Chemical composition and antibacterial activity of the essential oils of Ferula vesceritensis Coss et Dur. leaves, endemic in Algeria

Background The biological importance of members of genus Ferula promoted us to investigate the leaves of Ferula vesceritensis Coss et Dur. (endemic plant) previously not investigated. This study presents the chemical composition and antibacterial activities of the hydrodistilled oils. Results Volatile components of the leaves of F. vesceritensis have been studied by gas chromatography–mass spectrometry to afford 23 compounds. The major components were found to be 5,9-tetradecadiyne (24.72%), germacrene D (24.51%), farnesene (8.57%), and α-bisabolene (8.57%). The antimicrobial activities of the essential oils were evaluated by disk diffusion method and tested against Gram-positive and Gram-negative bacteria. The volatile oil showed a strong antibacterial activity against Staphylococcus aureus, Escherichia coli, and Klebsiella pneumonia. Conclusions These results reinforce the previous studies showing that the genus Ferula is considered as a good source of essential oils. The results presented here can be considered as the first information on the antimicrobial properties of F. vesceritensis.


Background
Since the middle ages, essential oils have widely been used for bactericidal, virucidal fungicidal, antiparasitical, insecticidal, medicinal, and cosmetic applications, especially nowadays in pharmaceutical, sanitary, cosmetic, and agricultural and food industries. Because of the mode of extraction, mostly by hydrodistillation from aromatic plants, they contain a variety of volatile molecules such as terpenes and terpenoids, phenol-derived aromatic components, and aliphatic components [1].
The exclusively old-world genus Ferula, belonging to the family Apiaceae, has some 130 species distributed throughout the Mediterranean area and Central Asia. These plants are often used as spices and in the preparation of local drugs. The resins are reported to be used for stomach disorders such as a febrifuge and carminative agent [2]. Some species are used in traditional medicine for the treatment of skin infections [3] and hysteria [2]. Previous study dealing with members of this genus revealed that the main constituents are sesquiterpenes and sesquiterpene coumarins. More than 70 species have been studied chemically leading to the fact that germacranes, humulanes, carotanes, himachalanes, and guaianes represent the main sesquiterpene constituents of the genus [4][5][6][7][8][9][10]. Ferula spp. are also known for their toxicity and pharmacology. Daucane esters from F. communis and Ferula arrigonii showed antiproliferative activity on human colon cancer lines [11] and calcium ionophoretic and apoptotic effects in the human jurkat T-cell line [12].
Ferula vesceritensis belongs to umbelifereae family, which widely spread in north Africa, this plant is abundant in south east of Algeria. The genus Ferula represented in Algeria by six species [13].
Ferula vesceritensis is indigenous to Algerian Sahara. According to ethnobotanical investigation, fruit decoction is used in folk medicine to treat headaches, fever, and throat infections, while the livestock avoids grazing it [14].
Our continuation of investigation carried out on F. vesceritensis [15][16][17]. The essential oils of the leaves of F. vesceritensis led to the identification of 23 compounds. Moreover, the evaluation of the antibacterial activity of the essential oils revealed a very important effect against some bacteria strains.

Methods
Gas chromatography/mass spectroscopy GC/MS analysis was carried out on a Thermoquest-Finnigan Trace GC/MS instrument equipped with a DB-1 fused silica column (30 m, 0.25 mm i.d., film thickness 0.25 m). The oven temperature was raised from 60 to 250°C at a rate of 5°C/min then held at 250°C for 10 min; transfer line temperature was adjusted at 250°C. Helium was used as the carrier gas at a flow rate of 1.1 mL/min with a split ratio of 1/50. Identification of the constituents of each oil was achieved by comparison of their mass spectra and retention times (Rt) with those reported in the literature, and those of authentic samples.

Antimicrobial activity
The antibacterial activity test was carried out on essential oils of the leaves of F. vesceritensis roots using disk diffusion method (NCCLS) against four human pathogenic bacteria, including Gram positive, Gram-negative bacteria.

Results and discussion
This study focused essentially on the phytochemical and antibacterial screening of F. vesceritensis. The specie has been screened for seven chemical groups. The analyses reveal the presence of volatile oils, flavonoids, saponins, tannins, carotenoids, and coumarins (Table 1).
Essential oils from the leaves of F. vesceritensis have been studied by GC-MS to afford 23 components. The yield was 1.82% on dry weight basis. In previous studies, the essential oil obtained from the roots of Ferula ferulaoides yielded 2.4-3.2% of essential oil from dry roots [18] and average 1,66-3.85% in the fruits of Ferula gummosa [19]. The essential oil components identified from F. vesceritensis are listed in Table 2; the major components were found to be 5,9-tetradecadiyne 24.72%, germacrene D (24.51%), farnesene (8.57%), α-bisabolene (8.57%). Some other compounds were only present in minor amounts.

Antibacterial activity
The antimicrobial activities and toxicity of essential oil have been documented, but their modes of action are complex and still in some cases unknown, considering the large number of different groups of chemical compounds present, this activity is due to the presence of active  constituents, mainly attributable to isoprenes such as monoterpenes, sesquiterpenes, and related alcohols, other hydrocarbons and phenols [1]. The diffusion test was applied to four microorganisms including Gram-positive, -negative bacteria. The results summarized in Table 3 showed that the volatile oil from F. vesceritensis prevented the growth of all the tested microorganisms and it has been revealed that the medium diameter of inhibition zone increase proportionally with the increase of concentrations.
The obtained inhibition zone varied from 6.00 to 27.00 mm with a highest inhibition zone recorded with Staphylococcus aureus at 8 mg mL and with 26 mm at E. coli in the same concentration. This results corresponding with those obtained on F. gummosa and F. latisecta [20]. It should be mentioned that there are no background antibacterial studies on F. vesceritensis.

Plant material
The leaves of F. vesceritensis were collected on May 2010 near Ghardaya Algeria. The plants were identified by Dr. M. Chahma, Faculty of Sciences, University of Ourgla, Algeria, voucher specimens were deposited at the Chemistry Department, University of Mentouri-Constantine under code number (AM#112).

Extraction
Essential oils were obtained by hydrodistillation of 150 g of dried aerial parts using a Clevenger-type apparatus for 3 h. Diethyl ether (10 mL) was used as the collector solvent as reported in literature. After evaporation of the solvent, the oil was dried over anhydrous sodium sulfate and stored in sealed vials protected from the light at −20°C before analyses. Three oil samples were obtained by hydrodistillation and subsequently analyzed by GC-MS.

Microorganism strains
All of the bacteria; standard strains E. coli ATCC 25922, P. aerugenosa ATCC 27853 and (clinical stains: S. aureus, K. pneumonia) were obtained from Bacteriology Laboratory Constantine Hospital University (C.H.U).
The bacterial strains were first grown on Muller Hinton medium (MHI) at 37°C for 24 h prior to seeding on to the nutrient agar. A sterile 6-mm-diameter filter disk (Whatman paper no. 3) was placed on the infusion agar seeded with bacteria, and each extract suspended in water was dropped on to each paper disk (40 μL per disk) for all of prepared concentrations (8, 4, 2, 1, 0.5, 0.25 mg/mL). The treated Petri dishes were kept at 4°C for 1 h, and incubated at 37°C for 24 h. The antibacterial activity was assessed by measuring the zone of growth inhibition surrounding the disks. Each experiment was carried out in triplicate.

Conclusions
Our study of the Algerian F. vesceritensis leaves led to the extraction and characterization of 23 compounds followed by the evaluation of antimicrobial activity for the first time. These results reinforce the previous studies showing that the genus Ferula is considered as a good source of essential oils. The results presented here can be considered as the first information on the antimicrobial properties of F. vesceritensis.