengardene. In the latter area, such compoundswere mainly represented by methional (Najjaa et al., 2007). It maybe suggested that meterological conditions would orchestrate themetabolic pathways.

Apart 1-oxa-4,6-diazacyclooctane-5-thione as major cyclic sul-

furous compound (3.9%), the 1,3-dithiane was also found atrelatively high rate (3,61%), while the 2-methoxy thiophene wasdetected at low rate (<1%). The first compound was also found inthe A. roseum essential oil collected from the South-East of Tunisia

176 S. Zouari et al. / Industrial Crops and Products 41 (2013) 172– 178

Table 3Mass spectral data of some newly isolated Allium roseum oil compounds.

Compound Retention time in min Percentage MW Structure m/z (%)

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

NH

O

S

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

Caprolactam 13.88 3.71 113

N H

O

113(95), 85(62), 67(13), 55(100), 42(47)

Elemicin 19.09 2.39 208CH3O

CH3O OCH3

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

Farnesol 21.83 6.92 222

OH

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

asorh

3

ma(rtisk2hcagiia2moan2

nd in Allium sativum bulb (Ejaz et al., 2003; Zouari et al., 2012). Theecond compound was evidenced for the first time in the essentialil of Allium genus with a relatively low level, and would characte-ize the essential oil of A. roseum L. var. odoratissimum chemotypearvested in Sfax area.

.1.2. Non-sulfurous compoundsThe A. roseum essential oil non-sulfurous compounds contained

ainly phenolic derivatives (butylated hydroxytoluene, eugenolnd its derivatives), representing the fourth of the total compoundsTable 2). Besides the butylated hydroxytoluene (10.03%), eugenolepresented the most dominant phenolic compound in this essen-ial oil, with a rate of 35% of the total phenolic derivatives. Eugenols used as a flavoring agent in cosmetic and in foods and has atrong antioxidant effect which may contribute to aldehydes andetones reduction (Chevance and Farmer, 1999; Fujisawa et al.,002). The second non-sulfurous compounds abundant group wasydrocarbons followed by fatty acids group at relatively high rates,onsidering that the sample was not methylated and hence fattycids would not be in the suitable conditions to be identified. Oxy-enated sesquiterpenes were also found at relatively high rate andncluded mainly farnesol (74%) of the total sesquiterpene. Farnesols a very interesting compound promoting epithelial cell defensegainst Candida albicans, a human pathogenic yeast (Décanis et al.,009). This compound was also evidenced to be a quorum sensingolecule produced by the dimorphic fungus C. albicans. More-

ver, farnesol acts to suppress C. albicans mycelia development in quorum-sensing manner and it was suggested as the first of aovel antifungal compounds class (Hornby et al., 2001; Gregus et al.,010). Interestingly, in the present work camphor, an oxygenated

monoterpene, was found at very low rate (0.41%), compared toA. roseum essential oil flowers harvested in the Tunisian South-East (13.4%) (Najjaa et al., 2007). Based on its concentration, theA. roseum flowers would be a safety food when wild grown in Sfax(Tunisia).

In Tunisia, the essential oils chemical profiles of the A. roseumflowers collected from the center (Sfax) and the South-East (Zouariet al., 2012) differed not only in the number of sulfurous com-pounds but also in their chemotypes (acyclic or cyclic structure) andrates. Indeed, in the Center the essential oil contained much morenon sulfurous compounds than those identified in the South-East,explaining the low sulfurous compounds rates found in Sfax andconsequently a lower number of sulfurous compounds. These couldbe attributed to several factors such as climate, soil compositionand 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 com-pounds and their relative area percentages are presented inTable 3. The newly sulfurous constituent was the 1-oxa-4,6-diazacyclooctane-5-thione with a retention time of 10.25 min andM+ ion at 146 represented the base peak in its mass spectrum. Frag-ments at m/z 73 and 61, could be attributed to [CH2NHCS]+• or[CH2OCH2CH2NH]+• and [HOCH2CH2NH2]+•. Moreover, the newlynon sulfurous compound, having a retention time of 13.88 min,

was characterized by a mass ion m/z 113 and identified ascaprolactam, with the fragment ion ([CH3CH CHCH2]+•) at m/z55 as the base peak. Caprolactam MS displayed also peaks atm/z 85 and 42, corresponding to the following fragment ions:

S. Zouari et al. / Industrial Crops and

Table 4Antiradical (DPPH) and reducing activity of Allium roseum leaves methanolic extract.

Compounds EC50 (�g/ml) DPPH IC50 (�g/ml) reducing power

A. roseum leavesmethanolic extract

240.4 ± 0.5 248.7 ± 0.5

[ctat

1figeoh

twt[wimeg

3

ptp

3

5stimslcmrfisdrewwoeia

3

d

BHA 14.1 ± 0.2 –Ascorbic acid – 6.7 ± 0.1

CH2CH2CH2CH2CH2NH]+• and [CH2CH2CH2]+•. It is noticeable thataprolactam, a hydrophobic organic compound, was recently iden-ified in leachate water samples from landfill site, and its MS was ingreement with the A. roseum mass spectral assignments and withheir good matching with library spectrum (Uchida et al., 2009).

Manfully, another interesting constituent was eluted at9.09 min and had M+• ion at m/z 208. This compound was identi-ed as elemicin, based on its mass spectral assignments and theirood matching with library spectrum. It should be mentioned thatlemicin was recently shown to exhibit dose-dependent inhibitionf the ADP-induced human platelet serotonin release and wouldave a health effect (Grice et al., 2010).

Finally, another interesting compound was identified at rela-ively high rate, with a retention time of 21.83 min and a moleculareight of 222. Its MS displayed peaks at m/z 136, 121 and 69, linked

o ion arising from loss of hydrogen and methyl of the fragment(CH3)2C CHCH2CH2C(CH3) CHCH2]+•, and [(CH3)2C CHCH2]+•

ith the peak at m/z 69 being the base peak. This compound wasdentified as farnesol, based on its mass spectrum and the spectral

atch with that of the standard. This compound of health ben-fit would attribute a neutraceutical value to this Allium species,rowing in the Centre of Tunisia under semi arid climate.

.2. Antioxidant activity determination

The A. roseum leaves methanolic extract was investigated forossible antioxidative capacities by using two complementaryests: DPPH radical-scavenging and reducing power essays, com-ared respectively to those of BHA and ascorbic acid.

.2.1. DPPH radical-scavenging activityDPPH is a stable free radical with a maximum absorbance at

17 nm. When DPPH radicals encounter a proton-donating sub-trate such as an antioxidant, the radicals would be scavenged andhe absorbance is reduced (Shimada et al., 1992). The decreasen absorbance would inform on radical-scavenging activity. This

ethod is widely used to investigate the scavenging activity ofome natural compounds (Hajji et al., 2010). The leaves methano-ic extract IC50 value was 17 times that of BHA (Table 4). Whenompared to the synthetic antioxidant BHA, the A. roseum leavesethanolic extract exhibited moderate antioxidant abilities to

educe DPPH radicals. Previous study on the antioxidant activity ofve Allium methanolic extracts species (Allium nevsehirense, Alliumivasicum, Allium dictyoprosum, Allium scrodoprosum subsp. rotun-um and Allium atroviolaceum), measured by DPPH, showed an IC50anged between 79 and 104 �g/ml with an efficiency of 3.95 (IC50xtract/IC50 BHT) (Tepe et al., 2005). For A. roseum, the efficiencyas equal to 17.05 (IC50 extract/IC50 BHA) and antioxidant activityas 4.5 times lower than that of the other Allium species previ-

usly studied. For further comparison with other species, Candant al. (2003) show that the essential oil of Achillea millefolium, richn eucalyptol and camphor, possesses strong antioxidative activitys evaluated by DPPH method.

.2.2. Reducing powerThe reducing power-essay is often used to evaluate the antioxi-

ant ability to donate electrons (Yildirim et al., 2000). The ability of

Products 41 (2013) 172– 178 177

the A. roseum leaves methanolic extract, and ascorbic acid as a posi-tive control, to reduce Fe3+ to Fe2+ is presented in Table 4. A directcorrelation between antioxidative activities and reducing powerof some bioactive compounds was exhibited by the conversion ofFe3+/ferricyanide complex to ferrous form (Shimada et al., 1992;Hajji et al., 2010).

The A. roseum leaves methanolic extract EC50 value of reducingpower ability was 37 times that of ascorbic acid, exhibiting a lowreducing power.

4. Conclusion

The results of this work describe the chemical composi-tion of essential oil of A. roseum growing wild in the semiarid region of Tunisia (central Tunisia) which showed the pre-sence of a wide range of compounds, accounting for more than90% of the whole oil constituents. Butylated hydroxytoluene,eugenol, farnesol, dimethyl trisulfide, methyl 2-propenyl trisul-fide, Z-9-octadecenoic acid and tricosane were the most abundantcomponents, constituting almost half the total oil. Moreover, 1-oxa-4,6-diazacyclooctane-5-thione, found at relatively high rate, wasisolated for the first time in the Tunisian A. roseum, wild growingin the Center of the country (Sfax City). The present data revealsthe characteristic composition of the essential oil of this specieswhich could be considered as a new chemotype specific to thespices growing in Sfax city, adapted to the climatic and the soilconditions of this region.

Since the antioxidant activities of the studied plant, evalu-ated by two complementary tests: DPPH radical-scavenging andreducing power-essays, have not been reported before, our findingdemonstrate that the methanolic leaves extract exhibited mode-rate antioxidant activities when compared to those of butylatedhydroxyl anisole and ascorbic acid. Therefore, it could be also con-cluded that the study presented herein is the first report on theessential oil composition and antioxydant activity of the A. roseumL. chemotype, growing wild in the semi arid region of Tunisia (Sfaxcity). Nevertheless, further investigations with other Allium speciesfrom this bioclimatic stage would be interesting to be considered,allowing the confirmation of the essential oil chemotype finger-print of the species.

Acknowledgment

The authors are very grateful to Mrs. Hela Chabouni Fourati, anEnglish teacher-trainer in the area of Sfax, for English Languagecorrection.

References

Angioni, A., Barra, A., Coroneo, V., Dessi, S., Cabras, P., 2006. Chemical composition,seasonal variability, and antifungal activity of Lavandula stoechas L. ssp. stoechasessential oils from stem/leaves and flowers. Journal of Agricultural and FoodChemistry 54, 4364–4370.

Ansorena, D., Gimeno, O., Astiasaran, I., Bello, J., 2001. Analysis of volatile compoundsby GC–MS of a dry fermented sausage: chorizo de Pamplona. Food ResearchInternational 34 (1), 67–75.

Arnault, I., Auger, J., 2006. Seleno-compounds in garlic and onion. Journal of Chro-matography A 1112, 23–30.

Asuming, W.A., Beauchamp, P.S., Descalzo, J.T., Dev, B.C., Dev, V., Frost, S., Ma, C.W.,2005. Essential oil composition of four Lomatium Raf. species and their chemo-taxonomy. Biochemical Systematics and Ecology 33, 17–26.

Auger, J., Yang, W., Arnault, I., Pannier, F., Potin-Gautier, M., 2004. High-performanceliquid chromatographic–inductively coupled plasma mass spectrometric evi-dence for Se-alliins in garlic and onion grown in Se-rich soil. Journal ofChromatography A 1032, 103–107.

Basar, S., Koch, A., Konig, W.A., 2001. A verticillane-type diterpene from Boswellia

carterii essential oil. Flavour and Fragrance Journal 16 (5), 315–318.

Ben Jannet, H., Sakka-Rouis, L., Neffati, M., Mighri, Z., 2007. Chemical compositionof flowers and stems essential oils from the Tunisian Allium roseum L. JournalEssential Oil Bearing Plant 10 (2), 151–156.

Block, E., 1985. The chemistry of garlic and onions. Scientific American 252, 94–99.

1 ps and

B

B

C

C

C

C

C

D

D

E

F

G

G

G

G

G

H

H

H

I

J

J

J

K

K

K

L

of Rhododendron with the help of chemometrics methods. Chemometrics and

78 S. Zouari et al. / Industrial Cro

lock, G., Patterson, B., Sapers, G.M., 1992. Varietal differences in distribution ofquercetin and kaempferol in onion (Allium cepa) tissue. Journal of Agriculturaland Food Chemistry 32, 274–276.

onaïti, C., Irlinger, F., Spinnler, H.E., Engel, E., 2005. An iterative sensory proce-dure to select odor-active associations in complex consortia of microorganisms:application to the construction of a cheese model. Journal of Dairy Science 88,1671–1684.

andan, F., Unlu, M., Tepe, B., Daferera, D., Polissiou, M., Sökmen, A., Akpulat, H.A.,2003. Antioxidant and antimicrobial activity of the essential oil and methanolextracts of Achillea millefolium subsp. millefolium Afan. (Asteraceae). Journal ofEthnopharmacology 87, 215–220.

hevance, F.F.V., Farmer, L.J., 1999. Identification of major volatile odor compoundsin frankfurters. Journal of Agricultural and Food Chemistry 47, 5151–5160.

ole, R.A., Haber, W.A., Setzer, W.N., 2007. Chemical composition of essential oils ofseven species of Eugenia from Monteverde, Costa Rica. Biochemical Systematicsand Ecology 35, 877–886.

onforti, F., Menichini, F., Formisano, C., Rigano, D., Senatore, F., Arnold, N.A., Piozzi,F., 2009. Comparative chemical composition, free radical-scavenging and cyto-toxic properties of essential oils of six Stachys species from different regions ofthe Mediterranean Area. Food Chemistry 116, 898–905.

uénod, A., 1954. Flore de la Tunisie. Office de l’Expérimentation et de la Vulgarisa-tion Agricole de Tunisie, 287 p.

écanis, N., Savignac, K., Rouabhia, M., 2009. Farnesol promotes epithelialcell defense against Candida albicans through Toll-like receptor 2 expres-sion, interleukin-6 and human �-defensin 2 production. Cytokine 45,132–140.

iplock, A.T., Charleux, J.L., Crozier-Willi, G., Kok, F.J., Rice-Evans, C., Roberfroid,M., Stahl, W., Vina-Ribes, J., 1998. Functional food science and defence againstreactive oxidative species. British Journal of Nutrition 80, 77–112.

jaz, S., Woong, L.C., Ejaz, A., 2003. Extraction of garlic (Allium sativum) in cancerchemoprevention. Experimental Oncology 25, 93–97.

ujisawa, S., Atsumi, T., Kadoma, Y., Sakagami, H., 2002. Antioxidant and prooxidantaction of eugenol-related compounds and their cytotoxicity. Toxicology 177,39–54.

o –devac, D., Vujisic, L., Mojovic, M., Ignjatovic, A., Spasojevic, I., Vajs, V., 2008. Eval-uation of antioxidant capacity of Allium ursinum L. volatile oil and its effect onmembrane fluidity. Food Chemistry 107, 1692–1700.

ourine, N., Yousfi, M., Bombarda, I., Nadjemi, B., Stocker, P., Gaydou, E.M., 2010.Antioxidant activities and chemical composition of essential oil of Pistaciaatlantica from Algeria. Industrial Crops and Products 31, 203–208.

regus, P., Vlckova, H., Buchta, V., Kestranek, J., Krivciková, L., Nováková, L., 2010.Ultra high performance liquid chromatography tandem mass spectrometryanalysis of quorum-sensing molecules of Candida albicans. Journal of Pharma-ceutical and Biomedical Analysis 53, 674–681.

rice, I.D., Rogers, K.L., Griffiths, L.R., 2010. Isolation of Bioactive Compounds thatRelate to the Anti-platelet Activity of Cymbopogon ambiguous, eCAM AdvanceAccess. Oxford University Press, pp. 1–8.

yawali, R., Seo, H.Y., Lee, H.J., Song, H.P., Kim, D.H., Byum, M.W., Kim, K.S., 2005.Effect of �-irradiation on volatile compounds of dried welsh onion (Allium fistu-losum L.). Radiation Physics and Chemistry 75, 322–328.

ajji, M., Masmoudi, O., Souissi, N., Triki, Y., Kammoun, S., Nasri, M., 2010. Chemicalcomposition, angiotensin I-converting enzyme (ACE) inhibitory, antioxidant andantimicrobial activities of the essential oil from Periploca laevigata root barks.Food Chemistry 121, 724–731.

elen, A., Krishnakumar, K., Vijayammal, P.L., Augusti, K.T., 2000. Antioxidant effectof onion oil (Allium cepa. Linn) on the damage induced by nicotine in rats ascompared to alpha-tocopherol. Toxicology Letters 116, 61–68.

ornby, J.M., Jensen, E.C., Lisec, A.D., Tasto, J.J., Jahnke, B., Shoemaker, R., Dussault, P.,Nickerson, K.W., 2001. Quorum sensing in the dimorphic fungus Candida albi-cans is mediated by farnesol. Applied and Environment Microbiology 67 (7),2982–2992.

nsausti, K., Goni, V., Petri, E., Gorraiz, C., Beriain, M.J., 2005. Effect of weight at slaugh-ter on the volatile compounds of cooked beef from Spanish cattle breeds. MeatScience 70, 83–90.

aillais, B., Cadoux, F., Auger, J., 1999. SPME-HPLC analysis of Allium lacrymatoryfactor and thiosulfinates. Talanta 50, 423–431.

arunrattanasri, A., Theerakulkait, C., Cadwallader, K.R., 2007. Aroma componentsof acid-hydrolyzed vegetable protein made by partial hydrolysis of rice branprotein. Journal of Agricultural and Food Chemistry 55, 3044–3050.

endoubi, R., Neffati, M., Henchi, B., Yobi, A., 2001. Système de reproduction et vari-abilité morpho-phénologique chez Allium roseum L. Plant Genetic ResourcesNewsletter 127, 29–34.

im, S.M., Kubota, K., Kobayashi, A., 1997. Antioxidative activity of sulfur-containingflavor compounds in garlic. Bioscience Biotechnology and Biochemistry 61,1482–1485.

irby, A.J., Schmidt, R.J., 1997. The antioxidant activity of Chinese herbs for eczemaand of placebo herbs. Journal of Ethnopharmacology 56, 103–110.

ovàts, E., 1958. Gas chromatographische charakterisierung organischer verbindun-gen teil 1 retentions indices aliphatischer halogenide, alkohole, aldehyde undketone (Characterization of organic compounds by gas chromatography. Part1. Retention indices of aliphatic halides, alcohols, aldehydes and ketones). Hel-

vetica Chimica Acta 41, 1915–1932.

ampe, J.W., 1999. Health effects of vegetables and fruit: assessing mechanisms ofaction in human experimental studies. American Journal of Clinical Nutrition70, 475S–490S.

Products 41 (2013) 172– 178

Ma, Y., Song, D., Wang, Z., Jiang, J., Jiang, T., Cui, F., Fan, X., 2011. Effect of ultrahighpressure treatment on volatile compounds in garlic. Journal of Food ProcessEngineering 34, 1915–1930.

Marcucci, R., Tornadore, N., 1997. Intraspecific variation of Allium roseum L. (Alli-aceae). Webbia 52, 137–154.

Mimica-Dukic, N., Kujundzic, S., Sokovic, M., Couladis, M., 2003. Essential oil com-position and antifungal activity of Foeniculum vulgare Mill, obtained by differentdistillation conditions. Phytotherapy Research 17, 368–371.

Najjaa, H., Neffati, M., Zouari, S., Ammar, E., 2007. Essential oil composition andantibacterial activity of different extracts of Allium roseum L., a North Africanendemic species. Compte Rendue Chimie 10, 820–826.

Najjaa, H., Zerria, K., Fattouch, S., Ammar, E., Neffati, M., 2011. Antioxidantand antimicrobial activities of Allium roseum Lazoul, a wild edible endemicspecies in North Africa. International Journal of Food Properties 14 (2),371–380.

Patro, B.S., Bauri, A.K., Mishra, S., Chattopadhyay, S., 2005. Antioxidant activity ofMyristica malabarica extracts and their constituents. Journal of Agricultural andFood Chemistry 53, 6912–6918.

Pérez, R.A., Navarro, T., de Lorenzo, C., 2007. HS-SPME analysis of the volatilecompounds from spices as a source of flavour in ‘Campo Real’ table olive prepa-rations. Flavour and Fragrance Journal 22, 265–273.

Prithiviraj, B., Vikram, A., Kushalappa, A.C., Yaylayan, V., 2004. Volatile metaboliteprofiling for the discrimination of onion bulbs infected by Erwinia carotovorassp. carotovora, Fusarium oxysporum and Botrytis allii. European Journal of PlantPathology 110, 371–377.

Pyun, M.S., Shin, S., 2006. Antifungal effects of the volatile oils from Allium plantsagainst Trichophyton species and synergism of the oils with ketoconazole. Phy-tomedecine 13, 394–400.

Quezel, S., Santa, S., 1963. Nouvelle flore de l’Algérie et des régions désertiquesméridionales, Tomes I and II. CNRS, Paris, 1170 p.

Ricroch, A., Yockteng, R., Brown, S.C., Nadot, S., 2005. Evolution of genome size acrosssome cultivated Allium species. Genome 48, 511–520.

Shimada, K., Fujikawa, K., Yahara, K., Nakamura, T., 1992. Antioxidative propertiesof xanthan on the autioxidation of soybean oil in cyclodextrin emulsion. Journalof Agricultural and Food Chemistry 40, 945–948.

Shon, M.-Y., Choi, S.-D., Kahng, G.-G., Nam, S.-H., Sung, N.-J., 2004. Antimu-tagenic, antioxidant and free radical scavenging activity of ethyl acetateextracts from white, yellow and red onions. Food and Chemical Toxicology 42,659–666.

Singh, B.N., Singh, B.R., Singh, R.L., Prakash, D., Singh, D.P., Sarma, B.K., Upadhyay,G., Singh, H.B., 2009. Polyphenolics from various extracts/fractions of red onion(Allium cepa) peel with potent antioxidant and antimutagenic activities. Foodand Chemical Toxicology 47, 1161–1167.

Skaltsa, H.D., Mavrommati, A., Constantinidis, T., 2001. A chemotaxonomic inves-tigation of volatiles constituents in stachys subsect. Swainsonianeae (Labiatae).Phytochemistry 57, 235–244.

Stearn, W.T., 1992. How many species of Allium are known? Kew Magazine 9,180–181.

Tepe, B., Sokmen, M., Akpulat, H.A., Sokmen, A., 2005. In vitro antioxidant activitiesof the methanol extracts of five Allium species from Turkey. Food Chemistry 92,89–92.

Teyeb, H., Zouari, S., Douki, W., Najjar, M.F., Neffati, M., 2010. Essential oils of leaves,flowers and fruits of Astragalus gombiformis (Pomel) Fabaceae. Proc. IS on MAP-SIPAM 2009. Acta Horticulturae 853, ISHS 2010.

Tounsi, M.S., Ouerghemmi, I., Wannes, W.A., Ksouri, R., Zemni, H., Marzouk, B.,Kchouk, M.E., 2009. Valorization of three varieties of grape. Industrial Cropsand Products 30, 292–296.

Tounsi, M.S., Wannes, W.A., Ouerghemmi, I., Msaadaa, K., Smaoui, A., Marzouk, B.,2011. Variation in essential oil and fatty acid composition in different organs ofcultivated and growing wild Ruta chalepensis L. Industrial Crops and Products33, 617–623.

Uchida, M., Hatayoshi, H., Syuku-Nobe, A., Shimoyama, T., Nakayama, T., Okuwaki, A.,Nishino, T., Hemmi, H., 2009. Polymerase chain reaction-denaturing gradient gelelectrophoresis analysis of microbial community structure in landfill leachate.Journal of Hazardous Materials 164, 1503–1508.

Xian, Q., Chen, H., Zou, H., Yin, D., 2006. Chemical composition of essential oils oftwo submerged macrophytes, Ceratophyllum demersum L. and Vallisneria spiralisL. Flavour and Fragrance Journal 21, 524–526.

Yildirim, A., Mavi, A., Oktay, M., Kara, A.A., Algur, Ö.F., Bilaloglu, V., 2000. Comparisonof antioxidant and antimicrobial activities of Tilia (Tilia argentea Desf Ex DC), sage(Salvia triloba L.) and black tea (Camellia sinensis) extracts. Journal of Agriculturaland Food Chemistry 48, 5030–5034.

Yildirim, A., Mavi, A., Kara, A., 2001. Determination of antioxidant and antimicrobialactivities of Rumex crispus L. extracts. Journal of Agricultural and Food Chemistry49, 4083–4089.

Zeng, Y.-X., Zhao, C.-X., Liang, Y.-Z., Yang, H., Fang, H.-Z., Yi, L.-Z., Zeng, Z.-D., 2007.Comparative analysis of volatile components from Clematis species growing inChina. Analytica Chimica Acta 595, 328–339.

Zhao, C.X., Li, X.N., Liang, Y.Z., Fang, H.Z., Huang, L.F., Guo, F.Q., 2006. Compara-tive analysis of chemical components of essential oils from different samples

Intelligent Laboratory Systems 82, 218–228.Zouari, S., Najjaa, H., Neffati, M., Ammar, E., 2012. A new essential oil chemotype

of Allium roseum analysed by an apolar column. International Journal of FoodProperties 15 (2), 385–397.