Food and Chemical Toxicology 48 (2010) 31443152

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Food and Chemical Toxicology

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Essential oils composition in two Rosmarinus ofcinalis L. varieties and incidence for antimicrobial and antioxidant activities
Yosr Zaouali a,, Taroub Bouzaine b, Mohamed Boussaid a,1
a b

National Institute of Applied Science and Technology, Department of Biology, Laboratory of Plant Biotechnology, B.P. 676, 1080 Tunis Cedex, Tunisia National Institute of Applied Science and Technology, Department of Biology, Laboratory of Ecology and Microbial Technology, B.P. 676, 1080 Tunis Cedex, Tunisia

a r t i c l e

i n f o

a b s t r a c t
The essential oil composition of Rosmarinus ofcinalis var. typicus and var. troglodytorum endemic to Tunisia, and growing wild in different bioclimates, was determined by GC and GCMS. Oils were assessed for their antimicrobial and antioxidant activity. A variation of the chemical composition attributed to varieties rather than to bioclimates was revealed. 1.8-Cineole (47.227.5%) and camphor (12.927.9%) were identied as the main constituents of var. typicus and var. troglodytorum, respectively. The principal component analysis performed on oil constituents for all the populations allowed the distinction of two distinct population groups in accordance to the varietal subdivision. Based on the determination of the diameter of inhibition and the determination of the minimum inhibitory concentration, a low to moderate antimicrobial activity according to oils was revealed against eight bacteria tested. However, oils from var. troglodytorum showed higher bactericidal effect than those from var. typicus. The oils antioxidant activity, determined by 1,1-diphenyl-1-picrylhydrazyl (DPPH) assay, ferric reducing (FRAP) assay and b-carotene bleaching test, was relatively high. The highest activity was found in oils from var. troglodytorum and in one population of var. typicus from the upper semi-arid bioclimate. 2010 Published by Elsevier Ltd.

Article history: Received 15 April 2010 Accepted 15 August 2010

Keywords: Rosmarinus ofcinalis L. var. typicus var. troglodytorum Essential oil Antibacterial Antioxidant

1. Introduction Plant secondary metabolites, such as essential oils and avonoids, have been widely studied for their antimicrobial, insecticidal, antifungal, antibacterial and cytotoxic activities (Faleiro et al., 1999). They were intensely screened and applied in the elds of pharmacology, medical and clinical microbiology, phytopathology and food preservation (Daferera et al., 2000). At present, there is an increasing interest in natural antioxidants, particularly for phenols intended to prevent not only the presumed deleterious effects of free radicals in the human body, but also the deterioration of fats and other constituents of foodstuffs (Rozman and Jersk, 2009). The antioxidant property of essential oils also has been veried in vitro by physicalchemical methods to promote their use as natural food additives (Ruberto and Baratta, 2000). The genus Rosmarinus L., widely assessed for the quality of its essential oils, includes ve species in the Mediterranean region: Rosmarinus ofcinalis L., Rosmarinus eriocalyx Jourdan and Fourr, Rosmarinus laxiorus (De No) Batt., Rosmarinus lavandulaceus Batt. and Rosmarinus tomentosus Huber-Morath and Maire (Martin and
Corresponding author.
E-mail addresses: yosrzaouali@yahoo.fr (Y. Zaouali), mohamed.boussaid@insat.rnu.tn (M. Boussaid). 1 Tel.: +216 71703829/929; fax: +216 71704329. 0278-6915/$ - see front matter 2010 Published by Elsevier Ltd. doi:10.1016/j.fct.2010.08.010

Bermejo, 2000; Angioni et al., 2004). R. ofcinalis L., a diploid (2n = 2x = 24) and allogamous species, mostly grows in the Mediterranean basin (Pottier-Alapetite, 1981). It is the most exploited species for its essential oil and phenol biological properties (Rozman and Jersk, 2009). Rosemarys oils from natural populations showed high variations in their antimicrobial and antioxidant activity (Janssen et al., 1987; Moreno et al., 2006). These variations were mostly correlated to differences in the chemical composition of oils according to regions (Chalchat et al., 1993; Elamrani et al., 2000; Pintore et al., 2002; Serrano et al., 2002; Angioni et al., 2004; Jamshidi et al., 2009), environmental and agronomic conditions (Moghtader and Afzali, 2009), the time of harvest (Celiktas et al., 2007), the stage of development of plants (Ruberto and Baratta, 2000) and the method of extraction (Lopez et al., 2005; Santoyo et al., 2005; Okoh et al., 2010). The relationship among the variation of morphological and/or genetic traits and differences in R. ofcinalis oils composition and their biological activity remain little reported (Falchi Delitala and Soccolini, 1998; Martin and Bermejo, 2000; Flamini et al., 2002; Stefanovits-Bnyai et al., 2003; Angioni et al., 2004). Most studies concerned whole wild population samples regardless the genetic diversity within the species. R. ofcinalis has been subdivided in a number of varieties and forms based on morphological descriptors (e.g. dimension of leaf, inorescence, calyx and corolla and the presence of glandular

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trichomes). However, the infraspecic delimitations among taxa remain uncertain because of their high morphological similarities and their high hybridization rate favored by the outcrossing mating system. Angioni et al. (2004) reported one variety in the Mediterranean region with three forms: erectus, humilis and albiorus. Quezel and Santa (1962) reported two species in Algeria: R. ofcinalis L. [synonym R. laxiorus De No] and R. Tournefortii De No [synonym R. ofcinalis L. var. lavandulaceus Batt.]. They hypothesized that R. ofcinalis L. var. lavandulaceus Batt. observed in the region of Oran is endemic to Algeria and may be an hybrid between R. tournefortii and R. ofcinalis. Pottier-Alapetite (1981) recognized for Tunisia one R. ofcinalis L. species including four varieties: var. typicus Batt., with up growth habit, short inorescences, pale blue corolla pinked purple (912 mm) and large puberulent calyx axillated by deciduous bracts. This variety is widely represented in Tunisia. var. laxiorus De No, with prostrate growth habit and white corolla (810 mm). Its distribution area is limited to several northeast coastal regions (e.g. Ghar El Melh). var. troglodytorum Maire, an endangered variety which is endemic to Tunisia. It is located only in the arid Matmata and Toujene Djebel Mountains (in the southeast of the country) at altitudes varying between 350 and 600 m. The corolla is long (1113 mm) and brightly blue to dark purple. Stamens are highly prominent (1920 mm). The calyx is large greenish and covered with dense branching hairs. var. lavandulaceum Batt. [synonym R. tournefortii De No], with glabrous stems, short inorescences and blue owers axillated by cordate persistent bracts. Stamens are prominent and the calyx is large, grayish and star-haired. This variety is mostly located in the Tunisian Dorsal Mountain and can overlap with var. typicus. Recently, Le Floch and Boulous (2008) grouped all Tunisian taxa into two species: R. eriocalyx Jourdan and Fourr. subsp eriocalyx [synonym R. ofcinalis var. tournefortii De No ex Murb, =R. tournefortii (Murb.) Maire, =R. ofcinalis var. lavandulaceum Batt.] and R. ofcinalis L. var. laxiorus (De No) Batt. [synonym R. laxiorus De No] and var. ofcinalis [synonym R. ofcinalis var. typicus Batt.]. R. ofcinalis var. troglodytorum has been reported as endemic to Tunisia with a mention advising its botanical revision. Our previous studies, on the morphological variation of Tunisian R. ofcinalis varieties, according to Pottier-Alapetite (1981), have not provided the sufcient resolution required for a detailed classication (Khiari and Boussaid, 2000). We detected only an evident distinction between R. ofcinalis var. troglodytorum and R. ofcinalis var. typicus. Recently isozymic and chemical markers (terpenoids), assessed in 19 Tunisian natural populations, have been used to elucidate the relationship between varieties. Both markers also allowed a clear distinction between var. typicus and

var. troglodytorum (Zaouali et al., 2003, 2005). Besides, the chemical population structure, based on terpenes, is concordant with that revealed by isozymes, and a signicant correlation between genetic and terpene data was shown (Zaouali and Boussaid, 2008). The present study reports the variation of the essential oils composition of two Tunisian R. ofcinalis varieties: var. typicus and var. troglodytorum collected in wild populations and compare their antimicrobial and antioxidant activity. It is complementary to that performed on genetic and chemical markers in order to suggest and promote appropriate use of taxa.
2. Materials and methods 2.1. Plant material Based on previous chemical and genetic data (Zaouali and Boussaid, 2008), samples were collected, in April 2008, in six natural populations (Table 1) belonging to different bioclimates according to Embergers pluviothermic coefcient Q2 (Emberger, 1966). Four populations of R. ofcinalis var. typicus, located in the sub-humid and the upper semi-arid bioclimates and two populations of R. ofcinalis var. troglodytorum from the upper arid zone were assessed. Three individuals from each population were collected at random during the vegetative stage. The considered number of individuals analysed per population was based on previous work showing that individual essential oil variation within the analysed populations was not signicant (Zaouali et al., 2005). Specimens were air-dried at 30 C in a shady place at room temperature for 10 days. Vouchers specimens are deposited in the herbarium of the National Institute of Applied Science and Technology.

2.2. Extraction of essential oil One hundred grams of dried leaves from each individual were submitted to hydro-distillation for 3 h using a Clevenger apparatus. Oils were recovered directly, using a micro-pipette from above the distillate without adding any solvent, and stored in dark vials at 4 C. Solutions of 1/50 (v/v) oil were prepared in n-hexane before GC and GCMS analyses.

2.3. Chromatographic analysis 2.3.1. Gas chromatography (GC) analysis GC analysis was carried out according to Zaouali et al. (2005) using an Agilant 6980 gas chromatograph equipped with a ame ionisation detector and split-splitless injector attached to HP-INNOWAX polyethylene glycol capillary column (30 m 0.25 mm). One micro-liter of the sample (dissolved in hexane as 1/50 v/ v) was injected into the system. The constituents were identied by comparing their relative retention times with those of authentic compounds injected in the same conditions.

2.3.2. Gas chromatography/mass spectrometry (GCMS) analysis The identication of the essential oil was performed using a Hewlett Packard HP5890 series II GCMS equipped with a HP5MS column (30 m x 0.25 mm). The carrier gas was helium at 1.2 ml/min. Each sample (1 ll) was injected in the split mode (1:20), the program used was isothermal at 70 C, followed by 50240 C at a rate of 5 C/min, then held at 240 C for 10 min. The mass spectrometer was an HP 5972 and the total electronic impact mode at 70 eV was used. The components were identied by comparing their relative retention times and mass spectra with the data from the library of essential oil constituents, Wiley, Mass-Finder and Adams GCMS libraries.

Table 1 Location, main ecological traits and essential oil yields (% v/w) of the analysed populations of Rosmarinus ofcinalis L. varieties. Variety Typicus Code of pop. 1 2 3 4 5 6 Locality Korbous Abderrahmane Dj. Mt. El Ayoun Chaambi Dj. Mt. Toujene Matmata Bioclimatica zone Sh Sh Usa Usa Ua Ua Latitude 36470 N 36490 N 35300 N 35110 N 33180 N 33280 N Longitude 10350 E 10450 E 8520 E 8450 E 10090 E 10040 E Altitude (m) 400 420 660 1000 600 350 Q2a 65.64 65.64 44.90 44.90 29.19 29.19 Rainfall (mm/year) 400500 500600 400500 400500 100150 100150 Yieldb (mean% SD) clevenger 1.17 0.03 1.26 0.05 1.58 0.08 1.52 0.1 2.7 0.25 2.33 0.15

Troglodytorum

Sh: sub-humid, Usa: upper semi-arid; Ua: upper arid; Dj. Mt.: Djebel Mountain. a Bioclimatic zones were dened according to Embergers (1966) pluviometric coefcient. Q2 = 2000P/(M2 m2) where P is the mean annual rainfall (mm), M is the average maximum temperatures (K) in the warmest month (June) and m (K) is the mean minimum temperatures in the coldest month (February). b Expressed as 100 g of dried weight. Mean% SD: represents mean standard deviation of three replicates.

3146 2.4. Antimicrobial activity

Y. Zaouali et al. / Food and Chemical Toxicology 48 (2010) 31443152 ture, 1.5 ml aliquot of the resulting emulsion was transferred into test tubes containing 150 ll of extract and the absorbance was measured at 470 nm against a blank consisting of an emulsion without b-carotene. The tubes were placed in a water bath at 50 C and the oxidation of the emulsion was monitored by measuring absorbance at 470 nm over a 60 min period using spectrophotometry. The same procedure was repeated with the synthetic antioxidant, butylated hydroxytoluene (BHT) as positive control. The antioxidant capacity (AA%) of the solutions tested was calculated as: AA% = (b-carotene content after 2 h assay/initial b-carotene content)100. An extract concentration providing 50% inhibition (IC50) was obtained plotting inhibition percentage versus extract solution concentrations. 2.6. Statistical analysis For each population from each variety, we calculated the mean percentage of compounds established on the three samples collected in each site. The mean percentage of each compound at the ecological group (each group included populations from the same bioclimate) and the species levels was also calculated. The results of antioxidant and antimicrobial activities were stated in mean standard deviation. The variations of oils among populations and of their biological activities were tested by a variance analysis (SAS, 1990; ANOVA procedure) at p < 0.001 and p < 0.05. The signicance of differences between means was determined by Duncans multiple range test (DMRT) at p < 0.05. The global variations of oils and of their antioxidant and antibacterial capacity among populations were assessed using a Principal Component Analysis (PCA) (MVSP 3.1 program) (Kovach, 1999) performed at three hierarchical levels: (i) on all populations using all identied compounds, (ii) on all bacteria using diameters of inhibition exhibited by the six oils and (iii) on all oils using as input values of the three antioxidant test systems.

2.4.1. Microbial strains The antimicrobial activity was tested using oils from each individual against Escherichia coli ATCC10536, Pseudomonas aeruginosa ATCC 9027, Klebsiella pneumoniae ATCC 10031, Staphylococcus aureus ATCC 6538, Bacillus subtilis ATCC 6633, Bacillus cereus ATCC 11778, Staphylococcus epidermis ATCC 12228 and Streptococcus feacalis ATCC 10541. Bacterial strains were cultured overnight at 37 C in nutrient broth (Scharlau Microbiology, Spain). 2.4.2. Antimicrobial activity assays The antimicrobial activity of oils was determined through the agar disc diffusion and the broth dilution methods. The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) were determinated. All tests were performed in duplicate. 2.4.2.1. Disc diffusion method. The disc diffusion method was made according to Sacchetti et al. (2005). Triptic soy agar (TSA) was distributed into sterilized Petri dishes with a diameter of 9 cm (15 ml). One hundred microliters of suspension of the tested microorganisms, containing 5 105 CFU/ml of bacterial strains was poured in TSA. The lter paper discs (6 mm in diameter) were individually impregnated with 10 ll of each oil and then placed onto the agar plates. Before incubation, all Petri dishes were kept in the refrigerator (4 C) for 2 h and incubated after at 37 C for 24 h for bacteria growth. After incubation, the diameters (mm) of the inhibition zones were measured including the diameter of discs. The antimicrobial potentials were estimated according to indices reported by Rodriguez Vaquero et al. (2007). Gentamycin (30 lg/disc) and DMSO served as a positive and negative control. 2.4.2.2. Determination of minimum inhibitory (MIC) and bactericidal (MBC) concentrations. Serial dilutions of 1/2, 1/4, 1/8 and 1/10 were made with dimethylsulphoxide (DMSO) and 10 ll of each dilution were put down on sterile paper discs (6 mm diameter) placed on the surface of inoculated Petri dishes. The MIC was dened as the lowest concentration of the total essential oil at which the microorganism does not demonstrate visible growth (Okeke et al., 2001). Referring to results of the MIC assay, the minimum bactericidal concentration (MBC) was determined. Fifty microliters from each dilution of essential oil, showing growth inhibition zone in disc diffusion method, were added to 5 ml of TSA broth tubes then incubated at 37 C for 24 h in an incubator shaker. From tubes without microbial growth, 0.1 ml of cells was spread on TSA agar plates. MBCs were determined as the highest dilution at which no growth occurred on the plates. 2.5. Antioxidant activity The antioxidant activity was assessed by 1,1-diphenyl-2-picrylhydrazyl (DPPH), ferric reducing ability (FRAP) and b-carotene bleaching method systems. Data collected for each assay was an average of three experiments. 2.5.1. Free radical-scavenging assay The free radical-scavenging activity of oils was evaluated with the DPPH assay (Brand et al., 1995). One milliliter of diluted oil was added to 3 ml of the methanolic DPPH solution (4 105 M). The mixture was then shaken and allowed to stand at room temperature in the dark. After 60 min, the decrease in absorbance at 517 nm was measured against a blank (methanol solution) by using a UVvis spectrophotometer. A mixture consisting of 1 ml of methanol and 3 ml of DPPH solution was used as the control. The radical-scavenging activity of samples, expressed as percentage inhibition of DPPH, was calculated according to the formula % inhibition = [(AB AA)/AB]100, where AB and AA are the absorbance values of the control and of the test sample, respectively. The extract concentration providing 50% inhibition (IC50) was calculated from the graph of inhibition percentage plotted against extract concentration (40, 20, 10, 5 and 2.5 ll/ml). 2.5.2. FRAP assay The ferric reducing ability was assessed following the method described by Benzie and Strain (1996). The FRAP reagent contained 2.5 ml of 10 mM of 2,4,6-tris(2pyridyl)-1,3,5-triazine (TPTZ) solution in 40 mM HCl plus 2.5 ml of 20 mM FeCl3 and 25 ml of 0.3 M acetate buffer, (pH 3.6), was warmed prior to the analysis. FRAP reagent (900 ll) was mixed with 90 ll distilled water and 30 ll of diluted extracts (1:10 v/v) and then was warmed to 37 C in a water bath. A standard curve was prepared using different concentrations of FeSO47H2O (2002000 lmol/l). Results were corrected for dilution and expressed in mmol Fe2+/l of plant extract. 2.5.3. b-Carotene bleaching assay The b-carotene method was carried out according to Wettasinghe and Shahidi (1999). Two milliliters of b-carotene solution (0.2 mg/ml in chloroform) were pipetted into a round-bottomed ask containing 20 ll linoleic acid and 200 ll Tween 20. The mixture was then evaporated at 40 C for 10 min to remove the solvent, the addition of distilled water (100 ml) followed immediately. After agitating the mix-

3. Results and discussion 3.1. Variation of the essential oil composition among populations The main ecological traits of the analysed populations, and their oil yields were reported in Table 1. Yields ranged between 1.17 (population 1, var. typicus from the sub-humid region) and 2.7 (population 5, var. troglodytorum from the upper arid zone). The highest yields were recorded for populations corresponding to var. troglodytorum. A total of 25 components accounting for 93.697.5% of the total oil, according to a population were identied. 1.8-Cineole (40.0%), camphor (17.9%), a-pinene (10.3%), and camphene (6.3%) were the main constituents at the species level (Table 2). The monotepenes hydrocarbons (64.0%), represented mainly by 1.8-cineole, apinene, camphene, formed the major group. Ketones constitute 18.0% and camphor was the major compound of this class. The amount of phenols, represented only by carvacrol, was very low (0.2%) (Table 2). At the species level, our results on the composition of Tunisian R. ofcinalis oils were in accordance with those previously reported for other Mediterranean Rosemary samples (Lawrence, 1997; Pintore et al., 2002). However, the variance analysis performed on the average of each compound showed signicant (p < 0.05) and high signicant differences (p < 0.001) among populations whether they belong to the same variety or not. Camphene (3.312.8%; populations 3 and 6, respectively), 1.8-cineole (26.0 to 51.2%; populations 6 and 2, respectively) and camphor (4.929.7%; populations 1 and 5, respectively) are the main compounds which distinguish the two varieties (Table 2). At the ecological group level, the results showed that the sub-humid populations 1 and 2 and the upper semi-arid populations 3 and 4 corresponding to R. ofcinalis var. typicus exhibited highly signicant concentrations of 1.8-cineole (45.6% and 49.4%, respectively). The two upper arid populations 5 and 6 representing R. ofcinalis var. troglodytorum were rich in camphor (27.54%). The principal component analysis performed on all populations using the amounts of all constituents showed that the three rst principal components accounted for 99.73% of the total variation. The rst axis (93.20% of the inertia) is mainly correlated to 1.8-cineole (loading, 0.89). The second axis accounted for 5.84% of the total variation, and camphor (loading, 0.85) mainly contributes to its denition. a-Pinene (loading, 0.45), camphene (loading, 0.47) and

Table 2 Mean percentage of the essential oil compounds at the population, ecological group and species levels. Compounds RI Populations 1 2 0.05c (00.09) 10.5ab (8.312.6) 3.3c (2.83.7) 2.4b (1.53.3) 1.2ab (0.91.4) 0.5a (0.40.5) 0.9bc (01.9) 44.1a (44.144.2) 0.7a (0.60.8) 0.8b (0.31.3) 0.5a (0.40.6) 3 0.2c (00.3) 10.7ab (8.912.6) 4.7c (1.27.8) 0.9c (0.31.1) 1.1ab (0.91.4) 0.4a (0.30.5) 1.0bc (02.2) 47.1a (30.059.1) 0.4a (0.30.6) 1.8ab (1.62.3) 0.2ab (0.20.3) 4 0.10c (0.090.11) 7.6b (7.37.9) 3.3c (3.13.6) 3.5a (3.53.6) 0.9bc (0.80.9) tra trc 51.2a (50.551.6) 0.2a (0.20.3) 1.5ab (1.21.7) trb 68.3 12.4cd (7.916.9) trb 12.4 0.8a (0.71.0) 0.9b (0.80.9) 3.3b (2.93.6) 3.9cd (3.74.2) 0.1a (00.2) 9.1 1.5a (1.21.8) 0.06b (00.1) 0.4a (0.30.5) 1.9 0.08a (00.16) 0.08 1.0a (0.91.0) 0.7ab (0.60.7) 0.1abc (0.10.2) 1.8 93.6 5 0.4b (0.30.4) 9.0ab (6.810.4) 8.9b (7.99.8) 0.5c (0.20.7) 0.8c (0.70.9) 0.7a (0.31.5) 2.5ab (2.32.8) 29.1b (20.543.2) 0.7a (0.21.4) 2.2a (1.73.0) 0.2ab (00.4) 6 0.6a (0.40.7) 11.9a (10.313.0) 12.8a (9.815.4) 0.3c (0.20.5) 0.8c (0.70.8) 0.2a (0.10.2) 2.9a (2.53.6) 26.0b (23.328.8) 0.1a (00.2) 2.0a (1.72.7) 0.06b (00.09) var. typicus var. troglodytorum Species F-stat level Sh (pop. 1 and 2) Usa (pop. 3 and 4) Ua (pop. 5 and 6) 0.09 11.24 3.95 1.77 1.23 0.46 1.03 45.26 0.60 1.07 0.34 0.14 9.14 4.03 2.19 1.01 0.21 0.51 49.14 0.32 1.63 0.10 68.42 14.40 0.06 14.46 0.79 0.95 4.05 3.07 0.11 8.97 0.91 0.36 0.35 1.62 0.09 0.09 1.08 0.71 0.18 1.97 95.53 0.48 10.48 10.85 0.41 0.79 0.45 2.70 27.54 0.39 2.14 0.13 56.37 27.89 0.04 27.93 0.24 0.83 2.20 4.42 0.04 7.73 1.51 0.33 0.05 1.89 0.36 0.36 1.19 0.38 0.34 1.91 96.20 0.2 10.3 6.3 1.5 1.0 0.4 1.4 40.7 0.4 1.6 0.2 64 17.9 0.04 18 0.6 1.5 3.2 5.2 0.05 10.5 1.0 0.3 0.2 1.5 0.2 0.2 1.3 0.6 0.2 2.1 96.0 16.65** 2.32 ns 14.99** 14.5** 5.08* 1.4 ns 4.54* 6.94* 1.41 ns 2.48 ns 4.34* 7.92* 3.52 ns 2.98 ns 8.40* 15.41** 51.74** 1.03 ns 1.48 ns 2.35 * 4.51 * 1.25 * 1.12 ns 7.43 * 4.16 *

a-Thujene a-Pinene
Camphene b-Pinene Myrcene a-Terpinene Limonene 1.8-Cineole Ocimene c-Terpinene p-Cymene Monoterpenes Camphor Verbenone Ketones Linalool Terpinene4-ol a-Terpeneol Borneol Geraniol Alcohols Linalyl acetate Geranyl acetate Methyl eugenol Esters Carvacrol

1024 1032 1076 1118 1174 1188 1203 1213 1232 1255 1280

0.14c (0.110.17) 12.0a (10.913.1) 4.6c (3.75.5) 1.1c (0.91.4) 1.3a (1.31.4) 0.5a (0.30.6) 1.10abc (02.21) 46.4a (46.146.7) 0.5a (0.40.6) 1.4ab (1.31.4) 0.22ab (0.20.3)

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69.3 1532 4.9d (1.87.9) 1209 0.04ab (00.08) 1536 1607 1696 1719 1857 4.9 0.7ab (0.31.1) 1.1b (02.1) 3.22b (3.23.3) 11.8a (6.117.5) 0.02a (00.03)

56.8 68.5 17.9bc (17.118.9) 16.4bc (6.026.9) b tr 0.1a (00.3) 11.5 0.8a (0.51.1) 4.5a (4.14.9) 3.2bc (2.93.3) 4.2c (4.24.2) tra 12.6 0.2a (0.10.2) trb 0.3abc (0.20.4) 0.5 0.09a (00.19) 0.09 0.5a (00.9) 0.7a (0.60.9) 0.07bc (00.1) 1.3 97.2 16.5 0.8a (0.11.6) 1.0b (0.24.5) 4.8a (3.66.1) 2.2e (1.43.2) 0.1a (00.2) 8.9 0.3a (01.5) 0.7a (0.31.2) 0.3ab (00.5) 1.3 0.11a (00.30) 0.11 1.2a (1.01.3) 0.8a (0.61.1) 0.2abc (00.6) 2.2 97.5

55.0 57.7 67.05 29.7a (24.337.3) 26.1ab (21.431.2) 11.42 ab b 0.08 (00.15) tr 0.02 29.8 0.4ab (0.20.6) 0.9b (0.31.5) 2.0d (1.92.1) 2.4de (1.54.1) 0.04a (00.06) 5.7 1.8a (0.43.2) 0.4ab (00.8) 0.03d (00.1) 2.26 0.2a (00.6) 0.2 1.4a (1.01.9) 0.4b (0.30.4) 0.3ab (0.20.3) 2.1 97.25 26.1 0.07b (00.16) 0.8b (0.41.6) 2.4cd (1.73.0) 6.5b (4.38.7) 0.05a (00.1) 6.7 1.2a (0.81.8) 0.24ab (0.20.3) 0.07cd (00.2) 1.5 0.5a (0.20.8) 0.5 1.0a (0.91.1) 0.4b (0.30.5) 0.4a (0.20.6) 1.7 95.17 11.44 0.73 2.75 3.18 8.00 0.01 14.68 0.51 0.08 0.21 0.80 0.08 0.08 1.48 0.78 0.03 2.30 96.36

16.8 1565 0.9a (0.31.4) 1765 0.2ab (00.3) 2003 0.1bcd (00.3) 1.2 2208 0.07a (00.14)

Phenols 0.07 b-Caryophyllene 1612 2.5a (0.74.3) a-Humelene 1684 0.8a (0.80.9) Caryophyllene oxide 2018 trc Sesquiterpenes All identied Components 3.3 95.6

RI: retention index in HP-INNOWAX capillary column. Numbers in parenthesis: minimum and maximum percentage of each compound. F-test of the variance analysis is highly signicant () at P < 0.001, signicant () at P < 0.05 and not signicant (ns) at P > 0.05. Numbers followed by the same letter are not signicant at P > 0.05 (Duncans multiple range test). tr: traces (<0.01%). Sh: sub-humid, Usa: upper semi-arid; Ua: upper arid.

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Fig. 1. Principal component analysis performed on the six populations of the two varieties using essential oil compounds. The length of each stick indicates the value of PCAs score of each population compared to the second axis. The position of each population against its relative stick indicates a positive (population below stick) or negative (population above stick) PCAs score value compared to axis 2. d, sub-humid; j, upper semi arid; , upper arid.

Table 3 Antibacterial activity estimated by diameter of inhibition of the six essential oils (EO1 to EO 6) of Rosmarinus ofcinalis isolated from the six populations of the two varieties. Bacteria Source no. Oils (10 ll/disc) var. typicus Sub-humid EO 1 Gram-negative Escherichia coli Pseudomonas aeruginosa Klebsiella pneumoniae Gram-positive Staphylococcus aureus Bacillus subtilis Bacillus cereus Staphylococcus epidermis Streptococcus feacalis ATCC10536 ATCC 9027 ATCC 10031 ATCC ATCC ATCC ATCC ATCC 6538 6633 11778 12228 10541 14.5 0.5b NAc 16.5 1a 14 1b 15.5 0.5b 13.25 0.5bc 11.5 1.5b 9.5 0.5b EO 2 16.5 1ab NAc 13 1b 10.5 1.5c 10.5 0.5d 13 1bc 10.5 0.5bc 9 1b Upper semi-arid EO 3 18 0.5a NAc 12 0.5b 13.5 0.5b 14 0.5bc 14 1bc 11 0.5bc 9 1b EO 4 17 0.5ab 7.5 0.5a 12.5 1.5b 9.5 0.5c 13.5 0.5c 15 0b 9 1c 7.5 0.5c var. troglodytorum Upper arid EO 5 15 1ab NAc 12 0b 10.5 0.5c 12.5 0.5c 12 0c 9 0c 9.5 0.5b EO 6 16 1ab NAc 16 1a 13.5 1.5b 10.5 0.5d 12.5 0.5c 9.5 0.5bc 10.5 0.5b 18 0a 12 0a 17 0a 21 0.5a 18 0.5a 20 0a 27 1a 12 0a Gentamycin (30 lg/disc)

No antimicrobial activity (NA). Values followed by the same letter under the same line are not signicantly different (Duncans multiple range test at P > 0.05). Values represent mean standard deviation of experiments in duplicate.

borneol (loading, 0.67) are more correlated to the third principal component (0.68% of variation). According to the rst three axes, the plot revealed two population groups (Fig. 1). The rst group includes the R. ofcinalis var. troglodytorum populations 5 and 6 characterized by a high amount of camphor (29.726.1%). The upper semi-arid populations 3 and 4 and the sub-humid populations 1 and 2 corresponding to R. ofcinalis var. typicus constitute the second group situated at the positive side of the axis 2. This group was mainly distinguished by high contents of 1.8-cineole. The populations 6 (var. troglodytorum) and 1 (var. typicus), situated at the positive side of the axis 3 also are characterized by high amounts of a-pinene, camphene and borneol. Differences in the Tunisian R. ofcinalis oils composition corroborate those previously reported for other regions where different chemotypes have been identied at both local and large scale spaces (Chalchat et al., 1993; Pintore et al., 2002; Angioni et al., 2004; Afzali et al., 2009; Jamshidi et al., 2009). Our work showed that Tunisian R. ofcinalis includes: (i) a 1.8-cineole chemotype corresponding to var. typicus, similar to that reported for samples such as from Morocco (Chalchat et al., 1993; Elamrani et al., 2000), Algeria (Boutekedjiret et al., 1998), Sardinia and Corsica (Pintore et al., 2002), Turkey (Celiktas et al., 2007) and (ii) a camphor chemotype detected in the var. troglydytorum similar to that reported in earlier published data

(ElAmrani et al., 2000; Pintore et al., 2002; Serrano et al., 2002). The distinction of the two Tunisian R. ofcinalis chemotypes based on essential oil composition is concordant with their systematical and genetic characteristics (Pottier-Alapetite, 1981; Khiari and Boussaid, 2000; Zaouali and Boussaid, 2008). Thus, the genotypical differences among the two R. ofcinalis samples may be responsible for the differences in their essential oil (Svoboda and Deans, 1992; Gachkar et al., 2007). Signicant differences both among and within bioclimates were observed for var. typicus. The sub-humid populations 1 and 2, particularly could be mainly distinguished by their contents in camphor and borneol. Differences may result from both local ecological (i.e. edaphic and topographical factors) and genetic factors. The cultivation of these populations under the same conditions should provide valuable insights into the maintenance of their chemical proles. 3.2. Variation of the antimicrobial activity The in vitro antimicrobial activity of the six essential oils estimated by the diameter of inhibition varied according to populations, varieties and bacteria strains (Table 3). The highest activity was observed against E. coli ATCC 10536 with the strongest inhibition zones (18, 17 and 16.5 mm) recorded for oils EO3 and EO4

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Fig. 2. Principal component analysis performed on all bacteria using the diameter of inhibition zone for oils of the 6 analysed populations according to the rst three axes. Symbols s and d indicates Gram negative and Gram positive bacteria, respectively. E.c: Escherichia coli, K.p: Klebsiella pneumonia, P.a: Pseudomonas aeruginosa, S. a: Staphylococcus aureus, B.s: Bacillus subtilis, B.c : Bacillus cereus, S.e: Staphylococcus epidermis, S.f: Streptococcus feacalis.

from the upper semi-arid populations 3 and 4, and EO1 from the sub-humid population 1 of R. ofcinalis var. typicus. Our results were similar to those previously reported by Santoyo et al. (2005) and Gachkar et al. (2007) against the same bacteria using oils of R. ofcinalis from Sardinia and Iran, respectively. P. aeruginosa ATCC 9027 was resistant to all oils except to that from the population 4 (EO4) with a weak inhibition zone (7.5 mm). K. pneumoniae ATCC 10031 is more sensitive to oils EO1 and EO6 from the sub-humid population 1 and the upper arid population 6 belonging to var. typicus and var. troglodytorum, respectively (growth inhibition zone 16.5 and 16 mm). These two populations exhibited high amounts of borneol (11.8% and 6.5%). Oils, rich in this compound were known to possess a substantial antimicrobial potential (Mourey and Canillac, 2002). Against B. subtilis ATCC 6633 and B. cereus ATCC 11778, oils exhibited a slight to moderate antimicrobial activity. However, these bacteria were more susceptible to EO1, EO2, EO3 and EO4 from var. typicus than to EO 5 and EO 6 from var. troglodytorum. Against S. aureus ATCC 6538 moderate activities (14, 13.5 and 13.5 mm) were observed for oils EO1, EO3 and EO6 extracted from the populations 1, 3 and 6 rich in apinene. S. epidermis ATCC 12228 and S. feacalis ATCC 10541 were relatively resistant; all oils showed a slight activity with a growth zone ranged from 7.5 to 11.5 mm. All tested bacteria were more susceptible to Gentamycin (1221 mm) than to the essential oils tested (Table 3); S. feacalis and P. aeruginosa being the most resistant to both Gentamycin and oils. The principal component analysis performed for all bacteria, using the diameter of inhibition zone induced by each oil, showed that the three rst principal components accounted for 99.66% of the total variation (Fig. 2). The rst axis (98.39%) is mainly correlated to oils EO5 and EO6 (loading, 0.37% and 0.41%, respectively) of var. troglodytorum coming from the populations 5 and 6. The second axis accounted for 0.95% of the total variation. EO4 (loading, 0.87%) from the population 4 belonging to var. typicus mainly contributes to its denition. Oils EO1, EO2 and EO3 from the var. typicus populations 1, 2 and 3 are correlated to the third principal component (loading, respectively, 0.67%, 0.42% and 0.53%). The plot established for all microorganisms according to axes 1 and 2 (99.34% of the inertia) showed two major bacteria aggregates (Fig. 2) situated at the positive side of the axis 1. The rst group is constituted by the most resistant bacteria P. aeruginosa to all oils. The second group is formed by the seven other bacteria. According

to the axis 2, E. coli, P. aeruginosa, B. subtilis and B. cereus, situated at the positive side were more sensitive to the oil EO4 of var. typicus. At the negative side of the axis 3, K. pneumoniae, S. aureus and B. subtilis were grouped according to their sensibility mainly to the oil EO1 from var. typicus rich in a-pinene, camphene, 1.8-cineole and borneol known to be highly active against these bacteria (Gachkar et al., 2007; Moghtader and Afzali, 2009; Okoh et al., 2010). The bacteriostatic and bactericidal effectiveness of the six Rosemary oils estimated by minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) respectively are shown in Table 4. The Results indicate a high variation of MICs and MBCs among R. ofcinalis oils and bacteria strains corroborating earlier published data (Lopez et al., 2005; Bozin et al., 2007). P. aeruginosa was found to be resistant to all investigated oils. All oils exhibited a low antibacterial activity (MIC = 10 ll/ml) against S. epidermis and S. feacalis (MICs 6 11.5 mm). Against E. coli all oils showed a low minimum inhibitory concentration (MICs = 1.25 and 2.5 ll/ml). For the four other bacteria the lowest MIC value was observed for the essential oil EO1 of var. typicus (Table 4). The most other oils exhibiting a moderate activity (MICs = 1.25 2.5 ll/ml) against these bacteria are EO3 of var. typicus, EO6 of var. troglydotorum followed by EO4 and EO5. Oils from var. typicus showed a low bactericidal effect (MBCs = 10 ll/ml, values not reported in Table 4). However, oils EO5 and EO6 from var. troglodytorum, showed a moderate bactericidal activity (MBC = 5 ll/ml) restricted to the gram negative bacteria E. coli and K. pneumoniae. P. aeruginosa, S. epidermis and S. feacalis were resistant for all oils. The relative bactericidal effectiveness of var. troglydotorum oils may come from the synergic effect of a-pinene and camphor highly represented in these oils. The levels of antimicrobial activity in Tunisian R. ofcinalis oils were similar to those reported by Gachkar et al. (2007) for R. ofcinalis from Iran, exhibiting a high antimicrobial activity against E. coli, S. aureus and Listeria monocytogenes (MBC varied from 2 to 4 ll/ ml) due mainly to the dominance of borneol followed by camphor and verbenone, respectively. The Turkish R. ofcinalis oil possesses a moderate antibacterial activity (MBCs varied from 2.5 to 20 ll/ ml) attributed to the high content of 1.8-cineole, the low content of camphor and verbenone, respectively (Celiktas et al., 2007). A weak activity was reported for samples from Sardinia dominated by a-pinene, camphene, verbenone, bornyl-acetate, camphor and borneol tested against S. aureus, Staphylococcus epidermidis,

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Table 4 Antimicrobial activity expressed as minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the Rosmarinus ofcinalis L. essentials oil from the two varieties. Microorganisms Source no. var. typicus EO 1 EO 2 EO 3 EO 4 var. troglodytorum EO 5 MIC (ll/ml) 1.25 5 1.25 2.5 2.5 10 10 1.25 5 5 2.5 1.25 10 10 2.5 2.5 5 5 2.5 10 10 MBC (ll/ml) 5 5 EO 6 MIC (ll/ml) 2.5 2.5 1.25 5 2.5 10 5 MBC (ll/ml) 5 5

MIC (ll/ml) Gram-negative Escherichia coli Pseudomonas aeruginosa Klebsiella pneumoniae Gram-positive Staphylococcus aureus Bacillus subtilis Bacillus cereus Staphylococcus epidermis Streptococcus feacalis ATCC 10536 ATCC 9027 ATCC 10031 ATCC ATCC ATCC ATCC ATCC 6538 6633 11778 12228 10541 2.5 2.5 1.25 1.25 1.25 10 10 1.25 5 5 5 2.5 10 10

Table 5 Total antioxidant capacity determined by FRAP, DPPH and b-carotene (BC) test systems for the six essential oils from populations of Rosmarinus ofcinalis L. var. typicus and var. troglodytorum. Variety Typicus Bioclimatic stage Sh Usa Troglodytorum Ua Code 1 2 3 4 5 6 Oils EO1 EO2 EO3 EO4 EO5 EO6 Antioxidant activity DPPH IC50 (ll/ml) 26.5 1.1 28.5 1.5a 7 0.5d 19 0.7c 6 0.5d 6 0.5d
b

FRAP (mmol L1) 17.76 1.2a 16.53 1.5a 18.78 1a 18.87 1.7a 21.65 1.2a 21.77 1.7a

BC IC50 (ll/ml) 10.1 0.5a 8.3 0.7b 3.2 0.2b 10.1 0.5a 1.1 0c 3.1 0.2b

Values not sharing common letters are signicantly different (P < 0.05).

Fig. 3. Principal component analysis for all populations performed on values of the three antioxidant systems (plot according to the rst three axes).

E. coli, P. aeruginosa, (Angioni et al., 2004; Lopez et al., 2005). Our work showed that the variation of antibacterial activity in Tunisian R. ofcinalis differed according to the quantitative variation of essential oil compounds attributed to both varietal and populational effects corroborating previous works (Deans and Ritchie, 1987; Kalamba and Kunicka, 2003; Gachkar et al., 2007). Considering the high number of the identied compounds in the analysed oils, it would be difcult to attribute the antibacterial activity differences to specic compounds (Gill et al., 2002). Our results, except for P. aeruginosa which was inhibited by all oils did not globally show clear differences between gram positive and gram negative strains as reported by Bozin et al. (2007). However, within the tested gram positive bacteria, S. epidermis and S. feacalis seemed to be less sensitive than the others bacteria.

3.3. Antioxidant activity Oils showed substantial antioxidant activity, although the three assays yielded quantitatively different values of antioxidant activity. Values determined by DPPH ranged between 6 and 28.5 ll/ml according to oils (Table 5). The best activities were observed for oils EO5 and EO6 from the upper arid populations 5 and 6 of var. troglodytorum (IC50 = 6 ll/ml). The oil EO3 from the upper semiarid population 3 of var. typicus exhibited a similar activity (IC50 = 7 ll/ml). EO1 and EO2 (sub-humid zones) and EO4 (upper semi-arid zone) from var. typicus showed a low antioxidant activity (IC50 19 and 28.5 ll/ml). The total antioxidant capacity, based on the FRAP assay, expressed as mmol Fe2+/l of essential oils did not vary markedly (p > 0.05) between oils according both to

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populations and varieties; values varied from 16.53 to 21.77 mmol/l with the lowest activity (16.5317.76 mmol/l) for oils EO1 and EO2 of var. typicus and the highest (21.65 21.77 mmol/l) for oils EO5 and EO6 of var. troglodytorum. The lipid peroxidation inhibitory activity of the essential oil tested with the b-carotene bleaching test (BC) was consistent with the data obtained from the DPPH test. The Essential oils extracted from populations 5 (1.1 ll/ml), 6 (3.1 ll/ml) of var. troglodytorum and 3 (3.2 ll/ml) of var. typicus showed the greatest activity. When compared with butylated hydroxytoluene (IC50 = 21 lg/ml), R. ofcinalis oils were less effective as reported by some authors (Prez et al., 2007; Wang et al., 2008). The PCA performed on oils of all the populations using jointly the values of the three antioxydant test systems, (DPPH, FRAP and b-carotene) showed that the total variance of the two principal components was equal to 99.68% (Fig. 3). The axis 1 was correlated to FRAP test (loading, 0.70). The axes 2 and 3 were correlated to the DPPH (loading, 0.67) and b-carotene tests (loading, 0.94) respectively. The plot according to axis 12 showed two groups of oils situated at the positive side of the axis 1 (Fig. 3) and oils from the same bioclimatic zone did not strictly grouped together. The rst group, constituted by oils EO5, EO6 and EO3, is situated at the negative side of the axis 2; it is characterized by a high antioxidant activity measured by the DPPH test. This nding is in accordance with results published about the scavenging activity of Rosmarinus ofcinilis extracts (Bozin et al., 2007; Gachkar et al., 2007; Prez et al., 2007). The high scavenging activity observed for oils EO5, EO6 and EO3 could be explained partially by the high amounts of camphor, linalyl acetate and a-thujene recorded for these oils (Lee et al., 1999; Bozin et al., 2007). However, its difcult to attribute the antioxidant effect of a total essential oil to one or few active compounds. Both minor and major compounds should make a signicant contribution to the oils activity (Wang et al., 2008). The second group, situated at the positive side of the axis 2, is formed by oils EO1, EO2 and EO4. Our study on Tunisian R. ofcinalis demonstrates a high variation in the composition of oils and their biological activity potentials according to varieties and populations. This points out the importance of the genetically and morphologically characterization of varieties within the species and of populations within varieties wherever, antioxidant and antimicrobial activities of oils were determined. Thus, the selection of suitable R. ofcinalis genotypes for desired chemical traits and/or biological properties can be achieved relatively easy. Thus, the relationship among differences of oils and their biological activities according to the infraspecic subdivisions of R. ofcinalis should be more assessed. Such information is important for the development of improvement and conservation programs. Tunisian populations of var. troglydoturum were chemically distinct and showed high yields of oils rich in camphor and a-pinene with higher antioxidant activity. They appear to be interest for selection via cutting or seeds to promote their culture. However, these populations occur in a restricted geographical distribution and were isolated from var. typicus populations. They are highly degraded and endangered because of the increasing anthropic pressure (overharvesting, overgrazing), the low soil quality and the insufciency of the rainfall. They should rst be protected by the preservation of their habitats by limiting human activities. The ex-situ conservation (in a gene bank or in a clonal park) of seeds or cuttings from these populations also constitutes an efcient strategy to preserve this variety.

Acknowledgements This research was supported by grants from the Tunisian Ministry of High Education. The authors were grateful to Professor Urdaci Maria from University of Bordeaux (France) for bacteria supplying.

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