Chemical characterization by GC-MS and in vitro activity against Candida albicans of volatile fractions prepared from Artemisia dracunculus, Artemisia abrotanum, Artemisia absinthium and Artemisia vulgaris

Background A large number of essential oils is reported to have significant activity against Candida albicans. But the different chemical composition influences the degree of their activity. The intention of this study was to investigate the chemical composition and the activity against Candida albicans of volatile oils obtained from Artemisia dracunculus, A. abrotanum, A. absinthium and A. vulgaris (Asteraceae). The aim of the study was to identify new chemical compounds that have effect against C. albicans. The essential oils were obtained by hydrodistillation or extraction with dichloromethane (a new procedure we developed trying to obtain better, more separated compounds) from air dried above ground plant material and analyzed by GC-MS. Additionally commercial essential oils from the same species were tested. The Candida albicans inhibition studies were carried out by the paper disc diffusion method. Results The essential oils shared common components but presented differences in composition and showed variable antifungal activity. Davanone and derivatives thereof, compounds with silphiperfolane skeleton, estragole, davanone oil, β-thujone, sabinyl acetate, herniarin, cis-chrysanthenyl acetate, 1,8-cineol, and terpineol were the main components of Artemisia volatiles. Conclusions Among the volatile fractions tested those from A. abrotanum containing davanone or silphiperfolane derivatives showed the highest antifungal activity. The in vitro tests revealed that the Artemisia oils are promising candidates for further research to develop novel anti-candida drugs.


Background
The genus Artemisia belongs to one of the largest and most widely distributed genera of the family Asteraceae (Compositae). It is a diverse and economically important genus and it has more than 500 species. Most plants within this genus have a great importance as medication, foodstuff, ornamentals or soil stabilizers, some are allergenic or toxic, and some are weeds growing in the fields [1][2][3][4].
The conditions that may separate these studies are the plant preparations put under investigation. The highest percentage of successful antifungal activity of Artemisia plants was obtained with essential oils; raw extracts generally showed less activity [5][6][7][8][9][10].
More information on the potential antifungal activity of Artemisia plants could stimulate research leading to their possible future use in the clinical treatment of candidiasis as natural antimycotic agents.

Results and discussion
In the present study, the chemical composition and the antifungal in vitro potency of seven oil preparations obtained from air dried plants and commercial essential oil samples of 4 different Artemisia species were put under investigation. Their chemical composition was evaluated by GC-MS. The antifungal activity was determined by the disk diffusion method test. The tests were done using the disc diffusion method following CLSI standard rules for antimicrobial susceptibility testing of impregnated disks [11].

Chemical composition
The volatile fractions studied showed a great chemical diversity. All four Artemisia species had distinct volatile patterns and there were also differences in the composition between the two different extracts, the distilled oil and the CH 2 Cl 2 extract, of the same species, as presented in Table 1.
A. abrotanum was characterized by the high content of the sesquiterpene davanone and its derivatives which could not all be identified as well as by the artedouglasia oxides A-D. Monoterpenes like 1-terpineol or trans-piperitol and the phenylpropanoid estragole were present in small amounts only in this species. All main compounds found in the distilled oil were also present in the CH 2 Cl 2 extract. The davanone derivatives were also the main compounds in the microdiostillates which also contained the artedouglasia oxides.
The main compound in the distilled oil of A. dracunculus was estragole accounting for approximately 80% of the oil. However, in the CH 2 Cl 2 extract this compound could not be identified. Small amounts of the phenylpropanoids chavicol and methyleugenol could be found. These extracts contained also some davanone and its derivatives. Both, the distilled oil and the extract contained the coumarin derivative herniarin. In the microdistillate, the main compounds were estragole (79.5%) followed by Z-β-ocimene (8.8%) and E-β-ocimene (7.2%).
From A. absinthium only a CH 2 Cl 2 extract was obtained. The main peak in the chromatogram could not be identified. Furthermore, the extract contained β-thujone and nerol as major compounds and estragole, spathulenol and α-bisabolol as further components. Herniarin was also present in this extract. The microdistillate was characterized by the presence of β-thujone (78.1%), sabinene (4.4%), and myrcene (4.4%).
The main volatiles in A. vulgaris were terpinen-4-ol, and borneol. One noticeable difference between the two volatile preparations was that 1,8-cineol, the main compound in the oil from the CH 2 Cl 2 extract, was not found in the distilled oil.

Test microorganisms
The anticandidal test with the commercial oils is presented in Table 3. All four oils showed anticandidal activity with A. abrotanum being the most efficient, followed by A. absinthium, A. dracunculus and A. vulgaris.
Cited literature [5,15] described an A. absinthium oil rich in thujone and in the aerial parts of A.vulgaris an oil with high proportions of 1,8-cineole, sabinene, thujone, and caryophyllene oxide.
Essential oils from A. vulgaris rich in camphor, 1,8 cineole or β-thujone have been described [14,[24][25][26]. These oils contain also chrysanthenyl acetate, borneol, methyleugenol, α-terpineol, t-verbenol, or β caryophyllene but owing to the  In this study, all volatile fractions isolated from all four Artemisia species showed some activity against Candida albicans. The highest activity was found for A. abrotanum where an inhibition zone larger than that produced by Nystatin could be observed. But it has to be considered that these fractions contain higher amounts of potentially active compounds than the fractions obtained from the other investigated species. Nevertheless, there are reports confirming the antimycotic activity of the main compounds identified by GC-MS in the present volatile fractions.
An extract from A. abrotanum grown in Sweden as well as the component davanone were effective against C. albicans [27]. Furthermore, davana oil from Artemisia pallens rich in davanone and the davana fraction derived from this oil were active against Candida albicans [28]. However in another study, davanone as single compound showed only very weak in vitro activity against C. albicans [29]. Candida albicans proved to be very susceptible against the essential oil from Ocimum selloi which contains more than 95% estragole (= methylchavicol) [30]. However, in another test series, estragole as single substance proved to be ineffective [31].
In the present study, 1,8-cineol, borneol, terpinen-4-ol, spathulenol, β-thujone, and α-bisabolol were major compounds in all of the volatile fractions of A. absinthium and A. vulgaris studied. The oils containing these compounds can be found in various plants and some of them were reported to inhibit the growth of C. albicans. An essential oil rich in α-pinene and α-bisabolol from Laserpitium zernyi showed a low activity against two strains of C. albicans [32]. The oil of A. annua containing 48% camphor as main compound was active against C. albicans [33]. A sage (Salvia officinalis) oil from Montenegro with the main compounds α-thujone (29.5%), camphor (22.5%) and 1,8-cineole (12.2%) had significant activity against C. albicans [34].
Oils from sage, myrtle, and laurel containing 1,8-cineol as main compound showed some activity against C. albicans [35], also an Eucalyptus oil with 85% of 1,8-cineole content [36]. However an Egyptian oil from Eucalyptus occidentalis that presumably contained 1,8 cineole had no influence on the growth of C. albicans [37].
Additionally, 7-methoxycoumarin (herniarin) isolated from twigs of Treculia obovoidea showed some activity against various Candida species [38]. Although reports on the antifungal activity of Artemisia species are inconsistent the results of this study confirm that Artemisia essential oils exert antimycotic effects and may represent good candidates to replace in the future allopathic treatments to which Candida has developed resistance. Under the conditions of this study, volatile fractions from A. abrotanum as well as the commercial oil from this plant had the strongest effect on Candida albicans although they differed in composition, having either davanone derivatives or silphiperfolene derivatives and 1,8-cineol as major compounds, respectively. The differences in activity and order in efficacy of the oils and volatile preparations most probably arises from differences in composition. So the own A. vulgaris preparations containing a mix of 1,8-cineol, terpinen-4-ol, borneol, camphor, and spathulenol were more active than the commercial oil with the thujones and camphor. The lower activity of the A. absinthium dichloromethane extract might be due to the lower overall concentration of active compounds in this preparation as compared to the commercial sample from the same species.

Experimental research Plant material
The A. abrotanum, A. dracunculus, and A. absinthium aerial parts were collected in the Botanical Garden of the University of Veterinary Medicine Vienna, Austria. A. vulgaris aerial parts were gathered in the surroundings of Timisoara (Bencecul de Sus), Romania. All plants were picked during the June-September period of 2010, while blooming. Voucher specimen of the collected plants were deposited in the Herbarium of the University of Vienna (WU-Generale, http:\\herbarium.univie.ac.at).

Test microorganisms
Isolates of Candida albicans (ATCC 10231) were obtained from the culture collection of the Department of Microbiology, Banat's University of Agricultural Sciences and Veterinary Medicine Timisoara.

Methods
The preparation of the essential oils was done using a modified standard procedure according to the European Pharmacopoeia [39]. The procedures were done at the same period of time. The plant material (15 g) was subjected to hydrodistillation using 150 ml distilled water for 4 hours at a constant rate. An average of 18.5 mg of essential oil was obtained.
In addition, an extraction with dichloromethane (CH 2 Cl 2 ) followed by hydrodistillation was carried out. 100 g of fresh plant material (leaves or flowers) were extracted 5 times with 100 ml of CH 2 Cl 2 in consecutive steps. The resulting extracts were combined and the solvent was removed in vacuo using a Rotavapor. The average yield was 2 g of crude extract; the pooled extract was hydrodistillated for 3 hours as mentioned above and stored at 4˚C until analysis. An average yield of 30 mg oil out of 2 g extract was obtained. For further analyses volatile oil from several preparations were combined.
Leaves of the plants collected from the botanical garden were also subjected to microdistillation using the automatic microdistillation unit MicroDistiller from Eppendorf (Hamburg, Germany) which is a gentle distillation method that allows investigating essential oil finger prints. The procedure was as follows: 0.2 to 0.3 g finely crushed dried plant material and 10 ml distilled water were filled into the sample vial. The collecting vial containing 1 ml water, 0.5 g NaCl and 300 μl n-hexane was connected with a capillary to the sample vial. The heating program applied to the sample vial was 15 min at 108°C followed by 45 min at 112°C. The collecting vial was kept at −2°C, where the volatiles were trapped in 0.3 ml n-hexane.

Analysis of the essential oils
The analysis of the volatiles was performed using a Hewlett-Packard 6890 GC linked to a Hewlett-Packard 5973 mass-selective detector. For the analysis a Zebron ZB-5MS, capillary column (27 m × 250 μm i. d., 0.25 μm film thickness) was used. The carrier gas was helium at 1.3 ml/ min in constant flow mode. The injector temperature was 250°C, the injection volume 1 μl, and the split ratio 1:20. The initial oven temperature of 60°C was held for 1 minute, then increased at a rate of 5°C/min up to 220°C, and subsequently at a rate of 15°C/min up to 280°C, and finally was held isothermal for 1 min. The transfer line to the MSD was set at 280°C and the scan conditions were: M/Z 40-300, at 1.75 scans/sec.
Prior to analysis 900 μl of the volatile fractions were mixed with 100 μl of biphenyl (2.0 mg/ml in hexane) as internal standard. The components of essential oils were identified by comparing their relative retention times and mass spectra with those of Registry of Mass Spectral Data and literature citations [40,41]. The amount of the individual compounds in the fractions was calculated using the Total Ion Current from the MSD signal and assuming the same response as for the internal standard biphenyl.
Inocula containing 10 6 cells/ml were spread on the medium (Sabouraud with added penicillin (40 units/ml) and streptomycin (2 mg/ml of medium)). The antifungal activity test was carried out by the disk diffusion method using sterilized 6 mm diameter filter paper disks. From each volatile oil preparation a quantity of 50 μl was submitted to testing and the tests were done in triplicate. The inoculated plates were incubated at 35°C for 24 h. Standard bio discs of Nystatin 100 units/disk (Himedia Laboratories Ltd., India) were used as positive control and blank sterilized filter papers as negative control.
After the incubation, the diameter of the inhibition zone for each essential oil was measured in millimeters (including the 6 mm diameter of the disk). The results are expressed as mean values of three determinations ± S.D.

Antifungal activity in vitro
The experimental protocols were approved by the Animal Ethics Committee of the Faculty of Veterinary Medicine Timisoara and conducted accordingly. All 7 Artemisia preparations were subjected to the disc diffusion test against Candida albicans (ATCC 10231). Inhibition zones observed ranged from 23.5 mm to 13 mm for the essential oils and volatile oils. An 20.5 mm inhibition zone was determined for Nystatin and 6 mm (the diameter of the biodisc) for the negative control (Table 4).
Highest antifungal activity was observed with A. abrotanum (both volatile fractions) followed by A. vulgaris, A. dracunculus, and A. absinthium.

Conclusions
Since all volatile fractions caused inhibition we conclude that compounds found in different quantities are responsible for the varying in vitro-activity against C. albicans, but it is difficult to attribute this effect of complex mixtures to a particular constituent. Possibly synergistic as well as antagonistic effects of compounds in the oil should also be taken into consideration as often stronger antifungal activity can be observed with complete essential oils in comparison to single oil components [42]. Our studies showed that different procedures for preparation of volatile fractions, starting from extracts or plant material, resulted in different chemical compositions. This phenomenon may contribute to the contradictory results published for the antimycotic effects of Artemisia. Under the conditions of this study, volatile fractions from Artemisia plants exert antifungal in vitro activity. Our results may open paths for the development of new phytotherapeutic products from the Artemisia species studied. Nystatin (positive control) 20.5 ± 4.04 Blank filter paper (negative control) 6.0 ± 0