Research Article

Received: 19 June 2012, Revised: 4 February 2013, Accepted: 24 February 2013 Published online in Wiley Online Library

(wileyonlinelibrary.com) DOI 10.1002/ffj.3160

Chemical variability and antioxidant activity of Eryngium maritimum L. essential oils from Corsica and Sardinia
Florent Darriet,a Stphane Andreani,a Marie-Ccile De Cian,b Jean Costaa and Alain Musellia*
ABSTRACT: The chemical compositions of Corsican and Sardinian Eryngium maritimum L. essential oils were investigated using column chromatography, gas chromatography with ame ionization detection and gas chromatographymass spectrometry in electron impact mode. Sixty-three compounds were identied accounting for 85.895.7% of the total amount. With germacrene D (13.745.9%), three uncommon oxygenated sesquiterpenes, 4bH-cadin-9-en-15-al (18.427.6%), 4bH-cadin-9en-15-ol (2.214.3%) and 4bH-muurol-9-en-15-al (4.39.3%), were identied as main components of the essential oils obtained from the plant aerial parts. Relative to these, essential oils from the roots differed drastically with high contents of 2,4,5-trimethylbenzaldehyde (39.8%), 2,3,6-trimethylbenzaldehyde (29.0%) and a-muurolene (23.5%). The chemical variability of Corsican and Sardinian E. maritimum essential oils was studied using statistical analysis. A direct correlation between the island of origin of the sample essential oils and their chemical compositions was assessed. Corsican essential oils exhibited higher amounts of hydrocarbon terpenes than Sardinian samples. Relative to other Eryngium species, Corsican and Sardinian E. maritimum essential oils exhibited original compositions with sesquiterpene aldehydes. These compounds, together with total essential oil, were tested for antioxidant properties using the DPPH and ABTS radical-scavenging activity tests. No meaningful activity could be attributed to sesquiterpene aldehydes and the corresponding alcohols but the total essential oil and the oxygenated fraction of E. maritimum both demonstrated strong antioxidant properties. Copyright 2013 John Wiley & Sons, Ltd. Keywords: Eryngium maritimum L.; essential oil; antioxidant activities; chemical variability; aldehyde sesquiterpenes

Introduction
The genus Eryngium belongs to the Apiaceae family and includes around 250 species that are widespread throughout the world.[1] Among them, several Eryngium species have been used as ornamental plants, condiments[2] or in traditional medicine.[3,4] Eryngium maritimum L., usually named sea holly in England or Panicaut des mers in France, is a perennial plant (3060 cm high) with mauve owers (blossoming time, JuneSeptember), growing wild on the sandy beaches of western Europe, the Mediterranean basin and the Black Sea.[1] The plant is one of the typical dune species implicated in the plant network that contributes to sand dune edication and restoration.[5,6] E. maritimum has also been reported to exhibit different therapeutic uses in folk medicine.[7] Several studies concerning the chemical compositions of solvent extracts from the Eryngium genus report triterpene saponines,[8,9] acetylenic compounds[10] and polyphenols[11] as the main constituents. Essential oils of more than 20 Eryngium species have already been studied and their chemical compositions are generally dominated by hydrocarbon sesquiterpenes such as germacrene D,[12,13] bicyclogermacrene,[14] g-muurolene[14] and trans-caryophyllene[15,16] or by non-terpenic oxygenated compounds such as trimethylbenzaldehyde and (E)-2-dodecenal.[1722] To our knowledge, only two poster communications deal with the chemical composition of E. maritimum essential oils. Both studies were incomplete in terms of sample origins, full chemical composition and relative percentages of the components; only the main oil components were reported. The rst study reports

germacrene D (43.1%) and 9-muurolene-15-aldehyde (22.4%) as main components of the essential oil from the aerial parts, and g-guaiene (40.2%), trimethylbenzaldehyde (24.5%) and germacrene D (10.6%) as main components of the essential oil from the roots.[23] The second study reports spathulenol, 1,5epoxysalvial-4(14)-ene, a-amorphene and caryophellene oxide as main compounds of the essential oil from the aerial parts.[24] Previous laboratory investigations of E. maritimum essential oil allowed the isolation of a known sesquiterpene (4bH-muurol-9-en15-al) and three new oxygenated sesquiterpenes (4bH-cadin-9-en15-al, 4bH-muurol-9-en-15-ol and 4bH-cadin-9-en-15-ol) which were efcient against Listeria monocytogenes and Echerichia coli.[25] The discovery of new natural antioxidants has been a chemical challenge in the last decade. Furthermore, toxic and/or mutagenic effects of many synthetic antioxidant components have suggested plant antioxidants as an interesting alternative. It is commonly assumed that almost all phenols can function as

* Correspondence to: Alain Muselli, Universit de Corse, UMR-CNRS 6134, Laboratoire Chimie des Produits Naturels, Campus Grimaldi, BP 52, 20250 Corte, France. E-mail: muselli@univ-corse.fr
a Universit de Corse, UMR CNRS 6134 SPE, Laboratoire Chimie des Produits Naturels, BP 52, 20250, Corte, France b Universit de Corse, UMR-CNRS 6134 SPE, Laboratoire de Gntique Molculaire, BP 52, 20250, Corte, France

Flavour Fragr. J. 2013

Copyright 2013 John Wiley & Sons, Ltd.

F. Darriet et al. antioxidants of lipid peroxidation, while the oxygenated terpenes had a high hydrogen-donating capacity towards radicals.[11] The aim of the present work was to obtain a better insight into the nature of the E. maritimum volatiles by: (1) using column chromatography (CC), gas chromatography (GC) and gas chromatographymass spectrometry (GC-MS) analysis of the essential oils obtained from aerial parts and roots; (2) investigating intra-species variations in the essential oils from six Corsican and six Sardinian sample locations and comparing the oils with those from literature data; and (3) determining the antioxidant activities by using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,20 -azinobis-(3ethylbenzothiazoline-6-sulfonic acid) (ABTS) methods with the entire oil and the separated uncommon oxygenated sesquiterpenes. University of Corsica, Corte, France. The fresh plant material was hydro-distilled (5 h) using a Clevenger-type apparatus according to the method recommended in the European Pharmacopoeia,[26] and the yield of essential oil was 0.060.13% based on fresh weight.

Oil Fractionation Sample C1 (2 g) was submitted to column chromatography (CC) on a silica gel (column diameter 2 cm, 30 g of 200500 mm silica, height 11 cm, ow 6 mL/min). A hydrocarbon fraction (HF; 599 mg) was obtained by elution with n-pentane and an oxygenated fraction (OF; 1250 mg) was obtained by using pure diethyl oxide. The hydrocarbon fraction (500 mg) was further chromatographed on silica gel impregnated with silver nitrate (column diameter 1 cm, 20 g of 63200 mm silica with 5 g of silver powder, height 22.5 cm, ow 2.5 mL/min), leading to 12 fractions by elution with npentane [HF1 (40 mg), HF2 (30 mg), HF3 (50 mg), HF4 (70 mg), HF5 (70 mg), HF6 (60 mg), HF7 (40 mg), HF8 (30 mg), HF9 (30 mg), HF10 (20 mg), HF11 (20 mg) and HF12 (20 mg)]. The oxygenated fraction (1202 mg) was further chromatographed on silica gel (column diameter 1 cm, 60 g of 63200 mm silica, height 46.5 cm, ow 2 mL/min) with a gradient pentanediethyl oxide (95/5) leading to six fractions [OF1 (300 mg), OF2 (180 mg), OF3 (130 mg), OF4 (130 mg), OF5 (200 mg) and OF6 (240 mg)].

Materials and Methods

Chemicals For measurement of response factors (RFs), the chemicals used were: neo-allo-ocimene, a-pinene, b-pinene, g-terpinene, limonene, b-caryophyllene, a-humulene, aromadendrene, nerol, lavandulol, (E)-hex-3-en-1-ol, cedrol, globulol, pentyl acetate, lavandulyl acetate, trans-myrtenyl acetate, cedryl acetate, artemisia ketone, camphor, jasmone, isoborneol methyl ether, carvacrol methyl ether, caryophyllene oxide, (E)-2-hexenal, (E,E)-2,4-decadienal and (E)-2-decenal. Authentic chemical samples were obtained from SigmaAldrich (Saint-Quentin Fallavier, France) and Fluka (SaintQuentin Fallavier, France) in the highest available purity. For the antioxidant screening, methanol, DPPH and ascorbic acid were purchased from SigmaAldrich. Plant Material and Isolation of the Essential Oil Aerial parts of Eryngium maritimum L. were collected in full bloom (June 2009) from six stations in Corsica (C1C6) and six stations in Sardinia (S1S6). The sample numbers, the localities of harvest and the GPS coordinates are given in Table 1. For the study of the chemical composition of the essential oil from E. maritimum separated organs, a supplementary harvest of stems, owers, leaves and roots was obtained from Quercioni location (C1). A voucher specimen was deposited in the herbarium of the

Reduction of Oxygenated Fraction 1 Fraction OF1 (300 mg) was dissolved in dry diethyl ether (40 mL) and carefully added to a suspension of aluminium lithium hydride (100 mg) in dry diethyl ether (60 mL) at 0 C. The mixture was stirred at room temperature and then reuxed for 3 h. The reaction mixture was hydrolysed by the addition of 15% sodium hydroxide solution (1 mL) and cold water. The organic layer was separated, washed with water to neutrality, dried over sodium sulfate and concentrated under vacuum. After purication on CC the alcohol-rich fraction (ROF1; 250 mg) contained 4bH-cadin-9-en-15-ol (63.8%) and 4bH-muurol-9-en-15-ol (33.5%) as major components.

Table 1. Data relative to the different samples of plant material No.a C1 C2 C3 C4 C5 C6 S1 S2 S3 S4 S5 S6

a b

Localityb Quercioni Pinia Marana Ostriconi Golfe de Ventilegne Calzarello La Caletta Isula di Caprera Marina di Sorso Golfo di S Ena Praxis Cagliari 41 42 42 42 41 41 40 41 40 39 39 39


GPS coordinates 560 4600 N, 9 240 4100 E 10 2100 N, 9 280 3000 E 390 1400 N, 9 270 1000 E 390 3600 N, 9 30 3400 E 280 3200 N, 9 40 4400 E 590 100 N, 9 260 900 E 350 3600 N, 9 450 2100 E 100 5900 N, 9 270 5200 E 490 4800 N, 8 330 2400 E 480 5400 N, 8 330 000 E 70 000 N, 9 310 1200 E 120 3100 N, 9 50 1400 E

Numbers associated with the samples. Localities of the harvests.

wileyonlinelibrary.com/journal/ffj

Copyright 2013 John Wiley & Sons, Ltd.

Flavour Fragr. J. 2013

Eryngium maritimum, essential oils, antioxidant activity Gas Chromatography with Flame Ionization Detection Gas chromatography with ame ionization detection (GC-FID) analyses were carried out using a PerkinElmer Clarus 600 GC apparatus (Walton, MA, USA) equipped with a single injector and two ame ionization detectors. The apparatus was used for simultaneous sampling to two fused-silica capillary columns (60 m 0.22 mm, lm thickness 0.25 mm) with different stationary phases: Rtx-1 (polydimethylsiloxane) and Rtx-Wax (polyethylene glycol). The temperature program was: 60230 C at 2 C/min and then held isothermal at 230 C for 30 min. The carrier gas was helium (1 mL/min). The injector and detector temperatures were held at 280 C. Split injection was conducted with a ratio split of 1:80. Injected volume was 0.1 mL. Gas ChromatographyMass Spectrometry in Electron Impact Mode The essential oils and the fractions obtained by CC were investigated using a PerkinElmer TurboMass quadrupole detector, directly coupled to a PerkinElmer AutoSystem XL (Walton, MA, USA) equipped with two fused-silica capillary columns (60 m 0.22 mm, lm thickness 0.25 mm), Rtx-1 (polydimethylsiloxane) and Rtx-Wax (polyethylene glycol). Both columns were used with the same MS detector. Oil analyses were consecutively carried out on the apolar and then on the polar column. For each sample, two reconstructed ionic chromatograms were provided and they have been investigated consecutively. Other GC conditions were the same as described above. Ion source temperature, 150 C; energy ionization, 70 eV; electron ionization mass spectra were acquired with a mass range of 35350 amu during a scan time 1 s. Oil injected volume, 0.1 mL; fraction injected volume, 0.2 mL. Component Identication Identication of individual components was based on: (1) a comparison of calculated retention indices, on polar and apolar columns, with those of authentic compounds or literature data;[27,28] and (2) on computer matching with commercial mass spectral libraries and comparison of mass spectra with those of our own library of authentic compounds or literature data.[2730] Component Quantication Component quantication of the E. maritimum essential oils was carried out using peak normalization, including RFs, with an internal standard, and expressed as normalized % abundances. The complexity of the oil, together with the lack of standards, makes determination of the RFs of all the components unrealistic. So, we used a solution introduced by Costa et al.[31] based on grouping the essential oil components by their functional groups and then by their chemical class. To calculate the RF of a compound for which a standard is not available, with another one, it is essential that the two compounds have the same empirical formula. RFs and calibration curves were determined by diluting each standard (see chemicals) in dichloromethane, at ve concentrations, with each specimen containing tridecane (nal concentration 0.7 g/100 g) as internal standard (IS); analyses were performed in triplicate. The response factors were calculated by using the equation RF = {Canalyte/[(Aabs,analyte/Aabs,IS)]} CIS, where Canalyte is the concentration of the standard compound, Aabs,analyte is its absolute peak area, Aabs,IS is the tridecane absolute peak area and CIS is its concentration (0.7 g/100 g). The average RFs obtained for each standard compound within a chemical class are used as a correction factor specic for each chemical class. This procedure gave RFs relative to tridecane (1.01 for monoterpene hydrocarbons, 1.0 for sesquiterpene hydrocarbons, 1.34 for alcohols, 1.55 for esters, 1.31 for ketones, 1.24 for ethers, 1.59 for oxides and 1.40 for aldehydes) and allows the determination of the normalized % abundances using the methodology reported by Bicchi et al.[32] Statistical Analysis In order to study the chemical variations in E. maritimum essential oils, a standardized data matrix was established from the chemical composition of the 12 Corsican and Sardinian samples studied in this present work. The data matrix displayed was analysed using Principal component analysis (PCA) and hierarchical ascending classication[33] with the aid of XLSTAT software (Version 2012.2.04; Addinsoft, Paris, France). PCA was made with a Pearson matrix and hierarchical ascending classication was made with a Euclidian matrix and Ward aggregation. Antioxidant Screening The 2,2-diphenyl-1-picrylhydrazyl radical-scavenging assay The hydrogen atoms or electron-donating ability of the total essential oil and corresponding fractions were measured from the bleaching of purple-coloured methanol solution of DPPH as described by Sharifar et al.[34] with some modications. A 100 mL aliquot of methanol containing amounts of E. maritimum samples ranging from 100 ng to 10 mg was added to 1 mL of a 0.2 mM methanol solution of DPPH. Ascorbic acid (125 mg/mL nal concentrations) was used as the antioxidant standard. The mixture was homogenized and incubated for 25 min at room temperature in the dark. The absorbance was measured at 515 nm against a blank using a 6405 UVvisible spectrophotometer (Jenway, Bibby Scientic Limited, Staffordshire, UK). Inhibition of the free radical, DPPH (I%) was calculated using the equation: I % = 100 (Ab As)/Ab, where Ab is the absorbance of the control reaction and As the absorbance of the sample. The sample concentration providing 50% inhibition (IC50) was calculated from the graph of inhibition percentage against sample concentration. Tests were carried out in triplicate. Ascorbic acid was used as a positive control. The 2,20 -azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) radicalscavenging assay The ability of total essential oil and fractions to bleach the 2,20 azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) radical (ABTS+) was evaluated according to the method of Re et al.[35] with modications. ABTS+ was produced by reaction of equal volumes of 2.4 mM ABTS solution and 2.4 mM potassium persulfate for 16 h, in the dark and at room temperature. The stock solution was then diluted in 0.2 M phosphate-buffered solution to achieve an absorbance of 0.80 0.05 at 734 nm. Then 900 ml of diluted ABTS+ solution was mixed with 0.110 mL of sample dissolved in 100 mL of methanol. The absorbance at 734 nm was taken 1 min after mixing. Inhibition of free-radical oxidation was expressed in % and calculated according to the same equation as used for DPPH scavenging assay.

Flavour Fragr. J. 2013

Copyright 2013 John Wiley & Sons, Ltd.

wileyonlinelibrary.com/journal/ffj

F. Darriet et al.

Results and Discussion

Chemical Composition of Corsican E. maritimum Essential Oil Due to the wide distribution of Eryngium maritimum along the sandy beaches of east Corsica, the station of Quercioni (C1) was chosen for detailed analysis. Preliminary analysis of the sample of essential oil from the aerial parts of E. maritimum allowed the identication of only 38 compounds, which accounted for 94.5% of the C1 sample oil. Investigation of all CC fractions and sub-fractions by GC-FID and GC/MS-EI led to the identication of 58 components which accounted for 94.5% of the sample oil (Table 2). The chemical composition of both hydrocarbon and oxygenated CC fractions (HF and OF, respectively) are reported in Table 2. Oil components included ve monoterpenes, 43 sesquiterpenes and 10 non-terpenic compounds. The total essential oil was dominated by 17 oxygenated sesquiterpenes (49.8%) and 26 sesquiterpenes hydrocarbon (42.2%), whereas the 15 other compounds represent only 2.5% of the oil. The main components were germacrene D, 34 (33.9%), 4bHcadin-9-en-15-al, 56 (26.1%), 4bH-cadin-9-en-15-ol, 58 (9.5%) and 4bH-muurol-9-en-15-al, 55 (5.2%). The essential oil was characterized by a large variety of sesquiterpene skeletons: acyclic (nerolidane), cyclic (bisabolane, bergamotane, germacrane and humulane), bi-cyclic (bicyclogermacrane, caryophyllane, cadinane and muurolane) and tri-cyclic (cubebane, copaane, bourbonane, ylangane and aromadendrane). In addition, leaves, stems, owers and roots of E. maritimum harvested from the same location (C1; Quercioni, Corsica) afforded essential oils with similar yields (0.060.09%). Integrated analysis of the four sample essential oils allowed identication of 58 compounds, which always accounted for more than 90.5% of the essential oils (Table 2). Essential oils from the leaves, stems, owers and full aerial parts were qualitatively similar and exhibited only few differences in the percentages of their main components. Relative to the essential oil from the aerial parts, the essential oil from E. maritimum roots displayed the simplest gas chromatogram. Only eight compounds, which accounted for 97.4% of the oil, were identied, and among them 2,4,5-trimethylbenzaldehyde, 14 (39.8%), 2,3,6-trimethylbenzaldehyde, 15 (29.0%) and a-muurolene, 38 (23.5%) were the three main components. Corsican root essential oil was quite different to the root essential oil reported by Kubeczka et al.[23] which contained trimethylbenzaldehyde (isomer not reported, 24.5%) and the sesquiterpene hydrocarbons g-guaiene (40.2%) and germacrene D (10.6%) as the main components.

Chemical Variability of E. maritimum Essential Oils and the Comparison with Literature Data Aerial parts of 12 E. maritimum oil samples, six from Corsica (C1C6) and six from Sardinia (S1S6), were hydrodistilled to afford essential oils with moderate yields: 0.060.13% of fresh material. The essential oils were analysed to obtain a better understanding of their chemical variability. GC-FID and GC/MS-EI analyses of the 12 essential oils showed some quantitative differences between sample essential oils from both islands and the occurrence of ve additional compounds, denoted 10 , 20 , 70 , 120 and 580 , in the Sardinian sample of essential oils (Table 3). The standardized essential oil matrix was statistically analysed employing hierarchical ascending classication and principal component analysis. The dendrogram and plot established

using the rst two axes, which accounted for 44.43% and 20.79% of the total variance, suggest the existence of two clusters (Figure 1 and Figure 2). Figure 2 shows the distribution of the discriminating volatile compounds germacrene D, 34, 4bH-cadin-9-en-15-ol, 58, aromadendrene oxide, 50, and the distribution of oil samples. The F1 axis is negatively correlated with oxygenated sesquiterpenes and positively correlated with sesquiterpene hydrocarbons. Cluster I and cluster II include the Corsican and Sardinian sample essential oils, respectively. The essential oils from both islands differed by the relative amounts of their main components, especially germacrene D, 34, 4bH-cadin-9-en-15-ol, 58, and aromadendrene oxide, 50, as seen on PCA. The main components of Sardinian E. maritimum samples were 4bH-cadin-9-en-15-al, 56 (19.726.1%), 4bH-muurol-9-en-15-al, 55 (6.39.2%), 4bH-cadin-9-en-15-ol, 58 (9.014.3%), germacrene D, 34 (13.723.8%) and aromadendrene oxide, 50 (1.25.0%). The main components of Corsican E. maritimum samples were germacrene D, 34 (33.145.9%), 4bHcadin-9-en-15-al, 56 (18.526.1%), 4bH-cadin-9-en-15-ol, 58 (5.29.5%) and 4bH-muurol-9-en-15-al,- 55 (5.2-8.3%). Sample oils of both clusters were discriminated by the proportions of (1) terpene hydrocarbons, which were lower in the Sardinian sample essential oils (21.638.2%) than in the Corsican sample essential oils (39.056.9%); and (2) oxygenated terpenes, which were higher in Sardinian sample essential oils (62.647.5%) than in Corsican sample essential oils (38.649.9%). It is noteworthy that Corsican E. maritimum oil was quite similar to that reported by Kubeczka et al.[23] except for 4bH-cadin-9-en-15-al, 56, and 4bH-cadin-9-en15-ol, 58, which were natural compounds identied for the rst time in E. maritimum in our previous work.[25] In addition, the Corsican oil sample was radically different to those reported by Aslan and Kartal[24] in which spathulenol, 1,5-epoxysalvial-4(14)ene, a-amorphene and caryophellene oxide were identied as the main components. Relative to essential oils from other Eryngium species previously studied,[1223,3656] samples of E. maritimum studied here show similarity with E. campestre (Egyptian sample)[40] and E. duriaei[43] in which sesquiterpene aldehydes are present in large amounts. They differed from the others. Eleven essential oil samples with high amounts of non-terpenic compounds, especially trimethylbenzaldehyde, linear aldehydes [such as (E)-2-dodecenal] and linear acids are reported in the literature. These Eryngium sample essential oils were distributed in four species: E. foetidum (seven samples),[17,4550] E. corniculatum (two samples),[21] E. creticum[39] and E. caucasicum (one sample).[41] Thirty-three essential oil samples from 18 various species are dominated by terpenic hydrocarbon compounds such as phyllocladene, a-pinene and germacrene D. It is the most represented essential oil pattern in the Eryngium genus in terms of species. The 17 species were distributed as: E. billardieri,[37] E. glaciale (two samples),[15] E. pandanifolium (two samples),[44] E. bourgatii (two samples),[14] E. serbicum,[51] E. campestre (Turkish and eight Spanish samples),[39,56] E. yuccifolium (two samples),[12] E. caeruleum,[42] E. thorifolium,[39] E. paludosum,[54] E. vesiculosum,[44] E. aqualifolium (two samples),[36] E. expansum,[44] E. rosulatum,[55] E. amethystinum (three samples),[13] E. planum (steam + leaf sample)[52] and E. caucasicum (one sample).[41] Finally, seven samples of essential oils which comprise ve species distributed as E. bungei,[38] E. palmatum,[51] E. rostratum (two samples),[44] E. paniculatum,[53] E. foetidum (Cuban sample)[20] and E. planum (one sample)[52] are dominated by terpene alcohols or oxides, such as spathulenol, carotol or aromadendrene oxide.

wileyonlinelibrary.com/journal/ffj

Copyright 2013 John Wiley & Sons, Ltd.

Flavour Fragr. J. 2013

Table 2. Chemical compositions of Corsican Eryngium maritimum essential oils (C1 location, Quercioni), column chromatography (CC) fractions and separated organs LRIb HF 39.8 29.0 tr 0.3 0.1 tr tr tr 0.2 tr 0.1 tr tr tr tr tr 0.6 2.8 0.1 0.1 0.1 0.1 0.6 0.9 tr tr 1.2 1.0 0.2 0.8 0.1 0.1 0.1 0.6 0.2 0.2 32.1 0.6 3.0 0.2 0.4 2.0 23.5 0.6 tr tr tr tr 0.1 tr 0.1 tr 0.1 tr 0.1 0.9 tr 0.1 0.7 tr 1.0 0.5 1.8 0.3 0.1 tr 0.1 tr tr 42.5 0.2 tr 2.2 tr 0.6 0.1 tr tr tr tr 0.1 tr tr tr 0.1 tr 0.1 tr 0.1 tr tr tr 0.5 tr tr 0.7 tr tr 0.7 0.7 0.2 tr 0.1 0.1 tr 0.5 tr 32.2 0.1 tr 1.2 1.5 0.1 OF Flowers Stems Leaves Roots RIAc RFse Total oil,f aerial parts CC fractionsf,g Separated organsf RIPd Identicationh

Flavour Fragr. J. 2013

Eryngium maritimum, essential oils, antioxidant activity

No.a

Compound

Copyright 2013 John Wiley & Sons, Ltd.

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

a-Pinene 6-Methylhept-5-ene-2-one 2-Pentyl-furan Octanal Myrcene 1,2,3-Trimethylbenzene Limonene cis-Verbenol trans-Verbenol (E)-Non-2-enal 4-Methylacetophenone Decanal 2,4,6-Trimethylbenzaldehyde 2,4,5-Trimethylbenzaldehyde 2,3,6-Trimethylbenzaldehyde a-Cubebene (Z)-b-Damascenone a-Copaene a-Ylangene b-Bourbonene b-Elemene b-Patchoulene b-Gurjunene a-Gurjunene cis-a-Bergamotene b-Ylangene a-Sesquiphellandrene b-Copaene trans-a-Bergamotene Aromadendrene a-Humulene g-Muurolene a-Curcumene Germacrene D b-Selinene 4-Epicubebol Bicyclogermacrene a-Muurolene b-Bisabolene g-Cadinene 936 972 981 981 987 1011 1025 1132 1132 1139 1156 1180 1280 1305 1314 1355 1343 1371 1372 1374 1384 1388 1404 1413 1414 1420 1428 1430 1434 1443 1455 1473 1474 1479 1486 1490 1494 1496 1503 1507 930 967 975 977 983 1006 1019 1126 1130 1144 1153 1182 1281 1297 1318 1346 1349 1373 1374 1375 1385 1394 1405 1411 1416 1420 1430 1431 1435 1440 1449 1468 1471 1478 1483 1487 1491 1494 1498 1505 994 1570 1201 1290 1130 1294 1166 1618 1637 1394 1731 1498 1827 1846 1935 1452 1820 1447 1476 1474 1555 1475 1591 1524 1480 1562 1765 1581 1580 1611 1665 1681 1682 1659 1712 1870 1979 1719 1720 1720 1.01 1.31 1.59 1.40 1.01 1.01 1.01 1.34 1.34 1.40 1.31 1.40 1.40 1.40 1.40 1.0 1.31 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.34 1.0 1.0 1.0 1.0 0.1 tr tr tr 0.2 tr 0.1 tr 0.1 0.1 0.1 tr tr 0.3 1.5 tr 0.1 0.6 tr 0.4 0.9 tr tr 0.2 1.0 0.6 0.1 tr 0.1 0.1 0.1 0.4 tr 33.9 tr tr 0.3 1.1 1.1 0.1 0.6 0.4 0.1 0.1 0.2 2.5 0.1 1.5 0.9 0.1 tr 0.2 4.3 2.1 0.4 2.5 tr 1.1 1.3 2.6 tr 47.7 0.4 1.9 2.6 1.7 3.2

0.2 tr tr tr 0.1 0.1 0.1 tr tr 0.5 1.9 0.2

RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS, Ref RI, MS RI, MS RI, MS RI, MS RI, MS, Ref RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS

wileyonlinelibrary.com/journal/ffj

Table 2. (Continued) LRIb HF OF Flowers Stems Leaves Roots RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS, Ref RI, MS RI, MS, Ref RI, MS, Ref RI, MS RI, MS RI, MS RI, MS RI, MS, Ref RI, MS RI, MS RI, MS RI, MS RIAc RFse Total oil,f aerial parts CC fractionsf,g Separated organsf RIPd Identicationh

wileyonlinelibrary.com/journal/ffj

No.a

Compound

41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 Total identiedf

Cubebol d-Cadinene Cadina-1,4-diene (E)-Nerolidol Spathulenol 4a-Hydroxygermacra-1,5-diene Caryophyllene oxide 4b-Hydroxygermacra-1,5-diene Muurola-4,10-dien-8a-ol Aromadendrene oxide t-Muurolol t-Cadinol a-Cadinol Eudesma-4,7-diene-1b-ol 4bH-Muurol-9-en-15-al 4bH-Cadin-9-en-15-al 4bH-Muurol-9-en-15-ol 4bH-Cadin-9-en-15-ol

1514 1520 1523 1553 1569 1571 1578 1580 1594 1623 1633 1633 1633 1671

1509 1513 1526 1549 1561 1565 1570 1571 1597 1618 1634 1638 1639 1669 1684 1684 1734 1742

1924 1700 1763 2037 2119 2296 1950 2042 2165 2002 2143 2163 2227 2354 2163 2173 2422 2452

1.34 1.0 1.0 1.34 1.34 1.34 1.59 1.34 1.34 1.59 1.34 1.34 1.34 1.34 1.40 1.40 1.34 1.34

Copyright 2013 John Wiley & Sons, Ltd.

Hydrocarbon compounds Oxygenated compounds Hydrocarbon monoterpenes Oxygenated monoterpenes Hydrocarbon sesquiterpenes Oxygenated sesquiterpenes Other oxygenated compounds Other Hydrocarbon compounds

Yields % (v/dw)

0.3 1.2 tr 0.5 0.5 1.1 0.5 tr 0.3 0.4 0.8 0.4 1.5 2.2 5.2 26.1 0.4 9.5 94.5 42.6 51.9 0.4 0.1 42.2 49.8 2.0 tr 0.08

13.4 0.5 1.2 1.1 94.6 92.3 2.3 1.1 91.2 2.3 0.1 0.4 1.1 1 2.6 0.5 0.8 2.4 0.9 3.2 5.6 16.3 34.7 0.6 20.9 94.1 94.1 0.1 91.2 2.8 tr 0.9 0.4 1.1 0.2 0.2 tr 0.2 0.5 0.4 0.5 2.3 tr 9.3 20.3 0.3 2.2 90.9 51.6 39.3 0.6 51.0 38.0 1.2 0.1 0.08 0.4 0.6 tr 0.2 0.2 1.2 0.7 tr 0.3 0.1 1.0 0.2 1.3 3.0 4.3 27.6 0.4 10.5 90.9 39.2 51.7 0.1 0.1 39.1 51.5 0.1 0.06

2.5 0.1 2.1 0.7 0.1 0.1 0.2 0.7 1.3 0.6 1.8 1.5 tr 5.2 18.4 0.2 4.5 90.5 49.0 41.5 0.4 tr 48.6 38.1 3.4 tr 0.09

1.6 1.2 97.4 25.8 71.6 25.8 2.8 68.8 0.09

Order of elution is given on the apolar column (Rtx-1). The main components are shown in bold type. Retention indices from literature on the apolar column: RIA, reported from literature.[27,28] c Retention indices on the Rtx-1 apolar column. d Retention indices on the Rtx-wax polar column. e Response factors (RFs). For the calculation mode, see text. f Percentages are given on the apolar column except for compounds with the same RIA (percentages are given on the polar column); tr, percentages below 0.05. g Fractions obtained by column chromatography: HF, hydrocarbon fraction, OF, oxygenated fraction (see experimental). h RI, retention indices; MS, mass spectra in electronic impact mode; Ref, compounds identied from commercial data libraries.[27]

F. Darriet et al.

Flavour Fragr. J. 2013

Table 3. Chemical compositions of Eryngium maritimum essential oils from Corsica and Sardinia LRIb Corsican samples C1 C2 C3 C4 C5 C6 S1 S2 S3 S4 S5 S6 Sardinian samples RIAc RFse RIPd Identicationg

Flavour Fragr. J. 2013

Eryngium maritimum, essential oils, antioxidant activity

No.a

Compound

Copyright 2013 John Wiley & Sons, Ltd.

wileyonlinelibrary.com/journal/ffj

10 1 2 20 3 4 5 6 7 70 8 9 10 11 12 120 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 909 936 972 973 981 981 987 10110 1025 1123 1132 1132 1139 1156 1180 1238 1280 1305 1314 1355 1343 1371 1372 1374 1384 1388 1404 1413 1414 1420 1428 1430 1434 1443 1455 1474 1473 1479 1486 1490 903 930 967 973 975 977 983 1006 1019 1123 1126 1130 1144 1153 1182 1234 1281 1297 1318 1346 1349 1373 1374 1375 1385 1394 1405 1411 1416 1420 1430 1431 1435 1440 1449 1468 1471 1478 1483 1487 1018 994 1570 1098 1201 1290 1130 1294 1166 1517 1618 1637 1394 1731 1498 1494 1827 1846 1935 1452 1820 1447 1476 1474 1555 1475 1591 1524 1480 1562 1765 1581 1580 1611 1665 1681 1682 1659 1712 1870

Santolinatriene a-Pinene 6-Methylhept-5-ene-2-one Sabinene 2-Pentyl-furane Octanal Myrcene 1,2,3-Trimethylbenzene Limonene Camphor cis-Verbenol trans-Verbenol (E)-Non-2-enal Methyl-4-acetophenone Decanal trans-Chrysanthenyl acetate 2,4,6-Trimethylbenzaldehyde 2,4,5-Trimethylbenzaldehyde 2,3,6-Trimethylbenzaldehyde a-Cubebene (Z)-b-Damascenone a-Copaene a-Ylangene b-Bourbonene b-Elemene b-Patchoulene b-Gurjunene a-Gurjunene cis-a-Bergamotene b-Ylangene a-Sesquiphellandrene b-Copaene trans-a-Bergamotene Aromadendrene a-Humulene g-Muurolene a-Curcumene Germacrene D b-Selinene 4-Epicubebol

1.01 tr 0.2 RI, MS 1.01 0.1 0.5 0.4 tr 0.4 0.1 0.1 0.2 0.4 0.4 0.2 1.3 RI, MS 1.31 tr 0.1 tr 0.1 0.1 0.1 0.2 0.1 0.2 tr 0.2 RI, MS 1.01 tr tr 0.1 0.2 tr 0.2 RI, MS 1.59 tr tr 0.2 tr tr tr tr tr tr tr tr 0.2 RI, MS 1.40 tr tr 1 0.1 tr 0.1 tr tr 1.8 RI, MS 1.01 0.2 0.6 tr tr 0.7 tr 0.1 0.2 tr 0.1 0.2 tr RI, MS 1.01 tr tr 0.1 tr tr tr 0.5 0.2 0.1 tr 0.1 0.4 RI, MS, Ref 1.01 0.1 0.1 0.2 tr 0.1 tr 1.0 0.2 0.1 0.9 0.4 0.2 RI, MS 1.31 0.6 RI, MS 1.34 tr tr tr tr tr tr tr tr 0.1 tr 0.1 0.2 RI, MS 1.34 0.1 tr 0.1 tr tr tr 0.2 tr 0.2 0.3 0.1 1.2 RI, MS 1.40 0.1 tr 0.1 0.2 tr 0.1 0.2 0.1 0.2 tr 0.1 0.5 RI, MS 1.31 0.1 tr 0.2 tr 0.1 0.1 0.2 0.2 0.2 0.1 0.2 0.7 RI, MS, Ref 1.40 tr 0.1 0.1 tr tr tr tr tr 0.1 tr tr 0.2 RI, MS 1.55 0.1 1.7 2.1 1.5 0.4 0.4 RI, MS 1.40 tr tr tr tr tr tr 0.1 0.2 0.4 0.1 0.3 tr RI, MS 1.40 0.3 0.3 0.4 0.5 0.2 0.3 0.2 0.3 0.5 0.2 0.5 1.2 RI, MS 1.40 1.5 2.0 1.4 2.6 1.0 1.1 2.8 1.8 2.0 2.5 1.9 6.2 RI, MS 1.0 tr tr tr tr tr tr tr tr tr tr tr tr RI, MS 1.31 0.1 tr 0.1 0.1 tr 0.1 0.1 0.1 0.1 0.2 0.1 tr RI, MS 1.0 0.6 0.5 0.8 0.6 0.9 0.8 0.7 0.4 0.8 0.4 0.9 0.7 RI, MS 1.0 tr tr tr tr tr tr tr tr tr tr tr tr RI, MS 1.0 0.4 0.1 0.2 0.1 0.1 0.2 0.2 0.3 0.3 0.1 0.4 RI, MS 1.0 0.9 0.9 1.1 1.0 1.3 1.3 0.5 0.4 1.2 0.9 1.0 0.8 RI, MS 1.0 tr tr tr tr tr 0.6 tr tr RI, MS 1.0 tr tr tr 0.1 0.1 tr 0.1 RI, MS 1.0 0.2 tr 0.6 0.7 0.7 0.6 0.9 1.1 0.8 1 0.9 RI, MS 1.0 1.0 tr 1.7 0.7 1.8 1.0 2.4 d 1.2 1.2 1.0 1.5 RI, MS 1.0 0.6 tr 1.0 0.2 0.7 0.9 0.1 0.2 0.2 0.9 RI, MS 1.0 0.1 tr 0.1 0.8 tr 1.0 0.2 0.1 0.2 0.1 0.1 0.3 RI, MS 1.0 tr 0.2 tr 0.5 tr tr RI, MS 1.0 0.1 0.2 tr 0.2 0.1 0.1 RI, MS 1.0 0.1 tr tr 0.2 0.2 0.2 0.1 0.2 0.4 0.4 0.2 RI, MS 1.0 0.1 tr 0.3 0.2 0.3 0.3 0.4 0.1 0.1 0.1 0.1 0.2 RI, MS 1.0 0.4 0.1 0.2 0.1 tr tr 0.1 0.3 0.3 0.1 tr tr RI, MS 1.0 tr tr tr tr tr tr 0.1 0.5 0.2 0.4 RI, MS 1.0 33.9 33.1 32.2 43.5 45.9 41.4 13.7 14.6 16.6 14.3 23.8 18.4 RI, MS 1.0 tr tr 0.2 tr tr 0.3 0.2 0.2 0.1 0.2 0.1 tr RI, MS 1.34 tr tr tr 0.3 0.2 tr 0.3 tr RI, MS

Table 3. (Continued) LRIb Corsican samples C1 1494 1496 1503 1507 1514 1520 1523 1553 1569 1571 1578 1580 1594 1623 1633 1633 1643 1671 1981 1491 1494 1498 1505 1509 1513 1526 1549 1561 1565 1570 1571 1597 1618 1634 1638 1645 1669 1684 1684 1734 1742 1980 1979 1719 1720 1720 1924 1700 1763 2037 2119 2296 1950 2042 2165 2002 2143 2163 2227 2354 2163 2173 2422 2452 2436 C2 C3 C4 C5 C6 S1 S2 S3 S4 S5 S6 RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS, Ref RI, MS RI, MS, Ref RI, MS, Ref RI, MS RI, MS RI, MS RI, MS RI, MS, Ref RI, MS RI, MS RI, MS RI, MS RI, MS, Ref Sardinian samples RIAc RFse RIPd Identicationg

wileyonlinelibrary.com/journal/ffj

No.a

Compound

Copyright 2013 John Wiley & Sons, Ltd.

37 Bicyclogermacrene 38 a-Muurolene 39 b-Bisabolene 40 g-Cadinene 41 Cubebol 42 d-Cadinene 43 Cadina-1,4-diene 44 (E)-Nerolidol 45 Spathulenol 46 4a-Hydroxygermacra-1,5-diene 47 Caryophyllene oxide 48 4b-Hydroxygermacra-1,5-diene 49 Muurola-4,10-dien-8a-ol 50 Aromadendrene oxide 51 t-Muurolol 52 t-Cadinol 53 a-Cadinol 54 Eudesma-4,7-diene-1b-ol 55 4bH-Muurol-9-en-15-al 56 4bH-Cadin-9-en-15-al 57 4bH-Muurol-9-en-15-ol 58 4bH-Cadin-9-en-15-ol 580 Isopimara-8,15-diene Total identiedf Hydrocarbon compounds Oxygenated compounds Hydrocarbon monoterpenes Oxygenated monoterpenes Hydrocarbon sesquiterpenes Oxygenated sesquiterpenes Other Hydrocarbon compounds Other oxygenated compounds Yields % (v/dw)

1.0 0.3 1.0 1.1 1.0 1.1 1.0 0.1 1.34 0.3 1.0 1.2 1.0 tr 1.34 0.5 1.34 0.5 1.34 1.1 1.59 0.5 1.34 tr 1.34 0.3 1.59 0.4 1.34 0.8 1.34 0.4 1.34 1.5 1.34 2.2 1.40 5.2 1.40 26.1 1.34 0.4 1.34 9.5 1.0 94.5 42.6 51.9 0.4 0.1 42.2 49.8 tr 2.1 0.08

1.5 0.4 1.2 0.2 1.5 0.3 0.2 0.2 0.7 tr 1.2 0.6 1.9 3.1 6.9 24.2 0.4 7.9 90.7 39.0 51.7 1.2 tr 37.8 49.3 tr 2.4 0.08

1.8 0.2 0.1 0.4 tr 1.4 0.1 0.8 0.3 2.2 1.4 tr 0.7 0.6 0.5 1.2 0.6 tr 7.4 18.5 0.4 8.5 90.3 43.5 46.8 0.6 0.1 42.8 43.2 0.1 3.5 0.13

2.3 0.1 0.1 0.4 tr 1.6 tr 0.5 0.1 0.6 0.1 tr 0.4 0.2 0.8 1.3 0.2 tr 8.3 20.4 0.3 5.2 94.8 53.6 42.2 tr tr 53.6 38.8 tr 3.4 0.08

2.3 0.1 tr 0.3 1.2 tr 0.1 0.9 0.4 tr 0.1 tr tr tr 0.6 tr 7.4 21.0 tr 6.7 95.7 56.9 38.8 1.2 0 55.7 37.4 tr 1.4 0.12

2.3 0.1 0.1 0.4 1.5 0.2 0.4 1.1 0.6 0.2 0.4 0.4 0.6 0.3 1.1 tr 7.5 19.1 tr 7.6 95.1 53.4 41.7 0.1 0.5 53.3 39.4 1.8 0.08

0.2 0.1 0.1 0.3 0.1 0.5 0.1 0.7 0.9 1.9 0.8 0.4 0.4 2.9 1.8 2.3 2.0 2.3 6.3 26.1 0.8 12.5 88.3 22.1 66.2 1.2 0.3 20.4 62.3 0.5 3.6 0.07

0.6 0.4 0.2 0.3 tr 0.6 0.3 0.5 1.3 1.1 0.6 tr 0.8 5.0 5.8 1.3 1.8 0.7 7.6 20.7 0.6 11.5 0.6 89.2 18.7 70.5 0.6 1.7 23.9 60.0 0.2 2.8 0.07

1.2 0.4 0.1 0.3 0.1 1.0 0.2 1.0 1.7 0.7 0.5 0.4 0.7 1.2 1.0 2.0 0.9 1.3 8.9 22.9 0.5 10.0 87.8 27.1 60.7 0.6 3.0 26.4 54.2 0.1 3.5 0.11

0.9 0.3 0.2 0.4 0.2 0.8 0.2 0.7 1.5 0.5 0.3 0.2 0.4 1.9 1.6 2.2 0.8 0.5 9.0 22.8 0.5 13.7 0.9 85.8 17.0 68.8 1.8 1.8 21.2 57.9 tr 3.1 0.10

1.2 0.2 0.1 0.2 tr 0.7 0.1 0.6 1.2 0.4 0.3 0.2 0.2 2.0 2.1 0.8 0.8 0.3 8.5 20.7 0.3 14.3 0.1 88.8 32.3 56.5 0.8 0.6 37.4 46.9 0.1 3.0 0.06

0.8 tr 0.2 0.3 1.1 1.5 0.1 3.6 2.1 0.1 1.2 0.6 0.9 0.8 0.6 9.2 19.7 0.4 9.0 91.9 29.3 62.6 1.7 1.8 27.2 49.8 0.4 11 0.10

Order of elution is given on the apolar coloumn (Rtx-1). Retention indices from literature on the apolar column; RIA, reported from literature.[27,28] c Retention indices on the Rtx-1 apolar column. d Retention indices on the Rtx-wax polar column. e Response factors (RFs). For the calculation mode, see text. f Percentages are given on the apolar column except for compounds with the same RIA (percentages are given on the polar column); tr, percentages below 0.1. g RI, retention indices; MS, mass spectra in electronic impact mode; Ref, compounds identied from commercial data libraries.[27]

F. Darriet et al.

Flavour Fragr. J. 2013

Eryngium maritimum, essential oils, antioxidant activity bicyclo[4.4.0]decene aldehydes have not been reported in other species of Eryngium genus essential oils and it would be interesting to check the presence of these compounds in the E. maritimum essential oils from different origins in order to consider them as chemical markers of the species.

Antioxidant Activity of E. maritimum Essential Oil and Fractions Antioxidant properties of E. maritimum essential oil and both hydrocarbon and oxygenated fractions obtained by column chromatography were tested regarding their scavenging activities on DPPH and ABTS radicals. Total essential oil showed a strong antioxidant activity, since IC50 values regarding DPPH and ABTS radical-scavenging abilities, respectively 5.9 0.1 mg/mL and 0.7 0.03 mg/mL, were equivalent or even lower than those registered for ascorbic acid, used as the antioxidant reference (6.1 0.04 mg/mL for DPPH and 2.66 0.01 mg/mL for ABTS radicals, Table 4). Total essential oil was thus separated into two fractions, containing oxygenated or hydrocarbon compounds. The best radical-scavenging effect was observed for the oxygenated fraction, with IC50 values of 8.8 0.01 mg/mL against DPPH and 1.34 0.02 mg/mL against the ABTS radical, similar to those found for ascorbic acid and total essential oil. No signicant antioxidant activity could be detected in the hydrocarbon fraction, since IC50 values of this fraction were more than 50-fold higher than those found for ascorbic acid, even though IC50 values regarding the ABTS radical were greater than those observed for the DPPH radical (Table 4 and Figure 3). Considering these results, we could infer that the antioxidant properties exhibited by E. maritimum essential oil are carried out by compounds of the oxygenated fraction. According to Table 1, the oxygenated fraction was dominated by two aldehydes and their corresponding alcohols (4bH-cadin-9-en-15-al, 56, 4bH-muurol-9-en-15-al, 55, 4bH-cadin-9en-15-ol, 58 and 4bH-muurol-9-en-15-ol, 57) representing, respectively, 34.7%, 16.3%, 20.9% and 0.6% of the fraction. In order to selectively examine the antioxidant properties of these four uncommon compounds, DPPH and ABTS radical-scavenging activity tests were also performed on: (1) the sesquiterpene aldehyde-rich fraction (OF1) with 55 (60.8%) and 56 (31.9%) obtained by CC, and (2) sesquiterpene alcohol-rich fraction (ROF1) with 57 (63.8%) and 58 (33.5%) obtained by reduction of OF1 (see experimental) (Table 4 and Figure 3). The aldehyde-rich fraction (OF1) showed moderate antioxidant activity on the ABTS radical (IC50 = 29.9 0.8 mg/mL) but its effect on the DPPH radical was less (IC50 = 111.3 31.3 mg/mL). The corresponding alcohol-

Figure 1. Dendrogram of essential oil chemical compositions of Corsican and Sardinian Eryngium maritimum samples

Figure 2. Principal component analysis of essential oil chemical compositions of Corsican and Sardinian Eryngium maritimum samples

As a concluding remark in this section, it is noticeable that quantitative chemical variability between Corsican and Sardinian E. maritimum samples oil reported here is probably linked to the harvest area. Moreover, it would be interesting to examine the genetic diversity of both Corsican and Sardinian populations in order to dene the origin of the essential oil variability. Compared to other species, E. maritimum sample essential oils were clearly discriminated by the higher amount of sesquiterpene aldehydes, such as 4bH-cadin-9-en-15-al, 56 (18.526.1%) and 4bH-muurol-9-en-15-al, 55 (5.29.2%). These uncommon

Table 4. Antioxidant activities of total essential oil and fractions of Eryngium maritimum in DPPH radical-scavenging activity and ABTS radical-scavenging activity Compound used in antioxidant screening DPPH ABTS IC50 value (mg/mL) SEM EO OF OF1 ROF1 HF AA

5.9 0.1 0.7 0.03

8.8 0.1 1.34 0.02

111 3 31.3 29.9 0.8

219 38 9 465.8 137.8

347.7 35.2 137.3 123.2

6.1 0.04 2.66 0.01

Results are given as mean SD (n = 3). EO, essential oil; OF, oxygenated fraction; OF1, fraction with uncommon aldehydes 5556; ROF1, fraction with uncommon alcohols 5758; HF, hydrocarbon fraction; AA, ascorbic acid (standard).

Flavour Fragr. J. 2013

Copyright 2013 John Wiley & Sons, Ltd.

wileyonlinelibrary.com/journal/ffj

F. Darriet et al.
7. R. Lisciani, E. Fattorusso, V. Surano, S. Cozzolino, M. Giannattasio, L. Sorrentino, J. Ethnopharmacol. 1984, 12, 263. 8. K. Hiller, M. Keipert, S. Pferifer, Pharmazie 1968, 23, 119. 9. M. Kartal, A. C. Mitaine-Offer, M. Abu-Asaker, Y. Miyamoto, L. Calis, H. Wagner, M. A. Lacaille-Dubois, Chem. Pharm. Bull. 2005, 53, 1318. 10. J. Lam, L. P. Christensen, T. Thomasen, Phytochemistry 1992 , 31 , 2881. 11. E. Le Claire, S. Schwaiger, B. Banaigs, H. Stuppner, F. Gafner, J. Agric. Food Chem. 2005, 53, 4367. 12. N. Ayoub, M. Al-Azizi, W. Knig, K. H. Kubeczka, Flavour Fragr. J. 2006, 21, 864. 13. G. Flamini, M. Tebano, P. L. Cioni, Food Chem. 2007, 107, 671. 14. J. Pala-Paul, M. J. Prez-Alonso, A. Velasco-Negueruela, J. Vadar, A. M. Villa, J. Sanz, J. J. Brophy, J. Chromatogr. A 2005a, 1074, 235. 15. J. Pala-Paul, M. J. Prez-Alonso, A. Velasco-Negueruela, J. Vadar, A. M. Villa, J. Sanz, J. J. Brophy, J. Chromatogr. A 2005b, 1094, 179. 16. J. Pala-Paul, J. J. Brophy, R. J. Goldsack, L. M. Copeland, M. J. Perez-Alonso, A. Velasco-Negueruela, Aust. J. Bot. 2003, 51, 497. 17. M. Cardozo, M. Rubio, L. B. Rojas, A. Usubillaga, J. Essent. Oil Res. 2004, 16, 33. 18. J. A. Pino, A. Rosado, V. Fuentes, J. Essent. Oil Res. 1997, 9, 467. 19. A. P. Martins, L. R. Salgueiro, A. P. Cunha, R. Vila, S. Canigueral, F. Tomi, J. Casanova, J. Essent. Oil Res. 2004, 15, 93. 20. J. A. Pino, A. Rosado, V. Fuentes, J. Essent. Oil Res. 1997, 9, 123. 21. J. Pala-Paul, J. J. Brophy, M. J. Prez-Alonso, J. Ursano, A. C. Soria, J. Chromatogr. A 2007, 1175, 289. 22. J. J. Brophy, R. J. Goldsack, L. M. Copeland, J. Pala-Paul, J. Essent. Oil Res. 2004, 15, 392. 23. K. H. Kubeczka, N. Ayoub, M. Grande, P. Torres, Poster at the 29th (ISEO) International Symposium od Essential Oils, Johann Wolfgang Goethe University: Frankfurt, Germany, 6-9 september 1998. 24. S. Aslan, M. Kartal, Poster at the 54th Annual Congress on Medical Plant Research, Thieme: Stuttgart, New York, 29/8-2/9 2006. 25. F. Darriet, M. Bendahou, J. M. Desjobert, J. Costa, A. Muselli, Planta Med. 2012, 78, 386. 26. Council of Europe, Pharmacope Europenne. Maisonneuve S.A, Sainte Rufne, 1996. 27. W. A. Knig, D. H. Hochmuth, D. Joulain, Terpenoids and Related Constituents of Essential Oils. Library of Mass Finder, Institute of Organic Chemistry, Hamburg, 2008. 28. National Institute of Standards and Technology (NIST), Spectral Database for Organic Compounds, NIST WebBook. Available: http:// webbook.nist.gov/chemistry, 2008. 29. R. P. Adams, Identication of Essential Oils by Capillary Gas Chromatography/Mass Spectroscopy. Allured Publishing, Carol Stream, IL, 2001. 30. National Institute of Standards and Technology (NIST). Perkin Elmer Corp, Norwalk, CT, 1999. 31. R. Costa, B. A. Zellner, M. L. Crupi, M. Fina, M. Valentino, P. Dugo, G. Dugo, L. Mondello, Flavour Fragr. J. 2008, 23, 40. 32. C. Bicchi, E. Liberto, M. Matteodo, B. Sgorbini, L. Mondello, B. A. Zellner, R. Costa, P. Rubiolo, Flavour Fragr. J. 2008, 23, 382. 33. R. G. Brereton, Chemometrics: Data Analysis for the Laboratory and Chemical Plant, Wiley Interscience, Wiley & Sons, Chichester, 2003. 34. F. Sharifar, V. Mozaffarian, S. Moradkhani, Pak. J. Biol. Sci. 2007, 10, 3895. 35. R. Re, N. Pellegrini, A. Proteggente, A. Pannala, M. Yang, C. Rice-Evans, Free Radical Biol. Med. 1999, 26, 1231. 36. J. Pala-Paul, J. Ursano, J. J. Brophy, M. J. Prez-Alonso, A. C. Soria, Nat. Prod. Commun. 2010, 5, 817. 37. F. Sedkon, M. Dabiri, A. Alamshahi, J. Essent. Oil Res. 2004, 16, 42. 38. K. Morteza-Semnani, J. Essent. Oil Res. 2005, 17, 485. 39. A. Celik, N. Aydinlik, I. Arslan, Chem. Biodivers. 2011, 8, 454. 40. A. R. Abd-Elmonem, N. G. Shehab, Bull. Faculty Pharm. Cairo Univ. 2006, 44, 53. 41. D. Hashemabadi, B. Kaviani, Plant Om. 2010, 3, 135. 42. F. Assadian, S. Masoudi, F. Nematollahi, A. Rustaiyan, K. Larijani, H. Mazloomifar, J. Essent. Oil Res. 2005, 17, 243. 43. C. Cavaleiro, M. J. Goncalves, D. Serra, G. Santoro, F. Tomi, A. Bighelli, L. Salgueiro, J. Casanova, J. Pharm. Biomed. Anal. 2011, 54, 619. 44. J. J. Brophy, R. J. Goldsack, L. M. Copeland, J. Pala-Paul, J. Essent. Oil Res. 2003, 15, 392. 45. J. U. Chowdhury, N. C.Nandi, M. Yusuf, Bangladesh J. Sci. Ind. Res. 2007, 42, 347. 46. L. Shunzhen, Z. Lixia, L. Hongxing, Huagong Jishu Yu Kaifa 2011, 40, 38. 47. B. C. Thakuri, C. S. Chanotiya, R. C. Padalia, C. S. Mathela, J. Essent. Oil Bearing Plants 2006, 9, 251.

Figure 3. Antioxidant activities of total essential oil and fractions of E. maritimum. Black bars, DPPH radical-scavenging activity; grey bars, ABTS radical-scavenging activity. Data are given as mean SD (n = 3). AA, ascorbic acid (standard); EO, essential oil; OF, oxygenated fraction; OF1, fraction with uncommon aldehydes, 5556; ROF1, fraction with uncommon alcohols, 5758; HF, hydrocarbon fraction

rich fraction (ROF1) exhibited no signicant antioxidant properties, considering that the IC50 values were more than 30-fold higher than those found for ascorbic acid (Table 4 and Figure 3). The antioxidant properties of E. maritimum essential oil was attributed to some oxygenated compounds contained in the oxygenated fraction but they do not seem to be directly correlated with the main sesquiterpenes, 5558. On the one hand, the intermediate antioxidant activity found for the aldehydes main compounds and the absence of activity found for their related primary alcohols when analysed separately could mean that antioxidant activity requires synergic associations between these main compounds and other (minor) compounds of the oxygenated fraction. On the other hand, this fraction also exhibited several tertiary alcohols, such as eudesma-4,7-diene-1b-ol, 54 (5.6%), a-cadinol, 53 (3.2%), 4a-hydroxygermacra-1,5-diene, 46 (2.6%) t-muurolol, 51 (2.4%) and spathulenol, 45 (1%) that could, even if in lower proportions, participate in the antioxidant activity of E. maritimum essential oil. Indeed, antioxidant properties have already been reported for essential oils displaying similar sesquiterpene composition[57] and the difference of the skeleton between tertiary and primary alcohols could explain their reactivity toward the ABTS and DPPH radicals.

Acknowledgements The authors are indebted to the Agence Economique de Dveloppement de la Corse (ADEC-CTC) and European Community (PIC INTERREG IIIA) for partial nancial support.

References
1. H. Coste, Flore descriptive et illustre de la France, de la Corse et des contres limitrophes II. Librairie Scientique et Technique Albert Blanchart, Paris, 1980, p. 627. 2. C. P. P. Rama, R. C. Sudhakar, S. H. Raza, C. B. S. Dutt, Fitoterapia 2008, 79, 458. 3. E. Kpeli, M. Kartal, S. Aslan, E. Yesilada, J. Ethnopharmacol. 2006, 107, 32. 4. Z. Yaniv, A. Dafni, J. Friedman, D. Palevitch, J. Ethnopharmacol. 1987, 19, 145. 5. M. H. Duffaud, Revue Forestiere Francaise 1998, 50, 328. 6. M. D. Lillis, L. Costanzo, P. M. Bianco, A. Tinelli, J. Coast. Conservat. 2004, 10, 93.

wileyonlinelibrary.com/journal/ffj

Copyright 2013 John Wiley & Sons, Ltd.

Flavour Fragr. J. 2013

Eryngium maritimum, essential oils, antioxidant activity

48. A. P. Martins, L. R. Salgueiro, A. Proenca da Cunha, R. Vila, S. Canigueral, F. Tomi, J. Casanova, J. Essent. Oil Res. 2003 , 15 , 93. 49. P. A. Leclercq, X. D. Nguyen, N. L. Vu, V. T. Nguyen, J. Essent. Oil Res. 1992, 4, 423. 50. K. C. Wong, M. C. Feng, T. W. Sam, G. L. Tan, J. Essent. Oil Res. 1994, 6, 369. 51. C. Capetanos, V. Saroglou, P. D. Marin, A. Simic, H. D. J. Skaltsa, J. Serb. Chem. Soc. 2007, 72, 961. 52. B. Thiem, M. Kikowska, A. Kurowska, D. Kalemba, Molecules 2011, 16, 7115. 53. M. I. Cobos, J. L. Rodriguez, A. De Petre, E. Spahn, J. Casermeiro, A. G. Lopez, J. A. Zygadlo, J. Essent. Oil Res. 2002, 14, 82. 54. J. Pala-Paul, L. M. Copeland, J. J. Brophy, R. J. Goldsack, J. Essent. Oil Res. 2008a, 20, 416. 55. J. Pala-Paul, L. M. Copeland, J. J. Brophy, R. J. Goldsack, Biochem. Sys. Ecol. 2006, 34, 796. 56. J. Pala-Paul, J. Usano-Alemany, A. C. Soria, M. J. Prez-Alonso, J. J. Brophy, Nat. Prod. Commun. 2008b, 3, 1121. 57. A. Augusti Boligon, T. G. Schwanz, M. Piana, R. V. Bandeira, J. Kieling Frohlich, T. Faccim de Brum, M. Zadra, M. Linde Athayde, Nat. Prod. Res. 2013, 27, 68-71 EAP.

Flavour Fragr. J. 2013

Copyright 2013 John Wiley & Sons, Ltd.

wileyonlinelibrary.com/journal/ffj