# Chemical Composition and Antioxidant and Antimicrobial Activities of Wormwood (Artemisia absinthium L.) Essential Oils and Phenolics

Authors: Kamel Msaada; Nidhal Salem; Olfa Bachrouch; Slim Bousselmi; Sonia Tammar; Abdulkhaleg Alfaify; Khaldoun Al Sane; Wided Ben Ammar; Sana Azeiz; Adel Haj Brahim; Majdi Hammami; Sawsen Selmi; Ferid Limam; Brahim Marzouk
Journal: Journal of Chemistry
 (2015)

---

## Abstract

The aim of this study was to determine the chemical variability of wormwood extracts as affected by the growing region. Antioxidant and antimicrobial activities were also investigated. The essential oil composition variability ofA. absinthium L. aerial parts collected from four different Tunisian regions was assessed by gas chromatography (GC/FID) and by gas chromatography mass spectrometry (GC/MS). In addition, total polyphenols, flavonoids, and condensed tannins as well as antioxidant, antibacterial, and antifungal activities of methanolic extract and essential oils were undertaken. Chromatographic analysis of wormwood essential oils showed the predominance of monoterpene hydrocarbons represented mainly by chamazulene. RP-HPLC analysis of wormwood methanolic extract revealed the predominance of phenolic acids. Antiradical activity was region-dependant and the methanolic extract of Bou Salem region has the strongest activity (CI
50
=
9.38
±
0.82 µg/mL). Concerning the reducing power, the methanolic extract of Bou Salem, Jérissa, and Boukornine regions was more active than the positive control. Obtained results of antimicrobial activities showed that wormwood essential oil is endowed with important antibacterial activity which was strongly related to the organoleptic quality of oil which appeared strongly region-dependant. A. absinthium L. EOs investigated are quite interesting from a pharmaceutical standpoint because of their biological activities.


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## Body

## 1. Introduction

Artemisia absinthium L. (Asteraceae), commonly known as wormwood in United Kingdom and absinthe in France, is an aromatic, perennial small shrub. It is locally known as “chajret mariem” in Tunisia. The herb has always been of a great botanical and pharmaceutical interest and is employed in folk medicine against various paints [1]. Wormwood essential oil has been widely used mainly for its neuroprotective [2], antifungal [3], antimicrobial [4], insecticidal [3], acaricidal [5], anthelmintic [6], antimalarial [7], hepatoprotective [8], and antidepressant [9] proprieties. In addition, the herb is used to make a tea for helping pregnant women during pain of labor and in treating leukaemia and sclerosis [10]. Moreover, the organic extract of this herb revealed toxic and antifeedant effect onLeptinotarsa decemlineata [11]. It was always used as a drink in France called absinthe which caused dementia. In the 18th century, alcoholic decoctions of wormwood and other plants were used as all-purpose remedies or “cure-alls,” but it was not until the beginning of the 19th century that the wormwood-flavored alcoholic extracts and distillates were seen not only as potent medicines, but also as aperitifs, and the large scale production of absinthe began. In addition,Artemisia absinthium L. was used freshly for the plant species, as well as for the alcoholic beverage.Aromatic and medicinal plants are an easily accessible and edible source of natural antioxidants. They have the ability to protect the organism from damage caused by free radical-induced oxidative stress such as cancer and cardiovascular and neurodegenerative diseases [12, 13].Various studies have reported previously that reactive oxygen species (ROS) are highly reactive and cause damage to protein lipids, enzymes, and DNA [14]. In this context, the use of plants and herbs in food, pharmaceutical, and cosmetic industries as a source of natural antioxidant and biologically active compounds [15, 16] has attracted a great deal of scientific interest. In addition, Pietta [17] reported that the antioxidant effect of plant products is mainly attributed to phenolic compounds.In the food industry, a large spectrum of microorganism leads to food spoilage. Therefore, preservation of food material from degradation mainly by oxidation processes and microorganism activity during production, storage, and marketing is an important stage. Also, Friedman et al. [18] reported the increase of some pathogens’ resistance to synthetic antibiotics which are also uncomfortable to patients due to their adverse drug reactions.That is why the use of plants and herbs as a source of natural products has attracted the interest of many researchers. They have been screened for their potential uses as alternative remedies for the treatment of many infections, as an antioxidant, and also as a natural food preservative [19, 20].Many authors have reported composition and antioxidant and antibacterial proprieties ofA. absinthium L. essential oil and extracts. Wright [21] has reported that wormwood stimulant propriety is caused by bitter substances as artabsin (sesquiterpene lactone) and absinthin (dimer of sesquiterpene lactone) present in plant extracts. In fact, Iranian wormwood essential oil was characterized by the predominance of β-pinene and β-thujone [22]. In the same context, essential oil ofA. absinthium L. collected from Morocco is essentially represented mainly by α-thujone (39.69%), sabinyl acetate (10.96%), and β-thujone (7.25%) [23]. Furthermore, Martín et al. [24] showed that the major compounds of wormwood found in the SFE extracts as well as in the hydrodistilled essential oils were Z-epoxyocimene, chrysanthenol, and chrysanthenyl acetate.To the best of our knowledge, there are no reports dealing with chemotypeA. absinthiumL. essential oil in Tunisia. In our investigation, we have studied essential oil and methanolic extracts composition of wild population of wormwood cultivated in Tunisia and harvested from four distinct regions (Jérissa, Bou Salem, Boukornine, and Kairouan). Furthermore, we have evaluated their antioxidant, antibacterial, and antifungal activities. The purpose of this study was to underline the variability of wormwood essential oil and methanolic extracts and their biological activities as affected by the collection site.


## 2. Material and Methods

### 2.1. Chemicals

All solvents used in the experiments (diethyl ether, acetonitrile, and water of high-performance liquid chromatography (HPLC) grade and ethanol, methanol, and ethyl acetate of analytical grade) were purchased from Merck (Darmstadt, Germany). Sodium hydroxide (NaOH), sodium phosphate (Na2HPO4), sodium monobasic phosphate anhydrous (NaH2PO4H2O), sodium carbonate (Na2CO3), sodium nitrite NaNO2, butylated hydroxytoluene (BHT), β-carotene, linoleic acid, ethylenediaminetetraacetic acid (EDTA), 3-(2-pyridyl)-5,6-bis(4-phenyl-sulphonic acid)-1,2,4-triazine (ferrozine), iron(II) chloride tetrahydrate (FeCl2·4H2O), iron(II) chloride (FeCl2), iron(III) chloride (FeCl3), 1,1-diphenyl-2-picrylhydrazyl (DPPH), polyvinylpolypyrrolidone Folin-Ciocalteu reagent, potassium ferricyanide (K3Fe(CN)6), aluminium chloride (AlCl3), homologous series of C6–C17 n-alkanes, and high-purity standards of essential oil were purchased from Sigma-Aldrich (Steinheim, Germany). Hydrochloric acid (HCl) (36–38%) and potassium hydroxide (KOH) were provided by J. T. Baker (Deventer, Netherlands). Trifluoroacetic acid (TFA) (99.9%) was purchased from ROMIL Ltd. (Cambridge, UK). Cyanidin 3-O-glucoside was purchased from Extrasynthese (Genay, France). Deionized water was used to prepare all solutions, unless otherwise indicated. These solutions were wrapped in aluminium foil and stored at 4°C. All other chemicals used were of analytical grade.


### 2.2. Plant Material

A. absinthium L. aerial parts were harvested randomly at flowering stage from different Tunisian regions. The choice of these sites was dictated by the geographical differences of the species studied (Table 1). The plant material was harvested from four regions (Jérissa, Bou Salem, Boukornine, and Kairouan). The harvested aerial parts were identified by Professor Abderrazek Smaoui (Borj Cédria Biotechnology Center, Tunisia) according to the Tunisian flora and a voucher specimen was deposited at the herbarium of the Laboratory of Bioactive Substances (Biotechnology Center of Borj Cédria) under the number Aab212.13. The harvested material was freeze-dried and then ground to fine powder by an electric mill and conserved in a dessicator at room temperature (~25°C) in darkness for further uses.Table 1
Geographical and bioclimatic collection sites parameters.
Longitude
Latitude
Elevation (m)
Bioclimatic stage
Boukornine
9°47′56.41′′E
36°42′25.33′′N
70
Superior semiarid
Jérissa
8°34′52.12′′E
35°54′38.34′′N
647
Superior semiarid
Bou Salem
8°59′2.39′′E
36°38′8.57′′N
247
Subhumid
Kairouan
9°47′56.41′′E
35°37′40.86′′N
191
Superior arid
E: east, N: north.


### 2.3. Essential Oil Extraction

The leaves, stems, and flowers were cut into small pieces and subjected to hydrodistillation type Clevenger for 90 min in accordance with European Pharmacopoeia method [25]. Essential oil extractions were done in triplicate for eachA. absinthium L. collection site. Yield percentage was calculated as mg of essential oil per 100 g of plant dry matter. All experiments were done in triplicate.


### 2.4. Gas Chromatography (GC) Analysis

Analysis ofA. absinthium L. essential oil volatile compounds by gas chromatography (GC) was carried out on a Hewlett-Packard 6890 gas chromatograph (Palo Alto, CA, USA) equipped with a flame ionization detector (FID) and an electronic pressure control (EPC) injector. A polar polyethylene glycol (PEG) HP-INNOWax and a 5% diphenyl and 95% dimethylpolysiloxane apolar HP-5 capillary columns (30 m × 0.25 mm, 0.25 mm film thickness; Hewlett-Packard, CA, USA) were used. The flow of the carrier gas (N2) was 1.6 mL/min. The split ratio was 60 : 1. The analysis was performed using the following temperature program: oven temperature kept isothermally at 35°C for 10 min, increased from 35 to 205°C at the rate of 3°C/min, and kept isothermally at 205°C for 10 min. Injector and detector temperatures were held, at 250 and 300°C, respectively. The individual peaks were identified by retention times and retention indices (relative to C6–C22 n-alkanes), compared with those of known compounds. Percentage composition of essential oils was computed from GC-FID peak areas without correction factor.


### 2.5. Gas Chromatography-Mass Spectrometry (GC/MS) Analysis

Volatile compounds analysis by GC/MS was performed on a gas chromatograph HP 5890 (II) interfaced with a HP 5972 mass spectrometer (Palo Alto, CA, USA) with electron impact ionization (70 eV). A HP-5 MS capillary column (30 m × 0.25 mm, coated with 5% phenyl methyl silicone and 95% dimethylpolysiloxane, 0.25 mm film thickness; Hewlett-Packard, CA, USA) was used. The column temperature was programmed to rise from 50 to 240°C at a rate of 5°C/min. The carrier gas was helium with a flow rate of 1.2 mL/min; split ratio was 60 : 1. Scan time and mass range were 1 s and 40–300 m/z, respectively.


### 2.6. Preparation of Plant Extracts

Sample extracts were obtained by stirring 1 g of dry aerial parts (leaves, stems, and flowers) powder with 10 mL of methanol according to the method of Mau et al. [26]. Obtained extracts were kept for 24 h at +4°C, filtered through a Whatman number 4 filter paper and freed of solvent under reduced pressure at 45°C, using a rotary evaporator, and then lyophilized. The dried crude concentrated extracts were stored at −20°C, until used for analyses.


### 2.7. Total Phenolic Content

The total phenolic content was assayed using the Folin-Ciocalteu reagent, following Singleton’s method, slightly modified by Dewanto et al. [27]. An aliquot (0.125 mL) of a suitable diluted methanol sample was added to 0.5 mL of deionized water and 0.125 mL of the Folin-Ciocalteu reagent. The mixture was shaken and allowed to stand for 6 min, before adding 1.25 mL of 7% sodium carbonate (Na2CO3) solution. The solution was then adjusted with deionized water to a final volume of 3 mL and mixed thoroughly. After incubation for 90 min at 23°C, the absorbance versus prepared blank was read at 760 nm. Total phenolic contents of leaves, stems, and flower (three replicates per treatment) were expressed as milligrams of gallic acid equivalents per gram of dry weight (mg of GAE/g of DW) through the calibration curve with gallic acid. The calibration curve range was 50–400 mg/mL (R
2
=
0.99). Triplicate measurements were taken for all samples.


### 2.8. Total Condensed Tannins Content

The total tannin content was measured using the modified vanillin assay described by Sun et al. [28]. A total of 3 mL of 4% methanol vanillin solution and 1.5 mL of concentrated H2SO4 were added to 50 μL of suitably diluted sample. The mixture was kept for 15 min, and the absorbance was measured at 500 nm against methanol as a blank. The amount of total condensed tannins was expressed as milligrams of (+)-catechin equivalent per gram of dry weight (mg of CE/g of DW) through the calibration curve with catechin. Triplicate measurements were taken for all samples.


### 2.9. Total Flavonoid Content

The total flavonoid content was measured according to Dewanto et al. [27]. A total of 250 μL of the sample appropriately diluted was mixed with 75 μL of 5% NaNO2 (sodium nitrite). After 6 min, 150 μL of 10% aluminum chloride (AlCl3) and 500 μL of 1 M NaOH were added to the mixture. Finally, the mixture was adjusted to 2.5 mL with distilled water. The absorbance versus prepared blank was read at 510 nm. Total flavonoid contents of aerial parts (three replicates per treatment) were expressed as milligrams of catechin equivalents per gram of dry weight (mg of CE/g of DW) through the calibration curve with catechin. The calibration curve range was 50–188 500 mg/mL. Triplicate measurements were taken for all samples.


### 2.10. Reversed-Phase- (RP-) HPLC Evaluation of Major Antioxidant Compounds

Phenolic compounds analysis was carried out using an Agilent Technologies 1100 series liquid chromatograph (RP-HPLC) coupled with an UV-Vis multiwavelength detector. The separation was carried out on 250 × 4.6 mm, 4μm Hypersil ODS C18 reversed phase column. The mobile phase consisted of acetonitrile (solvent A) and water with 0.2% sulphuric acid (solvent B). The flow rate was kept at 0.5 mL/min. The gradient programme was as follows: 15% A/85% B 0–12 min, 40% A/60% B 12–14 min, 60% A/40% B 14–18 min, 80% A/20% B 18–20 min, 90% A/10% B 20–24 min, and 100% A 24–28 min. The injected volume was 20 μL and peaks were monitored at 280 nm. Peaks were identified by congruent retention times compared with those of authentic standards.


### 2.11. Antioxidant Activities

#### 2.11.1. DPPH Assay

The electron donation ability of the aerial parts extracts was measured by bleaching of the purple-coloured solution of 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) according to the method of Hatano [29]. One-half mL of 0.2 mM DPPH methanolic solution was added to aerial parts extracts ofA. absinthium L. (2 mL, 10–1000 μg/mL). After an incubation period of 30 min at room temperature, the absorbance was read against a blank at 517 nm. The inhibition percentage of free radical DPPH (IP%) was calculated as follows: (1)
IP
%
=
A
blank
-
A
sample
A
blank
×
100
,where A
blank is the absorbance of the control reaction and A
sample is the absorbance in the presence of plant extract. Extract concentration providing 50% inhibition (IC50) was calculated from the regression equation prepared from the concentration of the extracts and the inhibition percentage. BHT was used as a positive control.


#### 2.11.2. Reducing Power

The method of Oyaizu [30] was used to assess the reducing power of aerial parts extracts ofA. absinthium L. These extracts (1 mL) were mixed with 2.5 mL of a 0.2 M sodium phosphate buffer (pH = 6.6) and 2.5 mL of 1% potassium ferricyanide (K3Fe(CN)6) and incubated in a water bath at 50°C for 20 min. Then, 2.5 mL of 10% trichloroacetic acid was added to the mixture that was centrifuged at 650 g for 10 min. The supernatant (2.5 mL) was then mixed with 2.5 mL distilled water and 0.5 mL of 0.1% ferric chloride solution. The intensity of the blue-green color was measured at 700 nm. The extract concentration at which the absorbance was 0.5 for the reducing power (EC50) was obtained from the linear regression equation prepared from the concentrations of the extracts and the absorbance values. High absorbance indicates high reducing power. Ascorbic acid was used as a positive control.


### 2.12. Screening of Antibacterial and Antifungal Activities

Antibacterial activity was analyzed by the disc diffusion method [31] against three human pathogenic bacteria includingStaphylococcus aureus 25923, Staphylococcusmethicillin-resistant,and Listeria monocytogenes. All bacteria were grown on LB plate at 30°C for 18–24 h previous inoculation onto the nutrient agar. A loop of bacteria from the agar slant stock was cultivated in nutrient broth overnight and spread with a sterile cotton swap onto Petri dishes containing 10 mL of API suspension medium and adjusted to the 0.5 McFarland turbidity standards with a Densimat (bioMérieux). Sterile filter paper discs (6 mm in diameter) impregnated with 20 μL of plant extract (10 mg/mL) were placed on the cultured plates. After 1-2 h at 4°C, the treated Petri dishes were incubated at 37°C for 18–24 h. The solvents, acetone/water, 2 : 8, and pure methanol without extracts served as negative controls and tetracycline was used as the positive one. The antimicrobial activity was evaluated by measuring the diameter of the growth inhibition zone around the discs. Each experiment was carried out in triplicate and the mean diameter of the inhibition zone was recorded.The same agar-disc diffusion method was used for screening the antifungal activity ofA. absinthium L. aerial parts extracts. Five yeast strains (Fusarium graminearum, Fusarium culmorum, Fusarium oxysporum, Sclerotinia,andRhizoctonia solani) were first grown on Sabouraud chloramphenicol agar plate at 30°C for 18–24 h. Several colonies of similar morphology of the clinical yeast were transferred into API suspension medium and adjusted to 2 McFarland turbidity standard with a Densimat (bioMérieux). The inocula of the respective yeast were streaked on to Sabouraud chloramphenicol agar plates at 30°C using a sterile swab and then dried. A sterilized 6 mm paper disc was loaded with 20 μL (10 mg/mL) of aerial parts extract. The treated Petri dishes were placed at 4°C for 1-2 h and then incubated at 37°C for 18–24 h. The inhibition of fungal growth was also evaluated by measuring the diameter of the transparent inhibition zone around each disc. Percent inhibition (%
I) was calculated as %
I
=
[
(
C
-
T
)
/
C
]
×
100, where C is the diameter of the control colonies and T is the diameter of the test colonies. The average of three measurements was taken. The susceptibility of the standard was determined using a disc paper containing Nystatin.


### 2.13. Data Analysis

All analyses were performed in triplicate and the results are expressed as mean values ± standard deviations (SD). The data were subjected to statistical analysis using statistical program package STATISTICA [32]. The one-way analysis of variance (ANOVA) followed by Duncan multiple range test was employed and the differences between individual mean values were deemed to be significant at P
<
0.05
.In addition, a principal component analysis (PCA) was performed in order to discriminate between different region on the basis of their essential oils and phenolic composition.


## 2.1. Chemicals

All solvents used in the experiments (diethyl ether, acetonitrile, and water of high-performance liquid chromatography (HPLC) grade and ethanol, methanol, and ethyl acetate of analytical grade) were purchased from Merck (Darmstadt, Germany). Sodium hydroxide (NaOH), sodium phosphate (Na2HPO4), sodium monobasic phosphate anhydrous (NaH2PO4H2O), sodium carbonate (Na2CO3), sodium nitrite NaNO2, butylated hydroxytoluene (BHT), β-carotene, linoleic acid, ethylenediaminetetraacetic acid (EDTA), 3-(2-pyridyl)-5,6-bis(4-phenyl-sulphonic acid)-1,2,4-triazine (ferrozine), iron(II) chloride tetrahydrate (FeCl2·4H2O), iron(II) chloride (FeCl2), iron(III) chloride (FeCl3), 1,1-diphenyl-2-picrylhydrazyl (DPPH), polyvinylpolypyrrolidone Folin-Ciocalteu reagent, potassium ferricyanide (K3Fe(CN)6), aluminium chloride (AlCl3), homologous series of C6–C17 n-alkanes, and high-purity standards of essential oil were purchased from Sigma-Aldrich (Steinheim, Germany). Hydrochloric acid (HCl) (36–38%) and potassium hydroxide (KOH) were provided by J. T. Baker (Deventer, Netherlands). Trifluoroacetic acid (TFA) (99.9%) was purchased from ROMIL Ltd. (Cambridge, UK). Cyanidin 3-O-glucoside was purchased from Extrasynthese (Genay, France). Deionized water was used to prepare all solutions, unless otherwise indicated. These solutions were wrapped in aluminium foil and stored at 4°C. All other chemicals used were of analytical grade.


## 2.2. Plant Material

A. absinthium L. aerial parts were harvested randomly at flowering stage from different Tunisian regions. The choice of these sites was dictated by the geographical differences of the species studied (Table 1). The plant material was harvested from four regions (Jérissa, Bou Salem, Boukornine, and Kairouan). The harvested aerial parts were identified by Professor Abderrazek Smaoui (Borj Cédria Biotechnology Center, Tunisia) according to the Tunisian flora and a voucher specimen was deposited at the herbarium of the Laboratory of Bioactive Substances (Biotechnology Center of Borj Cédria) under the number Aab212.13. The harvested material was freeze-dried and then ground to fine powder by an electric mill and conserved in a dessicator at room temperature (~25°C) in darkness for further uses.Table 1
Geographical and bioclimatic collection sites parameters.
Longitude
Latitude
Elevation (m)
Bioclimatic stage
Boukornine
9°47′56.41′′E
36°42′25.33′′N
70
Superior semiarid
Jérissa
8°34′52.12′′E
35°54′38.34′′N
647
Superior semiarid
Bou Salem
8°59′2.39′′E
36°38′8.57′′N
247
Subhumid
Kairouan
9°47′56.41′′E
35°37′40.86′′N
191
Superior arid
E: east, N: north.


## 2.3. Essential Oil Extraction

The leaves, stems, and flowers were cut into small pieces and subjected to hydrodistillation type Clevenger for 90 min in accordance with European Pharmacopoeia method [25]. Essential oil extractions were done in triplicate for eachA. absinthium L. collection site. Yield percentage was calculated as mg of essential oil per 100 g of plant dry matter. All experiments were done in triplicate.


## 2.4. Gas Chromatography (GC) Analysis

Analysis ofA. absinthium L. essential oil volatile compounds by gas chromatography (GC) was carried out on a Hewlett-Packard 6890 gas chromatograph (Palo Alto, CA, USA) equipped with a flame ionization detector (FID) and an electronic pressure control (EPC) injector. A polar polyethylene glycol (PEG) HP-INNOWax and a 5% diphenyl and 95% dimethylpolysiloxane apolar HP-5 capillary columns (30 m × 0.25 mm, 0.25 mm film thickness; Hewlett-Packard, CA, USA) were used. The flow of the carrier gas (N2) was 1.6 mL/min. The split ratio was 60 : 1. The analysis was performed using the following temperature program: oven temperature kept isothermally at 35°C for 10 min, increased from 35 to 205°C at the rate of 3°C/min, and kept isothermally at 205°C for 10 min. Injector and detector temperatures were held, at 250 and 300°C, respectively. The individual peaks were identified by retention times and retention indices (relative to C6–C22 n-alkanes), compared with those of known compounds. Percentage composition of essential oils was computed from GC-FID peak areas without correction factor.


## 2.5. Gas Chromatography-Mass Spectrometry (GC/MS) Analysis

Volatile compounds analysis by GC/MS was performed on a gas chromatograph HP 5890 (II) interfaced with a HP 5972 mass spectrometer (Palo Alto, CA, USA) with electron impact ionization (70 eV). A HP-5 MS capillary column (30 m × 0.25 mm, coated with 5% phenyl methyl silicone and 95% dimethylpolysiloxane, 0.25 mm film thickness; Hewlett-Packard, CA, USA) was used. The column temperature was programmed to rise from 50 to 240°C at a rate of 5°C/min. The carrier gas was helium with a flow rate of 1.2 mL/min; split ratio was 60 : 1. Scan time and mass range were 1 s and 40–300 m/z, respectively.


## 2.6. Preparation of Plant Extracts

Sample extracts were obtained by stirring 1 g of dry aerial parts (leaves, stems, and flowers) powder with 10 mL of methanol according to the method of Mau et al. [26]. Obtained extracts were kept for 24 h at +4°C, filtered through a Whatman number 4 filter paper and freed of solvent under reduced pressure at 45°C, using a rotary evaporator, and then lyophilized. The dried crude concentrated extracts were stored at −20°C, until used for analyses.


## 2.7. Total Phenolic Content

The total phenolic content was assayed using the Folin-Ciocalteu reagent, following Singleton’s method, slightly modified by Dewanto et al. [27]. An aliquot (0.125 mL) of a suitable diluted methanol sample was added to 0.5 mL of deionized water and 0.125 mL of the Folin-Ciocalteu reagent. The mixture was shaken and allowed to stand for 6 min, before adding 1.25 mL of 7% sodium carbonate (Na2CO3) solution. The solution was then adjusted with deionized water to a final volume of 3 mL and mixed thoroughly. After incubation for 90 min at 23°C, the absorbance versus prepared blank was read at 760 nm. Total phenolic contents of leaves, stems, and flower (three replicates per treatment) were expressed as milligrams of gallic acid equivalents per gram of dry weight (mg of GAE/g of DW) through the calibration curve with gallic acid. The calibration curve range was 50–400 mg/mL (R
2
=
0.99). Triplicate measurements were taken for all samples.


## 2.8. Total Condensed Tannins Content

The total tannin content was measured using the modified vanillin assay described by Sun et al. [28]. A total of 3 mL of 4% methanol vanillin solution and 1.5 mL of concentrated H2SO4 were added to 50 μL of suitably diluted sample. The mixture was kept for 15 min, and the absorbance was measured at 500 nm against methanol as a blank. The amount of total condensed tannins was expressed as milligrams of (+)-catechin equivalent per gram of dry weight (mg of CE/g of DW) through the calibration curve with catechin. Triplicate measurements were taken for all samples.


## 2.9. Total Flavonoid Content

The total flavonoid content was measured according to Dewanto et al. [27]. A total of 250 μL of the sample appropriately diluted was mixed with 75 μL of 5% NaNO2 (sodium nitrite). After 6 min, 150 μL of 10% aluminum chloride (AlCl3) and 500 μL of 1 M NaOH were added to the mixture. Finally, the mixture was adjusted to 2.5 mL with distilled water. The absorbance versus prepared blank was read at 510 nm. Total flavonoid contents of aerial parts (three replicates per treatment) were expressed as milligrams of catechin equivalents per gram of dry weight (mg of CE/g of DW) through the calibration curve with catechin. The calibration curve range was 50–188 500 mg/mL. Triplicate measurements were taken for all samples.


## 2.10. Reversed-Phase- (RP-) HPLC Evaluation of Major Antioxidant Compounds

Phenolic compounds analysis was carried out using an Agilent Technologies 1100 series liquid chromatograph (RP-HPLC) coupled with an UV-Vis multiwavelength detector. The separation was carried out on 250 × 4.6 mm, 4μm Hypersil ODS C18 reversed phase column. The mobile phase consisted of acetonitrile (solvent A) and water with 0.2% sulphuric acid (solvent B). The flow rate was kept at 0.5 mL/min. The gradient programme was as follows: 15% A/85% B 0–12 min, 40% A/60% B 12–14 min, 60% A/40% B 14–18 min, 80% A/20% B 18–20 min, 90% A/10% B 20–24 min, and 100% A 24–28 min. The injected volume was 20 μL and peaks were monitored at 280 nm. Peaks were identified by congruent retention times compared with those of authentic standards.


## 2.11. Antioxidant Activities

### 2.11.1. DPPH Assay

The electron donation ability of the aerial parts extracts was measured by bleaching of the purple-coloured solution of 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) according to the method of Hatano [29]. One-half mL of 0.2 mM DPPH methanolic solution was added to aerial parts extracts ofA. absinthium L. (2 mL, 10–1000 μg/mL). After an incubation period of 30 min at room temperature, the absorbance was read against a blank at 517 nm. The inhibition percentage of free radical DPPH (IP%) was calculated as follows: (1)
IP
%
=
A
blank
-
A
sample
A
blank
×
100
,where A
blank is the absorbance of the control reaction and A
sample is the absorbance in the presence of plant extract. Extract concentration providing 50% inhibition (IC50) was calculated from the regression equation prepared from the concentration of the extracts and the inhibition percentage. BHT was used as a positive control.


### 2.11.2. Reducing Power

The method of Oyaizu [30] was used to assess the reducing power of aerial parts extracts ofA. absinthium L. These extracts (1 mL) were mixed with 2.5 mL of a 0.2 M sodium phosphate buffer (pH = 6.6) and 2.5 mL of 1% potassium ferricyanide (K3Fe(CN)6) and incubated in a water bath at 50°C for 20 min. Then, 2.5 mL of 10% trichloroacetic acid was added to the mixture that was centrifuged at 650 g for 10 min. The supernatant (2.5 mL) was then mixed with 2.5 mL distilled water and 0.5 mL of 0.1% ferric chloride solution. The intensity of the blue-green color was measured at 700 nm. The extract concentration at which the absorbance was 0.5 for the reducing power (EC50) was obtained from the linear regression equation prepared from the concentrations of the extracts and the absorbance values. High absorbance indicates high reducing power. Ascorbic acid was used as a positive control.


## 2.11.1. DPPH Assay

The electron donation ability of the aerial parts extracts was measured by bleaching of the purple-coloured solution of 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) according to the method of Hatano [29]. One-half mL of 0.2 mM DPPH methanolic solution was added to aerial parts extracts ofA. absinthium L. (2 mL, 10–1000 μg/mL). After an incubation period of 30 min at room temperature, the absorbance was read against a blank at 517 nm. The inhibition percentage of free radical DPPH (IP%) was calculated as follows: (1)
IP
%
=
A
blank
-
A
sample
A
blank
×
100
,where A
blank is the absorbance of the control reaction and A
sample is the absorbance in the presence of plant extract. Extract concentration providing 50% inhibition (IC50) was calculated from the regression equation prepared from the concentration of the extracts and the inhibition percentage. BHT was used as a positive control.


## 2.11.2. Reducing Power

The method of Oyaizu [30] was used to assess the reducing power of aerial parts extracts ofA. absinthium L. These extracts (1 mL) were mixed with 2.5 mL of a 0.2 M sodium phosphate buffer (pH = 6.6) and 2.5 mL of 1% potassium ferricyanide (K3Fe(CN)6) and incubated in a water bath at 50°C for 20 min. Then, 2.5 mL of 10% trichloroacetic acid was added to the mixture that was centrifuged at 650 g for 10 min. The supernatant (2.5 mL) was then mixed with 2.5 mL distilled water and 0.5 mL of 0.1% ferric chloride solution. The intensity of the blue-green color was measured at 700 nm. The extract concentration at which the absorbance was 0.5 for the reducing power (EC50) was obtained from the linear regression equation prepared from the concentrations of the extracts and the absorbance values. High absorbance indicates high reducing power. Ascorbic acid was used as a positive control.


## 2.12. Screening of Antibacterial and Antifungal Activities

Antibacterial activity was analyzed by the disc diffusion method [31] against three human pathogenic bacteria includingStaphylococcus aureus 25923, Staphylococcusmethicillin-resistant,and Listeria monocytogenes. All bacteria were grown on LB plate at 30°C for 18–24 h previous inoculation onto the nutrient agar. A loop of bacteria from the agar slant stock was cultivated in nutrient broth overnight and spread with a sterile cotton swap onto Petri dishes containing 10 mL of API suspension medium and adjusted to the 0.5 McFarland turbidity standards with a Densimat (bioMérieux). Sterile filter paper discs (6 mm in diameter) impregnated with 20 μL of plant extract (10 mg/mL) were placed on the cultured plates. After 1-2 h at 4°C, the treated Petri dishes were incubated at 37°C for 18–24 h. The solvents, acetone/water, 2 : 8, and pure methanol without extracts served as negative controls and tetracycline was used as the positive one. The antimicrobial activity was evaluated by measuring the diameter of the growth inhibition zone around the discs. Each experiment was carried out in triplicate and the mean diameter of the inhibition zone was recorded.The same agar-disc diffusion method was used for screening the antifungal activity ofA. absinthium L. aerial parts extracts. Five yeast strains (Fusarium graminearum, Fusarium culmorum, Fusarium oxysporum, Sclerotinia,andRhizoctonia solani) were first grown on Sabouraud chloramphenicol agar plate at 30°C for 18–24 h. Several colonies of similar morphology of the clinical yeast were transferred into API suspension medium and adjusted to 2 McFarland turbidity standard with a Densimat (bioMérieux). The inocula of the respective yeast were streaked on to Sabouraud chloramphenicol agar plates at 30°C using a sterile swab and then dried. A sterilized 6 mm paper disc was loaded with 20 μL (10 mg/mL) of aerial parts extract. The treated Petri dishes were placed at 4°C for 1-2 h and then incubated at 37°C for 18–24 h. The inhibition of fungal growth was also evaluated by measuring the diameter of the transparent inhibition zone around each disc. Percent inhibition (%
I) was calculated as %
I
=
[
(
C
-
T
)
/
C
]
×
100, where C is the diameter of the control colonies and T is the diameter of the test colonies. The average of three measurements was taken. The susceptibility of the standard was determined using a disc paper containing Nystatin.


## 2.13. Data Analysis

All analyses were performed in triplicate and the results are expressed as mean values ± standard deviations (SD). The data were subjected to statistical analysis using statistical program package STATISTICA [32]. The one-way analysis of variance (ANOVA) followed by Duncan multiple range test was employed and the differences between individual mean values were deemed to be significant at P
<
0.05
.In addition, a principal component analysis (PCA) was performed in order to discriminate between different region on the basis of their essential oils and phenolic composition.


## 3. Results and Discussion

### 3.1. Essential Oil Yield

The hydrodistillation extraction of 100 g of wormwood dry aerial parts collected from four different regions yielded a dark blue essential oil. These yields were illustrated in Figure1. The results show that EO yield varies significantly (P
<
0.05) according to region factor. Thereby, the optimal yield was observed at the region of Bou Salem (1.46
±
0.06%) followed by the region of Kairouan (1.12
±
0.08%) and Boukornine and Jérissa regions (1.10
±
0.04% and 1.00
±
0.03%, resp.). Similar results were obtained by Orav et al. [33] at different European regions and the oil yields were ranged from 0.1 to 1.1% w/w. In addition, the EO yields detected in the present study were higher than those determined by Lopes-Lutz et al. [34] in the region of the central prairies of Alberta (Canada West) that they found (0.5%, w/w), which justifies the effect of region on the EO yield. The impact of other environmental factors on EO yield was also described by Msaada et al. [35] and Msaada et al. [36, 37]. In addition, seasonal and regional variations were observed in the EO yield of coriander [38]. The same authors also reported the effect of extraction techniques on the essential oils yield and composition [39].Figure 1
Essential oil yield of the aerial parts ofA. absinthium L. collected from four Tunisian regions. Values with different superscripts (a–c) are significantly different at P
<
0.05.


### 3.2. Variability of Chemical Composition of EO

The hydrodistillation of wormwood aerial parts collected from four different Tunisian regions provides an EO with blue-black color. This latter was due to the predominance of chamazulene component which gives the oil with a high organoleptic quality [33]. The results of chromatographic analysis by gas chromatography (GC/FID) and by coupling gas chromatography to mass spectrometry (GC/MS) are presented in Table 2.Table 2
ANOVA analysis, qualitative and quantitative essential oil composition (peak area % ± SD) ofA. absinthium L. aerial parts.
Compound*
RRIa
RRIb
Collection region
df
F
P
Jérissa
Boukornine
Kairouan
Bou Salem
Tricyclene
1014
927
1.00  ±  0.11a
0.02  ±  0.00d
0.74  ±  0.06b
0.23  ±  0.03c
3
9.6733
0.004***
α-Thujene
1032
939
0.05  ±  0.01c
0.05  ±  0.00c
3.28  ±  0.32a
0.23  ±  0.02b
3
10077.77
0.000***
α-Pinene
1035
931
0.10  ±  0.01c
0.02  ±  0.00b
0.62  ±  0.05a
0.20  ±  0.03b
3
65.81
0.000***
Camphene
1076
950
0.06  ±  0.01c
0.03  ±  0.00d
0.66  ±  0.07a
0.24  ±  0.03b
3
163.01
0.000***
β-Pinene
1118
980
1.20  ±  0.10a
0.25  ±  0.02c
0.02  ±  0.00d
0.27  ±  0.03b
3
251.96
0.000***
Sabinene
1132
976
0.28  ±  0.03c
1.87  ±  0.19a
0.02  ±  0.00d
1.67  ±  0.15b
3
3383.83
0.000***
Myrcene
1176
988
0.50  ±  0.04b
0.05  ±  0.01c
1.52  ±  0.14a
0.02  ±  0.00d
3
555.19
0.000***
α-Terpinene
1188
1018
0.03  ±  0.00c
0.44  ±  0.05a
0.08  ±  0.01b
0.03  ±  0.00c
3
247.78
0.000***
Limonene
1203
1026
0.10  ±  0.01d
0.95  ±  0.08a
0.14  ±  0.01c
0.40  ±  0.03b
3
39.49
0.000***
1,8-Cineole
1213
1033
0.03  ±  0.00c
0.03  ±  0.00c
0.35  ±  0.04a
0.08  ±  0.01b
3
83.14
0.000***
β-Phellandrene
1218
1031
0.10  ±  0.02b
0.04  ±  0.00d
0.06  ±  0.01c
0.26  ±  0.03a
3
25.44
0.000***
Z-β-Ocimene
1246
1040
0.20  ±  0.03c
1.59  ±  0.16a
0.26  ±  0.03b
0.05  ±  0.00d
3
538.84
0.000***
E-β-Ocimene
1266
1050
0.08  ±  0.01d
0.10  ±  0.02c
1.40  ±  0.16a
0.43  ±  0.05b
3
105.86
0.000***
γ-Terpinene
1267
1062
0.10  ±  0.02b
0.07  ±  0.01c
0.18  ±  0.02a
0.04  ±  0.00d
3
27.18
0.000***
p-Cymene
1280
1026
0.08  ±  0.01c
0.12  ±  0.01b
0.22  ±  0.03a
0.04  ±  0.00d
3
44.75
0.000***
Terpinolene
1290
1088
0.20  ±  0.03b
0.13  ±  0.01c
0.12  ±  0.01cd
0.62  ±  0.05a
3
57.94
0.000***
β-Thujone
1430
1089
17.16  ±  1.56c
21.02  ±  2.32b
22.09  ±  2.12a
6.72  ±  0.75d
3
590069.9
0.000***
α-Thujone
1430
1089
0.20  ±  0.02b
0.07  ±  0.01c
0.28  ±  0.03a
0.04  ±  0.00d
3
13.52
0.001***
trans-Linalool oxide
1450
1088
0.03  ±  0.00c
0.02  ±  0.00d
6.04  ±  0.77a
2.58  ±  0.25b
3
11876.62
0.000***
trans-Sabinene hydrate
1474
1053
12.07  ±  1.32c
12.58  ±  1.30b
5.32  ±  0.56d
21.78  ±  2.12a
3
242741.2
0.000***
cis-Linalool oxide
1478
1074
0.05  ±  0.00c
0.13  ±  0.01b
0.05  ±  0.01c
1.86  ±  0.16a
3
6371.93
0.000***
α-Copaene
1497
1379
0.50  ±  0.04b
4.07  ±  0.44a
0.33  ±  0.03c
0.22  ±  0.03d
3
3561.91
0.000***
Camphor
1532
1143
0.02  ±  0.00d
0.04  ±  0.00c
0.15  ±  0.01a
0.12  ±  0.01b
3
13.73
0.001***
Linalool
1553
1088
2.00  ±  0.23a
0.80  ±  0.07b
0.09  ±  0.01d
0.15  ±  0.02c
3
9.32
0.005**
cis-Sabinene hydrate
1556
1082
0.02  ±  0.00d
1.82  ±  0.15a
1.80  ±  0.20b
0.80  ±  0.07c
3
441.83
0.000***
Linalyl acetate
1565
1239
0.50  ±  0.06b
4.58  ±  0.51a
0.08  ±  0.01d
0.21  ±  0.03c
3
5308.55
0.000***
Bornyl acetate
1590
1270
1.10  ±  0.10a
0.04  ±  0.00c
0.36  ±  0.04b
0.02  ±  0.00d
3
268.07
0.000***
Terpinene-4-ol
1611
1176
3.00  ±  0.41a
0.15  ±  0.01c
0.05  ±  0.00d
1.10  ±  0.12b
3
22.16
0.000***
cis-Dihydrocarvone
1645
1193
0.48  ±  0.05b
0.94  ±  0.08a
0.11  ±  0.01c
0.03  ±  0.00d
3
942.9
0.000***
cis-Verbenol
1654
1132
2.34  ±  0.24b
0.02  ±  0.00d
0.31  ±  0.04c
3.57  ±  0.44a
3
15604.73
0.000***
Sabinyl acetate
1658
1291
0.29  ±  0.03b
5.01  ±  0.45a
0.11  ±  0.01c
0.08  ±  0.01d
3
100958.1
0.000***
trans-Sabinol
1666
1120
0.29  ±  0.03b
0.04  ±  0.00d
0.06  ±  0.01c
0.99  ±  0.08a
3
1582.13
0.000***
Lavandulol
1677
1150
2.71  ±  0.31c
0.28  ±  0.03d
3.61  ±  0.34b
5.54  ±  0.61a
3
26002.36
0.000***
α-Humulene
1687
1454
0.08  ±  0.01b
0.62  ±  0.05a
0.03  ±  0.00c
0.01  ±  0.00d
3
1699.33
0.000***
Neral
1694
1240
0.01  ±  0.00d
0.26  ±  0.03c
2.68  ±  0.27a
0.28  ±  0.03b
3
11106.53
0.000***
γ-Muurolene
1692
1474
0.28  ±  0.03b
1.09  ±  0.11a
0.15  ±  0.02c
0.05  ±  0.00d
3
871.32
0.000***
γ-Curcumene
1704
1475
1.00  ±  0.12c
2.93  ±  0.32a
0.57  ±  0.06d
1.95  ±  0.22b
3
52.19
0.000***
α-Terpinyl acetate
1705
1344
0.17  ±  0.01a
tr
0.14  ±  0.01b
0.03  ±  0.00c
3
91.11
0.000***
α-Terpineol
1706
1189
0.19  ±  0.02b
0.14  ±  0.01c
0.05  ±  0.01d
0.38  ±  0.04a
3
93.12
0.000***
Borneol
1719
1165
0.52  ±  0.06a
0.46  ±  0.05b
0.23  ±  0.03c
tr
3
397.35
0.000***
Germacrene D
1726
1480
0.04  ±  0.00d
0.10  ±  0.01c
1.58  ±  0.17a
0.52  ±  0.06b
3
1647.56
0.000***
Neryl acetate
1732
1356
0.20  ±  0.02c
0.26  ±  0.03b
0.03  ±  0.00d
0.51  ±  0.06a
3
42.73
0.000***
β-Selinene
1742
1486
4.72  ±  0.51c
4.50  ±  0.50d
5.29  ±  0.62b
6.67  ±  0.74a
3
45.48
0.000***
α-Bisabolene
1746
1494
0.88  ±  0.09b
0.11  ±  0.01c
1.25  ±  0.15a
0.02  ±  0.00d
3
15.32
0.001***
Carvone
1751
1242
2.00  ±  0.31a
0.14  ±  0.01c
0.46  ±  0.05b
0.05  ±  0.00d
3
9.88
0.004**
Geranyl acetate
1765
1383
0.08  ±  0.01b
0.61  ±  0.07a
0.03  ±  0.00c
0.02  ±  0.00d
3
2429
0.000***
γ-Cadinene
1776
1526
0.40  ±  0.05c
1.12  ±  0.15b
1.27  ±  0.13a
0.05  ±  0.00d
3
98.38
0.000***
Nerol
1797
1228
0.10  ±  0.01c
0.11  ±  0.01c
0.33  ±  0.04b
1.93  ±  0.31a
3
2118.79
0.000***
Geraniol
1857
1255
0.65  ±  0.07a
0.03  ±  0.00d
0.04  ±  0.00c
0.20  ±  0.02b
3
77.96
0.000***
p-Cymene-8-ol
1864
1183
tr
0.96  ±  0.08b
3.70  ±  0.41a
tr
3
3636.71
0.000***
α-Calacorene
1920
1527
0.11  ±  0.01d
0.16  ±  0.02b
0.12  ±  0.01c
0.52  ±  0.06a
3
355.3
0.000***
Caryophyllene oxide
2008
1578
0.05  ±  0.00b
0.06  ±  0.01a
0.06  ±  0.00a
tr
3
16.5
0.000***
cis-Nerolidol
2050
1553
0.27  ±  0.03a
0.06  ±  0.01c
0.07  ±  0.01b
0.01  ±  0.00d
3
92.88
0.000***
Eugenol
2192
1356
0.15  ±  0.02d
1.24  ±  0.14a
0.32  ±  0.04b
0.20  ±  0.03c
3
218.06
0.000***
α-Bisabolol
2232
1673
0.03  ±  0.00b
0.02  ±  0.00c
0.15  ±  0.02a
0.01  ±  0.00d
3
19.07
0.000***
Chamazulene
2430
1719
39.93  ±  4.56a
25.30  ±  3.12d
29.51  ±  3.26c
34.81  ±  4.11b
3
5324.71
0.000***
Chemical classes
Monoterpene hydrocarbons
56.02 ± 6.21b
45.31 ± 5.31d
45.73  ±  5.22c
62.08  ±  7.49a
3
26322.56
0.000***
Aromatic hydrocarbons
0.08 ± 0.01c
0.12 ± 0.01b
0.22  ±  0.03a
0.04  ±  0.00d
3
9563.42
0.000***
Monoterpene alcohols
12.22 ± 1.33b
4.29 ± 0.52d
8.86  ±  0.96c
14.07  ±  1.67a
3
912.67
0.000***
Monoterpene esters
2.34 ± 0.31b
10.05 ± 1.23a
0.75  ±  0.08d
0.87  ±  0.09c
3
567.01
0.000***
Monoterpene ketones
19.86 ± 2.13c
22.21 ± 2.11b
23.09  ±  3.10a
6.96  ±  7.41d
3
152.73
0.000***
Monoterpene aldehydes
0.01 ± 0.00d
0.26 ± 0.03c
2.68  ±  2.45a
0.28  ±  0.03b
3
4360.02
0.000***
Monoterpene ethers
0.11 ± 0.01d
0.18 ± 0.02c
6.44  ±  0.77a
4.52  ±  0.51b
3
4265.16
0.000***
Sesquiterpenes
8.09 ± 0.99d
14.78 ± 1.66a
10.80  ±  1.45b
10.02  ±  1.78c
3
1235.94
0.000***
Total identified
98.72 ± 10.23b
97.64 ± 9.78d
98.55  ±  9.88c
98.83  ±  9.76a
3
4581.23
0.000***
RRI: relative retention index; a: HP-INNOWax, b: HP-5.*Compounds in order of elution on HP-INNOWax; values of volatile essential oil percentages are the average three determinations (n = 3). These values with different letters (a–d) are significantly different at P < 0.05. nd: not detected. NS: not significant. **
P < 0.01. ***
P < 0.001.
df: degree of freedom,F: Fisher value, and P: probability.Essential oils analyzed are divided into eight classes based on their chemical functional groups. A total of 56 compounds were identified representing98.72
±
10.23%, 97.64
%
±
9.78, 98.55
±
9.88%, and 98.83
±
9.76% of total volatiles in the regions of Jérissa, Boukornine, Kairouan, and Bou Salem, respectively. These different identified compounds vary significantly (P
<
0.05) from region to another and are highly (P
<
0.001) affected by the regional factor (Table 2).The major contribution was attributed to the monoterpene hydrocarbons fraction which represents 56.02% ±6.21,45.31
±
5.31%, 45.73
±
5.22%, and 62.08
±
7.49% of all compounds detected in Jérissa, Boukornine, Kairouan, and Bou Salem regions, respectively. The latter fraction is dominated by chamazulene where its maximum rate (39.93
±
4.56%) was observed in the Jérissa region, followed by the regions of Bou Salem (34.81
±
4.11%), Kairouan (29.51
±
3.26%), and Boukornine (25.30
±
3.12%). These results were in agreement with the literature. However, Kordali et al. [40] showed that essential oils chemical composition of wormwood collected in Turkey was dominated by chamazulene (17.8%) followed by nuciferol butanoate (8.2%), nuciferol propionate (5.1%), and caryophyllene oxide (4.3%). In this work, β-thujone, a monoterpene ketone, was detected with significant percentages: 22.09
±
2.12% in the region of Kairouan, up to 6.72
±
0.75% in the region of Bou Salem. Meanwhile, the chemical composition of essential oils of wormwood growing in the USA shows that the β-thujone (17.5 to 42.3%) andcis-sabinyl acetate (15.1 to 53.4%) are the major compounds [41]. In agreement with our results, previous studies have shown that the rate of thujone in essential oils of wormwood is greater than 35% [42]. In addition, in the wormwood samples collected from different European countries (Greece, Spain, Ukraine, France, and Italy), α-thujone and β-thujone were detected as major components with percentages ranging from 4.5 to 38.7% [33].It should be noted that the sesquiterpene fraction which is represented byα-humulene, the γ-muurolene, the γ-curcumene, the α-copaene, the β-selinene, the α-bisabolene, the γ-cadinene, the germacrene D, the α-calacorene, caryophyllene oxide, and α-bisabolol ranges from 8 to 14% of total volatiles. This slight variation could be explained by similar environmental and genetic factors. An earlier study showed that the rate of sesquiterpenes detected was 2.2% [34], rate lower than that found in this work. Monoterpene alcohols (linalool, lavandulol, terpinen-4-ol, α-terpineol, nerol, sabinoltrans-p-cymene-8-ol, eugenol, borneol, and geraniol) were highly affected by the region factor (Table 2).Principal component analysis was carried out in order to determine the relationship between the different regions of plant collection on the basis of their essential oil composition. A better discrimination was revealed on the three-dimensional visualization of the plotted scores. Results obtained from the PCA (Figure2) showed the existence of one well-defined group represented by the regions Jérissa, Boukornine, and Kairouan suggesting similar compositions. The Bou Salem region was clearly distinguished from the latter group both in quality and in quantity.Figure 2
Relative position of the regions of plant collection based on their essential oils composition (Table2) in the space defined by the three principal components.In conclusion, theArtemisia absinthiumL. plants collected from four Tunisian habitats biosynthesized essential oils of one chemotype, chamazulene.


### 3.3. Total Polyphenols, Flavonoids, and Condensed Tannins Contents

Total phenol assay is used as a routing assay to assess phenolic antioxidants due to its simplicity and reproducibility. Phenolic compounds have the ability to reduce Folin-Ciocalteu reagent and the reaction may be followed by measuring a change in the color of the solution from intense yellow to blue spectrophotometrically. Quantitative and qualitative changes of the antioxidant compounds with function to region effect hold great significance from both dietary and nutritional points of view, while phenolic compounds are used as alternatives to synthetic drugs and were shown to be safe for use in processed foods [43].Phenolic quantification revealed a wide range of aerial parts’ polyphenol contents as function of the region as shown in Figure3. The total polyphenol contents varied significantly (P
≤
0.05) from one region to another and reached the maximum in the region of Kairouan (99.89
±
3.30 mg GAE/g DW), followed by the region of Bou Salem, Boukornine, and Jérissa where their levels were 83.70
±
1.31, 72.05
±
1.83, and 49.39
±
2.20 mg GAE/g DM, respectively. Monica et al. [44] showed that total polyphenol content of wormwood aerial parts collected in Romania was 98.20 mg/g DW. In addition, Ebrahimzadeh et al. [45] reported that the total polyphenol content of Iranian wormwood aerial parts was 194
±
9.7 mg EAG/g of extract which was significantly of greater value in comparison with our results. However, a lower content in total polyphenols (9.79 mg EAG/g extract) content was determined by Sengul et al. [46]. Other herbs with high levels of phenolics werePolygonum aviculare (11.2 mg GAE/100 g DW) andValeriana officinalis (11.1 mg GAE/100 g DW) [47]. In general, phenolic compounds are known for their role in the prevention of some diseases due to their richness in antioxidants and their antiradical effect, protecting cellular components against damage induced by free radicals. Due to the diversity of their chemical structures, they are likely to have very different antioxidant capacity [48]. Polyphenolic compounds are commonly found in both edible and inedible plants, and they have been reported to have multiple biological effects, including antioxidant activity [47]. The basic flavonoids structure is the flavan nucleus, which consists of 15 carbon atoms arranged in three rings (C6–C3–C6), labeled A, B, and C. Various classes of flavonoid differ in the level of oxidation and saturation of ring C, while individual compounds within a class differ in the substitution pattern of rings A and B. The differences in the structure and substitution will influence the phenoxyl radical stability and thereby the antioxidant properties of the flavonoids [49].Figure 3
Total polyphenols (TPP), total flavonoids (TF), and total condensed tannin (TCT) contents of different regions ofA. absinthium L. aerial parts. GAE: gallic acid equivalents; CE: catechin equivalents.The letters (a–d) indicate significant differences(P
<
0.05).As for total polyphenols, total flavonoids differ significantly(
P
≤
0.05
) depending on the region, but with a maximum in Kairouan region (126.40
±
2.32 mg CE/g DW) (Figure 3).Our results are in agreement with those reported by Wojdyło et al. [47] in that the most common flavonoids are mainly distributed in Compositae family. On the other hand,A. absinthiumL. aerial parts have been reported to contain flavonoids [50–52], thymol, and carvacrol as well as other phenolic compounds [53]. These pharmacophores have been shown to possess potent antioxidant and free radical scavenging activity [54]. In the present study, high levels of phenolic and flavonoid contents were estimated. For total condensed tannins, the maximum level was detected in the region Boukornine (152.35
±
4.59 mg CE/g DW) followed by Jérissa (83.50
±
6.69 mg CE/g DW), Bou Salem (73.94
±
2.19 mg CE/g DW), and Kairouan (37.19
±
1.16 mg CE/g DW) regions (Figure 3).


### 3.4. Individual Phenolic Compounds

For a clear understanding of the regional effect on metabolic changes especially on phenolics, it is necessary to investigate the profiles of individual phenolic compounds inA. absinthium L. Hence, changes of these phenolic compounds were different at four Tunisian regions (Table 3). The obtained results show that all the identified compounds were highly significant (P
<
0.001) by the region effect. Phenolic acids—tannic, gallic, hemihydrate chlorogenic, caffeic, vanillic, syringic, ferulic,p-coumaric, rosmarinic, andtrans-cinnamic acids—represent the major fraction of the polyphenols analyzed in regions of Bou Salem (34.96
±
3.56%), Kairouan (45.57
±
5.23%), and Jérissa (58.33
±
6.21%), while the flavones dominated at Boukornine region (38.61
±
4.13%). In the regions of Bou Salem, Kairouan, and Boukornine, the flavone was the predominant fraction (19.44
±
2.36%, 11.88
±
2.11%, and 32.47
±
4.23%, resp.), while, in the region of Jérissa, tannic acid was the predominant compound (37.92
±
4.02%). The quantification of total polyphenols of wormwood aerial parts by the Folin-Ciocalteu method gave different results in all studied regions. In the region of Bou Salem, total polyphenols measured by Folin-Ciocalteu method were estimated to be 83.7 mg GAE/g DW and 28.03
±
3.12 mg/g GAE DW by HPLC, in Kairouan were 99; 89 mg GAE/g DW by Folin-Ciocalteu and 15.91
±
1.65 mg GAE/g by HPLC, in Boukornine were 72.05 mg GAE/g DW by Folin-Ciocalteu and 48.23
±
5.21 mg GAE/g DM by HPLC, and in Jérissa were 49.39 mg GAE/g DW by Folin-Ciocalteu and 88.08
±
7.59 mg GAE/g DW by HPLC. These variations in the quantification showed a significant difference depending on the method used. An earlier study showed that the analysis by liquid chromatography (HPLC) revealed the presence of other flavonoid glycosides (isoquercitrin, quercetin-3-O-β-D-glucoside, quercetin-3-O-rhamnoglucoside, isorhamnetin-3-O-rhamnoglucoside, and isorhamnetin-3-glucoside) [50], which are characterized by important antioxidant activity. Gallic acid, widely used as an additive to prevent food spoilage, is renowned for its anticarcinogenic, anti-inflammatory, and antimutagenic activities [55].Table 3
Analysis of variance (ANOVA) and phenolic composition in % (w/w) and quantity (µg/g DW) of methanol extracts of the aerial parts of wormwood (A. absinthium L.).
Compound
Collection region
df
F
P
Bou Salem
Kairouan
Jérissa
Boukornine
µg/g DW
%
µg/g DW
%
µg/g DW
%
µg/g DW
%
Phenolic acids
Tannic acid
0.37  ±  0.04D
1.32  ±  0.11d
0.73  ±  0.08C
4.59  ±  0.51b
33.4  ±  3.45A
37.92  ±  4.02a
1.99  ±  2.01B
4.13  ±  0.44c
3
202865.9
0.000***
Gallic acid
0.38  ±  0.04D
1.36  ±  0.12c
0.41  ±  0.03C
2.58  ±  0.26b
4.75  ±  0.55A
5.39  ±  0.61a
0.63  ±  0.07B
1.31  ±  0.11d
3
29258.13
0.000***
Chlorogenic acid hemihydrate
3.36  ±  0.42A
11.99  ±  1.32a
1.49  ±  0.16C
9.37  ±  0.98b
2.33  ±  0.31B
2.65  ±  0.31c
0.81  ±  0.09D
1.68  ±  0.17d
3
89891.62
0.000***
Caffeic acid
nd
nd
1.52  ±  0.16A
9.55  ±  0.99a
1.43  ±  0.13B
1.62  ±  0.14b
nd
nd
3
86392.66
0.000***
Vanillic acid
0.47  ±  0.05B
1.68  ±  0.15b
0.94  ±  0.08A
5.91  ±  0.66a
nd
Nd
nd
nd
3
186787.8
0.000***
Syringic acid
nd
nd
nd
nd
1.26  ±  0.14
1.43  ±  0.12
nd
nd
3
6816.33
0.000***
Ferulic acid
0.35  ±  0.04D
1.25  ±  0.11d
0.68  ±  0.07B
4.27  ±  0.44a
1.17  ±  0.15A
1.33  ±  0.10c
0.65  ±  0.07C
1.35  ±  0.12b
3
4175.74
0.000***
p-Coumaric acid
3.06  ±  0.41A
10.92  ±  1.23a
0.55  ±  0.06D
3.46  ±  0.41b
2.17  ±  0.23B
2.46  ±  0.25c
0.59  ±  0.06C
1.22  ±  0.11d
3
47680.92
0.000***
Rosmarinic acid
0.91  ±  0.08C
3.25  ±  0.41a
0.33  ±  0.04D
2.07  ±  0.22d
2.52  ±  0.26A
2.86  ±  0.30b
1.30  ±  0.14B
2.70  ±  0.28c
3
217.56
0.000***
trans-Cinnamic acid
0.90  ±  0.08C
3.21  ±  0.34c
0.60  ±  0.05D
3.77  ±  0.36a
2.35  ±  0.25A
2.67  ±  0.24d
1.69  ±  0.17B
3.50  ±  0.36b
3
10.60
0.003**
Total
9.80 ±  0.88B
34.96 ±  3.56c
7.25 ±  0.81D
45.57 ±  5.23b
51.38 ±  4.36A
58.33 ±  6.21a
7.66 ±  0.80C
15.88 ±  1.66d
Flavanols
Catechin
0.26  ±  0.03C
0.93  ±  0.08c
0.50  ±  0.04B
3.14  ±  0.44b
4.57  ±  0.50A
5.19  ±  0.56a
nd
nd
3
5899.52
0.000***
Epicatechin
0.67  ±  0.08
2.39  ±  0.24
nd
nd
nd
Nd
nd
nd
3
171363.0
0.000***
Total
0.93 ±  0.08B
3.32 ±  0.35b
0.50 ±  0.06C
3.14 ±  0.29c
4.57 ±  0.52A
5.19 ±  0.62a
nd
nd
Flavanones
Naringenin
1.20  ±  0.11C
4.28  ±  0.51b
1.15  ±  0.12D
7.23  ±  0.77a
2.11  ±  0.22A
2.40  ±  0.25d
1.31  ±  0.14B
2.72  ±  0.28c
3
361.42
0.000***
Total
1.20 ±  0.10C
4.28 ±  0.45b
1.15 ±  0.10D
7.23 ±  0.77a
2.11 ±  0.23A
2.40 ±  0.26d
1.31 ±  0.12B
2.72 ±  0.28c
Flavonols
Myristin
0.87  ±  0.09C
3.10  ±  0.33d
0.59  ±  0.06D
3.71  ±  0.36c
3.45  ±  0.41A
3.92  ±  0.41b
3.04  ±  0.40B
6.30  ±  0.74a
3
235.83
0.000***
Quercetin dihydrate
0.94  ±  0.08C
3.35  ±  0.35a
0.33  ±  0.04D
2.07  ±  0.22b
1.06  ±  0.10B
1.20  ±  0.13c
1.61  ±  0.17A
3.34  ±  0.36a
3
296.86
0.000***
Campherol
0.57  ±  0.06D
2.03  ±  0.25c
1.26  ±  0.13C
7.92  ±  0.84a
1.35  ±  0.14B
1.53  ±  0.16d
1.55  ±  0.16A
3.21  ±  0.34b
3
44423.09
0.000***
Total
2.38 ±  0.24C
8.49 ±  0.99c
2.18 ±  0.22D
13.70 ±  1.45a
5.86 ±  0.66B
6.65 ±  0.76d
6.20 ±  0.65A
12.86 ±  1.42b
Flavones
Apigenin
1.40  ±  0.13C
4.99  ±  0.52b
0.41  ±  0.05D
2.58  ±  0.26d
2.78  ±  0.29B
3.16  ±  0.41c
2.96  ±  0.33A
6.14  ±  0.72a
3
12947.96
0.000***
Flavone
5.45  ±  0.61C
19.44  ±  2.36b
1.89  ±  0.17D
11.88  ±  2.11c
6.01  ±  0.59B
6.82  ±  0.73d
15.66  ±  1.72A
32.47  ±  4.23a
3
533454.7
0.000***
Total
7.38 ±  0.88C
26.33 ±  2.77b
2.30 ±  0.22D
14.46 ±  1.66c
8.79 ±  0.99B
9.98 ±  1.23d
18.62 ±  2.01A
38.61 ±  4.13a
Coumarins
Coumarin
4.59  ±  0.51B
16.38  ±  1.78b
1.67  ±  0.17C
10.50  ±  1.56d
13.34  ±  1.14A
15.15  ±  1.79c
13.31  ±  1.51A
27.60  ±  2.86a
3
618.87
0.000***
Total
4.59 ±  0.55B
16.38  ±  1.88b
1.67 ±  0.18C
10.50  ±  1.23d
13.34 ±  1.62A
15.15  ±  1.62c
13.31 ±  1.43A
27.60  ±  2.97a
Total
28.03 ±  3.12C
100 ±  9.23a
15.91 ±  1.65D
100 ±  8.46a
88.08 ±  7.59A
100 ±  9.87a
48.23 ±  5.21B
100 ±  8.99a
The values of the levels and percentages of phenolic compounds represent the average of three replicates (n = 3); nd: not detected. Letters (a–d (lower case): between percentages and A–D (capital letters) between amounts) indicate significant differences at P
<
0.05. ***Significant effect at P
<
0.001. df: degree of freedom, F: Fisher value, and P: probability.A principal component analysis (PCA) was performed in order to discriminate between different regions of collecting of absinthe based on the phenolic composition. This analysis revealed the existence of one group composed by Boukornine, Kairouan, and Bou Salem regions. But Jérissa is clearly distinct from this group (Figure4).Figure 4
Principal component analysis of different regions based on the phenolic composition ofA. absinthium L. (Table 3).A. absinthium L. can be considered a good source of naturally occurring antioxidant compounds, which have an application in food industry, due to its phenolic composition.


### 3.5. DPPH Test

As indicated in Table4, the methanol extracts are highly influenced by the regional effect (antiradical activity was region-dependent). Methanolic extract of Bou Salem region shows the highest antioxidant activity (9.38
±
0.82 mg/mL) which was stronger than that of the positive control: BHT (10.77
±
2.98 μg/mL). The lower activity was detected in the region of Boukornine (44.26
±
1.92 mg/mL). Mahmoudi et al. [9] have reported an antiradical of wormwood aerial parts activity with an IC50 of 612
±
30.6 μg/mL; this value was lower than our results. Meanwhile, Sengul et al. [46] showed that the antiradical activity of the methanol extracts of the aerial parts of wormwood collected in Turkey was 71.78% as compared to the positive control (BHA = 200 mg/L measuring 93.21%). Different results were found by Wojdyło et al. [47], who reported a highly significant positive correlation (R
=
0.8352, P
<
0.05) between the antiradical activity of Lamiaceae and total polyphenols demonstrating the importance of these compounds in the antioxidant activity of extracts of spices and their significant contribution to the total antioxidant activity.Table 4
DPPH test (IC50 µg/mL) and reducing power (EC50 µg/mL) of methanol extracts of wormwood aerial parts.
Bou Salem
Kairouan
Jérissa
Boukornine
BHT
Ascorbic acid
df
F
P
DPPH (IC50 µg/mL)
9.38  ±  0.82d
18.99  ±  0.38c
31.74  ±  1.23b
44.26  ±  1.92a
10.77  ±  2.98
3
56.46
0.000***
Reducing power(EC50 µg/mL)
2.36  ±  0.15b
4.03  ±  0.45d
2.46  ±  0.46a
2.16  ±  0.05c
4,20  ±  0,11
3
1.27
0.348NS
IC50 and EC50 values represent the mean of three replicates (n = 3); letters (a–d) indicate significant differences at P
<
0.05. ***Significant at P
<
0.001%. NS: not significant. df: degree of freedom, F: Fisher value, and P: probability.


### 3.6. Reducing Power

The ability of methanol extracts of wormwood aerial parts collected from four regions to reduce the metal ions was evaluated by determining the CE50 corresponding to the concentration of the extract to an absorbance of 0.5. The ascorbic acid has been used as a positive control. The results in Table 4 showed that the methanol extracts of wormwood aerial parts were characterized by EC50 values lower than that of the positive control: ascorbic acid (EC50 = 4.2
±
0.11 μg/mL). In addition, statistical analysis showed that there is no effect of region on the activities of the methanol extracts.


### 3.7. Antibacterial Activity

The test results of the antibacterial effect are summarized in Table5. The results show that the diameter of the inhibition zone is not affected by the region factor. The essential oils of wormwood at different regions showed an interesting antibacterial activity. Indeed, the highest activity was observed againstStaphylococcus aureus strain with a diameter of inhibition equal to 25
±
1.13 mm for the essential oil of Kairouan; this strain had a lower sensitivity to the EO of Bou Salem region (IZ = 18
±
1.13 mm). Essential oils of Boukornine and Jérissa regions show a moderately antibacterial activity (IZ = 20.66 mm). In agreement with our results, the EO of wormwood aerial parts growing in Canada has a high activity againstStaphylococcus aureus (IZ = 25
±
1.4 mm) which was greater than the positive control (methicillin activity IZ = 18
±
1.0 mm) [34]. Essential oils of Bou Salem and Kairouan have the same activity againstListeria monocytogenes (IZ = 20 mm), but the EO of Jérissa and Boukornine is less active (IZ = 18 mm). These results show that wormwood essential oil was endowed with a significant antibacterial activity that is closely related to the organoleptic quality of the oil, which in turn depends strongly on the collection region of plant material.Table 5
Antibacterial (IZ mm) and antifungal (%I) activities of wormwood essential oil.
Boukornine
Jérissa
Kairouan
Bou Salem
df
F
P
Tetracycline
Nystatin
Bacteria
Staphylococcus aureus 25923
20.66  ±  2.61b
20.66  ±  0.65b
25  ±  1.13a
18  ±  1.13c
3
2.05
0.184NS
25  ±  2.56
Staphylococcus methicillin-resistant
15.00  ±  1.13b
14.33  ±  0.65c
16  ±  1.30a
14.33  ±  0.65c
3
0.21
0.879NS
25  ±  2.54
Listeria monocytogenesATCC 19195
18.66  ±  2.35b
17.33  ±  1.72c
20.00  ±  1.95a
20.00  ±  1.13a
3
0.53
0.674NS
24  ±  2.42
Fungi
Fusarium graminearum
14.66  ±  0.57c
23.65  ±  0.57a
11.11  ±  0.12d
16.66  ±  0.57b
3
18.57
0.000***
25  ±  2.31
Fusarium culmorum
45.23  ±  3.11b
23.65  ±  0.57d
42.06  ±  0.57c
46.82  ±  0.57a
3
32.77
0.000***
25  ±  2.14
Fusarium oxysporum
19.04  ±  0.57c
27.58  ±  2.55a
17.94  ±  0.57d
25.28  ±  0.57b
3
7.79
0.009**
20  ±  1.67
Sclerotinia
nd
23.61  ±  2.12
nd
nd
3
185.81
0.000***
22  ±  2.01
Rhizoctonia solani
nd
25.39  ±  0.57
nd
nd
3
77.35
0.000***
20  ±  1.78
Results are the mean of three replications. The diameter of disc was 6 mm. Values with different superscripts (a–d) are significantly different atP
<
0.05. nd: not detected. NS: not significant. df: degree of freedom, F: Fisher value, and P: probability. *
*
P
<
0.01. *
*
*
P
<
0.001.


### 3.8. Antifungal Activity

The results of the antifungal activity of essential oils of absinthe are summarized in Table5. Wormwood EOs had a significant inhibitory activity against the three phytopathogenicF. graminearum,F. culmorum, andF. oxysporum while only EO of Jérissa proved to be active againstSclerotinia (23.61
±
2.12%) andRhizoctonia solani (25.39
±
0.57%). As for the antibacterial activity, antifungal activity could be attributed to the major component of essential oil of wormwood: chamazulene [56]. Meanwhile, it has been shown that the chamazulene possesses significant antifungal activity [57]. The antimicrobial effect of EOs of absinthe gives them an important role in the fields of food, cosmetics, and pharmaceutical industries. In additionA. absinthium L. EO from France containing(Z)-epoxyocimene and chrysanthenyl acetate as major components inhibited the growth of both the yeastsCandida albicans andSaccharomyces cerevisiae var. chevalieri [4]. Wormwood EO from a Turkish population, whose main components are camphor, 1,8-cineole, and chamazulene, has been described as fungicidal against 34 species of fungi includingF. solani andF. oxysporum [3]. Furthermore,A. absinthium EO from Uruguay rich in thujone showed antifungal effects againstAlternariasp. andBotrytis cinerea [58]. However, further studies are needed to identify the compound(s) responsible for the antifungal effects ofA. absinthium L. EOs.


## 3.1. Essential Oil Yield

The hydrodistillation extraction of 100 g of wormwood dry aerial parts collected from four different regions yielded a dark blue essential oil. These yields were illustrated in Figure1. The results show that EO yield varies significantly (P
<
0.05) according to region factor. Thereby, the optimal yield was observed at the region of Bou Salem (1.46
±
0.06%) followed by the region of Kairouan (1.12
±
0.08%) and Boukornine and Jérissa regions (1.10
±
0.04% and 1.00
±
0.03%, resp.). Similar results were obtained by Orav et al. [33] at different European regions and the oil yields were ranged from 0.1 to 1.1% w/w. In addition, the EO yields detected in the present study were higher than those determined by Lopes-Lutz et al. [34] in the region of the central prairies of Alberta (Canada West) that they found (0.5%, w/w), which justifies the effect of region on the EO yield. The impact of other environmental factors on EO yield was also described by Msaada et al. [35] and Msaada et al. [36, 37]. In addition, seasonal and regional variations were observed in the EO yield of coriander [38]. The same authors also reported the effect of extraction techniques on the essential oils yield and composition [39].Figure 1
Essential oil yield of the aerial parts ofA. absinthium L. collected from four Tunisian regions. Values with different superscripts (a–c) are significantly different at P
<
0.05.


## 3.2. Variability of Chemical Composition of EO

The hydrodistillation of wormwood aerial parts collected from four different Tunisian regions provides an EO with blue-black color. This latter was due to the predominance of chamazulene component which gives the oil with a high organoleptic quality [33]. The results of chromatographic analysis by gas chromatography (GC/FID) and by coupling gas chromatography to mass spectrometry (GC/MS) are presented in Table 2.Table 2
ANOVA analysis, qualitative and quantitative essential oil composition (peak area % ± SD) ofA. absinthium L. aerial parts.
Compound*
RRIa
RRIb
Collection region
df
F
P
Jérissa
Boukornine
Kairouan
Bou Salem
Tricyclene
1014
927
1.00  ±  0.11a
0.02  ±  0.00d
0.74  ±  0.06b
0.23  ±  0.03c
3
9.6733
0.004***
α-Thujene
1032
939
0.05  ±  0.01c
0.05  ±  0.00c
3.28  ±  0.32a
0.23  ±  0.02b
3
10077.77
0.000***
α-Pinene
1035
931
0.10  ±  0.01c
0.02  ±  0.00b
0.62  ±  0.05a
0.20  ±  0.03b
3
65.81
0.000***
Camphene
1076
950
0.06  ±  0.01c
0.03  ±  0.00d
0.66  ±  0.07a
0.24  ±  0.03b
3
163.01
0.000***
β-Pinene
1118
980
1.20  ±  0.10a
0.25  ±  0.02c
0.02  ±  0.00d
0.27  ±  0.03b
3
251.96
0.000***
Sabinene
1132
976
0.28  ±  0.03c
1.87  ±  0.19a
0.02  ±  0.00d
1.67  ±  0.15b
3
3383.83
0.000***
Myrcene
1176
988
0.50  ±  0.04b
0.05  ±  0.01c
1.52  ±  0.14a
0.02  ±  0.00d
3
555.19
0.000***
α-Terpinene
1188
1018
0.03  ±  0.00c
0.44  ±  0.05a
0.08  ±  0.01b
0.03  ±  0.00c
3
247.78
0.000***
Limonene
1203
1026
0.10  ±  0.01d
0.95  ±  0.08a
0.14  ±  0.01c
0.40  ±  0.03b
3
39.49
0.000***
1,8-Cineole
1213
1033
0.03  ±  0.00c
0.03  ±  0.00c
0.35  ±  0.04a
0.08  ±  0.01b
3
83.14
0.000***
β-Phellandrene
1218
1031
0.10  ±  0.02b
0.04  ±  0.00d
0.06  ±  0.01c
0.26  ±  0.03a
3
25.44
0.000***
Z-β-Ocimene
1246
1040
0.20  ±  0.03c
1.59  ±  0.16a
0.26  ±  0.03b
0.05  ±  0.00d
3
538.84
0.000***
E-β-Ocimene
1266
1050
0.08  ±  0.01d
0.10  ±  0.02c
1.40  ±  0.16a
0.43  ±  0.05b
3
105.86
0.000***
γ-Terpinene
1267
1062
0.10  ±  0.02b
0.07  ±  0.01c
0.18  ±  0.02a
0.04  ±  0.00d
3
27.18
0.000***
p-Cymene
1280
1026
0.08  ±  0.01c
0.12  ±  0.01b
0.22  ±  0.03a
0.04  ±  0.00d
3
44.75
0.000***
Terpinolene
1290
1088
0.20  ±  0.03b
0.13  ±  0.01c
0.12  ±  0.01cd
0.62  ±  0.05a
3
57.94
0.000***
β-Thujone
1430
1089
17.16  ±  1.56c
21.02  ±  2.32b
22.09  ±  2.12a
6.72  ±  0.75d
3
590069.9
0.000***
α-Thujone
1430
1089
0.20  ±  0.02b
0.07  ±  0.01c
0.28  ±  0.03a
0.04  ±  0.00d
3
13.52
0.001***
trans-Linalool oxide
1450
1088
0.03  ±  0.00c
0.02  ±  0.00d
6.04  ±  0.77a
2.58  ±  0.25b
3
11876.62
0.000***
trans-Sabinene hydrate
1474
1053
12.07  ±  1.32c
12.58  ±  1.30b
5.32  ±  0.56d
21.78  ±  2.12a
3
242741.2
0.000***
cis-Linalool oxide
1478
1074
0.05  ±  0.00c
0.13  ±  0.01b
0.05  ±  0.01c
1.86  ±  0.16a
3
6371.93
0.000***
α-Copaene
1497
1379
0.50  ±  0.04b
4.07  ±  0.44a
0.33  ±  0.03c
0.22  ±  0.03d
3
3561.91
0.000***
Camphor
1532
1143
0.02  ±  0.00d
0.04  ±  0.00c
0.15  ±  0.01a
0.12  ±  0.01b
3
13.73
0.001***
Linalool
1553
1088
2.00  ±  0.23a
0.80  ±  0.07b
0.09  ±  0.01d
0.15  ±  0.02c
3
9.32
0.005**
cis-Sabinene hydrate
1556
1082
0.02  ±  0.00d
1.82  ±  0.15a
1.80  ±  0.20b
0.80  ±  0.07c
3
441.83
0.000***
Linalyl acetate
1565
1239
0.50  ±  0.06b
4.58  ±  0.51a
0.08  ±  0.01d
0.21  ±  0.03c
3
5308.55
0.000***
Bornyl acetate
1590
1270
1.10  ±  0.10a
0.04  ±  0.00c
0.36  ±  0.04b
0.02  ±  0.00d
3
268.07
0.000***
Terpinene-4-ol
1611
1176
3.00  ±  0.41a
0.15  ±  0.01c
0.05  ±  0.00d
1.10  ±  0.12b
3
22.16
0.000***
cis-Dihydrocarvone
1645
1193
0.48  ±  0.05b
0.94  ±  0.08a
0.11  ±  0.01c
0.03  ±  0.00d
3
942.9
0.000***
cis-Verbenol
1654
1132
2.34  ±  0.24b
0.02  ±  0.00d
0.31  ±  0.04c
3.57  ±  0.44a
3
15604.73
0.000***
Sabinyl acetate
1658
1291
0.29  ±  0.03b
5.01  ±  0.45a
0.11  ±  0.01c
0.08  ±  0.01d
3
100958.1
0.000***
trans-Sabinol
1666
1120
0.29  ±  0.03b
0.04  ±  0.00d
0.06  ±  0.01c
0.99  ±  0.08a
3
1582.13
0.000***
Lavandulol
1677
1150
2.71  ±  0.31c
0.28  ±  0.03d
3.61  ±  0.34b
5.54  ±  0.61a
3
26002.36
0.000***
α-Humulene
1687
1454
0.08  ±  0.01b
0.62  ±  0.05a
0.03  ±  0.00c
0.01  ±  0.00d
3
1699.33
0.000***
Neral
1694
1240
0.01  ±  0.00d
0.26  ±  0.03c
2.68  ±  0.27a
0.28  ±  0.03b
3
11106.53
0.000***
γ-Muurolene
1692
1474
0.28  ±  0.03b
1.09  ±  0.11a
0.15  ±  0.02c
0.05  ±  0.00d
3
871.32
0.000***
γ-Curcumene
1704
1475
1.00  ±  0.12c
2.93  ±  0.32a
0.57  ±  0.06d
1.95  ±  0.22b
3
52.19
0.000***
α-Terpinyl acetate
1705
1344
0.17  ±  0.01a
tr
0.14  ±  0.01b
0.03  ±  0.00c
3
91.11
0.000***
α-Terpineol
1706
1189
0.19  ±  0.02b
0.14  ±  0.01c
0.05  ±  0.01d
0.38  ±  0.04a
3
93.12
0.000***
Borneol
1719
1165
0.52  ±  0.06a
0.46  ±  0.05b
0.23  ±  0.03c
tr
3
397.35
0.000***
Germacrene D
1726
1480
0.04  ±  0.00d
0.10  ±  0.01c
1.58  ±  0.17a
0.52  ±  0.06b
3
1647.56
0.000***
Neryl acetate
1732
1356
0.20  ±  0.02c
0.26  ±  0.03b
0.03  ±  0.00d
0.51  ±  0.06a
3
42.73
0.000***
β-Selinene
1742
1486
4.72  ±  0.51c
4.50  ±  0.50d
5.29  ±  0.62b
6.67  ±  0.74a
3
45.48
0.000***
α-Bisabolene
1746
1494
0.88  ±  0.09b
0.11  ±  0.01c
1.25  ±  0.15a
0.02  ±  0.00d
3
15.32
0.001***
Carvone
1751
1242
2.00  ±  0.31a
0.14  ±  0.01c
0.46  ±  0.05b
0.05  ±  0.00d
3
9.88
0.004**
Geranyl acetate
1765
1383
0.08  ±  0.01b
0.61  ±  0.07a
0.03  ±  0.00c
0.02  ±  0.00d
3
2429
0.000***
γ-Cadinene
1776
1526
0.40  ±  0.05c
1.12  ±  0.15b
1.27  ±  0.13a
0.05  ±  0.00d
3
98.38
0.000***
Nerol
1797
1228
0.10  ±  0.01c
0.11  ±  0.01c
0.33  ±  0.04b
1.93  ±  0.31a
3
2118.79
0.000***
Geraniol
1857
1255
0.65  ±  0.07a
0.03  ±  0.00d
0.04  ±  0.00c
0.20  ±  0.02b
3
77.96
0.000***
p-Cymene-8-ol
1864
1183
tr
0.96  ±  0.08b
3.70  ±  0.41a
tr
3
3636.71
0.000***
α-Calacorene
1920
1527
0.11  ±  0.01d
0.16  ±  0.02b
0.12  ±  0.01c
0.52  ±  0.06a
3
355.3
0.000***
Caryophyllene oxide
2008
1578
0.05  ±  0.00b
0.06  ±  0.01a
0.06  ±  0.00a
tr
3
16.5
0.000***
cis-Nerolidol
2050
1553
0.27  ±  0.03a
0.06  ±  0.01c
0.07  ±  0.01b
0.01  ±  0.00d
3
92.88
0.000***
Eugenol
2192
1356
0.15  ±  0.02d
1.24  ±  0.14a
0.32  ±  0.04b
0.20  ±  0.03c
3
218.06
0.000***
α-Bisabolol
2232
1673
0.03  ±  0.00b
0.02  ±  0.00c
0.15  ±  0.02a
0.01  ±  0.00d
3
19.07
0.000***
Chamazulene
2430
1719
39.93  ±  4.56a
25.30  ±  3.12d
29.51  ±  3.26c
34.81  ±  4.11b
3
5324.71
0.000***
Chemical classes
Monoterpene hydrocarbons
56.02 ± 6.21b
45.31 ± 5.31d
45.73  ±  5.22c
62.08  ±  7.49a
3
26322.56
0.000***
Aromatic hydrocarbons
0.08 ± 0.01c
0.12 ± 0.01b
0.22  ±  0.03a
0.04  ±  0.00d
3
9563.42
0.000***
Monoterpene alcohols
12.22 ± 1.33b
4.29 ± 0.52d
8.86  ±  0.96c
14.07  ±  1.67a
3
912.67
0.000***
Monoterpene esters
2.34 ± 0.31b
10.05 ± 1.23a
0.75  ±  0.08d
0.87  ±  0.09c
3
567.01
0.000***
Monoterpene ketones
19.86 ± 2.13c
22.21 ± 2.11b
23.09  ±  3.10a
6.96  ±  7.41d
3
152.73
0.000***
Monoterpene aldehydes
0.01 ± 0.00d
0.26 ± 0.03c
2.68  ±  2.45a
0.28  ±  0.03b
3
4360.02
0.000***
Monoterpene ethers
0.11 ± 0.01d
0.18 ± 0.02c
6.44  ±  0.77a
4.52  ±  0.51b
3
4265.16
0.000***
Sesquiterpenes
8.09 ± 0.99d
14.78 ± 1.66a
10.80  ±  1.45b
10.02  ±  1.78c
3
1235.94
0.000***
Total identified
98.72 ± 10.23b
97.64 ± 9.78d
98.55  ±  9.88c
98.83  ±  9.76a
3
4581.23
0.000***
RRI: relative retention index; a: HP-INNOWax, b: HP-5.*Compounds in order of elution on HP-INNOWax; values of volatile essential oil percentages are the average three determinations (n = 3). These values with different letters (a–d) are significantly different at P < 0.05. nd: not detected. NS: not significant. **
P < 0.01. ***
P < 0.001.
df: degree of freedom,F: Fisher value, and P: probability.Essential oils analyzed are divided into eight classes based on their chemical functional groups. A total of 56 compounds were identified representing98.72
±
10.23%, 97.64
%
±
9.78, 98.55
±
9.88%, and 98.83
±
9.76% of total volatiles in the regions of Jérissa, Boukornine, Kairouan, and Bou Salem, respectively. These different identified compounds vary significantly (P
<
0.05) from region to another and are highly (P
<
0.001) affected by the regional factor (Table 2).The major contribution was attributed to the monoterpene hydrocarbons fraction which represents 56.02% ±6.21,45.31
±
5.31%, 45.73
±
5.22%, and 62.08
±
7.49% of all compounds detected in Jérissa, Boukornine, Kairouan, and Bou Salem regions, respectively. The latter fraction is dominated by chamazulene where its maximum rate (39.93
±
4.56%) was observed in the Jérissa region, followed by the regions of Bou Salem (34.81
±
4.11%), Kairouan (29.51
±
3.26%), and Boukornine (25.30
±
3.12%). These results were in agreement with the literature. However, Kordali et al. [40] showed that essential oils chemical composition of wormwood collected in Turkey was dominated by chamazulene (17.8%) followed by nuciferol butanoate (8.2%), nuciferol propionate (5.1%), and caryophyllene oxide (4.3%). In this work, β-thujone, a monoterpene ketone, was detected with significant percentages: 22.09
±
2.12% in the region of Kairouan, up to 6.72
±
0.75% in the region of Bou Salem. Meanwhile, the chemical composition of essential oils of wormwood growing in the USA shows that the β-thujone (17.5 to 42.3%) andcis-sabinyl acetate (15.1 to 53.4%) are the major compounds [41]. In agreement with our results, previous studies have shown that the rate of thujone in essential oils of wormwood is greater than 35% [42]. In addition, in the wormwood samples collected from different European countries (Greece, Spain, Ukraine, France, and Italy), α-thujone and β-thujone were detected as major components with percentages ranging from 4.5 to 38.7% [33].It should be noted that the sesquiterpene fraction which is represented byα-humulene, the γ-muurolene, the γ-curcumene, the α-copaene, the β-selinene, the α-bisabolene, the γ-cadinene, the germacrene D, the α-calacorene, caryophyllene oxide, and α-bisabolol ranges from 8 to 14% of total volatiles. This slight variation could be explained by similar environmental and genetic factors. An earlier study showed that the rate of sesquiterpenes detected was 2.2% [34], rate lower than that found in this work. Monoterpene alcohols (linalool, lavandulol, terpinen-4-ol, α-terpineol, nerol, sabinoltrans-p-cymene-8-ol, eugenol, borneol, and geraniol) were highly affected by the region factor (Table 2).Principal component analysis was carried out in order to determine the relationship between the different regions of plant collection on the basis of their essential oil composition. A better discrimination was revealed on the three-dimensional visualization of the plotted scores. Results obtained from the PCA (Figure2) showed the existence of one well-defined group represented by the regions Jérissa, Boukornine, and Kairouan suggesting similar compositions. The Bou Salem region was clearly distinguished from the latter group both in quality and in quantity.Figure 2
Relative position of the regions of plant collection based on their essential oils composition (Table2) in the space defined by the three principal components.In conclusion, theArtemisia absinthiumL. plants collected from four Tunisian habitats biosynthesized essential oils of one chemotype, chamazulene.


## 3.3. Total Polyphenols, Flavonoids, and Condensed Tannins Contents

Total phenol assay is used as a routing assay to assess phenolic antioxidants due to its simplicity and reproducibility. Phenolic compounds have the ability to reduce Folin-Ciocalteu reagent and the reaction may be followed by measuring a change in the color of the solution from intense yellow to blue spectrophotometrically. Quantitative and qualitative changes of the antioxidant compounds with function to region effect hold great significance from both dietary and nutritional points of view, while phenolic compounds are used as alternatives to synthetic drugs and were shown to be safe for use in processed foods [43].Phenolic quantification revealed a wide range of aerial parts’ polyphenol contents as function of the region as shown in Figure3. The total polyphenol contents varied significantly (P
≤
0.05) from one region to another and reached the maximum in the region of Kairouan (99.89
±
3.30 mg GAE/g DW), followed by the region of Bou Salem, Boukornine, and Jérissa where their levels were 83.70
±
1.31, 72.05
±
1.83, and 49.39
±
2.20 mg GAE/g DM, respectively. Monica et al. [44] showed that total polyphenol content of wormwood aerial parts collected in Romania was 98.20 mg/g DW. In addition, Ebrahimzadeh et al. [45] reported that the total polyphenol content of Iranian wormwood aerial parts was 194
±
9.7 mg EAG/g of extract which was significantly of greater value in comparison with our results. However, a lower content in total polyphenols (9.79 mg EAG/g extract) content was determined by Sengul et al. [46]. Other herbs with high levels of phenolics werePolygonum aviculare (11.2 mg GAE/100 g DW) andValeriana officinalis (11.1 mg GAE/100 g DW) [47]. In general, phenolic compounds are known for their role in the prevention of some diseases due to their richness in antioxidants and their antiradical effect, protecting cellular components against damage induced by free radicals. Due to the diversity of their chemical structures, they are likely to have very different antioxidant capacity [48]. Polyphenolic compounds are commonly found in both edible and inedible plants, and they have been reported to have multiple biological effects, including antioxidant activity [47]. The basic flavonoids structure is the flavan nucleus, which consists of 15 carbon atoms arranged in three rings (C6–C3–C6), labeled A, B, and C. Various classes of flavonoid differ in the level of oxidation and saturation of ring C, while individual compounds within a class differ in the substitution pattern of rings A and B. The differences in the structure and substitution will influence the phenoxyl radical stability and thereby the antioxidant properties of the flavonoids [49].Figure 3
Total polyphenols (TPP), total flavonoids (TF), and total condensed tannin (TCT) contents of different regions ofA. absinthium L. aerial parts. GAE: gallic acid equivalents; CE: catechin equivalents.The letters (a–d) indicate significant differences(P
<
0.05).As for total polyphenols, total flavonoids differ significantly(
P
≤
0.05
) depending on the region, but with a maximum in Kairouan region (126.40
±
2.32 mg CE/g DW) (Figure 3).Our results are in agreement with those reported by Wojdyło et al. [47] in that the most common flavonoids are mainly distributed in Compositae family. On the other hand,A. absinthiumL. aerial parts have been reported to contain flavonoids [50–52], thymol, and carvacrol as well as other phenolic compounds [53]. These pharmacophores have been shown to possess potent antioxidant and free radical scavenging activity [54]. In the present study, high levels of phenolic and flavonoid contents were estimated. For total condensed tannins, the maximum level was detected in the region Boukornine (152.35
±
4.59 mg CE/g DW) followed by Jérissa (83.50
±
6.69 mg CE/g DW), Bou Salem (73.94
±
2.19 mg CE/g DW), and Kairouan (37.19
±
1.16 mg CE/g DW) regions (Figure 3).


## 3.4. Individual Phenolic Compounds

For a clear understanding of the regional effect on metabolic changes especially on phenolics, it is necessary to investigate the profiles of individual phenolic compounds inA. absinthium L. Hence, changes of these phenolic compounds were different at four Tunisian regions (Table 3). The obtained results show that all the identified compounds were highly significant (P
<
0.001) by the region effect. Phenolic acids—tannic, gallic, hemihydrate chlorogenic, caffeic, vanillic, syringic, ferulic,p-coumaric, rosmarinic, andtrans-cinnamic acids—represent the major fraction of the polyphenols analyzed in regions of Bou Salem (34.96
±
3.56%), Kairouan (45.57
±
5.23%), and Jérissa (58.33
±
6.21%), while the flavones dominated at Boukornine region (38.61
±
4.13%). In the regions of Bou Salem, Kairouan, and Boukornine, the flavone was the predominant fraction (19.44
±
2.36%, 11.88
±
2.11%, and 32.47
±
4.23%, resp.), while, in the region of Jérissa, tannic acid was the predominant compound (37.92
±
4.02%). The quantification of total polyphenols of wormwood aerial parts by the Folin-Ciocalteu method gave different results in all studied regions. In the region of Bou Salem, total polyphenols measured by Folin-Ciocalteu method were estimated to be 83.7 mg GAE/g DW and 28.03
±
3.12 mg/g GAE DW by HPLC, in Kairouan were 99; 89 mg GAE/g DW by Folin-Ciocalteu and 15.91
±
1.65 mg GAE/g by HPLC, in Boukornine were 72.05 mg GAE/g DW by Folin-Ciocalteu and 48.23
±
5.21 mg GAE/g DM by HPLC, and in Jérissa were 49.39 mg GAE/g DW by Folin-Ciocalteu and 88.08
±
7.59 mg GAE/g DW by HPLC. These variations in the quantification showed a significant difference depending on the method used. An earlier study showed that the analysis by liquid chromatography (HPLC) revealed the presence of other flavonoid glycosides (isoquercitrin, quercetin-3-O-β-D-glucoside, quercetin-3-O-rhamnoglucoside, isorhamnetin-3-O-rhamnoglucoside, and isorhamnetin-3-glucoside) [50], which are characterized by important antioxidant activity. Gallic acid, widely used as an additive to prevent food spoilage, is renowned for its anticarcinogenic, anti-inflammatory, and antimutagenic activities [55].Table 3
Analysis of variance (ANOVA) and phenolic composition in % (w/w) and quantity (µg/g DW) of methanol extracts of the aerial parts of wormwood (A. absinthium L.).
Compound
Collection region
df
F
P
Bou Salem
Kairouan
Jérissa
Boukornine
µg/g DW
%
µg/g DW
%
µg/g DW
%
µg/g DW
%
Phenolic acids
Tannic acid
0.37  ±  0.04D
1.32  ±  0.11d
0.73  ±  0.08C
4.59  ±  0.51b
33.4  ±  3.45A
37.92  ±  4.02a
1.99  ±  2.01B
4.13  ±  0.44c
3
202865.9
0.000***
Gallic acid
0.38  ±  0.04D
1.36  ±  0.12c
0.41  ±  0.03C
2.58  ±  0.26b
4.75  ±  0.55A
5.39  ±  0.61a
0.63  ±  0.07B
1.31  ±  0.11d
3
29258.13
0.000***
Chlorogenic acid hemihydrate
3.36  ±  0.42A
11.99  ±  1.32a
1.49  ±  0.16C
9.37  ±  0.98b
2.33  ±  0.31B
2.65  ±  0.31c
0.81  ±  0.09D
1.68  ±  0.17d
3
89891.62
0.000***
Caffeic acid
nd
nd
1.52  ±  0.16A
9.55  ±  0.99a
1.43  ±  0.13B
1.62  ±  0.14b
nd
nd
3
86392.66
0.000***
Vanillic acid
0.47  ±  0.05B
1.68  ±  0.15b
0.94  ±  0.08A
5.91  ±  0.66a
nd
Nd
nd
nd
3
186787.8
0.000***
Syringic acid
nd
nd
nd
nd
1.26  ±  0.14
1.43  ±  0.12
nd
nd
3
6816.33
0.000***
Ferulic acid
0.35  ±  0.04D
1.25  ±  0.11d
0.68  ±  0.07B
4.27  ±  0.44a
1.17  ±  0.15A
1.33  ±  0.10c
0.65  ±  0.07C
1.35  ±  0.12b
3
4175.74
0.000***
p-Coumaric acid
3.06  ±  0.41A
10.92  ±  1.23a
0.55  ±  0.06D
3.46  ±  0.41b
2.17  ±  0.23B
2.46  ±  0.25c
0.59  ±  0.06C
1.22  ±  0.11d
3
47680.92
0.000***
Rosmarinic acid
0.91  ±  0.08C
3.25  ±  0.41a
0.33  ±  0.04D
2.07  ±  0.22d
2.52  ±  0.26A
2.86  ±  0.30b
1.30  ±  0.14B
2.70  ±  0.28c
3
217.56
0.000***
trans-Cinnamic acid
0.90  ±  0.08C
3.21  ±  0.34c
0.60  ±  0.05D
3.77  ±  0.36a
2.35  ±  0.25A
2.67  ±  0.24d
1.69  ±  0.17B
3.50  ±  0.36b
3
10.60
0.003**
Total
9.80 ±  0.88B
34.96 ±  3.56c
7.25 ±  0.81D
45.57 ±  5.23b
51.38 ±  4.36A
58.33 ±  6.21a
7.66 ±  0.80C
15.88 ±  1.66d
Flavanols
Catechin
0.26  ±  0.03C
0.93  ±  0.08c
0.50  ±  0.04B
3.14  ±  0.44b
4.57  ±  0.50A
5.19  ±  0.56a
nd
nd
3
5899.52
0.000***
Epicatechin
0.67  ±  0.08
2.39  ±  0.24
nd
nd
nd
Nd
nd
nd
3
171363.0
0.000***
Total
0.93 ±  0.08B
3.32 ±  0.35b
0.50 ±  0.06C
3.14 ±  0.29c
4.57 ±  0.52A
5.19 ±  0.62a
nd
nd
Flavanones
Naringenin
1.20  ±  0.11C
4.28  ±  0.51b
1.15  ±  0.12D
7.23  ±  0.77a
2.11  ±  0.22A
2.40  ±  0.25d
1.31  ±  0.14B
2.72  ±  0.28c
3
361.42
0.000***
Total
1.20 ±  0.10C
4.28 ±  0.45b
1.15 ±  0.10D
7.23 ±  0.77a
2.11 ±  0.23A
2.40 ±  0.26d
1.31 ±  0.12B
2.72 ±  0.28c
Flavonols
Myristin
0.87  ±  0.09C
3.10  ±  0.33d
0.59  ±  0.06D
3.71  ±  0.36c
3.45  ±  0.41A
3.92  ±  0.41b
3.04  ±  0.40B
6.30  ±  0.74a
3
235.83
0.000***
Quercetin dihydrate
0.94  ±  0.08C
3.35  ±  0.35a
0.33  ±  0.04D
2.07  ±  0.22b
1.06  ±  0.10B
1.20  ±  0.13c
1.61  ±  0.17A
3.34  ±  0.36a
3
296.86
0.000***
Campherol
0.57  ±  0.06D
2.03  ±  0.25c
1.26  ±  0.13C
7.92  ±  0.84a
1.35  ±  0.14B
1.53  ±  0.16d
1.55  ±  0.16A
3.21  ±  0.34b
3
44423.09
0.000***
Total
2.38 ±  0.24C
8.49 ±  0.99c
2.18 ±  0.22D
13.70 ±  1.45a
5.86 ±  0.66B
6.65 ±  0.76d
6.20 ±  0.65A
12.86 ±  1.42b
Flavones
Apigenin
1.40  ±  0.13C
4.99  ±  0.52b
0.41  ±  0.05D
2.58  ±  0.26d
2.78  ±  0.29B
3.16  ±  0.41c
2.96  ±  0.33A
6.14  ±  0.72a
3
12947.96
0.000***
Flavone
5.45  ±  0.61C
19.44  ±  2.36b
1.89  ±  0.17D
11.88  ±  2.11c
6.01  ±  0.59B
6.82  ±  0.73d
15.66  ±  1.72A
32.47  ±  4.23a
3
533454.7
0.000***
Total
7.38 ±  0.88C
26.33 ±  2.77b
2.30 ±  0.22D
14.46 ±  1.66c
8.79 ±  0.99B
9.98 ±  1.23d
18.62 ±  2.01A
38.61 ±  4.13a
Coumarins
Coumarin
4.59  ±  0.51B
16.38  ±  1.78b
1.67  ±  0.17C
10.50  ±  1.56d
13.34  ±  1.14A
15.15  ±  1.79c
13.31  ±  1.51A
27.60  ±  2.86a
3
618.87
0.000***
Total
4.59 ±  0.55B
16.38  ±  1.88b
1.67 ±  0.18C
10.50  ±  1.23d
13.34 ±  1.62A
15.15  ±  1.62c
13.31 ±  1.43A
27.60  ±  2.97a
Total
28.03 ±  3.12C
100 ±  9.23a
15.91 ±  1.65D
100 ±  8.46a
88.08 ±  7.59A
100 ±  9.87a
48.23 ±  5.21B
100 ±  8.99a
The values of the levels and percentages of phenolic compounds represent the average of three replicates (n = 3); nd: not detected. Letters (a–d (lower case): between percentages and A–D (capital letters) between amounts) indicate significant differences at P
<
0.05. ***Significant effect at P
<
0.001. df: degree of freedom, F: Fisher value, and P: probability.A principal component analysis (PCA) was performed in order to discriminate between different regions of collecting of absinthe based on the phenolic composition. This analysis revealed the existence of one group composed by Boukornine, Kairouan, and Bou Salem regions. But Jérissa is clearly distinct from this group (Figure4).Figure 4
Principal component analysis of different regions based on the phenolic composition ofA. absinthium L. (Table 3).A. absinthium L. can be considered a good source of naturally occurring antioxidant compounds, which have an application in food industry, due to its phenolic composition.


## 3.5. DPPH Test

As indicated in Table4, the methanol extracts are highly influenced by the regional effect (antiradical activity was region-dependent). Methanolic extract of Bou Salem region shows the highest antioxidant activity (9.38
±
0.82 mg/mL) which was stronger than that of the positive control: BHT (10.77
±
2.98 μg/mL). The lower activity was detected in the region of Boukornine (44.26
±
1.92 mg/mL). Mahmoudi et al. [9] have reported an antiradical of wormwood aerial parts activity with an IC50 of 612
±
30.6 μg/mL; this value was lower than our results. Meanwhile, Sengul et al. [46] showed that the antiradical activity of the methanol extracts of the aerial parts of wormwood collected in Turkey was 71.78% as compared to the positive control (BHA = 200 mg/L measuring 93.21%). Different results were found by Wojdyło et al. [47], who reported a highly significant positive correlation (R
=
0.8352, P
<
0.05) between the antiradical activity of Lamiaceae and total polyphenols demonstrating the importance of these compounds in the antioxidant activity of extracts of spices and their significant contribution to the total antioxidant activity.Table 4
DPPH test (IC50 µg/mL) and reducing power (EC50 µg/mL) of methanol extracts of wormwood aerial parts.
Bou Salem
Kairouan
Jérissa
Boukornine
BHT
Ascorbic acid
df
F
P
DPPH (IC50 µg/mL)
9.38  ±  0.82d
18.99  ±  0.38c
31.74  ±  1.23b
44.26  ±  1.92a
10.77  ±  2.98
3
56.46
0.000***
Reducing power(EC50 µg/mL)
2.36  ±  0.15b
4.03  ±  0.45d
2.46  ±  0.46a
2.16  ±  0.05c
4,20  ±  0,11
3
1.27
0.348NS
IC50 and EC50 values represent the mean of three replicates (n = 3); letters (a–d) indicate significant differences at P
<
0.05. ***Significant at P
<
0.001%. NS: not significant. df: degree of freedom, F: Fisher value, and P: probability.


## 3.6. Reducing Power

The ability of methanol extracts of wormwood aerial parts collected from four regions to reduce the metal ions was evaluated by determining the CE50 corresponding to the concentration of the extract to an absorbance of 0.5. The ascorbic acid has been used as a positive control. The results in Table 4 showed that the methanol extracts of wormwood aerial parts were characterized by EC50 values lower than that of the positive control: ascorbic acid (EC50 = 4.2
±
0.11 μg/mL). In addition, statistical analysis showed that there is no effect of region on the activities of the methanol extracts.


## 3.7. Antibacterial Activity

The test results of the antibacterial effect are summarized in Table5. The results show that the diameter of the inhibition zone is not affected by the region factor. The essential oils of wormwood at different regions showed an interesting antibacterial activity. Indeed, the highest activity was observed againstStaphylococcus aureus strain with a diameter of inhibition equal to 25
±
1.13 mm for the essential oil of Kairouan; this strain had a lower sensitivity to the EO of Bou Salem region (IZ = 18
±
1.13 mm). Essential oils of Boukornine and Jérissa regions show a moderately antibacterial activity (IZ = 20.66 mm). In agreement with our results, the EO of wormwood aerial parts growing in Canada has a high activity againstStaphylococcus aureus (IZ = 25
±
1.4 mm) which was greater than the positive control (methicillin activity IZ = 18
±
1.0 mm) [34]. Essential oils of Bou Salem and Kairouan have the same activity againstListeria monocytogenes (IZ = 20 mm), but the EO of Jérissa and Boukornine is less active (IZ = 18 mm). These results show that wormwood essential oil was endowed with a significant antibacterial activity that is closely related to the organoleptic quality of the oil, which in turn depends strongly on the collection region of plant material.Table 5
Antibacterial (IZ mm) and antifungal (%I) activities of wormwood essential oil.
Boukornine
Jérissa
Kairouan
Bou Salem
df
F
P
Tetracycline
Nystatin
Bacteria
Staphylococcus aureus 25923
20.66  ±  2.61b
20.66  ±  0.65b
25  ±  1.13a
18  ±  1.13c
3
2.05
0.184NS
25  ±  2.56
Staphylococcus methicillin-resistant
15.00  ±  1.13b
14.33  ±  0.65c
16  ±  1.30a
14.33  ±  0.65c
3
0.21
0.879NS
25  ±  2.54
Listeria monocytogenesATCC 19195
18.66  ±  2.35b
17.33  ±  1.72c
20.00  ±  1.95a
20.00  ±  1.13a
3
0.53
0.674NS
24  ±  2.42
Fungi
Fusarium graminearum
14.66  ±  0.57c
23.65  ±  0.57a
11.11  ±  0.12d
16.66  ±  0.57b
3
18.57
0.000***
25  ±  2.31
Fusarium culmorum
45.23  ±  3.11b
23.65  ±  0.57d
42.06  ±  0.57c
46.82  ±  0.57a
3
32.77
0.000***
25  ±  2.14
Fusarium oxysporum
19.04  ±  0.57c
27.58  ±  2.55a
17.94  ±  0.57d
25.28  ±  0.57b
3
7.79
0.009**
20  ±  1.67
Sclerotinia
nd
23.61  ±  2.12
nd
nd
3
185.81
0.000***
22  ±  2.01
Rhizoctonia solani
nd
25.39  ±  0.57
nd
nd
3
77.35
0.000***
20  ±  1.78
Results are the mean of three replications. The diameter of disc was 6 mm. Values with different superscripts (a–d) are significantly different atP
<
0.05. nd: not detected. NS: not significant. df: degree of freedom, F: Fisher value, and P: probability. *
*
P
<
0.01. *
*
*
P
<
0.001.


## 3.8. Antifungal Activity

The results of the antifungal activity of essential oils of absinthe are summarized in Table5. Wormwood EOs had a significant inhibitory activity against the three phytopathogenicF. graminearum,F. culmorum, andF. oxysporum while only EO of Jérissa proved to be active againstSclerotinia (23.61
±
2.12%) andRhizoctonia solani (25.39
±
0.57%). As for the antibacterial activity, antifungal activity could be attributed to the major component of essential oil of wormwood: chamazulene [56]. Meanwhile, it has been shown that the chamazulene possesses significant antifungal activity [57]. The antimicrobial effect of EOs of absinthe gives them an important role in the fields of food, cosmetics, and pharmaceutical industries. In additionA. absinthium L. EO from France containing(Z)-epoxyocimene and chrysanthenyl acetate as major components inhibited the growth of both the yeastsCandida albicans andSaccharomyces cerevisiae var. chevalieri [4]. Wormwood EO from a Turkish population, whose main components are camphor, 1,8-cineole, and chamazulene, has been described as fungicidal against 34 species of fungi includingF. solani andF. oxysporum [3]. Furthermore,A. absinthium EO from Uruguay rich in thujone showed antifungal effects againstAlternariasp. andBotrytis cinerea [58]. However, further studies are needed to identify the compound(s) responsible for the antifungal effects ofA. absinthium L. EOs.



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*Source: 804658-2015-03-08.xml*