257 Advances in Environmental Biology, 5(2): 257-264, 2011 ISSN 1995-0756

This is a refereed journal and all articles are professionally screened and reviewed ORIGINAL ARTICLE

Essential and Composition of a Tunisian ( Carvi L.) Ecotype Cultivated under Water Deficit

1Bochra Laribi, 1Karima Kouki, 1Ali Sahli, 1Abdelaziz Mougou and 2Brahim Marzouk

1Institut National Agronomique de Tunis. 43, Av. Charles Nicolle-1082, Tunis, Tunisia. 2Laboratoire des substances bioactives, CBBC, BP 901, 2050- Hammam-Lif, Tunisia.

Bochra Laribi, Karima Kouki, Ali Sahli, Abdelaziz Mougou and Brahim Marzouk; and fatty acid composition of a Tunisian caraway (Carum carvi L.) seed ecotype cultivated under water deficit

ABSTRACT

The effects of water deficit on essential oil and fatty acid composition of Tunisian caraway (Carum carvi L.) were investigated. grown from of Menzel Temime ecotype were treated with different levels of water deficit: control (100% ETc), moderate water deficit (50% ETc) and severe water deficit (25% ETc). Essential oil extraction was performed by hydrodistillation. Total were extracted by a solvent mixture of chloroform/ / hexane (4:3:2, v/v/v). Analyses were carried out using gas chromatography-FID equipped with a polar column (polyethylene glycol). Fatty acid composition revealed that drought reduced significantly seed fatty acid content and mainly that of the petroselinic one which is reduced by 15 and 20.3% compared with the control under moderate and severe water deficit, respectively. Thus, the major variation consisted in a decrease of the unsatured fatty acids in favour of an increase in satured ones. Besides, the double bond index (DBI) showed significant decrease under the different drought levels which was more pronounced in plants conducted under severe water constraint. In addition, drought increased the essential oil yield. The main essential oil constituents were and . This last showed an increasing of its proportions under water deficit levels. Thus, water deficit induced a significant reduction in fatty acid content and an increase in the essential oil yield.

Key words: water deficit; Carum carvi L.; ecotype; essential oil; fatty acid

Introduction biomass, and such a reduction was mainly due to declining photosynthetic rates [3]. Thus, water deficit Water deficit is considered to be a major has a negative effect on growth and environmental factor affecting agriculture productivity development of many medicinal and aromatic plants worldwide and causing considerable crop yield like sweet (Ocimum basilicum L.), american reductions [1,2]. This is particularly true in the basil (Ocimum americanum L.) [4], Mediterranean Basin where the climate is typically (Petroselinum crispum Mill.) [5], sage (Salvia characterized by high potential evaporation and low officinalis L.) [6] etc. rainfall during the growing season. In the case of At the cellular level, it has been shown that, two agriculturally important Lamiaceae of the under water deficit, plants undergo various Mediterranean area, ( spicata L.) physiological and biochemical modifications including and (Rosmarinus officinalis L.), increasing changes in membrane composition [7]. These lasts levels of water deficit reduced progressively their are mainly constituted by fatty acids which are

Corresponding Author Bochra Laribi, Institut National Agronomique de Tunis. 43, Av. Charles Nicolle-1082, Tunis, Tunisia. E.mail: [email protected] Adv. Environ. Biol., 5(2): 257-264, 2011 258

important in maintaining membrane fluidity, integrity A completely randomized experimental design and functionality in front of external changes [8]. was conducted as a three factorial blocs with three Nevertheless, investigations dealing with the replications. In each bloc, three combinations of the water deficit effect on biochemical responses of caraway ecotype with water regimes were randomly aromatic and are scarce. Generally, distributed on three experimental plots. Plot area was water deficit has a positive effect in the biosynthesis of 2.8 m². Seeds were sown directly in the field on of secondary metabolites, enzyme activities and March 01, 2007 with a row spacing of 0.4m and by solute accumulation [9]. Thus, this constraint affects respecting a density of 125 plants m-2. Fertilization the essential oil yield and composition of various -1 consisted of 250, 200 and 100 Kg ha of P2O5, K2O aromatic species belonging to the family and N respectively, incorporated uniformly into the [5,10]. before sowing, and supplemented by 100 Kg ha- Among these species, caraway (Carum carvi L.) 1of N brought twice during the crop cycle. Drip is one of the most appreciate for its seed irrigation was made with a flow of 3.8 l h-1. Pre- essential oil richness and its plethora of biologically irrigation was done immediately after sowing for active compounds. Indeed, it has been shown that uniform emergence and establishment of seedlings Tunisian caraway essential oil is carvone chemotype before starting the water deficit treatment. [11]. This fact is of great economic interest due to Then, plants were subjected to three different the several applications of carvone in the alimentary water levels: 100% (control (C)), 50% (moderate and medicinal industries [12]. In addition, the fatty water deficit (MWD)) and 25% (severe water deficit acid composition revealed that Tunisian caraway seed (SWD)) of crop evapotranspiration (ETc) by means oil is rich in an unusual fatty acid - of the software MABIA, which is an irrigation - [11] which is of potential industrial significance. scheduling computer program [16,17,18]. The Use of this oil may also serve in improving human calculation procedures used by this model are based nutrition and health [13,14]. on dual crop coefficient approach according to the In a previous work dealing with the water deficit United Nations Food and Agriculture Organization effects on Tunisian caraway seed ecotype collected (FAO) Penman-Montheith equation [19]. In addition, from the region of Haouaria, we have demonstrated weeds were controlled by hand when needed. Harvest that this constraint induced a significant reduction in was on June 29, 2007. Seeds harvested were air-dried growth parameters and fatty acid contents but an and stored at 4°C until use for further analysis. increase in the proportions of essential oil constituents [15]. Reagents and standards Thus, the objective of this study was to determine the impact of water deficit on essential oil All solvents used in our experiments (hexane, and fatty acid composition of a Tunisian caraway ethanol and methanol) were purchased from Merck seed ecotype collected from the region of Menzel (Darmstadt, Germany). The homologous series of Temime and to compare the results to those obtained C7–C22 n-alkanes used for identification were obtained previously with the ecotype Haouaria. from Sigma Aldrich (Steinheim, Germany). Essential oil and fatty acid standards were purchased from Materials and methods Fluka (Riedel-de Haën, ) and Sigma Aldrich. Plant material and growth conditions Total lipids extraction Caraway seeds were collected from cultivated plants in the region of Menzel Temime (northeastern Total lipids from seeds were extracted by the Tunisia; latitude 36° 46’ 56’’ N; longitude 10° 59’ modified method of Bligh and Dyer, [20] according 15’’ E, altitude 38 m) on July 2006. The field to Marzouk and Cherif, [21]. Thus, 1 g air-dried seed experiment was carried out in the experimental were fixed in boiling water for 5 min and then station of the National Agronomic Institute of Tunis, ground manually with chloroform/methanol/hexane Tunisia (3 Km northwest Tunis; latitude 36° 55’ N; mixture (4:3:2, v/v/v). After washing with water of longitude 10°11’ E; altitude 10 m). fixation and decantation during 24 h at + 4 °C, the The site was characterized by a semi-arid climate organic phase containing total lipids was dried under with a mean annual precipitation of 500 mm (mainly a stream of nitrogen, dissolved in toluene-ethanol during the winter) and an average temperature of (4:1, v/v) and stored at -20 °C for further analyses. 18°C. The soil has a clayey-loamy texture with pH Total extraction was made in triplicate. 7.9 and consisted of 1.4% organic matter, 0.11% nitrogen, 0.034% phosphorus and 0.006% potassium. Fatty acid methylation

Experimental design and water deficit treatment Total fatty acids of total lipids were converted Adv. Environ. Biol., 5(2): 257-264, 2011 259

into their methyl using the sodium methylate ionization (70 eV). A HP-5MS capillary column (60 at 3 % (Sigma, Aldrich, St Louis, MO, USA) in m × 0.25 mm, 0.25 µm film thickness) was used. methanol according to the method described by The column temperature was programmed to rise Cecchi et al., [22]. Methyl heptadecanoate (C17:0) from 40 to 280°C at a rate of 5°C min-1. The carrier was used as an internal standard in order to quantify gas was helium with a flow rate of 1.2 ml min-1. fatty acids. Fatty acid methyl esters (FAMEs) Scan time and mass range were 1 s and 50-550 m/z, obtained were subsequently analysed. respectively. The injected volume was 1 µL.

Essential oil isolation Compounds identification

Whole air-dried seeds (50 g) were subjected to FAMEs were identified by comparison of their hydrodistillation for 90 min (time fixed after a retention times with those of authentic standards. The kinetic survey during 30, 60, 90 and 120 min. The identification of essential oil components was based optimal yield was obtained at 90 min). The on a comparison of their retention indices (RI) hydrodistillation was performed by a simple relative to (C7-C20) n-alkanes with those of literature laboratory Quik-fit apparatus which consisted of a or with those of authentic compounds available in 1000 ml steam generator flask, a distillation flask, a our laboratory. Further identification was made by condenser and a receiving vessel. Essential were matching their recorded mass spectra with those extracted from the distillate using diethyl-ether as stored in the Wiley/NBS mass spectral library of the solvent (v/v) dried over anhydrous sodium sulphate GC-MS data system and other published mass spectra then concentrated at + 35°C using a Vigreux column [23]. Quantitative data were obtained from the and stored at -20°C prior to analysis. All experiments electronic integration of the FID peak areas. were done in triplicates and results were expressed on the basis of dry matter weight (DMW). Statistical analyses

Gas chromatography (GC-FID) analyses Data were subjected to statistical analysis using statistical program package STATISTICA [24]. The Essential oil analysis was performed using a one-way and multivariate analysis of variance Hewlett-Packard 6890 gas chromatograph (Agilent (ANOVA) followed by Duncan’s multiple range tests Palo Alto, CA, USA) equipped with a flame were employed and the differences between ionization detector (FID) and an electronic pressure individual means were deemed to be significant at P control (EPC) injector. A polar HP Innowax (PEG) < 0.05. column and an apolar HP-5 one (30 m × 0.25 mm, 0.25 μm film thickness) were used. The carrier gas Results and discussion -1 (N2, U) flow was 1.6 ml min . The split ratio was 60:1. The analyses were performed using the Effect of water deficit on total fatty acids contents following temperature program: oven temperature isotherm at 35°C for 10 min, from 35 to 205°C at As shown in Fig.1, the total fatty acid content in the rate of 3°C min-1 and isotherm at 225°C during C. carvi seeds (mg g-1 DW) subjected to increasing 10 min. Injector and detector temperatures were held levels of water constraint declines significantly (P < at 250 and 300°C, respectively. 0.05) by about 29.01 and 47.53% under MWD and FAMEs were analyzed using the same apparatus SWD, respectively, as compared to control plants. previously described and separated on a RT-2560 These results are similar to those obtained in a capillary column (100 m × 0.25 mm, 0.20 mm film previous work with Tunisian caraway seed ecotype thickness). The oven temperature was kept at 170°C from Haouaria which seed total fatty acid content for 2 min, followed by a 3°C min-1 ramp to 240°C decreased by about 35.17 and 56.59% under MWD and finally held there for an additional 15 min and SWD, respectively, in comparison to the control. period. Nitrogen was used as carrier gas at a flow But, the decrease in the fatty acid content of this last rate of 1.2 ml min-1. The injector and detector ecotype is more pronounced [15]. temperatures were maintained at 225°C. In accordance with our findings, Bouchereau et al., [25] found a significant decrease in seed oil of Gas chromatography-mass spectrometry (GC-MS) Brassica napus L. genotypes. Besides, Rotundo and analyses Westgate, [26] noted that water constraint imposed during seed filling decreased soybean oil content by The GC-MS analyses were performed on a gas about 35%. These findings were also similar to those chromatograph HP 6890 (II) interfaced with a HP reported by Bettaieb et al., [6] who noted that 5973 mass spectrometer (Agilent Technologies, Palo drought decreased significantly the foliar fatty acid Alto, California, USA) with electron impact Adv. Environ. Biol., 5(2): 257-264, 2011 260

Fig. 1: Water deficit effect on total fatty acid contents (mg g-1DW) of Carum carvi seeds [C: control; MWD: moderate water deficit; SWD: severe water deficit; values with different superscripts (a-c) are significantly different at P < 0.05 (means of three replicates)].

content of sage ( L.) by about 34% 105.81 and 256.52% under SWD, respectively. Thus, in moderate stressed plants; nevertheless, it declines the major variation consisted in a decrease of the sharply in severely stressed plants representing only unsaturated acid proportion in favour of an increase 3.2% of the control. in saturated ones. Overall, these results showed that the reduction These results are in agreement with those in TFA content are thought to be attributed to reported by Hamrouni et al., [28] who showed that membrane lipid degradation under water deficit [27]. the major variation of safflower (Carthamus tinctorius L.) aerial part lipids submitted to water Effect of water deficit on total fatty acids proportion deficit, consisted in a decrease of polyunsaturated fatty acid proportions (18:2 and 18:3) in favour of Caraway seeds in control plants were the saturated ones (16:0 and 18:0). characterized by a high proportion of In the same context, Martins Junior et al., [29] monounsaturated fatty acids (MUFA) (56.82%) noted that leaf fatty acid composition of bean versus 27.41% of polyunsaturated (PUFA) and (Phaseolus vulgaris L.) was affected by drought. This 15.77% of saturated ones (SFA) (Table 1). last decrerased polyunsaturated fatty acid contents Petroselinic acid (C18:2n-12) was the major (linoleic and linolenic acids) but increased saturated compound reaching over 34% of TFA, followed by fatty acid ones (palmitic and stearic acids). linoleic (26.49%) and oleic (22.33%) acids. These results contrast with those of Bettaieb et In a previous work, we have found that the al., [6] who reported that water deficit decreased the fraction of monounsaturated fatty acid in three proportion of palmitic (16:0) and caused the Tunisian caraway seed ecotypes was mainly disappearance of palmitoleic (16:1), stearic (18:0) and dominated by petroselinic acid which ranged from arachidic (20:0) acids. 31.53 to 38.36% [11]. Furthermore, the DBIs (double bond indexes) Drought induced marked changes in fatty acid calculated according to Rie De Vos et al., [30] to composition of caraway seeds and mainly that of the evaluate the unsaturation degree of fatty acids pool, petroselinic one which is reduced by 15 and 20.3% decreased significantly (P<0.05) in comparison to the compared with the control under moderate and severe control (Table 1). So, these results showed that water water deficits, respectively. Linoleic and constraint tends to reduce the degree of unsaturation proportions decreased significantly (P < 0.05) by 6.95 of C.carvi seed fatty acids. The same results were and 11.37% respectively at MWD whereas under obtained by Hamrouni et al., [28] in safflower SWD, their proportions declined of 12.16 and (Carthamus tinctorius L.) and Bettaieb et al., [6] in 19.97%, respectively. sage (Salvia officinalis L.) when these plants were Thus, our present results are slightly higher than subjected to water deficit. those obtained in a previous work with Tunisian In summary, these findings showed that this caraway seed ecotype originated from Haouaria; for constraint tends to affect the quality and the stability example, we noted that petroselinic proportion of caraway seed oil and to reduce its degree of total decreased significantly by 12.7 and 18.47% under fatty acids unsaturation as we have demonstrated in MWD and SWD, respectively [15]. a previous work [15]. Indeed, water deficit causes In addition, we noted that water deficit increased degradative processes, an inhibition of lipid the saturated acid proportions .Thus, under MWD, biosynthesis [31] and a stimulation of lipolytic and myristic, palmitic and stearic acids increased up to peroxidative activities [32,33] that are associated with 59.44, 61.95 and 57.97%, respectively and 71.81, decreased membrane lipid content. Adv. Environ. Biol., 5(2): 257-264, 2011 261

Table 1: Effect of water deficit on fatty acid composition (%) and DBI changes of a Tunisian caraway (Carum carvi L.) seed ecotype [C: control, MWD: moderate water deficit, SWD: severe water deficit (Means of three replicates)]. CMWDSWD (C14:0) 10.43 ± 0.13c 16.63 ± 0.24b 17.92 ± 0.08a (C16:0) 3.96 ± 0.01c 6.73 ± 0.01b 8.15 ± 0.03a (C18:0) 1.38 ± 0.01c 2.18 ± 0.01b 4.92 ± 0.04a Oleic acid (C18:1 n-9) 22.33 ± 0.01a 19.79 ± 0.17b 17.87 ± 0.02c Petroselinic acid (C18:1 n-12) 34.49 ± 0.07a 29.32 ± 0.10b 27.49 ± 0.03c (C18:2) 26.49 ± 0.02a 24.65 ± 0.47b 23.27 ± 0.07b Linolenic acid (C18:3) 0.92 ± 0.00a 0.71 ± 0.03b 0.38 ± 0.00c SFA 15.77 ± 4.66c 25.54 ± 1.39b 30.99 ± 3.77a MUFA 56.82 ± 3.60a 49.11 ± 1.74b 45.36 ± 1.80c PUFA 27.41 ± 1.08a 25.36 ± 0.93b 23.65 ± 1.19b *DBI 1.13 ± 0.01a 1.01 ± 0.02b 0.93 ± 0.01c Values (means of three replicates ± SD) with different superscripts (a-c) are significantly different at P < 0.05. (SFA: saturated fatty acids; MUFA: monounsaturated fatty acids; PUFA: polyunsaturated fatty acids; DBI: double bond index).

Effect of water deficit on essential oil yield extraction methods (supercritical fluid extraction and steam distillation), harvest time and storage period Hydrodistillation of C. carvi seeds (control) [38,39]. offered an essential oil with an average yield of Water deficit induced an increase in the 0.38% (w/w the dry matter basis) (Fig.2). This yield limonene percentage, estimated by 80.85 and 95.38% is lower than that obtained with Tunisian caraway under MWD and SWD, respectively in comparison ecotype from Haouaria (0.47%) [15] and much lower to the control. Besides, drought induced a decrease than that reported by Bailer et al., [34] (2.8-3.3%) in the carvone proportion which was significantly and even lower than that reported by Arganosa et al., estimated by 7.43 and 9.61% at MWD and SWD, [35] for the same species. respectively compared to the control (Table 2). The essential oil yield is affected by the However, carvone remained the major compound of differents drought levels as we have found in a caraway seed essential oil. Thus, drought had not previous study [15]. Thus, it increased to 0.76 and affected the chemotype of caraway essential oil 0.94% under MWD and SWD, respectively, in which is in agreement with our previous study on comparison to the control (Fig.2). Tunisian caraway from Haouaria [15]. These results showed that water deficit enhances In spearmint (Mentha spicata L.), the content essential oil production in C.carvi seeds and this (µg g-1 DW) of the main compounds, which are effect is more pronounced with water constraint limonene and carvone, increased by 50% in severely sharpness. Since, MWD increased the essential oil stressed as compared to well-watered leaves at the yield by twice whereas this last was about 2.5 higher balsamic period. In rosemary (Rosmarinus officinalis under SWD compared with the control. L), the concentrations of α-pinene and camphor, the In agreement with our findings, this increase is main monoterpenes, were more than 100% higher in also observed in other aromatic plant species such as severely stressed leaves than in well-watered leaves Pimpinella anisum L. [36], Petroselinum crispum [5] at balsamic period. showed the highest and Salvia officinalis [6]. percentage increment respect to plant establishment Conversely, Fatima et al., [37] found that water [3]. deficit reduced essential oil content of two aromatic The seed essential oil was characterized by the grasses species, martinii and prevalence of ketones (86.88%) represented by Cymbopogon winterianus. This decrease in essential carvone, trans-dihydrocarvone and cis-dihydrocarvone. oil yield could result from the effect of water The monoterpene hydrocarbons (8.21%) constitued constraint on plant growth and differentiation rather the second main class and contained limonene as than a direct effect on essential oil biosynthesis. prominent constituent. The remaining fractions as aldehydes, oxygenated sesquiterpenes and esters Effect of water deficit on essential oil composition formed the minor classes. Hence, increasing levels of water deficit Essential oil compounds identified in C. carvi stimulated the biosynthesis of the monoterpene seeds are listed in Table 2. In control plants, 41 hydrocarbon class which proportion increased by compounds were identified out of which, carvone 73.45 and 92.69% under MWD and SWD, was the major compound (78.11%) followed by respectively while ketone class decreased by 7.26 and limonene (14.79%). Other minor constituents were 9.51% under MWD and SWD, respectively (Table noted such as β-pinene, β-myrcene, γ-terpinene etc. 2). Thus, water deficit induced changes which were Thus, carvone and limonene account for the main related to the relative proportions of C. carvi seed part and were commonly found in the essential oil of essential oil constituents but doesn’t cause the C. carvi seeds, but with different percentages appearance of new ones [15]. depending on type of cultivars, sampling technique, Adv. Environ. Biol., 5(2): 257-264, 2011 262

Fig. 2: Water deficit effect on essential oil yield (%) of Carum carvi seeds [C: control; MWD: moderate water deficit; SWD: severe water deficit; values with different superscripts (a-c) are significantly different at P < 0.05 (means of three replicates)].

Table 2: Effect of water deficit on essential oil composition (%) of a Tunisian caraway (Carum carvi L.) seed ecotype [C: control, MWD: moderate water deficit, SWD: severe water deficit (Means of three replicates)]. Compound* RIa RIb Identification Water deficit ------CMWDSWD α-Pinene 934 1032 GC/MS 0.25 ± 0.00a 0.18 ± 0.06b 0.16 ± 0.06c Camphene 951 1086 GC/MS.Co GC 0.13 ± 0.00b 0.08 ± 0.02c 0.24 ± 0.16a β-Pinene 980 1123 GC/MS.Co GC 0.09 ± 0.00b 0.05 ± 0.03c 0.18 ± 0.18a β-Myrcene 991 1166 GC/MS 0.08 ± 0.00b 0.10 ± 0.03a 0.12 ± 0.07a Limonene 1030 1206 GC/MS 7.57 ± 3.28c 13.69 ± 2.87b 14.79 ± 1.11a γ -Terpinene 1062 1255 GC/MS.Co GC 0.03 ± 0.03b 0.03 ± 0.01b 0.11± 0.01a (E)-β-Ocimene 1050 1266 GC/MS.Co GC 0.03 ± 0.03b 0.03 ± 0.01b 0.12 ± 0.02a p-Cymene 1026 1280 GC/MS.Co GC 0.02 ± 0.00b 0.04 ± 0.00a 0.02 ± 0.00b Terpinolene 1092 1290 GC/MS.Co GC 0.02 ± 0.02b 0.03 ± 0.01b 0.08 ± 0.01a Z-3-Hexenol 855 1370 GC/MS.Co GC 0.02 ± 0.02b 0.03 ± 0.01b 0.07 ± 0.01a Trans-limonene oxide 1136 1463 GC/MS 0.07 ± 0.07c 0.12 ± 0.01a 0.09 ± 0.02b Camphor 1143 1532 GC/MS.Co GC 0.02 ± 0.02b 0.04 ± 0.00a 0.03 ± 0.00ab Linalool 1100 1545 GC/MS.Co GC 0.02 ± 0.02b 0.07 ± 0.01a 0.06 ± 0.01a Linalyl acetate 1257 1556 GC/MS.Co GC 0.15 ± 0.03b 0.07 ± 0.00c 0.18 ± 0.02a β-Elemene 1594 1600 GC/MS.Co GC 0.02 ± 0.02c 0.06 ± 0.00b 0.19 ± 0.19a Terpinene-4-ol 1178 1611 GC/MS.Co GC 0.05 ± 0.05c 0.13 ± 0.04a 0.10 ± 0.01b β-Caryophyllene 1419 1612 GC/MS.Co GC 0.02 ± 0.02c 0.06 ± 0.00a 0.04 ± 0.04b Trans-dihydrocarvone 1204 1627 GC/MS 0.10 ± 0.10c 0.27 ± 0.02a 0.24 ± 0.06b Cis-dihydrocarvone 1197 1645 GC/MS 0.37 ± 0.06a 0.31 ± 0.03b 0.27 ± 0.08c Allo-aromadendrene 1474 1661 GC/MS.Co GC 0.27 ± 0.01a 0.21 ± 0.02b 0.18 ± 0.06c α-Terpineol 1189 1700 GC/MS.Co GC 0.02 ± 0.02b 0.04 ± 0.00a 0.02 ± 0.00b Germacrene-D 1480 1719 GC/MS.Co GC 0.15 ± 0.15c 0.46 ± 0.28a 0.24 ± 0.09b Dihydrocarveol 1253 1720 GC/MS 0.08 ± 0.01b 0.17 ± 0.03a 0.16 ± 0.00a Carvone 1241 1740 GC/MS 86.41 ± 2.04a 79.99 ± 2.51b 78.11 ± 1.13b β- Selinene 1481 1742 GC/MS.Co GC 0.22 ± 0.02b 0.40 ± 0.06a 0.20 ± 0.13b α -Selinene 1485 1745 GC/MS.Co GC 0.13 ± 0.13b 0.24 ± 0.07a 0.08 ± 0.08c α- Farnesene 1508 1755 GC/MS.Co GC 0.34 ± 0.02c 0.39 ± 0.04a 0.36 ± 0.04b Citronellol 1229 1766 GC/MS.Co GC 0.04 ± 0.00b 0.07± 0.00a 0.04 ± 0.00b δ- Cadinene 1517 1772 GC/MS.Co GC 0.43 ± 0.03a 0.38 ± 0.00b 0.31 ± 0.10c γ -Cadinene 1511 1776 GC/MS.Co GC 0.61 ± 0.07a 0.53 ± 0.02b 0.45 ± 0.07c Cuminaldhyde 1238 1785 GC/MS.Co GC 0.03 ± 0.00a 0.05 ± 0.01a 0.03 ± 0.00a -aldehyde 1272 1789 GC/MS.Co GC 0.27 ± 0.00a 0.23 ± 0.02b 0.17 ± 0.06c Nerol 1228 1797 GC/MS.Co GC 0.03 ± 0.03b 0.05 ± 0.00a 0.02 ± 0.02b Trans-carveol 1218 1841 GC/MS 0.27 ± 0.04b 0.29 ± 0.02b 0.52 ± 0.40a Cis-carveol 1230 1869 GC/MS 0.10 ± 0.02c 0.29 ± 0.02b 0.32 ± 0.19a Nonadecane 1900 1900 GC/MS.Co GC 0.11 ± 0.07a 0.05 ± 0.00b 0.07 ± 0.01b Perilla-alcool 1296 2001 GC/MS.Co GC 0.15 ± 0.04b 0.12 ± 0.02c 0.23 ± 0.16a Spathulenol 1575 2125 GC/MS.Co GC 0.27 ± 0.08b 0.29 ± 0.05b 0.32 ± 0.04a Eugenol 1356 2192 GC/MS.Co GC 0.49 ± 0.37a 0.10 ± 0.03c 0.42 ± 0.06b 1290 2198 GC/MS.Co GC 0.41 ± 0.17a 0.19 ± 0.13c 0.32 ± 0.11b Carvacrol 1296 2215 GC/MS.Co GC 0.42 ± 0.31a 0.07 ± 0.03c 0.33 ± 0.08b Chemical classes Adv. Environ. Biol., 5(2): 257-264, 2011 263

Table 2: Continue Monoterpene hydrocarbons (%) 8.21 ± 2.50b 14.24 ± 1.54a 15.82 ± 1.89a Aldehydes (%) 0.38 ± 0.12a 0.42 ± 0.09a 0.36 ± 0.06a Ketones (%) 86.88 ± 9.75a 80.57 ± 6.02b 78.62 ± 4.95b Oxygenated monoterpenes (%) 0.79 ± 0.08b 1.26 ± 0.09a 1.50 ± 0.07a Sesquiterpene hydrocarbons (%) 2.18 ± 0.19a 2.73 ± 0.17a 2.05 ± 0.13a Oxygenated sesquiterpenes (%) 0.58 ± 0.09a 0.66 ± 0.10c 0.91 ± 0.04b Esters (%) 0.15 ± 0.00a 0.07 ± 0.00c 0.18 ± 0.00a Others (%) 0.11 ± 0.00a 0.05 ± 0.00b 0.07 ± 0.00b *Components are listed in order of elution in polar column (HP-Innowax). Values with different letters (a-c) are significantly different at P < 0.05. Note: RIa and RIb: retention indices relatives to n-alcanes, on (a) apolar column (HP-5) and (b) polar column (HP-Innowax).

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