Antimicrobial activity of essential oil of Salvia officinalis L . collected in Syria

The essential oils of Salvia officinalis L. collected at two different altitudes in Syrian coastline were analyzed by gas chromatography. Plant’s development stage and the ecological factors had impact on the qualitative composition of S. officinalis essential oil. Although, the major components of the essential oils extracted from plants grown at both altitudes were 1,8-cineol, camphor, borneol, α-pinene, β-pinene, camphene, β-myrcene and caryophyllene, their percentage changed according to the altitude. S. officinalis essential oil was for its antibacterial activities by using Grampositive and negative bacteria. Both Staphylococcus aureus and Streptococcus group D were efficiently inhibited after 10 min of contact at oil concentration of 20 μl/ml. The inhibitory effect of the essential oil on Candida albicans was total and definitive within a minimum of contact time and oil concentration. But the essential oil showed a temporary bacteriostatic effect on Escherichia coli, Salmonella typhi, as well as Pseudomonas aeruoginosa. In comparison with most known antibiotics, the efficiency of S. officinalis essential oil was much better, especially against bacteria resistant to antibiotic.


INTRODUCTION
Salvia officinalis L. from the family Lamiaceae, is a worldwide cultivated aromatic herb that is endemic with Syria where it is known as Kasiin or Mariamia.It grows along the Syrian coastline at different altitudes up to 900 m above the sea level (Mouterde, 1983).As a result of the favorable conditions, 19 species of genus Salvia are found in the Syrian flora (Tohme and Tohme, 2002).Although S. officinalis has many different uses, essentially, it has been used as herbal remedy for a wide range of disorders and illnesses by applying it either internally or externally.It is employed as diuretic, tonic, menstruation's promoter, local styptic, antiseptic, antiinflammatory, antifungal and spasmodic pain relief (Ioannides, 2002).It is also used as treatment for dysentery, coughing, indigestion, ulcer, varicose veins, insect bites (Dweck, 2000;Izzo, 2005).On the other hand, it is used for treating nervous conditions, trembling, should be used carefully since large doses can be toxic (Jellin et al., 2000).The properties of S. officinalis were depression and to mitigate aging symptoms (Scholey et al., 2008).Moreover, it has a lot of cosmetic uses such as skin and hair care (Barnes and Phillipson, 2007).But in spite of its efficiency in herbal therapy, S. officinalis believed to be due to its volatile essential oil, whereas S. officinalis is considered to have the highest amount of this oil when compared to the other species within the genus Salvia (Giannouli and Kintzios, 2000).
The essential oil of S. officinalis has low viscosity and sharp herbal smell; the olfactive qualities of this oil are due to existence of caryophyllene (Chalcat et al., 1998).The content and composition of S. officinalis essential oil changes according to the surrounding ecological factors as well as to the plant's development stages (Perry et al., 1999;Santos-Gomes and Fernandes-Ferreira, 2001;Maksimovic et al., 2007).
This research aimed to investigate the composition of the essential oil extracted from S. officinalis grown in Syria; to evaluate the anti-microbial activities of S. officinalis oil against some Gram-positive and Gramnegative bacteria.

Study locations and preparation of plant materials
Samples of S. officinalis were collected from two sites in the Syrian coastline; Al-bahlolia (100 m) and Al-Hafa (500 m).Collecting process took place before and after plant's blossoming in June, July, August and September.
Fresh green leaves were collected in clean polythene bags and codified as follows: 'S1' leaves collected from Al-bahlolia, 'S2' leaves collected from Al-Hafa.Each set, consisting of six leaves mixed together, were washed with distilled water, dried at room temperature until the water content dropped to 4.8%.The weight of each sample was 40 g.

Extraction of essential oil
The air-dried samples were crushed, and then subjected to hydrodistillation for three hours by using Clevenger-type apparatus according to the standard procedure described in the European Pharmacopoeia (1997).In order to eliminate all water, the extracted oils were treated with anhydrous sodium sulphate (Na2SO4), then filtered and kept in the dark at 4°C until tested and analyzed.

Gas chromatography (GC) analysis conditions
Separating and analyzing the essential oil of S. officinalis samples was done by a Shimadzu GC-17A system equipped with fused silica capillary column (30 m × 0.25 mm), coated with 0.25 µm film Rtx-5MS.The injector and detector temperatures were set at 250 and 280°C, respectively.The applied oven temperature program was: 40°C for 5 min, rising at 4°C/min to 100°C, rising at 19°C/min to 280°C and held for 5 min.The control mode is split and split ratio 5:10.Carrier gas was helium with flow-rate of 1.5 ml/min.The mass spectra were recorded over a range of 30 to 1000 atomic mass unit at 0.5 s/scan.Solvent cut time was 3 min.Ionization energy was 70 eV.The inlet and ionization source temperature were 280°C.

Microbial strains
A panel of six microorganisms was used to access the antimicrobial activities of S. officinalis essential oil.Testing for all microbial strains was performed first by pre-culturing inocula of Escherichia coli O157:H7, Pseudomonas aeruoginosa (ATCC 27853), Salmonella typhi O:9,12, Vi -H: d, Staphylococcus aureus (ATCC 25923), Streptococcus group D and Candida albicans (ATCC 10239) in the following selective media: Nutrient broth, Mannitol agar, Salmonella Shigella (SS) agar, Mac Conkey agar, nutrient agar 1.5%, Bile-Esculine agar Kligler iron agar and Mueller Hinton agar (Biolife, Milano, Italy).The dishes were incubated overnight at 37°C in the case of the bacteria, while C. albicans was incubated overnight at 30°C.

Disc diffusion method
Only one essential oil specimen was chosen for the antimicrobial test.This choice was based on the more total components and the higher percentage of certain components such as 1,8-cineole and borneol.As a result, the essential oil extracted from plants grown at 100 m above the sea level was used in this study.
Petri dishes were kept for 2 h at 4°C, and then incubated for 24 h at 37°C in the case of bacteria, while the yeast was incubated at 30°C.Antimicrobial activity was assessed by measuring the diameter of the inhabitation area (including disc diameter of 6 mm) of the microorganism strain tested with the oil which was measured by a ruler and the number of germs inhibited by each antibiotic agent was calculated according to the following equation: Where, n is the number of inhibited germs/ml; a is the area of the inhibition disc (πr²) in cm²; N is the number of germs per ml; A is the area of the Petri dish (πR²) in cm².In order to have valid and reliable results, each test was repeated three times to eliminate any error.

Analysis of S. officinalis essential oil
Altitude had an impact on the essential oil's yield as it was 0.52 and 0.14% at altitudes 100 and 500 m, respectively.Moreover, altitude had effects on the quantitative and qualitative composition of the essential oil.The total components of oil extracted from plants collected at 100 and 500 m were 45 and 30, respectively; sabinene, α-terpinene and terpinolene only existed in essential oil extracted from plants collected at 500 m, while α-terpineol, geranyl acetate, phytol and farnesol existed only in essential oil extracted from plants collected at 100 m (Table 1).Table 1 revealed that the essential oils extracted from 100 and 500 m had the same major constituents, but their percentages differed due to the altitude.The major component in both oils was 1,8-cineol (62-55%), followed by camphor (8 to 10%), borneol (5 to 4.5%), α-pinene (3.7 to 4.5%), β-pinene (6 to 5.2%), camphene (2.6 to 5%), β-myrcene (3 to 3.5%), caryophyllene (2 to 1%); while α and β-thujone were in low concentration (0.72 to 1.5%).The concentrations of the major constituents in the essential oil of S. officinalis observed in this study were different from those obtained by Grella and Picci (1988), Marino et al. (2001) and Tucker et al. (1980).These differences can be attributed

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to the fact that essential oils are heterogeneous and their quality and quantity vary with the growth stages, ecological conditions and the extraction method (Kim et al., 1995;Ozcan and Erkmen, 2001).

Antimicrobial activities of S. officinalis essential oil
Many researches reported that the antimicrobial activities of the essential oils were due to the presence of some major and minor constituents.Ulubelen et al. (1994) observed strong antimicrobial activities of the essential oil of S. sclarea and attributed them to the existence of caryophyllene.Dorman and Deans (2000) stated that the minor components of essential oil such as a-pinene and borneol have antimicrobial activities.Also the antimicrobial effects of borneol were also reported by Vardar-Unlu et al. (2003).1,8-cineole and camphor are well-known chemicals that possess antifungal as well as antibacterial activities (Jalsenjak et al., 1987;Sur et al., 1991;Tzakou et al., 2001).
The essential oil extracted from plants collected at 100 m was used in this study as it had more total component than that of 500 m.Moreover, it had higher percentage of 1,8-cineol and borneol.The essential oil showed antimicrobial activities against the test microorganisms used in this study.The results presented in Table 2 shows that the essential oil of S. officinalis inhibited the growth of Gram-positive bacteria (S. aureus and Streptococcus group D) completely at concentration of 20 µl/ml with the minimum time of contact.At the lowest concentration (5 µl/ml), the inhabitation started after 10 min of contact and increased with time and concentration.
The results presented in Table 3 revealed that gramnegative bacteria (E.coli, S. typhi and P. aeruoginosa) were temporary bacteriostatic in relation to contact time and oil concentration: 1.In spite of the inhibitory effect of the essential oil against E. coli, the bacteria resumed their growth after 24 h at concentrations of 5 and 10 µl/ml.But the inhabitation was definitive at high oil concentration.2. Regarding S. typhi, the bacteria growth was inhibited after 1 h of contact with concentrations of 5 and 10 µl/ml, but the bacteria resumed their growth after 24 h even when the highest concentration was used.3.In the case of P. aeruginosa which was proven to be resistant bacteria to S. officinalis essential oil, the number of the inhibited bacteria after 10 min and 1 h of contact at any concentration of the essential oil was limited, and the optimum growth was reached after 24 h.
It was observed from this study that the antimicrobial activity of the essential oil was more definite against Gram-positive than against Gram-negative bacteria, that was also verified by Nostro et al. (2000).The higher resistance among Gram-negative bacteria might be due     to the presence of phospholipidic membrane which limits the effect of oil on the cell membrane (Nikaido and Vaara, 1985).Table 4 shows that C. albicans, was the most susceptible to the essential oil among all the test microorganisms.The inhibition was total and definitive within a minimum time of contact and a minimum concentration.Although the concentration of C. albicans was the highest, the remaining number of yeast after the inhabitation test was zero.This remarkable activity against C. albicans can be attributed to pinene (Dorman and Deans, 2000).
Results in Table 5 revealed that the numbers of bacteria inhibited by the essential oil were: E. coli (100), S. typhi (294), P. aeruginosa (18), S. aureus (470) and Streptococcus group D (44).The efficiency of the anti-microbial against the used bacteria were as follows: The highest efficiency among the anti-bacterial discs used was by chloramphenicol (30 µg) for E. coli; sulphamethoxazole (25 µg) and gentamicine (10 µg) for S. typhi, chloramphenicol (30 µg) for S. aureus; ampiciline (10 µg), chloramphenicol (30 µg) and sulphamethoxazole (25 µg) for Streptococcus group D; while P. aeruginosa was resistant to all antibiotics, it was obvious that the antibacterial activity of S. officinalis had strongly exceeded that of the usual antibiotics which inhibited the same numbers of bacteria but with the higher concentrations mentioned earlier.

Conclusion
The essential oil profile of S. officinalis was proven to be affected by the ecological and seasonal factors.But despite all factors, the predominance components were 1,8-cineol, camphor, borneol, α-pinene, β-pinene, camphene and β-myrcene and caryophyllene.The essential oil of S. officinalis proved to have antibacterial activity against Gram-positive and negative bacteria.This activity was more obvious against Gram-positive than negative bacteria, which might be due the existence of the outer phospholipid membrane of the gram-negative bacteria.In comparison with the commercialized antibiotics, the essential oil exhibited a better efficiency, especially against resistant bacteria to antibiotics.This made this essential oil a good alternative to the traditional antibiotics as well as food preservatives.

Table 1 .
Essential oil composition (% of major components) of S. officinalis collected in Syria.
*Compounds of essential oil extracted from fresh green leaves and flowering top.**S.officinalis L.: collected at 100 m above the sea level; ***S.officinalis L.: collected at 500 m above the sea level.

Table 2 .
Minimal and maximal inhibitory concentrations of S. officinalis essential oil against Gram-positive bacteria.

Table 3 .
Minimal and maximal bacteriostatic concentraions of S. officinalis essential oil against Gram-negative bacteria.

Table 4 .
Inhibitory concentrations of S. officinalis essential oil against yeast (C.albicans).

Table 5 .
Minimum inhibitory concentration (MIC) of S. officinalis essential oil and anti-microbial discs against Gram-negative and positive bacteria.