GC-MS Analysis and Antibacterial Activity of the Essential Oil Isolated from Wild Artemisia herba-alba Grown in South Jordan

Background: There is a high variability in chemical composition of essential oil from Artemisia herba-alba grown in different countries and different localities in the same country. This has led to the characterization of many oil-dependent chemotypes assigned to the plant. Only one report was published on the essential oil composition of Artemisia herba-alba grown in Jordan. Aim: The current study aims to determines the essential oil composition of Artemisia herba-alba grown wild in south Jordan and test their activity against clinical isolate antibiotics resistant bacteria. Methodology: The essential oils were isolated by hydrodistillation and analysed by Gas Chromatography-Mass Spectrometry (GC-MS). The screening for essential oil activity was carried out using disc diffusion method on methicillin-resistant Staphylococcus aureus , methicillin-sensitive Al-Shuneigat et al.; BJMMR, 5(3): 297-302, 2015; Article no. BJMMR.2015.032 Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pyogenes, Escherichia coli, Klebsiella pneumonia, Proteus mirabilis and Pseudomonas aeruginosa. Results: Fifty-eight components accounting for 98.8% of the oil were identified, with oxygenated monoterpenes accounting for about 75% of the total oil content. Major identified compounds were cis-chrysanthenol (13.83%), 1,8-cineole (12.84%), cis-limonene (12.57%), α -terpinenol (6.97%), and γ -muurolene (4.50%). The volatile fractions exhibited potent activity against all resistant strains except Pseudomonas aeruginosa. Conclusion: We report here a new chemotype of Artemisia herba-alba grown in Jordan characterized by the presence of chrysanthenol, 1, 8-cineole, cis-limonene, and α -terpinenol.


INTRODUCTION
Essential oils are volatile, natural compounds characterized by strong odor that are usually obtained by steam or hydro-distillation. Essential oils are very complex natural mixtures which can contain between 20 to 60 components although 2 to 3 components are found at higher concentrations (20-70%). These major components that determine the biological properties of the essential oil fall into two groups having different biosynthetical origins. The main group is composed of terpenes and the other is composed of aromatic and aliphatic compounds, all characterized by low molecular weight [1,2].
The genus Artemisia comprises more than 300 species. Artemisia herba-alba Asso (syn. A. inculta), known as desert wormwood, is a dwarf shrub growing wild throughout the Mediterranean basin and extending into the North-western Himalayas [3].
Artemisia herba-alba has been used in traditional medicine in Jordan for treatment against constipation, hypercholesterolemia, jaundice, abdominal pains, diabetes, parasitic worms, flatulence, inflammations, common cold, kidney sand and stones [4]. The strong and aromatic smell of some species of Artemisia genus is mainly due to high concentrations of volatile terpenes. Many studies have shown that Artemisia species display significant variations in terpene constituents of their essential oils. The quality and yield of essential oils from Artemisia species is influenced by the harvesting season, fertilizer and pH of soils, choice and stage of drying conditions, geographic location, chemotype, choice of plant part or genotype and extraction method.
Large variability in essential oil composition of the aerial parts of Artemisia herba-alba has been reported [5,2]. Diversity in oil composition from plants grown in different countries and from different localities in the same country has led to the assignment of many oil-dependent chemotypes to the plant [6]. For example, in Morocco seven chemotypes have been reported with monoterpenes as being the major components followed by sesquiterpene. The chemotypes are characterized by the presence of one major component such as camphor (40-70%), αor β-thujone (32-82%, chrysanthenone (51-70%), chrysanthenyl acetate (32-71%), or davanone (20-70%). One chemotype is characterized by the presence of camphor (34-55%) and α-thujone (26-37%) [7]. Data from Spain showed that monoterpene hydrocarbons and oxygenated monoterpene were the most abundant components in Artemisia herba-alba oil. Large amounts of sesquiterpenes were found for some populations with camphor, 1,8-cineole, p-cymene and davanone being the major components found [8,9].
In Jordan oxygenated monoterpenes were found to be the major oil components (39.3% of the oil), with αand β-thujones as being most predominant (24.7%). The other major identified components were: santolina alcohol (13.0%), artemisia ketone (12.4%), and trans-sabinyl acetate (5.4%) [6]. In Tunisian oil oxygenated monoterpenes were found to be the major components of Artemisia herba-alba oil extracted from aerial parts. The main compounds were βthujone and α-thujone, 1,8-cineole, camphor, chrysanthenone, trans-sabinyl acetate, transpinocarveol and borneol [5,10]. In the essential oil of Artemisia herba-alba growing in Pakistan, chrysanthenyl propionate and elixene were identified for the first time for any Artemisia species [11]. Two oil types were found for plants grown in Sinai Peninsula namely cineolthujanebornane type and the pinane type. The essential oil of all studied populations were found to contain 1,8-cineole although in varying concentrations [12]. More recently, it has been reported that a further five chemotypes could be distinguished in plants growing in Sinai based on variation in composition of the oil, suggesting the existence of a greater number of chemotypes in the region. For Algerian oil, monoterpenes were the major components with the main compounds being camphor, αand β-thujones, 1,8-cineole and chrysanthenyl derivatives [13,2].
The chemical composition of the oil of Artemisia herba-alba grown in south Jordan has never been reported. The aim of the present study was to report on the chemical composition of the essential oil from the aerial parts of Artemisia herba-alba grown in southern Jordan and its biological activity.

Collection and Authentication of Plants
Fresh amount of the Artemisia herba-alba was collected from Mutah, Alkarak, south Jordan, during the flowering period and the vegetative phase. The plant materials were taxonomically identified and authenticated by the Botanical Survey of Yarmouk University.

Isolation of Essential Oil
A fresh aerial parts (200 g) of Artemisia herbaalba was finely chopped and subjected to hydrodistillation for 4 h using a Clevenger-type apparatus, yielding 0.24% (v/wt), pale yellowish oil. Subsequently, oil was dried over anhydrous sodium sulfate and immediately stored in GCgrade hexane at 4ºC until the analysis by gas chromatography/mass spectrometry (GC/MS) was done.

GC-FID analysis
The oils were analyzed in an Agilent (Palo Alto, USA) 6890N gas chromatograph fitted with a 5% phenyl-95% methylsilicone (HP5, 30 m × 0.25 mm × 0.25 µm) fused silica capillary column. The oven temperature was programmed to run from 60ºC to 240ºC at 3ºC/min with hydrogen being used as the carrier gas (1.4 mL/min). 1.0 µL of a 1% solution of the oils in hexane was injected in split mode (1:50). The injector was kept at 250ºC and the flame ionization detector (FID) was kept at 280ºC. Concentrations (% contents) of oil ingredient for Artemisia herba-alba were determined using their relative area percentages obtained from GC chromatogram, assuming a unity response by all components.

GC-MS analysis
Chemical analysis of the essential oils was carried out using gas chromatography-mass spectrometry (Agilent (Palo Alto, USA) 6890N gas chromatograph). The chromatographic conditions were as follows: column oven program, 60ºC (1 min, isothermal) to 246ºC (3 min, isothermal) at 3ºC/min, the injector and detector temperatures were 250ºC and 300ºC, respectively. Helium was the carrier gas (flow rate 0.90 ml/min) and the ionization voltage was maintained at 70eV. A HP-5 MS capillary column (30 m × 0.25 mm i.d., 0.25 µm film thicknesses) was used. A hydrocarbon mixture of n-alkanes (C 8 -C 20 ) was analyzed separately by GC-MS under same chromatographic conditions using the same HP-5 column. Kovats Retention Indexes (KRIs) were calculated by injection of a series of n-alkanes (C 8 -C 20 ) in the same column and conditions as above for gas chromatography analyses.
Identification of the oil components were based on computer search using the library of mass spectral data and comparison of calculated Kovats retention index (KRI) with those of available authentic standards and literature data.

Disc Diffusion Assay
The antibacterial activity of the Artemisia herbaalba essential oil was determined by the disc diffusion method according to the National Committee for Clinical Laboratory Standards. Sterile paper discs of 6 mm in diameter were impregnated with 5 µL essential oil and deposited on the agar surface. Petri dishes were placed at 4ºC for 2 h to facilitate the dissemination of extract on the culture medium followed by incubation at 37ºC for 24 h. For each sample, negative water control and positive antibiotic disc control were used. At the end of the period, inhibition zones formed on the medium were evaluated in mm. Studies were performed in triplicate.
A study conducted by Hudaib and Aburjai [6] on the chemical composition of Artemisia herba-alba growing in south Amman Jordan, it was found that oxygenated monoterpenes were the major oil components (39.3% of the oil), with αand βthujones as being the principal components (24.7%). The other identified components as shown in Table 2 were: santolina alcohol (13.0%), artemisia ketone (12.4%), and αthujone (16.2%) [6]. To the best of our knowledge, this study was the only one to report on the chemical composition of Artemisia herbaalba essential oil grown in Jordan. We report here the existence of new chemotype of Artemisia herba-alba grown in Jordan. This new chemotype is characterized by the presence of chrysanthenol, 1,8-cineole, cis-limonene, and αterpinenol. This combination of components may also represent a new chemotype of Artemisia herba-alba.

Antimicrobial Activity
The results in Table 3

CONCLUSION
We report here a new chemotype of Artemisia herba-alba growing in Jordan characterized by the presence of chrysanthenol, 1,8-cineole, cislimonene, and α-terpinenol as a major ingredients. The results show a good activity of the oil against all tested pathogens except for Pseudomonas aeruginosa that remained resistant.

ETHICAL APPROVAL
Not applicable.