Evaluation of antibacterial activity of Laurus nobilis L., Rosmarinus officinalis L. and Ocimum basilicum L. from Northeast of Algeria

1 Laboratory of Cellular Toxicology, Department of Biology, Faculty of Sciences, Badji Mokhtar University of Annaba, 23000, B.P. 12, Algeria. 2 Laboratory of Medical Botany, Department of Pharmacy, Faculty of Medicine, Badji Mokhtar University of Annaba, 23000, B.P. 12, Algeria. 3 Laboratory of Biochemistry and environment Toxicology, Department of Biochemistry, Faculty of Sciences, Badji Mokhtar University of Annaba, 23000, B.P. 12, Algeria. 4 Laboratory of Microbiology, Department of Pharmacy, Faculty of Medicine, Badji Mokhtar University of Annaba, 23000, B. P. 12, Algeria.


INTRODUCTION
In recent years, multiple antibiotic resistances of pathogenic bacteria have been exacerbated by the excessive and inappropriate use of commercial antimicrobial drugs commonly used in the treatment of infectious diseases (Davis, 1994;Service, 1995).Renewed interest has grown in medicinal plants to counter resistance and find an alternative to antibiotics (Kalemba and Kunika, 2003;Juliani and Simson, 2002;Falerio et al, 2003).
Medicinal plants contain many phytochemicals composants such as flavonoids, alkaloids, tannins and essential oils.Among these plants, Rosmarinus officinalis, Ocimum basilicum and Laurus nobilis are widespread in the Mediterranean Basin (Quezel and Santa, 1963).
The three plants were harvested in El Kala National Park ranked Biosphere Reserve by UNESCO in 1990.Its area is 76,438 Ha (Aouadi, 1989).It has a rich flora of about 850 species (De Belair, 1990) and characterized by a sub-humid Mediterranean climate.
Ocimum basilicum and Rosmarinus officinalis belong to Lamiaceae that include the most commonly used medicinal plants in the world as a spice and as a source of extract with strong antibacterial and antioxidant properties.

Plant material
The leaves of Laurus nobilis L., Rosmarinus officinalis L. and Ocimum basilicum L. were collected in April 2012 at El Kala National Park located at latitude 36° 52' north and longitude 8° 27' East.Laurel is spontaneous in the park; however, Basil and Rosemary are cultivated.The plants were dried in the shade in order to preserve the integrity of their molecules.

Extraction of essential oils
The extraction of essential oils was carried out by hydro-distillation using Likens Nickerson apparatus for 2 h.We introduced 100 g of dry leaves in a flask filled with 3/4 distilled water and then heated to boil.The water and oil are separated during the condensation of vapour loaded onto the oils (Chiej, 1984).Essential oils have been recovered in small opaque bottles and kept away from light, at a temperature of 4°C.The yield was expressed in percentage.

Bacterial strains
The bacterial strains tested were provided by the Laboratory of Medical Microbiology, Faculty of Medicine Annaba.They are: Gram-positive Enterococcus faecalis ATCC 29212, Staphylococcus aureus ATCC 25923, MRSAATCC 31 (Méthicilino), Staphylococcus aureus, Staphylococcus epidermidis and Enterococcus avium.

Disk diffusion method
The antibacterial activity was tested using the disk diffusion method (Davis, 1994).Bacterial cultures were reactivated by sub culturing on nutrient agar and incubated for 24 h at 37°C.From these, pure cultures were prepared by releasing bacterial inoculum strains in physiological water.The homogeneous suspension was equivalent to 0.5 Mc Farland, so an OD of 0.08 to 0.10 was read at 625 nm.
Each essential oil was used at different concentrations: pure oil, diluted oil in DMSO (Dimethyl sulfoxide) to ratio 1/2, 1/4 and 1/8.Discs of 6 mm in diameter, previously sterilized, were used.10 μl of essential oils was put on each disc and placed on agar.A witness disc (soaked in DMSO) was incubated under the same conditions to ensure that DMSO was devoid of antibacterial activity.
After incubation for 24 h in an oven at 37°C, reading was done.The effect of essential oils on bacteria was estimated by the appearance of clear zones around the discs.The diameter of the halo of growth inhibition was measured and expressed in mm (including the diameter of the disc of 6 mm).

Determination of minimum inhibitory concentration (MIC)
The minimum inhibitory concentration (MIC) is the smallest concentration of essential oil, in which no growth is visible compared to the control without extract.It was evaluated on twelve tested strains by disc diffusion test.We used the dilution method on solid medium (incorporation) (Billerbeck et al., 2002;Marino et al., 2001).
Serial dilutions of essential oils were performed with DMSO for 2 h.Each dilution was incorporated into Mueller-Hinton medium, maintained, super cooled and poured into Petri dishes.The concentrations (in percent), of essential oils used are respectively: 1, 0.5, 0.25, 0.01, 0.125, 0.06 and 0.03.Witness discs containing culture medium and only DMSO were also prepared.
Seeding was done as a deposit of bacterial suspension.After incubation at 35°C for six days, the growth was compared to the control.

Essential oil yield
The yields of essential oils from the dry matter of Rosmarinus officinalis L., Laurus nobilis L. and Ocimum basilicum L. were respectively 0.36, 0.6 and 0.71%.These results are lower than those found in other regions of Algeria.The yield of Rosemary in Algiers found by Djeddi (2007) was 0.82% and Laurel in Tlemcen was 1.2% (Haddouchi et al., 2009).These differences may result from the high moisture that characterizes the study's area; because it is known that maximum yields are obtained by dry weather.Concerning Basil, the yield obtained is normal because it develops in its natural habitat.Harvesting for the three plants was conducted during the vegetative stage, which has generated a relatively low yield (Bruneton, 1993).

Antibiotic activity
The antibacterial activity of the three essential oils and MIC values are grouped in Tables 1 and 2.
All organisms are sensitive to the three oils except P. aeruginosa (Gram negative) which is more and more responsible for nosocomial infections.It has an intrinsic resistance to a wide range of antibiotics (April et al., 1992) and also to essential oils.This resistance is due to the impermeability of the wall of this bacterium (Djeddi et al., 2007;Dorman and Deans, 2000;Duke and Ayensu, 1985).
The essential oil of L. nobilis L. has demonstrated a strong activity on the majority of tested strains; the highest sensitivity was in Enterobacter sp. that has an inhibition diameter of 22.4 mm, 16.8 mm pure oil and 1/8 dilution.The most resistant strain was P. aeruginosa.These results are in concord with those of Dadalioglu and Evrendilek (2004).1,8 cineole has a part in this activity since it has antimicrobial activity against several strains such as E. coli, P. aeruginosa and Staphylococcus aureus (Sivropoulou et al., 1997).
Our results are in agreement with those found by other authors such as Santoyo et al. (2005), Faleiro et al. (2003) and Gachka ( 2007) Fiorini et al. (1998) with respect to the resistance of Pseudomonas aeruginosa against this oil.Celikta ( 2007) found a moderate activity against E. faecalis and Proteus sp., however Jiang et al. (2011) obtained pronounced antibacterial activity.Santoyo et al. (2005) and Graven (1992) attributed the antimicrobial properties of the essential oil of R. officinalis to the presence of α-pinene, 1,8-cineol, borneol and camphor.Even minor components have a significant contribution to the antibiotic activity (Wang et al., 2012).
Finally, with the essential oil of Ocimum basilicum, we found good inhibition zones.The most sensitive bacterial strain (Shigella sp.) presented an inhibition diameter ranging between 12.2 mm and 19.9 mm.Suppakul et al. (2003) reported, also, that Basil has good antimicrobial activity against a wide range of microorganisms.This activity is due in part to the presence of linalool (Koutsoudaki et al., 2005;Sartoratotto et al., 2004;Sokovic and Van Griensven, 2006;Suppakul et al., 2003).The MIC is equal to 9.5 (10 3 mg/ml).
This study shows that Gram negative bacteria and Gram positive bacteria are both sensitive to the three essential oils.
It is known that Gram negative bacteria are more resistant to essential oils than Gram positive bacteria (Loäpez et al., 2005;Marino et al., 2001).This resistance is due to the nature of these group of cellular membranes of bacteria, because their external structures make them to have highly hydrophobic surfaces (Smith-Palmer et al., 1998).One important characteristic of essential oils and their components is their hydrophobicity, which allows them to partition the lipids of the bacterial cell membrane and mitochondria, disturbing the cell structures and making them more permeable (Sikkema et al., 1994).Dorman and Deans (2000) indicate that the antimicrobial activity depends, not only, on the chemical composition of the essential oil, but also on lipophilic properties and power of functional groups or aqueous solubility.The mixture of compounds with different biochemical properties can improve the effectiveness of essential oils.The aim of this study was to evaluate the three essential oils against 20 bacterial strains.For 12 strains we have determined the minimum inhibitory concentration (MIC).The three oils showed good antibacterial activity against both Gram negative and Gram positive bacteria.Laurel oil is the most efficient, Shigella sp. has the highest sensitivity to the three oils and Pseudomonas aeruginosa is the most resistant to them.Among the three oils, Laurel gives the lowest MIC against E. faecalis ATCC 29212, Enterobacter sp., Shigella sp., S. aureus and S. Epermidis (0.25%).

Table 1 .
R. officinalis L., Laurus nobilis L. and O. basilicum L. are Diameter of inhibition of essential oils against the bacterial strains (mm).

Table 2 .
Activity of essential oils incorporated in the solid medium (MIC).No culture; +: presence of culture widespread herbs in Algeria.The samples used have been harvested in the National Park of El Kala where Basil and Rosemary are cultivated and Laurel is spontaneous.The essential oils from leaves of Rosemary, Laurel and Basil were extracted by hydro-distillation using Likens Nickerson apparatus for 2 h. -: