Essential Oil Composition and Antimicrobial Activity of Artemisia herba–alba Asso Grown in Algeria

In the recent decades, antimicrobial plant products have gained special interest because of the resistance to antibiotics that some micro-organisms have acquired. Aromatic and medicinal plants are an important source of bioactive molecules, especially in volatile extracts, that are considered among the most important antimicrobial agents present in these plants. Volatile components of Artemisia herba-alba Asso essential oil obtained by hydrodistillation growing in Algeria (Djelfacityof south Algeria) were investigated by GC/FID and GC-MS. The major components were found to be camphor (39.5%), chrysanthenone (10.38%), 1,8-cineole (8.6%), α-thujone (7.03%), Borneol (3.35%) and bornyl acetate (2.52%). The essential oil has been tested for antimicrobial activity against Gram-negative and Gram-positive bacteria. Inhibition of growth was tested by the agar diffusion method. The Minimal Inhibitory Concentration (MIC) was determined by the method of agar dilution.


Introduction
In the last few years, due to the misuse of antibiotics and an increasing incidence of immunodeficiency-related diseases, the development of microbial drug resistance has become more and more of a pressing problem [1]. Recently, natural products from aromatic and medicinal plants represent a fertile ground for the development of novel antibacterial agents [2]. Plants essential oils have come more into the focus of phytomedicine [2,3]. It is important to develop a better understanding in basic research applications, especially, of the anti-microbial activity of essential oils [4]. The Mediterranean region is relatively rich with plants (between 15,000 and 20,000 species). Algeria, a North African country with a large variety of soils (littoral, steppe, mountains and desert) and climates, possesses a rich flora (more than 3,000 species and 1,000 genders) [5].
In this context and in order to enhance our diverse and rich national heritage, we are interested in the study of Artemisia herbaalba Asso greenish-silver perennial herb, grows 20 to 40 cm in height and belongs to the daisy family Asteraceae [6]. In Algeria, this plant commonly known as the white wormwood in Arabic as "Chih" and in France as "Armoisebalnche" [7], is one of five spontaneous Artemisia species that were identified [8] and was used as aromatisant for tea. It has been used in folk medicine by many cultures since ancient times, to treat colds, coughing, bronchitis, intestinal disturbances, diarrhea, neuralgias arterial hypertension and/or diabetes [9][10][11]. Many researchers have reported various biological and/or pharmacological activities of Artemisia herba-alba Asso essential oil as an antimicrobial, antioxidant, antidiabetic, Antileishmanial, anthelmintic and antispasmodic agent [6,[12][13][14][15][16].
The aim of the present investigation was to study the antibacterial effect of Artemisia herba-alba Asso essential oil on different bacterial species. This was done through tow techniques: the agar diffusion assay as well as the agar dilution method was used for determination of Minimum Inhibitory Concentration (MIC). In addition, the compositions of volatile compounds were determined to use these data to deduce which components are likely to contribute to the activities of the whole oils and to determine any structural relationships between the components and their antibacterial activity.

Plant material
The aerial parts of Artemisia herba-alba Asso were collected on April 2013 from Djelfa, region with a semi-arid climate, located right in the heart of the steppe zone, 300 km south of Algiers. It is the last town before the Saharan Atlas and the desert. (Coordinates: Latitude 33°-35° N, longitude 2°-5° E). After been harvested, the fresh vegetable matter was first weighted and then dried on the shadow, until weight stability. Then the leaves were separated from stems.

Essential oil extraction
Air-dried leaves and flowers were submitted to hydro distillation for 3 h, using a Clevenger type apparatus [17], according to the European Pharmacopoeia [18]. The oil yield was expressed v/w vs. dry matter. The essential oil was dried over anhydrous sodium sulphate, filtered and stored in a sealed vial in the dark at +4°C before analysis and bioassays tests.

Mass spectrometry analysis
The oil was analyzed by Gas Chromatography-Mass Spectrometry (GC-MS) using a Hewlett Packard 6890 mass selective detector coupled with a Hewlett Packard 6890 gas chromatograph equipped with a 30 m × 0.25 mm HP-5 (cross-linked Phenyl-Methyl Siloxane) column with 0.25 µm film thickness (Agilent), Helium was used as carrier gas, the flow through the column was 1.4 ml/min, and the split less mode was used. The column temperature was programmed from 35 to 85°C at 20°C/min, increased from 85 to 300°C at rate of 5°C/min and finally held for 10 min.
α-thujone little present in our essential oils with 7.03%, it is the major component of the essential oil of Tunisia (BirElhfay) [6] and Jordan [31].
The MS operating parameters were as follows: ionization potential, 70 eV; ionization current, 2 A; ion source temperature, 200°C, resolution, 1000. Mass unit were monitored from 30 to 450 m/z. Identification of components in the oil was based on retention indices relatives to n-alkanes and computer matching with the WILLEY 275.L library, as well as by comparison of the fragmentation patterns of mass spectra with those reported in the literature Adams.

Antimicrobial activity
The essential oils were individually tested against Gram positive bacteria and Gram negative bacteria. Bacterial strains used in this study were obtained from American Type Culture Collection (ATCC), USA: Pseudomonas aeruginosa ATCC12228, Staphylococcus aureus ATCC 25923, Klebsiella pneumoniae (ATCC 13883), Bacillus cereus (ATCC, 11778) and Escherichia coli ATCC125922.

Antibacterial screening
Two techniques were used to test the microbial activity of Artmisiaherba-alba Asso: Agar diffusion method: Antibacterial activities of Artmisiaherbaalba Asso essential oil were assessed using the paper disk agar diffusion method according to Imelouane et al. [19] with some modifications. A 16-h culture was diluted with sterile physiological saline solution with reference to the MC Farland 0.5 standard to achieve an inoculum of approximately 10 6 CFU/ ml. A suspension was swabbed in three directions on 4 mm thick Mueller Hinton agar (MHA). Absorbent disk (Whatman disk N o . 4 of 6 mm diameter) containing 15 µl of filter sterilized test essential oil were applied on the surface of the plate (90 mm) inoculated with different microbial strains. The plates were then incubated for 24 h at 37°C. Negative control was prepared using a disk impregnated with sterile water. Finally, antimicrobial activity was evaluated by measuring the diameter (mm) of the growth inhibition zones including the 6 mm disk. The measurements of inhibition zones were carried out for three sample replications.

Agar dilution method: The Minimum Inhibitory Concentration
(MIC) of the tested essential oil was determined using the agar dilution method approved by Bansod and Rai [20] with the following modification: Tween-80 was incorporated into the agar after autoclaving to enhance oil solubility. Briefly, Petri plates containing various concentrations of essential oil 0.003 to 2.5% (v/v) were inoculated with each tested strain. Plates were dried at 35°C prior to inoculation with 1-2 ml spots containing approximately 10 5 CFU of each organism. Un-inoculated plates containing essential oil served as negative control. Tween-80 was used as a positive growth control. Test and control plates were then incubated for 24 h for pathogenic bacterial strains at 37°C. Plates were evaluated for the presence or the absence of colonies after incubation. For each treatment, the absence of colonies on all plates tested was considered as an inhibitory effect. The lowest concentration of essential oil required to completely inhibit the growth of the tested microorganism was designated as the MIC [21].

Results and Discussion
Hydro distillation of the leaves of Artemisia herba-alba Asso yielded yellow liquid oil with a strong penetrating pleasant herbaceous odor characteristic of the plant. The oil yield was 0.8%. The chemical composition of the oil was investigated using GC/ FID and GC/MS techniques. The percentages and the retention indices of the identified components are listed in Table 1 in the order of their elution on the HP-5MS column. From the data obtained, 31 compounds were identified, representing 92.84% of the oil.   Maximum activity was observed against S. aureus with MIC of 0.1 mg/ml to the oil. The essential oil was evaluated for antimicrobial activity against pathogenic strains of Gram positive (S. aureus) and Gram negative (E. coli, P. aeruginosa, K. pneumoniae) bacteria (Tables  2 and 3). It was found to be active against all the bacterial strains except for P. aeruginosa (no inhibition zone was observed). The essential oils evaluated in this work have a great variety of phytochemicals that could be considered as responsible for a larger or smaller part of the antimicrobial activity. The antimicrobial activity of Artemisia herbaalba Asso essential oil would be related to its oxygenated monoterpenes components [35] which constitute about 77.78% of the oil.
Research into the antimicrobial actions of monoterpenes suggests that they diffuse into and damage cell membrane structures [48].
Indeed, in essential oils, it was shown that monoterpenes hydrocarbons and oxygenated monoterpenes in essential oils are able to destroy cellular integrity resulting in respiration inhibition and permeability alteration [49]. Besides, the most abundant component in essential oil of A. herba alba, camphor, has been reported to exhibit bacteriostatic activity against P. aeruginosa [19], and this compound is a major constituent in a number of antibacterial essential oils [50,51]. The major components of Artemisia herba-alba Asso essential oils, such as the monoterpenoids thujone, camphor, 1-8 cineole, camphene, are known for their potential antimicrobial properties against both gram-positive and gram-negative bacteria [52][53][54].
In addition, other minor components such as borneol (4.88%) have been also reported to have antimicrobial potential [13]. In other studies, α-pinene, has been known to exhibit antimicrobial activity against the bacterial strains (E. coli, P. aeruginosa, S. aureus, bacillussubtilis) [1]. In fact, the biological effectiveness of essential oil is related to their different chemical constituents (major, minor and their mutual ratios) acting either synergistically or antagonistically with major components [55,56]. In general, the antimicrobial activity of the essential oils tested was more pronounced against Gram-positive than against Gramnegative bacteria [57].
This generally higher resistance among Gram-negative bacteria, According to Lambert [58], Harris [59] and Bezic et al. [60], could be ascribed to the structure of the cell wall of gram-negative bacteria primarily made up of a lipopolysaccharide that blocks the penetration of hydrophobic compounds and prevents the accumulation of essential oils in the membrane of target cells. The absence of this barrier in Gram-Similarly, Boukrich et al. [32] have shown that α-thujone is the main compound of wormwood from semi-arid and arid areas of Tunisia.
According Akrout et al. [33] and Mighri et al. [13], the essential oil of wormwood from the Beni-Khedache region (southern Tunisia) and the region of Kirchaou (southeast Tunisia) is characterized by the dominance of β-thujone. Moreover Zaim et al. [34] showed that the essential oil of Artemisia herba-alba Asso. South of Morocco (Ouarzazate) contains chrysanthenone as major compound. Cischrysanthenyl acetate was found to be the major component in some oils from Tunisia (Gafsa) and Algeria (Biskra) [35,36] (Table 1). Davanon was reported as constituting major in the essential oil of some chemo types of Spain and Algerian (Djelfa) species [37,38].
Similarly, Dahmani-Hamzaoui and Baaliouamer [39] identified the davanon in the essential oil of sagebrush Djelfa. Tilaoui et al. [40] showed that the oil sagebrush of Imilchil ErRachidia (Morocco) contains the verbenol as major compound. The eucalyptol is identified as the major constituent in the essential sagebrush oil from southern Spain (41% maximum) [37] and Egypt [41]. The chemical composition of Artemisia herba-alba Asso essential oils shows a large interspecies variability and, within the same species. Various compositions dominated either by a single component (α-thujone, camphor, chrysanthenone or transsabinyl acetate) or characterized by the occurrence, of two or more of these compounds at appreciable contents [32].
Chemical variability of Artemisia herba-alba Asso seems to depend on the genetic characteristics of the plant [42], geographical locations, consequently different climatic conditions under which it has grown [13,24], part of the plant, stadephénologique, and the method used to obtain the essential oil [43][44][45][46]. In fact, these factors influence the plant's biosynthetic pathways and, consequently, the relative proportion of the main characteristic compounds [47]. The in vitro antimicrobial activity of Artemisia herba-alba Asso essential oil against the microorganisms employed and its activity potentials were qualitatively and quantitatively assessed by the presence or absence of inhibition zones, zone diameters and MIC values. Table 2 reports the inhibition zone of essential oil determined for 24 of Gram positive or Gram negative bacteria using the diffusion technique on solid media.
The data shows that this oil had variable antimicrobial activity against all tested strains. The inhibition zones were in the range of 15-33 mm. Gram positive bacteria were shown to be more sensitive to the Artemisia herba-alba Asso essential oil. The data indicated that Gram-positive S. aureus was the most sensitive strain tested to the oil of Artemisia herba-alba Asso with the strongest inhibition zone (33.00 ± 0.45 mm). Among these, Gram-negative strains also displayed variable degree of susceptibility against investigated oil. Maximum activity was E. coli (19.00 ± 0.55). Modest activities were observed against K. pneumoniae, with inhibition zones of (15 ± 0.54 mm) ( Table 2). P. aeruginosa was considered resistant since no inhibition zone was observed. It is known to have high level of intrinsic resistance to virtually all known antimicrobials and antibiotics due to a combination of a very restrictive outer membrane barrier which is highly resistant even to synthetic drugs. The in vitro activity of Artemisia herba-alba Asso essential oil was evaluated by a broth micro dilution method using a panel of micro-organisms. Antimicrobial activity was expressed as Minimum Inhibitory Concentration (MIC). The results of the MIC are in Table 3. The data indicate that the oil exhibited varying levels of antimicrobial activity against the investigated food pathogens. The inhibitory properties of the oil were observed within a range of concentrations from 0.1 to 21.00 mg/ml. The essential oil was active against all the test strains. The Gram-negative P. aeruginosa seemed to be resistant to the investigated oil with a MIC of 21.00 mg/ml. positive bacteria allows the direct contact of the essential oils hydrophobic constituents with the phospholipid bilayer of her cell membrane, where they bring about their effect, causing either an increase of ion permeability and leakage of vital intracellular constituents, or impairment of the bacterial enzyme systems [61,62] (Table 3).
Although the antibacterial activity of essential oils from many plant species has been extensively surveyed, their antimicrobial mechanism has not been reported in great detail. Since the active antimicrobial compounds of essential oils are phenolics and terpenes, it seems reasonable that their mode of action might be similar to that of other phenolic compounds. According to Burt [63], given the large number of different groups of compounds present in essential oils, the antibacterial activity of essential oil is most likely not attributable to a specific mechanism but to several mechanisms related to various targets in the cell.
Most of the studies on the mechanism of phenolic compounds focused on their effects on cellular membranes, altering its response to antimicrobial challenge. These effects may develop as a result of membrane depolarization by altered ion transport or through changes in the membrane structure, inhibition of energy (ATP) generation by interference with glucose uptake or inhibition of enzymes involved in oxidative or substrate level phosphorylation. Increases in cytoplasmic membrane permeability appear to be a consequence of the loss of the cellular pH gradient, decreased ATP levels and, loss of the proton motive force, which lead to cell death [64].
The antimicrobial activity and, consequently, the minimum inhibitory concentration of essential oils can be influenced by the growing region of the plant, the extraction method used, the plant part used (leaf or whole plant), the method of preparation of the raw material (fresh or dry), the type of organism, the cultivation conditions (incubation time, temperature, oxygen), the culture medium, the concentration of the test substance and the solvents used to dilute the oil, among other factors [63,[65][66][67][68][69][70][71].

Conclusions
The composition of the essential oil of Artemisia herba-alba Asso growing in Algeria has been analyzed and its antimicrobial activity investigated. The results indicate that the oil may be used in the treatment of diseases caused by the micro-organisms tested. Further toxicological and clinical studies are required to prove the safety of the oil as a medicine.