Chemical Composition and Antimicrobial Activity of the Essential Oil of Algerian Phlomis bovei De Noé subsp . bovei

The chemical composition of essential oil obtained by steam distillation of dried aerial parts of Phlomis bovei De Noé subsp. bovei collected from Algeria, was analyzed by GC and GC/MS. Seventy five constituents (corresponding to 86.37% of the total weight) were identified. The main components were: germacrene D, β-caryophyllene, β-bournonene, thymol and hexahydrofarnesyl acetone. Furthermore, the antimicrobial activity of the oil was evaluated against six Gram (+/-) bacteria and three pathogenic fungi, using the agar dilution technique. It was found that the oil exhibited strong antimicrobial activity against most of the tested microorganisms.


Introduction
The plants of the genus Phlomis are native to Turkey, North Africa, Europe and Asia.Phlomis bovei De Noé, syn.Phlomis samia Desfontaines (Lamiaceae) is a rare Algerian endemic plant, commonly known as Kayat El Adjarah [1] in the Algerian dialect or variously named Farseouan, Tarseouan, Iniji, R'ilef and Azaref throughout the North of Africa [2].It is one among the nine endemic plants recorded in the 'Rapport National sur la Diversité Biologique' [1].P. bovei is a herbaceous perennial plant, which grows up to 0.8 m. and often develops a stout woody base.All parts are sticky, because of its dendroid stellate glandular hairs.Its basal leaves are green, heart-shaped, with scalloped margins, 6.5-25 x 4.5-20 cm and it has a petiole of between 4-18 cm in length.To date two subspecies have been recorded for P. bovei De Noé: P. bovei De Noé subsp.bovei and P. bovei De Noé subsp.maroccana Maire.The present study refers to the former, which to our knowledge has never been studied phytochemically before, whereas previous studies on the essentials oils of Phlomis species from around the Mediterranean have included: Phlomis fruticosa, P. cretica, P. samia, P. lanata, P. linearis, P. leucophracta, P. chimerae and P. grandiflora var.grandiflora.

Results and Discussion
The essential oil obtained by hydrodistillation of aerial parts of Phlomis bovei De Noé subsp.bovei was light yellow in color and possessed a distinct sharp odor.The yields were 0.22 % w/w.The analysis of the volatile constituents was carried out using two different GC-MS systems, equipped with two columns of different polarities (HP-5 and Aquawax, respectively).The chemical compositions are summarized in Tables 1 and 2. The identified components represented 86.37% of all the components found in the oil samples.These percentages were based on normalization of peak areas without application of the response correction factor.The major components included: germacrene D (21.45%), thymol (8.43%), β-caryophyllene (7.05%) and hexahydrofarnesyl acetone (5.84%).We should also note the presence in the essential oil of a total 6.03 % of normal saturated hydrocarbons (see Table 2).Although most of the identified constituents occurred in both methods of analysis, it was also noted that some chemical constituents occurring in appreciable amounts in HP-5 were absent in Aquawax and viceversa.This was due to the differences between the GC-MS instruments, the two columns and the absence of reference retention indexes for the second column.Thus the identification of the components for the second column was based on their mass spectra and by comparison of their retention times with those of authentic samples.For the essential oil obtained from the leaves of P. fruticosa collected in Montenegro (Table 3) the main constituents were: β-caryophyllene (12.0%), (E)-methyl-isoeugenol (15.3%), αasarone (10.9%), caryophyllene oxide (8.1%) and α-pinene (6.6%) [3].The antimutagenic activity of the essential oil and of the crude extract was evaluated by the same research group [4].Studies on the same plant, from the same region, have been conducted considering the antimicrobial and the antifungal activity of its essential oil, as well as its methanolic extract, with moderate results [5].Traditionally the infusion of P. fruticosa leaves is used in Greece as a tonic drink, whereas in Italy the dried leaves are used as a poultice on wounds [6].
The essential oil of Phlomis bovei De Noé subsp.bovei exhibited a wide profile of antimicrobial activity against most of the tested microorganisms, in comparison with the tested antibiotics and the standards β-caryophyllene and thymol (Table 4), while only K. pneumoniae appeared to be a microorganism displaying significant resistence.Considering the fact that β-caryophyllene possesses in general moderate antimicrobial activity, we conclude that the antimicrobial activity of the essential oil from P. bovei can be attributed, to a considerable degree, to the presence of germacrene D and thymol, which are well known to posses strong antimicrobial activity [13][14][15].

Conclusions
Our GC and GC/MS study of the essential oil from Algerian Phlomis bovei De Noé led to the identification of 75 constituents (corresponding to 86.37% of the total weight) among which germacrene D, β-caryophyllene, β-bournonene, thymol, and hexahydrofarnesyl acetone were the main ones.The oil exhibited a broad spectrum of strong antimicrobial activities and it possessed a much better antimicrobial activity in comparison with all previously tested and assayed samples from Greek Phlomis species [8], showing that this plant oil could have a commercial potential as an antiseptic agent, however, further investigation should be carried out against new series of pathogenic microorganisms.

Plant material and essential oil isolation
Aerial parts of Phlomis bovei De Noé were collected from the wild in July 2004 at ca. 1,550 m of altitude on Megriss Mountain (Eastern Algeria).The plants were authenticated by the staff of the Laboratory of Natural Resource Valorization by comparison with herbarium specimens.Voucher specimens are deposited in the Herbarium of the Institute of Biology, University of Setif, Algeria.The material was air-dried indoors prior to isolation of the essential oil.The dried aerial parts were subjected to hydro-distillation in 0.4 L of water in a Clevenger-type apparatus for 4 hrs, using a watercooled oil receiver to reduce formation of potential artifacts due to overheating during the hydrodistillation process [16].The essential oil was collected over water, dried over anhydrous sodium sulfate (Panreac Quimica S.A. Barcelona, Spain) and stored at 4 o -6 o C until it was analyzed.

Essential oil analysis
The oil was analysed by GC on a Perkin-Elmer 8500 gas chromatograph equipped with a FID, fitted with a Supelcowax-10 fused silica capillary column (30 m x 0.32 mm; film thickness, 0.25 µm).The column temperature was programmed from 75 o C to 200 o C at a rate of 2.5 o C/min.The injector and detector temperatures were programmed at 230 o C and 300 o C, respectively.Helium was used as carrier gas at flow rate of 0.6 mL/min.The GC-MS analysis was carried out using two different GC-MS systems.The first was a Hewlett Packard 5973-6890 GC-MS operating on EI mode (equipped with a HP 5MS 30 m x 0.25 mm x 0.25 µm film thickness capillary column).Helium (1 mL/min) was used as carrier gas.Temperature program: initial temperature of the column was 60 °C (for 5 min), then raised to 280 o C at 3 °C/min, and held there for 30 min (total time: 93.33 min).The compounds were identified by comparison of their retention indexes (RI) [17], retention times (RT) and mass spectra with those of authentic samples and/or the NIST/NBS, NIST02, Wiley 575 libraries spectra and the literature [18].The percentage composition of the essential oil is based on peak areas obtained without FID factor corrections.The second GC-MS system analysis was a Finnigan Trace GC Ultra system, operating on EI mode and equipped with AT™ Aquawax 30 m x 0.32 mm x 0.25 µm film thickness capillary column.Helium was used as the carrier gas, at a flow rate of 1.5 mL/min (constant flow) and a 1:10 split ratio.Temperature program: initial temperature of the column 60 °C (for 5 min), then raised to 235 o C at 3°C/min, and held there for 30 min (total time: 93.33 min).The MS parameters were as follows: source temperature, 200 °C; ionization energy, 70 eV; emission, 200 µA; mass range, 35-650 Da; scan time,1.25 s., scan rate (amu/s) 500.0;scans per second, 0.7974.

Antimicrobial activity
Antimicrobial activity of the essential oils against bacteria and fungi was determined by using the agar dilution technique.The microorganisms included two Gram-positive bacteria: Staphylococcus aureus (ATCC 25923) and Staphylococcus epidermidis (ATCC 12228); four Gram-negative bacteria: Escherichia coli (ATCC 25922), Enterobacter cloacae (ATCC 13047), Klebsiella pneumoniae (ATCC 13883) and Pseudomonas aeruginosa (ATCC 227853); and the pathogenic fungi Candida albicans (10231), C. tropicalis (13801) and C. glabrata (28838).Standard antibiotics (netilmicin and amoxicillin with clavulanic acid) were used as controls for the sensitivity of the tested bacteria and 5flucytocine, amphotericin B and itraconazole were used as controls for the tested fungi.The technical details have been described previously [19].Minimum inhibitory concentrations (MICs) were determined for oil samples and the standard pure compounds β-caryophyllene and thymol (Extrasynthese SAS, France), under identical conditions, for comparison purposes.Statistical analysis: data are expressed as means + S.D.

Table 1 .
Main components of the essential oil from the aerial parts of P. bovei De Noé.
* Compounds listed in order of elution from a HP-5 MS column.§ Retention indices (KI) on HP-5 MS capillary column.a= Retention time; b = Retention Index; c = Peak enrichment; d = mass spectra.

Table 2 .
Composition of P. bovei De Noé subsp.bovei essential oil by substance class.

Table 3 .
Main components of the essential oils from different Mediterranean Phlomis species.

Table 4 .
Antimicrobial activities (MIC mg/mL) of the studied Phlomis essential oils and its main components.