Chemical composition , antimicrobial activity , antioxidant and total phenolic content within the leaves essential oil of Artemisia absinthium L . growing wild in Iran

Water-distilled essential oil from the leaves of Artemisia absinthium L. collected from Ardabil, northwestern Iran, was analyzed by Gas chromatography and mass spectrometry (GC and GC-MS). In the leaf oil of A. absinthium, 19 components, which represented 100% of the total composition were identified. 1,8-Cineole (36.46%), borneol (25.99%) and camphor (10.20%) were the major components in this oil. The leaves of A. absinthium was investigated to analyze their antimicrobial activity, antioxidant activity and total phenolic content. The present study revealed that the leaf essential oil of A. absinthium indicated significant activity against Candida albicans. Killing kinetics of various microorganisms treated with leaf oil of A. absinthium indicated that C. albicans is the most vulnerable. The total phenol contents of the leaf oil of A. absinthium was determined to be 168.67 ± 9.50 μg gallic acid equivalent/mg sample. Antioxidative properties of the leaves essential oil of A. absinthium was determined by 3 methods: The Ferric-reducing antioxidant power (FRAP), radical-scavenging capacity of the oil or bleaching of 2,20-diphenylpicrylhydrazyl (DPPH) and β-Carotene-linoleic acid assay. The ferric reducing power of the essential oils was determined to be 10.67 ± 0.45 gallic acid equivalent (mg/g). The leaf essential oil of A. absinthium reduced the concentration of DPPH free radical (61.4 ± 1.4%, 10 mg/ml of essential oil) with an efficacy lower than that of reference oil Thymus x-porlock (69.3% inhibition). IC50 for DPPH radical-scavenging activity was 5.85 μg/ml. In β-carotene-linoleic acid test system, oxidation of linoleic acid was effectively inhibited by A. absinthium oil (58.56 ± 2.5%, amount of essential oil 0.625 mg/ml). The results suggest application of A. absinthium oil as a natural antioxidant agent.


INTRODUCTION
Artemisia is a genus of small herbs or shrubs found in northern temperate regions.It belongs to the important family compositae (Asteraceae) (Rechinger, 1986).Within this family, Artemisia is included into the trible Anthemideae and comprises itself of over 500 species (Mozaffarian, 1996).The genus Artemisia has always been of great botanical and pharmaceutical interest and is useful in traditional medicines for the treatment of a variety of diseases and complaints (Rustaiyan and Masoudi., 2011;Firouzni et al., 2008).Some Artemisia species has been investigated chemically and the presence of monoterpenes, sesquiterpenes, specially sesquiterpene lactones and chemical composition of essential oil reported.
The effectiveness of wormwood as an aromatic bitter and its antimicrobial properties come from the bitter compounds and its essential oil.Extracts of the plant have shown to exhibit strong antimicrobial activity, especially against Gram-positive pathogenic bacteria (Fiamegos et al., 2011).The oil of the plant can be used as a cardiac stimulant to improve blood circulation.Pure wormwood oil is very poisonous, but with proper dosage poses little or no danger (Lust, 1979).The oil is a potential source of novel agents for the treatment of leishmaniasis (Tariku et al., 2011).Although A. absinthium have been used as folk remedies to treat various ailments in medicine, as yet there has been little attempts made to study the antioxidant and antimicrobial potential of these plants against a wide range of microorganisms.As such, the aims of this study were to estimate the total phenolic content, antioxidant activity and antimicrobial activity of A. absinthium.The present study deals with the chemical composition, antibacterial, antioxidative and radical-scavenging properties of the essential oil of A. absinthium obtained by steamdistillation.

Isolation of the essential oil
The leaves of A. absinthium were dried at room temperature for several days.Air-dried leaves of A. absinthium (110 g) were separately subjected to hydrodistillation using a clevenger-type apparatus for 3 h.After decanting and drying of the oil over anhydrous sodium sulfate, the oil was recovered.Results showed that essential oil yield was 1.05% (w/w).

Analysis of the essential oil
The composition of the essential oil obtained by hydrodistillation from the leaves of A. absinthium was analyzed by GC and GC/MS.Identification of the constituents of oil was achieved by comparison of their mass spectra and retention indices with those reported in the literature and those of authentic samples (Adams, 2001).

Gas chromatography
GC analysis was performed on a Schimadzu 15 A gas chromatography equipped with a split/splitless injector (250°C) and a flame ionization detector (250°C).Nitrogen was used as carrier gas (1 ml/min) and the capillary column used was DB-5 (50 m × 0.2 mm, film thickness 0.32 μm).The column temperature was kept at 60°C for 3 min and then heated to 220°C with a 5°C/min rate and kept constant at 220°C for 5 min.Relative percentage amounts were calculated from peak area using a Schimadzu C-R4A chromatopac without the use of correction factors.

Gas chromatography-mass spectroscopy
GC-MS analysis was performed using a Hewlett-Packard 5973 with a HP-5MS column (30 m × 0.25 mm, film thickness 0.25 μm).The column temperature was kept at 60°C for 3 min and programmed to 220°C at a rate of 5°C/min and kept constant at 220°C/min for 5 min.The flow rate of Helium as carrier gas was 1 ml/min.MS were taken at 70 eV.The retention indices for all the components were determined according to the Van Den Dool method, using nalkanes as standards.The compounds were identified by (RRI, DB5) with those reported in the literature and by comparison of their mass spectra with the Wiley library or with the published mass spectra (Adams, 2001).

Oil dilution solvent
Bacterial strains were streaked on Mueller Hinton agar plates using sterile cotton swabs.Five microlitres of dimethylsulphoxide (DMSO), loaded on sterile blank disks, were placed on the agar plates and were incubated at 37°C for 24 h.There was no antibacterial activity on the plates and hence DMSO was selected as a safe diluting agent for the oil.Five microlitres from each oil dilution, followed by sterilization, using a 0.45 µm membrane filter, were added to sterile blank discs.The solvent also served as control.

Microbial strain and growth media
Escherichia coli (ATCC25922), Staphylococcus aureus (ATCC25923), Pseudomonas aeruginosa (ATCC8830), Candida albicans (ATCC 5027) and Acinetobacter baumannii (ATCC 17978) were employed in the study.Nutrient agar was used.Bacterial suspensions were made in brain heart infusion (BHI) broth to a concentration of approximately 10 8 cfu/ml.Subsequent dilutions were made from the above suspension, which were then used in the tests.

Oil sterility test
In order to ensure sterility of the oils, geometric dilutions ranging from 0.036 to 72.0 mg/ml of the essential oil, were prepared in a 96well microtitre plate, including one growth control (BHI + Tween 80) and one sterility control (BHI + Tween 80 + test oil).Plates were incubated under normal atmospheric conditions, at 37°C for 24 h.The contaminating bacterial growth, if at all, was indicated by the presence of a white ''pellet'' on the well bottom.

Disc diffusion method
The agar disc diffusion method was employed for the determination of antimicrobial activities of the essential oils in question.Briefly, 0.1 from 10 8 cfu/ml bacterial suspension was spread on the Mueller Hinton Agar (MHA) plates.Filter paper discs (6 mm in diameter) were impregnated with 5 µl of the undiluted oil and were placed on the inoculated plates.These plates, after remaining at 4°C for 2 h, were incubated at 37°C for 24 h.The diameters of the inhibition zones were measured in millimeters.All tests were performed in triplicate.

Determination of minimum inhibitory (MIC) and bactericidal (MBC) concentrations
All tests were performed in brain heart infusion (BHI) broth supplemented with Tween 80 detergent (final concentration of 0.5% (v/v)).Test strains were suspended in BHI broth to give a final density of 10 7 cfu/ml and these were confirmed by viable counts.The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were assessed according to our modified procedure (Rasooli and Mirmostafa, 2003).MIC was determined by a broth dilution method in test tubes as follows: 40 µl from each of various dilutions of the oils were added to 5 ml of brain heart infusion (BHI) both in tubes containing 10 7 cfu/ml of live bacterial cells.The tubes were then incubated on an incubator shaker to evenly disperse the oil throughout the broth in tubes.The highest dilution (lowest concentration), showing no visible growth, was regarded as the MIC.Cell suspensions (0.1 ml) from the tubes showing no growth were subcultured on BHI agar plates in triplicate to determine if the inhibition was reversible or permanent.MBC was determined as the highest dilution (lowest concentration) at which no growth occurred on the plates (Wayne, 2008;Akomo et al., 2009).

Bactericidal kinetics of the oil
Forty microlitres of each oil at the dilution determined by MBC was added to each 5 ml of brain heart infusion (BHI) broth in tubes containing bacterial suspension of 10 7 cfu/ml and were then incubated at 37°C in an incubator shaker.Samples (0.1 ml) were taken after 5, 10, 15, 20, 25, 30, 45, 90, 120, 150, 180, 210 and 240 min.The samples were immediately washed with sterile phosphate buffer, pH 7.0, centrifuged at 10,000 rpm/1 min, resuspended in the buffer and were then spread-cultured on BHI agar for 24 h at 37°C.Phosphate buffer was used as diluent when needed.Bactericidal experiments were performed three times.Microbial colonies were counted from triplicates after the incubation period and the mean total number of viable cells per ml was calculated.The mean total number of viable bacteria from bactericidal kinetics experiments at each time interval was converted to log 10 viable cells using routine mathematical formulae.The trend of bacterial death was plotted graphically (Yadegarinia et al., 2006)

Ferric-reducing antioxidant power (FRAP) assay of the oil
The FRAP assay was carried out according to the procedure employed by (Lim et al., 2009).One millilitre of the extract dilution was added to 2.5 ml of 0.2 M potassium phosphate buffer (pH 6.6) and 2.5 ml 1% potassium ferricyanide.The mixture was incubated for 20 min at 50°C, after which 2.5 ml of 10% trichloroacetic acid was added.The mixture was then separated into aliquots of 2.5 ml and mixed with 2.5 ml of deionised water.Then, 0.5 ml of 0.1% (w/v) FeCl 3 were added to each tube and allowed to stand for 30 min.Absorbance for each tube was measured at 700 nm.The FRAP was expressed as gallic acid equivalents (GAE) in mg/g of samples used (y = 16.66x+ 0.003; r 2 = 0.999).

Radical-scavenging capacity of the oil
The hydrogen atom or electron donation abilities of the corresponding extracts and some pure compounds were measured from the bleaching of the purple-coloured methanol solution of 2,2diphenylpicrylhydrazyl (DPPH).This spectrophotometric assay uses the stable radical DPPH as a reagent (Burits and Bucar, 2000;Cuendet et al., 1997).Fifty microlitres of 1:5 concentrations of the essential oils in methanol were added to 5 ml of a 0.004% methanol solution of DPPH.Trolox (1 mM) (Sigma-Aldrich), a stable antioxidant, was used as a synthetic reference.The essential oil from Thymus x-porlock was used as a natural reference.After a 30 min incubation period at room temperature, the absorbance was read against a blank at 517 nm.Inhibition of free radical by DPPH in percent (I%) was calculated in following way: Where Ablank is the absorbance of the control reaction (containing all reagents except the test compound), and Asample is the absorbance of the test compound.Tests were carried out in triplicate.

β-carotene-linoleic acid assay
Antioxidant activity of essential oils was determined using the βcarotene bleaching test (Taga et al., 1984).Approximately 10 mg of β-carotene (type I synthetic, Sigma-Aldrich) was dissolved in 10 ml of chloroform.The carotene-chloroform solution, 0.2 ml, was pipetted into a boiling flask containing 20 mg linoleic acid (Sigma-Aldrich) and 200 mg Tween 40 (Sigma-Aldrich).Chloroform was removed using a rotary evaporator at 40°C for 5 min and, to the residue, 50 ml of distilled water were added slowly with vigorous agitation to form an emulsion.Five millilitres of the emulsion were added to a tube containing 0.2 ml of essential oil solution prepared (Choi et al., 2000) and the absorbance was immediately measured at 470 nm against a blank consisting of an emulsion without βcarotene.The tubes were placed in a water bath at 50°C and the oxidation of the emulsion was monitored spectrophotometrically by measuring absorbance at 470 nm over a 60 min period.Control samples contained 10 µl of water instead of essential oils.Butylated hydroxy anisole (BHA; Sigma-Aldrich), a stable antioxidant, was used as a synthetic reference (Miraliakbari and Shahidi, 2008).The antioxidant activity was expressed as inhibition percentage with reference to the control after 60 min of incubation, using the following equation:

Total phenolic content assay
Total phenol content was estimated as gallic acid equivalents Taherkhani et al. 33 (GAE; mg gallic acid/g extract) as described earlier.In brief, a 100 μl aliquot of dissolved extract was transferred to a volumetric flask containing 46 ml distilled H 2 O, to which was subsequently added 1 ml Folin-Ciocalteu reagent.After 3 min, 3 ml of 2% Na 2 CO 3 was added.After 2 h of incubation at 25°C, the absorbance was measured at 760 nm.Gallic acid (Sigma Co, 0.2 to 1 mg/ml gallic acid) was used as the standard for the calibration curve, and the total phenolic contents were expressed as mg gallic acid equivalents per gram of tested extracts (Y = 0.001x + 0.0079; r 2 = 0.9967) (Kahkonen et al., 1999)

Chemical composition of the essential oil
In the leaf oil of A. absinthium collected from Ardabil, north-western Iran, 19 components, which represented 100% of the total composition were identified.1,8-Cineole (36.46%), borneol (25.99%) and camphor (10.20%) were the major components in this oil (Table 1).

Antibacterial Activity
As can be seen in

Total Phenolic and Content Antioxidant
The total phenol contents of the leaf oil of A. absinthium was determined to be 168.67 ± 9.50 μg gallic acid equivalent/mg sample.Antioxidative properties of the leaves essential oil of A. absinthium was determined by 3 methods: The Ferric-reducing antioxidant power (FRAP), Radical-scavenging capacity of the oil or bleaching of 2,20-diphenylpicrylhydrazyl (DPPH) and β-carotenelinoleic acid assay.The ferric reducing power of the essential oils was determined 10.67 ± 0.45 gallic acid equivalent (mg/g).The leaf essential oil of A. absinthium reduced the concentration of DPPH free radical (61.4 ± 1.4%, 10 mg/ml of essential oil) with an efficacy lower than that of reference oil T. x-porlock (69.3% inhibition).

Chemical composition of the essential oil
The composition of the essential oil obtained by hydrodistillation from the leaves of A. absinthium, from Iran analyzed by GC and GC/MS, is listed in Table 1.The percentage and retention indices of components are given.As it is shown in Table 1, in A. absinthium oil, 19 components represented about 100% of the total oil, were identified.1,8-Cineole (36.46%), borneol (25.99%), camphor (10.20%) were the major component in this oil.
The other main components of leaf oil of A. absinthium were p-menth-2-en-1-ol (6.20%), terpine-1-ol (4.42%) and 4-terpineol (2.72%).As it is shown in Table 1, the largest part of the leaf oil of A. absinthium essential oil was formed by oxygenated monoterpenes (99.51%).The results proved that chemotype of the studied wormwood essential oil was specific and different from other wormwood essential oil chemotypes, which have been reported so far.
In another investigation on chemical composition of essential oil from the leaves of A. absinthium collected from the region of Guigou and Errachidia, α-Thujone (39.69), sabinyl acetate (10.96) and β-thujone (7.25) were the major components in this oil (Derwich et al., 2009).On the other hand, the oils of A. absinthium of French origin contained (Z)-epoxyocimene and chrysanthenyl acetate as major components while the oils of croatian A. absinthium contained mainly (Z)-epoxyocimene and β-thujone (Juteau et al., 2003).Four chemotypes were found to be characteristic of A. absinthium growing in different geographical areas of Europe: sabinene and myrcene rich oil, α-and β-thujone rich oil, epoxyocimene rich oil, and (E)-sabinyl acetate rich oil.Some mixed chemotypes were also found (Orava et al., 2006).The essential oil of A. absinthium from western Canada was characterized by high amounts of myrcene (10.8%), trans-thujone (10.1%) and trans-sabinyl acetate (26.4%) (Lopes-Lutz et al., 2008).Bornyl acetate (23.02%) was the major constituent in the essential oil of A. absinthium collected from Cuban origin (Pino, 1997).On the other hand, sabinene (17.56%) was the main constituent in essential oil from the aerial part of A. absinthium collected from Turkey (Erel et al., 2012).
As shown, the concentration of the active constituents is seasonally and geographically different and some genotypes are characterised by particularly high contents of active essential oil constituents.Oxygenated monoterpenes (99.51%), were the main components in the the leaves of A. absinthium collected from Iran.

Antibacterial activity
The antibacterial activities of A. absinthium oil was assayed against five bacteria and results presented in Table 2.The antibacterial activities of the essential oils were evaluated by disc diffusion method using Muller-Hinton Agar for bacteria with determination of inhibition zones (IZ), minimal inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and decimal reduction time (D-value).The present study revealed the leaf essential oil of A. absinthium collected from Iran, indicated significant activity against C. albicans and moderate inhibitory activity against S. aureus.On the other hand, antimicrobial screening was performed on samples of French origin and showed that A. absinthium oil inhibited the growth of both tested yeasts C. albicans and Saccharomyces cerevisiae var.chevalieri (Juteau et al., 2003), while the extract of A. absinthium collected from Eastern Anatolia region of Turkey, also showed antibacterial activity against all tested microorganisms (6 to 19 mm inhibition zone), apart from Alcaligenes feacalis and Aspergillus niger (Erel et al., 2012).

Bactericidal kinetics of the oils
Table 2 shows reduction times of E. coli, S. aureus, P. aeruginosa, C. albicans and A. baumannii, respectively, after exposure to the MBC levels of the oils.It can be concluded that C. albicans is the most vulnerable to the oil under study.These values suggest the duration of time required for complete bactericidal effects of the oils.

Total phenol contents
As shown in Table 3, the total phenol contents (TPC) of the leaf essential oil of A. absinthium was determined to be 168.67 ± 9.50 μg gallic acid equivalent/mg sample (GAE/mg).Phytochemical investigation determined that extract of A. absinthium collected from Golestanak protege area central Elburz showed high phenolic and flavonoid contents (Mahmoudi et al., 2009).The high contents of total phenolic compounds (25.6 mg g −1 ) and total flavonoids (13.06 mg g −1 ) indicated that these compounds contribute to the antiradical and antioxidative activity (Canadanovic-Brunet et al., 2005).

Antioxidant activity
Antioxidative properties of the extract was determined by bleaching of β-carotene or 2,20-diphenylpicrylhydrazyl (DPPH).The Ferric-reducing antioxidant power (FRAP) was expressed as gallic acid equivalents or known Fe(II) concentration for A. absinthium essential oil.The DPPH Taherkhani et al. 35 radical-scavenging activities of the leaf essential oil of A. absinthium are shown in Table 3.The leaf essential oil of A. absinthium notably reduced the concentration of DPPH free radical.IC 50 for DPPH radical-scavenging activity was 5.85 μg/ml.In phytochemical investigation, the antiradical activity of A. absinthium, collected from Serbia, was tested by measuring their ability to reactive hydroxyl radical during the Fenton reaction trapped by 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), using electron spin resonance (ESR) spectroscopy.Results demonstrated that the antiradical activity depend on the type and concentration of applied extracts and increased in the order ethyl acetate > methanol > n-butanol > chloroform > petroleum ether > remaining water extracts (Canadanovic-Brunet et al., 2005).

Ferric-reducing antioxidant power (FRAP) assay of the oil
The FRAP assay was expressed as gallic acid equivalents (GAE) in mg/g of samples used (y = 16.66x+ 0.003; r 2 = 0.999).The ferric reducing power of the essential oils was determined 10.67 ± 0.45 gallic acid equivalent (mg/g).

Free radical-scavenging capacities of the oils
The DPPH radical-scavenging activities of the essential oil are shown in Table 3.The leaf essential oil of A. absinthium reduced the concentration of DPPH free radical (61.4 ± 1.4%, 10 mg/ml of essential oil) with an efficacy lower than that of reference oil T. x-porlock (69.3% inhibition).IC 50 for DPPH radical-scavenging activity was 5.85 μg/ml.

β-carotene-linoleic acid assay
The lipid peroxidation inhibitory activities of the essential oil was assessed by the β-carotene bleaching test.
Results of the reference oil (T.x-porlock) were almost consistent with data obtained from the DPPH test.In βcarotene-linoleic acid test system, oxidation of linoleic acid was effectively inhibited by A. absinthium oil (58.56 ± 2.5%, 0.625 mg/ml of essential oil).

AA = 100 (
DR C -DRS S ) / DR C Where AA = antioxidant activity, DR C = degradation rate of the control = [ln(a / b) / 60], DR S = degradation rate in presence of the sample = [ln(a / b) / 60], a = absorbance at time 0, b = absorbance at 60 min.
Artemisia absinthium L. were collected from Namin, Province of Ardabil, after Heyran ghaut, in north-western Iran in July 2011.Voucher specimens have been deposited at the Herbarium of the Research Institute of Forests and Rangelands (TARI), Tehran, Iran.
PlantThe leaves of

Table 1 .
Composition of the leaf oil of A. absinthium.
*RI, Retention indices were as determined on a DB-5 column using the homologous series of n-alkanes.

Table 2
, the essential oils were found to have good to moderate antimicrobial activities against all microorganisms tested.The leaf essential oil of A. absinthium indicated significant activity against Candida albicans and moderate inhibitory activity against Staphylococcus aureus.This oil has been reported to be weakly inhibitory against E. coli.Results from the disc diffusion method and determination of minimal inhibitory and bactericidal concentrations (MIC and MBC) indicate that C. albicans is the most sensitive microorganism, with the lowest MBC value (1 mg/ml).Other sensitive microorganism is S. aureus.Killing kinetics of various microorganisms treated with leaf oil of A. absinthium indicated that C. albicans is the most vulnerable while E. coli was found least vulnerable.

Table 2 .
Antimicrobial activities of the leaf oil of A. absinthium.

Table 3 .
Antioxidant activity and total phenolics of leaf essential oil of A. absinthium.