Effect of environmental conditions on chemical polymorphism and biological activities among Artemisia absinthium L. essential oil provenances grown in Tunisia

Industrial Crops and Products 66 (2015) 96–102

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Effect of environmental conditions on chemical polymorphism and biological activities among Artemisia absinthium L. essential oil provenances grown in Tunisia Leila Riahi a,b,∗ , Hanene Ghazghazi c , Besma Ayari d , Chedia Aouadhi e , Imen Klay b , Hnya Chograni f , Ameur Cherif b , Nejia Zoghlami a a

Laboratoire de Physiologie Moléculaire des Plantes, Centre de Biotechnologie Borj Cedria, B.P. 901, Hammam-Lif 2050, Tunisia LR Biotechnologie et Valorisation des Bio-Géo Ressources (LR11ES31) Institut Supérieur de Biotechnologie- Université de La Manouba Biotechpole de Sidi Thabet, Sidi Thabet 2020, Ariana, Tunisia c Laboratoire des Ressources Sylvo-Pastorales de Tabarka, Institut Sylvo-Pastoral de Tabarka, B.P. 345, Tabarka 8110, Tunisia d Laboratoire de Biochimie et de Biologie Moléculaire. Faculté des Sciences de Bizerte, Zarzouna 7021, Bizerte Tunisia e Laboratoire d’Epidémiologie et Microbiologie Vétérinaire, Groupes de Bactériologie et Développement Biotechnologique, Institut Pasteur de Tunis, B.P. 74, 13 Place Pasteur, Tunis 1002, Tunisia f National Institute of Applied Science and Technology, Department of Biology, Laboratory of Plant Biotechnology, B.P. 676, Tunis 1080, Tunisia b

a r t i c l e

i n f o

Article history: Received 11 July 2014 Received in revised form 15 December 2014 Accepted 18 December 2014 Keywords: Artemisia absinthium L. Essential oils Biological activities Environmental factors Tunisia

a b s t r a c t Variation on yields, chemical composition and biological activities of essential oils of Artemisia absinthium L. collected from four different bioclimatic areas ranging from Humid to Arid stage of Tunisia was investigated. The observed yields of essential oils increase significantly from arid to humid climate. A significant qualitative and quantitative variation of the chemical composition according to the studied localities was revealed. Plants collected from the Inferior Arid bioclimatic region (Gafsa) presented chamazulene, ␣-thujone and camphor as the main components of their essential oils. However, for Superior Arid (Kasserine) and Semi Arid (El Kef) bioclimatic regions, camphor and chamazulene are the dominant constituents followed by linalool for Kasserine and bornyl acetate for El Kef originated oils. The Humid bioclimatic zone (Ghar Dimaou) showed different chemotype and presented camphor, (Z)-sabinene hydrate and 1-terpinen-4-ol as the major compounds. Based on the two DPPH and ABTS tests, the investigated oils highlighted important in vitro antioxidant capacities which increase significantly from the humid (Ghar Dimaou) to the Inferior Arid bioclimatic zone (Gasfa). Even the investigated essential oils displayed an antimicrobial activity against all the tested bacterial and fungal strains with variable degrees, our findings did not reveal a clear correlation between the antimicrobial properties and the studied bioclimatic zones. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Essential oils and their components are gaining increasing interest in the food and pharmaceutical industries as natural antioxidants, because of their relatively safe status and their wide acceptance by consumers (Mothana et al., 2012). On the other hand, the use of essential oils to prevent the proliferation of pathogen

∗ Corresponding author at: Laboratoire de Physiologie Moléculaire des Plantes, Centre de Biotechnologie Borj Cedria, B.P. 901, Hammam-Lif 2050, Tunisia. Tel.: +216 70 527 882; fax: +216 70 527 882. E-mail address: [email protected] (L. Riahi). http://dx.doi.org/10.1016/j.indcrop.2014.12.036 0926-6690/© 2014 Elsevier B.V. All rights reserved.

microbial species in food preservation is less damaging to the human health regarding their limited toxicity and side effects (Isman, 2000; Mothana et al., 2012). With the increasing interest in the industrial exploitation of aromatic and medicinal species essential oils, phytochemical and biological investigations regarding those species are gaining more attention. The genus Artemisia L., comprising about 500 species distributed through the world (Bora and Sharma, 2011), is one of the most important genera of the Asteraceae family in the Mediterranean region. Among the different species of Artemisia L., Artemisia absinthium L. (wormwood) best known as the principal ingredient in the famous absinthe drink, is considered among the most important industrial crops. Wormwood essential oil has been reported

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to have different biological properties including antimicrobial (Juteau et al., 2003; Lopes-Lutz et al., 2008; Sengul et al., 2011), acaricidal (Chiasson et al., 2001), insecticidal (Kordali et al., 2006), anthelmintic, antiseptic, antispasmodic (Joshi, 2013) and antioxidant activities (Jasna et al., 2004; Riahi et al., 2013). Others findings suggested that A. absinthium is neuroprotective and may prove to be useful adjunct in the treatment of stroke (Bora and Sharma, 2010). In Tunisia, located in the Mediterranean basin area, about 25% of the total flora is considered for therapeutic uses (Le Floc’h, 1983). Artemisia species constitute an important component of the Tunisian shrub vegetation, especially in dry and arid environments. According to Leporatti and Ghedira (2009), this perennial plant species is traditionally used as an herbal medicine in Tunisia specifically to treat malaria. In some Tunisian regions, the popular use of A. absinthium as a medicinal plant is still familiar until now. Scientific reports concerning Tunisian Artemisia species highlighted their medicinal properties. Indeed, the essential oil of Agaricus campestris harvested from southern region of Tunisia, exhibited significant antitumor activity (Akrout et al., 2011). Findings by Mighri et al. (2010) showed an important antimicrobial capacity of Artemisia herba-alba essential oils originated from arid region of Tunisia. Moreover, Tunisian A. campestris and A. herbaalba essential oils showed antimutagenic properties (Neffati et al., 2008). The first phytochemical and biological investigation regarding A. absinthium L. in Tunisia was carried on by Riahi et al. (2013) based on one studied site (Bizerte). Last authors reported chamazulene (29.9–30.41%) and ␤-thujone (19.66–25.75%) as the major compounds of the investigated oils. Recent investigation of A. absinthium L. essential oils in Tunisia (Dhen et al., 2014), showed a strong fumigant toxicity against the two species Rhyzopertha dominica and Spodoptera littoralis considered among the most damaging agricultural pests. The diversity of biological activities of A. absinthium L. essential oils recorded in different regions of its distribution area in the world appeared to be a consequence of their rich chemical polymorphism. Chemical components of the essential oils are cited to vary qualitatively and quantitatively according to geographical location and environmental conditions (Bakkali et al., 2008). Thus, appropriate knowledge of the influence of environmental conditions on the variability of essential oils chemistry seems to be necessary for the optimization of a reproducible production quality (Formisano et al., 2015). In this context, the present study is a detailed investigation of the variation of essential oils yields, composition, antioxidant and antimicrobial activities among Artemisia absinthium L. essential oil extracted separately from four provenances belonging to four different bioclimatic areas of Tunisia characterized by contrasted pedo-climatic conditions. The detected variability will be interpreted and discussed according to environmental conditions of the studied sites which may highlight the effect of environment to diversify essential oil yield, phytochemical components and consequently their biological activities. Furthermore, we note that the antibacterial and antifungal properties of Tunisian wormwood essential oils were reported for the first time in this study.

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2. Material and methods 2.1. Plant material and characteristics of the studied sites Leaves of A. absinthium L. growing wild were collected separately from four regions (Ghar Dimaou, El Kef Kasserine and Gafsa) belonging to four different bioclimatic stages (ranging from humid to arid stage) of Tunisia. Before analyses, leaves were air-dried at room temperature in the shade for two weeks. After drying, the samples were ground to a fine powder, used for the extraction of essential oils. Considering the Mediterranean bioclimatic classification (Emberger, 1955), Tunisia has five bioclimatic stages, going from the driest to the wettest on the basis of rainfall. Four bioclimatic zones (arid, semiarid, sub-humid and humid), covering the North and Central regions of the country, have been defined in relation with increasing humidity and which are subject to the Mediterranean bioclimatic influence. The fifth bioclimatic zone (Saharan), where rainfall is less than 100 mm/year and spontaneous vegetation is almost absent, dominate the South of the country. The four studied sites in this investigation are belonging to four bioclimatic stages representing the distribution area of A. absinthium L. in Tunisia and exhibited contrasted geographical, bioclimatic and pedologic characteristics resumed in Table 1. 2.2. Essential oil extraction and identification For every site, three lots of 100 g of wormwood leaves were separately hydrodistilled for 3 h using a Clevenger-type apparatus. The essential oils were then stored in sterile tubes at 4 ◦ C until analyses. The essential oil composition was determined by GC–FID and GC–MS analyses as described by Riahi et al. (2013). The identification of oil components was assigned by comparison of their retention indices (RI) determined with reference to a homologous series of C9–C24 n-alkanes and with those of authentic standards. Identification was confirmed by comparison of their mass spectra with those recorded in NIST08 and W8N08 libraries. Component relative percentages were obtained from GC–FID peak areas without correction factors. 2.3. Antioxidant activities In this part of our work, the in vitro antioxidant activities of the leaves essential oils extracts stored at 4 ◦ C have been evaluated based on the two DPPH and ABTS tests. The DPPH radical scavenging capacity was measured according to Hanato et al. (1988) with modifications (Riahi et al., 2013). ABTS radical cation (ABTS+ ) was produced by reacting ABTS stock solution with 2.45 mM potassium persulfate and allowing the mixture to stand in the dark at room temperature for 24 h before use (Re et al., 1999). Afterwards, the ABTS+ solution was diluted with ethanol to an absorbance of 0.7 at 734 nm. After addition of 990 ml of diluted ABTS+ solution to 10 ml of sample, the absorbance reading was taken at 30 ◦ C exactly 6 mn after initial mixing, at 734 nm. The percentage inhibition of the ABTS cation radical by the samples was calculated according to the following formula: scavenging effect (%) = [(A0 − A1 )/A0 ] × 100, where A0 was the absorbance of the blank sample and A1 was the

Table 1 Locations, bioclimatic and pedologic characteristics of the studied sites. Site

Bioclimatic zone

Latitude

Longitude

Altitude (m)

Soil

Vegetation

Mean rainfall (mm/year)

Ghar Dimaou El Kef Kasserine Gafsa

Humid Semi Arid Superior Arid Inferior Arid

36◦ 36◦ 35◦ 34◦

8◦ 8◦ 8◦ 8◦

200 780 656 298

Clay-sandstone Marlstone-clay Limestone Sand-phosphateous

Agro-forestry-pastoral Agro-steppe Degraded steppe Rare

800–1200 400–600 200–400 100–200

26 11 10 25

N N N N

26 42 50 47

E E E E

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absorbance of the sample. Tests were carried out in triplicate. Sample concentrations providing 50% inhibition (IC50 ) were calculated. 2.4. Antimicrobial activity 2.4.1. Microbial strains The different essential oils of A. absinthium L. were tested for their antibacterial and antifungal activities against 10 indicators microorganisms including seven bacteria reference pathogenic (Escherichia coli ATCC 8739, Salmonella typhimurium NCTC 6017, Staphylococcus aureus ATCC 29213, Pseudomonas aeruginosa ATCC 27853, Aeromonas hydrophila EI (Trakhna et al., 2009), Listeria monocytogenes ATCC 7644, and Bacillus cereus ATCC 1247), and three fungi species (Aspergillus flavus, Aspergillus niger and Candida albicans ATCC 2091). 2.4.2. Disk diffusion assay determination of MIC and MBC The antibacterial and antifungal activities of A. absinthium L. essential oils were determined by disk diffusion assay (NCCLS, 1997). Antimicrobial activity was assessed by measuring the diameter of the growth-inhibition zone in millimeters for the test organisms comparing to the controls. The measurements of inhibition zones were carried out three times and values were the average of three replicates. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of each tested essential oil were determined using a broth dilution method as described by Cosentino et al. (1999) with some modifications (Ghazghazi et al., 2013). The MIC was the lowest concentration which resulted in a significant decrease in the inoculum viability (>90%), while the MBC was the concentration where 99.9% or more of the initial inoculum was killed. Each experiment was repeated three times and the modal MIC and MBC values were selected. 2.5. Data analysis All determinations were conducted in triplicates and results for each measured parameter were expressed as mean ± SD. Quantitative differences was assessed by ANOVA procedure (at P < 0.05) followed by Duncan’s multiple range test. Calculations were performed using the SAS v. 9.1.3 program (SAS, 1990). 3. Results and discussion 3.1. Essential oil yields and chemical polymorphism With the growing interest in the use of essential oils in both food and pharmaceutical industries, the yield of their extraction is an important parameter for their industrial exploitation. According to our results, the observed yield of essential oils increases significantly from arid to humid climate (Table 2). Indeed, the lowest yield of essential oil based on dry weight is recorded for the Inferior Arid bioclimatic zone (Gafsa: 1.24%) while the highest level is observed for the humid bioclimatic zone (Ghar Dimaou: 2.22%), with a mean value of 1.82%. Table 2 Leaves essential oil yields (%) of Tunisian Artemisia absinthium L. harvested from four different bioclimatic regions calculated based on dry weight. Site

Bioclimatic region

Essential oil yields (%)

Gafsa Kasserine El Kef Ghar Dimaou Mean value

Inferior Arid Superior Arid Semi Arid Humid –

1.24a 1.87b 1.95b 2.22c 1.82 ± 0.41

The essential oil compositions of Tunisian A. absinthium L. growing wild in the four cited different bioclimatic areas were determined by GC–MS analysis. A total of 50 compounds were identified with a significant variation of the essential oil chemical composition according to studied localities (Table 3). The significant chemical polymorphism recorded based on the species growing conditions is mainly quantitative but also qualitative. The chamazulene (42.96%), ␣-thujone (15.08%) and camphor (12.96%) were identified as the main components of the essential oils from plants growing in Inferior Arid zone (Gafsa). However for the Superior Arid harvested samples (Kasserine), camphor (33.29%) chamazulene (24.68%) and linalool (6.45%) are the major compounds. For the Semi Arid stage (El Kef), camphor (28.94%) and chamazulene (18.98%) are the dominant constituents followed by bornyl acetate (15.5%). Essential oils extracted from humid bioclimatic region (Ghar Dimaou) showed different chemotype and presented camphor (50.37%), (Z)-sabinene hydrate (11.83%) and 1-terpinen-4-ol (6.87%) as major compounds. Major compounds for the studied sites were followed by others components with lower percentages which are (Z)-sabinene sydrate (5.34%), germacrene D (3.91%) and ␣-curcumene (2.64%) for Gafsa; 1-terpinen-4-ol (6.14%), bornyl acetate (5.62%) and germacrene D (4.00%) for Kasserine. The main constituents observed for El Kef locality were followed by linalool (6.09%), 1-terpinen4-ol (5.03%) and germacrene D (4.14%). However, the second more important compounds observed in Ghar Dimaou essential oils are chamazulene (5.98%), linalool (5.45%) and germacrene D (1.94%). Furthermore, the studied oils share numerous others minor compounds with different relative concentrations and were characterized by some specific compounds (Table 3). Our results are comparable to the previous study regarding A. absinthium for Bizerte locality in the North East of Tunisia (Riahi et al., 2013). According to the last authors, leaves essential oils were shown to be dominated by chamazulene (30.41%), ␤thujone (25.75%), bornan-2-one (17.33%), bornyl acetate (10.12%), 1-terpinen-4-ol (4.75%) and p-cymene (3.41%). Based on our results, Tunisian wormwood essential oils showed a significant chemical polymorphism traduced by different chemotypes depending on the studied site. Thus, the contrasted bioclimatic and pedologic growing conditions seem to influence qualitatively and quantitavely the chemical composition of the studied essential oils. Additionally, genetic factors are not excluded to explain the observed variation in essential oils composition among the studied sites in Tunisia. Thus, camazulene and thujone secondary metabolite biosynthesis is significantly increased in the species samples growing in Gafsa site (Inferior Arid Bioclimatic zone), located in the Central West of Tunisia, where the mean rainfall do not exceed 100–200 mm/year and the soil is sand-phosphateous. The ␣-thujone present with high amount (15.08%) in Gafsa site is present in trace for Kasserine site (0.09%) and absent in El Kef and Ghar Dimaou localities. The region of Gafsa which presents the transition zone between the Mediterranean area and the Saharan area in Tunisia is characterized by a heat and drought stress. We can note that the most important camphor percentages were recorded for the others sites with the highest level (50.37%) for Ghar Dimaou region (Humid bioclimatic stage), presenting a mean rainfall of 800–1200 mm/year and a claysandstone soil rich in organic material. According to our results, the sites of Kasserine and El Kef which present the closest geographical locations, belonging respectively to the Superior Arid and Semi Arid bioclimatic zones, showed the closest essential oil compositions. El Kef site is characterized by a mean rainfall of 400–600 mm/year and a marlstone-clay soil, whereas Kasserine site is characterized by a mean rainfall of 200–400 mm/year and a limestone sol.

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Table 3 Chemical profiles of leaves essential oils of Tunisian Artemisia absinthium L. species harvested from four different bioclimatic regions. RI: retention index; Ps: mean percentage. Values in each table line followed by different letters are significantly different (P < 0.05). N◦

Compounds

RI

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50

␣-Thujene ␣-Pinene Sabinene ␤-Pinene ␤-Myrcene ␣-Phellandrene ␣-Terpinene p-Cymene ␣-Limonene 1,8-Cineole cis-Ocimene ␥-Terpinene (Z)-Sabinene hydrate Linalool ␣-Thujone ␤-Thujone P-Menth-2-en-1-ol ␣-Campholenal iso-Thujanol Norbornan-2-one Camphor Pinocarvone Borneol 1-Terpinen-4-ol trans-Myrtanol ␤-Fenchol 2-Methoxycarbonyl phenol Pulegone cis-Piperitol Pornyl acetate Thymol ␣-Cubebene Eugenol Geraniol acetate ␤-Bourbonene (Z)-Jasmone ␤-Elemene ␤-Caryophyllene ␣-Caryophyllene ␣-Amorphene Germacrene D ␤-Selinene Bicyclogermacrene ␣-Farnesene ␣-Muurolene ␤-Cadinene ␥-Cadinene ␤-Caryophyllene epoxide Chamazulene ␣-Curcumene

931 939 975 980 991 1002 1017 1025 1029 1031 1040 1062 1070 1097 1102 1114 1122 1126 1138 1140 1146 1165 1175 1177 1193 1208 1222 1238 1265 1285 1290 1345 1359 1381 1389 1393 1398 1419 1454 1475 1485 1490 1500 1443 1466 1472 1513 1646 1725 1776

Total (%)

95.12

Gafsa

Kasserine

El Kef

Ghar Dimaou

Ps

0.51a 5.34a 1.73a 15.08a 0.34a 0.14a – 0.11a – 12.96a 0.03a 0.30a 1.10a 0.07a 0.41a – – – 1.52a 0.08a 0.26a 0.21a 0.66a 0.12a – – 0.87a 0.11a – 3.91a – 0.15a 0.11a – 0.52a – 0.36a 42.96a 2.64a

0.07b 0.60b 0.12b 0.06bc 0.97b 0.39b 1.63b 0.71b 0.93b – – 2b 0.70b 6.45b 0.09b – 0.52b 0.08a – 0.07a 33.29b – 0.23b 6.14b 0.58b 0.76b – – 0.13a 5.62b 0.12b 0.50b 0.18b 0.30b 0.31b – 0.24a 0.16b – 0.03a 4.00a 0.08a 0.46b 0.26b 0.08a 0.21b 0.10a – 24.68b 1.07b

0.10c 0.81c 0.12b 0.07c 1.93c 0.29c 1.62b 0.50c 0.55c 0.14b 0.06a 2.11b 2.06c 6.09bc – – 0.54bc 0.07a – 0.27b 28.94c – 0.50c 5.03c 0.47c 0.56c 0.23a 0.64a 0.08b 15.50c – 0.46b – 0.27b 0.29b 0.02a – 0.68c – – 4.14a 0.07a 0.15a 0.14c – 0.08c – 0.14b 18.98c 1.79c

1.11d 0.61b 0.07c 0.05ac 1.03b 0.23d 1.35c 0.33d – 0.25c – 1.40c 11.83d 5.45c – 0.12b 0.62c – – – 50.37d – 0.91d 6.87b 0.30d 0.28d 0.10b – 0.10c 0.20d – 0.19c 0.04c 0.19c 0.11a – – 0.88a – – 1.94b 0.12b 0.07c 0.06d – 0.02d 0.13b 0.15b 5.98d 1.30d

0.33 0.58 0.20 0.05 1.15 0.23 1.26 0.42 0.43 0.12 0.01 1.50 4.98 4.93 3.79 0.11 0.45 0.04 0.03 0.08 31.39 0.01 0.48 4.78 0.35 0.50 0.08 0.16 0.08 5.71 0.05 0.35 0.11 0.35 0.21 0.01 0.06 0.65 0.03 0.01 3.50 0.07 0.21 0.14 0.02 0.21 0.06 0.16 23.15 1.70

94.92

96.49

94.76

0.03a 0.30a 0.48a 0.04a 0.68a 0.02a 0.46a 0.16a 0.24a 0.11a –

According to Jemâa (2014), changes in chemical composition among Artemisia species in Tunisia may be directly linked to climate and soil factors. Our findings confirmed those of Bailen et al. (2013) who reported a quantitative and qualitative variation in the chemical composition of essential oils from Spanish A. absinthium L. cultivated under different environmental conditions. The influence of environmental factors in the chemical composition of essential oils has also been reported in others Asteraceae species (Haider et al., 2004). According to Thompson et al. (2003), there is a relatively high correlation between soil type and chemotype structure. Variability in essential oil profiles according to geographical origin in A. absinthium species were highlighted by different previous studies. According to Rezaeinodehl and Khangholi (2008), the main components of A. absinthium essential oils in Iran were ␤pinene (23.8%) and ␤-thujone (18.6%). The oils of A. absinthium of French origin contain (Z)-epoxyocimene and chrysanthenyl acetate as major components while the oils of Croatian A. absinthium

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.52 0.21 0.19 0.01 0.54 0.16 0.55 0.23 0.4 0.1 0.03 0.73 4.96 2.17 7.52 0.16 0.21 0.04 0.05 0.13 15.38 0.01 0.3 2.57 0.22 0.21 0.11 0.32 0.05 6.92 0.06 0.15 0.1 0.21 0.11 0.01 0.12 0.34 0.05 0.01 1.04 0.05 0.17 0.08 0.04 0.22 0.07 0.15 15.35 0.69

contain mainly (Z)-epoxyocimene and beta-thujone (Juteau et al., 2003). In India, the major compounds of A. absinthium were borneol (16.7–18.7%), methyl hinokiate (11.9–12.9%) and isobornyl acetate (4.0–4.7%) (Joshi, 2013). 3.2. Antioxidant activities Studies concerning the natural products to replace the synthetic additives and preservatives in the food industries are growing intensively. The antioxidant activity of Artemisia oils suggests their possible role in providing protection against oxidative diseases and their use as a natural antioxidant in food, flavor, and pharmaceutical industry (Singh et al., 2010). In this work, the different leaves essential oils extracted from A. absinthium were screened for their antioxidant activity using DPPH and ABTS scavenging methods. Our results highlight an important in vitro antioxidant activity of the essential oils of A. absinthium in Tunisia which vary significantly according to the studied site (Table 4).

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Table 4 Antioxidant activities of Tunisian Artemisia absinthium L. essential oils extracted from leaves. Values are given as mean ± SD (n = 3). Means in each column followed by different letters are significantly different (P < 0.05). Site

DPPH (IC50 , ␮g/ml)

Ghar Dimaou El Kef Kasserine Gafsa

41.45 35.02 30.24 24.68

± ± ± ±

ABTS (IC50 , ␮g/ml)

1.11d 1.22c 1.05b 1.62a

14 12 10.26 9.16

± ± ± ±

1.01d 0.91c 0.88b 0.34a

For DPPH test, the highest antioxidant activity was observed for the site of Gasfa (IC50 = 24.68 ± 1.62 ␮g/ml) while the lowest antioxidant power was revealed for Ghar Dimaou site (IC50 = 41.45 ± 1.11 ␮g/ml). Results for ABTS test confirm findings by DPPH test. Thus, the higher antioxidant activity was observed for Gafsa locality essential oils (IC50 = 9.16 ± 0.34 ␮g/ml) while Ghar Dimaou site exhibited the lowest level (IC50 = 14 ± 1.01 ␮g/ml). These findings are comparable to previous reports which showed an important in vitro antioxidant activity of the essential oils of A. absinthium (Ramos et al., 2003; Baykan Erel et al., 2012; Riahi et al., 2013). Comparable results are obtained for Egyptian Artemisia judaica volatile oil which showed important antioxidative activity (El-Massry et al., 2002). Our results show that antioxidant capacities of the studies oils increase significantly from humid to arid bioclimatic areas. The variation of essential oil antioxidant capacity observed between the studied localities is attributed to their chemical polymorphism. The locality Gafsa which present the highest antioxidant activity differ from the others localities by the presence of ␣-thujone (15.08%) with high amount compared the others sites where this compound is absent or present in trace. The thujone is considered as the most important biologically active compound of absinthe essential oils and responsible for their toxicological properties (Patocka and Plucar, 2003). Recent study concerning Spanish A. absinthium showed that the strongest antifeedant effects were found for commercial oil samples rich in thujones (Bailen et al., 2013). Others report highlighted the use of essential oils containing thujone in herbal medicine (Mayer et al., 2009; Mueller et al., 2010). Moreover, Gafsa site is characterized by the highest amount of chamazulene (42.96%) which is present with lower amounts in the others localities. Essential oils that contain chamazulene are cited to have apparent antioxidant activity and for this reason are important in therapeutic applications (El Beyrouthy et al., 2011). It was reported that chamazulene is a potent hydroxyl radical scavenger and effectively inhibits lipid per-oxidation (Rekka et al., 1996). Additionally, Gafsa essential oils were characterized by some others specific minor compounds like iso-thujanol (0.11%), pinocarvone (0.03%) and ␣-caryophyllene (0.11%); those minor bioactive components could contribute to the final antioxidant activity of those oils.

Indeed, it is difficult to attribute the antioxidant effect of a total essential oil to one or few active compounds; both minor and major compounds should make a significant contribution to the oil’s activity which is the interaction result of their chemical composition (Wang et al., 2008). The caryophyllene is proved to have high inhibitory capacity on lipid peroxidation and showed high scavenging activities against hydroxyl radical and superoxide anion (Calleja et al., 2013). An antioxidant activity which is attributed to caryophyllene is confirmed by Legault and Pichette (2007). Variations of environmental factors (drought, chemical composition of soil. . .) among the four studied localities seem to have influenced the chemical polymorphism of the studied essential oils. This had clearly affected the antioxidant power of those oils. The highest antioxidant activities were recorded for Inferior Arid bioclimatic stage (Gafsa) where plants are facing heat and drought stress resulting in an antioxidant stress, and then they need to optimize their secondary metabolism to synthesize more antioxidant compounds (Larkindale and Huang, 2004). Essential oils originated from Humid bioclimatic stage (Ghar Dimaou) showed a significant increase in their antioxidant capacity compared to the others sites. 3.3. Antimicrobial activities The data obtained from the three used parameters (ID, MIC and MBC) indicated that the investigated essential oils displayed an antimicrobial activity against all tested strains with variable degrees which valorize Tunisian provenances of A. absinthium. Variations of antimicrobial activities did not show clear correlation with the studied locality but vary according both to the studied site and the tested microbial strain (Table 5). In the major cases Gram-positive bacterial strains showed more sensitivity against the studied oils. For Gram + bacteria strains, comparable levels of antibacterial activities were observed among the four studied oils and the highest activity was recorded against the bacteria P. aeruginosa; the highest level of activity is observed with Kasserine essential oils (ID: 18 mm, MIC: 6.25%, MBC: 12.5%). Concerning Gram- bacterial strains, Gafsa essential oils exhibited the highest antibacterial activities against S. aureus (ID: 12 mm, MIC: 12.5%, MBC: 25%). However, the three other sites presented their highest antibacterial potential against the species L. monocytogenes with the highest effect for Kasserine and Ghar Dimaou oils (ID: 19–20 mm, MIC: 6.25%, MBC: 12.5%). For fungal species the highest level of antifungal activity is observed against Asp. Niger for all the investigated oils (ID: 14–16 mm, MIC: 12.5%, MBC: 25%). Our results corroborate previous reports about the antibacterial and antifungal potential of wormwood essential oils which validate the traditional medicinal use of the studied species as an antiseptic. The essential oil of A. absinthium, had showed antibacterial activity against common human pathogens like E. coli, Salmonella enteritidis, Pseudomonas aeruginosa, Klebsiella pneumoniae and S.

Table 5 Antimicrobial activities of A. absinthium L. essential oils among the studied provenances. ID: diameter of inhibition (mm), MIC (% v/v): minimum inhibitory concentration, MBC (% v/v): minimum bactericidal concentration. G: Gafsa, K: Kasserine, EK: El Kef Gh: Ghar Dimaou. ID (mm)

Gram − Bacteria

Gram + Bacteria

Fungus

E. coli S. typhimurium A. hydrophila P. aeruginosa S. aureus L. monocytogenes B. cereus Asp. flavus Asp. niger C. albicans

MIC (% v/v)

MBC (% v/v)

G

K

EK

Gh

G

K

EK

Gh

G

K

EK

Gh

11 10 11 12 12 12 11 13 14 12

14 14 12 18 16 19 16 13 16 12

14 13 15 16 18 19 18 12 15 13

14 12 14 15 17 20 19 12 15 13

25 25 25 12.5 12.5 25 25 25 12.5 25

12.5 12.5 12.5 6.25 12.5 6.25 12.5 25 12.5 25

12.5 12.5 25 12.5 12.5 12.5 25 25 12.5 25

25 25 25 12.5 25 6.25 25 25 12.5 25

50 50 50 25 25 50 50 50 25 50

25 25 25 12.5 25 12.5 25 50 25 50

25 25 50 25 25 25 50 50 25 50

50 50 50 25 50 12.5 50 50 25 50

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aureus (Blagojevic et al., 2006). Antibacterial activities of the oil of A. absinthium in India have been reported by Joshi (2013). Antifungal activity of Spanish wormwood essential oils against three species of Fusarium was recorded (Bailen et al., 2013). A. absinthium oils from France inhibited the growth of both the yeasts C. albicans and Saccharomyces cerevisiae (Juteau et al., 2003). According to Kordali et al. (2005), Turkish Wormwood essential oil has been described as fungicidal against 34 species of fungi including Fusarium solani and Fusarium oxysporum. Furthermore, A. absinthium oils from Uruguay showed antifungal effects against Alternaria sp. and Botrytis cinerea (Umpiérrez et al., 2012). In this part of our work, the obtained results did not highlight a specific correlation between the studied site and the resulted essential oil antimicrobial capacities. The resulted antimicrobial activity of essential oils seems depend firstly on the interaction between the essential oil components for each site and secondly on the treated microorganism strains. This result is in agreement with report by Delaquis et al. (2002) who cited that the biological activity of different oils resulted of the interaction among their total chemical compounds and the action of some compounds can be inhibited by antagonistic effect. On the other hand the action mode of antimicrobial agents essentially depends on the type of the treated microorganism in relationships with their cell wall structure and the outer membrane arrangement (Shan et al., 2007). Considering the large number of different groups of chemical compounds present in essential oils, it is most likely that their antibacterial activity is not attributable to one specific mechanism but results from several damages in the cells (Sadaka et al., 2013). This is confirmed by results of Cha et al. (2005) who reported that the essential oils of Artemisia scoparia and Artemisia capillaris exhibited considerable inhibitory effects against all oral bacteria tested, while their major components demonstrated various degrees of growth inhibition. Generally, camphor, terpinen-4-ol and thujone rich oils have been shown to have strong antibacterial and fungicidal effects (Cermelli et al., 2008; Lopez-Lutz et al., 2008; Umpiérrez et al., 2012). 4. Conclusion Our findings revealed significant variations in the yields, chemical profiles, antioxidant and antimicrobial potentials of Tunisian wormwood essential oils between the four provenances characterized by different pedoclimatic factors. Thus the geographic origin and the plant growing conditions could significantly affect the chemical components and then the biological activities of its essential oils. Genetic factors cannot be excluded to explain this phytochemical polymorphism. Based on this study the essential oil yields increases significantly from arid to humid climate whereas antioxidant capacities of the studies oils increase significantly from humid to arid bioclimatic areas. Tunisian A. absinthium L. essential oils displayed an antimicrobial activity against all tested bacterial and fungal strains with variable degrees. However, our results did not show a clear correlation between the studied sites and the antimicrobial capacities. Acknowledgement The authors are grateful to the Tunisian Ministry of Higher Education, Scientific Research and Information and Communication Technologies for financial support. References Akrout, A., Alarcon Gonzalez, L., El Jani, H., Campra Madrid, P., 2011. Antioxidant and antitumor activities of Artemisia campestris and Thymelaea hirsute from southern Tunisia. Food Chem. Toxicol. 49 (2), 342–347.

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